U.S. patent application number 13/875066 was filed with the patent office on 2014-08-14 for peptides and methods for inducing cell death.
The applicant listed for this patent is Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Invention is credited to Eric P. BRASS, Michael R. YEAMAN, Nannette Y. YOUNT.
Application Number | 20140227767 13/875066 |
Document ID | / |
Family ID | 44483620 |
Filed Date | 2014-08-14 |
United States Patent
Application |
20140227767 |
Kind Code |
A2 |
YEAMAN; Michael R. ; et
al. |
August 14, 2014 |
PEPTIDES AND METHODS FOR INDUCING CELL DEATH
Abstract
The invention provide isolated peptides, protides and conjugates
having novel peptide sequences which are able to induce
antimicrobial, anti-cancer, anti-inflammatory, anti-proliferative
or programmed cell death activity. The invention also provides a
method of inducing programmed cell death in a cell by contacting
the cell with an isolated peptide, protide or conjugate described
herein. In some aspects, the method can be used in the diagnosis,
prevention, or treatment of a disease, such as an infection,
cancer, autoimmune disease, or inflammatory disease.
Inventors: |
YEAMAN; Michael R.; (Redondo
Beach, CA) ; YOUNT; Nannette Y.; (San Juan
Capistrano, CA) ; BRASS; Eric P.; (Palos Verdes,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical
Center |
Torrance |
CA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20140065695 A1 |
March 6, 2014 |
|
|
Family ID: |
44483620 |
Appl. No.: |
13/875066 |
Filed: |
May 1, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12932298 |
Feb 22, 2011 |
|
|
|
13875066 |
|
|
|
|
61338747 |
Feb 22, 2010 |
|
|
|
Current U.S.
Class: |
435/252.1 ;
435/254.1; 530/324; 530/326 |
Current CPC
Class: |
A61P 31/00 20180101;
C12N 7/00 20130101; C07K 14/4747 20130101; A01N 47/44 20130101;
C07K 14/005 20130101; A61K 47/6415 20170801; C12N 2795/10031
20130101; A61P 35/00 20180101; A61P 29/00 20180101; C07K 14/47
20130101 |
Class at
Publication: |
435/252.1 ;
435/254.1; 530/324; 530/326 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Claims
1. An isolated peptide comprising one or more amino acid sequence
selected from the group consisting of SEQ ID NOS: 3, 4, 6, 8, 10,
11, 13, 17, 18, 19, 21-25, 30, 31-36, 39-47, 49-52, 54-57, 59-63,
66-75, 84-93, 102-106, 108-121, 132-175, 179-187, 191-199, 205-209,
211-223, 227-235, 238-243, 245-247, 249-251, 253-256 and 260-263,
wherein the amino acid residue represented by (x) is a serine, a
threonine, a tryptophan, a H-bond donor residue or a H-bond
acceptor residue, wherein the amino acid residue represented by (b)
is a lysine, an arginine, an asparagine, a glutamine or a basic
residue, wherein the amino acid residue represented by (j) is a
cysteine or a thiol residue, wherein in the amino acid residue
represented by (o) is an anthrylalanine or other non-natural amino
acid and wherein said peptide induces antimicrobial, anti-cancer,
anti-inflammatory, anti-proliferative or programmed cell death
activity.
2. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS: 3,
4, 6, 8, 10, 11, 13, 264, 270 and 271.
3. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS: 17,
18, 19, 21-25, 269 and 272.
4. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS: 30,
31-36 and 273.
5. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
39-47.
6. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
49-52.
7. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
54-57.
8. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
59-63 and 274.
9. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
66-75.
10. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
84-93 and 275.
11. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
102-106, 108-121, 267, 276 and 277.
12. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
132-175.
13. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
179-187, 266 and 278.
14. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NO:
191-199.
15. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
205-209 and 211-223.
16. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
227-235.
17. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
238-243, 245-247, 265 and 279.
18. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
249-251.
19. The isolated protein of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
253-256.
20. The isolated peptide of claim 1 comprising one or more amino
acid sequence selected from the group consisting of SEQ ID NOS:
260-263 and 268.
21. An isolated peptide consisting of an amino acid sequence
selected from the group consisting of SEQ ID NOS: 1, 2, 5, 7, 9,
12, 14-16, 20, 26-29, 37, 38, 48, 53, 58, 64, 65, 72, 76-83,
94-101, 107, 114, 122-131, 170, 176-178, 188-190, 200-204, 210,
224-226, 236, 237, 244, 248, 252, 257-259 and 288-289, wherein said
peptide induces antimicrobial, anti-cancer, anti-inflammatory,
anti-proliferative or programmed cell death activity.
22. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS: 1, 2, 5,
7, 9 and 12.
23. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS: 14-16
and 20.
24. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS:
26-29.
25. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from SEQ ID NOS: 37 or 38.
26. The isolated peptide of claim 21 consisting of an amino acid
sequence of SEQ ID NO: 48.
27. The isolated peptide of claim 21 consisting of an amino acid
sequence of SEQ ID NO: 53.
28. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from SEQ ID NOS: 58 or 64.
29. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS: 65 or
72.
30. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS:
76-83.
31. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS: 94-101,
107 and 114.
32. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS: 122-131
and 170.
33. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS:
176-178.
34. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS:
188-190.
35. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS: 200-204
and 210.
36. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS:
224-226.
37. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS: 236, 237
and 244.
38. The isolated peptide of claim 21 consisting of an amino acid
sequence of SEQ ID NO: 248.
39. The isolated peptide of claim 21 consisting of an amino acid
sequence of SEQ ID NO: 252.
40. The isolated peptide of claim 21 consisting of an amino acid
sequence selected from the group consisting of SEQ ID NOS: 257-259
and 288-289.
41. The isolated peptide of any one of claims 1-40, wherein the
C-terminus of the peptide comprises a carboxamide.
42. A context-activated protide comprising at least one activator
site and two or more effectors, wherein at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 3, 4, 6, 8, 10, 11, 13, 17, 18, 19, 21-25, 30,
31-36, 39-47, 49-52, 54-57, 59-63, 66-75, 84-93, 102-106, 108-121,
132-175, 179-187, 191-199, 205-209, 211-223, 227-235, 238-243,
245-247, 249-251, 253-256 and 260-263, wherein the amino acid
residue represented by (x) is a serine, a threonine, a tryptophan,
a H-bond donor residue or a H-bond acceptor residue, wherein the
amino acid residue represented by (b) is a lysine, an arginine, an
asparagine, a glutamine or a basic residue, wherein the amino acid
residue represented by (j) is a cysteine or a thiol residue,
wherein the amino acid residue represented by (o) is an
anthrylalanine or other non-natural amino acid and wherein said at
least one effector induces antimicrobial, anti-cancer,
anti-inflammatory, anti-proliferative or programmed cell death
activity.
43. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 3, 4, 6,
8, 10, 11, 13, 264, 270 and 271.
44. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 17, 18,
19, 21-25, 269 and 272.
45. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 30,
31-36 and 273.
46. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
39-47.
47. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
49-52.
48. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
54-57.
49. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 59-63
and 274.
50. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
66-75.
51. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 84-93
and 275.
52. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 102-106,
108-121, 267, 276 and 277.
53. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
132-175.
54. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 179-187,
266 and 278.
55. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NO:
191-199.
56. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 205-209
and 211-223.
57. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
227-235.
58. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 238-243,
245-247, 265 and 279.
59. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
249-251.
60. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
253-256.
61. The protide of claim 42 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 260-263
and 268.
62. A context-activated protide comprising at least one activator
site and two or more effectors, wherein at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 1, 2, 5, 7, 9, 12, 14-16, 20, 26-29, 37, 38, 48, 53,
58, 64, 65, 72, 76-83, 94-101, 107, 114, 122-131, 170, 176-178,
188-190, 200-204, 210, 224-226, 236, 237, 244, 248, 252, 257-259
and 288-289, wherein said at least one effector induces
antimicrobial, anti-cancer, anti-inflammatory, anti-proliferative
or programmed cell death activity.
63. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 1, 2, 5, 7, 9 and 12.
64. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 14-16 and 20.
65. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 26-29.
66. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from SEQ ID NOS: 37 or
38.
67. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence of SEQ ID NO: 48.
68. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence of SEQ ID NO: 53.
69. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from SEQ ID NOS: 58 or
64.
70. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 65 or 72.
71. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 76-83.
72. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 94-101, 107 and 114.
73. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 122-131 and 170.
74. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 176-178.
75. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 188-190.
76. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 200-204 and 210.
77. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 224-226.
78. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 236, 237 and 244.
79. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence of SEQ ID NO: 248.
80. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence of SEQ ID NO: 252.
81. The protide of claim 62, wherein said at least one effector
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOS: 257-259 and 288-289.
82. The protide of any one of claims 42-81, wherein said activator
site is context-activated.
83. The protide of any one of claims 42-81, wherein
context-activation initiates programmed cell death.
84. The protide of any one of claims 42-81, wherein said
context-activation results from a physiological condition.
85. The protide of claim 84, wherein said physiological condition
is selected from the group consisting of acidity, alkalinity, ionic
strength and osmotic strength.
86. The protide of any one of claims 42-81, wherein said
context-activation results from association with an activator
molecule.
87. The protide of claim 86, wherein said activator molecule
modifies said activator site upon association.
88. The protide of claim 87, wherein said modification comprises
cleavage of said activator site.
89. The protide of claim 86, wherein said activator molecule is an
enzyme.
90. The protide of claim 89, wherein said activator molecule is
selected from the group consisting of protease, esterase and
lipase.
91. The protide of claim 90, wherein said activator molecule is a
protease.
92. The protide of claim 91, wherein said activator is expressed by
a pathogenic microorganism.
93. The protide of claim 92, wherein said pathogenic microorganism
is Staphylococcus aureus, Escherichia coli, Salmonella typhimurium,
Pseudomonas aeruginosa, Bacillus subtilis, Acinotobacter baumannii,
Acinotobacter calcoaceticus, Acinotobacter haemolyticus or Candida
albicans.
94. The protide of claim 86, wherein said activator is present in
the context of tumor cells.
95. The protide of claim 94, wherein said activator is a
tumor-specific protease.
96. The protide of claim 95, wherein said tumor-specific protease
is matrix bound.
97. The protide of claim 96, wherein said tumor-specific protease
is a matrix metalloproteinase.
98. The protide of claim 86, wherein said activator is present in
the context of an inflammatory response.
99. The protide of claim 86, wherein said activator is a peptide
selected from the group consisting of thrombin, bradykinin,
elastase and metalloproteinase.
100. A conjugate comprising one or more amino acid sequence
selected from the group consisting of SEQ ID NOS: 3, 4, 6, 8, 10,
11, 13, 17, 18, 19, 21-25, 30, 31-36, 39-47, 49-52, 54-57, 59-63,
66-75, 84-93, 102-106, 108-121, 132-175, 179-187, 191-199, 205-209,
211-223, 227-235, 238-243, 245-247, 249-251, 253-256 and 260-263
and a moiety, wherein the amino acid residue represented by (x) is
a serine, a threonine, a tryptophan, a H-bond donor residue or a
H-bond acceptor residue, wherein the amino acid residue represented
by (b) is a lysine, an arginine, an asparagine, a glutamine or a
basic residue, wherein the amino acid residue represented by (j) is
a cysteine or a thiol residue, wherein in the amino acid residue
represented by (o) is an anthrylalanine or other non-natural amino
acid and wherein said conjugate induces antimicrobial, anti-cancer,
anti-inflammatory, anti-proliferative or programmed cell death
activity.
101. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 3, 4, 6,
8, 10, 11, 13, 264, 270 and 271.
102. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 17, 18,
19, 21-25, 269 and 272.
103. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 30,
31-36 and 273.
104. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
39-47.
105. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
49-52.
106. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
54-57.
107. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 59-63
and 274.
108. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
66-75.
109. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 84-93
and 275.
110. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 102-106,
108-121, 267, 276 and 277.
111. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
132-175.
112. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 179-187,
266 and 278.
113. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NO:
191-199.
114. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 205-209
and 211-223.
115. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
227-235.
116. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 238-243,
245-247, 265 and 279.
117. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
249-251.
118. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS:
253-256.
119. The conjugate of claim 100 comprising one or more amino acid
sequence selected from the group consisting of SEQ ID NOS: 260-263
and 268.
120. A conjugate comprising one or more amino acid sequence
selected from the group consisting of SEQ ID NOS: 1, 2, 5, 7, 9,
12, 14-16, 20, 26-29, 37, 38, 48, 53, 58, 64, 65, 72, 76-83,
94-101, 107, 114, 122-131, 170, 176-178, 188-190, 200-204, 210,
224-226, 236, 237, 244, 248, 252, 257-259 and 288-289 and a moiety,
wherein said conjugate induces antimicrobial, anti-cancer,
anti-inflammatory, anti-proliferative or programmed cell death
activity.
121. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 1, 2, 5, 7, 9 and
12.
122. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 14-16 and 20.
123. The conjugate of claim 120 comprising of an amino acid
sequence selected from the group consisting of SEQ ID NOS:
26-29.
124. The conjugate of claim 120 comprising an amino acid sequence
selected from SEQ ID NOS: 37 or 38.
125. The conjugate of claim 120 comprising an amino acid sequence
of SEQ ID NO: 48.
126. The conjugate of claim 120 comprising an amino acid sequence
of SEQ ID NO: 53.
127. The conjugate of claim 120 comprising an amino acid sequence
selected from SEQ ID NOS: 58 or 64.
128. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 65 or 72.
129. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 76-83.
130. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 94-101, 107 and
114.
131. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 122-131 and
170.
132. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 176-178.
133. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 188-190.
134. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 200-204 and
210.
135. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 224-226.
136. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 236, 237 and
244.
137. The conjugate of claim 120 comprising an amino acid sequence
of SEQ ID NO: 248.
138. The conjugate of claim 120 comprising an amino acid sequence
of SEQ ID NO: 252.
139. The conjugate of claim 120 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS: 257-259 and
288-289.
140. The conjugate of any one of claims 100-139, wherein said
moiety comprises a therapeutic agent, a targeting peptide or a
label.
141. The conjugate of claim 140, wherein said therapeutic agent is
a cytotoxic agent.
142. The conjugate of claim 141, wherein said cytotoxic agent is an
antibiotic.
143. The conjugate of claim 140, wherein said therapeutic agent is
a chemotherapeutic agent.
144. The conjugate of claim 140, wherein said targeting peptide
selectively homes said conjugate to a microorganism.
145. The conjugate of claim 140, wherein said targeting peptide
selectively homes said conjugate to tumor tissue, tumor cell, or
tumor vasculature.
146. The conjugate of claim 140, wherein said targeting peptide
selectively homes said conjugate to an immune regulatory cell or an
immune effector cell.
147. The conjugate of claim 140, wherein said targeting peptide is
an antibody or a fragment thereof.
148. The conjugate of claim 140, wherein said label is a
radioisotope.
149. The conjugate of claim 140, wherein said label is a dye.
150. A method of inducing programmed cell death in a cell,
comprising contacting said cell with the isolated peptide of any
one of claims 1-40.
151. The method of claim 150, wherein said cell is a
microorganism.
152. The method of claim 151, wherein said microorganism is a
pathogenic microorganism.
153. The method of claim 152, wherein said pathogenic microorganism
is selected from the group consisting of Staphylococcus aureus,
Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa,
Bacillus subtilis, Acinotobacter baumannii, Acinotobacter
calcoaceticus, Acinotobacter haemolyticus, Pseudomonas aeruginosa
and Candida albicans.
154. The method of claim 150, wherein said cell is a tumor
cell.
155. The method of claim 154, wherein said tumor cell is a
malignant tumor cell.
156. The method of claim 150, wherein said cell is an immune
regulatory cell or an immune effector cell.
157. A method of inducing programmed cell death in a cell,
comprising contacting said cell with the protide of any one of
claims 62-81.
158. The method of claim 157, wherein said cell is a
microorganism.
159. The method of claim 158, wherein said microorganism is a
pathogenic microorganism.
160. The method of claim 159, wherein said pathogenic microorganism
is selected from the group consisting of Staphylococcus aureus,
Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa,
Bacillus subtilis, Acinotobacter baumannii, Acinotobacter
calcoaceticus, Acinotobacter haemolyticus, Pseudomonas aeruginosa
and Candida albicans.
161. The method of claim 157, wherein said cell is a tumor
cell.
162. The method of claim 161, wherein said tumor cell is a
malignant tumor cell.
163. The method of claim 157, wherein said cell is an immune
regulatory cell or an immune effector cell.
164. A method of inducing programmed cell death in a cell,
comprising contacting said cell with the conjugate of any one of
claims 100-139.
165. The method of claim 164, wherein said cell is a
microorganism.
166. The method of claim 165, wherein said microorganism is a
pathogenic microorganism.
167. The method of claim 166, wherein said pathogenic microorganism
is selected from the group consisting of Staphylococcus aureus,
Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa,
Bacillus subtilis, Acinotobacter baumannii, Acinotobacter
calcoaceticus, Acinotobacter haemolyticus, Pseudomonas aeruginosa
and Candida albicans.
168. The method of claim 164, wherein said cell is a tumor
cell.
169. The method of claim 168, wherein said tumor cell is a
malignant tumor cell.
170. The method of claim 164, wherein said cell is an immune
regulatory cell or an immune effector cell.
Description
[0001] This application is a continuation of U.S. Non-provisional
application Ser. No. 12/932,298, filed Feb. 22, 2011, which claims
the benefit of priority of U.S. Provisional application Ser. No.
61/338,747, filed Feb. 22, 2010, the entire contents of which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to compositions and
methods for inducing cell death, and more specifically to peptides
and compositions having antimicrobial, anti-cancer,
anti-inflammatory and/or anti-proliferative activity and methods of
using the peptides and compositions as therapeutics.
BACKGROUND OF THE INVENTION
[0003] Programmed cell death pathways are known to exist in most if
not all organisms on Earth, ranging from microbes to man. Proteins
that effect this function, also known as apoptosis, have been
identified in human, other mammals, plants, protozoa, fungi, and
bacteria, among other forms of life. In humans, these proteins
target the mitochondria, causing permeabilization, dissipation of
the membrane potential, activation of intracellular signaling
pathways, and ultimate death of the cell. Eukaryotic pathogens also
contain mitochondria, and mitochondria are now widely accepted by
evolutionary biologists to be decedents of specialized symbiotic
bacteria in eukaryotic cells.
[0004] Given these close parallels between mitochondria and
bacteria, it is contemplated that specific human, eukaryotic or
prokaryotic proteins have necessarily evolved to control
prokaryotic symbionts (eg. mitochondria, chloroplasts) or
competitors, and directly or indirectly prompt death of microbes or
infected or abnormal cells. These types of proteins exhibit
similarities in structures (eg., cationic helical domains) and
mechanisms of action (eg., membrane interaction or perturbation
that can lead to programmed cell death). Thus, such proteins may
serve as excellent templates for novel therapeutic molecules, and
reveal new insights into host-pathogen co-evolution, cancer
biology, and other disease prevention, pathogenesis and
treatment.
SUMMARY OF INVENTION
[0005] Embodiments of the invention provide isolated peptides,
protides and conjugates having novel peptide sequences which are
able to induce antimicrobial, anti-cancer, anti-inflammatory,
anti-proliferative or programmed cell death activity. Peptides,
protides and conjugates provided by the invention comprise, consist
essentially of, or consist of amino acid sequences represented by
SEQ ID NOS:1-263 and 288-289. In some aspects, peptides, protides
and conjugates described herein have conservative amino acid
substitutions or alternative residues at specific locations within
a peptide sequence. Non-limiting examples of such substitutions or
alternative residues include when the amino acid residue is
represented by (x) a serine, a threonine, a tryptophan, a H-bond
donor residue or a H-bond acceptor residue can be substituted, or
alternatively, when the amino acid residue is represented by (b) a
lysine, an arginine, an asparagine, a glutamine or a basic residue
can be substituted, or alternatively, when the amino acid residue
is represented by (j) a cysteine or a thiol residue can be
substituted, or alternatively, when the amino acid residue is
represented by (o) an anthrylalanine or other non-natural amino
acid can be substituted.
[0006] Embodiments of the invention also provide methods of
inducing programmed cell death in a cell by exposing the cell to an
isolated peptide, protide or conjugate described herein. In some
aspects, the methods can be used in the diagnosis, prevention, or
treatment of a disease, such as an infection, cancer, autoimmune
disease, or inflammatory disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0008] FIG. 1 shows the amino acid sequence of exemplary programmed
cell death/holin-like proteins (Dnm-1 (SEQ ID NO:280), Bax (SEQ IN
NO:281) and Bcl-2 (SEQ ID NO:282)) identified in Homo sapiens.
[0009] FIG. 2 shows the amino acid sequence of exemplary programmed
cell death proteins (CidA (SEQ ID NO:283) and LrgA (SEQ IN NO:284))
identified in Staphylococcus aureus.
[0010] FIG. 3 shows the amino acid sequence of exemplary candidate
proteins (Perforin 1 from Bos taurus (SEQ ID NO:285), Bcl-2 from
Homo sapiens (SEQ ID NO:286), and BCL-W from Homo sapiens (SEQ ID
NO:287)) used for the iterative primary structure analysis of the
protein databases (Blastp and/or equivalent thereof) available from
the National Center for Biotechnology Information utilizing the
basic local alignment sequence tool (BLAST).
[0011] FIG. 4 shows an exemplary data score table sorted by
alignment score using the multisequence alignment tool ClustalW
(Larkin et al., Bioinformatics 23(21): 2947-2948 (2007)) available
online from EMBL-EBI.
[0012] FIG. 5 shows exemplary multisequence alignments using the
multisequence alignment tool ClustalW (Larkin et al.,
Bioinformatics 23(21): 2947-2948 (2007)) available online from
EMBL-EBI.
[0013] FIG. 6 shows a phylogram of candidate programmed cell death
effector peptides using the multisequence alignment tool ClustalW
(Larkin et al., Bioinformatics 23(21): 2947-2948 (2007)) available
online from EMBL-EBI.
[0014] FIG. 7 shows a similarity alignment of helical region 1
(amino acids-.about.450-490) between candidate peptides identified
in the phylogram of FIG. 6 using the multisequence alignment tool
ClustalW (Larkin et al., Bioinformatics 23(21): 2947-2948 (2007))
available online from EMBL-EBI.
[0015] FIG. 8 shows a similarity alignment of helical region 2
(amino acids-.about.540-560) between candidate peptides identified
in the phylogram of FIG. 6 using the multisequence alignment tool
ClustalW (Larkin et al., Bioinformatics 23(21): 2947-2948 (2007))
available online from EMBL-EBI.
[0016] FIG. 9 shows a similarity alignment of helical region 3
(amino acids-.about.590-620) between candidate peptides identified
in the phylogram of FIG. 6 using the multisequence alignment tool
ClustalW (Larkin et al., Bioinformatics 23(21): 2947-2948 (2007))
available online from EMBL-EBI.
[0017] FIG. 10 shows a cladogram of candidate programmed cell death
effector molecule subset 1 using the multisequence alignment tool
ClustalW (Larkin et al., Bioinformatics 23(21): 2947-2948 (2007))
available online from EMBL-EBI.
[0018] FIG. 11 shows a similarity alignment of helical region
1/subset 1 (amino acids-.about.180-225) between candidate
programmed cell death effector molecules identified in the
cladogram of FIG. 10 using the multisequence alignment tool
ClustalW (Larkin et al., Bioinformatics 23(21): 2947-2948 (2007))
available online from EMBL-EBI.
[0019] FIG. 12 shows a similarity alignment of helical region
2/subset 1 (amino acids-.about.270-290) between candidate
programmed cell death effector molecules identified in the
cladogram of FIG. 10 using the multisequence alignment tool
ClustalW (Larkin et al., Bioinformatics 23(21): 2947-2948 (2007))
available online from EMBL-EBI.
[0020] FIG. 13 shows a similarity alignment of helical region
3/subset 1 (amino acids-.about.320-360) between candidate
programmed cell death effector molecules identified in the
cladogram of FIG. 10 using the multisequence alignment tool
ClustalW (Larkin et al., Bioinformatics 23(21): 2947-2948 (2007))
available online from EMBL-EBI.
[0021] FIG. 14 shows a similarity alignment of helical region
4/subset 1 (amino acids-.about.490-530) between candidate
programmed cell death effector molecules identified in the
cladogram of FIG. 10 using the multisequence alignment tool
ClustalW (Larkin et al., Bioinformatics 23(21): 2947-2948 (2007))
available online from EMBL-EBI.
[0022] FIG. 15 shows a secondary structure diagram of human Bcl-2,
isoform 1 or 2. Cylinders represent alpha helices. The colors are
green for alpha helices, orange for beta strands, and blue for
coils. The arrows on the helix cylinders point in the N-terminal to
C-terminal direction. The amino acid sequence NREIVMKYIHYKLS
(residues 1-14 of SEQ ID NO:48) of a peptide predicted to have
antimicrobial activity is shown in yellow.
[0023] FIG. 16 shows a secondary structure diagram of human Bcl-2,
isoform 1 or 2. Cylinders represent alpha helices. The colors are
green for alpha helices, orange for beta strands, and blue for
coils. The arrows on the helix cylinders point in the N-terminal to
C-terminal direction. The amino acid sequence HLALRQAGDDFSRRYR
having a peptide predicted to have antimicrobial activity (SEQ ID
NO:53) is shown in yellow.
[0024] FIG. 17 shows a secondary structure diagram of human Bcl-xL.
Cylinders represent alpha helices. The colors are green for alpha
helices, orange for beta strands, and blue for coils. The arrows on
the helix cylinders point in the N-terminal to C-terminal
direction. The amino acid sequence SQSNRELVVDFLSYKLSQK (SEQ ID
NO:288) of a peptide predicted to have antimicrobial activity is
shown in yellow. The amino acid sequence SQSNRELVVDFLSYKLSQK (SEQ
ID NO:288) has also been identified as being conserved in multiple
PCD-effector templates, including human Bcl-xL and Bcl-4, murine
Bcl-xy, and various related proteins.
[0025] FIG. 18 shows a secondary structure diagram of human Bcl-W.
Cylinders represent alpha helices. The colors are green for alpha
helices, orange for beta strands, and blue for coils. The arrows on
the helix cylinders point in the N-terminal to C-terminal
direction. The amino acid sequence TRALVADFVGYKLRQK (residues 1-16
of SEQ ID NO:14) of a peptide predicted to have antimicrobial
activity is shown in yellow.
[0026] FIG. 19 shows a secondary structure diagram of human Bax.
Cylinders represent alpha helices. The colors are green for alpha
helices, orange for beta strands, and blue for coils. The arrows on
the helix cylinders point in the N-terminal to C-terminal
direction. The amino acid sequence RVVALFYFASKLVLKALCTK (residues
1-20 of SEQ ID NO:7) of a peptide predicted to have antimicrobial
activity is shown in yellow.
[0027] FIG. 20 shows a secondary structure diagram of human CTL
Granulysin. Cylinders represent alpha helices. The colors are green
for alpha helices, orange for beta strands, and blue for coils. The
arrows on the helix cylinders point in the N-terminal to C-terminal
direction. The amino acid sequence RDYRTCLTIVQKLKKM having a
peptide predicted of have antimicrobial activity (residues 3-17 of
SEQ ID NO:224) is shown in yellow.
[0028] FIG. 21 shows secondary structure diagram of human CTL
Granulysin. Cylinders represent alpha helices. The colors are green
for alpha helices, orange for beta strands, and blue for coils. The
arrows on the helix cylinders point in the N-terminal to C-terminal
direction. The amino acid sequence QKLKKMVDKPTQRSVSN (SEQ ID
NO:289) of a peptide predicted to have antimicrobial activity is
shown in yellow.
[0029] FIG. 22 shows a secondary structure ribbon diagram of human
Bax (1F16) protein. Helix-1, residues 104-129 are represented in
red.
[0030] FIG. 23 shows a secondary structure ribbon diagram of human
Bax Helix-1, residues 104-129. Structure A shows the location of
positive residues in blue. Structure B shows the most hydrophilic
residues in blue and the most hydrophobic in brown.
[0031] FIG. 24 shows a secondary structure ribbon diagram of human
Bax (1F16) protein. Helix-2, residues 168-190 are represented in
red.
[0032] FIG. 25 shows a secondary structure ribbon diagram of human
Bax Helix-2, residues 168-190. Structure A shows the location of
positive residues in blue. Structure B shows the most hydrophilic
residues as represented in blue and the most hydrophobic residues
as represented in brown.
[0033] FIG. 26, panels A-D, show three dimensional alignments
between human Bax Helix-1, residues 104-129 vs. IL-8 helix,
residues 55-72. For the alignment analyses, comparative sequences
of X and Y length are entered, and the computation prioritizes
which span of those length are most comparable. Panel A shows a
sequence alignment based on the following structural alignment.
Panel B shows a horizontal view of a ribbon diagram alignment
between Bax, residues 104-129 (Red) and IL-8, residues 56-72
(Blue), whereas panel C shows an axial view of the same alignment.
Panel D shows the same ribbon alignment as panel B, wherein the
most hydrophilic residues are represented in blue and the most
hydrophobic residues are represented in brown. Also included in the
figure are the root mean square deviation (RMSD) score and other
results from the alignment.
[0034] FIG. 27, panels A-D, show three dimensional alignments
between human Bax helix, residues 168-190 vs. IL-8 helix, residues
55-72. For the alignment analyses, comparative sequences of X and Y
length are entered, and the computation prioritizes which span of
those length are most comparable. Panel A shows a sequence
alignment based on the following structural alignment. Panel B
shows a horizontal view of a ribbon diagram alignment between Bax,
residues 168-190 (Red) and IL-8, residues 55-72 (Blue), whereas
panel C shows an axial view of the same alignment. Panel D shows
the same ribbon alignment as panel B, wherein the most hydrophilic
residues are represented in blue and the most hydrophobic residues
are represented in brown. Also included in the figure are the root
mean square deviation (RMSD) score and other results from the
alignment.
[0035] FIG. 28, panels A-D, show three dimensional alignments
between human Bax helix, residues 104-129 vs. magainin residues
1-16. For the alignment analyses, comparative sequences of X and Y
length are entered, and the computation prioritizes which span of
those length are most comparable. Panel A shows a sequence
alignment based on the following structural alignment. Panel B
shows a horizontal view of a ribbon diagram alignment between Bax,
residues 104-129 (Red) and magainin, residues 1-16 (Blue), whereas
panel C shows an axial view of the same alignment. Panel D shows
the same ribbon alignment as panel B, wherein the most hydrophilic
residues are represented in blue and the most hydrophobic residues
are represented in brown. Also included in the figure are the root
mean square deviation (RMSD) score and other results from the
alignment.
[0036] FIG. 29, panels A-D, show three dimensional alignments
between human Bax helix, residues 168-190 vs. magainin residues
1-16. For the alignment analyses, comparative sequences of X and Y
length are entered, and the computation prioritizes which span of
those length are most comparable. Panel A shows a sequence
alignment based on the following structural alignment. Panel B
shows a horizontal view of a ribbon diagram alignment between Bax,
residues 168-190 (Red) and magainin, residues 1-16 (Blue), whereas
panel C shows an axial view of the same alignment. Panel D shows
the same ribbon alignment as panel B, wherein the most hydrophilic
residues are represented in blue and the most hydrophobic residues
are represented in brown. Also included in the figure are the root
mean square deviation (RMSD) score and other results from the
alignment.
[0037] FIG. 30 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4, LrgA-1-4 and CidA-II-12 against
pathogenic bacteria and fungi at pH 7.5. The size of the complete
zone of inhibition (ZOI) is represented by a blue bar, whereas the
size of the partial (ZOI) is represented by a red bar. See Tables
22 and 23 for peptide and microorganism designations.
[0038] FIG. 31 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4, LrgA-1-4 and CidA-II-12 against
pathogenic bacteria and fungi at pH 5.5. The size of the complete
zone of inhibition (ZOI) is represented by a blue bar, whereas the
size of the partial (ZOI) is represented by a red bar. See Tables
22 and 23 for peptide and microorganism designations.
[0039] FIG. 32 shows a histogram of the antimicrobial spectra of
exemplary peptides Dnm2-II-4, Dnm1-IV-2, Ncl-VIII-6 and Mfn1-II-2
against pathogenic bacteria and fungi at pH 7.5. The size of the
complete zone of inhibition (ZOI) is represented by a blue bar,
whereas the size of the partial (ZOI) is represented by a red bar.
See Tables 22 and 23 for peptide and microorganism
designations.
[0040] FIG. 33 shows a histogram of the antimicrobial spectra of
exemplary peptides Dnm2-II-4, Dnm1-IV-2, Ncl-VIII-6 and Mfn1-II-2
against pathogenic bacteria and fungi at pH 5.5. The size of the
complete zone of inhibition (ZOI) is represented by a blue bar,
whereas the size of the partial (ZOI) is represented by a red bar.
See Tables 22 and 23 for peptide and microorganism
designations.
[0041] FIG. 34 shows a histogram of the antimicrobial spectra of
exemplary peptides BclWP-1-4, Csp3-II-12, BclXb-1-2 and BaxP-1-18
against pathogenic bacteria and fungi at pH 7.5. The size of the
complete zone of inhibition (ZOI) is represented by a blue bar,
whereas the size of the partial (ZOI) is represented by a red bar.
See Tables 22 and 23 for peptide and microorganism
designations.
[0042] FIG. 35 shows a histogram of the antimicrobial spectra of
exemplary peptides BclWP-1-4, Csp3-II-12, BclXb-1-2 and BaxP-1-18
against pathogenic bacteria and fungi at pH 5.5. The size of the
complete zone of inhibition (ZOI) is represented by a blue bar,
whereas the size of the partial (ZOI) is represented by a red bar.
See Tables 22 and 23 for peptide and microorganism
designations.
[0043] FIG. 36 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Pseudomonas aeruginosa CRM27853 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0044] FIG. 37 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Pseudomonas aeruginosa PA 01 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0045] FIG. 38 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Pseudomonas aeruginosa XEN 5 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0046] FIG. 39 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii 19606 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0047] FIG. 40 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii 17978 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0048] FIG. 41 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter calcoaceticus 23055 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0049] FIG. 42 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter haemolyticus 17906 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0050] FIG. 43 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii HUMC #ATCC at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0051] FIG. 44 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii HUMC #1 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0052] FIG. 45 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii HUMC #6 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0053] FIG. 46 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii HUMC #12 at pH 5.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0054] FIG. 47 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Pseudomonas aeruginosa CRM27853 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0055] FIG. 48 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Pseudomonas aeruginosa PA 01 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0056] FIG. 49 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Pseudomonas aeruginosa XEN 5 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0057] FIG. 50 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii 19606 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0058] FIG. 51 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii 17978 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0059] FIG. 52 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter calcoaceticus 23055 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0060] FIG. 53 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter haemolyticus 17906 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0061] FIG. 54 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii HUMC #ATCC at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0062] FIG. 55 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii HUMC #1 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0063] FIG. 56 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii HUMC #6 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
[0064] FIG. 57 shows a histogram of the antimicrobial spectra of
exemplary peptides Hol-III-4 (SEQ ID NO. 268) and NC1-VIII-6 (SEQ
ID NO. 152) against Acinetobacter baumannii HUMC #12 at pH 7.5.
Positive control peptides RP1, 6W-RP1, IK and PMP-2, whereas the
negative control of double-distilled water (DDH2O) are also shown.
The size of the complete zone of inhibition (ZOI) is represented by
a blue bar, whereas the size of the partial (ZOI) is represented by
a red bar.
DETAILED DESCRIPTION OF THE INVENTION
[0065] Over the last several years, unforeseen structural,
functional, and evolutionary relationships among host defenses and
other proteins across all kingdoms of life have been discovered. In
the course of the studies described herein, unifying themes among
polypeptides based on sequence formulae, functional mechanisms,
and/or 3D structures have been identified. Embodiments provided by
the invention are based in part on the observations that 1)
programmed cell death and apoptosis pathway proteins contain
archetype sequences that confer membrane interacting/modifying
domains similar to those of antimicrobial or other host defense
peptides; 2) such sequences encode helical or other sequence and/or
3D structural signatures; and 3) such peptides exert antimicrobial
and anti-cancer cell activities. Without being bound by theory,
such peptides induce or regulate programmed cell death or related
responses in target cells (e.g. microbial pathogens, cancer cells,
etc.) leading to death of these cells. It is also contemplated that
the mechanisms of action of peptides based on programmed cell death
may activate archetypal apoptosis pathways in target cells, thus
killing the target cell and circumventing resistance to existing
antimicrobial, anti-cancer, or other preventive or therapeutic
agents.
[0066] Embodiments of the invention provide that novel
antimicrobial, anti-cancer, anti-inflammatory and/or
anti-proliferative activity peptides reside in peptide sequences of
programmed cell death effector proteins by virtue of the
evolutionary necessity for control of microbial and cancer cell
survival by increasingly complex eukaryotic systems/symbionts. Thus
mitochondrial, chloroplast, and/or nuclear-encoded proteins capable
of activating and/or modulating programmed cell death pathways are
contemplated to be evolutionary relatives/descendents of
polypeptides that originally provided a survival advantage in the
face of microbial or neoplastic challenge.
[0067] The peptides, protides and conjugates described herein have
the potential to create, augment, or improve several existing
therapeutic, prophylactic, diagnostic, and basic research problems.
For example, therapeutically, these peptides, protides and
conjugates can address the problem of antibiotic-resistant
infections and antineoplastic-resistant cancers. Likewise, the
peptides, protides and conjugates may serve as immunotherapeutic
agents to enhance or restore efficacy of endogenous host defenses.
As adjunctive agents, these peptides, protides and conjugates will
increase efficacy of conventional agents (such as antibiotics or
anti-neoplastic agents), enhance immune functions, and activate or
inactivate apoptotic mechanisms of cell regulation associated with
aging or other degenerative conditions, and many other potential
applications. The scope and diversity of other uses for these
peptides, protides and conjugates are considerable. For example,
the peptides, protides and conjugates described herein can be used
as diagnostic probes in isotopic or non-isotopic forms to localize
or characterize diseases or conditions containing signatures such
as those characteristic of microbial, neoplastic, necrotic,
apoptotic, or other tissues or cells. Additionally, extensions of
the above concepts are applicable to the construction, design,
delivery, and use of such peptides as research reagents.
[0068] As will be clear to those skilled in the art, the above
novel concepts relating to structure-activity relationships in
programmed cell death proteins enabled the design of novel
antimicrobial, anti-cancer, anti-inflammatory and
anti-proliferative peptides, protides and conjugates. These
peptides and compositions are useful as diagnostic, prophylactic,
and/or therapeutic agents that exploit programmed cell death
pathways in pathogens, cancer cells, autoimmune cells, and other
disease-caused cells and tissues. Specific examples of peptides,
variants, congeners, and mimetics of these molecules are included
herein. Embodiments of the invention provide conjugates in which
one given molecule can represent or include one or more
antimicrobial, anti-cancer, anti-inflammatory, immunomodulatory
peptide and one or more non-peptide functional motifs or domains,
or combinations of these. Embodiments of the invention also provide
protides which are multifunctional and context-activated
polypeptides that have two or more effectors with individually
distinct biological functions and one or more corresponding
activator sites that can each initiate or amplify the biological
function of one or more effectors upon context-activation.
Therefore, peptides, protides and conjugates exemplified herein are
relevant to Antibiotide, Immodulotide, Antineotide, Apoptide,
and/or Cascatide class peptides.
[0069] The novel concepts, peptide design strategies, and
exemplifying peptides encompass conceptual as well as material
inventions. Moreover, variations upon these fundamental themes are
applicable to novel therapeutic agents and strategies in virtually
any area of medicine, including, but not limited to diagnosis,
prevention, and therapy of infectious diseases, cancer and
cancer-like diseases, immune and autoimmune disorders, cardiology,
aging, and/or other conditions or disease states. Furthermore, the
novel peptides based on programmed cell death effectors described
herein represent agents and strategies to treat human, animal, and
agricultural diseases. Other applications include their use in
diagnosis, prevention, or research of diseases, or as research
tools to investigate pathogenesis, apoptosis, or related biological
phenomena.
[0070] Embodiments of the invention, herein provide an isolated
peptide comprising one or more amino acid sequences selected from
the group consisting of SEQ ID NOS: 3, 4, 6, 8, 10, 11, 13, 17, 18,
19, 21-25, 30, 31-36, 39-47, 49-52, 54-57, 59-63, 66-75, 84-93,
102-106, 108-121, 132-175, 179-187, 191-199, 205-209, 211-223,
227-235, 238-243, 245-247, 249-251, 253-256 and 260-263, wherein
the amino acid residue represented by (x) is a serine, a threonine,
a tryptophan, a H-bond donor residue or a H-bond acceptor residue,
wherein the amino acid residue represented by (b) is a lysine, an
arginine, an asparagine, a glutamine or a basic residue, wherein
the amino acid residue represented by (j) is a cysteine or a thiol
residue, wherein in the amino acid residue represented by (o) is an
anthrylalanine or other non-natural amino acid and wherein the
peptide induces antimicrobial, anti-cancer, anti-inflammatory,
anti-proliferative or programmed cell death activity.
