U.S. patent application number 13/256112 was filed with the patent office on 2012-05-03 for kinase protein binding inhibitors.
This patent application is currently assigned to University of Florida. Invention is credited to William G. Cance, Steven N. Hochwald, Elena Kurenova, David A. Ostrov.
Application Number | 20120107323 13/256112 |
Document ID | / |
Family ID | 42728999 |
Filed Date | 2012-05-03 |
United States Patent
Application |
20120107323 |
Kind Code |
A1 |
Hochwald; Steven N. ; et
al. |
May 3, 2012 |
KINASE PROTEIN BINDING INHIBITORS
Abstract
The invention provides compounds capable of treating a subject
suffering from or being susceptible to a cell proliferative
disorder (especially, cancer), methods of identifying and using the
compounds, pharmaceutical compositions and kits thereof.
Inventors: |
Hochwald; Steven N.;
(Gainesville, FL) ; Ostrov; David A.;
(Gainesville, FL) ; Cance; William G.; (Orchard
Park, NY) ; Kurenova; Elena; (West Falls,
NY) |
Assignee: |
University of Florida
Gainesville
FL
|
Family ID: |
42728999 |
Appl. No.: |
13/256112 |
Filed: |
March 12, 2010 |
PCT Filed: |
March 12, 2010 |
PCT NO: |
PCT/US2010/000754 |
371 Date: |
December 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61210053 |
Mar 12, 2009 |
|
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|
Current U.S.
Class: |
424/141.1 ;
424/649; 424/94.6; 514/108; 514/110; 514/19.3; 514/27; 514/34;
514/468; 514/49; 514/52; 514/572; 514/81; 536/26.7; 544/244;
549/461; 562/102; 562/12; 600/1 |
Current CPC
Class: |
A61K 31/194 20130101;
A61P 35/04 20180101; A61K 31/7076 20130101; A61K 31/663 20130101;
A61P 43/00 20180101; A61K 31/194 20130101; A61K 31/343 20130101;
A61P 35/00 20180101; A61K 31/343 20130101; A61K 31/663 20130101;
A61K 31/7068 20130101; A61K 31/661 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/661
20130101; A61K 31/7076 20130101; Y10T 428/2933 20150115; A61K 45/06
20130101; A61K 31/7068 20130101 |
Class at
Publication: |
424/141.1 ;
514/81; 514/52; 514/572; 514/108; 514/468; 544/244; 536/26.7;
549/461; 562/102; 562/12; 424/94.6; 514/19.3; 424/649; 514/110;
514/34; 514/27; 514/49; 600/1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7064 20060101 A61K031/7064; A61K 31/194
20060101 A61K031/194; A61K 31/663 20060101 A61K031/663; A61K 31/343
20060101 A61K031/343; C07F 9/6561 20060101 C07F009/6561; C07H 19/14
20060101 C07H019/14; C07D 307/91 20060101 C07D307/91; C07C 309/17
20060101 C07C309/17; C07F 9/38 20060101 C07F009/38; A61K 38/50
20060101 A61K038/50; A61K 38/14 20060101 A61K038/14; A61K 33/24
20060101 A61K033/24; A61K 31/704 20060101 A61K031/704; A61K 31/7048
20060101 A61K031/7048; A61K 31/7068 20060101 A61K031/7068; A61P
35/00 20060101 A61P035/00; A61P 35/04 20060101 A61P035/04; A61N
5/00 20060101 A61N005/00; A61K 31/675 20060101 A61K031/675 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] This work was supported in part by a National Institutes of
Health/NCI Grant, Grant No. CA 113766 (S.N.H.). The government has
certain rights in the invention.
Claims
1-12. (canceled)
13. A method of treating a subject suffering from or susceptible to
cancer, said method comprising administering to said subject
thereof an effective amount of a compound selected from the group
consisting of a)
2-(hydroxymethyl)-6-imino-2,3,3a,9a-tetrahydro-6H-furo[2,3:4,5][1,3]oxazo-
lo[3,2-a]pyrimidin-3-yl dihydrogen phosphate; b)
4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine}; c)
1,1'-(1,7,9-Trihydroxy-8,9b-dimethyl-3-oxo-4-a-(phenylthio)-3,4,-
4a,9b-tetrahydrodibenzo-[b,d]furan-2,6-diyl)diethanone; d)
3-Methyl-2,4-disulfopentanedioic acid; and e)
1-Aminopropane-1,3-diyldiphosphonic acid; or a pharmaceutically
acceptable salt thereof.
14. The method of claim 13, wherein said compound is
4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine}, or a pharmaceutically acceptable salt thereof.
15. The method of claim 13, wherein said cancer is pancreatic
cancer, melanoma cancer, or esophageal cancer.
16. The method of claim 13, wherein said method further comprises
administering to said subject an additional therapeutic agent.
17. The method of claim 13, wherein said method further comprises
treating said subject thereof with surgery, chemotherapy,
radiation, immunotherapy, monoclonal antibody therapy or epidermal
growth factor receptor therapies.
18-33. (canceled)
34. A kit for use in treating a subject suffering from or
susceptible to a cell proliferative disorder, said kit comprising
an effective amount of a compound capable of modulating binding
interactions between FAK and IGF-1R.
35. The kit of claim 34, wherein said compound is selected from the
group consisting of a)
2-(Hydroxymethyl)-6-imino-2,3,3a,9a-tetrahydro-6H-furo[2,3:4,5][1,3]oxazo-
lo[3,2-a]pyrimidin-3-yl dihydrogen phosphate; b)
4-(Methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine}; c)
1,1'-(1,7,9-Trihydroxy-8,9b-dimethyl-3-oxo-4-a-(phenylthio)-3,4,-
4a,9b-tetrahydrodibenzo-[b,d]furan-2,6-diyl)diethanone; d)
3-Methyl-2,4-disulfopentanedioic acid; and e)
1-Aminopropane-1,3-diyldiphosphonic acid; or a pharmaceutically
acceptable salt thereof.
36. The kit of claim 35, wherein said compound is
4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine} or a pharmaceutically acceptable salt thereof.
37. The kit of claim 34, wherein said cell proliferative disorder
is a cancer.
38. The kit of claim 37, wherein said cancer is a cancer of the
breast, respiratory tract, brain, reproductive organs, digestive
tract, urinary tract, eye, liver, skin, head and neck, thyroid,
parathyroid or a distant metastasis of a solid tumor.
39. The kit of claim 38, wherein said cancer is pancreatic cancer,
melanoma cancer, or esophageal cancer.
40. The kit of claim 39, wherein said cancer is pancreatic
cancer.
41. The kit of claim 34, further comprising an additional
therapeutic agent.
42. The kit of claim 41, wherein said additional therapeutic agent
is selected from the group consisting of asparaginase, bleomycin,
calcein-AM, carboplatin, carmustine, chlorambucil, cisplatin,
colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin,
daunorubicin, docetaxel, doxorubicin (adriamycine), epirubicin,
etoposide, ET-743, erlotinib, 5-fluorouracil, gemcitabine,
gefitinib, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan,
leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna,
methotrexate, mitomycin C, mitoxantrone, NVP-AEW541, paclitaxel,
prednisolone, prednisone, procarbazine, raloxifen, rhodamine-123,
streptozocin, TAE226, tamoxifen, thioguanine, topotecan,
vinblastine, vincristine, vindesine, and zalypsis.
43. A pharmaceutical composition for treating a subject suffering
from or susceptible to cancer, said composition comprising an
effective amount of a compound capable of modulating binding
interactions between FAK and IGF-1R, and a pharmaceutically
acceptable carrier or diluent.
44. The pharmaceutical composition of claim 43, wherein said
compound is selected from the group consisting of a)
2-(Hydroxymethyl)-6-imino-2,3,3a,9a-tetrahydro-6H-furo[2,3:4,5][1,3]oxazo-
lo[3,2-a]pyrimidin-3-yl dihydrogen phosphate; b)
4-(Methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine}; c)
1,1'-(1,7,9-Trihydroxy-8,9b-dimethyl-3-oxo-4-a-(phenylthio)-3,4,-
4a,9b-tetrahydrodibenzo-[b,d]furan-2,6-diyl)diethanone; d)
3-Methyl-2,4-disulfopentanedioic acid; and e)
1-Aminopropane-1,3-diyldiphosphonic acid; or a pharmaceutically
acceptable salt thereof.
45. The pharmaceutical composition of claim 44, wherein said
compound is
4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine} or a pharmaceutically acceptable salt thereof.
46. The pharmaceutical composition of claim 43, wherein said cancer
is pancreatic cancer, melanoma cancer, or esophageal cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of the following U.S.
Provisional Application No. 61/210,053, which was filed on Mar. 12,
2009, the contents of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Cell proliferative disorders are disorders involving the
undesired or uncontrolled proliferation of a cell. A particular
example of the cell proliferative disorders is cancer. Cancer is a
serious health issue all around the world. Cancer affects people at
all ages, even fetuses. As reported by the World Health
Organization in 2007, cancer causes about 13% of all deaths. About
7.6 million people died from cancer in the world during 2007.
According to the American Cancer Society, it is estimated that
425,000 new cases of these cancers will be diagnosed each year in
the United States alone.
[0004] Cancer can develop in a wide variety of different organs,
tissues and cell types. The term "cancer" refers to a collection of
over a thousand different diseases. One example is pancreatic
cancer, which is a malignant tumor of the pancreas. Pancreatic
cancer is a lethal disease accounting for the fourth leading cause
of cancer death in USA. The treatment of pancreatic cancer,
especially, a locally advanced pancreatic cancer, represents a
clinical challenge, with a median survival of approximately 10-12
months. The standard therapeutic strategy includes radiation and/or
chemotherapy. Unfortunately, local control is poor, with 1- and
2-year local progression rates estimated at 36% and 62%,
respectively, and the median time to local progression of 6.4
months. Failure to control the primary tumor is associated with
symptoms such as pain, gastric outlet and duodenal obstruction, and
upper gastrointestinal ulceration and bleeding. Therefore, there is
an urgent need in discovering a therapeutic approach that can
achieve improved patient outcomes (e.g., overall survival,
disease-free survival, local control, adverse effects and quality
of life).
[0005] Focal adhesion kinase (FAK) is a nonreceptor protein
tyrosine kinase that is localized at contact points (focal
adhesions) between cells and their extra-cellular matrix and is a
point of convergence of a number of signaling pathways from
integrins, growth factors and kinases (see McLean G W et al. Nature
Reviews 2005; 5(7):505-15). FAK plays an important role in
mediating essential cellular processes, such as cell growth,
survival, and migration. FAK is expressed at low levels in normal
tissues but is over-expressed in many cancer types, for example,
the majority of tumors from pancreatic cancer patients (see Liu W.
et al. Carcinogenesis, 2008; 29(6): 1096-107; and WO 2005/049852).
It has been shown that silencing of the FAK gene facilitates
apoptosis and suppresses metastasis in pancreatic cancer cells and
xenograft models (see Liu W. et al. Carcinogenesis, 2008; 29(6):
1096-107). Thus, FAK is a viable target for a cancer treatment. The
development of drugs targeting FAK would be a natural complement to
many existing cancer therapies.
[0006] The Insulin-like Growth Factor 1 Receptor (IGF-1R) is a
receptor tyrosine kinase playing a major role in cell proliferation
and has also been linked to tumorigenesis (see, Vincent A M, et
al., Growth Hormone and IGF Research. 2002; 12:193-197). This
receptor mediates the effects of IGF-1, which is a polypeptide
protein hormone similar in molecular structure to insulin.
Over-expressed in a variety of human cancers, IGF-1R stimulates
cell proliferation, enables oncogenic transformation, and
suppresses apoptosis. The IGF-1/IGF-1R autocrine loop is expressed
in a variety of human tumor cells and activation of this axis in
several tumor types, including pancreatic cancer, has been shown to
promote metastasis. Further, it has been shown that inhibition of
IGF-1R signaling leads to suppression of tumor growth in many
animal models.
[0007] Nevertheless, despite the fact that emerging data in the
field suggests that FAK and/or IGF-1R may be viable targets for
developing cancer therapeutics, kinase inhibitors with desired
specificity are yet to be obtained. In particular, sparse
information is available in medical field regarding contribution of
FAK and IGF-1R to the malignant behavior of pancreatic cancer.
[0008] Thus, there is an unmet clinical need for the development of
novel cancer therapeutics with desired specificity and of novel
strategies in treating cancer.
SUMMARY OF THE INVENTION
[0009] One aspect of the invention provides a method of treating a
subject suffering from or susceptible to cancer; the method
comprises administering to the subject a compound capable of
modulating binding interactions between FAK and IGF-1R.
[0010] In one embodiment, the compound is capable of modulating
binding interactions between FAK-NT and IGF-1R. Another embodiment
provides that the compound is capable of modulating binding
interactions between FAK-NT2 and IGF-1R.
[0011] Certain embodiments provide that the compound is a)
2-(hydroxymethyl)-6-imino-2,3,3a,9a-tetrahydro-6H-furo[2,3:4,5][1,3]oxazo-
lo[3,2-a]pyrimidin-3-yl dihydrogen phosphate; b)
4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine}; c)
1,1'-(1,7,9-trihydroxy-8,9b-dimethyl-3-oxo-4-a-(phenylthio)-3,4,-
4a,9b-tetrahydrodibenzo-[b,d]furan-2,6-diyl)diethanone; d)
3-methyl-2,4-disulfopentanedioic acid; or e)
1-aminopropane-1,3-diyldiphosphonic acid; or a pharmaceutically
acceptable salt, ester or prodrug thereof. A particular example is
4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine}, or a pharmaceutically acceptable salt, ester or prodrug
thereof.
[0012] The invention also provides a method of treating a subject
suffering from or susceptible to cancer by administering to the
subject thereof an effective amount of a compound selected from the
group consisting of a)
2-(hydroxymethyl)-6-imino-2,3,3a,9a-tetrahydro-6H-furo[2,3:4,5][1,3]oxazo-
lo[3,2-a]pyrimidin-3-yl dihydrogen phosphate; b)
4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine}; c)
1,1'-(1,7,9-trihydroxy-8,9b-dimethyl-3-oxo-4-a-(phenylthio)-3,4,-
4a,9b-tetrahydrodibenzo-[b,d]furan-2,6-diyl)diethanone; d)
3-methyl-2,4-disulfopentanedioic acid; and e)
1-aminopropane-1,3-diyldiphosphonic acid; or a pharmaceutically
acceptable salt, ester or prodrug thereof. A particular example of
the compound is
4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine}, or a pharmaceutically acceptable salt, ester or prodrug
thereof.
[0013] In an embodiment, the cancer is a cancer of the breast,
respiratory tract, brain, reproductive organs, digestive tract,
urinary tract, eye, liver, skin, head and neck, thyroid,
parathyroid or a distant metastasis of a solid tumor. Certain
embodiments provide that the cancer is pancreatic cancer, melanoma
cancer, or esophageal cancer. In an embodiment, the cancer is
pancreatic cancer.
[0014] In one embodiment, a method of the invention further
comprises administering to the subject an additional therapeutic
agent. One embodiment provides that the additional therapeutic
agent is a chemotherapeutic agent. Certain embodiments provide that
the additional therapeutic agent is selected from the group
consisting of 5-fluorouracil (5-FU), gemcitabine,
fluoropyrimidines, nucleoside cytidine analogues, NVP-AEW541,
platinum analogues, TAE226, topoisomerase inhibitors,
antimicrotubule agents, phosphatidylinositol 3 kinase inhibitors
(PI3 kinase inhibitors), proteasome inhibitors, vitamin D
analogues, arachidonic acid pathway inhibitors, histone
deacytylator inhibitors, and farnesyltransferase inhibitors.
Certain embodiments provide that the additional therapeutic agent
is TAE226, NVP-AEW541, wortmannin, or LY294002.
[0015] In certain embodiments, the additional therapeutic agent is
asparaginase, bleomycin, calcein-AM, carboplatin, carmustine,
chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine,
dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin
(adriamycine), epirubicin, etoposide, ET-743, erlotinib,
5-fluorouracil, gemcitabine, gefitinib, hexamethylmelamine,
hydroxyurea, ifosfamide, irinotecan, leucovorin, lomustine,
mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin
C, mitoxantrone, NVP-AEW541, paclitaxel, prednisolone, prednisone,
procarbazine, raloxifen, rhodamine-123, streptozocin, TAE226,
tamoxifen, thioguanine, topotecan, vinblastine, vincristine,
vindesine, or zalypsis.
[0016] In another embodiment, a method of the invention further
includes treating the subject in need thereof with at least one
additional therapy. The therapy can be, but not limited to,
surgery, chemotherapy, radiation, immunotherapy, monoclonal
antibody therapy and epidermal growth factor receptor
therapies.
[0017] In one embodiment, the additional therapy is chemotherapy.
Another embodiment provides that the additional therapy is
radiation. In an embodiment, the method includes treating the
subject with a combination of chemotherapy and radiation.
[0018] Another aspect of the invention provides a method of
treating a subject suffering from or susceptible to cancer; the
method comprises administering to the subject in need thereof a
compound capable of decreasing IGF-1R and AKT phosphorylation and
inducing apoptosis of cancer cells. Exemplary compounds are
delineated herein.
[0019] Yet another aspect of the invention presents a method of
modulating uncontrolled proliferation of cells. The method includes
contacting a cell undergoing uncontrolled proliferation with a
compound identified as capable of modulating binding interactions
between FAK and IGF-1R. In one embodiment, the compound is a
compound delineated herein.
[0020] In one aspect, the invention provides a method of treating a
subject suffering from or susceptible to a cell proliferative
disorder. The method comprises administering to the subject an
effective amount of a compound capable of modulating the binding
interactions between FAK and IGF-1R. One embodiment provides that
the compound is a compound delineated herein.
[0021] The invention also provides a method of modulating binding
interactions between FAK and IGF-1R by contacting FAK with a
compound capable of binding to or associating with FAK or specific
domains thereof. In one embodiment, the compound is capable of
inhibiting tyrosine phosphorylation of FAK, thereby disrupting the
binding interactions between FAK and IGF-1R. In one embodiment, the
compound is a compound delineated herein. Another embodiment
provides that the compound is capable of binding to or associating
with a FAK amino terminus fragment (NT2).
[0022] In another aspect, a method of modulating binding
interactions between FAK and IGF-1R includes contacting IGF-1R with
a compound capable of binding to or associating with IGF-1R or
specific domains thereof. In one embodiment, the compound is
capable of inhibiting tyrosine phosphorylation of IGF-1R, thereby
disrupting the binding interactions between FAK and IGF-1R.
Exemplary compounds are delineated herein. One embodiment provides
that the compound is capable of binding to or associating with the
kinase domain of IGF-1R.
[0023] The invention also provides a kit for use in treating a
subject suffering from or susceptible to a cell proliferative
disorder. In particular, the kit includes an effective amount of a
compound capable of modulating the binding interactions between FAK
and IGF-1R. In one embodiment, the compound included in the kit is
a compound delineated herein.
[0024] In one embodiment, the cell proliferative disorder is a
cancer. In certain embodiments, the cancer is a cancer of the
breast, respiratory tract, brain, reproductive organs, digestive
tract, urinary tract, eye, liver, skin, head and neck, thyroid,
parathyroid or a distant metastasis of a solid tumor. Certain
embodiments provide that the cancer is pancreatic cancer, melanoma
cancer, or esophageal cancer. A particular example is pancreatic
cancer.
[0025] In certain embodiments, the kit further includes an
additional therapeutic agent. Certain embodiments present that the
additional therapeutic agent is an agent delineated supra.
[0026] The invention also provides a pharmaceutical composition for
treating a subject suffering from or susceptible to cancer. In
particular, the composition includes an effective amount of a
compound capable of modulating binding interactions between FAK and
IGF-1R, together with a pharmaceutically acceptable carrier or
diluent. In an embodiment, the cancer is pancreatic cancer,
melanoma cancer, or esophageal cancer. In another embodiment, the
compound a compound delineated herein. The pharmaceutical
composition may further include an additional therapeutic agent.
Examples of the additional therapeutic agent are discussed
supra.
[0027] The invention also provides methods for designing,
evaluating and identifying compounds which bind to FAK, IGF-1R, or
specific domains thereof, or compounds capable of modulating the
binding interactions between FAK and IGF-1R. Other embodiments of
the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows the results of protein pull down assay between
FAK and IGF-1R protein constructs;
[0029] FIG. 2 depicts immunoprecipitation and western blot results
on cell lystate (from C8161 melanoma cancer cells) treated with NSC
344553 for 24 hours;
[0030] FIG. 3 depicts immunoprecipitation and western blot results
of 75 .mu.M of NSC 344553 tested on C8161 melanoma cancer
cells;
[0031] FIG. 4 depicts western blot results for C8161 melanoma
cancer cells treated with 1 .mu.M of NSC 344553;
[0032] FIG. 5 depicts western blot results for C8161 melanoma
cancer cells treated with 5 .mu.M of NSC 344553 and PI3 Kinase
inhibitors for 24 hours;
[0033] FIG. 6 depicts cell viability assay results for pancreatic
and melanoma cells treated with NSC 344553 for 72 hours;
[0034] FIG. 7 depicts the effects of 2 .mu.M NSC 344553 treatment
on IGF-1R +//- cells;
[0035] FIG. 8 depicts the effects of NSC 344553 treatment on FAK
+//- cells;
[0036] FIG. 9 depicts western blot analysis for Panc-1 cancer cells
treated with NSC 344553.
