U.S. patent application number 10/791628 was filed with the patent office on 2004-09-02 for specifically targeted catalytic antagonists and uses thereof.
Invention is credited to Bott, Richard R., Davis, Benjamin G., Estell, David Aaron, Jones, John Bryan, Sanford, Karl John.
Application Number | 20040170618 10/791628 |
Document ID | / |
Family ID | 26829404 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040170618 |
Kind Code |
A1 |
Davis, Benjamin G. ; et
al. |
September 2, 2004 |
Specifically targeted catalytic antagonists and uses thereof
Abstract
This invention provides chimeric molecules that are catalytic
antagonists of a target molecule. The catalytic antagonists of this
invention preferably comprise a targeting moiety attached to an
enzyme that degrades the molecule specifically bound by the
targeting moiety. The catalytic antagonists of this invention thus
bind to a target recognized by the targeting moiety (e.g., a
receptor) the enzyme component of the chimera then degrades all or
part of the target. This typically results in a reduction or loss
of activity of the target and release of the chimeric molecule. The
chimeric molecule is then free to attack and degrade another target
molecule.
Inventors: |
Davis, Benjamin G.; (Durham,
GB) ; Jones, John Bryan; (Lakefield, CA) ;
Bott, Richard R.; (Burlingame, CA) ; Sanford, Karl
John; (Cupertino, CA) ; Estell, David Aaron;
(San Mateo, CA) |
Correspondence
Address: |
Genencor International, Inc.
925 Page Mill Road
Palo Alto
CA
94034-1013
US
|
Family ID: |
26829404 |
Appl. No.: |
10/791628 |
Filed: |
March 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10791628 |
Mar 1, 2004 |
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09566466 |
May 8, 2000 |
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6707470 |
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60161362 |
Oct 26, 1999 |
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Current U.S.
Class: |
424/94.6 ;
435/219 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 11/08 20180101; A61P 3/06 20180101; A61P 9/12 20180101; A61P
25/24 20180101; A61P 5/26 20180101; A61P 25/04 20180101; A61K
47/6898 20170801; A61P 5/30 20180101; A61P 3/10 20180101; A61P
21/02 20180101; A61P 25/18 20180101; A61P 9/10 20180101; A61P 25/08
20180101; A61P 11/02 20180101; A61P 9/06 20180101; B82Y 5/00
20130101; A61P 25/16 20180101; A61P 31/04 20180101; A61K 47/665
20170801; A61P 31/12 20180101; A61P 37/00 20180101; A61P 43/00
20180101; A61K 47/51 20170801; A61P 7/02 20180101; A61P 5/28
20180101 |
Class at
Publication: |
424/094.6 ;
435/219 |
International
Class: |
A61K 038/46; C12N
009/50 |
Claims
What is claimed is:
1. A catalytic antagonist of a target molecule, said antagonist
comprising a targeting moiety that specifically binds to said
target molecule said targeting moiety being attached to an enzyme
that degrades said target molecule to reduce binding of the target
molecule to its cognate ligand and to said targeting moiety thereby
resulting in the release of said antagonist thereby allowing said
antagonist to bind and degrade another target molecule.
2. The antagonist of claim 1, wherein said targeting moiety is
joined to said enzyme through the sulfur group on a cysteine.
3. The antagonist of claim 2, wherein said cysteine is a cysteine
that is substituted for a native amino acid other than cysteine in
said enzyme.
4. The antagonist of claim 3, wherein said cysteine is a cysteine
that is substituted for a native amino acid other than cysteine in
or near a subsite comprising a substrate binding site of said
enzyme.
5. The antagonist of claim 4, wherein said cysteine is a cysteine
that is substituted for an amino acid forming a substrate binding
site.
6. The antagonist of claim 3, wherein said enzyme is an enzyme
selected from the group consisting of a protease, an esterase, an
amidase, a peptidase, a lactamase, a cellulase, an oxidase, an
oxidoreductase, a reductase, a transferase, a hydrolase, an
isomerase, a ligase, a lipase, a phospholipase, a phosphatase, a
kinase, a sulfatase, a lysozyme, a glycosidase, a nuclease, an
aldolase, a ketolase, a lyase, a cyclase, a reverse transcriptase,
a hyaluronidase, an amylase, a cerebrosidase and a chitinase.
7. The antagonist of claim 6, wherein said enzyme is a serine
hydrolase.
8. The antagonist of claim 5, wherein said enzyme is a
subtilisin-type serine hydrolase and said cysteine is substituted
for an amino acid in or near a subsite selected from the group
consisting of an S1 subsite, an S1' subsite, and an S2 subsite.
9. The antagonist of claim 8, wherein said enzyme is a Bacillus
lentus subtilisin.
10. The antagonist of claim 8, wherein said cysteine is substituted
for an amino acid corresponding to a reference residue in a
Bacillus lentus subtilisin, where said reference residue is at or
near a residue selected from the group consisting of residue 156,
residue 166, residue 217, residue 222, residue 62, residue 96,
residue 104, residue 107, residue 189, and residue 209.
11. The antagonist of claim 6, wherein said enzyme is a
chymotrypsin-type serine protease and said cysteine is substituted
for the amino acid corresponding to a reference residue in a mature
trypsin (Protein Data Bank entry 1TPP), wherein said reference
residue is at or near a residue selected from the group consisting
of Tyr94, Leu99, Gln175, Asp189, Ser190, Gln192, Phe41, Lys60,
Tyr151, Ser214, and Lys224.
12. The antagonist of claim 7, wherein said enzyme is an alpha/beta
type serine hydrolase and said cysteine is substituted for the
amino acid corresponding to a reference residue in a Candida
antartica lipase (Protein Data Bank entry 1TCA), where the
reference residue is at or near a residue selected from the group
consisting of Trp104, Leu140, Leu144, Val154, Glu188, Ala 225,
Leu278 and Ile285.
13. The antagonist of claim 7, wherein said enzyme is an aspartyl
protease.
14. The antagonist of claim 13, wherein said enzyme is a
pepsin-type protease and said cysteine is substituted for the amino
acid corresponding to a reference residue in the mature human
pepsin (Protein Data Bank entry 1PSN), where the reference residue
is at or near a residue selected from the group consisting of Tyr9,
Met12, Glu13, Gly76, Thr77, Phe111, Phe117, Ile128, Ser130, Tyr189,
Ile213, Glu239, Met245, Gln287, Met289, Leu291, and Glu294.
15. The antagonist of claim 6, wherein said enzyme is an cysteine
protease.
16. The antagonist of claim 15, wherein said enzyme is a papain and
said cysteine is substituted for the amino acid corresponding to a
reference residue in a mature papain (Protein Data Bank entry
1BQI), where the reference residue is at or near a residue selected
from the group consisting of Asn18, Ser21, Asn64, Tyr67, Trp69,
Gln112, Gln142, Asp158, Trp177, and Phe207.
17. The antagonist of claim 6, wherein said enzyme is a
metalloprotease.
18. The antagonist of claim 17, wherein said enzyme is a
metalloprotease and said cysteine is substituted for the amino acid
corresponding to a reference residue in the mature human matrix
metalloprotease (Protein Data Bank entry 830C), where the reference
residue is at or near a residue selected from the group consisting
of Leu111, Phe175, Tyr176, Ser182, Leu184, Phe189, Tyr214, Asp231,
Lys234, and Ile243.
19. The antagonist of claim 1, wherein said target is a molecule
present on the surface of a cell.
20. The antagonist of claim 19, wherein said molecule present on
the surface of a cell is a molecule forming a receptor.
21. The antagonist of claim 19, wherein said molecule present on
the surface of a cell is a ligand.
22. The antagonist of claim 19, wherein said molecule present on
the surface of a cell is a component of a cell wall.
23. The antagonist of claim 19, wherein said molecule present on
the surface of a cell is a component of a cell membrane.
24. The antagonist of claim 1, wherein said targeting moiety is
selected from the group consisting of a protein, an antigen, a
carbohydrate, a nucleic acid, a lipid, a coordination complex, a
sugar, a vitamin, a dendrimer, and a crown ether.
25. The antagonist of claim 24, wherein said targeting moiety is a
cognate ligand for a receptor or an enzyme.
26. The antagonist of claim 24, wherein said targeting moiety is an
inhibitor for a receptor or an enzyme.
27. The antagonist of claim 1, wherein said enzyme is a protease
and said targeting moiety is a ligand selected from the group
consisting of a carbohydrate, a vitamin or vitamin analog, an
enzyme inhibitor, a peptide, a pharmaceutical that is a small
organic molecule, and biotin.
28. The antagonist of claim 1, wherein said enzyme is a protease
and said targeting moiety is a receptor.
29. The antagonist of claim 27, wherein said enzyme is a
subtilisin.
30. The antagonist of claim 29, wherein said targeting moiety is an
enzyme inhibitor that is a pyrazole.
31. The antagonist of claim 29, wherein said targeting moiety is a
biotin.
32. The antagonist of claim 29, wherein said targeting moiety is a
ligand that binds a lectin.
33. The antagonist of claim 32, wherein said lectin is concanavalin
A.
34. The antagonist of claim 33, wherein targeting moiety is a
carbohydrate.
35. The antagonist of claim 34, wherein said targeting moiety is
-thioethyl D-mannopyranoside.
36. The antagonist of claim 33, wherein said targeting moiety
specifically binds to a soil and said enzyme degrades a component
of said soil.
37. A method of degrading a target molecule, said method comprising
contacting said target molecule with a catalytic antagonist
comprising a targeting moiety that specifically binds to said
target molecule said targeting moiety being attached to an enzyme
that degrades said target molecule resulting in the release of said
antagonist thereby allowing said antagonist to bind and degrade
another target molecule.
38. The method of claim 37, wherein said targeting moiety is joined
to said enzyme through the sulfur group on a cysteine.
39. The method of claim 38, wherein said cysteine is a cysteine
that is substituted for a native amino acid other than cysteine in
said enzyme.
40. The method of claim 39, wherein said cysteine is a cysteine
that is substituted for a native amino acid other than cysteine in
or near a subsite comprising a substrate binding site of said
enzyme.
41. The method of claim 39, wherein said enzyme is an enzyme
selected from the group consisting of a protease, an esterase, an
amidase, a peptidase, a lactamase, a cellulase, an oxidase, an
oxidoreductase, a reductase, a transferase, a hydrolase, an
isomerase, a ligase, a lipase, a phospholipase, a phosphatase, a
kinase, a sulfatase, a lysozyme, a glycosidase, a
glycosyltransferase, a nuclease, an aldolase, a ketolase, a lyase,
a cyclase, a reverse transcriptase, a hyaluronidase, an amylase, a
cerebrosidase and a chitinase.
42. The method of claim 41, wherein said enzyme is a serine
hydrolase.
43. The method of claim 42, wherein said enzyme is a
subtilisin.
44. The antagonist of claim 39, wherein said cysteine is a cysteine
that is substituted for an amino acid forming a substrate binding
site.
45. The method of claim 44, wherein said enzyme is a
subtilisin-type serine hydrolase and said cysteine is substituted
for an amino acid in or near a subsite selected from the group
consisting of an S1 subsite, an S1' subsite, and an S2 subsite.
46. The method of claim 45, wherein said enzyme is a Bacillus
lentus subtilisin.
47. The method of claim 45, wherein said cysteine is substituted
for an amino acid corresponding to a reference residue in a
Bacillus lentus subtilisin, where said reference residue is at or
near a residue selected from the group consisting of residue 156,
residue 166, residue 217, residue 222, residue 62, residue 96,
residue 104, residue 107, residue 189, and residue 209.
48. The method of claim 41, wherein said enzyme is a
chymotrypsin-type serine protease and said cysteine is substituted
for an amino acid corresponding to a reference residue in a mature
trypsin (Protein Data Bank entry 1TPP), wherein said reference
residue is at or near a residue selected from the group consisting
of Tyr94, Leu99, Gln175, Asp189, Ser190, Gln192, Phe41, Lys60,
Tyr151, Ser214, and Lys224.
49. The method of claim 41, wherein said enzyme is an alpha/beta
type serine hydrolase and said cysteine is substituted for an amino
acid corresponding to a reference residue in a Candida antartica
lipase (Protein Data Bank entry 1TCA), where the reference residue
is at or near a residue selected from the group consisting of
Trp104, Leu140, Leu144, Val154, Glu188, Ala225, Leu278 and
Ile285.
50. The method of claim 41, wherein said enzyme is an aspartyl
protease.
51. The method of claim 50, wherein said enzyme is a pepsin-type
protease and said cysteine is substituted for the amino acid
corresponding to a reference residue in the mature human pepsin
(Protein Data Bank entry 1PSN), where the reference residue is at
or near a residue selected from the group consisting of Tyr9,
Met12, Glu13, Gly76, Thr77, Phe111, Phe117, Ile128, Ser130, Tyr189,
Ile213, Glu239, Met245, Gln287, Met289, Leu291, and Glu294.
52. The method of claim 41, wherein said enzyme is an cysteine
protease.
53. The method of claim 52, wherein said enzyme is a papain and
said cysteine is substituted for the amino acid corresponding to a
reference residue in a mature papain (Protein Data Bank entry
Asn18, Ser21, Asn64, Tyr67, Trp69, Gln112, Gln142, Asp158, Trp177,
and Phe207.
54. The method of claim 41, wherein said enzyme is a
metalloprotease.
55. The method of claim 54, wherein said enzyme is a
metalloprotease and said cysteine is substituted for the amino acid
corresponding to a reference residue in the mature human matrix
metalloprotease (Protein Data Bank entry 830C), where the reference
residue is at or near a residue selected from the group consisting
of Leu111, Phe175, Tyr176, Ser182, Leu184, Phe189, Tyr214, Asp231,
Lys234, and Ile243.
56. The method of claim 37, wherein said target is a molecule
present on the surface of a cell.
57. The method of claim 56, wherein said molecule present on the
surface of a cell is a molecule forming a receptor.
58. The method of claim 56, wherein said molecule present on the
surface of a cell is a ligand.
59. The method of claim 56, wherein said-molecule present on the
surface of a cell is component of a cell wall.
60. The method of claim 56, wherein said molecule present on the
surface of a cell is component of a cell membrane.
61. The method of claim 37, wherein said targeting moiety is
selected from the group consisting of a protein, an antigen, a
carbohydrate, a nucleic acid, a lipid, a coordination complex, a
sugar, a vitamin, a dendrimer, and a crown ether.
62. The method of claim 61, wherein said targeting moiety is a
cognate ligand for a receptor or an enzyme.
63. The method of claim 61, wherein said targeting moiety is an
inhibitor for a receptor or an enzyme.
64. The method of claim 37, wherein said enzyme is a protease and
said targeting moiety is a ligand selected from the group
consisting of a carbohydrate, a vitamin or vitamin analog, an
enzyme inhibitor, a peptide, a pharmaceutical that is a small
organic molecule, and biotin.
65. The antagonist of claim 37, wherein said enzyme is a protease
and said targeting moiety is a receptor.
66. The method of claim 64, wherein said enzyme is a
subtilisin.
67. The method of claim 66, wherein said targeting moiety is an
enzyme inhibitor that is a pyrazole.
68. The method of claim 66, wherein said targeting moiety is an
biotin.
69. The method of claim 66, wherein said targeting moiety is a
ligand that binds a lectin.
70. The method of claim 69, wherein said lectin is concanavalin
A.
71. The method of claim 70, wherein targeting moiety is a
carbohydrate.
72. The method of claim 70, wherein said targeting moiety is
-thioethyl D-mannopyranoside.
73. The method of claim 66, wherein said targeting moiety
specifically binds to a soil and said enzyme degrades a component
of said soil.
74. An enzyme having altered substrate specificity said enzyme
comprising a targeting moiety attached to a subsite comprising the
substrate binding site of said enzyme.
75. The enzyme of claim 74, wherein said targeting moiety is
coupled to said enzyme through to a sulfur of a cysteine in said
subsite of said enzyme.
76. The enzyme of claim 75, wherein said cysteine is substituted
for a native amino acid that is not cysteine in said subsite of
said enzyme.
77. The enzyme of claim 75, wherein said enzyme is an enzyme
selected from the group consisting of a protease, an esterase, an
amidase, a peptidase, a lactamase, a cellulase, an oxidase, an
oxidoreductase, a reductase, a transferase, a hydrolase, an
isomerase, a ligase, a lipase, a phospholipase, a phosphatase, a
kinase, a sulfatase, a lysozyme, a glycosidase, a
glycosyltransferase, a nuclease, an aldolase, a ketolase, a lyase,
a cyclase, a reverse transcriptase, a hyaluronidase, an amylase, a
cerebrosidase and a chitinase.
78. The enzyme of claim 77, wherein said enzyme is a serine
hydrolase.
79. The enzyme of claim 78, wherein said enzyme is a
subtilisin.
80. The enzyme of claim 76, wherein said cysteine is a cysteine
that is substituted for an amino acid forming a substrate binding
site.
81. The enzyme of claim 80, wherein said enzyme is a
subtilisin-type serine hydrolase and said cysteine is substituted
for an amino acid in or near a subsite selected from the group
consisting of an S1 subsite, an S1' subsite, and an S2 subsite.
82. The enzyme of claim 81, wherein said enzyme is a Bacillus
lentus subtilisin.
83. The enzyme of claim 81, wherein said cysteine is substituted
for an amino acid corresponding to a reference residue in a
Bacillus lentus subtilisin, where said reference residue is at or
near a residue selected from the group consisting of residue 156,
residue 166, residue 217, residue 222, residue 62, residue 96,
residue 104, residue 107, residue 189, and residue 209.
84. The enzyme of claim 77, wherein said enzyme is a
chymotrypsin-type serine protease and said cysteine is substituted
for an amino acid corresponding to a reference residue in a mature
trypsin (Protein Data Bank entry 1TPP), wherein said reference
residue is at or near a residue selected from the group consisting
of Tyr94, Leu99, Gln175, Asp189, Ser190, Gln192, Phe41, Lys60,
Tyr151, Ser214, and Lys224.
85. The enzyme of claim 77, wherein said enzyme is an alpha/beta
type serine hydrolase and said cysteine is substituted for an amino
acid corresponding to a reference residue in a Candida antartica
lipase (Protein Data Bank entry 1TCA), where the reference residue
is at or near a residue selected from the group consisting of
Trp104, Leu140, Leu144, Val154, Glu188, Ala225, Leu278 and
Ile285.
86. The enzyme of claim 77, wherein said enzyme is an aspartyl
protease.
87. The enzyme of claim 86, wherein said enzyme is a pepsin-type
protease and said cysteine is substituted for the amino acid
corresponding to a reference residue in the mature human pepsin
(Protein Data Bank entry 1PSN), where the reference residue is at
or near a residue selected from the group consisting of Tyr9,
Met12, Glu13, Gly76, Thr77, Phe111, Phe117, Ile128, Ser130, Tyr189,
Ile213, Glu239, Met245, Gln287, Met289, Leu291, and Glu294.
88. The enzyme of claim 77, wherein said enzyme is an cysteine
protease.
89. The enzyme of claim 88, wherein said enzyme is a papain and
said cysteine is substituted for the amino acid corresponding to a
reference residue in a mature papain (Protein Data Bank entry
Asn18, Ser21, Asn64, Tyr67, Trp69, Gln112, Gln142, Asp158, Trp177,
and Phe207.
90. The enzyme of claim 77, wherein said enzyme is a
metalloprotease.
91. The enzyme of claim 90, wherein said enzyme is a
metalloprotease and said cysteine is substituted for the amino acid
corresponding to a reference residue in the mature human matrix
metalloprotease (Protein Data Bank entry 830C), where the reference
residue is at or near a residue selected; from the group consisting
of Leu111, Phe175, Tyr176, Ser182, Leu184, Phe189, Tyr214, Asp231,
Lys234, and Ile243.
92. The enzyme of claim 37, wherein said target is a molecule
present on the surface of a cell.
93. The enzyme of claim 75, wherein said targeting moiety is
selected from the group consisting of a protein, an antigen, a
carbohydrate, a nucleic acid, a lipid, a coordination complex, a
sugar, a vitamin, a dendrimer, and a crown ether.
94. The enzyme of claim 75, wherein said targeting moiety is a
cognate ligand for a receptor or an enzyme.
95. The enzyme of claim 75, wherein said targeting moiety is an
inhibitor for a receptor or an enzyme.
96. The enzyme of claim 75, wherein said targeting moiety is
selected from the group consisting of a growth factor, a cytokine,
and a receptor ligand.
97. The enzyme of claim 75, wherein said enzyme is a protease and
said targeting moiety is a ligand selected from the group
consisting of a carbohydrate, a vitamin or vitamin analog, an
enzyme inhibitor, a peptide, a pharmaceutical that is a small
organic molecule, and biotin.
98. The enzyme of claim 75, wherein said enzyme is a protease and
said targeting moiety is a receptor.
99. The enzyme of claim 97, wherein said enzyme is a
subtilisin.
100. The enzyme of claim 99, wherein said targeting moiety is an
enzyme inhibitor that is a pyrazole.
101. The enzyme of claim 99, wherein said targeting moiety is an
biotin.
102. The enzyme of claim 99, wherein said targeting moiety is a
ligand that binds a lectin.
103. The enzyme of claim 102, wherein said lectin is concanavalin
A.
104. The enzyme of claim 103, wherein targeting moiety is a
carbohydrate.
105. The enzyme of claim 103, wherein said targeting moiety is
-thioethyl D-mannopyranoside.
106. The enzyme of claim 99, wherein said targeting moiety
specifically binds to a soil and said enzyme degrades a component
of said soil.
107. A method of directing the activity of an enzyme to a specific
target, said method comprising providing an enzyme having altered
substrate specificity said enzyme comprising a targeting moiety
attached to a subsite within the substrate binding region of said
enzyme; and contacting said target with said enzyme, whereby said
enzyme specifically binds to said target thereby localizing the
activity of said enzyme at said target.
108. The method of claim 107, wherein said targeting moiety is
coupled to said enzyme through to a sulfur of a cysteine in said
subsite of said enzyme.
109. The method of claim 108, wherein said cysteine is substituted
for a native amino acid that is not cysteine in said subsite of
said enzyme.
110. The method of claim 108, wherein said method further comprises
substituting an amino acid in said subsite with a cysteine and
chemically coupling said targeting moiety to said cysteine.
111. The method of claim 107,-wherein said enzyme is an enzyme
selected from the group consisting of a protease, an esterase, an
amidase, a peptidase, a lactamase, a cellulase, an oxidase, an
oxidoreductase, a reductase, a transferase, a hydrolase, an
isomerase, a ligase, a lipase, a phospholipase, a phosphatase, a
kinase, a sulfatase, a lysozyme, a glycosidase, a
glycosyltransferase, a nuclease, an aldolase, a ketolase, a lyase,
a cyclase, a reverse transcriptase, a hyaluronidase, an amylase, a
cerebrosidase and a chitinase.
112. The method of claim 111, wherein said enzyme is a serine
hydrolase.
113. The method of claim 112, wherein said enzyme is a
subtilisin.
114. The method of claim 109, wherein said cysteine is a cysteine
that is substituted for an amino acid forming a substrate binding
site.
115. The method of claim 114, wherein said enzyme is a
subtilisin-type serine hydrolase and said cysteine is substituted
for an amino acid in or near a subsite selected from the group
consisting of an S1 subsite, an S1' subsite, and an S2 subsite.
116. The method of claim 112, wherein said enzyme is a Bacillus
lentus subtilisin.
117. The method of claim 112, wherein said cysteine is substituted
for an amino acid corresponding to a reference residue in a
Bacillus lentus subtilisin, where said reference residue is at or
near a residue selected from the group consisting of residue 156,
residue 166, residue 217, residue 222, residue 62, residue 96,
residue 104, residue 107, residue 189, and residue 209.
118. The method of claim 111, wherein said enzyme is a
chymotrypsin-type serine protease and said cysteine is substituted
for the amino acid corresponding to a reference residue in a mature
trypsin (Protein Data Bank entry 1TPP), wherein said reference
residue is at or near a residue selected from the group consisting
of Tyr94, Leu99, Gln175, Asp189, Ser190, Gln192, Phe41, Lys60,
Tyr151, Ser214, and Lys224.
119. The method of claim 111, wherein said enzyme is an alpha/beta
type serine hydrolase and said cysteine is substituted for the
amino acid corresponding to a reference residue in a Candida
antartica lipase (Protein Data Bank entry 1TCA), where the
reference residue is at or near a residue selected from the group
consisting of Trp104, Leu140, Leu144, Val154, Glu188, Ala 225,
Leu278 and Ile285.
120. The method of claim 111, wherein said enzyme is an aspartyl
protease.
121. The method of claim 120, wherein said enzyme is a pepsin-type
protease and said cysteine is substituted for the amino acid
corresponding to a reference residue in the mature human pepsin
(Protein Data Bank entry 1PSN), where the reference residue is at
or near a residue selected from the group consisting of Tyr9,
Met12, Glu13, Gly76, Thr77, Phe111, Phe117, Ile128, Ser130, Tyr189,
Ile213, Glu239, Met245, Gln287, Met289, Leu291, and Glu294.
122. The method of claim 111, wherein said enzyme is an cysteine
protease.
123. The method of claim 122, wherein said enzyme is a papain and
said cysteine is substituted for the amino acid corresponding to a
reference residue in a mature papain (Protein Data Bank entry 1
BQI), where the reference residue is at or near a residue selected
from the group consisting of Asn18, Ser21, Asn64, Tyr67, Trp69,
Gln112, Gln142, Asp158, Trp177, and Phe207.
124. The method of claim 111, wherein said enzyme is a
metalloprotease.
125. The method of claim 124, wherein said enzyme is a
metalloprotease and said cysteine is substituted for the amino acid
corresponding to a reference residue in the mature human matrix
metalloprotease (Protein Data Bank entry 830C), where the reference
residue is at or near a residue selected from the group consisting
of Leu111, Phe175, Tyr176, Ser182, Leu184, Phe189, Tyr214, Asp231,
Lys234, and Ile243.
126. The method of claim 75, wherein said targeting moiety is
selected from the group consisting of a protein, an antigen, a
carbohydrate, a nucleic acid, a lipid, a coordination complex, a
metal, a sugar, a vitamin, a dendrimer, and a crown ether.
127. The method of claim 107, wherein said targeting moiety is a
cognate ligand for a receptor or an enzyme.
128. The method of claim 107, wherein said targeting moiety is an
inhibitor for a receptor or an enzyme.
129. The method of claim 107, wherein said targeting moiety is
selected from the group consisting of a growth factor, and a
cytokine.
130. The method of claim 107, wherein said enzyme is a protease and
said targeting moiety is a ligand selected from the group
consisting of a carbohydrate, a vitamin or vitamin analog, an
enzyme inhibitor, a peptide, a pharmaceutical that is a small
organic molecule, and biotin.
131. The antagonist of claim 107, wherein said enzyme is a protease
and said targeting moiety is a receptor.
132. The method of claim 130, wherein said enzyme is a
subtilisin.
133. The method of claim 132, wherein said targeting moiety is an
enzyme inhibitor that is a pyrazole.
134. The method of claim 132, wherein said targeting moiety is an
biotin.
135. The method of claim 132, wherein said targeting moiety is a
ligand that binds a lectin.
136. The method of claim 135, wherein said lectin is concanavalin
A.
137. The method of claim 136, wherein targeting moiety is a
carbohydrate.
138. The method of claim 136, wherein said targeting moiety is
-thioethyl D-mannopyranoside.
139. The method of claim 132, wherein said targeting moiety
specifically binds to a soil and said enzyme degrades a component
of said soil.
140. A method of enhancing the activity of a drug that acts as an
inhibitor of a receptor or an enzyme, said method comprising:
coupling a serine hydrolase to said drug such that when said drug
binds said receptor or enzyme, the serine hydrolase degrades the
receptor or enzyme.
141. The method of claim 140, wherein said method increases the
dosage therapeutic window of said drug.
142. The method of claim 140, wherein said serine hydrolase is a
subtilisin.
143. A method of inhibiting an enzyme or a receptor, said method
comprising contacting the enzyme or receptor with a chimeric
molecule comprising a ligand that binds said enzyme or receptor
attached to an enzyme that degrades the cognate ligand of said
enzyme or receptor.
144. The method of claim 143, wherein said chimeric molecule
comprises a protease attached to an inhibitor of said enzyme or
receptor.
145. The method of claim 144, wherein said protease is selected
from the group consisting of a serine protease, a cysteine
protease, an aspartyl protease, a pepsin-type protease, and a
metalloprotease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of U.S. Ser.
No. 60/131,362, filed on Apr. 28, 1999, as provided for under 35
U.S.C. .sctn.119 and/or 35 U.S.C. .sctn.120, as appropriate. U.S.
Ser. No. 60/131,362 is incorporated herein by reference in its
entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] [Not Applicable ]
FIELD OF THE INVENTION
[0003] This invention relates to the field of chimeric molecules.
In particular this invention provides novel chimeric molecules that
act as catalytic antagonists of targets (e.g. receptors, enzymes,
lectins, etc.).
BACKGROUND OF THE INVENTION
[0004] In a chimeric molecule, two or more molecules that exist
separately in their native state are joined together to form a
single molecule having the desired functionality of all of its
constituent molecules. Frequently, one of the constituent molecules
of a chimeric molecule is a "targeting molecule". The targeting
molecule is a molecule such as an antibody that specifically binds
to its corresponding target and, by virtue of the targeting
molecule, the chimeric molecule will specifically bind (target)
cells and tissues bearing the target (e.g. the epitope) to which
the targeting moiety is directed.
[0005] Another constituent of the chimeric molecule may be an
"effector molecule". The effector molecule refers to a molecule
that is to be specifically transported to the target to which the
chimeric molecule is specifically directed.
[0006] Chimeric molecules comprising a targeting moiety attached to
an effector moiety have been used in a wide variety of contexts.
Thus, for example, chimeric molecules comprising a targeting moiety
joined to a cytotoxic "effector molecule" have frequently been used
to target and kill tumor cells (see, e.g., Pastan et al., Ann. Rev.
Biochem., 61: 331-354 (1992). Other chimeric molecules comprising a
targeting moiety attached to angiogenesis inhibitors have been used
to inhibit tumor growth and/or proliferation. Conversely,
angiogenesis inducers) have been proposed for the treatment of
atherosclerosis. Other uses of chimeric molecules have involved the
delivery of intrabodies, intracellularly expressed antibodies that
then bind to an intracellular protein, the specific delivery of
vectors (e.g. for gene therapy), or the creation of tissue-specific
liposomes.
[0007] Typically, the target recognized by the targeting moiety is
not the desired site of action of the effector molecule. Thus, for
example, in the case of chimeric cytotoxins used to treat cancers
(e.g. IL4-PE, B1FvPE38, etc., see, e.g., Benhar & Pastan (1995)
Clin. Canc. Res., 1: 1023-1029, Thrush et al. (1996) Ann. Rev.
Immunol., 14: 49-71, etc.) the targeting moiety specifically binds
to a target on the surface of the cell. The chimeric molecule is
then internalized into the cell and the effector molecule (e.g.,
ricin, abrin, Diptheria toxin, Pseudomonas exotoxin) is transported
to the cytosol of the cell where it exerts its characteristic
activity (e.g. ADP ribosylation in the case of Pseudomonas
exotoxin).
[0008] Similarly, targeted liposomes are typically internalized
through a receptor-mediated process or through the action of the
lipid. Targeted intrabodies and gene therapy vectors are also
internalized for expression within the cell. In addition, a common
goal in the design of targeted chimeric molecules has been the
increase of binding specificity and avidity. It is generally
believed that, by increasing avidity and specificity the
concentration of the chimeric molecule to achieve a given result
will decrease. Thus, release of the chimeric molecule from its
target is generally viewed as undesirable.
[0009] Because the chimeric molecule is typically internalized (in
the case of targeted cells) and the activity of the effector
molecule is directed to a molecule other than the specifically
recognized target, chimeric molecules typically act in a
"stoichiometric" manner. That is, each chimeric molecule is
essentially consumed upon interaction with its "substrate" and
activity of the chimeric molecule is unavailable for subsequent
reactions. As a consequence chimeric molecules must be maintained
at relatively high level for efficacy and a recurring problem of
chimeric moieties, particularly in in vivo applications is the
inability to maintain elevated serum levels of the chimeric
molecule over therapeutically significant periods of time and the
increased (e.g. non-specific) toxicity caused by the high dosages
that must be utilized.
[0010] Attempts at solving these problems have focused on reducing
the immunogenicity of the chimera (e.g. by using humanized
antibodies, antibody fragments, small fusion proteins, etc.) or
"masking" the chimeric molecule (e.g. "stealth" liposomes). In
particular, the impetus to reduced immunogenicity, improved tumor
penetration, and the like, has led to the increasing use of fusion
proteins instead of chemically coupled moieties in chimeric
molecules (see, e.g., Pastan, (1992) Ann. Rev. Biochem., 61:
331-354; Thrush (1996) Ann. Rev. Immunol., 14: 49-71; Brinkmann and
Pastan (1994) Biochem. Biophys. Acta, 1198: 27-45, etc.), but have
not addressed the actual stoichiometry or kinetics of the
chimera.
SUMMARY OF THE INVENTION
[0011] This invention provides a novel approach to the design of
chimeric molecules. In one embodiment, the molecules of this
invention specifically bind to a target molecule and degrade that
bound molecule. In preferred embodiments, this results in a loss of
activity (e.g. biological activity) of the target molecule and also
results in the release of the chimeric molecule so that it is free
to find and degrade another target. In this manner the chimeric
molecule is "regenerated" and essentially catalytic. Because a
single chimeric molecule can attack and degrade an essentially
limitless number of targets, the so called "catalytic antagonists"
of this invention are highly effective at relatively low
dosages.
[0012] Thus, in one embodiment, this invention provides a catalytic
antagonist of a target molecule (e.g. an enzyme, a receptor, etc.).
The antagonist comprises a targeting moiety that specifically binds
to the target molecule and the targeting moiety is attached to an
enzyme that degrades the target molecule to reduce binding of the
target molecule to its cognate ligand. In particularly preferred
embodiments, the degradation of the target molecule also reduces
binding of the antagonist to the target molecule. Thus, in these
embodiments, the antagonist is released from the target thereby
allowing the antagonist to bind and degrade another target
molecule.
[0013] In particularly preferred embodiments the targeting moiety
is joined to the enzyme through the sulfur group on a cysteine and
the cysteine is a naturally occurring cysteine in the enzyme or a
cysteine introduced into the enzyme (e.g. substituted for a native
amino acid other than cysteine in the enzyme). In certain preferred
embodiments, the cysteine is a cysteine that is substituted for a
native amino acid other than cysteine in or near a subsite
comprising a substrate binding site of the enzyme. In some
embodiments, the cysteine is a cysteine that is substituted for an
amino acid forming a substrate binding site.
[0014] Preferred enzymes include, but are not limited to a
protease, an esterase, an amidase, a peptidase, a lactamase, a
cellulase, an oxidase, an oxidoreductase, a reductase, a
transferase, a hydrolase, an isomerase, a ligase, a lipase, a
phospholipase, a phosphatase, a kinase, a sulfatase, a lysozyme, a
glycosidase, a nuclease, an aldolase, a ketolase, a lyase, a
cyclase, a reverse transcriptase, a hyaluronidase, an amylase, a
cerebrosidase, and a chitinase. In a particularly preferred
embodiment, the enzyme is a serine hydrolase. In an even more
preferred embodiment, the enzyme is a subtilisin-type serine
hydrolase (e.g. a Bacillus lentus subtilisin) and said cysteine is
substituted for an amino acid in or near a subsite selected from
the group consisting of an S1 subsite, an S1' subsite, and an S2
subsite.
[0015] In a particularly preferred embodiment the enzyme is a
Bacillus lentus subtilisin. In preferred embodiments, the cysteine
is substituted for an amino acid in a subtillisin, where the amino
acid corresponds to a reference residue in a Bacillus lentus
subtilisin, where the reference residue is at or near a residue
selected from the group consisting of residue 156, residue 166,
residue 217, residue 222, residue 62, residue 96, residue 104,
residue 107, residue 189, and residue 209.
[0016] In another embodiment the enzyme is a chymotrypsin-type
serine protease and the cysteine is substituted for the amino acid
corresponding to a reference residue in a mature trypsin (Protein
Data Bank entry 1TPP), wherein said reference residue is at or near
a residue selected from the group consisting of Tyr94, Leu99,
Gln175, Asp189, Ser190, Gln192, Phe41, Lys60, Tyr151, Ser214, and
Lys224.
[0017] In still another embodiment the enzyme is an alpha/beta type
serine hydrolase and the cysteine is substituted for the amino acid
corresponding to a reference residue in a Candida antartica lipase
(Protein Data Bank entry 1TCA), where the reference residue is at
or near a residue selected from the group consisting of Trp104,
Leu140, Leu144, Val154, Glu188, Ala 225, Leu278 and Ile285.
[0018] In yet another embodiment the enzyme is an aspartyl
protease. More preferably the enzyme is a pepsin-type protease and
the cysteine is substituted for the amino acid corresponding to a
reference residue in the mature human pepsin (Protein Data Bank
entry 1PSN), where the reference residue is at or near a residue
selected from the group consisting of Tyr9, Met12, Glu13, Gly76,
Thr77, Phe111, Phe117, Ile128, Ser130, Tyr189Ile213, Glu239,
Met245, Gln287, Met289, Leu291, and Glu294.
