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dc.contributor.authorGogate, Priyanka Nikhil
dc.date.accessioned2016-04-05T19:34:25Z
dc.date.available2016-04-05T19:34:25Z
dc.date.issued2014
dc.identifier.isbn9781321262964
dc.identifier.other1627179041
dc.identifier.urihttp://hdl.handle.net/10477/51096
dc.description.abstractFAK functions as both, a critical signaling kinase and a scaffolding protein to collectively mediate cell proliferation, survival, adhesion, migration, invasion, and angiogenesis. The scaffolding function of FAK is mediated by its large N- and C- terminal domains which recruit multiple proteins that promote tumorigenesis. VEGFR3 is a receptor tyrosine kinase that acts as a key modulator of survival signals and lymphangiogenesis. FAK and VEGFR3 have stirred considerable interest in recent years as attractive anti-cancer targets, owing to their high expression across several tumor types and relatively low levels in normal tissues. Our lab has shown that VEGFR3 binds to the C-terminal FAT domain of FAK and the physical interaction between the two proteins confers a significant survival advantage to cancer cells. We developed a platform for the discovery of small molecules that specifically target the FAK C-terminal scaffold and identified small molecule C4. Although in vitro and in vivo studies with C4 are promising, it exerts anti-tumor effects at moderate concentrations. This prompted us to design C4 analogs that display enhanced potency, minimum toxicity, and high specificity for the target site. By using C4 as the parent moiety, we synthesized a series of C4 analogs on the basis of structure activity relationship (SAR). Based on the preliminary results from in vitro screening and in vivo toxicity and efficacy assays, we selected analogs 13 and 29 for further analyses. To test target specificity, we used an isogenic cell system, immunoprecipitation, and fluorescence polarization assays and found that analogs 13 and 29 successfully disrupt the FAK-VEGFR3 interaction. Binding of 13 and 29 to the FAT domain of FAK was confirmed with biolayer interferometry. Induction of apoptosis after treatment with 13 and 29 was confirmed via caspase 3/7 activation, caspase-3 and PARP cleavage. Down-regulation of tyrosine phosphorylated residues on FAK and VEGFR3 and phosphorylated forms of Akt, Erk1/2, Grb2 and paxillin was observed in a dose and time dependent manner after treatment with 13 and 29. These drugs also inhibited migration and invasion of pancreatic cancer cells. In vivo, 30 days treatment with low doses of 13 and 29 led to massive reduction in tumor growth when treated as single agents. Immunohistochemistry of pancreatic tumors obtained after treatment with 13 and 29 as single agents revealed a significant decrease in microvessel density for both blood (CD31) and lymphatic (LYVE1) vessels. Since FAK and VEGFR3 have been implicated in tumor angiogenesis, we also investigated the potential anti-angiogenic effects of the lead analogs. 13 and 29 dramatically decreased HUVEC motility and capillary network formation by reducing total branching points, total loops, and total tube length in a dose and time dependent manner. Directed in vivo angiogenesis assay (DIVAA(TM)) confirmed that 13 and 29 significantly reduced the formation of endothelial blood vessels as compared to untreated groups. Furthermore, 13 and 29 significantly decreased interstitial fluid pressure. We further explored the combination treatment of the lead analogs with HPPH-PDT in a glioblastoma tumor model and found a marked decrease in cancer cell survival in the combination treated group as compared to drug alone groups. Thus, in this study we have described a detailed analysis of two new chemical entities, 13 and 29 that specifically target the FAK C-terminal scaffold. Developing novel small molecules that target the FAK scaffold represents a unique approach towards anti-cancer treatment options.
dc.languageEnglish
dc.sourceDissertations & Theses @ SUNY Buffalo,ProQuest Dissertations & Theses Global
dc.subjectPure sciences
dc.subjectBiological sciences
dc.subjectFocal adhesion kinase
dc.subjectPancreatic cancer
dc.subjectScaffold
dc.subjectSmall molecule inhibitor
dc.subjectVascular endothelial growth factor receptor 3
dc.titleDevelopment of small molecule inhibitors targeting the C-terminal scaffold of focal adhesion kinase
dc.typeDissertation/Thesis


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