[0037] FIG. 10 depicts MTT cell titer 96 assay results of NSC
344553 treatment on FAK wild type and null cells and IGF-1R
wildtype and null cells;
[0038] FIG. 11 depicts cell viability results for A375 and C8161
melanoma cancer cells treated with NSC 344553 using MTT assay;
[0039] FIG. 12 depicts immunoprecipitation and western blot results
for pancreatic (MiaPaCa-2) cells treated with NSC 128687 for 24
hours;
[0040] FIG. 13 shows dose response curve of MiaPaCa-2 cancer cells
to a 72-hour NSC 250435 treatment;
[0041] FIG. 14 shows western blot results for melanoma (A375 and
C8161) and pancreatic (Panc-1 and MiaPaCa-2) cancer cells treated
with NSC 344553.
[0042] FIG. 15 depicts in silico modeling of FAK and IGF-1R
interaction, the structure of INT2-31 (NSC 344553) and disruption
of the interaction of FAK and IGF-1R. A. The proposed site of
interaction of FAK and IGF-1R is demonstrated based on
computational modeling. INT2-31 is modeled in the pocket on FAK (aa
127-243) corresponding to the site of FAK interaction with IGF-1R.
Structure of INT2-31 is demonstrated on the top right. B. With
increasing doses of INT2-31, GST-FAK-NT2 pulldown of IGF-1R.beta.
is diminished. C. With increasing doses of INT2-31,
coimmunoprecipitation of FAK and IGF-1R is decreased in C8161
melanoma cells. D. With increasing doses of INT2-31,
coimmunoprecipitation of FAK and IGF-1R is decreased in A375
melanoma cells. Densitometry showing the ratio of IGF-1R to FAK is
shown below the Western blots in FIGS. 15B, 15C and 15D. Figures
are representative of experiments performed in triplicate.
[0043] FIG. 16 depicts the effects of INT2-31 on the viability and
proliferation of melanoma cells. A. INT2-31 inhibited the cell
viability of normal melanocytes and three melanoma cell lines in a
dose dependent fashion over 72 h. B. Expression of FAK, IGF-1R, Akt
and ERK in the three melanoma cell lines and melanocytes. C. CSFE
cell proliferation assay with A375 melanoma cells (left) and C8161
melanoma cells (right) in the presence of increasing doses of
INT2-31 or TAE 226 (dual FAK and IGF-1R kinase inhibitor). D.
C.8161 melanoma cell counts in the presence of INT2-31 or TAE 226.
Figures are representative of experiments performed in
triplicate.
[0044] FIG. 17 demonstrates that the effects of INT2-31 are FAK and
IGF-1R specific. A. Western blot showing knockdown of FAK with FAK
shRNA. B. MTT assay showing a decreased sensitivity to INT2-31
treatment in FAK knockdown C8161 cells compared to parental and
mock transfected cells. C. FAK specificity. MTT assay showing the
increased effect of INT2-31(31) or NVP AEW541 (IGF-1R kinase
inhibitor, NVP) on FAK wildtype compared to null fibroblasts.
*p<0.05 D. IGF1R specificity. MTT assay showing the increased
effect of INT2-31(31) or NVP AEW541 (IGF-1R kinase inhibitor, NVP)
on IGF-1R wildtype compared to null fibroblasts. *p<0.05.
Figures are representative of experiments performed in
triplicate.
[0045] FIG. 18 demonstrates that INT2-31 induces detachment and
apoptosis. A. There is a small but not significant increase in
detachment in cells treated with 5 .mu.M INT2-31 at 72 h. Greater
effects are observed with TAE (TAE 226) at 48 h and 72 h
(*p<0.05 vs control). B. Hoescht staining of INT2-31 treated
cells. C. Activated caspase 3/7 detection with 48 h of treatment of
INT2-31 or TAE 226. D. Western blot analysis of biochemical markers
of the apoptotic pathway. Figures are representative of experiments
performed in triplicate.
[0046] FIG. 19 demonstrates that INT2-31 disrupts
FAK-IGF-1R-dependent signaling and abrogates IGF dependent Akt
activation without inhibiting kinase activity. Effect of increasing
doses of INT2-31 in the presence and absence of IGF-1 stimulation
on signaling in C8161 A. A375 B. and SK-MEL-28 C. cells after 24 h.
Figures are representative of experiments performed in triplicate.
D. INT2-31 did not significantly inhibit the kinase activity of
these 12 kinases. E. C8161 cells were plated into a 6-well plate
and treated with 5 .mu.M INT2-31 for 24, 48, and 72 hours. F. and
G. Overexpression of FAK-NT2 fragment reduces IGF-1 induced
phosphorylation of AKT. C8161 cells transfected with 3 GFP
fragments of the FAK N-terminus (FAK NT1, FAK NT2 and FAK NT3).
[0047] FIG. 20 demonstrates that INT2-31 decreases tumor p-Akt and
growth in melanoma xenografts. Animals were inoculated
subcutaneously with A. C8161 or B. A375 tumor cells and were
treated with 15 mg/kg of INT2-31 vs PBS via intraperitoneal
injection. Animal weights are shown below growth curves
(*p<0.05). Tumor growth figures are representative of
experiments performed in triplicate. C. Ki67 staining of C8161
tumors treated with INT2-31, 15 mg/kg, vs PBS control. The
percentage of reactive cells is shown in the left upper graph. The
intensity of staining is shown in the lower left graph
(*p<0.05). TUNEL staining of excised tumors at the completion of
the experiment is shown on the right (*p<0.05). D. The effect of
INT2-31 (15 mg/kg) on the phosphorylation of AKT in vivo.
Densitometric analysis of the ratio of p-Akt/Akt/GAPDH is shown
below the figure. This demonstrates a significantly decreased ratio
of p-Akt/Akt in tumors from animals treated with INT2-31 vs PBS
control. E. The effect of INT2-31 on the coimmunoprecipitation of
FAK and IGF-1R from tumor specimens. The lower graph shows the
densitometry of the ratio of the IGF-1R to FAK signal.
[0048] FIG. 21 INT2-31 sentisized esophageal cancer cells to
chemotherapy. MTT assay showing the viability of A) KYSE70 and B)
KYSE140 esophageal cancer cell lines treated with increasing
concentrations of INT2-31, 5-FU or combination for 72 hours.
[0049] FIG. 22 INT2-31 sentisized pancreatic cancer cells to
chemotherapy. A)MTT assay showing the viability of Panc-1 cell
lines treated with increasing concentrations of INT2-31, 5-FU or
combination for 72 hours.
[0050] FIG. 23 Effects of INT2-31 on direct esophageal cancer
patient #5 specimen. A) MTT assay showing that increasing
concentrations of INT2-31 inhibited the cell viability of
esophageal patient #5 cells. B) Esophageal patient #5 xenografts
were treated with 50 mg/kg of INT2-31 vs PBS via intraperitoneal
injection. Treatment was started on day 10 after tumor
implantation. Animal weights are shown below growth curves.
*p<0.05 C) The percentage of reactive cells stained with Ki67
antibody is shown in the treatment vs control xenografts.
*p<0.05.
[0051] FIG. 24 Effects of INT2-31 on orthotopic pancreatic mice
model. A) Miapaca-2 xenografts were treated with 50 mg/kg of
INT2-31 vs PBS via intraperitoneal injection. Treatment was started
on day 7 after tumor implantation. B) Panc-1 xenografts were
treated with 15 mg/kg of INT2-31 vs PBS via subcutaneous injection.
Treatment was started on day 15 after tumor implantation.
DETAILED DESCRIPTION OF THE INVENTION
[0052] The invention provides compounds as novel cancer
therapeutics capable of inhibiting the viability of cancer cells or
increasing apoptosis. One aspect is that the compounds of the
invention are capable of modulating (e.g., inhibiting) the binding
interactions between FAK and IGF-1R. Another aspect is that the
compounds of the invention are capable of targeting (e.g.,
associating with or binding to) the interaction site(s) of FAK
and/or IGF-1R, thereby disrupting the binding interactions between
FAK and IGF-1R. The invention also provides a compound capable of
inhibiting tyrosine phosphorylation of FAK and/or IGF-1R, and
thereby disrupting the binding interactions between FAK and
IGF-1R.
[0053] The invention also provides methods of using the compounds
of the invention to treat a subject suffering from or susceptible
to a cell proliferative disorder. In certain embodiments, the cell
proliferative disorder is cancer.
[0054] The invention is based on, at least in part, on the
discovery that FAK physically interacts with IGF-1R in cancer cells
(e.g. pancreatic cancer cells). It is now believed that the
interactions between FAK and IGF-1R depend on the phosphorylation
status of both kinases, and that inhibition of tyrosine
phosphorylation of either kinase disrupts the interaction (See W.
Liu et al., Carcinogenesis, 29, 6, 2008, 1096-1107).
[0055] Non-cell based assays with GST and HIS tagged purified
proteins have been performed. The results demonstrate that direct
physical interaction exists between a FAK amino terminus fragment
(NT) (more specifically, NT2) and the kinase domain of IGF-1R (FIG.
1). It is believed that such interactions between FAK and IGF-1R
provide essential survival signals for cancer cells, including
pancreatic cancer cells.
[0056] In accordance with the invention, computer modeling together
with structural analysis has been performed. Following Lipinski
rules, about 250,000 small-molecule compounds with known precise
structures were docked into the site of interaction between FAK and
IGF-1R in 100 different orientations using the DOCK5.1 computer
program. Small molecules with the highest probability of binding to
FAK NT2 and disrupting the interaction with IGF-1R were then
obtained for functional testing from the National Cancer Institute
Developmental Therapeutics Program (NCI/DTP). Lead compounds that
target the interaction site of FAK and IGF-1R were identified. The
lead compounds were then evaluated in multiple cell-based assays on
their ability to disrupt the binding between purified FAK and
IGF-1R proteins, to inhibit cancer cell viability, to decrease
IGF-1R and AKT phosphorylation, and/or to induce apoptosis. Several
cancer cell lines, including esophageal (KYSE 140), pancreatic
(Panc-1), and melanoma (C8161) were tested with some lead
compounds. Preliminary results demonstrate that a lead compound,
4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine}, inhibits tumor cell viability, alters FAK and IGF-1R
signaling, and inhibits tumor growth in vivo.
[0057] The invention provides small molecule inhibitors as novel
cancer therapeutic agents. These inhibitors are capable of
modulating (e.g. inhibiting or disrupting) the binding interactions
between FAK and IGF-1R. The invention also provides a novel and
effective therapeutic strategy to treat cancer.
[0058] In one embodiment the inhibitors of the invention have at
least one of the following functions: reducing the viability of
cancer cells (for example melanoma cells), inhibiting cancer cell
proliferation (for example melanoma cells), inducing apoptosis,
decreasing activation of Akt without inhibiting kinase activity,
decreasing tumor p-Akt, and decreasing growth in melanoma
xenografts.
I. DEFINITIONS
[0059] Before further description of the invention, and in order
that the invention may be more readily understood, certain terms
are first defined and collected here for convenience.
[0060] The term "administration" or "administering" includes routes
of introducing the compound of the invention to a subject to
perform their intended function. Examples of routes of
administration that may be used include injection (subcutaneous,
intravenous, parenterally, intraperitoneally, intrathecal), oral,
inhalation, rectal and transdermal. The pharmaceutical preparations
may be given by forms suitable for each administration route. For
example, these preparations are administered in tablets or capsule
form, by injection, inhalation, eye lotion, ointment, suppository,
etc. administration by injection, infusion or inhalation; topical
by lotion or ointment; and rectal by suppositories. Oral
administration is preferred. The injection can be bolus or can be
continuous infusion. Depending on the route of administration, the
compound of the invention can be coated with or disposed in a
selected material to protect it from natural conditions which may
detrimentally effect its ability to perform its intended function.
The compound of the invention can be administered alone, or in
conjunction with either another agent as described above or with a
pharmaceutically-acceptable carrier, or both. The compound of the
invention can be administered prior to the administration of the
other agent, simultaneously with the agent, or after the
administration of the agent. Furthermore, the compound of the
invention can also be administered in a pro-drug form which is
converted into its active metabolite, or more active metabolite in
vivo.
[0061] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. The term alkyl further includes alkyl groups, which can
further include oxygen, nitrogen, sulfur or phosphorous atoms
replacing one or more carbons of the hydrocarbon backbone, e.g.,
oxygen, nitrogen, sulfur or phosphorous atoms. In certain
embodiments, a straight chain or branched chain alkyl has 30 or
fewer carbon atoms in its backbone (e.g., C1-C30 for straight
chain, C3-C30 for branched chain), 26 or fewer, 20 or fewer, or 4
or fewer. Likewise, cycloalkyls may have from 3-10 (e.g. 3, 4, 5 or
6) carbon atoms in their ring structure in the ring structure.
[0062] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, or one to six, or one to four
carbon atoms in its backbone structure, which may be straight or
branched-chain. Examples of lower alkyl groups include methyl,
ethyl, n-propyl, i-propyl, tert-butyl, hexyl, heptyl, octyl and so
forth. In an embodiment, the term "lower alkyl" includes a straight
chain alkyl having 4 or fewer carbon atoms in its backbone, e.g.,
C1-C4 alkyl.
[0063] The term "apoptosis" refers to the process of programmed
cell death (PCD) that may occur in multicellular organisms.
Programmed cell death involves a series of biochemical events
leading to a characteristic cell morphology and death, more
specifically, a series of biochemical events that lead to a variety
of morphological changes, for example, changes to the cell membrane
such as loss of membrane asymmetry and attachment, cell shrinkage,
nuclear fragmentation, chromatin condensation, and chromosomal DNA
fragmentation.
[0064] The term "associating with" refers to a condition of
proximity between a chemical entity or compound, or portions
thereof, and a binding pocket or binding site on a protein. The
association may be non-covalent (wherein the juxtaposition is
energetically favored by hydrogen bonding or van der Waals or
electrostatic interactions) or it may be covalent.
[0065] The term "binding pocket", as used herein, refers to a
region of a molecule or molecular complex, that, as a result of its
shape, favorably associates with another chemical entity or
compound.
[0066] The term "biological activities" of a compound of the
invention includes all activities elicited by compound of the
inventions in a responsive cell. It includes genomic and
non-genomic activities elicited by these compounds.
[0067] "Biological composition" or "biological sample" refers to a
composition containing or derived from cells or biopolymers.
Cell-containing compositions include, for example, mammalian blood,
red cell concentrates, platelet concentrates, leukocyte
concentrates, blood cell proteins, blood plasma, platelet-rich
plasma, a plasma concentrate, a precipitate from any fractionation
of the plasma, a supernatant from any fractionation of the plasma,
blood plasma protein fractions, purified or partially purified
blood proteins or other components, serum, semen, mammalian
colostrum, milk, saliva, placental extracts, a cryoprecipitate, a
cryosupernatant, a cell lysate, mammalian cell culture or culture
medium, products of fermentation, ascites fluid, proteins induced
in blood cells, and products produced in cell culture by normal or
transformed cells (e.g., via recombinant DNA or monoclonal antibody
technology). Biological compositions can be cell-free. In an
embodiment, a suitable biological composition or biological sample
is a red blood cell suspension. In some embodiments, the blood cell
suspension includes mammalian blood cells. The blood cells can be
obtained from a human, a non-human primate, a dog, a cat, a horse,
a cow, a goat, a sheep or a pig. In certain embodiments, the blood
cell suspension includes red blood cells and/or platelets and/or
leukocytes and/or bone marrow cells.
[0068] The term "cancer" refers to a class of diseases in which a
group of cells display uncontrolled growth, invasion, and
metastasis. The term is meant to include, but not limited to, a
cancer of the breast, respiratory tract, brain, reproductive
organs, digestive tract, urinary tract, eye, liver, skin, head and
neck, thyroid, parathyroid or a distant metastasis of a solid
tumor. Some specific examples of cancers include, but not limited
to, breast cancer, bladder cancer, colon and rectal cancer,
colorectal cancer, cutaneous melanoma, endometrial cancer, kidney
cancer, lung cancer, ovarian cancer, pancreatic cancer,
osteosarcoma, prostate cancer, lymphoma, leukemia, skin cancer,
thyroid cancer and sarcoma.
[0069] The term "cell proliferative disorder" includes disorders
involving the undesired or uncontrolled proliferation of a cell.
Examples of such disorders include, but are not limited to, tumors
or cancers (e.g., lung (small cell and non-small cell), thyroid,
prostate, pancreatic, breast or colon), sarcoma or melanoma.
[0070] The term of "chemotherapy" refers to treatment of disease by
chemicals that kill cells, specifically those of micro-organisms or
cancer. In the present application, this term refers to anti-cancer
therapeutic agents used to treat cancer or the combination of these
drugs into a cytotoxic standardized treatment regimen.
[0071] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner.
[0072] The term "cytotoxicity" and "toxicity" refers to the quality
of being toxic to cells. A toxic agent can be a chemical substance,
an immune cell or some types of venom.
[0073] The term "diastereomers" refers to stereoisomers with two or
more centers of dissymmetry and whose molecules are not mirror
images of one another.
[0074] The term "distant metastasis" means the spread of a disease
from one organ or part to another non-adjacent organ or parts via
lymph or blood. For the purposes of the present application, the
term "metastasis" refers to cancer cells that can spread from a
primary tumor, enter lymphatic and blood vessels, circulate through
the bloodstream, and settle down to grow within normal tissues
elsewhere in the body.
[0075] The term "an effective amount" refers to "a therapeutically
effective anti-proliferative amount" or "a prophylactically
effective anti-proliferative amount." The term includes an amount
effective, at dosages and for periods of time necessary, to achieve
the desired result, e.g., sufficient to treat a cell proliferative
disorder. An effective amount of compound of the invention may vary
according to factors such as the disease state, age, and weight of
the subject, and the ability of the compound of the invention to
elicit a desired response in the subject. Dosage regimens may be
adjusted to provide the optimum therapeutic response. An effective
amount is also one in which any toxic or detrimental effects (e.g.,
side effects) of the compound of the invention are outweighed by
the therapeutically beneficial effects.
[0076] A therapeutically effective amount of compound of the
invention (i.e., an effective dosage) may range from about 0.001 to
100 mg/Kg body weight. Certain examples are about 0.01 to 30 mg/kg
body weight, about 0.1 to 20 mg/kg body weight, about 1 to 10
mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg
body weight. The skilled artisan will appreciate that certain
factors may influence the dosage required to effectively treat a
subject, including but not limited to, the type of the disease or
disorder the subject has or susceptible to, the stage of the
disease or disorder, the severity of the disease or disorder,
previous therapeutic treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a compound of
the invention can include a single treatment or, can include a
series of treatments. One example is that a subject is treated with
a compound of the invention at a dosage in the range of between
about 0.001 to about 100 mg/Kg body weight, once per day. It will
also be appreciated that the effective dosage of a compound of the
invention used for treatment may increase or decrease over the
course of a particular treatment.
[0077] The term "enantiomers" refers to two stereoisomers of a
compound which are non-superimposable mirror images of one another.
An equimolar mixture of two enantiomers is called a "racemic
mixture" or a "racemate."
[0078] The term "epidermal growth factor receptor therapy" refers
to a cancer therapy that targets the epidermal growth factor
receptor (EGFR). EGFR is a receptor tyrosine kinase receptor that
is frequently expressed in epithelial tumors. Certain anti-EGFR
agents available in the clinic include, for example, gefitinib and
erlotinib.
[0079] The language "FAK binding partner" refers to a protein
recruited into complex with FAK (e.g., full length, N-terminus,
C-terminus, carboxy terminus, kinase domain, FERM domain, FAT
domain).
[0080] The term "homeostasis" is art-recognized to mean maintenance
of static, or constant, conditions in an internal environment.
[0081] The term "isomers" or "stereoisomers" refers to compounds
which have identical chemical constitution, but differ with regard
to the arrangement of the atoms or groups in space.
[0082] The term "immunotherapy" refers to a therapy to treat cancer
by modulating the immune system of the subject being treated. In
certain embodiments, immunotherapy used in cancer treatment aims to
stimulate tumor specific adaptive immune responses within the body
of the subject.
[0083] The language "improved biological properties" refers to any
activity inherent in a compound of the invention that enhances its
effectiveness in vivo. In one embodiment, this term refers to any
qualitative or quantitative improved therapeutic property of a
compound of the invention, such as reduced cytotoxicity.
[0084] The term "mitotic catastrophe" refers to a form of cell
death occurring during mitosis as a result of DNA damage or
deranged spindle formation. This is coupled with the dysregulation
of different checkpoint mechanisms (most notably, p53) that would
normally arrest progression into mitosis, and hence, suppress
catastrophic events until repair has been achieved. This results in
micronucleation and nuclear segmentation, which leads to cell
death.