[0071] In one aspect, the isolated peptide comprises one or more
amino acid sequence, identified from the Bax protein, which are
represented by amino acid sequences of SEQ ID NOS: 3, 4, 6, 8, 10,
11, 13, 264, 270 and 271. In one aspect, the isolated peptide
comprises one or more amino acid sequence, identified from the
Bcl-W protein, which are represented by amino acid sequences of SEQ
ID NOS: 17, 18, 19, 21-25, 269 and 272. In one aspect, the isolated
peptide comprises one or more amino acid sequence, identified from
the Bcl-x.beta. protein, which are represented by amino acid
sequences of SEQ ID NOS: 30, 31-36 and 273. In one aspect, the
isolated peptide comprises one or more amino acid sequence,
identified from the Bak protein, which are represented by amino
acid sequences of SEQ ID NOS: 39-47. In one aspect, the isolated
peptide comprises one or more amino acid sequence, identified from
the Bcl-2 protein, which are represented by amino acid sequences of
SEQ ID NOS: 49-52. In one aspect, the isolated peptide comprises
one or more amino acid sequence, identified from the Bcl-2 isoform
1 protein, which are represented by amino acid sequences of SEQ ID
NOS: 54-57. In one aspect, the isolated peptide comprises one or
more amino acid sequence, identified from the Mfn-1 protein, which
are represented by amino acid sequences of SEQ ID NOS: 59-63 and
274. In one aspect, the isolated peptide comprises one or more
amino acid sequence, identified from the Mfn-2 protein, which are
represented by amino acid sequences of SEQ ID NOS: 66-75. In one
aspect, the isolated peptide comprises one or more amino acid
sequence, identified from the Dnm-1 protein, which are represented
by amino acid sequences of SEQ ID NOS: 84-93 and 275. In one
aspect, the isolated peptide comprises one or more amino acid
sequence, identified from the Dnm-2 protein, which are represented
by amino acid sequences of SEQ ID NOS: 102-106, 108-121, 267, 276
and 277. In one aspect, the isolated peptide comprises one or more
amino acid sequence, identified from the Ncl protein, which are
represented by amino acid sequences of SEQ ID NOS: 132-175. In one
aspect, the isolated peptide comprises one or more amino acid
sequence, identified from the Csp3 protein, which are represented
by amino acid sequences of SEQ ID NOS: 179-187, 266 and 278. In one
aspect, the isolated peptide comprises one or more amino acid
sequence, identified from the Bad protein, which are represented by
amino acid sequences of SEQ ID NOS: 191-199. In one aspect, the
isolated peptide comprises one or more amino acid sequence,
identified from the Prf-1 protein, which are represented by amino
acid sequences of SEQ ID NOS: 205-209 and 211-223. In one aspect,
the isolated peptide comprises one or more amino acid sequence,
identified from the Granulysin protein, which are represented by
amino acid sequences of SEQ ID NOS: 227-235. In one aspect, the
isolated peptide comprises one or more amino acid sequence,
identified from the CidA protein, which are represented by amino
acid sequences of SEQ ID NOS: 238-243, 245-247, 265 and 279. In one
aspect, the isolated peptide comprises one or more amino acid
sequence, identified from the LrgA protein, which are represented
by amino acid sequences of SEQ ID NOS: 249-251. In one aspect, the
isolated peptide comprises one or more amino acid sequence,
identified from the Lambda S21 protein, which are represented by
amino acid sequences of SEQ ID NOS: 253-256. In one aspect, the
isolated peptide comprises one or more amino acid sequence,
identified from the Holin protein, which are represented by amino
acid sequences of SEQ ID NOS: 260-263 and 268.
[0072] Embodiments of the invention provide an isolated peptide
consisting of an amino acid sequence selected from the group
consisting of SEQ ID NOS: 1, 2, 5, 7, 9, 12, 14-16, 20, 26-29, 37,
38, 48, 53, 58, 64, 65, 72, 76-83, 94-101, 107, 114, 122-131, 170,
176-178, 188-190, 200-204, 210, 224-226, 236, 237, 244, 248, 252,
257-259 and 288-289, wherein the peptide induces antimicrobial,
anti-cancer, anti-inflammatory, anti-proliferative or programmed
cell death activity.
[0073] In one aspect, the isolated peptide consists of one or more
amino acid sequence, identified from the Bax protein, which are
represented by amino acid sequences of SEQ ID NOS: 1, 2, 5, 7, 9
and 12. In one aspect, the isolated peptide consists of one or more
amino acid sequence, identified from the Bcl-W protein, which are
represented by amino acid sequences of SEQ ID NOS: 14-16 and 20. In
one aspect, the isolated peptide consists of one or more amino acid
sequence, identified from the Bcl-x.beta. protein, which are
represented by amino acid sequences of SEQ ID NOS: 26-29. In one
aspect, the isolated peptide consists of one or more amino acid
sequence, identified from the Bak protein, which are represented by
amino acid sequences of SEQ ID NOS: 37 or 38. In one aspect, the
isolated peptide consists of the amino acid sequence, identified
from the Bcl-2 protein, which is represented by amino acid sequence
of SEQ ID NO: 48. In one aspect, the isolated peptide consists of
the amino acid sequence, identified from the Bcl-2 isoform 1
protein, which is represented by amino acid sequence of SEQ ID NOS:
53. In one aspect, the isolated peptide consists of one or more
amino acid sequence, identified from the Mfn-1 protein, which are
represented by amino acid sequences of SEQ ID NOS: 58 or 64. In one
aspect, the isolated peptide consists of one or more amino acid
sequence, identified from the Mfn-2 protein, which are represented
by amino acid sequences of SEQ ID NOS: 65 or 72. In one aspect, the
isolated peptide consists of one or more amino acid sequence,
identified from the Dnm-1 protein, which are represented by amino
acid sequences of SEQ ID NOS: 76-83. In one aspect, the isolated
peptide consists of one or more amino acid sequence, identified
from the Dnm-2 protein, which are represented by amino acid
sequences of SEQ ID NOS: 94-101, 107 and 114. In one aspect, the
isolated peptide consists of one or more amino acid sequence,
identified from the Ncl protein, which are represented by amino
acid sequences of SEQ ID NOS: 122-131 and 170. In one aspect, the
isolated peptide consists of one or more amino acid sequence,
identified from the Csp3 protein, which are represented by amino
acid sequences of SEQ ID NOS: 176-178. In one aspect, the isolated
peptide consists of one or more amino acid sequence, identified
from the Bad protein, which are represented by amino acid sequences
of SEQ ID NOS: 188-190. In one aspect, the isolated peptide
consists of one or more amino acid sequence, identified from the
Prf-1 protein, which are represented by amino acid sequences of SEQ
ID NOS: 200-204 and 210. In one aspect, the isolated peptide
consists of one or more amino acid sequence, identified from the
Granulysin protein, which are represented by amino acid sequences
of SEQ ID NOS: 224-226. In one aspect, the isolated peptide
consists of one or more amino acid sequence, identified from the
CidA protein, which are represented by amino acid sequences of SEQ
ID NOS: 236, 237 and 244. In one aspect, the isolated peptide
consists of the amino acid sequence, identified from the LrgA
protein, which is represented by amino acid sequence of SEQ ID NOS:
248. In one aspect, the isolated peptide consists of the amino acid
sequence, identified from the Lambda S21 protein, which is
represented by amino acid sequences of SEQ ID NOS: 252. In one
aspect, the isolated peptide consists of one or more amino acid
sequence, identified from the Holin protein, which are represented
by amino acid sequences of SEQ ID NOS: 257-259. In one aspect, the
isolated peptide consists of one or more amino acid sequence,
identified from human Bcl-xL protein, which is represented by the
amino acid sequence SEQ ID NO: 288 or human CTL Granulysin, which
is represented by the amino acid sequence SEQ ID NO: 289. In one
aspect, an isolated peptide as described herein has a C-terminus
comprising a carboxamide.
[0074] Embodiments of the invention are intended to be used as in
ways similar to antibiotic, anti-cancer, or similar medical
administration either as local (e.g. topical, oral rinse, inhaled,
nebulized, etc.) or systemic (oral ingestion, intravenous,
intramuscular, etc) agents. Additionally, the peptides may be used
as research tools for basic molecular biology, microbiology,
biochemistry or other disciplines as they relate broadly to
cellular or molecular biology, infection and immunity, cell
regulation and apoptosis, gene expression, signal transduction, or
any other area of investigation in which a concept, approach, or
specific peptide or may be used.
[0075] In certain embodiments, the invention provides novel
isolated peptides having one or more continuous amino acids
sequences. As used herein, a "peptide" generally has from about 3
to about 100 amino acids, whereas a polypeptide or protein has
about 100 or more amino acids, up to a full length sequence
translated from a gene. Additionally, as used herein a peptide can
be a subsequence or a portion of a polypeptide or protein. In
certain embodiments the size of at least one peptide may comprise,
but is not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100
amino acid residues.
[0076] As used herein, an "amino acid residue" refers to any
naturally or non-naturally occurring amino acid, any amino acid
derivative or any amino acid mimic known in the art. In certain
embodiments, the residues of the peptide are sequential, without
any non-amino acid interrupting the sequence of amino acid
residues. In other embodiments, the sequence may comprise one or
more non-amino acid moieties. In particular embodiments, the
sequence of residues of the peptide may be interrupted by one or
more non-amino acid moieties. Accordingly, the term peptide
encompasses amino acid sequences comprising at least one of the
common amino acids found in naturally occurring proteins, or at
least one modified or unusual or non-natural amino acid, including,
but not limited to, Anthrylalanine, 2 Aminoadipic acid (Aad), N
Ethylasparagine (EtAsn), 3 Aminoadipic acid (Baad), Hydroxylysine
(Hyl), .beta. alanine, .beta. Amino propionic acid (Bala), allo
Hydroxylysine (AHyl), 2 Aminobutyric acid (Abu), 3 Hydroxyproline
(3Hyp), 4 Aminobutyric acid (4Abu), 4 Hydroxyproline (4Hyp), 6
Aminocaproic acid (Acp), Isodesmosine (Ide), 2 Aminoheptanoic acid
(Ahe), allo Isoleucine (AIle), 2 Aminoisobutyric acid (Aib), N
Methylglycine (MeGly), 3 Aminoisobutyric acid (Baib), N
Methylisoleucine (MeIle), 2 Aminopimelic acid (Apm), 6 N
Methyllysine (MeLys), 2,4 Diaminobutyric acid (Dbu), N Methylvaline
(MeVal), Desmosine (Des), Norvaline (Nva), 2,2'Diaminopimelic acid
(Dpm), Norleucine (Nle), 2,3 Diaminopropionic acid (Dpr), Ornithine
(Orn), or N Ethylglycine (EtGly).
[0077] A peptide containing one or more mimetic structures having a
similar charge and spatial or steric arrangements as the reference
amino acid residues is included within the definition of the term
so long as the peptide containing the mimetic portion exhibits a
similar or enhanced activity as compared with the reference
peptide. It is thus understood that a peptide described herein
includes such mimetics as chemically modified peptides,
peptide-like molecules containing non-naturally occurring amino
acids, and peptoids, which are peptide-like molecules resulting
from oligomeric assembly of N-substituted glycines, with similar or
enhanced activity as compared with the reference protide upon which
the mimetic is derived or having any other property desired by the
user, for example, enhanced biostability (see, for example, Goodman
and Ro, Peptidomimetics for Drug Design, in "Burger's Medicinal
Chemistry and Drug Discovery" Vol. 1 (ed. M. E. Wolff; John Wiley
& Sons 1995), pages 803 861), which is incorporated herein by
reference in its entirety. Mimetics also include
constrained-structures so as to maintain optimal spacing and charge
interactions of the amino acid or of the amino acid functional
groups. Those skilled in the art know or can determine what
structures constitute functionally equivalent amino acid analogs
and amino acid mimetics useful for preparation of a peptide
described herein.
[0078] Specific examples of amino acid analogs and mimetics can be
found described in, for example, Roberts and Vellaccio, The
Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meinhofer,
Vol. 5, p. 341, Academic Press, Inc., New York, N.Y. (1983), the
entire volume of which is incorporated herein by reference. Other
examples include peralkylated amino acids, particularly
permethylated amino acids. See, for example, Combinatorial
Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, John Wiley
& Sons Inc., New York, N.Y. (1997), which is incorporated
herein by reference in its entirety. Yet other examples include
amino acids whose amide portion and, therefore, the amide backbone
of the resulting peptide, has been replaced, for example, by a
sugar ring, steroid, benzodiazepine or carbo cycle. See, for
example, Burger's Medicinal Chemistry and Drug Discovery, supra,
Ch. 15, pp. 619 620, which is incorporated herein by reference in
its entirety. Methods for synthesizing peptides, polypeptides,
peptidomimetics and proteins are well known in the art (see, for
example, U.S. Pat. No. 5,420,109; Bodanzsky, Principles of Peptide
Synthesis (1st ed. & 2d rev. ed.), Springer-Verlag, New York,
N.Y. (1984 & 1993), see Chapter 7; Stewart and Young, Solid
Phase Peptide Synthesis, (2d ed.), Pierce Chemical Co., Rockford,
Ill. (1984), each of which is incorporated-herein by reference in
its entirety).
[0079] In one aspect, the peptide, protide or conjugate can
comprise conservatively substituted sequences or alternative
residues at specifically identified positions described herein, for
example, residues identified in SEQ ID NOS: 3, 4, 6, 8, 10, 11, 13,
17, 18, 19, 21-25, 30, 31-36, 39-47, 49-52, 54-57, 59-63, 66-75,
84-93, 102-106, 108-121, 132-175, 179-187, 191-199, 205-209,
211-223, 227-235, 238-243, 245-247, 249-251, 253-256 and 260-263.
In general, a conservative substitution refers to replacement of a
given amino acid residue with a residue having similar
physiochemical characteristics. Examples of conservative
substitutions include (1) non-polar amino acids (Gly, Ala, Val,
Leu, and Ile); (2) polar neutral amino acids (Cys, Met, Ser, Thr,
Asn, and Gln); (3) polar acidic amino acids (Asp and Glu); (4)
polar basic amino acids (Lys, Arg and His); and (5) aromatic amino
acids (Phe, Trp, Tyr, and His). Other such conservative
substitutions, for example, include substitutions of entire regions
having similar hydrophobicity characteristics or substitution of
one H-bond donor/acceptor with another H-bond donor/acceptor. An
alternative residue refers to a residue that may not be
traditionally considered a conservative substitution, but when
substituted at the designated position does not adversely effect
the functional characteristics of the peptide.
[0080] Hydrogen bonding (H-bond) is a non-covalent type of bonding
between molecules or within them, intermolecularly or
intramolecularly, and in the context of the peptides described
herein include H-bond between amino acids. The H-bond donor is the
molecule that has a hydrogen atom bonded to a highly
electronegative, small atom with available valence. For example,
H--O, H--N, and H--F bonds are extremely polar and as a result, the
electron density is easily withdrawn from the hydrogen atom towards
the electronegative atom. The partially positive hydrogen in one
molecule attracts to partially negative lone pair of the
electronegative atom on the other molecule, i.e. an H-bond
acceptor, and thus a H-bond forms as a result of such an
interaction.
[0081] A "basic" residue refer to an amino acid residue which has a
second basic group, which can be, but is not limited to, an amino
group (i.e. lysine), a guanidine group (i.e. arginine), or an
imidazole ring (i.e. histidine).
[0082] A "thiol" residue refers to an amino acid residue which has
a functional sulfur-hydrogen present in the side chain (i.e.
cysteine or methionine). A thiol residue, such as cysteine, can
also play an important role in the folding and stability of some
peptides and proteins through the formation of disulfide bonds.
[0083] A pathological condition appropriate for treatment with a
peptide, protide or conjugate described here can be a symptomatic
disease or other abnormal condition or injury of a mammalian cell
or tissue. Such pathological conditions include, for example,
hyperproliferative and unregulated neoplastic cell growth,
degenerative conditions, inflammatory diseases, autoimmune diseases
and infectious diseases. Hyperplastic and cancer cells proliferate
in an unregulated manner, causing destruction of tissues and
organs. Specific examples of hyperplasias include benign prostatic
hyperplasia and endometrial hyperplasia.
[0084] Abnormal cellular growth can also result from infectious
diseases in which foreign organisms cause excessive growth. For
example, human papilloma viruses can cause abnormal growth of
tissues. The growth of cells infected by a pathogen is abnormal due
to the alteration of the normal condition of a cell resulting from
the presence of a foreign organism. Specific examples of infectious
diseases include DNA and RNA viral diseases, bacterial diseases,
fungal diseases, and protozoal or parasitic diseases. Similarly,
the cells mediating autoimmune and inflammatory diseases are
aberrantly regulated which results in, for example, the continued
proliferation and activation of immune mechanisms with the
destruction of tissues and organs. Accordingly, "anti-inflammatory
activity" refers to a cellular response to a substance or treatment
that reduces inflammation and "anti-proliferative activity" refers
to a cellular response to a substance that prevents the
proliferation or uncontrolled dividing of cells. Specific examples
of autoimmune diseases include, for example, rheumatoid arthritis
and systemic lupus erythmatosis. Specific examples of degenerative
disease include osteoarthritis and Alzheimer's disease. Similarly,
the terms infectious diseases, degenerative diseases, autoimmune
diseases and inflammatory diseases are intended to include all
classes and types of these pathological conditions. Those skilled
in the art will know the various classes and types of
proliferative, neoplastic, infectious, autoimmune and inflammatory
diseases.
[0085] By specific mention of the above categories of pathological
conditions, those skilled in the art will understand that such
terms include all classes and types of these pathological
conditions. For example, the term cancer is intended to include all
known cancers, whether characterized as malignant, benign, soft
tissue or solid tumors, or hematologic tumors relating to cells in
circulation, such as Anal Cancer, Basal Cell Carcinoma, Bladder
Cancer, Bone Cancer (Osteosarcoma and Malignant Fibrous
Histiocytoma), Brain Tumor, Breast Cancer, Bronchial Tumors,
Burkitt Lymphoma, Central Nervous System Lymphoma, Cervical Cancer,
Childhood Cancers, Colon Cancer, Colorectal Cancer, Eye Cancer
(Intraocular Melanoma or Retinoblastoma), Gallbladder Cancer,
Gastric (Stomach) Cancer, Germ Cell Tumor, Head and Neck Cancer,
Kidney (Renal Cell) Cancer, Lip and Oral Cavity Cancer, Liver
Cancer, Lung Cancer (Non-Small Cell), Lung Cancer (Small Cell),
Neuroblastoma, Oral Cancer (Oropharyngeal Cancer), Ovarian
Epithelial Cancer, Pancreatic Cancer, Paranasal Sinus and Nasal
Cavity Cancer, Parathyroid Cancer, Penile Cancer, Prostate Cancer,
Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Skin Cancer
(Nonmelanoma), Skin Cancer (Melanoma), Skin Carcinoma (Merkel
Cell), Testicular Cancer, Throat Cancer, Thymoma and Thymic
Carcinoma, Thyroid Cancer, Urethral Cancer and Vaginal Cancer.
Accordingly, "anti-cancer activity" refers to a cellular response
to a substance that kills or inhibits the growth of a cancer cell.
Cancer cells typically display uncontrolled growth (division beyond
the normal limits), invasion (intrusion on and destruction of
adjacent tissues), and sometimes metastasis (spread to other
locations in the body via lymph or blood). Peptides, protides and
conjugates described herein which have anti-cancer activity can
kill the cancer cell or prevent the invasion or metastasis of the
cancer cell into other tissues. It is contemplated that the
mechanism of action through which this activity occurs is through
the programmed cell death or related responses in the cells.
[0086] "Antimicrobial activity" refers to a cellular response to a
substance that kills or inhibits the growth of a microorganism,
such as bacteria, fungi or protozoans. Peptides described herein
which have antimicrobial activity can either kill the microorganism
(microbicidal) or prevent the growth of the microorganism
(microbistatic). In some aspects, the peptides, protides and
conjugates described herein show antimicrobial activity again
pathogenic microorganisms. A "pathogenic microorganism" refers to a
microorganism that causes a disease, disorder or condition, which
is commonly referred to as an infection. Pathogenic microorganisms
are well known to one of skill in the art and include pathogenic
bacteria such as Acinotobacter baumannii, Acinotobacter
calcoaceticus, Acinotobacter haemolyticus, Bordetella pertussis,
Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella
melitensis, Brucella suis, Campylobacter jejuni, Chlamydia
pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Clostridium
botulinum, Clostridium difficile, Clostridium perfringens,
Clostridium tetani, Corynebacterium diphtheriae, Enterococcus
faecalis, Enterococcus faecum, Escherichia coli, Francisella
tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella
pneumophila, Leptospira interrogans, Listeria monocytogenes,
Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma
pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides,
Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi,
Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus,
Staphylococcus epidermidis, Staphylococcus saprophyticus,
Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus
pyogenes, Treponema pallidum, Vibrio cholerae and Yersinia pestis,
pathogenic fungi such as Cryptococcus neoformans, Aspergillus
fumigatus, Aspergillus flavus, Aspergillus clavatus, Cryptococcus
neoformans, Cryptococcus laurentii, Crytococcus albidus,
Histoplasma capsulatum, Pneumocystis jirovecii, Stachybotrys
chartarum and several members of the Canidida species, such as C.
albicans, C. glabrata, C. tropicalis, C. stellatoidea, C. glabrata,
C. Krusei, C. parapsilosis, C. guilliermondii, C. viswanathii and
C. lusitaniae. Additional examples of pathogenic microorganisms are
described by Jorgensen and Pfaller in "A Clinician's Dictionary of
Pathogenic Microorganisms" ASM Press (2004), which is herein
incorporated by reference in its entirety.
[0087] Human infections due to antibiotic-resistant bacteria and
fungi are increasing in frequency and severity. Microbial pathogens
exhibiting resistance to one or more antibiotics can now commonly
be found in community and nosocomial settings. Antibiotic resistant
pathogens currently of the greatest concern are methicillin
(multiple) resistant Staphylococcus aureus (MRSA), vancomycin
resistant Enterococcus faecalis and Enterococcus faecium (VRE),
multi-drug-resistant Streptococcus pneumoniae (MDRSPn) or
Streptococcus pyogenes (MDRBRSPy), Pseudomonas aeruginosa (MDRA),
and Candida albicans (MDRCA).
[0088] "Programmed cell death" or "PCD" is death of a cell in any
form, mediated by an intracellular program. In contrast to other
types of cell death, such as necrosis, PCD is carried out as a
regulated process which generally confers advantages during an
organism's life-cycle. A strategic advantage of apoptosis is that
it typically does not induce broader inflammatory responses, which
can be injurious and/or delay wound healing, whereas necrosis often
induces considerable inflammation. PCD is commonly categorized by
two types, apoptosisin (type I cell-death) or autophagic (type II
cell death). Apoptosisin, also known as apoptosis, is a series of
biochemical events leading to a characteristic cell morphology
including blebbing, loss of membrane asymmetry and attachment, cell
shrinkage, nuclear fragmentation, chromatin condensation and
chromosomal DNA fragmentation known as laddering. Autophagic PCD is
a catabolic process involving the degradation of a cell's own
components and organelles through the lysosomal machinery prior to
the nucleus being destroyed. Additionally, in some aspects, PCD
refers to other pathways that have been described including
non-apoptotic (i.e. caspase-independent) programmed cell-death,
necrosis-like programmed cell death, anoikis, excitotoxicity and
Wallerian degeneration.
[0089] Embodiments of the invention provide a context-activated
protide having at least one activator site and two or more
effectors, wherein at least one effector comprises an amino acid
sequence selected from the group consisting of SEQ ID NOS: 3, 4, 6,
8, 10, 11, 13, 17, 18, 19, 21-25, 30, 31-36, 39-47, 49-52, 54-57,
59-63, 66-75, 84-93, 102-106, 108-121, 132-175, 179-187, 191-199,
205-209, 211-223, 227-235, 238-243, 245-247, 249-251, 253-256 and
260-263, wherein the amino acid residue represented by (x) is a
serine, a threonine, a tryptophan, a H-bond donor residue or a
H-bond acceptor residue, wherein the amino acid residue represented
by (b) is a lysine, an arginine, an asparagine, a glutamine or a
basic residue, wherein the amino acid residue represented by (j) is
a cysteine or a thiol residue, wherein the amino acid residue
represented by (o) is an anthrylalanine or other non-natural amino
acid and wherein the at least one effector induces antimicrobial,
anti-cancer, anti-inflammatory, anti-proliferative or programmed
cell death activity. Embodiments of the invention also provide a
context-activated protide comprising at least one activator site
and two or more effectors, wherein at least one effector comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 1, 2, 5, 7, 9, 12, 14-16, 20, 26-29, 37, 38, 48, 53, 58, 64,
65, 72, 76-83, 94-101, 107, 114, 122-131, 170, 176-178, 188-190,
200-204, 210, 224-226, 236, 237, 244, 248, 252, 257-259 and
288-289, wherein the at least one effector induces antimicrobial,
anti-cancer, anti-inflammatory, anti-proliferative or programmed
cell death activity
[0090] In one aspect of the invention, the protide has at least one
effector comprising an amino acid sequence, identified from the Bax
protein, referenced by the amino acid sequence of SEQ ID NOS: 1-13,
264, 270 and 271. In one aspect of the invention, the protide has
at least one effector comprising an amino acid sequence, identified
from the Bcl-W protein, referenced by the amino acid sequence of
SEQ ID NOS: 14-25, 269 and 272. In one aspect of the invention, the
protide has at least one effector comprising an amino acid
sequence, identified from the Bcl-x.beta. protein, referenced by
the amino acid sequence of SEQ ID NOS: 26-36 and 273. In one aspect
of the invention, the protide has at least one effector comprising
an amino acid sequence, identified from the Bak protein, referenced
by the amino acid sequence of SEQ ID NOS: 37-47. In one aspect of
the invention, the protide has at least one effector comprising an
amino acid sequence, identified from the Bcl-2 protein, referenced
by the amino acid sequence of SEQ ID NOS: 48-52. In one aspect of
the invention, the protide has at least one effector comprising an
amino acid sequence, identified from the Bcl-2 isoform 1 protein,
referenced by the amino acid sequence of SEQ ID NOS: 53-57. In one
aspect of the invention, the protide has at least one effector
comprising an amino acid sequence, identified from the Mfn-1
protein, referenced by the amino acid sequence of SEQ ID NOS: 58-64
and 274. In one aspect of the invention, the protide has at least
one effector comprising an amino acid sequence, identified from the
Mfn-2 protein, referenced by the amino acid sequence of SEQ ID NOS:
65-75. In one aspect of the invention, the protide has at least one
effector comprising an amino acid sequence, identified from the
Dnm-1 protein, referenced by the amino acid sequence of SEQ ID NOS:
76-93 and 275. In one aspect of the invention, the protide has at
least one effector comprising an amino acid sequence, identified
from the Dnm-2 protein, referenced by the amino acid sequence of
SEQ ID NOS: 94-121, 267, 276 and 277. In one aspect of the
invention, the protide has at least one effector comprising an
amino acid sequence, identified from the Ncl protein, referenced by
the amino acid sequence of SEQ ID NOS: 122-175. In one aspect of
the invention, the protide has at least one effector comprising an
amino acid sequence, identified from the Csp3 protein, referenced
by the amino acid sequence of SEQ ID NOS: 176-187, 266 and 278. In
one aspect of the invention, the protide has at least one effector
comprising an amino acid sequence, identified from the Bad protein,
referenced by the amino acid sequence of SEQ ID NOS: 188-199. In
one aspect of the invention, the protide has at least one effector
comprising an amino acid sequence, identified from the Prf-1
protein, referenced by the amino acid sequence of SEQ ID NOS:
200-223. In one aspect of the invention, the protide has at least
one effector comprising an amino acid sequence, identified from the
Granulysin protein, referenced by the amino acid sequence of SEQ ID
NOS: 224-235. In one aspect of the invention, the protide has at
least one effector comprising an amino acid sequence, identified
from the CidA protein, referenced by the amino acid sequence of SEQ
ID NOS: 236-247, 265 and 279. In one aspect of the invention, the
protide has at least one effector comprising an amino acid
sequence, identified from the LrgA protein, referenced by the amino
acid sequence of SEQ ID NOS: 248-251. In one aspect of the
invention, the protide has at least one effector comprising an
amino acid sequence, identified from the Lambda S21 protein,
referenced by the amino acid sequence of SEQ ID NOS: 252-256. In
one aspect of the invention, the protide has at least one effector
comprising an amino acid sequence, identified from the Holin
protein, referenced by the amino acid sequence of SEQ ID NOS:
257-263 and 268. In one aspect, the protide has at least one
effector comprising an amino acid sequence, identified from human
Bcl-xL protein, which is represented by the amino acid sequence SEQ
ID NO: 288 or human CTL Granulysin, which is represented by the
amino acid sequence SEQ ID NO: 289.
[0091] In one aspect, the activator site is context-activated. The
invention also provides that upon context-activation, the protide
initiate programmed cell death of the target cells. In some
aspects, the context-activation results from a physiological
condition, such as, but not limited to acidity, alkalinity, ionic
strength or osmotic strength. In some aspects, the
context-activation results from association with an activator
molecule. The activator molecules can modify the activator site
upon association. In some aspects, modification of the activator
site includes cleavage of the activator site. In some aspects, the
activator molecule is an enzyme, such as a protease, esterase or
lipase. In one aspect, the activator is expressed by a pathogenic
microorganism as described herein. In another aspect, the activator
is present in the context of a tumor cell, such as a tumor-specific
protease. An example of a tumor-specific protease is a matrix bound
protein such as matrix metalloproteinase. In another aspect, the
activator is present in the context of an inflammatory response,
wherein activators such as thrombin, bradykinin, elastase and
metalloproteinase are expressed.
[0092] The term "protide," as used herein, refers to a mosaic
molecule composed of two or more peptide or non-peptide functional
domains, referred to as effectors, and one or more corresponding
activator sites. A protide can consist of an indefinite number of
effector and activator domains that can vary in function,
activation, position, continuity, or sequence. Additional examples
of protide compositions and designs are described in U.S. Patent
Application Publication 2006-0074016, 2006-0135416 and U.S. Pat.
No. 7,067,621, which are herein incorporated by reference.
[0093] The protides described herein have two or more distinct
biological functions and are designed to be activated within a
defined or characteristic context. The protides described herein
have at least one activator site and two or more effectors, wherein
at least one of the effectors has and amino acid sequence of a
peptide described herein. Protides have the advantage of designs
that can be customized, engineered, chosen, or combined to allow
for highly selective correspondence to or association with or
unique to a specific pathological condition or etiology. The
distinct biological functions can further be associated with
distinct functional aims, for example, therapy, prevention,
amplification and detoxification. As described herein, a
multifunctional, context-activated protide can be designed to be
activated in any context desired by the user, a feature which makes
the protides useful to applications in many areas of medicine and
biomedical research, including, for example, diagnosis, imaging,
detection, speciation or other specification,
prevention/prophylaxis, and therapy of a wide range of pathological
conditions such as infectious diseases, neoplastic diseases, immune
and autoimmune disorders, cardiovascular conditions, disorders in
metabolism or physiology, diseases of inheritance or genetic
abnormality, a variety of pathological conditions associated with
gene expression, mitochondrial dysfunction or regulation, as well
as cell death and/or cellular senescence.
[0094] As described herein, in addition to their direct
antimicrobial efficacies, the peptides, protides and conjugate
described herein are useful based on their ability to circumvent or
minimize conventional resistance mechanisms by pathogens or tumor
cells. For example, this can be the result of activation by
activators that are present outside of the target cell such that
the peptides, protides or conjugate need not necessarily enter the
target cell to be activated and to achieve subsequent efficacy,
thus minimizing the likelihood for resistance due to reduced target
access or increased efflux of the peptides, protides or conjugate.
Furthermore, in many conventional resistance mechanisms, resistance
can be induced by the presence of the anti-infective agent itself.
In particular, protides can be designed to be activated by such
microbial counter-responses or virulence factors. Thus, the more of
the activator that is made by the organism, the more protide
activation results, yielding an expected amplification of the
anti-pathogenic efficacy of the protide. Conversely, decreased
production of the activators can translate in turn to decreased
presence or function of these same activators such as virulence
factors or mediators of pathogenesis, in essence turning off the
pathogenic potential of the target cell, or reducing its ability to
protect itself from otherwise normal host defenses. Similarly,
protides can be beneficial by reconstituting tumor cell or
microbial pathogen susceptibility to conventional therapeutic
agents, to which these pathogenic cells would otherwise be
resistant. Thus, the protides can either be activated from
upregulation of resistance- or virulence factor expression, or can
impact efficacy by effecting the downregulation of virulence factor
expression by pathogenic cells or organisms.
[0095] In applications of the methods described herein, involving
an established infection or a host response to infection,
activators can be present or generated. An activator useful for
activation of a protide of the invention can be advantageously
selected based on a high concentration in the immediate proximity
of the infection locus so as to allow for activation of the
majority of protides in the desired context. One skilled in the art
will be able to select an activator that represents the desired
activation context. For applications of the invention methods in
the arena of microbial infection, context-activation can be
designed to specifically occur in the local context of infection so
as to effect optimal relative protide effector concentrations in
specific contexts of infection. In addition to context activation
that maximizes efficacy, the protides and methods of the invention
also minimize the potential for inadvertent host cytotoxicity in
areas that do not represent the context. Therefore, in the absence
of infection, the protide activators are either absent or are
present at concentrations insufficient for effective protide
activation, thereby minimizing inadvertent or indiscriminant acute
toxicity.
[0096] In addition to specific pathogen or host molecules that can
serve as activators as described above, protides can also be
designed to become activated to diagnose, prevent, or treat
infection in unique and/or specific biochemical or physiological
contexts associated with microbial pathogens. Examples of such
biochemical or environmental contexts include ionic, osmotic, pH,
oxidation/reduction, or other conditions that are unique to,
characteristic of, or present in the context of infection or
disease processes that occur upon infection, or host responses to
these events. For example, a protide can be designed to require the
influence of protonation, conformation change, or other
modification that occurs uniquely or disproportionately in the
context of acidic pH, to activate the protide or its ensuing
effectors by altering their structure-activity relationship(s) from
inactive to active. As one example, genitourinary tissues, such as
renal-tissues or genitourinary mucosa, can exhibit pH values that
are decreased normally, or in the setting of infection. A protide
designed to be activated only under such acidic conditions could be
designed to either be vulnerable to activation in these conditions,
or directly activated by these conditions, and thus would be
predicted to be active only in such contexts. Alternatively,
protides can be designed to be inactive in particular contexts or
conditions, such as conditions of relatively high osmotic strength
or relatively high pH, so as to minimize or prevent untoward or
toxic effects such as nephro- or hepatotoxicity. By way of a
further example, activation as well as leukocyte accumulation are
conditions associated with infection. Moreover, a fundamental
strategy of host defense phagocytes is to phagocytize the microbial
pathogen, subjecting it to the harsh environment of the acidic
phagolysosome. The compartment so created can become acidified to
pH values of 5.5 or lower as the leukocyte responds to the
pathogen. Therefore, a protide can be designed that is activated or
has amplified or antimicrobial activities, for example, by pH,
phagolysosomal enzymes or reactants, or a combination of these
conditions, or can amplify or potentiate the antimicrobial
mechanisms of leukocytes or other host cells within such settings,
so as to inhibit or kill pathogens that enter such cells.
[0097] Protide activation also can include conformational,
oxidation or reduction-mediated changes in disulfide array,
assembly into multimers of two or more homomeric (identical) or
heteromeric (non-identical) effectors, or other modifications of
the protide and/or its subsequent effectors. In a particular
embodiment, protide activation is triggered as a result of protide
accumulation, or its resulting effector components, so as to
achieve or surpass threshold concentrations required to optimize or
catalyze activation or activity through multimerization or other
modification in structure or function of the protide or its
effectors.
[0098] It is understood, that activation can involve combinations
of the protide activation strategies described above. For example,
a protide can be designed that is not responsive to an activator
unless both the protide and the activator are present within a
context associated with or resulting from infection or other
disease.
[0099] The term "context-activated," as used herein in reference to
a protide of the invention, refers to the initiation, activation or
amplification of a biological or other desired, for example,
diagnostic or prophylactic function of one or more protide
effectors in a particular temporal, spatial, pathological and/or
biochemical context. Context-activation can be initiated by direct
or indirect interaction between a protide activator site and a
corresponding activator that is selectively associated with the
particular context. As used herein, context-activation encompasses
activation in a wide variety of contexts that can include, for
example, local, regional, systemic, and/or temporal proximity; as
well as the presence or absence of an etiological agent, pathologic
condition, or characteristic components thereof.
[0100] Thus, context need not be limited to a place, time or
quality, but also can be the presence or absence of an activator,
for example, an enzyme elaborated by an organism such as, for
example, a specific strain of bacteria. The context for activation
can consequently be of any breadth desired by the user, for
example, can target a class of organisms or cell types, for
example, by using an activator that is ubiquitous to the targeted
class, or can alternatively have a more narrow focus by using an
activator that represents a more narrowly defined target, for
example, a particular genus, organism, species, subspecies, strain,
or cell or tissue type. The context can be associated with a
pathological condition, but also can be selected to represent a
non-pathological environment, for example, in prophylactic
applications of the invention practiced to preserve a normal or
homeostatic condition.
[0101] As used herein, the term "effector" refers to the peptide or
non-peptide functional domains of a protide provide herein that
have specific individual functions, which are initiated or
amplified upon activation and achieve specific functions relating
to the diagnosis, prevention, or treatment of a disease. As
described herein, a protide has at least two effector domains with
distinct, complementary and/or synergistic biological functions. An
effector is inactive or exhibits relatively reduced or attenuated
biological activity unless an activator, by virtue of either its
presence or absence, alters or facilitates or allows the altering
of its corresponding activator site and, as a result, initiates or
amplifies the diagnostic, prophylactic, therapeutic, or other
biological function(s) of the effector(s). Multiple effectors can
be induced by the same activator site. Peptide and non-peptide
effectors can be present in the same protide, which can be referred
to as a hybrid protide. Similarly, a protide can consist
exclusively of peptide effectors, also referred to as a peptide
protide. Similarly, a protide of the invention can consist
exclusively of non-peptidic effectors. The biological function(s)
of an effector that corresponds to a protide described herein can
be, for example, antimicrobial, immunomodulatory, pro- or
anti-inflammatory, tumoricidal, pro- or anti-apoptotic, pro- and
anti-angiogenic and/or hemolytic.
[0102] As described herein, a protide of the invention can be
bifunctional or multifunctional, with two or more unique
complementary effectors, and one or more activators as determined
by specific effector and activator site domains engineered into the
mosaic protide, which can be activated by specific molecules or
conditions present in unique or strategic contexts of interest.
Examples of such effectors can include one or more antimicrobial,
anti-neoplastic, anti-inflammatory, immunomodulatory, or other
peptide or non-peptide functional domains, or combinations
thereof.
[0103] As used herein, the term "activator site" when used in
reference to a protide of the invention, refers to a domain of the
protide that, in the presence of an activator, initiates, promotes,
amplifies or modulates the specific biological function of one or
more effectors. As described herein, an activator site can be
modified, cleaved, processed or otherwise altered in the presence
of an activator. In addition, an activator site can be sensitive
either to the absolute presence or absence of an activator as well
as can be sensitive to a threshold concentration of an activator
rather than its mere presence.
[0104] An activator site useful in the invention can include one or
more sites for cleavage, modification, processing or other
triggering by strategic activators, which can be, for example,
proteases, esterases, lipases, or other endogenous enzymatic
activators or cascades generated by or associated with a specific
condition such as, for example, the presence of pathogenic
microorganisms, damaged or inflamed tissues, or hematologic or
solid neoplastic or pre-neoplastic cells or tumors. Such an
activator site also can be selected to exploit contexts associated
with biochemical or physical conditions such as requisite acidity
or alkalinity, for example, acidic phagolysomes containing
intracellular bacteria or fungi; or ionic or osmotic strength, for
example, in a renal context, that represent a specific pathologic
or non-pathologic context. Furthermore, an activator site can be
selected to exploit normal rather than a pathologic context.
[0105] An activator site can be subject to proteolytic as well as
non-proteolytic activation. For example, the activator site can be
located within the peptide moiety, and require a protease
activator. In other embodiments, the non-proteolytic activator can
target a non-proteinaceous substrate component of the protide. For
example, a protide of the invention can include an esterase
activator and can link peptide and/or non-peptide moieties (eg. a
protide consisting of peptide and conventional antibiotic
effectors) by means of an ester bond. Other biochemically relevant
bonds or linkages that can serve as activation sites in an
invention protide can include, for example, lipase-(lipid
cleaving), nuclease-(nucleic acid cleaving), and kinase or
phosphatase-(phosphate addition or removal) sensitive activators
that target substrates other than peptides. For example, certain
microbial pathogens or tumor cells can express, or abnormally
express restriction enzymes that can provide a suitable basis for
design of a protide that could be activated only-within the target
cell, further reducing indiscriminant host cytotoxicity.
[0106] As used herein, the term "activator" refers to a molecule or
condition that, by altering the activator site, causes the
liberation or onset of a specific diagnostic or biological function
of effector(s). As described herein, an activator can be a normal
or abnormal exogenous or endogenous cell, structure or molecule, a
condition or milieu (normal or abnormal), or a combination thereof
that is associated with a specific context in which activation of
the protide is desired. Thus, an activator can be selected based on
its presence in a temporal, spatial, or physiological context,
which can be normal or abnormal, that is associated with the
desired context for protide activation. An activator can
consequently include physiological conditions including, for
example, acidity, alkalinity, conditions of oxidation or reduction,
and/or ionic and/or osmotic strength, that are associated with a
particular context, and modulate protide activation. Alternatively,
an activator can be a structure or molecule, for example, an
enzyme, that is present in a particular spatial, temporal or
pathological context. The activator molecule can modify the
activator site upon association, for example, by cleavage or other
modification that results in activation in the particular context,
or can facilitate interaction between protide and activator(s). The
activator molecule can be an enzyme including, for example,
protease, esterase, lipase, nucleases or peptidase.
[0107] In one embodiment of the invention, an activator site can
encompass one or more domains for cleavage, modification,
processing or any other type of liberation by an activator, for
example, a protease, esterase, lipase or other endogenous or
exogenous enzymatic activator or cascade. The choice of one or more
activator sites that correspond to specific activators depends
directly on the desired context for activation. Thus, an activator
can be a particular pathologic setting or condition that is chosen
based on its association with a particular etiological agent or
host response. In the presence of the activator, one or more
effectors are liberated so as to achieve a specific function
relating to, for example, the treatment, prevention, or diagnosis
of a targeted disease. An activator site can thus be strategically
designed to become activated in temporal and spatial proximity to
activator expression, thereby allowing the activation of a protide
to be targeted to a particular context and over time so as to
maximize the desired therapeutic or prophylactic effect, while
minimizing untoward or undesirable toxicities or other side
effects.
