US20120107323A1

US20120107323A1 - Kinase protein binding inhibitors

Info

Publication number: US20120107323A1
Application number: US13/256,112
Authority: US
Inventors: Steven N. Hochwald; David A. Ostrov; William G. Cance; Elena Kurenova
Original assignee: University of Florida
Current assignee: University of Florida
Priority date: 2009-03-12
Filing date: 2010-03-12
Publication date: 2012-05-03
Also published as: BRPI1013623A2; WO2010104598A3; EP2413938A2; CA2755191A1; KR20110132446A; AU2010223058A1; EA201190210A1; WO2010104598A2; CN102427814A; US8051947B2; US20100230203A1; EP2413938A4; JP2012520299A

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.

Description

CROSS-REFERENCE TO RELATED APPLICATION

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.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

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.

BACKGROUND OF THE INVENTION

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.
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).
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.
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.
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.
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

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.
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.
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]oxazolo[3,2-a]pyrimidin-3-yl dihydrogen phosphate; b) 4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimidine}; 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]pyrimidine}, or a pharmaceutically acceptable salt, ester or prodrug thereof.
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]oxazolo[3,2-a]pyrimidin-3-yl dihydrogen phosphate; b) 4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimidine}; 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]pyrimidine}, or a pharmaceutically acceptable salt, ester or prodrug thereof.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
In certain embodiments, the kit further includes an additional therapeutic agent. Certain embodiments present that the additional therapeutic agent is an agent delineated supra.
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.
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

FIG. 1 shows the results of protein pull down assay between FAK and IGF-1R protein constructs;

FIG. 2 depicts immunoprecipitation and western blot results on cell lystate (from C8161 melanoma cancer cells) treated with NSC 344553 for 24 hours;

FIG. 3 depicts immunoprecipitation and western blot results of 75 μM of NSC 344553 tested on C8161 melanoma cancer cells;

FIG. 4 depicts western blot results for C8161 melanoma cancer cells treated with 1 μM of NSC 344553;

FIG. 5 depicts western blot results for C8161 melanoma cancer cells treated with 5 μM of NSC 344553 and PI3 Kinase inhibitors for 24 hours;

FIG. 6 depicts cell viability assay results for pancreatic and melanoma cells treated with NSC 344553 for 72 hours;

FIG. 7 depicts the effects of 2 μM NSC 344553 treatment on IGF-1R +//− cells;

FIG. 8 depicts the effects of NSC 344553 treatment on FAK +//− cells;

FIG. 9 depicts western blot analysis for Panc-1 cancer cells treated with NSC 344553.

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;

FIG. 11 depicts cell viability results for A375 and C8161 melanoma cancer cells treated with NSC 344553 using MTT assay;

FIG. 12 depicts immunoprecipitation and western blot results for pancreatic (MiaPaCa-2) cells treated with NSC 128687 for 24 hours;

FIG. 13 shows dose response curve of MiaPaCa-2 cancer cells to a 72-hour NSC 250435 treatment;

FIG. 14 shows western blot results for melanoma (A375 and C8161) and pancreatic (Panc-1 and MiaPaCa-2) cancer cells treated with NSC 344553.

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-1Rβ 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.

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.

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.

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 μM 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.

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 μM 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).

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.

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.

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.

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.

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

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.
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.
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).
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.
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]pyrimidine}, inhibits tumor cell viability, alters FAK and IGF-1R signaling, and inhibits tumor growth in vivo.
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.
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

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.
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.
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.
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.
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.
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.
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.
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.
“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.
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.
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.
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.
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.
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.
The term “diastereomers” refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.
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.
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.
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.
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.”
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.
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).
The term “homeostasis” is art-recognized to mean maintenance of static, or constant, conditions in an internal environment.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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

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.
In one embodiment, compounds of the invention include compounds specifically delineated herein:

1). 2-(Hydroxymethyl)-6-imino-2,3,3a,9a-tetrahydro-6H-furo[2,3:4,5][1,3]oxazolo[3,2-a]pyrimidin-3-yl dihydrogen phosphate (also as “NSC 128687”):

2). 4-(Methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimidine} (also as “NSC 344553”):

3). 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 (also as “NSC 250435”):

4). 3-Methyl-2,4-disulfopentanedioic acid (also as “NSC 243620”):

5). 1-Aminopropane-1,3-diyldiphosphonic acid (also as “NSC 133881”):

The chemical name and structure of each of the afore-mentioned compounds expressly include all diastereomers of the compound.
The invention also provides the pharmaceutically acceptable salts, esters, hydrates, solvates, clathrates, polymorphs, and prodrugs of a compound of the invention.
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.
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.
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^rdEd., 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.
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.
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.
The invention also provides a compound that is capable of decreasing IGF-1R and AKT phosphorylation, and inducing apoptosis of cancer cells.
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.
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.
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