[0085] The term "modulate" refers to an increase or decrease, e.g.,
in the ability of a cell to proliferate in response to exposure to
a compound of the invention, e.g., the inhibition of proliferation
of at least a sub-population of cells in an animal such that a
desired end result is achieved, e.g., a therapeutic result.
[0086] The term "monoclonal antibody therapy" refers to a therapy
using monoclonal antibodies (or mAb) to specifically target cancer
cells. The goal is to stimulate the subject's immune system to
attack the malignant tumor cells and the prevention of tumor growth
by blocking specific cell receptors. Examples of this therapy
include radioimmunotherapy, antibody-directed enzyme prodrug
therapy, drug and gene therapy using immuno-liposomes. Certain
therapeutic monoclonal antibodies include, but are not limited to,
alemtuzumab, bevacizumab, cetuximab, efalizumab, ibritumomab
tiuxetan, 111in-capromab, imciromab, panitumumab, gemtuzumab
ozogamicin, rituximab, tositumomab, and trastuzumab.
[0087] The term "obtaining" as in languages like "obtaining a
compound capable of modulating (or inhibiting) the binding
interactions between FAK and IGF-1R" is intended to include
purchasing, synthesizing or otherwise acquiring the compound.
[0088] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticulare, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0089] The term "prodrug" or "pro-drug" includes compounds with
moieties that can be metabolized in vivo. Generally, the prodrugs
are metabolized in vivo by esterases or by other mechanisms to
active drugs. Examples of prodrugs and their uses are well known in
the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J.
Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during
the final isolation and purification of the compounds, or by
separately reacting the purified compound in its free acid form or
hydroxyl with a suitable esterifying agent. Hydroxyl groups can be
converted into esters via treatment with a carboxylic acid.
Examples of prodrug moieties include substituted and unsubstituted,
branch or unbranched lower alkyl ester moieties, (e.g., propionoic
acid esters), lower alkenyl esters, di-lower alkyl-amino
lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino
lower alkyl esters (e.g., acetyloxymethyl ester), acyloxy lower
alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl
ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted
(e.g., with methyl, halo, or methoxy substituents) aryl and
aryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkyl
amides, and hydroxy amides. In certain embodiments, prodrug
moieties are propionoic acid esters and acyl esters. Prodrugs which
are converted to active forms through other mechanisms in vivo are
also included.
[0090] The language "a prophylactically effective amount" of a
compound refers to an amount of a compound of the invention or
otherwise described herein which is effective, upon single or
multiple dose administration to a subject identified as in need, in
preventing or treating a cell proliferative disorder.
[0091] The term "radiation therapy" (or "radiotherapy") refers to
the medical use of ionizing radiation as part of a therapeutic
treatment to control malignant cells. Certain examples provide that
a radiotherapy is used for curative or adjuvant cancer
treatment.
[0092] The language "reduced toxicity" is intended to include a
reduction in any undesired side effect elicited by a compound of
the invention when administered in vivo.
[0093] The term "pharmaceutically acceptable salt" refers to a
relatively non-toxic, inorganic or organic acid addition salt of a
compound of the invention. For example, see S. M. Berge, et al.
"Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19.
Pharmaceutically acceptable salts include those obtained by
reacting the main compound, functioning as a base, with an
inorganic or organic acid to form a salt, for example, salts of
hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonic
acid, camphor sulfonic acid, oxalic acid, maleic acid, succinic
acid and citric acid. Pharmaceutically acceptable salts also
include those in which the main compound functions as an acid and
is reacted with an appropriate base to form, e.g., sodium,
potassium, calcium, magnesium, ammonium, and chorine salts. Those
skilled in the art will further recognize that acid addition salts
of the claimed compounds may be prepared by reaction of the
compounds with the appropriate inorganic or organic acid via any of
a number of known methods. Alternatively, alkali and alkaline earth
metal salts of acidic compounds of the invention are prepared by
reacting the compounds of the invention with the appropriate base
via a variety of known methods.
[0094] Representative salts of the compounds of the invention
include the conventional non-toxic salts and the quaternary
ammonium salts which are formed, for example, from inorganic or
organic acids or bases by means well known in the art. For example,
such acid addition salts include acetate, adipate, alginate,
ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate,
butyrate, citrate, camphorate, camphorsulfonate, cinnamate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, itaconate, lactate, maleate,
mandelate, methane sulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oxalate, pamoate, pectinate, persulfate,
3-phenylpropionate, picrate, pivalate, propionate, succinate,
sulfonate, tartrate, thiocyanate, tosylate, and undecanoate. Base
salts include alkali metal salts such as potassium and sodium
salts, alkaline earth metal salts such as calcium and magnesium
salts, and ammonium salts with organic bases such as
dicyclohexylamine and N-methyl-D-glucamine. Additionally, basic
nitrogen containing groups may be quaternized with such agents as
lower alkyl halides such as methyl, ethyl, propyl, and butyl
chlorides, bromides and iodides; dialkyl sulfates like dimethyl,
diethyl, and dibutyl sulfate; and diamyl sulfates, long chain
halides such as decyl, lauryl, myristyl and strearyl chlorides,
bromides and iodides, aralkyl halides like benzyl and phenethyl
bromides and others.
[0095] The term "solvate" is meant to encompass a complex of a
solvent and a compound of the invention in the solid state.
Exemplary solvates would include, but are not limited to, complexes
of a compound of the invention with ethanol or methanol. Hydrates
are a specific form of solvate wherein the solvent is water.
[0096] The term "subject" includes organisms which are capable of
suffering from a cell proliferative disorder or who could otherwise
benefit from the administration of a compound of the invention of
the invention, such as human and non-human animals. Preferred
humans include human patients suffering from or prone to suffering
from a cell proliferative disorder or associated state, as
described herein. The term "non-human animals" of the invention
includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice,
and non-mammals, such as non-human primates, e.g., sheep, dog, cow,
chickens, amphibians, reptiles, etc.
[0097] The term "susceptible to a cell proliferative disorder" is
meant to include subjects at risk of developing disorder of cell
proliferation, e.g., cancer, i.e., subjects suffering from viral
infection with cancer viruses, subjects that have been exposed to
ionizing radiation or carcinogenic compounds, subjects having a
family or medical history of cancer, and the like.
[0098] The phrases "systemic administration," "administered
systemically", "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound
of the invention(s), drug or other material, such that it enters
the patient's system and, thus, is subject to metabolism and other
like processes, for example, subcutaneous administration.
[0099] The language "therapeutically effective amount" of a
compound of the invention of the invention refers to an amount of
an agent which is effective, upon single or multiple dose
administration to the patient, in inhibiting cell proliferation
and/or symptoms of a cell proliferative disorder, or in prolonging
the survivability of the patient with such a cell proliferative
disorder beyond that expected in the absence of such treatment.
[0100] With respect to the nomenclature of a chiral center, terms
"d" and "1" configuration are as defined by the IUPAC
Recommendations. As to the use of the terms, diastereomer,
racemate, epimer and enantiomer will be used in their normal
context to describe the stereochemistry of preparations.
II. COMPOUNDS OF THE INVENTION
[0101] In one aspect, the invention provides compounds capable of
treating a subject suffering from or susceptible to a cell
proliferative disorder, especially cancer. In an embodiment, the
cancer is pancreatic cancer, melanoma cancer, or esophageal cancer.
A compound of the invention is believed to be capable of modulating
(e.g., inhibiting) FAK and/or IGF-1R activity either directly or
indirectly. In an embodiment, the invention provides a compound
capable of modulating the binding interaction between FAK and
IGF-1R; and pharmaceutically acceptable esters, salts, and prodrugs
thereof.
[0102] In one embodiment, compounds of the invention include
compounds specifically delineated herein: [0103] 1).
2-(Hydroxymethyl)-6-imino-2,3,3a,9a-tetrahydro-6H-furo[2,3:4,5][1,3]oxazo-
lo[3,2-a]pyrimidin-3-yl dihydrogen phosphate (also as "NSC
128687"):
[0103] ##STR00001## [0104] 2).
4-(Methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimi-
dine} (also as "NSC 344553"):
[0104] ##STR00002## [0105] 3).
1,1'-(1,7,9-Trihydroxy-8,9b-dimethyl-3-oxo-4-a-(phenylthio)-3,4,4a,9b-tet-
rahydrodibenzo-[b,d]furan-2,6-diyl)diethanone (also as "NSC
250435"):
[0105] ##STR00003## [0106] 4). 3-Methyl-2,4-disulfopentanedioic
acid (also as "NSC 243620"):
[0106] ##STR00004## [0107] 5). 1-Aminopropane-1,3-diyldiphosphonic
acid (also as "NSC 133881"):
##STR00005##
[0107] The chemical name and structure of each of the
afore-mentioned compounds expressly include all diastereomers of
the compound.
[0108] The invention also provides the pharmaceutically acceptable
salts, esters, hydrates, solvates, clathrates, polymorphs, and
prodrugs of a compound of the invention.
[0109] The compounds of the invention may 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 expressly included in the invention. The compounds of this
invention may also be represented in multiple tautomeric forms, in
such instances, the invention expressly includes all tautomeric
forms of the compounds described herein. All such isomeric forms of
such compounds are expressly included in the invention. All crystal
forms of the compounds described herein are expressly included in
the invention.
[0110] Naturally occurring or synthetic isomers can be separated in
several ways known in the art. Methods for separating a racemic
mixture of two enantiomers include chromatography using a chiral
stationary phase (see, e.g., "Chiral Liquid Chromatography," W. J.
Lough, Ed. Chapman and Hall, New York (1989)). Enantiomers can also
be separated by classical resolution techniques. For example,
formation of diastereomeric salts and fractional crystallization
can be used to separate enantiomers. For the separation of
enantiomers of carboxylic acids, the diastereomeric salts can be
formed by addition of enantiomerically pure chiral bases such as
brucine, quinine, ephedrine, strychnine, and the like.
Alternatively, diastereomeric esters can be formed with
enantiomerically pure chiral alcohols such as menthol, followed by
separation of the diastereomeric esters and hydrolysis to yield the
free, enantiomerically enriched carboxylic acid. For separation of
the optical isomers of amino compounds, addition of chiral
carboxylic or sulfonic acids, such as camphorsulfonic acid,
tartaric acid, mandelic acid, or lactic acid can result in
formation of the diastereomeric salts.
[0111] Methods of obtaining a compound of the invention include
purchasing, synthesizing or otherwise acquiring the compound.
Synthesizing a compound of the invention is within the means of
chemists of ordinary skill in the art. Methods for optimizing
reaction conditions, if necessary minimizing competing by-products,
are known in the art. The methods may also additionally include
steps, either before or after the steps described specifically
herein, to add or remove suitable protecting groups in order to
ultimately allow synthesis of the compounds herein. In addition,
various synthetic steps may be performed in an alternate sequence
or order to give the desired compounds. Synthetic chemistry
transformations and protecting group methodologies (protection and
deprotection) useful in synthesizing the applicable compounds are
known in the art and include, for example, those described in R.
Larock, Comprehensive Organic Transformations, VCH Publishers
(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 3.sup.rd Ed., John Wiley and Sons (1999); L.
Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic
Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,
Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons
(1995) and subsequent editions thereof.
[0112] In another aspect, the invention provides compounds which
associate with or bind to FAK or specific domains thereof, thereby
interrupting the binding interactions between FAK and IGF-1R. In
one embodiment, the compound is capable of inhibiting tyrosine
phosphorylation of FAK, thereby disrupting the binding interactions
between FAK and IGF-1R.
[0113] In another aspect, the invention provides compounds which
associate with or bind to IGF-1R or specific domains thereof, which
thereby interrupts the binding interactions between FAK and IGF-1R.
In one embodiment, the compound is capable of inhibiting tyrosine
phosphorylation of IGF-1R, thereby disrupting the binding
interactions between FAK and IGF-1R.
[0114] The invention also provides a compound that is capable of
decreasing IGF-1R and AKT phosphorylation, and inducing apoptosis
of cancer cells.
[0115] The invention also provides polypeptides useful in screening
compounds for treating cell proliferative disorders. Such
polypeptides include, for example, FAK, domains of FAK, domains of
IGF-1R. An embodiment provides that the FAK domains include FAK-NT.
Another embodiment provides that the FAK domain is FAK-NT2. In a
separate embodiment, the domains of IGF-1R comprises the kinase
domain of IGF-1R.
[0116] Such polypeptides can be a fusion protein, e.g., a binding
pocket moiety fused with a detectable reporter moiety such as green
fluorescent protein, or labeled with a detectable tag such as a
fluorescent label, a radiolabel, and the like. Such a fusion
protein can be used in screening for compounds capable of
modulating the binding interactions between FAK and IGF-1R.
[0117] Without wishing to be bound by any theory, a compound of the
invention is capable of inhibiting the viability of cancer cells,
thereby treating a subject suffering from or susceptible to
cancer.
III. USES OF THE COMPOUNDS OF THE INVENTION
[0118] The invention provides a method of treating a subject
suffering from or susceptible to cancer. The method includes
administering to the subject in need thereof an effective amount of
a compound of the invention. In certain embodiments, the cancer is
a cancer of the breast, respiratory tract, brain, reproductive
organs, digestive tract, urinary tract, eye, liver, skin, head and
neck, thyroid, parathyroid, or a distant metastasis of a solid
tumor. Specific examples are pancreatic cancer, melanoma cancer,
and esophageal cancer.
[0119] One aspect of the invention provides a methods for using a
compound of the invention and compositions thereof, for treating a
subject suffering from or susceptible to a cell proliferative
disorder. One specific example of the cell proliferative disorder
is cancer. In one embodiment, a method of the invention includes
administering to a subject in need thereof an effective amount of a
compound capable of directly or indirectly modulating the binding
interactions between FAK and IGF-1R; thereby treating the subject
suffering from or susceptible to unwanted or undesired cell
proliferation or a cell proliferative disorder.
[0120] The effective amount of a compound of the invention is an
amount sufficient to reduce (the incidence or severity of) the
disease/disorder in the subject. An effective amount of a compound
of this invention can be provided in one or a series of
administrations (or doses). The effective amount of a compound of
this invention is generally determined by the physician on a
case-by-case basis and is within the skill of one in the art.
[0121] The administration may be by any route of administering
known in the pharmaceutical arts. The subject may have been
diagnosed with (e.g., cancer), may be at risk of developing a cell
proliferative disorder, may be exhibiting symptoms of a cell
proliferative disorder, or may need prophylactic treatment prior to
anticipated or unanticipated exposure to a conditions capable of
increasing susceptibility to a cell proliferative disorder. The
identification of those subjects that are in need of treatment for
cell proliferative disorders (e.g., cancer) is well within the
ability and knowledge of one skilled in the art. Certain of the
methods for identification of subjects that are at risk of
developing cell proliferative disorders which can be treated by the
method(s) of the invention are appreciated in the medical arts,
such as family history, and the presence of risk factors associated
with the development of that disease/disorder state in the subject.
A clinician skilled in the art can readily identify such candidate
subjects, by the use of, for example, clinical tests, physical
examination and medical/family history.
[0122] In certain embodiments, the subject is a mammal, e.g., a
primate, e.g., a human.
[0123] Certain embodiments provides that the cancer is a cancer of
the breast, respiratory tract, brain, reproductive organs,
digestive tract, urinary tract, eye, liver, skin, head and neck,
thyroid, parathyroid or a distant metastasis of a solid tumor. In
one embodiment, the cancer is pancreatic cancer, melanoma cancer,
or esophageal cancer.
[0124] The invention also provides a method of assessing or
monitoring the efficacy of a treatment in a subject includes
determining the pre-treatment extent of a cell proliferative
disorder (especially, cancer) by methods well known in the art
(e.g., determining tumor size or screening for tumor markers where
the cell proliferative disorder is cancer) and then administering
an effective amount of a compound of the invention to the subject.
After an appropriate period of time after the administration of the
compound (e.g., 1 day, 1 week, 2 weeks, one month, six months), the
extent of the condition is determined again. The modulation (e.g.,
decrease) of the extent or severity of the disease/disorder
indicates efficacy of the treatment. The extent or severity of the
disorder may be determined periodically throughout treatment. For
example, the extent or severity of the condition may be checked
every few hours, days or weeks to assess the further efficacy of
the treatment. A decrease in extent or severity of the
disease/disorder indicates that the treatment is efficacious. The
method described may be used to screen or select patients that may
benefit from treatment with a compound of the invention.
[0125] If the modulation of the status indicates that the subject
may have a favorable clinical response to the treatment, the
subject may be treated with the compound. For example, the subject
can be administered therapeutically effective dose or doses of the
compound.
[0126] The methods of the invention may include administering to a
subject identified as in need thereof an effective amount of a
compound of the invention in combination with one or more
additional therapeutic agents. Examples of these therapeutic agents
include drugs known to treat cancer, e.g., anticancer agents,
antiproliferative agents, and chemotherapeutic agents.
[0127] In one embodiment, the therapeutic agent is a
chemotherapeutic agent. Another embodiment provides that the agents
include 5-fluorouracil (5-FU), gemcitabine, fluoropyrimidines,
nucleoside cytidine analogues, NVP-AEW541, platinum analogues,
TAE226, topoisomerase inhibitors, antimicrotubule agents, PI3
kinase inhibitors, proteasome inhibitors, vitamin D analogues,
arachidonic acid pathway inhibitors, histone deacytylator
inhibitors, and farnesyltransferase inhibitors.
[0128] Examples of the therapeutic agents include, but are not
limited to, asparaginase, bleomycin, calcein-AM, carboplatin,
carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide,
cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel,
doxorubicin (adriamycine), epirubicin, etoposide, ET-743,
erlotinib, 5-fluorouracil, gemcitabine, gefitinib,
hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan,
leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna,
methotrexate, mitomycin C, mitoxantrone, NVP-AEW541, paclitaxel,
prednisolone, prednisone, procarbazine, raloxifen, rhodamine-123,
streptozocin, TAE226, tamoxifen, thioguanine, topotecan,
vinblastine, vincristine, vindesine, and zalypsis.
[0129] Other therapeutic agents that may be used can be found in
Harrison's Principles of Internal Medicine, 17th Edition, Eds. T.
R. Harrison et al. McGraw-Hill N.Y., NY; and the Physicians Desk
Reference 62nd Edition 2008, Oradell New Jersey, Medical Economics
Co., the complete contents of which are expressly incorporated
herein by reference. The compound of the invention and the
additional therapeutic agent(s) may be administered to the subject
in the same pharmaceutical composition or in different
pharmaceutical compositions (at the same time or at different
times).
[0130] A method of the invention may further include treating the
subject with one or more anti-cell proliferation therapies. In
particular, the therapy is a cancer therapy. Conventional cancer
therapies include, but are not limited to, surgery, chemotherapy,
radiation, immunotherapy, monoclonal antibody therapy and epidermal
growth factor receptor therapies. One example of the cancer therapy
is radiation. Another example is chemotherapy. An embodiment
provides that the method includes treating the subject with a
combination of chemotherapy and radiation.
[0131] The methods of the invention can be performed on cells in
culture, e.g. in vitro or ex vivo, or on cells present in an animal
subject, e.g., in vivo. Compounds of the inventions can be
initially tested in vitro using primary cultures of proliferating
cells, e.g., transformed cells, tumor cell lines, and the like.
Alternatively, the effects of compound of the invention can be
characterized in vivo using animals models.
[0132] The invention also provides a method to modulate (e.g.,
inhibit) uncontrolled proliferation of cells. The method includes
contacting a compound of the invention with a cell undergoing
uncontrolled proliferation. The compound thereof may either
directly or indirectly modulate the activity of FAK, the activity
of IGF-1R, or the binding interactions between FAK and IGF-1R. Such
contacting between a compound of the invention and the cell
inhibits cell proliferation or induce apoptosis. Contacting cells
or administering the compounds of the invention to a subject is one
method of treating a cell or a subject suffering from or
susceptible to a cell proliferative disorder.
[0133] In one embodiment, the contacting may be in vitro, e.g., by
addition of the compound to a fluid surrounding the cells, for
example, to the growth media in which the cells are living or
existing. The contacting may also be by directly contacting the
compound to the cells. Alternately, the contacting may be in vivo,
e.g., by passage of the compound through a subject; for example,
after administration, depending on the route of administration, the
compound may travel through the digestive tract or the blood stream
or may be applied or administered directly to cells in need of
treatment.
[0134] The invention also presents a method to identify a compound
capable of treating a subject suffering from or being susceptible
to cancer. In particular, the compound is capable of modulating the
binding interaction between FAK and IGF-1R.
[0135] In another aspect, the invention provides a method of
modulating binding interactions between FAK and IGF-1R by
contacting FAK and/or IGF-1R with a compound of the invention.
Certain embodiments provide that the compound is capable of
inhibiting tyrosine phosphorylation of FAK and/or IGF-1R, thereby
disrupting the binding interactions between FAK and IGF-1R. Another
embodiment provides that the method further includes using a
dominant-negative construct (FAK-CD) or small interfering RNA.
[0136] Certain embodiments provide that the compound of the
invention is capable of associating with or binding to FAK or
specific domains thereof, thereby interrupting the binding
interactions between FAK and IGF-1R. An embodiment provides that
the compound is capable of inhibiting tyrosine phosphorylation of
FAK. An embodiment provides that the compound is capable of binding
to or associating with a FAK amino terminus fragment (NT2).