[0019] In still yet another embodiment the enzyme is a cysteine
protease. More preferably the enzyme is a papain and the cysteine
is substituted for the amino acid corresponding to a reference
residue in a mature papain (Protein Data Bank entry 1BQI), where
the reference residue is at or near a residue selected from the
group consisting of Asn18, Ser21, Asn64, Tyr67, Trp69, Gln112,
Gln142, Asp158, Trp177, and Phe207.
[0020] In certain embodiments the enzyme is a metalloprotease and
the cysteine is substituted for the amino acid corresponding to a
reference residue in the mature human matrix metalloprotease
(Protein Data Bank entry 830C), where the reference residue is at
or near a residue selected from the group consisting of Leu111,
Phe175, Tyr176, Ser182, Leu184, Phe189, Tyr214, Asp231, Lys234, and
Ile243.
[0021] In certain embodiments the catalytic antagonist targeting
moiety is directed against a target where the target is a molecule
present on the surface of a cell (e.g., a. molecule forming a
receptor, a ligand, a component of a cell wall, a component of a
cell membrane, etc.). In certain embodiments the targeting moiety
includes, but is not limited to an antigen, a carbohydrate, a
nucleic acid, a lipid, a coordination complex, a sugar, a vitamin,
a dendrimer, and a crown ether. In a particularly preferred
embodiment the targeting moiety is a cognate ligand for a receptor
or an enzyme. In another particularly preferred embodiment the
targeting moiety is an inhibitor for a receptor or an enzyme.
[0022] In certain preferred embodiments, the enzyme is a protease
(e.g. a papain, a subtilisin, a pepsin, a trypsin, a
metalloprotease, etc.) and the targeting moiety is a ligand
selected from the group consisting of a carbohydrate, a vitamin or
vitamin analog, an enzyme inhibitor, a peptide, a pharmaceutical
that is a small organic molecule, and biotin. In another embodiment
the enzyme is a protease and said targeting moiety is a
receptor.
[0023] In certain preferred embodiments, the enzyme is a protease
(e.g. a papain, a subtilisin, a pepsin, a trypsin, a
metalloprotease, etc.) and the targeting moiety is an enzyme
inhibitor that is a pyrazole, a biotin, a ligand that binds a
lectin (e.g. concanavalin A), a carbohydrate (e.g. thioethyl
D-mannopyranoside). In one particularly preferred embodiment the
targeting moiety specifically binds to a soil and the enzyme
degrades a component of the soil.
[0024] In another embodiment this invention provides a method of
degrading a target molecule. The method involves contacting the
target molecule with a catalytic antagonist comprising a targeting
moiety that specifically binds to the target molecule the targeting
moiety being attached to an enzyme that degrades the target
molecule. In a preferred embodiment the degradation of the target
molecule releases the antagonist thereby allowing the antagonist to
bind and degrade another target molecule. In preferred embodiments,
the targeting moiety is joined to the enzyme through the sulfur
group on a cysteine. Preferred antagonist molecules include, but
are not limited to the catalytic antagonist molecules described
above.
[0025] In still another embodiment, this invention provides an
enzyme having altered substrate specificity (i.e. a "redirected
enzyme). The enzyme preferably comprises a targeting moiety
attached to a subsite comprising the substrate binding site of said
enzyme. In preferred embodiments, the targeting moiety is coupled
to said enzyme through to a sulfur of a cysteine in said subsite of
said enzyme. The cysteine may be a native cysteine or a cysteine is
substituted for a native amino acid that is not cysteine in the
subsite of the enzyme. Preferred enzymes include, but are not
limited to a protease, an esterase, an amidase, a peptidase, a
lactamase, a cellulase, an oxidase, an oxidoreductase, a reductase,
a transferase, a hydrolase, an isomerase, a ligase, a lipase, a
phospholipase, a phosphatase, a kinase, a sulfatase, a lysozyme, a
glycosidase, a glycosyltransferase, a nuclease, an aldolase, a
ketolase, a lyase, a cyclase, a reverse transcriptase, a
hyaluronidase, an amylase, a cerebrosidase and a chitinase.
[0026] In particularly preferred embodiments, the enzyme is a
serine hydrolase (e.g., a subtilisin). In a subtilisin, the
cysteine is preferably subsitited for amino acids at or near a
subsite selected from the group consisting of an S1 subsite, an S1'
subsite, and an S2 subsite. Particularly preferred sites for
substitution of the cysteine in various enzymes include, but are
not limited to those identified above. Similarly, particularly
preferred targets and targeting moieties include those identified
above. In certain embodiments the targeting moiety is an inhibitor
for a receptor or an enzyme, in other embodiments the targeting
moiety is selected from the group consisting of a growth factor, a
cytokine, and a receptor ligand. In certain embodiments, the enzyme
is a protease and the targeting moiety is a ligand selected from
the group consisting of a carbohydrate, a vitamin or vitamin
analog, an enzyme inhibitor, a peptide, a pharmaceutical that is a
small organic molecule, and biotin. In one particularly preferred
embodiment the enzyme is a protease (e.g. a subtilisin, a papain, a
pepsin, etc.) and the targeting moiety is a receptor, enzyme
inhibitor that is a pyrazole, a biotin, a ligand that binds a
lectin (e.g. concanavalin A), or a carbohydrate (e.g. thioethyl
D-mannopyranoside). In one embodiment the targeting moiety
specifically binds to a soil and said enzyme degrades a component
of the soil.
[0027] In still yet another embodiment this invention provides
methods of directing the activity of an enzyme to a specific
target. The methods comprise providing an enzyme having altered
substrate specificity said enzyme comprising a targeting moiety
attached to a subsite within the substrate binding region of said
enzyme; and contacting the target with the enzyme, whereby the
enzyme specifically binds to the target thereby localizing the
activity of the enzyme at the target. Preferred enzymes include,
but are not limited to, the "redirected" enzymes described
above.
[0028] This invention also provides methods of enhancing the
activity of a drug that acts as an inhibitor of a receptor or an
enzyme. The methods involve coupling a hydrolase to said drug such
that when said drug binds said receptor or enzyme, the hydrolase
degrades the receptor or enzyme. In preferred embodiments, the
method increases the dosage therapeutic window of said drug. In one
particularly preferred embodiments the hydrolase is a serine
hydrolase (e.g. a subtilisin). In certain preferred embodiments,
the hydrolase is a metalloprotease, a cysteine protease, an
aspartyl protease, and the like.
[0029] This invention also provides a method of inhibiting an
enzyme or a receptor. The method comprises contacting the enzyme or
receptor with a chimeric molecule comprising a ligand that binds
the enzyme or receptor attached to an enzyme that degrades the
cognate ligand of the enzyme or receptor. The enzyme thus becomes
linked to the enzyme or receptor where it is free to degrade the
cognate ligand thereby preventing the cognate ligand from
activating the receptor or acting as a substrate for the enzyme. In
a preferred embodiment the chimeric molecule comprises a hydrolase
(e.g. a protease) attached to an inhibitor of the enzyme or
receptor. Preferred hydrolases include, but are not limited to a
serine protease, a cysteine protease, an aspartyl protease, a
pepsin-type protease, and a metalloprotease.
[0030] In certain embodiments, this invention does not include
catalytic antibodies, e.g. as described by Hifumi et al. (1999) J.
Bioscience and Bioengineering, 88: 323.
Definitions
[0031] The term "catalytic antagonist", as used herein refers to an
enzyme that can inhibit the activity of a molecule that has a
particular biological activity and/or simply degrade a molecule
that has no particular biological activity. The inhibition can be a
blocking or destroying of the function of the "target" molecule. In
preferred embodiments, the inhibition or blockage is by partial or
complete degradation of the target molecule. The "catalytic
antagonist" is catalytic by virtue of the fact that the antagonist
is not itself consumed or significantly altered (i.e., permanently
changed) by its interaction with the target molecule. Thus, in
preferred embodiments, the degradation of the target molecule
ultimately results in the release of the catalytic antagonist so
that it is free to attack another target molecule. The reaction is
preferably sub-stoichiometric (ratio of catalytic antagonist to
target is less than 1) and a single catalytic antagonist is free to
degrade any number of target molecules.
[0032] A "target molecule" refers to a molecule that is
specifically bound by the catalytic antagonist or specifically
directed enzymes described herein. Where a catalytic antagonist is
employed the target molecule is partially or completely degraded by
that antagonist.
[0033] A "targeting moiety" refers to a moiety in the chimeric
molecule that that specifically binds to the target molecule. Prior
to coupling the targeting moiety to the enzyme, the targeting
moiety is a targeting molecule. In preferred embodiments, the
targeting moiety is one of a pair of cognate binding partners.
[0034] The term "specifically binds", when referring to the
interaction of a targeting moiety and its cognate binding partner
refers to a binding reaction which is determinative of the presence
of the targeting moiety or the cognate molecule in the presence of
a heterogeneous population of molecules (e.g., proteins and other
biologics). Thus, for example, in the case of a receptor/ligand
binding pair the ligand would specifically and/or preferentially
select its receptor from a complex mixture of molecules, or vice
versa. The binding may be by one or more of a variety of mechanisms
including, but not limited to ionic interactions, covalent
interactions, hydrophobic interactions, van der Waals interactions,
etc.
[0035] The terms "binding partner", or a member of a "binding
pair", or "cognate ligand" refers to molecules that specifically
bind other molecules to form a binding complex such as
antibody/antigen, lectin/carbohydrate, nucleic acid/nucleic acid,
receptor/receptor ligand (e.g. IL-4 receptor and IL-4),
avidin/biotin, etc.
[0036] The term ligand is used to refer to a molecule that
specifically binds to another molecule. Commonly a ligand is a
soluble molecule, e.g. a hormone or cytokine, that binds to a
receptor. The decision as to which member of a binding pair is the
ligand and which the "receptor" is often a little arbitrary when
the broader sense of receptor is used (e.g., where there is no
implication of transduction of signal). In these cases, typically
the smaller of the two members of the binding pair is called the
ligand. Thus, in a lectin-sugar interaction, the sugar would be the
ligand (even if it is attached to a much larger molecule,
recognition is of the saccharide).
[0037] The terms "polypeptide", "oligopeptide", "peptide" and
"protein" are used interchangeably herein to refer to a polymer of
amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residue is an artificial chemical
analogue of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers. The term also
includes variants on the traditional peptide linkage joining the
amino acids making up the polypeptide. Proteins also include
glycoproteins (e.g. histidine-rich glycoprotein (HRG), Lewis Y
antigen (Le.sup.Y), and the like.).
[0038] The terms "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein refer to at least two nucleotides covalently
linked together. A nucleic acid of the present invention is
preferably single-stranded or double stranded and will generally
contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et al.
(1993) Tetrahedron 49(10):1925) and references therein; Letsinger
(1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J.
Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acids Res. 14:
3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988)
J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) Chemica
Scripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic
Acids Res. 19:1437; and U.S. Pat. No. 5,644,048),
phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111 :2321,
O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides
and Analogues: A Practical Approach, Oxford University Press), and
peptide nucleic acid backbones and linkages (see Egholm (1992) J.
Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl.
31: 1008; Nielsen (1993) Nature, 365: 566; Carlsson et al. (1996)
Nature 380: 207). Other analog nucleic acids include those with
positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA
92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684,
5,602,240, 5,216,141 and 4,469,863; Letsinger et al. (1988) J. Am.
Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside &
Nucleotide 13:1597; Chapters 2 and 3, ACS Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic &
Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular
NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribose
backbones, including those described in U.S. Pat. Nos. 5,235,033
and 5,034,506, and Chapters 6 and 7, ACS Symposium Series 580,
Carbohydrate Modifications in Antisense Research, Ed. Y. S. Sanghui
and P. Dan Cook. Nucleic acids containing one or more carbocyclic
sugars are also included within the definition of nucleic acids
(see Jenkins et al. (1995), Chem. Soc. Rev. pp169-176). Several
nucleic acid analogs are described in Rawls, C & E News Jun. 2,
1997 page 35. These modifications of the ribose-phosphate backbone
may be done to facilitate the addition of additional moieties such
as labels, or to increase the stability and half-life of such
molecules in physiological environments.
[0039] The term "residue" as used herein refers to natural,
synthetic, or modified amino acids.
[0040] The term enzyme includes proteins that are capable of
catalyzing chemical changes in other substances without being
permanently changed themselves. The enzymes can be wild-type
enzymes or variant enzymes. Enzymes within the scope of the present
invention include, but are not limited to, proteases, esterases,
amidases, peptidases, lactamases, cellulases, oxidases,
oxidoreductases, reductases, transferases, hydrolases, isomerases,
ligases, lipases, phospholipases, phosphatases, kinases,
sulfatases, lysozymes, glycosidases, glycosyltransferases,
nucleases, aldolases, ketolases, lyases, cyclases, reverse
transcriptases, hyaluronidases, amylases, cerebrosidases,
chitinases, and the like.
[0041] A "mutant enzyme" is an enzyme that has been changed by
replacing an amino acid residue with a cysteine (or other)
residue.
[0042] A "chemically modified" enzyme is an enzyme that has been
derivatized to bear a substituent not normally found at that
location in the enzyme. The derivatization typically is of a post
translational modification, occasionally performed in vivo, but
more typically performed ex vivo.
[0043] A "chemically modified mutant enzyme" or "CMM" is an enzyme
in which an amino acid residue has been replaced with another amino
acid residue (preferably a cysteine) and the replacement residue is
chemically derivatized to bear a substituent not normally found on
that residue.
[0044] The term "thiol side chain group", "thiol containing group",
and "thiol side chain" are terms that can be used interchangeably
and include groups that are used to replace the thiol hydrogen of a
cysteine. Commonly the thiol side chain group includes a sulfur
atom through which the thiol side chain group that is attached to
the thiol sulfur of the cysteine. The "substituent" typically
refers to the group remains attached to the cysteine through a
disulfide linkage formed by reacting the cysteine with a
methanesulfonate reagent as described herein. While the term
substituent preferably refers just to the group that remains
attached (excluding its thiol group), the substituent can also
refer to the entire thiol side chain group. The difference will be
clear from the context.
[0045] The "binding site of an enzyme" consists of a series of
subsites across the substrate binding surface of the enzyme (Berger
& Schechter (1970) Phil. Trans. Roy Soc. Lond. B 257: 249-264).
The substrate residues that correspond to the subsites are labeled
P and the subsites are labeled S. By convention, the subsites are
labeled S.sub.1, S.sub.2, S.sub.3, S.sub.4, S.sub.1', and S.sub.2'.
A discussion of subsites can be found in Siezen et al. (1991)
Protein Engineering, 4: 719-737, and Fersht (1985) Enzyme Structure
and Mechanism, 2nd ed. Freeman, New York, 29-30. The preferred
subsites include S.sub.1, S.sub.1', and S.sub.2.
[0046] The phrase "amino acid ##" or "amino acid ## in the XX
subsite" is intended to include the amino acid at the referenced
position (e.g. amino acid 156 of B. lentus subtilisin which is in
the S.sub.1 subsite) and the amino acids at the corresponding
(homologous) position in related enzymes.
[0047] A residue (amino acid) of an enzyme is equivalent to a
residue of a referenced enzyme (e.g. B. amyloliquefaciens
subtilisin) if it is either homologous (i.e., corresponding in
position in either primary or tertiary structure) or analogous to a
specific residue or portion of that residue in B. amyloliquefaciens
subtilisin (i.e., having the same or similar finctional capacity to
combine, react, or interact chemically).
[0048] In order to establish homology to primary structure, the
amino acid sequence of the subject enzyme (e.g. a serine hydrolase,
cysteine protease, aspartyl protease, metalloprotease, etc.) is
directly compared to a reference enzyme (e.g. B. amyloliquefaciens
subtilisin in the case of a subtilisin type serine protease)
primary sequence and particularly to a set of residues known to be
invariant in all enzymes of that family (e.g. subtilisins) for
which sequence is known. After aligning the conserved residues,
allowing for necessary insertions and deletions in order to
maintain alignment (i.e., avoiding the elimination of conserved
residues through arbitrary deletion and insertion), the residues
equivalent to particular amino acids in the primary sequence of the
reference enzyme (e.g. B. amyloliquefaciens subtilisin) are
defined. Alignment of conserved residues preferably should conserve
100% of such residues. However, alignment of greater than 75% or as
little as 50% of conserved residues is also adequate to define
equivalent residues. Conservation of the catalytic triad, (e.g.,
Asp32/His64/Ser221) should be maintained for serine hydrolases.
[0049] The conserved residues may be used to define the
corresponding equivalent amino acid residues in other related
enzymes. For example, the two (reference and "target") sequences
are aligned in order to produce the maximum homology of conserved
residues. There may be a number of insertions and deletions in the
"target" sequence as compared to the reference sequence. Thus, for
example, a number of deletions are seen in the thermitase sequence
as compared to B. amyloliquefaciens subtilisin (see, e.g. U.S. Pat.
No. 5,972,682). Thus, the equivalent amino acid of Tyr217 in B.
amyloliquefaciens subtilisin in thermitase is the particular lysine
shown beneath Tyr217 in FIG. 5B-2 of the U.S. Pat. No.
5,972,682.
[0050] The particular "equivalent" resides may be substituted by a
different amino acid to produce a mutant carbonyl hydrolase since
they are equivalent in primary structure.
[0051] Equivalent residues homologous at the level of tertiary
structure for a particular enzyme whose tertiary structure has been
determined by x-ray crystallography, are defined as those for which
the atomic coordinates of 2 or more of the main chain atoms of a
particular amino acid residue of the reference sequence (e.g. B.
amyloliquefaciens subtilisin) and the sequence in question (target
sequence) (N on N, CA on CA, C on C, and O on O) are within 0.13 nm
and preferably 0.1 nm after alignment. Alignment is achieved after
the best model has been oriented and positioned to give the maximum
overlap of atomic coordinates of non-hydrogen protein atoms of the
enzyme in question to the reference sequence. The best model is the
crystallographic model giving the lowest R factor for experimental
diffraction data at the highest resolution available. 1 R = h Fo (
h ) - Fc ( h ) h Fo ( h )
[0052] Equivalent residues which are functionally analogous to a
specific residue of a reference sequence (e.g. B. amyloliquefaciens
subtilisin) are defined as those amino acids sequence in question
(e.g. related subtilisin) which may adopt a conformation such that
they will alter, modify or contribute to protein structure,
substrate binding or catalysis in a manner defined and attributed
to a specific residue of the reference sequence as described
herein. Further, they are those residues of the sequence in
question (for which a tertiary structure has been obtained by x-ray
crystallography), which occupy an analogous position to the extent
that although the main chain atoms of the given residue may not
satisfy the criteria of equivalence on the basis of occupying a
homologous position, the atomic coordinates of at least two of the
side chain atoms of the residue lie with 0.13 nm of the
corresponding side chain atoms of the reference sequence
residue(s). The three dimensional structures would be aligned as
outlined above. For an illustration of this procedure see U.S. Pat.
No. 5,972,682.
[0053] A "reference residue" refers to a residue that is specified
in a particular enzyme and which serves as a "reference point" for
identifying, e.g., as described above, equivalent residues in other
members of the family of which the reference enzyme is a member.
Thus, the phrase "the amino acid corresponding to a reference
residue in the mature human protein X"refers to residues equivalent
(or homologous) to the reference residue of protein X in other
members of the same protein family. In addition, where the subject
protein is protein X, the phrase refers to the reference residue
itself.
[0054] A "serine hydrolase" is a hydrolytic enzyme utilizing an
active serine side chain to serve as a nucleophile in a hydrolytic
reaction. This term includes native and synthetic serine hydrolases
as well as enzymes engineered to perform the reverse reaction,
e.g., for synthetic purposes. The family of serine peptidases is
characterized by Bartlett and Rawlings (1994) Meth. Enzymol., 244:
19-61, Academic Press, S.D.
[0055] The "alpha/beta serine hydrolases" are a family of serine
hydrolyases based on structural homology to enzymes including wheat
germ serine carboxypeptidase's II (see, e.g., Liam et al. (1992)
Biochemistry 31: 9796-9812; Ollis et al. (1992) Protein
Engineering, 5: 197-211).
[0056] The term "aspartyl proteases", also known as aspartic
proteases, are proteases that are directly dependent on aspartic
acid residues for catalytic activity. The family of aspartyl
proteases is characterized in a number of publications known to
those of skill in the art (see, e.g.,. Rawlings and Barrett, (1995)
Meth. Enzymology, 248: 105-120, Academic Press, S.D.).
[0057] The term "cysteine proteases" is used herein consistently
with conventional usage of those of skill in the art. The family of
cysteine proteases is characterized in a number of publications
known to those of skill in the art (see, e.g., Rawlings and
Barrett, (1994) Meth. Enzymology, 224: 461-486, Academic Press,
S.D.).
[0058] The term "metalloproteases" is used herein consistently with
the conventional usage of those of skill in the art. The family of
metalloproteases is characterized in a number of publications known
to those of skill in the art (see, e.g., Rawlings and Barrett,
(1995) Meth. Enzymology, 248: 183-228, Academic Press, S.D.)
[0059] The "subtilisin type serine proteases" refer to a family of
serine hydrolyases based on structural homology to enzymes derived
from Bacillus subtilus, including subtilisin BPN' (Bott et al.
(1988) J. Biol. Chem. 263: 7895-7906; Siezen and Louise (1997)
Protein Science 6: 501-523; Bartlett and Rawlings (1994) Meth.
Enzymol., 244: 19-61, Academic Press, S.D.). Subtilisins are
bacterial or fungal proteases which generally act to cleave peptide
bonds of proteins or peptides. As used herein, "subtilisin" means a
naturally-occurring subtilisin or a recombinant subtilisin. A
series of naturally-occurring subtilisins is known to be produced
and often secreted by various microbial species. Amino acid
sequences of the members of this series are not entirely
homologous. However, the subtilisins in this series exhibit the
same or similar type of proteolytic activity. This class of serine
proteases shares a common amino acid sequence defining a catalytic
triad which distinguishes them from the chymotrypsin related class
of serine proteases. The subtilisins and chymotrypsin related
serine proteases have a catalytic triad comprising aspartate,
histidine and serine. In the subtilisin related proteases the
relative order of these amino acids, reading from the amino to
carboxy terminus, is aspartate-histidine-serine. In the
chymotrypsin related proteases, the relative order, however, is
histidine-aspartate-serine. Thus, subtilisin herein refers to a
serine protease having the catalytic triad of subtilisin related
proteases.
[0060] The "chymotrypsin serine protease family" refers to a family
of serine hydrolyases based on structural homology to enzymes
including gamma chymotrypsin (Birktoft and Blow. (1972) J.
Molecular Biology 68: 187-240).
[0061] A "dendritic polymer" is a polymer exhibiting regular
dendritic branching, formed by the sequential or generational
addition of branched layers to or from a core. The term dendritic
polymer encompasses "dendrimers", which are characterized by a
core, at least one interior branched layer, and a surface branched
layer (see, e.g., Petar et al. Pages 641-645 In Chem. in Britain,
(August 1994). A "dendron" is a species of dendrimer having
branches emanating from a focal point which is or can be joined to
a core, either directly or through a linking moiety to form a
dendrimer. Many dendrimers comprise two or more dendrons joined to
a common core. However, the term dendrimer is used broadly to
encompass a single dendron.
[0062] Dendritic polymers include, but are not limited to,
symmetrical and unsymmetrical branching dendrimers, cascade
molecules, arborols, dense star polymers, and the like. The PAMAM
dense star dendrimers (disclosed in U.S. Pat. No. 5,714,166) are
symmetric, in that the branch arms are of equal length. The
branching occurs at the nitrogen atom of a terminal amine group on
a preceding generation branch. The lysine-based dendrimers are
unsymmetric, in that the branch arms are of a different length. One
branch occurs at the epsilon nitrogen of the lysine molecule, while
another branch occurs at the alpha nitrogen, adjacent to the
reactive carboxy group which attaches the branch to a previous
generation branch.
[0063] Even though not formed by regular sequential addition of
branched layers, hyperbranched polymers, e.g., hyperbranched
polyols, may be equivalent to a dendritic polymer where the
branching pattern exhibits a degree of regularity approaching that
of a dendrimer.
[0064] As used herein, an "antibody" refers to a protein or
glycoprotein consisting of one or more polypeptides substantially
encoded by immunoglobulin genes or fragments of immunoglobulin
genes. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. A typical immunoglobulin (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"
(about 25 kD) and one "heavy" chain (about 50-70 kD). The
N-terminus of each chain defines a variable region of about 100 to
110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (VL) and variable heavy
chain (VH) refer to these light and heavy chains respectively.
[0065] Antibodies exist as intact immunoglobulins or as a number of
well characterized fragments produced by digestion with various
peptidases. Thus, for example, pepsin digests an antibody below
(i.e. toward the Fc domain) the disulfide linkages in the hinge
region to produce F(ab)'2, a dimer of Fab which itself is a light
chain joined to V.sub.H--C.sub.Hl by a disulfide bond. The F(ab)'2
may be reduced under mild conditions to break the disulfide linkage
in the hinge region thereby converting the (Fab')2 dimer into an
Fab' monomer. The Fab' monomer is essentially a Fab with part of
the hinge region (see, Paul (1993) Fundamental Immunology, Raven
Press, N.Y. for a more detailed description of other antibody
fragments). While various antibody fragments are defined in terms
of the digestion of an intact antibody, one of skill will
appreciate that such fragments may be synthesized de novo either
chemically, by utilizing recombinant DNA methodology, or by "phage
display" methods (see, e.g., Vaughan et al. (1996) Nature
Biotechnology, 14(3): 309-314, and PCT/US96/10287). Preferred
antibodies include single chain antibodies, e.g., single chain Fv
(scFv) antibodies in which a variable heavy and a variable light
chain are joined together (directly or through a peptide linker) to
form a continuous polypeptide.
[0066] The term "carbohydrate" includes mono-, oligo- and
poly-saccharides as well as substances derived from monosaccharides
by reduction of the carbonyl group (alditols), by oxidation of one
or more terminal groups to carboxylic acids, or by replacement of
one or more hydroxy group(s) by an hydrogen atom, an amino group, a
thiol group or similar heteroatomic groups. It also includes
derivatives of these compounds. The term "sugar" is frequently
applied to monosaccharides and lower oligosaccharides. Parent
monosaccharides are polyhydroxy aldehydes H--[CHOH].sub.n--CHO or
polyhydroxy ketones H--[CHOH].sub.n--CO--[CHOH].sub.m--H with three
or more carbon atoms. The generic term "monosaccharide" (as opposed
to oligosaccharide or polysaccharide) denotes a single unit,
without glycosidic connections to other such units. It also
includes aldoses, dialdoses, aldoketoses, ketoses and diketoses, as
well as deoxy sugars and amino sugars, and their derivatives,
provided that the parent compound has a (potential) carbonyl group
(see, e.g., McNaught (1996) Pure Appl. Chem. 68: 1919-2008)]. The
smallest are monosaccharides like glucose, ribose and threose.
Carbohydrates also include, but are not limited to,
oligosaccharides and polysaccharides (e.g. starch, cellulose,
glycogen) and carbohydrate analogues (e.g., those in which OH have
been replaced by H, F, NH.sub.2 or NHC(O)CH.sub.3).
[0067] The term "soil" or "stain" refers to the accumulation of
foreign material on a substrate of interest (e.g. a textile). The
"soil" or "stain" may have no biological activity, but may serve to
discolor, and/or degrade the underlying substrate. The "soil" need
not be visible to the naked eye. Deposition of foreign materials
that, while not visible to the naked eye, but that create odors or
support bacterial growth are also considered "soils" in the context
of this application. Typical stains or soils include, but are not
limited to grass stains, blood stains, milk stains, egg, egg white,
and the like.
[0068] The term "small organic molecule" refers to a molecule of a
size comparable to those organic molecules generally used in
pharmaceuticals. The term excludes biological macromolecules (e.g.,
proteins, nucleic acids, etc.). Preferred small organic molecules
range in size up to about 5000 Da, more preferably up to 2000 Da,
and most preferably up to about 1000 Da.
[0069] The term "near" or "adjacent to", when used to indicate a
location with respect to a particular amino acid residue (e.g.
"adjacent to residue 149") refers to a residue covalently attached
to the "reference residue", either preceding or following that
residue, or in van der Waals contact with the reference
residue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] FIG. 1 illustrates a variety of chimeric molecules of this
invention utilizing dendrimers as targeting moieties.
[0071] FIG. 2 illustrates SBL targeting an enzyme with an
inhibitor.
[0072] FIG. 3 illustrates scheme 11 for synthesis of MTS-pyrazole
4.
[0073] FIG. 4 illustrates results of HLADH targeting assay for
SBL-pyrazole chimeric molecules.
[0074] FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D illustrate results of
HLADH degradation assay for SBL-pyrazole chimeric molecules.
[0075] FIG. 6 shows HLADH activity for HLADH/AP mixtures with and
without S166C- pyrazole.
[0076] FIG. 7 shows AP activity for HLADH/AP mixtures with and
without S166C-pyrazole.
[0077] FIG. 8 shows HLADH activity for HLADH/AP mixtures with and
without S166C-pyrazole.
[0078] FIG. 9 shows AP activity for HLADH/AP mixtures with and
without S166C-pyrazole.
[0079] FIG. 10 shows HLADH degradation by substoichiometric
pyrazole-CMMs.
[0080] FIG. 11 shows HLADH degradation by pyrazole-CMMs in the
presence of alkaline phosphatase
[0081] FIG. 12 illustrates alkaline phosphatase degradation by
pyrazole-CMMs in the presence of HLADH.
[0082] FIG. 13 shows 11 mono- and disaccharide
methanethiosulfonates that were prepared.
[0083] FIG. 14A, FIG. 14B, and FIG. 14C illustrate selective lectin
degradation by sugar-modified GG36-WT.
[0084] FIG. 15A, FIG. 15B, FIG. 15B, and FIG. 15D illustrate time
course plots of the formation of <3000 MW protein fragments
during a lectin assay.
[0085] FIG. 16 illustrates synthesis scheme 7 for the synthesis of
biotin-MTS reagent 1.
[0086] FIG. 17 illustrates a standard enzyme linked immunosorbent
assay (ELISA)-technique for assaying targeting of biotinylated CMMs
to anti-biotin.
[0087] FIG. 18 illustrates a targeting assay for anti-biotin using
using hapten modified subtilisins in a 96-well plate.
[0088] FIG. 19 plot of anti-biotin degradation by biotin-CMM as a
function of time.
DETAILED DESCRIPTION
I. Catalytic Antagonists
[0089] This invention provides novel chimeric molecules that
exploit a fundamentally different mode of activity to avoid
problems of dosage, activity and persistence problems often
associated with the activity of chimeric molecules. In one
embodiment, the chimeric molecules are catalytic antagonists of a
target molecule. The catalytic antagonists of this invention
preferably comprise a targeting moiety attached to an enzyme that
degrades the molecule specifically bound by the targeting moiety.
The catalytic antagonists of this invention thus bind to a target
recognized by the targeting moiety (e.g. a receptor) the enzyme
component of the chimera then degrades all or part of the target.
This preferably resulting in a reduction or loss of activity of the
target and release of the chimeric molecule. The chimeric molecule
is then free to attack and degrade another target molecule.
[0090] Thus, unlike typical, chimeric molecules in which the
chimeric molecule is effective only once (e.g. due to expenditure
of the effector activity, and/or internalization and/or lysis of
the chimera) a chimeric molecule of this invention is free to
attack and degrade essentially a limitless number of targets. In
preferred embodiments, the antagonists of this invention are thus
catalytic in nature being effectively regenerated (rendered
available again) after degrading each substrate molecule (target).
The activity of the catalytic antagonists of this invention is thus
essentially sub-stoichiometric.
[0091] As a consequence, the catalytic antagonists of this
invention are effective in far lower concentrations than chimeric
molecules or traditional inhibitors. Consequently formulations
(e.g. detergents) comprising the catalytic inhibitors of this
invention can utilize significantly lower concentrations of
inhibitor and can be fabricated at lower cost. In in vivo
applications, the catalytic inhibitors of this invention because
they offer greater activity at lower concentration, are expected to
show longer effective serum half-life and lower toxicities than
"traditional" chimeric molecules.
[0092] The catalytic antagonists of this invention are useful in a
wide variety of contexts where it is desired to degrade a target
molecule and/or inhibit the activity of that target molecule. Thus,
for example, in ex vivo applications, the catalytic antagonists can
be used to specifically target and degrade a particular molecule.
Thus, for example, in cleaning operations, the chimeric molecules
of this invention can be utilized to specifically target and
degrade a component of a soil (e.g. a protein component, a lipid
component, etc.). In chemical synthetic processes, or biochemical
synthetic processes (e.g. in analytic or industrial preparations,
in bioreactors, etc.) to specifically degrade particular
preselected molecules. Thus, for example, where it is desired to
eliminate a particular enzymatic activity in a bioreactor (e.g. a
glycosylation) the catalytic antagonist of this invention
comprises, as a targeting moiety, a substrate for the enzyme
mediating the activity (e.g. a glycosyltransferase). The enzyme
(receptor) in the reactor binds the targeting moiety and the
enzymatic component of the chimera (e.g. a hydrolase) degrades the
enzyme reducing or eliminating its activity and also freeing itself
from the enzyme binding site whereby it is free to attack another
target enzyme.
[0093] The chimeric molecules of this invention having, e.g.
targeting moieties directed against lectins present on bacterial
surfaces attached to, e.g. lipases, or hydrolases, are effective
antimicrobial agents and can be used in a wide variety of
disinfectants.
[0094] In biological systems (e.g. in vitro or in vivo) the
chimeric molecules of this invention can be used to bind and
antagonize/inhibit a wide variety of receptors and/or enzymes,
and/or intermediary signaling molecules. A wide variety of drugs
act by inhibiting the activity of cellular receptors. Thus, for
example, antiestrogens (e.g. tamoxifen) bind to and block estrogen
receptors, beta blockers (e.g. digoxin) are used in the management
of hypertension and post myocardial infarction, histamine H2
receptor antagonists (cimetidine, ranitidine) are used in the
treatment of esophageal reflux disease, selective serotonin (5-HT)
reuptake inhibitors (e.g. Prozac, Zoloft, Paxil, etc.) are used in
the treatment of depression, and so forth. Chimeric catalytic
antagonists of this invention comprising, e.g. a cognate ligand
bound by the target receptor or a non-cognate ligand (e.g., a
mimetic or drug bound by the receptor), as targeting moiety
attached to an enzyme that can degrade the receptor act as
effective receptor antagonists.
[0095] Unlike a simple competitive inhibitor that "temporarily"
blocks the target receptor(s), the catalytic antagonists of this
invention effectively degrade the receptor. Thus, once bound and
degraded, the receptor is unlikely to function again, absent some
repair mechanism. Thus at equal concentrations, the catalytic
antagonists of this invention will produce a far greater degree of
activity and/or duration of activity than "traditional" competitive
inhibitors. It will also be appreciated that, in this context, an
enzyme (e.g. an intracellular enzyme) can also be regarded as a
receptor for its cognate substrate. Thus, catalytic antagonists of
this invention can be used to degrade target enzymes as well. In
this instance, it is preferably to use, as the targeting moiety, a
molecule that is not degraded or altered by the target enzyme.
Known competitive inhibitors of enzymes make good targeting
moieties in this context.
[0096] In still another embodiment this invention include chemical
antagonists (e.g. of receptors and/or enzymes) comprising a
targeting moiety that binds to the receptor or enzyme attached to
an enzyme that degrades the cognate ligand that binds to that
enzyme and/or receptor. The inhibitor of the enzyme or receptor
binds and anchors the enzyme comprising the chimeric molecule to
the target enzyme or receptor. When the cognate ligand of the
receptor or enzyme approaches, the enzyme degrades is and thereby
blocks its activity on the receptor. Again, the process is
"catalytic" with no permanent change to the chimeric molecule.
[0097] In preferred embodiments, the catalytic antagonists of this
invention are chemically coupled chimeric molecules. The targeting
moiety preferably coupled, directly or through a linker, to either
terminus of the enzyme (the amino or carboxyl terminus or through
an R group of the terminal amino acid), or more preferably, is
coupled, directly or through a linker, to a non-terminal amino acid
in the enzyme. In certain particularly preferred embodiments, the
catalytic antagonists of this invention comprise a chemically
modified mutant (CMM) enzyme. A chemically modified mutant enzyme
is an enzyme in which a native amino acid residue is replaced with
a different amino acid residue (e.g. cysteine, affording a reactive
site suitable for coupling the targeting moiety. Thus, preferred
chimeric molecules of this invention are chemically coupled
molecules rather than fusion proteins.
[0098] The use of chemically coupled targeting moieties in this
invention affords a number of advantages. The targeting moiety is
not limited to a peptide or protein, but rather can be any of a
number of ligands including, but not limited to, known drugs,
vitamins, carbohydrates, lectins, and the like. Because the
targeting moieties are typically smaller than proteins, they are
less immunogenic and show greater tissue penetration. In addition,
because the targeting moieties are often various small organic
molecules, they retain their conformation and specificity in a
physiological context and are typically less subject to degradation
in vivo. The chimeric molecules of this invention offer a number of
other advantages. Because they are chemically conjugated using a
"standard" chemistry, they are easier to make and/or to vary. In
addition, the molecules are smaller than typical "therapeutic"
fusion proteins (e.g. immunotoxins) and are expected to have
increased serum half-life. In addition, because, in certain
embodiments, the molecules actually destroy/degrade existing
receptors and/or enzymes, a single dosage is expected to have a
longer-lasting effect since the subject organisms must actually
replace the receptor and/or enzyme to restore that
functionality.
