Date of Award

8-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Wei Li, Ph.D.

Committee

Duane D. Miller, Ph.D. Bernd Meibohm, Ph.D. Andrzej Slominski, M.D., Ph.D. Frank Park, Ph.D.

Keywords

BRAF inhibitor, combination therapy, inhibitor of apoptosis, melanoma, survivin inhibitor, tubulin inhibitor

Abstract

The first part (Chapter 1 and 2) of this dissertation presents a novel combination study of melanoma therapy. Acquired clinical resistance to vemurafenib, a selective BRAFV600E inhibitor, arises frequently after short term chemotherapy. Since the inhibitions of targets in the RAFMEK-ERK pathway result in G0/G1 cell cycle arrest, vemurafenib-resistant cancer cells are expected to escape this cell cycle arrest and progress to subsequent G2/M phase. We hypothesized that a combined therapy using vemurafenib with a G2/M phase blocking agent will trap resistant cells and overcome vemurafenib resistance. To test this hypothesis, we first determined the combination index (CI) values of our novel tubulin inhibitor ABI-274 and vemurafenib on parental human A375 and MDA-MB-435 melanoma cell lines to be 0.32 and 0.1, respectively, suggesting strong synergy for the combination. We then developed an A375RF21 subline with significant acquired resistance to vemurafenib and confirmed the strong synergistic effect. Next we studied the potential mechanisms of overcoming vemurafenib resistance. Flow cytometry confirmed that the combination of ABI-274 and vemurafenib synergistically arrested cells in G1/G2/M phase, and significantly increased apoptosis in both parental A375 and the vemurafenib-resistant A375RF21 cells. Western blot analysis revealed that the combination treatment effectively reduced the level of phosphorylated and total AKT, activated the apoptosis cascade, and increased cleaved caspase-3 and cleaved PARP, but had no significant influence on the level of ERK phosphorylation. Finally, in vivo co-administration of vemurafenib with ABI-274 showed strong synergistic efficacy in the vemurafenib-resistant xenograft model in nude mice. Overall, these results offer a rational combination strategy to significantly enhance the therapeutic benefit in melanoma patients who inevitably become resistant to current BRAF inhibition therapy.

The second part (Chapter 3 to 5) of this dissertation focuses on the discovery of a series of small molecule survivin inhibitors. Inhibitors of apoptosis (IAP) proteins are widely considered as promising cancer drug targets, especially for drug-resistant tumors. Mimicking the IAP-binding motif of second mitochondria-derived activator of caspases (Smac) is a rational strategy to design potential IAP inhibitors. In this report, we used the bioactive conformation of AVPI tetrapeptide in the N-terminus of Smac as a template and performed a shape-based virtual screening against a drug-like compound library to identify novel IAP inhibitors. Top hits were subsequently docked to available IAP crystal structures as a secondary screening followed by validation using in vitro biological assays. Four novel hit compounds were identified that potently inhibited cell growth in two human melanoma (A375 and M14) and two human prostate (PC-3 and DU145) cancer cell lines. The best compound, UC-112, has IC50 values ranging from 0.7 to 3.4 µM. UC-112 also potently inhibits the growth of P-glycoprotein (Pgp) overexpressed multidrug-resistant cancer cells, strongly activates capase-3/7 and caspase-9 activities, and selectively down-regulates survivin level at a concentration as low as 1 µM. Co-incubation of UC-112 with a known proteasome inhibitor (MG132) rescued survivin inhibition, consistent with the anticipated mechanism of action for UC-112. As a single agent, UC-112 strongly inhibits tumor growth and reduces both XIAP and survivin levels in an A375 human melanoma xenograft model in vivo. Three analogs generated from UC-112 structural modification along with template compound UC-112 were submitted to NCI-60 cancer cell line screening. The results indicated that structural modification of UC-112 to give our best compound MX106 has improved activity by four fold (2.2 µM for UC-112 vs. 0.5 µM for MX106, average GI50 values over all cancer cell lines in the NCI-60 panel).Western blot analyses demonstrated the new compounds maintained high selectivity for survivin inhibition over other members in the inhibitiors of apoptosis protein family. When tested in an A375 human melanoma xenograft model, the most active compound MX106 effectively suppressed tumor growth and strongly induced cancer cell apoptosis in tumor tissues. Taken together, this novel scaffold is promising for the development of selective survivin inhibitors as potential anticancer agents.

DOI

10.21007/etd.cghs.2015.0341

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