[0137] Another embodiment provides that the compound of the
invention is capable of associating with or binding to IGF-1R or
specific domains thereof, which thereby interrupts the binding
interactions between FAK and IGF-1R. An embodiment provides that
the compound is capable of inhibiting tyrosine phosphorylation of
IGF-1R. One embodiment provides that the compound is capable of
binding to or associating with the kinase domain of IGF-1R.
[0138] The methods of the invention may further include using a
dominant-negative construct (FAK-CD) or small interfering RNA.
[0139] The invention also provides a method of identification of a
compound that is capable of decreasing IGF-1R and AKT
phosphorylation, and inducing apoptosis of cancer cells.
[0140] The methods may include obtaining crystal structures of FAK,
IGF-1R, or specific domains thereof (optionally apo form or
complexed) or obtaining the information relating to the crystal
structure of FAK, IGF-1R, or specific domains thereof (optionally
apo form or complexed), in the presence and/or absence of the test
compound. Examples of these specific domains include FAK NT2 and
the kinase domain of IGF-1R. Compounds may then be computer modeled
into binding sites of the crystal structures of FAK, IGF-1R, or
specific domains thereof to predict stabilization of the
interaction between the test compound and the FAK, IGF-1R, or
specific domains thereof. Once potential modulating compounds are
identified, the compounds may be screened using cellular assays,
such as the ones identified herein and competition assays known in
the art. Compounds identified in this manner are useful as
therapeutic agents.
[0141] In one embodiment, the method further includes evaluating a
test compound that comprises 1) contacting FAK, IGF-1R, or specific
domains thereof with a test compound (complex), and 2) evaluating
the binding interaction following contact, wherein a change in the
stability of the complex relative to a reference value is an
indication that the test compound modulates the stability of the
complex.
[0142] In an embodiment, the complex of FAK, IGF-1R, or specific
domains thereof, may be modeled in silico, or may be a complex
within a cell, isolated from a cell, recombinantly expressed,
purified or isolated from a cell or recombinant expression system,
or partially purified or isolated from a cell or recombinant
expression system.
[0143] In yet another aspect, the invention provides the use of a
compound of the invention, alone or together with one or more
additional therapeutic agents in the manufacture of a medicament,
either as a single composition or as separate dosage forms, for
treatment or prevention in a subject of a disease, disorder or
condition set forth herein. Another aspect of the invention is a
compound of the invention for use in the treatment or prevention in
a subject of a disease, disorder or condition thereof delineated
herein.
[0144] Methods delineated herein include those wherein the subject
is identified as in need of a particular stated treatment.
Identifying a subject in need of such treatment can be in the
judgment of a subject or a health care professional and can be
subjective (e.g. opinion) or objective (e.g. measurable by a test
or diagnostic method).
IV. DOSAGES
[0145] Determination of a suitable dosage of the compound of the
invention can be readily made by the physician or veterinarian (the
"attending clinician"), as one skilled in the art, by the use of
known techniques and by observing results obtained under analogous
circumstances.
[0146] The dosages may be varied depending upon the requirements of
the patient in the judgment of the attending clinician; the
severity of the condition being treated, the stage of the condition
being treated and the particular compound being employed. In
determining the therapeutically effective anti-proliferative amount
or dose, and the prophylactically effective anti-proliferative
amount or dose, a number of factors are considered by the attending
clinician, including, but not limited to: the specific cell
proliferative disorder involved; pharmacodynamic characteristics of
the particular agent and its mode and route of administration; the
desired time course of treatment; the species of mammal; its size,
age, and general health; the specific disease involved; the degree
of or involvement or the severity of the disease; the response of
the individual patient; the particular compound administered; the
mode of administration; the bioavailability characteristics of the
preparation administered; the dose regimen selected; the kind of
concurrent treatment (i.e., the interaction of the compound of the
invention with other co-administered therapeutics); and other
relevant circumstances.
[0147] Standard texts, such as Remington: The Science and Practice
of Pharmacy, 17th edition, Mack Publishing Company, and the
Physician's Desk Reference, each of which are incorporated herein
by reference, can be consulted to prepare suitable compositions and
doses for administration. A determination of the appropriate dosage
is within the skill of one in the art given the parameters for use
described herein.
[0148] Treatment can be initiated with smaller dosages. The dosage
may then be increased by small increments until the optimum effect
under the circumstances is reached. For convenience, the total
dosage may be divided and administered in portions during the
administration period if desired.
[0149] The dosage of a compound of the invention can vary from
about 0.01 mg to about 5,000 mg per day. In some instances, the
dosage varies from about 100 mg to about 4000 mg per day, or about
1000 mg to about 3000 mg per day. Ascertaining dosage ranges is
well within the skill of one in the art. In certain embodiments,
the dosage of a compound of the invention can range from about
0.001 to about 100 mg/Kg of body weight. Certain ranges are about
0.01 to about 30 mg/kg body weight, about 0.1 to 20 mg/kg body
weight, about 1 to 10 mg/kg, about 2 to 9 mg/kg, about 3 to 8
mg/kg, about 4 to 7 mg/kg, or about 5 to 6 mg/kg body weight. Such
dosages may vary, for example, depending on whether multiple
administrations are given, tissue type and route of administration,
the condition of the individual, the desired objective and other
factors known to those of skill in the art. Administrations can be
conducted frequently, for example, on a regular daily or weekly
basis, until a desired, measurable parameter is detected, such as
diminution of disease symptoms. Administration can then be
diminished, such as to a biweekly or monthly basis
[0150] Compounds determined to be effective for the prevention or
treatment of cell proliferative disorders in animals, e.g., dogs,
chickens, and rodents, may also be useful in treatment of tumors in
humans. Those skilled in the art of treating tumors in humans will
know, based upon the data obtained in animal studies, the dosage
and route of administration of the compound to humans.
V. PHARMACEUTICAL COMPOSITIONS AND DOSAGE FORMS
[0151] The invention also provides pharmaceutical compositions
containing an effective amount of a compound of the invention. The
pharmaceutical compositions may also comprise a pharmaceutically
acceptable carrier or diluent. The composition may be formulated
for treating a subject suffering from or susceptible to a cell
proliferative disorder (e.g. cancer), and packaged with
instructions to treat a subject suffering from or susceptible to
the disease/disorder. The effective amount is effective to treat
the disease/disorder as described previously.
[0152] In an embodiment, the compound of the invention is
administered to the subject using a pharmaceutically-acceptable
formulation, e.g., a pharmaceutically-acceptable formulation that
provides sustained delivery of the compound of the invention to a
subject for at least 12 hours, 24 hours, 36 hours, 48 hours, one
week, two weeks, three weeks, or four weeks after the
pharmaceutically-acceptable formulation is administered to the
subject.
[0153] In certain embodiments, these pharmaceutical compositions
are suitable for topical or oral administration to a subject. In
other embodiments, as described in detail below, the pharmaceutical
compositions of the present invention may be specially formulated
for administration in solid or liquid form, including those adapted
for the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets,
boluses, powders, granules, pastes; (2) parenteral administration,
for example, by subcutaneous, intramuscular or intravenous
injection as, for example, a sterile solution or suspension; (3)
topical application, for example, as a cream, ointment or spray
applied to the skin; (4) intravaginally or intrarectally, for
example, as a pessary, cream or foam; or (5) aerosol, for example,
as an aqueous aerosol, liposomal preparation or solid particles
containing the compound.
[0154] The phrase "pharmaceutically acceptable" refers to those
compound of the inventions of the present invention, compositions
containing such compounds, and/or dosage forms which are, within
the scope of sound medical judgment, suitable for use in contact
with the tissues of human beings and animals without excessive
toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk
ratio.
[0155] The phrase "pharmaceutically-acceptable carrier" includes
pharmaceutically-acceptable material, composition or vehicle, such
as a liquid or solid filler, diluent, excipient, solvent or
encapsulating material, involved in carrying or transporting the
subject chemical from one organ, or portion of the body, to another
organ, or portion of the body. Each carrier is "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not injurious to the patient. Some examples of
materials which can serve as pharmaceutically-acceptable carriers
include: (1) sugars, such as lactose, glucose and sucrose; (2)
starches, such as corn starch and potato starch; (.sub.3)
cellulose, and its derivatives, such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; (4) powdered
tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such
as cocoa butter and suppository waxes; (9) oils, such as peanut
oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil
and soybean oil; (10) glycols, such as propylene glycol; (11)
polyols, such as glycerin, sorbitol, mannitol and polyethylene
glycol; (12) esters, such as ethyl oleate and ethyl laurate;
(1.sub.3) agar; (14) buffering agents, such as magnesium hydroxide
and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water;
(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol;
(20) phosphate buffer solutions; and (21) other non-toxic
compatible substances employed in pharmaceutical formulations.
[0156] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0157] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0158] Compositions containing a compound of the invention include
those suitable for oral, nasal, topical (including buccal and
sublingual), rectal, vaginal, aerosol and/or parenteral
administration. The compositions may conveniently be presented in
unit dosage form and may be prepared by any methods well known in
the art of pharmacy. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will vary depending upon the host being treated, the particular
mode of administration. The amount of active ingredient which can
be combined with a carrier material to produce a single dosage form
will generally be that amount of the compound which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 1 percent to about ninety-nine percent
of active ingredient, preferably from about 5 percent to about 70
percent, more preferably from about 10 percent to about 30
percent.
[0159] Methods of preparing these compositions include the step of
bringing into association a compound of the invention with the
carrier and, optionally, one or more accessory ingredients. In
general, the formulations are prepared by uniformly and intimately
bringing into association a compound of the invention with liquid
carriers, or finely divided solid carriers, or both, and then, if
necessary, shaping the product.
[0160] Compositions of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
invention as an active ingredient. A compound may also be
administered as a bolus, electuary or paste.
[0161] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically-acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (.sub.3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, acetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0162] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered active ingredient moistened with an inert
liquid diluent.
[0163] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0164] Liquid dosage forms for oral administration of the compound
of the invention include pharmaceutically-acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0165] In addition to inert diluents, the oral compositions can
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0166] Suspensions, in addition to the active compound of the
invention may contain suspending agents as, for example,
ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and
sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar and tragacanth, and mixtures
thereof.
[0167] Pharmaceutical compositions of the invention for rectal or
vaginal administration may be presented as a suppository, which may
be prepared by mixing one or more compound of the invention with
one or more suitable nonirritating excipients or carriers
comprising, for example, cocoa butter, polyethylene glycol, a
suppository wax or a salicylate, and which is solid at room
temperature, but liquid at body temperature and, therefore, will
melt in the rectum or vaginal cavity and release the active
agent.
[0168] Compositions of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0169] Dosage forms for the topical or transdermal administration
of a compound of the invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound of the invention may be mixed under sterile
conditions with a pharmaceutically-acceptable carrier, and with any
preservatives, buffers, or propellants which may be required.
[0170] The ointments, pastes, creams and gels may contain, in
addition to compound of the invention, excipients, such as animal
and vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc and zinc oxide, or mixtures thereof.
[0171] Powders and sprays can contain, in addition to a compound of
the invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0172] The compound of the invention can be alternatively
administered by aerosol. This is accomplished by preparing an
aqueous aerosol, liposomal preparation or solid particles
containing the compound. A nonaqueous (e.g., fluorocarbon
propellant) suspension could be used. Sonic nebulizers are
preferred because they minimize exposing the agent to shear, which
can result in degradation of the compound.
[0173] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the agent together with
conventional pharmaceutically-acceptable carriers and stabilizers.
The carriers and stabilizers vary with the requirements of the
particular compound, but typically include nonionic surfactants
(Tweens, Pluronics, or polyethylene glycol), innocuous proteins
like serum albumin, sorbitan esters, oleic acid, lecithin, amino
acids such as glycine, buffers, salts, sugars or sugar alcohols.
Aerosols generally are prepared from isotonic solutions.
[0174] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the invention to the body.
Such dosage forms can be made by dissolving or dispersing the agent
in the proper medium. Absorption enhancers can also be used to
increase the flux of the active ingredient across the skin. The
rate of such flux can be controlled by either providing a rate
controlling membrane or dispersing the active ingredient in a
polymer matrix or gel.
[0175] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
the invention.
[0176] Pharmaceutical compositions of the invention suitable for
parenteral administration comprise one or more compound of the
invention in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.
[0177] Examples of suitable aqueous and nonaqueous carriers, which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0178] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0179] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0180] Injectable depot forms are made by forming microencapsule
matrices of compound of the invention in biodegradable polymers
such as polylactide-polyglycolide. Depending on the ratio of drug
to polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0181] When the compound of the invention are administered as
pharmaceuticals, to humans and animals, they can be given per se or
as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of active ingredient in
combination with a pharmaceutically-acceptable carrier.
[0182] Regardless of the route of administration selected, the
compound of the invention, which may be used in a suitable hydrated
form, and/or the pharmaceutical compositions of the present
invention, are formulated into pharmaceutically-acceptable dosage
forms by conventional methods known to those of skill in the
art.
[0183] Actual dosage levels and time course of administration of
the active ingredients in the pharmaceutical compositions of the
invention may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic
response for a particular patient, composition, and mode of
administration, without being toxic to the patient. An exemplary
dose range is from 0.1 to 10 mg per day.
[0184] The suitable dose of a compound of the invention is the
maximum that a patient can tolerate and not develop serious side
effects. For example, a compound of the invention is administered
at a concentration of about 0.001 mg to about 100 mg per kilogram
of body weight, about 0.001--about 10 mg/kg or about 0.001
mg--about 100 mg/kg of body weight. Other examples for the dose
range are discussed supra.
[0185] The pharmaceutical compositions of the invention may further
include additional therapeutic agent as previously discussed. One
embodiment provides that the additional therapeutic agent is a
chemotherapeutic agent. Another embodiment provides that the
additional therapeutic agent is selected from the group consisting
of 5-fluorouracil (5-FU), gemcitabine, fluoropyrimidines,
nucleoside cytidine analogues, NVP-AEW541, platinum analogues,
TAE226, topoisomerase inhibitors, antimicrotubule agents, PI3
kinase inhibitors, proteasome inhibitors, vitamin D analogues,
arachidonic acid pathway inhibitors, histone deacytylator
inhibitors, and farnesyltransferase inhibitors.
[0186] Certain examples of the additional therapeutic agent
include, but are not limited to, asparaginase, bleomycin,
calcein-AM, carboplatin, carmustine, chlorambucil, cisplatin,
colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin,
daunorubicin, docetaxel, doxorubicin (adriamycine), epirubicin,
etoposide, ET-743, erlotinib, 5-fluorouracil, gemcitabine,
gefitinib, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan,
leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna,
methotrexate, mitomycin C, mitoxantrone, NVP-AEW541, paclitaxel,
prednisolone, prednisone, procarbazine, raloxifen, rhodamine-123,
streptozocin, TAE226, tamoxifen, thioguanine, topotecan,
vinblastine, vincristine, vindesine, and zalypsis.
[0187] When a compound of the invention is administered as
pharmaceuticals, to humans and animals, it can be given per se or
as a pharmaceutical composition containing, for example, 0.1 to
99.5% (or 0.5 to 90%) of active ingredient in combination with a
pharmaceutically-acceptable carrier.
[0188] Regardless of the route of administration selected, the
compound of the invention, which may be used in a suitable hydrated
form, and/or the pharmaceutical compositions of the invention, are
formulated into pharmaceutically-acceptable dosage forms by
conventional methods known to those of skill in the art.
[0189] Actual dosage levels and time course of administration of
the active ingredients in the pharmaceutical compositions of the
invention may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic
response for a particular subject, composition, and mode of
administration, without being toxic to the subject.
VI. KITS
[0190] The invention also provides kits for treating
disorders/diseases delineated herein. A typical kit of the
invention includes a compound, a pharmaceutical formulation or a
combination described in this document, and instructions for use.
The instructions for use may include information on dosage, method
of delivery, storage of the kit, etc. Certain embodiments provide
that the kit includes instructions for administering the compound,
formulation or combination of the invention.
[0191] A kit may include instructions and/or information for
identification of a subject in need for treatment. In certain
embodiments, the kit may include instructions to treat a subject
suffering from or susceptible to a cell proliferative disorder. In
one embodiment, the disorder is cancer. Certain examples provide
that the cancer is a cancer of the breast, respiratory tract,
brain, reproductive organs, digestive tract, urinary tract, eye,
liver, skin, head and neck, thyroid, parathyroid or a distant
metastasis of a solid tumor. Specific examples are pancreatic
cancer, melanoma cancer, and esophageal cancer.
[0192] In one embodiment, the kit further includes instructions for
use to treat or prevent a cell proliferative disorder in a subject.
The instructions for use may include information on dosage, method
of delivery, storage of the kit, etc.
[0193] The effective amount of the compound included in the kit is
as above discussed. Typically, the effective amount of a compound
of the invention is a dosage lower than that is required to develop
serious side effects in the subject being treated. Certain examples
provide that the kit includes a compound of the invention at a dose
of between about 0.001 mg/Kg and about 100 mg/Kg.
[0194] Some embodiments provide that the kit further includes an
additional therapeutic agent. In one embodiment, the additional
therapeutic agent is a chemotherapeutic agent. Another embodiment
provides that the additional therapeutic agent is selected from the
group consisting of 5-fluorouracil (5-FU), gemcitabine,
fluoropyrimidines, nucleoside cytidine analogues, NVP-AEW541,
platinum analogues, topoisomerase inhibitors, TAE226,
antimicrotubule agents, PI3 kinase inhibitors, proteasome
inhibitors, vitamin D analogues, arachidonic acid pathway
inhibitors, histone deacytylator inhibitors, and
farnesyltransferase inhibitors. Certain embodiments provide that
the additional therapeutic agent is TAE226, NVP-AEW541, wortmannin,
or LY294002.
[0195] Examples of the additional therapeutic agent include, but
are not limited to, asparaginase, bleomycin, calcein-AM,
carboplatin, carmustine, chlorambucil, cisplatin, colaspase,
cyclophosphamide, cytarabine, dacarbazine, dactinomycin,
daunorubicin, docetaxel, doxorubicin (adriamycine), epirubicin,
etoposide, ET-743, erlotinib, 5-fluorouracil, gemcitabine,
gefitinib, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan,
leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna,
methotrexate, mitomycin C, mitoxantrone, NVP-AEW541, paclitaxel,
prednisolone, prednisone, procarbazine, raloxifen, rhodamine-123,
streptozocin, TAE226, tamoxifen, thioguanine, topotecan,
vinblastine, vincristine, vindesine, and zalypsis.
[0196] The kits may also include, reagents, for example, test
compounds, buffers, media (e.g., cell growth media), cells, etc.
Test compounds may include known compounds or newly discovered
compounds, for example, combinatorial libraries of compounds.
[0197] Kits of the invention can further comprise devices that are
used to administer a compound of the invention. Examples of such
devices include, but are not limited to, intravenous cannulation
devices, syringes, drip bags, patches, topical gels, pumps,
containers that provide protection from photodegredation,
autoinjectors, and inhalers.
[0198] Kits of the invention can also comprise pharmaceutically
acceptable vehicles that can be used to administer one or more
active ingredients. For example, if an active ingredient is
provided in a solid form that must be reconstituted for parenteral
administration, the kit can comprise a sealed container of a
suitable vehicle in which the active ingredient can be dissolved to
form a particulate-free sterile solution that is suitable for
parenteral administration. Examples of pharmaceutically acceptable
vehicles include, but are not limited to: Water for Injection USP;
aqueous vehicles such as, but not limited to, Sodium Chloride
Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodium Chloride Injection, and Lactated Ringer's Injection;
water-miscible vehicles such as, but not limited to, ethyl alcohol,
polyethylene glycol, and polypropylene glycol; and non-aqueous
vehicles such as, but not limited to, corn oil, cottonseed oil,
peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and
benzyl benzoate.
[0199] One or more of the kit of the invention may be packaged
together, for example, a kit for assessing the efficacy of an
treatment for a cell proliferative disorder (e.g. cancer) may be
packaged with a kit for monitoring the progress of a subject being
treated for a cell proliferative disorder according to the
invention.
VII. SCREENING METHODS AND SYSTEMS
[0200] In another aspect, the invention provides a machine readable
storage medium which comprises the structural coordinates of either
one or both of the binding pockets identified herein, or similarly
shaped, homologous binding pockets. Such storage medium encoded
with these data are capable of displaying a three-dimensional
graphical representation of a molecule or molecular complex which
comprises such binding pockets on a computer screen or similar
viewing device.
[0201] The invention also provides methods for designing,
evaluating and identifying compounds which bind to the
afore-mentioned binding pockets. Thus, the computer produces a
three-dimensional graphical structure of a molecule or a molecular
complex which comprises a binding pocket.
[0202] In another embodiment, the invention provides a computer for
producing a three-dimensional representation of a molecule or
molecular complex defined by structure coordinates of FAK, IGF-1R,
or specific domains thereof, or a three-dimensional representation
of a homologue of said molecule or molecular complex, wherein said
homologue comprises a binding pocket that has a root mean square
deviation from the backbone atoms of said amino acids of not more
than 2.0 (more preferably not more than 1.5) angstroms. In one
embodiment, the structure used coordinates to a FAK amino terminus
fragment (NT), or the kinase domain of IGF-1R. In another
embodiment, the structure used coordinates to FAK-NT2.