II. Retargeting Enzymatic Activity
[0099] In many applications, the catalytic antagonists of this
invention can be regarded as enzymes that have been "redirected" so
that they either act on a non-native substrate (for the enzymatic
component) or, more typically, so that the enzymatic activity is
localized at the site of the target molecule. Thus, in some
embodiments, this invention provides an enzyme having altered
substrate specificity where the enzyme is a component of a chimeric
molecule comprising a targeting moiety attached to a subsite
comprising the substrate binding site of the enzyme.
[0100] Traditionally targeted chimeric molecules are designed to
position the targeting moiety/domain some distance away from active
sites of interest in the effector moiety. It was generally believed
that a targeting moiety located too close to an active site of the
effector moiety would interfere with proper functioning of the
effector (e.g. via steric hindrance).
[0101] It was a surprising discovery of this invention that
targeting moieties comprising the chimeric molecules of this
invention can be coupled to amino acid residues comprising a
substrate binding site of the enzyme. Moreover, attachment of the
targeting moiety to an amino acid residue in the substrate binding
site of the enzyme results in the substrate binding site being
closely juxtaposed to the target bound by the targeting moiety.
[0102] Using chemically conjugated mutants according to the methods
of this invention, provides a versatile method of directing a
single enzyme to any target simply by changing the chemical moiety.
This is a substantial advantage over traditional methods where
extensive modification (e.g. by mutagenesis techniques) was
required to make a particular target-specific enzyme.
[0103] The activity of the enzyme is thus "redirected" in one or
both of two ways: First the activity of the enzyme can be
"spatially localized" by binding of the targeting moiety to a
particular preselected target. Thus, the enzyme may be specifically
directed to a particular cell type, a particular enzyme, a
particular receptor, etc. Second, by virtue of alterations in the
enzyme produced by the presence of the targeting molecule and/or by
virtue of the fact that the targeting molecule brings the substrate
binding site in close proximity to the target, the enzyme can show
significant activity against a target that is not its usual
substrate.
[0104] The redirected enzymes are useful in a wide variety of
contexts. For example, the targeting moiety can be selected to
redirect/localize the enzyme to a particular target for selective
degradation. For example, in the case of a detergent, the targeting
moiety can be selected to specifically bind to a particular class
of "soil" (e.g. egg) and thereby direct and appropriate degradative
enzyme (e.g. a protease) to that substrate.
[0105] In pharmaceutical applications, in some embodiments, the
retargeted enzyme can comprise a targeting moiety that directs the
enzyme to a particular target cell (e.g. a tumor cell) where the
retargeted enzyme (e.g. a thymidine kinase (tk)) activates a
particular drug (e.g. a cytotoxin such as ganclovir). In other
embodiments the enzyme may be retargeted to a cell that contains an
overabundance of a particular metabolite (e.g. as in storage
diseases such as Tay Sachs disease). At the new site, the
redirected enzyme affords the "missing" enzymatic activity thereby
treating the condition. These examples are merely illustrative,
and, along with others are discussed in greater detail below.
[0106] It will be appreciated in view of the teachings provided
herein that catalytic antagonists can be retargeted enzymes, but
are not necessarily so. Conversely, retargeted enzymes may act as
catalytic antagonists, but there are retargeted enzymes that are
not necessarily catalytic antagonists.
[0107] In any case, in preferred embodiments, the retargeted
enzymes and catalytic antagonists are created by selecting a
targeting moiety, selecting an enzyme (an effector moiety) and
chemically conjugating the two to form a chimeric molecule.
[0108] Selection of targeting moieties, enzymes and conjugating
such is described in detail below.
II. Selection of Targets and Targeting Moieties
[0109] In preferred embodiments, virtually any cognate binding
partner of a target (e.g. a receptor and/or an enzyme and/or a
lectin) can be used as a targeting moiety in the molecules of this
invention. In addition, molecules that are not cognate binding
partners, but that are specifically bound by the target molecules
(e.g. receptors or an enzymes) can also be used as targeting
moieties in the chimeric molecules of this invention.
[0110] The selection of the targeting moiety depends on the
application for which the chimeric molecule (e.g. catalytic
antagonist) is to be utilized. Targeting moieties can be grouped
and/or identified according to a wide variety of classification
schemes. Thus, for example they can be grouped according to type of
molecule, e.g. a peptide, an oligopeptide, a peptidomimetic, an
antibody, a hapten, an epitope, a carbohydrate, a monosaccharide,
an oligosaccharide, a polysaccharide, a glycomimetic, a nucleic
acid, a gene, a lipid, a coordination complex, a metal, a sugar, an
enzyme, a zymogen, a coenzyme, a cofactor, a coenzyme analog, a
cofactor analog, a vitamin, a vitamin analog, a crown ether, a
crown ether analog, mono and polycyclic ligands, heterocyclic
ligands, chiral ligands, enantiomerically enriched ligands, and any
multivalent or dendrimeric variation of the above or,
alternatively, they can be grouped according to the nature of the
target.
[0111] Preferred targets include, but are not limited to receptors,
enzymes, and lectins. In some instances it is simple to refer to
the targeting moiety that binds to one of these targets. Thus, for
example serotonin or a serotonin analogue may be a targeting
moiety. Similarly targeting moieties can be referred to/identified
by the target to which they bind. Thus a serotonin analogue as
targeting moiety is encompassed by a drug or compound that
specifically binds to a serotonin receptor.
[0112] By way of illustration, some preferred targets in the above
categories are discussed below. The described embodiments, however,
are illustrative in nature and not intended to be limiting.
[0113] A) Targeting Moieties for Receptors
[0114] Receptors provide highly effective targets, particularly for
the catalytic antagonists of this invention. Receptors typically
specifically bind a cognate ligand and are involved in a wide
variety of biological processes. Typically, receptors mediate
signaling or the influx or efflux of molecules from a cell.
Particularly as transducers of signals, receptors are involved in a
wide variety of processes including, but not limited to regulation
of growth and morphology/differentiation, gene expression and
production of particular molecules, cell proliferation, elements of
the immune response, various biological cascades (e.g. the
inflammatory response, the clotting response, etc.) and the
like.
[0115] As a consequence, receptors have long been recognized as
good targets for drugs and a wide variety of drugs are agonists
and/or antagonists of particular receptor activity (see, e.g.,
Table 1). Typically these drugs are relatively small organic
molecule and, as such, are good candidates as targeting moieties
for the chimeric molecules of this invention.
1TABLE 1 Typical pharmacological agents and their mode of activity.
Such pharmaceuticals make useful targeting moieties to specifically
direct a catalytic antagonist of this invention to a target
receptor. Activity Drug 5-HT1 Receptor Agonist Amerge 5-HT2
Receptor Antagonist Nexopamil (LU 49938) Ca2+ Channel blocker ACE
Inhibitor Mavik ACE Inhibitor Prinivil ACE Inhibitor/Diuretic
Accuretic Adjunct in Affective Disorders Management Alti-Tryptophan
Alpha 1A Adrenoceptor Blocker Flomax Alpha Adrenergic Receptor
Agonist Alphagan (relatively selective) Analgesic Nu-Mefenamic
Analgesic-Antipyretic Advil Analgesic-Antipyretic Asaphen E.C.
Analgesic-Antipyretic Motrin (Children's)
Analgesic-Antipyretic-Decongestant Tylenol Decongestant
Analgesic-Antitussive Codeine 15 mg & 30 mg
Analgesic-Decongestant-Antihistamine Sinutab Nightime Extra
Strength Androgen Testosterone Enanthate Injection Androgen
Testosterone Propionate Injection Anesthetic-Sedative Propofol
Injection Angiotensin II AT1 Receptor Blocker Avapro Angiotensin II
AT1 Receptor Blocker Diovan Angiotensin-converting Enzyme Inhibitor
Captril Antiandrogen Novo-Cyproterone Antiandrogen-estrogen
Combination Diane - 35 Hormone Antianginal Agent Nitrolingual
Pumpspray Antianginal Agent pms-Nifedipine Antianginal Agent
Trinipatch 0.2, 0.4 & 0.6 Antiarrhythmic Nu-Sotalol
Antiarrhythmic Rho-Sotalol Antiarrhythmic Sotzmol Anticholinergic
Nu-Oxybutyn Anticholinergic-Antispa- smodic agent Gen-Oxybutynin
Anticoagulant Viprinex Anticoagulant-Low Molecular Weight
Fraxiparine Heparin Anticonvulsant Deproic Anticonvulsant
Novo-Clobazam Anticonvulsant Nu-Clonazepam Anticonvulsant
Rho-Clonazepam Anticonvulsant Taro-Carbamazepine CR Antidepressant
Apo-Moclobemide Antidepressant Effexor XR Antidepressant
Gen-Nortriptyline Antidepressant Gen-Trazodone Antidepressant
Novo-Nortriptyline Antidepressant Nu-Desipramine Antidepressant
Nu-Nortriptyline Antidepressant Wellbutrin SR
Antidepressant-Antiobsessional-Antibulimic Nu-Fluoxetine
Antiepileptic Agent Cerebyx Antihistamine Benadryl Junior Strength
Chewable Tablets Antihistamine-Decongestant Dimetapp Quick Dissolve
Antihistamine-Decongestant Tavist-D Antihyperglycemic Nu-Metformin
Antihyperglycemic Rho-Metformin Antihyperlipidemic Nu-Fenofibrate
Antihypertensive Novo-Terazosin Antihypertensive Nu-Nifedipine-PA
Antihypertensive Tarka Antihypertensive-Antianginal Gen-Diltiazem
SR Antihypertensive-BPH Treatment Apo-Terazosin
Antihypertensive-BPH Treatment Nu-Terazosin Antiparkinson Mirapex
Antiparkinson Nu-Levocarb Antiparkinson Nu-Selegiline Antiparkinson
Requip Antiparkinson Tasmar Antipsychotic Apo-Loxapine
Antipsychotic Nu-Loxapine Antipsychotic Seroquel Antiretroviral
Zerit Antirheumatic Ridaura Antispasmodic Ditropan Antithrombotic
Lovenox Antitussive Benylin DM 12 Hour Antitussive
Broncho-grippol-DM Anxiolytic Buspirex Anxiolytic Bustab Anxiolytic
Gen-Buspirone Anxiolytic Novo-Poxide Anxiolytic Nu-Buspirone
Anxiolytic-Sedative Nu-Bromazepam Beta2-adrenergic Stimulant
Airomir Beta2-adrenergic Stimulant Nu-Salbutamol Solution
Beta-adrenergic Blocker Nu-Acebutolol Beta-adrenergic Blocker
pms-Atenolol Beta-adrenergic Blocker pms-Metoprolol-L
Beta-adrenergic Blocker Rho-Atenolol Bronchodilator Gen-Salbutamol
Respirator Solution Bronchodilator Nu-Ipratropium Bronchodilator
Oxeze Turbuhaler Bronchodilator Quibron-T Calcium Channel Blocker
Chronovera Carbonic Anhydrase Inhibitor Neptazane Chimeric
Monoclonal Antiplatelet Antibody Reopro Corticosteroid Flovent
Diskus Corticosteroid Novo-Flunisolide Corticosteroid Triamcinolone
Diacetate Injectable Suspension Diuretic Demadex Diuretic
Nu-Indapamide Estrogen Climara Gastrointestinal anti-inflammatory
Salofalk Glucocorticoid Methylpred nisolone Sodium Succinate for
Injection USP Hematinic Ferodan Hematopoietic Acti-B12
Hematopoietic Heracline Histamine H1 Receptor Antagonist Claritin
Histamine H2 Receptor Antagonist Maalox H2 Acid Controller
Histamine H2 Receptor Antagonist Ulcidine Histamine H2 Receptor
Antagonist Zantac 75 Human Gonadotropin Pregnyl Human Gonadotropin
Puregon Hypnotic Gen-Temazepam Hypnotic Nu-Temazepam Hypnotic
Nu-Zopiclone Hypnotic pms-Temazepam Hypnotic-Anticonvulsant
Rho-Nitrazepam Hypoglycemic Euglucon Hypoglycemic Novo-Gliclazide
Hypoglycemic pms-Glyburide Leukotriene Receptor Antagonist Accolate
Leukotriene Receptor Antagonist Singulair Lipid Metabolism
Regulator Baycol Lipid Metabolism Regulator Lescol Muscle Relaxant
Flexitec Muscle Relaxant Gen-Cycloprine Muscle Relaxant Liotec
Muscle Relaxant Nu-Cyclobenzaprine Muscle Relaxant-Analgesic
Acetazone Forte Muscle Relaxant-Analgesic Acetazone Forte C8 Muscle
Relaxant-Analgesic Methoxacet Muscle Relaxant-Analgesic Methoxacet
C1/8 Muscle Relaxant-Analgesic Methoxisal Muscle Relaxant-Analgesic
Methoxisal-C Neuroleptic pms-Methotrimeprazine
Neuroleptic-Antiemetic Droperidol Injection Neuromuscular Blocking
Agent Atracurium Besylate Injection Neuromuscular Blocking Agent
Atracurium Besylate Injection Nonsteroidal Anti-inflammatory Drug
Apo-Etodolac Nonsteroidal Anti-inflammatory Drug Diclotec
Nonsteroidal Anti-inflammatory Drug Fexicam Nonsteroidal
Anti-inflammatory Drug Novo-Ketorolac Nonsteroidal
Anti-inflammatory Drug Nu-Diclo-SR Nonsteroidal Anti-inflammatory
Drug Nu-Ketoprofen-SR Nonsteroidal Anti-inflammatory Drug
Nu-Tiaprofenic Nonsteroidal Anti-inflammatory Drug pms-Diclofenac
Nonsteroidal Anti-inflammatory Drug Rhodiaprox Opioid Analgesic
Pethidine Injection BP Oral Contraceptive Alesse 21 and 28
Pediculicide Para Platelet Aggregation Inhibitor Apo-Ticlopidine
Platelet Aggregation Inhibitor Nu-Ticlopidine Platelet Aggregation
Inhibitor Plavix Progestogen Nu-Megestrol Proton Pump Inhibitor
Losec Retinoid Rejuva-A Selective Estrogen Receptor Modulator
Evista Somatostatin Analogue Sandostatin Type II Alpha-reducatase
Inhibitor Propecia Upper Gastrointestinal Motility Modifier
Apo-Domperidone Upper Gastrointestinal Motility Modifier
Novo-Domperidone Upper Gastrointestinal Motility Modifier
Nu-Domperidone Upper Gastrointestinal Motility Modifier
pms-Domperidone Vasoactive Agent Nu-Pentoxifylline-SR Vitamin &
Mineral Supplement Calcium D 500 Vitamin & Mineral Supplement
Caltrate Plus Vitamin & Mineral Supplement Hemarexin Vitamin
Supplement Hormodausse Vitamin Supplement Sopalamine/3B Vitamin
Supplement Sopalamine/3B Plus C
[0116] The targeting moiety, however, need not be a known
pharmaceutical. There are a number of receptors for which
inhibitors or agonists are known where the inhibitors or agonists
are not approved pharmaceuticals.
[0117] There is, as yet, no uniform classification for receptors.
However, as indicated above, a great many receptors are signal
transduction receptors and within this group signal-transduction
receptors fall into three general classes:
[0118] The first class includes receptors that penetrate the plasma
membrane and have intrinsic enzymatic activity. Such receptors
include, but are not limited to, those that are tyrosine kinases
(e.g. PDGF, insulin, EGF and FGF receptors), tyrosine phosphatases
(e.g. CD45 [cluster determinant-45] protein of T cells and
macrophages), guanylate cyclases (e.g. natriuretic peptide
receptors), , and serine/threonine kinases (e.g. are cAMP-dependent
protein kinase (PKA), protein kinase C (PKC), MAP kinases, activin
and TGF-.beta. receptors). Additionally, several families of
receptors lack intrinsic enzyme activity, yet are coupled to
intracellular tyrosine kinases by direct protein-protein
interactions.
[0119] The proteins encoding receptor tyrosine kinases (RTKS)
typically contain four major domains: an extracellular ligand
binding domain, an intracellular tyrosine kinase domain, an
intracellular regulatory domain, and a transmembrane domain. The
amino acid sequences of the tyrosine kinase domains of RTKs are
highly conserved with those of cAMP-dependent protein kinase (PKA)
within the ATP binding and substrate binding regions. Some RTKs
have an insertion of non-kinase domain amino acids into the kinase
domain termed the kinase insert. RTK proteins are classified into
families based upon structural features in their extracellular
portions (as well as the presence or absence of a kinase insert)
which include the cysteine rich domains, immunoglobulin-like
domains, leucine-rich domains, Kringle domains, cadherin domains,
fibronectin type III repeats, discoidin I-like domains, acidic
domains, and EGF-like domains. Based upon the presence of these
various extracellular domains the RTKs have been sub-divided into
at least 14 different families. Representative RTKs include, but
are not limited to I EGF receptor, NEU/HER2, HER3, insulin
receptor, IGF-1 receptor, PDGF receptors, c-Kit, FGF receptors,
vascular endothelial cell growth factor (VEGF) receptor, hepatocyte
growth factor (HGF) and scatter factor (SC) receptors, the
neurotrophin receptor family (trkA, trkB, trkC) and NGF receptor,
and the like.
[0120] The second class includes receptors that are coupled, inside
the cell, to GTP-binding and hydrolyzing proteins (termed
G-proteins). The G-protein coupled receptors (GPCRs) are a
superfamily of integral membrane proteins that are typically
characterized by seven hydrophobic domains which are of sufficient
length (typically 20-28 amino acid residues) to span the plasma
membrane. Examples of this class include, but are not limited to
the -adrenergic receptors, odorant receptors and receptors for
peptide hormones (e.g. glucagon, angiotensin, vasopressin and
bradykinin).
[0121] The third class includes receptors that are found
intracellularly and that, upon ligand binding, migrate to the
nucleus where the ligand-receptor complex directly affects gene
transcription. These receptors include, but are not limited to
steroid/thyroid hormone receptor superfamily (e.g. glucocorticoid,
vitamin D, retinoic acid and thyroid hormone receptors). This is a
class of proteins that reside in the cytoplasm and bind the
lipophilic steroid/thyroid hormones. Upon binding ligand the
hormone-receptor complex translocates to the nucleus and binds to
specific DNA sequences termed hormone response elements (HREs). The
binding of the complex to an HRE results in altered transcription
rates of the associated gene.
[0122] Ligands that bind such receptors are well known to those of
skill in the art. These include, but are not limited to A.sub.2
receptor agonists (see, e.g., U.S. Pat. No. 6,026,317), 5HT1
receptor agonists or antagonists (see, e.g., U.S. Pat. No.
6,025,374 and 6,025,367), N-methyl-D-aspartate (NMDA) receptor
blockers for the prevention of atherosclerosis (see, e.g., U.S.
Pat. No. 6,025,369), modulators of peroxisome proliferator
activated receptor-gamma (see, e.g., U.S. Pat. No. 6,022,897),
endothelin receptor antagonists (see, e.g., U.S. Pat. Nos.
6,022,886, 6,020,348), human growth hormone variants having
enhanced affinity for human growth hormone receptor at site 1 (see,
e.g., U.S. Pat. No. 6,022,711), antagonists of the human neuronal
nicotinic acetylcholine receptor (see, e.g, U.S. Pat. No.
6,020,335), platelet GPIIb/IIIa receptor antagonists (see, e.g.,
U.S. Pat. No. 6,022,523), adenosine receptor agonists (see, e.g.,
U.S. Pat. No. 6,020,321), interleukin receptor (e.g. IL-2R, IL4R,
IL-6R, IL-8R, IL-10R, IL-13R, etc.) antagonists, binding agents
specific for growth factor receptors (e.g. EGF, TGF and analogues
or mimetics thereof), binding agents specific for IgA receptor
(see, e.g., U.S. Pat. No. 6,018,031), agonists of the strychnine
insensitive glycine modulatory site of the N-methyl-D-aspartate
receptor complex (see, e.g., U.S. Pat. No. 6,017,957), integrin
receptor antagonists (see, e.g., U.S. Pat. No. 6,017,926), androgen
receptor modulator compounds (see, e.g., U.S. Pat. No. 6,017,924),
PCP receptor ligands (see, e.g., U.S. Pat. No. 6,017,910), azole
peptidomimetics as thrombin receptor antagonists (see, e.g., U.S.
Pat. No. 6,017,890), NPY Y2-receptor agonists (see, e.g., U.S. Pat.
No. 6,017,879), receptor activators of NF-.kappa.B (see, e.g., U.S.
Pat. No. 6,017,729), antagonists of the TNF receptor, somatostatin
receptor-binding agents (see, e.g., U.S. Pat. No. 6,017,509), human
histamine H.sub.2 receptor, bradykinin binding agents (see, e.g.,
U.S. Pat. No. 6,015,812), glutamate receptor antagonist (see, e.g.,
U.S. Pat. No. 6,015,800), imidazoline receptors, transferrin
receptors, benzodiazepine receptor binding agents (see, e.g., U.S.
Pat. No. 6,015,544), gaba brain receptor ligands (see, e.g., U.S.
Pat. No. 6,013,799), neurotensin NT1 and NT2 receptors, CXCR2
receptors, CCR5 receptors, macrophage mannose receptors, and the
like.
[0123] Other receptors that provide good targets for the chimeric
molecules of this invention include but are not limited to, SP-K
receptor, substance K receptors, tachykinin 2 receptors,
.alpha.1-adrenoceptors subtype A, .alpha.1-adrenoceptors subtype B,
.alpha.2-Adrenoceptors subtype A, .beta.1-, .beta.2-,
.beta.3-adrenoceptors .delta. receptors, .kappa. receptors, .mu.
receptors, ACTH receptors, angiotensin receptors, adenosine
receptors, bombesin receptors, gastrin-releasing peptide receptors,
bradykinin receptors, C5a receptors, Calcitonin gene-related
peptide receptors, calcitonin receptors, CCK-A receptors.
corticotropin releasing factor receptors, dopamine receptors, EP2
receptors, EP3 receptors, ETA receptors , ETB receptors, FSH
receptors, GABA receptors, galanin receptors, glucagon receptors,
glucagon-like peptide-1 receptors, gonadotropin receptors, growth
hormone-releasing hormone receptors, histamine H1 receptors,
histamine H2 receptors, leukotriene B4 receptors, melatonin
receptors, MSH receptors, muscarinic M1, M2, M3, and M4 receptors,
neurotensin receptors, parathyroid hormone receptors, pituitary
adenylate cyclase-activating polypeptide receptors,
platelet-activating factor receptors, prostacyclin receptors, P2U
purinoceptors, P2Y purinoceptors, rhodopsins, secretin receptors,
somatostatin receptors, SSTR receptors, VIP receptors, vasopressin
receptors, estrogen receptors, neuropeptide receptors, T-cell
receptors, and the like.
[0124] B) Targeting Moieties for Enzymes and Antibodies.
[0125] In other embodiments, the targeting moieties used in the
chimeric molecules of this invention are moieties specifically
bound by enzymes or antibodies. A wide variety of enzymes, their
substrates and competitive inhibitors thereof are known to those of
skill in the art. Moreover, many of these enzymes provide good
targets for drug in a wide variety of pathologies.
[0126] For example, caspases are a remarkable and intricately
regulated network of enzymes that can trigger cell suicide in
animals from yeast and worms to humans. Caspases are known to
mediate programmed cell death in a number of diseases, including
ischemic brain injury, or stroke. It is believed that the cardiac
cell death that occurs during heart "attack" is caused by
activation of several caspases. In addition, it has been
demonstrated administration of an experimental caspase inhibitor
known as YVAD-cmk blocks this biochemical cascade and also protects
heart tissue, dramatically reducing the amount of myocardial deaths
by over 30 percent. Catalytic antagonists of this invention
comprising caspase-specific agents as targeting moieties attached
to a protease (enzyme) can specifically target and degrade the
caspase. It is expected this will offer protection of heart tissue
during and after myocardial infarction and brain tissue during and
after stroke. Agents that specifically bind to caspases (e.g.
YVAD-cmk, and various protected caspase substrates) are known to
those of skill in the art.
[0127] In another example, the enzyme GARFT (Glycinamide
Ribonucleotide Formyl Transferase) is an enzyme in a biochemical
pathway through which tumor cells synthesize purines, essential
components of DNA. Blocking the action of GARFT inhibits purine
synthesis and subsequent tumor DNA molecule construction. With the
exception of liver cells, all normal human tissues can obtain
purines via an alternative pathway (purine salvage pathway).
Inhibitors of GARFT will show selectivity for tumor cells and less
significant bone marrow toxicity than other chemotherapeutic
agents. A catalytic antagonist of this invention comprising a GARFT
targeting moiety attached to a protease capable of degrading GARFT
is expected to show similar tumor selectivity. One suitable
targeting moiety is AG2037 (produced by Agouron) which is in
preclinical studies. AG2037 has been engineered using
structure-based design to exhibit potent and selective inhibition
of GARFT but to avoid binding to mFBP, a membrane protein believed
to be important in the side-effects of earlier GARFT inhibitors.
AG2037 is well tolerated in a variety of mouse cancer models and
demonstrates broad-spectrum antitumor efficacy, at least equal to
that of paclitaxel when studied in the same tumors grown in
mice.
[0128] In still another example, the intracellular enzyme,
dihydroorotate dehydrogenase (DHODH) provides a good target. DHODH
is the fourth sequential enzyme involved in the de novo
biosynthesis of uridylate (UMP). Since activated T cells require
rapid de novo pyrimidine biosynthesis, this enzyme is known to be
critical for the activation of the immune response, making it a
good target for intervention in transplantation and autoimmune
disease. One compound that targets this enzyme, Leflunomide
(Hoechst), has been approved by the U.S. Food and Drug
Administration (FDA) for the treatment of active rheumatoid
arthritis in adults leflunomide, or related DHODH-specific agents
can be used as a targeting moiety attached to an enzyme that
degrades the DHODH enzyme and provides a similar therapeutic
result. Another known inhibitor, Brequinar sodium, has shown
efficacy in many animal models of immunosuppression, but was not
successful in clinical trials for transplantation, apparently due
to a narrow therapeutic window. When used as a targeting moiety in
a catalytic antagonist of this invention, it is expected that the
therapeutic window will be improved because the molecule will be
effective in lower dosages. In general, it is believed that
conversion of drugs that act as competitive inhibitors into
catalytic antagonists in accordance with this invention will show
an improved therapeutic window due to their higher efficacy at
lower concentration.
[0129] In still yet another example, the catalytic antagonists of
this invention are useful in the treatment of hereditary emphysema.
The inherited form of emphysema is called alpha-1 proteinase
inhibitor deficiency or "alpha-one" for short. People with this
disease have a deficiency in a major protein, alpha-1 proteinase
inhibitor. Alpha-1 proteinase inhibitor is a major protein in the
blood and is produced primarily in the liver cells but also by some
white blood cells. It protects the lung by blocking the effects of
powerful enzymes called elastases. Elastase is normally carried in
white blood cells and protects the delicate tissue of the lung by
killing bacteria and neutralizing tiny particles inhaled into the
lung. Once the protective work of this enzyme is finished, further
action is blocked by the alpha-1 proteinase inhibitor. Without
alpha-1 proteinase inhibitor, elastase can destroy the air sacs of
the lung.
[0130] Thus, catalytic antagonists of this invention comprising an
alpha-1 proteinase binding moiety attached (e.g. the drug called
Prolastin) to, e.g. a protease, will degrade alpha-1 proteinase
affording similar or better therapeutic benefit.
[0131] Antibodies also provide useful targets for the catalytic
antagonists of this invention. Thus, for example, a catalytic
antagonist that targets and antagonizes (e.g. degrades) .alpha.-Gal
epitope specific antibodies is expected to significantly reduce an
immune response (e.g. to a xenotransplant). In one embodiment,
then, the .alpha.-Gal epitope can be used as a targeting moiety in
a chimera of this invention. It may be attached to a protease (e.g.
a subtilisin, a pepsin, etc.) and when it is bound by the antibody
it will degrade that antibody thereby inhibiting the
antibody-mediated immune response.
[0132] Similarly particularly where the xenotransplant is from a
different species, the catalytic antagonist can use as a targeting
moiety the MHC (or component thereof) of the xenotransplant. If the
enzymatic component is a hydrolase (e.g. a protease), the catalytic
inhibitor will specifically digest the receptor on effector cells
of the immune system (e.g. cytotoxic T lymphocytes (CTLs) only on
those cells specifically directed against the xenotransplant. The
catalytic antagonists will thus confer specific tolerance to the
xenograft without generally compromising the host immune
system.
[0133] Other antibodies that are good targets for the catalytic
inhibitors of this invention are antibodies produced in auto-immune
responses and/or other allergic responses. In these instances, the
targeting moiety is a molecule bearing an epitope recognized by the
antibodies mediating the autoimmune or allergic response.
Degradation of the antibody by, e.g. a hydrolase attached to the
targeting moiety will reduce the pathologic symptoms associated
with the autoimmune response. Specific allergens and substrates
recognized by antibodies in various autoimmune responses are well
known to those of skill in the art and can readily incorporated
into the chimeric molecules of this invention.
[0134] While many of the illustrations provided above are directed
to in vivo applications, the invention is not so limited. It is
well recognized that it is desirable to inhibit enzymes and/or
antibodies in a wide variety of ex vivo applications. These
include, but are not limited to various cell cultures, bioreactors
or fermentation systems, or various ex vivo synthetic processes
(e.g. laboratory processes and/or commercial processes),
antimicrobials/disinfectants, and the like.
[0135] C) Targeting Moieties for Lectins and Sugars.
[0136] In still other embodiments, targeting moieties are selected
that bind to particular lectins. Lectins are proteins obtained from
many plant, animal, and bacterial sources that have binding sites
for specific mono or oligosaccharides. Lectins such as concanavalin
A and wheat germ agglutinin are widely used as analytical and
preparative agents in the study of glycoproteins.
[0137] Lectins are also present on the surfaces of eukaryotic and
bacterial cells. In eukaryotic cells, lectins are often involved in
cell-cell interactions. In bacterial cells, lectins often mediate
adhesion of the bacterium to its target/host and, in many cases,
such adhesion is required for the bacterium to infect the host
cell.
[0138] In addition, bacterial adhesion and contamination of
non-biological surfaces are serious problems in the medical, dental
and food science fields. The most detrimental effects are
encountered in medicine, where the failure of implanted or
transdermal medical devices primarily results from
surface-associated bacterial infections. Bacterial
interaction/adhesion to a surface is often mediated by lectins
(often referred to as adhesins). Bacteria adhering onto a surface
frequently secrete an exopolysaccharide matrix in order to cement
themselves to the surface. This slimy layer of bacteria embedded in
a polysaccharide matrix is known as a biofilm. At the present time,
there is no cure for biofilm infections in vivo because the
bacteria are resistant to any anti-microbial or antibiotic
treatment.
[0139] Biofilm formation is also problematic in a wide variety of
commercial synthetic systems. Often fermentation vessels and other
bioreactors are contaminated by biofilms. Biofilms also growing in
and contaminate apparatus used for many chemical processes
particularly those involving "digestible" organic reagents. Thus,
biofilms often contaminate filters, conduits, separators and other
devices.
[0140] In certain embodiments, the catalytic antagonists of this
invention can be used to inhibit/degrade various lectins. Thus, for
example, a catalytic antagonist comprising a sugar or
oligosaccharide attached to a hydrolase will specifically target
and degrade a lectin that binds the target molecule. Such a
catalytic antagonist is illustrated in the examples.
[0141] Degradation of lectins/adhesins on a bacterial surface will
interfere with the bacteria's ability to bind to a surface and
thereby prevent the bacterium from entering a host cell or from
forming a biofilm.
[0142] In a related embodiment, it is known that many bacteria and
bacterial toxins bind to ganglioside, an acid glycosphingolipid and
invade host cells. The best known of these is the cholera toxin, an
enterotoxin produced by. Vibrio cholerae, which is known to bind
ganglioside GM1. Other ganglioside-binding bacterial toxins include
Tetanus toxin (GD1b), botulinum toxins (GT1b and GQ1b) and delta
toxin produced by Clostridium perfringens (GM2). Shiga toxin
produced by Shigella dysenteriae and Vero toxin produced by
enterohaemorrhagic E. coli bind to neutral glycosphingolipids
having an alpha-1,4 galabiose moiety in the sugar chain, such as
galabioside (Ga2Cer) and ceramide trihexoside (Gb3Cer).
[0143] Many pathogenic bacteria also bind to glycosphingolipids of
host cell surface for colonization and infection. Thus,
uropathogenic E. coli which cause urinary tract infections can bind
to glycosphingolipids having an alpha-1,4 galabiose moiety at the
non-reducing end of the sugar chain (Gb3Cer, etc). Uropathogenic E.
coli can also bind to globoside (Gb4Cer) and Forssman glycolipid,
both of which have an alpha-1,4 galabiose moiety internally in a
sugar chain. E. coli binds to glycosphingolipids by pili that exist
on the bacterial cell surface and are similar to fibers or hairs.
On the top of pili, there is an adhesin characterized as a lectin.
Several types of adhesin, with respect to their sugar specificity,
have been identified. They are the type I adhesin of E. coli that
are mannose specific, type P adhesin, also of E. coli, specific for
alpha-1,4 galabiose moiety, and type S adhesin of E. coli, specific
for sialylgalactose moiety. It was reported that the amino acid
sequence of P-adhesin is similar as that of Shiga toxin because
both recognize alpha-1,4 galabiose moiety of glycosphingolipid.
Propionibacterium, which causes skin disease, recognizes the
lactosyl moiety of glycosphingolipids as a binding epitope. These
bacteria can bind strongly to lactosylceramide and also bind to
isoreceptors such as asialo GM1 (GA1) and asialo GM2 (GA2). Because
almost all glycosphingolipids contain a common lactosyl moiety,
Propionibacterium may be assumed to bind almost all
glycosphingolipids. However, the bacteria cannot bind to any
glycosphingolipids composed of a dihydroxy base and nonhydroxy
fatty acid in ceramide, even though these contain a lactosyl
moiety. This fact indicates that the binding epitope of the
bacteria also depends on the ceramide structure in addition to the
lactosyl moiety in sugar chain. Neisseria gonorrhoeae, which cause
gonorrhoea, also bind glycosphingolipids having a lactosyl
moiety.
[0144] In view of the foregoing, catalytic antagonists having
targeting moieties that specifically bind various
glycosphingolipids and/or various adhesins (e.g. mannose specific
type I adhesin of E. coli, alpha-1,4 galabiose specific type P
adhesin of E. coli, and sialylgalactose type S adhesin of E. coli)
attached to enzymes that degrade the glycosphingolipids (e.g.
glycosidases, cerebrosidases, etc.) will act to prevent bacterial
infections and are expected to provide effective therapeutics to
block acute effects of bacterial-produced toxins (e.g. cholera
toxin).
[0145] Lectins, particularly membrane glycoproteins, are also
implicated in various inflammatory processes (e.g. inflammatory
processes associated with rheumatoid arthritis, arthritis, septic
shock, myocardial infarction., etc.). For example Lec-CAMs or
selectins are expressed on the surfaces of endothelium, leukocytes
and platelets and influence leukocyte-endothelial adhesion at sites
of inflammation. GMP-140 (P-selectin) stored in Weibel-Palade
bodies of endothelial cells and a platelet granules, when
stimulated by TNF-.alpha.IL-1 is transported within minutes to the
cell surface and participates in interactions between endothelium,
platelets, neutrophils. ELAM-1 (E-selectin) is synthesized de novo
by stimulated endothelium e.g. by TNF-.alpha./IL-1, and enhances
later recruitment of leukocytes. LAM-1 (L-selectin) regulates
lymphocyte binding to high endothelial lymph node venules, the
surface of neutrophils and lymphocytes to localize these cells to
injury.
[0146] In addition, the integrins, a family of adhesion molecules
composed of heterodimers of .alpha. and .beta. subunits; act in
regulation of cell-matrix and cell-cell adhesion. These molecules
are transmembrane in structure, thus linking or "integrating"
exterior/surface stimuli to the internal cell cytoskeleton. .beta.2
integrins: also known as CD11/CD18 molecules confer adhesion
specificity, mediate activation of phagocytic cells by chemotactic
stimuli.
[0147] The surface expression of integrins e.g. MO-1, leukocyte
function antigen-1 (LFA-1) and gp150,95; assist in localization of
phagocytes to injury sites; deficiency states result in enhanced
susceptibility to bacterial infection.
[0148] The intercellular adhesion molecule-1 (ICAM-1): assists in
localization of leukocytes to tissue injury; expressed on surface
of cytokine stimulated endothelium and leukocytes; binds to LFA-1
and MO-1 present on cell membranes of neutrophils and macrophages.
The vascular cell adhesion molecule-1 (VCAM-1): binds VLA-4
leukocyte receptor on lymphocytes, monocytes, eosinophils,
basophils.
[0149] All of these molecules also offer suitable targets for the
catalytic antagonists of this invention. The forgoing illustrations
of lectin-directed catalytic antagonists of this invention are
intended to be illustrative and not limiting. Numerous other lectin
targets will be known to those of skill in the art.
[0150] It will be appreciated that the target and targeting
moieties described herein (and others) can be reversed. Thus,
instead of a sugar, the targeting moiety can be a lectin that will
specifically direct the catalytic antagonist (or redirected enzyme)
to a sugar or sugar-bearing target. Thus, the molecules can be
directed to the sugars present on and characteristic of particular
bacteria. In one embodiment, the sugar-targeted catalytic
antagonist will make an effective microbicide.