[0108] As described herein, an activator site is selected based on
its correspondence and/or association with the context in which the
two or more protide effectors are to be liberated so as to initiate
or potentiate their functions. Therefore, as long as an activator
is associated with the context, the invention can be practiced with
any context desired. Those skilled in the art will appreciate that,
given the versatility of activators useful for practicing the
invention as described herein, a protide can be designed based on
virtually any context desired, including, for example, vascular
injury, presence of a neoplasm or cancer, infection, and
inflammation.
[0109] In one embodiment, the protide is an antimicrobial protide,
which also can be referred to as an antimicrotide. Cleavage sites
for strategic proteases can be engineered into multifunctional
antimicrobial protides so as to represent the activator site of the
protide. Upon activation of the protease in the localized or
generalized context of tissue injury or infection, as selected by
the user, the inactive protide is cleaved, liberating independent
and active molecules to effect their respective biological
functions. Prior to and beyond the setting of activation of the
strategic protease representing the activator, the mosaic protide
construct is relatively inactive both with respect to antimicrobial
function and host cell toxicity. A mosaic protide construct can
consist of an indefinite number (1 through n) of effector and
activator domains that can vary in function, activation, position,
continuity, or sequence. Effectors corresponding to one or more
protides activated by the same or distinct activators also can
function synergistically, and/or can recombine in a manner
facilitating their complementary functions. As an example, in the
context of vascular injury, a protide activator can be selected
that specifically represents this particular context, for example,
a clotting cascade protease such as thrombin, or a complement
fixing protease such as a C3 convertase, for example, C4B2A or
C3bBb. Similarly, as another example, a protide activator can be
selected that represents a broader constellation of symptoms or
conditions, such as sepsis, in which corresponding activators can
include serine proteases associated with systemic inflammation,
sepsis, or injury, such as activated protein C.
[0110] A further embodiment of the invention encompasses
anti-neoplastic protides, which also are referred to as
antineotides. Many tumor cells produce or overexpress
characteristic activators, such as matrix metalloproteinases (MMP)
or other enzymes that are not expressed by, or at levels much
higher than normal cells. Consequently, the activator can be a
tumor-specific protease, for example, a matrix metalloproteinase or
thymidylate synthase (TS), which is overexpressed in the majority
of cancers. A tumor-specific protease also can be associated with a
more narrow neoplastic context, such as a serine protease that is
specifically expressed in prostate cells, for example, PSA, human
kallikrein-2 (hK2), human kallikrein-11 (hK11) and TMPRSS2.
[0111] Embodiment of the invention provide a conjugate having one
or more amino acid sequence selected from the group consisting of
SEQ ID NOS: 3, 4, 6, 8, 10, 11, 13, 17, 18, 19, 21-25, 30, 31-36,
39-47, 49-52, 54-57, 59-63, 66-75, 84-93, 102-106, 108-121,
132-175, 179-187, 191-199, 205-209, 211-223, 227-235, 238-243,
245-247, 249-251, 253-256 and 260-263 and a moiety, wherein the
amino acid residue represented by (x) is a serine, a threonine, a
tryptophan, a H-bond donor residue or a H-bond acceptor residue,
wherein the amino acid residue represented by (b) is a lysine, an
arginine, an asparagine, a glutamine or a basic residue, wherein
the amino acid residue represented by (j) is a cysteine or a thiol
residue, wherein in the amino acid residue represented by (o) is an
anthrylalanine or other non-natural amino acid and wherein the
conjugate induces antimicrobial, anti-cancer, anti-inflammatory,
anti-proliferative or programmed cell death activity. Embodiments
of the invention also provide a conjugate having one or more amino
acid sequence selected from the group consisting of SEQ ID NOS: 1,
2, 5, 7, 9, 12, 14-16, 20, 26-29, 37, 38, 48, 53, 58, 64, 65, 72,
76-83, 94-101, 107, 114, 122-131, 170, 176-178, 188-190, 200-204,
210, 224-226, 236, 237, 244, 248, 252, 257-259 and 288-289 and a
moiety, wherein said conjugate induces antimicrobial, anti-cancer,
anti-inflammatory, anti-proliferative or programmed cell death
activity.
[0112] In one aspect of the invention, the conjugate has at least
one or more amino acid sequence, identified from the Bax protein,
referenced by the amino acid sequence of SEQ ID NOS: 1-13, 264, 270
and 271. In one aspect of the invention, the conjugate has at least
one or more amino acid sequence, identified from the Bcl-W protein,
referenced by the amino acid sequence of SEQ ID NOS: 14-25, 269 and
272. In one aspect of the invention, the conjugate has at least one
or more amino acid sequence, identified from the Bcl-x.beta.
protein, referenced by the amino acid sequence of SEQ ID NOS: 26-36
and 273. In one aspect of the invention, the conjugate has at least
one or more amino acid sequence, identified from the Bak protein,
referenced by the amino acid sequence of SEQ ID NOS: 37-47. In one
aspect of the invention, the conjugate has at least one or more
amino acid sequence, identified from the Bcl-2 protein, referenced
by the amino acid sequence of SEQ ID NOS: 48-52. In one aspect of
the invention, the conjugate has at least one or more amino acid
sequence, identified from the Bcl-2 isoform 1 protein, referenced
by the amino acid sequence of SEQ ID NOS: 53-57. In one aspect of
the invention, the conjugate has at least one or more amino acid
sequence, identified from the Mfn-1 protein, referenced by the
amino acid sequence of SEQ ID NOS: 58-64 and 274. In one aspect of
the invention, the conjugate has at least one or more amino acid
sequence, identified from the Mfn-2 protein, referenced by the
amino acid sequence of SEQ ID NOS: 65-75. In one aspect of the
invention, the conjugate has at least one or more amino acid
sequence, identified from the Dnm-1 protein, referenced by the
amino acid sequence of SEQ ID NOS: 76-93 and 275. In one aspect of
the invention, the conjugate has at least one or more amino acid
sequence, identified from the Dnm-2 protein, referenced by the
amino acid sequence of SEQ ID NOS: 94-121, 267, 276 and 277. In one
aspect of the invention, the conjugate has at least one or more
amino acid sequence, identified from the Ncl protein, referenced by
the amino acid sequence of SEQ ID NOS: 122-175. In one aspect of
the invention, the conjugate has at least one or more amino acid
sequence, identified from the Csp3 protein, referenced by the amino
acid sequence of SEQ ID NOS: 176-187, 266 and 278. In one aspect of
the invention, the conjugate has at least one or more amino acid
sequence, identified from the Bad protein, referenced by the amino
acid sequence of SEQ ID NOS: 188-199. In one aspect of the
invention, the conjugate has at least one or more amino acid
sequence, identified from the Prf-1 protein, referenced by the
amino acid sequence of SEQ ID NOS: 200-223. In one aspect of the
invention, the conjugate has at least one or more amino acid
sequence, identified from the Granulysin protein, referenced by the
amino acid sequence of SEQ ID NOS: 224-235. In one aspect of the
invention, the conjugate has at least one or more amino acid
sequence, identified from the CidA protein, referenced by the amino
acid sequence of SEQ ID NOS: 236-247, 265 and 279. In one aspect of
the invention, the conjugate has at least one or more amino acid
sequence, identified from the LrgA protein, referenced by the amino
acid sequence of SEQ ID NOS: 248-251. In one aspect of the
invention, the conjugate has at least one or more amino acid
sequence, identified from the Lambda S21 protein, referenced by the
amino acid sequence of SEQ ID NOS: 252-256. In one aspect of the
invention, the conjugate has at least one or more amino acid
sequence, identified from the Holin protein, referenced by the
amino acid sequence of SEQ ID NOS: 257-263 and 268. In one aspect,
the conjugate has at least one or more amino acid sequence,
identified from human Bcl-xL protein, which is represented by the
amino acid sequence SEQ ID NO: 288 or human CTL Granulysin, which
is represented by the amino acid sequence SEQ ID NO: 289.
[0113] In one aspect, the moiety comprises a therapeutic agent, a
targeting peptide or a label. In one aspect, therapeutic agent is a
cytotoxic agent, such as an antibiotic or a chemotherapeutic agent.
In one aspect, the targeting peptide selectively homes a conjugate
described herein to a microorganism, a tumor tissue, tumor cell or
tumor vasculature. In one aspect, the targeting peptide selectively
homes the conjugate to an immune regulatory cell or an immune
effector cell. In one aspect, the conjugate described herein has a
targeting peptide, such as, but not limited to, an antibody or a
fragment thereof. In another aspect, the moiety of the conjugate
described herein is a label, such as a radioisotope or a dye.
[0114] As used herein, the term "conjugate" refers to a peptide
having an amino acid sequence as described herein linked to a
moiety. A "moiety" is used broadly to mean a physical, chemical, or
biological material that is linked to a peptide for the purpose of
targeting the peptide to a select organ, tissue or cell type or
providing an additional functional group to the peptide. In
particular, a moiety is a biologically useful moiety such as
therapeutic moiety, a diagnostic moiety or a drug delivery vehicle.
Thus, a moiety can be a therapeutic agent, for example, a cancer
chemotherapeutic agent. Cancer chemotherapeutic agents are well
known to one of skill in the art and include, without limitation,
alkylating agents such as cyclophosphamide, mechlorethamine,
chlorambucil and melphalan, anthracyclines such as daunorubicin,
doxorubicin, epirubicin, idarubicin, mitoxantrone and valrubicin,
cytoskeletal disrupters such as paclitaxel and docetaxel,
epothilones such as epothilones A through F, inhibitors of
topoisomerase II such as etoposide, teniposide and tafluposide,
nucleotide analogs and precursor analogs such as azacitidine,
azathioprine, capecitabine, cytarabine, doxifluridine,
fluorouracil, gemcitabine, mercaptopurine, methotrexate, and
tioguanine, peptide antibiotics, such as bleomycin, platinum-based
agents such as carboplatin, cisplatin and oxaliplatin, retinoids
such as tretinoin, and vinca alkaloids or their derivatives such as
vinblastine, vincristine, vindesine and vinorelbine. Such a moiety
when linked to a peptide, provides a conjugate useful for treating
a cancer in a subject. In addition, a moiety can be a drug delivery
vehicle such as a chambered microdevice, a cell, a liposome or a
virus, which can contain an agent such as a drug or a nucleic
acid.
[0115] A moiety also can be a targeting peptide or nucleic acid, to
which a peptide as described herein is grafted for the purpose of
directing the peptide to a selected organ, tissue, tumor or cell
(Smith et al., J. Biol. Chem. 269:32788-32795 (1994); Goldman et
al., Cancer Res. 15:1447-1451 (1997) and U.S. Pat. No. 6,576,239,
each of which is incorporated herein by reference). For example, a
targeting peptide or nucleic acid can be expressed as a fusion
protein with a desired peptide such that the peptide or nucleic
acid targets the grafted peptide to a selected tumor tissue, tumor
cell or tumor vasculature. Such a desired peptide, which is grafted
to the tumor homing peptide, can be a polypeptide involved in
initiating a programmed cell death pathway as described herein or
inducing any other cellular response resulting in anti-cancer
activity. Additionally, targeting peptides, which can be grafted to
a peptide as described herein having antimicrobial activity,
include peptides that selectively home to a microorganism. For
example, peptide sequences have been identified that selectively
bind to surface molecules of fugal pathogens such as invasive
Aspergillus species as described in U.S. Patent Application
2005-0187161. Still further, the invention provides a conjugate
wherein the targeting peptide selectively homes the desired peptide
to a cell involved in the immune response, including immune
regulatory cells such as lymphocytes or immune effector cells such
as macrophages or granulocytes. Conjugates provided herein include
these and other exemplary peptide or nucleic acid sequences grafted
to a peptide described herein. tumor tissue, tumor cell, tumor
vasculature, immune regulatory cell or immune effector cell.
[0116] A "targeting peptide" is a peptide comprising a contiguous
sequence of amino acids, which is characterized by selective
localization to an organ, tissue, or cell type. Selective
localization may be determined, for example, by methods disclosed
below, wherein the putative targeting peptide sequence is
incorporated into a protein that is displayed on the outer surface
of a phage. Administration to a subject of a library of such phage
that have been genetically engineered to express a multitude of
such targeting peptides of different amino acid sequence is
followed by collection of one or more organs, tissues, or cell
types from the subject and identification of phage found in that
organ, tissue, or cell type. A phage expressing a targeting peptide
sequence is considered to be selectively localized to a tissue or
organ if it exhibits greater binding in that tissue or organ
compared to a control tissue or organ. Preferably, selective
localization of a targeting peptide should result in a two-fold or
higher enrichment of the phage in the target organ, tissue, or cell
type, compared to a control organ, tissue, or cell type. Selective
localization resulting in at least a three-fold, four-fold,
five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold or
higher enrichment in the target organ compared to a control organ,
tissue or cell type is more preferred. Alternatively, a phage
expressing a targeting peptide sequence that exhibits selective
localization preferably shows an increased enrichment in the target
organ compared to a control organ when phage recovered from the
target organ are reinjected into a second host for another round of
screening. Further enrichment may be exhibited following a third
round of screening. Another alternative means to determine
selective localization is that phage expressing the putative target
peptide preferably exhibit a two-fold, more preferably a three-fold
or higher enrichment in the target organ or tissue compared to
control phage that express a non-specific peptide or that have not
been genetically engineered to express any putative target
peptides. Another means to determine selective localization is that
localization to the target organ or tissue of phage expressing the
target peptide is at least partially blocked by the
co-administration of a synthetic peptide containing the target
peptide sequence. "Targeting peptide" and "homing peptide" are used
synonymously herein.
[0117] A targeting peptide is useful, for example, for targeting a
desired peptide to the selected tumor as discussed above. In
addition, a targeting peptide in conjunction with a detectable
label can be used to identify the delivery of a desired peptide to
a sample. As used herein, the term "sample" is used in its broadest
sense to mean a cell, tissue, organ or portion thereof, including a
tumor, that is isolated from the body. A sample can be, for
example, a histologic section or a specimen obtained by biopsy or
cells that are placed in or adapted to tissue culture.
[0118] The term "antibody" is well-known in the art and refers to a
protein functionally defined as a binding protein and structurally
defined as comprising an amino acid sequence that is recognized by
one of skill in the art as having variable and constant regions. A
typical antibody structural unit is known to comprise a tetramer.
Each tetramer is composed of two identical pairs of polypeptide
chains, each pair having one "light" and one "heavy" chain. The
N-terminal portion of each chain defines the variable region of
about 100 to about 110 amino acids, which are primarily responsible
for antigen recognition and binding. The terms variable heavy chain
(V.sub.H) and variable light chain (V.sub.L) regions refer to these
light and heavy chains, respectively. The variable region includes
the segments of Framework 1 (FR1), CDR1, Framework 2 (FR2), CDR2,
Framework 3, CDR3 and Framework 4 (FR4). Antibodies are typically
divided into five major classes, IgM, IgG, IgA, IgD, and IgE, based
on their constant region structure and immune function. The
constant region is identical in all antibodies of the same isotype,
but differs in antibodies of different isotypes. Heavy chains
.gamma., .alpha. and .delta. have a constant region composed of
three tandem (in a line) Ig domains, and a hinge region for added
flexibility; heavy chains .mu. and .epsilon. have a constant region
composed of four immunoglobulin domains. Antibody classes can also
be divided into subclasses, for example, there are four IgG
subclasses IgG1, IgG2, IgG3 and IgG4. The structural
characteristics that distinguish these subclasses from each other
are known to those of skill in the art and can include the size of
the hinge region and the number and position of the interchain
disulfide bonds between the heavy chains. The constant region also
determines the mechanism used to destroy the bound antigen. A light
chain has two successive regions: one constant region, which are
designated as .kappa. and .lamda., and one variable region.
[0119] As used herein, the term "functional fragment" when used in
reference to the antibodies described herein is intended to refer
to a portion of the antibody including heavy or light chain
polypeptides which still retains some or all or the binding
activity of the antibody. Such functional fragments can include,
for example, antibody functional fragments such as Fab, F(ab).sub.2
Fv, and single chain Fv (scFv). Other functional fragments can
include, for example, heavy or light chain polypeptides, variable
region polypeptides or CDR polypeptides or portions thereof so long
as such functional fragments retain binding activity, specificity,
inhibitory and activation activity. The term is also intended to
include polypeptides encompassing, for example, modified forms of
naturally occurring amino acids such as D-stereoisomers,
non-naturally occurring amino acids, amino acid analogues and
mimetics so long as such polypeptides retain functional activity as
defined above.
[0120] A moiety can be a detectable label such a radiolabel or can
be a cytotoxic agent, including a toxin such as ricin or a drug
such as a chemotherapeutic agent or can be a physical, chemical or
biological material such as a liposome, microcapsule, micropump or
other chambered microdevice, which can be used, for example, as a
drug delivery system. Generally, such microdevices, should be
nontoxic and, if desired, biodegradable. Various moieties,
including microcapsules, which can contain an agent, and methods
for linking a moiety, including a chambered microdevice, to a
molecule of the invention are well known in the art and
commercially available (see, for example, "Remington's
Pharmaceutical Sciences" 18th ed. (Mack Publishing Co. 1990),
chapters 89-91; Harlow and Lane, Antibodies: A laboratory manual
(Cold Spring Harbor Laboratory Press 1988), each of which is
incorporated herein by reference; see, also, Hermanson,
Bioconjugate Techniques (Academic Press 1996)).
[0121] A "label" refers a compound has at least one element,
isotope or chemical compound attached to enable the detection of
the compound. In general, labels fall into three classes: a)
isotopic labels, which may be radioactive or heavy isotopes; b)
immune labels, which may be antibodies or antigens; and c) colored
or fluorescent dyes. The labels may be incorporated into a HIPK1
nucleic acids, proteins and antibodies at any position. For
example, the label should be capable of producing, either directly
or indirectly, a detectable signal. The detectable moiety may be a
radioisotope, such as .sup.3H, .sup.14C, .sup.32P, .sup.35S, or
.sup.125I, a fluorescent or chemiluminescent compound, such as
fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme,
such as alkaline phosphatase, beta-galactosidase or horseradish
peroxidase. Any method known in the art for conjugating the
antibody to the label may be employed, including those methods
described by Hunter et al., Nature, 144:945 (1962); David et al.,
Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,
40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407
(1982).
[0122] Peptides, protides and conjugates, which are identified
herein, can be synthesized in required quantities using routine
methods of solid state peptide synthesis or can be purchased from
commercial sources (for example, Anaspec; San Jose Calif.) and a
desired moiety can be linked to the peptide. Several methods useful
for linking a moiety to a peptide are known in the art, depending
on the particular chemical characteristics of the molecule. For
example, methods of linking haptens to carrier proteins as used
routinely in the field of applied immunology (see, for example,
Harlow and Lane, supra, 1988; Hermanson, supra, 1996).
[0123] A moiety such as a therapeutic or diagnostic agent can be
conjugated to a peptide using, for example, carbodiimide
conjugation (Bauminger and Wilchek, Meth. Enzymol. 70:151-159
(1980), which is incorporated herein by reference). Carbodiimides
comprise a group of compounds that have the general formula
R--N.dbd.C.dbd.N--R', where R and R' can be aliphatic or aromatic,
and are used for synthesis of peptide bonds. The preparative
procedure is simple, relatively fast, and is carried out under mild
conditions. Carbodiimide compounds attack carboxylic groups to
change them into reactive sites for free amino groups. Carbodiimide
conjugation has been used to conjugate a variety of compounds to
carriers for the production of antibodies.
[0124] The water soluble carbodiimide,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is particularly
useful for conjugating a moiety to a peptide and was used to
conjugate doxorubicin to tumor homing peptides (U.S. Patent
Application Publication 2004-0131623). The conjugation of
doxorubicin and a tumor homing peptide requires the presence of an
amino group, which is provided by doxorubicin, and a carboxyl
group, which is provided by the peptide.
[0125] In addition to using carbodiimides for the direct formation
of peptide bonds, EDC also can be used to prepare active esters
such as N-hydroxysuccinimide (NHS) ester. The NHS ester, which
binds only to amino groups, then can be used to induce the
formation of an amide bond with the single amino group of a moiety.
The use of EDC and NHS in combination is commonly used for
conjugation in order to increase yield of conjugate formation
(Bauminger and Wilchek, supra, 1980).
[0126] Other methods for conjugating a moiety to a peptide can also
be used. For example, sodium periodate oxidation followed by
reductive alkylation of appropriate reactants can be used, as can
glutaraldehyde crosslinking. However, it is recognized that,
regardless of which method of producing a conjugate of the
invention is selected, a determination may be needed to confirm
that the peptide described herein maintains its antimicrobial,
anti-cancer, anti-inflammatory, anti-proliferative or programmed
cell death activity and that the moiety maintains its relevant
function. Methods for determining the activity of the conjugates
are well know to one of skill in the art.
[0127] An agar radial diffusion assay has been used as described
herein to determine antimicrobial activities of proteins against
microbial pathogens in vitro. One million CFU will be mixed into 10
ml (i.e., 1.times.10.sup.5 CFU/ml) of melted 1% agarose (in 10 mM
NaHPO.sub.4 and cooled to 42.degree. C.) containing minimal
nutrient and adjusted to either pH 5.5 or pH 7.2. The agar is
solidified in culture dishes, and sample wells are formed. Peptides
at various concentrations are dissolved in 10 .mu.l of 0.01% acetic
acid buffer (pH 5.5 or 7.2), loaded into individual wells, and
incubated at 37.degree. C. for three hours. The plate is then
overlayed with 1% agarose containing nutrients and incubated
(37.degree. C., for at least 24 hours). Peptides purified by
RP-HPLC lacking antimicrobial activity are tested in parallel as
controls. Zones of inhibition are measured to quantify
antimicrobial activity. This assay will not distinguish between
microbicidal and microbiostatic actions, but is highly sensitive to
peptides with one or both functions.
[0128] Minimum inhibitory (MIC) and microbiocidal concentration
(MMC) assays can also be performed, and may include a microvolume
assay which is used to quantitatively screen peptides for
antimicrobial activities. In this assay, suspensions of bacteria or
fungi in appropriate media are placed in 100-200 .mu.l final
volumes in microtiter plates. Poly-L-lysine coated or otherwise
positively charged plates are used for these assays, since cationic
peptides may bind to anionic surfaces. Purified peptides are then
serially diluted, descending from 100 .mu.g/ml. Organisms are
inoculated into wells to a concentration of 1.times.10.sup.5
CFU/ml, and plates incubated (37.degree. C., for at least 24
hours). Well turbidities are then assessed visually and by
spectrophotometry to quantify growth inhibition versus wells
containing no peptide. MMCs are then determined by quantitative
culture of MIC wells exhibiting no visible growth.
[0129] Microbicidal kinetics of purified peptides are assessed by
resuspending the peptides in 0.01% acetic acid buffer (pH 5.5 or
7.2), and organisms are resuspended to a concentration of
1.times.10.sup.5 CFU/ml in 50-250 .mu.l of sterile buffer
containing peptide concentrations from 0 to 40 .mu.g/ml. Controls
contain buffer alone or non-antimicrobial proteins and organism as
above. Mixtures are incubated at 37.degree. C. for up to 48 hours,
after which aliquots are quantitatively cultured and incubated for
24 to 48 hours. Killing is expressed as decrease in
logarithm.sub.10 surviving CFU/ml. The limit of sensitivity in
microbicidal assays is considered to be a 1 log reduction in viable
cells.
[0130] Flow cytometry can also be used to examine kinetics and
mechanisms of the action of the peptides on bacterial membrane
integrity and energetics. Peptides which differ in activity or
specificity for their ability to depolarize and/or permeabilize
microbial membranes can also be compared by analysis of membrane
depolarization, and permeabilization. DiOC.sub.5 is a charged
lipophilic dye which partitions into the cytoplasm, and is
dependent on intact transmembrane potential (.DELTA..PSI.) for
intracellular retention. Organisms prepared as above are labeled in
darkness for 30 minutes at about 20.degree. C. in PBS containing
0.05 .mu.M DiOC.sub.5 Organisms are resuspended to a concentration
of 5.times.10.sup.8 CFU/ml in K.sup.+ MEM containing an individual
peptide, and incubated at 37.degree. C. For flow cytometry,
organisms are washed, sonicated, counted, and resuspended in
K.sup.+ MEM buffer. Reductions in mean DiOC.sub.5 fluorescence
relative to controls are interpreted to represent loss of
DiOC.sub.5, indicating membrane depolarization. Positive control
cells exposed to valinomycin, as well as control cells not exposed
to any peptides, are analyzed for DiOC.sub.5 fluorescence in
parallel.
[0131] Propidium iodide is excluded from cells with normal membrane
integrity, but enters cells permealized to molecules .gtoreq.2 nm
in diameter, and can be stimulated to emit fluorescence at >620
nm. Organisms prepared as above are resuspended to a concentration
of 5.times.10.sup.8 CFU/ml in K.sup.+ MEM containing a selected
peptide, and incubated for pre-selected times (ranging from zero up
to about 120 minutes) at 37.degree. C. Cells are washed in fresh
K.sup.+ MEM, sonicated, counted, and resuspended in K.sup.+ MEM
buffer containing 20 .mu.M propidium iodide. Control cells exposed
to ethanol (positive control for permeabilization) are assessed for
propidium iodide uptake in parallel. Increases in mean propidium
iodide fluorescence relative to control cells are interpreted to
indicate increases in permeability.
[0132] Erythrocyte permeabilizing and hemolytic activities of
peptides exhibiting potent microbicidal activity are also studied
as indicators of potential in vivo toxicity. Four-percent (vol/vol)
of washed human erythrocytes (in PBS alone, or in PBS plus 10%
heat-inactivated PNHS are incubated with selected peptides ranging
in concentration up to 100 times greater than geometric mean MICs.
After 24 hours of incubation at 37.degree. C., erythrocyte
permeabilization and hemolysis are determined
spectrophotometrically. Permeabilization and hemolysis will be
compared to buffers alone, and with a triton X-100 control (100%
hemolysis).
[0133] Endothelial cell injury due to peptides can also be measured
using a standard .sup.51Cr release assay, described in Filler et
al., J Infect Dis., 164:928-935 (1991); Filler, et al., Infect
Immun. 62:1064-1069 (1994); Filler et al., Infect Immun. 63:976-983
(1995). Briefly, endothelial cells in 96 well tissue culture plates
are incubated with Na.sup.5l CrO.sub.4 overnight. The following
day, the unincorporated isotope tracer is removed by rinsing, and
peptides in 0.01% acetic acid buffer are added to the endothelial
cells. Control wells are exposed to buffer alone. After a
predetermined incubation period, the medium is aspirated and the
amount of .sup.51Cr released into the medium is measured by
scintillation. This approach facilitates toxicity screening of
multiple peptides simultaneously, and minimizes the amount of
peptide necessary for assessment.
[0134] Each antimicrobial and toxicity assay described above is
performed independently a minimum of two times, and
means.+-.standard error is calculated for each peptide under
varying exposure conditions (concentration or pH) as compared with
control samples. Statistical analyses of microbicidal data are
performed using Student t test or Kruskall-Wallis rank sum analysis
for non-parametric data, and corrected for multiple comparisons as
appropriate.
[0135] The yield of moiety/peptide conjugate formed is determined
using routine methods. For example, HPLC or capillary
electrophoresis or other qualitative or quantitative method can be
used (see, for example, Liu et al., J. Chromatogr. 735:357-366
(1996); Rose et al., J. Chromatogr. 425:419-412 (1988), each of
which is incorporated herein by reference). In particular, the
skilled artisan will recognize that the choice of a method for
determining yield of a conjugation reaction depends, in part, on
the physical and chemical characteristics of the specific moiety
and peptide. Following conjugation, the reaction products can be
desalted to remove any free peptide and free drug.
[0136] Embodiments of the invention provide a method of inducing
programmed cell death in a cell, including contacting the cell with
an isolated peptide, protide or conjugate described herein. In one
aspect, the cell is a microorganism, or in some aspects a
pathogenic microorganism. In some aspects, the pathogenic
microorganism is Staphylococcus aureus, Escherichia coli,
Salmonella typhimurium, Pseudomonas aeruginosa, Bacillus subtilis,
Acinotobacter baumannii, Acinotobacter calcoaceticus, Acinotobacter
haemolyticus, Pseudomonas aeruginosa, Candida albicans, or any of
the pathogenic microorganisms described herein. In another aspect,
the cell is a tumor cell, including both a malignant and
non-malignant tumor cell, or in a preferred aspect a malignant
cell. In yet another aspect, the cell is an immune regulatory cell
or an immune effector cell.
[0137] A peptide, protide or conjugate of the invention useful for
practicing the methods of the invention can be formulated and
administered by those skilled in the art in a manner and in an
amount appropriate for the pathological condition to be treated,
for example, an infection, neoplastic disorder, inflammation; the
rate or amount of inflammation; the weight, gender, age and health
of the individual; the biochemical nature, bioactivity,
bioavailability and side effects of the particular compound; and in
a manner compatible with concurrent treatment regimens. An
appropriate amount and formulation for decreasing the severity of a
pathological condition in humans can be extrapolated from credible
animal models known in the art of the particular disorder. It is
understood, that the dosage of a therapeutic substance has to be
adjusted based on the binding affinity of the substance, such that
a lower dose of a substance exhibiting significantly higher binding
affinity can be administered compared to the dosage necessary for a
substance with lower binding affinity. For a peptide, protide or
conjugate described herein several factors can be taken into
account when determining the proper dosage. For example, for a
protide, the nature of the protide effectors and their bioactivity
upon activation, the anticipated concentration of activator and the
responsiveness of the activator site to presence of the activator,
may be taken into account.
[0138] The total amount of peptide, protide or conjugate can be
administered as a single dose or by infusion over a relatively
short period of time, or can be administered in multiple doses
administered over a more prolonged period of time. Such
considerations will depend on a variety of case-specific factors
such as, for example, whether the disease category is characterized
by acute episodes or gradual or chronic deterioration. For an
individual affected with an acute infection or inflammatory
response, for example, as associated with a bacterial infection,
the substance can be administered as a single dose or by infusion
of several large doses in a relatively short period of time. For an
individual affected with chronic deterioration, for example, as
associated with a neuroinflammatory disorder, the substance can be
administered in a slow-release matrice, which can be implanted for
systemic delivery or at the site of the target tissue, which means
an area proximal to the desired context. Contemplated matrices
useful for controlled release of therapeutic compounds are well
known in the art, and include materials such as DepoFoam.TM.,
biopolymers, micropumps, and the like.
[0139] The peptides, protides and conjugates administered in the
methods of the invention can be administered to the individual by
any number of routes known in the art including, for example,
systemically, such as intravenously or intraarterially. A
therapeutic peptide, protide or conjugate can be provided in the
form of isolated and substantially purified polypetides in
pharmaceutically acceptable formulations using formulation methods
known to those of ordinary skill in the art. These formulations can
be administered by standard routes, including for example, topical,
transdermal, intraperitoneal, intracranial,
intracerebroventricular, intracerebral, intravaginal, intrauterine,
oral, rectal or parenteral such as intravenous, intraspinal,
intrathecal, subcutaneous or intramuscular routes. Intrathecal
administration of a therapeutic peptide, protide or conjugate into
the intradural or subarachnoid space can be an appropriate route
for decreasing the severity of a neuroinflammatory condition.
Intravenous administration of a terhapeutic substance containing a
peptide, protide or conjugate also is a preferred route for
practicing the invention. In addition, a therapeutic substance
administered in the methods of the invention can be incorporated
into biodegradable polymers allowing for sustained release of the
substance useful for prophylactic and reconstitutive applications
described above. Biodegradable polymers and their use are
described, for example, in Brem et al., J. Neurosurg. 74:441 446
(1991), which is incorporated herein by reference.
[0140] The methods for treating a particular pathological condition
additionally can be practiced in conjunction with other therapies.
For example, for treating cancer, the methods of the invention can
be practiced prior to, during, or subsequent to conventional cancer
treatments such as surgery, chemotherapy, including administration
of cytokines and growth factors, radiation or other methods known
in the art. Similarly, for treating pathological conditions which
include infectious disease, the methods of the invention can be
practiced prior to, during, or subsequent to conventional
treatments, such as antibiotic administration, against infectious
agents or other methods known in the art. Treatment of pathological
conditions of autoimmune disorders also can be accomplished by
combining the methods of the invention for inducing an immune
response with conventional treatments for the particular autoimmune
diseases. Conventional treatments include, for example,
chemotherapy, steroid therapy, insulin and other growth factor and
cytokine therapy, passive immunity and inhibitors of T cell
receptor binding. The peptides, protides and conjugates of the
invention can be administered in conjunction with these or other
methods known in the art and at various times prior, during or
subsequent to initiation of conventional treatments. For a
description of treatments for pathological conditions characterized
by aberrant cell growth see, for example, The Merck Manual,
Sixteenth Ed, (Berkow, R., Editor) Rahway, N.J., 1992.
[0141] As described above, administration of a peptide, protide or
conjugate can be, for example, simultaneous with or delivered in
alternative administrations with the conventional therapy,
including multiple administrations. Simultaneous administration can
be, for example, together in the same formulation or in different
formulations delivered at about the same time or immediately in
sequence. Alternating administrations can be, for example,
delivering a peptide, protide or conjugate formulation and a
conventional therapeutic treatment in temporally separate
administrations. Temporally separate administrations of a peptide,
protide or conjugate and conventional therapy can use different
modes of delivery and routes.
[0142] A therapeutic peptide, protide or conjugate containing
substance administered in the methods of the invention also can be
administered as a solution or suspension together with a
pharmaceutically acceptable medium. Such a pharmaceutically
acceptable medium can include, for example, sterile aqueous
solvents such as sodium phosphate buffer, phosphate buffered
saline, normal saline or Ringer's solution or other physiologically
buffered saline, or other solvent or vehicle such as a glycol,
glycerol, an oil such as olive oil or an injectable organic ester.
A pharmaceutically acceptable medium can additionally contain
physiologically acceptable compounds that act, for example,
stabilize the neutralizing agent, increase its solubility, or
increase its absorption. Such physiologically acceptable compounds
include, for example, carbohydrates such as glucose, sucrose or
dextrans; antioxidants such as ascorbic acid or glutathione;
receptor mediated permeabilizers, which can be used to increase
permeability of the blood-brain barrier; chelating agents such as
EDTA, which disrupts microbial membranes; divalent metal ions such
as calcium or magnesium; low molecular weight proteins; lipids or
liposomes; or other stabilizers or excipients. Those skilled in the
art understand that the choice of a pharmaceutically acceptable
carrier depends on the route of administration of the compound
containing the protides and on its particular physical and chemical
characteristics.
[0143] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions such as the
pharmaceutically acceptable mediums described above. The solutions
can additionally contain, for example, buffers, bacteriostats and
solutes which render the formulation isotonic with the blood of the
intended recipient. Other formulations include, for example,
aqueous and non-aqueous sterile suspensions which can include
suspending agents and thickening agents. The formulations can be
presented in unit-dose or multi-dose containers, for example,
sealed ampules and vials, and can be stored in a lyophilized
condition requiring, for example, the addition of the sterile
liquid carrier, immediately prior to use. Extemporaneous injection
solutions and suspensions can be prepared from sterile powders,
granules and tablets of the kind previously described.
[0144] For applications that require the peptide, protide, or
conjugate containing compounds to cross the blood-brain barrier,
formulations that increase the lipophilicity of the compound are
particularly desirable. For example, the neutralizing agent can be
incorporated into liposomes (Gregoriadis, Liposome Technology,
Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla. (1993)).
Liposomes, which consist of phospholipids or other lipids, are
nontoxic, physiologically acceptable and metabolizable carriers
that are relatively simple to make and administer.
[0145] A therapeutic peptide, protide or conjugate containing
substance administered in the methods of the invention can also be
prepared as nanoparticles. Adsorbing peptide compounds onto the
surface of nanoparticles has proven effective in delivering peptide
drugs to the brain (see Kreuter et al., Brain Research 674:171 174
(1995)). Exemplary nanoparticles are colloidal polymer particles of
poly-butylcyanoacrylate with a therapeutic protide-containing
substance to be administered in the methods of the invention
adsorbed onto the surface and then coated with polysorbate 80.
[0146] Image-guided ultrasound delivery of a therapeutic peptide,
protide or conjugate containing substance administered in the
methods of the invention through the blood-brain barrier to
selected locations in the brain can be utilized as described in
U.S. Pat. No. 5,752,515. Briefly, to deliver a therapeutic
substance past the blood-brain barrier a selected location in the
brain is targeted and ultrasound used to induce a change detectable
by imaging in the central nervous system (CNS) tissues and/or
fluids at that location. At least a portion of the brain in the
vicinity of the selected location is imaged, for example, via
magnetic resonance imaging (MRI), to confirm the location of the
change. An therapeutic substance administered in the methods of the
invention into the patient's bloodstream can be delivered to the
confirmed location by applying ultrasound to effect opening of the
blood-brain barrier at that location and, thereby, to induce uptake
of the substance.
[0147] In addition, polypeptides called receptor mediated
permeabilizers (RMP) can be used to increase the permeability of
the blood-brain barrier to molecules such as therapeutic,
prophylactic or diagnostic substances as described in U.S. Pat.
Nos. 5,268,164; 5,506,206; and 5,686,416. These receptor mediated
permeabilizers can be intravenously co-administered to a host with
molecules whose desired destination is the cerebrospinal fluid
compartment of the brain, for example, in the treatment of a
neuroinflammatory condition. The permeabilizer polypeptides or
conformational analogues thereof allow therapeutic substances to
penetrate the blood-brain barrier and arrive at their target
destination which can be selected based on its proximity to the
desired activation context. Such polypeptides can be designed as
part of strategic invention protides.
[0148] In current treatment regimes for most diseases, more than
one compound is often administered to an individual for management
of the same or different aspects of the disease. Similarly, in the
methods of the invention for treating neoplastic condition,
microbial infection, a condition associated with decreased cell
death or inflammatory condition, a therapeutic peptide, protide or
conjugate containing substance can advantageously be formulated
with a second therapeutic compound such as an anti-inflammatory
compound, antimicrobial compound, chemotherapeutic compound,
immunosuppressive compound or any other compound that manages the
same or different aspects of the particular disease. As an example,
for treatment of an infectious disease a therapeutic substance can
advantageously be formulated with a second therapeutic compound
such as an antibiotic. Contemplated methods of treating a
pathological condition by administering to a subject a
therapeutically effective amount of a peptide, protide or conjugate
therefore include administering a therapeutic substance useful in
the methods of the invention alone, in combination with, or in
sequence with, such other compounds. Alternatively, combination
therapies can consist of fusion proteins, where a therapeutic
substance useful for treating a particular pathological condition
is linked to a heterologous protein, such as an invention
protide.
[0149] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also provided within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
Example I
Antimicrobial, Anti-cancer, and other Therapeutic Applications of
Peptides Designed from Programmed Cell Death Effector Proteins
[0150] Candidate proteins representing a variety of nuclear- or
mitochondrial-encoded programmed cell death effector and/or
modulatory molecules from eukaryotic sources were identified for
comparison. When candidate proteins were compared, similarities in
sequences and helical domains that have amphipathic and cationic
characteristics were identified. Analogues were also identified
where possible from prokaryotic sources.
[0151] Template proteins were initially chosen based on the
following: 1) mammalian or other eukaryotic cell nuclear-encoded
peptides with structure-activity relationships to antimicrobial
peptide sequences that afford interaction/control/inhibition of
mitochondria or chloroplast symbionts, or host cell apoptosis
(examples include Bax, Bcl-W, dynamin, mitofusin, nucleolin, and
other programmed cell death effector proteins); and 2) structural
and/or functional homologues of such proteins in prokaryotes
(examples include CidA/B, LrgA/B, and other holin-like or
programmed cell death effector proteins). The identification of
candidate peptide sequences was achieved through an iterative
process, which included a search of protein databases for
additional candidate proteins, multisequence alignments of the
identified candidates, followed by an integration and analysis of
candidate sequences.
[0152] Upon identification of an initial group of template proteins
having the above-identified characteristics, additional candidate
proteins were identified using primary sequence similarity searches
of protein databases using the basic local alignment search tool
(BLAST) available from the National Center for Biotechnology
Information. The searches utilized the amino acid sequences of
candidate template proteins having the desired characteristics as
query sequences. FIG. 3 shows exemplary amino acid sequence used as
the query sequences.
[0153] Once all additional candidate template proteins were
identified, a compiled set of query sequences were submitted for
analysis to the modeling server ClustalW (Larkin et al.,
Bioinformatics 23(21): 2947-2948 (2007)) available online from
EMBL-EBI. Utilizing the modeling server, multisequence alignments
were performed to identify regions of sequence homology within the
candidate sequences or with known host defense or antimicrobial
peptide sequences. For example, candidate proteins were ranked
based on their alignment score (FIG. 4) and/or analyzed for
conserved residues through multisequence alignments (FIG. 5).
Additionally, phylogenetic and cladogenetic analyses were conducted
between the candidate proteins, followed by multisequence
alignments of identified putative helical domains (FIGS. 6-14).
[0154] The results from the above processes were integrated to
analyze and prioritize candidate sequences. The criteria for their
prioritization included: 1) conservation of sequence homology or
motif(s); 2) homology to known antimicrobial or anti-cancer
peptides; 3) similarity to known or recognized antimicrobial
peptide structure-activity relationships (SAR; including presence,
periodicity, and distribution of cationic, hydrophobic, and
aromatic residues); and 4) visual inspection. Candidate sequences
that were identified included mitochondria-targeting proteins, such
as Bcl-2, Bcl-W, Bax, and Mitofusin; NK/Tcyto cell effectors, such
as Granulysin, Granzyme H, Perforin-1 and Azurocidin (CAP37);
apoptosis/cell signaling proteins, such as Fas ligand, Caspase 7
and Dynamin 1; and other related proteins, such as Serpin B9
(CytoPro3), Fractalkine (CX3CL1), CXCL3 and Atrophin 1.
