U.S. patent application number 12/378047 was filed with the patent office on 2009-11-05 for therapeutic peptidomimetic macrocycles.
This patent application is currently assigned to AILERON Therapeutics, Inc.. Invention is credited to David Allen Annis, Jiawen Han, Rosana Kapeller-Libermann, Noriyuki Kawahata, Huw M. Nash, Tomi K. Sawyer.
Application Number | 20090275519 12/378047 |
Document ID | / |
Family ID | 40952614 |
Filed Date | 2009-11-05 |
United States Patent
Application |
20090275519 |
Kind Code |
A1 |
Nash; Huw M. ; et
al. |
November 5, 2009 |
Therapeutic peptidomimetic macrocycles
Abstract
The present invention provides biologically active
peptidomimetic macrocycles for the treatment of cell proliferative
disorders such as cancer and immunoproliferative disease.
Inventors: |
Nash; Huw M.; (Concord,
MA) ; Annis; David Allen; (Cambridge, MA) ;
Kapeller-Libermann; Rosana; (Chestnut Hill, MA) ;
Sawyer; Tomi K.; (Southborough, MA) ; Kawahata;
Noriyuki; (Somerville, MA) ; Han; Jiawen;
(Newton, MA) |
Correspondence
Address: |
WILSON, SONSINI, GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Assignee: |
AILERON Therapeutics, Inc.
Cambridge
MA
|
Family ID: |
40952614 |
Appl. No.: |
12/378047 |
Filed: |
February 9, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61027326 |
Feb 8, 2008 |
|
|
|
61120380 |
Dec 5, 2008 |
|
|
|
Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 3/00 20180101; C07K 7/64 20130101; A61K 38/12 20130101; A61P
35/02 20180101; A61K 38/1761 20130101; A61K 45/06 20130101 |
Class at
Publication: |
514/13 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of treating cancer in a human patient in need thereof
comprising administering to the patient a peptidomimetic
macrocycle, wherein the cancer is selected from the group
consisting of ovarian cancer, prostate cancer, renal cancer, breast
cancer, pancreatic cancer, and Ph+ acute lymphocytic leukemia.
2. The method of claim 1, wherein the peptidomimetic macrocycle
comprises an .alpha.-helix.
3. The method of claim 1, wherein the peptidomimetic macrocycle
comprises a BH3 domain.
4. The method of claim 1, wherein the peptidomimetic macrocycle is
a BIM polypeptide.
5. The method of claim 4, wherein an amino acid sequence of said
BIM polypeptide is more than about 60% identical to an amino acid
sequence IWIAQELR*IGD*FNAYYARR (SEQ ID NO: 1) and wherein * is a
tethered amino acid.
6. The method of claim 4, wherein an amino acid sequence of said
BIM polypeptide is more than about 80% identical to an amino acid
sequence IWIAQELR*IGD*FNAYYARR (SEQ ID NO: 1) and wherein * is a
tethered amino acid.
7. The method of claim 4, wherein an amino acid sequence of said
BIM polypeptide is more than about 95% identical to an amino acid
sequence IWIAQELR*IGD*FNAYYARR (SEQ ID NO: 1) and wherein * is a
tethered amino acid.
8. The method of claim 1, wherein the cancer is breast cancer.
9. The method of claim 8, wherein the breast cancer is an invasive
breast carcinoma.
10. The method of claim 9, wherein the invasive breast carcinoma is
invasive ductal carcinoma.
11. The method of claim 1, wherein the cancer is prostate
cancer.
12. The method of claim 1, wherein the cancer is ovarian
cancer.
13. The method of claim 1, wherein the cancer is pancreatic
cancer.
14. The method of claim 1, wherein the cancer is renal cancer.
15. The method of claim 1, wherein the cancer is leukemia.
16. The method of claim 1, wherein the cancer is Ph+ acute
lymphocytic leukemia.
17. A method of treating cancer in a human patient in need thereof
comprising administering to the patient a peptidomimetic
macrocycle, wherein the cancer is colon cancer.
18. The method of claim 17, wherein the peptidomimetic macrocycle
comprises an .alpha.-helix.
19. The method of claim 17, wherein the peptidomimetic macrocycle
comprises a BH3 domain.
20. The method of claim 19, wherein the peptidomimetic macrocycle
is a BID polypeptide.
21. The method of claim 20, wherein an amino acid sequence of said
BID polypeptide is more than about 60% identical to a sequence
DIIRNIARHLA*VGD*NleDRSI (SEQ ID NO: 2) and wherein * is a tethered
amino acid and Nle is norleucine.
22. The method of claim 20, wherein an amino acid sequence of said
BID polypeptide is more than about 80% identical to a sequence
DIIRNIARHLA*VGD*NleDRSI (SEQ ID NO: 2) and wherein * is a tethered
amino acid and Nle is norleucine.
23. The method of claim 20, wherein an amino acid sequence of said
BID polypeptide is more than about 95% identical to a sequence
DIIRNIARHLA*VGD*NleDRSI (SEQ ID NO: 2) and wherein * is a tethered
amino acid and Nle is norleucine.
24. A method of treating cancer in a human patient in need thereof
comprising administering to the patient a peptidomimetic macrocycle
wherein said peptidomimetic macrocycle shows an EC.sub.50 lower
than 5 .mu.M when tested in an in vitro cell viability assay
against a cell line derived from said cancer.
25. The method of claim 24, wherein the EC.sub.50 is lower than 3
.mu.M.
26. The method of claim 24, wherein the cancer is selected from the
group consisting of ovarian cancer, skin cancer, prostate cancer,
renal cancer, breast cancer, pancreatic cancer, small-cell lung
cancer, colon cancer, liver cancer, Multiple myeloma, Burkitt's
lymphoma, acute lymphocytic leukemia (ALL) of T cell lineage or B
cell lineage or mixed lineage, Chronic lymphocytic leukemia (CLL),
Cutaneous T cell lymphoma (CTCL), Acute myelocytic leukemia (AML),
Chronic Myelocytic leukemia, and follicular lymphoma.
27. The method of claim 26, wherein the peptidomimetic macrocycle
comprises an .alpha.-helix.
28. The method of claim 26, wherein the peptidomimetic macrocycle
comprises a BH3 domain.
29. The method of claim 26, wherein the peptidomimetic macrocycle
is a BIM polypeptide.
30. The method of claim 29, wherein the BIM polypeptide is more
than about 60% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR (SEQ ID NO: 1) and wherein * is a tethered
amino acid.
31. The method of claim 29, wherein the BIM polypeptide is more
than about 80% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR (SEQ ID NO: 1) and wherein * is a tethered
amino acid.
32. The method of claim 29, wherein the BIM polypeptide is more
than about 95% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR (SEQ ID NO: 1) and wherein * is a tethered
amino acid.
33. The method of claim 24, wherein the cancer is selected from the
group consisting of colon cancer, small-cell lung cancer, liver
cancer, ovarian cancer, skin cancer, prostate cancer, renal cancer,
breast cancer, pancreatic cancer, Multiple myeloma, Burkitt's
lymphoma, acute lymphocytic leukemia (ALL) of T cell lineage or B
cell lineage or mixed lineage, Chronic lymphocytic leukemia (CLL),
Cutaneous T cell lymphoma (CTCL), Acute myelocytic leukemia (AML),
Chronic Myelocytic leukemia and follicular lymphoma.
34. The method of claim 33, wherein the peptidomimetic macrocycle
comprises an .alpha.-helix.
35. The method of claim 33, wherein the peptidomimetic macrocycle
comprises a BH3 domain.
36. The method of claim 33, wherein the peptidomimetic macrocycle
is a BID polypeptide.
37. The method of claim 36, wherein the BID polypeptide is more
than about 60% identical to a sequence DIIRNIARHLA*VGD*NleDRSI (SEQ
ID NO: 2 and wherein * is a tethered amino acid and Nle is
norleucine.
38. The method of claim 36, wherein the BID polypeptide is more
than about 80% identical to a sequence DIIRNIARHLA*VGD*NleDRSI (SEQ
ID NO: 2) and wherein * is a tethered amino acid and Nle is
norleucine.
39. The method of claim 36, wherein the BID polypeptide is more
than about 95% identical to a sequence DIIRNIARHLA*VGD*NleDRSI (SEQ
ID NO: 2) and wherein * is a tethered amino acid and Nle is
norleucine.
40. A method of treating a disorder in a human patient in need
thereof comprising: a) preparing a peptidomimetic macrocycle by
introducing a cross-link between two amino acid residues of a
polypeptide; b) testing the peptidomimetic macrocycle for the
presence or absence of an immunogenic response; and c)
administering the peptidomimetic macrocycle to a patient if said
immunogenic response does not cause a substantial side-effect.
41. The method of claim 40, wherein said immunogenic response is
evidenced as minimal antibody response in an in vivo assay in
rodents.
42. The method of claim 40, wherein the disorder is cancer.
43. The method of claim 40, wherein the disorder is a metabolic
disorder.
44. The method of claim 40, wherein the peptidomimetic macrocycle
comprises an .alpha.-helix.
45. The method of claim 40, wherein the peptidomimetic macrocycle
comprises a BH3 domain.
46. A method of treating an immunoproliferative disorder in a human
patient in need thereof comprising administering to the patient a
peptidomimetic macrocycle.
47. The method of claim 46, wherein the peptidomimetic macrocycle
comprises an .alpha.-helix.
48. The method of claim 46, wherein the peptidomimetic macrocycle
comprises a BH3 domain.
49. The method of claim 46, wherein the peptidomimetic macrocycle
is a BID polypeptide.
50. The method of claim 49, wherein the BID polypeptide is more
than about 60% identical to a sequence DIIRNIARHLA*VGD*NleDRSI (SEQ
ID NO: 2) and wherein * is a tethered amino acid and Nle is
norleucine.
51. The method of claim 49, wherein the BID polypeptide is more
than about 80% identical to a sequence DlIRNIARHLA*VGD*NleDRSI (SEQ
ID NO: 2) and wherein * is a tethered amino acid and Nle is
norleucine.
52. The method of claim 49, wherein the BID polypeptide is more
than about 95% identical to a sequence DIIRNIARHLA*VGD*NleDRSI (SEQ
ID NO: 2) and wherein * is a tethered amino acid and Nle is
norleucine.
53. The method of claim 46, wherein the peptidomimetic macrocycle
is a BIM polypeptide.
54. The method of claim 53, wherein the BIM polypeptide is more
than about 60% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR (SEQ ID NO: 1) and wherein * is a tethered
amino acid.
55. The method of claim 53, wherein the BIM polypeptide is more
than about 80% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR (SEQ ID NO: 1) and wherein * is a tethered
amino acid.
56. The method of claim 53, wherein the BIM polypeptide is more
than about 95% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR (SEQ ID NO: 1) and wherein * is a tethered
amino acid.
57. The method of claim 46, wherein the peptidomimetic macrocycle
reduces activated hPBL proliferation.
58. The method of claim 57, wherein the peptidomimetic macrocycle
reduces activated hPBL proliferation by more than 20% in an in
vitro BrdU incorporation assay.
59. The method of claim 46, wherein said immunoproliferative
disease is a lymphoproliferative disorder.
60. The method of claim 1, 17, 24, 40 or 46 wherein an
.alpha.-carbon atom in said peptidomimetic macrocycle is
additionally substituted with independent substituents of formula
R--, wherein R-- is alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-.
61. The method of claim 60, wherein an .alpha.-carbon atom to which
the crosslinker is attached is additionally substituted with a
substituent of formula R--, wherein R-- is alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,
unsubstituted or substituted with halo-.
62. The method of claim 60, wherein an .alpha.-carbon atom to which
the crosslinker is not attached is additionally substituted with a
substituent of formula R--, wherein R-- is alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,
unsubstituted or substituted with halo-.
63. The method of claim 1, 17, 24, 40 or 46, wherein two
.alpha.-carbon atoms in said peptidomimetic macrocycle are
additionally substituted with independent substituents of formula
R--, wherein R-- is alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-.
64. The method of claim 63, wherein two .alpha.-carbon atoms to
which the crosslinker is attached are additionally substituted with
independent substituents of formula R--, wherein R-- is alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-.
65. The method of claim 63, wherein two .alpha.-carbon atoms to
which the crosslinker is not attached are additionally substituted
with independent substituents of formula R--, wherein R-- is alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-.
66. The method of claim 60 or 63, wherein R-- is alkyl.
67. The method of claim 60 or 63, wherein R-- is methyl.
68. The method of claim 60 or 63, wherein the crosslinker connects
two .alpha.-carbon atoms.
69. The method of claim 60 or 63, wherein R-- and any portion of
the crosslinker taken together form a cyclic structure.
70. The method of claim 60 or 63, wherein the crosslinker is formed
of consecutive carbon-carbon bonds.
71. The method of claim 60 or 63, wherein the crosslinker contains
about 9 consecutive bonds.
72. The method of claim 60 or 63, wherein the crosslinker contains
about 12 consecutive bonds.
73. The method of claim 60 or 63, wherein the crosslinker comprises
at least about 6 carbon atoms.
74. The method of claim 60 or 63, wherein the crosslinker comprises
at least about 9 carbon atoms.
75. The method of claim 1, 17, 24, 40 or 46 wherein the
peptidomimetic macrocycle is administered in conjunction with a
standard method of care.
76. The method of claim 75, wherein the standard method of care is
chemotherapy.
77. The method of claim 75, wherein the standard method of care is
radiation therapy.
78. The method of claim 75, wherein the standard method of care is
surgery.
79. The method of claim 1, 17, 24, 40 or 46, wherein the
peptidomimetic macrocycle is cell permeable.
80. The method of claim 24, wherein the assay is performed in the
presence of 10% serum.
81. The method of claim 80, wherein the serum is human serum.
82. The method of claim 24, wherein the peptidomimetic macrocycle
possesses an affinity of less than 10 .mu.M for Mcl-1.
83. The method of claim 82, wherein the peptidomimetic macrocycle
antagonizes the interaction between Mcl-1 and a pro-apoptotic
protein.
84. The method of claim 24, wherein the cancer is resistant to a
compound that possesses an affinity greater than 10 .mu.M for
Mcl-1.
85. The method of claim 84, wherein the cancer is resistant to
ABT-737 or an analog thereof.
86. A method of treating ABT-737 resistant small cell lung cancer
in a human patient in need thereof comprising administering to the
patient a peptidomimetic macrocycle, wherein the peptidomimetic
macrocycle comprises a BH3 domain.
87. A method of treating prostate cancer in a human patient in need
thereof comprising administering to the patient a peptidomimetic
macrocycle, wherein the peptidomimetic macrocycle comprises a BH3
domain.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/027,326 filed 8 Feb. 2008 and U.S. Provisional
Application No. 61/120,380 filed 5 Dec. 2008, each of which
applications is incorporated herein in its entirety by
reference.
BACKGROUND OF THE INVENTION
[0002] Uncontrolled cell proliferation is implicated in a wide
number of disorders ranging from cancer to immunoproliferative
diseases. For example, in the U.S. alone, cancer surpasses heart
disease as the leading cause of death for the largest fraction of
the population (Journal of the National Cancer Institute, Vol. 97,
No. 5, Mar. 2, 2005, p. 330) and contributes to more than 500,000
deaths annually. Despite decades of intense research efforts in
this area, the treatment of cell proliferative disorders remains a
challenge.
[0003] Therapeutic methods for cancer such as surgery or
chemotherapy are still limited in terms of efficacy, side effect
profile and cost. In particular, the efficacy and applicability of
the available therapeutic options varies greatly by the specific
type of tumor and disease. Thus, there remains a need for
compositions and methods of treating cell proliferative disorders
and other diseases.
SUMMARY OF THE INVENTION
[0004] The present invention addresses this and other needs. The
invention provides compositions and methods of treatment based on
the surprising finding that certain peptidomimetic macrocycles
exhibit unexpected specificity, efficacy and potency when used for
treatment of cell proliferative disorders.
[0005] In one aspect, the present invention provides a method of
treating cancer in a human patient in need thereof comprising
administering to the patient a peptidomimetic macrocycle, wherein
the cancer is selected from the group consisting of small cell lung
carcinoma, melanoma, ovarian cancer, prostate cancer, renal cancer,
breast cancer, pancreatic cancer, and Ph+ acute lymphocytic
leukemia (Ph+ ALL). In one embodiment, the peptidomimetic
macrocycle comprises an c-helix. In another embodiment, the
peptidomimetic macrocycle comprises a BH3 domain. The
peptidomimetic macrocycle can be, for example, a BIM polypeptide.
In some cases, an amino acid sequence of the BIM polypeptide is
more than about 60% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
Alternatively, the amino acid sequence of the BIM polypeptide is
more than about 80% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
Furthermore, an amino acid sequence of said BIM polypeptide may be
more than about 95% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid. In some
embodiments, the cancer is at least 2-fold less sensitive to
treatment using a corresponding cross-linked BID polypeptide as
measured in an in vitro cell viability assay. In other embodiments,
the cancer is at least 5-fold less sensitive to treatment using a
corresponding cross-linked BID polypeptide as measured in an in
vitro cell viability assay. In yet other embodiments, the cancer is
at least 8-fold less sensitive to treatment using a corresponding
cross-linked BID polypeptide as measured in an in vitro cell
viability assay.
[0006] In selected embodiments, the cancer is breast cancer, for
example an invasive breast carcinoma such as an invasive ductal
carcinoma. Alternatively, the cancer is prostate cancer. In other
embodiments, the cancer is ovarian cancer. In still other
embodiments, the cancer is pancreatic cancer. In further
embodiments, the cancer is renal cancer. Alternatively, the cancer
is Ph+ acute lymphocytic leukemia (Ph+ ALL).
[0007] The invention also provides a method of treating cancer in a
human patient in need thereof comprising administering to the
patient a peptidomimetic macrocycle, wherein the cancer is colon
cancer. In one embodiment, the peptidomimetic macrocycle comprises
an .alpha.-helix. In another embodiment, the peptidomimetic
macrocycle comprises a BH3 domain. The peptidomimetic macrocycle
can be, for example, a BID polypeptide. In some cases, an amino
acid sequence of the BID polypeptide is more than about 60%
identical to a sequence DIIRNIARHLA*VGD*NleDRSI and wherein * is a
tethered amino acid and Nle is norleucine. Alternatively, an amino
acid sequence of the BID polypeptide is more than about 80%
identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a
tethered amino acid and Nle is norleucine. Furthermore, an amino
acid sequence of said BID polypeptide may be more than about 95%
identical to a sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a
tethered amino acid and Nle is norleucine. In some embodiments, the
cancer is at least 2-fold less sensitive to treatment using a
corresponding cross-linked BIM polypeptide as measured in an in
vitro cell viability assay. In other embodiments, the cancer is at
least 5-fold less sensitive to treatment using a corresponding
cross-linked BIM polypeptide as measured in an in vitro cell
viability assay. In yet other embodiments, the cancer is at least
8-fold less sensitive to treatment using a corresponding
cross-linked BIM polypeptide as measured in an in vitro cell
viability assay.
[0008] Also provided is a method of treating cancer in a human
patient in need thereof comprising administering to the patient a
peptidomimetic macrocycle wherein said peptidomimetic macrocycle
shows an EC.sub.50 lower than about 5 .mu.M when tested in an in
vitro cell viability assay against a cell line derived from said
cancer. In some embodiments, the EC.sub.50 may be lower than about
4 .mu.M. In other embodiments, the EC.sub.50 may be lower than
about 3 .mu.M. In yet other embodiments, the EC.sub.50 may be lower
than about 2 .mu.M. In yet other embodiments, the EC.sub.50 may be
lower than about 1 .mu.M. In some embodiments, the in vitro assay
is performed in the presence of serum. For example, the assay may
be performed in 10% human serum. In some aspects, the cancer is
selected from the group consisting of ovarian cancer, skin cancer,
prostate cancer, renal cancer, breast cancer, pancreatic cancer,
small-cell lung cancer, colon cancer, multiple myeloma, Burkitt's
lymphoma, acute lymphocytic leukemia (ALL) of T cell lineage or B
cell lineage or mixed lineage, chronic lymphocytic leukemia (CLL),
cutaneous T cell lymphoma (CTCL), acute myelocytic leukemia (AML),
chronic myelocytic leukemia, and follicular lymphoma.
[0009] In one aspect, the present invention provides a method of
treating cancer in a human patient in need thereof comprising
administering to the patient a peptidomimetic macrocycle, wherein
the cancer is selected from the group consisting of ovarian cancer,
prostate cancer, renal cancer, breast cancer, pancreatic cancer,
and Ph+ acute lymphocytic leukemia. In one embodiment, the
peptidomimetic macrocycle comprises an .alpha.-helix. In another
embodiment, the peptidomimetic macrocycle comprises a BH3 domain.
The peptidomimetic macrocycle can be, for example, a BIM
polypeptide. In some cases, an amino acid sequence of the BIM
polypeptide is more than about 60% identical to an amino acid
sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
Alternatively, the amino acid sequence of the BIM polypeptide is
more than about 80% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
Furthermore, an amino acid sequence of said BIM polypeptide may be
more than about 95% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
[0010] In some embodiments, the cancer is selected from the group
consisting of colon cancer, small-cell lung cancer, liver cancer,
ovarian cancer, skin cancer, prostate cancer, renal cancer, breast
cancer, pancreatic cancer, glioma, multiple myeloma, Burkitt's
lymphoma, acute lymphocytic leukemia (ALL) of T cell lineage or B
cell lineage or mixed lineage, chronic lymphocytic leukemia (CLL),
cutaneous T cell lymphoma (CTCL), acute myelocytic leukemia (AML),
chronic myelocytic leukemia and follicular lymphoma. In one
embodiment, the peptidomimetic macrocycle comprises an
.alpha.-helix. In another embodiment, the peptidomimetic macrocycle
comprises a BH3 domain. The peptidomimetic macrocycle can be, for
example, a BID polypeptide. In some cases, an amino acid sequence
of the BID polypeptide is more than about 60% identical to a
sequence DIIRNIARHLA*VGD*NleDRSI and wherein * is a tethered amino
acid and Nle is norleucine. Alternatively, an amino acid sequence
of the BID polypeptide is more than about 80% identical to a
sequence DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid
and Nle is norleucine. Furthermore, an amino acid sequence of said
BID polypeptide may be more than about 95% identical to a sequence
DIIRNIARHLA*VGD*NleDRSI wherein * is a tethered amino acid and Nle
is norleucine.
[0011] The present invention additionally provides a method of
treating a disorder in a human patient in need thereof comprising
administering to the patient a peptidomimetic macrocycle,
comprising a) preparing a peptidomimetic macrocycle by introducing
a cross-link between two amino acid residues of a polypeptide; b)
testing the peptidomimetic macrocycle for the presence or absence
of an immunogenic response; and c) administering the peptidomimetic
macrocycle to a patient if said immunogenic response does not cause
a substantial side-effect. The non-immunogenicity may be evidenced
as minimal antibody response in an in vivo assay in rodents such as
mice, in non-human primates, or in humans. When administered to a
human patient, a compound which is nonimmunogenic may induce no
substantial or minimal side-effects related to its immunogenicity
in the patient. The disorder may be, for example, cancer, a
metabolic disease, cardiovascular disease, inflammatory disease or
a degenerative disease. In one embodiment, the peptidomimetic
macrocycle comprises an .alpha.-helix. In another embodiment, the
peptidomimetic macrocycle comprises a BH3 domain. The
peptidomimetic macrocycle can be, for example, a BID polypeptide.
In some cases, an amino acid sequence of the BID polypeptide is
more than about 60% identical to a sequence DIIRNIARHLA*VGD*NleDRSI
and wherein * is a tethered amino acid and Nle is norleucine.
Alternatively, an amino acid sequence of the BID polypeptide is
more than about 80% identical to a sequence DIIRNIARHLA*VGD*NleDRSI
wherein * is a tethered amino acid and Nle is norleucine.
Furthermore, an amino acid sequence of said BID polypeptide may be
more than about 95% identical to a sequence DIIRNIARHLA*VGD*NleDRSI
wherein * is a tethered amino acid and Nle is norleucine. The
peptidomimetic macrocycle may also be, for example, a BIM
polypeptide. In some cases, an amino acid sequence of the BIM
polypeptide is more than about 60% identical to an amino acid
sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
Alternatively, the amino acid sequence of the BIM polypeptide is
more than about 80% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
Furthermore, an amino acid sequence of said BIM polypeptide may be
more than about 95% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
[0012] In another aspect, the invention provides a method of
treating an immunoproliferative disorder in a human patient in need
thereof comprising administering to the patient a peptidomimetic
macrocycle. The peptidomimetic macrocycle may reduce activated hPBL
proliferation by more than about 5%, 10%, 20%, 30%, 40%, or 50% in
an in vitro BrdU incorporation assay. The immunoproliferative
disease may be, for example, a lymphoproliferative disorder, or an
autoimmune disease, for example, systemic lupus erythematosus. The
peptidomimetic macrocycle can be, for example, a BID polypeptide.
In some cases, an amino acid sequence of the BID polypeptide is
more than about 60% identical to a sequence DIIRNIARHLA*VGD*NleDRSI
and wherein * is a tethered amino acid and Nle is norleucine.
Alternatively, an amino acid sequence of the BID polypeptide is
more than about 80% identical to a sequence-DIIRNIARHLA*VGD*NleDRSI
wherein * is a tethered amino acid and Nle is norleucine.
Furthermore, an amino acid sequence of said BID polypeptide may be
more than about 95% identical to a sequence DIIRNIARHLA*VGD*NleDRSI
wherein * is a tethered amino acid and Nle is norleucine. The
peptidomimetic macrocycle may also be, for example, a BIM
polypeptide. In some cases, an amino acid sequence of the BIM
polypeptide is more than about 60% identical to an amino acid
sequence IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
Alternatively, the amino acid sequence of the BIM polypeptide is
more than about 80% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
Furthermore, an amino acid sequence of said BIM polypeptide may be
more than about 95% identical to an amino acid sequence
IWIAQELR*IGD*FNAYYARR wherein * is a tethered amino acid.
[0013] For any of the peptidomimetic macrocyles disclosed above, an
.alpha.-carbon atom in said peptidomimetic macrocycle may be
additionally substituted with independent substituents of formula
R--, wherein R-- is alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or
substituted with halo-. In some embodiments, an .alpha.-carbon atom
to which the crosslinker is attached is additionally substituted
with a substituent of formula R--. In other embodiments, an
.alpha.-carbon atom to which the crosslinker is not attached is
additionally substituted with a substituent of formula R--.
Alternatively, two .alpha.-carbon atoms in a peptidomimetic
macrocycle are additionally substituted with independent
substituents of formula R--. In some embodiments, two
.alpha.-carbon atoms to which the crosslinker is attached are
additionally substituted with independent substituents of formula
R--. In other embodiments, two .alpha.-carbon atoms to which the
crosslinker is not attached are additionally substituted with
independent substituents of formula R--. R-- may be, for example,
alkyl such as methyl, ethyl, propyl or isopropyl. The crosslinker
may connect two .alpha.-carbon atoms. In some embodiments, R-- and
any portion of the crosslinker taken together form a cyclic
structure. In other embodiments, the crosslinker is formed of
consecutive carbon-carbon bonds. In still other embodiments, the
crosslinker contains about 6, 7, 8, 9, 10, 11, 12 or 13 consecutive
bonds. In yet other embodiments, the crosslinker comprises at least
about 5, 6, 7, 8, or 9 carbon atoms.
[0014] Also provided is a method of treating cancer in a human
patient in need thereof comprising administering to the patient a
peptidomimetic macrocycle wherein said peptidomimetic macrocycle
interacts with Mcl-1. In some embodiments, the peptidomimetic
macrocycle antagonizes the interaction between Mcl-1 and
pro-apoptotic proteins such as Bid, Bim, Bax or Bak. In other
embodiments, the peptidomimetic macrocycles of the invention are
used to treat cancer in a human patient wherein the cancer is
resistant to ABT-737 or an analog thereof, or is resistant to a
compound that possesses an affinity greater than 1, 2, 5 or 10
.mu.M for Mcl-1.
[0015] The invention further provides a method of treating ABT-737
resistant small cell lung cancer in a human patient in need thereof
comprising administering to the patient a peptidomimetic
macrocycle, wherein the peptidomimetic macrocycle comprises a BH3
domain. The invention also provides a method of treating prostate
cancer in a human patient in need thereof comprising administering
to the patient a peptidomimetic macrocycle, wherein the
peptidomimetic macrocycle comprises a BH3 domain.
[0016] In any of the methods of treatment indicated herein, the
peptidomimetic macrocycle is administered in conjunction with a
standard method of care. The standard method of care may, for
example, be chemotherapy. Alternatively, the standard method of
care may be radiation therapy. In a further embodiment, the
standard method of care is surgery.
INCORPORATION BY REFERENCE
[0017] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0019] FIG. 1 shows the sensitivity of 24 different tumor cell
lines to treatment with 20 .mu.M SP-1.
[0020] FIG. 2 shows the sensitivity of 7 human leukemia/lymphoma
cell lines to treatment with 5 .mu.M of either SP-1 or SP-4.
[0021] FIG. 3 shows the sensitivity of twelve human solid tumor
lines to treatment with 20 .mu.M of either SP-1 or SP-4.
[0022] FIG. 4 shows EC.sub.50 curves for SP-1 or SP-4 tested
against a variety of cell lines.
[0023] FIGS. 5-15 describe EC.sub.50 curves for SP-1, SP-2, SP-3,
SP-4, SP-5 and SP-6 tested against several individual cell
lines.
[0024] FIG. 16 indicates that SP-1 does not induce programmed cell
death of resting human peripheral lymphocytes (hPBLs).
[0025] FIG. 17 exemplifies that SP-1 is as effective as rapamycin
in blocking the proliferation of hPBLs activated by
PMA+ionomycin+LPS treatment.
[0026] FIGS. 18 and 19 show that SP-1 decreases tumor burden in a
SEMK2 human leukemia xenograft model.
[0027] FIG. 20 shows that SP-1 does not elicit an antibody response
in rodents.
[0028] FIG. 21 illustrates that SP-1 is highly stable, and
maintains its helical conformation as temperature increases up to
64.degree. C.
[0029] FIG. 22 describes the human plasma stability of a series of
peptidomimetic macrocycles of the invention.