[0151] As indicated above, such molecules can be targeted by using
simple sugars, or oligosaccharides and the like as targeting
moieties in the chimeric molecules of this invention. In addition,
dendrimers can also be used as targeting moieties. Thus, multiple
functionalization of the enzyme (e.g. either a catalytic antagonist
or a redirected enzyme) can be achieved using dendrimeric targeting
moieties whereby multiple branched linking structures can be
employed to create a polyfunctionalized enzyme (chimeric
molecule).
[0152] For instance, multiple glycosylation, including multiple
mannose-containing chimeras and varied sugar moieties can be
created. This could confer the benefit of increased affinity for,
and increased binding affinity between, lectins and the targeted
enzyme (e.g. a hydrolase). This would also permit multiple
concurrent targeting of sites, for instance by incorporating
multiple biotin molecules into a targeting moiety that would elicit
multiple concurrent biotin-avidin interactions. The dendrimer
targeting moieties (before coupling to the enzyme) would preferably
include methanethiosulfonates with simple branching such as: 1
[0153] derived from pentaerythritol, to very complex branched
dendrimer reagents as illustrated in FIG. 1. Highly branched
molecules or dendrimers were first synthesized by Vogtle in 1978
(Buhleier et al (1978) Synthesis, 178). The attachment of identical
building blocks that contain branching sites to a central core may
be achieved with a high degree of homogeneity and control Each
branch contains a functional group which, after chemical
alteration, may be connected to yet another branching building
block. In this manner, layer after layer of branching rapidly
generates highly functionalized molecules (Bosman et al (1999)
Chem. Rev., 99: 1665-1688).
[0154] D) Targeting other "Miscellaneous Ligands.
[0155] Using the teaching provided herein, a wide variety of other
moieties for targeting by the chimeric molecules of this invention
will be apparent to those of skill in the art. For example, the
redirected enzymes describe herein can be used in a variety of drug
delivery strategies. The targeting moiety can be directed to
specifically bind to a particular cell type or tissue (e.g. a tumor
cell). The enzymatic component can be selected for an activity that
converts a (e.g. non-toxic prodrug) to an active form (e.g. a
cytotoxin). The retargeted enzyme of this invention thus localizes
the activity of the prodrug/drug to the cells bound by the chimeric
molecule. Numerous cell-specific markers are known to those of
skill in the art. These include, but are not limited to the LewisY
antigen (tumor cells), the G250 antigen (renal cell carcinoma
cells), the IL-13 receptor (tumor cells) and the like. One example
of a suitable enzyme for use in this application is a thymidine
kinase (e.g., Herpes simplex thymidine kinase (HSVTK) or Varicella
zoster thymidine kinase (VZVTK)). thymidine kinase assists in
metabolizing antiviral nucleoside analogues to their active form
are therefore useful in activating nucleoside analogue precursors
(e.g., AZT or ddC) into their active form. In addition,
tk-containing cells are killed when contacted with ganclovir.
[0156] Thus, in one embodiment, the redirected enzyme of this
invention can be a thymidine kinase targeted, for example to a cell
expressing a CCR5 and/or a CCR3 receptor (and hence likely to be
susceptible to HIV infection). The tk re-directed to these cells
can activate AZT or ddC precursors into their active form.
[0157] In another embodiment, the tk enzyme can be directed to a
tumor cell (e.g. via a tumor specific antigen). Treatment with
ganclovir then results in death of the tumor cell.
[0158] Other examples of prodrugs that can be converted to their
active form using the redirected enzymes of this invention include,
but are not limited to prodrugs of 5-FU or inhibitors of
dihydropyrimidine dehydrogenase (DPD) (GW 776C85).
[0159] Still another example is the prodrug phosphenytoin, a
relatively soluble prodrug that is converted by phosphatase to
relatively insoluble phenytoin an active. anticonvulsant.
Similarly, depivefrin is converted by esterase to epinephrine, an
adrenergic, useful in the treatment of glaucoma.
[0160] The re-directed enzymes of this invention can also act as
"self-protected" polypeptides particularly when utilized as in vivo
therapeutics. In such an embodiment, the organic molecule (e.g. the
targeting moiety) component of the chimeric molecule can sterically
shield and protect the effector (enzyme) component of the chimeric
molecule. The idea of this is that bulky groups attached near to
key positions on the chimeric molecule would hinder the attack of
another reagent on, e.g. cleavage sites in the remainder of the
chimera and therefore prolong its lifetime. For example, sugars on
proteins increase their stability to proteinases, i.e., the
proteinase can't get in to cleave its preferred amide bond because
a sugar is blocking it (see, e.g., Rudd et al. (1994) Biochemistry,
33: 17-22). In certain embodiments, the organic molecule can
perform "double duty" providing both a targeting functionality as
well as a protective function.
[0161] In still another embodiment, the re-directed enzymes of this
invention can be utilized in enzyme replacement therapy, particular
in the treatment of storage diseases. Storage diseases are caused
by the increased accumulation of metabolic products (e.g., lipids,
proteins, and complex carbohydrates) due to either the inactivity
of an enzyme that degrades the products or the hyperactivity of an
enzyme that creates the products. Storage disease include but are
not limited to glycogen storage disease I, GM1 gangliosidoses, MPS
IV B (Morquio B), GM2 gangliosidoses (O, B, AB, B1 variants),
Niemann-Pick disease (A, B, and C), Metachromatic leukodystrophy
(arylsulfatase A and SAP-1 deficient), Krabbe disease, Fabry
disease, Gaucher disease, Farber disease, Wolman disease
(cholesterol ester storage disease), MPS I (Hurler and Scheie
syndromes), MPS II (Hunter syndrome), MPS III A, C, and D
(Sanfilippo A, C, and D), PS III B (Sanfilippo B), MPS IV A
(Morquio A), MPS VI (Maroteaux-Lamy syndrome), MPS VII
(beta-glucuronidase deficiency), Multiple sulfatase deficiency,
Mucolipidosis I (Sialidosis), Mucolipidosis II & III,
alpha-Mannosidosis, beta-Mannosidosis, Fucosidosis, Sialic acid
storage disease, Galactosialidosis, Aspartylglucosaminuria
Cystinosis. Storage diseases can be treated by supplementing the
"missing" enzymatic activity.
[0162] For example, Gaucher's disease can be treated by use of a
glucocerebrosidase targeted to spleen cells. Similarly, superoxide
dismutase can be targeted to the liver as an anti-oxidant, and so
forth.
III. Selection of Enzymes (Effector Molecules)
[0163] Virtually any enzyme can be utilized in the chimeric
molecules of this invention. Where the chimeric molecule is a
catalytic antagonist, enzymes are selected that are capable of
degrading the substrate specifically bound by the targeting moiety.
Such enzymes include, but are not limited to proteases, cellulases,
nucleases (exo- and endo-), amylases, lipases, aldolases,
ketolases, glycosidases, and the like.
[0164] Where the chimeric molecule is an enzyme whose activity is
directed to a new location and/or substrate, the enzyme can be ,
but need not necessarily be an enzyme that degrades its substrate.
Thus, in addition to proteases, cellulases, nucleases (exo- and
endo-), amylases, lipases, aldolases, ketolases, glycosidases, and
the like, the redirected enzymes can also include enzymes such as
isomerases, oxidases, oxidoreductases, ligases, transferases, and
the like.
[0165] Preferred enzymes for use in the catalytic antagonists of
this invention are the hydrolases. Particularly preferred enzymes
for use in the catalytic antagonists of this invention are the
serine hydrolases. The serine hydrolases are a class of hydrolytic
enzymes characterized by a hydrolytic enzymes that posses a
catalytic triad composed of a serine, histidine and a carboxylate
amino acid (either aspartic or glutamic acid), and which catalyze
the hydrolysis, and microscopic reverse reactions thereof, of
carboxylic acid derivatives including, but not restricted to,
esters, peptides and amides.
[0166] Preferred serine hydrolases comprising this invention
include the trypsin-chymotrypsin proteases, the subtilisin
proteases, and the alpha/beta hydrolases. In a particularly
preferred embodiment the enzyme is protease, more preferably a
subtilisin (e.g. a Bacillus lentis subtilisin). Subtilisin is a
serine endoprotease (MW .about.27,500) which is secreted in large
amounts from a wide variety of Bacillus species. The protein
sequence of subtilisin has been determined from at least four
different species of Bacillus (see, e.g., Markland et al. (1971)
pages 561-608 In: The Enzymes, ed. Boyer P. D., Acad Press, New
York, Vol. III, pp.; Nedkov et al. (1983) Hoppe-Seyler's Z.
Physiol. Chem. 364: 1537-1540). The three-dimensional
crystallographic structure of subtilisin BPN' (from B.
amyloligoefaciens) to 2.5 .ANG. resolution has also been reported
(Wright et al. (1969) Nature 221, 235-242; Drenth et al. (1972)
Eur. J. Biochem. 26: 177-181. These studies indicate that although
subtilisin is genetically unrelated to the mammalian serine
proteases, it has a similar active site structure. The x-ray
crystal structures of subtilisin containing covalently bound
peptide inhibitors (Robertus, et al. (1972) Biochemistry 11:
2439-2449), product complexes (Robertus et al. (1972) Biochemistry
11: 4293-4303), and transition state analogs (Matthews et al (1975)
J. Biol. Chem. 250: 7120-7126; Poulos et al. (1976) J. Biol. Chem.
251, 1097-1103), which have been reported have also provided
information regarding the active site and putative substrate
binding cleft of subtilisin. In addition, a large number of kinetic
and chemical modification studies have been reported for subtilisin
(Philipp et al. (1983) Mol. Cell. Biochem. 51:5-32; Svendsen (1976)
Carlsbera Res. Comm. 41: 237-291; Markland, Id.) as well as at
least one report wherein the side chain of methionine at residue
222 of subtilisin was converted by hydrogen peroxide to
methionine-sulfoxide (Stauffer et al. (1965) J. Biol. Chem. 244:
5333-5338).
[0167] Other particularly preferred hydrolases for use in this
invention include, but are not limited to .alpha./.beta.
hydrolases, trypsin/chymotryspsin families of serine hydrolase
enzymes, aspartyl proteases, cysteine proteases, metalloproteases,
lysozymes and other glycosidases etc.
IV. Construction of Chimeric Molecules
[0168] In preferred embodiments, the chimeric catalytic antagonists
and/or redirected enzymes of this invention are made by chemically
conjugating the desired enzyme (directly or through a linker) to
the targeting moiety. While many strategies are known for preparing
chemically conjugated chimeric molecules (see, e.g., European
Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958,
4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; 4,589,071;
4,545,985 and 4,894,443; Borlinghaus et al. (1987) Cancer Res. 47:
40714075; Thorpe et al. (1991) Monoclonal Antibody-Toxin
Conjugates: Aiming the Magic Bullet, Thorpe et al., (1982)
Monoclonal Antibodies in Clinical Medicine, Academic Press, pp.
168-190; Waldmann (1991) Science, 252: 1657, and the like), in a
preferred embodiment, the targeting moiety is
derivatized/functionalized with a reactive group that can react
with an available R group (e.g. NH.sub.2, N, NH, OH, COOH, SH,
etc.) on an amino acid residue comprising the enzyme. In
particularly preferred embodiments, the targeting moiety is
derivatized as a methanethiosulfonate reagent that can then react
with the --SH in a cysteine to provide the targeting moiety coupled
in place of the thiol hydrogen on the cysteine. The coupling can be
direct or through a linker.
[0169] In certain embodiments, the cysteine to which the targeting
moiety is attached is a native cysteine in the enzyme, however, in
preferred embodiments the cysteine is a cysteine substituted for a
different native amino acid residue in the enzyme. The enzyme so
modified is, optionally, referred to as a mutant enzyme. Chimeric
molecules of this invention in which the targeting molecule is
chemically coupled to a mutant enzyme are, optionally, referred to
as Chemically Modified Mutants (CMMs).
[0170] Typically, once the targeting moiety and the enzyme that are
to be coupled are selected, e.g., as described above, the location
(residue) in the enzyme for attachment of the targeting moiety is
identified. Where this residue is not already a cysteine, a
cysteine is substituted for the native residue. The targeting
moiety, or a linker attached thereto, is derivatized as a
methanethiosulfonate which can then be reacted with the cysteines
--SH group as described herein. Detailed protocols for the
preparation of mutant enzymes and the coupling of a targeting
moiety are provided below and in the examples.
[0171] A) Production of Mutant Enzymes for Chemical
Modification.
[0172] 1) Selection of Residues for Modification.
[0173] In general, virtually any residue of the enzyme can be
selected for mutagenesis (e.g. substitution of a cysteine) and
chemical modification to introduce a targeting moiety, as long as
the modification retains the desired level of activity of the
subject enzyme. Typically this is accomplished by making the
substitution at a location that does not block critical substrate
interactions or drastically alter folding/conformation of the
subject enzyme.
[0174] Suitable sites for introduction of a targeting moiety can be
determined by substituting cysteine, and optionally an attached
targeting moiety, and assaying the enzymes for the desired
activity. With the current advances in combinatorial chemistry and
high throughput screening systems such modifications and screening
can be accomplished with only routine experimentation.
[0175] In a preferred embodiment, however, residues for
modification/substitution in the enzyme (e.g. serine hydrolase) are
rationally selected. Preferred sites include sites not in critical
conformation determining regions and sites disposed away from the
subsite(s) of the enzyme. However, in other preferred embodiments,
particularly where it is desired to enhance, or otherwise alter,
substrate specificity and/or activity, preferred amino acid
residues selected for modification include residues expected to be
important discriminatory sites near, adjacent to or within the
substrate binding region of the enzyme. Such residues are
determined from mutagenesis experiments where the subsite residues
are systematically mutagenized and the effect of such mutagenesis
on binding specificity and/or enzymatic activity is determined. In
addition, important residues can be identified from inspection of
crystal structures of the enzyme alone or in complex with subtrate,
substrate analogues or inhibitors and/or from predicted protein
folding or protein-protein interactions determined using protein
modeling software (e.g., Quanta, Cerius, Insight (Molecular
Simulations Inc.) and Frodo (academic software). Side chains
situated to alter interaction at subsites defined by Berger and
Schechter can be selected based on the crystallographic models of
the enzymes and extrapolated to homologous enzymes if necessary if
structural information on a specific enzyme is unavailable. In B.
lentus subtilisin sites 62, 156, 166, 217 and 222 are important
substrate specificity determining sites. Additional related sites
include position 96, 104, 107, 189 and 209 in subtilisin and
homologous positions in related enzymes. In preferred embodiments,
such residues typically lie in the S1, S2, S4, S1', S2', or S3'
subsites although it will be appreciated that in certain cases,
alteration of residues in other subsites can also produce dramatic
effects.
[0176] In one particularly preferred embodiment, where the serine
hydrolase is a subtilisin-type serine hydrolase, preferred residues
for mutation include, but are not limited to residues at or near
residues 156 and 166 in the S1 subsite, residues 217 and 222 in the
S1' subsite, residue 62 in the S2 subsite, and Leu96, Val104,
Ile107, Phe189 and Tyr209 or residues at or near homologous
positions other subtilisin-type serine proteases (preferably
positions within subsites).
[0177] In another preferred embodiment, where the serine hydrolase
is a trypsin-chymotrypsin type serine hydrolase, preferred residues
for mutation include, but are not limited to, residues at or near
residues Tyr94, Leu99, Gln175, Asp189, Ser190, Gln192, Leu111,
Phe175, Tyr176, Ser182, Leu184, Phe189, Tyr214, Asp231, Lys234, and
Ile243 of trypsin (Protein Databank Entry 1TPP) or residues at or
near homologous positions of other chymotrypsin-type
(trypsin-chymotrypsin-type) serine proteases (preferably positions
within subsites).
[0178] In still another preferred embodiment, where the serine
hydrolase is an alpha/beta serine hydrolase, preferred residues for
mutation include, but are not limited to, residues at or near the
following residues: Trp104, Thr138, Leu144, Val154, Ile189, Ala
225, Leu278 and Ile185, where these are residues of Candida
antartica lipase (Protein Data Bank entry 1tca) or residues at
homologous positions of other alpha/beta type serine hydrolases
(preferably positions within subsites).
[0179] Preferably the amino acids replaced in the enzyme by
cysteines are selected from the group consisting of asparagine,
leucine, methionine, or serine. More preferably the amino acid to
be replaced is located in or near a subsite of the enzyme
preferably the S1, S1' or S2 subsites. More preferably, in a
subtilisin the amino acids to be replaced are N62, L217, M222,
S156,S166, site 104, site 107 (S4), site 96 (S2), site 189(S2') and
site 209 (S1'/S3') or their homologues where the numbered position
corresponds to naturally occurring subtilisin from Bacilus
amyloliquefaciens or to equivalent amino acid residues in other
subtilisins such as Bacillus lentus subtilisin.
[0180] The chimeric molecules of this invention are not limited to
serine hydrolases. In addition to other enzymes, in particularly
preferred embodiments, this invention includes other chimeric
proteases. Such proteases include, but are not limited to aspartyl
proteases, cysteine proteases, metalloproteases, and the like.
[0181] Where the protease is aspartyl protease such as pepsin,
preferred residues for mutation include, but are not limited to,
amino acid(s) corresponding (e.g. at a homologous position) to a
residue at or near the following residues Tyr9, Met12, Gln13,
Gly76, Thr77, Phe111, Phe 117, Ser127, Ile 128, Ser130, Tyr189,
Ile213, Glu239, Met245, Gln 287, Met 289, Asn290, Leu291, and
Glu294, where these "reference" residues are residues in the mature
human pepsin (Protein Data Bank entry 1PSN).
[0182] Where the protease is cysteine protease, preferred residues
for mutation include, but are not limited to, amino acid(s)
corresponding (e.g. at a homologous position) to a residue at or
near the following residues Asn18, Ser21, Asn64, Tyr67, Trp69,
Gln112, Gln142. Asp158, Trp177, and Phe207, where these reference
residues are residues in the mature papain (Protein Data Bank entry
1BQI).
[0183] Where the protease is metalloprotease, preferred residues
for mutation include, but are not limited to, amino acid(s)
corresponding (e.g. at a homologous position) to a residue at or
near the following residues Leu111, Phe175, Tyr176, Ser182, Leu184,
Phe189, Tyr214, Asp231, Lys234, and Ile243, where these reference
residues are residues in the mature human matrix metalloprotease
(Protein Data Bank entry 830C).
[0184] 2) Introduction of Cysteine.
[0185] The substitution of a cysteine for one or more native
residue(s) in the enzyme (e.g. serine hydrolase) can be
accomplished using routine methods well known to those of ordinary
skill in the art. In one preferred embodiment, the mutants
described herein are most efficiently prepared by site-directed
mutagenesis of the DNA encoding the wild-type enzyme of interest
(e.g. Bacillus lentis subtilisin). Techniques for performing
site-directed mutagenesis or non-random mutagenesis are known in
the art. Such methods include, but are not limited to alanine
scanning mutagenesis (Cunningham and Wells (1989) Science, 244,
1081-1085), oligonucleotide-mediated mutagenesis (Adellman et al.
(1983) DNA, 2, 183), cassette mutagenesis (Wells et al. (1985)
Gene, 344: 315) and binding mutagenesis (Ladner et al. WO
88/06630).
[0186] In one embodiment of the present invention, the substitute
amino acid residue (e.g. cysteine) is introduced into the selected
position by oligonucleotide-mediated mutagenesis using the
polymerase chain reaction technique. In this approach, the gene
encoding the desired native enzyme (e.g. subtilisin) is carried by
a suitable plasmid. More preferably, the plasmid is an expression
vector, e.g., a plasmid from the pBR, pUC, pUB, pET or pHY4 series.
The plasmid can be chosen by persons skilled in the art for
convenience or as desired.
[0187] For site-directed mutagenesis, the fragment containing the
selected mutation site is cleaved from the gene encoding the
subject enzyme by restriction endonucleases and is used as the
template in a modified PCR technique (see, Higuchi et al. (1988)
Nucleic Acid Res., 16, 7351-7367). For each target substitution, an
oligonucleotide containing the desired mutation is used as a
mismatch primer to initiate chain extension between 5' and 3 PCR
flanking primers. The process includes two PCR reactions. In the
first PCR, the mismatch primer and the 5' primer are used to
generate a DNA fragment containing the desired base substitution.
The fragment is separated from the primers by electrophoresis.
After purification, it is then used as the new 5' primer in a
second PCR with the 3' primer to generate the complete fragment
containing the desired base substitution. After confirmation of the
mutation by sequencing, the mutant fragment is then inserted back
to the position of the original fragment.
[0188] In another approach, a cassette mutagenesis method may be
used to facilitate the construction and identification of the
cysteine mutants of the present invention. First, the gene encoding
the serine hydrolase is obtained and sequenced in whole or in part.
Then the point(s) at which it is desired to make a mutation of one
or more amino acids in the expressed enzymes is identified. The
sequences flanking these points are evaluated for the presence of
restriction sites for replacing a short segment of the gene with an
oligonucleotide which when expressed will encode the desired
mutants. Such restriction sites are preferably unique sites within
the serine hydrolase gene so as to facilitate the replacement of
the gene segment. However, any convenient restriction site which is
not overly redundant in the hydrolase gene may be used, provided
the gene fragments generated by restriction digestion can be
reassembled in proper sequence. If restriction sites are not
present at locations within a convenient distance from the selected
point (e.g., from 10 to 15 nucleotides), such sites are generated
by substituting nucleotides in the gene in such a fashion that
neither the reading frame nor the amino acids encoded are changed
in the final construction. The task of locating suitable flanking
regions and evaluating the needed changes to arrive at two
convenient restriction site sequences is made routine by the
redundancy of the genetic code, a restriction enzyme map of the
gene and the large number of different restriction enzymes. If
convenient flanking restriction site is available, the above method
need be used only in connection with the flanking region which does
not contain a site.
[0189] Mutation of the gene in order to change its sequence to
conform to the desired sequence is accomplished e.g., M13 primer
extension in accord with generally known methods. Once the gene is
cloned, the restriction sites flanking the sequence to be mutated
are digested with the cognate restriction enzymes and the end
termini-complementary oligonucleotide cassette(s) are ligated into
the gene. The mutagenesis is enormously simplified by this method
because all of the oligonucleotides can be synthesized so as to
have the same restriction sites, and no synthetic linkers are
necessary to create the restriction sites.
[0190] A suitable DNA sequence computer search program simplifies
the task of finding potential 5' and 3' convenient flanking sites.
In preferred embodiments, any mutation introduced in creation of
the restriction site(s) are silent to the final construction amino
acid coding sequence. For a candidate restriction site 5' to the
target codon a sequence preferably exists in the gene that contains
at least all the nucleotides but for one in the recognition
sequence 5' to the cut of the candidate enzyme. For example, the
blunt cutting enzyme SmaI (CCC/GGG) would be a good 5' candidate if
a nearby 5' sequence contained NCC, CNC, or CCN. Furthermore, if N
needs to be altered to C this alteration preferably leaves the
amino acid coding sequence intact. In cases where a permanent
silent mutation is necessary to introduce a restriction site one
may want to avoid the introduction of a rarely used codon. A
similar situation of SmaI would apply for 3' flanking sites except
the sequence NGG, GNG, or GGN must exist. The criteria for locating
candidate enzymes are most relaxed for blunt cutting enzymes and
most stringent for 4 base overhang enzymes. In general many
candidate sites are available.
[0191] A particularly preferred of method of introducing cysteine
mutants into the enzyme of interest is illustrated with respect to
the subtilisin gene from Bacillus lentus ("SBL"). In a preferred
embodiment, the gene for SBL is cloned into a bacteriophage vector
(e.g. M13mp19 vector) for mutagenesis (see, e.g. U.S. Pat. No.
5,185,258). Oligonucleotide-directed mutagenesis is performed
according to the method described by Zoller et al. (1983) Meth.
Enzymol., 100: 468-500. The mutated sequence is then cloned,
excised, and reintroduced into an expression plasmid (e.g. plasmid
GG274) in the B. subtilis host. PEG (50%) is added as a
stabilizer.
[0192] The crude protein concentrate thus obtained is purified by
first passing through a Sephadex.TM. G-25 desalting matrix with a
pH 5.2 buffer (e.g. 20 mM sodium acetate, 5 mM CaCl.sub.2) to
remove small molecular weight contaminants. Pooled fractions from
the desalting column are then applied to a strong cation exchange
column (e.g. SP Sepharose.TM. FF) in the sodium acetate buffer
described above and the SBL is eluted with a one step gradient of
0-200 mM NaCl acetate buffer, pH 5.2. Salt-free enzyme powder is
obtained following dialysis of the eluent against Millipore
purified water and subsequent lyophilization.
[0193] The purity of the mutant and wild-type enzymes, which are
denatured by incubation with a 0.1 M HCl at 0.degree. C. for 30
minutes is ascertained by SDS-PAGE on homogeneous gels (e.g. using
the Phast.TM. system from Pharmacia, Uppsala, Sweden). The
concentration of SBL is determined using the Bio-Rad (Hercules,
Calif.) dye reagent kit which is based on the method of Bradford
(1976) Anal. Biochem., 72: 248-254). Specific activity of the
enzymes is determined as described below and in the examples.
[0194] One of ordinary skill in the art will appreciate that the
protocol described above can be routinely modified, if necessary,
for use with other enzymes. Other protocols for site-directed
modification of proteins are well know to those of skill in the art
and can be found, for example, in U.S. Pat. Nos. 5,932,419 and
5,789,166, 5,705,479, 5,635,475, 5,556,747, 5,354,670, 5,352,779,
5,284,760, and 5,071,743.
[0195] In addition, kits for site-directed mutagenesis are
commercially available (see, e.g. Transfomer.TM. Site-Directed
Mutagenesis Kit available from Toyobo).
[0196] 3) Optimization of Coupling Site.
[0197] A number of particularly preferred sites for introduction of
the cysteine and coupling the targeting moiety are indicated
herein. The positions are indicated with respect to a "reference"
enzyme and "homologous" sites in related enzymes in the same family
can be determined, e.g. as described herein. It may, however, be
desired to optimize, coupling sites for a particular enzyme,
targeting moiety combination.
[0198] Because this invention utilizes chemically coupled targeting
moieties this can be accomplished with relative ease and, at most,
routine experimentation. Cysteines can be introduced, e.g. into
positions near the reference site of interest, and then the
targeting moiety can be readily conjugated as described herein. The
resulting chimera can then be tested for the desired activity.
[0199] The entire protein need not be re-engineered for each
variation and, because particularly preferred sites are already
taught herein, only a relatively few positions need be explored to
optimize any particular chimeric molecule.
[0200] 4) Other Coupling Strategies.
[0201] In preferred embodiments, chemical coupling of the targeting
moiety is to a cysteine, either naturally occurring in the subject
enzyme or introduced (e.g. via site-directed mutagenesis. The
chimeric molecules of this invention, however, need not be limited
to molecules conjugated through cysteines. In certain embodiments
the conjugation can be through virtually any other amino acid
(e.g., a serine, a glycine, a tyrosine, etc.). The conjugation can
be through the existing R group (using other coupling chemistries),
or alternatively a sulfhydryl group (SH) can be introduced (linked)
to the R group and the targeting moiety, derivatized as a
methanethiosulfonate reagent, can be coupled, e.g. as illustrated
in the examples.
[0202] 5) Expression of the Mutated Enzyme.
[0203] In a preferred embodiment, the mutated enzyme is expressed
from a heterologous nucleic acid in a host cell. The expressed
enzyme is then isolated and, if necessary, purified. The choice of
host cell and expression vectors will to a large extent depend upon
the enzyme of choice and its source.
[0204] A useful expression vector contains an element that permits
stable integration of the vector into the host cell genome or
autonomous replication of the vector in a host cell independent of
the genome of the host cell, and preferably one or more phenotypic
markers that permit easy selection of transformed host cells. The
expression vector may also include control sequences encoding a
promoter, ribosome binding site, translation initiation signal,
and, optionally, a repressor gene, a selectable marker or various
activator genes. To permit the secretion of the expressed protein,
nucleotides encoding a signal sequence may be inserted prior to the
coding sequence of the gene. For expression under the direction of
control sequences, a gene or cDNA encoding a mutated enzyme to be
used according to the invention is operably linked to the control
sequences in the proper reading frame.
[0205] Vectors containing the mutant genes obtained by
site-directed mutagenesis are then used respectively to transform
suitable host cells and expressed. Suitable host cells include
bacteria such as E. coli or Bacillus, yeast such as S. cerevisiae,
mammalian cells such as mouse fibroblast cell, or insect cells.
Preferably, a bacterial expression system is used. Most preferably,
the host is Bacillus. Protein expression is performed by processes
well known in the art according to factors such as the selected
host cell and the expression vector to culture the transformed host
cell under conditions favorable for a high-level expression of the
foreign plasmid.
[0206] Methods of cloning and expression of peptides are well known
to those of skill in the art. See, for example, Sambrook, et al.
(1989) Molecular Cloning: a Laboratory Manual (2nd Ed., Vols. 1-3,
Cold Spring Harbor Laboratory), Berger and Kimmel (1987) Methods in
Enzymology, Vol. 152: Guide to Molecular Cloning Techniques,
Academic Press, Inc. San Diego, or Ausubel et al. (1987) Current
Protocols in Molecular Biology, Greene Publishing and
Wiley-Interscience, New York.
[0207] As indicated above, one particularly preferred expression
system is plasmid GG274 which is then expressed in a B. subtilis
host.
[0208] B) Coupling the Targeting Moiety to the Enzyme.
[0209] 1) Selection of Substitutents for Modifying Mutated
Residues.
[0210] A wide variety of targeting moieties can be coupled to the
cysteine(s) introduced into the subject enzyme (e.g. serine
hydrolase). As indicated above, the targeting moiety is selected
depending on the desired use of the enzyme. As further indicated
above, suitable targeting moieties include, but are not limited to,
moieties that are bound by receptors, targeting moieties that are
bound by antibodies and enzymes, targeting moieties that are bound
by lectins, and various other targeting moieties. In certain
particularly preferred embodiments, the targeting moieties are
drugs or prodrugs that are specifically bound by a receptor and/or
an enzyme.
[0211] 2) Coupling Targeting Moieties to the Cysteine.
[0212] The R group on cysteines provides a convenient relatively
reactive thiol group (--SH) that can be exploited for coupling a
desired targeting moiety to the cysteine. In a preferred
embodiment, the targeting moiety of interest is provided,
derivatized as a methanethiosulfonate reagent which, when reacted
with the cysteine, results in the substituent of interest
covalently coupled to the cysteine by a disulfide linkage
(--S--S--).
[0213] In a preferred embodiment, chemical modification with the
methanethiosulfonate reagent(s) is carried out as described by
Berglund et al. (1997) J. Am. Chem. Soc., 119: 5265-5255 and
DeSantis et al. (1998) Biochemistry, 37: 5968-5973. Briefly, 200
.mu.L of a 1 M solution of the methanethiosulfonate (MTS) reagent
is added to a solution (5-10 mg/mL, 3.5 mL) of the cysteine mutant
in 70 mM CHES, 5 mM MES, 2 mM CaCl.sub.2, pH 9.5. The MTS reagent
is added in two portions over 30 minutes. Reaction mixtures are
kept at 20.degree. C. with continuous end-over-end mixing.
Reactions are monitored by following the specific activity (e.g.
with suc-AAPF-pNA) and by tests for residual free thiol (e.g. with
Ellman's reagent). Once the reaction is complete, the reaction
mixture is loaded on a Sephadex.TM. PD-10 G25 column with 5 mM MES
and 2 mM CaCl2, pH 6.5. The protein fraction is then dialyzed
against 1 mM CaCl2 and the dialysate is lyophilized. In a
particulary preferred embodiment the fraction is dialyzed against
pH 6.5 MES then flash frozen.
[0214] In certain instances, where the targeting moiety that is to
be coupled to the cysteine, bears reactive groups the reactive
groups may be derivatized with, appropriate blocking/protecting
groups to prevent undesired reactions during the coupling.
Similarly, where the serine hydrolase contains one or more
cysteines that are not to be derivatized, the cysteines may be
replaced with other amino acids (e.g. via site directed
mutagenesis) and/or the thiol group(s) on these cysteines may be
derivatized with appropriate protecting groups (e.g. (e.g. benzyl,
trityl, tert-butyl, MOM, acetyl, thiocarbonate, thiocarbamate, and
others). The use of blocking/protecting groups is well know to
those of skill in the art (see, e.g., Protective Groups in Organic
Synthesis" Theodora W. Greene and Peter G. M. Wuts Third Edition,
Wiley-Interscience, Toronto, (1999), pp 454-493.)
[0215] While in particularly preferred embodiments, a cysteine is
introduced/substituted into the enzyme, in certain embodiments,
other amino acids (e.g. lysine, histadine, etc.) may be introduced,
and in certain embodiments, the targeting moiety may be coupled to
these residues.
[0216] Derivatization of a number of targeting moieties and their
coupling to mutant enzymes is illustrated in the examples provided
herein.
[0217] C) Screening Chemically Chimeric Colecules for Desired
Activity.
[0218] The chimeric molecules of this invention are typically
screened for the activity or activities of interest. The activity
of interest depends on the desired use of the chimeric molecule.
Thus, for example, in the case of catalytic antagonists of this
invention, the chimeric molecule may be assayed for two properties:
1) The ability to reduce or eliminate the activity of the target,
e.g., where the target is biologically active, or simply to
partially or fully degrade the target, e.g. where the target is not
biologically active; and 2) the ability to release from the target
after the target is degraded and to bind and degrade another
target. Alternatively, the chimieric molecule may simply be assayed
for activity (e.g. the ability to perform degradations) in a
substoichiometric manner.
[0219] The details of the particular assay, will vary with the
target of the chimeric molecule. Many assays for the degradation of
various molecules (e.g. proteins, carbohydrates, nucleic acids,
etc.) and/or the inhibition of various receptors and/or antibodies
are well known to those of skill in the art. For example, in one
embodiment, activity of a molecule on a cell surface receptor can
be determined by providing a cell expressing the receptor and
measuring the activity of the receptor in the presence or absence
of the chimeric molecule. Receptor assays are commonly performed in
oocytes (e.g. Xenopus oocytes) into which an RNA encoding the
subject receptor is inserted. Receptor activity is monitored by
measuring electrochemical activity (e.g. via patch clamps, etc.),
uptake of receptor substrates, and the like. Such methods are well
known to those of skill in the art and described in detail, for
example, Racke et al. (1993) FEBS Letters 333(1,2):132-136. Assays
for ligand binding, alteration of enzyme activity, and the like are
also well known to those of skill in the art. In addition, a number
of suitable assays are provided in the examples.
V. Illustrative Uses of Catalytic Antagonists and/or Redirected
Enzymes
[0220] From the foregoing discussion myriad applications/uses of
the chimeric molecules of this invention will be apparent to one of
ordinary skill in the art. Moreover, a number of specific
embodiments and applications are described in the discussion of
targeting moieties. By way of further illustration, however, a
number of specific, particularly preferred embodiments are
discussed below.
[0221] A) Therapeutics Based on Targeted Destruction
[0222] As indicated above, the catalytic antagonists of this
invention can be used as therapeutic in a wide number of
pathologies including, but not limited to inhibitors of viral
infection and/or replication, inhibitors of bacterial infection
and/or biofilm formation, modulators of an immune response,
modulators of an autoimmune response, inhibitors of an inflammatory
response, and the like. More generally, as indicated above, the
catalytic antagonists of this invention can be used to replace
existing pharmaceuticals where the pharmaceutical acts by
inhibiting and/or antagonizing a receptor.
[0223] It was explained above, that a wide variety of
pharmaceuticals act as antagonists of receptors or receptor
mediated activity. These pharmaceuticals typically specifically
bind a particular receptor and or enzyme. The use of such
pharmaceuticals as targeting moieties in catalytic antagonists of
this invention where they are attached to an enzyme (e.g. a serine
hydrolase) that degrades the target receptor and/or enzyme
essentially renders the drug catalytic. Thus, instead of acting in
a stoichiometric manner (a single drug molecule blocks/antagonizes
a single receptor), when converted into catalytic antagonists, the
new drug acts in a substoichiometic manner (a single molecule can
antagonize an essentially unlimited number of receptors). Moreover,
in contrast to the drug alone (where the receptor often regains
activity when the drug is released), a receptor bound by a
catalytic antagonist of this invention is degraded (thereby
releasing the catalytic antagonist to act on another receptor).
Because the receptor is degraded it does not recover its activity.
The catalytic antagonists are thus expected to provide greater
efficacy at a lower dosage and to provide longer lasting activity
at a particular dosage.
[0224] Thus, in one embodiment, this invention provides methods of
improving the activity of a drug. The methods involve attaching the
drug to an enzyme capable of degrading the target (e.g. receptor)
to which the drug binds. Preferred enzymes in this context include
hydrolases and even more preferably include proteases (e.g. serine
proteases, metalloproteases, cysteine proteases, aspartyl
proteases, etc.).