[0155] Following the above interactive primary structure analysis,
a secondary (2.degree.) structure analysis was conducted. This
analysis included visualization, qualitative, and/or quantitative
analyses of candidate sequences identified above. This analysis,
including 3D visualization of target sequences, conformation 3D
homology, qualitative 3D analysis of target sequences, quantitative
3D analysis of target sequences and comparative 3D refinement of
target sequences.
[0156] 3D visualization of target sequences was achieved using Cn3D
software available through PubMed. Exemplary 3D visualization of
identified candidate peptides within the native total protein are
shown in FIG. 15-21. These results identified specific sequences as
novel targets for further analysis/design. The conformational 3D
homology of target sequences to known antimicrobial or anti-cancer
template polypeptides was assessed using the threading and 3D
homology fold recognition server Protein Homology/analogy
Recognition Engine (PHYRE) "Protein structure prediction on the
web: a case study using the Phyre server" (Kelley and Sternberg
Nature Protocols 4:363-371 (2009). Statistical e values were used
to guide prioritization of molecules for further analysis.
[0157] Priority target sequences identified above were visualized
for qualitative analysis of distribution of 3D physicochemical
attributes using the public domain UCSF software package Chimera.
Next, priority target sequences were quantitatively evaluated for
structural homology and/or structure-activity relationships to
known antimicrobial and/or anti-cancer peptides/proteins using a
combinatorial extension method of Shindyalov and Bourne, Protein
Engineering 11:739-747 (1998). The results from these analyses
provided quantitative alignment of compositional elements,
including charged and hydrophobic residues (FIGS. 22-29). The
results included root mean square deviation (RMSD) scores as
quantitative data that allowed further prioritization of target
sequences. The priority target sequences emanating from the process
described above were then used as novel templates for 3D analyses
using VAST and/or 3Dpssm software to identify homologous sequences
and discover other novel target sequences.
[0158] As a final step, a computation simulation of the
antimicrobial activity of the selected sequences was conducted.
Selected target sequences emerging from the above process were
subjected to a computational assessment tool which integrates
multiple physical and biochemical attributes of polypeptide
sequences to generate a predicted minimal inhibitory concentration
(MIC) based on the inverse of the target sequence calculated
hydrophobic moment (1/MH; see U.S. Pat. No. 6,743,769).
[0159] Based on the above process, specific peptide sequences were
identified, which have the desired primary and secondary structure
within the candidate proteins (Tables 1-20). These identified
peptides are predicted to have antimicrobial and anti-cancer
activity. Exemplifying predictive accuracy, candidate peptides have
been synthesized and antimicrobial efficacy has been demonstrated
in vitro against a panel of Gram-positive and Gram-negative
bacteria and fungi (see Example II). Furthermore, specific amino
acid substitutions were identified for several peptide sequences
(Tables 1-19). The identified peptide residues were
prioritized/ranked utilizing to above process (Table 21).
TABLE-US-00001 TABLE 1 Engineered peptides based on Bax protein
(pro- apoptotic protein/human/nuclear-encoded). SEQ ID NO: Amino
Acid Sequence Native Sequence Domains 1.
.sup.104nfnwgrvvalfyfasklvlkalctkv.sup.129 2.
.sup.168twqtvtifvagvltasltiwkk.sup.190 Synthetic Peptides 1.
H.sub.2N-nfnwgrvvalfyfasklvlkalctkv-COOH 1.
H.sub.2N-nfnwgrvvalfyfasklvlkalctkv-CONH.sub.2 3.
H.sub.2N-nfnwgrvvalfyfasklvlkalxtkv-COOH 3.
H.sub.2N-nfnwgrvvalfyfasklvlkalxtkv-CONH.sub.2 4.
H.sub.2N-nfnwgrvvalfyfasklvlkalbtjv-COOH 4.
H.sub.2N-nfnwgrvvalfyfasklvlkalbtjv-CONH.sub.2 5.
H.sub.2N-wgrvvalfyfasklvlkalctkv-COOH 5.
H.sub.2N-wgrvvalfyfasklvlkalctkv-CONH.sub.2 6.
H.sub.2N-wgrvvalfyfasklvlkalxtkv-COOH 6.
H.sub.2N-wgrvvalfyfasklvlkalxtkv-CONH.sub.2 7.
H.sub.2N-rvvalfyfasklvlkalctkv-COOH 7.
H.sub.2N-rvvalfyfasklvlkalctkv-CONH.sub.2 8.
H.sub.2N-rvvalfyfasklvlkalxtkv-COOH 8.
H.sub.2N-rvvalfyfasklvlkalxtkv-CONH.sub.2 9.
H.sub.2N-alfyfasklvlkalctkv-COOH 9.
H.sub.2N-alfyfasklvlkalctkv-CONH.sub.2 10.
H.sub.2N-alfyfasklvlkalxtkv-COOH 10.
H.sub.2N-alfyfasklvlkalxtkv-CONH.sub.2 2.
H.sub.2N-twqtvtifvagvltasltiwkk-COOH 2.
H.sub.2N-twqtvtifvagvltasltiwkk-CONH.sub.2 11.
H.sub.2N-twqtvtifvabvltasltiwkk-COOH 11.
H.sub.2N-twqtvtifvabvltasltiwkk-CONH.sub.2 12.
H.sub.2N-tvtifvagvltasltiwkk-COOH 12.
H.sub.2N-tvtifvagvltasltiwkk-CONH.sub.2 13.
H.sub.2N-tvtifvabvltasltiwkk-COOH 13.
H.sub.2N-tvtifvabvltasltiwkk-CONH.sub.2 Underlined residues
indicate substitutions; x = s, t, y, other natural/non-natural
H-bond donor/acceptor residue; b = k, r, n, q, other
natural/non-natural basic residue; j = cysteine or other
natural/non-natural thiol residue.
TABLE-US-00002 TABLE 2 Engineered peptides based on Bcl-W protein
(pro-apoptotic protein/human/nuclear-encoded). SEQ ID NO: Amino
Acid Sequence Native Sequence Domains 14.
.sup.10tralvadfvgyklrqkgyv.sup.28 15.
.sup.55trfrrtfsdlaaqlhvt.sup.71 16.
.sup.159arrlregnwasvrtvltgavalgalvtvgaffask.sup.193 Synthetic
Peptides 14. H.sub.2N-tralvadfvgyklrqkgyv-COOH 14.
H.sub.2N-tralvadfvgyklrqkgyv-CONH.sub.2 17.
H.sub.2N-tralvabfvgyklrqkgyv-COOH 17.
H.sub.2N-tralvabfvgyklrqkgyv-CONH.sub.2 15.
H.sub.2N-trfrrtfsdlaaqlhvt-COOH 15.
H.sub.2N-trfrrtfsdlaaqlhvt-CONH.sub.2 18.
H.sub.2N-trfrbtfsdlaaqlhvt-COOH 18.
H.sub.2N-trfrbtfsdlaaqlhvt-CONH.sub.2 19.
H.sub.2N-trfrrtfsblaaqlhvt-COOH 19.
H.sub.2N-trfrrtfsblaaqlhvt-CONH.sub.2 20.
H.sub.2N-rrlregnwasvrtvltgavalgalvtvgaffask-COOH 20.
H.sub.2N-rrlregnwasvrtvltgavalgalvtvgaffask-CONH.sub.2 21.
H.sub.2N-rrlrbgnwasvrtvltgavalgalvtvgaffask-COOH 21.
H.sub.2N-rrlrbgnwasvrtvltgavalgalvtvgaffask-CONH.sub.2 22.
H.sub.2N-rrlregnwasvrtvltbavalgalvtvgaffask-COOH 22.
H.sub.2N-rrlregnwasvrtvltbavalgalvtvgaffask-CONH.sub.2 23.
H.sub.2N-rrlregnwasvrtvltgavalbalvtvgaffask-COOH 23.
H.sub.2N-rrlregnwasvrtvltgavalbalvtvgaffask-CONH.sub.2 24.
H.sub.2N-rrlregnwasvrtvltgavalgalvtvbaffask-COOH 24.
H.sub.2N-rrlregnwasvrtvltgavalgalvtvbaffask-CONH.sub.2 25.
H.sub.2N-rrlrbgnwasvrtvltbavalbalvtvbaffask-COOH 25.
H.sub.2N-rrlrbgnwasvrtvltbavalbalvtvbaffask-CONH.sub.2 Underlined
residues indicate substitutions; b = k, r, n, q, other
natural/non-natural basic residue.
TABLE-US-00003 TABLE 3 Engineered peptides based on Bc1-x.beta.
protein(apoptotic protein/human/ nuclear-encoded). SEQ ID NO: Amino
Acid Sequence Native Sequence Domains 26.
.sup.12flsyklsqkgyswsqfs.sup.28 27.
.sup.99lryrrafsdltsqlhitpgtayqsf.sup.122 28.
.sup.187gwvrtkplvcpfslasgqrs.sup.196 29.
.sup.203gqrsptalllylfllcwvivg.sup.223 Synthetic Peptides 26.
H.sub.2N-flsyklsqkgyswsqfs-COOH 26.
H.sub.2N-flsyklsqkgyswsqfs-CONH.sub.2 27.
H.sub.2N-lryrrafsdltsqlhitpgtayqsf-COOH 27.
H.sub.2N-lryrrafsdltsqlhitpgtayqsf-CONH.sub.2 30.
H.sub.2N-lryrrafsbltsqlhitpgtayqsf-COOH 30.
H.sub.2N-lryrrafsbltsqlhitpgtayqsf-CONH.sub.2 28.
H.sub.2N-gwvrtkplvcpfslasgqrs-COOH 28.
H.sub.2N-gwvrtkplvcpfslasgqrs-CONH.sub.2 31.
H.sub.2N-gwvrtkplvxpfslasgqrs-COOH 31.
H.sub.2N-gwvrtkplvxpfslasgqrs-CONH.sub.2 32.
H.sub.2N-gwvrtkplvxpfslasbqrs-COOH 32.
H.sub.2N-gwvrtkplvxpfslasbqrs-CONH.sub.2 29.
H.sub.2N-gqrsptalllylfllcwvivg-COOH 29.
H.sub.2N-gqrsptalllylfllcwvivg-CONH.sub.2 33.
H.sub.2N-gqrsptalxlylfllcwvivg-COOH 33.
H.sub.2N-gqrsptalxlylfllcwvivg-CONH.sub.2 34.
H.sub.2N-gqrsptalllylfllxwvivg-COOH 34.
H.sub.2N-gqrsptalllylfllxwvivg-CONH.sub.2 35.
H.sub.2N-gqrsptalllylfllcwvivb-COOH 35.
H.sub.2N-gqrsptalllylfllcwvivb-CONH.sub.2 36.
H.sub.2N-gqrsptalxlylfllxwvivb-COOH 36.
H.sub.2N-gqrsptalxlylfllxwvivb-CONH.sub.2 Underlined residues
indicate substitutions; b = k, r, n, q, other natural/non-natural
basic residue x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue.
TABLE-US-00004 TABLE 4 Engineered peptides based on Bak protein
(apoptotic protein/human/ nuclear-encoded). SEQ ID NO: Amino Acid
Sequence Native Sequence Domains 37.
.sup.127rvvallgfgyrlalhvyq.sup.144 38.
.sup.187ilnvlvvlgvvllgqfvvrrffks.sup.211 Synthetic Peptides 37.
H.sub.2N-rvvallgfgyrlalhvyq-COOH 37.
H.sub.2N-rvvallgfgyrlalhvyq-CONH.sub.2 39.
H.sub.2N-rvvallbfgyrlalhvyq-COOH 39
H.sub.2N-rvvallbfgyrlalhvyq-CONH.sub.2 40.
H.sub.2N-rvvallgfbyrlalhvyq-COOH 40.
H.sub.2N-rvvallgfbyrlalhvyq-CONH.sub.2 41.
H.sub.2N-rvvallbfbyrlalhvyq-COOH 41.
H.sub.2N-rvvallbfbyrlalhvyq-CONH.sub.2 42.
H.sub.2N-rvvalygfgyrlalhvyq-COOH 42.
H.sub.2N-rvvalygfgyrlalhvyq-CONH.sub.2 43.
H.sub.2N-rvvalwgfgyrlalhvyq-COOH 43.
H.sub.2N-rvvalwgfgyrlalhvyq-CONH.sub.2 44.
H.sub.2N-rvvalybfbyrlalhvyq-COOH 44.
H.sub.2N-rvvalybfbyrlalhvyq-CONH.sub.2 45.
H.sub.2N-rvvalwbfbyrlalhvyq-COOH 45.
H.sub.2N-rvvalwbfbyrlalhvyq-CONH.sub.2 38.
H.sub.2N-ilnvlvvlgvvllgqfvvrrffks-COOH 38.
H.sub.2N-ilnvlvvlgvvllgqfvvrrffks-CONH.sub.2 46.
H.sub.2N-ilnvlvxlgvvllgqfvvrrffks-COOH 46.
H.sub.2N-ilnvlvxlgvvllgqfvvrrffks-CONH.sub.2 47.
H.sub.2N-ilnvlvblvlgqfvrfks-COOH 47.
H.sub.2N-ilnvlvblvlgqfvrfks-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue.
TABLE-US-00005 TABLE 5 Engineered peptides based on Bcl-2 protein
(apoptotic regulator/human). SEQ ID NO: Amino Acid Sequence Native
Sequence Domain 48. .sup.11nreivmkyihyklsqrgy.sup.28 Synthetic
Peptides 48. H.sub.2N-nreivmkyihyklsqrgy-COOH 48.
H.sub.2N-nreivmkyihyklsqrgy-CONH.sub.2 49.
H.sub.2N-nrbivmkyihyklsqrgy-COOH 49.
H.sub.2N-nrbivmkyihyklsqrgy-CONH.sub.2 50.
H.sub.2N-nreivxkyihyklsqrgy-COOH 50.
H.sub.2N-nreivxkyihyklsqrgy-CONH.sub.2 51.
H.sub.2N-nreivmkyibyklsqrgy-COOH 51.
H.sub.2N-nreivmkyibyklsqrgy-CONH.sub.2 52.
H.sub.2N-nrbivxkyibyklsqrgy-COOH 52.
H.sub.2N-nrbivxkyibyklsqrgy-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond donor
residue.
TABLE-US-00006 TABLE 6 Engineered peptides based on Bcl-2 isoform 1
protein (apoptotic regulator/human). SEQ ID NO: Amino Acid Sequence
Native Sequence Domain 53. .sup.54lalrqagddfsrryrg.sup.69 Synthetic
Peptides 53. H.sub.2N-lalrqagddfsrryrg-COOH 53.
H.sub.2N-lalrqagddfsrryrg-CONH.sub.2 54.
H.sub.2N-lalrqagbdfsrryrg-COOH 54.
H.sub.2N-lalrqagbdfsrryrg-CONH.sub.2 55.
H.sub.2N-lalrqagdbfsrryrg-COOH 55.
H.sub.2N-lalrqagdbfsrryrg-CONH.sub.2 56.
H.sub.2N-lalrqagbxfsrryrg-COOH 56.
H.sub.2N-lalrqagbxfsrryrg-CONH.sub.2 57.
H.sub.2N-lalrqaobxfsrryrg-COOH 57.
H.sub.2N-lalrqaobxfsrryrg-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue; o = anthrylalanine or other non-natural
amino acid.
TABLE-US-00007 TABLE 7 Engineered peptides based on Mfn-1 protein
(mitofusin-1; human mitochondrial regulator). SEQ ID NO: Amino Acid
Sequence Native Sequence Domain 58.
.sup.699keidqlekiqnnskllrnkavqlenelenftkqfl.sup.734 Synthetic
Peptides 58. H.sub.2N-keidqlekiqnnskllrnkavqlenelenftkqfl-COOH 58.
H.sub.2N-keidqlekiqnnskllrnkavqlenelenftkqfl-CONH.sub.2 59.
H.sub.2N-keibqlekiqnnskllrnkavqlenelenftkqfl-COOH 59
H.sub.2N-keibqlekiqnnskllrnkavqlenelenftkqfl-CONH.sub.2 60.
H.sub.2N-keibqlbkiqnnskllrnkavqlbnelenftkqfl-COOH 60.
H.sub.2N-keibqlbkiqnnskllrnkavqlbnelenftkqfl-CONH.sub.2 61.
H.sub.2N-keibqlbkiqnnskllrnkavqlbnelenftkqfl-COOH 61.
H.sub.2N-keibqlbkiqnnskllrnkavqlbnelenftkqfl-CONH.sub.2 62.
H.sub.2N-keibqlbkiqnnskllrnkavqlbnblenftkqfl-COOH 62.
H.sub.2N-keibqlbkiqnnskllrnkavqlbnblenftkqfl-CONH.sub.2 63.
H.sub.2N-keibqlbkiqnnskllrnkavqlbnblbnftkqfl-COOH 63.
H.sub.2N-keibqlbkiqnnskllrnkavqlbnblbnftkqfl-CONH.sub.2 64.
H.sub.2N-kiqnnskllrnkavql-COOH 64.
H.sub.2N-kiqnnskllrnkavql-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue.
TABLE-US-00008 TABLE 8 Engineered peptides based on Mfn-2 protein
(mitofusin-1; human mitochondrial regulator). SEQ ID NO: Amino Acid
Sequence Native Sequence Domain 65.
.sup.718nkkievldslqskakllrnkagwldselnmfthqylqpsr.sup.757 Synthetic
Peptides 65. H.sub.2N-nkkievldslqskakllrnkagwldselnmfthqylqpsr-
COOH 65. H.sub.2N-nkkievldslqskakllrnkagwldselnmfthqylqpsr-
CONH.sub.2 66. H.sub.2N-nkkibvldslqskakllrnkagwldselnmfthqylqpsr-
COOH 66. H.sub.2N-nkkibvldslqskakllrnkagwldselnmfthqylqpsr-
CONH.sub.2 67. H.sub.2N-nkkievlbslqskakllrnkagwldselnmfthqylqpsr-
COOH 67. H.sub.2N-nkkievlbslqskakllrnkagwldselnmfthqylqpsr-
CONH.sub.2 68. H.sub.2N-nkkievldslqskakllrnkagwlbselnmfthqylqpsr-
COOH 68. H.sub.2N-nkkievldslqskakllrnkagwlbselnmfthqylqpsr-
CONH.sub.2 69. H.sub.2N-nkkievldslqskakllrnkagwldsblnmfthqylqpsr-
COOH 69. H.sub.2N-nkkievldslqskakllrnkagwldsblnmfthqylqpsr-
CONH.sub.2 70. H.sub.2N-nkkievldslqskakllrnkagwldselnxfthqylqpsr-
COOH 70. H.sub.2N-nkkievldslqskakllrnkagwldselnxfthqylqpsr-
CONH.sub.2 71. H.sub.2N-nkkibvlbslqskakllrnkagwlbsblnxfthqylqpsr-
COOH 71. H.sub.2N-nkkibvlbslqskakllrnkagwlbsblnxfthqylqpsr-
CONH.sub.2 72. H.sub.2N-kkievldslqskakllrnkagwl-COOH 72.
H.sub.2N-kkievldslqskakllrnkagwl-CONH.sub.2 73.
H.sub.2N-kkibvldslqskakllrnkagwl-COOH 73.
H.sub.2N-kkibvldslqskakllrnkagwl-CONH.sub.2 74.
H.sub.2N-kkievlbslqskakllrnkagwl-COOH 74.
H.sub.2N-kkievlbslqskakllrnkagwl-CONH.sub.2 75.
H.sub.2N-kkibvlbslqskakllrnkagwl-COOH 75.
H.sub.2N-kkibvlbslqskakllrnkagwl-CONH.sub.2 Underlined residues
indicate substitutions; b = k, r, n, q, other natural/non-natural
basic residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue.
TABLE-US-00009 TABLE 9 Engineered peptides based on Dnm-1 protein
(dynamin-1; human mitochondrial regulator). SEQ ID NO: Amino Acid
Sequence Native Sequence Domains 76.
.sup.223kllplrrgyigvvnrsqk.sup.240 77.
.sup.256rkfflshpsyrhla.sup.269 78. .sup.294glrnklqsqllsiek.sup.309
79. .sup.323arktkallqmvqqfavdf.sup.340 80.
.sup.411atvkkqvqklk.sup.501 81.
.sup.519virkgwltinnigimkggsk.sup.539 82.
.sup.569nlklrdvekgfmsskhifalfnteqrnvyk.sup.598 83.
.sup.617kasflragvypervgdk.sup.633 Synthetic Peptides 76.
H.sub.2N-kllplrrgyigvvnrsqk-COOH 76.
H.sub.2N-kllplrrgyigvvnrsqk-CONH.sub.2 77.
H.sub.2N-rkfflshpsyrhla-COOH 77. H.sub.2N-rkfflshpsyrhla-CONH.sub.2
78. H.sub.2N-glrnklqsqllsiek-COOH 78.
H.sub.2N-glrnklqsqllsiek-CONH.sub.2 84.
H.sub.2N-glrnklqsqllsibk-COOH 84.
H.sub.2N-glrnklqsqllsibk-CONH.sub.2 79.
H.sub.2N-arktkallqmvqqfavdf-COOH 79.
H.sub.2N-arktkallqmvqqfavdf-CONH.sub.2 85.
H.sub.2N-arktkallqmvqqfavbf-COOH 85.
H.sub.2N-arktkallqmvqqfavbf-CONH.sub.2 80.
H.sub.2N-atvkkqvqklk-COOH 80. H.sub.2N-atvkkqvqklk-CONH.sub.2 81.
H.sub.2N-virkgwltinnigimkggsk-COOH 81.
H.sub.2N-virkgwltinnigimkggsk-CONH.sub.2 82.
H.sub.2N-nlklrdvekgfmsskhifalfnteqrnvyk-COOH 82.
H.sub.2N-nlklrdvekgfmsskhifalfnteqrnvyk-CONH.sub.2 86.
H.sub.2N-nlklrbvekgfmsskhifalfnteqrnvyk-COOH 86.
H.sub.2N-nlklrbvekgfmsskhifalfnteqrnvyk-CONH.sub.2 87.
H.sub.2N-nlklrdvbkgfmsskhifalfnteqrnvyk-COOH 87.
H.sub.2N-nlklrdvbkgfmsskhifalfnteqrnvyk-CONH.sub.2 88.
H.sub.2N-nlklrdvekgfmsskhifalfntbqrnvyk-COOH 88.
H.sub.2N-nlklrdvekgfmsskhifalfntbqrnvyk-CONH.sub.2 89.
H.sub.2N-nlklrbvbkgfmsskhifalfnteqrnvyk-COOH 89.
H.sub.2N-nlklrbvbkgfmsskhifalfnteqrnvyk-CONH.sub.2 90.
H.sub.2N-nlklrbvbkgfmsskhifalfntbqrnvyk-COOH 90.
H.sub.2N-nlklrbvbkgfmsskhifalfntbqrnvyk-CONH.sub.2 83.
H.sub.2N-kasflragvypervgdk-COOH 83.
H.sub.2N-kasflragvypervgdk-CONH.sub.2 91.
H.sub.2N-kasflragvypbrvgdk-COOH 91.
H.sub.2N-kasflragvypbrvgdk-CONH.sub.2 92.
H.sub.2N-kasflragvypervgbk-COOH 92.
H.sub.2N-kasflragvypervgbk-CONH.sub.2 93.
H.sub.2N-kasflragvypbrvgbk-COOH 93.
H.sub.2N-kasflragvypbrvgbk-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue.
TABLE-US-00010 TABLE 10 Engineered peptides based on Dnm-2 protein
(dynamin-2; human mitochondrial regulator). SEQ ID NO: Amino Acid
Sequence Native Sequence Domains 94.
.sup.187klakevdpqglrtigvitkl.sup.206 95.
.sup.295alrsklqsqllslek.sup.309 96.
.sup.323trktkallqmvqqfgv.sup.338 97. .sup.420aivkkqvvklk.sup.421
98. .sup.499aqqrstqlnkkraipnqg.sup.516 99.
.sup.559kekkymlpldnlkirdvekgfmsnkhvfaifnteqrnvyk.sup.598 100.
.sup.569nlkirdvekgfmsnkhvfaifnteqrnvyk.sup.598 101.
.sup.615swkasflragvypekdqa.sup.632 Synthetic Peptides 94.
H.sub.2N-klakevdpqglrtigvitkl-COOH 94.
H.sub.2N-klakevdpqglrtigvitkl-CONH.sub.2 102.
H.sub.2N-klakbvdpqglrtigvitkl-COOH 102.
H.sub.2N-klakbvdpqglrtigvitkl-CONH.sub.2 103.
H.sub.2N-klakevbpqglrtigvitkl-COOH 103.
H.sub.2N-klakevbpqglrtigvitkl-CONH.sub.2 104.
H.sub.2N-klakbvbpqglrtigvitkl-COOH 104.
H.sub.2N-klakbvbpqglrtigvitkl-CONH.sub.2 95.
H.sub.2N-alrsklqsqllslek-COOH 95.
H.sub.2N-alrsklqsqllslek-CONH.sub.2 105.
H.sub.2N-alrsklqsqllslbk-COOH 105
H.sub.2N-alrsklqsqllslbk-CONH.sub.2 96.
H.sub.2N-trktkallqmvqqfgv-COOH 96.
H.sub.2N-trktkallqmvqqfgv-CONH.sub.2 97. H.sub.2N-aivkkqvvklk-COOH
97. H.sub.2N-aivkkqvvklk-CONH.sub.2 106. H.sub.2N-oivkkqvvklk-COOH
106. H.sub.2N-oivkkqvvklk-CONH.sub.2 98.
H.sub.2N-aqqrstqlnkkraipnqg-COOH 98.
H.sub.2N-aqqrstqlnkkraipnqg-CONH.sub.2 99.
H.sub.2N-kekkymlpldnlkirdvekgfmsnkhvfaifnteqrnvyk- COOH 99.
H.sub.2N-kekkymlpldnlkirdvekgfmsnkhvfaifnteqrnvyk- CONH.sub.2 107.
H.sub.2N-kekkymlpldnlkir-COOH 107.
H.sub.2N-kekkymlpldnlkir-CONH.sub.2 108.
H.sub.2N-kxkkymlpldnlkir-COOH 108.
H.sub.2N-kxkkymlpldnlkir-CONH.sub.2 109.
H.sub.2N-kekkyxlpldnlkir-COOH 109.
H.sub.2N-kekkyxlpldnlkir-CONH.sub.2 110.
H.sub.2N-kekkymlplbnlkir-COOH 110.
H.sub.2N-kekkymlplbnlkir-CONH.sub.2 111.
H.sub.2N-kekkymlplxnlkir-COOH 111.
H.sub.2N-kekkymlplxnlkir-CONH.sub.2 112.
H.sub.2N-kxkkymlplbnlkir-COOH 112.
H.sub.2N-kxkkymlplbnlkir-CONH.sub.2 113.
H.sub.2N-kxkkyxlplbnlkir-COOH 113.
H.sub.2N-kxkkyxlplbnlkir-CONH.sub.2 114.
H.sub.2N-kgfmsnkhvfaifnteqrnvyk-COOH 114.
H.sub.2N-kgfmsnkhvfaifnteqrnvyk-CONH.sub.2 115.
H.sub.2N-kxkkymlpldnlkirdvekgfmsnkhvfaifnteqrnvyk- COOH 115.
H.sub.2N-kxkkymlpldnlkirdvekgfmsnkhvfaifnteqrnvyk- CONH.sub.2 116.
H.sub.2N-kekkyxlpldnlkirdvekgfmsnkhvfaifnteqrnvyk- COOH 116.
H.sub.2N-kekkyxlpldnlkirdvekgfmsnkhvfaifnteqrnvyk- CONH.sub.2 117.
H.sub.2N-kekkymlpldnlkirdvekgfxsnkhvfaifnteqrnvyk- COOH 117.
H.sub.2N-kekkymlpldnlkirdvekgfxsnkhvfaifnteqrnvyk- CONH.sub.2 100.
H.sub.2N-nlkirdvekgfmsnkhvfaifnteqrnvyk-COOH 100.
H.sub.2N-nlkirdvekgfmsnkhvfaifnteqrnvyk-CONH.sub.2 118.
H.sub.2N-nlkirbvekgfmsnkhvfaifnteqrnvyk-COOH 118.
H.sub.2N-nlkirbvekgfmsnkhvfaifnteqrnvyk-CONH.sub.2 119.
H.sub.2N-nlkirdvbkgfmsnkhvfaifnteqrnvyk-COOH 119.
H.sub.2N-nlkirdvbkgfmsnkhvfaifnteqrnvyk-CONH.sub.2 120.
H.sub.2N-nlkirbvbkgfmsnkhvfaifntbqrnvyk-COOH 120.
H.sub.2N-nlkirbvbkgfmsnkhvfaifntbqrnvyk-CONH.sub.2 101.
H.sub.2N-swkasflragvypekdqa-COOH 101.
H.sub.2N-swkasflragvypekdqa-CONH.sub.2 121.
H.sub.2N-swkasflragvypbkdqa-COOH 121.
H.sup.2N-swkasflragvypbkdqa-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; o = anthrylalanine or other non-natural amino acid; x = s,
t, y, other natural/non-natural H-bond donor/acceptor residue.
TABLE-US-00011 TABLE 11 Engineered peptides based on Ncl protein
(nucleolin; human mitochondrial regulator). SEQ ID NO: Amino Acid
Sequence Native Sequence Domains 122.
.sup.50qkkgkkaaatsakkvvvs.sup.67 123.
.sup.69tkkvavatpakkaavt.sup.84 124.
.sup.124kkgaaipakgakngknakk.sup.142 125.
.sup.216akgkkaakvvpvkaknva.sup.233 126.
.sup.273vkeapgkrkkemakqkaa.sup.290 127.
.sup.362kaleltglkvfgneiklek.sup.380 128.
.sup.382kgkdskkerdartllaknlpykvtq.sup.406 129.
.sup.419irlvskdgkskgiayi.sup.434 130.
.sup.467kgqnqdyrggknstwsgesktivlsnlsysat.sup.498 131.
.sup.508katfikvpqnqngkskgyafi.sup.528 Synthetic Peptides 122.
H.sub.2N-qkkgkkaaatsakkvvvs-COOH 122.
H.sub.2N-qkkgkkaaatsakkvvvs-CONH.sub.2 132.
H.sub.2N-qkkgkkaxatsakkvyvs-COOH 132.
H.sub.2N-qkkgkkaxatsakkvyvs-CONH.sub.2 133.
H.sub.2N-qkkgkkaaatsakkvyvs-COOH 133.
H.sub.2N-qkkgkkaaatsakkvyvs-CONH.sub.2 134.
H.sub.2N-qkkgkkaaatsakkvwvs-COOH 134.
H.sub.2N-qkkgkkaaatsakkvwvs-CONH.sub.2 135.
H.sub.2N-qkkgkkaxatsakkvyvs-COOH 135.
H.sub.2N-qkkgkkaxatsakkvyvs-CONH.sub.2 136.
H.sub.2N-qkkgkkaxatsakkvwvs-COOH 136.
H.sub.2N-qkkgkkaxatsakkvwvs-CONH.sub.2 123.
H.sub.2N-tkkvavatpakkaavt-COOH 123.
H.sub.2N-tkkvavatpakkaavt-CONH.sub.2 124.
H.sub.2N-kkgaaipakgakngknakk-COOH 124.
H.sub.2N-kkgaaipakgakngknakk-CONH.sub.2 137.
H.sub.2N-kkgaxipakgakngknakk-COOH 137.
H.sub.2N-kkgaxipakgakngknakk-CONH.sub.2 125.
H.sub.2N-akgkkaakvvpvkaknva-COOH 125.
H.sub.2N-akgkkaakvvpvkaknva-CONH.sub.2 138.
H.sub.2N-akgkkaakvvxvkaknva-COOH 138.
H.sub.2N-akgkkaakvvxvkaknva-CONH.sub.2 126.
H.sub.2N-vkeapgkrkkemakqkaa-COOH 126.
H.sub.2N-vkeapgkrkkemakqkaa-CONH.sub.2 139.
H.sub.2N-vkbapgkrkkemakqkaa-COOH 139
H.sub.2N-vkbapgkrkkemakqkaa-CONH.sub.2 140.
H.sub.2N-vkeapgkrkkbmakqkaa-COOH 140.
H.sub.2N-vkeapgkrkkbmakqkaa-CONH.sub.2 141.
H.sub.2N-vkeapgkrkkexakqkaa-COOH 141.
H.sub.2N-vkeapgkrkkexakqkaa-CONH.sub.2 142.
H.sub.2N-vkbapgkrkkbxakqkaa-COOH 142.
H.sub.2N-vkbapgkrkkbxakqkaa-CONH.sub.2 127.
H.sub.2N-kaleltglkvfgneiklek-COOH 127.
H.sub.2N-kaleltglkvfgneiklek-CONH.sub.2 143.
H.sub.2N-kalbltglkvfgneiklek-COOH 143.
H.sub.2N-kalbltglkvfgneiklek-CONH.sub.2 144.
H.sub.2N-kaleltglkvfgnbiklek-COOH 144.
H.sub.2N-kaleltglkvfgnbiklek-CONH.sub.2 145.
H.sub.2N-kaleltglkvfgneiklbk-COOH 145.
H.sub.2N-kaleltglkvfgneiklbk-CONH.sub.2 146.
H.sub.2N-kalbltglkvfgnbiklbk-COOH 146.
H.sub.2N-kalbltglkvfgnbiklbk-CONH.sub.2 128.
H.sub.2N-kgkdskkerdartllaknlpykytq-COOH 128.
H.sub.2N-kgkdskkerdartllaknlpykvtq-CONH.sub.2 147.
H.sub.2N-kgkxskkerdartllaknlpykytq-COOH 147.
H.sub.2N-kgkxskkerdartllaknlpykvtq-CONH.sub.2 148.
H.sub.2N-kgkdskkxrdartllaknlpykytq-COOH 148.
H.sub.2N-kgkdskkxrdartllaknlpykvtq-CONH.sub.2 149.
H.sub.2N-kgkdskkerbartllaknlpykytq-COOH 149.
H.sub.2N-kgkdskkerbartllaknlpykvtq-CONH.sub.2 150.
H.sub.2N-kgkxskkbrbartllaknlpykytq-COOH 150.
H.sub.2N-kgkxskkbrbartllaknlpykvtq-CONH.sub.2 129.
H.sub.2N-irlvskdgkskgiayi-COOH 129.
H.sub.2N-irlvskdgkskgiayi-CONH.sub.2 151.
H.sub.2N-irlvskfgkskgiayi-COOH 151.
H.sub.2N-irlvskfgkskgiayi-CONH.sub.2 152.
H.sub.2N-irlvskygkskgiayi-COOH 152.
H.sub.2N-irlvskygkskgiayi-CONH.sub.2 153.
H.sub.2N-irlvskwgkskgiayi-COOH 153
H.sub.2N-irlvskwgkskgiayi-CONH.sub.2 154.
H.sub.2N-irlvsklwgkskgiayi-COOH 154
H.sub.2N-irlvsklwgkskgiayi-CONH.sub.2 155.
H.sub.2N-irlvskdgkskg-COOH 155. H.sub.2N-irlvskdgkskg-CONH.sub.2
156. H.sub.2N-irlvskfgkskgi-COOH 156.
H.sub.2N-irlvskfgkskg-CONH.sub.2 157. H.sub.2N-irlvskygkskg-COOH
157. H.sub.2N-irlvskygkskg-CONH.sub.2 158.
H.sub.2N-irlvskwgkskg-COOH 158. H.sub.2N-irlvskwgkskg-CONH.sub.2
159. H.sub.2N-irlvsklwgkskg-COOH 159.
H.sub.2N-irlvsklwgkskgi-CONH.sub.2 130.
H.sub.2N-kgqnqdyrggknstwsgesktlvlsnlsysat-COOH 130.
H.sub.2N-kgqnqdyrggknstwsgesktlvlsnlsysat-CONH.sub.2 160.
H.sub.2N-kgqnqbyrggknstwsgesktlvlsnlsysat-COOH 160.
H.sub.2N-kgqnqbyrggknstwsgesktlvlsnlsysat-CONH.sub.2 161.
H.sub.2N-kgqnqdyrggknstwsgbsktlvlsnlsysat-COOH 161.
H.sub.2N-kgqnqdyrggknstwsgbsktlvlsnlsysat-CONH.sub.2 162.
H.sub.2N-kgqnqbyrggknstwsgbsktlvlsnlsysat-COOH 162
H.sub.2N-kgqnqbyrggknstwsgbsktlvlsnlsysat-CONH.sub.2 163.
H.sub.2N-kgbnqdyrlgknstwsgbsktlvlsnlsysat-COOH 163.
H.sub.2N-kgbnqdyrlgknstwsgbsktlvlsnlsysat-CONH.sub.2 164.
H.sub.2N-kgbnqdyrlgknstwsgbskt-COOH 164.
H.sub.2N-kgbnqdyrlgknstwsgbskt-CONH.sub.2 131.
H.sub.2N-katfikvpqnqngkskgyafi-COOH 131.
H.sub.2N-katfikvpqnqngkskgyafi-CONH.sub.2 165.
H.sub.2N-katfikvpqnqnxkskgyafi-COOH 165.
H.sub.2N-katfikvpqnqnxkskgyafi-CONH.sub.2 166.
H.sub.2N-katfikvpqnqnlkskgyafi-COOH 166.
H.sub.2N-katfikvpqnqnlkskgyafi-CONH.sub.2 167.
H.sub.2N-katfikvpqnqnykskgyafi-COOH 167.
H.sub.2N-katfikvpqnqnykskgyafi-CONH.sub.2 168.
H.sub.2N-katfikvpqnqnfkskgyafi-COOH 168.
H.sub.2N-katfikvpqnqnfkskgyafi-CONH.sub.2 169.
H.sub.2N-katfikvpqnqnwkskgyafi-COOH 169.
H.sub.2N-katfikvpqnqnwkskgyafi-CONH.sub.2 170.
H.sub.2N-katfikvpqnqngkskgy-COOH 170.
H.sub.2N-katfikvpqnqngkskgy-CONH.sub.2 171.
H.sub.2N-katfikvpqnqnxkskgy-COOH 171.
H.sub.2N-katfikvpqnqnxkskgy-CONH.sub.2 172.
H.sub.2N-katfikvpqnqnlkskgy-COOH 172.
H.sub.2N-katfikvpqnqnlkskgy-CONH.sub.2 173.
H.sub.2N-katfikvpqnqnykskgy-COOH 173.
H.sub.2N-katfikvpqnqnykskgy-CONH.sub.2 174.
H.sub.2N-katfikvpqnqnfkskgy-COOH 174.
H.sub.2N-katfikvpqnqnfkskgy-CONH.sub.2 175.
H.sub.2N-katfikvpqnqnwkskgy-COOH 175.
H.sub.2N-katfikvpqnqnwkskgy-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue.
TABLE-US-00012 TABLE 12 Engineered peptides based on Csp3 protein
(caspase 3; apoptosis effector/nuclear encoded). SEQ ID NO: Amino
Acid Sequence Native Sequence Domains 176.
.sup.10sksiknlepkiihgs.sup.24 177.
.sup.136lkkitnffrgdrcrsltgkpklfiiqacrgt.sup.166 178.
.sup.215fiqslcamlkqyadklefmhiltrvnrkvat.sup.245 Synthetic Peptides
176. H.sub.2N-sksiknlepkiihgs-COOH 176.
H.sub.2N-sksiknlepkiihgs-CONH.sub.2 179.
H.sub.2N-sksiknlbpkiihgs-COOH 179.
H.sub.2N-sksiknlbpkiihgs-CONH.sub.2 180.
H.sub.2N-sksiknlepkiiygs-COOH 180.
H.sub.2N-sksiknlepkiiygs-CONH.sub.2 181.
H.sub.2N-sksiknlepkiiybs-COOH 181.
H.sub.2N-sksiknlepkiiybs-CONH.sub.2 182.
H.sub.2N-sksiknlbpkiiybs-COOH 182.
H.sub.2N-sksiknlbpkiiybs-CONH.sub.2 177.
H.sub.2N-lkkitnffrgdrcrsltgkpklfiiqacrgt-COOH 177.
H.sub.2N-lkkitnffrgdrcrsltgkpklfiiqacrgt-CONH.sub.2 183.
H.sub.2N-lkkitnffrgbrcrsligkpklfiiqacrgt-COOH 183.
H.sub.2N-lkkitnffrgbrcrsltgkpklfiiqacrgt-CONH.sub.2 184.
H.sub.2N-lkkitnffrgdrxrsltgkpklfiiqacrgt-COOH 184.
H.sub.2N-lkkitnffrgdrxrsltgkpklfiiqacrgt-CONH.sub.2 185.
H.sub.2N-lkkitnffrgdrcrsltgkpklfiiqaxrgt-COOH 185.
H.sub.2N-lkkitnffrgdrcrsltgkpklfiiqaxrgt-CONH.sub.2 186.
H.sub.2N-lkkitnfrgbrxrsligkpklfiiqaxrgt-COOH 186.
H.sub.2N-lkkitnfrgbrxrsltgkpklfiiqaxrgt-CONH.sub.2 187.
H.sub.2N-lkkitnfrgbrxrsligk-COOH 187.
H.sub.2N-lkkitnfrgbrxrsltgk-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue.
TABLE-US-00013 TABLE 13 Engineered peptides based on Bad protein
(apoptotic protein/human/nuclear-encoded). SEQ ID NO: Amino Acid
Sequence Native Sequence Domains 188.
.sup.93frgrsrsappnlwaaqrygrelrr.sup.116 189.
.sup.115rrmsdefvdsfkkglprpksagtatq.sup.140 190.
.sup.121fvdsikkglprpksagtatq.sup.140 Synthetic Peptides 188.
H.sub.2N-frgrsrsappnlwaaqrygrelrr-COOH 188.
H.sub.2N-frgrsrsappnlwaaqrygrelrr-CONH.sub.2 191.
H.sub.2N-frgrsrsappnlwaaqrygrblrr-COOH 191.
H.sub.2N-frgrsrsappnlwaaqrygrblrr-CONH.sub.2 189.