[0203] In exemplary embodiments, the computer or computer system
can include components which are conventional in the art, e.g., as
disclosed in U.S. Pat. Nos. 5,978,740 and/or 6,183,121
(incorporated herein by reference). For example, a computer system
can includes a computer comprising a central processing unit
("CPU"), a working memory (which may be, e.g., RAM (random-access
memory) or "core" memory), a mass storage memory (such as one or
more disk drives or CD-ROM drives), one or more cathode-ray tube
(CRT) or liquid crystal display (LCD) display terminals, one or
more keyboards, one or more input lines, and one or more output
lines, all of which are interconnected by a conventional system
bus.
[0204] Machine-readable data of this invention may be inputted to
the computer via the use of a modem or modems connected by a data
line. Alternatively or additionally, the input hardware may include
CD-ROM drives, disk drives or flash memory. In conjunction with a
display terminal, a keyboard may also be used as an input
device.
[0205] Output hardware coupled to the computer by output lines may
similarly be implemented by conventional devices. By way of
example, output hardware may include a CRT or LCD display terminal
for displaying a graphical representation of a binding pocket of
this invention using a program such as QUANTA or PYMOL. Output
hardware might also include a printer, or a disk drive to store
system output for later use.
[0206] In operation, the CPU coordinates the use of the various
input and output devices, coordinates data accesses from the mass
storage and accesses to and from working memory, and determines the
sequence of data processing steps. A number of programs may be used
to process the machine-readable data of this invention, including
commercially-available software.
[0207] A magnetic storage medium for storing machine-readable data
according to the invention can be conventional. A magnetic data
storage medium can be encoded with a machine-readable data that can
be carried out by a system such as the computer system described
above. The medium can be a conventional floppy diskette or hard
disk, having a suitable substrate which may be conventional, and a
suitable coating, which may also be conventional, on one or both
sides, containing magnetic domains whose polarity or orientation
can be altered magnetically. The medium may also have an opening
(not shown) for receiving the spindle of a disk drive or other data
storage device.
[0208] The magnetic domains of the medium are polarized or oriented
so as to encode in manner which may be conventional, machine
readable data such as that described herein, for execution by a
system such as the computer system described herein.
[0209] An optically-readable data storage medium also can be
encoded with machine-readable data, or a set of instructions, which
can be carried out by a computer system. The medium can be a
conventional compact disk read only memory (CD-ROM) or a rewritable
medium such as a magneto-optical disk which is optically readable
and magneto-optically writable.
[0210] In the case of CD-ROM, as is well known, a disk coating is
reflective and is impressed with a plurality of pits to encode the
machine-readable data. The arrangement of pits is read by
reflecting laser light off the surface of the coating. A protective
coating, which preferably is substantially transparent, is provided
on top of the reflective coating.
[0211] In the case of a magneto-optical disk, as is well known, a
data-recording coating has no pits, but has a plurality of magnetic
domains whose polarity or orientation can be changed magnetically
when heated above a certain temperature, as by a laser. The
orientation of the domains can be read by measuring the
polarization of laser light reflected from the coating. The
arrangement of the domains encodes the data as described above.
[0212] Structure data, when used in conjunction with a computer
programmed with software to translate those coordinates into the
3-dimensional structure of a molecule or molecular complex
comprising a binding pocket may be used for a variety of purposes,
such as drug discovery.
[0213] In an embodiment, DOT is the software used for prediction of
molecules or molecular complexes. DOT performs a systematic,
rigid-body search of one molecule translated and rotated about a
second molecule. The intermolecular energies for all configurations
generated by this search are calculated as the sum of electrostatic
and van der Waals energies. These energy terms are evaluated as
correlation functions, which are computed efficiently with Fast
Fourier Transforms. In a typical run, energies for about 108
billion configurations of two molecules can be calculated in a few
hours.
[0214] For example, the structure encoded by the data may be
computationally evaluated for its ability to associate with
chemical entities. Chemical entities that associate with a binding
pocket of FAK, IGF-1R, or specific domains thereof, and are
potential drug candidates. Cerain FAK domains include FAK-NT. In an
embodiment, the structure encoded by the data coordinates to
FAK-NT2, or the kinase domain of IGF-1R. In another embodiment, the
structure encoded by the data coordinates to FAK aa 126-243 or
IGF-1R aa 959-1266. The structure encoded by the data may be
displayed in a graphical three-dimensional representation on a
computer screen. This allows visual inspection of the structure, as
well as visual inspection of the structure's association with
chemical entities.
[0215] Thus, according to another embodiment, the invention relates
to a method for evaluating the potential of a chemical entity to
associate with a) a molecule or molecular complex comprising a
binding pocket of FAK, IGF-1R, or specific domains thereof, or b) a
homologue of said molecule or molecular complex, wherein said
homologue comprises a binding pocket that has a root mean square
deviation from the backbone atoms of said amino acids of not more
than 2.0 (more preferably 1.5) angstroms.
[0216] This method comprises the steps of:
[0217] i) employing computational means to perform a fitting
operation between the chemical entity and a binding pocket of the
molecule or molecular complex; and
[0218] ii) analyzing the results of the fitting operation to
quantify the association between the chemical entity and the
binding pocket. The term "chemical entity", as used herein, refers
to chemical compounds, complexes of at least two chemical
compounds, and fragments of such compounds or complexes.
[0219] According to this invention, the design of compounds that
bind to or associate with an interaction site between FAK, and
IGF-1R, or that bind to or inhibit FAK, IGF-1R, or specific domains
thereof generally involves consideration of several factors. First,
the entity must be capable of physically and structurally
associating with parts or all of the FAK, IGF-1R, or specific
domains thereof, or a site of interaction between FAK and IGF-1R.
Non-covalent molecular interactions important in this association
include hydrogen bonding, van der Waals interactions, hydrophobic
interactions and electrostatic interactions. Second, the entity
must be able to assume a conformation that allows it to associate
with the FAK, IGF-1R, or specific domains thereof, or a site of
interaction between FAK and IGF-1R directly. Although certain
portions of the entity will not directly participate in these
associations, those portions of the entity may still influence the
overall conformation of the molecule. This, in turn, may have a
significant impact on potency. Such conformational requirements
include the overall three-dimensional structure and orientation of
the chemical entity in relation to all or a portion of the binding
pocket, or the spacing between functional groups of an entity
comprising several chemical entities that directly interact with
the binding pocket or homologues thereof.
[0220] The potential inhibitory or binding effect of a chemical
entity on the FAK, IGF-1R, or specific domains thereof, or a site
of interaction between FAK and IGF-1R may be analyzed prior to its
actual synthesis and testing by the use of computer modeling
techniques. If the theoretical structure of the given entity
suggests insufficient interaction and association between it and
the target binding pocket, testing of the entity is obviated.
However, if computer modeling indicates a strong interaction, the
molecule may then be synthesized and tested for its ability to bind
to the FAK, IGF-1R, or specific domains thereof, or associate with
a site of interaction between FAK and IGF-1R. In an embodiment, the
molecule may be tested for its ability to bind to FAK-NT (or
FAK-NT2), or the kinase domain of IGF-1R. In another embodiment,
the compound is selected for its ability to bind to FAK aa 126-243
and/or IGF-1R aa 959-1266. This may be achieved, e.g., by testing
the ability of the molecule to inhibit the activity of the FAK,
IGF-1R, or specific domains thereof, or modulate the binding
interaction between FAK and IGF-1R, e.g., using assays described
herein or known in the art. In this manner, synthesis of
inoperative compounds may be avoided.
[0221] A potential inhibitor of a FAK, IGF-1R, or specific domains
thereof, or of the binding interaction between FAK and IGF-1R may
be computationally evaluated by means of a series of steps in which
chemical entities or fragments are screened and selected for their
ability to associate with the FAK, IGF-1R, or specific domains
thereof, or a site of interaction between FAK and IGF-1R. In an
embodiment, the potential inhibitor may be evaluated for its
ability to associate with the FAK-NT (or FAK-NT2) domain, or the
kinase domain of IGF-1R. As an example, FAK aa 126-243 and/or
IGF-1R aa 959-1266 can be utilized in this process.
[0222] One skilled in the art may use one of several methods to
screen chemical entities or fragments for their ability to
associate with the FAK, IGF-1R, or specific domains thereof, or a
site of interaction between FAK and IGF-1R. This process may begin
by visual inspection of, for example, a FAK, IGF-1R, or specific
domains thereof (e.g., FAK NT2 domain, or the kinase domain of
IGF-1R), or a site of interaction between FAK and IGF-1R on the
computer screen based on structure of a FAK, IGF-1R, specific
domains thereof, or complex of FAK and IGF-1R, or other coordinates
which define a similar shape generated from the machine-readable
storage medium. Selected fragments or chemical entities may then be
positioned in a variety of orientations, or docked, within that
binding pocket as defined supra. Docking may be accomplished using
software such as Quanta and DOCK, followed by energy minimization
and molecular dynamics with standard molecular mechanics force
fields, such as CHARMM and AMBER.
[0223] Specialized computer programs (e.g., as known in the art
and/or commercially available and/or as described herein) may also
assist in the process of selecting fragments or chemical
entities.
[0224] Once suitable chemical entities or fragments have been
selected, they can be assembled into a single compound or complex.
Assembly may be preceded by visual inspection of the relationship
of the fragments to each other on the three-dimensional image
displayed on a computer screen in relation to the structure
coordinates of the target binding pocket.
[0225] Instead of proceeding to build an inhibitor of a binding
pocket in a step-wise fashion one fragment or chemical entity at a
time as described above, inhibitory or other binding compounds may
be designed as a whole or "de novo" using either an empty binding
site or optionally including some portion(s) of a known
inhibitor(s). There are many de novo ligand design methods known in
the art, some of which are commercially available (e.g., LeapFrog,
available from Tripos Associates, St. Louis, Mo.).
[0226] Other molecular modeling techniques may also be employed in
accordance with this invention [see, e.g., N. C. Cohen et al.,
"Molecular Modeling Software and Methods for Medicinal Chemistry,
J. Med. Chem., 33, pp. 883-894 (1990); see also, M. A. Navia and M.
A. Murcko, "The Use of Structural Information in Drug Design",
Current Opinions in Structural Biology, 2, pp. 202-210 (1992); L.
M. Balbes et al., "A Perspective of Modern Methods in
Computer-Aided Drug Design", in Reviews in Computational Chemistry,
Vol. 5, K. B. Lipkowitz and D. B. Boyd, Eds., VCH, New York, pp.
337-380 (1994); see also, W. C. Guida, "Software For
Structure-Based Drug Design", Curr. Opin. Struct. Biology, 4, pp.
777-781 (1994)].
[0227] Once a compound has been designed or selected, the
efficiency with which that entity may bind to a binding pocket may
be tested and optimized by computational evaluation.
[0228] Specific computer software is available in the art to
evaluate compound deformation energy and electrostatic
interactions. Examples of programs designed for such uses include:
AMBER; QUANTA/CHARMM (Accelrys, Inc., Madison, Wis.) and the like.
These programs may be implemented, for instance, using a
commercially-available graphics workstation. Other hardware systems
and software packages will be known to those skilled in the
art.
[0229] Another technique involves the in silico screening of
virtual libraries of compounds, e.g., as described herein. Many
thousands of compounds can be rapidly screened and the best virtual
compounds can be selected for further screening (e.g., by synthesis
and in vitro testing). Small molecule databases can be screened for
chemical entities or compounds that can bind, in whole or in part,
to a binding pocket in FAK, IGF-1R, or specific domains thereof, or
associate with a site of interaction between FAK and IGF-1R. In
this screening, the quality of fit of such entities to the binding
site may be judged either by shape complementarity or by estimated
interaction energy.
EXAMPLES
[0230] The invention is further illustrated by the following
examples which are intended to illustrate but not limit the scope
of the invention.
Materials
[0231] Cell lines and culture--Panc-1 and MiaPaca-2 cells were
obtained from American Type Culture Collection. Panc-1 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum (FBS), and 1 .mu.g/ml
penicillin/streptomycin. MiaPaca-2 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2.5%
horse serum, and 1 .mu.g/ml penicillin/streptomycin. The L.3.6 pl
cell lines were obtained from the University of Texas MD Anderson
Cancer Center, and were maintained in modified Eagle's medium
supplemented with 10% FBS, 1 .mu.g/ml penicillin/streptomycin,
vitamins, 1 mmol/l sodium pyruvate, 2 mmol/l L-glutamine, and
non-essential amino acids. All cell lines were incubated at
37.degree. C. in a 5% CO.sub.2 humidified incubator.
[0232] Recombinant adenovirus carrying the LacZ or the
dominant-negative FAK construct coding for amino acids 693-1052 of
FAK (Ad-FAK-CD) are propagated by the Gene Therapy Center Virus
Vector Core Facility of the University of North Carolina.
[0233] A375, SK-MEL-28 cells were obtained from American Type
Culture Collection (Rockville, Md.). The C8161, FAK +/+ and FAK -/-
mouse embryonic fibroblast (MEF) cell lines which were kindly
provided by Dr. William Cance (Roswell Park Cancer Institute,
Buffalo, N.Y.). IGF-1R+/+ and -/- MEFs were kindly provided by
Renato Baserga (Thomas Jefferson University, Philadelphia, Pa.).
Melanocytes were obtained from Lifeline Cell Technology and
maintained in DermaLife.RTM. M Melanocyte Culture Medium (Lifeline
Cell Technology, Walkersville, Md.).
[0234] Esophageal Cancer Cell Lines
[0235] TE and KYSE group cell lines were kindly provided by Dr.
Yutaka Shimada (University of Toyama, Toyama, Japan). Esophageal
cancer lines were maintained in RPMI 1640 supplemented with 10%
FBS, 1 .mu.g/ml penicillin-streptomycin. All cell lines were
incubated at 37.degree. C. in a 5% CO.sub.2 humidified
incubator.
Pancreatic Cancer Cell Lines
[0236] As-PC1, Bx-PC3, Panc-1 and MiaPaca-2 cells were obtained
from American Type Culture Collection (Rockville, Md.). Panc-1
cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (FBS) and 1 .mu.g/ml
penicillin-streptomycin. MiaPaca-2 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2.5%
horse serum and 1 .mu.g/ml penicillin-streptomycin. The As-PC1 and
Bx-PC3 cell lines were maintained in RPMI 1640 supplemented with
10% FBS, 1 .mu.g/ml penicillin-streptomycin. Human pancreatic duct
epithelial (HPDE) cells were kindly provided by Dr. Carol Otey
(University of North Carolina, Chapel Hill, N.C.) and maintained in
Keratinocyte-SFM Serum free medium (Gibco/Invitrogen, Carlsbad,
Calif.) supplemented with L-Glutamine, EGF&BPE and soy bean
trypsin inhibitor (Gibco/Invitrogen, Carlsbad, Calif.). All cell
lines were incubated at 37.degree. C. in a 5% CO.sub.2 humidified
incubator.
Other Cell Lines
[0237] FAK knockout mouse embryonic fibroblast cells (FAK -/- MEFs)
were kindly provided by Dr. William Cance (Roswell Park, Buffalo,
N.Y.) and maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (FBS) and 1 .mu.g/ml
penicillin-streptomycin. IGF-1R knockout mouse embryonic fibroblast
cells (IGF-1R-/- MEF) were kindly provided by Dr. Renato Baserga
(Kimmel Cancer Center, Thomas Jefferson University, Philadelphia,
Pa.) and maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (FBS) and 1 .mu.g/ml
penicillin-streptomycin. IGF-1R-/- clones were selected by using
200 mg/ml of Hygromycin B. MCF7, MCF10A and BT474 cells were
purchased from American Type Culture Collection (ATCC, Rockville,
Md.). BT474 were maintained in RPMI-1640 with 10% fetal bovine
serum and insulin 250 .mu.g/ml. MCF7 cells were maintained with
Modified minimum Eagle's media with 10% fetal bovine serum,
1.times. non-essential amino acids (Cellgro, Herndon, Va.), 1 mM
sodium pyruvate, and 500 .mu.g/ml insulin. MCF10A, an immortalized
human mammary epithelial cell line was cultured in a 1:1 mixture of
Dulbecco's modified Eagle's medium and F12 medium (DMEM-F12)
supplemented with 5% horse serum, hydrocortisone (0.5 .mu.g/ml),
insulin (10 .mu.g/ml), epidermal growth factor (20 ng/ml), and
penicillin-streptomycin (100 .mu.g/ml each). Reagents and
antibodies: FAK siRNA was purchased from Dharmacon RNA Technologies
(Lafayette, Colo.). NVP-AEW 541 and TAE226 were obtained from
Novartis (East Hanover, N.J.). Anti-FAK monoclonal (4.47) and
anti-phospho-tyrosine monoclonal (4G10) antibodies were obtained
from Upstate (Lake Placid, N.Y.). Anti-IGF-IR antibody was from
Calbiochem (San Diego, Calif.). Anti-phospho-FAK (Tyr397) and
anti-phospho-Src antibody were from Biosource (Camarillo, Calif.).
Anti-phospho-EGFR, anti-EGFR, anti-phospho-Akt, anti-Akt,
anti-phospho-ERK1/2, anti-ERK1/2, anti-cycin B1 and anti-Aurora B
were from Cell Signaling Technology (Beverly, Mass.). Anti-caspase
3 and anti-PARP antibodies were from BD Biosciences (San Jose,
Calif., catalogue #611038). Anti-actin antibodies were from Sigma
(St Louis, Mo.). Anti-glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) antibodies were from Advanced ImmunoChemical (Long Beach,
Calif.). And, different protein constructs of IGF-1R and FAK used
in the experiments were made in the lab of the inventors.
[0238] TAE226 was obtained from Novartis (East Hanover, N.J.).
Anti-FAK (4.47) and anti-phospho-tyrosine monoclonal (4G10)
antibodies from Upstate (Lake Placid, N.Y.). Anti-FAK (C20) and
anti-IGF-1R.beta. antibody (C20) from Santa Cruz Biotechnology
(Santa Cruz, Calif.). Anti-His and anti-GST antibodies from Sigma
(Saint Louis, Miss.). Anti-phospho-IGF-1R and anti-IGF-1R
antibodies, from Calbiochem (San Diego, Calif.). Anti-phospho-FAK
(Tyr397) from Biosource (Camarillo, Calif.). Anti-caspase 8,
anti-caspase 9, anti-phospho-Akt, anti-Akt, anti-phospho-ERK1/2,
anti-ERK1/2 antibodies from Cell Signaling Technology (Beverly,
Mass.). Anti-caspase 3/7 and anti-PARP antibodies from BD
Biosciences (Catalogue #611038, San Jose, Calif., recognizes the
full length, uncleaved form of PARP). Anti-.beta.-actin antibody
from Sigma (St Louis, Mo.). Anti-glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) antibody from Advanced ImmunoChemical (Long
Beach, Calif.).
[0239] MTT reagent was purchased from Promega (Madison, Wis.). CFSE
was purchased from Molecular Probes (Eugene, Oreg.). TAE226 was
obtained from Novartis (East Hanover, N.J.). Gemcitabine (Gemzar)
was purchased from Eli Lilly (Indianapolis, Ind., USA).
5-Fluorouracil (5-FU) was supplied by Sigma-Aldrich Chemical
(Poole, UK). Recombinant Human IGF-I was purchased from R&D
(Minneapolis, Minn.). Anti-FAK monoclonal (4.47) and
anti-phospho-tyrosine monoclonal (4G10) antibodies were obtained
from Upstate (Lake Placid, N.Y.). Anti-FAK (C20) antibody and
anti-IGF-1R.beta. antibody (C20) were obtained from Santa Cruz
Biotechnology (Santa Cruz, Calif.). Anti-His antibody and anti-GST
antibody were obtained from Sigma (Saint Louis, Miss.).
Anti-phospho-IGF-1R and anti-IGF-1R antibodies were from Calbiochem
(San Diego, Calif.). Anti-phospho-FAK (Tyr397) and anti-phospho-Src
antibody were from Biosource (Camarillo, Calif.). Anti-src,
anti-caspase 8, anti-caspase 9, anti-phospho-Akt, anti-Akt,
anti-phospho-ERK1/2, anti-ERK1/2, were from Cell Signaling
Technology (Beverly, Mass.). Anti-caspase 3/7 and anti-PARP
antibodies were from BD Biosciences (Catalogue #611038, San Jose,
Calif.). This PARP antibody recognized the full length, uncleaved
form of PARP. Anti-.beta.-actin antibodies were from Sigma (St
Louis, Mo.). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
antibody was from Advanced ImmunoChemical (Long Beach, Calif.).
Cell viability (MTT) and CFSE Proliferation assay: Cells were
plated in 96-well plates and let adhere overnight. After cell
treatment, cell viability was measured by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay (CellTiter 96.RTM. Aqueous, Promega, Madison, Wis.).
[0240] In detachment assays, detached and attached cells were
harvested separately and counted in a hemocytometer. The percentage
of detachment was calculated by dividing the number of detached
cells by the total number of cells.