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.
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.
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.
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.
In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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).
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.
The methods of the invention may further include using a dominant-negative construct (FAK-CD) or small interfering RNA.
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.
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.
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.
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.
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.
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

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.
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.
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.
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.
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
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

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.
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.
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.
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.
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; (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; (1₃) 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.
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.
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.
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.
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.
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.
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; (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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of the invention.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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

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.
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.
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.
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.
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.
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.
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.
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.
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.
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

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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
This method comprises the steps of:
i) employing computational means to perform a fitting operation between the chemical entity and a binding pocket of the molecule or molecular complex; and
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.
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.
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.
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.
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.
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.
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.
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.).
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)].
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.
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.
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

The invention is further illustrated by the following examples which are intended to illustrate but not limit the scope of the invention.

Materials

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 μg/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 μg/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 μg/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° C. in a 5% CO₂humidified incubator.
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.
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® M Melanocyte Culture Medium (Lifeline Cell Technology, Walkersville, Md.).
Esophageal Cancer Cell Lines
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 μg/ml penicillin-streptomycin. All cell lines were incubated at 37° C. in a 5% CO₂humidified incubator.

Pancreatic Cancer Cell Lines

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 μg/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 μg/ml penicillin-streptomycin. The As-PC1 and Bx-PC3 cell lines were maintained in RPMI 1640 supplemented with 10% FBS, 1 μg/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° C. in a 5% CO₂humidified incubator.

Other Cell Lines

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 μg/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 μg/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 μg/ml. MCF7 cells were maintained with Modified minimum Eagle's media with 10% fetal bovine serum, 1× non-essential amino acids (Cellgro, Herndon, Va.), 1 mM sodium pyruvate, and 500 μg/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 μg/ml), insulin (10 μg/ml), epidermal growth factor (20 ng/ml), and penicillin-streptomycin (100 μg/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.
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-1Rβ 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-β-actin antibody from Sigma (St Louis, Mo.). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody from Advanced ImmunoChemical (Long Beach, Calif.).
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-1Rβ 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-β-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® Aqueous, Promega, Madison, Wis.).
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.
For staining with CFSE (Molecular Probes, Eugene, Oreg.)., 1×10⁷/ml cells were suspended in PBS and incubated at 37° C. for 5 min with the 10 μM 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° 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 μg of GST-FAK fusion protein immobilized on the glutathione-Sepharose 4B beads for 1 h at 4° C. and then washed 3× with PBS. Equal amounts of GST-fusion proteins were used for each binding assay. Bound proteins were boiled in 6× Laemmli buffer and analyzed by SDS-PAGE and Western blotting.
Immunoprecipitation and western blotting: Cells were washed twice with ice cold 1×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™ Protease Inhibitor, Roche, N.J.) and phosphatase inhibitors (Calbiochem, Calif.). For immunoprecipitation, 100-200 μg of total cell extract was used for each sample. The extracts were incubated with 1 μg of antibody overnight at 4° C. Next, 25 μl of protein A/G-agarose beads were added and the samples were incubated for 2 h at 4° C. The precipitates were washed 4× with lysis buffer and samples containing 30 μg 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 (2×10⁵cells/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

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

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.
Pancreatic cancer cell lines were trypsinized and counted. The cells were then plated to 24-well trays and incubated at 37° C., humidified 5% CO₂-95% air until 60-80% confluent. In each well, a volume of 10 μl 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° C. in a humidified incubator in an atmosphere of 5% CO₂, 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 μg/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
In addition, apoptotic cells were also analyzed by Hoechst staining. To the prepared cells as described above, Hoechst 33342 (1 μg/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

For detection of activated caspase 3/7 enzymes, as a confirmation of apoptosis in the treated cells, Apo-ONE® 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 μL 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× oil objective.
Kinase Profiler Screening: Kinase specificity screening was performed with Invitrogen's SelectScreen® Kinase Profiling Services http://www.invitrogen.com/site/us/en/home/Products-and-Services/Services/Discoverv-Research/SelectScreen-Profiling-Service/SelectScreen-Kinase-Profiling-Service.html. The screening was performed with 1 μM compound, INT2-31, 10 μM ATP, and kinase substrates against ten recombinant kinases according to Z′-LYTET™ Kinase Assay. For PI3 kinase activity, 100 μM ATP, and kinase substrate were utilized with the Invitrogen Adapta® 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, 5×10⁶cells, subcutaneously. When the tumor size reached 100 mm³, 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 mm³was calculated using the formula [(width)²×length]/2. At the end of experiment, tumor weight and volume were determined.