H.sub.2N-rrmsdefvdsfkkglprpksagtatq-COOH 189.
H.sub.2N-rrmsdefvdsfkkglprpksagtatq-CONH.sub.2 192.
H.sub.2N-rrmsbefvdsfkkglprpksagtatq-COOH 192.
H.sub.2N-rrmsbefvdsfkkglprpksagtatq-CONH.sub.2 193.
H.sub.2N-rrmsdbfvdsfkkglprpksagtatq-COOH 193.
H.sub.2N-rrmsdbfvdsfkkglprpksagtatq-CONH.sub.2 194.
H.sub.2N-rrmsdefvbsfkkglprpksagtatq-COOH 194.
H.sub.2N-rrmsdefvbsfkkglprpksagtatq-CONH.sub.2 195.
H.sub.2N-rrmsbbfvbsfkkglprpksagtatq-COOH 195.
H.sub.2N-rrmsbbfvbsfkkglprpksagtatq-CONH.sub.2 196.
H.sub.2N-rrxsbbfvbsfkkglprpksagtatq-COOH 196.
H.sub.2N-rrxsbbfvbsfkkglprpksagtatq-CONH.sub.2 190.
H.sub.2N-fvdsfkkglprpksagtatq-COOH 190.
H.sub.2N-fvdsfkkglprpksagtatq-CONH.sub.2 197.
H.sub.2N-fvbsfkkglprpksagtatq-COOH 197.
H.sub.2N-fvbsfkkglprpksagtatq-CONH.sub.2 198.
H.sub.2N-fvbsfkkglxrpksag-COOH 198.
H.sub.2N-fvbsfkkglxrpksag-CONH.sub.2 199.
H.sub.2N-fvbsfkkglyrpksag-COOH 199.
H.sub.2N-fvbsfkkglyrpksag-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue.
TABLE-US-00014 TABLE 14 Engineered peptides based on Prf-1 protein
(perforin-1/apoptotic/ human/nuclear-encoded). SEQ ID NO: Amino
Acid Sequence Native Sequence Domains 200.
.sup.32krshkfvpgawlag.sup.45 201. .sup.49vtslrrsgsfpvdtqrflr.sup.68
202. .sup.123rsirndwkvgldvtpk.sup.138 203.
.sup.356rrealrralsqyltdrarwr.sup.375 204.
.sup.520nlnhghlkfryhar.sup.534 Synthetic Peptides 200.
H.sub.2N-krshkfvpgawlag-COOH 200.
H.sub.2N-krshkfvpgawlag-CONH.sub.2 205.
H.sub.2N-krshkfvxgawlag-COOH 205.
H.sub.2N-krshkfvxgawlag-CONH.sub.2 201.
H.sub.2N-vtslrrsgsfpvdtqrflr-COOH 201.
H.sub.2N-vtslrrsgsfpvdtqrflr-CONH.sub.2 206.
H.sub.2N-vtslrrsgsfxvdtqrflr-COOH 206.
H.sub.2N-vtslrrsgsfxvdtqrflr-CONH.sub.2 207.
H.sub.2N-vtslrrsgsfpvbtqrflr-COOH 207.
H.sub.2N-vtslrrsgsfpvbtqrflr-CONH.sub.2 208.
H.sub.2N-vtslrrsgsfxvbtqrflr-COOH 208.
H.sub.2N-vtslrrsgsfxvbdtqrflr-CONH.sub.2 202.
H.sub.2N-rsirndwkvgldvtpk-COOH 202.
H.sub.2N-rsirndwkvgldvtpk-CONH.sub.2 209.
H.sub.2N-rsirnbwkvgldvtpk-COOH 209.
H.sub.2N-rsirnbwkvgldvtpk-CONH.sub.2 210.
H.sub.2N-rsirndwkvgldvt-COOH 210.
H.sub.2N-rsirndwkvgldvt-CONH.sub.2 211.
H.sub.2N-rsirnbwkvgldvt-COOH 211.
H.sub.2N-rsirnbwkvgldvt-CONH.sub.2 203.
H.sub.2N-rrealrralsqyltdrarwr-COOH 203.
H.sub.2N-rrealrralsqyltdrarwr-CONH.sub.2 212.
H.sub.2N-rrbalrralsqyltdrarwr-COOH 212.
H.sub.2N-rrbalrralsqyltdrarwr-CONH.sub.2 213.
H.sub.2N-rrealrralsqyltbrarwr-COOH 213.
H.sub.2N-rrealrralsqyltbrarwr-CONH.sub.2 214.
H.sub.2N-rrbalrralsqyltbrarwr-COOH 214.
H.sub.2N-rrbalrralsqyltbrarwr-CONH.sub.2 215.
H.sub.2N-rxbalrralsqyltbrarwr-COOH 215.
H.sub.2N-rxbalrralsqyltbrarwr-CONH.sub.2 204
H.sub.2N-nlnhghlkfryhar-COOH 204.
H.sub.2N-nlnhghlkfryhar-CONH.sub.2 216.
H.sub.2N-nlnbghlkfryhar-COOH 216.
H.sub.2N-nlnbghlkfryhar-CONH.sub.2 217.
H.sub.2N-nlnbgblkfryhar-COOH 217.
H.sub.2N-nlnbgblkfryhar-CONH.sub.2 218.
H.sub.2N-nlnbgblkfrybar-COOH 218.
H.sub.2N-nlnbgblkfrybar-CONH.sub.2 219.
H.sub.2N-nlnxghlkfryhar-COOH 219.
H.sub.2N-nlnxghlkfryhar-CONH.sub.2 220.
H.sub.2N-nlnbgxlkfryhar-COOH 220.
H.sub.2N-nlnbgxlkfryhar-CONH.sub.2 221.
H.sub.2N-nlnbgblkfryxar-COOH 221.
H.sub.2N-nlnbgblkfryxar-CONH.sub.2 222.
H.sub.2N-olnbgblkfrybar-COOH 222.
H.sub.2N-olnbgblkfrybar-CONH.sub.2 223.
H.sub.2N-olnbgblkfryxar-COOH 223.
H.sub.2N-olnbgblkfryxar-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue; o = anthrylalanine or other non-natural
amino acid.
TABLE-US-00015 TABLE 15 Engineered peptides based on Granulysin
protein (granulysin-1/apoptotic/human/ nuclear-encoded). SEQ ID NO:
Amino Acid Sequence Native Sequence Domains 224.
.sup.62lgrdyrtcltivqklkk.sup.78 225.
.sup.82kptqrsysnaatrvcrtgrsrwr.sup.104 226.
.sup.101srwrrryqsrvtqglvag.sup.125 Synthetic Peptides 224.
H.sub.2N-lgrdyrtcltivqklkk-COOH 224.
H.sub.2N-lgrdyrtcltivqklkk-CONH.sub.2 227.
H.sub.2N-lgrbyrtcltivqklkk-COOH 227.
H.sub.2N-lgrbyrtcltivqklkk-CONH.sub.2 228.
H.sub.2N-lgrdyrtxltivqklkk-COOH 228.
H.sub.2N-lgrdyrtxltivqklkk-CONH.sub.2 229.
H.sub.2N-lgrbyrtxltivqklkk-COOH 229.
H.sub.2N-lgrbyrtxltivqklkk-CONH.sub.2 225.
H.sub.2N-kptqrsvsnaatrvcrtgrsrwr-COOH 225.
H.sub.2N-kptqrsvsnaatrvcrtgrsrwr-CONH.sub.2 230.
H.sub.2N-kptqrsvsnaatrvxrtgrsrwr-COOH 230.
H.sub.2N-kptqrsvsnaatrvxrtgrsrwr-CONH.sub.2 231.
H.sub.2N-kptqrsvsnaatrvxrtg-COOH 231.
H.sub.2N-kptqrsvsnaatrvxrtg-CONH.sub.2 232.
H.sub.2N-kptqrsvsnyatrvxrtg-COOH 232.
H.sub.2N-kptqrsvsnyatrvxrtg-CONH.sub.2 233.
H.sub.2N-kptqrsvsnfatrvxrtg-COOH 233.
H.sub.2N-kptqrsvsnfatrvxrtg-CONH.sub.2 226.
H.sub.2N-srwrrryqsrvtqglvag-COOH 226.
H.sub.2N-srwrrryqsrvtqglvag-CONH.sub.2 234.
H.sub.2N-srwrryqsrvtqylvag-COOH 234.
H.sub.2N-srwrryqsrvtqylvag-CONH.sub.2 235.
H.sub.2N-orwrryqsrvtqylvag-COOH 235.
H.sub.2N-orwrryqsrvtqylvag-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue.
TABLE-US-00016 TABLE 16 Engineered peptides based on CidA protein
(pro-programmed cell death protein/ S. aureus). SEQ ID NO: Amino
Acid Sequence Native Sequence Domains 236.
.sup.28qkifhlplagsivglflfylllqfkiv.sup.54 237.
.sup.88eitlnyilffaviiigtcivalssgyiaekmsykhkqrkgi.sup.127 Synthetic
Peptides 236. H.sub.2N-qkifhlplagsivglflfylllqfkiv-COOH 236.
H.sub.2N-qkifhlplagsivglflfylllqfkiv-CONH.sub.2 238.
H.sub.2N-qkifhlplabsivglflfylllqfkiv-COOH 238.
H.sub.2N-qkifhlplabsivglflfylllqfkiv-CONH.sub.2 239.
H.sub.2N-qkifhlplagsivglflfylglqfkiv-COOH 239.
H.sub.2N-qkifhlplagsivglflfylglqfkiv-CONH.sub.2 240.
H.sub.2N-qkifhlplabsivglflfylglqfkiv-COOH 240.
H.sub.2N-qkifhlplabsivglflfylglqfkiv-CONH.sub.2 241.
H.sub.2N-labsivglflfylglqfkiv-COOH 241.
H.sub.2N-labsivglflfylglqfkiv-CONH.sub.2 242.
H.sub.2N-labsivblflfylglqfkiv-COOH 242.
H.sub.2N-labsivblflfylglqfkiv-CONH.sub.2 237.
H.sub.2N-eitlnyilffaviiigtcivalssgyiaekmsvkhkqrkgi- COOH 237.
H.sub.2N-eitlnyilffaviiigtcivalssgyiaekmsvkhkqrkgi- CONH.sub.2 243.
H.sub.2N-eitlnyilffaviiigtxivalssgyiaekxsvkhkqrkgi- COOH 243.
H.sub.2N-eitlnyilffaviiigtxivalssgyiaekxsvkhkqrkgi- CONH.sub.2 244.
H.sub.2N-aekmsvkhkqrkgi-COOH 244.
H.sub.2N-aekmsvkhkqrkgi-CONH.sub.2 245.
H.sub.2N-abkmsvkhkqrkgi-COOH 245.
H.sub.2N-abkmsvkhkqrkgi-CONH.sub.2 246.
H.sub.2N-alkmsvkhkqrkgi-COOH 246.
H.sub.2N-alkmsvkhkqrkgi-CONH.sub.2 247.
H.sub.2N-alkxsvkhkqrkgi-COOH 247.
H.sub.2N-alkxsvkhkqrkgi-COOH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue.
TABLE-US-00017 TABLE 17 Engineered peptides based on LrgA protein
(anti-programmed cell death protein/ S. aureus). SEQ ID NO: Amino
Acid Sequence Native Sequence Domains 248.
.sup.123kvtsrskgdkvtkkiki.sup.139 Synthetic Peptides 248.
H.sub.2N-kvtsrskgdkvtkkiki-COOH 248.
H.sub.2N-kvtsrskgdkvtkkiki-CONH.sub.2 249.
H.sub.2N-kvtsrskgdkvtkwiki-COOH 249.
H.sub.2N-kvtsrskgdkvtkwiki-CONH.sub.2 250.
H.sub.2N-kvtsrskgdkvtkziki-COOH 250.
H.sub.2N-kvtsrskgdkvtkziki-CONH.sub.2 251.
H.sub.2N-kvtsrskgdkvtkxiki-COOH 251.
H.sub.2N-kvtsrskgdkvtkxiki-CONH.sub.2 Underlined residues indicate
substitutions; z = d, e, other anionic residue; x = s, t, y, other
natural/non-natural H-bond donor/acceptor residue.
TABLE-US-00018 TABLE 18 Engineered peptides based on Lambda S21
protein (lytic regulator protein/.lamda.21 phage). SEQ ID NO: Amino
Acid Sequence Native Sequence Domains 252.
.sup.44slvlgfltyltnlyfkiredrrkaarge.sup.71 Synthetic Peptides 252.
H.sub.2N-slvlgfltyltnlyfkiredrrkaarge-COOH 252.
H.sub.2N-slvlgfltyltnlyfkiredrrkaarge-CONH.sub.2 253.
H.sub.2N-slvlgfltyltnlyfkirbdrrkaarge-COOH 253.
H.sub.2N-slvlgfltyltnlyfkirbdrrkaarge-CONH.sub.2 254.
H.sub.2N-slvlgfltyltnlyfkirebrrkaarge-COOH 254.
H.sub.2N-slvlgfltyltnlyfkirebrrkaarge-CONH.sub.2 255.
H.sub.2N-slvlgfltyltnlyfkirxxrrkaarge-COOH 255.
H.sub.2N-slvlgfltyltnlyfkirxxrrkaarge-CONH.sub.2 256.
H.sub.2N-lyfkirxxrrkaarg-COOH 256.
H.sub.2N-lyfkirxxrrkaarg-CONH.sub.2 Underlined residues indicate
substitutions; b = k, r, n, q, other natural/non-natural basic
residue; x = s, t, y, other natural/non-natural H-bond
donor/acceptor residue.
TABLE-US-00019 TABLE 19 Engineered peptides based on Holin protein
(lytic regulatory protein/ Enterobacteria .lamda. phage). SEQ ID
NO: Amino Acid Sequence Native Sequence Domains 257.
.sup.27aylrgrynggaftktvi.sup.44 258.
.sup.84sigslikrfaakkagvedgrnq.sup.105 259.
.sup.84sigslikrfaakkagv.sup.100 Synthetic Peptides 257.
H.sub.2N-aylrgrynggaftktvi-COOH 257.
H.sub.2N-aylrgrynggaftktvi-CONH.sub.2 260.
H.sub.2N-oylrgrynggaftktvi-COOH 260.
H.sub.2N-oylrgrynggaftktvi-CONH.sub.2 258.
H.sub.2N-sigslikrfaakkagvedgrnq-COOH 258.
H.sub.2N-sigslikrfaakkagvedgrnq-CONH.sub.2 261.
H.sub.2N-sigslikrfaakkagvbdgrnq-COOH 261.
H.sub.2N-sigslikrfaakkagvbdgrnq-CONH.sub.2 262.
H.sub.2N-sigslikrfaakkagvebgrnq-COOH 262.
H.sub.2N-sigslikrfaakkagvebgrnq-CONH.sub.2 259
H.sub.2N-sigslikrfaakkagv-COOH 259.
H.sub.2N-sigslikrfaakkagv-CONH.sub.2 263.
H.sub.2N-sigslikrfaxkkagv-COOH 263.
H.sub.2N-sigslikrfaxkkagv-CONH.sub.2 Underlined residues indicate
substitutions; o = anthrylalanine or other non-natural amino acid;
b = k, r, n, q, other natural/non-natural basic residue; x = s, t,
y, other natural/non-natural H-bond donor/acceptor residue.
TABLE-US-00020 TABLE 20 Additional engineered peptides based on
programmed cell death effector proteins. SEQ ID Native Protein NO:
Amino Acid Sequence Human Bcl-xL 288
H.sub.2N-SQSNRELVVDFLSYKLSQK-COOH Human Bcl-xL 288
H.sub.2N-SQSNRELVVDFLSYKLSQK-CONH.sub.2 Human CTL 289
H.sub.2N-QKLKKMVDKPTQRSVSN-COOH Granulysin Human CTL 289
H.sub.2N-QKLKKMVDKPTQRSVSN-CONH.sub.2 Granulysin
TABLE-US-00021 TABLE 21 Novel Therapeutic Peptide Designs Based on
Programmed Cell Death Effector Domains. SEQ ID ID Name Sequence NO:
Length Priority BaxP-I-18 H.sub.2N-alfyfasklvlkalytkv-CONH.sub.2
264. 18 1 CidA-II-12 H.sub.2N-alkysvkhkqrkgi-CONH.sub.2 265. 14 2
Ncl-VIII-6 H.sub.2N-irlvskygkskgiayi-CONH.sub.2 152. 16 3
Csp3-II-12 H.sub.2N-lkkitnfrgkryrsltgk-CONH.sub.2 266. 18 4
Dnm2-II-4 H.sub.2N-alrsklqsqllslrk-CONH.sub.2 267. 15 5 Dnm1-IV-2
H.sub.2N-atvkkqvqklk-CONH.sub.2 80. 11 6 BclXb-I-2
H.sub.2N-flsyklsqkgyswsqfs-CONH.sub.2 26. 17 7 Hol-III-4
H.sub.2N-sigslikrfaykkagv-CONH.sub.2 268. 16 8 Mfn1-II-2
H.sub.2N-kiqnnskllrnkavql-CONH.sub.2 64. 16 9 BclWP-I-4
H.sub.2N-tralvakfvgyklrqkgyv-CONH.sub.2 269. 19 10 LrgA-I-4
H.sub.2N-kvtsrskgdkvtkwiki-CONH.sub.2 249. 17 11 BaxP-I-1
H.sub.2N-nfnwgrvvalfyfasklvlkalytkv-CONH.sub.2 270. 26 BaxP-II-8
H.sub.2N-tvtifvakvltasltiwkk-CONH.sub.2 271. 19 BclWP-II-6
H.sub.2N-trfrrtfsklaaqlhvt-CONH.sub.2 272. 17 BclXb-IV-10
H.sub.2N-gqrsptalslylfllywvivk-CONH.sub.2 273. 21 Mfn1-II-20
H.sub.2N-kkievlkslqskakllrnkagwl-CONH.sub.2 274. 23 Dnm1-III-4
H.sub.2N-glrnklqsqllsikk-CONH.sub.2 275. 15 Dnm2-I-4
H.sub.2N-klakkvdpqglrtigvitkl-CONH.sub.2 276. 21 Dnm2-VI-16
H.sub.2N-kskkytlplknlkir-CONH.sub.2 277. 15 Csp3-I-10
H.sub.2N-sksiknlkpkiiyks-CONH.sub.2 278. 15 CidA-I-12
H.sub.2N-laksivrlflfylglqfkiv-CONH.sub.2 279. 20
Example II
In Vitro Antimicrobial Assay
[0160] The following assay is designed to measure the relative
antimicrobial activity of peptides by determining zones of growth
inhibition.
[0161] The top eleven prioritized target sequences identified in
Example I (see Table 21) were synthesized by solid-phase chain
extension synthesis using conventional techniques. Each synthetic
peptide was purified by RP-HPLC, and authenticated for purity and
correct sequence by mass spectroscopy. Stock concentrations of the
synthetic peptides were prepared at 1 mg/mL in 0.01% acetic acid
and adjusted to pH 7.2. Synthetic peptides were assessed for
antimicrobial efficacy, spectra, and conditional optima (pH 5.5 or
7.5) using the following modified radial diffusion assay, as
detailed in Yount and Yeaman, PNAS 1010:7363-7368 (2004).
[0162] Media Preparation
[0163] Molecular grade agarose (1.0%) in 10 mM
NaH.sub.2PO.sub.4H.sub.2O was prepared, pH adjusted to 7.5 or 5.5,
and autoclaved for 15 minutes at 121.degree. C., then held in a
waterbath set at 48.degree. C. until used. Mueller Hinton II
overlay agarose was prepared by adding molecular grade agarose to
Mueller Hinton II Broth at a final concentration of 1.0%, pH
adjusted to 7.5 or 5.5, autoclaved for 10 minutes at 121.degree.
C., and then held at 48.degree. C. until used.
[0164] Inoculum Preparation
[0165] Trypticase Soy Broth (TSB) (10 mL) we inoculated with an
overnight growth of the test organism and incubated three to six
hours until the organism reached log phase. The cells were
collected by centrifugation, washed in PBS, then 0.01% acetic acid
adjusted to pH 7.2. The pellet was resuspended in TSB and
standardized to a 0.5 McFarland turbidity standard. A 10 .mu.l
aliquot of the inoculum is added to 10 mL of the pH-adjusted 1.0%
molecular grade agarose cooled to 48.degree. C. resulting in a
final inoculum concentration of 5.times.10.sup.5 CFU/mL. The
suspension is poured into a 15.times.100 mm Petri dish and allowed
to solidify.
[0166] After solidification had occurred, five 4 mm diameter wells
were bored into the agarose. The central well was used as the
acetic acid control while 10 .mu.l of peptide stock solution was
added to each of the other wells resulting in a final concentration
of 10 .mu.g peptide/well. The plates were incubated upright for
three hours at 37.degree. C., then overlaid with 10 mL of Mueller
Hinton II agarose. After the overlay solidified, the plates were
inverted and incubated overnight at 37.degree. C.
[0167] Activity Determination
[0168] The synthetic peptides identified in Table 22 were assayed
for antimicrobial activity against known pathogenic microorganisms.
These pathogenic microorganisms included five species of bacteria
(Staphylococcus aureus, Escherichia coli, Salmonella typhimurium,
Pseudomonas aeruginosa and Bacillus subtilis) and one species of
fungi (Candida albicans) (see Table 23). Zones of growth inhibition
were measured and were grouped as complete and/or partial growth
zones for the assayed microorganism. Zones were considered complete
clearance when there was no visible growth (i.e. completely clear
or free of growth). Zones were considered partial clearance when
growth was impeded or partially cleared (i.e. reduction in
microbial density as compared to adjacent confluent growth). The
larger the zone size, the greater the antimicrobial activity of the
test peptide. The lack of a zone is an indication of no
antimicrobial activity of the test peptide against the target
organism under the conditions tested.
TABLE-US-00022 TABLE 22 Peptide key for antimicrobial assay (FIGS.
30-35). SEQ ID Template Identifier* Design Sequence NO: Length
Holin protein Hol-III-4 H.sub.2N-sigslikrfaykkagv-CONH.sub.2 268.
16 Dynamin-2 Dnm2-II-4 H.sub.2N-alrsklqsqllslrk-CONH.sub.2 267. 15
BclW protein BclWP-I-4 H.sub.2N-tralvakfvgyklrqkgyv-CONH.sub.2 269.
19 Caspase-3 Csp3-II-12 H.sub.2N-lkkitnfrgkryrsltgk-CONH.sub.2 266
18 LrgA protein LrgA-I-4 H.sub.2N-kvtsrskgdkvtkwiki-CONH.sub.2 249.
17 Dynamin-1 Dnm1-IV-2 H.sub.2N-atvkkqvqklk-CONH.sub.2 80. 11 BclXb
protein BclXb-I-2 H.sub.2N-flsyklsqkgyswsqfs-CONH.sub.2 26. 17
Nucleolin Ncl-VIII-6 H.sub.2N-irlvskygkskgiayi-CONH.sub.2 152. 16
Mitofusin-1 Mfn1-II-2 H.sub.2N-kiqnnskllrnkavql-CONH.sub.2 64. 16
Bax protein BaxP-I-18 H.sub.2N-alfyfasklvlkalytkv-CONH.sub.2 264.
18 CidA protein CidA-II-12 H.sub.2N-alkysvkhkqrkgi-CONH.sub.2 265.
14 *Note: identifier formula = [Template]-[Model Domain]-[Design
No.]
TABLE-US-00023 TABLE 23 Microorganism key for antimicrobial assay
(FIG. 30-35). Genus/Species Identifier Strain Bacteria
Staphylococcus aureus SAISP479C ISP479C Escherichia coli ECML-35
ML-35 Salmonella typhimurium ST14028 14028 Pseudomonas aeruginosa
PA01 01 Bacillus subtilis BS6633 ATCC 6633 Fungi Candida albicans
CA36082S 36082S
Results
[0169] 85% (46/54) of all peptide and microorganism combinations
tested at neutral pH showed antimicrobial activity (FIGS. 30, 32
and 34). The majority of peptides also maintained antimicrobial
activity under acidic conditions, i.e. pH 5.5 (FIGS. 31, 33 and
35), albeit at a reduced level. The acidic pH reduced the
antimicrobial activity of all peptides when tested against
Staphylococcus aureus. Additionally, 64% (7/11), 73% (8/11) and 73%
(8/11) of the peptides showed lesser antimicrobial activity when
tested against Escherichia coli, Pseudomonas aeruginosa and
Salmonella typhimurium, respectively (FIGS. 31, 33 and 35) at pH
5.5 versus pH 7.5. However, the acidic conditions did not appear to
affect the activity of peptides Hol-III-4 (SEQ ID NO:268),
Ncl-VIII-6 (SEQ ID NO:152) or BaxP-1-18 (SEQ ID NO:264) against
Bacillus subtilis, Escherichia coli, Salmonella typhimurium, and
Pseudomonas aeruginosa. Alternatively, the antimicrobial activity
was increased for 82% (9/11) of the peptides when tested against
the fungal pathogen Candida albicans under acidic conditions.
Notably, all peptides assayed showed significant and consistently
high antimicrobial activity against Bacillus subtilis regardless of
the acidity of the agarose media (FIGS. 30-35, lane designation
BS6633).
[0170] The above results show that the designed peptides exerted
consistent in vitro efficacy against Bacillus. These results
supports the concept that these molecules exploit a targetable
evolutionary relationship between prokaryotic organisms and
eukaryotic mitochondria. Evidence underscoring the
bacterium-to-mitochondrial evolution is consistent with this
concept (see Herrmann, TRENDS Micro 11(2):74-79 (2003)). For
example, proteins that are believed to mediate ion-permeability
transition in mitochondria are likely to have evolved from membrane
targeting motifs such as helical pre-sequences present in ancestral
Gram-positive organisms such as Bacillus, or other prokaryotes (see
von Heijne, EMBO Journal 5:1335-1342 (1986)). Such molecules can
target mitochondria, are often comprised of 20-60 amino acids, have
the potential to form amphipathic .alpha.-helices that segregate
hydrophobic and hydrophilic facets, and have one facet that is
positively charged. The peptides described herein are highly
consistent with such molecules. Therefore, the peptides described
herein which have antimicrobial activity will also have
anti-cancer, anti-inflammatory, anti-rheumatologic and other
efficacy by virtue of their likelihood to target mitochondria and
induce or cause dysfunctions in programmed cell death circuits.
Example III
Antimicrobial Activity against Pseudomonas aeruginos and
Acinetobacter spp. strains
[0171] Further to the methods disclosed in Example II, utilizing
the same antimicrobial assay above, the antimicrobial activity of
peptides Hol-III-4 (SEQ ID NO: 268) and Ncl-VIII-6 (SEQ ID NO: 152
was determined by identifying zones of growth inhibition.
[0172] Hol-III-4 and Ncl-VIII-6 peptides were assayed for
antimicrobial activity against a panel of drug-resistant
Gram-negative bacterial pathogens, specifically Pseudomonas
aeruginosa, and various Acinetobacter spp. strains. The efficacies
of these peptides were tested in the context of pH 5.5 (FIGS.
36-46) and pH 7.5 (FIGS. 47-57) conditions, and compared with other
peptides known to have antimicrobial activity (e.g. RP-1, 6W-RP-1
(a 6-Trp variant of RP-1), IK, and PMP-2), in the radial diffusion
assay. The RP-1 peptide is well known in the art to have
antimicrobial activity, as illustrated in Yeaman et al.,
Antimicrobial Agents and Chemotherapy, 46(12):3883-3891 (2002). The
6W-RP-1 peptide is a 6-Trp variant of RP-1, which also has
antimicrobial activity, as illustrated in Kilelee et al,
Antimicrobial Agents and Chemotherapy 54(10):4476-4479 (2010). PMP
is the C-terminal helix of the consensus molecule cPMP, as shown in
Table 1 (bottom row; N-AALYKKKIIKKLLES-C; as shown in Yeaman et
al., Bichimica et Biophysica Acta, 1768:609-619 (2007). The IK
peptide is designed to have a nearly maximal polar angle (maximum
angle is approximately 180.degree.). The results of the
antimicrobial assay show that both Hol-III-4 and Ncl-VIII-6
consistently showed significant antimicrobial activity against all
Pseudomonas aeruginosa, and all Acinetobacter spp. strains tested.
Additionally, the antimicrobial activity of both Hol-III-4 and
Ncl-VIII-6 against the various Acinetobacter baumannii isolates
tested appeared to be pH dependent (see FIGS. 39, 40, 43-46 vs.
FIGS. 50, 51, 54-57)
[0173] These results expand on and further substantiate the results
shown in Example II regarding the antimicrobial efficacy of the
peptides disclosed here. Additionally, the current data supports
the conclusion that the PCD peptides may have a unique mechanism of
action, but achieve generally equivalent efficacy as RP-1-like
peptides against most organisms tested. The is evident by the
following observations. The RP-1 and related antimicrobial peptides
appear to target microbial cells enriched with electronegative
constituents (e.g. phosphatidylglycerol, cardiolipin, etc.), and/or
those having electronegative transmembrane potential. These
features, in addition to conformational plasticity, inhibition of
intracellular functions (e.g. macromolecular synthesis) and
possibly superstructural assembly, are believed to participate in
the preferential microbial targeting and antimicrobial effects of
such peptides. By comparison, the current peptides are designed
from programmed cell death effector or modulating proteins. Without
being bound by theory, one hypothesis is that such peptides induce
microbial and other target cell death by inducing or dysregulating
programmed cell death. Thus, it is possible that these peptides
function via a mechanism that is not identical to the RP-1-like
peptides. In balance, it is also possible that PCD peptides evolved
(e.g. diverged) from antimicrobial peptide sequences, based on the
view that mitochondria are modern day "bacteria"; this is one
hypothesis we posed in the patent. If so, then the mechanisms may
be conserved among antimicrobial helices, even if the helices come
from proteins believed to have vastly divergent functions (e.g.
helices from PMPs, PCD proteins, chemokines, etc).
[0174] Throughout this application various publications have been
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference in this application
in order to more fully describe the state of the art to which this
invention pertains. Although the invention has been described with
reference to the examples provided above, it should be understood
that various modifications can be made without departing from the
spirit of the invention.
