Biology

Title

Structural Studies of the Oncogenic Co-Transcriptional Regulator C-Terminal Binding Protein (CtBP): On the Road to Rational Drug Design

Date of Award

2017

Degree Type

Thesis

Degree Name

Master of Science in Biochemistry & Molecular Biology

Department

Biology

Chief Instructor

William E. Royer

Second Reader

Donald E. Spratt

Third Reader

David L. Thurlow

Abstract

C-terminal Binding Proteins 1 and 2 (CtBP1 and CtBP2) are paralogous co-transcriptional regulators that induce anti-epithelial and anti-apoptotic programs. When aberrantly regulated, CtBP is powerfully oncogenic, repressing the expression of cell-cell adhesion molecules including E-cadherin, pro-apoptotic genes such as BAX, tumor suppressors including p16INK4a, and activates the oncogene TIAM1. CtBP is linked to progression and metastasis in multiple carcinomas. Higher levels of CtBP in breast cancer are correlated with lower survival. CtBP is unique among co-transcriptional regulators in that it includes a D-isomer specific 2-hydroxyacid dehydrogenase (D2HDH) domain, which reduces or oxidizes substrates utilizing NAD+/NADH. The D2HDH substrate-binding pocket is a viable target for structure-based drug design (SBDD). Crystal structures of CtBP in complex with 4-Methylthio 2-oxobutyric acid (MTOB), the putative substrate, and the observation that high concentrations of MTOB reduce co-transcriptional activity have provided the foundation for SBDD with CtBP. However, all known inhibitors have low potency as anti-neoplastic agents. Therefore, the identification of additional druggable structural features outside of the substrate-binding pocket is necessary. The binding of NAD(H) induces oligomerization and is necessary for co-transcriptional activity. A dimeric form of CtBP identified crystallographically has been assumed to be the biologically active assembly, however new evidence suggests that the tetramer could be the functional unit. Here, multi-angle light scattering (MALS) data showing NADH-induced tetramerization are reported. Furthermore, FPLC, MALS, and site-directed mutagenesis were used to identify residues and interactions that are necessary for tetramerization. The crystal structures of CtBP1 WT 28-378 and the tetramer-destabilizing mutant CtBP1 A123V 28-378 are reported. Structural changes induced by the mutation give insights into the development of inhibitors that could bind in the substrate-binding pocket and disrupt the tetramer with an adjunct moiety. Lastly, the co-crystal structure of CtBP1 28-353 and [5,5'-Bithiazolidine]-2,2',4,4' tetrone is reported. This structure provides proof-of-concept that in silico approaches are viable for identifying high-affinity CtBP inhibitors.

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