Melanoma Xenograft

For the melanoma study, the University of Florida IACUC approved the following protocol (IACUC Study #200801077). Melanoma cells were injected, 5×10⁶cells, subcutaneously. When the tumor size reached 100 mm³, 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 mm³was calculated using the formula [(width)²×length]/2. At the end of experiment, tumor weight and volume were determined.

Patient Subjects and Xenograft

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.
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 (0.3×0.3×0.3 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 (0.3×0.3×0.3 cm), and transplanted subcutaneously into another 10 mice. When tumors reached ˜100 mm³, mice were randomized in the following 2 groups, with 5 mice in each group:

- Group 1: Control: no treatment.
- Group 2: INT2-31 (Compound 31): 50 mg/kg/day in 50 μL by i.p administration for 21 days. This drug has been previously tested by our laboratory and has no measurable toxicity at this dose.

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×width×height×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).
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).
Orthotopic Model of Pancreatic Cancer
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 5×10⁶tumor 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.
When tumors reach ˜100 mm³, mice were randomized in the following 4 groups, with 3 mice in each group:

- Group 1: Control: no treatment.
- Group 2: Gemcitabine: 40 mg/kg in 50 μL treated every 5 days for three weeks by intraperitoneal (i.p) administration.
- Group 3: INT2-31: 15 mg/kg/day in 50 μL by i.p administration
- Group 4: Combination of Gemcitabine and INT2-31 treatments

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

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 μL 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.
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° C. The tissues were stained with the chromogen DAB and counterstained with hematoxylin and 1% TBS.
For assessment of apoptotic cells, staining was performed utilizing the Dead End™ 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

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.
In the first case, 96-microtiter plate wells were coated with purified GST-fused FERM domain of FAK in 50 μl of PBS (NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄4.3 mM, KH₂PO₄1.4 mM) overnight at 4° C. Wells were then rinsed with wash buffer (PBS, 0.05% Tween) and blocked with 200 μl of blocking buffer (PBS, 1% BSA) for 3 h at 37° C. After rinsing three times with wash buffer, with or without the compounds, 100 μl of binding buffer (PBS, 0.05% Tween, 1% BSA) containing 0.2 μM of purified IGF-1R whole protein was added to the wells and allowed to react for 1 h at 37° C. Wells were rinsed again three times and 100 μl 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° C. After three additional rinsings, 100 μl of the same buffer containing a secondary HRP anti-rabbit antibody was added and incubated for another hour at 37° C. Finally, 100 μl 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.
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

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.
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

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.
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 μl/min. Anti-mouse antibody surfaces were prepared using amine-coupling chemistry at 30° C. and at a flow rate of 10 μl/min. NHS/EDC was injected for 15 min to activate the surface, 100 μg/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 μg/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 μl/min. This immobilization procedure yielded 15000 to 20000 resonance units (RU) of immobilized primary antibody. Finally, 200 μg/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 μl/min. This immobilization yielded 30000 to 40000 resonance units (RU) of immobilized FAK-NT.

Capture of IGF-1R

Aliquots of IGF-1R were kept frozen at −80° C. until use. A volume of freshly prepared, 200 μg/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 MgCl₂, pH 8.0) twice. The capture procedure yielded typically to densities of 2000-4000 RU) onto a FAK-NT surface at 25° C. A primary antibody bound surface served as the reference.

Preparation of Analyte Solutions

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 μl of this secondary stock to 1 ml of 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl₂, 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

At each temperature, five buffer blanks were first injected to equilibrate the instrument fully. Using a flow rate of 50 μl/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° C., the surface was regenerated with 10 100-s pulses of 60% ethylene glycol; at 16 to 18° C., 40% ethylene glycol; at 22 to 28° C., 30% ethylene glycol; and at 32 to 39° C., 50 mM Tris-HCl, 150 mM NaCl, 10% ethylene glycol, 15 mM ATP, 15 mM MgCl₂, 5% DMSO, and 0.1% Tween 20 (pH 8.0). The data collection rate was 10 Hz.

Data Analysis

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)=ΔH°/RT−ΔS°/R. (Although the use of integrated forms van't Hoff equation that includes a term for ΔCp° was considered, the lack of curvature in the ln(KD) versus 1/T plots indicated that using this approach was unnecessary.) Values for ΔH° and ΔS° were obtained directly using the Solver macro in Microsoft Excel. ΔH° and ΔS° 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 ΔH°/R and −ΔS°/R, respectively. Fitting errors for ΔH° and ΔS° from Solver were obtained using a downloadable macro called SolverAid (http://www.bowdoin.edu/˜rdelevie/excellaneous). Errors for the parameters ΔH° and ΔS° 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 Δz2=(∂f/∂x)2Δx2+(∂f/∂y)2Δy2+ . . . in Excel. Programmed formulas were first checked using the downloadable macro Propagate (also available at http://www.bowdoin.edu/˜rdelevie/excellaneous).