Sequence CWU 1
1
428126PRTArtificial SequenceEngineered peptides based on Bax
protein 1Asn Phe Asn Trp Gly Arg Val Val Ala Leu Phe Tyr Phe Ala
Ser Lys1 5 10 15 Leu Val Leu Lys Ala Leu Cys Thr Lys Val 20 25
222PRTArtificial SequenceEngineered peptides based on Bax protein
2Thr Trp Gln Thr Val Thr Ile Phe Val Ala Gly Val Leu Thr Ala Ser1 5
10 15 Leu Thr Ile Trp Lys Lys 20 326PRTArtificial
SequenceEngineered peptides based on Bax protein 3Asn Phe Asn Trp
Gly Arg Val Val Ala Leu Phe Tyr Phe Ala Ser Lys1 5 10 15 Leu Val
Leu Lys Ala Leu Xaa Thr Lys Val 20 25 426PRTArtificial
SequenceEngineered peptides based on Bax protein 4Asn Phe Asn Trp
Gly Arg Val Val Ala Leu Phe Tyr Phe Ala Ser Lys1 5 10 15 Leu Val
Leu Lys Ala Leu Xaa Thr Xaa Val 20 25 523PRTArtificial
SequenceEngineered peptides based on Bax protein 5Trp Gly Arg Val
Val Ala Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu1 5 10 15 Lys Ala
Leu Cys Thr Lys Val 20 623PRTArtificial SequenceEngineered peptides
based on Bax protein 6Trp Gly Arg Val Val Ala Leu Phe Tyr Phe Ala
Ser Lys Leu Val Leu1 5 10 15 Lys Ala Leu Xaa Thr Lys Val 20
721PRTArtificial SequenceEngineered peptides based on Bax protein
7Arg Val Val Ala Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala1 5
10 15 Leu Cys Thr Lys Val 20 821PRTArtificial SequenceEngineered
peptides based on Bax protein 8Arg Val Val Ala Leu Phe Tyr Phe Ala
Ser Lys Leu Val Leu Lys Ala1 5 10 15 Leu Xaa Thr Lys Val 20
918PRTArtificial SequenceEngineered peptides based on Bax protein
9Ala Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala Leu Cys Thr1 5
10 15 Lys Val1018PRTArtificial SequenceEngineered peptides based on
Bax protein 10Ala Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala
Leu Xaa Thr1 5 10 15 Lys Val1122PRTArtificial SequenceEngineered
peptides based on Bax protein 11Thr Trp Gln Thr Val Thr Ile Phe Val
Ala Xaa Val Leu Thr Ala Ser1 5 10 15 Leu Thr Ile Trp Lys Lys 20
1219PRTArtificial SequenceEngineered peptides based on Bax protein
12Thr Val Thr Ile Phe Val Ala Gly Val Leu Thr Ala Ser Leu Thr Ile1
5 10 15 Trp Lys Lys1319PRTArtificial SequenceEngineered peptides
based on Bax protein 13Thr Val Thr Ile Phe Val Ala Xaa Val Leu Thr
Ala Ser Leu Thr Ile1 5 10 15 Trp Lys Lys1419PRTArtificial
SequenceEngineered peptides based on Bcl-W protein 14Thr Arg Ala
Leu Val Ala Asp Phe Val Gly Tyr Lys Leu Arg Gln Lys1 5 10 15 Gly
Tyr Val1517PRTArtificial SequenceEngineered peptides based on Bcl-W
protein 15Thr Arg Phe Arg Arg Thr Phe Ser Asp Leu Ala Ala Gln Leu
His Val1 5 10 15 Thr1635PRTArtificial SequenceEngineered peptides
based on Bcl-W protein 16Ala Arg Arg Leu Arg Glu Gly Asn Trp Ala
Ser Val Arg Thr Val Leu1 5 10 15 Thr Gly Ala Val Ala Leu Gly Ala
Leu Val Thr Val Gly Ala Phe Phe 20 25 30 Ala Ser Lys 35
1719PRTArtificial SequenceEngineered peptides based on Bcl-W
protein 17Thr Arg Ala Leu Val Ala Xaa Phe Val Gly Tyr Lys Leu Arg
Gln Lys1 5 10 15 Gly Tyr Val1817PRTArtificial SequenceEngineered
peptides based on Bcl-W protein 18Thr Arg Phe Arg Xaa Thr Phe Ser
Asp Leu Ala Ala Gln Leu His Val1 5 10 15 Thr1917PRTArtificial
SequenceEngineered peptides based on Bcl-W protein 19Thr Arg Phe
Arg Arg Thr Phe Ser Xaa Leu Ala Ala Gln Leu His Val1 5 10 15
Thr2034PRTArtificial SequenceEngineered peptides based on Bcl-W
protein 20Arg Arg Leu Arg Glu Gly Asn Trp Ala Ser Val Arg Thr Val
Leu Thr1 5 10 15 Gly Ala Val Ala Leu Gly Ala Leu Val Thr Val Gly
Ala Phe Phe Ala 20 25 30 Ser Lys2134PRTArtificial
SequenceEngineered peptides based on Bcl-W protein 21Arg Arg Leu
Arg Xaa Gly Asn Trp Ala Ser Val Arg Thr Val Leu Thr1 5 10 15 Gly
Ala Val Ala Leu Gly Ala Leu Val Thr Val Gly Ala Phe Phe Ala 20 25
30 Ser Lys2234PRTArtificial SequenceEngineered peptides based on
Bcl-W protein 22Arg Arg Leu Arg Glu Gly Asn Trp Ala Ser Val Arg Thr
Val Leu Thr1 5 10 15 Xaa Ala Val Ala Leu Gly Ala Leu Val Thr Val
Gly Ala Phe Phe Ala 20 25 30 Ser Lys2334PRTArtificial
SequenceEngineered peptides based on Bcl-W protein 23Arg Arg Leu
Arg Glu Gly Asn Trp Ala Ser Val Arg Thr Val Leu Thr1 5 10 15 Gly
Ala Val Ala Leu Xaa Ala Leu Val Thr Val Gly Ala Phe Phe Ala 20 25
30 Ser Lys2434PRTArtificial SequenceEngineered peptides based on
Bcl-W protein 24Arg Arg Leu Arg Glu Gly Asn Trp Ala Ser Val Arg Thr
Val Leu Thr1 5 10 15 Gly Ala Val Ala Leu Gly Ala Leu Val Thr Val
Xaa Ala Phe Phe Ala 20 25 30 Ser Lys2534PRTArtificial
SequenceEngineered peptides based on Bcl-W protein 25Arg Arg Leu
Arg Xaa Gly Asn Trp Ala Ser Val Arg Thr Val Leu Thr1 5 10 15 Xaa
Ala Val Ala Leu Xaa Ala Leu Val Thr Val Xaa Ala Phe Phe Ala 20 25
30 Ser Lys2617PRTArtificial SequenceEngineered peptides based on
Bcl-xBetta protein 26Phe Leu Ser Tyr Lys Leu Ser Gln Lys Gly Tyr
Ser Trp Ser Gln Phe1 5 10 15 Ser2725PRTArtificial
SequenceEngineered peptides based on Bcl-xBetta protein 27Leu Arg
Tyr Arg Arg Ala Phe Ser Asp Leu Thr Ser Gln Leu His Ile1 5 10 15
Thr Pro Gly Thr Ala Tyr Gln Ser Phe 20 25 2820PRTArtificial
SequenceEngineered peptides based on Bcl-xBetta protein 28Gly Trp
Val Arg Thr Lys Pro Leu Val Cys Pro Phe Ser Leu Ala Ser1 5 10 15
Gly Gln Arg Ser 20 2921PRTArtificial SequenceEngineered peptides
based on Bcl-xBetta protein 29Gly Gln Arg Ser Pro Thr Ala Leu Leu
Leu Tyr Leu Phe Leu Leu Cys1 5 10 15 Trp Val Ile Val Gly 20
3025PRTArtificial SequenceEngineered peptides based on Bcl-xBetta
protein 30Leu Arg Tyr Arg Arg Ala Phe Ser Xaa Leu Thr Ser Gln Leu
His Ile1 5 10 15 Thr Pro Gly Thr Ala Tyr Gln Ser Phe 20 25
3120PRTArtificial SequenceEngineered peptides based on Bcl-xBetta
protein 31Gly Trp Val Arg Thr Lys Pro Leu Val Xaa Pro Phe Ser Leu
Ala Ser1 5 10 15 Gly Gln Arg Ser 20 3220PRTArtificial
SequenceEngineered peptides based on Bcl-xBetta protein 32Gly Trp
Val Arg Thr Lys Pro Leu Val Xaa Pro Phe Ser Leu Ala Ser1 5 10 15
Xaa Gln Arg Ser 20 3321PRTArtificial SequenceEngineered peptides
based on Bcl-xBetta protein 33Gly Gln Arg Ser Pro Thr Ala Leu Xaa
Leu Tyr Leu Phe Leu Leu Cys1 5 10 15 Trp Val Ile Val Gly 20
3421PRTArtificial SequenceEngineered peptides based on Bcl-xBetta
protein 34Gly Gln Arg Ser Pro Thr Ala Leu Leu Leu Tyr Leu Phe Leu
Leu Xaa1 5 10 15 Trp Val Ile Val Gly 20 3521PRTArtificial
SequenceEngineered peptides based on Bcl-xBetta protein 35Gly Gln
Arg Ser Pro Thr Ala Leu Leu Leu Tyr Leu Phe Leu Leu Cys1 5 10 15
Trp Val Ile Val Xaa 20 3621PRTArtificial SequenceEngineered
peptides based on Bcl-xBetta protein 36Gly Gln Arg Ser Pro Thr Ala
Leu Xaa Leu Tyr Leu Phe Leu Leu Xaa1 5 10 15 Trp Val Ile Val Xaa 20
3718PRTArtificial SequenceEngineered peptides based on Bak protein
37Arg Val Val Ala Leu Leu Gly Phe Gly Tyr Arg Leu Ala Leu His Val1
5 10 15 Tyr Gln3824PRTArtificial SequenceEngineered peptides based
on Bak protein 38Ile Leu Asn Val Leu Val Val Leu Gly Val Val Leu
Leu Gly Gln Phe1 5 10 15 Val Val Arg Arg Phe Phe Lys Ser 20
3918PRTArtificial SequenceEngineered peptides based on Bak protein
39Arg Val Val Ala Leu Leu Xaa Phe Gly Tyr Arg Leu Ala Leu His Val1
5 10 15 Tyr Gln4018PRTArtificial SequenceEngineered peptides based
on Bak protein 40Arg Val Val Ala Leu Leu Gly Phe Xaa Tyr Arg Leu
Ala Leu His Val1 5 10 15 Tyr Gln4118PRTArtificial
SequenceEngineered peptides based on Bak protein 41Arg Val Val Ala
Leu Leu Xaa Phe Xaa Tyr Arg Leu Ala Leu His Val1 5 10 15 Tyr
Gln4218PRTArtificial SequenceEngineered peptides based on Bak
protein 42Arg Val Val Ala Leu Tyr Gly Phe Gly Tyr Arg Leu Ala Leu
His Val1 5 10 15 Tyr Gln4318PRTArtificial SequenceEngineered
peptides based on Bak protein 43Arg Val Val Ala Leu Trp Gly Phe Gly
Tyr Arg Leu Ala Leu His Val1 5 10 15 Tyr Gln4418PRTArtificial
SequenceEngineered peptides based on Bak protein 44Arg Val Val Ala
Leu Tyr Xaa Phe Xaa Tyr Arg Leu Ala Leu His Val1 5 10 15 Tyr
Gln4518PRTArtificial SequenceEngineered peptides based on Bak
protein 45Arg Val Val Ala Leu Trp Xaa Phe Xaa Tyr Arg Leu Ala Leu
His Val1 5 10 15 Tyr Gln4624PRTArtificial SequenceEngineered
peptides based on Bak protein 46Ile Leu Asn Val Leu Val Xaa Leu Gly
Val Val Leu Leu Gly Gln Phe1 5 10 15 Val Val Arg Arg Phe Phe Lys
Ser 20 4718PRTArtificial SequenceEngineered peptides based on Bak
protein 47Ile Leu Asn Val Leu Val Xaa Leu Val Leu Gly Gln Phe Val
Arg Phe1 5 10 15 Lys Ser4818PRTArtificial SequenceEngineered
peptides based on Bcl-2 protein 48Asn Arg Glu Ile Val Met Lys Tyr
Ile His Tyr Lys Leu Ser Gln Arg1 5 10 15 Gly Tyr4918PRTArtificial
SequenceEngineered peptides based on Bcl-2 protein 49Asn Arg Xaa
Ile Val Met Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg1 5 10 15 Gly
Tyr5018PRTArtificial SequenceEngineered peptides based on Bcl-2
protein 50Asn Arg Glu Ile Val Xaa Lys Tyr Ile His Tyr Lys Leu Ser
Gln Arg1 5 10 15 Gly Tyr5118PRTArtificial SequenceEngineered
peptides based on Bcl-2 protein 51Asn Arg Glu Ile Val Met Lys Tyr
Ile Xaa Tyr Lys Leu Ser Gln Arg1 5 10 15 Gly Tyr5218PRTArtificial
SequenceEngineered peptides based on Bcl-2 protein 52Asn Arg Xaa
Ile Val Xaa Lys Tyr Ile Xaa Tyr Lys Leu Ser Gln Arg1 5 10 15 Gly
Tyr5316PRTArtificial SequenceEngineered peptides based on Bcl-2
isoform 1 protein 53Leu Ala Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg
Arg Tyr Arg Gly1 5 10 15 5416PRTArtificial SequenceEngineered
peptides based on Bcl-2 isoform 1 protein 54Leu Ala Leu Arg Gln Ala
Gly Xaa Asp Phe Ser Arg Arg Tyr Arg Gly1 5 10 15 5516PRTArtificial
SequenceEngineered peptides based on Bcl-2 isoform 1 protein 55Leu
Ala Leu Arg Gln Ala Gly Asp Xaa Phe Ser Arg Arg Tyr Arg Gly1 5 10
15 5616PRTArtificial SequenceEngineered peptides based on Bcl-2
isoform 1 protein 56Leu Ala Leu Arg Gln Ala Gly Xaa Xaa Phe Ser Arg
Arg Tyr Arg Gly1 5 10 15 5716PRTArtificial SequenceEngineered
peptides based on Bcl-2 isoform 1 protein 57Leu Ala Leu Arg Gln Ala
Xaa Xaa Xaa Phe Ser Arg Arg Tyr Arg Gly1 5 10 15 5835PRTArtificial
SequenceEngineered peptides based on Mfn-1 protein 58Lys Glu Ile
Asp Gln Leu Glu Lys Ile Gln Asn Asn Ser Lys Leu Leu1 5 10 15 Arg
Asn Lys Ala Val Gln Leu Glu Asn Glu Leu Glu Asn Phe Thr Lys 20 25
30 Gln Phe Leu 35 5935PRTArtificial SequenceEngineered peptides
based on Mfn-1 protein 59Lys Glu Ile Xaa Gln Leu Glu Lys Ile Gln
Asn Asn Ser Lys Leu Leu1 5 10 15 Arg Asn Lys Ala Val Gln Leu Glu
Asn Glu Leu Glu Asn Phe Thr Lys 20 25 30 Gln Phe Leu 35
6035PRTArtificial SequenceEngineered peptides based on Mfn-1
protein 60Lys Glu Ile Xaa Gln Leu Xaa Lys Ile Gln Asn Asn Ser Lys
Leu Leu1 5 10 15 Arg Asn Lys Ala Val Gln Leu Xaa Asn Glu Leu Glu
Asn Phe Thr Lys 20 25 30 Gln Phe Leu 35 6135PRTArtificial
SequenceEngineered peptides based on Mfn-1 protein 61Lys Glu Ile
Xaa Gln Leu Xaa Lys Ile Gln Asn Asn Ser Lys Leu Leu1 5 10 15 Arg
Asn Lys Ala Val Gln Leu Xaa Asn Glu Leu Glu Asn Phe Thr Lys 20 25
30 Gln Phe Leu 35 6235PRTArtificial SequenceEngineered peptides
based on Mfn-1 protein 62Lys Glu Ile Xaa Gln Leu Xaa Lys Ile Gln
Asn Asn Ser Lys Leu Leu1 5 10 15 Arg Asn Lys Ala Val Gln Leu Xaa
Asn Xaa Leu Glu Asn Phe Thr Lys 20 25 30 Gln Phe Leu 35
6335PRTArtificial SequenceEngineered peptides based on Mfn-1
protein 63Lys Glu Ile Xaa Gln Leu Xaa Lys Ile Gln Asn Asn Ser Lys
Leu Leu1 5 10 15 Arg Asn Lys Ala Val Gln Leu Xaa Asn Xaa Leu Xaa
Asn Phe Thr Lys 20 25 30 Gln Phe Leu 35 6416PRTArtificial
SequenceEngineered peptides based on Mfn-1 protein 64Lys Ile Gln
Asn Asn Ser Lys Leu Leu Arg Asn Lys Ala Val Gln Leu1 5 10 15
6540PRTArtificial SequenceEngineered peptides based on Mfn-2
protein 65Asn Lys Lys Ile Glu Val Leu Asp Ser Leu Gln Ser Lys Ala
Lys Leu1 5 10 15 Leu Arg Asn Lys Ala Gly Trp Leu Asp Ser Glu Leu
Asn Met Phe Thr 20 25 30 His Gln Tyr Leu Gln Pro Ser Arg 35 40
6640PRTArtificial SequenceEngineered peptides based on Mfn-2
protein 66Asn Lys Lys Ile Xaa Val Leu Asp Ser Leu Gln Ser Lys Ala
Lys Leu1 5 10 15 Leu Arg Asn Lys Ala Gly Trp Leu Asp Ser Glu Leu
Asn Met Phe Thr 20 25 30 His Gln Tyr Leu Gln Pro Ser Arg 35 40
6740PRTArtificial SequenceEngineered peptides based on Mfn-2
protein 67Asn Lys Lys Ile Glu Val Leu Xaa Ser Leu Gln Ser Lys Ala
Lys Leu1 5 10 15 Leu Arg Asn Lys Ala Gly Trp Leu Asp Ser Glu Leu
Asn Met Phe Thr 20 25 30 His Gln Tyr Leu Gln Pro Ser Arg 35 40
6840PRTArtificial SequenceEngineered peptides based on Mfn-2
protein 68Asn Lys Lys Ile Glu Val Leu Asp Ser Leu Gln Ser Lys Ala
Lys Leu1 5 10 15 Leu Arg Asn Lys Ala Gly Trp Leu Xaa Ser Glu Leu
Asn Met Phe Thr 20 25 30 His Gln Tyr Leu Gln Pro Ser Arg 35 40
6940PRTArtificial SequenceEngineered peptides based on Mfn-2
protein 69Asn Lys Lys Ile Glu Val Leu Asp Ser Leu Gln Ser Lys Ala
Lys Leu1 5 10 15 Leu Arg Asn Lys Ala Gly Trp Leu Asp Ser Xaa Leu
Asn Met Phe Thr 20 25 30 His Gln Tyr Leu Gln Pro Ser Arg
35 40 7040PRTArtificial SequenceEngineered peptides based on Mfn-2
protein 70Asn Lys Lys Ile Glu Val Leu Asp Ser Leu Gln Ser Lys Ala
Lys Leu1 5 10 15 Leu Arg Asn Lys Ala Gly Trp Leu Asp Ser Glu Leu
Asn Xaa Phe Thr 20 25 30 His Gln Tyr Leu Gln Pro Ser Arg 35 40
7140PRTArtificial SequenceEngineered peptides based on Mfn-2
protein 71Asn Lys Lys Ile Xaa Val Leu Xaa Ser Leu Gln Ser Lys Ala
Lys Leu1 5 10 15 Leu Arg Asn Lys Ala Gly Trp Leu Xaa Ser Xaa Leu
Asn Xaa Phe Thr 20 25 30 His Gln Tyr Leu Gln Pro Ser Arg 35 40
7223PRTArtificial SequenceEngineered peptides based on Mfn-2
protein 72Lys Lys Ile Glu Val Leu Asp Ser Leu Gln Ser Lys Ala Lys
Leu Leu1 5 10 15 Arg Asn Lys Ala Gly Trp Leu 20 7323PRTArtificial
SequenceEngineered peptides based on Mfn-2 protein 73Lys Lys Ile
Xaa Val Leu Asp Ser Leu Gln Ser Lys Ala Lys Leu Leu1 5 10 15 Arg
Asn Lys Ala Gly Trp Leu 20 7423PRTArtificial SequenceEngineered
peptides based on Mfn-2 protein 74Lys Lys Ile Glu Val Leu Xaa Ser
Leu Gln Ser Lys Ala Lys Leu Leu1 5 10 15 Arg Asn Lys Ala Gly Trp
Leu 20 7523PRTArtificial SequenceEngineered peptides based on Mfn-2
protein 75Lys Lys Ile Xaa Val Leu Xaa Ser Leu Gln Ser Lys Ala Lys
Leu Leu1 5 10 15 Arg Asn Lys Ala Gly Trp Leu 20 7618PRTArtificial
SequenceEngineered peptides based on Dnm-1 protein 76Lys Leu Leu
Pro Leu Arg Arg Gly Tyr Ile Gly Val Val Asn Arg Ser1 5 10 15 Gln
Lys7714PRTArtificial SequenceEngineered peptides based on Dnm-1
protein 77Arg Lys Phe Phe Leu Ser His Pro Ser Tyr Arg His Leu Ala1
5 10 7815PRTArtificial SequenceEngineered peptides based on Dnm-1
protein 78Gly Leu Arg Asn Lys Leu Gln Ser Gln Leu Leu Ser Ile Glu
Lys1 5 10 15 7918PRTArtificial SequenceEngineered peptides based on
Dnm-1 protein 79Ala Arg Lys Thr Lys Ala Leu Leu Gln Met Val Gln Gln
Phe Ala Val1 5 10 15 Asp Phe8011PRTArtificial SequenceEngineered
peptides based on Dnm-1 protein 80Ala Thr Val Lys Lys Gln Val Gln
Lys Leu Lys1 5 10 8120PRTArtificial SequenceEngineered peptides
based on Dnm-1 protein 81Val Ile Arg Lys Gly Trp Leu Thr Ile Asn
Asn Ile Gly Ile Met Lys1 5 10 15 Gly Gly Ser Lys 20
8230PRTArtificial SequenceEngineered peptides based on Dnm-1
protein 82Asn Leu Lys Leu Arg Asp Val Glu Lys Gly Phe Met Ser Ser
Lys His1 5 10 15 Ile Phe Ala Leu Phe Asn Thr Glu Gln Arg Asn Val
Tyr Lys 20 25 30 8317PRTArtificial SequenceEngineered peptides
based on Dnm-1 protein 83Lys Ala Ser Phe Leu Arg Ala Gly Val Tyr
Pro Glu Arg Val Gly Asp1 5 10 15 Lys8415PRTArtificial
SequenceEngineered peptides based on Dnm-1 protein 84Gly Leu Arg
Asn Lys Leu Gln Ser Gln Leu Leu Ser Ile Xaa Lys1 5 10 15
8518PRTArtificial SequenceEngineered peptides based on Dnm-1
protein 85Ala Arg Lys Thr Lys Ala Leu Leu Gln Met Val Gln Gln Phe
Ala Val1 5 10 15 Xaa Phe8630PRTArtificial SequenceEngineered
peptides based on Dnm-1 protein 86Asn Leu Lys Leu Arg Xaa Val Glu
Lys Gly Phe Met Ser Ser Lys His1 5 10 15 Ile Phe Ala Leu Phe Asn
Thr Glu Gln Arg Asn Val Tyr Lys 20 25 30 8730PRTArtificial
SequenceEngineered peptides based on Dnm-1 protein 87Asn Leu Lys
Leu Arg Asp Val Xaa Lys Gly Phe Met Ser Ser Lys His1 5 10 15 Ile
Phe Ala Leu Phe Asn Thr Glu Gln Arg Asn Val Tyr Lys 20 25 30
8830PRTArtificial SequenceEngineered peptides based on Dnm-1
protein 88Asn Leu Lys Leu Arg Asp Val Glu Lys Gly Phe Met Ser Ser
Lys His1 5 10 15 Ile Phe Ala Leu Phe Asn Thr Xaa Gln Arg Asn Val
Tyr Lys 20 25 30 8930PRTArtificial SequenceEngineered peptides
based on Dnm-1 protein 89Asn Leu Lys Leu Arg Xaa Val Xaa Lys Gly
Phe Met Ser Ser Lys His1 5 10 15 Ile Phe Ala Leu Phe Asn Thr Glu
Gln Arg Asn Val Tyr Lys 20 25 30 9030PRTArtificial
SequenceEngineered peptides based on Dnm-1 protein 90Asn Leu Lys
Leu Arg Xaa Val Xaa Lys Gly Phe Met Ser Ser Lys His1 5 10 15 Ile
Phe Ala Leu Phe Asn Thr Xaa Gln Arg Asn Val Tyr Lys 20 25 30
9117PRTArtificial SequenceEngineered peptides based on Dnm-1
protein 91Lys Ala Ser Phe Leu Arg Ala Gly Val Tyr Pro Xaa Arg Val
Gly Asp1 5 10 15 Lys9217PRTArtificial SequenceEngineered peptides
based on Dnm-1 protein 92Lys Ala Ser Phe Leu Arg Ala Gly Val Tyr
Pro Glu Arg Val Gly Xaa1 5 10 15 Lys9317PRTArtificial
SequenceEngineered peptides based on Dnm-1 protein 93Lys Ala Ser
Phe Leu Arg Ala Gly Val Tyr Pro Xaa Arg Val Gly Xaa1 5 10 15
Lys9420PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 94Lys Leu Ala Lys Glu Val Asp Pro Gln Gly Leu Arg Thr Ile
Gly Val1 5 10 15 Ile Thr Lys Leu 20 9515PRTArtificial
SequenceEngineered peptides based on Dnm-2 protein 95Ala Leu Arg
Ser Lys Leu Gln Ser Gln Leu Leu Ser Leu Glu Lys1 5 10 15
9616PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 96Thr Arg Lys Thr Lys Ala Leu Leu Gln Met Val Gln Gln Phe
Gly Val1 5 10 15 9711PRTArtificial SequenceEngineered peptides
based on Dnm-2 protein 97Ala Ile Val Lys Lys Gln Val Val Lys Leu
Lys1 5 10 9818PRTArtificial SequenceEngineered peptides based on
Dnm-2 protein 98Ala Gln Gln Arg Ser Thr Gln Leu Asn Lys Lys Arg Ala
Ile Pro Asn1 5 10 15 Gln Gly9940PRTArtificial SequenceEngineered
peptides based on Dnm-2 protein 99Lys Glu Lys Lys Tyr Met Leu Pro
Leu Asp Asn Leu Lys Ile Arg Asp1 5 10 15 Val Glu Lys Gly Phe Met
Ser Asn Lys His Val Phe Ala Ile Phe Asn 20 25 30 Thr Glu Gln Arg
Asn Val Tyr Lys 35 40 10030PRTArtificial SequenceEngineered
peptides based on Dnm-2 protein 100Asn Leu Lys Ile Arg Asp Val Glu
Lys Gly Phe Met Ser Asn Lys His1 5 10 15 Val Phe Ala Ile Phe Asn
Thr Glu Gln Arg Asn Val Tyr Lys 20 25 30 10118PRTArtificial
SequenceEngineered peptides based on Dnm-2 protein 101Ser Trp Lys
Ala Ser Phe Leu Arg Ala Gly Val Tyr Pro Glu Lys Asp1 5 10 15 Gln
Ala10220PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 102Lys Leu Ala Lys Xaa Val Asp Pro Gln Gly Leu Arg Thr Ile
Gly Val1 5 10 15 Ile Thr Lys Leu 20 10320PRTArtificial
SequenceEngineered peptides based on Dnm-2 protein 103Lys Leu Ala
Lys Glu Val Xaa Pro Gln Gly Leu Arg Thr Ile Gly Val1 5 10 15 Ile
Thr Lys Leu 20 10420PRTArtificial SequenceEngineered peptides based
on Dnm-2 protein 104Lys Leu Ala Lys Xaa Val Xaa Pro Gln Gly Leu Arg
Thr Ile Gly Val1 5 10 15 Ile Thr Lys Leu 20 10515PRTArtificial
SequenceEngineered peptides based on Dnm-2 protein 105Ala Leu Arg
Ser Lys Leu Gln Ser Gln Leu Leu Ser Leu Xaa Lys1 5 10 15
10611PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 106Xaa Ile Val Lys Lys Gln Val Val Lys Leu Lys1 5 10
10715PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 107Lys Glu Lys Lys Tyr Met Leu Pro Leu Asp Asn Leu Lys Ile
Arg1 5 10 15 10815PRTArtificial SequenceEngineered peptides based
on Dnm-2 protein 108Lys Xaa Lys Lys Tyr Met Leu Pro Leu Asp Asn Leu
Lys Ile Arg1 5 10 15 10915PRTArtificial SequenceEngineered peptides
based on Dnm-2 protein 109Lys Glu Lys Lys Tyr Xaa Leu Pro Leu Asp
Asn Leu Lys Ile Arg1 5 10 15 11015PRTArtificial SequenceEngineered
peptides based on Dnm-2 protein 110Lys Glu Lys Lys Tyr Met Leu Pro
Leu Xaa Asn Leu Lys Ile Arg1 5 10 15 11115PRTArtificial
SequenceEngineered peptides based on Dnm-2 protein 111Lys Glu Lys
Lys Tyr Met Leu Pro Leu Xaa Asn Leu Lys Ile Arg1 5 10 15
11215PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 112Lys Xaa Lys Lys Tyr Met Leu Pro Leu Xaa Asn Leu Lys Ile
Arg1 5 10 15 11315PRTArtificial SequenceEngineered peptides based
on Dnm-2 protein 113Lys Xaa Lys Lys Tyr Xaa Leu Pro Leu Xaa Asn Leu
Lys Ile Arg1 5 10 15 11422PRTArtificial SequenceEngineered peptides
based on Dnm-2 protein 114Lys Gly Phe Met Ser Asn Lys His Val Phe
Ala Ile Phe Asn Thr Glu1 5 10 15 Gln Arg Asn Val Tyr Lys 20
11540PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 115Lys Xaa Lys Lys Tyr Met Leu Pro Leu Asp Asn Leu Lys Ile
Arg Asp1 5 10 15 Val Glu Lys Gly Phe Met Ser Asn Lys His Val Phe
Ala Ile Phe Asn 20 25 30 Thr Glu Gln Arg Asn Val Tyr Lys 35 40
11640PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 116Lys Glu Lys Lys Tyr Xaa Leu Pro Leu Asp Asn Leu Lys Ile
Arg Asp1 5 10 15 Val Glu Lys Gly Phe Met Ser Asn Lys His Val Phe
Ala Ile Phe Asn 20 25 30 Thr Glu Gln Arg Asn Val Tyr Lys 35 40
11740PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 117Lys Glu Lys Lys Tyr Met Leu Pro Leu Asp Asn Leu Lys Ile
Arg Asp1 5 10 15 Val Glu Lys Gly Phe Xaa Ser Asn Lys His Val Phe
Ala Ile Phe Asn 20 25 30 Thr Glu Gln Arg Asn Val Tyr Lys 35 40
11830PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 118Asn Leu Lys Ile Arg Xaa Val Glu Lys Gly Phe Met Ser Asn
Lys His1 5 10 15 Val Phe Ala Ile Phe Asn Thr Glu Gln Arg Asn Val
Tyr Lys 20 25 30 11930PRTArtificial SequenceEngineered peptides
based on Dnm-2 protein 119Asn Leu Lys Ile Arg Asp Val Xaa Lys Gly
Phe Met Ser Asn Lys His1 5 10 15 Val Phe Ala Ile Phe Asn Thr Glu
Gln Arg Asn Val Tyr Lys 20 25 30 12030PRTArtificial
SequenceEngineered peptides based on Dnm-2 protein 120Asn Leu Lys
Ile Arg Xaa Val Xaa Lys Gly Phe Met Ser Asn Lys His1 5 10 15 Val
Phe Ala Ile Phe Asn Thr Xaa Gln Arg Asn Val Tyr Lys 20 25 30
12118PRTArtificial SequenceEngineered peptides based on Dnm-2
protein 121Ser Trp Lys Ala Ser Phe Leu Arg Ala Gly Val Tyr Pro Xaa
Lys Asp1 5 10 15 Gln Ala12218PRTArtificial SequenceEngineered
peptides based on Ncl protein 122Gln Lys Lys Gly Lys Lys Ala Ala
Ala Thr Ser Ala Lys Lys Val Val1 5 10 15 Val Ser12316PRTArtificial
SequenceEngineered peptides based on Ncl protein 123Thr Lys Lys Val
Ala Val Ala Thr Pro Ala Lys Lys Ala Ala Val Thr1 5 10 15
12419PRTArtificial SequenceEngineered peptides based on Ncl protein
124Lys Lys Gly Ala Ala Ile Pro Ala Lys Gly Ala Lys Asn Gly Lys Asn1
5 10 15 Ala Lys Lys12518PRTArtificial SequenceEngineered peptides
based on Ncl protein 125Ala Lys Gly Lys Lys Ala Ala Lys Val Val Pro
Val Lys Ala Lys Asn1 5 10 15 Val Ala12618PRTArtificial
SequenceEngineered peptides based on Ncl protein 126Val Lys Glu Ala
Pro Gly Lys Arg Lys Lys Glu Met Ala Lys Gln Lys1 5 10 15 Ala
Ala12719PRTArtificial SequenceEngineered peptides based on Ncl
protein 127Lys Ala Leu Glu Leu Thr Gly Leu Lys Val Phe Gly Asn Glu
Ile Lys1 5 10 15 Leu Glu Lys12825PRTArtificial SequenceEngineered
peptides based on Ncl protein 128Lys Gly Lys Asp Ser Lys Lys Glu
Arg Asp Ala Arg Thr Leu Leu Ala1 5 10 15 Lys Asn Leu Pro Tyr Lys
Val Thr Gln 20 25 12916PRTArtificial SequenceEngineered peptides
based on Ncl protein 129Ile Arg Leu Val Ser Lys Asp Gly Lys Ser Lys
Gly Ile Ala Tyr Ile1 5 10 15 13032PRTArtificial SequenceEngineered
peptides based on Ncl protein 130Lys Gly Gln Asn Gln Asp Tyr Arg
Gly Gly Lys Asn Ser Thr Trp Ser1 5 10 15 Gly Glu Ser Lys Thr Leu
Val Leu Ser Asn Leu Ser Tyr Ser Ala Thr 20 25 30 13121PRTArtificial
SequenceEngineered peptides based on Ncl protein 131Lys Ala Thr Phe
Ile Lys Val Pro Gln Asn Gln Asn Gly Lys Ser Lys1 5 10 15 Gly Tyr
Ala Phe Ile 20 13218PRTArtificial SequenceEngineered peptides based
on Ncl protein 132Gln Lys Lys Gly Lys Lys Ala Xaa Ala Thr Ser Ala
Lys Lys Val Tyr1 5 10 15 Val Ser13318PRTArtificial
SequenceEngineered peptides based on Ncl protein 133Gln Lys Lys Gly
Lys Lys Ala Ala Ala Thr Ser Ala Lys Lys Val Tyr1 5 10 15 Val
Ser13418PRTArtificial SequenceEngineered peptides based on Ncl
protein 134Gln Lys Lys Gly Lys Lys Ala Ala Ala Thr Ser Ala Lys Lys
Val Trp1 5 10 15 Val Ser13518PRTArtificial SequenceEngineered
peptides based on Ncl protein 135Gln Lys Lys Gly Lys Lys Ala Xaa
Ala Thr Ser Ala Lys Lys Val Tyr1 5 10 15 Val Ser13618PRTArtificial
SequenceEngineered peptides based on Ncl protein 136Gln Lys Lys Gly
Lys Lys Ala Xaa Ala Thr Ser Ala Lys Lys Val Trp1 5 10 15 Val
Ser13719PRTArtificial SequenceEngineered peptides based on Ncl
protein 137Lys Lys Gly Ala Xaa Ile Pro Ala Lys Gly Ala Lys Asn Gly
Lys Asn1 5 10 15 Ala Lys Lys13818PRTArtificial SequenceEngineered
peptides based on Ncl protein 138Ala Lys Gly Lys Lys Ala Ala Lys
Val Val Xaa Val Lys Ala Lys Asn1 5 10 15 Val Ala13918PRTArtificial
SequenceEngineered peptides based on Ncl protein 139Val Lys Xaa Ala
Pro Gly Lys Arg Lys Lys Glu Met Ala Lys Gln Lys1 5 10 15 Ala
Ala14018PRTArtificial SequenceEngineered peptides based on Ncl
protein 140Val Lys Glu Ala Pro Gly Lys Arg Lys Lys Xaa Met Ala Lys
Gln Lys1 5 10 15 Ala Ala14118PRTArtificial SequenceEngineered
peptides based on Ncl protein 141Val Lys Glu Ala Pro Gly Lys Arg
Lys Lys Glu Xaa Ala Lys Gln Lys1 5 10 15 Ala Ala14218PRTArtificial
SequenceEngineered peptides based on Ncl protein 142Val Lys Xaa Ala
Pro Gly Lys Arg Lys Lys Xaa Xaa Ala Lys Gln Lys1 5 10 15 Ala
Ala14319PRTArtificial SequenceEngineered peptides based on Ncl
protein 143Lys Ala Leu Xaa Leu Thr Gly Leu Lys Val Phe Gly Asn Glu
Ile Lys1 5 10 15 Leu Glu Lys14419PRTArtificial SequenceEngineered
peptides based on Ncl protein 144Lys Ala Leu Glu Leu Thr Gly Leu
Lys Val Phe Gly Asn Xaa Ile Lys1 5 10 15 Leu Glu
Lys14519PRTArtificial SequenceEngineered peptides based on Ncl
protein 145Lys Ala Leu Glu Leu Thr Gly Leu Lys Val Phe Gly Asn Glu
Ile Lys1
5 10 15 Leu Xaa Lys14619PRTArtificial SequenceEngineered peptides
based on Ncl protein 146Lys Ala Leu Xaa Leu Thr Gly Leu Lys Val Phe
Gly Asn Xaa Ile Lys1 5 10 15 Leu Xaa Lys14725PRTArtificial
SequenceEngineered peptides based on Ncl protein 147Lys Gly Lys Xaa
Ser Lys Lys Glu Arg Asp Ala Arg Thr Leu Leu Ala1 5 10 15 Lys Asn
Leu Pro Tyr Lys Val Thr Gln 20 25 14825PRTArtificial
SequenceEngineered peptides based on Ncl protein 148Lys Gly Lys Asp
Ser Lys Lys Xaa Arg Asp Ala Arg Thr Leu Leu Ala1 5 10 15 Lys Asn
Leu Pro Tyr Lys Val Thr Gln 20 25 14925PRTArtificial
SequenceEngineered peptides based on Ncl protein 149Lys Gly Lys Asp
Ser Lys Lys Glu Arg Xaa Ala Arg Thr Leu Leu Ala1 5 10 15 Lys Asn
Leu Pro Tyr Lys Val Thr Gln 20 25 15025PRTArtificial
SequenceEngineered peptides based on Ncl protein 150Lys Gly Lys Xaa
Ser Lys Lys Xaa Arg Xaa Ala Arg Thr Leu Leu Ala1 5 10 15 Lys Asn
Leu Pro Tyr Lys Val Thr Gln 20 25 15116PRTArtificial
SequenceEngineered peptides based on Ncl protein 151Ile Arg Leu Val
Ser Lys Phe Gly Lys Ser Lys Gly Ile Ala Tyr Ile1 5 10 15
15216PRTArtificial SequenceEngineered peptides based on Ncl protein
152Ile Arg Leu Val Ser Lys Tyr Gly Lys Ser Lys Gly Ile Ala Tyr Ile1
5 10 15 15316PRTArtificial SequenceEngineered peptides based on Ncl
protein 153Ile Arg Leu Val Ser Lys Trp Gly Lys Ser Lys Gly Ile Ala
Tyr Ile1 5 10 15 15417PRTArtificial SequenceEngineered peptides
based on Ncl protein 154Ile Arg Leu Val Ser Lys Leu Trp Gly Lys Ser
Lys Gly Ile Ala Tyr1 5 10 15 Ile15512PRTArtificial
SequenceEngineered peptides based on Ncl protein 155Ile Arg Leu Val
Ser Lys Asp Gly Lys Ser Lys Gly1 5 10 15613PRTArtificial
SequenceEngineered peptides based on Ncl protein 156Ile Arg Leu Val
Ser Lys Phe Gly Lys Ser Lys Gly Ile1 5 10 15712PRTArtificial
SequenceEngineered peptides based on Ncl protein 157Ile Arg Leu Val
Ser Lys Tyr Gly Lys Ser Lys Gly1 5 10 15812PRTArtificial
SequenceEngineered peptides based on Ncl protein 158Ile Arg Leu Val
Ser Lys Trp Gly Lys Ser Lys Gly1 5 10 15913PRTArtificial
SequenceEngineered peptides based on Ncl protein 159Ile Arg Leu Val
Ser Lys Leu Trp Gly Lys Ser Lys Gly1 5 10 16032PRTArtificial
SequenceEngineered peptides based on Ncl protein 160Lys Gly Gln Asn
Gln Xaa Tyr Arg Gly Gly Lys Asn Ser Thr Trp Ser1 5 10 15 Gly Glu
Ser Lys Thr Leu Val Leu Ser Asn Leu Ser Tyr Ser Ala Thr 20 25 30
16132PRTArtificial SequenceEngineered peptides based on Ncl protein
161Lys Gly Gln Asn Gln Asp Tyr Arg Gly Gly Lys Asn Ser Thr Trp Ser1
5 10 15 Gly Xaa Ser Lys Thr Leu Val Leu Ser Asn Leu Ser Tyr Ser Ala
Thr 20 25 30 16232PRTArtificial SequenceEngineered peptides based
on Ncl protein 162Lys Gly Gln Asn Gln Xaa Tyr Arg Gly Gly Lys Asn
Ser Thr Trp Ser1 5 10 15 Gly Xaa Ser Lys Thr Leu Val Leu Ser Asn
Leu Ser Tyr Ser Ala Thr 20 25 30 16332PRTArtificial
SequenceEngineered peptides based on Ncl protein 163Lys Gly Xaa Asn
Gln Asp Tyr Arg Leu Gly Lys Asn Ser Thr Trp Ser1 5 10 15 Gly Xaa
Ser Lys Thr Leu Val Leu Ser Asn Leu Ser Tyr Ser Ala Thr 20 25 30
16421PRTArtificial SequenceEngineered peptides based on Ncl protein
164Lys Gly Xaa Asn Gln Asp Tyr Arg Leu Gly Lys Asn Ser Thr Trp Ser1
5 10 15 Gly Xaa Ser Lys Thr 20 16521PRTArtificial
SequenceEngineered peptides based on Ncl protein 165Lys Ala Thr Phe
Ile Lys Val Pro Gln Asn Gln Asn Xaa Lys Ser Lys1 5 10 15 Gly Tyr
Ala Phe Ile 20 16621PRTArtificial SequenceEngineered peptides based
on Ncl protein 166Lys Ala Thr Phe Ile Lys Val Pro Gln Asn Gln Asn
Leu Lys Ser Lys1 5 10 15 Gly Tyr Ala Phe Ile 20 16721PRTArtificial
SequenceEngineered peptides based on Ncl protein 167Lys Ala Thr Phe
Ile Lys Val Pro Gln Asn Gln Asn Tyr Lys Ser Lys1 5 10 15 Gly Tyr
Ala Phe Ile 20 16821PRTArtificial SequenceEngineered peptides based
on Ncl protein 168Lys Ala Thr Phe Ile Lys Val Pro Gln Asn Gln Asn
Phe Lys Ser Lys1 5 10 15 Gly Tyr Ala Phe Ile 20 16921PRTArtificial
SequenceEngineered peptides based on Ncl protein 169Lys Ala Thr Phe
Ile Lys Val Pro Gln Asn Gln Asn Trp Lys Ser Lys1 5 10 15 Gly Tyr
Ala Phe Ile 20 17018PRTArtificial SequenceEngineered peptides based
on Ncl protein 170Lys Ala Thr Phe Ile Lys Val Pro Gln Asn Gln Asn
Gly Lys Ser Lys1 5 10 15 Gly Tyr17118PRTArtificial
SequenceEngineered peptides based on Ncl protein 171Lys Ala Thr Phe
