Doctor of Philosophy
Keith C. Ellis
Human C-terminal binding proteins (CtBP1 and CtBP2) are transcriptional coregulators of multiple genes in the human genome, including tumor suppressor genes (e.g., Bik, PTEN, BRCA1, and E-cadherin) as well as oncogenes (e.g. MDR1 and Tiam1). Both homologues of CtBP are overexpressed in many types of cancer, including breast cancer (92%), ovarian cancer (83%), colorectal cancer (64%), hepatocellular carcinoma (60%), gastric cancer, prostate cancer, and pancreatic adenocarcinoma. Further, expression levels of CtBP correlate with worse prognostic outcomes and more aggressive tumor features because it promotes proliferation, epithelial-mesenchymal transition, and cancer stem cell self-renewal activity.
Our laboratory has identified a lead inhibitor of CtBP, 2-hydroxyimino-3-phenyl propanoic acid (HIPP), with a binding affinity of 370 nM for CtBP1. Data from the co-crystal structure of CtBP1 complexed with HIPP and NADH (PDBID: 4U6Q) revealed essential interactions between HIPP and residues Arg97, Arg266, His315, and Trp318. Importantly, the aromatic ring of HIPP forms a π-stacking interaction with Trp318. HIPP has been shown to displace CtBP from transcriptional promoter regions, restore expression of tumor suppressing genes, and induce apoptosis. However, high doses of HIPP are required to induce these anti-oncogenic effects in cell and animal models.
The first part of this work describes the rational design and computational evaluation of new, heteroaromatic HIPP analogues. A small library of compounds (36 total) was constructed in silico and docked into the co-crystal structure of HIPP-CtBP. The best scoring compounds were synthesized and the binding affinity of these compounds was measured utilizing isothermal titration calorimetry (ITC). We identified 2 analogues that bind to CtBP with higher affinity than HIPP. We found that replacement of the carboxylic acid on 4-Cl HIPP with an ethyl ester increases its potency in A2780 cells 50-fold, and the same substitution increases the potency of HIPP in HCT116 cells 15-fold. We also discovered 5 analogues that were more potent than the HIPP ester in cells.
The second part of this work describes the design and development of HIPP-based CtBP PROteolysis TArgeting Chimeras (PROTACs) for in vivo degradation of CtBP using the endogenous Ubiquitin/Proteasome System. The PROTACs were synthesized in a 9-step synthetic route, and the degradation of CtBP was quantified by western blot. We found that CtBP1/2 can be successfully and effectively degraded in cells with HIPP-based PROTACs.
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