Example 1

Database of Small Molecules

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_—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

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.
For the model of selecting compounds as FAK and/or IGF-1R inhibitors, the following sequences were utilized.

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

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.
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

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

Adenoviral infections: Cells were plated at a density of 6×10³or 2×10⁵into 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

siRNA transfection assay: Cells were plated at a density of 6×10³cells for 60 mm diameter or 2×10⁵cells 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

Immunoprecipitation and western blotting: Cells were washed twice with ice cold 1× 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™ 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° C. and the supernatants were analyzed. Protein concentration was determined by using Bio-Rad Protein Assay.
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° 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° C. The precipitates were washed three times with lysis buffer, resuspended in 40 μl Laemmli buffer and 35 μl was removed for western blotting.
Western blotting: boiled samples containing 30 μg 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™ 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 μM of NSC 344553. Immunoprecipitation and western blotting analysis is depicted in FIG. 12 for MiaPaCa-2 pancreatic cancer cells treated with NSC 128687.
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).
FIG. 5 depicts the effects on C8161 melanoma cancer cells treated with 5 μM 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

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® AQueous, Promega, Madison, Wis.). Briefly, 20 μl of the tetrazolium compound was added to each well. The cells were then incubated at 37° 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 μM). FIG. 6 shows that NSC 344553 inhibits the cell viability of pancreatic (Panc-1 and MiaPaCa-2) and melanoma (C8161 and A375) cancer cells.
FIG. 10 demonstrates that a 72-hour treatment of 0.05 μM 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.
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 μM) with associated PARP cleavage. It was also observed that 0.05 μM 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.
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

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 ≧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

To measure cell movement, the transwell migration assay is performed in two-well Boyden-type chambers. 1×10⁵cells/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

Cells were fixed in 3.7% paraformaldehyde in 1×PBS for 10 min and permeabilized with 0.5% Triton X-100 for 5 minutes. Cells were then washed with 1×PBS, blocked with 25% normal goat serum in 1×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×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×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×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

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

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

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

In the study of DNA DSB and repair, the phosphorylated form of the histone variant H2AX (termed γH2AX) has been adopted for its relationship with DNA double strand breaks. Specifically, γH2AX 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 γH2AX (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 γH2AX 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

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 C₁₂H₁₆N₃O₇PS. 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

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-1Rβ with an average IC₅₀of 3.96 μM (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 IC₅₀of 2.72 and 3.17 μM, respectively) as demonstrated by immunoprecipitation using an antibody against FAK (FIGS. 15C and D).
In addition, the effect of INT2-31 on cell viability of esophageal, pancreatic and breast cancer cell lines was analyzed and the IC₅₀value for each cell line was determined, as shown in Table 1. To get the average IC₅₀value, each cell line was treated with increasing concentrations of the compound for 72 hours in triplicate and the average of IC₅₀values 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 1

IC₅₀of INT2-31 for cancer cell lines

		[INT2-31]
	Cell Lines	μM

	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

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 IC₅₀value 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

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).
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

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 μM 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 μM dose (FIG. 18B). This was confirmed by analysis of caspase 3/7 activation following treatment for 72 h with 1 μM and 5 μM 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

INT2-31 Decreases Activation of Akt without Inhibiting Kinase Activity
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 μM and 5-10 μM, 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).
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 μM, 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.
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

INT2-31 Decreases Tumor p-Akt and Growth in Melanoma Xenografts
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

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 μM 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 μM concentrations (FIGS. 21 and 22).

Example 20

In Vitro and In Vivo Inhibition of Esophageal Cancer Viability and Proliferation with INT2-31 Treatment
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 IC₅₀value of 2.18 μM.
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 (0.3×0.3×0.3 cm) from a fresh esophageal human adenocarcinoma tumor sample were implanted subcutaneously into 2 mice. When one of the tumors reached 1.5 cc³, it was excised and cut into small pieces of (0.3×0.3×0.3 cm), and transplanted subcutaneously into another 10 mice. When tumors reached ˜100 mm³, 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

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 5×10⁶tumor 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 ˜100 mm³, 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

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

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.
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.
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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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]oxazolo[3,2-a]pyrimidin-3-yl dihydrogen phosphate;

b) 4-(methylthio)-7-(5-O-phosphono-D-ribofuranosyl)-{7H-Pyrrolo[2,3-d]pyrimidine}; 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]pyrimidine}, 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

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]pyrimidine} 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

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]pyrimidine} or a pharmaceutically acceptable salt thereof.

46. The pharmaceutical composition of claim 43, wherein said cancer is pancreatic cancer, melanoma cancer, or esophageal cancer.