[0030] FIG. 23 describes the mouse plasma stability of a series of
peptidomimetic macrocycles of the invention.
[0031] FIG. 24 shows the pharmacokinetic properties of SP-1 and
SP-4 exhibit in rats.
[0032] FIG. 25 describes the induction of programmed cell death in
Jurkat tumor cells by peptidomimetic macrocycles of the invention
in the absence of human serum and compares the potency of BH3
peptidomimetic macrocycles of the invention to
BCL-2/BCL-XL-specific antagonists such as ABT-737.
[0033] FIG. 26 describes the induction of programmed cell death in
Jurkat tumor cell line by peptidomimetic macrocycles of the
invention in the presence of 10% human serum and compares the
potency of BH3 peptidomimetic macrocycles of the invention to
BCL-2/BCL-XL-specific antagonists such as ABT-737.
[0034] FIG. 27 compares the binding affinity of several BH3
peptidomimetic macrocycles of the invention and ABT-737 to
Bcl-X.sub.L, Bcl-2, and Mcl-1.
[0035] FIG. 28 shows efficacy of peptidomimetic macrocycles to a
number of hematological malignancies.
[0036] FIG. 29 illustrates the varied Bcl-2 family protein
expression profile of cell lines sensitive to treatment by
compositions of the invention.
[0037] FIG. 30 compares efficacy of peptidomimetic macrocycles with
ABT-737 in Raji, an ABT-737 resistant Burkitt's lymphoma cell
line.
[0038] FIG. 31 illustrates efficacy of BH3 peptidomimetic
macrocycles against a variety of solid tumor cell lines.
[0039] FIG. 32 depicts induction of programmed cell death by a
peptidomimetic macrocycle of the invention in an ABT-737 resistant
small cell lung cancer line (NCI-H82).
[0040] FIG. 33 shows suppression of SEMK-2 tumor progression in
NOD-SCID mice by compounds of the invention.
[0041] FIG. 34 describes induction of programmed cell death by
peptidomimetic macrocycles of the invention in an ovarian tumor
cell line (OVCAR8), treated in the absence of serum.
[0042] FIG. 35 depicts induction of programmed cell death by
peptidomimetic macrocycles of the invention in an ovarian tumor
cell line (OVCAR8), treated in the presence of 2% human serum.
[0043] FIG. 36 depicts induction of programmed cell death by
peptidomimetic macrocycles of the invention in a melanoma cell line
(A375), treated in the absence of serum.
[0044] FIG. 37 depicts induction of programmed cell death by
peptidomimetic macrocycles of the invention in a melanoma cell line
(A375), treated in the presence of 2% human serum.
[0045] FIG. 38 depicts induction of programmed cell death by
peptidomimetic macrocycles of the invention in a breast tumor cell
line (MDA-MD-231-Met), treated in the absence of serum.
[0046] FIG. 39 depicts induction of programmed cell death by
peptidomimetic macrocycles of the invention in a breast tumor cell
line (MDA-MD-231-Met), treated in 2% human serum.
[0047] FIG. 40 depicts induction of programmed cell death by
peptidomimetic macrocycles of the invention in a prostate tumor
cell line (PC3), treated in the absence of serum.
[0048] FIG. 41 depicts induction of programmed cell death by
peptidomimetic macrocycles of the invention in a prostate tumor
cell line (PC3), treated in 2% human serum.
[0049] FIG. 42 depicts induction of programmed cell death by
peptidomimetic macrocycles of the invention in a small cell lung
cancer cell line (NCI-H-82), treated in the absence of serum.
[0050] FIG. 43 depicts a Western Blot showing variable expression
of various BCL-family proteins in cancers that are sensitive to
peptidomimetic macrocycles of the invention.
[0051] FIG. 44 depicts a timeline for mouse treatment in a prostate
cancer orthotopic xenograft model.
[0052] FIG. 45 depicts efficacy of a peptidomimetic macrocycle of
the invention in a prostate cancer orthotopic xenograft model.
[0053] FIG. 46 describes induction of programmed cell death by
peptidomimetic macrocycles of the invention in a T-cell leukemia
cell line (Jurkat), treated in the absence of serum.
[0054] FIG. 47 describes induction of programmed cell death by
peptidomimetic macrocycles of the invention in a mixed-lineage
T/B-cell leukemia cell line (SEMK2), treated in the absence of
serum.
[0055] FIG. 48 describes induction of programmed cell death by
peptidomimetic macrocycles of the invention in a T-cell leukemia
cell line (MOLT-4), treated in the absence of serum.
[0056] FIG. 49 describes induction of programmed cell death by
peptidomimetic macrocycles of the invention in a diffuse large
B-cell lymphoma cell line (DHL-6), treated in the absence of
serum.
[0057] FIG. 50 describes induction of programmed cell death by
peptidomimetic macrocycles of the invention in a mixed-lineage
T/B-cell leukemia cell line (RS4;11), treated in the absence of
serum.
[0058] FIG. 51 describes induction of programmed cell death by
peptidomimetic macrocycles of the invention in a Burkitt's lymphoma
cell line (Raji), treated in the absence of serum.
[0059] FIG. 52 describes induction of programmed cell death by
peptidomimetic macrocycles of the invention in a multiple myeloma
cell line (MM1S), treated in the absence of serum.
[0060] FIG. 53 depicts efficacy of a peptidomimetic macrocycle of
the invention in a SEMK2 leukemia orthotopic xenograft model, as
measured by reduced tumor burden in treated animals.
[0061] FIG. 54 depicts efficacy of a peptidomimetic macrocycle of
the invention in a SEMK2 leukemia orthotopic xenograft model, as
measured by reduced tumor burden in treated animals.
[0062] FIG. 55 depicts efficacy of a peptidomimetic macrocycle of
the invention in a SEMK2 leukemia orthotopic xenograft model, as
measured by increased survival of treated animals.
[0063] FIG. 56 shows sequence-specific and structure-specific
binding of peptidomimetic macrocycles of the invention to the
pro-apoptotic target protein BAX in multiple myeloma (MM1S) cell
lysates, as demonstrated by immunoprecipitation with the stapled
SP-4 peptide.
DETAILED DESCRIPTION OF THE INVENTION
[0064] As used herein, the terms "treating" and "to treat", mean to
alleviate symptoms, eliminate the causation either on a temporary
or permanent basis, or to prevent or slow the appearance of
symptoms. The term "treatment" includes alleviation, elimination of
causation (temporary or permanent) of, or prevention of symptoms
and disorders associated with any condition. The treatment may be a
pre-treatment as well as a treatment at the onset of symptoms.
[0065] The term "standard method of care" refers to any therapeutic
or diagnostic method, compound, or practice which is part of the
standard of care for a particular indication. The "standard of
care" may be established by any authority such as a health care
provider or a national or regional institute for any diagnostic or
treatment process that a clinician should follow for a certain type
of patient, illness, or clinical circumstance. Exemplary standard
of care methods for various type of cancers are provided for
instance by the National Cancer Institute.
[0066] As used herein, the term "cell proliferative disorder"
encompasses cancer, hyperproliferative disorders, neoplastic
disorders, immunoproliferative disorders and other disorders. A
"cell proliferative disorder" relates to cells having the capacity
for autonomous growth, i.e., an abnormal state or condition
characterized by rapidly proliferating cell growth.
Hyperproliferative and neoplastic disease states may be categorized
as pathologic, i.e., characterizing or constituting a disease
state, or may be categorized as non-pathologic, i.e., a deviation
from normal but not associated with a disease state. The term is
meant to include all types of cancerous growths or oncogenic
processes, metastatic tissues or malignantly transformed cells,
tissues, or organs, irrespective of histopathologic type or stage
of invasiveness. A metastatic tumor can arise from a multitude of
primary tumor types, including but not limited to those of breast,
lung, liver, colon and ovarian origin. "Pathologic
hyperproliferative" cells occur in disease states characterized by
malignant tumor growth and immunoproliferative diseases. Examples
of non-pathologic hyperproliferative cells include proliferation of
cells associated with wound repair. Examples of cellular
proliferative and/or differentiative disorders include cancer,
e.g., carcinoma, sarcoma, or metastatic disorders.
[0067] The term "derived from" in the context of the relationship
between a cell line and a related cancer signifies that the cell
line may be established from any cancer in a specific broad
category of cancers.
[0068] As used herein, the term "macrocycle" refers to a molecule
having a chemical structure including a ring or cycle formed by at
least 9 covalently bonded atoms.
[0069] As used herein, the term "peptidomimetic macrocycle",
"crosslinked polypeptide" or "stapled peptide" refers to a compound
comprising a plurality of amino acid residues joined by a plurality
of peptide bonds and at least one macrocycle-forming linker which
forms a macrocycle between a first naturally-occurring or
non-naturally-occurring amino acid residue (or analog) and a second
naturally-occurring or non-naturally-occurring amino acid residue
(or analog) within the same molecule. Peptidomimetic macrocycles
include embodiments where the macrocycle-forming linker connects
the .alpha. carbon of the first amino acid residue (or analog) to
the .alpha. carbon of the second amino acid residue (or analog).
The peptidomimetic macrocycles optionally include one or more
non-peptide bonds between one or more amino acid residues and/or
amino acid analog residues, and optionally include one or more
non-naturally-occurring amino acid residues or amino acid analog
residues in addition to any which form the macrocycle.
[0070] As used herein, the term "stability" refers to the
maintenance of a defined secondary structure in solution by a
peptidomimetic macrocycle of the invention as measured by circular
dichroism, NMR or another biophysical measure, or resistance to
proteolytic degradation in vitro or in vivo. Non-limiting examples
of secondary structures contemplated in this invention are
.alpha.-helices, .beta.-turns, and .beta.-pleated sheets.
[0071] As used herein, the term "helical stability" refers to the
maintenance of .alpha. helical structure by a peptidomimetic
macrocycle of the invention as measured by circular dichroism or
NMR. For example, in some embodiments, the peptidomimetic
macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75 or
2-fold increase in .alpha.-helicity as determined by circular
dichroism compared to a corresponding uncrosslinked
polypeptide.
[0072] The term "amino acid" or simply "amino acid" refers to a
molecule containing both an amino group and a carboxyl group bound
to a carbon which is designated the .alpha.-carbon. Suitable amino
acids include, without limitation, both the D- and L-isomers of the
naturally-occurring amino acids, as well as non-naturally occurring
amino acids prepared by organic synthesis or other metabolic
routes. Unless the context specifically indicates otherwise, the
term amino acid, as used herein, is intended to include amino acid
analogs.
[0073] The term "naturally occurring amino acid" refers to any one
of the twenty amino acids commonly found in peptides synthesized in
nature, and known by the one letter abbreviations A, R, N, C, D, Q,
E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
[0074] The term "amino acid analog" or "non-natural amino acid"
refers to a molecule which is structurally similar to an amino acid
and which can be substituted for an amino acid in the formation of
a peptidomimetic macrocycle. Amino acid analogs include, without
limitation, compounds which are structurally identical to an amino
acid, as defined herein, except for the inclusion of one or more
additional methylene groups between the amino and carboxyl group
(e.g., .alpha.-amino .beta.-carboxy acids), or for the substitution
of the amino or carboxy group by a similarly reactive group (e.g.,
substitution of the primary amine with a secondary or tertiary
amine, or substitution or the carboxy group with an ester).
[0075] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of a polypeptide (e.g., a
BH3 domain or the p53 MDM2 binding domain) without abolishing or
substantially altering its essential biological or biochemical
activity (e.g., receptor binding or activation). An "essential"
amino acid residue is a residue that, when altered from the
wild-type sequence of the polypeptide, results in abolishing or
substantially abolishing the polypeptide's essential biological or
biochemical activity.
[0076] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., K, R, H), acidic side
chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S,
T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W),
beta-branched side chains (e.g., T, V, I) and aromatic side chains
(e.g., Y, F, W, H). Thus, a predicted nonessential amino acid
residue in a BH3 polypeptide, for example, is preferably replaced
with another amino acid residue from the same side chain family.
Other examples of acceptable substitutions are substitutions based
on isosteric considerations (e.g. norleucine for methionine) or
other properties (e.g. 2-thienylalanine for phenylalanine).
[0077] The term "member" as used herein in conjunction with
macrocycles or macrocycle-forming linkers refers to the atoms that
form or can form the macrocycle, and excludes substituent or side
chain atoms. By analogy, cyclodecane, 1,2-difluoro-decane and
1,3-dimethyl cyclodecane are all considered ten-membered
macrocycles as the hydrogen or fluoro substituents or methyl side
chains do not participate in forming the macrocycle.
[0078] The symbol when used as part of a molecular structure refers
to a single bond or a trans or cis double bond.
[0079] The term "amino acid side chain" refers to a moiety attached
to the .alpha.-carbon in an amino acid. For example, the amino acid
side chain for alanine is methyl, the amino acid side chain for
phenylalanine is phenylmethyl, the amino acid side chain for
cysteine is thiomethyl, the amino acid side chain for aspartate is
carboxymethyl, the amino acid side chain for tyrosine is
4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino
acid side chains are also included, for example, those that occur
in nature (e.g., an amino acid metabolite) or those that are made
synthetically (e.g., an .alpha.,.alpha. di-substituted amino
acid).
[0080] The term ".alpha.,.alpha. di-substituted amino" acid refers
to a molecule or moiety containing both an amino group and a
carboxyl group bound to a carbon (the .alpha.-carbon) that is
attached to two natural or non-natural amino acid side chains.
[0081] The term "polypeptide" encompasses two or more naturally or
non-naturally-occurring amino acids joined by a covalent bond
(e.g., an amide bond). Polypeptides as described herein include
full length proteins (e.g., fully processed proteins) as well as
shorter amino acid sequences (e.g., fragments of
naturally-occurring proteins or synthetic polypeptide
fragments).
[0082] The term "macrocyclization reagent" or "macrocycle-forming
reagent" as used herein refers to any reagent which may be used to
prepare a peptidomimetic macrocycle of the invention by mediating
the reaction between two reactive groups. Reactive groups may be,
for example, an azide and alkyne, in which case macrocyclization
reagents include, without limitation, Cu reagents such as reagents
which provide a reactive Cu(I) species, such as CuBr, CuI or CuOTf,
as well as Cu(II) salts such as Cu(CO.sub.2CH.sub.3).sub.2,
CuSO.sub.4, and CuCl.sub.2 that can be converted in situ to an
active Cu(I) reagent by the addition of a reducing agent such as
ascorbic acid or sodium ascorbate. Macrocyclization reagents may
additionally include, for example, Ru reagents known in the art
such as Cp*RuCl(PPh.sub.3).sub.2, [Cp*RuCl].sub.4 or other Ru
reagents which may provide a reactive Ru(II) species. In other
cases, the reactive groups are terminal olefins. In such
embodiments, the macrocyclization reagents or macrocycle-forming
reagents are metathesis catalysts including, but not limited to,
stabilized, late transition metal carbene complex catalysts such as
Group VIII transition metal carbene catalysts. For example, such
catalysts are Ru and Os metal centers having a +2 oxidation state,
an electron count of 16 and pentacoordinated. Additional catalysts
are disclosed in Grubbs et al., "Ring Closing Metathesis and
Related Processes in Organic Synthesis" Acc. Chem. Res. 1995, 28,
446-452, and U.S. Pat. No. 5,811,515. In yet other cases, the
reactive groups are thiol groups. In such embodiments, the
macrocyclization reagent is, for example, a linker functionalized
with two thiol-reactive groups such as halogen groups.
[0083] The term "halo" or "halogen" refers to fluorine, chlorine,
bromine or iodine or a radical thereof.
[0084] The term "alkyl" refers to a hydrocarbon chain that is a
straight chain or branched chain, containing the indicated number
of carbon atoms. For example, C.sub.1-C.sub.10 indicates that the
group has from 1 to 10 (inclusive) carbon atoms in it. In the
absence of any numerical designation, "alkyl" is a chain (straight
or branched) having 1 to 20 (inclusive) carbon atoms in it.
[0085] The term "alkylene" refers to a divalent alkyl (i.e.,
--R--).
[0086] The term "alkenyl" refers to a hydrocarbon chain that is a
straight chain or branched chain having one or more carbon-carbon
double bonds. The alkenyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group has from 2 to 10 (inclusive) carbon atoms in it. The term
"lower alkenyl" refers to a C.sub.2-C.sub.6 alkenyl chain. In the
absence of any numerical designation, "alkenyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0087] The term "alkynyl" refers to a hydrocarbon chain that is a
straight chain or branched chain having one or more carbon-carbon
triple bonds. The alkynyl moiety contains the indicated number of
carbon atoms. For example, C.sub.2-C.sub.10 indicates that the
group has from 2 to 10 (inclusive) carbon atoms in it. The term
"lower alkynyl" refers to a C.sub.2-C.sub.6 alkynyl chain. In the
absence of any numerical designation, "alkynyl" is a chain
(straight or branched) having 2 to 20 (inclusive) carbon atoms in
it.
[0088] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon
bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of
each ring are substituted by a substituent. Examples of aryl groups
include phenyl, naphthyl and the like. The term "arylalkyl" or the
term "aralkyl" refers to alkyl substituted with an aryl. The term
"arylalkoxy" refers to an alkoxy substituted with aryl.
[0089] "Arylalkyl" refers to an aryl group, as defined above,
wherein one of the aryl group's hydrogen atoms has been replaced
with a C.sub.1-C.sub.5 alkyl group, as defined above.
Representative examples of an arylalkyl group include, but are not
limited to, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl,
2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl, 2-propylphenyl,
3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3-butylphenyl,
4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl,
2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,
2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl,
2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec-butylphenyl,
2-t-butylphenyl, 3-t-butylphenyl and 4-t-butylphenyl.
[0090] "Arylamido" refers to an aryl group, as defined above,
wherein one of the aryl group's hydrogen atoms has been replaced
with one or more --C(O)NH.sub.2 groups. Representative examples of
an arylamido group include 2-C(O)NH.sub.2-phenyl,
3-C(O)NH.sub.2-phenyl, 4-C(O)NH.sub.2-phenyl,
2-C(O)NH.sub.2-pyridyl, 3-C(O)NH.sub.2-pyridyl, and
4-C(O)NH.sub.2-pyridyl,
[0091] "Alkylheterocycle" refers to a C.sub.1-C.sub.5 alkyl group,
as defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a heterocycle. Representative
examples of an alkylheterocyclo group include, but are not limited
to, --CH.sub.2CH.sub.2-morpholine, --CH.sub.2CH.sub.2-piperidine,
--CH.sub.2CH.sub.2CH.sub.2-morpholine, and
--CH.sub.2CH.sub.2CH.sub.2-imidazole.
[0092] "Alkylamido" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a --C(O)NH.sub.2 group.
Representative examples of an alkylamido group include, but are not
limited to, --CH.sub.2--C(O)NH.sub.2,
--CH.sub.2CH.sub.2--C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O)NH.sub.2,
--CH.sub.2CH(C(O)NH.sub.2)CH.sub.3,
--CH.sub.2CH(C(O)NH.sub.2)CH.sub.2CH.sub.3,
--CH(C(O)NH.sub.2)CH.sub.2CH.sub.3,
--C(CH.sub.3).sub.2CH.sub.2C(O)NH.sub.2,
CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--C(O)--CH.sub.3--CH.sub.3, and
--CH.sub.2--CH.sub.2--NH--C(O)--CH.dbd.CH.sub.2.
[0093] "Alkanol" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a hydroxyl group.
Representative examples of an alkanol group include, but are not
limited to, --CH.sub.2OH, --CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH,
--CH.sub.2CH.sub.2CH.sub.2 CH.sub.2CH.sub.2OH,
--CH.sub.2CH(OH)CH.sub.3, --CH.sub.2CH(OH)CH.sub.2CH.sub.3,
--CH(OH)CH.sub.3 and --C(CH.sub.3).sub.2CH.sub.2OH.
[0094] "Alkylcarboxy" refers to a C.sub.1-C.sub.5 alkyl group, as
defined above, wherein one of the C.sub.1-C.sub.5 alkyl group's
hydrogen atoms has been replaced with a--COOH group. Representative
examples of an alkylcarboxy group include, but are not limited to,
--CH.sub.2COOH, --CH.sub.2CH.sub.2COOH,
--CH.sub.2CH.sub.2CH.sub.2COOH,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH, --CH.sub.2CH(COOH)CH.sub.3,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2COOH,
--CH.sub.2CH(COOH)CH.sub.2CH.sub.3, --CH(COOH)CH.sub.2CH.sub.3 and
--C(CH.sub.3).sub.2CH.sub.2COOH.
[0095] The term "cycloalkyl" as employed herein includes saturated
and partially unsaturated cyclic hydrocarbon groups having 3 to 12
carbons, preferably 3 to 8 carbons, and more preferably 3 to 6
carbons, wherein the cycloalkyl group additionally is optionally
substituted. Some cycloalkyl groups include, without limitation,
cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,
cyclohexenyl, cycloheptyl, and cyclooctyl.
[0096] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring are
substituted by a substituent. Examples of heteroaryl groups include
pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl,
thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the
like.
[0097] The term "heteroarylalkyl" or the term "heteroaralkyl"
refers to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0098] The term "heteroarylalkyl" or the term "heteroaralkyl"
refers to an alkyl substituted with a heteroaryl. The term
"heteroarylalkoxy" refers to an alkoxy substituted with
heteroaryl.
[0099] The term "heterocyclyl" refers to a nonaromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring are
substituted by a substituent. Examples of heterocyclyl groups
include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl,
tetrahydrofuranyl, and the like.
[0100] The term "substituent" refers to a group replacing a second
atom or group such as a hydrogen atom on any molecule, compound or
moiety. Suitable substituents include, without limitation, halo,
hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl,
aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido,
carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.
[0101] In some embodiments, the compounds of this invention contain
one or more asymmetric centers and thus occur as racemates and
racemic mixtures, single enantiomers, individual diastereomers and
diastereomeric mixtures. All such isomeric forms of these compounds
are included in the present invention unless expressly provided
otherwise. In some embodiments, the compounds of this invention are
also represented in multiple tautomeric forms, in such instances,
the invention includes all tautomeric forms of the compounds
described herein (e.g., if alkylation of a ring system results in
alkylation at multiple sites, the invention includes all such
reaction products). All such isomeric forms of such compounds are
included in the present invention unless expressly provided
otherwise. All crystal forms of the compounds described herein are
included in the present invention unless expressly provided
otherwise.
[0102] As used herein, the terms "increase" and "decrease" mean,
respectively, to cause a statistically significantly (i.e.,
p<0.1) increase or decrease of at least 5%.
[0103] As used herein, the recitation of a numerical range for a
variable is intended to convey that the invention may be practiced
with the variable equal to any of the values within that range.
Thus, for a variable which is inherently discrete, the variable is
equal to any integer value within the numerical range, including
the end-points of the range. Similarly, for a variable which is
inherently continuous, the variable is equal to any real value
within the numerical range, including the end-points of the range.
As an example, and without limitation, a variable which is
described as having values between 0 and 2 takes the values 0, 1 or
2 if the variable is inherently discrete, and takes the values 0.0,
0.1, 0.01, 0.001, or any other real values .gtoreq.0 and .ltoreq.2
if the variable is inherently continuous.
[0104] As used herein, unless specifically indicated otherwise, the
word "or" is used in the inclusive sense of "and/or" and not the
exclusive sense of "either/or."
[0105] The term "on average" represents the mean value derived from
performing at least three independent replicates for each data
point.
[0106] The term "biological activity" encompasses structural and
functional properties of a macrocycle of the invention. Biological
activity is, for example, structural stability, alpha-helicity,
affinity for a target, resistance to proteolytic degradation, cell
penetrability, intracellular stability, in vivo stability, or any
combination thereof.
[0107] The details of one or more particular embodiments of the
invention are set forth in the accompanying drawings and the
description below. Other features, objects, and advantages of the
invention will be apparent from the description and drawings, and
from the claims.
Design of the Peptidomimetic Macrocycles of the Invention
[0108] Any protein or polypeptide with a known primary amino acid
sequence which contains a helical structure believed to impart
biological activity is the subject of the present invention. For
example, the sequence of the polypeptide can be analyzed and amino
acid analogs containing groups reactive with macrocyclization
reagents can be substituted at the appropriate positions. The
appropriate positions are determined by ascertaining which
molecular surface(s) of the secondary structure is (are) required
for biological activity and, therefore, across which other
surface(s) the macrocycle forming linkers of the invention can form
a macrocycle without sterically blocking the surface(s) required
for biological activity. Such determinations are made using methods
such as X-ray crystallography of complexes between the secondary
structure and a natural binding partner to visualize residues (and
surfaces) critical for activity; by sequential mutagenesis of
residues in the secondary structure to functionally identify
residues (and surfaces) critical for activity; or by other methods.
By such determinations, the appropriate amino acids are substituted
with the amino acids analogs and macrocycle-forming linkers of the
invention. For example, for an .alpha.-helical secondary structure,
one surface of the helix (e.g., a molecular surface extending
longitudinally along the axis of the helix and radially
45-135.degree. about the axis of the helix) may be required to make
contact with another biomolecule in vivo or in vitro for biological
activity. In such a case, a macrocycle-forming linker is designed
to link two .alpha.-carbons of the helix while extending
longitudinally along the surface of the helix in the portion of
that surface not directly required for activity.
[0109] In some embodiments of the invention, the peptide sequence
is derived from the BCL-2 family of proteins. The BCL-2 family is
defined by the presence of up to four conserved BCL-2 homology (BH)
domains designated BH1, BH2, BH3, and BH4, all of which include
.alpha.-helical segments (Chittenden et al. (1995), EMBO 14:5589;
Wang et al. (1996), Genes Dev. 10:2859). Anti-apoptotic proteins,
such as BCL-2 and BCL-X.sub.L, display sequence conservation in all
BH domains. Pro-apoptotic proteins are divided into "multidomain"
family members (e.g., BAK, BAX), which possess homology in the BH1,
BH2, and BH3 domains, and "BH3-domain only" family members (e.g.,
BID, BAD, BIM, BIK, NOXA, PUMA), that contain sequence homology
exclusively in the BH3 amphipathic .alpha.-helical segment. BCL-2
family members have the capacity to form homo- and heterodimers,
suggesting that competitive binding and the ratio between pro- and
anti-apoptotic protein levels dictates susceptibility to death
stimuli. Anti-apoptotic proteins function to protect cells from
pro-apoptotic excess, i.e., excessive programmed cell death.
Additional "security" measures include regulating transcription of
pro-apoptotic proteins and maintaining them as inactive conformers,
requiring either proteolytic activation, dephosphorylation, or
ligand-induced conformational change to activate pro-death
functions. In certain cell types, death signals received at the
plasma membrane trigger apoptosis via a mitochondrial pathway. The
mitochondria can serve as a gatekeeper of cell death by
sequestering cytochrome c, a critical component of a cytosolic
complex which activates caspase 9, leading to fatal downstream
proteolytic events. Multidomain proteins such as BCL-2/BCL-X.sub.L
and BAK/BAX play dueling roles of guardian and executioner at the
mitochondrial membrane, with their activities further regulated by
upstream BH3-only members of the BCL-2 family. For example, BID is
a member of the BH3-domain only family of pro-apoptotic proteins,
and transmits death signals received at the plasma membrane to
effector pro-apoptotic proteins at the mitochondrial membrane. BID
has the capability of interacting with both pro- and anti-apoptotic
proteins, and upon activation by caspase 8, triggers cytochrome c
release and mitochondrial apoptosis. Deletion and mutagenesis
studies determined that the amphipathic .alpha.-helical BH3 segment
of pro-apoptotic family members may function as a death domain and
thus may represent a critical structural motif for interacting with
multidomain apoptotic proteins. Structural studies have shown that
the BH3 helix can interact with anti-apoptotic proteins by
inserting into a hydrophobic groove formed by the interface of BH1,
2 and 3 domains. Activated BID can be bound and sequestered by
anti-apoptotic proteins (e.g., BCL-2 and BCL-X.sub.L) and can
trigger activation of the pro-apoptotic proteins BAX and BAK,
leading to cytochrome c release and a mitochondrial apoptosis
program. BAD is also a BH3-domain only pro-apoptotic family member
whose expression triggers the activation of BAX/BAK. In contrast to
BID, however, BAD displays preferential binding to anti-apoptotic
family members, BCL-2 and BCL-X.sub.L. Whereas the BAD BH3 domain
exhibits high affinity binding to BCL-2, BAD BH3 peptide is unable
to activate cytochrome c release from mitochondria in vitro,
suggesting that BAD is not a direct activator of BAX/BAK.
Mitochondria that over-express BCL-2 are resistant to BID-induced
cytochrome c release, but co-treatment with BAD can restore BID
sensitivity. Induction of mitochondrial apoptosis by BAD appears to
result from either: (1) displacement of BAX/BAK activators, such as
BID and BID-like proteins, from the BCL-2/BCL-XL binding pocket, or
(2) selective occupation of the BCL-2/BCL-XL binding pocket by BAD
to prevent sequestration of BID-like proteins by anti-apoptotic
proteins. Thus, two classes of BH3-domain only proteins have
emerged, BID-like proteins that directly activate
mitochondrial/apoptosis, and BAD-like proteins, that have the
capacity to sensitize mitochondria to BID-like pro-apoptotics by
occupying the binding pockets of multidomain anti-apoptotic
proteins. Various .alpha.-helical domains of BCL-2 family member
proteins amenable to the methodology disclosed herein have been
disclosed (Walensky et al. (2004), Science 305:1466; and Walensky
et al., U.S. Patent Publication No. 2005/0250680, the entire
disclosures of which are incorporated herein by reference).