Ile Lys Val Pro Gln Asn Gln Asn Xaa Lys Ser Lys1 5 10 15 Gly
Tyr17218PRTArtificial SequenceEngineered peptides based on Ncl
protein 172Lys Ala Thr Phe Ile Lys Val Pro Gln Asn Gln Asn Leu Lys
Ser Lys1 5 10 15 Gly Tyr17318PRTArtificial SequenceEngineered
peptides based on Ncl protein 173Lys Ala Thr Phe Ile Lys Val Pro
Gln Asn Gln Asn Tyr Lys Ser Lys1 5 10 15 Gly Tyr17418PRTArtificial
SequenceEngineered peptides based on Ncl protein 174Lys Ala Thr Phe
Ile Lys Val Pro Gln Asn Gln Asn Phe Lys Ser Lys1 5 10 15 Gly
Tyr17518PRTArtificial SequenceEngineered peptides based on Ncl
protein 175Lys Ala Thr Phe Ile Lys Val Pro Gln Asn Gln Asn Trp Lys
Ser Lys1 5 10 15 Gly Tyr17615PRTArtificial SequenceEngineered
peptides based on Csp3 protein 176Ser Lys Ser Ile Lys Asn Leu Glu
Pro Lys Ile Ile His Gly Ser1 5 10 15 17731PRTArtificial
SequenceEngineered peptides based on Csp3 protein 177Leu Lys Lys
Ile Thr Asn Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu1 5 10 15 Thr
Gly Lys Pro Lys Leu Phe Ile Ile Gln Ala Cys Arg Gly Thr 20 25 30
17831PRTArtificial SequenceEngineered peptides based on Csp3
protein 178Phe Ile Gln Ser Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp
Lys Leu1 5 10 15 Glu Phe Met His Ile Leu Thr Arg Val Asn Arg Lys
Val Ala Thr 20 25 30 17915PRTArtificial SequenceEngineered peptides
based on Csp3 protein 179Ser Lys Ser Ile Lys Asn Leu Xaa Pro Lys
Ile Ile His Gly Ser1 5 10 15 18015PRTArtificial SequenceEngineered
peptides based on Csp3 protein 180Ser Lys Ser Ile Lys Asn Leu Glu
Pro Lys Ile Ile Tyr Gly Ser1 5 10 15 18115PRTArtificial
SequenceEngineered peptides based on Csp3 protein 181Ser Lys Ser
Ile Lys Asn Leu Glu Pro Lys Ile Ile Tyr Xaa Ser1 5 10 15
18215PRTArtificial SequenceEngineered peptides based on Csp3
protein 182Ser Lys Ser Ile Lys Asn Leu Xaa Pro Lys Ile Ile Tyr Xaa
Ser1 5 10 15 18331PRTArtificial SequenceEngineered peptides based
on Csp3 protein 183Leu Lys Lys Ile Thr Asn Phe Phe Arg Gly Xaa Arg
Cys Arg Ser Leu1 5 10 15 Thr Gly Lys Pro Lys Leu Phe Ile Ile Gln
Ala Cys Arg Gly Thr 20 25 30 18431PRTArtificial SequenceEngineered
peptides based on Csp3 protein 184Leu Lys Lys Ile Thr Asn Phe Phe
Arg Gly Asp Arg Xaa Arg Ser Leu1 5 10 15 Thr Gly Lys Pro Lys Leu
Phe Ile Ile Gln Ala Cys Arg Gly Thr 20 25 30 18531PRTArtificial
SequenceEngineered peptides based on Csp3 protein 185Leu Lys Lys
Ile Thr Asn Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu1 5 10 15 Thr
Gly Lys Pro Lys Leu Phe Ile Ile Gln Ala Xaa Arg Gly Thr 20 25 30
18630PRTArtificial SequenceEngineered peptides based on Csp3
protein 186Leu Lys Lys Ile Thr Asn Phe Arg Gly Xaa Arg Xaa Arg Ser
Leu Thr1 5 10 15 Gly Lys Pro Lys Leu Phe Ile Ile Gln Ala Xaa Arg
Gly Thr 20 25 30 18718PRTArtificial SequenceEngineered peptides
based on Csp3 protein 187Leu Lys Lys Ile Thr Asn Phe Arg Gly Xaa
Arg Xaa Arg Ser Leu Thr1 5 10 15 Gly Lys18824PRTArtificial
SequenceEngineered peptides based on Bad protein 188Phe Arg Gly Arg
Ser Arg Ser Ala Pro Pro Asn Leu Trp Ala Ala Gln1 5 10 15 Arg Tyr
Gly Arg Glu Leu Arg Arg 20 18926PRTArtificial SequenceEngineered
peptides based on Bad protein 189Arg Arg Met Ser Asp Glu Phe Val
Asp Ser Phe Lys Lys Gly Leu Pro1 5 10 15 Arg Pro Lys Ser Ala Gly
Thr Ala Thr Gln 20 25 19020PRTArtificial SequenceEngineered
peptides based on Bad protein 190Phe Val Asp Ser Phe Lys Lys Gly
Leu Pro Arg Pro Lys Ser Ala Gly1 5 10 15 Thr Ala Thr Gln 20
19124PRTArtificial SequenceEngineered peptides based on Bad protein
191Phe Arg Gly Arg Ser Arg Ser Ala Pro Pro Asn Leu Trp Ala Ala Gln1
5 10 15 Arg Tyr Gly Arg Xaa Leu Arg Arg 20 19226PRTArtificial
SequenceEngineered peptides based on Bad protein 192Arg Arg Met Ser
Xaa Glu Phe Val Asp Ser Phe Lys Lys Gly Leu Pro1 5 10 15 Arg Pro
Lys Ser Ala Gly Thr Ala Thr Gln 20 25 19326PRTArtificial
SequenceEngineered peptides based on Bad protein 193Arg Arg Met Ser
Asp Xaa Phe Val Asp Ser Phe Lys Lys Gly Leu Pro1 5 10 15 Arg Pro
Lys Ser Ala Gly Thr Ala Thr Gln 20 25 19426PRTArtificial
SequenceEngineered peptides based on Bad protein 194Arg Arg Met Ser
Asp Glu Phe Val Xaa Ser Phe Lys Lys Gly Leu Pro1 5 10 15 Arg Pro
Lys Ser Ala Gly Thr Ala Thr Gln 20 25 19526PRTArtificial
SequenceEngineered peptides based on Bad protein 195Arg Arg Met Ser
Xaa Xaa Phe Val Xaa Ser Phe Lys Lys Gly Leu Pro1 5 10 15 Arg Pro
Lys Ser Ala Gly Thr Ala Thr Gln 20 25 19626PRTArtificial
SequenceEngineered peptides based on Bad protein 196Arg Arg Xaa Ser
Xaa Xaa Phe Val Xaa Ser Phe Lys Lys Gly Leu Pro1 5 10 15 Arg Pro
Lys Ser Ala Gly Thr Ala Thr Gln 20 25 19720PRTArtificial
SequenceEngineered peptides based on Bad protein 197Phe Val Xaa Ser
Phe Lys Lys Gly Leu Pro Arg Pro Lys Ser Ala Gly1 5 10 15 Thr Ala
Thr Gln 20 19816PRTArtificial SequenceEngineered peptides based on
Bad protein 198Phe Val Xaa Ser Phe Lys Lys Gly Leu Xaa Arg Pro Lys
Ser Ala Gly1 5 10 15 19916PRTArtificial SequenceEngineered peptides
based on Bad protein 199Phe Val Xaa Ser Phe Lys Lys Gly Leu Tyr Arg
Pro Lys Ser Ala Gly1 5 10 15 20014PRTArtificial SequenceEngineered
peptides based on Prf-1 protein 200Lys Arg Ser His Lys Phe Val Pro
Gly Ala Trp Leu Ala Gly1 5 10 20119PRTArtificial SequenceEngineered
peptides based on Prf-1 protein 201Val Thr Ser Leu Arg Arg Ser Gly
Ser Phe Pro Val Asp Thr Gln Arg1 5 10 15 Phe Leu
Arg20216PRTArtificial SequenceEngineered peptides based on Prf-1
protein 202Arg Ser Ile Arg Asn Asp Trp Lys Val Gly Leu Asp Val Thr
Pro Lys1 5 10 15 20320PRTArtificial SequenceEngineered peptides
based on Prf-1 protein 203Arg Arg Glu Ala Leu Arg Arg Ala Leu Ser
Gln Tyr Leu Thr Asp Arg1 5 10 15 Ala Arg Trp Arg 20
20414PRTArtificial SequenceEngineered peptides based on Prf-1
protein 204Asn Leu Asn His Gly His Leu Lys Phe Arg Tyr His Ala Arg1
5 10 20514PRTArtificial SequenceEngineered peptides based on Prf-1
protein 205Lys Arg Ser His Lys Phe Val Xaa Gly Ala Trp Leu Ala Gly1
5 10 20619PRTArtificial SequenceEngineered peptides based on Prf-1
protein 206Val Thr Ser Leu Arg Arg Ser Gly Ser Phe Xaa Val Asp Thr
Gln Arg1 5 10 15 Phe Leu Arg20719PRTArtificial SequenceEngineered
peptides based on Prf-1 protein 207Val Thr Ser Leu Arg Arg Ser Gly
Ser Phe Pro Val Xaa Thr Gln Arg1 5 10 15 Phe Leu
Arg20819PRTArtificial SequenceEngineered peptides based on Prf-1
protein 208Val Thr Ser Leu Arg Arg Ser Gly Ser Phe Xaa Val Xaa Thr
Gln Arg1 5 10 15 Phe Leu Arg20916PRTArtificial SequenceEngineered
peptides based on Prf-1 protein 209Arg Ser Ile Arg Asn Xaa Trp Lys
Val Gly Leu Asp Val Thr Pro Lys1 5 10 15 21014PRTArtificial
SequenceEngineered peptides based on Prf-1 protein 210Arg Ser Ile
Arg Asn Asp Trp Lys Val Gly Leu Asp Val Thr1 5 10
21114PRTArtificial SequenceEngineered peptides based on Prf-1
protein 211Arg Ser Ile Arg Asn Xaa Trp Lys Val Gly Leu Asp Val Thr1
5 10 21220PRTArtificial SequenceEngineered peptides based on Prf-1
protein 212Arg Arg Xaa Ala Leu Arg Arg Ala Leu Ser Gln Tyr Leu Thr
Asp Arg1 5 10 15 Ala Arg Trp Arg 20 21320PRTArtificial
SequenceEngineered peptides based on Prf-1 protein 213Arg Arg Glu
Ala Leu Arg Arg Ala Leu Ser Gln Tyr Leu Thr Xaa Arg1 5 10 15 Ala
Arg Trp Arg 20 21420PRTArtificial SequenceEngineered peptides based
on Prf-1 protein 214Arg Arg Xaa Ala Leu Arg Arg Ala Leu Ser Gln Tyr
Leu Thr Xaa Arg1 5 10 15 Ala Arg Trp Arg 20 21520PRTArtificial
SequenceEngineered peptides based on Prf-1 protein 215Arg Xaa Xaa
Ala Leu Arg Arg Ala Leu Ser Gln Tyr Leu Thr Xaa Arg1 5 10 15 Ala
Arg Trp Arg 20 21614PRTArtificial SequenceEngineered peptides based
on Prf-1 protein 216Asn Leu Asn Xaa Gly His Leu Lys Phe Arg Tyr His
Ala Arg1 5 10 21714PRTArtificial SequenceEngineered peptides based
on Prf-1 protein 217Asn Leu Asn Xaa Gly Xaa Leu Lys Phe Arg Tyr His
Ala Arg1 5 10 21814PRTArtificial SequenceEngineered peptides based
on Prf-1 protein 218Asn Leu Asn Xaa Gly Xaa Leu Lys Phe Arg Tyr Xaa
Ala Arg1 5 10 21914PRTArtificial SequenceEngineered peptides based
on Prf-1 protein 219Asn Leu Asn Xaa Gly His Leu Lys Phe Arg Tyr His
Ala Arg1 5 10 22014PRTArtificial SequenceEngineered peptides based
on Prf-1 protein 220Asn Leu Asn Xaa Gly Xaa Leu Lys Phe Arg Tyr His
Ala Arg1 5 10 22114PRTArtificial SequenceEngineered peptides based
on Prf-1 protein 221Asn Leu Asn Xaa Gly Xaa Leu Lys Phe Arg Tyr Xaa
Ala Arg1 5 10 22214PRTArtificial SequenceEngineered peptides based
on Prf-1 protein 222Xaa Leu Asn Xaa Gly Xaa Leu Lys Phe Arg Tyr Xaa
Ala Arg1 5 10 22314PRTArtificial SequenceEngineered peptides based
on Prf-1 protein 223Xaa Leu Asn Xaa Gly Xaa Leu Lys Phe Arg Tyr Xaa
Ala Arg1
5 10 22417PRTArtificial SequenceEngineered peptides based on
Granulysin protein 224Leu Gly Arg Asp Tyr Arg Thr Cys Leu Thr Ile
Val Gln Lys Leu Lys1 5 10 15 Lys22523PRTArtificial
SequenceEngineered peptides based on Granulysin protein 225Lys Pro
Thr Gln Arg Ser Val Ser Asn Ala Ala Thr Arg Val Cys Arg1 5 10 15
Thr Gly Arg Ser Arg Trp Arg 20 22618PRTArtificial
SequenceEngineered peptides based on Granulysin protein 226Ser Arg
Trp Arg Arg Arg Tyr Gln Ser Arg Val Thr Gln Gly Leu Val1 5 10 15
Ala Gly22717PRTArtificial SequenceEngineered peptides based on
Granulysin protein 227Leu Gly Arg Xaa Tyr Arg Thr Cys Leu Thr Ile
Val Gln Lys Leu Lys1 5 10 15 Lys22817PRTArtificial
SequenceEngineered peptides based on Granulysin protein 228Leu Gly
Arg Asp Tyr Arg Thr Xaa Leu Thr Ile Val Gln Lys Leu Lys1 5 10 15
Lys22917PRTArtificial SequenceEngineered peptides based on
Granulysin protein 229Leu Gly Arg Xaa Tyr Arg Thr Xaa Leu Thr Ile
Val Gln Lys Leu Lys1 5 10 15 Lys23023PRTArtificial
SequenceEngineered peptides based on Granulysin protein 230Lys Pro
Thr Gln Arg Ser Val Ser Asn Ala Ala Thr Arg Val Xaa Arg1 5 10 15
Thr Gly Arg Ser Arg Trp Arg 20 23118PRTArtificial
SequenceEngineered peptides based on Granulysin protein 231Lys Pro
Thr Gln Arg Ser Val Ser Asn Ala Ala Thr Arg Val Xaa Arg1 5 10 15
Thr Gly23218PRTArtificial SequenceEngineered peptides based on
Granulysin protein 232Lys Pro Thr Gln Arg Ser Val Ser Asn Tyr Ala
Thr Arg Val Xaa Arg1 5 10 15 Thr Gly23318PRTArtificial
SequenceEngineered peptides based on Granulysin protein 233Lys Pro
Thr Gln Arg Ser Val Ser Asn Phe Ala Thr Arg Val Xaa Arg1 5 10 15
Thr Gly23417PRTArtificial SequenceEngineered peptides based on
Granulysin protein 234Ser Arg Trp Arg Arg Tyr Gln Ser Arg Val Thr
Gln Tyr Leu Val Ala1 5 10 15 Gly23517PRTArtificial
SequenceEngineered peptides based on Granulysin protein 235Xaa Arg
Trp Arg Arg Tyr Gln Ser Arg Val Thr Gln Tyr Leu Val Ala1 5 10 15
Gly23627PRTArtificial SequenceEngineered peptides based on CidA
protein 236Gln Lys Ile Phe His Leu Pro Leu Ala Gly Ser Ile Val Gly
Leu Phe1 5 10 15 Leu Phe Tyr Leu Leu Leu Gln Phe Lys Ile Val 20 25
23741PRTArtificial SequenceEngineered peptides based on CidA
protein 237Glu Ile Thr Leu Asn Tyr Ile Leu Phe Phe Ala Val Ile Ile
Ile Gly1 5 10 15 Thr Cys Ile Val Ala Leu Ser Ser Gly Tyr Ile Ala
Glu Lys Met Ser 20 25 30 Val Lys His Lys Gln Arg Lys Gly Ile 35 40
23827PRTArtificial SequenceEngineered peptides based on CidA
protein 238Gln Lys Ile Phe His Leu Pro Leu Ala Xaa Ser Ile Val Gly
Leu Phe1 5 10 15 Leu Phe Tyr Leu Leu Leu Gln Phe Lys Ile Val 20 25
23927PRTArtificial SequenceEngineered peptides based on CidA
protein 239Gln Lys Ile Phe His Leu Pro Leu Ala Gly Ser Ile Val Gly
Leu Phe1 5 10 15 Leu Phe Tyr Leu Gly Leu Gln Phe Lys Ile Val 20 25
24027PRTArtificial SequenceEngineered peptides based on CidA
protein 240Gln Lys Ile Phe His Leu Pro Leu Ala Xaa Ser Ile Val Gly
Leu Phe1 5 10 15 Leu Phe Tyr Leu Gly Leu Gln Phe Lys Ile Val 20 25
24120PRTArtificial SequenceEngineered peptides based on CidA
protein 241Leu Ala Xaa Ser Ile Val Gly Leu Phe Leu Phe Tyr Leu Gly
Leu Gln1 5 10 15 Phe Lys Ile Val 20 24220PRTArtificial
SequenceEngineered peptides based on CidA protein 242Leu Ala Xaa
Ser Ile Val Xaa Leu Phe Leu Phe Tyr Leu Gly Leu Gln1 5 10 15 Phe
Lys Ile Val 20 24341PRTArtificial SequenceEngineered peptides based
on CidA protein 243Glu Ile Thr Leu Asn Tyr Ile Leu Phe Phe Ala Val
Ile Ile Ile Gly1 5 10 15 Thr Xaa Ile Val Ala Leu Ser Ser Gly Tyr
Ile Ala Glu Lys Xaa Ser 20 25 30 Val Lys His Lys Gln Arg Lys Gly
Ile 35 40 24414PRTArtificial SequenceEngineered peptides based on
CidA protein 244Ala Glu Lys Met Ser Val Lys His Lys Gln Arg Lys Gly
Ile1 5 10 24514PRTArtificial SequenceEngineered peptides based on
CidA protein 245Ala Xaa Lys Met Ser Val Lys His Lys Gln Arg Lys Gly
Ile1 5 10 24614PRTArtificial SequenceEngineered peptides based on
CidA protein 246Ala Leu Lys Met Ser Val Lys His Lys Gln Arg Lys Gly
Ile1 5 10 24714PRTArtificial SequenceEngineered peptides based on
CidA protein 247Ala Leu Lys Xaa Ser Val Lys His Lys Gln Arg Lys Gly
Ile1 5 10 24817PRTArtificial SequenceEngineered peptides based on
LrgA protein 248Lys Val Thr Ser Arg Ser Lys Gly Asp Lys Val Thr Lys
Lys Ile Lys1 5 10 15 Ile24917PRTArtificial SequenceEngineered
peptides based on LrgA protein 249Lys Val Thr Ser Arg Ser Lys Gly
Asp Lys Val Thr Lys Trp Ile Lys1 5 10 15 Ile25017PRTArtificial
SequenceEngineered peptides based on LrgA protein 250Lys Val Thr
Ser Arg Ser Lys Gly Asp Lys Val Thr Lys Xaa Ile Lys1 5 10 15
Ile25117PRTArtificial SequenceEngineered peptides based on LrgA
protein 251Lys Val Thr Ser Arg Ser Lys Gly Asp Lys Val Thr Lys Xaa
Ile Lys1 5 10 15 Ile25228PRTArtificial SequenceEngineered peptides
based on Lambda S21 protein 252Ser Leu Val Leu Gly Phe Leu Thr Tyr
Leu Thr Asn Leu Tyr Phe Lys1 5 10 15 Ile Arg Glu Asp Arg Arg Lys
Ala Ala Arg Gly Glu 20 25 25328PRTArtificial SequenceEngineered
peptides based on Lambda S21 protein 253Ser Leu Val Leu Gly Phe Leu
Thr Tyr Leu Thr Asn Leu Tyr Phe Lys1 5 10 15 Ile Arg Xaa Asp Arg
Arg Lys Ala Ala Arg Gly Glu 20 25 25428PRTArtificial
SequenceEngineered peptides based on Lambda S21 protein 254Ser Leu
Val Leu Gly Phe Leu Thr Tyr Leu Thr Asn Leu Tyr Phe Lys1 5 10 15
Ile Arg Glu Xaa Arg Arg Lys Ala Ala Arg Gly Glu 20 25
25528PRTArtificial SequenceEngineered peptides based on Lambda S21
protein 255Ser Leu Val Leu Gly Phe Leu Thr Tyr Leu Thr Asn Leu Tyr
Phe Lys1 5 10 15 Ile Arg Xaa Xaa Arg Arg Lys Ala Ala Arg Gly Glu 20
25 25615PRTArtificial SequenceEngineered peptides based on Lambda
S21 protein 256Leu Tyr Phe Lys Ile Arg Xaa Xaa Arg Arg Lys Ala Ala
Arg Gly1 5 10 15 25717PRTArtificial SequenceEngineered peptides
based on Holin protein 257Ala Tyr Leu Arg Gly Arg Tyr Asn Gly Gly
Ala Phe Thr Lys Thr Val1 5 10 15 Ile25822PRTArtificial
SequenceEngineered peptides based on Holin protein 258Ser Ile Gly
Ser Leu Ile Lys Arg Phe Ala Ala Lys Lys Ala Gly Val1 5 10 15 Glu
Asp Gly Arg Asn Gln 20 25916PRTArtificial SequenceEngineered
peptides based on Holin protein 259Ser Ile Gly Ser Leu Ile Lys Arg
Phe Ala Ala Lys Lys Ala Gly Val1 5 10 15 26017PRTArtificial
SequenceEngineered peptides based on Holin protein 260Xaa Tyr Leu
Arg Gly Arg Tyr Asn Gly Gly Ala Phe Thr Lys Thr Val1 5 10 15
Ile26122PRTArtificial SequenceEngineered peptides based on Holin
protein 261Ser Ile Gly Ser Leu Ile Lys Arg Phe Ala Ala Lys Lys Ala
Gly Val1 5 10 15 Xaa Asp Gly Arg Asn Gln 20 26222PRTArtificial
SequenceEngineered peptides based on Holin protein 262Ser Ile Gly
Ser Leu Ile Lys Arg Phe Ala Ala Lys Lys Ala Gly Val1 5 10 15 Glu
Xaa Gly Arg Asn Gln 20 26316PRTArtificial SequenceEngineered
peptides based on Holin protein 263Ser Ile Gly Ser Leu Ile Lys Arg
Phe Ala Xaa Lys Lys Ala Gly Val1 5 10 15 26418PRTArtificial
SequenceNovel therapeutic peptide designs based on programmed cell
death effector domains 264Ala Leu Phe Tyr Phe Ala Ser Lys Leu Val
Leu Lys Ala Leu Tyr Thr1 5 10 15 Lys Val26514PRTArtificial
SequenceNovel therapeutic peptide designs based on programmed cell
death effector domains 265Ala Leu Lys Tyr Ser Val Lys His Lys Gln
Arg Lys Gly Ile1 5 10 26618PRTArtificial SequenceNovel therapeutic
peptide designs based on programmed cell death effector domains
266Leu Lys Lys Ile Thr Asn Phe Arg Gly Lys Arg Tyr Arg Ser Leu Thr1
5 10 15 Gly Lys26715PRTArtificial SequenceNovel therapeutic peptide
designs based on programmed cell death effector domains 267Ala Leu
Arg Ser Lys Leu Gln Ser Gln Leu Leu Ser Leu Arg Lys1 5 10 15
26816PRTArtificial SequenceNovel therapeutic peptide designs based
on programmed cell death effector domains 268Ser Ile Gly Ser Leu
Ile Lys Arg Phe Ala Tyr Lys Lys Ala Gly Val1 5 10 15
26919PRTArtificial SequenceNovel therapeutic peptide designs based
on programmed cell death effector domains 269Thr Arg Ala Leu Val
Ala Lys Phe Val Gly Tyr Lys Leu Arg Gln Lys1 5 10 15 Gly Tyr
Val27026PRTArtificial SequenceNovel therapeutic peptide designs
based on programmed cell death effector domains 270Asn Phe Asn Trp
Gly Arg Val Val Ala Leu Phe Tyr Phe Ala Ser Lys1 5 10 15 Leu Val
Leu Lys Ala Leu Tyr Thr Lys Val 20 25 27119PRTArtificial
SequenceNovel therapeutic peptide designs based on programmed cell
death effector domains 271Thr Val Thr Ile Phe Val Ala Lys Val Leu
Thr Ala Ser Leu Thr Ile1 5 10 15 Trp Lys Lys27217PRTArtificial
SequenceNovel therapeutic peptide designs based on programmed cell
death effector domains 272Thr Arg Phe Arg Arg Thr Phe Ser Lys Leu
Ala Ala Gln Leu His Val1 5 10 15 Thr27321PRTArtificial
SequenceNovel therapeutic peptide designs based on programmed cell
death effector domains 273Gly Gln Arg Ser Pro Thr Ala Leu Ser Leu
Tyr Leu Phe Leu Leu Tyr1 5 10 15 Trp Val Ile Val Lys 20
27423PRTArtificial SequenceNovel therapeutic peptide designs based
on programmed cell death effector domains 274Lys Lys Ile Glu Val
Leu Lys Ser Leu Gln Ser Lys Ala Lys Leu Leu1 5 10 15 Arg Asn Lys
Ala Gly Trp Leu 20 27515PRTArtificial SequenceNovel therapeutic
peptide designs based on programmed cell death effector domains
275Gly Leu Arg Asn Lys Leu Gln Ser Gln Leu Leu Ser Ile Lys Lys1 5
10 15 27620PRTArtificial SequenceNovel therapeutic peptide designs
based on programmed cell death effector domains 276Lys Leu Ala Lys
Lys Val Asp Pro Gln Gly Leu Arg Thr Ile Gly Val1 5 10 15 Ile Thr
Lys Leu 20 27715PRTArtificial SequenceNovel therapeutic peptide
designs based on programmed cell death effector domains 277Lys Ser
Lys Lys Tyr Thr Leu Pro Leu Lys Asn Leu Lys Ile Arg1 5 10 15
27815PRTArtificial SequenceNovel therapeutic peptide designs based
on programmed cell death effector domains 278Ser Lys Ser Ile Lys
Asn Leu Lys Pro Lys Ile Ile Tyr Lys Ser1 5 10 15 27920PRTArtificial
SequenceNovel therapeutic peptide designs based on programmed cell
death effector domains 279Leu Ala Lys Ser Ile Val Arg Leu Phe Leu
Phe Tyr Leu Gly Leu Gln1 5 10 15 Phe Lys Ile Val 20 280780PRTHomo
sapiensprogrammed cell death / holin-like proteins - Dnm1 (Fig. 1)
280Met Gly Asn Arg Gly Met Glu Asp Leu Ile Pro Leu Val Asn Arg Leu1
5 10 15 Gln Asp Ala Phe Ser Ala Ile Gly Gln Asn Ala Asp Leu Asp Leu
Pro 20 25 30 Gln Ile Ala Val Val Gly Gly Gln Ser Ala Gly Lys Ser
Ser Val Leu 35 40 45 Glu Asn Phe Val Gly Arg Asp Phe Leu Pro Arg
Gly Ser Gly Ile Val 50 55 60 Thr Arg Arg Pro Leu Val Leu Gln Leu
Val Asn Ala Thr Thr Glu Tyr65 70 75 80 Ala Glu Phe Leu His Cys Lys
Gly Lys Lys Phe Thr Asp Phe Glu Glu 85 90 95 Val Arg Leu Glu Ile
Glu Ala Glu Thr Asp Arg Val Thr Gly Thr Asn 100 105 110 Lys Gly Ile
Ser Pro Val Pro Ile Asn Leu Arg Val Tyr Ser Pro His 115 120 125 Val
Leu Asn Leu Thr Leu Val Asp Leu Pro Gly Met Thr Lys Val Pro 130 135
140 Val Gly Asp Gln Pro Pro Asp Ile Glu Phe Gln Ile Arg Asp Met
Leu145 150 155 160 Met Gln Phe Val Thr Lys Glu Asn Cys Leu Ile Leu
Ala Val Ser Pro 165 170 175 Ala Asn Ser Asp Leu Ala Asn Ser Asp Ala
Leu Lys Val Ala Lys Glu 180 185 190 Val Asp Pro Gln Gly Gln Arg Thr
Ile Gly Val Ile Thr Lys Leu Asp 195 200 205 Leu Met Asp Glu Gly Thr
Asp Ala Arg Asp Val Leu Glu Asn Lys Leu 210 215 220 Leu Pro Leu Arg
Arg Gly Tyr Ile Gly Val Val Asn Arg Ser Gln Lys225 230 235 240 Asp
Ile Asp Gly Lys Lys Asp Ile Thr Ala Ala Leu Ala Ala Glu Arg 245 250
255 Lys Phe Phe Leu Ser His Pro Ser Tyr Arg His Leu Ala Asp Arg Met
260 265 270 Gly Thr Pro Tyr Leu Gln Lys Val Leu Asn Gln Gln Leu Thr
Asn His 275 280 285 Ile Arg Asp Thr Leu Pro Gly Leu Arg Asn Lys Leu
Gln Ser Gln Leu 290 295 300 Leu Ser Ile Glu Lys Glu Val Glu Glu Tyr
Lys Asn Phe Arg Pro Asp305 310 315 320 Asp Pro Ala Arg Lys Thr Lys
Ala Leu Leu Gln Met Val Gln Gln Phe 325 330 335 Ala Val Asp Phe Glu
Lys Arg Ile Glu Gly Ser Gly Asp Gln Ile Asp 340 345 350 Thr Tyr Glu
Leu Ser Gly Gly Ala Arg Ile Asn Arg Ile Phe His Glu 355 360 365 Arg
Phe Pro Phe Glu Leu Val Lys Met Glu Phe Asp Glu Lys Glu Leu 370 375
380 Arg Arg Glu Ile Ser Tyr Ala Ile Lys Asn Ile His Gly Ile Arg
Thr385 390 395 400 Gly Leu Phe Thr Pro Asp Leu Ala Phe Glu Ala Thr
Val Lys Lys Gln 405 410 415 Val Gln Lys Leu Lys Glu Pro Ser Ile Lys
Cys Val Asp Met Val Val 420 425 430 Ser Glu Leu Thr Ala Thr Ile Arg
Lys Cys Ser Glu Lys Leu Gln Gln 435 440 445 Tyr Pro Arg Leu Arg Glu
Glu Met Glu Arg Ile Val Thr Thr His Ile 450 455 460 Arg Glu Arg Glu
Gly Arg Thr Lys Glu Gln Val Met Leu Leu Ile Asp465 470 475 480 Ile
Glu Leu Ala Tyr Met Asn Thr Asn His Glu Asp Phe Ile Gly Phe 485 490
495 Ala Asn Ala Gln Gln Arg Ser Asn Gln Met Asn Lys Lys Lys Thr Ser
500 505 510 Gly Asn Gln Asp Glu Ile Leu Val Ile Arg Lys Gly Trp Leu
Thr Ile 515 520 525 Asn Asn Ile Gly Ile Met Lys Gly Gly Ser Lys Glu
Tyr Trp Phe Val 530
535 540 Leu Thr Ala Glu Asn Leu Ser Trp Tyr Lys Asp Asp Glu Glu Lys
Glu545 550 555 560 Lys Lys Tyr Met Leu Ser Val Asp Asn Leu Lys Leu
Arg Asp Val Glu 565 570 575 Lys Gly Phe Met Ser Ser Lys His Ile Phe
Ala Leu Phe Asn Thr Glu 580 585 590 Gln Arg Asn Val Tyr Lys Asp Tyr
Arg Gln Leu Glu Leu Ala Cys Glu 595 600 605 Thr Gln Glu Glu Val Asp
Ser Trp Lys Ala Ser Phe Leu Arg Ala Gly 610 615 620 Val Tyr Pro Glu
Arg Val Gly Asp Lys Glu Lys Ala Ser Glu Thr Glu625 630 635 640 Glu
Asn Gly Ser Asp Ser Phe Met His Ser Met Asp Pro Gln Leu Glu 645 650
655 Arg Gln Val Glu Thr Ile Arg Asn Leu Val Asp Ser Tyr Met Ala Ile
660 665 670 Val Asn Lys Thr Val Arg Asp Leu Met Pro Lys Thr Ile Met
His Leu 675 680 685 Met Ile Asn Asn Thr Lys Glu Phe Ile Phe Ser Glu
Leu Leu Ala Asn 690 695 700 Leu Tyr Ser Cys Gly Asp Gln Asn Thr Leu
Met Glu Glu Ser Ala Glu705 710 715 720 Gln Ala Gln Arg Arg Asp Glu
Met Leu Arg Met Tyr His Ala Leu Lys 725 730 735 Glu Ala Leu Ser Ile
Ile Gly Asp Ile Asn Thr Thr Thr Val Ser Thr 740 745 750 Pro Met Pro
Pro Pro Val Asp Asp Ser Trp Leu Gln Val Gln Ser Val 755 760 765 Pro
Ala Gly Arg Arg Ser Pro Thr Ser Ser Pro Thr 770 775 780
281191PRTHomo sapiensprogrammed cell death / holin-like proteins -
Bax (Fig. 1) 281Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gly Gly Pro
Thr Ser Ser1 5 10 15 Glu Gln Ile Met Lys Thr Gly Ala Leu Leu Leu
Gln Gly Phe Ile Gln 20 25 30 Asp Arg Ala Gly Arg Met Gly Gly Glu
Ala Pro Glu Leu Ala Leu Asp 35 40 45 Pro Val Pro Gln Asp Ala Ser
Thr Lys Lys Leu Ser Glu Cys Leu Lys 50 55 60 Arg Ile Gly Asp Glu
Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile65 70 75 80 Ala Ala Val
Asp Thr Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala 85 90 95 Ala
Asp Met Phe Ser Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala 100 105
110 Leu Phe Tyr Phe Ala Ser Lys Val Leu Lys Ala Leu Cys Thr Lys Val
115 120 125 Pro Glu Leu Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe
Leu Arg 130 135 140 Glu Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly
Trp Asp Gly Leu145 150 155 160 Leu Ser Tyr Phe Gly Thr Pro Thr Trp
Gln Thr Val Thr Ile Phe Val 165 170 175 Ala Gly Val Leu Thr Ala Ser
Leu Thr Ile Trp Lys Lys Met Gly 180 185 190 282166PRTHomo
sapiensprogrammed cell death / holin-like proteins - Bcl-2 (Fig. 1)
282Met Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Arg Glu Ile Val Met1
5 10 15 Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg Gly Tyr Glu Trp Asp
Ala 20 25 30 Gly Asp Asp Val Glu Glu Asn Arg Thr Glu Ala Pro Glu
Gly Thr Glu 35 40 45 Ser Glu Val Val His Leu Ala Leu Arg Gln Ala
Gly Asp Asp Phe Ser 50 55 60 Arg Arg Tyr Arg Gly Asp Phe Ala Glu
Met Ser Ser Gln Leu His Leu65 70 75 80 Thr Pro Phe Thr Ala Arg Gly
Arg Phe Ala Thr Val Val Glu Glu Leu 85 90 95 Phe Arg Asp Gly Val
Asn Trp Gly Arg Ile Val Ala Phe Phe Glu Phe 100 105 110 Gly Gly Val
Met Cys Val Glu Ser Val Asn Arg Glu Met Ser Pro Leu 115 120 125 Val
Asp Asn Ile Ala Leu Trp Met Thr Glu Tyr Leu Asn Arg His Leu 130 135
140 His Thr Trp Ile Gln Asp Asn Gly Gly Trp Asp Ala Phe Val Glu
Leu145 150 155 160 Tyr Gly Pro Ser Met Xaa 165
283131PRTStaphylococcus aureusprogrammed cell death / holin-like
proteins - CidA (Fig. 2) 283Met His Lys Val Gln Leu Ile Ile Lys Leu
Leu Leu Gln Leu Gly Ile1 5 10 15 Ile Ile Val Ile Thr Tyr Ile Gly
Thr Glu Ile Gln Lys Ile Phe His 20 25 30 Leu Pro Leu Ala Gly Ser
Ile Val Gly Leu Phe Leu Phe Tyr Leu Leu 35 40 45 Leu Gln Phe Lys
Ile Val Pro Leu Thr Trp Val Glu Asp Gly Ala Asn 50 55 60 Phe Leu
Leu Lys Thr Met Val Phe Phe Phe Ile Pro Ser Val Val Gly65 70 75 80
Ile Met Asp Val Ala Ser Glu Ile Thr Leu Asn Tyr Ile Leu Phe Phe 85
90 95 Ala Val Ile Ile Ile Gly Thr Cys Ile Val Ala Leu Ser Ser Gly
Tyr 100 105 110 Ile Ala Glu Lys Met Ser Val Lys His Lys Gln Arg Lys
Gly Ile Asp 115 120 125 Ala Tyr Glu 130 284147PRTStaphylococcus
aureusprogrammed cell death proteins - LrgA (Fig. 2) 284Met Val Val
Lys Gln Gln Lys Asp Ala Ser Lys Pro Ala His Phe Phe1 5 10 15 His
Gln Val Ile Val Ile Ala Leu Val Leu Phe Val Ser Lys Ile Ile 20 25
30 Glu Ser Phe Met Pro Ile Pro Met Pro Ala Ser Val Ile Gly Leu Val
35 40 45 Leu Leu Phe Val Leu Leu Cys Thr Gly Ala Val Lys Leu Gly
Glu Val 50 55 60 Glu Lys Val Gly Thr Thr Leu Thr Asn Asn Ile Gly
Leu Leu Phe Val65 70 75 80 Pro Ala Gly Ile Ser Val Val Asn Ser Leu
Gly Val Ile Ser Gln Ala 85 90 95 Pro Phe Leu Ile Ile Gly Leu Ile
Ile Val Ser Thr Ile Leu Leu Leu 100 105 110 Ile Cys Thr Gly Tyr Val
Thr Gln Ile Ile Met Lys Val Thr Ser Arg 115 120 125 Ser Lys Gly Asp
Lys Val Thr Lys Lys Ile Lys Ile Glu Glu Ala Gln 130 135 140 Ala His
Asp145 285309PRTBos taurusPerforin 1 protein (Fig. 3) 285Asp Thr
Gln Arg Phe Leu Arg Pro Asp Gly Thr Cys Thr Leu Cys Arg1 5 10 15
Asn Ala Leu Gln Lys Asp Val Leu Gln Arg Leu Pro Leu Ala Ile Thr 20
25 30 Asp Trp Arg Ala His Gly Ala Gly Cys Lys Arg Arg Val Val Lys
Leu 35 40 45 Glu Gly Arg Ser Thr Glu Asp Val Ala Gly Glu Ala Ala
Asn Arg Ile 50 55 60 Arg Asn Asp Trp Gln Val Gly Leu Asp Val Ser
Pro Lys Pro Asn Ala65 70 75 80 Asn Val Arg Val Thr Val Ala Gly Ser
His Ser Glu Asp Ala Asn Phe 85 90 95 Ala Ala Gln Lys Thr His Gln
Asp Asn Tyr Arg Phe Ser Met Asp Leu 100 105 110 Val Glu Cys Arg Phe
Tyr Ser Phe His Leu Val His Thr Pro Pro Val 115 120 125 His Pro Glu
Phe Lys Arg Ala Leu Lys Thr Leu Pro Pro His Phe Asn 130 135 140 Thr
Ser Thr Lys Pro Asp Tyr His Arg Leu Ile Ser Ser Tyr Gly Thr145 150
155 160 His Phe Ile Arg Ser Met Glu Leu Gly Gly Arg Ile Ser Ala Leu
Thr 165 170 175 Ala Leu Arg Thr Cys Glu Leu Ala Leu Glu Gly Leu Thr
Ala Ser Glu 180 185 190 Val Glu Asp Cys Leu Ala Val Glu Ala Glu Val
Ser Ile Ser Asp Arg 195 200 205 Ala Ser Ala Ser Pro Ser Phe Lys Ala
Cys Glu Glu Lys Lys Lys Asn 210 215 220 His Lys Val Gly Thr Ser Phe
His Gln Ala Tyr Arg Glu Arg His Ser225 230 235 240 Asn Val Asp Gly
Gly His His Ser Thr Met His Asp Leu Leu Phe Gly 245 250 255 Ser Gln
Ala Gly Pro Glu Gln Phe Ser Ala Trp Val Ala Ser Leu Gln 260 265 270
Asp Ser Pro Gly Leu Val Asp Tyr Thr Leu Glu Pro Leu His Met Leu 275
280 285 Val Glu Ser Gln Asp Pro Arg Arg Glu Ala Leu Arg Gln Ala Val
Ser 290 295 300 Lys Tyr Val Thr Asp305 286239PRTHomo sapiensBcl-2
protein (Fig. 3) 286Met Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Arg
Glu Ile Val Met1 5 10 15 Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg
Gly Tyr Glu Trp Asp Ala 20 25 30 Gly Asp Val Gly Ala Ala Pro Pro
Gly Ala Ala Pro Ala Pro Gly Ile 35 40 45 Phe Ser Ser Gln Pro Gly
His Thr Pro His Pro Ala Ala Ser Arg Asp 50 55 60 Pro Val Ala Arg
Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala65 70 75 80 Ala Ala
Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu Thr 85 90 95
Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Arg Asp Phe 100
105 110 Ala Glu Met Ser Ser Gln Leu His Leu Thr Pro Phe Thr Ala Arg
Gly 115 120 125 Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg Asp Gly
Val Asn Trp 130 135 140 Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gly
Val Met Cys Val Glu145 150 155 160 Ser Val Asn Arg Glu Met Ser Pro
Leu Val Asp Asn Ile Ala Leu Trp 165 170 175 Met Thr Glu Tyr Leu Asn
Arg His Leu His Thr Trp Ile Gln Asp Asn 180 185 190 Gly Gly Trp Asp
Ala Phe Val Glu Leu Tyr Gly Pro Ser Met Arg Pro 195 200 205 Leu Phe
Asp Phe Ser Trp Leu Ser Leu Lys Thr Leu Leu Ser Leu Ala 210 215 220
Leu Val Gly Ala Cys Ile Thr Leu Gly Ala Tyr Leu Gly His Lys225 230
235 287193PRTHomo sapiensBCL-W protein (Fig. 3) 287Met Ala Thr Pro
Ala Ser Ala Pro Asp Thr Arg Ala Leu Val Ala Asp1 5 10 15 Phe Val
Gly Tyr Lys Leu Arg Gln Lys Gly Tyr Val Cys Gly Ala Gly 20 25 30
Pro Gly Glu Gly Pro Ala Ala Asp Pro Leu His Gln Ala Met Arg Ala 35
40 45 Ala Gly Asp Glu Phe Glu Thr Arg Phe Arg Arg Thr Phe Ser Asp
Leu 50 55 60 Ala Ala Gln Leu His Val Thr Pro Gly Ser Ala Gln Gln
Arg Phe Thr65 70 75 80 Gln Val Ser Asp Glu Leu Phe Gln Gly Gly Pro
Asn Trp Gly Arg Leu 85 90 95 Val Ala Phe Phe Val Phe Gly Ala Ala
Leu Cys Ala Glu Ser Val Asn 100 105 110 Lys Glu Met Glu Pro Leu Val
Gly Gln Val Gln Glu Trp Met Val Ala 115 120 125 Tyr Leu Glu Thr Arg
Leu Ala Asp Trp Ile His Ser Ser Gly Gly Trp 130 135 140 Ala Glu Phe
Thr Ala Leu Tyr Gly Asp Gly Ala Leu Glu Glu Ala Arg145 150 155 160
Arg Leu Arg Glu Gly Asn Trp Ala Ser Val Arg Thr Val Leu Thr Gly 165
170 175 Ala Val Ala Leu Gly Ala Leu Val Thr Val Gly Ala Phe Phe Ala
Ser 180 185 190 Lys28819PRTArtificial Sequenceengineered peptide
based on programmed cell death effector proteins 288Ser Gln Ser Asn
Arg Glu Leu Val Val Asp Phe Leu Ser Tyr Lys Leu1 5 10 15 Ser Gln
