Abstract C124: Development of novel FAK inhibitors targeting the FAK-VEGFR3 protein-protein interaction site.
Authors: Agharkar Priyanka A. , Ethirajan Manivannan , Liao Jianqun , Yemma Michael , Magis Andrew , Cance William , Pandey Ravindra , Kurenova Elena 
Published: June 2012

Introduction: FAK and VEGFR-3 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 VEGFR-3 binds to the FAT domain of FAK and the physical interaction between the two proteins confers a significant survival advantage to tumor cells. We developed a platform for the discovery of small molecules that specifically target the FAK-VEGFR-3 interaction site, and identified small molecule C4. Although in vitro and in vivo studies with C4 are promising, it exerts anti-tumor effects at moderate concentrations. We next sought to discover 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). In this study, we have shown that some C4 analogs disrupt FAK-VEGFR3 interaction and inhibit pancreatic tumor growth at low concentrations as single agents.

Methods: The structure of C4 was modified at various positions by substituting alkyl and aryl moieties. The resulting analogs were screened for anti-cancer inhibitory effects in different cancer cell lines by MTT assay. Effects of C4 analogs on six pancreatic cancer cell lines were determined by MTT assay (viability and IC50), Clonogenecity assay (colony formation) and Western blot (phosphorylation and apoptosis). Target specificity and binding confirmation of C4 analogs was done using immunoprecipitation and Forte-Bio's Octet Red Technology. Subcutaneous xenograft mouse model of pancreatic cancer was used to determine anti-tumor effects of C4 analogs in vivo.

Results: After in vitro screening of C4 analogs, C9, C10, C9A and C9B was selected for further analyses. All four analogs revealed dramatic decrease in cell viability in a panel of six pancreatic cancer cell lines. Analog C9 showed highest cytotoxic effects in pancreatic cancer cell lines and inhibited colony formation of Panc-1 cells at 1μM concentration. Treatment with C9 leads to apoptosis with dose dependent cleavage of caspase-3 and PARP. It caused decrease in phosphorylation levels of Akt and Erk, which are critical downstream targets of FAK that mediate survival signaling. Disruption of FAK-VEGFR3 interaction was seen after 24h treatment with 1μM C9. With Octet method we determined that at 20μM C9, KD (M) = 1.29×10(−7). In vivo, 30 days treatment with low doses of C9, C10 and C9A as single agents led to 40% to 45% reduction in tumor volume (P&lt;0.05).

Conclusions: Our results demonstrate that targeting the FAK-VEGFR-3 interaction site inhibited the survival function of these two tyrosine kinases, thus representing a unique approach for molecular-targeted highly specific cancer therapeutics. The significance of this study lies in the discovery of an oral-based small molecule inhibitor that might impact the therapeutic management for pancreatic cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C124.