[0225] It was explained above, however, that the targeting moieties
need not be limited to drugs. A wide variety of other targeting
moieties are suitable as well and provide catalytic antagonists
useful in a wide variety of therapeutic contexts. Thus, for
example, in one embodiment, the targeting moiety can be a molecule
that specifically binds to the CCR5 and/or CXCR2 receptors,
commonly found on lymphocytes (e.g. T-cells). CCR5 and CXCR2
receptors are implicated in the infection of a cell by HIV and
persons defective in one or more of these receptors typically
demonstrate resistance to HIV infection. Targeted
destruction/inhibition of either or both of these receptors, e.g.
by a catalytic antagonist comprising a CCR5 and/or CXCR2 specific
targeting agent attached to a suitable hydrolase (e.g. subtilisin)
will increase the target cell's resistance to HIV infection.
[0226] In another embodiment, glycosidases involved in N-linked
protein glycosylation can be specifically targeted (e.g. using an
Aza-sugar targeting moiety) attached to a suitable hydrolase (e.g.
subtilisin, pepsin, etc.). Such a catalytic antagonist will be of
use in the treatment of HIV, herpes, other viruses, and various
cancers.
[0227] Catalytic antagonists comprising a sugar targeting moiety
attached to a suitable hydrolase (e.g. subtilisin, pepsin, etc.)
will be useful in the treatment of a wide variety of conditions,
including inflammatory responses (e.g. associated with arthritis,
septic shock, myocardial infarction, etc.), bacterial binding to
cells and subsequent infection (e.g. H. pylori infection associated
with ulcers). Where the targeting moiety is a Gal.alpha.(1,4)Gal
pathogenic E. coli infections can be blocked. In addition,
lectin-directed catalytic antagonists can be used to inhibit
biofilm formation in vivo or ex vivo.
[0228] In still another embodiment the targeting moiety can be a
sialic acid sugar (or mimetics such as Rilenza (Glaxo-Wellcome) or
the like). When attached to a suitable hydrolase (e.g. subtilisin)
the catalytic antagonist can specifically target the sialidase
activity of viruses such as influenza.
[0229] The chimeric catalytic antagonists of this invention can
also be used to specifically target hyperproliferative and/or
invasive cells (e.g. metastatic cells). Various enzymes (e.g.
especially matrix metalloproteases) are known to be highly active
in invasive cells. These enzymes can be specifically targeted using
a number of targeting moieties (e.g. crown ethers). When attached
to suitable hydrolase (e.g. a subtilisin) the catalytic antagonist
will degrade the target enzyme (e.g. metalloprotease) and thereby
interfere with or eliminate the ability of a cell to invade, e.g. a
basement membrane, thereby slowing the progression of a cancer.
[0230] The catalytic antagonists of this invention can also be used
to target and alter a variety of immune processes. Thus, for
example, by using a target directed at a T-cell receptor, e.g. by
using a MHC complex from a xenotransplant, it is possible to
inhibit immune cells that mount an immune response directed against
that antigen. In a related embodiment, alpha-Gal epitope
disaccharide (Gal.alpha.(1,3)Gal) can be used as a targeting moiety
and (e.g. when attached to a subtilisin) can bind to and
specifically inhibit alpha-Gal epitope specific antibodies thereby
mitigating host rejection of a xenograft. Similarly, known
allergens (e.g. various pollens or epitopes present on such
allergens) can be used as targeting moieties to target and inhibit
T-cells and/or antibodies that specifically recognize such
antigens. This will mitigate allergic reactions against such
allergens.
[0231] B) Drug Delivery Strategies
[0232] As indicated above, the chimeric molecules can be used in
various drug delivery strategies to specifically target a
therapeutic activity to a cell, organ, or tissue of interest. In
certain embodiments, as described above, enzymes capable of
converting prodrugs to their active form are attached to a
targeting moiety (e.g. a tumor cell specific moiety) that localizes
them to the site of desired activity (e.g. a tumor cell), various
prodrugs are known to those of skill in the art and include, but
are not limited to ganclovir activated by thymidine kinase,
phosphenytoin converted to phenytoin by a phosphatase, depivefrin
is converted to epinephrine by an esterase and the like.
[0233] Another example of a "drug delivery" strategy uses the
targeted chimeric molecule to deliver an enzyme having an activity
that supplements an absent, typically endogenous activity. Thus,
for example, glucocerebrosidase may be directed, e.g. to spleen
cells in the treatment of Gaucher's or Tay-Sachs disease. Similarly
superoxide dismutase may be attached to liver cell specific
targeting moieties so that it is targeted to liver tissue where it
can provide anti-oxidant activity.
[0234] C) Other Embodiments.
[0235] In other particularly preferred embodiments, the chimeric
molecules of this invention can be used to target and destroy
particular preselected molecules whether or not they have a
biological activity. Thus, for example, components of various soils
or stains (e.g. milk, blood, eggs, grass stains, oil stains, etc.)
can be specifically targeted. For example, avidin/egg protein can
be specifically targeted by using a biotin as a targeting moiety to
specifically directed, e.g. a protease to the site. The stain is
degraded/digested and thereby released from the underlying
substrate. Such specifically targeted chimeras are particularly in
various cleaning formulations.
VI. Pharmaceutical Formulations
[0236] The therapeutic chimeric molecules of this invention, (e.g.
the therapeutic catalytic antagonists) are useful for intravenous,
parenteral, topical, oral, or local administration (e.g., by
aerosol or transdermally). Particularly preferred modes of
administration include intra-arterial injection, more preferably
intra-peritoneal intra-hepatic artery injection or, where it is
desired to deliver a composition to the brain, (e.g., for treatment
of brain tumors) a carotid artery or an artery of the carotid
system of arteries (e.g., occipital artery, auricular artery,
temporal artery, cerebral artery, maxillary artery, etc.). The
chimeric molecules are typically combined with a pharmaceutically
acceptable carrier (excipient) to form a pharmacological
composition. Pharmaceutically acceptable carriers can contain a
physiologically acceptable compound that acts, for example, to
stabilize the composition or to increase or decrease the absorption
of the agent. Physiologically acceptable compounds can include, for
example, carbohydrates, such as glucose, sucrose, or dextrans,
antioxidants, such as ascorbic acid or glutathione, chelating
agents, low molecular weight proteins, compositions that reduce the
clearance or hydrolysis of the anti-mitotic agents, or excipients
or other stabilizers and/or buffers.
[0237] Other physiologically acceptable compounds include wetting
agents, emulsifying agents, dispersing agents or preservatives
which are particularly useful for preventing the growth or action
of microorganisms. Various preservatives are well known and
include, for example, phenol and ascorbic acid. One skilled in the
art would appreciate that the choice of a pharmaceutically
acceptable carrier, including a physiologically acceptable compound
depends, for example, on the route of administration of the
chimeric molecule and on the particular physio-chemical
characteristics of the agent.
[0238] The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. For example, unit dosage forms suitable for oral
administration include powder, tablets, pills, capsules and
lozenges. It is recognized that the chimeric molecules, when
administered orally, are preferably protected from digestion. This
is typically accomplished either by complexing the subject molecule
with a composition to render it resistant to acidic and enzymatic
hydrolysis or by packaging the chimeric molecule agent in an
appropriately resistant carrier such as a liposome. Means of
protecting compounds from digestion are well known in the art (see,
e.g., U.S. Pat. No. 5,391,377 describing lipid compositions for
oral delivery of therapeutic agents).
[0239] Certain chimeric molecules of this invention may be only
marginally soluble in aqueous solutions. In a preferred embodiment,
these compositions are either delivered directly to the desired
site (e.g. by injection, cannulization, or direct application
during a surgical procedure) or they are solubilized in an
acceptable excipient.
[0240] The pharmaceutical compositions of this invention are useful
for topical administration e.g., in surgical wounds to treat
incipient tumors, neoplastic and metastatic cells and their
precursors. In another embodiment, the compositions are useful for
parenteral administration, such as intravenous administration or
administration into a body cavity or lumen of an organ. The
compositions for administration will commonly comprise a solution
of the chimeric molecule agent dissolved in a pharmaceutically
acceptable carrier, preferably an aqueous carrier for water-soluble
chimeric molecules. A variety of carriers can be used, e.g.,
buffered saline and the like. These solutions are sterile and
generally free of undesirable matter. These compositions may be
sterilized by conventional, well known sterilization techniques.
The compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions such
as pH adjusting and buffering agents, toxicity adjusting agents and
the like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like.
[0241] The concentration of chimeric molecule in these formulations
can vary widely, and will be selected primarily based on fluid
volumes, viscosities, body weight and the like in accordance with
the particular mode of administration selected and the patient's
needs. Actual methods for preparing administrable compositions will
be known or apparent to those skilled in the art and are described
in more detail in such publications as Remington's Pharmaceutical
Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
[0242] Dosages for typical chemotherapeutics are well known to
those of skill in the art. Moreover, such dosages are typically
advisorial in nature and may be adjusted depending on the
particular therapeutic context, patient tolerance, etc. Single or
multiple administrations of the compositions may be administered
depending on the dosage and frequency as required and tolerated by
the patient. In any event, the composition should provide a
sufficient quantity of the proteins of this invention to
effectively treat the patient.
[0243] In the case of therapeutic chimeric molecules dosage for a
typical pharmaceutical composition for intravenous administration
would be about 0.01 to per patient per day. Dosages from 0.1 up to
about 1000 mg per patient per day may be used, particularly when
the drug is administered to a secluded site and not into the blood
stream, such as into a body cavity or into a lumen of an organ.
VII. Kits
[0244] In certain embodiments, this invention provides kits for the
creation and/or use of the chimeric molecules of this invention. In
one embodiment the kits comprise one or more containers containing
one or more targeting moieties derivatized as methanesulfonates for
coupling to a cysteine in an enzyme. In addition or alternatively
the kits may comprise one or more enzymes, more preferably mutant
enzymes having an inserted cysteine ready for coupling to a
methanesulfonate derivatized targeting moiety. When provided in
this manner the kits enable one or ordinary skill in the art to
assemble the desired chimeric molecule for a particular use. Thus,
for example one typically kit may include a multiplicity of
methanesulfonate derivatized targeting moieties and one or more
enzymes suitable for coupling. The desired enzyme is then reacted
(as described herein) with the desired targeting moiety to produce
the desired chimeric molecule. Such kits may additional comprise
one or more of the reagents utilized in a typical coupling
reaction.
[0245] In another embodiment, this invention provides one or more
chimeric molecules (e.g. catalytic antagonists and/or redirected
enzymes) of this invention. The chimeric molecules can be provided
as a dry (e.g. lyophilized powder) or in solution and/or as an
emulsion. In certain embodiments the chimeric molecules are
provided in, or along with, a pharmacological excipient and,
optionally, may be provided in a unit dosage format.
[0246] The kits may optionally include any reagents and/or
apparatus to facilitate the uses described herein. Such reagents
include, but are not limited to buffers, organic solvents, labels,
labeled antibodies, bioreactors, cells, etc.
[0247] In addition, the kits may include instructional materials
containing directions (i.e., protocols) for the assembly of
chimeric molecules of this invention and/or for the use thereof.
While the instructional materials typically comprise written or
printed materials they are not limited to such. Any medium capable
of storing such instructions and communicating them to an end user
is contemplated by this invention. Such media include, but are not
limited to electronic storage media (e.g., magnetic discs, tapes,
cartridges, chips), optical media (e.g., CD ROM), and the like.
Such media may include addresses to internet sites that provide
such instructional materials.
EXAMPLES
[0248] The following examples are offered to illustrate, but not to
limit the claimed invention. The following examples detail the
construction and evaluation of a number of chimeric molecules of
this invention. In particular, Examples 1-4 demonstrates the highly
specific selectivity of a catalytic antagonist of this invention in
which the targeting moiety is a known enzyme inhibitor. Examples 5
through 7 detail the construction and evaluation of chimeric
molecules in which the chimeric molecules are targeted to the
binding protein lectin concanavalin A. Examples 8 through 10 detail
the construction and evaluation of chimeric molecules in which the
chimeric molecules are targeted to the binding protein avidin.
Examples 11 and 12 detail the construction and evaluation of
chimeric molecules in which the chimeric molecules are targeted to
a monoclonal anti-biotin antibody IgG. Example 13 details the
respective stoichiometry of these examples.
Example 1
Targeting Enzymes with Inhibitors
Synthesis and Attachment of an HLADH Inhibitor to SBL and
Characterization of the Resulting SBL-S-Pyrazole CMMs
[0249] In order to direct SBL towards various enzyme targets for
their degradation, we decided to attach specific inhibitors for
those enzymes to SBL by our combined site directed mutagenesis
chemical modification (CMM) approach FIG. 2).
[0250] In a preferred embodiment the inhibitor(s) chosen as
targeting moieties for this approach are strong
inhibitor(s)/degraders of the target enzyme, but are poor
inhibitors of the CMM. In this example, alcohol dehydrogenase
(ADH), which is strongly inhibited by 4-pyrazole derivatives, was
chosen as the target enzyme and the inhibitors chosen as targeting
were pyrazoles known to inhibit ADH. The modified CMM in this case
was a subtilisin (SBL).s
[0251] In one embodiment, horse liver alcohol dehydrogenase (HLADH)
was chosen as the target enzyme. Several 4-substituted pyrazoles
are described as reasonable selective inhibitors of HLADH (Theorell
and Yonetani (1963) Biochem. Z., 388: 537-553; Theorell et al.
(1969) Acta Chem. Scand., 23: 255-260; Tolf et al. (1979) Acta
Chem. Scand. B 33: 483-487; Tolf et al. (1982) Acta Chem. Scand. B,
36: 101-107). Pyrazole derivatives with long hydrophobic alkyl
substituents in 4-position inhibit HLADH activity especially
strongly. The binding affinity of this substituent and hence the
inhibitory power of the 4-pyrazole increases as the alkyl chain
length increases up to six carbon atoms. 4-hexylpyrazole is known
to inhibit horse liver alcohol dehydrogenase (LADH) with
K.sub.I=0.5 nm. Therefore, we decided to synthesize a pyrazole-MTS
reagent bearing a methanesulfonyl-hexyl side chain at the
4-position. The synthesis of the MTS-pyrazole reagent. is
illustrated by scheme 11 (FIG. 3).
[0252] Synthesis of the MTS-Pyrazole
[0253] Halogen-metal exchange of 4-iodopyrazole (1) with n-BuLi,
and subsequent coupling with an excess of 1,6-dibromohexane (2)
provided 4-(6-bromo)-hexylpyrazole (3). Attempts to purify compound
3 were only partially successful. Reaction of crude bromide 3 with
sodium methanethiosulfonate furnished the MTS-pyrazole 4 in 16%
overall yield. Although the overall yield was low, no attempts
towards optimization were made as this would have probably required
the use of protecting groups. Therefore, the MTS-pyrazole 4 can be
synthesized in a straightforward two step reaction sequence as
outlined in scheme 11 (FIG. 3).
[0254] Inhibition of SBL-WT by Pyrazole
[0255] To determine how much modification with a pyrazole will
influence the catalytic activity of SBL we carried out K.sub.I
measurements for SBL-WT using the method of Waley (Waley (1982)
Biochem. J., 205: 631) with our standard substrate suc-AAPFpNA and
standard conditions (pH 8.6, 0.1 M Tris with 0.005% Tween 80, 1%
DMSO). Unsubstituted pyrazole does not significantly inhibit SBL-WT
(K.sub.M=0.73.+-.0.05 mM, k.sub.cat=153.+-.4 s.sup.-1,
k.sub.cat/K.sub.M=209.+-.15 mM s.sup.-1, K.sub.I=97.2.+-.7.2 mM).
Attempts to use the same method for the determination of the
K.sub.I of SBL-WT with the MTS-pyrazole 4 failed due to the
insolubility of the pyrazole compound.
[0256] Modification and Characterization of the Pyrazole-CMMs
[0257] N62C, L217C, S166C, and S156C mutants were modified with the
MTS-pyrazole reagent 4 by reaction at pH 9.5 following the standard
protocol. In all cases the resulting enzymes were active after
modification and the data for amidase kinetics (substrate
suc-AAPFpNA) and ESMS are shown in Table 2.
2TABLE 2 vKinetic constants for pyrazole-CMMs Pyrazole- Amidase
Kinetics ESMS CMM k.sub.cat K.sub.M k.sub.cat/K.sub.M Calc. Found
S166C 10.4 .+-. 0.2 0.46 .+-. 0.03 22.7 .+-. 1.4 26896 26900 S156C
59.8 .+-. 1.5 0.65 .+-. 0.05 92.2 .+-. 7.2 26896 26897 N62C 97.9
.+-. 2.3 0.90 .+-. 0.05 109 .+-. 6.6 26869 26868 L217C 61.3 .+-.
1.0 0.87 .+-. 0.04 70.2 .+-. 3.1 26870 ???-.sup.a GG36-WT 153 .+-.
4 0.73 .+-. 0.05 209 .+-. 15 26698 26694 .sup.aCould not be
obtained until now due to measurement problems. Kinetic constants
determined in duplicate by method of initial rates in 0.1 M TRIS
buffer, pH 8.6, 0.005% Tween 80, 1% DMSO. [S] = 0.125 mM to 3 mM,
[E] = 1.5 .times. 10.sup.-8 M to 9.0 .times. 10.sup.-8 M.
[0258] Although it has the smallest K.sub.M value among all
pyrazole-CMMs, the S166C-S5-Pyrazole CMM shows the lowest
k.sub.cat/K.sub.M; about 9 times smaller than for SBL-WT.
[0259] The k.sub.cat of the S156- and L217C-S-Pyrazole CMM were
both very similar and about 2.5 times smaller than for the WT
enzyme. Their substrate binding properties, however, were fairly
different: S156C-S-Pyrazole bound better than SBL-WT whereas the
K.sub.M of L217C-S-Pyrazole is larger than that of the WT
enzyme.
[0260] N62C-S-Pyrazole is slightly more active than the other
pyrazole CMMs and its k.sub.cat is just 1.5 times smaller compared
to SBL-WT. However it had the largest K.sub.M among all
pyrazole-CMMs and its k.sub.cat/K.sub.M was about 2 times smaller
compared to SBL-WT.
Experimental Details
[0261] 4-(6-Methanethiosulfonyl)hexylpyrazole (MTS-Pyrazole)
(4)
[0262] n-Butyl lithium (2.5 M solution in hexanes, 12.4 mL, 30.9
mmol) was added dropwise to a solution of 4-iodopyrazole (1) (2.00
g, 10.3 mmol) in THF (40 mL) at -78.degree. C. under N.sub.2. After
0.5 h a solution of 1,6-dibromohexane (2) (5.03 g, 20.6 mmol) in
THF (40 mL) was added slowly. When the addition was complete, the
reaction mixture was allowed to warm up to rt and was stirred
overnight. Water (50 mL) was added, the layers were separated and
the aqueous layer was extracted with AcOEt (4.times.50 mL). The
combined organic layers were washed with brine (50 mL) and dried
(MgSO.sub.4). Evaporation of the solvent and subsequent drying
under vacuum furnished 5.03 g crude product as a brown oil.
Separation by flash chromatography (silica gel, hexanes:AcOEt,
gradient elution, 95:5 to 55:45) provided 0.810 g (34%)
4-(6-bromo)-hexylpyrazole (3) as a yellow oil; .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta.1.35-1.48 (m, 4H), 1.51-1.63 (m, 2H),
1.79-1.90 (m, 2H), 2.50 (t, J.sub.1',2'7.5 Hz, 2H, H-1'), 3.40 (t,
J.sub.5',6' 7.0 Hz, 2H, H-6'), 7.42 (s, 2H, H-3, H-5), 11.5 (s, 1H,
NH); .sup.13C NMR (CDCl.sub.3, 75.5 MHz) .delta. 23.9, 28.0, 28.4,
30.8, 32.8, 34.1 [(CH.sub.2).sub.6], 120.9 (C-4) 132.6* (C-3, C-5),
*signal has double intensity. Both NMR spectra contain additional
signals due to impurities. Bromo-compound 3 was not further
purified and characterized. Furthermore 2.41 g (48%) dibromide 2
could be reisolated.
[0263] NaSSO.sub.2Me (0.319 g, 2.38 mmol) was added to a solution
of crude bromide 2 (0.400 g, max. 1.73 mmol) in DMF (10 mL) and the
resulting solution heated under nitrogen at 50.degree. C. After 16
h water (10 mL) and AcOEt (10 mL) were added, the layers were
separated and the aqueous layer was extracted with AcOEt
(4.times.10 mL). The combined organic layers were washed with brine
(10 mL) and dried (MgSO.sub.4). Evaporation of the solvent and
subsequent drying under vacuum furnished 0.350 g crude product as a
yellow oil. Separation by flash chromatography (silica gel, hexanes
: AcOEt, gradient elution, 8:2 to 0:1) furnished 0.215 g (16% over
two steps) 4-(6-methanethiosulfonyl)-hexylpyrazole (4) as a
colourless solid, mp 68-70; IR (KBr) 3170, 3165 (NH), 3065, 3024
(arC--H), 2932, 2854 (C--H), 1306, 1122 (S--SO.sub.2) cm.sup.-1;
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 1.35-1.47 (m, 4H),
1.52-1.62 (m, 2H), 1.70-1.79 (m, 2H), 2.48 (t, J.sub.1',2' 7.5 Hz,
2H, H-1'), 3.14 (t, J.sub.5',6' 7.5 Hz 2H, H-6'), 3.30 (s, 3H,
CH.sub.3SO.sub.2), 7.40 (s, 2H, H-3, H-5), 11.5 (s, 1H, NH);
.sup.13C NMR (CDCl.sub.3, 75.5 MHz) .delta. 23.6, 28.0, 28.2, 30.4,
36.1 [(CH.sub.2).sub.5], 50.3 (CH.sub.3SO.sub.2), 120.4 (C-4),
132.3* (C-3, C-5), *signal has double intensity; HRMS m/z (EI)
Found 263.0882 (M+H.sup.+); C.sub.10H.sub.19N.sub.2O.sub.2S.sub.2
requires 263.0888.
[0264] Standard Modification Protocol
[0265] General Procedure for Modification of SBL Mutants Stored as
Flash-frozen Solutions:
[0266] A 1.25 mL frozen aliquot of the mutant enzyme (N62C, L217C
or S166C) containing approximately 25 mg of enzyme was thawed and
added to 1.25 mL of Modifying Buffer (see below) in a polypropylene
test-tube. To this solution was added 100 .mu.L of a 0.2 M MTS
reagent solution The mixture was sealed, vortexed and placed on an
end-over-end rotator at room temperature. When the modification was
complete (determined by a specific activity assay, using
succinyl-AlaAlaProPhe-p-nitroanilide [.epsilon..sub.410=8800
M.sup.-1 cm.sup.-1](Bonneau et al. (1991) J Am. Chem. Soc. 119:
1026-1030.) as substrate in 0.1 M Tris-HCl buffer containing 0.005%
Tween 80, 1% DMSO, pH 8.6 showing constant activity and titration
with Ellman's reagent (Ellman et al. (1961) Biochem. Pharmacol. 7:
88-95.)(.epsilon..sub.412=13600 M.sup.-1cm.sup.-1) showing no free
thiol present in solution), a further 50 .mu.L of the modifying
reagent solution was added and the mixture placed back on the
end-over-end rotator for a further 10 minutes. The reaction was
poured onto a pre-packed, pre-equilibrated G-25 Sephadex.RTM. PD10
column and eluted with 3.5 mL Quench Buffer (see below). The eluant
was dialysed at 4.degree. C. against 10 mM MES, 1 mM CaCl.sub.2 pH
5.8 (2.sub.13 1L, 2.sub.13 45 min). The resulting dialysate was
flash frozen in liquid nitrogen and stored at -18.degree. C.
[0267] Modifying Buffer: pH 9.5: 140 mM CHES, 2 mM CaCl.sub.2
[0268] pH 7.5: 140 mM HEPES, 2 mM CaCl.sub.2
[0269] pH 5.5: 140mM MES, 2 mM CaCl.sub.2
[0270] Quench Buffer: Reactions at pH 7.5-9.5: 5 mM MES 1 mM
CaCl.sub.2 pH 6.5
[0271] Reactions at pH 5.5: 5 mM MES 1 mM CaCl.sub.2 pH 5.5
[0272] The free thiol content of all CMMs, was determined
spectrophotometrically by titration with Ellman's reagent in
phosphate buffer 0.25 M, pH 8.0. In all cases no free thiol was
detected. Modified enzymes were analyzed by nondenaturing gradient
(8-25%) gels at pH 4.2, run towards the cathode, on the Pharmacia
Phast-system and appeared as a single band. Prior to ES-MS analysis
CMMs were purified by FPLC (BioRad, Biologic System) on a Source 15
RPC matrix (17-0727-20 from Pharmacia) with 5% acetonitrile, 0.01%
TFA as the running buffer and eluted with 80% acetonitrile, 0.01%
TFA in a one step gradient.
[0273] General Procedure for Modification of SBL Mutants Stored as
Lyophilized Powders:
[0274] This procedure is only used with S156C, which is stored as a
lyophilized powder to prevent dimerization. Into a polypropylene
test tube was weighed about 25-30 mg of lyophilized S156C. This was
dissolved in the following modifying buffers (2.5 mL):
[0275] pH 9.5: 70 mM CHES, 2 mM CaCl.sub.2
[0276] pH 7.5: 70 mM HEPES, 2 mM CaCl.sub.2
[0277] pH 5.5: 70 mM MES, 2 mM CaCl.sub.2
[0278] MTS reagent was added and the reaction then proceeded as for
flash-frozen mutant solutions, using the appropriate quench
buffer.
[0279] General Method for Amidase Kinetics Analysis of SBL
Conjugates
[0280] Michaelis-Menten constants were measured at
25(.+-.0.2).degree. C. by curve fitting (GraFit.RTM. 3.03) of the
initial rate data determined at nine concentrations (0.125 mM-3.0
mM) of succinyl-AAPF-pNA substrate in 0.1 M Tris-HCl buffer
containing 0.005% Tween 80, 1% DMSO, pH 8.6 (.epsilon..sub.410=8800
M.sup.-1cm.sup.-1)(Bonneau et al. (1991) J. Am. Chem. Soc. 119:
1026-1030).
[0281] General Method for Esterase Kinetics Analysis of SBL
Conjugates
[0282] Specificity constants determined using the low substrate
approximation were measured indirectly using Ellman's reagent
(.epsilon..sub.412=13600 M.sup.-1cm.sup.-1) using 15 or 30 .mu.M
succinyl-AAPF-SBn as substrate in 0.1 M Tris.HCl, containing 0.005
vol % Tween-80, 1 vol % 37.5 mM Ellman's reagent in DMSO, pH
8.6.
[0283] Michaelis-Menten constants were measured at 25.degree. C. by
curve fitting (Grafit.RTM. 3.03) of the initial rate data
determined at eight concentrations (31.25 .mu.M-2.0 mM) of the
succinyl-AAPF-SBn substrate, followed indirectly using Ellman's
reagent in 0.1 M Tris.HCl, containing 0.005 vol % Tween-80, 1 vol %
37.5 mM Ellman's reagent in DMSO, pH 8.6.
Example 2
Initial HLADH Targeting Assay
Assessing the Targetting of HLADH by Suprastoichiometric
Pyrazole-CMMs and then the Degradation
[0284] Targeting of pyrazole-CMMs to HLADH will be evident from
reduction in ADH activity due to inhibition by the pyrazole moiety
of the CMM.
[0285] Hence, the catalytic activity of HLADH in absence and
presence of the pyrazole-CMMs was investigated. Controls were
carried out with SBL-WT. The amount of CMM and WT used was
calculated for equal amounts of active enzyme as determined by PMSF
titration. Cyclohexanol was used as HLADH substrate and NAD.sup.+
as cofactor. FIG. 4 shows the results of this "Targeting
Assay".
[0286] As expected, SBL-WT did not influence the activity of HLADH
significantly, whereas all pyrazole-CMMs inhibit HLADH. The most
efficient inhibition was caused by S156C-S-Pyrazole, the only CMM
with surface exposed side chain. N62C-S-Pyrazole and
L217C-S-Pyrazole demonstrated very similar inhibition power.
Surprisingly, S166C-S-Pyrazole inhibited HLADH quite strongly, even
though its modified side chain is buried in the S.sub.1 pocket.
This may be rationalized in terms of the pyrazole moiety adopting a
conformation where it bends outside the binding pocket. These
results clearly demonstrate the ability of our modified enzymes to
target another enzyme via an inhibitor.
[0287] Targeted association of CMMs with HLADH via the pyrazole
inhibitor should lead to selective hydrolysis. If hydrolysis of the
HLADH takes place, the oxidoreductase activity of the HLADH should
be diminished or eradicated after a certain time of incubation with
our CMMs. To demonstrate this, the "Targeting Assay" as described
above was carried out again after 4 h incubation. Remaining HLADH
activity was determined by addition of cyclohexanol as substrate.
The results are shown in FIG. 5.
[0288] Experimental for HLADH Targeting Assay
[0289] Six cuvettes were filled as shown in Table 3.
3TABLE 3 Setup for HLADH targeting assay. Cuvette Buffer.sup.a/
Cyclohexane.sup.b/ NAD.sup.+c/ HLADH.sup.d/ Inhibitor-Enzyme.sup.e/
No. .mu.L .mu.L .mu.L .mu.L .mu.L 1 2535 300 150 15 -- 2 2435 300
150 15 100 of SBL-WT.sup.f (1.072 mg/mL, 64%) 3 2435 300 150 15 100
of S156C-S-Pyrazole (0.686 mg/mL) 4 2496 300 150 15 39.0 of
S166C-S-Pyrazole (1.76 mg/mL) 5 2508 300 150 15 27.2 of
N62C-S-Pyrazole (2.52 mg/mL) 6 2483 300 150 15 52.0 of
L217C-S-Pyrazole (1.32 mg/mL) .sup.aAssay buffer: 0.1 M
Glycine-NaOH, pH 9.0. .sup.bSolution (10 mg/mL) in Assay buffer.
.sup.cSolution (33.2 mg/mL) in Assay buffer. .sup.dSolution (10
mg/mL, 52.4% activity) in TRIS-HCl buffer (0.05 M TRIS, pH 7.4).
.sup.eThe amounts are calculated for equal concentrations of active
enzyme (as determined by initial rate kinetics with succ-AAPFpNA).
.sup.fLyophylized enzyme dissolved in MES buffer (10 mM MES, 1 mM
CaCl.sub.2, pH 5.8).
[0290] Before addition of HLADH the cuvette was equilibrated in the
spectrophotometer for two minutes. HLADH was added and A.sub.340
was measured every 20 s over a period of 300 s. The measurements
were carried out in duplicate. The results ar own in Table 4.
4TABLE 4 Results for HLADH targeting assay. Time [s] control SBL-WT
S156CMM S166CMM N62CMM L217CMM 10 0.058 0.053 0.050 0.046 0.055
0.050 30 0.150 0.145 0.121 0.122 0.145 0.138 50 0.239 0.234 0.185
0.191 0.230 0.221 70 0.326 0.320 0.242 0.254 0.311 0.300 90 0.409
0.403 0.294 0.314 0.389 0.377 110 0.489 0.483 0.343 0.370 0.463
0.450 130 0.567 0.561 0.388 0.423 0.535 0.520 150 0.642 0.636 0.431
0.474 0.604 0.588 170 0.716 0.709 0.472 0.522 0.671 0.654 190 0.787
0.780 0.511 0.569 0.736 0.717 210 0.856 0.849 0.547 0.614 0.799
0.779 230 0.924 0.916 0.583 0.658 0.860 0.839 250 0.990 0.982 0.617
0.700 0.919 0.898 270 1.054 1.046 0.650 0.741 0.977 0.955 290 1.117
1.108 0.681 0.781 1.034 1.011 310 1.178 1.169 0.712 0.821 1.089
1.065
[0291] Experimental for HLADH Targeting Assay
[0292] Six eppendorf vials were filled as shown in Table 5.
5TABLE 5 Setup for HLADH targeting assay. Vial Buffer.sup.a/
NAD.sup.+b/ HLADH.sup.c/ Inhibitor-Enzyme.sup.e/ Number .mu.L .mu.L
.mu.L Decoy.sup.d .mu.L 1 535 150 15 -- -- 2 435 150 15 100 -- 3
435 150 15 -- 100 of SBL-WT.sup.f (1.072 mg/mL, 64%) 4 335 150 15
100 100 of SBL-WT.sup.f (1.072 mg/mL, 64%) 5 435 150 15 -- 100 of
S156C-S-Pyrazole (0.686 mg/mL) 6 335 150 15 100 100 of
S156C-S-Pyrazole (0.686 mg/mL) 7 496 150 15 -- 39.0 of
S166C-S-Pyrazole (1.76 mg/mL) 8 396 150 15 100 39.0 of
S166C-S-Pyrazole (1.76 mg/mL) 9 508 150 15 -- 27.2 of
N62C-S-Pyrazole (2.52 mg/mL) 10 408 150 15 100 27.2 of
N62C-S-Pyrazole (2.52 mg/mL) 11 483 150 15 -- 52.0 of
L217C-S-Pyrazole (1.32 mg/mL) 12 383 150 15 100 52.0 of
L217C-S-Pyrazole (1.32 mg/mL) .sup.aAssay buffer: 0.1 M
Glycine-NaOH, pH 9.0. .sup.bSolution (33.2 mg/mL) in Assay buffer.
.sup.cSolution (10 mg/mL) in TRIS-HCl buffer (0.05 M TRIS, pH 7.4).
.sup.d0.5 mg/mL solution of Ribonuclease A with Scrambled Disulfide
Bonds (Sigma) in Milli-Q water. .sup.eThe amounts are calculated
for equal concentrations of active enzyme (as determined by PMSF
titration). .sup.fLyophylized enzyme dissolved in MES buffer (10 mM
MES, 1 mM CaCl.sub.2, pH 5.8).
[0293] These solutions were incubated at 35.degree. C. in a
thermostat-controlled water bath for 4 h 650 .mu.L solution of each
eppendorf vial was mixed with 2 mL Assay buffer in a cuvette. After
two minutes of equilibration in the spectrophotometer and
autozeroing, cyclohexanol (300 .mu.L) was added and A.sub.340 was
measured every 20 s over a period of 300 s. The measurements were
carried out in duplicate and are shown in Table 6.
6TABLE 6 Results for HLADH targeting assay. SBL-WT S156CMM S166CMM
N62CMM L217CMM time [s] Control.sup.a 1.sup.b 2.sup.c 1.sup.b
2.sup.c 1.sup.b 2.sup.c 1.sup.b 2.sup.c 1.sup.b 2.sup.c 10 0.053
0.032 0.037 -0.011 -0.010 -0.010 -0.008 0.009 0.014 0.013 0.028 30
0.140 0.097 0.104 -0.010 -0.010 -0.010 -0.009 0.024 0.046 0.051
0.081 50 0.224 0.159 0.169 -0.010 -0.009 -0.009 -0.008 0.040 0.075
0.088 0.135 70 0.304 0.219 0.231 -0.010 -0.008 -0.008 -0.006 0.056
0.106 0.126 0.184 90 0.382 0.280 0.292 -0.009 -0.008 -0.006 -0.005
0.072 0.136 0.162 0.234 110 0.456 0.338 0.351 -0.008 -0.007 -0.005
-0.004 0.088 0.165 0.198 0.283 130 0.528 0.392 0.408 -0.009 -0.006
-0.004 -0.003 0.103 0.195 0.233 0.332 150 0.598 0.448 0.466 -0.007
-0.005 -0.002 -0.002 0.119 0.224 0.268 0.378 170 0.667 0.501 0.520
-0.006 -0.004 -0.001 0.000 0.134 0.253 0.301 0.423 190 0.734 0.554
0.574 -0.005 -0.004 0.001 0.002 0.149 0.282 0.336 0.469 210 0.797
0.607 0.625 -0.005 -0.003 0.002 0.004 0.164 0.310 0.368 0.515 230
0.860 0.655 0.676 -0.004 -0.002 0.003 0.005 0.179 0.338 0.401 0.558
250 0.921 0.705 0.727 -0.003 -0.001 0.005 0.007 0.194 0.366 0.433
0.601 270 0.983 0.753 0.778 -0.002 -0.000 0.007 0.008 0.209 0.393
0.466 0.644 290 1.041 0.801 0.822 -0.002 0.001 0.009 0.011 0.224
0.420 0.497 0.687 310 1.096 0.845 0.871 -0.000 0.002 0.011 0.012
0.238 0.448 0.528 0.725 .sup.aAverage of control measurements
without and with decoy protein. .sup.bWithout decoy protein.
.sup.cWith decoy protein.
Example 3
Targeting and Destroying HLADH in the Presence of Alkaline
Phosphatase
[0294] In order to direct subtilisins (e.g. SBL) towards various
enzyme targets for their degradation we decided to attach specific
inhibitors for those enzymes to the subtilisin by our combined site
directed mutagenesis chemical modification (CMM) approach as
illustrated in FIG. 3.
[0295] Our preliminary target has been HLADH, which is inhibited by
pyrazoles. The ability of pyrazole-CMMs to selectively destroy
HLADH in the presence, and in the absence, of the decoy protein,
scrambled RNase A, was also explored (see examples below).
[0296] This example describes further experiments in the presence
of an active enzyme, alkaline phosphatase (AP), as a potential
decoy protein. This mimics the in vivo situation where several
enzymes are present in a cell, and where we are targeting the
destruction of one enzyme (HLADH) while leaving the others (e.g.