[0110] A non-limiting exemplary list of suitable peptide sequences
for use in the present invention is given below:
TABLE-US-00001 TABLE 1 Name BH3 peptides Sequence (bold = critical
residues) Cross-linked Sequence (X = x-link residue) BID-BH3
QEDIIRNIARHLAQVGDSMDRSIPP QEDIIRNIARHLAXVGDXMDRSIPP BIM-BH3
DNRPEIWIAQELRRIGDEFNAYYAR DNRPEIWIAQELRXIGDXFNAYYAR BAD-BH3
NLWAAQRYGRELRRMSDEFVDSFKK NLWAAQRYGRELRXMSDXFVDSFKK PUMA-BH3
EEQWAREIGAQLRRMADDLNAQYER EEQWAREIGAQLRXMADXLNAQYER Hrk-BH3
RSSAAQLTAARLKALGDELHQRTM RSSAAQLTAARLKXLGDXLHQRTM NOXAA-BH3
AELPPEFAAQLRKIGDKVYCTW AELPPEFAAQLRXIGDXVYCTW NOXAB-BH3
VPADLKDECAQLRRIGDKVNLRQKL VPADLKDECAQLRXIGDXVNLRQKL BMF-BH3
QHRAEVQIARKLQCIADQFHRLHT QHRAEVQIARKLQXIADXFHRLHT BLK-BH3
SSAAQLTAARLKALGDELHQRT SSAAQLTAARLKXLGDXLHQRT BIK-BH3
CMEGSDALALRLACIGDEMDVSLRA CMEGSDALALRLAXIGDXMDVSLRA Bnip3
DIERRKEVESILKKNSDWTWDWSS DIERRKEVESILKXNSDXIWDWSS BOK-BH3
GRLAEVCAVLLRLGDELEMIRP GRLAEVCAVLLXLGDXLEMIRP BAX-BH3
PQDASTKKSECLKRIGDELDSNMEL PQDASTKKSECLKXIGDXLDSNMEL BAK-BH3
PSSTMGQVGRQLAIIGDDINRR PSSTMGQVGRQLAXIGDXINRR BCL2L1-BH3
KQALREAGDEFELR KQALRXAGDXFELR BCL2-BH3 LSPPVVHLALALRQAGDDFSRR
LSPPVVHLALALRXAGDXFSRR BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRY
EVIPMAAVKQALRXAGDXFELRY BCL-W-BH3 PADPLHQAMRAAGDEFETRF
PADPLHQAMRXAGDXFETRF MCL1-BH3 ATSRKLETLRRVGDGVQRNHETA
ATSRKLETLRXVGDXVQRNHETA MTD-BH3 LAEVCTVLLRLGDELEQIR
LAEVCTVLLXLGDXLEQIR MAP-1-BH3 MTVGELSRALGHENGSLDP
MTVGELSRALGXENGXLDP NIX-BH3 VVEGEKEVEALKKSADWVSDWS
VVEGEKEVEALKXSADXVSDWS 4ICD(ERBB4)-BH3 SMARDPQRYLVIQGDDRMKL
SMARDPQRYLVXQGDXRMKL
Table 1 lists human sequences which target the BH3 binding site and
are implicated in cancers, autoimmune disorders, metabolic diseases
and other human disease conditions.
TABLE-US-00002 TABLE 2 Name BH3 peptides Sequence (bold = critical
residues) Cross-linked Sequence (X = x-link residue) BID-BH3
QEDIIRNIARHLAQVGDSMDRSIPP QEDIIRNIXRHLXQVGDSMDRSIPP BIM-BH3
DNRPEIWIAQELRRIGDEFNAYYAR DNRPEIWIXQELXRIGDEFNAYYAR BAD-BH3
NLWAAQRYGRELRRMSDEFVDSFKK NLWAAQRYXRELXRMSDEFVDSFKK PUMA-BH3
EEQWAREIGAQLRRMADDLNAQYER EEQWAREIXAQLXRMADDLNAQYER Hrk-BH3
RSSAAQLTAARLKALGDELHQRTM RSSAAQLTXARLXALGDELHQRTM NOXAA-BH3
AELPPEFAAQLRKIGDKVYCTW AELPPEFXAQLXKIGDKVYCTW NOXAB-BH3
VPADLKDECAQLRRIGDKVNLRQKL VPADLKDEXAQLXRIGDKVNLRQKL BMF-BH3
QHRAEVQIARKLQCIADQFHRLHT QHRAEVQIXRKLXCIADQFHRLHT BLK-BH3
SSAAQLTAARLKALGDELHQRT SSAAQLTXARLXALGDELHQRT BIK-BH3
CMEGSDALALRLACIGDEMDVSLRA CMEGSDALXLRLXCIGDEMDVSLRA Bnip3
DIERRKEVESILKKNSDWIWDWSS DIERRKEVXSILXKNSDWIWDWSS BOK-BH3
GRLAEVCAVLLRLGDELEMIRP GRLAEVXAVLXRLGDELEMIRP BAX-BH3
PQDASTKKSECLKRIGDELDSNMEL PQDASTKKXECLXRIGDELDSNMEL BAK-BH3
PSSTMGQVGRQLAIIGDDINRR PSSTMGQVXRQLXIIGDDINRR BCL2L1-BH3
KQALREAGDEFELR XQALXEAGDEFELR BCL2-BH3 LSPPVVHLALALRQAGDDFSRR
LSPPVVHLXLALXQAGDDFSRR BCL-XL-BH3 EVIPMAAVKQALREAGDEFELRY
EVIPMAAVXQALXEAGDEFELRY BCL-W-BH3 PADPLHQAMRAAGDEFETRF
PADPLXQAMXAAGDEFETRF MCL1-BH3 ATSRKLETLRRVGDGVQRNHETA
ATSRKXETLXRVGDGVQRNHETA MTD-BH3 LAEVCTVLLRLGDELEQIR
LAEVXTVLXRLGDELEQIR MAP-1-BH3 MTVGELSRALGHENGSLDP
MTVGELXRALXHENGSLDP NIX-BH3 VVEGEKEVEALKKSADWVSDWS
VVEGEKEXEALXKSADWVSDWS 4ICD(ERBB4)-BH3 SMARDPQRYLVIQGDDRMKL
SMARDPXRYLXIQGDDRMKL
Table 2 lists human sequences which target the BH3 binding site and
are implicated in cancers, autoimmune disorders, metabolic diseases
and other human disease conditions.
Peptidomimetic Macrocycles of the Invention
[0111] In some embodiments of the method, a polypeptide of the
invention contains one crosslink. In other embodiments of the
method, said polypeptide contains two cross-links. In some
embodiments of the method, one crosslink connects two
.alpha.-carbon atoms. In other embodiments of the method, one
.alpha.-carbon atom to which one crosslink is attached is
substituted with a substituent of formula R--. In another
embodiment of the method, two .alpha.-carbon atoms to which one
crosslink is attached are substituted with independent substituents
of formula R--. In one embodiment of the methods of the invention,
R-- is alkyl. For example, R-- is methyl. Alternatively, R-- and
any portion of one crosslink taken together can form a cyclic
structure. In another embodiment of the method, one crosslink is
formed of consecutive carbon-carbon bonds. For example, one
crosslink may comprise at least 8, 9, 10, 11, or 12 consecutive
bonds. In other embodiments, one crosslink may comprise at least 7,
8, 9, 10, or 11 carbon atoms.
[0112] In another embodiment of the method, the crosslinked
polypeptide comprises an .alpha.-helical domain of a BCL-2 family
member. For example, the crosslinked polypeptide comprises a BH3
domain. In other embodiments, the crosslinked polypeptide comprises
at least 60%, 70%, 80%, 85%, 90% or 95% of any of the sequences in
Tables 1, 2, 3 and 4. In some embodiments of the method, the
crosslinked polypeptide penetrates cell membranes by an
energy-dependent process and binds to an intracellular target.
[0113] In some embodiments, said helical polypeptide contains one
crosslink. In other embodiments, said helical polypeptide contains
two cross-links.
[0114] In some embodiments, one crosslink connects two
.alpha.-carbon atoms. In other embodiments, one .alpha.-carbon atom
to which one crosslink is attached is substituted with a
substituent of formula R--. In another embodiment, two a carbon
atoms to which one crosslink is attached are substituted with
independent substituents of formula R--. In one embodiment of the
invention, R-- is alkyl. For example, R-- is methyl. Alternatively,
R-- and any portion of one crosslink taken together can form a
cyclic structure. In another embodiment, one crosslink is formed of
consecutive carbon-carbon bonds. For example, one crosslink may
comprise at least 8, 9, 10, 11, or 12 consecutive bonds. In other
embodiments, one crosslink may comprise at least 7, 8, 9, 10, or 11
carbon atoms.
[0115] In another embodiment, the crosslinked polypeptide comprises
an .alpha.-helical domain of a BCL-2 family member. For example,
the crosslinked polypeptide comprises a BH3 domain. In other
embodiments, the crosslinked polypeptide comprises at least 60%,
70%, 80%, 85%, 90% or 95% of any of the sequences in Tables 1, 2, 3
and 4. In some embodiments, the crosslinked polypeptide penetrates
cell membranes by an energy-dependent process and binds to an
intracellular target.
[0116] In some embodiments, the peptidomimetic macrocycles of the
invention have the Formula (I):
##STR00001##
[0117] wherein:
[0118] each A, C, D, and E is independently a natural or
non-natural amino acid;
[0119] B is a natural or non-natural amino acid, amino acid
analog,
##STR00002##
[0120] [--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0121] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-;
[0122] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0123] L is a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-;
[0124] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5;
[0125] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0126] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0127] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0128] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0129] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0130] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue; each of v and w is
independently an integer from 1-1000; each of x, y, and z is
independently an integer from 0-10; u is an integer from 1-10;
and
[0131] n is an integer from 1-5.
[0132] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0133] In some embodiments of the invention, x+y+z is at least 3.
In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle
or macrocycle precursor of the invention is independently selected.
For example, a sequence represented by the formula [A].sub.x, when
x is 3, encompasses embodiments where the amino acids are not
identical, e.g. Gln-Asp-Ala as well as embodiments where the amino
acids are identical, e.g. Gln-Gln-Gln. This applies for any value
of x, y, or z in the indicated ranges.
[0134] In some embodiments, the peptidomimetic macrocycle of the
invention comprises a secondary structure which is an .alpha.-helix
and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid. In other
embodiments, at least one of A, B, C, D or E is
##STR00003##
[0135] In other embodiments, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the peptidomimetic
macrocycle including, but not necessarily limited to, those between
the first C.alpha. to a second C.alpha.
[0136] In one embodiment, the peptidomimetic macrocycle of Formula
(I) is:
##STR00004##
wherein each R.sub.1 and R.sub.2 is independently --H, alkyl,
alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl,
heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with
halo-.
[0137] In related embodiments, the peptidomimetic macrocycle of
Formula (I) is:
##STR00005##
[0138] In other embodiments, the peptidomimetic macrocycle of
Formula (I) is a compound of any of the formulas shown below:
##STR00006## ##STR00007## ##STR00008## ##STR00009##
wherein "AA" represents any natural or non-natural amino acid side
chain and is [D].sub.v, [E].sub.w as defined above, and n is an
integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some
embodiments, n is 0. In other embodiments, n is less than 50.
[0139] Exemplary embodiments of the macrocycle-forming linker L are
shown below.
##STR00010## [0140] where X, Y=--CH.sub.2--, O, S, or NH [0141] m,
n, o, p=0-10
[0141] ##STR00011## [0142] where X, Y=CH.sub.2--, O, S, or NH
[0143] m, n, o, p=0-10
[0143] ##STR00012## [0144] where X, Y=--CH.sub.2--, O, S, or NH
[0145] m, n, o, p=0-10
[0146] R=H, alkyl, other substituent
##STR00013## [0147] where X, Y=--CH.sub.2--, O, S, or NH [0148] m,
n, o=0-10
[0149] Exemplary embodiments of peptidomimetic macrocycles of the
invention are shown below:
##STR00014## ##STR00015##
Other embodiments of peptidomimetic macrocycles of the invention
include analogs of the macrocycles shown above.
[0150] In some embodiments, the peptidomimetic macrocycles of the
invention have the Formula (II):
##STR00016##
[0151] wherein:
[0152] each A, C, D, and E is independently a natural or
non-natural amino acid;
[0153] B is a natural or non-natural amino acid, amino acid
analog,
##STR00017##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0154] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-;
[0155] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0156] L is a macrocycle-forming linker of the formula
##STR00018##
[0157] L.sub.1, L.sub.2 and L.sub.3 are independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5;
[0158] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0159] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0160] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0161] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0162] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with a D residue;
[0163] R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with R.sub.5,
or part of a cyclic structure with an E residue;
[0164] each of v and w is independently an integer from 1-1000;
[0165] each of x, y, and z is independently an integer from 0-10; u
is an integer from 1-10; and
[0166] n is an integer from 1-5.
[0167] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0168] In some embodiments of the invention, x+y+z is at least 3.
In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle
or macrocycle precursor of the invention is independently selected.
For example, a sequence represented by the formula [A].sub.x, when
x is 3, encompasses embodiments where the amino acids are not
identical, e.g. Gln-Asp-Ala as well as embodiments where the amino
acids are identical, e.g. Gln-Gln-Gln. This applies for any value
of x, y, or z in the indicated ranges.
[0169] In some embodiments, the peptidomimetic macrocycle of the
invention comprises a secondary structure which is an .alpha.-helix
and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid. In other
embodiments, at least one of A, B, C, D or E is
##STR00019##
[0170] In other embodiments, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the peptidomimetic
macrocycle including, but not necessarily limited to, those between
the first C.alpha. to a second C.alpha.
[0171] Exemplary embodiments of the macrocycle-forming linker L are
shown below.
##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029##
##STR00030##
[0172] In other embodiments, the invention provides peptidomimetic
macrocycles of Formula (III):
##STR00031##
wherein: each A, C, D, and E is independently a natural or
non-natural amino acid; B is a natural or non-natural amino acid,
amino acid analog,
##STR00032##
[--NH-L.sub.4-CO--], [--NH-L.sub.4-SO.sub.2--], or
[--NH-L.sub.4-];
[0173] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-; R.sub.3
is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or
heterocycloaryl, unsubstituted or substituted with R.sub.5;
L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are independently alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, cycloarylene, heterocycloarylene or
[--R.sub.4--K--R.sub.4-].sub.n, each being unsubstituted or
substituted with R.sub.5;
K is O, S, SO, SO.sub.2, CO, CO.sub.2, or CONR.sub.3;
[0174] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene; each R.sub.5 is independently halogen, alkyl,
--OR.sub.6, --N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6,
--SO.sub.2R.sub.6, --CO.sub.2R.sub.6, a fluorescent moiety, a
radioisotope or a therapeutic agent; each R.sub.6 is independently
--H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,
heterocycloalkyl, a fluorescent moiety, a radioisotope or a
therapeutic agent; R.sub.7 is --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl,
heterocycloalkyl, cycloaryl, or heterocycloaryl, unsubstituted or
substituted with R.sub.5, or part of a cyclic structure with a D
residue; R.sub.8 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, unsubstituted or substituted with
R.sub.5, or part of a cyclic structure with an E residue; each of v
and w is independently an integer from 1-1000; each of x, y, and z
is independently an integer from 0-10; u is an integer from 1-10;
and n is an integer from 1-5.
[0175] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0176] In some embodiments of the invention, x+y+z is at least 3.
In other embodiments of the invention, x+y+z is 3, 4, 5, 6, 7, 8, 9
or 10. Each occurrence of A, B, C, D or E in a macrocycle or
macrocycle precursor of the invention is independently selected.
For example, a sequence represented by the formula [A].sub.x, when
x is 3, encompasses embodiments where the amino acids are not
identical, e.g. Gln-Asp-Ala as well as embodiments where the amino
acids are identical, e.g. Gln-Gln-Gln. This applies for any value
of x, y, or z in the indicated ranges.
[0177] In some embodiments, the peptidomimetic macrocycle of the
invention comprises a secondary structure which is an .alpha.-helix
and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid. In other
embodiments, at least one of A, B, C, D or E is
##STR00033##
[0178] In other embodiments, the length of the macrocycle-forming
linker [-L.sub.1-S-L.sub.2-S-L.sub.3-] as measured from a first
Ca.alpha. a second C.alpha. is selected to stabilize a desired
secondary peptide structure, such as an .alpha.-helix formed by
residues of the peptidomimetic macrocycle including, but not
necessarily limited to, those between the first C.alpha. to a
second C.alpha..
[0179] Macrocycles or macrocycle precursors are synthesized, for
example, by solution phase or solid-phase methods, and can contain
both naturally-occurring and non-naturally-occurring amino acids.
See, for example, Hunt, "The Non-Protein Amino Acids" in Chemistry
and Biochemistry of the Amino Acids, edited by G. C. Barrett,
Chapman and Hall, 1985. In some embodiments, the thiol moieties are
the side chains of the amino acid residues L-cysteine, D-cysteine,
.alpha.-methyl-L cysteine, .alpha.-methyl-D-cysteine,
L-homocysteine, D-homocysteine, .alpha.-methyl-L-homocysteine or
.alpha.-methyl-D-homocysteine. A bis-alkylating reagent is of the
general formula X-L.sub.2-Y wherein L.sub.2 is a linker moiety and
X and Y are leaving groups that are displaced by --SH moieties to
form bonds with L.sub.2. In some embodiments, X and Y are halogens
such as I, Br, or Cl.
[0180] In other embodiments, D and/or E in the compound of Formula
I, II or III are further modified in order to facilitate cellular
uptake. In some embodiments, lipidating or PEGylating a
peptidomimetic macrocycle facilitates cellular uptake, increases
bioavailability, increases blood circulation, alters
pharmacokinetics, decreases immunogenicity and/or decreases the
needed frequency of administration.
[0181] In other embodiments, at least one of [D] and [E] in the
compound of Formula I, II or III represents a moiety comprising an
additional macrocycle-forming linker such that the peptidomimetic
macrocycle comprises at least two macrocycle-forming linkers. In a
specific embodiment, a peptidomimetic macrocycle comprises two
macrocycle-forming linkers.
[0182] In the peptidomimetic macrocycles of the invention, any of
the macrocycle-forming linkers described herein may be used in any
combination with any of the sequences shown in Tables 14 and also
with any of the R-- substituents indicated herein.
[0183] In some embodiments, the peptidomimetic macrocycle comprises
at least one .alpha.-helix motif. For example, A, B and/or C in the
compound of Formula I, II or III include one or more
.alpha.-helices. As a general matter, .alpha.-helices include
between 3 and 4 amino acid residues per turn. In some embodiments,
the .alpha.-helix of the peptidomimetic macrocycle includes 1 to 5
turns and, therefore, 3 to 20 amino acid residues. In specific
embodiments, the .alpha.-helix includes 1 turn, 2 turns, 3 turns, 4
turns, or 5 turns. In some embodiments, the macrocycle-forming
linker stabilizes an .alpha.-helix motif included within the
peptidomimetic macrocycle. Thus, in some embodiments, the length of
the macrocycle-forming linker L from a first C.alpha. to a second
C.alpha. is selected to increase the stability of an .alpha.-helix.
In some embodiments, the macrocycle-forming linker spans from 1
turn to 5 turns of the .alpha.-helix. In some embodiments, the
macrocycle-forming linker spans approximately 1 turn, 2 turns, 3
turns, 4 turns, or 5 turns of the .alpha.-helix. In some
embodiments, the length of the macrocycle-forming linker is
approximately 5 .ANG. to 9 .ANG. per turn of the .alpha.-helix, or
approximately 6 .ANG. to 8 .ANG. per turn of the .alpha.-helix.
Where the macrocycle-forming linker spans approximately 1 turn of
an .alpha.-helix, the length is equal to approximately 5
carbon-carbon bonds to 13 carbon-carbon bonds, approximately 7
carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9
carbon-carbon bonds. Where the macrocycle-forming linker spans
approximately 2 turns of an .alpha.-helix, the length is equal to
approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds,
approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or
approximately 12 carbon-carbon bonds. Where the macrocycle-forming
linker spans approximately 3 turns of an .alpha.-helix, the length
is equal to approximately 14 carbon-carbon bonds to 22
carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20
carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where
the macrocycle-forming linker spans approximately 4 turns of an
.alpha.-helix, the length is equal to approximately 20
carbon-carbon bonds to 28 carbon-carbon bonds, approximately 22
carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24
carbon-carbon bonds. Where the macrocycle-forming linker spans
approximately 5 turns of an .alpha.-helix, the length is equal to
approximately 26 carbon-carbon bonds to 34 carbon-carbon bonds,
approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or
approximately 30 carbon-carbon bonds. Where the macrocycle-forming
linker spans approximately 1 turn of an .alpha.-helix, the linkage
contains approximately 4 atoms to 12 atoms, approximately 6 atoms
to 10 atoms, or approximately 8 atoms. Where the macrocycle-forming
linker spans approximately 2 turns of the .alpha.-helix, the
linkage contains approximately 7 atoms to 15 atoms, approximately 9
atoms to 13 atoms, or approximately 11 atoms. Where the
macrocycle-forming linker spans approximately 3 turns of the
.alpha.-helix, the linkage contains approximately 13 atoms to 21
atoms, approximately 15 atoms to 19 atoms, or approximately 17
atoms. Where the macrocycle-forming linker spans approximately 4
turns of the .alpha.-helix, the linkage contains approximately 19
atoms to 27 atoms, approximately 21 atoms to 25 atoms, or
approximately 23 atoms. Where the macrocycle-forming linker spans
approximately 2 turns of the .alpha.-helix, the linkage contains
approximately 25 atoms to 33 atoms, approximately 27 atoms to 31
atoms, or approximately 29 atoms. Where the macrocycle-forming
linker spans approximately 1 turn of the .alpha.-helix, the
resulting macrocycle forms a ring containing approximately 17
members to 25 members, approximately 19 members to 23 members, or
approximately 21 members. Where the macrocycle-forming linker spans
approximately 2 turns of the .alpha.-helix, the resulting
macrocycle forms a ring containing approximately 29 members to 37
members, approximately 31 members to 35 members, or approximately
33 members. Where the macrocycle-forming linker spans approximately
3 turns of the .alpha.-helix, the resulting macrocycle forms a ring
containing approximately 44 members to 52 members, approximately 46
members to 50 members, or approximately 48 members. Where the
macrocycle-forming linker spans approximately 4 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 59 members to 67 members, approximately 61 members to
65 members, or approximately 63 members. Where the
macrocycle-forming linker spans approximately 5 turns of the
.alpha.-helix, the resulting macrocycle forms a ring containing
approximately 74 members to 82 members, approximately 76 members to
80 members, or approximately 78 members.
[0184] In other embodiments, the invention provides peptidomimetic
macrocycles of Formula (IV) or (IVa):
##STR00034##
[0185] wherein:
[0186] each A, C, D, and E is independently a natural or
non-natural amino acid;
[0187] B is a natural or non-natural amino acid, amino acid
analog,
##STR00035##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0188] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-, or part
of a cyclic structure with an E residue;
[0189] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0190] L is a macrocycle-forming linker of the formula
-L.sub.1-L.sub.2-;
[0191] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5;
[0192] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0193] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0194] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0195] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0196] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0197] v is an integer from 1-1000;
[0198] w is an integer from 1-1000;
[0199] x is an integer from 0-10;
[0200] y is an integer from 0-10;
[0201] z is an integer from 0-10; and
[0202] n is an integer from 1-5.
[0203] In one example, at least one of R.sub.1 and R.sub.2 is
alkyl, unsubstituted or substituted with halo-. In another example,
both R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl.
[0204] In some embodiments of the invention, x+y+z is at least 3.
In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle
or macrocycle precursor of the invention is independently selected.
For example, a sequence represented by the formula [A].sub.x, when
x is 3, encompasses embodiments where the amino acids are not
identical, e.g. Gln Asp-Ala as well as embodiments where the amino
acids are identical, e.g. Gln-Gln-Gln. This applies for any value
of x, y, or z in the indicated ranges.
[0205] In some embodiments, the peptidomimetic macrocycle of the
invention comprises a secondary structure which is an .alpha.-helix
and R.sub.8 is --H, allowing intrahelical hydrogen bonding. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid. In other
embodiments, at least one of A, B, C, D or E is
##STR00036##
[0206] In other embodiments, the length of the macrocycle-forming
linker L as measured from a first C.alpha. to a second C.alpha. is
selected to stabilize a desired secondary peptide structure, such
as an .alpha.-helix formed by residues of the peptidomimetic
macrocycle including, but not necessarily limited to, those between
the first C.alpha. to a second C.alpha..
[0207] Exemplary embodiments of the macrocycle-forming linker L are
shown below.
##STR00037## [0208] where X, Y=--CH.sub.2--, O, S, or NH [0209] m,
n, o, p=0-10
[0209] ##STR00038## [0210] where X, Y=--CH.sub.2--, O, S, or NH
[0211] m, n, o, p=0-10
[0211] ##STR00039## [0212] where X, Y=--CH.sub.2--, O, S, or NH
[0213] m, n, o, p=0-10 [0214] m, n, o, p=0-10 [0215] R=H, alkyl,
other substituent
[0215] ##STR00040## [0216] where X, Y=--CH.sub.2--, O, S, or NH
[0217] m, n, o=0-10
Preparation of Peptidomimetic Macrocycles
[0218] Peptidomimetic macrocycles of the invention may be prepared
by any of a variety of methods known in the art. For example, any
of the residues indicated by "X" in Tables 1, 2, 3 or 4 may be
substituted with a residue capable of forming a crosslinker with a
second residue in the same molecule or a precursor of such a
residue.
[0219] Various methods to effect formation of peptidomimetic
macrocycles are known in the art. For example, the preparation of
peptidomimetic macrocycles of Formula I is described in
Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000);
Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005);
Walensky et al., Science 305:1466-1470 (2004); and U.S. Pat. No.
7,192,713. The .alpha.,.alpha.-disubstituted amino acids and amino
acid precursors disclosed in the cited references may be employed
in synthesis of the peptidomimetic macrocycle precursor
polypeptides. Following incorporation of such amino acids into
precursor polypeptides, the terminal olefins are reacted with a
metathesis catalyst, leading to the formation of the peptidomimetic
macrocycle.
[0220] In other embodiments, the peptidomimetic macrocyles of the
invention are of Formula IV or IVa. Methods for the preparation of
such macrocycles are described, for example, in U.S. Pat. No.
7,202,332.
[0221] In some embodiments, the synthesis of these peptidomimetic
macrocycles involves a multi-step process that features the
synthesis of a peptidomimetic precursor containing an azide moiety
and an alkyne moiety; followed by contacting the peptidomimetic
precursor with a macrocyclization reagent to generate a
triazole-linked peptidomimetic macrocycle. Macrocycles or
macrocycle precursors are synthesized, for example, by solution
phase or solid-phase methods, and can contain both
naturally-occurring and non-naturally-occurring amino acids. See,
for example, Hunt, "The Non-Protein Amino Acids" in Chemistry and
Biochemistry of the Amino Acids, edited by G. C. Barrett, Chapman
and Hall, 1985.
[0222] In some embodiments, an azide is linked to the
.alpha.-carbon of a residue and an alkyne is attached to the
.alpha.-carbon of another residue. In some embodiments, the azide
moieties are azido-analogs of amino acids L-lysine, D-lysine,
alpha-methyl-L-lysine, alpha-methyl-D-lysine, L-ornithine,
D-ornithine, alpha-methyl-L-ornithine or alpha-methyl-D-ornithine.
In another embodiment, the alkyne moiety is L-propargylglycine. In
yet other embodiments, the alkyne moiety is an amino acid selected
from the group consisting of L-propargylglycine,
D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,
(R)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-2-methyl-5-hexynoic acid,
(R)-2-amino-2-methyl-5-hexynoic acid,
(S)-2-amino-2-methyl-6-heptynoic acid,
(R)-2-amino-2-methyl-6-heptynoic acid,
(S)-2-amino-2-methyl-7-octynoic acid,
(R)-2-amino-2-methyl-7-octynoic acid,
(S)-2-amino-2-methyl-8-nonynoic acid and
(R)-2-amino-2-methyl-8-nonynoic acid.
[0223] In some embodiments, the invention provides a method for
synthesizing a peptidomimetic macrocycle, the method comprising the
steps of contacting a peptidomimetic precursor of Formula V or
Formula VI:
##STR00041##
with a macrocyclization reagent; wherein v, w, x, y, z, A, B, C, D,
E, R.sub.1, R.sub.2, R.sub.7, R.sub.8, L.sub.1 and L.sub.2 are as
defined for Formula (II); R.sub.12 is --H when the macrocyclization
reagent is a Cu reagent and R.sub.12 is --H or alkyl when the
macrocyclization reagent is a Ru reagent; and further wherein said
contacting step results in a covalent linkage being formed between
the alkyne and azide moiety in Formula III or Formula IV. For
example, R.sub.12 may be methyl when the macrocyclization reagent
is a Ru reagent.
[0224] In the peptidomimetic macrocycles of the invention, at least
one of R.sub.1 and R.sub.2 is alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,
unsubstituted or substituted with halo-. In some embodiments, both
R.sub.1 and R.sub.2 are independently alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-. In some
embodiments, at least one of A, B, C, D or E is an
.alpha.,.alpha.-disubstituted amino acid. In one example, B is an
.alpha.,.alpha.-disubstituted amino acid. For instance, at least
one of A, B, C, D or E is 2-aminoisobutyric acid.
[0225] For example, at least one of R.sub.1 and R.sub.2 is alkyl,
unsubstituted or substituted with halo-. In another example, both
R.sub.1 and R.sub.2 are independently alkyl, unsubstituted or
substituted with halo-. In some embodiments, at least one of
R.sub.1 and R.sub.2 is methyl. In other embodiments, R.sub.1 and
R.sub.2 are methyl. The macrocyclization reagent may be a Cu
reagent or a Ru reagent.
[0226] In some embodiments, the peptidomimetic precursor is
purified prior to the contacting step. In other embodiments, the
peptidomimetic macrocycle is purified after the contacting step. In
still other embodiments, the peptidomimetic macrocycle is refolded
after the contacting step. The method may be performed in solution,
or, alternatively, the method may be performed on a solid
support.
[0227] Also envisioned herein is performing the method of the
invention in the presence of a target macromolecule that binds to
the peptidomimetic precursor or peptidomimetic macrocycle under
conditions that favor said binding. In some embodiments, the method
is performed in the presence of a target macromolecule that binds
preferentially to the peptidomimetic precursor or peptidomimetic
macrocycle under conditions that favor said binding. The method may
also be applied to synthesize a library of peptidomimetic
macrocycles.