Lys28917PRTArtificial Sequenceengineered peptide based on
programmed cell death effector proteins 289Gln Lys Leu Lys Lys Met
Val Asp Lys Pro Thr Gln Arg Ser Val Ser1 5 10 15 Asn29076PRTHomo
sapiensHelix 1 of human Bcl-2 290Met Ala His Ala Gly Arg Thr Gly
Tyr Asp Asn Arg Glu Ile Val Met1 5 10 15 Lys Tyr Ile His Tyr Lys
Leu Ser Gln Arg Gly Tyr Glu Trp Asp Ala 20 25 30 Gly Asp Asp Val
Glu Glu Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu 35 40 45 Ser Glu
Val Val His Leu Thr Leu Arg Gln Ala Gly Asp Asp Phe Ser 50 55 60
Arg Arg Tyr Arg Arg Asp Phe Ala Glu Met Ser Ser65 70 75
29176PRTHomo sapiensHelix 2 of human Bcl-2 291Gly Asp Asp Val Glu
Glu Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu1 5 10 15 Ser Glu Val
Val His Leu Ala Leu Arg Gln Ala Gly Asp Asp Phe Ser 20 25 30 Arg
Arg Tyr Arg Gly Asp Phe Ala Glu Met Ser Ser Gln Leu His Leu 35 40
45 Thr Pro Phe Thr Ala Arg Gly Arg Phe Ala Thr Val Val Glu Glu Leu
50 55 60 Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala65 70 75
29277PRTHomo sapiensHelix 1 of human Bcl-X1 292Met Ser Gln Ser Asn
Arg Glu Leu Val Val Asp Phe Leu Ser Tyr Lys1 5 10 15 Leu Ser Gln
Lys Gly Tyr Ser Trp Ser Gln Phe Ser Asp Val Glu Glu 20 25 30 Asn
Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu Met Glu Thr Pro 35 40
45 Ser Ala Ile Asn Gly Asn Pro Ser Trp His Lys Leu Ala Asp Ser Pro
50 55 60 Ala Val Asn Gly Ala Thr Gly His Ser Ser Ser Leu Asp65 70
75 29377PRTHomo sapiensHelix 2 of human Bcl-W 293Gly Pro Leu Gly
Ser Met Ala Thr Pro Ala Ser Ala Pro Asp Thr Arg1 5 10 15 Ala Leu
Val Ala Asp Phe Val Gly Tyr Lys Leu Arg Gln Lys Gly Tyr 20 25 30
Val Cys Gly Ala Gly Pro Gly Glu Gly Pro Ala Ala Asp Pro Leu His 35
40 45 Gln Ala Met Arg Ala Ala Gly Asp Glu Phe Glu Thr Arg Phe Arg
Arg 50 55 60 Thr Phe Ser Asp Leu Ala Ala Gln Leu His Val Thr Pro65
70 75 29474PRTHomo sapiensHelix 4 of human Bax 294Pro Arg Glu Val
Phe Phe Arg Val Ala Ala Asp Met Phe Ser Asp Gly1 5 10 15 Asn Phe
Asn Trp Gly Arg Val Val Ala Leu Phe Tyr Phe Ala Ser Lys 20 25 30
Leu Val Leu Lys Ala Leu Cys Thr Lys Val Pro Glu Leu Ile Arg Thr 35
40 45 Ile Met Gly Trp Thr Leu Asp Phe Leu Arg Glu Arg Leu Leu Gly
Trp 50 55 60 Ile Gln Asp Gln Gly Gly Trp Asp Gly Leu65 70
29574PRTHomo sapiensHelix 1 of human CTL Granulysin 295Gly Arg Asp
Tyr Arg Thr Cys Leu Thr Ile Val Gln Lys Leu Lys Lys1 5 10 15 Met
Val Asp Lys Pro Thr Gln Arg Ser Val Ser Asn Ala Ala Thr Arg 20 25
30 Val Cys Arg Thr Gly Arg Ser Arg Trp Arg Asp Val Cys Arg Asn Phe
35 40 45 Met Arg Arg Tyr Gln Ser Arg Val Ile Gln Gly Leu Val Ala
Gly Glu 50 55 60 Thr Ala Gln Gln Ile Cys Glu Asp Leu Arg65 70
29674PRTHomo sapiensHelix 1-2 span of human CTL Granulysin 296Gly
Arg Asp Tyr Arg Thr Cys Leu Thr Ile Val Gln Lys Leu Lys Lys1 5 10
15 Met Val Asp Lys Pro Thr Gln Arg Ser Val Ser Asn Ala Ala Thr Arg
20 25 30 Val Cys Arg Thr Gly Arg Ser Arg Trp Arg Asp Val Cys Arg
Asn Phe 35 40 45 Met Arg Arg Tyr Gln Ser Arg Val Ile Gln Gly Leu
Val Ala Gly Glu 50 55 60 Thr Ala Gln Gln Ile Cys Glu Asp Leu
Arg65
70 29718PRTHomo sapiens 297Phe Thr Ala Arg Gly Arg Phe Ala Thr Val
Val Glu Glu Leu Phe Arg1 5 10 15 Asp Gly29818PRTHomo sapiens 298Gly
Ser Ala Gln Gln Arg Phe Thr Gln Val Ser Asp Glu Leu Phe Gln1 5 10
15 Gly Gly29918PRTHomo sapiens 299Ser Pro Arg Glu Val Phe Phe Arg
Val Ala Ala Asp Met Phe Ser Asp1 5 10 15 Gly Asn30050PRTHomo
sapiens 300Glu Glu Val Tyr Lys Arg Pro Leu Phe Leu Gln Pro Thr Tyr
Arg Tyr1 5 10 15 His Arg Leu Pro Leu Pro Glu Gln Gly Ser Pro Leu
Glu Ala Gln Leu 20 25 30 Asp Ala Phe Val Ser Val Leu Arg Glu Thr
Pro Ser Leu Leu Gln Leu 35 40 45 Arg Asp 50 30119PRTHomo sapiens
301Glu Gly Pro Gln Gly Asp Leu Leu Thr Lys Thr Gln Glu Leu Gly Arg1
5 10 15 Asp Tyr Arg30226PRTHomo sapiens 302Glu Val Arg Ala Gln Leu
Leu Glu Leu Pro Tyr Ala Arg Lys Glu Leu1 5 10 15 Ser Leu Leu Val
Leu Leu Pro Asp Asp Gly 20 25 30320PRTHomo sapiens 303Leu Ile Ser
Ser Tyr Gly Thr His Phe Ile Arg Ser Met Glu Leu Gly1 5 10 15 Gly
Arg Ile Ser 20 30420PRTHomo sapiens 304Leu Gln Asn Ala Thr Val Glu
Ala Gly Thr Arg Cys Gln Val Ala Gly1 5 10 15 Trp Gly Ser Gln 20
30520PRTHomo sapiens 305Ser Ser Lys Ala Gln Val Lys Pro Gly Gln Leu
Cys Ser Val Ala Gly1 5 10 15 Trp Gly Tyr Val 20 30620PRTHomo
sapiens 306Ser Ala Pro His Gln Pro Gly Pro Ser Leu Trp Ala Glu Ala
Lys Thr1 5 10 15 Ser Glu Ala Pro 20 30721PRTHomo sapiens 307Lys Asp
Gly Val Thr Pro Ile Lys Asp Leu Thr Ala His Phe Arg Gly1 5 10 15
Asp Arg Cys Lys Thr 20 30819PRTHomo sapiens 308Pro Pro Pro Glu Lys
Lys Glu Leu Arg Lys Val Ala His Leu Thr Gly1 5 10 15 Lys Ser
Asn30928PRTHomo sapiens 309Val Asn Trp Gly Arg Ile Val Ala Phe Phe
Glu Phe Gly Gly Val Met1 5 10 15 Cys Val Glu Ser Val Asn Arg Glu
Met Ser Pro Leu 20 25 31028PRTHomo sapiens 310Pro Asn Trp Gly Arg
Leu Val Ala Phe Phe Val Phe Gly Ala Ala Leu1 5 10 15 Cys Ala Glu
Ser Val Asn Lys Glu Met Glu Pro Leu 20 25 31128PRTHomo sapiens
311Phe Asn Trp Gly Arg Val Val Ala Leu Phe Tyr Phe Ala Ser Lys Leu1
5 10 15 Val Leu Lys Ala Leu Cys Thr Lys Val Pro Glu Leu 20 25
31250PRTHomo sapiens 312Ala His Gly Pro Pro Pro Ala Leu Val Phe Ser
Cys Gln Met Gly Val1 5 10 15 Gly Arg Thr Asn Leu Gly Met Val Leu
Gly Thr Leu Ile Leu Leu His 20 25 30 Arg Ser Gly Thr Thr Ser Gln
Pro Glu Ala Ala Pro Thr Gln Ala Lys 35 40 45 Pro Leu 50
31328PRTHomo sapiens 313Thr Cys Leu Thr Ile Val Gln Lys Leu Lys Lys
Met Val Asp Lys Pro1 5 10 15 Thr Gln Arg Ser Val Ser Asn Ala Ala
Thr Arg Val 20 25 31431PRTHomo sapiens 314Val Glu Leu Ser Thr Val
Glu Lys Ser Leu Thr Phe Glu Lys Leu Thr1 5 10 15 Ala Trp Thr Lys
Pro Asp Cys Met Lys Ser Thr Glu Val Glu Val 20 25 30 31531PRTHomo
sapiens 315Ala Leu Thr Ala Leu Arg Thr Cys Glu Leu Ala Leu Glu Gly
Leu Thr1 5 10 15 Ala Ser Glu Val Glu Asp Cys Leu Ala Val Glu Ala
Glu Val Ser 20 25 30 31628PRTHomo sapiens 316Arg Ser Gly Gly Arg
Leu Ser Arg Phe Pro Arg Phe Val Asn Val Thr1 5 10 15 Val Thr Pro
Glu Asp Gln Cys Arg Pro Asn Asn Val 20 25 31725PRTHomo sapiens
317Ser Met Ser Thr Leu Ala Thr Thr Leu Gln Glu Val Leu Leu Thr Val1
5 10 15 Gln Lys Asp Cys Gln Cys Glu Arg Leu 20 25 31828PRTHomo
sapiens 318Ser Thr Gln Asp Pro Ser Thr Gln Ala Ser Thr Ala Ser Ser
Pro Ala1 5 10 15 Pro Glu Glu Asn Ala Pro Ser Glu Gly Gln Arg Val 20
25 31929PRTHomo sapiens 319Leu Leu Glu Lys Pro Lys Leu Phe Phe Ile
Gln Ala Cys Arg Gly Thr1 5 10 15 Glu Leu Asp Asp Gly Ile Gln Ala
Asp Ser Gly Pro Ile 20 25 32027PRTHomo sapiens 320Ser Arg Ser Met
Pro Leu Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu1 5 10 15 Leu Ser
Gly Val Lys Tyr Lys Lys Gly Gly Leu 20 25 32115PRTHomo sapiens
321Val Asp Asn Ile Ala Leu Trp Met Thr Glu Tyr Leu Asn Arg His1 5
10 15 32215PRTHomo sapiens 322Val Gly Gln Val Gln Glu Trp Met Val
Ala Tyr Leu Glu Thr Arg1 5 10 15 32315PRTHomo sapiens 323Ile Arg
Thr Ile Met Gly Trp Thr Leu Asp Phe Leu Arg Glu Arg1 5 10 15
32450PRTHomo sapiens 324Pro Met Glu Gln Phe Gln Val Ile Gln Ser Phe
Leu Arg Met Val Pro1 5 10 15 Gln Gly Arg Arg Met Val Glu Glu Val
Asp Arg Ala Ile Thr Ala Cys 20 25 30 Ala Glu Leu His Asp Leu Lys
Glu Val Val Leu Glu Asn Gln Lys Lys 35 40 45 Leu Glu 50
32515PRTHomo sapiens 325Cys Arg Thr Gly Arg Ser Arg Trp Arg Asp Val
Cys Arg Asn Phe1 5 10 15 32621PRTHomo sapiens 326Leu Leu Pro Lys
Phe Lys Leu Gln Glu Asp Tyr Asp Met Glu Ser Val1 5 10 15 Leu Arg
His Leu Gly 20 32724PRTHomo sapiens 327Ile Ser Asp Arg Ala Ser Ala
Ser Pro Ser Phe Lys Ala Cys Glu Glu1 5 10 15 Lys Lys Lys Asn His
Lys Val Gly 20 32815PRTHomo sapiens 328Cys Thr Gly Val Leu Thr Arg
Arg Gly Gly Ile Cys Asn Gly Asp1 5 10 15 32924PRTHomo sapiens
329Phe His Gly Asn Tyr Ser Arg Ala Thr Glu Ile Cys Val Gly Asp Pro1
5 10 15 Lys Lys Thr Gln Thr Gly Phe Lys 20 33025PRTHomo sapiens
330Trp Gly Gln Gly Gln Ser Pro Arg Pro Glu Asn Ser Leu Glu Arg Glu1
5 10 15 Glu Met Gly Pro Val Pro Ala His Thr 20 25 33122PRTHomo
sapiens 331Asn Asp Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu
Ala Asp1 5 10 15 Phe Leu Phe Ala Tyr Ser 20 33225PRTHomo sapiens
332Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser Lys Val Tyr Phe1
5 10 15 Arg Gly Gln Ser Cys Asn Asn Leu Pro 20 25 33353PRTHomo
sapiens 333Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro
Ala Leu1 5 10 15 Ser Pro Val Pro Pro Val Val His Leu Thr Leu Arg
Gln Ala Gly Asp 20 25 30 Asp Phe Ser Arg Arg Tyr Arg Arg Asp Phe
Ala Glu Met Ser Ser Gln 35 40 45 Leu His Leu Thr Pro 50
33434PRTHomo sapiens 334Ala Asp Pro Leu His Gln Ala Met Arg Ala Ala
Gly Asp Glu Phe Glu1 5 10 15 Thr Arg Phe Arg Arg Thr Phe Ser Asp
Leu Ala Ala Gln Leu His Val 20 25 30 Thr Pro33538PRTHomo sapiens
335Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys1
5 10 15 Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu Gln Arg Met
Ile 20 25 30 Ala Ala Val Asp Thr Asp 35 33682PRTHomo sapiens 336Glu
Pro Val Leu Phe Leu Arg Ala Asp Glu Asp Phe Val Ser Tyr Thr1 5 10
15 Pro Arg Asp Lys Gln Asn Leu His Glu Asn Leu Gln Gly Leu Gly Pro
20 25 30 Gly Val Arg Val Glu Ser Leu Glu Leu Ala Ile Arg Lys Glu
Ile His 35 40 45 Asp Phe Ala Gln Leu Ser Glu Asn Thr Tyr His Val
Tyr His Asn Thr 50 55 60 Glu Asp Leu Trp Gly Glu Pro His Ala Val
Ala Ile His Gly Glu Asp65 70 75 80 Asp Leu33729PRTHomo sapiens
337Leu Val Phe Ser Arg Leu Ser Pro Glu Tyr Tyr Asp Pro Ala Arg Ala1
5 10 15 His Leu Arg Asp Gly Glu Lys Ser Cys Pro Cys Gly Gln 20 25
33861PRTHomo sapiens 338Gly His Glu Ala Phe Leu Leu Thr Glu Gly Ser
Glu Glu Lys Arg Ser1 5 10 15 Ala Lys Thr Val Asn Gln Leu Ala His
Ala Leu His Gln Asp Lys Gln 20 25 30 Leu His Ala Gly Ser Leu Val
Ser Val Met Trp Pro Asn Ser Lys Cys 35 40 45 Pro Leu Leu Lys Asp
Asp Leu Val Leu Met Asp Ser Pro 50 55 60 33947PRTHomo sapiens
339Pro Val Ser Ile Ser Ser Ala Leu Ala Met Val Leu Leu Gly Ala Lys1
5 10 15 Gly Asn Thr Ala Thr Gln Met Ala Gln Ala Leu Ser Leu Asn Thr
Glu 20 25 30 Glu Asp Ile His Arg Ala Phe Gln Ser Leu Leu Thr Glu
Val Asn 35 40 45 34077PRTHomo sapiens 340Ile Leu Ala Val Ser Pro
Ala Asn Ser Asp Leu Ala Asn Ser Asp Ala1 5 10 15 Leu Lys Val Ala
Lys Glu Val Asp Pro Gln Gly Gln Arg Thr Ile Gly 20 25 30 Val Ile
Thr Lys Leu Asp Leu Met Asp Glu Gly Thr Asp Ala Arg Asp 35 40 45
Val Leu Glu Asn Lys Leu Leu Pro Leu Arg Arg Gly Tyr Ile Gly Val 50
55 60 Val Asn Arg Ser Gln Lys Asp Ile Asp Gly Lys Lys Asp65 70 75
34185PRTHomo sapiens 341Pro Ser Leu Pro Ser Gln Ala Val Trp Ser Gln
Gly Pro Pro Pro Pro1 5 10 15 Pro Pro Tyr Gly Arg Leu Leu Ala Asn
Ser Asn Ala His Pro Gly Pro 20 25 30 Phe Pro Pro Ser Thr Gly Ala
Gln Ser Thr Ala His Pro Pro Val Ser 35 40 45 Thr His His His His
His Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 50 55 60 Gln Gln Gln
Gln Gln Gln Gln Gln Gln His His Gly Asn Ser Gly Pro65 70 75 80 Pro
Pro Pro Gly Ala 85 34252PRTHomo sapiens 342Leu Cys Leu Leu Val Met
Phe Phe Met Val Leu Val Ala Leu Val Gly1 5 10 15 Leu Gly Leu Gly
Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala 20 25 30 Glu Leu
Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu 35 40 45
Lys Gln Ile Gly 50 34358PRTHomo sapiens 343Phe Ala Ala Gln Lys Thr
His Gln Asp Asn Tyr Arg Phe Ser Met Asp1 5 10 15 Leu Val Glu Cys
Arg Phe Tyr Ser Phe His Leu Val His Thr Pro Pro 20 25 30 Val His
Pro Glu Phe Lys Arg Ala Leu Lys Thr Leu Pro Pro His Phe 35 40 45
Asn Thr Ser Thr Lys Pro Asp Tyr His Arg 50 55 34458PRTHomo sapiens
344Met Asp Glu Ser Val Val Leu Glu Pro Glu Ala Thr Gly Glu Ser Ser1
5 10 15 Ser Leu Glu Pro Thr Pro Ser Ser Gln Glu Ala Gln Arg Ala Leu
Gly 20 25 30 Thr Ser Pro Glu Leu Pro Thr Gly Val Thr Gly Ser Ser
Gly Thr Arg 35 40 45 Leu Pro Pro Thr Pro Lys Ala Gln Asp Gly 50 55
34544PRTHomo sapiens 345Ala Tyr Asp Leu Arg Arg Arg Glu Arg Gln Ser
Arg Gln Thr Phe Ser1 5 10 15 Ile Ser Ser Met Ser Glu Asn Gly Tyr
Asp Pro Gln Gln Asn Leu Asn 20 25 30 Asp Leu Met Leu Leu Gln Leu
Asp Arg Glu Ala Asn 35 40 34644PRTHomo sapiens 346Ala His Asn Ile
Lys Glu Gln Glu Arg Thr Gln Gln Phe Ile Pro Val1 5 10 15 Lys Arg
Pro Ile Pro His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn 20 25 30
Asp Ile Met Leu Leu Gln Leu Glu Arg Lys Ala Lys 35 40 34763PRTHomo
sapiens 347Glu Asp Asp Glu Glu Glu Asp Glu Phe Glu Pro Pro Ile Val
Lys Gly1 5 10 15 Val Lys Pro Ala Lys Ala Ala Pro Ala Ala Pro Ala
Ser Glu Asp Glu 20 25 30 Glu Asp Asp Glu Asp Glu Asp Asp Glu Glu
Asp Asp Asp Glu Glu Glu 35 40 45 Glu Asp Asp Ser Glu Glu Glu Val
Met Glu Ile Thr Thr Ala Lys 50 55 60 34847PRTHomo sapiens 348Phe
Asp Val Ile Val Tyr Asn Asp Cys Ser Cys Ala Lys Met Gln Asp1 5 10
15 Leu Leu Lys Lys Ala Ser Glu Glu Asp His Thr Asn Ala Ala Cys Phe
20 25 30 Ala Cys Ile Leu Leu Ser His Gly Glu Glu Asn Val Ile Tyr
Gly 35 40 45 34918PRTHomo sapiens 349Phe Thr Ala Arg Gly Arg Phe
Ala Thr Val Val Glu Glu Leu Phe Arg1 5 10 15 Asp Gly35018PRTHomo
sapiens 350Gly Ser Ala Gln Gln Arg Phe Thr Gln Val Ser Asp Glu Leu
Phe Gln1 5 10 15 Gly Gly35118PRTHomo sapiens 351Ser Pro Arg Glu Val
Phe Phe Arg Val Ala Ala Asp Met Phe Ser Asp1 5 10 15 Gly
Asn35270PRTHomo sapiens 352Val Ala Ile His Gly Glu Asp Asp Leu His
Val Thr Glu Glu Val Tyr1 5 10 15 Lys Arg Pro Leu Phe Leu Gln Pro
Thr Tyr Arg Tyr His Arg Leu Pro 20 25 30 Leu Pro Glu Gln Gly Ser
Pro Leu Glu Ala Gln Leu Asp Ala Phe Val 35 40 45 Ser Val Leu Arg
Glu Thr Pro Ser Leu Leu Gln Leu Arg Asp Ala His 50 55 60 Gly Pro
Pro Pro Ala Leu65 70 35319PRTHomo sapiens 353Glu Gly Pro Gln Gly
Asp Leu Leu Thr Lys Thr Gln Glu Leu Gly Arg1 5 10 15 Asp Tyr
Arg35444PRTHomo sapiens 354Gly Ile Asp Val Thr Thr Glu Leu Asp Ser
Trp Ile Asp Lys Phe Cys1 5 10 15 Leu Asp Ala Asp Val Phe Val Leu
Val Ala Asn Ser Glu Ser Thr Leu 20 25 30 Met Gln Thr Glu Lys His
Phe Phe His Lys Val Ser 35 40 35519PRTHomo sapiens 355Lys Ala Gly
Thr Gln Tyr Leu Leu Arg Thr Ala Asn Arg Leu Phe Gly1 5 10 15 Glu
Lys Thr35657PRTHomo sapiens 356Gln Lys Asp Ile Asp Gly Lys Lys Asp
Ile Thr Ala Ala Leu Ala Ala1 5 10 15 Glu Arg Lys Phe Phe Leu Ser
His Pro Ser Tyr Arg His Leu Ala Asp 20 25 30 Arg Met Gly Thr Pro
Tyr Leu Gln Lys Val Leu Asn Gln Gln Leu Thr 35 40 45 Asn His Ile
Arg Asp Thr Leu Pro Gly 50 55 35770PRTHomo sapiens 357Asn Ser Gly
Pro Pro Pro Pro Gly Ala Phe Pro His Pro Leu Glu Gly1 5 10 15 Gly
Ser Ser His His Ala His Pro Tyr Ala Met Ser Pro Ser Leu Gly 20 25
30 Ser Leu Arg Pro Tyr Pro Pro Gly Pro Ala His Leu Pro Pro Pro His
35 40 45 Ser Gln Val Ser Tyr Ser Gln Ala Gly Pro Asn Gly Pro Pro
Val Ser 50 55 60 Ser Ser Ser Asn Ser Ser65 70 35822PRTHomo sapiens
358His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg Lys Val Ala His1
5 10 15 Leu Thr Gly Lys Ser Asn 20 35920PRTHomo sapiens 359Leu Ile
Ser Ser Tyr Gly Thr His Phe Ile Arg Ser Met Glu Leu Gly1
5 10 15 Gly Arg Ile Ser 20 36018PRTHomo sapiens 360Gly Pro Val Gly
Thr Glu Leu Phe Arg Val Pro Pro Val Ser Thr Ala1 5 10 15 Ala
Thr36120PRTHomo sapiens 361Leu Thr Ser Ser Val Thr Ile Leu Pro Leu
Pro Leu Gln Asn Ala Thr1 5 10 15 Val Glu Ala Gly 20 36222PRTHomo
sapiens 362Leu Glu Arg Lys Ala Lys Trp Thr Thr Ala Val Arg Pro Leu
Arg Leu1 5 10 15 Pro Ser Ser Lys Ala Gln 20 36323PRTHomo sapiens
363Gly Lys Lys Thr Pro Ala Lys Val Val Pro Met Lys Ala Lys Ser Val1
5 10 15 Ala Glu Glu Glu Asp Asp Glu 20 36421PRTHomo sapiens 364Lys
Asp Gly Val Thr Pro Ile Lys Asp Leu Thr Ala His Phe Arg Gly1 5 10
15 Asp Arg Cys Lys Thr 20 36543PRTHomo sapiens 365Val Asn Trp Gly
Arg Ile Val Ala Phe Phe Glu Phe Gly Gly Val Met1 5 10 15 Cys Val
Glu Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile 20 25 30
Ala Leu Trp Met Thr Glu Tyr Leu Asn Arg His 35 40 36643PRTHomo
sapiens 366Pro Asn Trp Gly Arg Leu Val Ala Phe Phe Val Phe Gly Ala
Ala Leu1 5 10 15 Cys Ala Glu Ser Val Asn Lys Glu Met Glu Pro Leu
Val Gly Gln Val 20 25 30 Gln Glu Trp Met Val Ala Tyr Leu Glu Thr
Arg 35 40 36743PRTHomo sapiens 367Phe Asn Trp Gly Arg Val Val Ala
Leu Phe Tyr Phe Ala Ser Lys Leu1 5 10 15 Val Leu Lys Ala Leu Cys
Thr Lys Val Pro Glu Leu Ile Arg Thr Ile 20 25 30 Met Gly Trp Thr
Leu Asp Phe Leu Arg Glu Arg 35 40 36888PRTHomo sapiens 368Leu Arg
Asp Ala His Gly Pro Pro Pro Ala Leu Val Phe Ser Cys Gln1 5 10 15
Met Gly Val Gly Arg Thr Asn Leu Gly Met Val Leu Gly Thr Leu Ile 20
25 30 Leu Leu His Arg Ser Gly Thr Thr Ser Gln Pro Glu Ala Ala Pro
Thr 35 40 45 Gln Ala Lys Pro Leu Pro Met Glu Gln Phe Gln Val Ile
Gln Ser Phe 50 55 60 Leu Arg Met Val Pro Gln Gly Arg Arg Met Val
Glu Glu Val Asp Arg65 70 75 80 Ala Ile Thr Ala Cys Ala Glu Leu 85
36943PRTHomo sapiens 369Thr Cys Leu Thr Ile Val Gln Lys Leu Lys Lys
Met Val Asp Lys Pro1 5 10 15 Thr Gln Arg Ser Val Ser Asn Ala Ala
Thr Arg Val Cys Arg Thr Gly 20 25 30 Arg Ser Arg Trp Arg Asp Val
Cys Arg Asn Phe 35 40 37049PRTHomo sapiens 370Glu Arg Leu Ser Arg
Pro Asn Ile Phe Ile Leu Asn Asn Arg Trp Asp1 5 10 15 Ala Ser Ala
Ser Glu Pro Glu Tyr Met Glu Glu Val Arg Arg Gln His 20 25 30 Met
Glu Arg Cys Thr Ser Phe Leu Val Asp Glu Leu Gly Val Val Asp 35 40
45 Arg37146PRTHomo sapiens 371Cys Gln Phe Leu Ser Thr Phe Lys Glu
Ser Cys Leu Gln Phe Tyr His1 5 10 15 Ala Glu Leu Lys Glu Leu Ser
Phe Ile Arg Ala Ala Glu Glu Ser Arg 20 25 30 Lys His Ile Asn Thr
Trp Val Ser Lys Lys Thr Glu Gly Lys 35 40 45 37251PRTHomo sapiens
372Leu Arg Asn Lys Leu Gln Ser Gln Leu Leu Ser Ile Glu Lys Glu Val1
5 10 15 Glu Glu Tyr Lys Asn Phe Arg Pro Asp Asp Pro Ala Arg Lys Thr
Lys 20 25 30 Ala Leu Leu Gln Met Val Gln Gln Phe Ala Val Asp Phe
Glu Lys Arg 35 40 45 Ile Glu Gly 50 37389PRTHomo sapiens 373Gly Pro
Pro Val Ser Ser Ser Ser Asn Ser Ser Ser Ser Thr Ser Gln1 5 10 15
Gly Ser Tyr Pro Cys Ser His Pro Ser Pro Ser Gln Gly Pro Gln Gly 20
25 30 Ala Pro Tyr Pro Phe Pro Pro Val Pro Thr Val Thr Thr Ser Ser
Ala 35 40 45 Thr Leu Ser Thr Val Ile Ala Thr Val Ala Ser Ser Pro
Ala Gly Tyr 50 55 60 Lys Thr Ala Ser Pro Pro Gly Pro Pro Pro Tyr
Gly Lys Arg Ala Pro65 70 75 80 Ser Pro Gly Ala Tyr Lys Thr Ala Ile
85 37444PRTHomo sapiens 374Ser Arg Ser Met Pro Leu Glu Trp Glu Asp
Thr Tyr Gly Ile Val Leu1 5 10 15 Leu Ser Gly Val Lys Tyr Lys Lys
Gly Gly Leu Val Ile Asn Glu Thr 20 25 30 Gly Leu Tyr Phe Val Tyr
Ser Lys Val Tyr Phe Arg 35 40 37546PRTHomo sapiens 375Ala Leu Thr
Ala Leu Arg Thr Cys Glu Leu Ala Leu Glu Gly Leu Thr1 5 10 15 Ala
Ser Glu Val Glu Asp Cys Leu Ala Val Glu Ala Glu Val Ser Ile 20 25
30 Ser Asp Arg Ala Ser Ala Ser Pro Ser Phe Lys Ala Cys Glu 35 40 45
37646PRTHomo sapiens 376Trp Gln Ser Ser Ala Pro His Gln Pro Gly Pro
Ser Leu Trp Ala Glu1 5 10 15 Ala Lys Thr Ser Glu Ala Pro Ser Thr
Gln Asp Pro Ser Thr Gln Ala 20 25 30 Ser Thr Ala Ser Ser Pro Ala
Pro Glu Glu Asn Ala Pro Ser 35 40 45 37742PRTHomo sapiens 377Thr
Arg Cys Gln Val Ala Gly Trp Gly Ser Gln Arg Ser Gly Gly Arg1 5 10
15 Leu Ser Arg Phe Pro Arg Phe Val Asn Val Thr Val Thr Pro Glu Asp
20 25 30 Gln Cys Arg Pro Asn Asn Val Cys Thr Gly 35 40 37858PRTHomo
sapiens 378Arg Pro Leu Arg Leu Pro Ser Ser Lys Ala Gln Val Lys Pro
Gly Gln1 5 10 15 Leu Cys Ser Val Ala Gly Trp Gly Tyr Val Ser Met
Ser Thr Leu Ala 20 25 30 Thr Thr Leu Gln Glu Val Leu Leu Thr Val
Gln Lys Asp Cys Gln Cys 35 40 45 Glu Arg Leu Phe His Gly Asn Tyr
Ser Arg 50 55 37947PRTHomo sapiens 379Glu Glu Asp Glu Asp Asp Glu
Asp Glu Asp Asp Glu Glu Glu Asp Asp1 5 10 15 Glu Asp Asp Asp Glu
Glu Glu Glu Glu Glu Glu Pro Val Lys Ala Ala 20 25 30 Pro Gly Lys
Arg Lys Lys Glu Met Thr Lys Gln Lys Glu Ala Pro 35 40 45
38045PRTHomo sapiens 380Leu Leu Glu Lys Pro Lys Leu Phe Phe Ile Gln
Ala Cys Arg Gly Thr1 5 10 15 Glu Leu Asp Asp Gly Ile Gln Ala Asp
Ser Gly Pro Ile Asn Asp Thr 20 25 30 Asp Ala Asn Pro Arg Tyr Lys
Ile Pro Val Glu Ala Asp 35 40 45 38141PRTHomo sapiens 381Gly Pro
Ala Leu Ser Pro Val Pro Pro Val Val His Leu Thr Leu Arg1 5 10 15
Gln Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Arg Asp Phe Ala Glu 20
25 30 Met Ser Ser Gln Leu His Leu Thr Pro 35 40 38231PRTHomo
sapiens 382Leu His Gln Ala Met Arg Ala Ala Gly Asp Glu Phe Glu Thr
Arg Phe1 5 10 15 Arg Arg Thr Phe Ser Asp Leu Ala Ala Gln Leu His
Val Thr Pro 20 25 30 38331PRTHomo sapiens 383Thr Lys Lys Leu Ser
Glu Cys Leu Lys Arg Ile Gly Asp Glu Leu Asp1 5 10 15 Ser Asn Met
Glu Leu Gln Arg Met Ile Ala Ala Val Asp Thr Asp 20 25 30
38458PRTHomo sapiens 384Val Ser Tyr Thr Pro Arg Asp Lys Gln Asn Leu
His Glu Asn Leu Gln1 5 10 15 Gly Leu Gly Pro Gly Val Arg Val Glu
Ser Leu Glu Leu Ala Ile Arg 20 25 30 Lys Glu Ile His Asp Phe Ala
Gln Leu Ser Glu Asn Thr Tyr His Val 35 40 45 Tyr His Asn Thr Glu
Asp Leu Trp Gly Glu 50 55 38529PRTHomo sapiens 385Leu Val Phe Ser
Arg Leu Ser Pro Glu Tyr Tyr Asp Pro Ala Arg Ala1 5 10 15 His Leu
Arg Asp Gly Glu Lys Ser Cys Pro Cys Gly Gln 20 25 38644PRTHomo
sapiens 386Phe Lys Gly Lys Trp Asn Glu Pro Phe Asp Glu Thr Tyr Thr
Arg Glu1 5 10 15 Met Pro Phe Lys Ile Asn Gln Glu Glu Gln Arg Pro
Val Gln Met Met 20 25 30 Tyr Gln Glu Ala Thr Phe Lys Leu Ala His
Val Gly 35 40 38748PRTHomo sapiens 387Tyr Arg Phe Ser Met Asp Leu
Val Glu Cys Arg Phe Tyr Ser Phe His1 5 10 15 Leu Val His Thr Pro
Pro Val His Pro Glu Phe Lys Arg Ala Leu Lys 20 25 30 Thr Leu Pro
Pro His Phe Asn Thr Ser Thr Lys Pro Asp Tyr His Arg 35 40 45
38839PRTHomo sapiens 388Ile Ser Ser Met Ser Glu Asn Gly Tyr Asp Pro
Gln Gln Asn Leu Asn1 5 10 15 Asp Leu Met Leu Leu Gln Leu Asp Arg
Glu Ala Asn Leu Thr Ser Ser 20 25 30 Val Thr Ile Leu Pro Leu Pro 35
38940PRTHomo sapiens 389Val Lys Arg Pro Ile Pro His Pro Ala Tyr Asn
Pro Lys Asn Phe Ser1 5 10 15 Asn Asp Ile Met Leu Leu Gln Leu Glu
Arg Lys Ala Lys Trp Thr Thr 20 25 30 Ala Val Arg Pro Leu Arg Leu
Pro 35 40 39048PRTHomo sapiens 390Gly Thr Ser Pro Glu Leu Pro Thr
Gly Val Thr Gly Ser Ser Gly Thr1 5 10 15 Arg Leu Pro Pro Thr Pro
Lys Ala Gln Asp Gly Gly Pro Val Gly Thr 20 25 30 Glu Leu Phe Arg
Val Pro Pro Val Ser Thr Ala Ala Thr Trp Gln Ser 35 40 45
39140PRTHomo sapiens 391Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu
Lys Lys Ala Ser Glu1 5 10 15 Glu Asp His Thr Asn Ala Ala Cys Phe
Ala Cys Ile Leu Leu Ser His 20 25 30 Gly Glu Glu Asn Val Ile Tyr
Gly 35 40 39237PRTHomo sapiens 392Leu Gly Met Phe Gln Leu Phe His
Leu Gln Lys Glu Leu Ala Glu Leu1 5 10 15 Arg Glu Ser Thr Ser Gln
Met His Thr Ala Ser Ser Leu Glu Lys Gln 20 25 30 Ile Gly His Pro
Ser 35 39318PRTHomo sapiens 393Phe Thr Ala Arg Gly Arg Phe Ala Thr
Val Val Glu Glu Leu Phe Arg1 5 10 15 Asp Gly39418PRTHomo sapiens
394Gly Ser Ala Gln Gln Arg Phe Thr Gln Val Ser Asp Glu Leu Phe Gln1
5 10 15 Gly Gly39518PRTHomo sapiens 395Ser Pro Arg Glu Val Phe Phe
Arg Val Ala Ala Asp Met Phe Ser Asp1 5 10 15 Gly Asn39655PRTHomo
sapiens 396Phe Leu Gln Pro Thr Tyr Arg Tyr His Arg Leu Pro Leu Pro
Glu Gln1 5 10 15 Gly Ser Pro Leu Glu Ala Gln Leu Asp Ala Phe Val
Ser Val Leu Arg 20 25 30 Glu Thr Pro Ser Leu Leu Gln Leu Arg Asp
Ala His Gly Pro Pro Pro 35 40 45 Ala Leu Val Phe Ser Cys Gln 50 55
39719PRTHomo sapiens 397Glu Gly Pro Gln Gly Asp Leu Leu Thr Lys Thr
Gln Glu Leu Gly Arg1 5 10 15 Asp Tyr Arg39826PRTHomo sapiens 398Glu
Val Arg Ala Gln Leu Leu Glu Leu Pro Tyr Ala Arg Lys Glu Leu1 5 10
15 Ser Leu Leu Val Leu Leu Pro Asp Asp Gly 20 25 39920PRTHomo
sapiens 399Leu Ile Ser Ser Tyr Gly Thr His Phe Ile Arg Ser Met Glu
Leu Gly1 5 10 15 Gly Arg Ile Ser 20 40020PRTHomo sapiens 400Leu Gln
Asn Ala Thr Val Glu Ala Gly Thr Arg Cys Gln Val Ala Gly1 5 10 15
Trp Gly Ser Gln 20 40120PRTHomo sapiens 401Ser Ser Lys Ala Gln Val
Lys Pro Gly Gln Leu Cys Ser Val Ala Gly1 5 10 15 Trp Gly Tyr Val 20
40220PRTHomo sapiens 402Ser Ala Pro His Gln Pro Gly Pro Ser Leu Trp
Ala Glu Ala Lys Thr1 5 10 15 Ser Glu Ala Pro 20 40321PRTHomo
sapiens 403Lys Asp Gly Val Thr Pro Ile Lys Asp Leu Thr Ala His Phe
Arg Gly1 5 10 15 Asp Arg Cys Lys Thr 20 40419PRTHomo sapiens 404Pro
Pro Pro Glu Lys Lys Glu Leu Arg Lys Val Ala His Leu Thr Gly1 5 10
15 Lys Ser Asn40542PRTHomo sapiens 405Val Asn Trp Gly Arg Ile Val
Ala Phe Phe Glu Phe Gly Gly Val Met1 5 10 15 Cys Val Glu Ser Val
Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile 20 25 30 Ala Leu Trp
Met Thr Glu Tyr Leu Asn Arg 35 40 40643PRTHomo sapiens 406Pro Asn
Trp Gly Arg Leu Val Ala Phe Phe Val Phe Gly Ala Ala Leu1 5 10 15
Cys Ala Glu Ser Val Asn Lys Glu Met Glu Pro Leu Val Gly Gln Val 20
25 30 Gln Glu Trp Met Val Ala Tyr Leu Glu Thr Arg 35 40
40743PRTHomo sapiens 407Phe Asn Trp Gly Arg Val Val Ala Leu Phe Tyr
Phe Ala Ser Lys Leu1 5 10 15 Val Leu Lys Ala Leu Cys Thr Lys Val
Pro Glu Leu Ile Arg Thr Ile 20 25 30 Met Gly Trp Thr Leu Asp Phe
Leu Arg Glu Arg 35 40 40872PRTHomo sapiens 408Ala Leu Val Phe Ser
Cys Gln Met Gly Val Gly Arg Thr Asn Leu Gly1 5 10 15 Met Val Leu
Gly Thr Leu Ile Leu Leu His Arg Ser Gly Thr Thr Ser 20 25 30 Gln
Pro Glu Ala Ala Pro Thr Gln Ala Lys Pro Leu Pro Met Glu Gln 35 40
45 Phe Gln Val Ile Gln Ser Phe Leu Arg Met Val Pro Gln Gly Arg Arg
50 55 60 Met Val Glu Glu Val Asp Arg Ala65 70 40943PRTHomo sapiens
409Thr Cys Leu Thr Ile Val Gln Lys Leu Lys Lys Met Val Asp Lys Pro1
5 10 15 Thr Gln Arg Ser Val Ser Asn Ala Ala Thr Arg Val Cys Arg Thr
Gly 20 25 30 Arg Ser Arg Trp Arg Asp Val Cys Arg Asn Phe 35 40
41053PRTHomo sapiens 410Val Glu Leu Ser Thr Val Glu Lys Ser Leu Thr
Phe Glu Lys Leu Thr1 5 10 15 Ala Trp Thr Lys Pro Asp Cys Met Lys
Ser Thr Glu Val Glu Leu Val 20 25 30 Leu Leu Pro Lys Phe Lys Leu
Gln Glu Asp Tyr Asp Met Glu Ser Val 35 40 45 Leu Arg His Leu Gly 50
41155PRTHomo sapiens 411Ala Leu Thr Ala Leu Arg Thr Cys Glu Leu Ala
Leu Glu Gly Leu Thr1 5 10 15 Ala Ser Glu Val Glu Asp Cys Leu Ala
Val Glu Ala Glu Val Ser Ile 20 25 30 Ser Asp Arg Ala Ser Ala Ser
Pro Ser Phe Lys Ala Cys Glu Glu Lys 35 40 45 Lys Lys Asn His Lys
Val Gly 50 55 41243PRTHomo sapiens 412Arg Ser Gly Gly Arg Leu Ser
Arg Phe Pro Arg Phe Val Asn Val Thr1 5 10 15 Val Thr Pro Glu Asp
Gln Cys Arg Pro Asn Asn Val Cys Thr Gly Val 20 25 30 Leu Thr Arg
Arg Gly Gly Ile Cys Asn Gly Asp 35 40 41349PRTHomo sapiens 413Ser
Met Ser Thr Leu Ala Thr Thr Leu Gln Glu Val Leu Leu Thr Val1 5 10
15 Gln Lys Asp Cys Gln Cys Glu Arg Leu Phe His Gly Asn Tyr Ser Arg
20 25 30 Ala Thr Glu Ile Cys Val Gly Asp Pro Lys Lys Thr Gln Thr
Gly Phe 35 40 45 Lys41452PRTHomo sapiens 414Ser Thr Gln Asp Pro Ser
Thr Gln Ala Ser Thr Ala Ser Ser Pro
Ala1 5 10 15 Pro Glu Glu Asn Ala Pro Ser Glu Gly Gln Arg Val Trp
Gly Gln Gly 20 25 30 Gln Ser Pro Arg Glu Asn Ser Leu Glu Arg Glu
Glu Met Gly Pro Val 35 40 45 Pro Ala His Thr 50 41551PRTHomo
sapiens 415Leu Leu Glu Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg
Gly Thr1 5 10 15 Glu Leu Asp Asp Gly Ile Gln Ala Asp Ser Gly Pro
Ile Asn Asp Thr 20 25 30 Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val
Glu Ala Asp Phe Leu Phe 35 40 45 Ala Tyr Ser 50 41652PRTHomo
sapiens 416Ser Arg Ser Met Pro Leu Glu Trp Glu Asp Thr Tyr Gly Ile
Val Leu1 5 10 15 Leu Ser Gly Val Lys Tyr Lys Lys Gly Gly Leu Val
Ile Asn Glu Thr 20 25 30 Gly Leu Tyr Phe Val Tyr Ser Lys Val Tyr
Phe Arg Gly Gln Ser Cys 35 40 45 Asn Asn Leu Pro 50 41747PRTHomo
sapiens 417Gly Gly Trp Asp Ala Phe Val Glu Leu Tyr Gly Pro Ser Met
Arg Pro1 5 10 15 Leu Phe Asp Phe Ser Trp Leu Ser Leu Lys Thr Leu
Leu Ser Leu Ala 20 25 30 Leu Val Gly Ala Cys Ile Thr Leu Gly Ala
Tyr Leu Gly His Lys 35 40 45 41852PRTHomo sapiens 418Gly Gly Trp
Ala Glu Phe Thr Ala Leu Tyr Gly Asp Gly Ala Leu Glu1 5 10 15 Glu
Ala Arg Arg Leu Arg Glu Gly Asn Trp Ala Ser Val Arg Thr Val 20 25
30 Leu Thr Gly Ala Val Ala Leu Gly Ala Leu Val Thr Val Gly Ala Phe
35 40 45 Phe Ala Ser Lys 50 41963PRTHomo sapiens 419Gly Gly Trp Val
Arg Leu Leu Lys Pro Pro His Pro His His Arg Ala1 5 10 15 Leu Thr
Thr Ala Pro Ala Pro Pro Ser Leu Pro Pro Ala Thr Pro Leu 20 25 30
Gly Pro Trp Ala Phe Trp Ser Arg Ser Gln Trp Cys Pro Leu Pro Ile 35
40 45 Phe Arg Ser Ser Asp Val Val Tyr Asn Ala Phe Ser Leu Arg Val
50 55 60 42070PRTHomo sapiens 420Ala Gly Pro Val Ala Pro Arg Asp
Leu Ile Ala Arg Gly Ser Leu Arg1 5 10 15 Glu Asp Asp Leu Val Ser
Pro Asp Ala Leu Ser Thr Val Arg Glu Met 20 25 30 Asp Val Ala Asn
Phe Arg Arg Val Pro Arg Met Pro Ile Tyr Gly Thr 35 40 45 Ala Gln
Pro Ser Ala Lys Ala Leu Gly Ser Ile Leu Ala Tyr Leu Thr 50 55 60
Asp Ala Lys Arg Arg Ile65 70 42135PRTHomo sapiens 421Met Arg Arg
Tyr Gln Ser Arg Val Ile Gln Gly Leu Val Ala Gly Glu1 5 10 15 Thr
Ala Gln Gln Ile Cys Glu Asp Leu Arg Leu Cys Ile Pro Ser Thr 20 25
30 Gly Pro Leu 35 42254PRTHomo sapiens 422Ala Asp Leu Ser Ala Met
Ser Ala Glu Arg Asp Leu Cys Leu Ser Lys1 5 10 15 Phe Val His Lys
Ser Phe Val Glu Val Asn Glu Glu Gly Thr Glu Ala 20 25 30 Ala Ala
Ala Ser Ser Cys Phe Val Val Ala Glu Cys Cys Met Glu Ser 35 40 45
Gly Pro Arg Phe Cys Ala 50 42354PRTHomo sapiens 423Glu Arg His Ser
Asn Val Asp Gly Gly His His Ser Thr Met His Asp1 5 10 15 Leu Leu
Phe Gly Ser Gln Ala Gly Pro Glu Gln Phe Ser Ala Trp Val 20 25 30
Ala Ser Leu Gln Asp Ser Pro Gly Leu Val Asp Tyr Thr Leu Glu Pro 35
40 45 Leu His Met Leu Val Glu 50 42451PRTHomo sapiens 424Gly Gly
Thr Pro Leu Val Cys Glu Gly Leu Ala His Gly Val Ala Ser1 5 10 15
Phe Ser Leu Gly Pro Cys Gly Arg Gly Pro Asp Phe Phe Thr Arg Val 20
25 30 Ala Leu Phe Arg Asp Trp Ile Asp Gly Val Leu Asn Asn Pro Gly
Pro 35 40 45 Gly Pro Ala 50 42547PRTHomo sapiens 425Gly Asp Ser Gly
Gly Pro Leu Val Cys Lys Asp Val Ala Gln Gly Ile1 5 10 15 Leu Ser
Tyr Gly Asn Lys Lys Gly Thr Pro Pro Gly Val Tyr Ile Lys 20 25 30
Val Ser His Phe Leu Pro Trp Ile Lys Arg Thr Met Lys Arg Leu 35 40
45 42657PRTHomo sapiens 426Met Ala His Val Ser Val Val Pro Val Ser
Ser Glu Gly Thr Pro Ser1 5 10 15 Arg Glu Pro Val Ala Ser Gly Ser
Trp Thr Pro Lys Ala Glu Glu Pro 20 25 30 Ile His Ala Thr Met Asp
Pro Gln Arg Leu Gly Val Leu Ile Thr Pro 35 40 45 Val Pro Asp Ala
Gln Ala Ala Thr Arg 50 55 42754PRTHomo sapiens 427Thr Val Pro Gly
Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp1 5 10 15 Phe Val
Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu 20 25 30
Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His 35
40 45 Phe Glu Ser Gln Ser Asp 50 42853PRTHomo sapiens 428Leu Ser
His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu1 5 10 15
Val Met Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Met Trp 20
25 30 Ala Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala
Asp 35 40 45 His Leu Tyr Val Asn 50
* * * * *