[0241] For staining with CFSE (Molecular Probes, Eugene, Oreg.).,
1.times.10.sup.7/ml cells were suspended in PBS and incubated at
37.degree. C. for 5 min with the 10 .mu.M of CFSE. Stained cells
were cultured with medium alone or with inhibitor for 24, 48, and
72 hours, fixed and analyzed by a FACS Calibur cytometer (Becton
Dickinson, San Jose, Calif.).
Computational Docking The crystal structures of the N-terminal
domain of FAK (PDB code 2AL6) (22) and the kinase domain of
IGF-1R(PDB 1P40A) (23) were utilized for in silico molecular
modeling of their interaction as previously described. The
three-dimensional coordinates of compound NSC344553 (INT2-31),
obtained from the database of the National Cancer Institute,
Developmental Therapeutics Program (NCI/DTP), were docked onto the
predicted interface of the amino-terminus of FAK (amino acids
127-243) with the intracytoplasmic portion of IGF-1R (21). All
docking calculations were performed with the University of
California-San Francisco DOCK 5.1 program, using a clique-matching
algorithm to orient small molecule structures with sets of spheres
that describe the target sites on FAK (37). 100 orientations were
created for NSC344553 in the target site and were scored using the
computer program grid-based scoring function. Docking calculations
were performed on the University of Florida High Performance
Computing supercomputing cluster (http:hpc.ufl.edu). The
intermolecular energies for all configurations of NSC 344553 in
binding to FAK-NT2 were calculated as the sum of electrostatic and
van der Waals energies. These energy terms were evaluated as
correlation functions, which were computed efficiently with Fast
Fourier Transforms. Production of GST-fusion proteins: The FAK-GST
plasmid constructs (pGEX vector) were kindly provided by Dr. Elena
Kurenova (Roswell Park Cancer Institute, Buffalo, N.Y.). His-tagged
IGF-1R protein was purchased from Blue Sky Biotechnology
(Worchester, Mass.). The GST-fusion proteins (FAK fragments) were
expressed in BL21 (DE3) Escherichia coli bacteria by incubation
with 0.2 mM isopropyl b-D-galactopyranoside (IPTG) for 6 h at
37.degree. C. The bacteria were lysed by sonication, and the fusion
proteins were purified with glutathione-Sepharose 4B beads (GE
Healthcare, NJ). Pull-down assay: For the pull-down binding assay,
His-tagged IGF-1R fragment protein (200 ng) were precleared with
GST immobilized on glutathione-Sepharose 4B beads. The precleared
His-tagged protein was incubated with 0.2 .mu.g of GST-FAK fusion
protein immobilized on the glutathione-Sepharose 4B beads for 1 h
at 4.degree. C. and then washed 3.times. with PBS. Equal amounts of
GST-fusion proteins were used for each binding assay. Bound
proteins were boiled in 6.times. Laemmli buffer and analyzed by
SDS-PAGE and Western blotting. Immunoprecipitation and western
blotting: Cells were washed twice with ice cold 1.times.PBS and
lysed in buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 1%
NP-40, 5 mM EDTA acid, protease inhibitors (Complete.TM. Protease
Inhibitor, Roche, N.J.) and phosphatase inhibitors (Calbiochem,
Calif.). For immunoprecipitation, 100-200 .mu.g of total cell
extract was used for each sample. The extracts were incubated with
1 .mu.g of antibody overnight at 4.degree. C. Next, 25 .mu.l of
protein A/G-agarose beads were added and the samples were incubated
for 2 h at 4.degree. C. The precipitates were washed 4.times. with
lysis buffer and samples containing 30 .mu.g of protein were
resolved by SDS-PAGE. The intensity of the bands in the western
blots was measured with scion image analysis software program.
Short hairpin RNA Transfection of cells: Control shRNA (mock) and
FAK shRNAs was obtained from Open Biosystems. The sequences of
short hairpin RNAs against human FAK were:
(5'-CCGGCCGATTGGAAACCAACATATACTCGAGTATATGTTGGTT TCCAATCGTTTTG-3';
5'-CCGGGCCCAGAAGAAGGAATCAGTTCTCGAG AACTGATTCTTCTTCTGGGCTTTTTG-3')
and control shRNA (mock)
(5'-TCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGA-3'). For the
transfection of cells (2.times.10.sup.5 cells/well) were seeded
into 6-well plates in 2 ml medium one day prior to transfection.
According to the protocols of the manufacturer, cells were
transfected using Lipofectamine 2000 reagent (Invitrogen, CA).
GFP-Fused FAK Constructs and Transfection of Cells
[0242] FAK-NT1 (a.a. 1-126), FAK-NT2 (a.a. 127-243), and FAK-NT3
(a.a. 244-415) were amplified by PCR using gene specific primers
and cloned into the pEGFP-C2 vector (Clonetech, Mountain View,
Calif.). All sequences were confirmed by automatic sequencing (ICBR
Sequencing Facility, University of Florida). To over-express FAK
fragments, plasmids pEGFP-FAK-NT1, pEGFP-FAK-NT2 and pEGFP-FAK-NT3
were transfected into cells with Lipofectamine 2000 (Invitrogen,
CA) according to instructions from the provider.
Stable Transduction of Cell Lines
[0243] Infection of pancreatic cancer cell lines, Panc-1 and Mia
paca-2 was done in the laboratory of Dr. Lung-ji Chang. The
lentiviral vectors for luciferase expression were registered on
RD-0637 and RD-0633 protocol at the University of Florida.
[0244] Pancreatic cancer cell lines were trypsinized and counted.
The cells were then plated to 24-well trays and incubated at
37.degree. C., humidified 5% CO.sub.2-95% air until 60-80%
confluent. In each well, a volume of 10 .mu.l of firefly luciferase
and red fluorescent protein (RFP) containing lentivirus particles
were added to the medium. After gently swirling the plate to mix,
cells were incubated at 37.degree. C. in a humidified incubator in
an atmosphere of 5% CO.sub.2, to allow the optimal transduction
efficiency. Four hours later, viral containing medium replaced with
fresh medium. Based on expression of RFP protein and flow
cytometric sorting of the cells, the pure population of transduced
cell was obtained.
Detachment assay: Cells were plated with and without compound for
24, 48, and 72 h, and detached and attached cells were counted in a
hemocytometer. The percent of detachment was calculated by dividing
the number of detached cells by the total number of cells and
experiments performed in triplicate. Apoptosis assays: After
treatment, attached and detached cells were collected, counted and
prepared for terminal uridine deoxynucleotidyl transferase (TUNEL)
assay by utilizing an APO-BRDU kit (BD Pharmingen, San Diego,
Calif.) and cells analyzed with a FACSCalibur cytometer (Becton
Dickinson, San Jose, Calif.). In addition, apoptotic cells were
analyzed with Hoechst 33342 staining (1 .mu.g/ml). The percent of
apoptotic cells was calculated as the ratio of apoptotic cells to
total number of cells. For caspase 3/7 activation detection, 2000
cells were plated onto glass bottom, 2% gelatin coated plates and
fluorescent activation evaluated by confocal microscopy.Hoechst
Staining
[0245] In addition, apoptotic cells were also analyzed by Hoechst
staining. To the prepared cells as described above, Hoechst 33342
(1 .mu.g/ml) was added, incubated in the dark room temperature for
10 minutes, and the specimens were mounted on glass coverslips. The
slides were viewed under the Zeiss microscope for apoptotic nuclei.
The percent of apoptotic cells was calculated as the ratio of
apoptotic cells to total number of cells. Over 300 cells per sample
were analyzed.
Caspase 3/7 Apoptosis Assay
[0246] For detection of activated caspase 3/7 enzymes, as a
confirmation of apoptosis in the treated cells, Apo-ONE.RTM.
Caspase-3/7 Reagent kit was used (Promega, Madison, Wis.). 2000
cells were plated into a 96 well glass bottom plate, and treated
with different concentrations of the compound. 24, 48 and 72 h
after the treatment, cells were incubated with 10 .mu.L of a
profluorescent caspase-3/7 consensus substrate, rhodamine 110
bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-aspartic acid amide)
(Z-DEVD-R110), for 30 minutes in the dark at room temperature. Upon
cleavage on the C-terminal side of the aspartate residue in the
DEVD peptide substrate sequence by caspase-3/7 enzymes, the
rhodamine 110 becomes fluorescent when excited at a wavelength of
498 nm. The emission maximum is 521 nm. The amount of fluorescent
product generated is representative of the amount of active
caspase-3/7 present in the sample. Imaging was with a Leica TCS SP5
laser-scanning confocal microscope with LAS-AF imaging software,
using a 40.times. oil objective.
Kinase Profiler Screening: Kinase specificity screening was
performed with Invitrogen's SelectScreen.RTM. Kinase Profiling
Services
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Services/-
Discoverv-Research/SelectScreen-Profiling-Service/SelectScreen-Kinase-Prof-
iling-Service.html. The screening was performed with 1 .mu.M
compound, INT2-31, 10 .mu.M ATP, and kinase substrates against ten
recombinant kinases according to Z'-LYTET.TM. Kinase Assay. For PI3
kinase activity, 100 .mu.M ATP, and kinase substrate were utilized
with the Invitrogen Adapta.RTM. Universal Kinase Assay protocol.
Tumor Growth in Nude Mice in vivo: Six week old athymic, female
nude mice were purchased from Harlan Laboratory. The mice were
maintained in the animal facility, and all experiments were
performed in compliance with NIH animal-use guidelines and under an
IACUC approved protocol. Melanoma cells were injected,
5.times.10.sup.6 cells, subcutaneously. When the tumor size reached
100 mm.sup.3, the INT2-31 was introduced by intraperitoneal
injection at a dose of 15 mg/kg daily for 21 days. Tumor diameters
were measured with calipers, and tumor volume in mm.sup.3 was
calculated using the formula [(width).sup.2.times.length]/2. At the
end of experiment, tumor weight and volume were determined.
Melanoma Xenograft
[0247] For the melanoma study, the University of Florida IACUC
approved the following protocol (IACUC Study #200801077). Melanoma
cells were injected, 5.times.10.sup.6 cells, subcutaneously. When
the tumor size reached 100 mm.sup.3, the INT2-31 was introduced by
intraperitoneal injection at a dose of 15 mg/kg daily. Tumor
diameters were measured with calipers, and tumor volume in mm.sup.3
was calculated using the formula [(width).sup.2.times.length]/2. At
the end of experiment, tumor weight and volume were determined.
Patient Subjects and Xenograft
[0248] The use of human subjects in this study was for the sole
purpose of the procurement of solid esophageal and pancreatic tumor
tissue for studies reviewed and the specific approval of the
University of Florida Health Center Institution Review Board (IRB)
under protocols #276-2008 and 321-2005 has already been
obtained.
[0249] For tumor samples from human patients with esophageal or
pancreatic cancer, the University of Florida IACUC approved the
following protocol (IACUC Study #2000902767). A total of 25
patients, 10 with pancreatic cancer and 15 with esophageal cancer
have been identified and implanted into nude mice. Initially small
pieces (0.3.times.0.3.times.0.3 cm) from fresh pancreatic and
esophageal human tumor samples were obtained from surgical
specimens of patients operated at the University of Florida Shands
Hospital, and implanted subcutaneously in-group of 2 mice for each
patient. For esophageal cancer specimens, when one of them has
reached 1.5 cc, it was excised and was cut into small pieces of
(0.3.times.0.3.times.0.3 cm), and transplanted subcutaneously into
another 10 mice. When tumors reached .about.100 mm.sup.3, mice were
randomized in the following 2 groups, with 5 mice in each group:
[0250] Group 1: Control: no treatment. [0251] Group 2: INT2-31
(Compound 31): 50 mg/kg/day in 50 .mu.L by i.p administration for
21 days. This drug has been previously tested by our laboratory and
has no measurable toxicity at this dose.
[0252] Mice were euthanized 30-40 days after tumor innoculation and
tumor and tissue collected. For inhibition of tumor growth in our
subcutaneous model, tumor volumes
(length.times.width.times.height.times.p/6) and body weights were
determined daily including weekends and holidays, to monitor tumor
growth and evaluate overall clinical condition, taking into account
weight loss and indications of pain, distress, or abnormal behavior
and physiology. Experiments were terminated when the mean control
tumor volume was 1.5 cc (approximately 30-40 days).
[0253] Antitumor activity was expressed as T/C % (mean increase of
tumor volumes of treated animals divided by the mean increase of
tumor volumes of control animals multiplied by 100).
[0254] Orthotopic Model of Pancreatic Cancer
[0255] For the orthotopic model of pancreatic cancer, the
University of Florida IACUC approved the following protocol (IACUC
Study #2000801506). The pancreatic cancer cell lines, Mia paca-2
and Panc-1 cells were stably transfected using luciferase-RFP (red
fluorescent protein) reporter gene for in vivo imaging of the
xenografts. Following expansion and sorting of RFP positive cells,
cells were expanded in culture and 5.times.10.sup.6 tumor cells
were implanted into the pancreas of 20 mice. For intra-pancreatic
implantation of cells, mice were anesthetized with Isoflurane using
the ACS provided and maintained rodent anesthesia machine. Under
sterille surgical conditions, via 1.0 cm incision of the skin,
abdominal wall and peritonium, the spleen was retracted and cells
were injected in 304 volume into the tail of the pancreas using a
29-gauge needle. The abdominal wall and peritoneum was sutured
using 5.0 absorbable surgical sutures and the skin was closed with
medical glue (dermabond). Postoperative analgesia was 0.05 mg/kg of
buprenorphine subcutaneously per 8-12 hours postoperatively.
[0256] When tumors reach .about.100 mm.sup.3, mice were randomized
in the following 4 groups, with 3 mice in each group: [0257] Group
1: Control: no treatment. [0258] Group 2: Gemcitabine: 40 mg/kg in
50 .mu.L treated every 5 days for three weeks by intraperitoneal
(i.p) administration. [0259] Group 3: INT2-31: 15 mg/kg/day in 50
.mu.L by i.p administration [0260] Group 4: Combination of
Gemcitabine and INT2-31 treatments
[0261] Mice were hand restrained prior to intraperitoneal
injections. Mice were euthanized 6 weeks after tumor innoculation
and tumor and tissue collected. As described below, mice were
imaged weekly with the IVIS lumina imager and tumor size was
estimated by the bioluminescent signal.
In Vivo Imaging of Mice
[0262] Noninvasive imaging was performed in all tumor-bearing mice
expressing bioluminescent tags. The IVIS lumina platform was used
with tumors that express a luciferase reporter gene. To accomplish
the imaging, mice were anesthetized with Isoflurane using the ACS
provided and maintained rodent anesthesia machine. A cryogenically
cooled IVIS Imaging System (Xenogen) with Living Image acquisition
and analysis software (Version 2.11, Xenogen) was used to detect
the bioluminescence signals in mice. For mice bearing tumors
expressing a luciferase reporter gene, prior to imaging, mice were
injected intraperitoneally or subcutaneously with 150 mg of
luciferin (Xenogen Corp., Alameda, Calif.) per kg of body weight in
100 .mu.L using a 25-27 g needle. The area of injection was cleaned
using standard surgical disinfectant, all solutions were sterile
and satisfied the drug policy of the University of Florida. After
10 min, the mice were anesthetized as described above and placed on
a heated sample shelf. The imaging system first took a photographic
image in the chamber under dim illumination; this was followed by
luminescent image acquisition. An integration time of 1 min was
used for luminescent image acquisition for all mouse tumor models.
Living Image software was used to integrate the total
bioluminescence signals (in terms of photon counts) obtained from
mice. The in vitro detection limit of the IVIS Imaging System is
1,000 ES-2/luc cells.
[0263] Each animal was studied no more than weekly over a six week
period. Based on the luminescent signal, the tumor size was easily
estimated. On day 42 or when the tumor size reached 1.5 cc in size,
the mice were euthanized.
Immunohistochemistry: Xenograft tumor tissue was fixed in 10%
formalin and embedded in paraffin. For Ki-67 staining, samples
underwent deparaffinization and antigen retrieval and incubated
with the primary antibody, Ki-67 (Dako M7240), at 1:200
concentration overnight at 4.degree. C. The tissues were stained
with the chromogen DAB and counterstained with hematoxylin and 1%
TBS.
[0264] For assessment of apoptotic cells, staining was performed
utilizing the Dead End.TM. Calorimetric TUNEL System (Promega,
Madison, Wis.) according to instructions from the manufacturer.
Percent apoptotic cells were determined from counting at least 400
cells in a high power field.
Statistical Analyses Student's t-test was performed to determine
significance. The difference between data with p<0.05 was
considered significant.
ELISA Test
[0265] Two different enzyme-linked immunosorbent assays were
performed to study binding between IGF-1R beta subunit and FAK-NT.
The first assay involved interaction of IGF-1R with immobilized
FAK-NT; the second assay involved interaction of FAK-NT with
immobilized IGF-1R.
[0266] In the first case, 96-microtiter plate wells were coated
with purified GST-fused FERM domain of FAK in 50 .mu.l of PBS (NaCl
137 mM, KCl 2.7 mM, Na.sub.2HPO.sub.4 4.3 mM, KH.sub.2PO.sub.4 1.4
mM) overnight at 4.degree. C. Wells were then rinsed with wash
buffer (PBS, 0.05% Tween) and blocked with 200 .mu.l of blocking
buffer (PBS, 1% BSA) for 3 h at 37.degree. C. After rinsing three
times with wash buffer, with or without the compounds, 100 .mu.l of
binding buffer (PBS, 0.05% Tween, 1% BSA) containing 0.2 .mu.M of
purified IGF-1R whole protein was added to the wells and allowed to
react for 1 h at 37.degree. C. Wells were rinsed again three times
and 100 .mu.l of binding buffer containing 200 ng/ml of a primary
antibody anti-IGF-1R (sc-613 Santa Cruz) was added and incubated
for 1 h at 37.degree. C. After three additional rinsings, 100 .mu.l
of the same buffer containing a secondary HRP anti-rabbit antibody
was added and incubated for another hour at 37.degree. C. Finally,
100 .mu.l of ABTS substrate (2,2'-azinobis
[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was
applied and the plate was kept in the dark until the color
intensity of the positive controls was maximum and the negative
controls did not develop nonspecific reactions (6-10 min). The
ELISA plate was scanned in a Biotech ELISA reader at 450 nm.
[0267] For the second assay, the same method was applied, but
IGF-1R was immobilized in the wells and incubated with FAK-NT.
Primary antibody anti-FAK-4.47 (05-537, Upstate) was used to reveal
the binding reaction.
BIACORE Analysis
[0268] Biacore T100 technology was used in conjunction with ELISA
analysis to characterize the thermodynamic binding parameters of
small-molecule compounds targeting the interaction site of FAK and
IGF-1R.
[0269] All experiments were performed using a Biacore T100 optical
biosensor (http://www.biacore.com). Series S CM5 sensor chips,
N-hydroxysuccinimide (NHS), N-ethyl-N'-(3-dimethylaminopropyl)
carbodiimide (EDC), ethanolamine HCl, and instrument-specific
consumables and accessories were provided by ICBR at the University
of Florida.
FAK-NT Immobilization
[0270] In order to reuse the sensor chip for both FAK and IGF-1R,
anti-mouse secondary antibody was immobilized to the sensor chip
surface. This allowed the primary antibody to be used to immobilize
the ligand protein on the surface and also eliminated the
possibility of masking the interaction site of proteins during
immobilization of protein on the chip surface.
[0271] Immobilization procedures were performed using
Hepes-buffered saline (HBS: 10 mM Hepes and 150 mM NaCl, pH 7.4) as
the running buffer. Sensor chip surfaces were first preconditioned
with two 6-s pulses each of 100 mM HCl, 50 mM NaOH, and 0.1% sodium
dodecyl sulfate (SDS) at a flow rate of 100 .mu.l/min. Anti-mouse
antibody surfaces were prepared using amine-coupling chemistry at
30.degree. C. and at a flow rate of 10 .mu.l/min. NHS/EDC was
injected for 15 min to activate the surface, 100 .mu.g/ml antibody
(dissolved in 10 mM sodium acetate, pH 4.5) was injected for 10
min, and finally ethanolamine was injected for 7 min to block
residual activated groups. This immobilization procedure yielded
5000 to 7000 resonance units (RU) of immobilized antibody. After
immobilization, the instrument was primed extensively with the
analysis running buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2,
0.1% Tween 20, 0.1% Brij-35, and 5% dimethyl sulfoxide [DMSO], pH
8.0). After immobilization of anti-mouse antibody, 100 .mu.g/ml
mouse-anti-FAK 4.47 antibody (05-537, Upstate) (dissolved in 10 mM
sodium acetate, pH 4.5) was injected for 10 min, and sensor chip
surfaces were washed to remove unbound antibodies with three 5-s
pulses each of 100 mM HCl, 50 mM NaOH, and 0.1% sodium dodecyl
sulfate (SDS) at a flow rate of 100 .mu.l/min. This immobilization
procedure yielded 15000 to 20000 resonance units (RU) of
immobilized primary antibody. Finally, 200 .mu.g/ml FAK-NT
(dissolved in 10 mM sodium acetate, pH 4.5) was injected for 10 min
and sensor chip surfaces were washed with three 5-s pulses each of
100 mM HCl, 50 mM NaOH, and 0.1% sodium dodecyl sulfate (SDS) at a
flow rate of 100 .mu.l/min. This immobilization yielded 30000 to
40000 resonance units (RU) of immobilized FAK-NT.
