Active-site tryptophan, the target of anti-neoplastic CtBP inhibitors, mediates inhibitor disruption of CtBP oligomerization and transcription coregulatory activities

C-terminal Binding Proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (eg. PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (EMT; ex. CDH1); and activating expression of genes that promote migratory and invasive properties of cancer cells (eg. TIAM1) and genes responsible for enhanced drug resistance (eg. MDR1). CtBP9s transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtB9s transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP9s oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated W3249s contribution to CtBP29s biochemical and physiological activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP9s association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.