[0228] In some embodiments, the alkyne moiety of the peptidomimetic
precursor of Formula V or Formula VI is a sidechain of an amino
acid selected from the group consisting of L-propargylglycine,
D-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,
(R)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-2-methyl-5-hexynoic acid,
(R)-2-amino-2-methyl-5-hexynoic acid,
(S)-2-amino-2-methyl-6-heptynoic acid,
(R)-2-amino-2-methyl-6-heptynoic acid,
(S)-2-amino-2-methyl-7-octynoic acid,
(R)-2-amino-2-methyl-7-octynoic acid,
(S)-2-amino-2-methyl-8-nonynoic acid, and
(R)-2-amino-2-methyl-8-nonynoic acid. In other embodiments, the
azide moiety of the peptidomimetic precursor of Formula V or
Formula VI is a sidechain of an amino acid selected from the group
consisting of .epsilon.-azido-L-lysine, .epsilon.-azido-D-lysine,
.epsilon.-azido-.epsilon.-methyl-L-lysine,
.epsilon.-azido-.alpha.-methyl-D-lysine,
.delta.-azido-.alpha.-methyl-L-ornithine, and
.delta.-azido-.alpha.-methyl-D-ornithine.
[0229] In some embodiments, x+y+z is 3, and A, B and C are
independently natural or non-natural amino acids. In other
embodiments, x+y+z is 6, and A, B and C are independently natural
or non-natural amino acids.
[0230] In some embodiments of peptidomimetic macrocycles of the
invention, [D].sub.v and/or [E].sub.w comprise additional
peptidomimetic macrocycles or macrocyclic structures. For example,
[D].sub.v may have the formula:
##STR00042##
[0231] wherein each A, C, D', and E' is independently a natural or
non-natural amino acid;
[0232] B is a natural or non-natural amino acid, amino acid
analog
##STR00043##
[--NH-L.sub.3-CO--], [--NH-L.sub.3-SO.sub.2--], or
[--NH-L.sub.3-];
[0233] R.sub.1 and R.sub.2 are independently --H, alkyl, alkenyl,
alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or
heterocycloalkyl, unsubstituted or substituted with halo-, or part
of a cyclic structure with an E residue;
[0234] R.sub.3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl,
heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0235] L.sub.1 and L.sub.2 are independently alkylene, alkenylene,
alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene,
cycloarylene, heterocycloarylene, or
[--R.sub.4--K--R.sub.4--].sub.n, each being optionally substituted
with R.sub.5;
[0236] each R.sub.4 is alkylene, alkenylene, alkynylene,
heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or
heteroarylene;
[0237] each K is O, S, SO, SO.sub.2, CO, CO.sub.2, or
CONR.sub.3;
[0238] each R.sub.5 is independently halogen, alkyl, --OR.sub.6,
--N(R.sub.6).sub.2, --SR.sub.6, --SOR.sub.6, --SO.sub.2R.sub.6,
--CO.sub.2R.sub.6, a fluorescent moiety, a radioisotope or a
therapeutic agent;
[0239] each R.sub.6 is independently --H, alkyl, alkenyl, alkynyl,
arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety,
a radioisotope or a therapeutic agent;
[0240] R.sub.7 is --H, alkyl, alkenyl, alkynyl, arylalkyl,
cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl,
cycloaryl, or heterocycloaryl, optionally substituted with
R.sub.5;
[0241] v is an integer from 1-1000;
[0242] w is an integer from 1-1000; and
[0243] x is an integer from 0-10.
[0244] In another embodiment, [E].sub.w has the formula:
##STR00044##
wherein the substituents are as defined in the preceding
paragraph.
[0245] In some embodiments, the contacting step is performed in a
solvent selected from the group consisting of protic solvent,
aqueous solvent, organic solvent, and mixtures thereof. For
example, the solvent may be chosen from the group consisting of
H.sub.2O, THF, THF/H.sub.2O, tBuOH/H.sub.2O, DMF, DIPEA, CH.sub.3CN
or CH.sub.2Cl.sub.2, ClCH.sub.2CH.sub.2Cl or a mixture thereof. The
solvent may be a solvent which favors helix formation.
[0246] Alternative but equivalent protecting groups, leaving groups
or reagents are substituted, and certain of the synthetic steps are
performed in alternative sequences or orders to produce the desired
compounds. Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds described herein include, for example, those such as
described in Larock, Comprehensive Organic Transformations, VCH
Publishers (1989); Greene and Wuts, Protective Groups in Organic
Synthesis, 2d. Ed., John Wiley and Sons (1991); Fieser and Fieser,
Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and
Sons (1994); and Paquette, ed., Encyclopedia of Reagents for
Organic Synthesis, John Wiley and Sons (1995), and subsequent
editions thereof.
[0247] The peptidomimetic macrocycles of the invention are made,
for example, by chemical synthesis methods, such as described in
Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed.
Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Hence,
for example, peptides are synthesized using the automated
Merrifield techniques of solid phase synthesis with the amine
protected by either tBoc or Fmoc chemistry using side chain
protected amino acids on, for example, an automated peptide
synthesizer (e.g., Applied Biosystems (Foster City, Calif.), Model
430A, 431, or 433).
[0248] One manner of producing the peptidomimetic precursors and
peptidomimetic macrocycles described herein uses solid phase
peptide synthesis (SPPS). The C-terminal amino acid is attached to
a cross-linked polystyrene resin via an acid labile bond with a
linker molecule. This resin is insoluble in the solvents used for
synthesis, making it relatively simple and fast to wash away excess
reagents and by-products. The N-terminus is protected with the Fmoc
group, which is stable in acid, but removable by base. Side chain
functional groups are protected as necessary with base stable, acid
labile groups.
[0249] Longer peptidomimetic precursors are produced, for example,
by conjoining individual synthetic peptides using native chemical
ligation. Alternatively, the longer synthetic peptides are
biosynthesized by well known recombinant DNA and protein expression
techniques. Such techniques are provided in well-known standard
manuals with detailed protocols. To construct a gene encoding a
peptidomimetic precursor of this invention, the amino acid sequence
is reverse translated to obtain a nucleic acid sequence encoding
the amino acid sequence, preferably with codons that are optimum
for the organism in which the gene is to be expressed. Next, a
synthetic gene is made, typically by synthesizing oligonucleotides
which encode the peptide and any regulatory elements, if necessary.
The synthetic gene is inserted in a suitable cloning vector and
transfected into a host cell. The peptide is then expressed under
suitable conditions appropriate for the selected expression system
and host. The peptide is purified and characterized by standard
methods.
[0250] The peptidomimetic precursors are made, for example, in a
high-throughput, combinatorial fashion using, for example, a
high-throughput polychannel combinatorial synthesizer (e.g.,
Thuramed TETRAS multichannel peptide synthesizer from CreoSalus,
Louisville, Ky. or Model Apex 396 multichannel peptide synthesizer
from AAPPTEC, Inc., Louisville, Ky.).
[0251] The following synthetic schemes are provided solely to
illustrate the present invention and are not intended to limit the
scope of the invention, as described herein. To simplify the
drawings, the illustrative schemes depict azido amino acid analogs
.epsilon.-azido-.alpha.-methyl-L-lysine and
.epsilon.-azido-.alpha.-methyl-D-lysine, and alkyne amino acid
analogs L-propargylglycine, (S)-2-amino-2-methyl-4-pentynoic acid,
and (S)-2-amino-2-methyl-6-heptynoic acid. Thus, in the following
synthetic schemes, each R.sub.1, R.sub.2, R.sub.7 and R.sub.8 is
--H; each L.sub.1 is --(CH.sub.2).sub.4--; and each L.sub.2 is
--(CH.sub.2)--. However, as noted throughout the detailed
description above, many other amino acid analogs can be employed in
which R.sub.1, R.sub.2, R.sub.7, R.sub.8, L.sub.1 and L.sub.2 can
be independently selected from the various structures disclosed
herein.
##STR00045## ##STR00046## ##STR00047##
[0252] Synthetic Scheme 1 describes the preparation of several
compounds of the invention. Ni(II) complexes of Schiff bases
derived from the chiral auxiliary
(S)-2-[N--(N'-benzylpropyl)amino]benzophenone (BPB) and amino acids
such as glycine or alanine are prepared as described in Belokon et
al. (1998), Tetrahedron Asymm. 9:42494252. The resulting complexes
are subsequently reacted with alkylating reagents comprising an
azido or alkynyl moiety to yield enantiomerically enriched
compounds of the invention. If desired, the resulting compounds can
be protected for use in peptide synthesis.
##STR00048## ##STR00049##
[0253] In the general method for the synthesis of peptidomimetic
macrocycles shown in Synthetic Scheme 2, the peptidomimetic
precursor contains an azide moiety and an alkyne moiety and is
synthesized by solution-phase or solid-phase peptide synthesis
(SPPS) using the commercially available amino acid
N-.alpha.-Fmoc-L-propargylglycine and the N-.alpha.-Fmoc-protected
forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic
acid, N-methyl-.epsilon.-azido-L-lysine, and
N-methyl-.epsilon.-azido-D-lysine. The peptidomimetic precursor is
then deprotected and cleaved from the solid-phase resin by standard
conditions (e.g., strong acid such as 95% TFA). The peptidomimetic
precursor is reacted as a crude mixture or is purified prior to
reaction with a macrocyclization reagent such as a Cu(I) in organic
or aqueous solutions (Rostovtsev et al. (2002), Angew. Chem. Int.
Ed. 41:2596-2599; Tornoe et al. (2002), J. Org. Chem. 67:3057-3064;
Deiters et al. (2003), J. Am. Chem. Soc. 125:11782-11783; Punna et
al. (2005), Angew. Chem. Int. Ed. 44:2215-2220). In one embodiment,
the triazole forming reaction is performed under conditions that
favor .alpha.-helix formation. In one embodiment, the
macrocyclization step is performed in a solvent chosen from the
group consisting of H.sub.2O, THF, CH.sub.3CN, DMF , DIPEA, tBuOH
or a mixture thereof. In another embodiment, the macrocyclization
step is performed in DMF. In some embodiments, the macrocyclization
step is performed in a buffered aqueous or partially aqueous
solvent.
##STR00050## ##STR00051##
[0254] In the general method for the synthesis of peptidomimetic
macrocycles shown in Synthetic Scheme 3, the peptidomimetic
precursor contains an azide moiety and an alkyne moiety and is
synthesized by solid-phase peptide synthesis (SPPS) using the
commercially available amino acid N-.alpha.-Fmoc-L-propargylglycine
and the N-.alpha.-Fmoc-protected forms of the amino acids
(S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic
acid, (S)-2-amino-2-methyl-6-heptynoic acid,
N-methyl-.epsilon.-azido-L-lysine, and
N-methyl-.epsilon.-azido-D-lysine. The peptidomimetic precursor is
reacted with a macrocyclization reagent such as a Cu(I) reagent on
the resin as a crude mixture (Rostovtsev et al. (2002), Angew.
Chem. Int. Ed. 41:2596-2599; Tomoe et al. (2002), J. Org. Chem.
67:3057-3064; Deiters et al. (2003), J. Am. Chem. Soc.
125:11782-11783; Punna et al. (2005), Angew. Chem. Int. Ed.
44:2215-2220). The resultant triazole-containing peptidomimetic
macrocycle is then deprotected and cleaved from the solid-phase
resin by standard conditions (e.g., strong acid such as 95% TFA).
In some embodiments, the macrocyclization step is performed in a
solvent chosen from the group consisting of CH.sub.2Cl.sub.2,
ClCH.sub.2CH.sub.2Cl, DMF, THF, NMP, DIPEA, 2,6-lutidine, pyridine,
DMSO, H.sub.2O or a mixture thereof. In some embodiments, the
macrocyclization step is performed in a buffered aqueous or
partially aqueous solvent.
##STR00052## ##STR00053##
[0255] In the general method for the synthesis of peptidomimetic
macrocycles shown in Synthetic Scheme 4, the peptidomimetic
precursor contains an azide moiety and an alkyne moiety and is
synthesized by solution-phase or solid-phase peptide synthesis
(SPPS) using the commercially available amino acid
N-.alpha.-Fmoc-L-propargylglycine and the N-.alpha.-Fmoc-protected
forms of the amino acids (S)-2-amino-2-methyl-4-pentynoic acid,
(S)-2-amino-6-heptynoic acid, (S)-2-amino-2-methyl-6-heptynoic
acid, N-methyl-.epsilon.-azido-L-lysine, and
N-methyl-.epsilon.-azido-D-lysine. The peptidomimetic precursor is
then deprotected and cleaved from the solid-phase resin by standard
conditions (e.g., strong acid such as 95% TFA). The peptidomimetic
precursor is reacted as a crude mixture or is purified prior to
reaction with a macrocyclization reagent such as a Ru(II) reagents,
for example Cp*RuCl(PPh.sub.3).sub.2 or [Cp*RuCl].sub.4 (Rasmussen
et al. (2007), Org. Lett. 9:5337-5339; Zhang et al. (2005), J. Am.
Chem. Soc. 127:15998-15999). In some embodiments, the
macrocyclization step is performed in a solvent chosen from the
group consisting of DMF, CH.sub.3CN and THF.
##STR00054## ##STR00055##
[0256] In the general method for the synthesis of peptidomimetic
macrocycles shown in Synthetic Scheme 5, the peptidomimetic
precursor contains an azide moiety and an alkyne moiety and is
synthesized by solid-phase peptide synthesis (SPPS) using the
commercially available amino acid N-.alpha.-Fmoc-L-propargylglycine
and the N-.alpha.-Fmoc-protected forms of the amino acids
(S)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-6-heptynoic
acid, (S)-2-amino-2-methyl-6-heptynoic acid,
N-methyl-.beta.-azido-L-lysine, and N-methyl-.beta.-azido-D-lysine.
The peptidomimetic precursor is reacted with a macrocyclization
reagent such as a Ru(II) reagent on the resin as a crude mixture.
For example, the reagent can be Cp*RuCl(PPh.sub.3).sub.2 or
[Cp*RuCl].sub.4 (Rasmussen et al. (2007), Org. Lett. 9:5337-5339;
Zhang et al. (2005), J. Am. Chem. Soc. 127:15998-15999). In some
embodiments, the macrocyclization step is performed in a solvent
chosen from the group consisting of CH.sub.2Cl.sub.2,
ClCH.sub.2CH.sub.2Cl, CH.sub.3CN, DMF, and THF.
[0257] Several exemplary peptidomimetic macrocycles are shown in
Table 5. "Nle" represents norleucine and replaces a methionine
residue. It is envisioned that similar linkers are used to
synthesize peptidomimetic macrocycles based on the polypeptide
sequences disclosed in Table 1 through Table 4.
TABLE-US-00003 TABLE 5 ##STR00056## MW = 2464 ##STR00057## MW =
2464 ##STR00058## MW = 2464 ##STR00059## MW = 2464 ##STR00060## MW
= 2478 ##STR00061## MW = 2478 ##STR00062## MW = 2478 ##STR00063##
MW = 2478 ##STR00064## MW = 2492 ##STR00065## MW = 2492
##STR00066## MW = 2492 ##STR00067## MW = 2492
Table 5 shows exemplary peptidommimetic macrocycles of the
invention. "Nle" represents norleucine.
[0258] The present invention contemplates the use of
non-naturally-occurring amino acids and amino acid analogs in the
synthesis of the peptidomimetic macrocycles described herein. Any
amino acid or amino acid analog amenable to the synthetic methods
employed for the synthesis of stable triazole containing
peptidomimetic macrocycles can be used in the present invention.
For example, L-propargylglycine is contemplated as a useful amino
acid in the present invention. However, other alkyne-containing
amino acids that contain a different amino acid side chain are also
useful in the invention. For example, L-propargylglycine contains
one methylene unit between the .alpha.-carbon of the amino acid and
the alkyne of the amino acid side chain. The invention also
contemplates the use of amino acids with multiple methylene units
between the .alpha.-carbon and the alkyne. Also, the azido-analogs
of amino acids L-lysine, D-lysine, alpha-methyl-L-lysine, and
alpha-methyl-D-lysine are contemplated as useful amino acids in the
present invention. However, other terminal azide amino acids that
contain a different amino acid side chain are also useful in the
invention. For example, the azido-analog of L-lysine contains four
methylene units between the .alpha.-carbon of the amino acid and
the terminal azide of the amino acid side chain. The invention also
contemplates the use of amino acids with fewer than or greater than
four methylene units between the .alpha.-carbon and the terminal
azide. Table 6 shows some amino acids useful in the preparation of
peptidomimetic macrocycles of the invention.
TABLE-US-00004 TABLE 6 ##STR00068## ##STR00069## ##STR00070##
##STR00071## ##STR00072## ##STR00073## ##STR00074## ##STR00075##
##STR00076## ##STR00077## ##STR00078## ##STR00079## ##STR00080##
##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085##
##STR00086## ##STR00087##
[0259] Table 6 shows exemplary amino acids useful in the
preparation of peptidomimetic macrocycles of the invention.
[0260] In some embodiments the amino acids and amino acid analogs
are of the D-configuration. In other embodiments they are of the
L-configuration. In some embodiments, some of the amino acids and
amino acid analogs contained in the peptidomimetic are of the
D-configuration while some of the amino acids and amino acid
analogs are of the L-configuration. In some embodiments the amino
acid analogs are .alpha.,.alpha.-disubstituted, such as
.alpha.-methyl-L-propargylglycine,
.alpha.-methyl-D-propargylglycine,
.alpha.-azido-alpha-methyl-L-lysine, and
.epsilon.-azido-alpha-methyl-D-lysine. In some embodiments the
amino acid analogs are N-alkylated, e.g.,
N-methyl-L-propargylglycine, N-methyl-D-propargylglycine,
N-methyl-.epsilon.-azido-L-lysine, and
N-methyl-.epsilon.-azido-D-lysine.
[0261] In some embodiments, the --NH moiety of the amino acid is
protected using a protecting group, including without limitation
-Fmoc and -Boc. In other embodiments, the amino acid is not
protected prior to synthesis of the peptidomimetic macrocycle.
[0262] In other embodiments, peptidomimetic macrocycles of Formula
III are synthesized. The following synthetic schemes describe the
preparation of such compounds. To simplify the drawings, the
illustrative schemes depict amino acid analogs derived from L- or
D-cysteine, in which L.sub.1 and L.sub.3 are both --(CH.sub.2)--.
However, as noted throughout the detailed description above, many
other amino acid analogs can be employed in which L.sub.1 and
L.sub.3 can be independently selected from the various structures
disclosed herein. The symbols "[AA].sub.m", "[AA].sub.n",
"[AA].sub.o" represent a sequence of amide bond-linked moieties
such as natural or unnatural amino acids. As described previously,
each occurrence of "AA" is independent of any other occurrence of
"AA", and a formula such as "[AA].sub.m" encompasses, for example,
sequences of non-identical amino acids as well as sequences of
identical amino acids.
##STR00088## ##STR00089##
[0263] In Scheme 6, the peptidomimetic precursor contains two --SH
moieties and is synthesized by solid-phase peptide synthesis (SPPS)
using commercially available N-.alpha.-Fmoc amino acids such as
N-.alpha.-Fmoc-S-trityl-L-cysteine or
N-.alpha.-Fmoc-5-trityl-D-cysteine. Alpha-methylated versions of
D-cysteine or L-cysteine are generated by known methods (Seebach et
al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and
references therein) and then converted to the appropriately
protected N-.alpha.-Fmoc-S-trityl monomers by known methods
("Bioorganic Chemistry: Peptides and Proteins", Oxford University
Press, New York: 1998, the entire contents of which are
incorporated herein by reference). The precursor peptidomimetic is
then deprotected and cleaved from the solid-phase resin by standard
conditions (e.g., strong acid such as 95% TFA). The precursor
peptidomimetic is reacted as a crude mixture or is purified prior
to reaction with X-L.sub.2-Y in organic or aqueous solutions. In
some embodiments the alkylation reaction is performed under dilute
conditions (i.e. 0.15 mmol/L) to favor macrocyclization and to
avoid polymerization. In some embodiments, the alkylation reaction
is performed in organic solutions such as liquid NH.sub.3 (Mosberg
et al. (1985), J. Am. Chem. Soc. 107:2986-2987; Szewczuk et al.
(1992), Int. J. Peptide Protein Res. 40:233-242), NH.sub.3/MeOH, or
NH.sub.3/DMF (Or et al. (1991), J. Org. Chem. 56:3146-3149). In
other embodiments, the alkylation is performed in an aqueous
solution such as 6M guanidinium HCL, pH 8 (Brunel et al. (2005),
Chem. Commun. (20):2552-2554). In other embodiments, the solvent
used for the alkylation reaction is DMF or dichloroethane.
##STR00090## ##STR00091##
[0264] In Scheme 7, the precursor peptidomimetic contains two or
more --SH moieties, of which two are specially protected to allow
their selective deprotection and subsequent alkylation for
macrocycle formation. The precursor peptidomimetic is synthesized
by solid-phase peptide synthesis (SPPS) using commercially
available N-.alpha.-Fmoc amino acids such as
N-.alpha.-Fmoc-S-p-methoxytrityl-L-cysteine or
N-.alpha.-Fmoc-S-p-methoxytrityl-D-cysteine. Alpha-methylated
versions of D-cysteine or L-cysteine are generated by known methods
(Seebach et al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748,
and references therein) and then converted to the appropriately
protected N-.alpha.-Fmoc-S-p-methoxytrityl monomers by known
methods (Bioorganic Chemistry: Peptides and Proteins, Oxford
University Press, New York: 1998, the entire contents of which are
incorporated herein by reference). The Mmt protecting groups of the
peptidomimetic precursor are then selectively cleaved by standard
conditions (e.g., mild acid such as 1% TFA in DCM). The precursor
peptidomimetic is then reacted on the resin with X-L.sub.2-Y in an
organic solution. For example, the reaction takes place in the
presence of a hindered base such as diisopropylethylamine. In some
embodiments, the alkylation reaction is performed in organic
solutions such as liquid NH.sub.3 (Mosberg et al. (1985), J. Am.
Chem. Soc. 107:2986-2987; Szewczuk et al. (1992), Int. J. Peptide
Protein Res. 40 :233-242), NH.sub.3/MeOH or NH.sub.3/DMF (Or et al.
(1991), J. Org. Chem. 56:3146-3149). In other embodiments, the
alkylation reaction is performed in DMF or dichloroethane. The
peptidomimetic macrocycle is then deprotected and cleaved from the
solid-phase resin by standard conditions (e.g., strong acid such as
95% TFA).
##STR00092##
[0265] In Scheme 8, the peptidomimetic precursor contains two or
more --SH moieties, of which two are specially protected to allow
their selective deprotection and subsequent alkylation for
macrocycle formation. The peptidomimetic precursor is synthesized
by solid-phase peptide synthesis (SPPS) using commercially
available N-.alpha.-Fmoc amino acids such as
N-.alpha.-Fmoc-S-p-methoxytrityl-L-cysteine,
N-.alpha.-Fmoc-S-p-methoxytrityl-D-cysteine,
N-.alpha.-Fmoc-S--S-t-butyl-L-cysteine, and
N-.alpha.-Fmoc-S--S-t-butyl-D-cysteine. Alpha-methylated versions
of D-cysteine or L-cysteine are generated by known methods (Seebach
et al. (1996), Angew. Chem. Int. Ed. Engl. 35:2708-2748, and
references therein) and then converted to the appropriately
protected N-.alpha.-Fmoc-S-p-methoxytrityl or
N-.alpha.-Fmoc-S--S-t-butyl monomers by known methods (Bioorganic
Chemistry: Peptides and Proteins, Oxford University Press, New
York: 1998, the entire contents of which are incorporated herein by
reference). The S--S-tButyl protecting group of the peptidomimetic
precursor is selectively cleaved by known conditions (e.g., 20%
2-mercaptoethanol in DMF, reference: Galande et al. (2005), J.
Comb. Chem. 7:174-177). The precursor peptidomimetic is then
reacted on the resin with a molar excess of X-L.sub.2-Y in an
organic solution. For example, the reaction takes place in the
presence of a hindered base such as diisopropylethylamine. The Mmt
protecting group of the peptidomimetic precursor is then
selectively cleaved by standard conditions (e.g., mild acid such as
1% TFA in DCM). The peptidomimetic precursor is then cyclized on
the resin by treatment with a hindered base in organic solutions.
In some embodiments, the alkylation reaction is performed in
organic solutions such as NH.sub.3/MeOH or NH.sub.3/DMF (Or et al.
(1991), J. Org. Chem. 56:3146-3149). The peptidomimetic macrocycle
is then deprotected and cleaved from the solid-phase resin by
standard conditions (e.g., strong acid such as 95% TFA).
##STR00093##
[0266] In Scheme 9, the peptidomimetic precursor contains two
L-cysteine moieties. The peptidomimetic precursor is synthesized by
known biological expression systems in living cells or by known in
vitro, cell-free, expression methods. The precursor peptidomimetic
is reacted as a crude mixture or is purified prior to reaction with
X-L.sub.2-Y in organic or aqueous solutions. In some embodiments
the alkylation reaction is performed under dilute conditions (i.e.
0.15 mmol/L) to favor macrocyclization and to avoid polymerization.
In some embodiments, the alkylation reaction is performed in
organic solutions such as liquid NH.sub.3 (Mosberg et al. (1985),
J. Am Chem. Soc. 107:2986-2987; Szewczuk et al. (1992), Int. J.
Peptide Protein Res. 40 :233-242), NH.sub.3/MeOH, or NH.sub.3/DMF
(Or et al. (1991), J. Org. Chem. 56:3146-3149). In other
embodiments, the alkylation is performed in an aqueous solution
such as 6M guanidinium HCL, pH 8 (Brunel et al. (2005), Chem.
Commun. (20):2552-2554). In other embodiments, the alkylation is
performed in DMF or dichloroethane. In another embodiment, the
alkylation is performed in non-denaturing aqueous solutions, and in
yet another embodiment the alkylation is performed under conditions
that favor .alpha.-helical structure formation. In yet another
embodiment, the alkylation is performed under conditions that favor
the binding of the precursor peptidomimetic to another protein, so
as to induce the formation of the bound .alpha.-helical
conformation during the alkylation.
[0267] Various embodiments for X and Y are envisioned which are
suitable for reacting with thiol groups. In general, each X or Y is
independently be selected from the general category shown in Table
5. For example, X and Y are halides such as --Cl, --Br or --I. Any
of the macrocycle-forming linkers described herein may be used in
any combination with any of the sequences shown in Tables 14 and
also with any of the R-- substituents indicated herein.
TABLE-US-00005 TABLE 7 Examples of Reactive Groups Capable of
Reacting with Thiol Groups and Resulting Linkages (1) X or Y (2)
Resulting Covalent Linkage (3) acrylamide (4) Thioether (5) halide
(e.g. alkyl or aryl halide) (6) Thioether (7) sulfonate (8)
Thioether (9) aziridine (10) Thioether (11) epoxide (12) Thioether
(13) haloacetamide (14) Thioether (15) maleimide (16) Thioether
(17) sulfonate ester (18) Thioether
[0268] Table 8 shows exemplary macrocycles of the invention.
"N.sub.L" represents norleucine and replaces a methionine residue.
It is envisioned that similar linkers are used to synthesize
peptidomimetic macrocycles based on the polypeptide sequences
disclosed in Table 1 through Table 4.
TABLE-US-00006 TABLE 8 Examples of Peptidomimetic Macrocycles of
the Invention ##STR00094## MW = 2477 ##STR00095## MW = 2463
##STR00096## MW = 2525 ##STR00097## MW = 2531 ##STR00098## MW =
2475 ##STR00099## MW = 2475 For the examples shown in this table,
"N.sub.L" represents norleucine.
[0269] The present invention contemplates the use of both
naturally-occurring and non-naturally-occurring amino acids and
amino acid analogs in the synthesis of the peptidomimetic
macrocycles of Formula (III). Any amino acid or amino acid analog
amenable to the synthetic methods employed for the synthesis of
stable bis-sulfhydryl containing peptidomimetic macrocycles can be
used in the present invention. For example, cysteine is
contemplated as a useful amino acid in the present invention.
However, sulfur containing amino acids other than cysteine that
contain a different amino acid side chain are also useful. For
example, cysteine contains one methylene unit between the
.alpha.-carbon of the amino acid and the terminal --SH of the amino
acid side chain. The invention also contemplates the use of amino
acids with multiple methylene units between the .alpha.-carbon and
the terminal --SH. Non-limiting examples include
.alpha.-methyl-L-homocysteine and .alpha.-methyl-D-homocysteine. In
some embodiments the amino acids and amino acid analogs are of the
D-configuration. In other embodiments they are of the
L-configuration. In some embodiments, some of the amino acids and
amino acid analogs contained in the peptidomimetic are of the
D-configuration while some of the amino acids and amino acid
analogs are of the L-configuration. In some embodiments the amino
acid analogs are .alpha.,.alpha.-disubstituted, such as
.alpha.-methyl-L-cysteine and .alpha.-methyl-D-cysteine.
[0270] The invention includes macrocycles in which
macrocycle-forming linkers are used to link two or more --SH
moieties in the peptidomimetic precursors to form the
peptidomimetic macrocycles of the invention. As described above,
the macrocycle-forming linkers impart conformational rigidity,
increased metabolic stability and/or increased cell penetrability.
Furthermore, in some embodiments, the macrocycle-forming linkages
stabilize the .alpha.-helical secondary structure of the
peptidomimetic macrocycles. The macrocycle-forming linkers are of
the formula X-L.sub.2-Y, wherein both X and Y are the same or
different moieties, as defined above. Both X and Y have the
chemical characteristics that allow one macrocycle-forming linker
-L.sub.2- to bis alkylate the bis-sulfhydryl containing
peptidomimetic precursor. As defined above, the linker -L.sub.2-
includes alkylene, alkenylene, alkynylene, heteroalkylene,
cycloalkylene, heterocycloalkylene, cycloarylene, or
heterocycloarylene, or --R.sub.4--K--R.sub.4--, all of which can be
optionally substituted with an R.sub.5 group, as defined above.
Furthermore, one to three carbon atoms within the
macrocycle-forming linkers -L.sub.2-, other than the carbons
attached to the --SH of the sulfhydryl containing amino acid, are
optionally substituted with a heteroatom such as N, S or O.