AP) unaffected
[0297] Digestion experiments were performed using S166C-pyrazole as
a representative CMM. The concentrations of species in the
digestion mixtures (when present) were:
7 HLADH 2.62 .mu.M (1.0 eq. of active sites*) AP 2.74 .mu.M (1.05
eq. of active sites*) S166C-pyrazole 3.40 .mu.M (1.3 eq. of active
sites) It is noted that HLADH and AP are both dimers. MWs: HLADH:
39492 Da/subunit AP: 57099 Da/subunit]
[0298] HLADH activity was monitored by periodically withdrawing a
portion of the digestion mixture, and assessing the ability of the
aliquot to oxidize cyclohexanol to cyclohexanone. The reaction
course was monitored by observing the change of NAD.sup.+ to NADH
at 340 nm as cyclohexanol was oxidized.
[0299] Alkaline phosphatase (AP) activity was monitored by
periodically withdrawing a portion of the digestion mixture, and
assessing the ability of the aliquot to hydrolyze p-nitrophenyl
phosphate to inorganic phosphate and p-nitrophenolate. The reaction
course was monitored by observing the appearance of
p-nitrophenolate at 405 nm.
[0300] Results
[0301] Six vials were prepared containing S166C-pyrazole, AP and/or
HLADH. The HLADH (Table 7) and AP (Table 8) activities of each vial
(where applicable) were periodically assayed (see
experimental).
8TABLE 7 HLADH activity after incubation relative to initial
activity. HLADH activity after incubation* % HLADH activity as % of
initial (0 h) value vial 0 h 1 h 2 h 3 h 72 h AP + HLADH + no
S166C- 100 117 108 109 114 pyrazole AP + HLADH + S166C-pyrazole 100
22 19 16 2 HLADH alone 100 108 74 69 89 HLADH + S166C-pyrazole 100
54 33 30 3 *HLADH activity was assessed by monitoring the NAD.sup.+
to NADH conversion at 340 nm as cyclohexanol was oxidized (see
experimental). Incubation at 35.degree. C.
[0302]
9TABLE 8 Alkaline phosphatase activity after incubation relative to
initial value. AP activity after incubation* AP activity as % of
initial (0 h) value vial 0 h 1 h 2 h 3 h 72 h AP + HLADH + no
S166C- 100 89 77 91 88 pyrazole AP + HLADH + S166C-pyrazole 100 66
82 74 94 AP alone 100 94 94 # # AP + S166C-pyrazole 100 85 88 # #
*AP activity was assessed by monitoring p-nitropheloate release
from p-nitrophenyl phosphate at 405 nm (see experimental).
Incubation at 35.degree. C. # AP and AP + S166C-pyrazole
experiments were not performed for 3 h and 72 h time points.
[0303] The data are also represented graphically in FIG. 6, FIG. 7,
FIG. 8, and FIG. 9.
[0304] The data (Table 7), FIG. 6, and FIG. 8) show that HLADH
activity remains constant when assayed alone or in the presence of
AP. Also, AP activity is basically unaffected in the presence of
S166C-pyrazole CMM (as predicted; Table 2, FIG. 3 and FIG. 5). But
the activity of HLADH in the presence of S166C-pyrazole CMM is
rapidly and totally lost (Table 1, FIG. 2, and FIG. 4).
[0305] Furthermore, AP and HLADH do not interfere with each others
activities or catalytic functions (Table 7, Table 8, FIG. 6, FIG.
7, FIG. 8, and FIG. 9)
Experimental Details
[0306] Materials/
[0307] pH 9.0 0.1 M Glycine-NaOH Buffer with 1 mM Mg.sup.2+ and 0.1
mM Zn.sup.2+(pH 9.0 Assay Buffer):
[0308] Glycine (0.1 mol) was dissolved in water (ca. 800 mL), and
MgCl.sub.2 (1 mL of a 1 M solution in MQ water) and ZnCl.sub.2 (1
mL of a 0.1 M solution in MQ water) were added. The pH was adjusted
to 9.0 with ca. 5 M NaOH solution, and the mixture was made up to 1
L.
[0309] pH 7.4 0.05 M TRIS-HCl Buffer (pH 7.4 TRIS):
[0310] A solution of TRIS was neutralized to pH 7.4, and was then
diluted to 0.05 M.
[0311] pH 7.8 0.05 M Triethanolamine-HCl Buffer with 3 M NaCl, 0.1
mM Mg.sup.2+ and 0.01 mM Zn.sup.2+(pH 7.8 Buffer):
[0312] Triethanolamine (0.05 moles, 7.5 g), NaCl (3 moles, 175.5
g), MgCl2 (0.1 mL of 1 M solution) and ZnCl2 (0.1 mL of 1M
solution) were dissolved in MQ water (ca. 900 mL). The pH was
adjusted to 7.4 with ca 2N HCl and the resulting solution made up
to 1 L.
[0313] pH 8.6 ca. 0.1 M TRIS-HCl Buffer with 0.05% Tween, 1 mM
Mg.sup.2+ and 0.1 mM Zn.sup.2+(pH 8.6 Buffer):
[0314] MgCl.sub.2 (0.1 mL of a 1 M solution in MQ water) and
ZnCl.sub.2 (0.1 mL of a 0.1 M solution in MQ water) were added to a
100 mL volumetric flask, and the flask was made up to the mark with
pH 8.6 0.1 M TRIS-HCl buffer containing 0.05% Tween.
[0315] HLADH Solution:
[0316] Horse liver alcohol dehydrogenase (Sigma A-9589, EC 1.1.1.1,
8 mg of ca. 50% w/w protein) was dissolved in pH 7.4 TRIS (0.8 mL)
to give a 10 mg/mL solution.
[0317] Alkaline Phosphatase (AP) Solution:
[0318] Alkaline phosphatase (Boehringer Mannheim 713 023, EC
3.1.3.1, ca. 950 .mu.L as received in 50% w/v glycerol: buffer) was
diluted with pH 7.8 buffer (ca. 15 mL), and was concentrated at
4.degree. C. to 10-20% of its original volume using a Centriprep
concentrator. A further 15 mL of pH 7.8 buffer was added, and the
sample was concentrated once more. This process was repeated a
further 3 times using pH 9.0 assay buffer for dilutions. After the
third concentration the concentrate (ca. 1.85 mL) was collected and
was stored on ice. This procedure was necessary to remove glycerol,
which is a substrate for HLADH.
[0319] NAD.sup.+ Solution:
[0320] 33.2 mg/mL of NAD.sup.+ was dissolved in pH 9.0 assay
buffer.
[0321] Cyclohexanol Solution:
[0322] 10 mg/mL of cyclohexanol in pH 9.0 assay buffer.
[0323] p-Nitrophenyl Phosphate Solution (PNPP Solution):
[0324] A tablet containing 20 mg of p-nitrophenyl phosphate (Sigma
N-2765) was dissolved in pH 8.6 buffer (20 mL).
[0325] Pyrazole-CMM:
[0326] S166C-pyrazole (1.76 mg/mL) in MES storage buffer (p H 5.8
10 mM MES, 2 mM CaCl.sub.2).
[0327] Assaying HLADH Activity
[0328] Six eppendorf vials were filled as shown in Table 9:
10TABLE 9 Setup for HLADH activity assay. Assay pyrazole- vial
Contents label buffer/.mu.L NAD.sup.+/.mu.L HLADH/.mu.L AP/.mu.L
CMM/.mu.L 1 AP + HLADH no CMM 100 150 15 435 0.0 2 AP + HLADH + CMM
61 150 15 435 39.0 3 AP only 26.5 0 0 43.5 0.0 4 HLADH only 535 150
15 0 0.0 5 HLADH + CMM 496 150 15 0 39.0 6 AP + CMM 22.6 0 0 43.5
39.0 CMM was S166C-pyrazole.
[0329] The vials were incubated at 35.degree. C. for the times
indicated in the tables below. Aliquots were periodically withdrawn
in order to assay the HLADH and alkaline phosphatase activities as
time progressed.
[0330] A portion of solution (65 .mu.L) was withdrawn from an
incubation vial and was then injected into a micro-cuvette
containing pH 9.0 assay buffer (200 .mu.L). The cuvette was
incubated at 25.degree. C. for 2 minutes, and then cyclohexanol
solution (30 .mu.L) was added. The absorbance at 340 nm was then
monitored for 300 s, and the O.D. change per second up to 0.2
absorbance units was recorded.
[0331] Assaying Alkaline Phosphatase Activity
[0332] A portion (20 .mu.L) was withdrawn from an incubation vial
and was then injected into pH 8.6 buffer (980 .mu.L). The mixture
was vortexed. 10 .mu.L was the removed from the mixture, and was
injected into a cuvette containing 990 .mu.L of PNPP solution
incubated at 25.degree. C. The absorbance change at 405 nm was
monitored for 150 s, and the O.D. change per second up to 1
Absorbance unit was recorded.
Example 4
Targeting HLADH with Substoichiometric Pyrazole-CMMs
[0333] Stoichiometry:
[0334] Experiments were performed using 2 eq. HLADH dimer (4 eq.
active sites) to 1 eq. pyrazole-CMM or WT-SBL as illustrated in
Table 10.
11TABLE 10 Stoichiometry. HLADH (ca. 79 kD for the dimer) SBL or
pyrazole-CMM (ca. 27 kD) 2 eq. dimer (4 eq. active sites) 1 eq.
(SBL/CMM is a monomer) 1.42 .mu.M dimer (2.84 .mu.M active sites)
0.71 .mu.M (SBL/CMM is a monomer)
[0335] Conditions:
[0336] The reactions were performed at pH 9.0, 0.1 M glycine-NaOH
with 0.005% Tween 80, 35.degree. C.
[0337] Results:
[0338] HLADH solutions were incubated in the presence of WT-SBL,
S166C-pyrazole or S156C-pyrazole. A control experiment was
performed in the absence of any SBL-based enzyme (HLADH alone). The
HLADH activities of the four mixtures were periodically assayed
(see experimental)--see Table 11.
12TABLE 11 HLADH activities after incubation with or without
pyrazole-CMMs. % HLADH activity compared to the initial "HLADH
alone" value* HLADH + HLADH + HLADH + HLADH WT S166C- S156C- time/h
alone SBL pyrazole pyrazole 0 100 97 89 67 1 99 89 65 21 3 98 82 49
12 20 93 70 17 5 *HLADH activity was assessed by monitoring the
conversion of NAD.sup.+ to NADH at 340 nm as cyclohexanol was
oxidized at 25.degree. C., pH 9.0 (see experimental).
[0339] Discussion:
[0340] The initial drop in HLADH activity caused on addition of the
pyrazole-CMMs reflects the ability of the CMMs to target and thus
inhibit HLADH. S156C-pyrazole and, to a lesser extent,
S166C-pyrazole clearly cause dramatic reductions in HLADH activity
on incubation. The pyrazole-CMMs were used in less than
stoichiometric amounts with respect to HLADH--4 eq. HLADH active
sites: 1 eq. pyrazole-CMM-but they rapidly caused a greater than
25% diminution of HLADH activity. Indeed, in the case of
S156C-pyrazole, HLADH activity is seen to drop from 67% to 5% over
20 h, representing a 13.5-fold reduction of HLADH activity
over-and-above the maximum inhibitory effect of the pyrazole
moiety. WT-SBL causes a mere 1.4-fold reduction of HLADH activity
over the same 20 h period, despite its enhanced amidase specific
activity when compared to the pyrazole-CMMs.
[0341] Summary:
[0342] Pyrazole-CMMs are seen to target and to catalytically
destroy HLADH.
Experimental Materials:
[0343] pH 9.0 0.1 M Glycine-NaOH Buffer with 0.005% Tween 80 (pH
9.0 Assay Buffer):
[0344] Glycine (0.1 mol) was dissolved in water (ca. 800 mL). A
solution of Tween 80 (50 mL of a 0.1% v/v in MQ water) was added,
and the pH was adjusted to 9.0 with ca. 5 M NaOH solution. The
mixture was made up to 1 L with MQ water.
[0345] pH 7.4 0.05 M TRIS-HCl Buffer (pH 7.4 TRIS):
[0346] TRIS (302.9 mg, 2.5 mmol) was dissolved in MQ water (ca. 40
mL). The pH was adjusted to 7.4 with ca. 1 M HCl solution, and the
volume of the mixture was made up to 50 mL with MQ water.
[0347] HLADH Solution:
[0348] Horse liver alcohol dehydrogenase (Sigma A-9589, Lot
58H7004, EC 1.1.1.1, 8.45 mg of 52.4% w/w protein--according to
manufacturer's Biuret titration) was dissolved in pH 7.4 TRIS
(0.845 mL) to give a 5.24 mg/mL solution of active protein.
[0349] Checking HLADH Concentration:
[0350] HLADH (50 .mu.L) solution was added to pH 7.4 TRIS (450
.mu.L) to give a tenfold diluted solution. Bradford (Bio-Rad)
protein determination was performed on this diluted sample, and
yielded a protein concentration of 0.616 mg/mL. This translates to
a concentration of 6.16 mg/mL in the original HLADH stock. We
assume the lower value of 5.24 mg/mL to be correct in order to
ensure that protein concentration is more likely to be under-rather
than overestimated.
[0351] NAD.sup.+ Solution:
[0352] NAD.sup.+ (332 mg) was dissolved in pH 9.0 assay buffer (10
mL) to give a 33.2 mg/mL solution.
[0353] Cyclohexanol Solution:
[0354] Cyclohexanol (100 mg) was dissolved in pH 9.0 assay buffer
(10 mL) to give a 10 mg/mL solution.
[0355] Subtilisin Solutions:
[0356] WT-SBL (1.88 mg of dry powder, 73% w/w active protein) was
dissolved in pH 5.8, 10 mM MES, 2 mM CaCl.sub.2 "storage buffer"
(500 .mu.L) to give a 2.74 mg/mL solution of active WT-SBL.
S156C-pyrazole and S166C-pyrazole were previously titrated with
PMSF: their concentrations were 2.5 mg/mL and 3.62 mg/mL
respectively.
Experimental Details
[0357] HLADH Hydrolysis Assay:
[0358] Four 5 mL falcon tubes were filled according to Table 12
13TABLE 12 Preparation of reaction mixtures. Tube pH 9.0 assay
NAD.sup.+ solution.sup.b HLADH SBL/ no. buffer.sup.a (.mu.L)
(.mu.L) solution.sup.c (.mu.L) CMM.sup.d 1 2140 600 60 not added 2
2120 600 60 WT-SBL 19.52 .mu.L 3 2125 600 60 S166C- pyrazole 14.80
.mu.L 4 2119 600 60 S156C- pyrazole 21.4 .mu.L .sup.a0.1 M
glycine-NaOH with 0.005% Tween 80. .sup.b33.2 mg/mL in pH 9.0 assay
buffer. .sup.c5.24 mg/mL active HLADH. .sup.dConcentrations:
WT-SBL, 2.74 mg/mL; S166C-pyrazole, 3.62 mg/mL; S156C-pyrazole, 2.5
mg/mL.
[0359] The tubes were kept on ice until the HLADH activity of each
tube had been assayed in order to give a "time zero" value for each
tube (see below for assay protocol). The tubes were then incubated
on a water bath at 35.degree. C. Periodically, 700 .mu.L of
reaction mixture with withdrawn from each falcon tube, the aliquots
were placed in individual eppendorf tubes, and the eppendorf tubes
were stored on ice. The content of each eppendorf tube was then
assayed for HLADH activity (see below).
[0360] Assaying HLADH Activity:
[0361] A portion of reaction mixture (650 .mu.L) was injected into
a cuvette containing pH 9.0 assay buffer (2.00 mL). The cuvette was
incubated at 25.degree. C. for 2 minutes, and then cyclohexanol
solution (300 .mu.L) was added. After a 10 s delay, the absorbance
at 340 nm was monitored for 300 s. Tthe O.D. change per second up
to 0.2 absorbance units was used to calculate an initial rate.
[0362] Results.
[0363] The results are summarized in Table 13.
14TABLE 13 HLADH activities during HLADH hydrolysis experiment (raw
data). (slope at 340 nm in units of O.D. units per second) .times.
1000* HLADH + HLADH + HLADH + HLADH WT S166C- S156C- incubation
alone SBL pyrazole pyrazole time/h (tube 1) (tube 2) (tube 3) (tube
4) 0 5.14 4.99 4.56 3.43 1 5.07 4.56 3.36 1.05 3 5.05 4.22 2.53
0.61 20 4.76 3.58 0.88 0.26 *The O.D. change up to 0.2 absorbance
units was used to calculate these numbers.
Example 5
Targeting HLADH in the Presence of Alkaline Phosphatase Using
S156C- and S166C-Pyrazole-CMMs at substoichiometric levels of
CMMs
[0364] Stoichiometry
[0365] Experiments were performed using 2 eq. HLADH dimer (4 eq.
active sites) to 1 eq. pyrazole-CMM or WT-SBL; alkaline phosphatase
from calf intestine was used as an "active-enzyme" decoy protein in
all experiments. Alkaline phosphatase from calf intestine is
composed of two isozymes of molecular weights 66 and 68 kD per
subunit. Both isozymes are dimers, thus we assume an approximate
molecular weight of 134 kD for each dimer in our calculations. The
stoichiometries used are shown in Table 14.
15TABLE 14 Stoichiometries used HLADH targeting assay. HLADH (ca.
79 kD Alkaline WT-SBL or Pyrazole-CMM for the dimer) phosphatase
(AP) (ca. 27 kD) (ca. 134 kD for each dimer) 2 eq. dimer (4 eq. 2
eq. dimer (4 eq. 1 eq. (SBL/CMM is a active sites) active sites)
monomer) 1.42 .mu.M dimer 1.42 .mu.M dimer 0.71 .mu.M (SBL/CMM is a
(2.84 .mu.M active sites) (2.84 .mu.M active monomer) sites)
[0366] Conditions.
[0367] pH 9.0, 0.1 M glycine-NaOH with 0.005% Tween 80, 1 MM
MgCl.sub.2 and 0.1mM ZnCl.sub.2, 3 5.degree. C.
[0368] Four experiments were performed simultaneously: all four
experiments were performed with HLADH and AP present in each of the
four vials (i.e. they are in direct competition as substrates for
hydrolysis). In addition, each vial contained one of: buffer (no
SBL added), WT-SBL, S156C-pyrazole or S166C-pyrazole.
[0369] Results:
[0370] Vials that each contained a mixture of HLADH and alkaline
phosphatase were incubated in the presence of WT-SBL,
S166C-pyrazole or S156C-pyrazole. A control experiment was
performed in the absence of any SBL-based enzyme (no SBL). The
HLADH and Alkaline phosphatase activities of the four mixtures were
periodically assayed in order to determine the fidelity of the CMMs
toward HLADH vs. Alkaline phosphatase (see experimental)--see Table
15, Table 16, FIG. 11 and FIG. 12. (Data are also presented
relative to "no SBL added" time=0 h values in the appendix to
demonstrate the inhibitory effects of the pyrazole CMMs on
HLADH)
16TABLE 15 HLADH activities after incubation with or without
pyrazole-CMMs. % HLADH activity* compared to the time = 0 h value
for each experiment SBL derivative (if added) time/h no SBL WT-SBL
S156C-pyrazole S166C-pyrazole 100 100 100 100 1 109 103 45 93 3 112
99 33 46 19.5 95 79 2 41 *HLADH activity was assessed by monitoring
the conversion of NAD.sup.+ to NADH at 340 nm as cyclohexanol was
oxidized at 25.degree. C., pH 9.0 (see experimental).
[0371]
17TABLE 16 AP activities after incubation with or without
pyrazole-CMMs. % alkaline phosphatase activity* compared to the
time = 0 h value for each experiment SBL derivative (if added)
time/h no SBL WT-SBL S156C-pyrazole S166C-pyrazole 0 100 100 100
100 1 98 100 93 104 3 94 97 96 99 19.5 88 91 85 90 alkaline
phosphatase activity was assessed by monitoring p-nitropheloate
release from p-nitrophenyl phosphate at 405 nm (see
experimental).
[0372] Discussion.
[0373] Alkaline phosphatase is clearly not very susceptible to
hydrolysis by WT-SBL or Pyrazole-CMMs. HLADH activity is not
significantly diminished on incubation in the absence of SBL or in
the presence of WT-SBL. However, in the presence of S156C-pyrazole
or S166C-pyrazole HLADH activity is seen to diminish rapidly.
[0374] Summary.
[0375] Pyrazole-CMMs are seen to target and to catalytically
destroy HLADH in the presence of alkaline phosphatase. Alkaline
phosphatase is unaffected by the hydrolytic action of WT-SBL and
Pyrazole-CMMs.
Experimental
[0376] Materials
[0377] pH 9.0 0.1 M Glycine-NaOH Buffer with 0.005% Tween 80, 1 mM
Mg.sup.2+ and 0.1 mM Zn.sup.2+ (pH 9.0 Assay Buffer with Tween,
Mg.sup.2+ and Zn.sup.2+):
[0378] Glycine (0.1 mol) was dissolved in water (ca. 800 mL).
Magnesium chloride solution (1 mL of a 1 M solution) and zinc
chloride solution (1 mL of a 0.1 M solution) were added to the
glycine solution. A solution of Tween 80 (50 mL of a 0.1% v/v in MQ
water) was added to the mixture, and the pH was adjusted to 9.0
with ca. 5 M NaOH solution. The mixture was made up to 1 L with MQ
water.
[0379] pH 9.0 0.1 M Glycine-NaOH Buffer with 1 mM Mg2+ and 0.1 mM
Zn2+(pH 9.0 Dialysis Buffer).
[0380] Glycine (0.1 mol) was dissolved in water (ca. 800 mL).
Magnesium chloride solution (1 mL of a 1 M solution) and zinc
chloride solution (1 mL of a 0.1 M solution) were added to the
glycine solution, and the of the mixture pH was adjusted to 9.0
with ca. 5 M NaOH solution. The mixture was made up to 1 L with MQ
water.
[0381] pH 7.4 0.05 M TRIS-HCl Buffer (pH 7.4 TRIS)
[0382] TRIS (302.9 mg, 2.5 mmol) was dissolved in MQ water (ca. 40
mL). The pH was adjusted to 7.4 with ca. 1 M HCl solution, and the
volume of the mixture was made up to 50 mL with MQ water.
[0383] pH 7.4 0.05 M TRIS-HCl Buffer with 1 mM Mg2+ and 0.1 mM
Zn2+(pH 7.4 Dialysis Buffer)
[0384] TRIS (6.057 g, 0.05 mol) was dissolved in MQ water (ca. 800
mL). Magnesium chloride solution (1 mL of a 1 M solution) and zinc
chloride solution (1 mL of a 0.1 M solution) were added to the TRIS
solution, and the of the mixture pH was adjusted to 7.4 with ca. 1
M HCl solution. The mixture was made up to 1 L with MQ water.
[0385] pH 8.6 ca. 0.1 M TRIS-HCl Buffer with 0.05% Tween, 1 mM Mg2+
and 0.1 mM Zn2+(PH 8.6 Buffer)
[0386] MgCl.sub.2 (0.1 mL of a 1 M solution in MQ water) and
ZnCl.sub.2 (0.1 mL of a 0.1 M solution in MQ water) were added to a
100 mL volumetric flask, and the flask was made up to the mark with
pH 8.6 0.1 M TRIS-HCl buffer containing 0.05% Tween (standard
amidase kinetics buffer).
[0387] HLADH Solution
[0388] Horse liver alcohol dehydrogenase (Sigma A-9589, Lot
58H7004, EC 1.1.1.1, 3.77 mg of 52.4% w/w protein--according to
manufacturer's Biuret titration) was dissolved in pH 7.4 TRIS
(0.377 mL) to give a 5.24 mg/mL solution of active protein.
[0389] NAD+ Solution
[0390] NAD.sup.+ (39.15 mg) was dissolved in pH 9.0 assay buffer
(1.179 ML) to give a 33.2 mg/mL solution.
[0391] Cyclohexanol Solution
[0392] Cyclohexanol (100 mg) was dissolved in pH 9.0 assay buffer
(10 mL) to give a 10 mg/mL solution.
[0393] Subtilisin Solutions.
[0394] WT-SBL (1.88 mg of dry powder, 73% w/w active protein) was
dissolved in pH 5.8, 10 nM MES, 2 mM CaCl.sub.2 "storage buffer"
(500 .mu.L) to give a 2.74 mg/mL solution of active WT-SBL. This
solution was diluted four-fold with pH 5.8, 10 mM MES, 2 mM
CaCl.sub.2 "storage buffer" to give a 0.685 mg/mL solution.
[0395] S156C-pyrazole and S166C-pyrazole were previously titrated
with PMSF: their concentrations were 2.5 mg/mL and 3.62 mg/mL
respectively. These stock solutions were diluted four-fold with pH
5.8, 10 mM MES, 2 mM CaCl.sub.2 "storage buffer" to give 0.63mg/mL
and 0.91 mg/mL solutions of S156C-pyrazole and S166C-pyrazole,
respectively.
[0396] p-Nitrophenyl Phosphate Solution (PNPP Solution).
[0397] Two tablets, each containing 20 mg of p-nitrophenyl
phosphate (Sigma N-2765), were dissolved in pH 8.6 buffer (40 mL).
The solution was stored on ice.
[0398] Dialysis of Alkaline Phosphatase.
[0399] Two vials of Calf intestinal alkaline phosphatase (Sigma
P-7923, Lots 128H1210 and 17H0204) were mixed with pH 7.4 dialysis
buffer (0.5 mL). The mixture was dialysed against 2.times.500 mL pH
7.4 dialysis buffer (1.times.4 h then 1.times. overnight) and then
2.times.500 mL pH 9.0 dialysis buffer (2.times.2 h). The total
protein concentration was then determined using the Bradford
technique (Bio-Rad), and was found to be 2.36 mg/mL.
[0400] Experimental Details
[0401] Four 1.5 mL Eppendorf tubes were filled according to Table
22.
18TABLE 17 Preparation of reaction mixtures. NAD.sup.+ HLADH AP
SBL/ pH 9.0 assay solution.sup.b solution.sup.c solution.sup.d
CMM.sup.e - Tube no. buffer.sup.a (.mu.L) (.mu.L) (.mu.L) (.mu.L)
if added 1 479 150 15 56.4 Nothing added 2 459 150 15 56.4 WT- SBL
19.52 .mu.L 3 457 150 15 56.4 S156C- pyrazole 21.4 .mu.L 4 464 150
15 56.4 S166C- pyrazole 14.80 .mu.L .sup.a0.1 M glycine-NaOH with
0.005% Tween 80, 1 mM MgCl.sub.2 and 0.1 mM ZnCl.sub.2. .sup.b33.2
mg/mL in pH 9.0 assay buffer with Tween, Mg.sup.2+ and Zn.sup.2+.
.sup.c5.24 mg/mL active HLADH. .sup.d2.36 mg/mL alkaline
phosphatase (Bradford). .sup.eConcentrations: WT-SBL, 0.685 mg/mL;
S166C-pyrazole, 0.91 mg/mL; S156C-pyrazole, 0.63 mg/mL.
[0402] The tubes were kept on ice until aliquots had been withdrawn
from each tube to establish initial HLADH and alkaline phosphatase
activities. These activities were used to give "time zero" values
for each tube (see below for assay protocols). The tubes were then
incubated on a water bath at 35.degree. C. Periodically, aliquots
of reaction mixture were withdrawn from each eppendorf tube in
order to assay HLADH and alkaline phosphatase activities.
[0403] Assaying HLADH Activity
[0404] A portion of solution (65 .mu.L) was withdrawn from an
incubation vial and was then injected into a micro-cuvette
containing pH 9.0 assay buffer (200 .mu.L). The cuvette was
incubated at 25.degree. C. for 2 minutes, and then cyclohexanol
solution (30 .mu.L) was added. The absorbance at 340 nm was then
monitored for 120 s, and the O.D. change per second up to 0.2
absorbance units was recorded.
[0405] Assaying Alkaline Phosphatase Activity
[0406] A portion (10 .mu.L) was withdrawn from an incubation vial
and was then injected into pH 8.6 buffer (490 .mu.L). The mixture
was vortexed. 10 .mu.L was the rremoved from the mixture, and was
injected into a cuvette containing 990 .mu.L of PNPP solution
incubated at 25.degree. C. The absorbance change at 405 nm was
monitored for 120 s, and the O.D. change per second up to 1.0
Absorbance unit was recorded.
[0407] Results.
[0408] The results are illustrated in Table 18 and Table 19
19TABLE 18 HLADH activities during HLADH/alkaline phosphatase
competitive hydrolysis experiments (raw data). (slope at 340 nm in
units of O.D. incubation units per second) .times. 1000* time/h no
SBL WT-SBL S156C-pyrazole S166C-pyrazole 0 5.13 5.09 2.76 4.58 1
5.57 5.25 1.25 4.25 3 5.73 5.05 0.90 2.09 19.5 4.88 4.01 0.06 1.88
*The O.D. change up to 0.2 absorbance units was used to calculate
these numbers.
[0409]
20TABLE 19 Alkaline phosphatase activities during HLADH/alkaline
phosphatase competitive hydrolysis experiments (raw data).
incubation (slope at 405 nm in units of O.D. units per second)
.times. 1000* time/h no SBL WT-SBL S156C-pyrazole S166C-pyrazole 0
8.04 7.78 8.05 7.48 1 7.85 7.79 7.52 7.77 3 7.56 7.52 7.76 7.41
19.5 7.11 7.09 6.83 6.77 *The O.D. change up to 1.0 absorbance
units was used to calculate these numbers.
Example 5
Synthesis of Carbohydrate Modified Serine Hydrolases
[0410] The contamination of animal feed by certain lectins
substantially reduces their nutritional value (Gatel (1994) Animal
Feed Sci. Technl45: 317-348; Mogridge et al. (1996) J. Animal Sci.
74: 1897-1904; Pusztai et al. (1997) G. Brit. J. Nutrition 77,
933-945). In particular contamination of soy-based feeds by
mannose-binding lectins prevents the effective use of crude feed
without substantial purification.
[0411] With the aim of preparing glycosylated CMMs useful for the
we have prepared 11 mono- and disaccharide methanethiosulfonates
(FIG. 13) bearing different carbohydrates which allow the
preparation of a large number glycosylated CMMs for use, e.g. as
lectin-directed proteases. A number of chemically modified enzymes
having chemically conjugated carbohydrate moieties are described in
PCT Application WO 00001712 entitled "Chemically modified proteins
with a carbohydrate moiety.
Example 6
Targeted Lectin Degradation Assay Using Mannosylated-SBL
[0412] This example describes a highly effective lectin assay that
has allowed us to start a screen of the ability of sugar-modified
CMMs to degrade the lectin Concanavalin A in the manner shown
schematically below (FIG. 14A, FIG. 14B, and FIG. 14C).
[0413] S156C-sugar CMMs which contain surface exposed sugar groups
were chosen initially. For each assay, biotinylated lectin was
incubated with glyco-CMM and compared with samples incubated with
GG36-WT. To allow comparison, equal amounts of active enzyme were
used. These samples were also incubated both with and without the
decoy protein disulfide scrambled-RNaseA, in order to measure the
selectivity of these enzymes for the lectin over the decoy.
[0414] Small protein fragments (<3000 Da), the products of
proteolysis, were separated from larger proteins using a
size-exclusion membrane. Fragments of lectin are labeled with
biotin whereas non-lectin fragments are unlabelled. By monitoring
both the levels of biotinylated fragments released, using a
HABA/Avidin test, and total protein fragment concentration, using
A.sub.280, we can qualitatively judge both the amount of lectin
degradation and selectivity for lectin over decoy. The results of
initial screens are shown in FIG. 15A and FIG. 15B, FIG. 15C, and
FIG. 15D.
[0415] It is clear that both GG36-WT and the two CMMs S156C-S-EtMan
(FIG. 15A and FIG. 15B) and S156C-S-EtMan(Ac).sub.4 (FIG. 15C, and
FIG. 15D are able to rapidly degrade lectin concanavalin A. The
higher rate of hydrolysis by GG36-WT is consistent with its higher
k.sub.cat/K.sub.M value towards Suc-AAPF-pNA (k.sub.cat/K.sub.M for
GG36 of 209 s.sup.-1mM.sup.-1, as compared with 112 and 85
s.sup.-1mM.sup.-1 for S156C-S-Et-Man and S156C-S-EtMan(Ac).sub.4,
respectively).
[0416] A more detailed examination of FIG. 15A-FIG. 15D reveals
that
[0417] a) Released Biotin levels (indicating lectin degradation)
are similar to each other. The presence of decoy reduces slightly
both the level of GG36-WT and S156C-S-EtMan degradation.
[0418] b) GG36-WT in the presence of decoy produces 18% more total
protein after 210 min. than without. In contrast, S166C-S-EtMan in
the presence of decoy produces only 7% more total
protein--therefore the greater selectivity of S156C-S-EtMan reduces
total protein absorption changes by 11%.
[0419] c) Again, released Biotin levels (indicating lectin
degradation) are similar to each other.
[0420] d) Both GG36-WT and S156C-S-EtManAc produce more total
protein after 210 min. in the presence of decoy than without (under
these conditions, 7% more for WT and 5% more for S156C-S-EtManAc).
These similar levels indicate little or no selectivity of
S156C-S-EtManAc for Concanavalin A.
[0421] This slight but exciting creation of selectivity of
S156C-S-EtMan is consistent with the introduction of an unprotected
mannose group--since this is the natural ligand of concanavalin A.
The lower/lack of selectivity shown by fully protected
S156C-S-EtMan(Ac).sub.4 is consistent with the importance of the
unprotected hydroxyl groups of mannose for correct recognition by
lectins.
[0422] Experimental
[0423] Ten disposable eppendorf vials were filled as shown in Table
20.
21TABLE 20 Lectin assay design. Lectin- Assay Concanavalin Vial
Buffer.sup.a/ A.sup.b/ Decoy Protein.sup.c/ Enzyme/ Number .mu.L
.mu.L .mu.L .mu.L 1 900 100 -- -- 2 890 100 10 -- 3 900 100 -- 10
of glyco-CMM 4 900 100 -- 10 of glyco-CMM 5 890 100 10 10 of
glyco-CMM 6 890 100 10 10 of glyco-CMM 7 900 100 -- 10 of WT.sup.d
8 900 100 -- 10 of WT.sup.d 9 890 100 10 10 of WT.sup.d 10 890 100
10 10 of WT.sup.d .sup.aLectin-Assay Buffer: 20 mM Tris.HCl, 2 mM
CaCl.sub.2, pH 8.6. .sup.b5 mg/mL solution of Biotinylated
Concanavalin A (Vector Laboratories) in Milli Q water. .sup.c5
mg/mL solution of Ribonuclease A with Scrambled Disulfide Bonds
(Sigma) in Milli Q water. .sup.dSolution of lyophilized GG36-WT
diluted to the same concentration as the glyco-CMM (as determined
by PMSF) in 20 mM MES, 1 mM CaCl.sub.2, pH 5.5.
[0424] These solutions were warmed to 35.degree. C. in a
thermostat-controlled water bath. After the indicated incubation
time, the contents of the appropriate vials were each placed in the
top of a Centricon-SR3 Concentrator (Amicon, MWCO 3000, previously
cleaned by 2 mL of Milli Q water centrifuged at 3750 rpm for 90
min.) and centrifuged at 3750 rpm for 60 min. The resulting
filtrates were then assayed as shown in Table 21 and Table 22.
22TABLE 21 Assay of filtrates for S156C-S-EtMan (Enzyme
Concentrations 2.58 mg/mL) HABA/ HABA/ HABA/ % of Max Avidin Avidin
Avidin Incub. Biotin Total % of Max Total Vial Before.sup.a/
After.sup.b/ Diff./ Time/ Release/ Protein/ Biotin/ Protein/ No.
Abs Abs Abs min A.sub.280.sup.c Abs.sup.d Abs Abs Abs 1 1.006 0.726
0.28 60 0.003 -- -- -- -- 2 1.004 0.719 0.285 60 0.003 -- -- -- --
3 1.012 0.665 0.347 60 0.033 0.062 0.03 45 54 4 1.009 0.597 0.412
210 0.042 0.127 0.039 93 70 5 1.01 0.662 0.348 60 0.022 0.063 0.019
46 34 6 1 0.6 0.4 210 0.046 0.115 0.043 84 77 7 1.014 0.649 0.365
60 0.028 0.08 0.025 58 45 8 0.998 0.576 0.422 210 0.049 0.137 0.046
100 82 9 1.012 0.659 0.353 60 0.035 0.068 0.032 50 57 10 1.007
0.591 0.416 210 0.059 0.131 0.056 96 100 .sup.a800 .mu.L of
HABA/Avidin Reagent (Sigma) prepared with 1 mL of Milli Q water.
.sup.bAfter addition of 200 .mu.L of lectin-assay filtrate.
.sup.cValue for 300 .mu.L of lectin-assay filtrate diluted with 700
.mu.L of Milli Q as compared with Milli Q water blank (1 mL).
.sup.dCalculated from difference between HABA/Avidin drop in Abs.
for sample and the drop in Abs. caused by dilution alone
(controls).
[0425]
23TABLE 22 Assay of filtrates for S156C-S-EtMan(Ac).sub.4 (Enzyme
Concentration 2.40 mg/mL) HABA/ HABA/ HABA/ % of Max Avidin Avidin
Avidin Incub. Biotin Total % of Max Total Vial Before.sup.a/
After.sup.b/ Diff./ Time/ Release/ Protein/ Biotin/ Protein/ No.
