Crystal structures of human CtBP in complex with substrate MTOB reveal active site features useful for inhibitor design

Abstract

The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD+ revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD+ phosphate. Neither feature is present in other D2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors.

Small Molecule, NSC95397, Inhibits the CtBP1-Protein Partner Interaction and CtBP1-Mediated Transcriptional Repression

“…CtBP1 is capable of reducing MTOB through the oxidation of NADH to NAD+, with a maximum activity of approximately 91.0 ± 1.9 nmol/min/mg protein 16 . The ability of MTOB to serve as a substrate of CtBP1, which leads to NADH to NAD+ conversion, was thought to be one possible mechanism of inhibition 25 . To determine if NSC95397 is also a substrate of CtBP1, we performed an enzymatic assay monitoring the levels of NADH using its characteristic absorption at 340 nm with increasing concentrations of NSC95397 or MTOB.…”

“…Because the oxidation of NADH to NAD+ has been shown to decrease CtBP1’s affinity for E1A, it is possible that the reduction of a substrate and conversion of NADH to NAD+ could disrupt the CtBP1-E1A interaction, which is one proposed mechanism of action for MTOB 25 . However, the effect of MTOB on the CtBP-E1A interaction has never been directly evaluated.…”

“…This suggests that although NSC95397 is a weak substrate of CtBP1, its mechanism of action as a protein interaction inhibitor is likely separate from its role as a substrate and it disrupts CtBP1-binding partner interaction in a manner distinct from MTOB. Recently, the crystal structure of CtBP1 and 2 in complex with MTOB has been solved 25 . The co-crystal structures showed no significant change in the overall tertiary and quaternary conformation of both CtBP proteins, suggesting MTOB must work through a mechanism other than an induced conformational change.…”

Structure-Guided Design of a High Affinity Inhibitor to Human CtBP

“…MTOB bound CtBP1 does not differ from apo CtBP1. 23 Therefore, the position of the MTOB complex α-carbons were used for comparison with the HIPP (B) and PPy (C and D) structures. Small differences may be the result of different crystallization conditions relative to the MTOB structure.…”

“…23 The structures revealed two particularly attractive properties, a dominant tryptophan (318 in CtBP1) and a hydrophilic channel filled by four ordered water molecules, that are not shared by other members of the D2-HDH protein family, and thus offer a potential basis for the development of highly specific inhibitors. 23 Here we present structural and thermodynamic data on two compounds that exploit these active site features. Both compounds bind with favorable ring stacking to W318 and with significantly higher affinity than MTOB, including one compound that binds more than 1000 times more tightly.…”