Capture of IGF-1R
[0272] Aliquots of IGF-1R were kept frozen at -80.degree. C. until
use. A volume of freshly prepared, 200 .mu.g/ml IGF-1R (dissolved
in 10 mM sodium acetate, pH 4.5) was injected for 10 min and
unbound protein was removed by passing the solution over a fast
desalting column (equilibrated with 50 mM Tris-HCl, 150 mM NaCl,
and 10 mM MgCl.sub.2, pH 8.0) twice. The capture procedure yielded
typically to densities of 2000-4000 RU) onto a FAK-NT surface at
25.degree. C. A primary antibody bound surface served as the
reference.
Preparation of Analyte Solutions
[0273] For stock solutions, the compounds were dissolved in 100%
DMSO to a concentration of 10 mM; further dilutions of the compound
stocks into DMSO and/or running buffer were performed immediately
prior to analysis. To match precisely the DMSO content of the
analytes and running buffer, a secondary stock of lower
concentration was prepared by diluting the compound in DMSO to a
concentration such that the addition of 50 .mu.l of this secondary
stock to 1 ml of 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl.sub.2,
0.1% Tween 20, and 0.1% Brij-35 (pH 8.0) yielded a compound
concentration that was nine times greater than the high
concentration chosen for analysis. This starting concentration was
diluted ninefold in analysis running buffer to yield the high
concentration. An additional ninefold dilution of this sample
produced the low concentration. The propagated errors in the
concentrations of the high and low analyte concentrations were
calculated to be approximately 3.0%.
Analysis Parameters
[0274] At each temperature, five buffer blanks were first injected
to equilibrate the instrument fully. Using a flow rate of 50
.mu.l/min, compounds were injected for 30 to 60 s and dissociation
was monitored for 1 to 20 min. (The selected injection and
dissociation times were determined in preliminary binding tests.)
For the tightly bound complexes, a regeneration step was required.
At 4 to 11.degree. C., the surface was regenerated with 10 100-s
pulses of 60% ethylene glycol; at 16 to 18.degree. C., 40% ethylene
glycol; at 22 to 28.degree. C., 30% ethylene glycol; and at 32 to
39.degree. C., 50 mM Tris-HCl, 150 mM NaCl, 10% ethylene glycol, 15
mM ATP, 15 mM MgCl.sub.2, 5% DMSO, and 0.1% Tween 20 (pH 8.0). The
data collection rate was 10 Hz.
Data Analysis
[0275] Biosensor data, processed and analyzed using Scrubber 2
(BioLogic Software, Australia), were fit to either a simple 1:1
model (A+B=AB) or a 1:1 interaction model that included a mass
transport term (Ao=A, A+B=AB). Equilibrium dissociation constants
determined in Scrubber were fit to the van't Hoff equation
ln(KD)=.DELTA.H.degree./RT-.DELTA.S.degree./R. (Although the use of
integrated forms van't Hoff equation that includes a term for
.DELTA.Cp.degree. was considered, the lack of curvature in the
ln(KD) versus 1/T plots indicated that using this approach was
unnecessary.) Values for .DELTA.H.degree. and .DELTA.S.degree. were
obtained directly using the Solver macro in Microsoft Excel.
.DELTA.H.degree. and .DELTA.S.degree. values were also determined
indirectly via linear regression analysis of ln(KD) versus 1/T
plots using the Regression function in Excel, where the slope and
intercept corresponded to .DELTA.H.degree./R and
-.DELTA.S.degree./R, respectively. Fitting errors for
.DELTA.H.degree. and .DELTA.S.degree. from Solver were obtained
using a downloadable macro called SolverAid
(http://www.bowdoin.edu/.about.rdelevie/excellaneous). Errors for
the parameters .DELTA.H.degree. and .DELTA.S.degree. from the
Regression routine were obtained directly from a statistical
readout in Microsoft Excel. The values obtained from both methods
agreed well. Standard errors were propagated according to the
general formula
.DELTA.z2=(.differential.f/.differential.x)2.DELTA.x2+(.differential.f/.d-
ifferential.y)2.DELTA.y2+ . . . in Excel. Programmed formulas were
first checked using the downloadable macro Propagate (also
available at
http://www.bowdoin.edu/.about.rdelevie/excellaneous).
Example 1
Database of Small Molecules
[0276] The NCI/DTP maintains a repository of approximately 250,000
samples (i.e., the plated compound set) which are non-proprietary
and offered to the research community for discovery and development
of new agents for the treatment of cancer, AIDS, or opportunistic
infections afflicting subjects with cancer or AIDS. The
three-dimensional coordinates for the NCI/DTP plated compound set
is obtained in the MDL SD format
(http://www.chm.tu-dresden.de/edv/vamp65/REFERS/vr.sub.--03d.htm)
and converted to the mol2 format by the DOCK utility program
SDF2MOL2. Partial atomic charges, solvation energies and van der
Waals parameters for the ligands are calculated using SYBDB and
added to the plated compound set mol2 files.
Example 2
Database Screening to Identify Potential Small Molecule Inhibitors
of FAK/IGF-1R
[0277] In lieu of conducting high-throughput screening, a
structure-based approach combining molecular docking in silico with
functional testing is used. A large chemical library of compounds
with known three-dimensional structure is positioned in the
structural pocket selected by SPHGEN (UCSF) on the crystal
structure of human FAK (PDB code 1K05). 250,000 small molecule
compounds with drug-like characteristics (following the Lipinski
rules) were docked into the site of interaction between FAK and
IGF-1R in 100 different orientations using the DOCK5.1 computer
program (UCSF). The general features of DOCK include rigid
orienting of ligands to receptor spheres, AMBER energy scoring,
GB/SA solvation scoring, contact scoring, internal nonbonded energy
scoring, ligand flexibility, and both rigid and torsional simplex
minimization.
[0278] For the model of selecting compounds as FAK and/or IGF-1R
inhibitors, the following sequences were utilized.
TABLE-US-00001 FAK aa 126-243: ssvr ekyelahppe ewkyelriry
lpkgflnqft edkptlnffy qqvksdymle iadqvdqeia lklgcleirr sywemrgnal
ekksnyevle kdvglkrffp kslldsvkak tlr IGF-1R aa 959-1266:
hrkrnnsrlgng vlyasvnpey fsaadvyvpd ewevarekit msrelgqgsf gmvyegvakg
vvkdepetry aiktvneaas mrerieflne asvmkefnch hvvrllgvvs qgqptivime
lmtrgdlksy lrslrpemen npvlappsls kmiqmageia dgmaylnank fvhrdlaarn
cmvaedftvk igdfgmtrdi yetdyyrkgg kgllpvrwms peslkdgvft tysdvwsfgv
vlweiatlae qpyqglsneq vlrfvmeggl ldkpdncpdm lfelmrmcwq ynpkmrpsfl
eiissi
[0279] The predicted binding energies of interaction between each
compound and the interaction site are estimated, with the top
scoring compound given a DOCK score of -17.7 kcal per mol. The top
scoring compounds with the highest scores are requested for
functional testing from the NCI/DTP. Selected small molecules were
evaluated in cell-based proliferation and apoptosis assays in
esophageal (KYSE 140), melanoma (C8161, A375) and pancreatic
(Panc-1) cancer cells.
[0280] The three-dimensional coordinates for the NCI/DTP plated
compound set was obtained in the MDL SD format and converted to the
mol2 format by the DOCK utility program SDF2MOL2. Partial atomic
charges, solvation energies, and van der Waals parameters for the
ligands were calculated using SYBDB and added to the plated
compound set mol2 file.
Example 3
[0281] Cell proliferation assay: Cell proliferation assay (Promega)
using CellTiter 96 aqueous one solution was performed by adding a
small amount of the One Solution Reagent directly to culture wells,
incubating for 1-4 hours and then recording absorbance at 490 nm
with a spectrophotometric plate reader. The quantity of formazan
product as measured by the amount of 490 nm absorbance was directly
proportional to the number of living cells in culture.
Example 4
[0282] Adenoviral infections: Cells were plated at a density of
6.times.10.sup.3 or 2.times.10.sup.5 into culture plates and
allowed to attach for 24 h. The cells were then infected with
adenovirus at a viral concentration of 50-500 multiplicity of
infection or focus-forming units (FFU) per cell (See Golubovskaya,
V. et al., J. Biol. Chem., 277, 2002, 38978-38987). This optimal
viral titer was determined by infecting cells with various doses of
Ad-GFP and visualizing the percent infection by fluorescent
microscopy. Treatment with 100 FFU of Ad-GFP per cell resulted in
0.95% infection rate. Cells were used 48 or 72 h after infection
for further experiments.
Example 5
[0283] siRNA transfection assay: Cells were plated at a density of
6.times.10.sup.3 cells for 60 mm diameter or 2.times.10.sup.5 cells
for 100 mm diameter culture plates and allowed to attach for 24 h.
The cells were then transfected with 1-10 nM of FAK siRNA or
non-specific siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad,
Calif.) according to the manufacturer's protocol. Several FAK siRNA
sequences were utilized to screen for knock down of FAK. The
sequences of FAK siRNA utilized in cell lines were
5'-GAAGUUGGGUUGUCUAGAAUU-3' and 5'-GGUUCAAGCUGGAUUAUUUUU-3'. Cells
were then incubated 48-72 h after transfection and then used for
experiments. FAK inhibition by siRNA was verified with western
blotting. The experiments were done in triplicate.
Example 6
[0284] Immunoprecipitation and western blotting: Cells were washed
twice with ice cold 1.times. phosphate-buffered saline (PBS) and
lysed on ice for 30 min in buffer containing 20 mM Tris, pH 7.4,
150 mM NaCl, 1% NP-40, 5 mM ethylenediaminetetraacetic acid,
protease inhibitors (Complete.TM. Protease Inhibitor Cocktail from
Roche, Nutley, N.J.) and phosphatase inhibitors (Phosphatase
Inhibitor Cocktail Set I and Set II from Calbiochem). The lysates
were centrifuged at 10 000 r.p.m. for 30 min at 4.degree. C. and
the supernatants were analyzed. Protein concentration was
determined by using Bio-Rad Protein Assay.
[0285] Immunoprecipitation: 1 mg of total cell extract was used for
each sample. The extracts were incubated with 1 lg of the
appropriate antibody overnight at 4.degree. C. Twenty-five
microliters of protein A/G-agarose beads (Oncogene Research
Products, La Jolla, Calif.) were added and the samples were
incubated with rocking for an additional 2 h at 4.degree. C. The
precipitates were washed three times with lysis buffer, resuspended
in 40 .mu.l Laemmli buffer and 35 .mu.l was removed for western
blotting.
[0286] Western blotting: boiled samples containing 30 .mu.g of
protein were resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis followed by transferring to polyvinylidene
difluoride membrane (Bio-Rad, Hercules, Calif.). Western blotting
was carried out according to the protocol supplied with each
antibody. The immunoblots were developed with the Western
Lightning.TM. Chemiluminescence Reagent Plus (PerkinElmer Life
Sciences, Waltham, Mass.). The intensity of the bands in the
western blots was measured with an image analysis software program
(image J).
Results: Immunoprecipitation and western blotting analysis on cell
lystate treated with NSC 344553 are depicted in FIGS. 2 and 3. FIG.
2 demonstrates the effects of NSC 344553 on FAK-IGF-1R interaction,
illustrated by immunoprecipitation and western blotting analysis
for cell lysates from C8161 melanoma cancer cells tested with
various doses of NSC 344553. FIG. 3 shows the effects on C8161
melanoma cancer cells treated with 75 .mu.M of NSC 344553.
Immunoprecipitation and western blotting analysis is depicted in
FIG. 12 for MiaPaCa-2 pancreatic cancer cells treated with NSC
128687.
[0287] Western blot analysis were performed on melanoma (C8161 and
A375) and pancreatic (Panc-1 and MiaPaca-2) cancer cells treated
with IGF-1, NSC 344553, PI3 kinase inhibitors, or NVP-AEW541
("NVP") (see FIGS. 4, 5, 7, 8, 9, and 14).
[0288] FIG. 5 depicts the effects on C8161 melanoma cancer cells
treated with 5 .mu.M of NSC 344553 or PI3 Kinase inhibitors. FIG. 7
shows that the most significant effects were observed in the
decrease of p-AKT in 24-h treated and 30-min IGF-1 stimulated group
in mouse embryo fibroblasts that were wildtype and null for IGF-1R.
FIG. 8 demonstrates the western blot analysis for FAK wildtype and
null fibroblasts. The most significant effect is seen in the
decrease of p-AKT in 24-h treated and 30-min IGF-1 stimulated
group. FIG. 9 shows the effects on Panc-1 cancer cells when treated
with NSC 344553.
Example 7
[0289] Cell viability and detachment assays: After cells were
treated with siRNA transfection, cell viability was measured by
3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay (CellTiter 96.RTM. AQueous, Promega, Madison, Wis.). Briefly,
20 .mu.l of the tetrazolium compound was added to each well. The
cells were then incubated at 37.degree. C. for 1 h. The plate was
read at 490 nm with a plate reader to determine the viability. In
detachment assays, detached and attached cells were harvested
separately and counted in a hemocytometer. The percentage of
detachment was calculated by dividing the number of detached cells
by the total number of cells. Apoptosis assays: After treatment,
attached and detached cells were collected, counted and prepared
for terminal uridine deoxynucleotidyl transferase (TUNEL) assay by
utilizing an APO-BRDU kit (BD Pharmingen, San Diego, Calif.)
according to the manufacturer's instructions. Stained cells were
analyzed with a fluorescence-activated cell sorting-Calibur flow
cytometer (BD Biosciences). Calculation of the percentage of
apoptotic cells in the sample was completed with CellQuest software
(BD Biosciences). Apoptotic cells were also analyzed by Hoechst
staining. Hoechst 33342 (1 lg/rill) was added to the fixed cells
and the specimens were mounted on glass coverslips. The slides were
viewed under the Zeiss microscope for apoptotic nuclei. The percent
of apoptotic cells was calculated as the ratio of apoptotic cells
to total number of cells. Results: MTT assay (Cell titer 96) assay
results on NSC 344553 were depicted in FIG. 11, which demonstrates
that treatments on A375 and C8161 melanoma cancer cells with NSC
344553 led inhibited cell viability in a dose-dependent manner
(range 0.05-25 .mu.M). FIG. 6 shows that NSC 344553 inhibits the
cell viability of pancreatic (Panc-1 and MiaPaCa-2) and melanoma
(C8161 and A375) cancer cells.
[0290] FIG. 10 demonstrates that a 72-hour treatment of 0.05 .mu.M
of NSC 344553 on FAK wild type and null cells and IGF-1R wildtype
and null cells. The results show that NSC 344553 treatment reduced
the proliferation of FAK+/+ and IGF-1R+/+ fibroblast cells, but had
no effect on FAK-/- and IGF-1R-/- cells.
[0291] MTT assay results demonstrate that treatment with NSC 344553
led to decrease of phosphorylation of AKT and inhibited cell
viability in a dose-dependent manner (range 0.05-100 .mu.M) with
associated PARP cleavage. It was also observed that 0.05 .mu.M of
NSC 344553 reduced the proliferation of FAK+/+ and IGF-1R+/+
fibroblast cells, but had no effect on FAK-/- and IGF-1R-/- cells.
More importantly, intraperitoneal injection of 15 mg/kg of NSC
344553 for 5 days effectively (p<0.05) caused melanoma tumor
regression in nude mice.
[0292] MTT assay (Cell titer 96) assay results on NSC 250435 were
depicted in FIG. 13, which demonstrates that NSC 250435 inhibited
cell viability of A375 and C8161 melanoma cancer cells.
Example 8
[0293] Clonogenic assay: Pancreatic cancer cell lines Panc-1,
MiaPaca-2, Panc 2.03 and Panc 3.27 are used in the experiments. To
define cell survival, the clonogenic assay is performed to evaluate
for cellular reproductive integrity (See Chinnaiyan P. et al.,
Clin. Cancer Res. 2008; 14(17): 5410-5). The clonogenic assay
detects all forms of radiation-induced cell death and is thus
considered the "gold standard" for radiosensitivity analysis. Two
drug concentrations are used, including 1) the minimum dose
required to abrogate the FAK/IGF-1R pathway (based on western blot)
and 2) the concentration which demonstrates sub-maximal activity
(20%-50% decrease in cell survival). Cells are exposed to the
FAK/IGF-1R inhibitor 24 hours prior to radiation (based upon the
time required for inhibition of FAK/IGF-1R activation as determined
by western blot). Drug containing media is replaced with fresh
media (without drug) 24 hours post-radiation. Colonies (as defined
by .gtoreq.50 cells) are then stained and counted 10-14 days
following irradiation.
Example 9
The Effects of Inhibiting the FAK/IGF-1R Pathway on
Radiation-Induced Cell Migration
[0294] To measure cell movement, the transwell migration assay is
performed in two-well Boyden-type chambers. 1.times.10.sup.5
cells/well are plated in their respective media in the upper
chamber of 5-uM pore (24-well) transwells and allowed to adhere for
30 min. The cells include control cells and cells pre-exposed to
the FAK/IGF-1R inhibitor for 24 hrs. Treatment conditions include
untreated, RT alone, FAK/IGF-1R inhibited alone, and the RT and
FAK/IGF-1R inhibitor combination. The cells are exposed to graded
doses of radiation (2 Gy, 4 Gy, 6 Gy, or 8 Gy) and returned to the
incubator for 24 hr, rinsed, fixed. Cells remaining on the top of
the polycarbonate membrane are removed with cotton swabs. The cells
that have migrated through pores to the lower surface are stained
with ethanol-based crystal violet. The membranes are then mounted
on microslides and counted.
Example 10
Immunofluorescent Staining and Confocal Microscopy
[0295] Cells were fixed in 3.7% paraformaldehyde in 1.times.PBS for
10 min and permeabilized with 0.5% Triton X-100 for 5 minutes.
Cells were then washed with 1.times.PBS, blocked with 25% normal
goat serum in 1.times.PBS for 20 min and incubated with primary
antibody (1:200 dilution in 25% goat serum) for 30 min at room
temperature. After washing three times with 1.times.PBS, cells were
incubated with a Texas Red-conjugated secondary antibody (1:400
dilution in 25% goat serum) for 30 min at room temperature and
washed another three times with 1.times.PBS before observed under
the microscope. For coimmunostaining experiments, cells were
incubated with another primary antibody diluted 1:100 in 25% goat
serum for 1 h. After washing three times with 1.times.PBS, a
fluorescein isothiocyanate-conjugated secondary antibody (1:100
dilution) was applied to the coverslip. Cells immunostained with
FAK and IGF-IR antibodies were evaluated for colocalization with a
Leica confocal microscope and the MRC-1024 confocal laser scanning
system. Cells treated with FAK-CD or FAK siRNA with or without test
compound were stained with FAK antibody and evaluated for
displacement of FAK from the focal adhesions with a Zeiss
microscope. Results: Confocal microscopy assay demonstrates that
there is colocalization of the FAK-NT and FAK-NT2 constructs with
IGF-1R. The percentage of overlapping is high for the FAK-NT and
FAK-NT2 transfection, which shows that it is FAK-NT, more
specifically FAK-NT2, that colocalizes with IGF-1R. Colocalization
is low for FAK-NT1, FAK-NT3 and FAK-CD. When cells are transfected
in the presence of NSC 250435, the percentage of interaction
between FAK-NT, more specifically FAK-NT2, and IGF-1R is
significantly decreased to around 30%. The results demonstrate that
NSC 250435 disrupts the interaction between FAK-NT2 and IGF-1R.
Example 11
Radiation-Induced Activation of the FAK/IGF-1R Pathway
[0296] Cells are collected in a time-course manner following
exposure to 2 Gy and 10 Gy of radiation. Lysates are collected,
immunoprecipated for both FAK and IGF-1R. Western blot is performed
on the cells for their respective phosphorylated forms (See Liu W.
et al., Carcinogenesis, 2008; 29(6): 1096-1107). Similar studies
are performed after pre-treating the cells with the FAK/IGF-1R
kinase and small molecule inhibitors. Mass spectroscopy is
performed to define proteins associated with FAK/IGF-1R following
RT. Identified proteins which are bound to FAK-IGF-1R complexes in
the presence of radiation treatment are then targeted using siRNA
to determine its relative role on FAK/IGF-1R activation and
downstream signaling.
Mitotic Catastrophe
[0297] Mitotic catastrophe is then evaluated by both
cellular/nuclear morphology and abrogated G2/M checkpoint
activation (Xu B. et al., Molecular and Cell Biology, 2002; 22(4):
1049-59). Microscopic determination of mitotic catastrophe is
performed using Hoechst staining and quantified by the percentage
of multi-nucleated cells (Castedo M., et al., Oncogene, 2004;
23(16): 2825-37).
Cell Cycle Checkpoint Activation
[0298] Abrogated G2/M phase arrest in cells exposed to FAK/IGF-1R
inhibitors following radiation is determined using flow cytometry.