Abs Abs Abs min A.sub.280.sup.c Abs.sup.d Abs Abs Abs 1 0.983 0.717
0.266 60 0.002 -- -- -- -- 2 0.982 0.711 0.271 60 0.009 -- -- -- --
3 0.98 0.633 0.347 60 0.027 0.079 0.021 35 31 4 1.002 0.559 0.443
210 0.06 0.175 0.054 78 79 5 0.991 0.628 0.363 60 0.034 0.095 0.028
42 41 6 1.004 0.548 0.456 210 0.063 0.188 0.057 84 84 7 0.998 0.606
0.392 60 0.041 0.124 0.035 55 51 8 1.003 0.511 0.492 210 0.069
0.224 0.063 100 93 9 1.001 0.651 0.35 60 0.038 0.082 0.032 37 47 10
1.013 0.54 0.473 210 0.074 0.205 0.068 92 100 .sup.a,b,c,das
above.
Example 7
Lectin Degradation Assay Using Other Glycosylated-CMMs and of
Mannosylated-SBL with Higher Decoy Protein Concentrations
[0426] In addition to S156C-S-EtMan and S156C-S-EtMan(Ac).sub.4
reported earlier the lectin assay was performed for other
glycosylated CMMs. Furthermore the selectivity of S156C-S-EtMan at
higher levels of decoy protein was investigated. The data showed
that as for S-EtManAc the other sugars glucose, galactose and
lactose showed little or no selectivity. By challenging
S156C-S-EtMan with 5-fold higher levels of decoy protein the
selectivity of this mannosylated CMM was decreased to approximately
a difference of about 12% in total protein levels with and without
decoy.
Experimental
[0427] Lectin Assay Method 1.
[0428] Ten disposable eppendorf vials were filled as shown in Table
23:
24TABLE 23 Lectin assay method 1 Lectin-Assay Decoy Vial
Buffer.sup.a/ Concanavalin A.sup.b/ Protein.sup.c/ Enzyme/ No.
.mu.L .mu.L .mu.L .mu.L 1 900 100 -- -- 2 890 100 10 -- 3 900 100
-- 10 of glyco-CMM 4 900 100 -- 10 of glyco-CMM 5 890 100 10 10 of
glyco-CMM 6 890 100 10 10 of glyco-CMM 7 900 100 -- 10 of WT.sup.d
8 900 100 -- 10 of WT.sup.d 9 890 100 10 10 of WT.sup.d 10 890 100
10 10 of WT.sup.d .sup.aLectin-Assay Buffer: 20 mM Tris.HCl, 2 mM
CaCl.sub.2, pH 8.6. .sup.b5 mg/mL solution of Biotinylated
Concanavalin A (Vector Laboratories) in Milli Q water. .sup.c5
mg/mL solution of Ribonuclease A with Scrambled Disulfide Bonds
(Sigma) in Milli Q water. .sup.dSolution of lyophilized GG36-WT
diluted to the same concentration as the glyco-CMM (as determined
by PMSF) in 20 mM MES, 1 mM CaCl.sub.2, pH 5.5.
[0429] These solutions were warmed to 35.degree. C. in a
thermostat-controlled water bath. After the indicated incubation
time, the contents of the appropriate vials were each placed in the
top of a Centricon-SR3 Concentrator (Amicon, MWCO 3000, previously
cleaned by 2 mL of Milli Q water centrifuged at 3750 rpm for 90
min.) and centrifuged at 3750 rpm for 60 min. The resulting
filtrates were then assayed as shown in Table 24, Table 25, and
Table 26.
25TABLE 24 Assay of filtrate (S156C-S-Et.beta.Glc (Enzyme
Concentration 2.29 mg/mL)) HABA/ HABA/ HABA/ % of Max Avidin Avidin
Avidin Incub. Biotin Total % of Max Total Vial Before.sup.a/
After.sup.b/ Diff./ Time/ Release/ Protein/ Biotin/ Protein/ No.
Abs Abs Abs min A.sub.280.sup.c Abs.sup.d Abs Abs Abs 1 0.989 0.779
0.21 60 0.003 -- -- -- -- 2 1.011 0.789 0.222 60 0.009 -- -- -- --
3 0.992 0.637 0.355 60 0.025 0.14 0.019 53 29 4 1.024 0.545 0.479
210 0.068 0.264 0.062 100 95 5 1.011 0.63 0.381 60 0.038 0.166
0.032 63 49 6 0.995 0.566 0.429 210 0.068 0.214 0.062 81 95 7 0.999
0.604 0.395 60 0.036 0.18 0.03 68 46 8 1.003 0.554 0.449 210 0.062
0.234 0.056 89 86 9 1.037 0.648 0.389 60 0.042 0.174 0.036 66 55 10
0.996 0.568 0.428 210 0.071 0.213 0.065 81 100 .sup.a800 .mu.L of
HABA/Avidin Reagent (Sigma) prepared with 1 mL of Milli Q water.
.sup.bAfter addition of 200 .mu.L of lectin-assay filtrate.
.sup.cValue for 300 .mu.L of lectin-assay filtrate diluted with 700
.mu.L of Milli Q as compared with Milli Q water blank (1 mL).
.sup.dCalculated from difference between HABA/Avidin drop in Abs.
for sample and the drop in Abs. caused by dilution alone
(controls).
[0430]
26TABLE 25 Assay of filtrate (S156C-S-EtGal (Enzyme Concentration
1.73 mg/mL)) % of HABA/ HABA/ HABA/ % of Max Avidin Avidin Avidin
Incub. Biotin Total Max Total Vial Before.sup.a/ After.sup.b/
Diff./ Time/ Release/ Protein/ Biotin/ Protein/ No. Abs Abs Abs min
A.sub.280.sup.c Abs.sup.d Abs Abs Abs 1 0.995 0.77 0.225 60 -0.004
-- -- -- -- 2 1.002 0.768 0.234 60 -0.007 -- -- -- -- 3 1.011 0.629
0.382 60 0.02 0.152 0.026 67 37 4 0.975 0.531 0.444 210 0.055 0.214
0.061 94 87 5 1.005 0.621 0.384 60 0.028 0.154 0.034 68 49 6 0.978
0.543 0.435 210 0.063 0.205 0.069 90 99 7 1.018 0.652 0.366 60 0.01
0.136 0.016 60 23 8 1.01 0.553 0.457 210 0.048 0.227 0.054 100 77 9
1.011 0.666 0.345 60 0.014 0.115 0.02 51 29 10 0.997 0.563 0.434
210 0.064 0.204 0.07 90 100 .sup.a,b,c,das above.
[0431]
27TABLE 26 Assay of filtrate S156C-S-EtLac (Enzyme Concentration
2.25 mg/mL). % of HABA/ HABA/ HABA/ % of Max Avidin Avidin Avidin
Incub. Biotin Total Max Total Vial Before.sup.a/ After.sup.b/
Diff./ Time/ Release/ Protein/ Biotin/ Protein/ No. Abs Abs Abs min
A.sub.280.sup.c Abs.sup.d Abs Abs Abs 1.006 0.77 0.236 60 -0.006 --
-- -- -- 2 0.994 0.762 0.232 60 -0.002 -- -- -- -- 3 0.993 0.684
0.309 60 0.008 0.075 0.012 35 15 4 1.023 0.584 0.439 210 0.04 0.205
0.044 95 56 5 0.994 0.676 0.318 60 0.013 0.084 0.017 39 22 6 1
0.578 0.422 210 0.068 0.188 0.072 87 91 7 1.014 0.655 0.359 60
0.012 0.125 0.016 58 20 8 1.012 0.562 0.45 210 0.047 0.216 0.051
100 65 9 1.001 0.674 0.327 60 0.014 0.093 0.018 43 23 10 1.006 0.57
0.436 210 0.075 0.202 0.079 94 100 .sup.a,b,c,das above.
[0432] Control Without Lectin.
[0433] The assay was performed as for method 1 except 100 .mu.L
aliquots of concanavalin replaced by 100 .mu.L of Milli Q water.
Results are shown in Table 27.
28TABLE 27 Assay of filtrate. Control without lectin. HABA/ HABA/
HABA/ % of Max Avidin Avidin Avidin Incub. Biotin Total % of Max
Total Vial Before.sup.a/ After.sup.b/ Diff./ Time/ Release/
Protein/ Biotin/ Protein/ No. Abs Abs Abs min A.sub.280.sup.c
Abs.sup.d Abs Abs Abs 1 1.004 0.768 0.236 60 -0.005 -- -- -- -- 2 1
0.77 0.23 60 -0.008 -- -- -- -- 3 0.998 0.768 0.23 60 -0.008 -0.003
-0.001 -1 -1 4 1 0.774 0.226 210 -0.007 -0.007 0 -3 0 5 1.023 0.78
0.243 60 0.004 0.01 0.011 5 14 6 1.02 0.783 0.237 210 0.007 0.004
0.014 2 18 7 1.018 0.774 0.244 60 -0.005 0.011 0.002 5 3 8 1.001
0.774 0.227 210 -0.002 -0.006 0.005 -3 6 9 1.006 0.769 0.237 60
0.008 0.004 0.015 2 19 10 1.004 0.771 0.233 210 0.012 -2.8E-17
0.019 0 24 .sup.a,b,c,das above.
[0434] Lectin Assay Method 2.
[0435] Ten disposable eppendorf vials were filled as shown in Table
28.
29TABLE 28 Design of lectin assay 2. Lectin- Vial Assay
Concanavalin A.sup.b/ Decoy Protein.sup.c/ Enzyme/ No.
Buffer.sup.a/.mu.L .mu.L .mu.L .mu.L 1 900 100 -- -- 2 850 100 50
-- 3 900 100 -- 10 of glyco-CMM 4 900 100 -- 10 of glyco-CMM 5 850
100 50 10 of glyco-CMM 6 850 100 50 10 of glyco-CMM 7 900 100 -- 10
of WT.sup.d 8 900 100 -- 10 of WT.sup.d 9 850 100 50 10 of WT.sup.d
10 850 100 50 10 of WT.sup.d .sup.aLectin-Assay Buffer: 20 mM
Tris.HCl, 2 mM CaCl.sub.2, pH 8.6. .sup.b5 mg/mL solution of
Biotinylated Concanavalin A (Vector Laboratories) in Milli Q water.
.sup.c5 mg/mL solution of Ribonuclease A with Scrambled Disulfide
Bonds (Sigma) in Milli Q water. .sup.dSolution of lyophilized
GG36-WT diluted to the same concentration as the glyco-CMM (as
determined by PMSF) in 20 mM MES, 1 mM CaCl.sub.2, pH 5.5.
[0436] All further determinations were carried out as for Method 1
and the results are shown in Table 29.
30TABLE 29 Assay of filtrate S156C-S-EtMan (Enzyme Concentrations
2.58 mg/mL) HABA/ HABA/ HABA/ % of Max Avidin Avidin Avidin Incub.
Biotin Total % of Max Total Vial Before.sup.a/ After.sup.b/ Diff./
Time/ Release/ Protein/ Biotin/ Protein/ No. Abs Abs Abs min
A.sub.280.sup.c Abs.sup.d Abs Abs Abs 1 0.969 0.76 0.209 60 -0.003
-- -- -- -- 2 0.982 0.756 0.226 60 0.005 -- -- -- -- 3 0.975 0.649
0.326 60 0.018 0.108 0.017 52 21 4 0.97 0.562 0.408 210 0.048 0.19
0.047 92 57 5 0.99 0.685 0.305 60 0.03 0.087 0.029 42 35 6 0.988
0.589 0.399 210 0.068 0.181 0.067 87 82 7 0.983 0.637 0.346 60
0.028 0.128 0.027 62 33 8 0.974 0.549 0.425 210 0.053 0.207 0.052
100 63 9 0.978 0.659 0.319 60 0.037 0.101 0.036 49 44 10 0.982
0.599 0.383 210 0.083 0.165 0.082 80 100 .sup.a,b,c,das for Method
1.
Example 8
Synthesis of Biotin-MTS
[0437] In order to exploit the powerful binding of biotin to avidin
as a model system to clearly demonstrate the targeting strategy the
biotin-MTS reagent 1 was synthesized.
[0438] In the synthetic strategy chosen (FIG. 16, scheme 7) we
chose the carboxylic acid group of biotin as the point at which to
introduce methanethiosulfonate as previous studies have shown that
functionalization of this part of the molecule preserves affinity
for avidin (Green (1975) Adv. Protein Chem. 29: 85-133; Green
(1990) Meth. Enzymol. 184: 51-67).
[0439] Initial attempts to reduce the N-hydroxysuccinamide ester 3
using NaBH.sub.4 (Islam et al. (1994) J. Med. Chem., 37: 293-304.),
synthesized from (+)-biotin (2) in 61% yield according to
literature methods (Chaturvedi et al. (1984) J. Med. Chem., 27:
1406-1410), gave only a poor 15% yield of (+)-biotinol (4). In
contrast, direct reduction of (+)-biotin (2) with LiAlH.sub.4 gave
4 in a reasonable 69% yield (Flaster and Kohn (1981) Heterocycl.
Chem. 18: 1425-1436). The use of ether as a solvent is crucial to
the success of this reduction as THF gave only a very low yield of
(+)-biotinol (4).
[0440] Biotinol (4) was elaborated, according to our established
preparative procedure, to the target biotin-MTS via the
corresponding primary mesylate and bromide. The use of MsCl led to
only a moderate yield of mesylate as a result of competing
formation of primary chloride. Consequently, biotinol (4) was
treated with mesylic anhydride in pyridine/DCM, then LiBr in
refluxing acetone and finally NaSSO.sub.2Me in DMF to give target
biotin-MTS1 in 54% yield over 3 steps (37% overall yield from
(+)-biotin (2)). Attempts to scale up this synthesis gave reduced
yields.
Experimental
[0441] (+)-Biotinol (4) via Hydroxysuccinamide Ester (3).
[0442] 1,1'-Dicarbonylimidazole (360 mg, 2.22 mmol) was added to a
stirred solution of (+)-biotin (2) (540 mg, 2.2 mmol) in DMF (10
mL) under N.sub.2 and the resulting solution heated until evolution
of CO.sub.2 ceased (approx 30 min.). The solution was cooled to RT
and stirred for a further 2 h, during which time a white solid
precipitated from solution. A solution of N-hydroxysuccinimide (260
mg, 2.26 mmol) in DMF (10 mL) was added and the mixture stirred.
After a further 6 h, the reaction solvent was removed and the
residue recrystallized first from propan-2-ol (mp 187-187.degree.
C.) and then DMF/propan-2-ol to give 3 (457 mg, 61%) as a white
solid.; mp 197-201.degree. C. (DMF/propan2-ol) [lit., Becker et al.
(1971) Proc. Natl. Acad. Sci., USA, 68: 2604-2607, mp
196-200.degree. C.; lit., Parameswaran (1990) Org. Prep. Proc.
Intl. 22: 119-121, mp 210.degree. C.]; .sup.1H NMR (d.sup.6-DMSO,
200 MHz) 1.43-1.70 (m, 4H), 2.52-2.90 (m, 9H), 3.07-3.14 (m, 4H,
H-4), 4.17 (dd, J 6 Hz, J 4 Hz, 1H, H-3a), 4.32 (dd, J 6 Hz, J 7
Hz, 1H, H-6a), 6.39, 6.45 (s.sub.--2, 1H.sub.--2, H-1, H-3).
[0443] NaBH.sub.4 (50 mg, 1.32 mmol) was added to a suspension of 3
(170 mg, 0.5 mmol) stirred in THF/HMPA (40: 1, 41 mL) under
nitrogen. After 4.5 h the volume of reaction solvent was reduced
and the resulting residue quenched with water. The residue was
dried further under vacuum and purified by flash chromatography
(MeOH: CHCl.sub.3, 1: 19) to give 4 (17 mg, 15%) as a white
solid.
[0444]
5-([3aS-(3a.alpha.,4.beta.,6a.alpha.)]-Hexahydro-2-oxo-1H-thieno[3,-
4-d]imidazol-4-yl)pentyl methanethiosulfonate [(+)-Biotin MTS]
(1).
[0445] (+)-Biotin (2) (196 mg, 0.8 mmol) was dissolved in pyridine
(5 mL) by careful warming at 80.degree. C. under nitrogen. The
resulting solution was added dropwise to a suspension of
LiAlH.sub.4 (196 mg, 5.15 mmol) in freshly distilled dry ether (25
mL) under nitrogen. After 30 min. the resulting mixture was heated
to reflux. After a further 40 min., tlc (MeOH:CHCl.sub.3, 1:9)
showed the formation of a major product (R.sub.f 0.35) from
starting material (R.sub.f 0.45). The reaction was cooled and
remaining LiAlH.sub.4 quenched by the dropwise addition of water.
After effervescence had ceased more water (100 mL) was added and
the solvent removed. The residue was dried overnight under vacuum
and then purified by flash chromatography (MeOH:CHCl.sub.3, 1:19)
to give (+)-biotinol
[3aS-(3a.alpha.,4.beta.,6a.alpha.)]-Tetrahydro-4-(5-hydroxyp-
entyl)-1H-thieno[3,4-d]imidazol-2(3H)-one (4) (128 mg, 69%)
[53906-36-8] as a white solid; mp 168-172 [lit., U.S. Pat. No.
2,489,237, mp 174.5-175.5 (MeOH)]; [.alpha.].sup.28.sub.D=+91.2 (c
0.43, MeOH) [lit., [.alpha.].sup.25.sub.D=+84.7 (c 1, MeOH)];
.sup.1H NMR (CD3OD, 400 MHz) .delta. 1.42-1.48 (m, 4H), 1.52-1.63
(m, 3H), 1.72-1.75 (m, 1H), 2.71 (d, J.sub.6,6'12.6 Hz, 1H, H-6),
2.93 (dd, J.sub.6',6a 4.9 Hz, J.sub.6,6 '12.8 Hz, 1H, H-6'), 3.32
(qu, J 4.8 Hz, 1H, H-4), 3.56 (t, J 6.5 Hz, 2H, CH.sub.2OH), 4.31
(dd, J.sub.3a,4 4.4 Hz, J.sub.3a,6a 7.8 Hz, 1H, H-3a), 4.51 (dd,
J.sub.6',6a 4.9 Hz, J.sub.3a,6a 7.9 Hz, 1H, H-6a).
[0446] Ms.sub.2O (78 mg, 0.45 mmol) was added to a solution of 4
(80 mg, 0.35 mmol) in pyridine/DCM (1:1, 4 mL) under nitrogen.
After 14 h the solvent was removed. The residue was dissolved in
CHCl.sub.3 (30 mL), washed (water (10 mL), brine (10 mL)), dried
(MgSO.sub.4), filtered and the solvent removed. The residue was
purified by flash chromatography (MeOH:CHCl.sub.3, 1:50) to give
the mesylate [3aS-(3a.alpha.,4.beta.,6a.a-
lpha.)]-Tetrahydro-4-[5-(methanesulfonyl)pentyl]-1H-thieno[3,4-d]imidazol--
2(3H)-one (83 mg, 77%) as a yellow oil; a scale up provides the
mesylate as a pale yellow solid; mp 134-136; IR (film) 3432 (NH),
1702 (amide I), 1636 (amide II) cm.sup.-1; .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 1.45-1.47 (m, 4H), 1.67-1.79 (m, 4H), 2.75 (d,
J.sub.6,6'12.8 Hz, 1H, H-6), 2.92 (br d, J 9.4 Hz, 1H, H-6'), 3.02
(s, 3H, CH.sub.3SO.sub.2-), 3.15-3.19 (m, 1H, H-4), 4.24 (t, J 6.4
Hz, 2H, CH.sub.2OMs), 4.32 (dd, J.sub.3a,4 4.8 Hz, J.sub.3a,6a 7.3
Hz, 1H, H-3a), 4.51 (m, 1H, H-6a); .sup.13C NMR (CDCl.sub.3, 50
MHz) .delta. 25.4, 28.3, 28.5, 28.8, 37.4, 40.5, 55.5
((CH.sub.2).sub.4, C-4, C-6, CH.sub.3SO.sub.2-), 60.3, 62.1 (C-3a,
C-6a), 70.1 (CH.sub.2OMs), 163.6 (C-2).
[0447] LiBr (80 mg, 0.92 mmol) was added to a solution of mesylate
(40mg, 0.13 numol) in acetone (2 mL) under nitrogen and the
resulting solution heated under reflux. After 14 h, tlc
(MeOH:CHCl.sub.3, 1:9) showed the conversion of starting material
(R.sub.f 0.3) to product (R.sub.f 0.45). The solvent was removed
and the residue partitioned between ether (30 mL) and water (10
mL). The aqueous fraction was further extracted with CHCl.sub.3 (30
mL.sub.--2) in which the bromide is more soluble. The organic
fractions were combined, dried (MgSO.sub.4), filtered and the
solvent removed to give the crude bromide
[3aS-(3a.alpha.,4.beta.,6a.alph-
a.)]-Tetrahydro-4-(5-bromopentyl)-1H-thieno[3,4-d]imidazol-2(3H)-one
(28 mg, 74%) as a yellow oil, which was used directly in the next
step. A scale up provides the product as a pale yellow solid; mp
157-159.
[0448] NaSSO.sub.2Me (15 mg, 0.11 mmol) was added to a solution of
crude bromide (24 mg, 0.08 mmol) in DMF (2 mL) and the resulting
solution heated under nitrogen at 50.degree. C. After 19 h, tlc
(MeOH:CHCl.sub.3, 1:9) showed the formation of a major product
(R.sub.f 0.35) from starting material (R.sub.f 0.45). The solvent
was removed and the residue purified by repeated flash
chromatography (MeOH:CHCl.sub.3 1:19 then 3:97) to give 1 (25 mg,
94%, 37% from (+)-biotin (2)) as an amorphous solid; a scale up
provides 1 as a pale yellow solid; [.alpha.].sub.D.sup.26+42.1 (c,
0.62 in CHCl.sub.3); IR (film) 3215 (NH), 1699 (C.dbd.O), 1310,
1129 (S--SO.sub.2) cm.sup.-1; .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 1.45-1.47 (m, 4H), 1.62-1.70 (m, 2H), 1.72-1.80 (m, 2H),
2.75 (d, J.sub.6,6' 13.0 Hz, 1H, H-6), 2.93 (br d, J 9.3 Hz, 1H,
H-6'), 3.13-3.19 (m, 3H, H-4, --CH.sub.2S--), 3.34 (s, 3H,
CH.sub.3SO.sub.2--), 4.33 (dd, J.sub.3a,4 4.2 Hz, J.sub.3a,6a 7.1
Hz, 1H, H-3a), 4.53 (dd, J.sub.6',6a 4.9 Hz, J.sub.3a,6a 6.8 Hz,
1H, H-6a); .sup.13C NMR (CDCl.sub.3, 100 MHz) .delta. 28.4, 28.5,
28.6, 29.4, 36.4, 40.6, 55.6 ((CH.sub.2).sub.4, C-4, C-6,
--CH.sub.2S--), 50.9 (CH.sub.3SO.sub.2--), 60.6, 62.4 (C-3a, C-6a),
163.5 (C-2); HRMS m/z (FAB+): Found 325.0755 (M+H.sup.+);
C.sup.11H.sub.20N.sub.2O.sub.3S.sub.3 requires 325.0714.
Example 9
Preparation and Characterization of Biotin-CMMs
[0449] Preparation of the Biotin-CMMs
[0450] Biotin-MTS reagent 1 was used to prepare the biotinylated
CMMs of N62C, L217C, S166C, and S156C mutants, by reaction at pH
9.5 following the standard protocol. In all cases the resulting
enzymes are active after modification.
[0451] Amidase Kinetics of the Biotin-CMMs
[0452] The data for amidase kinetics and ESMS are shown in Table
30.
31TABLE 30 Kinetic Constants for Biotin-CMMs. (+)-Biotin- Amidase
Kinetics ESMS CMM k.sub.cat K.sub.M k.sub.cat/K.sub.M Calc. Found
S166C 56.3 1.4 1.00 0.05 56.1 3.3 26958 26967 S156C 75.4 2.2 0.83
0.06 91.1 7.2 26958 26968 N62C 122 1.8 1.06 0.04 115 4.4 26931
26936 L217C 60.3 0.8 0.72 0.02 83.4 3.1 26932 26936 GG36-WT 153 4
0.73 0.05 209 15 26698 26694 Kinetic constants determined in
duplicate by method of initial rates in 0.1 M TRIS buffer, pH 8.6,
0.005% Tween 80, 1% DMSO. [S] = 0.125 mM to 3 mM, [E] = 1.4
10.sup.-8 M to 2.4 10.sup.-8 M.
[0453] All biotin-CMMs had a smaller catalytic activity than SBL-WT
(k.sub.cat/K.sub.M=209 15).
[0454] The values for the S156C-S-Biotin CMM and the L217C-S-Biotin
CMM were similar and showed a decreased k.sub.cat compared to
SBL-WT, whereas the change in K.sub.M is negligible.
[0455] Modification of the S166C mutant with the biotin-MTS reagent
gave, compared to SBL-WT, a four times lower k.sub.cat/K.sub.M. The
S166C-S-Biotin CMM also had the lowest k.sub.cat/K.sub.M of all the
biotinylated CMMs. Both k.sub.cat and K.sub.M are altered for this
CMM.
[0456] The N62C-S-Biotin CMM had a slightly decreased k.sub.cat
compared to SBL-WT and has the highest K.sub.M of all the
biotin-CMMs, however it was still the most active of these
biotinylated CMMs.
[0457] Esterase Kinetics of the Biotin-CMMs
[0458] Esterase kinetics was carried out for the biotin-CMMs
according to the standard protocol with suc-AAPF-SBn as substrate.
The results are shown in Table 31.
32TABLE 31 Esterase Kinetics for Biotin-CMMs (+)-Biotin-Esterase
Kinetics CMM k.sub.cat K.sub.M k.sub.cat/K.sub.M S166C 489 41.0
0.59 0.14 830 212 S156C 825 42.7 0.68 0.10 1221 187 N62C 422 27.3
0.21 0.05 1973 497 L217C 432 52.5 0.35 0.15 1229 559 GG36-WT 1940
180 0.54 0.07 3560 540 Kinetic constants determined in duplicate by
method of initial rates in 0.1 M TRIS buffer, pH 8.6, 0.005% Tween
80, 1% DMSO. [S] = 0.015 mM to 3 mM, [E] = 0.8 .times. 10.sup.-9 M
to 1.2 .times. 10.sup.-9 M.
[0459] The k.sub.cat/K.sub.M results for esterase activity follow
the same trend compared to amidase kinetics.
[0460] The S1 66C-S-Biotin CMM shows the smallest
k.sub.cat/K.sub.M, which is about four times lower than for
SBL-WT.
[0461] The biotin-CMMs have an approximately four fold lower
k.sub.cat compared to SBL-WT with the S156C-S-Biotin CMM as the
only exception. The k.sub.cat of the S156C-S-Biotin CMM is about
two fold lower than for SBL-WT and therefore two fold higher
compared to the other biotin-CMMs. However, S156C-S-Biotin CMM is
not the most active of all biotinylated CMMs since it has also the
highest K.sub.M value. The k.sub.cat/K.sub.M of S156C-S-Biotin CMM
and L217C-S-Biotin CMM are very similar, about 3-fold lower than
for SBL-WT, although k.sub.cat and K.sub.M show big
differences.
[0462] The K.sub.M values of the biotin-CMMs were slightly higher
for the S166C-S-Biotin CMM and the S156C-S-Biotin CMM compared to
SBL-WT. Whereas, the values for the N62C-S-Biotin CMM and the
L217C-S-Biotin CMM are about two times smaller compared to
SBL-WT.
[0463] The N62C-S-Biotin CMM has the lowest K.sub.M of all the
biotinylated CMMs, and is 2.6 fold lower than SBL-WT. Although it
also has the lowest k.sub.cat of the biotin-CMMs, it has the
highest catalytic activity, which is still 1.8 fold lower then
SBL-WT.
Example 10
[0464] Targetting a Binding Protein--Targeting and Hydrolysis of
Avidin with Biotin-CMMs
[0465] It is known from the literature that biotinylated proteins
will bind to avidin only when the biotin is separated from the
surface of the macromolecule to which it is covalently linked by at
least five methylene groups (Green (1970) Meth. Enzymol. 18A:
418-424). Furthermore, Wilchek et al. observed that proteolytic
enzymes are not able to cleave avidin. Even when the proteases is
biotinylated, avidin is not cleaved (Bayer et al. (1990)
Biochemistry, 29: 11274-11279).
[0466] Our goal in this project is not only to establish targeting
of our new biotin-CMMs to avidin but also to demonstrate that the
CMMs are capable of catalyzing avidin proteolysis. SBL-WT, which it
is not able to complex avidin but may hydrolyze avidin in an
unselective process, is used for comparison.
[0467] The colorimetric method previously used to demonstrate
lectin degradation with glycosylated CMMs was adapted, to assay the
ability of the synthesized biotin-CMMs to target avidin. We
measured the release of HABA from HABA/avidin reagent which was
detected by an increase of absorbance at 500 nm. All biotinylated
CMMs were examined and the amount of CMM used was corrected for
equal catalytic activity compared to each other based on
k.sub.cat/K.sub.M with suc-AAPF-pNA. Therefore, differences between
the capability of our biotin-CMMs to target to avidin can be
discussed.
[0468] In order to investigate the ability of our biotin-CMMs not
only to target avidin but also to hydrolyze, we decided to separate
the assay for targeting and the assay for hydrolysis of avidin with
our biotinylated CMMs.
[0469] As already mentioned, targeting can be clearly proven by
measuring HABA release from a HABA/avidin solution at 500 nm. Since
we were now only interested in using this method as a targeting
assay for avidin we measured the HABA release over a 5 min period.
Despite the fact that only for S156C-S-Biotin the introduced
biotin-side-chain is surface exposed and therefore easily
accessible for binding to avidin, surprisingly all biotin-CMMs
caused immediate HABA release when added to a buffered solution of
HABA/avidin. Due to its surface exposure S156C-S-Biotin resulted in
the highest HABA release compared to the other CMMs.
[0470] We controlled the targeting process by comparison with
solutions of SBL-WT (concentration calculated for same catalytic
activity) and addition of (+)-biotin [diluted to the same
concentration as expected in each one of the biotin-CMMs (by
determination of PMSF-value).
[0471] Since, for steric reasons, (+)-biotin should be better
available to bind to avidin than the biotinylated side-chain of our
CMMs we expected a smaller or, for the best case, the same HABA
release for all biotin-CMMs compared to the biotin/WT mixture.
[0472] S166C-, N62C- and L217C-S-Biotin confirmed our predictions,
the S156C CMM however gave a drastically higher HABA release.
[0473] We determined therefore the whole protein amount of the
S156C-S-Biotin solution by lyophilization and calculated the biotin
amount again (B2a). In this case the HABA release for a biotin/WT
mixture is higher than for the S156C CMM, and we suggest that there
might be inactive enzyme bearing the biotin group in the CMM
solution which also binds avidin and causes therefore additional
HABA release.
[0474] For determination of avidin hydrolysis catalyzed by SBL-WT
and the biotin-CMMs, respectively, we adopted the lectin assay
method and measured A.sub.280 for hydrolysis fragments <3000 Da.
The assay was carried out for S156C-S-Biotin since this CMM proved
to be the best enzyme in the targeting assay. To allow comparison
and demonstrate unselective hydrolysis, the SBL-WT was used as a
solution diluted to the same catalytic activity as the biotin-CMM
solution.
[0475] The initial measurements with HABA/avidin and the enzyme
revealed anomalies presumably caused by HABA, therefore we decided
to do this assay with avidin alone. To determine the selectivity of
avidin hydrolysis, a decoy protein, disulfide scrambled-RNAse A,
was used, similar to the lectin assay described above. The
solutions were incubated for 1 h and 4 h, respectively, and small
protein fragments (<3000 Da), the products of hydrolysis, were
separated using a size-exclusion membrane. Measurement of A.sub.280
furnished the total protein fragment concentration and is therefore
an indicator of avidin hydrolysis.
[0476] Both SBL-WT and S156C-S-Biotin are able to hydrolyze avidin.
Since the enzyme concentrations were calculated for equal catalytic
activity with the standard amidase substrate suc-AAPF-pNA the
hydrolysis values can be compared directly. Therefore we are able
to demonstrate not only that avidin is hydrolyzed by SBL-proteases
but is also more efficiently hydrolyzed by a biotinylated protease.
S156C-S-Biotin produces 45% more protein fragments after 240 min
than GG36-WT. In the presence of decoy protein [0.05 mg, Decoy (1)]
the amount of total protein produced increased drastically for the
WT enzyme 27% after 240 min) whereas the production of total
protein did not change significantly for the biotin-CMM. However a
3-fold higher level of decoy protein [0.15 mg, Decoy (2)] resulted
also for S156C-S-Biotin in increasing production of total protein
(38% after 240 min) which indicates a decreased selectivity for
avidin. In studies run with controls for GG36-WT with different
amounts of biotin added, the differences were fairly small.
Experimental
[0477] Avidin Targeting Assay (Displacement of HABA)
[0478] 12 disposable cuvettes were filled as shown in Table 32
(before biotin and enzyme addition measurement of A.sub.500):
33TABLE 32 Avidin targeting assay d-Biotin/ Cuvette Buffer.sup.a/
HABA/Avidin.sup.b/ .mu.L (conc. Enzyme/ Number .mu.L .mu.L [mg/mL])
.mu.L (conc. [mg/mL]) 1 400 400 -- --.sup.c 2 400 400 -- --.sup.c 3
400 400 -- --.sup.c 4 390 400 10 (0.060).sup.d --.sup.c 5 400 400
-- 200 of S156C- S-Biotin (0.329) 6 400 400 -- 200 of S166C-
S-Biotin (0.534) 7 400 400 -- 200 of N62C- S-Biotin (0.260) 8 400
400 -- 200 of L217C- S-Biotin (0.359) 9 400 400 -- 200 of
WT(0.143).sup.e 10 390 400 10 (0.060).sup.d 200 of WT (0.143).sup.e
11 390 400 10 (0.097).sup.f 200 of WT (0.143).sup.e 12 390 400 10
(0.047).sup.g 200 of WT (0.143).sup.e 13 390 400 10 (0.065).sup.h
200 of WT (0.143).sup.e 14 390 400 10 (0.649).sup.i 200 of WT
(0.143).sup.e .sup.aAssay Buffer: 20 mM Tris.HCl, 2 mM CaCl.sub.2,
pH 8.6. .sup.bHABA/avidin reagent (Sigma) prepared with 10 mL of
Milli-Q water. .sup.c200 .mu.L of MES buffer (10 mM MES, 1 mM
CaCl.sub.2, pH 5.8). .sup.dSolution of d-biotin (Sigma) diluted to
the same concentration (in 10 .mu.L) as in 200 .mu.L of active
S156C-S-Biotin (0.329 mg/mL) in Assay Buffer. .sup.eSolution of
lyophilized GG36-WT in 10 mM MES, 1 mM CaCl.sub.2, pH 5.8 (PMSF
corrected). .sup.fSolution of d-biotin (Sigma) diluted to the same
concentration (in 10 .mu.L) as in 200 .mu.L of active
S166C-S-Biotin (0.534 mg/mL) in Assay Buffer. .sup.gSolution of
d-biotin (Sigma) diluted to the same concentration (in 10 .mu.L) as
in 200 .mu.L of active N62C-S-Biotin (0.260 mg/mL) in Assay Buffer.
.sup.hSolution of d-biotin (Sigma) diluted to the same
concentration (in 10 .mu.L) as in 200 .mu.L of active
L217C-S-Biotin (0.359 mg/mL) in Assay Buffer. .sup.iSolution of
d-biotin (Sigma) diluted to the same concentration (in 10 .mu.L) as
for protein amount of 200 .mu.L of S156C-S-Biotin (as determined by
lyophilization) in Assay Buffer.
[0479] Before addition of biotin and enzyme the cuvette was
equilibrated in the spectrophotometer until A.sub.500 stabilized
(5-10 min). Biotin and enzyme, respectively, were added and
A.sub.500 was measured over a period of 5 min. Table 33 shows the
assay results:
34TABLE 33 Results of assay. HABA/ % of Max HABA/Avidin HABA/Avidin
Avidin HABA HABA Cuvette Before.sup.a/ After.sup.b/ Diff..sup.b/
Release/ Release.sup.b/ Number Abs Abs Abs Abs.sup.b,c Abs 1 0.535
0.382 0.153 -- -- 2 0.487 0.344 0.143 -- -- 3 0.482 0.353 0.129 --
-- 4 0.563 0.304 0.259 0.117 40 5 0.545 0.121 0.424 0.282 95 6
0.547 0.267 0.280 0.138 47 7 0.545 0.347 0.198 0.056 19 8 0.545
0.331 0.214 0.072 24 9 0.493 0.349 0.144 0.002 1 10 0.513 0.272
0.242 0.100 34 11 0.522 0.218 0.304 0.162 55 12 0.513 0.289 0.224
0.082 28 13 0.501 0.255 0.246 0.104 35 14 0.522 0.084 0.438 0.296
100 .sup.aMixture of HABA/avidin and buffer before addition of
enzyme (and biotin). .sup.bValue 5 min after addition of enzyme
(and biotin). .sup.cCalculated from difference between HABA/Avidin
drop in Abs. for sample and the drop in Abs. caused by dilution
alone (controls).
[0480] Avidin Hydrolysis Assay (Via A.sub.280
Mesaurement)--Measured for S156C-biotin Only
[0481] Twenty disposable eppendorf vials were filled as shown in
Table 34.