[0271] The L.sub.2 component of the macrocycle-forming linker
X-L.sub.2-Y may be varied in length depending on, among other
things, the distance between the positions of the two amino acid
analogs used to form the peptidomimetic macrocycle. Furthermore, as
the lengths of L.sub.1 and/or L.sub.3 components of the
macrocycle-forming linker are varied, the length of L.sub.2 can
also be varied in order to create a linker of appropriate overall
length for forming a stable peptidomimetic macrocycle. For example,
if the amino acid analogs used are varied by adding an additional
methylene unit to each of L.sub.1 and L.sub.3, the length of
L.sub.2 are decreased in length by the equivalent of approximately
two methylene units to compensate for the increased lengths of
L.sub.1 and L.sub.3.
[0272] In some embodiments, L.sub.2 is an alkylene group of the
formula --(CH.sub.2).sub.n--, where n is an integer between about 1
and about 15. For example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In
other embodiments, L.sub.2 is an alkenylene group. In still other
embodiments, L.sub.2 is an aryl group.
[0273] Table 9 shows additional embodiments of X-L.sub.2-Y
groups.
TABLE-US-00007 TABLE 9 Exemplary L-L.sub.2-Y groups of the
invention. ##STR00100## ##STR00101## ##STR00102## ##STR00103##
##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108##
##STR00109## ##STR00110## ##STR00111## ##STR00112## ##STR00113##
##STR00114## ##STR00115## ##STR00116## ##STR00117## ##STR00118##
##STR00119## ##STR00120## ##STR00121## ##STR00122## ##STR00123##
##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128##
##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133##
##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138##
##STR00139## ##STR00140## ##STR00141## ##STR00142##
[0274] Each X and Y in this table, is, for example, independently
Cl--, Br-- or I--.
[0275] Additional methods of forming peptidomimetic macrocycles
which are envisioned as suitable to perform the present invention
include those disclosed by Mustapa, M. Firouz Mohd et al., J. Org.
Chem. (2003), 68, pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem.
Lett. (2004), 14, pp. 1403-1406; U.S. Pat. No. 5,364,851; U.S. Pat.
No. 5,446,128; U.S. Pat. No. 5,824,483; U.S. Pat. No. 6,713,280;
and U.S. Pat. No. 7,202,332. In such embodiments, aminoacid
precursors are used containing an additional substituent R-- at the
alpha position. Such aminoacids are incorporated into the
macrocycle precursor at the desired positions, which may be at the
positions where the crosslinker is substituted or, alternatively,
elsewhere in the sequence of the macrocycle precursor. Cyclization
of the precursor is then effected according to the indicated
method.
Assays
[0276] The properties of the peptidomimetic macrocycles of the
invention are assayed, for example, by using the methods described
below.
Assay to Determine .alpha.-Helicity.
[0277] In solution, the secondary structure of polypeptides with
.alpha.-helical domains will reach a dynamic equilibrium between
random coil structures and .alpha.-helical structures, often
expressed as a "percent helicity". Thus, for example, unmodified
pro-apoptotic BH3 domains are predominantly random coils in
solution, with .alpha.-helical content usually under 25%.
Peptidomimetic macrocycles with optimized linkers, on the other
hand, possess, for example, an alpha-helicity that is at least
two-fold greater than that of a corresponding uncrosslinked
polypeptide. In some embodiments, macrocycles of the invention will
possess an alpha-helicity of greater than 50%. To assay the
helicity of peptidomimetic macrocyles of the invention, such as BH3
domain-based macrocycles, the compounds are dissolved in an aqueous
solution (e.g. 50 mM potassium phosphate solution at pH 7, or
distilled H.sub.2O, to concentrations of 25-50 .mu.M). Circular
dichroism (CD) spectra are obtained on a spectropolarimeter (e.g.,
Jasco J-710) using standard measurement parameters (e.g.
temperature, 20.degree. C.; wavelength, 190-260 nm; step
resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response,
1 sec; bandwidth, 1 nm; path length, 0.1 cm). The .alpha.-helical
content of each peptide is calculated by dividing the mean residue
ellipticity (e.g. .PHI.222obs) by the reported value for a model
helical decapeptide (Yang et al. (1986), Methods Enzymol.
130:208)).
Assay to Determine Melting: Temperature (Tm).
[0278] A peptidomimetic macrocycle of the invention comprising a
secondary structure such as an .alpha.-helix exhibits, for example,
a higher melting temperature than a corresponding uncrosslinked
polypeptide. Typically peptidomimetic macrocycles of the invention
exhibit Tm of >60.degree. C. representing a highly stable
structure in aqueous solutions. To assay the effect of macrocycle
formation on melting temperature, peptidomimetic macrocycles or
unmodified peptides are dissolved in distilled H.sub.2O (e.g. at a
final concentration of 50 .mu.M) and the Tm is determined by
measuring the change in ellipticity over a temperature range (e.g.
4 to 95.degree. C.) on a spectropolarimeter (e.g., Jasco J-710)
using standard parameters (e.g. wavelength 222 nm; step resolution,
0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec;
bandwidth, 1 nm; temperature increase rate: 1.degree. C./min; path
length, 0.1 cm).
Protease Resistance Assay.
[0279] The amide bond of the peptide backbone is susceptible to
hydrolysis by proteases, thereby rendering peptidic compounds
vulnerable to rapid degradation in vivo. Peptide helix formation,
however, typically buries the amide backbone and therefore may
shield it from proteolytic cleavage. The peptidomimetic macrocycles
of the present invention may be subjected to in vitro trypsin
proteolysis to assess for any change in degradation rate compared
to a corresponding uncrosslinked polypeptide. For example, the
peptidomimetic macrocycle and a corresponding uncrosslinked
polypeptide are incubated with trypsin agarose and the reactions
quenched at various time points by centrifugation and subsequent
HPLC injection to quantitate the residual substrate by ultraviolet
absorption at 280 nm. Briefly, the peptidomimetic macrocycle and
peptidomimetic precursor (5 mcg) are incubated with trypsin agarose
(Pierce) (S/E .about.125) for 0, 10, 20, 90, and 180 minutes.
Reactions are quenched by tabletop centrifugation at high speed;
remaining substrate in the isolated supernatant is quantified by
HPLC-based peak detection at 280 nm. The proteolytic reaction
displays first order kinetics and the rate constant, k, is
determined from a plot of ln [S] versus time (k=-1Xslope).
Ex Vivo Stability Assay.
[0280] Peptidomimetic macrocycles with optimized linkers possess,
for example, an ex vivo half-life that is at least two-fold greater
than that of a corresponding uncrosslinked polypeptide, and possess
an ex vivo half-life of 12 hours or more. For ex vivo serum
stability studies, a variety of assays may be used. For example, a
peptidomimetic macrocycle and/or a corresponding uncrosslinked
polypeptide (2 mcg) are each incubated with fresh mouse, rat and/or
human serum (e.g. 1-2 mL) at 37.degree. C. for 0, 1, 2, 4, 8, and
24 hours. Samples of differing macrocycle concentration may be
prepared by serial dilution with serum. To determine the level of
intact compound, the following procedure may be used: The samples
are extracted by transferring 100 .mu.l of sera to 2 ml centrifuge
tubes followed by the addition of 10 .mu.L of 50% formic acid and
500 .mu.L acetonitrile and centrifugation at 14,000 RPM for 10 min
at 4.+-.2.degree. C. The supernatants are then transferred to fresh
2 ml tubes and evaporated on Turbovap under N.sub.2<10 psi,
37.degree. C. The samples are reconstituted in 100 .mu.L of 50:50
acetonitrile:water and submitted to LC-MS/MS analysis. Equivalent
or similar procedures for testing ex vivo stability are known and
may be used to determine stability of macrocycles in serum.
In Vitro Binding Assays.
[0281] To assess the binding and affinity of peptidomimetic
macrocycles and peptidomimetic precursors to acceptor proteins, a
fluorescence polarization assay (FPA) is used, for example. The FPA
technique measures the molecular orientation and mobility using
polarized light and fluorescent tracer. When excited with polarized
light, fluorescent tracers (e.g., FITC) attached to molecules with
high apparent molecular weights (e.g. FITC-labeled peptides bound
to a large protein) emit higher levels of polarized fluorescence
due to their slower rates of rotation as compared to fluorescent
tracers attached to smaller molecules (e.g. FITC-labeled peptides
that are free in solution).
[0282] For example, fluoresceinated peptidomimetic macrocycles (25
mM) are incubated with the acceptor protein (25-1000 nM) in binding
buffer (140 mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room
temperature. Binding activity is measured, for example, by
fluorescence polarization on a luminescence spectrophotometer (e.g.
Perkin-Elmer LS50B). Kd values may be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.). A peptidomimetic
macrocycle of the invention shows, in some instances, similar or
lower Kd than a corresponding uncrosslinked polypeptide.
[0283] Acceptor proteins for BH3-peptides such as BCL-2,
BCL-X.sub.L, BAX or MCL1 may, for example, be used in this assay.
Additional methods to perform such assays are described in the
Example section below.
In Vitro Displacement Assays to Characterize Antagonists of
Peptide-Protein Interactions.
[0284] To assess the binding and affinity of compounds that
antagonize the interaction between a peptide (e.g. a BH3 peptide or
a p53 peptide) and an acceptor protein, a fluorescence polarization
assay (FPA) utilizing a fluoresceinated peptidomimetic macrocycle
derived from a peptidomimetic precursor sequence is used, for
example. The FPA technique measures the molecular orientation and
mobility using polarized light and fluorescent tracer. When excited
with polarized light, fluorescent tracers (e.g., FITC) attached to
molecules with high apparent molecular weights (e.g. FITC-labeled
peptides bound to a large protein) emit higher levels of polarized
fluorescence due to their slower rates of rotation as compared to
fluorescent tracers attached to smaller molecules (e.g.
FITC-labeled peptides that are free in solution). A compound that
antagonizes the interaction between the fluoresceinated
peptidomimetic macrocycle and an acceptor protein will be detected
in a competitive binding FPA experiment.
[0285] For example, putative antagonist compounds (1 nM to 1 mM)
and a fluoresceinated peptidomimetic macrocycle (25 nM) are
incubated with the acceptor protein (50 nM) in binding buffer (140
mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room
temperature. Antagonist binding activity is measured, for example,
by fluorescence polarization on a luminescence spectrophotometer
(e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear
regression analysis using, for example, Graphpad Prism software
(GraphPad Software, Inc., San Diego, Calif.).
[0286] Any class of molecule, such as small organic molecules,
peptides, oligonucleotides or proteins can be examined as putative
antagonists in this assay. Acceptor proteins for BH3-peptides such
as BCL2, BCL-XL, BAX or MCL1 can be used in this assay. Additional
methods to perform such assays are described in the Example section
below.
Binding Assays in Cell Lysates or Intact Cells.
[0287] It is possible to measure binding of peptides or
peptidomimetic macrocycles to their natural acceptors in cell
lysates or intact cells by immunoprecipitation and pull-down
experiments. For example, intact cells are incubated with
fluoresceinated (FITC-labeled) or biotinylated compounds for 4 hrs
in the absence of serum, followed by serum replacement and further
incubation that ranges from 4-18 hrs. Alternatively, cells can be
incubated for the duration of the experiment in Opti-MEM
(Invitrogen). Cells are then pelleted and incubated in lysis buffer
(50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and protease inhibitor
cocktail) for 10 minutes at 4.degree. C. 1% NP-40 or Triton X-100
may be used instead of CHAPS. Extracts are centrifuged at 14,000
rpm for 15 minutes and supernatants collected and incubated with 10
.mu.l goat anti-FITC antibody or streptavidin-coated beads for 2
hrs, rotating at 4.degree. C. followed by further 2 hrs incubation
at 4.degree. C. with protein A/G Sepharose (50 .mu.l of 50% bead
slurry)). No secondary step is necessary if using streptavidin
beads to pull down biotinylated compounds. Alternatively
FITC-labeled or biotinylated compounds are incubated with cell
lysates, prepared as described above, for 2 hrs, rotating at
4.degree. C. followed by incubation with 10 .mu.l goat anti-FITC
antibody or streptavidin-coated beads for 2 hrs, rotating at
4.degree. C. followed by further 2 hrs incubation at 4.degree. C.
with protein A/G Sepharose (50 .mu.l of 50% bead slurry), no
secondary step is necessary if using streptavidin beads to pull
down biotinylated compounds. After quick centrifugation, the
pellets may be washed in lysis buffer containing increasing salt
concentration (e.g., 150, 300, 500 mM of NaCl). The beads may be
then re-equilibrated at 150 mM NaCl before addition of
SDS-containing sample buffer and boiling. The beads and cell
lysates may be electrophoresed using 4%-12% gradient Bis-Tris gels
followed by transfer into Immobilon-P membranes. After blocking,
blots may be incubated with an antibody that detects FITC or
biotin, respectively and also with one or more antibodies that
detect proteins that bind to the peptidomimetic macrocycle,
including BCL2, MCL1, BCL-XL, A1, BAX, and BAK. The lysate blots
are also probed with anti-Hsc-70 for loading control.
Alternatively, after electrophoresis the gel may be silver stained
to detect proteins that come down specifically with FITC-labeled or
biotinylated compounds.
Cellular Penetrability Assays.
[0288] A peptidomimetic macrocycle is, for example, more cell
permeable compared to a corresponding uncrosslinked polypeptide. In
some embodiments, the peptidomimetic macrocycles are more cell
permeable than a corresponding uncrosslinked polypeptides.
Peptidomimetic macrocycles with optimized linkers possess, for
example, cell penetrability that is at least two-fold greater than
a corresponding uncrosslinked polypeptide, and often 20% or more of
the applied peptidomimetic macrocycle will be observed to have
penetrated the cell after 4 hours. To measure the cell
penetrability of peptidomimetic macrocycles and corresponding
uncrosslinked polypeptides, intact cells are incubated with
fluoresceinated peptidomimetic macrocycles or corresponding
uncrosslinked polypeptides (10 .mu.M) for 4 hrs in serum free media
at 37.degree. C., washed twice with media and incubated with
trypsin (0.25%) for 10 min at 37.degree. C. The cells are washed
again and resuspended in PBS. Cellular fluorescence is analyzed,
for example, by using either a FACSCalibur flow cytometer or
Cellomics' KineticScan.RTM. HCS Reader. Additional methods of
quantitating cellular penetration may be used. A particular method
is described in more details in the Examples provided.
Cellular Efficacy Assays.
[0289] The efficacy of certain peptidomimetic macrocycles is
determined, for example, in cell-based killing assays using a
variety of tumorigenic and non-tumorigenic cell lines and primary
cells derived from human or mouse cell populations. Cell viability
is monitored, for example, over 24-96 hrs of incubation with
peptidomimetic macrocycles (0.5 to 50 .mu.M) to identify those that
kill at EC.sub.50<10 .mu.M. In this context, EC.sub.50 refers to
the half maximal effective concentration, which is the
concentration of peptidomimetic macrocycle at which 50% the
population is viable. Several standard assays that measure cell
viability are commercially available and are optionally used to
assess the efficacy of the peptidomimetic macrocycles. In addition,
assays that measure Annexin V and caspase activation are optionally
used to assess whether the peptidomimetic macrocycles kill cells by
activating the apoptotic machinery. For example, the Cell Titer-glo
assay is used which determines cell viability as a function of
intracellular ATP concentration.
In Vivo Stability Assay.
[0290] To investigate the in vivo stability of the peptidomimetic
macrocycles, the compounds are, for example, administered to mice
and/or rats by IV, IP, SC, PO or inhalation routes at
concentrations ranging from 0.1 to 50 mg/kg and blood specimens
withdrawn at 0', 5', 15', 30', 1 hr, 4 hrs, 8 hrs, 12 hrs, 24 hrs
and 48 hrs post-injection. Levels of intact compound in 25 .mu.L of
fresh serum are then measured by LC-MS/MS as described herein.
In Vivo Efficacy in Animal Models.
[0291] To determine the anti-oncogenic activity of peptidomimetic
macrocycles of the invention in vivo, the compounds are, for
example, given alone (IP, IV, SC, PO, by inhalation or nasal
routes) or in combination with sub-optimal doses of relevant
chemotherapy (e.g., cyclophosphamide, doxorubicin, etoposide). In
one example, 5.times.10.sup.6 SEMK2 cells (established from the
bone marrow of a patient with acute lymphoblastic leukemia) that
stably express luciferase are injected by tail vein in NOD-SCID,
SCID-beige or NOD.IL2rg KO mice 3 hrs after they have been
subjected to total body irradiation. Non-radiated mice may also be
used for these studies. If left untreated, this form of leukemia is
fatal in 3 weeks in this model. The leukemia is readily monitored,
for example, by injecting the mice with D-luciferin (60 mg/kg) and
imaging the anesthetized animals (e.g., Xenogen In Vivo Imaging
System, Caliper Life Sciences, Hopkinton, Mass.). Total body
bioluminescence is quantified by integration of photonic flux
(photons/sec) by Living Image Software (Caliper Life Sciences,
Hopkinton, Mass.). Peptidomimetic macrocycles alone or in
combination with sub-optimal doses of relevant chemotherapeutics
agents are, for example, administered to leukemic mice (8-10 days
after injection/day 1 of experiment, in bioluminescence range of
14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to
50 mg/kg for 7 to 21 days. Optionally, the mice are imaged
throughout the experiment every other day and survival monitored
daily for the duration of the experiment. Expired mice are
optionally subjected to necropsy at the end of the experiment.
Another animal model is implantation into NOD-SCID mice of DoHH2, a
cell line derived from human follicular lymphoma, that stably
expresses luciferase. These in vivo tests optionally generate
preliminary pharmacokinetic, pharmacodynamic and toxicology
data.
Clinical Trials.
[0292] To determine the suitability of the peptidomimetic
macrocycles of the invention for treatment of humans, clinical
trials are performed. For example, patients diagnosed with cancer
and in need of treatment are selected and separated in treatment
and one or more control groups, wherein the treatment group is
administered a peptidomimetic macrocycle of the invention, while
the control groups receive a placebo, a known anti-cancer drug, or
the standard of care. The treatment safety and efficacy of the
peptidomimetic macrocycles of the invention can thus be evaluated
by performing comparisons of the patient groups with respect to
factors such as survival and quality-of-life. In this example, the
patient group treated with a peptidomimetic macrocycle show
improved long-term survival compared to a patient control group
treated with a placebo or the standard of care.
Pharmaceutical Compositions and Routes of Administration
[0293] Methods of administration include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, oral, sublingual,
intracerebral, intravaginal, transdermal, rectal, by inhalation, or
topical by application to ears, nose, eyes, or skin.
[0294] The peptidomimetic macrocycles of the invention also include
pharmaceutically acceptable derivatives or prodrugs thereof. A
"pharmaceutically acceptable derivative" means any pharmaceutically
acceptable salt, ester, salt of an ester, pro-drug or other
derivative of a compound of this invention which, upon
administration to a recipient, is capable of providing (directly or
indirectly) a compound of this invention. For example,
pharmaceutically acceptable derivatives may increase the
bioavailability of the compounds of the invention when administered
to a mammal (e.g., by increasing absorption into the blood of an
orally administered compound) or which increases delivery of the
active compound to a biological compartment (e.g., the brain or
lymphatic system) relative to the parent species. Some
pharmaceutically acceptable derivatives include a chemical group
which increases aqueous solubility or active transport across the
gastrointestinal mucosa.
[0295] In some embodiments, the peptidomimetic macrocycles of the
invention are modified by covalently or non-covalently joining
appropriate functional groups to enhance selective biological
properties. Such modifications include those which increase
biological penetration into a given biological compartment (e.g.,
blood, lymphatic system, central nervous system), increase oral
availability, increase solubility to allow administration by
injection, alter metabolism, and alter rate of excretion.
[0296] Pharmaceutically acceptable salts of the compounds of this
invention include those derived from pharmaceutically acceptable
inorganic and organic acids and bases. Examples of suitable acid
salts include acetate, adipate, benzoate, benzenesulfonate,
butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate,
glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride,
hydrobromide, hydroiodide, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
palmoate, phosphate, picrate, pivalate, propionate, salicylate,
succinate, sulfate, tartrate, tosylate and undecanoate. Salts
derived from appropriate bases include alkali metal (e.g., sodium),
alkaline earth metal (e.g., magnesium), ammonium and
N-(alkyl).sub.4.sup.+ salts.
[0297] For preparing pharmaceutical compositions from the compounds
of the present invention, pharmaceutically acceptable carriers
include either solid or liquid carriers. Solid form preparations
include powders, tablets, pills, capsules, cachets, suppositories,
and dispersible granules. A solid carrier can be one or more
substances, which also acts as diluents, flavoring agents, binders,
preservatives, tablet disintegrating agents, or an encapsulating
material. Details on techniques for formulation and administration
are well described in the scientific and patent literature, see,
e.g., the latest edition of Remington's Pharmaceutical Sciences,
Maack Publishing Co, Easton Pa.
[0298] In powders, the carrier is a finely divided solid, which is
in a mixture with the finely divided active component. In tablets,
the active component is mixed with the carrier having the necessary
binding properties in suitable proportions and compacted in the
shape and size desired.
[0299] Suitable solid excipients are carbohydrate or protein
fillers include, but are not limited to sugars, including dextrose,
lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat,
rice, potato, or other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
and gums including arabic and tragacanth; as well as proteins such
as gelatin and collagen. If desired, disintegrating or solubilizing
agents are added, such as the cross-linked polyvinyl pyrrolidone,
agar, alginic acid, or a salt thereof, such as sodium alginate.
[0300] Liquid form preparations include solutions, suspensions, and
emulsions, for example, water or water/propylene glycol solutions.
For parenteral injection, liquid preparations can be formulated in
solution in aqueous polyethylene glycol solution. The term
"parenteral" as used herein refers modes of administration
including intravenous, intraarterial, intramuscular,
intraperitoneal, intrasternal, and subcutaneous.
[0301] The pharmaceutical preparation is preferably in unit dosage
form. In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of preparation, such as packeted tablets,
capsules, and powders in vials or ampoules. Also, the unit dosage
form can be a capsule, tablet, cachet, or lozenge itself, or it can
be the appropriate number of any of these in packaged form.
[0302] When the compositions of this invention comprise a
combination of a peptidomimetic macrocycle and one or more
additional therapeutic or prophylactic agents, both the compound
and the additional agent should be present at dosage levels of
between about 1 to 100%, and more preferably between about 5 to 95%
of the dosage normally administered in a monotherapy regimen. In
some embodiments, the additional agents are administered
separately, as part of a multiple dose regimen, from the compounds
of this invention. Alternatively, those agents are part of a single
dosage form, mixed together with the compounds of this invention in
a single composition.
Methods of Use
[0303] In one aspect, the present invention provides novel
peptidomimetic macrocycles that are useful in competitive binding
assays to identify agents which bind to the natural ligand(s) of
the proteins or peptides upon which the peptidomimetic macrocycles
are modeled. For example, in the BH3/BCL-X.sub.L anti-apoptotic
system labeled peptidomimetic macrocycles based on BH3 can be used
in a BCL-X.sub.L binding assay along with small molecules that
competitively bind to BCL-X.sub.L. Competitive binding studies
allow for rapid in vitro evaluation and determination of drug
candidates specific for the BH3/BCL-X.sub.L system. The invention
further provides for the generation of antibodies against the
peptidomimetic macrocycles. In some embodiments, these antibodies
specifically bind both the peptidomimetic macrocycle and the BH3
peptidomimetic precursors upon which the peptidomimetic macrocycles
are derived. Such antibodies, for example, disrupt the
BH3/BCL-X.sub.L systems, respectively.
[0304] In other aspects, the present invention provides for both
prophylactic and therapeutic methods of treating a subject at risk
of (or susceptible to) a disorder or having a disorder associated
with aberrant (e.g., insufficient or excessive) BCL-2 family member
expression or activity (e.g., extrinsic or intrinsic apoptotic
pathway abnormalities). It is believed that some BCL-2 type
disorders are caused, at least in part, by an abnormal level of one
or more BCL-2 family members (e.g., over or under expression), or
by the presence of one or more BCL-2 family members exhibiting
abnormal activity. As such, the reduction in the level and/or
activity of the BCL-2 family member or the enhancement of the level
and/or activity of the BCL-2 family member, is used, for example,
to ameliorate or reduce the adverse symptoms of the disorder.
[0305] In one embodiment, the compounds of the invention are used
to treat disorders associated with expression or overexpression of
Mcl-1. Mcl-1 has been shown to be expressed in many tissues and
neoplastic cell lines and is thought to participate in the
development of malignancies (Thallinger et al. (2004) Clin. Cancer
Res. 10:4185-4191). The peptidomimetic macrocycles of the invention
may be used for the treatment of such malignancies.
[0306] In one embodiment, the disorder being treated (e.g. cancer)
is differentially responsive to the peptidomimetic macrocycles of
the invention. In some embodiments, the cancer is treated with a
BIM peptidomimetic macrocycle and is at least 2-fold less sensitive
to treatment using a BID polypeptide (such as a BID peptidomimetic
macrocycle or uncrosslinked polypeptide) as measured in an in vitro
cell viability assay. In other embodiments, the cancer is at least
5-fold less sensitive to treatment using a BID polypeptide as
measured in an in vitro cell viability assay. In yet other
embodiments, the cancer is at least 8-fold less sensitive to
treatment using a BID polypeptide as measured in an in vitro cell
viability assay. In other embodiments, the cancer is treated with a
BID peptidomimetic macrocycle and is at least 2-fold less sensitive
to treatment using a BIM polypeptide (such as a BIM peptidomimetic
macrocycle or uncrosslinked polypeptide) as measured in an in vitro
cell viability assay. In other embodiments, the cancer is at least
5-fold less sensitive to treatment using a BIM polypeptide as
measured in an in vitro cell viability assay. In yet other
embodiments, the cancer is at least 8-fold less sensitive to
treatment using a BIM polypeptide as measured in an in vitro cell
viability assay.
[0307] In another embodiment, a method of treating a human patient
is provided comprising performing an assay to evaluate the levels
of a BCL-family protein and administering to the patient a
peptidomimetic macrocycle if an abberant or irregular level of
expression of the BCL-family protein is detected. BCL-family
proteins include, for example, BCL-2, BCL-XL, MCL-1, Bfl1/A1,
BOO/DIVA, NRH/NR13, BAX, BAD, BAK, BOK, BIK, PUMA, BIM, BMF, BLK,
BNIP3, HRK, NIX, SPIKE, and Noxa. In one embodiment, a method of
treating a human patient is provided comprising performing an assay
to evaluate the levels of BCL-2 in the patient and administering to
the patient a peptidomimetic macrocycle if an abberant or irregular
level of expression of BCL-2 is detected. In another embodiment, a
method of treating a human patient is provided comprising
performing an assay to evaluate the levels of BCL-XL in the patient
and administering to the patient a peptidomimetic macrocycle if an
abberant or irregular level of expression of BCL-XL is detected. In
another embodiment, a method of treating a human patient is
provided comprising performing an assay to evaluate the levels of
MCL-1 in the patient and administering to the patient a
peptidomimetic macrocycle if an abberant or irregular level of
expression of MCL-1 is detected. In another embodiment, a method of
treating a human patient is provided comprising performing an assay
to evaluate the levels of BAX in the patient and administering to
the patient a peptidomimetic macrocycle if an abberant or irregular
level of expression of BAX is detected. In another embodiment, a
method of treating a human patient is provided comprising
performing an assay to evaluate the levels of BAD in the patient
and administering to the patient a peptidomimetic macrocycle if an
abberant or irregular level of expression of BAD is detected. In
another embodiment, a method of treating a human patient is
provided comprising performing an assay to evaluate the levels of
BAK in the patient and administering to the patient a
peptidomimetic macrocycle if an abberant or irregular level of
expression of BAK is detected. In another embodiment, a method of
treating a human patient is provided comprising performing an assay
to evaluate the levels of PUMA in the patient and administering to
the patient a peptidomimetic macrocycle if an abberant or irregular
level of expression of PUMA is detected. In another embodiment, a
method of treating a human patient is provided comprising
performing an assay to evaluate the levels of Noxa in the patient
and administering to the patient a peptidomimetic macrocycle if an
abberant or irregular level of expression of Noxa is detected. In
another embodiment, a method of treating a human patient is
provided comprising performing an assay to evaluate the levels of
Noxa in the patient and administering to the patient a
peptidomimetic macrocycle if an abberant or irregular level of
expression of Noxa is detected. In another embodiment, a method of
treating a human patient is provided comprising performing an assay
to evaluate the levels of Bfl1/A1 in the patient and administering
to the patient a peptidomimetic macrocycle if an abberant or
irregular level of expression of Bfl1/A1 is detected. In another
embodiment, a method of treating a human patient is provided
comprising performing an assay to evaluate the levels of BOO/DIVA
in the patient and administering to the patient a peptidomimetic
macrocycle if an abberant or irregular level of expression of
BOO/DIVA is detected. In another embodiment, a method of treating a
human patient is provided comprising performing an assay to
evaluate the levels of NRH/NR13 in the patient and administering to
the patient a peptidomimetic macrocycle if an abberant or irregular
level of expression of NRH/NR13 is detected. In another embodiment,
a method of treating a human patient is provided comprising
performing an assay to evaluate the levels of BOK in the patient
and administering to the patient a peptidomimetic macrocycle if an
abberant or irregular level of expression of BOK is detected. In
another embodiment, a method of treating a human patient is
provided comprising performing an assay to evaluate the levels of
BIK in the patient and administering to the patient a
peptidomimetic macrocycle if an abberant or irregular level of
expression of BIK is detected. In another embodiment, a method of
treating a human patient is provided comprising performing an assay
to evaluate the levels of BMF in the patient and administering to
the patient a peptidomimetic macrocycle if an abberant or irregular
level of expression of BMF is detected. In another embodiment, a
method of treating a human patient is provided comprising
performing an assay to evaluate the levels of BLK in the patient
and administering to the patient a peptidomimetic macrocycle if an
abberant or irregular level of expression of BLK is detected. In
another embodiment, a method of treating a human patient is
provided comprising performing an assay to evaluate the levels of
BNIP3 in the patient and administering to the patient a
peptidomimetic macrocycle if an abberant or irregular level of
expression of BNIP3 is detected. In another embodiment, a method of
treating a human patient is provided comprising performing an assay
to evaluate the levels of HRK in the patient and administering to
the patient a peptidomimetic macrocycle if an abberant or irregular
level of expression of HRK is detected. In another embodiment, a
method of treating a human patient is provided comprising
performing an assay to evaluate the levels of Nix in the patient
and administering to the patient a peptidomimetic macrocycle if an
abberant or irregular level of expression of Nix is detected. In
another embodiment, a method of treating a human patient is
provided comprising performing an assay to evaluate the levels of
SPIKE in the patient and administering to the patient a
peptidomimetic macrocycle if an abberant or irregular level of
expression of SPIKE is detected.
[0308] As used herein, the term "treatment" is defined as the
application or administration of a therapeutic agent to a patient,
or application or administration of a therapeutic agent to an
isolated tissue or cell line from a patient, who has a disease, a
symptom of disease or a predisposition toward a disease, with the
purpose to cure, heal, alleviate, relieve, alter, remedy,
ameliorate, improve or affect the disease, the symptoms of disease
or the predisposition toward disease.
[0309] In some embodiments, the peptidomimetics macrocycles of the
invention is used to treat, prevent, and/or diagnose cancers and
neoplastic conditions. As used herein, the terms "cancer",
"hyperproliferative" and "neoplastic" refer to cells having the
capacity for autonomous growth, i.e., an abnormal state or
condition characterized by rapidly proliferating cell growth.
Hyperproliferative and neoplastic disease states may be categorized
as pathologic, i.e., characterizing or constituting a disease
state, or may be categorized as non-pathologic, i.e., a deviation
from normal but not associated with a disease state. The term is
meant to include all types of cancerous growths or oncogenic
processes, metastatic tissues or malignantly transformed cells,
tissues, or organs, irrespective of histopathologic type or stage
of invasiveness. A metastatic tumor can arise from a multitude of
primary tumor types, including but not limited to those of breast,
lung, liver, colon and ovarian origin. "Pathologic
hyperproliferative" cells occur in disease states characterized by
malignant tumor growth. Examples of non-pathologic
hyperproliferative cells include proliferation of cells associated
with wound repair. Examples of cellular proliferative and/or
differentiative disorders include cancer, e.g., carcinoma, sarcoma,
or metastatic disorders. In some embodiments, the peptidomimetics
macrocycles are novel therapeutic agents for controlling breast
cancer, ovarian cancer, colon cancer, lung cancer, metastasis of
such cancers and the like.
[0310] Examples of cancers or neoplastic conditions include, but
are not limited to, a fibrosarcoma, myosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer,
pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer,
cancer of the head and neck, skin cancer, brain cancer, squamous
cell carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, testicular cancer, small cell lung
carcinoma, non-small cell lung carcinoma, bladder carcinoma,
epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma,
acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi
sarcoma.
[0311] Examples of proliferative disorders include hematopoietic
neoplastic disorders. As used herein, the term "hematopoietic
neoplastic disorders" includes diseases involving
hyperplastic/neoplastic cells of hematopoietic origin, e.g.,
arising from myeloid, lymphoid or erythroid lineages, or precursor
cells thereof. Preferably, the diseases arise from poorly
differentiated acute leukemias, e.g., erythroblastic leukemia and
acute megakaryoblastic leukemia. Additional exemplary myeloid
disorders include, but are not limited to, acute promyeloid
leukemia (APML), acute myelogenous leukemia (AML) and chronic
myelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit. Rev.
Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are
not limited to acute lymphoblastic leukemia (ALL) which includes
B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia
(CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and
Waldenstrom's macroglobulinemia (WM). Additional forms of malignant
lymphomas include, but are not limited to non-Hodgkin lymphoma and
variants thereof, peripheral T cell lymphomas, adult T cell
leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large
granular lymphocytic leukemia (LGF), Hodgkin's disease and
Reed-Sternberg disease.
[0312] Examples of cellular proliferative and/or differentiative
disorders of the breast include, but are not limited to,
proliferative breast disease including, e.g., epithelial
hyperplasia, sclerosing adenosis, and small duct papillomas;
tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor,
and sarcomas, and epithelial tumors such as large duct papilloma;
carcinoma of the breast including in situ (noninvasive) carcinoma
that includes ductal carcinoma in situ (including Paget's disease)
and lobular carcinoma in situ, and invasive (infiltrating)
carcinoma including, but not limited to, invasive ductal carcinoma,
invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms. Disorders in the male breast
include, but are not limited to, gynecomastia and carcinoma.
[0313] Examples of cellular proliferative and/or differentiative
disorders of the lung include, but are not limited to, bronchogenic
carcinoma, including paraneoplastic syndromes, bronchioloalveolar
carcinoma, neuroendocrine tumors, such as bronchial carcinoid,
miscellaneous tumors, and metastatic tumors; pathologies of the
pleura, including inflammatory pleural effusions, noninflammatory
pleural effusions, pneumothorax, and pleural tumors, including
solitary fibrous tumors (pleural fibroma) and malignant
mesothelioma.
[0314] Examples of cellular proliferative and/or differentiative
disorders of the colon include, but are not limited to,
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0315] Examples of cellular proliferative and/or differentiative
disorders of the liver include, but are not limited to, nodular
hyperplasias, adenomas, and malignant tumors, including primary
carcinoma of the liver and metastatic tumors.
[0316] Examples of cellular proliferative and/or differentiative
disorders of the ovary include, but are not limited to, ovarian
tumors such as, tumors of coelomic epithelium, serous tumors,
mucinous tumors, endometrioid tumors, clear cell adenocarcinoma,
cystadenofibroma, Brenner tumor, surface epithelial tumors; germ
cell tumors such as mature (benign) teratomas, monodermal
teratomas, immature malignant teratomas, dysgerminoma, endodermal
sinus tumor, choriocarcinoma; sex cord-stomal tumors such as,
granulosa-theca cell tumors, thecomafibromas, androblastomas, hill
cell tumors, and gonadoblastoma; and metastatic tumors such as
Krukenberg tumors.
Breast Cancer
[0317] In one aspect, the invention provides methods of treating
breast cancer by administering the peptidomimetic macrocycles of
the invention. Breast cancer includes invasive breast carcinomas,
such as invasive ductal carcinoma, invasive lobular carcinoma,
tubular carcinoma, invasive cribriform carcinoma, medullary
carcinoma, mucinous carcinoma and other tumours with abundant
mucin, cystadenocarcinoma, columnar cell mucinous carcinoma, signet
ring cell carcinoma, neuroendocrine tumours (including solid
neuroendocrine carcinoma, atypical carcinoid tumour, small cell/oat
cell carcinoma, or large cell neuroendocrine carcinoma), invasive
papillary carcinoma, invasive micropapillary carcinoma, apocrine
carcinoma, metaplastic carcinomas, pure epithelial metaplastic
carciomas, mixed epithelial/mesenchymal metaplastic carcinomas,
lipid-rich carcinoma, secretory carcinoma, oncocytic carcinoma,
adenoid cystic carcinoma, acinic cell carcinoma, glycogen-rich
clear cell carcinoma, sebaceous carcinoma, inflammatory carcinoma
or bilateral breast carcinoma; mesenchymal tumors such as
haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatous
stromal hyperplasia, myofibroblastoma, fibromatosis (aggressive),
inflammatory myofibroblastic tumour, lipoma, angiolipoma, granular
cell tumour, neurofibroma, schwannoma, angiosarcoma, liposarcoma,
rhabdomyosarcoma, osteosarcoma, leiomyoma, or leiomysarcoma;
myoepithelial lesions such as myoepitheliosis, adenomyoepithelial
adenosis, adenomyoepithelioma, or malignant myoepithelioma;
fibroepithelial tumours such as fibroadenoma, phyllodes tumour, low
grade periductal stromal sarcoma, or mammary hamartoma; and tumours
of the nipple such as nipple adenoma, syringomatous adenoma, or
Paget's disease of the nipple.
[0318] Treatment of breast -cancer may be effected in conjunction
with any additional therapy, such as a therapy that is part of the
standard of care. A surgical technique such as lumpectomy or
mastectomy may be performed prior to, during, or following
treatment with the peptidomimetic macrocycles of the invention.
Alternatively, radiation therapy may be used for the treatment of
breast cancer in conjunction with the peptidomimetic macrocycles of
the invention. In other cases, the peptidomimetic macrocycles of
the invention are administered in combination with a second
therapeutic agent. Such an agent may be a chemotherapeutic agent
such as an individual drug or combination of drugs and therapies.
For example, the chemotherapeutic agent can be an adjuvant
chemotherapeutic treatment such as CMF (cyclophosphamide,
methotrexate, and 5-fluorouracil); FAC or CAF (5-fluorouracil,
doxorubicin, cyclophosphamide); AC or CA (doxorubicin and
cyclophosphamide); AC-Taxol (AC followed by paclitaxel); TAC
(docetaxel, doxorubicin, and cyclophosphamide); FEC
(5-fluorouracil, epirubicin and cyclophosphamide); FECD (FEC
followed by docetaxel); TC (docetaxel and cyclophosphamide). In
addition to chemotherapy, trastu mab may also be added to the
regimen depending on the tumor characteristics (i.e. HER2/neu
status) and risk of relapse. Hormonal therapy may also be
appropriate before, during or following chemotherapeutic treatment.
For example, tamoxifen may be administered or a compound in the
category of aromatase inhibitors including, but not limited to
aminogluthetimide, anastrozole, exemestane, formestane, letrozole,
or vorozole. In other embodiments, an antiangiogenic agent may be
used in combination therapy for the treatment of breast cancer. The
antiangiogenic agent may be an anti-VEGF agent including, but not
limited to bevacizumab.
Ovarian Cancer
[0319] In another aspect, the peptidomimetic macrocycles of the
invention may be used to treat ovarian cancer. Ovarian cancers
include ovarian tumors such as, tumors of coelomic epithelium,
serous tumors, mucinous tumors, endometrioid tumors, clear cell
adenocarcinoma, cystadenofibroma, Brenner tumor, surface epithelial
tumors; germ cell tumors such as mature (benign) teratomas,
monodermal teratomas, immature malignant teratomas, dysgerminoma,
endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors
such as, granulosa-theca cell tumors, thecomafibromas,
androblastomas, hill cell tumors, and gonadoblastoma; and
metastatic tumors such as Krukenberg tumors.
[0320] The peptidomimetic macrocycles of the invention may be
administered in conjunction with a second therapy such as a therapy
that is part of the standard of care. Surgery, immunotherapy,
chemotherapy, hormone therapy, radiation therapy, or a combination
thereof are some possible treatments available for ovarian cancer.
Some possible surgical procedures include debulking, and a
unilateral or bilateral oophorectomy and/or a unilateral or
bilateral salpigectomy.
[0321] Anti-cancer drugs that may be used include cyclophosphamide,
etoposide, altretamine, and ifosfamide. Hormone therapy with the
drug tamoxifen may be used to shrink ovarian tumors. Radiation
therapy may be external beam radiation therapy and/or
brachytherapy.
Prostate Cancer
[0322] In another aspect, the peptidomimetic macrocycles of the
invention may be used to treat prostate cancer. Prostate cancers
include adenocarcinomas and metastasized adenocarcinomas. The
peptidomimetic macrocycles of the invention may be administered in
conjunction with a second therapy such as a therapy that is part of
the standard of care. Treatment for prostate cancer may involve
surgery, radiation therapy, High Intensity Focused Ultrasound
(HIFU), chemotherapy, cryosurgery, hormonal therapy, or any
combination thereof. Surgery may involve prostatectomy, radical
perineal prostatectomy, laparoscopic radical prostatectomy,
transurethral resection of the prostate or orchiectomy. Radiation
therapy may include external beam radiation therapy and/or
brachytherapy. Hormonal therapy may include orchiectomy;
administration of antiandrogens such as flutamide, bicalutamide,
nilutamide, or cyproterone acetate; medications which inhibit the
production of adrenal androgens such as DHEA, such as ketoconazole
and aminoglutethimide; and GnRH antagonists or agonists such as
Abarelix (Plenaxis.RTM.), Cetrorelix (Cetrotide.RTM.), Ganirelix
(Antagon.RTM.), leuprolide, goserelin, triptorelin, or buserelin.
Treatment with an anti-androgen agent, which blocks androgen
activity in the body, is another available therapy. Such agents
include flutamide, bicalutamide, and nilutamide. This therapy is
typically combined with LHRH analog administration or an
orchiectomy, which is termed a combined androgen blockade (CAB).
Chemotherapy includes, but is not limited to, administration of
docetaxel, for example with a corticosteroid such as prednisone.
Anti-cancer drugs such as doxorubicin, estramustine, etoposide,
mitoxantrone, vinblastine, paclitaxel, carboplatin may also be
administered to slow the growth of prostate cancer, reduce symptoms
and improve the quality of life. Additional compounds such as
bisphosphonate drugs may also be administered.
Renal Cancer
[0323] In another aspect, the peptidomimetic macrocycles of the
invention may be used to treat renal cancer. Renal cancers include,
but are not limited to, renal cell carcinomas, metastases from
extra-renal primary neoplasms, renal lymphomas, squamous cell
carcinomas, juxtaglomerular tumors (reninomas), transitional cell
carcinomas, angiomyolipomas, oncocytomas and Wilm's tumors. The
peptidomimetic macrocycles of the invention may be administered in
conjunction with a second therapy such as a therapy that is part of
the standard of care. Treatment for renal cancer may involve
surgery, percutaneous therapies, radiation therapies, chemotherapy,
vaccines, or other medication. Surgical techniques useful for
treatment of renal cancer in combination with the peptidomimetic
macrocycles of the invention include nephrectomy, which may include
removal of the adrenal gland, retroperitoneal lymph nodes, and any
other surrounding tissues affected by the invasion of the tumor.
Percutaneous therapies include, for example, image-guided therapies
which may involve imaging of a tumor followed by its targeted
destruction by radiofrequency ablation or cryotherapy. In some
cases, other chemotherapeutic or other medications useful in
treating renal cancer may be alpha-interferon, interleukin-2,
bevacizumab, sorafenib, sunitib, temsirolimus or other kinase
inhibitors.
Pancreatic Cancer
[0324] In other aspects, the invention provides methods of treating
pancreatic cancer by administering peptidomimetic macrocycles of
the invention, such as a pancreatic cancer selected from the
following: an epitheliod carcinoma in the pancreatic duct tissue
and an adenocarcinoma in a pancreatic duct. The most common type of
pancreatic cancer is an adenocarcinoma, which occurs in the lining
of the pancreatic duct. Possible treatments available for
pancreatic cancer include surgery, immunotherapy, radiation
therapy, and chemotherapy. Possible surgical treatment options
include a distal or total pancreatectomy and a
pancreaticoduodenectomy (Whipple procedure). Radiation therapy may
be an option for pancreatic cancer patients, specifically external
beam radiation where radiation is focused on the tumor by a machine
outside the body. Another option is intraoperative electron beam
radiation administered during an operation. Chemotherapy may also
be used to treat pancreatic cancer patients. Suitable anti-cancer
drugs include, but are not limited to, 5-fluorouracil (5-FU),
mitomycin, ifosfamide, doxorubicin, streptozocin, chlorozotocin,
and combinations thereof. The methods provided by the invention can
provide a beneficial effect for pancreatic cancer patients, by
administration of a polypeptide of the invention or a combination
of administration of a peptidomimetic macrocycle and surgery,
radiation therapy, or chemotherapy.
Colon Cancer
[0325] In one aspect, peptidomimetic macrocycles of the invention
may be used for the treatment of colon cancer, including but not
limited to non-neoplastic polyps, adenomas, familial syndromes,
colorectal carcinogenesis, colorectal carcinoma, and carcinoid
tumors. Possible treatments available for colon cancer that may be
used in conjunction with the peptidomimetic macrocycles of the
invention include surgery, chemotherapy, radiation therapy or
targeted drug therapy.
[0326] Radiation therapy may include external beam radiation
therapy and/or brachytherapy. Chemotherapy may be used to reduce
the likelihood of metastasis developing, shrink tumor size, or slow
tumor growth. Chemotherapy is often applied after surgery
(adjuvant), before surgery (neo-adjuvant), or as the primary
therapy if surgery is not indicated (palliative). For example,
exemplary regimens for adjuvant chemotherapy involve the
combination of infusional 5-fluorouracil, leucovorin, and
oxaliplatin (FOLFOX). First line chemotherapy regimens may involve
the combination of infusional 5-fluorouracil, leucovorin, and
oxaliplatin (FOLFOX) with a targeted drug such as bevacizumab,
cetuximab or panitumumab or infusional 5-fluorouracil, leucovorin,
and irinotecan (FOLFIRI) with targeted drug such as bevacizumab,
cetuximab or panitumumab. Other chemotherapeutic agents that may be
useful in the treatment or prevention of colon cancer in
combination with the peptidomimetic macrocycles of the invention
are Bortezomib (Velcade.RTM.), Oblimersen (Genasense.RTM., G3139),
Gefitinib and Erlotinib (Tarceva.RTM.) and Topotecan
(Hycamtin.RTM.).
Lung Cancer
[0327] Some embodiments provide methods for the treatment of lung
cancer using the peptidomimetic macrocycles of the invention.
Examples of cellular proliferative and/or differentiative disorders
of the lung include, but are not limited to, bronchogenic
carcinoma, including paraneoplastic syndromes, bronchioloalveolar
carcinoma, neuroendocrine tumors, such as bronchial carcinoid,
miscellaneous tumors, and metastatic tumors; pathologies of the
pleura, including inflammatory pleural effusions, noninflammatory
pleural effusions, pneumothorax, and pleural tumors, including
solitary fibrous tumors (pleural fibroma) and malignant
mesothelioma.
[0328] The most common type of lung cancer is non-small cell lung
cancer (NSCLC), which accounts for approximately 80-85% of lung
cancers and is divided into squamous cell carcinomas,
adenocarcinomas, and large cell undifferentiated carcinomas. Small
cell lung cancer, e.g. small cell lung carcinomas, accounts for
15-20% of lung cancers. Treatment options for lung cancer include
surgery, immunotherapy, radiation therapy, chemotherapy,
photodynamic therapy, or a combination thereof. Some possible
surgical options for treatment of lung cancer are a segmental or
wedge resection, a lobectomy, or a pneumonectomy. Radiation therapy
may be external beam radiation therapy or brachytherapy. Some
anti-cancer drugs that may be used in chemotherapy to treat lung
cancer in combination with the peptidomimetic macrocycles of the
invention include cisplatin, carboplatin, paclitaxel, docetaxel,
gemcitabine, vinorelbine, irinotecan, etoposide, vinblastine,
gefitinib, ifosfamide, methotrexate, or a combination thereof.
Photodynamic therapy (PDT) may be used to treat lung cancer
patients. The methods described herein can provide a beneficial
effect for lung cancer patients, by administration of a
peptidomimetic macrocycle or a combination of administration of a
peptidomimetic macrocycle and surgery, radiation therapy,
chemotherapy, photodynamic therapy, or a combination thereof.
[0329] Examples of cellular proliferative and/or differentiative
disorders of the liver include, but are not limited to, nodular
hyperplasias, adenomas, and malignant tumors, including primary
carcinoma of the liver and metastatic tumors.
Immunoproliferative Disorders
[0330] Immunoproliferative disorders (also known as
"immunoproliferative diseases" or "immunoproliferative neoplasms")
are disorders of the immune system that are characterized by the
abnormal proliferation of the primary cells of the immune system,
which includes B cells, T cells and Natural Killer (NK) cells, or
by the excessive production of immunoglobulins (also known as
antibodies). Such disorders include the general categories of
lymphoproliferative disorders, hypergammaglobulinemias, and
paraproteinemias. Examples of such disorders include, but are not
limited to, X-linked lymphoproliferative disorder, autosomal
lymphoproliferative disorder, Hyper-IgM syndrome, heavy chain
disease, and cryoglobulinemia. Other immunoproliferative disorders
can be graft versus host disease (GVHD); psoriasis; immune
disorders associated with graft transplantation rejection; T cell
lymphoma; T cell acute lymphoblastic leukemia; testicular
angiocentric T cell lymphoma; benign lymphocytic angiitis; and
autoimmune diseases such as lupus erythematosus, Hashimoto's
thyroiditis, primary myxedema, Graves' disease, pernicious anemia,
autoimmune atrophic gastritis, Addison's disease, insulin dependent
diabetes mellitis, good pasture's syndrome, myasthenia gravis,
pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune
uveitis, multiple sclerosis, autoimmune hemolytic anemia,
idiopathic thrombocytopenia, primary biliary cirrhosis, chronic
action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatoid
arthritis, polymyositis, scleroderma, and mixed connective tissue
disease.
Combination Treatments
[0331] In one embodiment, peptidomimetic macrocycles of the
invention may be used for the treatment of cancer in conjunction
with alkylating and alkylating-like agents. Such agents include,
for example, nitrogen mustards such as chlorambucil, chlormethine,
cyclophosphamide, ifosfamide, and melphalan; nitrosoureas such as
carmustine, fotemustine, lomustine, and streptozocin; platinum
therapeutic agents such as carboplatin, cisplatin, oxaliplatin,
BBR3464, and satraplatin; or other agents, including but not
limited to busulfan, dacarbazine, procarbazine, temozolomide,
thiotepa, treosulfan, or uramustine.
[0332] In another embodiment, peptidomimetic macrocycles of the
invention may be used in conjunction with an antineoplastic agent
which is an antimetabolite. For example, such an antineoplastic
agent may be a folic acid such as aminopterin, methotrexate,
pemetrexed, or raltitrexed. Alternatively, the antineoplastic agent
may be a purine, including but not limited to cladribine,
clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine.
In further embodiments, the antineoplastic agent may be a
pyrimidine such as capecitabine, cytarabine, fluorouracil,
floxuridine, and gemcitabine.
[0333] In still other embodiments, peptidomimetic macrocycles of
the invention may be used in conjunction with an antineoplastic
agent which is an spindle poison/mitotic inhibitor. Agents in this
category include taxanes, for example docetaxel and paclitaxel; and
vinca alkaloids such as vinblastine, vincristine, vindesine, and
vinorelbine. In yet other embodiments, peptidomimetic macrocycles
of the invention may be used in combination with an antineoplastic
agent which is a cytotoxic/antitumor antibiotic from the
anthracycline family such as daunorubicin, doxorubicin, epirubicin,
idarubicin, mitoxantrone, pixantrone, or valrubicin; an antibiotic
from the streptomyces family such as actinomycin, bleomycin,
mitomycin, or plicamycin; or hydroxyurea. Alternatively, agents
used for combination therapy may be topoisomerase inhibitors
including, but not limited to camptothecin, topotecan, irinotecan,
etoposide, or teniposide.
[0334] Alternatively, the antineoplastic agent may be an antibody
or antibody-derived agent. For example, a receptor tyrosine
kinase-targeted antibody such as cetuximab, panitumumab, or
trastuzumab may be used Alternatively, the antibody may be an
anti-CD20 antibody such as rituximab or tositumomab, or any other
suitable antibody including but not limited to alemtuzumab,
bevacizumab, and gemtuzumab. In other embodiments, the
antineoplastic agent is a photosensitizer such as aminolevulinic
acid, methyl aminolevulinate, porfimer sodium, or verteporfin. In
still other embodiments, the antineoplastic agent is a tyrosine
kinase inhibitor such as dediranib, dasatinib, erlotinib,
gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, or
vandetanib. Other neoplastic agents suitable in the use of the
invention include, for example, alitretinoin, tretinoin,
altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase
(pegaspargase), bexarotene, bortezomib, denileukin diftitox,
estramustine, ixabepilone, masoprocol, or mitotane.
[0335] In other or further embodiments, the peptidomimetics
macrocycles described herein are used to treat, prevent or diagnose
conditions characterized by overactive cell death or cellular death
due to physiologic insult, etc. Some examples of conditions
characterized by premature or unwanted cell death are or
alternatively unwanted or excessive cellular proliferation include,
but are not limited to hypocellular/hypoplastic,
acellular/aplastic, or hypercellular/hyperplastic conditions. Some
examples include hematologic disorders including but not limited to
fanconi anemia, aplastic anemia, thalaessemia, congenital
neutropenia, myelodysplasia
[0336] In other or further embodiments, the peptidomimetics
macrocycles of the invention that act to decrease apoptosis are
used to treat disorders associated with an undesirable level of
cell death. Thus, in some embodiments, the anti-apoptotic
peptidomimetics macrocycles of the invention are used to treat
disorders such as those that lead to cell death associated with
viral infection, e.g., infection associated with infection with
human immunodeficiency virus (HIV). A wide variety of neurological
diseases are characterized by the gradual loss of specific sets of
neurons, and the anti-apoptotic peptidomimetics macrocycles of the
invention are used, in some embodiments, in the treatment of these
disorders. Such disorders include Alzheimer's disease, Parkinson's
disease, amyotrophic lateral sclerosis (ALS) retinitis pigmentosa,
spinal muscular atrophy, and various forms of cerebellar
degeneration. The cell loss in these diseases does not induce an
inflammatory response, and apoptosis appears to be the mechanism of
cell death. In addition, a number of hematologic diseases are
associated with a decreased production of blood cells. These
disorders include anemia associated with chronic disease, aplastic
anemia, chronic neutropenia, and the myelodysplastic syndromes.
Disorders of blood cell production, such as myelodysplastic
syndrome and some forms of aplastic anemia, are associated with
increased apoptotic cell death within the bone marrow. These
disorders could result from the activation of genes that promote
apoptosis, acquired deficiencies in stromal cells or hematopoietic
survival factors, or the direct effects of toxins and mediators of
immune responses. Two common disorders associated with cell death
are myocardial infarctions and stroke. In both disorders, cells
within the central area of ischemia, which is produced in the event
of acute loss of blood flow, appear to die rapidly as a result of
necrosis. However, outside the central ischemic zone, cells die
over a more protracted time period and morphologically appear to
die by apoptosis.
Other Methods of Use
[0337] In other or further embodiments, the anti-apoptotic
peptidomimetics macrocycles of the invention are used to treat all
such disorders associated with undesirable cell death.
[0338] Some examples of immunologic disorders that are treated with
the peptidomimetics macrocycles described herein include but are
not limited to organ transplant rejection, arthritis, lupus, IBD,
Crohn's disease, asthma, multiple sclerosis, diabetes, etc.
[0339] Some examples of neurologic disorders that are treated with
the peptidomimetics macrocycles described herein include but are
not limited to Alzheimer's Disease, Down's Syndrome, Dutch Type
Hereditary Cerebral Hemorrhage Amyloidosis, Reactive Amyloidosis,
Familial Amyloid Nephropathy with Urticaria and Deafness,
Muckle-Wells Syndrome, Idiopathic Myeloma;
Macroglobulinemia-Associated Myeloma, Familial Amyloid
Polyneuropathy, Familial Amyloid Cardiomyopathy, Isolated Cardiac
Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,
Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the
Thyroid, Familial Amyloidosis, Hereditary Cerebral Hemorrhage With
Amyloidosis, Familial Amyloidotic Polyneuropathy, Scrapie,
Creutzfeldt-Jacob Disease, Gerstmann Straussler-Scheinker Syndrome,
Bovine Spongiform Encephalitis, a prion-mediated disease, and
Huntington's Disease.
[0340] Some examples of endocrinologic disorders that are treated
with the peptidomimetics macrocycles described herein include but
are not limited to diabetes, hypothyroidism, hypopituitarism,
hypoparathyroidism, hypogonadism, etc.
[0341] Examples of cardiovascular disorders (e.g., inflammatory
disorders) that are treated or prevented with the peptidomimetics
macrocycles of the invention include, but are not limited to,
atherosclerosis, myocardial infarction, stroke, thrombosis,
aneurism, heart failure, ischemic heart disease, angina pectoris,
sudden cardiac death, hypertensive heart disease; non-coronary
vessel disease, such as arteriolosclerosis, small vessel disease,
nephropathy, hypertriglyceridemia, hypercholesterolemia,
hyperlipidemia, xanthomatosis, asthma, hypertension, emphysema and
chronic pulmonary disease; or a cardiovascular condition associated
with interventional procedures ("procedural vascular trauma"), such
as restenosis following angioplasty, placement of a shunt, stent,
synthetic or natural excision grafts, indwelling catheter, valve or
other implantable devices. Preferred cardiovascular disorders
include atherosclerosis, myocardial infarction, aneurism, and
stroke.
EXAMPLES
[0342] The following section provides illustrative examples of the
present invention.
Example 1
Synthesis of Peptidomimetic Macrocycles of the Invention
[0343] .alpha.-helical BID and BIM peptidomimetic macrocycles were
synthesized, purified and analyzed as previously described
(Walensky et al (2004) Science 305:1466-70; Walensky et al (2006)
Mol Cell 24:199-210) and as indicated below. The macrocycles used
in this study are shown below. The corresponding uncrosslinked
polypeptides are indicated as "WT Sequence" and represent the
natural counterparts of the peptidomimetic macrocycles of the
invention.
TABLE-US-00008 Calculat- Calcu- Found Macro- WT ed m/z lated m/z
m/z cycle Sequence Sequence (M + H) (M + 3H) (M + 3H) SP-1 BID-BH3
Ac-DIIRNIARHLA$VGD$NleDRSI-NH2 2438.40 813.47 813.7 SP-2 BID-BH3
Ac-DIIRNIARHLA$VED$NleDRSI-NH2 2510.42 837.48 837.25 SP-3 BID-BH3
Ac-DIIRNIARHLAQVGDSNleDRSI-NH2 2403.32 801.78 801.89 SP-4 BIM-BH3
Ac-IWIAQELR$IGD$FNAYYARR-NH2 2646.43 882.82 883.15 SP-5 BIM-BH3
Ac-IWIAQELR$IED$FNAYYARR-NH2 2718.45 906.82 906.9 SP-6 BIM-BH3
Ac-IWIAQELRRIGDEFNAYYARR-NH2 2681.41 894.47 894.69 SP-7 BID-BH3
Pr-RNIARHLA$VAibD$NleDRSI-NH2 2139.25 713.76 713.79 SP-8 BID-BH3
Pr-RNIARHLAib$VAibD$NleDRSI-NH2 2153.27 718.43 718.56 SP-9 BID-BH3
Pr-RNIARHLA$VAibD$FARSI-NH2 2129.25 710.42 710.3 SP-10 BID-BH3
Pr-RNIARHLA$VGD$NleAibRSI-NH2 2081.25 694.42 694.42 SP-11 BIM-BH3
Ac-IWIAQALR$IGD$FNAYYARR-NH2 2588.43 863.48 863.85 SP-12 BIM-BH3
Ac-RWIAQALR$IGNle$FNAYYARR-NH2 2629.5 877.17 877.8 SP-13 BIM-BH3
Pr-RNChgARHLA$VAibD$FNAYYARR-NH2 2622.45 874.82 875.22 SP-14
BIM-BH3 Ac-IWIAQALR$IGD$FNAibYYARR-NH2 2602.44 868.15 868.54 SP-15
BIM-BH3 Ac-RWIAQALR$IGD$FNAFYARR-NH2 2615.45 872.49 872.64 SP-16
BIM-BH3 Ac-RWIAQALR$IGA$FNAYYARR-NH2 2587.45 863.16 863.39 SP-17
BIM-BH3 Ac-IWIAQAibLR$IGD$FNAibYYARR-NH2 2616.46 872.82 872.91
SP-18 BIM-BH3 Ac-IWIAQQLR$IGD$FNAYYARR-NH2 2645.45 882.49 882.62
SP-19 BIM-BH3 Ac-RWIAQQLR$IGD$FNAYYARR-NH2 2688.46 896.83 896.84
SP-20 BIM-BH3 Ac-IWIAQALR$IGD$FNARRA-NH2 2262.3 754.77 755.08 SP-21
BIM-BH3 Ac-IWIAQALR$IGD$FNAYKA-NH2 2241.26 747.76 748.12 SP-22
BIM-BH3 Ac-IWIAQALR$IGD$FNAYK-NH2 2170.22 724.08 724.35 SP-23
BIM-BH3 Ac-RWIAQALR$IGN$FNAYYARR-NH2 2630.45 877.48 877.36 SP-24
BIM-BH3 Ac-IWIAQAAR$DIG$ANAYYARR-NH2 2470.34 824.11 824.10 SP-25
BIM-BH3 Ac-IWIAQALR$IGN$FNAYYARR-NH2 2587.43 863.14 863.00 SP-26
BIM-BH3 Ac-IWIAQALRRIGDEFNAYYARR-NH2 2623.39 875.13 874.97
[0344] Alpha, alpha-disubstituted non-natural amino acids
containing olefinic side chains were synthesized according to
Williams et al. (1991) J. Am. Chem. Soc. 113:9276; and Schafmeister
et al. (2000) J. Am. Chem. Soc. 122:5891. BID-BH3 and BIM-BH3
peptidomimetic macrocycles were designed by replacing two naturally
occurring amino acids with the corresponding synthetic amino acids.
Substitutions were made at the i and i+4 positions. BID BH3 and
BIM-BH3 macrocycles were generated by solid phase peptide synthesis
followed by olefin metathesis-based crosslinking of the synthetic
amino acids via their olefin-containing side chains. The control
sequences for BID and BIM peptidomimetic macrocycles, as well as
specific sequence mutations generated are shown above.
[0345] In the sequences shown, "Nle" represents norleucine, "Aib"
represents 2-aminoisobutyric acid, "Chg" represents
cyclohexylglycine, "Ac" represents acetyl and "Pr" represents
propionyl. Amino acids represented as $ connect an all-carbon
crosslinker comprising one double bond and wherein each
.alpha.-carbon atom to which the crosslinker is attached is
additionally substituted with a methyl group. In all cases, the
crosslinker is a linear all-carbon crosslinker comprising eight
carbon atoms between the alpha carbons of each amino acid. If a
double bond is present, it is positioned between the fourth and
fifth carbon atom.
[0346] The non-natural amino acids (R and S enantiomers of the
5-carbon olefinic amino acid and the S enantiomer of the 8-carbon
olefinic amino acid) were characterized by nuclear magnetic
resonance (NMR) spectroscopy (Varian Mercury 400) and mass
spectrometry (Micromass LCT). Peptide synthesis was performed
either manually or on an automated peptide synthesizer (Applied
Biosystems, model 433A), using solid phase conditions, rink amide
AM resin (Novabiochem), and Fmoc main-chain protecting group
chemistry. For the coupling of natural Fmoc-protected amino acids
(Novabiochem), 10 equivalents of amino acid and a 1:1:2 molar ratio
of coupling reagents HBTU/HOBt (Novabiochem)/DIEA were employed.
Non-natural amino acids (4 equiv) were coupled with a 1:1:2 molar
ratio of HATU (Applied Biosystems)/HOBt/DIEA. Olefin metathesis was
performed in the solid phase using 10 mM Grubbs catalyst
(Blackewell et al. 1994 supra) (Strem Chemicals) dissolved in
degassed dichloromethane and reacted for 2 hours at room
temperature. Isolation of metathesized compounds was achieved by
trifluoroacetic acid-mediated deprotection and cleavage, ether
precipitation to yield the crude product, and high performance
liquid chromatography (HPLC) (Varian ProStar) on a reverse phase
C18 column (Varian) to yield the pure compounds. Chemical
composition of the pure products was confirmed by LC/MS mass
spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC
system) and amino acid analysis (Applied Biosystems, model
420A).
Cell Lines:
[0347] Cell lines used in this study are indicated in the table
below:
TABLE-US-00009 Cells Type Source Jurkat human acute T cell leukemia
ATCC K562 human chronic myelogeneous leukemia ATCC Karpas299 human
T cell lymphoma ATCC MOLT4 human T cell leukemia NCI RPMI8226 human
B lymphoblastoma NCI Ramos human B cell lymphoma ATCC Raji human B
cell lymphoma ATCC HL-60 Human myeloid leukemia NCI Malme-3M lung
malignant melanoma NCI SKMEL2 human malignant melanoma NCI SKMEL5
human malignant melanoma NCI PC3 human prostate adenocarcinoma NCI
Caki1 human kidney clear cell carcinoma NCI HCT116 human colorectal
carcinoma NCI HT-29 Colorectal adenocarcinoma NCI HEPG2 human
hepatocellular carcinoma ATCC MDAMB231 human breast adenocarcinoma
NCI MCF7 human breast adenocarcinoma NCI A549 human non-small cell
lung carcinoma NCI H460 human non-small cell lung carcinoma NCI
NCI-H220 human small cell lung carcinoma NCI NCI-H146 human small
cell lung carcinoma NCI NCI-H128 human small cell lung cancer ATCC
SKOV3 human ovary adenocarcinoma NCI Panc-1 human pancreas
carcinoma ATCC U251 Human glioblastoma ATCC NCI-H82 Human small
cell lung carcinoma Supt1 Human T cell lymphoma DHL-6 Human B cell
lymphoma RS4; 11 Human lymphoblastic leukemia MM1S Human multiple
myeloma SEMK2 Human mixed lineage leukemia A375 Human malignant
melanoma OVCAR8 Human ovarian carcinoma
Example 2
Cell Viability Assays
[0348] Cell viability assays shown in FIGS. 1-32 were performed
according to the following protocol. Tumor cell lines were grown in
specific serum-supplemented media (growth media) as necessary. A
day prior to the initiation of the study, cells were plated at
optimal cell density (15,000 to 25,000 cells/well) in 200 .mu.l
growth media in microtiter plates. The next day, cells were washed
twice in serum-free/phenol red-free RPMI complete media (assay
buffer) and a final volume of 100 .mu.l assay buffer was added to
each well. Human peripheral blood lymphocytes (hPBLs) were isolated
from Buffy coats (San Diego Blood Bank) using Ficoll-Paque gradient
separation and plated on the day of the experiment at 25,000
cells/well.
[0349] Peptidomimetic macrocycles were diluted from 1 mM stocks
(100% DMSO) in sterile water to prepare 400 .mu.M working
solutions. The peptidomimetic macrocycles and controls were then
diluted 10 or 40 fold or alternatively serially two-fold diluted in
assay buffer in dosing plates to provide concentrations of either
40 and 20 .mu.M or between 1.2 and 40 .mu.M, respectively. 100
.mu.L of each dilution was then added to the appropriate wells of
the test plate to achieve final concentrations of the
peptidomimetic macrocycles equal to 20 or 5 .mu.M, or between 0.6
to 20 .mu.M, respectively. Controls included wells without
peptidomimetic macrocycles containing the same concentration of
DMSO as the wells containing the peptidomimetic macrocycles, wells
containing 0.1% Triton X-100, wells containing a chemo cocktail
comprised of 1 .mu.M Velcade, 100 .mu.M Etoposide and 20 .mu.M
Taxol and wells containing no cells. Plates were incubated for 4
hours at 37.degree. C. in humidified 5% CO.sub.2 atmosphere.
[0350] Towards the end of the 4 hour incubation time, 22 .mu.l FBS
was added to each well for a total concentration of 10% FBS. After
addition of serum, the plates were incubated for an additional 44
hours at 37.degree. C. in humidified 5% CO.sub.2 atmosphere. At the
end of the incubation period, MTT assay was performed according to
manufacturer's instructions (Sigma, catalog #M2128) and absorbance
was measured at 560 nm using Dynex Opsys MR Plate reader.
[0351] In FIGS. 1-3, the values were plotted as percent
cytotoxicity, i.e. as a percentage of the positive control
corresponding to 100% cell death. In FIGS. 4-15, 25 and 26, values
were plotted as percent viable, i.e. as percent of negative control
corresponding to 100% viable cells. All assays were performed in
quadruplicates.
[0352] FIG. 1 shows human tumor cell lines treated with SP-1 (20
.mu.M) and assessed for cell viability by an MTT assay 48 hrs post
test article addition. All leukemia/lymphoma lines tested were
sensitive to SP-1. In addition, SP-1 also induced apoptosis of
several solid tumor lines including three small cell lung carcinoma
(SCLC) lines, NCI-H220, NCI-H128 and NCI-H1146. Conversely, there
were several solid tumor lines resistant to SP-1 including
non-small cell lung carcinoma (NSCLC) lines A-549 and H460, MCF7
(breast cancer) and U251 (glioma). FIG. 2 shows seven
leukemia/lymphoma human cell lines were treated with 5 .mu.M of
either SP-1 or SP-4 for 48 hrs and assessed for cell viability. As
shown, all cell lines exhibited similar sensitivity to both
macrocycles at this concentration.
[0353] FIG. 3 shows twelve human solid tumor lines tested for
sensitivity to either SP-1 or SP-4 (20 .mu.M). As shown, there
seems to be a cell-specific difference of sensitivity for each
macrocycle tested. EC.sub.50 curves for SP-1, SP-2, SP-3, SP-4,
SP-5 and SP-6 for individual cell lines are shown in FIGS.
4-15.
[0354] Cell viability assays shown in FIGS. 34-52 were performed
according to a similar protocol. Cells were split at optimal cell
density a day prior to the initiation of the study. The next day,
cells were washed twice in serum-free Opti-MEM media and 4000
cells/well were added to each well in a final volume of 100 .mu.l
Opti-MEM. For serum-free experiments, macrocycles were diluted from
2 mM stocks (100% DMSO) in sterile water to prepare 400 .mu.M
working solutions. A 40 .mu.M solution was then generated by
ten-fold dilution in assay buffer. The macrocycle and controls were
then serially diluted two-fold in assay buffer in dosing plates to
provide concentrations between 1.2 and 40 .mu.M, respectively. For
experiments in 2% human serum, macrocycles were diluted from 10 mM
stocks (100% DMSO) in sterile water to prepare 1 mM working
solutions. A 100 .mu.M solution was generated by ten-fold dilution
in assay buffer. The macrocycles and controls were then serially
diluted two-fold in assay buffer in dosing plates to provide
concentrations between 3 and 100 .mu.M, respectively. 50 .mu.L of
each dilution was then added to the appropriate wells of the test
plate to achieve final concentrations of the macrocycle between 0.6
to 20 .mu.M (for serum free experiment) or 1.5 to 50 .mu.M (for 2%
serum experiment), respectively. Controls included wells without
macrocycles containing the same concentration of DMSO as the
macrocycle-containing wells, wells containing 0.1% Triton X-100,
and wells containing no cells. Plates were incubated for 24 hours
at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere. At the end
of a 24 hr incubation period, a CellTiter-Glo Luminescent Cell
Viability Assay was performed according to manufacturer's
instructions (Promega, catalog #G7571) and the luminescence was
measured using a BIO-TEK synergy HT Plate reader. Values were
plotted as percent viable, i.e. as a percentage of the negative
control value (derived from cells exposed to DMSO only). All assays
were performed in duplicate.
[0355] The following tables summarize the EC.sub.50 values (.mu.M)
observed with peptidomimetic macrocycles of the invention in
various cell lines:
TABLE-US-00010 EC.sub.50 values in serum-free media MDA- NCI-H82
MB231- (SCLC- Compound Met A375 PC3 OVCAR8 MCL1+) SP-1 9.8 11 20 20
2.5 SP-2 >20 >20 ND >20 >20 SP-9 2.9 4.1 5.7 7.1 4.2
SP-10 3.1 5.2 6 8.1 5.7 SP-4 8.5 8 5 20 4.9 SP-11 2 4.3 4.6 3.2 1.2
SP-15 0.9 0.8 2.1 1.8 ND SP-23 0.6 1 2 0.9 0.6 SP-12 0.5 0.7 1.9
0.7 0.9 SP-24 >20 >20 >20 >20 >20 SP-25 1 1 0.6 1.4
0.5
TABLE-US-00011 EC.sub.50 values in 2% human serum MDA- MB231-
Compound Met A375 PC3 OVCAR8 SP-9 ND ND 25.5 18.4 SP-10 ND ND 24.4
19.1 SP-4 40 27 >50 >50 SP-11 ND ND 23.4 10.7 SP-15 2.6 2.7
9.3 6.4 SP-23 3.8 2.7 4.2 4 SP-24 >50 >50 >50 >50 SP-25
4.3 3.8 9.5 6.9 SP-26 >50 >50 >50 >50
TABLE-US-00012 EC.sub.50 values for various liquid tumors Com-
pound Jurkat SEMK2 Molt-4 RS4; 11 Raji DHL-6 MM1S SP-1 2.5 10 4.3
>20 13 7 3.5 SP-2 >20 >20 >20 >20 >20 ND >20
SP-3 >20 >20 >20 >20 >20 >20 >20 SP-9 1.9 8.1
1 4.3 5.7 2.7 5.5 SP-10 1.9 8.2 1.9 3.5 5 1.9 2.5 SP-4 1.6 4 2.2 10
9 3.8 4.9 SP-11 0.9 2.6 1.6 3.7 2.7 0.9 1.9 SP-15 0.4 0.8 0.7 1.7 1
0.5 0.6 SP-23 0.9 1 0.6 1 1.8 0.5 0.7 SP-12 0.4 0.5 0.7 1.71 0.3 ND
ND SP-24 >20 ND ND ND ND ND ND SP-25 0.5 0.9 0.9 1 1.8 0.6
0.6
Example 3
BrdU Cell Proliferation Assay
[0356] hPBLs isolated from two different donors were stimulated or
not with 5 .mu.g/ml PHA, 1 .mu.M Ionomycin and 1 .mu.g/ml LPS and
treated with either 5 or 20 .mu.M of SP-1 in assay buffer. 1 .mu.M
Rapamycin was used as a positive control to inhibit BrdU
incorporation. The cells were incubated for 48 hrs under the
conditions indicated in FIG. 17. BrdU incorporation was assayed by
ELISA according to manufacturer's instructions (Roche, catalog
number 11444611001). In FIG. 7, the Y axis shows OD=Absorbance
(A.sub.405 nm/A.sub.492 nm.)
Example 4
Efficacy of Peptidomimetic Macrocycles in a Human Leukemia
Xenograft Model
[0357] SEMK2-LN cells stably expressing luciferase were generated
as previously described (Armstrong et al (2003) Cancer Cell
3:173-83). 6-8 week old female NOD-SCID mice (Jackson Laboratory)
were injected with 5.times.10.sup.6 SEMK2-LN cells by tail vein.
The animals were imaged as described (Walensky et al., Science
305:1466-1470 (2004)) using Xenogen's In Vivo Imaging System
(Caliper Life Sciences) and total body bioluminescence quantified
by integration of photonic flux (photons/sec) (Living Imaging
Software for Xenogen In Vivo Imaging System, Caliper Life
Sciences). Animals were imaged on days 8 and 12, post-injection of
leukemic cells, to identify animals with established leukemia. On
day 12, prior to the initiation of treatment (treatment day 1),
animals were divided into cohorts with statistically equivalent
bioluminescence. Leukemic mice received a daily tail vein injection
of peptidomimetic macrocycle at 3 or 10 mg/Kg/day for 21 days or 30
mg/Kg/day for 12 days. Animals were imaged at days 1, 3, 5, 7, 9,
13 and 17 during treatment, and the resulting tumor reduction is
shown in FIG. 18.
Example 5
Immunogenicity Determination
[0358] 6-8 week old Balb/c or KM female mice were immunized with
unconjugated SP-1 or SP-4. Sera were collected pre-immunization and
25 .mu.g of each peptide in D5W were injected by tail vein using
the following immunization schedule: sera were collected seven days
following the first two immunizations at days 1 and 14 and 14 days
following the third and fourth immunizations. Antibody titers were
determined by indirect ELISA. In brief, microtiter plates were
coated with either SP-1 or SP-3 (5 .mu.g/ml) overnight at 4.degree.
C. The next day, the plates were washed 5 times with PBS/0.05%
Tween20 (PBST) and blocked with 5% non-fat milk/PBST for one hour
at 37.degree. C., followed by additional washing with PBST. The
anti-sera was serially diluted and added to the coated plates for 1
hr at 37.degree. C. The plates were then washed extensively with
PBST and further incubated with either HRP-conjugated anti-mouse
IgG or IgM for 1 hr at 37.degree. C. and washed 5 times with PBST
prior to the addition of HRP substrate. Plates were incubated at
room temperature for 10 minutes and the reaction stopped with 0.5 M
oxalic acid solution. Absorbance was read at 450 nm in an ELISA
microplate reader. The OD values were determined for both control-
and anti-sera at 1:100 dilution. The graphs in FIG. 20 are plotted
as the ratio of OD anti-sera/OD control sera. A ratio below 4 was
considered negative.
Example 6
Melting temperature (Tm) Determination
[0359] Lyophilized SP-1 was dissolved in ddH.sub.2O to a final
concentration of 50 .mu.M. Tm was determined by measuring the
circular dichroism (CD) spectra in a Jasco-810 spectropolarimeter
at a fixed wavelength of 222 nm between the temperatures of
5-95.degree. C. The following parameters were used for the
measurement: data pitch, 0.1.degree. C.; bandwidth, 1 nm and path
length, 0.1 cm averaging the signal for 16 seconds. The results are
shown in FIG. 21.
Example 7
Sample Preparation for Plasma Stability Determination
[0360] For ex-vivo plasma stability studies 10 .mu.M of SP-1, SP-3,
SP-4 and SP-6 were incubated with pre-cleared human and mouse
plasma at 37.degree. C. for 0, 15 and 120 minutes. At the end of
each incubation time, 100 .mu.L of sample was removed, placed in a
fresh low retention eppendorf tube with 300 .mu.l of ice cold MeOH.
The samples were centrifuged at 10,000 rpm, the supernatant removed
and placed in a fresh low retention eppendorf tube and 200 .mu.l of
water was added to each sample. Samples were then analyzed by
LC-MS/MS as indicated below. The results are shown in FIGS. 22 and
23.
Example 8
Intravenous Pharmacokinetic Analysis
[0361] The IV dose formulation was prepared by dissolving SP-1 or
SP-4 in 5% DMSO/D5W to achieve a 10 mg/Kg/dose. Canulated
Crl:CD.RTM. (SD) male rats (7-8 weeks old, Charles River
Laboratories) were used in these studies. Intravenous doses were
administered via the femoral cannula and the animals were dosed at
10 mL/kg per single injection. Blood for pharmacokinetic analysis
was collected at 10 time points (0.0833, 0.25, 0.5, 1, 2, 4, 6, 8,
12 and 24 hrs post-dose). Animals were terminated (without
necropsy) following their final sample collection.
[0362] The whole blood samples were centrifuged
(.about.1500.times.g) for 10 min at .about.4.degree. C. Plasma was
prepared and transferred within 30 min of blood
collection/centrifugation to fresh tubes that were frozen and
stored in the dark at -70.degree. C. until they were prepared for
LC-MS/MS analysis.
[0363] Sample extraction was achieved by adding 10 .mu.L of 50%
formic acid to 100 .mu.L plasma (samples or standards), following
by vortexing for 10 seconds. 500 .mu.L acetonitrile was added to
the followed by vortexing for 2 minutes and centrifuged at 14,000
rpm for 10 minutes at .about.4.degree. C. Supernatants were
transferred to clean tubes and evaporated on trubovap <10 psi at
37.degree. C. Prior to LC-MS/MS analysis samples were reconstituted
with 100 .mu.L of 50:50 acetonitrile:water.
[0364] The peak plasma concentration (C.sub.max), the time required
to achieve the peak plasma concentration (t.sub.max), the plasma
terminal half-life (t.sub.1/2), the area under the plasma
concentration time curve (AUC), the clearance and volume of
distribution were calculated from the plasma concentration data.
All pharmacokinetic calculations were done using WinNonlin version
4.1 (Pharsight Corp) by non-compartmental analysis. FIG. 24
summarizes the observed results.
[0365] The following LC-MS/MS method was used. In brief, the
LC-MS/MS instruments used was an API 365 (Applied Biosystems). The
analytical column was a Phenomenex Synergi (4.mu., Polar-RP, 50
mm.times.2 mm) and mobile phases A (0.1% formic acid in water) and
B (0.1% formic acid in methanol) were pumped at a flow rate of 0.4
ml/min to achieve the following gradient:
TABLE-US-00013 Time (min) % B 0 15 0.5 15 1.5 95 4.5 95 4.6 15 8.0
Stop
[0366] MRM: 814.0 to 374.2 (positive ionization)
Example 9
FACS Analysis of Detection of FITC-Labeled Peptidomimetic
Macrocycles in Treated Cells
[0367] Cells (e.g. Jurkat cells) were cultured in suspension in
RPMI 1640 medium with 2 mM L-Glutamine (Invitrogen) and
supplemented with 10% FBS and 1% penicillin-Streptomycin. Cells
were subcultured a day prior to the day of experiment to keep them
in an exponential growing phase. To analyze the uptake of
FITC-labeled peptidomimetic macrocycles by FACS, exponentially
growing Jurkat cells were seeded in 0.9 ml of serum-free medium at
density of 1.times.10.sup.6 cells. Cells were allowed to settle
down until compounds were diluted. Test compounds were diluted to 2
mM stock in DMSO, followed by dilution to 400 .mu.M in sterile
water; further dilution to 100 .mu.M was done using OptiMEM. Thus
100 .mu.l of 100 uM FITC-labeled peptidomimetic macrocycle was then
added to appropriate wells to achieve a final concentration of 10
.mu.M in 1 ml volume. Plates were returned to 37.degree. C., 5%
CO.sub.2 incubators for designated time points. At the end of each
time point, the cell suspension was diluted with media, washed
twice and subjected to trypsin (0.25%) for 15 min at 37.degree. C.
Cells were then washed with OptiMem and finally resuspended in 500
.mu.l of PBS. Cellular fluorescence was measured using Beckman
Coulter FACS instrument counting at least 20000 events per sample.
Analysis was done using Summit version 4, Dako Colorado, Inc.
Example 10
Protein-Ligand Binding Experiments
[0368] Protein-ligand binding experiments were conducted according
to the following representative procedure outlined for a
system-wide control experiment using 1 .mu.M SP-4 plus 5 .mu.M
Bcl-x.sub.L. A 1 .mu.L DMSO aliquot of a 40 .mu.M stock solution of
peptidomimetic macrocycle was dissolved in 19 .mu.L of PBS
(Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer
containing 150 mM NaCl). The resulting solution was mixed by
repeated pipetting and clarified by centrifugation at 10 000 g for
10 min. To a 4 .mu.L aliquot of the resulting supernatant was added
4 .mu.L of 10 .mu.M BCL-x.sub.L in PBS. Each 8.0 .mu.L experimental
sample thus contained 40 .mu.mol (1.5 .mu.g) of protein at 5.0
.mu.M concentration in PBS plus 1 .mu.M peptidomimetic macrocycle
and 2.5% DMSO. Duplicate samples thus prepared for each
concentration point were incubated for 60 min at room temperature,
and then chilled to 4.degree. C. prior to size-exclusion
chromatography-LC-MS analysis of 5.0 .mu.L injections. Samples
containing a target protein, protein-ligand complexes, and unbound
compounds were injected onto an SEC column, where the complexes
were separated from non-binding component by a rapid SEC step. The
SEC column eluate was monitored using UV detectors to confirm that
the early-eluting protein fraction, which elutes in the void volume
of the SEC column, was well resolved from unbound components that
are retained on the column. After the peak containing the protein
and protein-ligand complexes elutes from the primary UV detector,
it entered a sample loop where it was excised from the flow stream
of the SEC stage and transferred directly to the LC-MS via a
valving mechanism. The (M+3H).sup.3+ ion of ALRN-0034 is observed
by ESI-MS at m/z 883.8, confirming the detection of the
protein-ligand complex.
Example 11
Competitive Binding Experiments
[0369] A mixture of ligands at 40 .mu.M per component was prepared
by combining 2 .mu.L aliquots of 400 .mu.M stocks of each of the
three compounds with 14 .mu.L of DMSO. Then, 1 .mu.L aliquots of
this 40 .mu.M per component mixture were combined with 1 .mu.L DMSO
aliquots of a serially diluted stock solution of titrant
peptidomimetic macrocycle (10, 5, 2.5, . . . , 0.078 mM). These 2
.mu.L samples were dissolved in 38 .mu.L of PBS. The resulting
solutions were mixed by repeated pipetting and clarified by
centrifugation at 10 000 g for 10 min. To 4.0 .mu.L aliquots of the
resulting supernatants was added 4.0 .mu.L of 10 .mu.M BCL-x.sub.L
in PBS. Each 8.0 .mu.L experimental sample thus contained 40
.mu.mol (1.5 .mu.g) of protein at 5.0 .mu.M concentration in PBS
plus 0.5 .mu.M ligand, 2.5% DMSO, and varying concentrations (125,
62.5, . . . , 0.98 .mu.M) of the titrant peptidomimetic macrocycle.
Duplicate samples thus prepared for each concentration point were
incubated at room temperature for 60 min, then chilled to 4.degree.
C. prior to SEC-LC-MS analysis of 2.0 .mu.L injections. Additional
details on these and other methods are provided in "A General
Technique to Rank Protein-Ligand Binding Affinities and Determine
Allosteric vs. Direct Binding Site Competition in Compound
Mixtures." Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.;
Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in
"ALIS: An Affinity Selection-Mass Spectrometry System for the
Discovery and Characterization of Protein-Ligand Interactions" D.
A. Annis, C.-C. Chuang, and N. Nazef. In Mass Spectrometry in
Medicinal Chemistry. Edited by Wanner K, Hofner G: Wiley-VCH;
2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors):
Methods and Principles in Medicinal Chemistry.
Example 12
Quantitative Analysis of FITC-Labeled Peptidomimetic Macrocycle
Uptake Using Fluorimetry
[0370] Cells (e.g. INA-6 or Jurkat cells) were cultured in
suspension in RPMI 1640 medium with 2 mM L-Glutamine and
(Invitrogen) supplemented with 10% FBS and 1%
penicillin-Streptomycin as well as 1 ng/ml of recombinant human
IL-6 supplements in the case of INA-6 cells. Cells were subcultured
a day prior to the day of experiment to keep them in an exponential
growing phase. To analyze the uptake of FITC-labeled peptidomimetic
macrocycles in cells, exponentially growing cells were seeded in
0.9 ml of serum-free medium at density of 0.5.times.10.sup.6 cells.
Cells were allowed to settle down until compounds were diluted.
Test compounds were diluted to 2 mM stock in DMSO, followed by
dilution to 400 .mu.M in sterile water; further dilution to 100
.mu.M was done using OptiMEM. Thus 100 .mu.l of 100 uM FITC-labeled
peptidomimetic macrocycle was then added to appropriate wells to
achieve a final concentration of 10 .mu.M in 1 ml volume. Plates
were returned to 37.degree. C., 5% CO.sub.2 incubators for
designated time points. If needed, further dilutions of test
compounds were also prepared in OptiMEM. At the end of each time
point, cells were harvested, washed twice with RPMI supplemented
with FBS, and washed once with PBS+0.5% BSA. Pelleted cells were
resuspended and incubated with 0.25% Trypsin-EDTA for 15 min at
37.degree. C., 5% CO.sub.2. Post-incubation, cells were washed with
media containing serum once and twice with PBS with 0.5% BSA. At
the end of washes, cells were lysed with Triton X-100-containing
cell lysis buffer from Cell Signaling Technologies. Fluorescence
intensity was measured on a BioTek Synergy 4 instrument. Dilutions
of FITC-labeled peptidomimetic macrocycles for the standard curves
made in cell lysis buffer and were used for quantitation of the
amount of peptidomimetic macrocycles in cells. Analysis was done
using Gen 5 software provided by Biotek Inc.
Example 13
Efficacy of Peptidomimetic Macrocycles in an Orthotopic Prostate
Tumor Model
[0371] Experiments were conducted using a Bioware.RTM. Cell Line
(PC-3M-Luc-C6) and using 5.times.10.sup.6 cells/mouse /100 .mu.l. A
total of 70 nu/nu male mice were used (7-10 weeks old). Male nu/nu
mice were anesthetized and incisions along the posterior midline of
their abdomens, right above the prostate, were created. The bladder
was retracted and pressed lightly to expose the prostate.
PC-3M-luc-C6 cells (5.times.10.sup.5) were slowly injected into
either dorsal prostatic lobe. The peritoneal incision was sutured
and the skin was closed. The mice were given buprenorphine (0.1
mg/kg in 50 .mu.l) subcutaneously after the surgical procedure.
Animals were first imaged on day 7 after wound healing. The final
50 mice were grouped into 5 groups (10 mice per group) based on BLI
before the start of the treatment. The experimental mice were
imaged twice weekly starting from day 14 for 2.5 weeks. At the end
of the experiment, the tumors were dissected and weighed. The
tumors were then cut into two pieces for snap freeze and fixed in
10% formalin. Test compound treatment was initiated after two
stable or increasing bioluminescent signals were registered from
the tumor cell inoculation site. Several test groups were used:
Group 1 (Vehicle, IV daily dosing), Group 2 (test compound, IV
daily dosing at 10 mg/kg), Group 3 (Vehicle, i.p. daily dosing),
Group 4 (test compound, i.p. daily dosing at 10 mg/kg), and Group 5
(Taxotere, IV weekly dosing for two doses at 30 mg/kg). The test
peptidomimetic macrocycles were formulated as follows. Only low
retention/siliconized plastic tubes and tips were used. A 60 mg/mL
stock solution of each peptidomimetic macrocycle was prepared by
dissolving 140 mg of each macrocycle into 2.3 mL of 100% DMSO. The
stock solution was divided into 10 aliquots of 0.23 mL for daily
dosing (14 mg per vial) and keep frozen at -20 C. One vial was
thawed on each day of dosing. Working concentrations (2 mg/mL) of
each macrocycle were prepared by diluting one aliquot of the stock
solution into 6.5 mL of filter sterilized 5% dextrose. The DMSO
stock was added dropwise into the D5W with constant stirring. The
solution was adjusted to a final volume of 7 mL with 5% dextrose,
without filter sterilizing the final dose formation. The dosing
volume was 5 .mu.L/g (125 .mu.L for a 25 g mouse). The dose is
delivered to the mouse by slow bolus (over 30 seconds). FIG. 45
shows a time treatment for the prostate cancer orthotopic xenograft
model. The bioluminescence of the prostate region of each
experimental animal was measured and expressed as photons/second.
The in vivo tumor growth kinetics were graphed and two-way ANOVA
for repeated measure (using time and treatment as two main factors)
were used. The kinetic mouse images from representative mice from
each group were obtained and are shown in FIG. 43.
Example 14
Orthotopic Xenograft Tumor Model Using Ovarian Cancer (SKOV3-Luc)
Tumors
[0372] 1.times.10.sup.6 SKOV3-Luc cells stably expressing firefly
luciferase are injected into the ovarian bursa of anesthetized
SCID-beige mice (9 weeks old, female). The animals are monitored
weekly by bioluminescent imaging (BLI). Test compound treatment is
initiated after two stable or increasing bioluminescent signals are
registered from the tumor cell inoculation site (up to 9 weeks in
this model). Prior to the initiation of the treatment animals are
randomized into control and treatment groups (10 mice/group).
Animals are treated by daily injection (IP, IV or SC) of test
compound (low, med, high doses) and vehicle control for 10, 14
and/or 21 days, as needed. Efficacy is determined by comparison of
the tumor burden between peptidomimetic macrocycle and vehicle
control treated animals. Tumor growth/volume is monitored by BLI
after IP injection of 150 mg/kg D-luciferin and imaged by IVIS
Imaging System both dorsally and ventrally. Metastatic lesions can
be imaged by shielding the primary tumor bioluminescence. At the
conclusion of the experiment, the animals are humanely euthanized
and the ovarian tumor is excised, weighed and prepared for
subsequent analysis.
Example 15
Orthotopic Xenograft Tumor Model Using Breast Cancer
(MDA-MB-231-Luc) Tumors
[0373] 1.times.10.sup.6 MDA-MB-231-Luc cells stably expressing
firefly luciferase are injected into the breast tissue of
anesthetized SCID-beige mice (9 weeks old, female). The animals are
monitored weekly by bioluminescent imaging. Test compound treatment
is initiated after two stable or increasing bioluminescent signals
are registered from the tumor cell inoculation site. Prior to the
initiation of the treatment animals are randomized into control and
treatment groups (10 mice/group). Animals are treated by daily
injection (IP, IV or SC) of test compound (low, med, high doses)
and vehicle control for 10, 14 and/or 21 days, as needed. Efficacy
is determined by comparison of the tumor burden between test
compound and vehicle control treated animals. Tumor growth/volume
is monitored by bioluminescent imaging (BLI) after IP injection of
150 mg/kg D-luciferin and imaged by IVIS Imaging System both
dorsally and ventrally. Metastatic lesions can be imaged by
shielding the primary tumor bioluminescence. At the conclusion of
the experiment, the animals are humanely euthanized and the ovarian
tumor is excised, weighed and prepared for subsequent analysis.
Example 16
Subcutaneous Xenograft Tumor Model Using Melanoma (A375) or Small
Cell Lung Cancer (NCI-H-82) tumors
[0374] An optimized amount of tumor cells is injected into the
flank of anesthetized NOD/SCID or nu/nu mice, as required, by
subcutaneous injection. When the tumors reach an average volume of
20-50 mm.sup.3, the animals are sorted into control and treatment
groups (10 mice/group). Animals are treated by daily injection (IP,
IV or SC) of test compound (low, medium, and high doses) and
vehicle control for 10, 14 and/or 21 days, as needed. Efficacy is
determined by comparison of the tumor volume between test compound
and vehicle control treated animals. Tumor growth/volume is
monitored by external caliper measurement (L.times.W.times.D). At
the conclusion of the experiment, the animals are humanely
euthanized and the tumor is excised, weighed and prepared for
subsequent analysis.
Example 17
Metastatic Tumor Model Using Metastatic Breast Cancer
(MDA-MB-231-Met-Luc) Tumors
[0375] Anesthetized NOD/SCID mice (9 weeks old, female) are
injected with the optimized amount of MDA-MB-213-MET-Luc cells
stably expressing firefly luciferase into the left ventricle of the
heart (hence directly into arterial system). A successful
intracardiac injection is indicated by day zero images showing a
systemic bioluminescence distributed throughout the animals and
only mice with evidence of a satisfactory injection will remain in
the experiment. The animals are sorted into control and treatment
groups (10 mice/group). Animals are treated by daily injection (IP,
IV or SC) of test compound (low, medium, and high doses) and
vehicle control for 10, 14 and/or 21 days, as needed. The
development of subsequent metastasis is monitored twice a week in
vivo by BLI after IP injection of 150 mg/kg D-luciferin and imaged
by IVIS Imaging System both dorsally and ventrally. Lung and bone
metastases in particular are monitored. At the conclusion of the
experiment, the animals are humanely euthanized and tissues of
interest are excised and prepared for ex vivo imaging and
subsequent analysis.
[0376] The above tumor models are described in more detail in
Jenkins, D. E. et al., Clin. & Exp. Metastasis. 2003, 20,
745-756; Scatena C. D. et al., Prostate 2004, 59, 292-303;
Greenaway J. Et al., Mol. Cancer. Ther. 2009, 8, 64-74; Guan, J. et
al., Cancer Chemo Pharma. 2008, Online Pub Dec. 24; and Lelekakis,
M. et al., Clin & Exp Metastasis. 1999, 163-170.
[0377] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
120121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ile Trp Ile Ala Gln Glu Leu Arg Xaa Ile Gly Asp
Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg 20221PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Asp
Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10
15Xaa Asp Arg Ser Ile 20325PRTHomo sapiens 3Gln Glu Asp Ile Ile Arg
Asn Ile Ala Arg His Leu Ala Gln Val Gly1 5 10 15Asp Ser Met Asp Arg
Ser Ile Pro Pro 20 25425PRTHomo sapiens 4Asp Asn Arg Pro Glu Ile
Trp Ile Ala Gln Glu Leu Arg Arg Ile Gly1 5 10 15Asp Glu Phe Asn Ala
Tyr Tyr Ala Arg 20 25525PRTHomo sapiens 5Asn Leu Trp Ala Ala Gln
Arg Tyr Gly Arg Glu Leu Arg Arg Met Ser1 5 10 15Asp Glu Phe Val Asp
Ser Phe Lys Lys 20 25625PRTHomo sapiens 6Glu Glu Gln Trp Ala Arg
Glu Ile Gly Ala Gln Leu Arg Arg Met Ala1 5 10 15Asp Asp Leu Asn Ala
Gln Tyr Glu Arg 20 25724PRTHomo sapiens 7Arg Ser Ser Ala Ala Gln
Leu Thr Ala Ala Arg Leu Lys Ala Leu Gly1 5 10 15Asp Glu Leu His Gln
Arg Thr Met 20822PRTHomo sapiens 8Ala Glu Leu Pro Pro Glu Phe Ala
Ala Gln Leu Arg Lys Ile Gly Asp1 5 10 15Lys Val Tyr Cys Thr Trp
20925PRTHomo sapiens 9Val Pro Ala Asp Leu Lys Asp Glu Cys Ala Gln
Leu Arg Arg Ile Gly1 5 10 15Asp Lys Val Asn Leu Arg Gln Lys Leu 20
251024PRTHomo sapiens 10Gln His Arg Ala Glu Val Gln Ile Ala Arg Lys
Leu Gln Cys Ile Ala1 5 10 15Asp Gln Phe His Arg Leu His Thr
201122PRTHomo sapiens 11Ser Ser Ala Ala Gln Leu Thr Ala Ala Arg Leu
Lys Ala Leu Gly Asp1 5 10 15Glu Leu His Gln Arg Thr 201225PRTHomo
sapiens 12Cys Met Glu Gly Ser Asp Ala Leu Ala Leu Arg Leu Ala Cys
Ile Gly1 5 10 15Asp Glu Met Asp Val Ser Leu Arg Ala 20
251324PRTHomo sapiens 13Asp Ile Glu Arg Arg Lys Glu Val Glu Ser Ile
Leu Lys Lys Asn Ser1 5 10 15Asp Trp Ile Trp Asp Trp Ser Ser
201422PRTHomo sapiens 14Gly Arg Leu Ala Glu Val Cys Ala Val Leu Leu
Arg Leu Gly Asp Glu1 5 10 15Leu Glu Met Ile Arg Pro 201525PRTHomo
sapiens 15Pro Gln Asp Ala Ser Thr Lys Lys Ser Glu Cys Leu Lys Arg
Ile Gly1 5 10 15Asp Glu Leu Asp Ser Asn Met Glu Leu 20
251622PRTHomo sapiens 16Pro Ser Ser Thr Met Gly Gln Val Gly Arg Gln
Leu Ala Ile Ile Gly1 5 10 15Asp Asp Ile Asn Arg Arg 201714PRTHomo
sapiens 17Lys Gln Ala Leu Arg Glu Ala Gly Asp Glu Phe Glu Leu Arg1
5 101822PRTHomo sapiens 18Leu Ser Pro Pro Val Val His Leu Ala Leu
Ala Leu Arg Gln Ala Gly1 5 10 15Asp Asp Phe Ser Arg Arg
201923PRTHomo sapiens 19Glu Val Ile Pro Met Ala Ala Val Lys Gln Ala
Leu Arg Glu Ala Gly1 5 10 15Asp Glu Phe Glu Leu Arg Tyr
202020PRTHomo sapiens 20Pro Ala Asp Pro Leu His Gln Ala Met Arg Ala
Ala Gly Asp Glu Phe1 5 10 15Glu Thr Arg Phe 202123PRTHomo sapiens
21Ala Thr Ser Arg Lys Leu Glu Thr Leu Arg Arg Val Gly Asp Gly Val1
5 10 15Gln Arg Asn His Glu Thr Ala 202219PRTHomo sapiens 22Leu Ala
Glu Val Cys Thr Val Leu Leu Arg Leu Gly Asp Glu Leu Glu1 5 10 15Gln
Ile Arg2319PRTHomo sapiens 23Met Thr Val Gly Glu Leu Ser Arg Ala
Leu Gly His Glu Asn Gly Ser1 5 10 15Leu Asp Pro2422PRTHomo sapiens
24Val Val Glu Gly Glu Lys Glu Val Glu Ala Leu Lys Lys Ser Ala Asp1
5 10 15Trp Val Ser Asp Trp Ser 202520PRTHomo sapiens 25Ser Met Ala
Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp Asp1 5 10 15Arg Met
Lys Leu 202625PRTHomo sapiensMOD_RES(14)..(14)Cross linked amino
acid residue 26Gln Glu Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala
Xaa Val Gly1 5 10 15Asp Xaa Met Asp Arg Ser Ile Pro Pro 20
252725PRTHomo sapiensMOD_RES(14)..(14)Cross linked amino acid
residue 27Asp Asn Arg Pro Glu Ile Trp Ile Ala Gln Glu Leu Arg Xaa
Ile Gly1 5 10 15Asp Xaa Phe Asn Ala Tyr Tyr Ala Arg 20
252825PRTHomo sapiensMOD_RES(14)..(14)Cross linked amino acid
residue 28Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu Arg Xaa
Met Ser1 5 10 15Asp Xaa Phe Val Asp Ser Phe Lys Lys 20
252925PRTHomo sapiensMOD_RES(14)..(14)Cross linked amino acid
residue 29Glu Glu Gln Trp Ala Arg Glu Ile Gly Ala Gln Leu Arg Xaa
Met Ala1 5 10 15Asp Xaa Leu Asn Ala Gln Tyr Glu Arg 20
253024PRTHomo sapiensMOD_RES(14)..(14)Cross linked amino acid
residue 30Arg Ser Ser Ala Ala Gln Leu Thr Ala Ala Arg Leu Lys Xaa
Leu Gly1 5 10 15Asp Xaa Leu His Gln Arg Thr Met 203122PRTHomo
sapiensMOD_RES(13)..(13)Cross linked amino acid residue 31Ala Glu
Leu Pro Pro Glu Phe Ala Ala Gln Leu Arg Xaa Ile Gly Asp1 5 10 15Xaa
Val Tyr Cys Thr Trp 203225PRTHomo sapiensMOD_RES(14)..(14)Cross
linked amino acid residue 32Val Pro Ala Asp Leu Lys Asp Glu Cys Ala
Gln Leu Arg Xaa Ile Gly1 5 10 15Asp Xaa Val Asn Leu Arg Gln Lys Leu
20 253324PRTHomo sapiensMOD_RES(14)..(14)Cross linked amino acid
residue 33Gln His Arg Ala Glu Val Gln Ile Ala Arg Lys Leu Gln Xaa
Ile Ala1 5 10 15Asp Xaa Phe His Arg Leu His Thr 203422PRTHomo
sapiensMOD_RES(13)..(13)Cross linked amino acid residue 34Ser Ser
Ala Ala Gln Leu Thr Ala Ala Arg Leu Lys Xaa Leu Gly Asp1 5 10 15Xaa
Leu His Gln Arg Thr 203525PRTHomo sapiensMOD_RES(14)..(14)Cross
linked amino acid residue 35Cys Met Glu Gly Ser Asp Ala Leu Ala Leu
Arg Leu Ala Xaa Ile Gly1 5 10 15Asp Xaa Met Asp Val Ser Leu Arg Ala
20 253624PRTHomo sapiensMOD_RES(14)..(14)Cross linked amino acid
residue 36Asp Ile Glu Arg Arg Lys Glu Val Glu Ser Ile Leu Lys Xaa
Asn Ser1 5 10 15Asp Xaa Ile Trp Asp Trp Ser Ser 203722PRTHomo
sapiensMOD_RES(12)..(12)Cross linked amino acid residue 37Gly Arg
Leu Ala Glu Val Cys Ala Val Leu Leu Xaa Leu Gly Asp Xaa1 5 10 15Leu
Glu Met Ile Arg Pro 203825PRTHomo sapiensMOD_RES(14)..(14)Cross
linked amino acid residue 38Pro Gln Asp Ala Ser Thr Lys Lys Ser Glu
Cys Leu Lys Xaa Ile Gly1 5 10 15Asp Xaa Leu Asp Ser Asn Met Glu Leu
20 253922PRTHomo sapiensMOD_RES(14)..(14)Cross linked amino acid
residue 39Pro Ser Ser Thr Met Gly Gln Val Gly Arg Gln Leu Ala Xaa
Ile Gly1 5 10 15Asp Xaa Ile Asn Arg Arg 204014PRTHomo
sapiensMOD_RES(6)..(6)Cross linked amino acid residue 40Lys Gln Ala
Leu Arg Xaa Ala Gly Asp Xaa Phe Glu Leu Arg1 5 104122PRTHomo
sapiensMOD_RES(14)..(14)Cross linked amino acid residue 41Leu Ser
Pro Pro Val Val His Leu Ala Leu Ala Leu Arg Xaa Ala Gly1 5 10 15Asp
Xaa Phe Ser Arg Arg 204223PRTHomo sapiensMOD_RES(14)..(14)Cross
linked amino acid residue 42Glu Val Ile Pro Met Ala Ala Val Lys Gln
Ala Leu Arg Xaa Ala Gly1 5 10 15Asp Xaa Phe Glu Leu Arg Tyr
204320PRTHomo sapiensMOD_RES(11)..(11)Cross linked amino acid
residue 43Pro Ala Asp Pro Leu His Gln Ala Met Arg Xaa Ala Gly Asp
Xaa Phe1 5 10 15Glu Thr Arg Phe 204423PRTHomo
sapiensMOD_RES(11)..(11)Cross linked amino acid residue 44Ala Thr
Ser Arg Lys Leu Glu Thr Leu Arg Xaa Val Gly Asp Xaa Val1 5 10 15Gln
Arg Asn His Glu Thr Ala 204519PRTHomo sapiensMOD_RES(10)..(10)Cross
linked amino acid residue 45Leu Ala Glu Val Cys Thr Val Leu Leu Xaa
Leu Gly Asp Xaa Leu Glu1 5 10 15Gln Ile Arg4619PRTHomo
sapiensMOD_RES(12)..(12)Cross linked amino acid residue 46Met Thr
Val Gly Glu Leu Ser Arg Ala Leu Gly Xaa Glu Asn Gly Xaa1 5 10 15Leu
Asp Pro4722PRTHomo sapiensMOD_RES(13)..(13)Cross linked amino acid
residue 47Val Val Glu Gly Glu Lys Glu Val Glu Ala Leu Lys Xaa Ser
Ala Asp1 5 10 15Xaa Val Ser Asp Trp Ser 204820PRTHomo
sapiensMOD_RES(12)..(12)Cross linked amino acid residue 48Ser Met
Ala Arg Asp Pro Gln Arg Tyr Leu Val Xaa Gln Gly Asp Xaa1 5 10 15Arg
Met Lys Leu 204925PRTHomo sapiensMOD_RES(9)..(9)Cross linked amino
acid residue 49Gln Glu Asp Ile Ile Arg Asn Ile Xaa Arg His Leu Xaa
Gln Val Gly1 5 10 15Asp Ser Met Asp Arg Ser Ile Pro Pro 20
255025PRTHomo sapiensMOD_RES(9)..(9)Cross linked amino acid residue
50Asp Asn Arg Pro Glu Ile Trp Ile Xaa Gln Glu Leu Xaa Arg Ile Gly1
5 10 15Asp Glu Phe Asn Ala Tyr Tyr Ala Arg 20 255125PRTHomo
sapiensMOD_RES(9)..(9)Cross linked amino acid residue 51Asn Leu Trp
Ala Ala Gln Arg Tyr Xaa Arg Glu Leu Xaa Arg Met Ser1 5 10 15Asp Glu
Phe Val Asp Ser Phe Lys Lys 20 255225PRTHomo
sapiensMOD_RES(9)..(9)Cross linked amino acid residue 52Glu Glu Gln
Trp Ala Arg Glu Ile Xaa Ala Gln Leu Xaa Arg Met Ala1 5 10 15Asp Asp
Leu Asn Ala Gln Tyr Glu Arg 20 255324PRTHomo
sapiensMOD_RES(9)..(9)Cross linked amino acid residue 53Arg Ser Ser
Ala Ala Gln Leu Thr Xaa Ala Arg Leu Xaa Ala Leu Gly1 5 10 15Asp Glu
Leu His Gln Arg Thr Met 205422PRTHomo sapiensMOD_RES(8)..(8)Cross
linked amino acid residue 54Ala Glu Leu Pro Pro Glu Phe Xaa Ala Gln
Leu Xaa Lys Ile Gly Asp1 5 10 15Lys Val Tyr Cys Thr Trp
205525PRTHomo sapiensMOD_RES(9)..(9)Cross linked amino acid residue
55Val Pro Ala Asp Leu Lys Asp Glu Xaa Ala Gln Leu Xaa Arg Ile Gly1
5 10 15Asp Lys Val Asn Leu Arg Gln Lys Leu 20 255624PRTHomo
sapiensMOD_RES(9)..(9)Cross linked amino acid residue 56Gln His Arg
Ala Glu Val Gln Ile Xaa Arg Lys Leu Xaa Cys Ile Ala1 5 10 15Asp Gln
Phe His Arg Leu His Thr 205722PRTHomo sapiensMOD_RES(8)..(8)Cross
linked amino acid residue 57Ser Ser Ala Ala Gln Leu Thr Xaa Ala Arg
Leu Xaa Ala Leu Gly Asp1 5 10 15Glu Leu His Gln Arg Thr
205825PRTHomo sapiensMOD_RES(9)..(9)Cross linked amino acid residue
58Cys Met Glu Gly Ser Asp Ala Leu Xaa Leu Arg Leu Xaa Cys Ile Gly1
5 10 15Asp Glu Met Asp Val Ser Leu Arg Ala 20 255924PRTHomo
sapiensMOD_RES(9)..(9)Cross linked amino acid residue 59Asp Ile Glu
Arg Arg Lys Glu Val Xaa Ser Ile Leu Xaa Lys Asn Ser1 5 10 15Asp Trp
Ile Trp Asp Trp Ser Ser 206022PRTHomo sapiensMOD_RES(7)..(7)Cross
linked amino acid residue 60Gly Arg Leu Ala Glu Val Xaa Ala Val Leu
Xaa Arg Leu Gly Asp Glu1 5 10 15Leu Glu Met Ile Arg Pro
206125PRTHomo sapiensMOD_RES(9)..(9)Cross linked amino acid residue
61Pro Gln Asp Ala Ser Thr Lys Lys Xaa Glu Cys Leu Xaa Arg Ile Gly1
5 10 15Asp Glu Leu Asp Ser Asn Met Glu Leu 20 256222PRTHomo
sapiensMOD_RES(9)..(9)Cross linked amino acid residue 62Pro Ser Ser
Thr Met Gly Gln Val Xaa Arg Gln Leu Xaa Ile Ile Gly1 5 10 15Asp Asp
Ile Asn Arg Arg 206314PRTHomo sapiensMOD_RES(1)..(1)Cross linked
amino acid residue 63Xaa Gln Ala Leu Xaa Glu Ala Gly Asp Glu Phe
Glu Leu Arg1 5 106422PRTHomo sapiensMOD_RES(9)..(9)Cross linked
amino acid residue 64Leu Ser Pro Pro Val Val His Leu Xaa Leu Ala
Leu Xaa Gln Ala Gly1 5 10 15Asp Asp Phe Ser Arg Arg 206523PRTHomo
sapiensMOD_RES(9)..(9)Cross linked amino acid residue 65Glu Val Ile
Pro Met Ala Ala Val Xaa Gln Ala Leu Xaa Glu Ala Gly1 5 10 15Asp Glu
Phe Glu Leu Arg Tyr 206620PRTHomo sapiensMOD_RES(6)..(6)Cross
linked amino acid residue 66Pro Ala Asp Pro Leu Xaa Gln Ala Met Xaa
Ala Ala Gly Asp Glu Phe1 5 10 15Glu Thr Arg Phe 206723PRTHomo
sapiensMOD_RES(6)..(6)Cross linked amino acid residue 67Ala Thr Ser
Arg Lys Xaa Glu Thr Leu Xaa Arg Val Gly Asp Gly Val1 5 10 15Gln Arg
Asn His Glu Thr Ala 206819PRTHomo sapiensMOD_RES(5)..(5)Cross
linked amino acid residue 68Leu Ala Glu Val Xaa Thr Val Leu Xaa Arg
Leu Gly Asp Glu Leu Glu1 5 10 15Gln Ile Arg6919PRTHomo
sapiensMOD_RES(7)..(7)Cross linked amino acid residue 69Met Thr Val
Gly Glu Leu Xaa Arg Ala Leu Xaa His Glu Asn Gly Ser1 5 10 15Leu Asp
Pro7022PRTHomo sapiensMOD_RES(8)..(8)Cross linked amino acid
residue 70Val Val Glu Gly Glu Lys Glu Xaa Glu Ala Leu Xaa Lys Ser
Ala Asp1 5 10 15Trp Val Ser Asp Trp Ser 207120PRTHomo
sapiensMOD_RES(7)..(7)Cross linked amino acid residue 71Ser Met Ala
Arg Asp Pro Xaa Arg Tyr Leu Xaa Ile Gln Gly Asp Asp1 5 10 15Arg Met
Lys Leu 207221PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 72Asp Ile Ile Arg Asn Ile Ala Arg His
Leu Ala Xaa Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile
207321PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 73Ile Trp Ile Ala Gln Glu Leu Arg Xaa Ile Gly Asp
Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg 207421PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Arg
Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly Asp Xaa Phe Asn Ala1 5 10
15Phe Tyr Ala Arg Arg 207521PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 75Arg Trp Ile Ala Gln Ala Leu
Arg Xaa Ile Gly Asn Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg
207621PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 76Ile Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly Asn
Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg 207721PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 77Asp
Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10
15Xaa Asp Arg Ser Ile 207821PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 78Asp Ile Ile Arg Asn Ile Ala
Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile
207921PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 79Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa
Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile 208021PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Asp
Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10
15Xaa Asp Arg Ser Ile 208121PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 81Asp Ile Ile Arg Asn Ile Ala
Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile
208221PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 82Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa
Val Gly Asp Xaa1 5 10
15Xaa Asp Arg Ser Ile 208321PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 83Asp Ile Ile Arg Asn Ile Ala
Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile
208421PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 84Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa
Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile 208521PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Asp
Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10
15Xaa Asp Arg Ser Ile 208621PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 86Asp Ile Ile Arg Asn Ile Ala
Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile
208721PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 87Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa
Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile 208821PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 88Asp
Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10
15Xaa Asp Arg Ser Ile 208921PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 89Asp Ile Ile Arg Asn Ile Ala
Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile
209021PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa
Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile 209121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 91Asp
Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10
15Xaa Asp Arg Ser Ile 209221PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 92Asp Ile Ile Arg Asn Ile Ala
Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile
209321PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 93Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa
Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile 209421PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Asp
Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10
15Xaa Asp Arg Ser Ile 209521PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 95Asp Ile Ile Arg Asn Ile Ala
Arg His Leu Ala Xaa Val Gly Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile
209621PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 96Asp Ile Ile Arg Asn Ile Ala Arg His Leu Ala Xaa
Val Glu Asp Xaa1 5 10 15Xaa Asp Arg Ser Ile 209721PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 97Asp
Ile Ile Arg Asn Ile Ala Arg His Leu Ala Gln Val Gly Asp Ser1 5 10
15Xaa Asp Arg Ser Ile 209821PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 98Ile Trp Ile Ala Gln Glu Leu
Arg Xaa Ile Gly Asp Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg
209921PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 99Ile Trp Ile Ala Gln Glu Leu Arg Xaa Ile Glu Asp
Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg 2010021PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 100Ile
Trp Ile Ala Gln Glu Leu Arg Arg Ile Gly Asp Glu Phe Asn Ala1 5 10
15Tyr Tyr Ala Arg Arg 2010118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 101Arg Asn Ile Ala Arg His
Leu Ala Xaa Val Xaa Asp Xaa Xaa Asp Arg1 5 10 15Ser
Ile10218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 102Arg Asn Ile Ala Arg His Leu Xaa Xaa Val Xaa
Asp Xaa Xaa Asp Arg1 5 10 15Ser Ile10318PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 103Arg
Asn Ile Ala Arg His Leu Ala Xaa Val Xaa Asp Xaa Phe Ala Arg1 5 10
15Ser Ile10418PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 104Arg Asn Ile Ala Arg His Leu Ala Xaa
Val Gly Asp Xaa Xaa Xaa Arg1 5 10 15Ser Ile10521PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 105Ile
Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly Asp Xaa Phe Asn Ala1 5 10
15Tyr Tyr Ala Arg Arg 2010621PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 106Arg Trp Ile Ala Gln Ala
Leu Arg Xaa Ile Gly Xaa Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg
2010721PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107Arg Asn Xaa Ala Arg His Leu Ala Xaa Val Xaa
Asp Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg
2010821PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Ile Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly
Asp Xaa Phe Asn Xaa1 5 10 15Tyr Tyr Ala Arg Arg
2010921PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 109Arg Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly
Asp Xaa Phe Asn Ala1 5 10 15Phe Tyr Ala Arg Arg
2011021PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Arg Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly
Ala Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg
2011121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 111Ile Trp Ile Ala Gln Xaa Leu Arg Xaa Ile Gly
Asp Xaa Phe Asn Xaa1 5 10 15Tyr Tyr Ala Arg Arg
2011221PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Ile Trp Ile Ala Gln Gln Leu Arg Xaa Ile Gly
Asp Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg
2011321PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Arg Trp Ile Ala Gln Gln Leu Arg Xaa Ile Gly
Asp Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg
2011419PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 114Ile Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly
Asp Xaa Phe Asn Ala1 5 10 15Arg Arg Ala11519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 115Ile
Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly Asp Xaa Phe Asn Ala1 5 10
15Tyr Lys Ala11618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 116Ile Trp Ile Ala Gln Ala Leu Arg Xaa
Ile Gly Asp Xaa Phe Asn Ala1 5 10 15Tyr Lys11721PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 117Arg
Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly Asn Xaa Phe Asn Ala1 5 10
15Tyr Tyr Ala Arg Arg 2011821PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 118Ile Trp Ile Ala Gln Ala
Ala Arg Xaa Asp Ile Gly Xaa Ala Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg
2011921PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 119Ile Trp Ile Ala Gln Ala Leu Arg Xaa Ile Gly
Asn Xaa Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg
2012021PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Ile Trp Ile Ala Gln Ala Leu Arg Arg Ile Gly
Asp Glu Phe Asn Ala1 5 10 15Tyr Tyr Ala Arg Arg 20
* * * * *