To separate cells in G2/M phase (4n) into the individual M- and
G2-phase components, dual labeling is performed with propidium
iodide and phosphorylated histone H3, which is specifically
expressed during the mitotic phase. This analysis provides a
measure of the progression of G2 cells into M phase and of the
influence of the FAK/IGF-1R pathway on the activation of the G2
checkpoint.
DNA Damage/Repair
[0299] In the study of DNA DSB and repair, the phosphorylated form
of the histone variant H2AX (termed .gamma.H2AX) has been adopted
for its relationship with DNA double strand breaks. Specifically,
.gamma.H2AX foci can be detected within minutes of radiation by
immunofluorescence, and this has been directly related to double
strand breaks. It has been shown previously that the residual level
of .gamma.H2AX (or conversely, foci dispersion) measured 24 hours
after irradiation correlates to radiation sensitivity (Banath J P,
et al., Cancer Res., 2004; 64(19): 7144-9). The influence of
FAK/IGF-1R pathway inhibition on DNA DSB repair is determined by
defining .gamma.H2AX foci kinetics (See Chinnaiyan P. et al., Clin.
Cancer Res. 2008; 14(17): 5410-5).
Example 12
Structure-Based in Silico Molecular Modeling and Computational
Docking
[0300] Previous studies have demonstrated that the amino terminus
of FAK (aa 127-243, FAK-NT2) directly binds with a portion of the
intracytoplasmic portion of IGF-1R (aa 959-1266) (21). Compounds
from the database of the NCI Developmental Therapeutics Program
were analyzed using the DOCK 5.1 program, to identify those that
putatively bind to FAK-FERM on the predicted FAK-NT2/IGF-1R
interface (FIG. 15A). Compounds with high probability of binding to
the interface were screened for their ability to inhibit the
interaction of FAK and IGF-1R. Subsequently, INT2-31 (NSC 344553)
was identified as the most potent FAK/IGF-1R binding inhibitor.
This compound has a molecular weight of 377.31 g/mol and a
molecular formula of C.sub.12H.sub.16N.sub.3O.sub.7PS. The
structure is demonstrated in FIG. 15A. The intermolecular energies
for all configurations of INT2-31 in binding to FAK-NT2 were
calculated as the sum of electrostatic and van der Waals energies
and the predicted lowest energies of interaction with FAK-NT2
include a predicted score of -50.12 with a van der Waals charge of
-16.28 and an electrostatic charge of -33.84.
Example 13
INT2-31 Disrupts the Interaction of FAK and IGF-1R
[0301] The potency of INT2-31 to disrupt the protein-protein
interactions of FAK and IGF-1R was evaluated in pulldown assays
using tagged purified protein constructs. INT2-31 caused a dose
dependent decrease in binding between purified GST-FAK-NT2 and
IGF-1R.beta. with an average IC.sub.50 of 3.96 .mu.M (FIG. 15B). To
characterize the effects of the drug in vitro two melanoma cell
lines were evaluated. INT2-31 disrupted binding in C8161 and A375
melanoma cancer cells at low micromolar concentrations (average
IC.sub.50 of 2.72 and 3.17 .mu.M, respectively) as demonstrated by
immunoprecipitation using an antibody against FAK (FIGS. 15C and
D).
[0302] In addition, the effect of INT2-31 on cell viability of
esophageal, pancreatic and breast cancer cell lines was analyzed
and the IC.sub.50 value for each cell line was determined, as shown
in Table 1. To get the average IC.sub.50 value, each cell line was
treated with increasing concentrations of the compound for 72 hours
in triplicate and the average of IC.sub.50 values from three
separate experiments was calculated. Similar to melanoma results,
INT2-31 inhibits viability more in cancer cells compared to normal
cells. Sensitivity of the cells to INT2-31 varied and directly
correlated to the FAK and IGF-1R expression level of the cells.
TABLE-US-00002 TABLE 1 IC.sub.50 of INT2-31 for cancer cell lines
[INT2-31] Cell Lines .mu.M Melanoma Melonocyte 97.3 A375 2.7 C8161
0.5 SK-MEL-28 22.1 Esophageal Cancer TE3 5.6 TE7 3.2 TE9 3.6 KYSE70
4.6 KYSE140 2.5 KYSE180 19.8 Pancreatic Cancer HPDE Panc-1 6.7
Miapaca-2 4.73 AsPC1 16.9 BxPC3 45.6 Breast Cancer MCF10A 100 MCF7
0.03 BT474 2.39
Example 14
INT2-31 Reduces the Viability of Melanoma Cells
[0303] To determine the effect on melanoma cell viability, three
human melanoma cell lines were exposed to increasing doses of
INT2-31 for 72 h and the results compared to human melanocytes. As
shown in FIG. 16A, INT2-31 inhibits viability in cancer cells more
than normal cells. Each cell line was treated with increasing
concentrations of the compound for 72 h in triplicate and the
average IC.sub.50 value calculated from three separate experiments.
All three melanoma cell lines had upregulated FAK and IGF-1R
expression and increased sensitivity to INT2-31 compared to normal
human melanocytes (FIG. 16B). The effects of INT2-31 varied in the
three cell lines and was possibly related to constitutive FAK and
IGF-1R activation with the least sensitive cell line (SK-MEL-28)
having the greatest expression of FAK and IGF-1R.
Example 15
INT2-31 Inhibits Melanoma Cell Proliferation and has Effects
Dependent on the Presence of FAK and IGF-1R
[0304] To assess the effects of INT2-31 on cell proliferation, a
CSFE cell distribution assay was performed. As shown in FIG. 16C,
INT2-31 inhibited cell proliferation in both C8161 and A375 cells,
but the effect was greater in C8161 cells. Evaluation of cell
numbers with INT2-31 treatment demonstrated a potent time and dose
dependent inhibition of the growth of C8161 melanoma cells (FIG.
16D). These results were similar to the findings seen by MTT assay
(FIG. 16A).
[0305] To show that the effect of INT2-31 was specific for cells
expressing FAK, C8161 cells were transfected with FAK shRNA
constructs resulting in transient knockdown of FAK (FIG. 17A). FAK
shRNA1 was utilized for MTT assay due to greater efficiency of FAK
knockdown compared to FAK shRNA2. C8161 cells expressing FAK shRNA
were significantly less sensitive to the effects of INT2-31 than
parental and mock transfected cells (FIG. 17B). These findings were
confirmed with the use of FAK wildtype and null fibroblasts. FAK
wildtype fibroblasts were significantly more sensitive to the
effects of INT2-31 than FAK null fibroblasts (FIG. 17C).
Specificity for IGF-1R was also shown during the treatment of
IGF-1R proficient and deficient fibroblasts. IGF-1R -/- fibroblasts
were significantly less sensitive to the effects of INT2-31 than
IGF-1R+/+ cells (p<0.05, FIG. 17D)
Example 16
INT2-31 Induces Apoptosis
[0306] The effect of INT2-31 on detachment of treated cells was
determined. Detachment of C8161 melanoma cells was determined in
the presence of increasing concentrations of INT2-31. As shown in
FIG. 18A, only 7% of C8161 cells detached from the plate after 72 h
of treatment with 5 .mu.M of INT2-31. The effect of INT2-31 was
significantly less than the dual FAK and IGF-1R kinase inhibitor,
TAE 226 (Novartis, Basel). The effect of INT2-31 on apoptosis was
marked with a greater than 50% induction of apoptosis as indicated
by the detection of Hoescht positive cells after 72 h of treatment
with a 5 .mu.M dose (FIG. 18B). This was confirmed by analysis of
caspase 3/7 activation following treatment for 72 h with 1 .mu.M
and 5 .mu.M of INT2-31 detected by confocal microscopy (FIG. 18C).
Finally, the effect of INT2-31 was evaluated by Western blot. FIG.
18D depicts PARP and caspase-9 cleavage after 48 hours of treatment
with INT2-31. There was no significant effect of INT2-31 on caspase
8 levels.
Example 17
[0307] INT2-31 Decreases Activation of Akt without Inhibiting
Kinase Activity
[0308] The effect of INT2-31 on FAK and IGF-1R pathway effectors
was analyzed in three melanoma cell lines at different
concentration and treatment times (FIG. 19). INT2-31 treatment
resulted in a consistent inhibition of constitutive and IGF-1
induced signaling to AKT. Of note, there was no significant effect
of INT2-31 on the constitutive phosphorylation of FAK or the
constitutive or IGF-1 induced phosphorylation of IGF-1R. In
addition, while there was a pronounced effect on Akt, the effects
on signaling to ERK were less with a slight decrease in p-ERK in
all cell lines with higher doses. The effects of INT2-31 on p-Akt
correlated with the effects on cell growth, viability and apoptosis
with C8161 cells having significant inhibition of p-Akt with of
treatment, while higher doses of INT2-31 were necessary to
significantly decrease p-Akt in A375 and SK-MEL-28 cells (1-5 .mu.M
and 5-10 .mu.M, respectively).
The analysis of the time course of the INT2-31 treatment on Akt
phosphorylation revealed some dephosphorylation after 24 hours of
treatment with a sustained effect at 72 hours (FIG. 19E).
[0309] Subsequently, the effect of this compound on the kinase
activity of FAK, IGF-1R, insulin receptor, VEGFR-1, AKT-1, EGFR,
VEGFR-2, c-MET, PDGFRa, p70S6K, Src and PI3Kinase was determined
(FIG. 19D). Ata dose of 1 .mu.M, this compound did not inhibit the
kinase activity of FAK or IGF-1R and did not inhibit any of the
other protein kinases by more than 22%. Therefore, INT2-31
disrupted binding of FAK and IGF-1R without inhibiting their kinase
activity and inhibited melanoma cell viability in a dose and time
dependent fashion.
[0310] Furthermore, to confirm that INT2-31 specifically binds to
the NT2 (aa 127-243) region of FAK to disrupt interaction with
IGF-1R and decreases phosphorylation of Akt, C8161 cells were
transfected with 3 GFP fragments of the FAK N-terminus (FAK-NT1,
FAK-NT2 and FAK-NT3). As shown in FIGS. 19F and 19G, overexpression
of FAK-NT2 fragment reduced the IGF-1 induced phosphorylation of
AKT compared to FAK-NT1 and NT3 overexpressed cells.
Example 18
[0311] INT2-31 Decreases Tumor p-Akt and Growth in Melanoma
Xenografts
[0312] As demonstrated in FIGS. 20A and 20B, daily intraperitoneal
injection of 15 mg/kg of INT2-31 for 21 days resulted in a
significant decrease in C8161 and A375 subcutaneous tumor growth
compared to mice receiving PBS control injections
(p<0.05). At this concentration the drug did not have serious
toxic effects as there was no significant difference in body
weights between animals in each group. To assess the in vivo
effects of INT2-31 on cell proliferation, C8161 xenografts were
stained with Ki67 antibody (FIG. 20C). The percent of cells
reactive to Ki67 and the intensity of Ki67 staining were
significantly decreased in the tumors from mice treated with
INT2-31 vs those treated with PBS (control). In addition, the
percent of cells undergoing apoptosis was significantly increased
in the tumors treated with INT2-31 compared to control (FIG. 20C,
p<0.05). This confirmed in vitro data demonstrating that INT2-31
decreases proliferation and increases apoptosis of cancer cells.
The effect of INT2-31 on the in vivo interaction of FAK and IGF-1R
in C8161 tumors was analyzed by immunoprecipitation of FAK from
treated and untreated tumor. Western blot for IGF-1R demonstrates a
decrease in the co-immunoprecipitation of FAK and IGF-1R.
Densitometry of the ratio of IGF-1R to FAK in each tumor showed a
decreased mean ratio in INT2-31 treated (0.78+/-0.16) compared to
PBS treated (0.98+/-0.11, p=0.09) tumor samples. Finally, tumor
analysis for AKT activation was performed and the level of p-AKT
was detected by Western blot. Analysis demonstrated a decrease in
phosphorylation of AKT in animals treated with INT2-31 vs PBS
control (FIG. 20D). Therefore, our lead compound, INT2-31,
decreases in vivo tumor growth, disrupts the in vivo interaction of
FAK and IGF-1R and results in a decrease in phosphorylation of
AKT.
Example 19
INT2-31 Sensitized Cancer Cells to Chemotherapy
[0313] To evaluate and correlate the effect of INT2-31 on Akt
de-phosphorylation with the sensitivity of cells to conventional
chemotherapy, esophageal and pancreatic cancer cell lines were
analyzed for the effects of combination therapies on cell viability
and apoptosis. Both KYSE 70 and 140 esophageal cancer cells were
sensitive to INT2-31 and 5-FU treatment and 0.5 and 1 .mu.M INT2-31
had synergistic effects with 5-FU (FIG. 21). In our pancreatic
cancer cells, while the effect on cell viability of INT2-31 was
only additive when combined with gemcitabine (data not shown),
INT2-31 had synergistic effects with 5-FU chemotherapy at 1 .mu.M
concentrations (FIGS. 21 and 22).
Example 20
[0314] In Vitro and In Vivo Inhibition of Esophageal Cancer
Viability and Proliferation with INT2-31 Treatment
[0315] Esophageal cancer has been shown to overexpress FAK and
IGF-1R. To assimilate the effects of targeting the interaction of
these proteins in direct patient specimens, a system was developed
in which direct esophageal cancer specimens were grown in mice and
tissue culture plates to allow fresh human tissue for
experimentation. More than 20 tumors and corresponding normal
tissue specimens have been obtained from cancer patients.
Immunohistochemical and western blot analysis of the samples also
demonstrated increased level of FAK and IGF-1R in tumor samples
compared to the normal tissue. To evaluate the in vitro effects of
INT2-31 on patient specimens, we utilized MTT assay of cells grown
in a tissue culture plate maximum up to eight passages were
utilized. A representative result of MTT assay of esophageal
patient #5 shown in FIG. 23A. Increasing concentrations of INT1-31
effectively decreased the viability of cells with an average
IC.sub.50 value of 2.18 .mu.M.
[0316] Subsequently, we evaluated the inhibition of in vivo tumor
growth of esophageal patient #5 specimen was evaluated. As
described in the methods section, small pieces
(0.3.times.0.3.times.0.3 cm) from a fresh esophageal human
adenocarcinoma tumor sample were implanted subcutaneously into 2
mice. When one of the tumors reached 1.5 cc.sup.3, it was excised
and cut into small pieces of (0.3.times.0.3.times.0.3 cm), and
transplanted subcutaneously into another 10 mice. When tumors
reached .about.100 mm.sup.3, mice were randomized in the 2 groups,
with 5 mice in each group. As demonstrated in FIG. 23B, daily
intraperitoneal injection of 50 mg/kg of INT2-31 for 21 days
resulted in a significant decrease in fresh esophageal
adenocarcinoma tumor growth compared to mice receiving PBS control
injections (p<0.05). At this concentration, the drug did not
have serious toxic effects, as there was no significant difference
in body weights between animals in each group. To assess the in
vivo effects of INT2-31 on cell proliferation, we stained
esophageal patient #5 tumor specimen xenografts were stained with
Ki67 antibody. As shown in FIG. 23C, immunohistochemical staining
of tumors demonstrated that the percent of cells reactive to Ki67
were significantly decreased in the tumors from mice treated with
INT2-31 compared to PBS group. This confirmed our in vitro data
that the drug decreases proliferation of cancer cells and in vivo
data for the melanoma model.
Example 21
[0317] Inhibition of Orthotopic Pancreatic Xenografts with INT2-31
Treatment To further validate the activity and specificity of
INT2-31, orthotopic mouse models were employed. The pancreatic
cancer cell lines, Mia paca-2 and Panc-1 cells were stably
transfected using luciferase-RFP (red fluorescent protein) reporter
gene for in vivo imaging of the xenografts. Following expansion and
sorting of RFP positive cells, cells were expanded in culture and
5.times.10.sup.6 tumor cells were implanted into the pancreas of 14
mice. As described in the materials and methods section, mice were
imaged weekly with the IVIS lumina imager and tumor size was
estimated by the bioluminescent signal. When tumors reached
.about.100 mm.sup.3, mice were randomized in the following 2
groups, with 7 mice in each group: Control and 15 mg/kg INT2-31. As
shown in FIG. 24, daily intraperitoneal 50 mg/kg treatment of
Miapaca2 and subcutaneous 15 mg/kg injection of INT2-31 for 21 days
sufficiently reduced the growth of the orthotopic pancreatic
xenografts without any significant side effects as measured by body
weights and the appearances of the animals.
INCORPORATION BY REFERENCE
[0318] The contents of all references (including literature
references, issued patents, published patent application, and
co-pending patent applications) cited throughout this application
are hereby expressly incorporated in their entireties by
reference.
EMBODIMENTS AND EQUIVALENTS
[0319] The recitation of a listing of chemical groups herein
includes definitions of any single group or combination of listed
groups. The recitation of an embodiment herein includes that
embodiment as any single embodiment or in combination with any
other embodiments or portions thereof.
[0320] Although the invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of the invention may be devised by others skilled in the
art without departing from the true spirit and scope of the
invention. The claims are intended to be construed to include such
embodiments and equivalent variations.
[0321] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
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Sequence CWU 1
1
8156DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1ccggccgatt ggaaaccaac atatactcga
gtatatgttg gtttccaatc gttttg 56257DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 2ccgggcccag
aagaaggaat cagttctcga gaactgattc ttcttctggg ctttttg
57343DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 3tccgaacgtg tcacgttctc ttgaaacgtg
acacgttcgg aga 4344PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 4Asp Glu Val Asp15117PRTHomo sapiens
5Ser Ser Val Arg Glu Lys Tyr Glu Leu Ala His Pro Pro Glu Glu Trp1 5
10 15Lys Tyr Glu Leu Arg Ile Arg Tyr Leu Pro Lys Gly Phe Leu Asn
Gln 20 25 30Phe Thr Glu Asp Lys Pro Thr Leu Asn Phe Phe Tyr Gln Gln
Val Lys 35 40 45Ser Asp Tyr Met Leu Glu Ile Ala Asp Gln Val Asp Gln
Glu Ile Ala 50 55 60Leu Lys Leu Gly Cys Leu Glu Ile Arg Arg Ser Tyr
Trp Glu Met Arg65 70 75 80Gly Asn Ala Leu Glu Lys Lys Ser Asn Tyr
Glu Val Leu Glu Lys Asp 85 90 95Val Gly Leu Lys Arg Phe Phe Pro Lys
Ser Leu Leu Asp Ser Val Lys 100 105 110Ala Lys Thr Leu Arg
1156308PRTHomo sapiens 6His Arg Lys Arg Asn Asn Ser Arg Leu Gly Asn
Gly Val Leu Tyr Ala1 5 10 15Ser Val Asn Pro Glu Tyr Phe Ser Ala Ala
Asp Val Tyr Val Pro Asp 20 25 30Glu Trp Glu Val Ala Arg Glu Lys Ile
Thr Met Ser Arg Glu Leu Gly 35 40 45Gln Gly Ser Phe Gly Met Val Tyr
Glu Gly Val Ala Lys Gly Val Val 50 55 60Lys Asp Glu Pro Glu Thr Arg
Val Ala Ile Lys Thr Val Asn Glu Ala65 70 75 80Ala Ser Met Arg Glu
Arg Ile Glu Phe Leu Asn Glu Ala Ser Val Met 85 90 95Lys Glu Phe Asn
Cys His His Val Val Arg Leu Leu Gly Val Val Ser 100 105 110Gln Gly
Gln Pro Thr Leu Val Ile Met Glu Leu Met Thr Arg Gly Asp 115 120
125Leu Lys Ser Tyr Leu Arg Ser Leu Arg Pro Glu Met Glu Asn Asn Pro
130 135 140Val Leu Ala Pro Pro Ser Leu Ser Lys Met Ile Gln Met Ala
Gly Glu145 150 155 160Ile Ala Asp Gly Met Ala Tyr Leu Asn Ala Asn
Lys Phe Val His Arg 165 170 175Asp Leu Ala Ala Arg Asn Cys Met Val
Ala Glu Asp Phe Thr Val Lys 180 185 190Ile Gly Asp Phe Gly Met Thr
Arg Asp Ile Tyr Glu Thr Asp Tyr Tyr 195 200 205Arg Lys Gly Gly Lys
Gly Leu Leu Pro Val Arg Trp Met Ser Pro Glu 210 215 220Ser Leu Lys
Asp Gly Val Phe Thr Thr Tyr Ser Asp Val Trp Ser Phe225 230 235
240Gly Val Val Leu Trp Glu Ile Ala Thr Leu Ala Glu Gln Pro Tyr Gln
245 250 255Gly Leu Ser Asn Glu Gln Val Leu Arg Phe Val Met Glu Gly
Gly Leu 260 265 270Leu Asp Lys Pro Asp Asn Cys Pro Asp Met Leu Phe
Glu Leu Met Arg 275 280 285Met Cys Trp Gln Tyr Asn Pro Lys Met Arg
Pro Ser Phe Leu Glu Ile 290 295 300Ile Ser Ser
Ile305721RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 7gaaguugggu ugucuagaau u
21821RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 8gguucaagcu ggauuauuuu u 21
* * * * *
References