35TABLE 34 Avidin hydrolysis assay. Decoy Vial Buffer.sup.a/
Avidin.sup.b/ Protein.sup.c/ Biotin/ Enzyme/ Number .mu.L .mu.L
.mu.L .mu.L .mu.L 1 700 100 -- -- --.sup.d 2 600 100 100 --
--.sup.d 3 700 100 -- -- 200 of WT.sup.e 4 700 100 -- -- 200 of
WT.sup.e 5 600 100 100 -- 200 of WT.sup.e 6 600 100 100 -- 200 of
WT.sup.e 7 690 100 -- 10.sup.f 200 of WT.sup.e 8 690 100 --
10.sup.f 200 of WT.sup.e 9 590 100 100 10.sup.f 200 of WT.sup.e 10
590 100 100 10.sup.f 200 of WT.sup.e 11 690 100 -- 10.sup.g 200 of
WT.sup.e 12 690 100 -- 10.sup.g 200 of WT.sup.e 13 590 100 100
10.sup.g 200 of WT.sup.e 14 590 100 100 10.sup.g 200 of WT.sup.e 15
700 100 -- -- 200 of S156CMM 16 700 100 -- -- 200 of S156CMM 17 600
100 100 -- 200 of S156CMM 18 600 100 100 -- 200 of S156CMM 19 400
100 300 -- 200 of S156CMM 20 400 100 300 -- 200 of S156CMM
.sup.aAssay Buffer: 20 mM Tris.HCl, 2 mM CaCl.sub.2, pH 8.6.
.sup.b5 mg/mL solution of avidin (Sigma) in Milli-Q water.
.sup.c0.5 mg/mL solution of Ribonuclease A with Scrambled Disulfide
Bonds (Sigma) in Milli-Q water. .sup.d200 .mu.L of MES buffer (10
mM MES, 1 mM CaCl.sub.2, pH 5.8). .sup.eSolution of lyophilized
GG36-WT diluted to the same catalytic activity as the biotin-CMM
(as determined by initial rate kinetics with sAAPFpNA) in 10 mM
MES, 1 mM CaCl.sub.2, pH 5.8. .sup.fSolution of d-biotin (Sigma)
diluted to the same concentration (in 10 .mu.L) as in 200 .mu.L of
active S156C-S-Biotin (0.329 mg/mL) in Assay Buffer. .sup.gSolution
of d-biotin (Sigma) diluted to the same concentration (in 10 .mu.L)
as for protein amount of 200 .mu.L of S156C-S-Biotin (as determined
by lyophilization) in Assay Buffer.
[0482] These solutions were incubated at 35.degree. C. in a
thermostat-controlled water bath for the indicated time. The
contents of the appropriate vials were then each placed in the top
of a Centricon-SR3 Concentrator (Amicon, MWCO 3000, previously
cleaned by 2 mL of Milli-Q water centrifuged at 3750 rpm for 90
min) and centrifuged at 3750 rpm for 60 min. The resulting
filtrates were then assayed as shown in Table 35.
36TABLE 35 Assay. S156C-S-d-Biotin (0.329 mg/mL = 2.4 .mu.M) Vial
Incub. % of Max Total Number Time/min A.sub.280.sup.a Total
Protein/Abs Protein/Abs 1 60 0.002 -- -- 2 60 0.014 -- -- 3 60
0.013 0.005 4 4 240 0.028 0.020 17 5 60 0.040 0.032 27 6 240 0.059
0.051 44 7 60 0.015 0.007 6 8 240 0.030 0.022 19 9 60 0.043 0.035
30 10 240 0.059 0.051 44 11 60 0.013 0.005 4 12 240 0.029 0.021 18
13 60 0.035 0.027 23 14 240 0.046 0.038 32 15 60 0.054 0.046 39 16
240 0.081 0.073 62 17 60 0.058 0.050 43 18 240 0.080 0.072 62 19 60
0.103 0.095 81 20 240 0.125 0.117 100 .sup.aValue for 700 .mu.L of
Avidin Hydrolysis Assay filtrate diluted with 300 .mu.L of Milli-Q
as compared with Milli-Q water blank (1 mL).
[0483] Controls without Avidin
[0484] Assay performed as Avidin Hydrolysis Assay except 100 .mu.L
aliquots of avidin replaced by 100 .mu.L of Milli-Q water; the
measurement for S156C-S-Biotin and a higher decoy protein amount
[0.15 mg, Decoy (2)] was not repeated as control without avidin
(see Table 36).
37TABLE 36 Control hydrolysis assay without avidin. Vial Incub.
Total Protein/ % of Number Time/min A.sub.280.sup.a Abs Max Total
Protein - Abs 1 60 -0.001 -- -- 2 60 0.018 -- -- 3 60 0.000 -0.008
-7 4 240 0.009 0.001 0.9 5 60 0.020 0.012 10 6 240 0.035 0.027 23 7
60 0.003 -0.005 -4 8 240 0.010 0.002 2 9 60 0.024 0.016 14 10 240
0.029 0.021 18 11 60 0.001 -0.007 -6 12 240 0.012 0.004 3 13 60
0.026 0.018 15 14 240 0.029 0.021 18 15 60 0.028 0.020 17 16 240
0.024 0.016 14 17 60 0.039 0.031 26 18 240 0.052 0.044 38 .sup.aas
above.
[0485] Avidin Hydrolysis Assay (Via A.sub.280 Mesaurement) Using
S166C-biotin, L217C-biotin and N62C-biotin
[0486] The Avidin Hydrolysis Assay was also performed for
N62C-S-Biotin and S166C-S-Biotin in addition to S156C-S-Biotin
reported earlier. For comparison, we report the results for the
S156C CMM again.
[0487] S156C-S-Biotin produces 72% more protein fragments after 240
min than SBL-WT. In the presence of decoy protein [0.05 mg] the
amount of total protein produced increases drastically for the WT
enzyme (23% after 240 min) whereas the production of total protein
does not change significantly for the biotin-CMM.
[0488] N62C-S-Biotin provides nearly the same amount of protein
fragments as SBL-WT after 240 min. However, in the presence of a
decoy the N62C CMM gives only 5 % more protein release after 240
min and is therefore clearly more selective than SBL-WT (23% more
protein fragments after 240 min). Those results suggests that the
biotin side-chain of the N62C CMM is less available since overall
protein hydrolysis is less effective by this CMM compared to S156C
CMM which contains a surface exposed biotin moiety. However, since
in the N62 CMM the biotin side-chain is adjacent to the catalytic
center its avidin hydrolysis selectivity is nearly as effective as
for S156C-S-Biotin.
[0489] The S166C CMM gives a 7% higher protein release compared to
SBL-WT but is fairly unselective in the presence of a decoy protein
[18% protein fragments compared to 23% for SBL-WT after 240 min].
Although this enzyme proved to be the second best of the
biotin-CMMs in the "Avidin Targeting Assay", it is less selective
than the N62C CMM with respect to the "Avidin Hydrolysis Assay".
Presumably the biotin side chain buried in the S.sub.1 pocket is
available for avidin targeting but conformationally not very
favorable for the effective and selective catalysis of avidin
hydrolysis.
Experimental
[0490] Fourteen disposable eppendorf vials were filled as shown in
Table 37.
38TABLE 37 Setup for avidin hydrolysis assay. Decoy Vial
Buffer.sup.a/ Avidin.sup.b/ Protein.sup.c/ Biotin/ Enzyme/ Number
.mu.L .mu.L .mu.L .mu.L .mu.L 1 700 100 -- -- --.sup.d 2 600 100
100 -- --.sup.d 3 700 100 -- -- 200 of WT.sup.e 4 700 100 -- -- 200
of WT.sup.e 5 600 100 100 -- 200 of WT.sup.e 6 600 100 100 -- 200
of WT.sup.e 7 700 100 -- -- 200 of biotin-CMM.sup.f 8 700 100 -- --
200 of biotin-CMM.sup.f 9 600 100 100 -- 200 of biotin-CMM.sup.f 10
600 100 100 -- 200 of biotin-CMM.sup.f 11 690 100 -- 10.sup.g 200
of WT.sup.e 12 690 100 -- 10.sup.g 200 of WT.sup.e 13 590 100 100
10.sup.g 200 of WT.sup.e 14 590 100 100 10.sup.g 200 of WT.sup.e
.sup.aAssay Buffer: 20 mM Tris.HCl, 2 mM CaCl.sub.2, pH 8.6.
.sup.b5 mg/mL solution of avidin (Sigma) in Milli-Q water.
.sup.c0.5 mg/mL solution of Ribonuclease A with Scrambled Disulfide
Bonds (Sigma) in Milli-Q water. .sup.d200 .mu.L of MES buffer (10
mM MES, 1 mM CaCl.sub.2, pH 5.8). .sup.eSolution of lyophilized
GG36-WT diluted to the same catalytic activity as the biotin-CMMs
(as determined by initial rate kinetics with succ-AAPF-pNA) in 10
mM MES, 1 mM CaCl.sub.2, pH 5.8 (concentrations of biotin-CMMs
see.sup.f). .sup.fS156C-S-Biotin (0.329 mg/mL), N62C-S-Biotin
(0.260 mg/mL), S166C-S-Biotin (0.534 mg/mL); concentrations of
biotin-CMMs calculated for same catalytic activity (as determined
by initial rate kinetics with succ-AAPF-pNA); solutions in 10 mM
MES, 1 mM CaCl.sub.2, pH 5.8. .sup.gSolution of d-biotin (Sigma)
diluted to the same concentration (in 10 .mu.L) as in 200 .mu.L of
active biotin-CMM (0.060 mg/mL biotin for S156C-S-Biotin, 0.047
mg/mL biotin for N62C-S-Biotin, 0.097 mg/mL biotin for
S166C-S-Biotin) or as for protein amount of 200 .mu.L of
S156C-S-Biotin (as determined by lyophilization; 0.649 mg/mL
biotin) in Assay Buffer.
[0491] These solutions were incubated at 35.degree. C. in a
thermostat-controlled water bath for the indicated time. The
contents of the appropriate vials were then each placed in the top
of a Centricon SR3 or Centricon YM-3 Concentrator (Amicon, MWCO
3000, previously cleaned with 2 mL of Milli-Q water centrifuged at
3750 rpm for 90 min) and centrifuged at 3750 rpm for 60 min. The
resulting filtrates were then assayed and the results are shown in
:Table 38, Table 39, Table 40, Table 41, and Table 42.
39TABLE 38 Results for avidin hydrolysis assay for controls and WT
Vial Incub. % of Number Time/min A.sub.280.sup.a Total Protein/Abs
Max Total Protein/Abs 1 60 0.004 -- -- 2 60 0.013 -- -- 3 60 0.023
0.014 19 4 240 0.029 0.020 28 5 60 0.035 0.026 36 6 240 0.046 0.037
51 .sup.aValue determined in triplicate for 700 .mu.L of Avidin
Hydrolysis Assay filtrate diluted with 300 .mu.L of Milli-Q as
compared with Milli-Q water blank (1 mL).
[0492]
40TABLE 39 Results of avidin hydrolysis assay for S156C-S-Biotin.
Vial Incub. % of Number Time/min A.sub.280.sup.a Total Protein/Abs
Max Total Protein/Abs 7 60 0.054 0.045 62 8 240 0.081 0.072 100 9
60 0.058 0.049 68 10 240 0.080 0.071 99 .sup. 9.sup.b 60 0.103
0.094 131 10.sup.b 240 0.125 0.116 161 .sup.aValue for 700 .mu.L of
Avidin Hydrolysis Assay filtrate diluted with 300 .mu.L of Milli-Q
as compared with Milli-Q water blank (1 mL). .sup.b300 .mu.L
instead of 100 .mu.L of decoy protein used.
[0493]
41TABLE 40 Results of avidin hydrolysis assay for N62C-S-Biotin.
Vial Incub. % of Number Time/min A.sub.280.sup.a Total Protein/Abs
Max Total Protein/Abs 7 60 0.018 0.009 12 8 240 0.032 0.023 32 9 60
0.029 0.020 28 10 240 0.036 0.027 37 .sup.aAs above.
[0494]
42TABLE 41 Results of avidin hydrolysis assay for S166C-S-Biotin
Vial Incub. Total Protein/ % of Max Total Number Time/min
A.sub.280.sup.a Abs Protein/Abs 7 60 0.026 0.017 24 8 240 0.034
0.025 35 9 60 0.033 0.024 33 10 240 0.047 0.038 53 .sup.aAs
above.
[0495]
43TABLE 42 Controls of avidin hydrolysis by SBL-WT with different
amounts of biotin added Vial Incub. Total Protein/ % of Max Number
Time/min A.sub.280.sup.a Abs Total Protein/Abs 11.sup.b 60 0.015
0.006 8 12.sup.b 240 0.030 0.021 29 13.sup.b 60 0.045 0.036 50
14.sup.b 240 0.051 0.042 58 11.sup.c 60 0.014 0.005 7 12.sup.c 240
0.026 0.017 24 13.sup.c 60 0.030 0.021 29 14.sup.c 240 0.039 0.030
42 11.sup.d 60 0.021 0.012 17 12.sup.d 240 0.032 0.023 32 13.sup.d
60 0.036 0.027 37 14.sup.d 240 0.047 0.038 53 11.sup.e 60 0.013
0.004 5 12.sup.e 240 0.029 0.020 28 13.sup.e 60 0.035 0.026 36
14.sup.e 240 0.046 0.037 51 .sup.aAs above. .sup.bSame biotin
amount as in active S156C-S-Biotin. .sup.cSame biotin amount as in
active N62C-S-Biotin. .sup.dSame biotin amount as in active
S166C-S-Biotin. .sup.eSame biotin amount as in S156C-S-Biotin
calculated for whole protein amount (active and inactive
enzyme).
Example 11
Targeting Antibodies Using a Hapten Modified Subtilisin
[0496] As an extension of the targeted degradation of enzymes, we
have now focused on hapten directed degradation of antibodies by
SBL. This example demonstrates antibody targeting using an
anti-biotin antibody/biotin system.
[0497] Preparation of biotin-MTS and Biotinylated CMMs
[0498] In order to target anti-biotin with SBL, we have attached
the biotin-MTS reagent to our mutant enzymes. The synthesis of the
biotin-MTS was accomplished as outlined in Example 8. Each of the
CMMs was prepared according to standard protocol, e.g., as
described in Example 9. All CMMs were characterized using MALDI
technique which has an method-dependent error in the magnitude of
.+-.0.2-0.5%. The results are shown in Table 43 and Table 44.
44TABLE 43 MALDI-MS for biotinylated CMMs Enzyme Calculated mass
Found % error N62C-Biotin 26931 26943.7 0.04 S156C-Biotin 26958
26980.4 0.08 S166C-Biotin 26958 26977.5 0.07 L217C-Biotin 26932
26971.7 0.14
[0499] The amidase activities of the new biotinylated CMMs were
determined and show the same trend as described above in Example 9.
The results are shown in Table 36.
45TABLE 44 Amidase Activity for Biotinylated CMMs Amidase activity
Enzyme k.sub.cat K.sub.M k.sub.cat/K.sub.M WT.sup.a 153 0.73 209
N62C-Biotin 108.86 0.96 113.40 S156C-Biotin 59.46 0.78 76.46
S166C-Biotin 46.00 0.999 45.83 L217C-Biotin 55.35 1.07 51.73
[0500] Targeting Assay for Biotinylated CMMs to Anti-biotin
[0501] Antibodies to biotin (anti-Biotin) are commercially
available as either free antibody or as an enzyme-conjugate. We
chose an anti-biotin conjugated to alkaline phosphatase as our
model target antibody. Using the standard Enzyme Linked
Immuno-sorbent Assay (ELISA)-technique, we could demonstrate the
ability of our CMMs to target the antibody. The experiment is
outlined schematically in FIG. 17.
[0502] Our first assay series was carried out using the
ELISA-technique (FIG. 17) with polystyrene 96-well plates (Harlow
and Lane (1988) Antibodies: A laboratory Manual: Cold Spring Habour
Laboratory, USA, p. 564-597). The CMM was immobilized on the plate
surface overnight at 4.degree. C. [typical binding of protein to a
polystyrene plate is approximately 100 ng/well (300 ng/cm.sup.2).
In our case, we attached less protein as we did not fully fill each
well with enzyme solution]. After blocking the remaining binding
sites on the plate with BSA, unbound protein (loosely antibody and
BSA) was washed out twice with phosphate-buffered saline containing
Tween, pH 7.2 (PBST). (Tween was used in this buffer to prevent the
unbound polystyrene surface from the attachment of antibody.) Then
the anti-Biotin was added to each well and incubated at 4.degree.
C. for 2 h. The plate was washed with PBST (4 times) to remove the
loosely, unbound protein, and then with glycine buffer pH 10.4 (2
times) to wash away the phosphate buffer and to optimize the pH for
alkaline phosphatase activity. To assay the biotin-CMM/anti-biotin
binding the enzyme activity of the antibody-linked alkaline
phosphatase was used. The phosphatase substrate [a solution of
p-nitrophenylphosphate disodium salt (PNPP) in glycine buffer pH
10.4] was added and the reaction was carried out at 4.degree. C.
The release of p-nitrophenolate was determined visually using a
96-well plate (FIG. 17).
[0503] From the 96-well results, we found that the amount of PNPP
substrate is very important for demonstrating the differences of
the targeting abilities of our biotin-CMMs to anti-biotin. Using a
high amount of substrate we were unable to distinguish between
different CMMs, as all gave a bright yellow colour within 30 min.
However, we were still able to discern the location of the
S156C-biotin on the plate since the color change was extremely
rapid in this case.
[0504] We also carried out a control reaction, using PMSF as an
inhibitor for WT-and CMM-SBL (as SBL might hydrolyze anti-biotin
leading to a negative result). No difference between the reactions
with and without PMSF could be found. Therefore we excluded this
reagent from our following experiments.
[0505] Using the same protocol and conditions as for the 96-well
plate experiments, later experiments were carried out in
polystyrene cuvettes and the absorption (A.sub.405) of released
p-nitrophenolate from PNPP (150 .mu.L in 1 mL) was monitored
spectrometrically (Table 45). Because of the high dilution of PNPP
used, the reaction had to be monitored for a long time. It should
be noted that the results of the assay using cuvettes are not
always as consistent as those from 96-well plates. This was
probably due to the fact that 96-well plates have been developed
for protein attachment. The assay results are shown in Table
37.
46TABLE 45 A.sub.405 assay (p-nitrophenolate release, low PNPP
concentration) A.sub.405 S166C- L217C- time (h) WT N62C-Biotin
S156C-Biotin Biotin Biotin 18 0.145 1.018 1.111 0.483 0.292 24
0.173 1.191 1.322 0.569 0.348 27 0.222 1.522 1.689 0.736 0.451 30
0.259 1.739 1.915 1.065 0.526
[0506] All CMMs caused a higher release of p-nitrophenolate as
compared to WT. S156C-biotin was found to induce the greatest
p-nitrophenolate release (up to 86.5% more than WT after 30 h),
showing it to be the most proficient CMM for Anti-biotin IgG
targeting.
[0507] Further experiments suggested that the concentration of PNPP
can be increased (300 .mu.L in 1 mL) to shorten the observation
time. The trend of the p-nitrophenolate release is the same as in
the low PNPP concentration assay. The results are shown in Table
46.
47TABLE 46 A.sub.405 Assay (p-Nitrophenolate Release, high PNPP
concentration) A.sub.405 S156C- S166C- L217C- time (h) WT
N62C-Biotin Biotin Biotin Biotin 6 0.214 0.667 1.040 0.647 0.483 12
0.398 0.969 1.400 0.908 0.577
[0508] All biotinylated CMMs gave an increased p-nitrophenolate
release compared to WT. Therefore binding of the biotin-CMMs to
anti-biotin must have been occured. The assay results (Tables 37
and 38) clearly demonstrate the ability of our biotinylated CMMs to
target a biotin-antibody.
[0509] "Hydrolysis" Assay of Anti-iotin by Biotinylated CMMs
[0510] Next, we were interested in demonstrating the ability of our
CMMs to hydrolyze the anti-biotin selectively. We adopted the
approach used successfully to monitor the release of protein
fragments during SBL-mediated avidin hydrolysis. It should be noted
that equal concentrations of active enzymes (as determined by PMSF
titration) were used in these experiments. The enzymes and
anti-biotin IgG were incubated in Tris buffer (20 mM Tris HCl, 2 mM
CaCl.sub.2, pH 8.6) at 35.degree. C. for 60 and 240 min. The
protein fragments were separated from the crude hydrolysate using
size exclusion-membranes as reported previously. Measurement of the
absorption at 280 nm afforded the concentration of the released
protein fragments. The results are shown in Table 47.
48TABLE 47 Anti-biotin hydrolysis assay (A.sub.280). Enzyme
A.sub.280 (60 min) A.sub.280 (240 min) anti-Biotin + Tris (blank)
0.004 0.008 WT 0.086 0.087 N62C-Biotin 0.102 0.112 S156C-Biotin
0.140 0.177 S166C-Biotin 0.115 0.127 L217C-Biotin 0.069 0.125
[0511] The results, except for one (L217C-Biotin), demonstrate
clearly that our CMMs are able to hydrolyze anti-Biotin better than
WT. These results also correspond to the targeting results
(A.sub.405 assay for p-nitrophenolate release) which show that all
biotinylated CMMs target anti-biotin and therefore give a higher
p-nitrophenolate release than WT.
[0512] To determine whether or not the hydrolysis was specifically
towards anti-biotin, we adopted the biotin assay experiment again
which uses RNAase as a decoy protein. The results for each CMM
hydrolysis compared to WT are shown in Table 48, Table 49, Table
50, and Table 51.
49TABLE 48 Assay (A.sub.280) for selective hydrolysis of
anti-biotin by biotin-CMM N62C-biotin and by WT.sup.a A.sub.280
time WT + N62C- N62C- (min) WT decoy difference Biotin Biotin +
decoy difference 60 0.040 0.148 0.108 0.045 0.068 0.023 240 0.040
0.148 0.108 0.052 0.075 0.023
[0513]
50TABLE 49 Assay (A.sub.280) for selective hydrolysis of
anti-biotin by biotin-CMM S156C-biotin and by WT.sup.a A.sub.280
time WT + S156C- S156C- (min) WT decoy difference Biotin Biotin +
decoy difference 60 0.040 0.148 0.108 0.110 0.112 0.002 240 0.040
0.148 0.108 0.134 0.148 0.014
[0514]
51TABLE 50 Assay (A.sub.280) for selective hydrolysis of
anti-biotin by biotin-CMM S166C-biotin and by WT.sup.a A.sub.280
time WT + S166C- S166C- (min) WT decoy difference Biotin Biotin +
decoy difference 60 0.040 0.148 0.108 0.074 0.085 0.011 240 0.040
0.148 0.108 0.087 0.090 0.003
[0515]
52TABLE 51 Assay (A.sub.280) for selective hydrolysis of
anti-biotin by biotin-CMM L217C-biotin and by WT.sup.a A.sub.280
time WT + L217C- L217C- (min) WT decoy difference Biotin Biotin +
decoy difference 60 0.040 0.148 0.108 0.037 0.060 0.023 240 0.040
0.148 0.108 0.053 0.065 0.012 .sup.aThe numbers in the table are
corrected with A.sub.280 of the background reaction.
[0516] The experiments reveal that the differences in extend of
hydrolysis between the reactions carried out in the presence and
absence of decoy protein for CMMs catalyzed reactions are much
smaller than for WT catalyzed reactions. Hence, as expected, our
biotinylated CMMs hydrolyze anti-biotin more specifically than WT.
Control experiments were carried out without anti-biotin (only
SBL-CMM and RNAase) as well as with SBL-CMM and Tris buffer alone
(without RNAase and anti-Biotin). The background absorption was
insignificant in all cases.
Experimental
[0517]
5-([3aS-(3a.alpha.,4.beta.,6a.alpha.)]-Hexahydro-2-oxo-1H-thieno[3,-
4-d]imidazol-4-yl)pentyl methanethiosulfonate [(+)-Biotin-MTS]
[0518] The Biotin-MTS was prepared according to the procedure
described in Example 8.
[0519] Materials
[0520] Phosphate buffered saline (PBS) solution was prepared from 8
g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4, 0.24 g
KH.sub.2PO.sub.4 in 1 L water, adjust pH to 7.2 with 1N HCl and was
stored at room temperature. 10% Sodium azide solution was prepared
from 10 g NaN.sub.3 in 100 mL water and was stored at room
temperature. 3% BSA in PBS solution was prepared from 3 g bovine
serum albumin (fraction V) in 100 mL water, then 0.2 mL 10%
NaN.sub.3 solution added and was stored at 4.degree. C. Phosphate
buffered saline with Tween (PBST) solution was prepared from 0.5 mL
of Tween 80 in 1 L PBS solution. Anti-biotin solution (1:30,000)
solution was prepared from 16.67 .mu.L anti-biotin (Sigma A-6561
clone BN-34, conc. 1.15 mg/mL) in 50 mL 3% BSA/PBS and was stored
at 4.degree. C. 0.1 M glycine buffer pH 10.4 solution was prepared
from 7.51 g glycine, 203 mg MgCl.sub.2 H.sub.2O, 136 mg ZnCl.sub.2
in 1 L water. The pH was adjusted to pH 10.4 with 10 N NaOH.
p-Nitrophenylphosphate 1 mg/mL solution was prepared from I tablet
PNPP (Sigma N-2765) (20 mg) in 20 mL of 0.1 M glycine buffer and
was stored at 4.degree. C. 0.06 M PMSF solution was prepared from
47.2 mg .alpha.-toluenesulfonyl fluoride (PMSF) in 449.2 .mu.L EtOH
and was stored at 0.degree. C. The following enzymes were used: WT
(1mg/mL), N62C-Biotin (0.97 mg/mL), S156C-Biotin (0.71 mg/mL),
S166C-Biotin (1.09 mg/mL) and L217C-Biotin (1.0 mg/mL). All enzymes
were dissolved in MES buffer (20 mM MES, 1 mM CaCl.sub.2, pH 5.8).
0.1 M Tris Buffer pH 8.6 consisted of 1.21 mg Tris in 100 mL water.
The pH was adjusted to pH 8.6 with conc. HCl. Ribonuclease A, 5
mg/mL consisted of 5 mg of ribonuclease A (with scrambled disulfide
bonds, Sigma R-2638) in 1 mL water.
[0521] Targeting Assay of CMM-SBL to Anti-biotin Using Polystyrene
96-Well Plate
[0522] 50 .mu.L of each enzyme solution was added to each well of a
polystyrene 96-well plate as shown in FIG. 18. All reactions were
conducted twice to verity the reproducibility of the results.
[0523] The plate was incubated at 4.degree. C. overnight. The
remaining enzyme solutions were removed using a pipette. Then,
BSA/PBS (3%, 100 .mu.L) was added to each well and the plate was
incubated at RT for 1 h. The BSA solution was removed and the plate
was dried by flicking and banging it on layers of paper towel. PMSF
(10 .mu.L) was added to columns 1, 3, 5, 7, 9, 11 row A and B.
After this procedure the plate was washed with PBST (2.times.) and
dried as described previously. Anti-Biotin solution (30 .mu.L) was
added to each well, except for 1, 3, 5, 7, 9, 11 C and D, and the
plate was kept for 2 h at 4.degree. C. to minimize the proteolysis
of antibody by the enzymes. The anti-Biotin solution was removed,
the plate was washed with PBST (4.times.) to remove unbound
antibody, and was dried in order to remove unbound antibody. The
phosphate buffer was removed and the pH was adjusted for alkaline
phosphatase activity by washing the plate with glycine buffer (0.1
M, pH 10.4) (2.times.) and drying afterwards. Then, glycine buffer
and PNPP solution were added to the wells in the following
manner:
[0524] 1. Column 1, 3, 5, 7, 9, 11 row A and B (already treated
with 10 .mu.L PMSF solution) were filled with 40 .mu.L 1 mg/mL
PNPP.
[0525] 2. Column 2, 4, 6, 8, 10, 12 row A and B were filled with 50
.mu.L 1 mg/mL PNPP.
[0526] 3. Column 2, 4, 6, 8, 10, 12 row C and D were filled with 25
.mu.L 1 mg/mL PNPP+25 .mu.L glycine buffer.
[0527] 4. Column 2, 4, 6, 8, 10, 12 row E and F were filled with
12.5 .mu.L 1 mg/mL PNPP+37.5 .mu.L glycine buffer.
[0528] 5. Column 2, 4, 6, 8, 10, 12 row G and H were filled with 6
.mu.L 1 mg/mL PNPP+44 .mu.L glycine buffer.
[0529] 6. Column 1,3,5,7,9, 11 row C and D were filled with 50
.mu.L 1 mg/mL PNPP.
[0530] The assay results can be summarized as follows:
[0531] The reactions in columns 1, 2 and 3 turned yellow in color
almost instantly for all enzymes. After. 15 min, the reactions in 4
for S156C-Biotin started to visibly change their color to pale
yellow. S166C- started changing color after 20 min. N62C- started
changing color after 1 h. After 1.5 h, there was no color change
for WT and L217C-. The reactions in 6 for S156C- gave a color
change after 2 h. All the reactions had strong yellow color after
incubation overnight at 4.degree. C.
[0532] Targeting Assay of CMM-SBL to Anti-Biotin Using Polystyrene
Cuvettes
[0533] The immobilization of the enzymes and anti-biotin and the
washing processes for the cuvettes were conducted in the manner
similar to that described for the 96-well plate. Two different
concentrations of PNPP were used. The experiments were carried out
in duplicate and the data shown in Table 52 below are the average
results:
53TABLE 52 Targeting assay in polystyrene cuvettes Glycine
A.sub.405.sup.a PNPP Buffer time (h) Enzyme .mu.L .mu.L 6 12 18 24
27 30 WT 150 850 -- -- 0.145 0.173 0.222 0.259 N62C- 150 850 -- --
1.108 1.191 1.522 1.739 S156C- 150 850 -- -- 1.111 1.322 1.689
1.915 S166C- 150 850 -- -- 0.483 0.569 0.736 1.065 L217C- 150 850
-- -- 0.292 0.348 0.451 0.526 WT 300 700 0.214 0.398 -- -- -- N62C-
300 700 0.667 0.969 -- -- -- S156C- 300 700 1.040 1.400 -- -- --
S166C- 300 700 0.647 0.908 -- -- -- L217C- 300 700 0.483 0.577 --
-- -- .sup.aGlycine buffer was used to autozero the background
absorption.
[0534] "Hydrolysis Assay" of Anti-biotin with Biotin-CMMs and
WT
[0535] Eppendorf vials were filled according to Table 43. The vials
were then incubated at 35.degree. C. in a thermostat-controlled
water bath for 60 and 240 min. The contents of each vial were then
placed in the top of a Centricon YM-3 filter (Amicon, MWCO 3000,
pre-rinsed with 2 mL Milli-Q water centrifuged at 3750 rpm for 90
min) and centrifuged at 3750 rpm for 60 min. The filtrates were
then assayed by measuring A.sub.280 (zeroed against with Milli-Q
water). The results are as shown in Table 53.
54TABLE 53 Hydrolysis assay of anti-niotin with niotin-CMMs and WT
A.sub.280.sup.b Anti-Biotin Tris Buffer RNAase time (min)
Enzyme.sup.a .mu.L .mu.L .mu.L 60 240 -- 50 940 -- 0.004 0.008 --
50 930 10 0.005 0.007 WT -- 990 -- 0.046 0.050 N62C- -- 990 --
0.057 0.060 S156C- -- 990 -- 0.028 0.029 S166C- -- 990 -- 0.041
0.040 L217C- -- 990 -- 0.072 0.072 WT -- 980 10 0.025 0.036 N62C-
-- 980 10 0.055 0.060 S156C- -- 980 10 0.045 0.054 S166C- -- 980 10
0.040 0.045 L217C- -- 980 10 0.051 0.059 WT 50 940 -- 0.086 0.087
N62C- 50 940 -- 0.102 0.112 S156C- 50 940 -- 0.140 0.117 S166C- 50
940 -- 0.115 0.177 L217C- 50 940 -- 0.069 0.125 WT 50 930 10 0.173
0..184 N62C- 50 930 10 0.123 0.135 S156C- 50 930 10 0.155 0.188
S166C- 50 930 10 0.125 0.135 L217C- 50 930 10 0.114 0.124 .sup.a1
mg of active enzyme was used for each experiment (WT = 11.5 .mu.L,
N62C- = 11.8 .mu.L, S156C- = 16.2 .mu.L, S166C- = 10.6 .mu.L,
L217C- = 11.5 .mu.L). The concentration of each enzyme as
determined by PMSF titration is described in the Materials Section.
.sup.bThe data show the actual absorption (data are corrected by
subtracting the actual absorption from the corresponding background
absorption).
Example 12
Assay for Residual Binding Capabilities of Anti-Biotin After
Exposure to Biotinyl-CMMs
[0536] Anti-biotin Degradation
[0537] The ability of Si156C-biotin to destroy anti-biotin IgG was
examined using micro-partition experiments. Two control experiments
were also performed to validate the data.
[0538] Aliquots of an antibody-CMM (1:1, molecule:molecule) mixture
were periodically withdrawn from an incubated vial (pH 8.6,
35.degree. C.), and biotin solution was added to the aliquot. It
was anticipated that only one CMM molecule would bind to each
antibody molecule, as the steric bulk of the CMM would block the
approach of a second CMM molecule to the other available binding
site of the antibody. However, molecular biotin should be
sufficiently small to allow it to bind to the second antibody
binding site. As the antibody binding sites are degraded, fewer
binding sites will be available resulting in more unbound biotin.
Using a 3000 MWCO membrane, unbound biotin was separated from the
macromolecules, and the biotin concentration was assayed using the
HABA/avidin system.
[0539] Additionally, control experiments were performed containing
biotin (hapten) only. Experiments where the antibody had been
pre-incubated with biotin (hapten) before addition of the CMM were,
also studied. Reduction in the observed value of A.sub.500
corresponds to diminution of the antibody's binding ability (FIG.
19).
[0540] Assay to Prove Destruction of Anti-biotin Binding Sites
Experimental
[0541] Monoclonal anti-biotin IgG (clone BN-34, Sigma,
immunoglobulin concentration 1.3 mg/mL) conjugated to alkaline
phosphatase was used for all studies. The TRIS buffer employed for
these studies was the standard buffer employed for our amidase
kinetic studies (0.1 M, pH 8.6, 0.005% Tween).
[0542] A solution of biotin (1.94 mg/mL) in TRIS (pH 8.6) was
prepared. 10 .mu.L of this solution was added to 779.5 .mu.L of
TRIS (this solution will be referred to as B/79 throughout). An
Eppendorf vial containing TRIS (700.5 .mu.L) and anti-biotin (37.5
.mu.L, 1.3 mg/mL immunoglobulin, alkaline phosphatase labelled) was
prepared. S156C-biotin. (12.15 .mu.L, 0.71 mg/mL) was added to the
vial. The vial was vortexed, and was placed in a water bath
(35.degree. C.). Aliquots (170 .mu.L) were periodically withdrawn
from the vial, B/79 (20 .mu.L) was added to each aliquot, and the
mixture was then transferred to a pre-rinsed Centricon filter. The
filters were centrifuged (2.times.1 h, 3750 rpm, 4.degree. C.), and
the filtrate was collected. The filtrate was assayed for biotin
content using the HABA/avidin system: 70 .mu.L of the filtrate was
added to 70 .mu.L of HABA/avidin reagent, and the A.sub.500 reading
of the mixture was observed using a 130 .mu.L cuvette. The results
are tabulated below in Table 54.
55TABLE 54 A.sub.500 results from anti-biotin binding site
degradation assay. Incubation A.sub.500 time/h CMM then hapten 0
0.3172 1 0.31 2 0.2945 3 0.2907
[0543] Control Experiments
[0544] A solution of biotin (1.94 mg/mL) in TRIS (pH 8.6) was
prepared. The solution was diluted twenty-fold (referred to
throughout as B/20). Eppendorfyvials were filled according to Table
55, and incubated for varying times at 35.degree. C.
56TABLE 55 Volumes used for control experiments (each prepared in
quadruplicate) Contents of each vial for each time point
anti-biotin S156C- B/20 Experiment TRIS (.mu.L) (.mu.L) biotin
(.mu.L) (.mu.L) Ab then B/20. Incubate 693 37.5 0 20 Ab then B/20,
then CMM. 681 37.5 12.15 20 Incubate
[0545] After incubation, the contents of each vial were transferred
to a pre-rinsed Centricon filter (MWCO 3000), and the filters were
centrifuged. The filtrates were assayed spectrophotometrically
using the HABA/avidin reagent: 500 .mu.L of filtrate was added to
500 .mu.L of HABA/avidin reagent, and the A.sub.500 value was
determined using a 1 mL cuvette. The results are shown in Table
56.
57TABLE 56 A.sub.500 results for control experiments Incubation
time/h hapten then CMM hapten only 0 0.252 0.2687 1 0.2466 0.2659 2
0.2531 0.2704 3 0.2517 0.2702
Example 13
Stoichiometry of the Targeted Degradation Examples Above
[0546] In various experiments the following catalytic antagonists
were found to function substoichoimetrically: Pyrazole CMM-HLADH
(Example 2), pyrazole CMM-HLADH in the presence of AP (Example 3),
substoichiometric pyrazole CMM-HLADH (Example 4), sugar
CMM-biotinylated Con A (Examples 7 and 8), biotin CMM-Avidin, and
biotin CMM-anti-biotin IgG.
[0547] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *