Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP)

Abstract

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.

Keywords: C-terminal Binding Protein; CtBP; Dehydrogenase; Hydroxyimine; MTOB; Oxime; Transcriptional co-repressor; Tumor suppressor gene.

Figure 1

a. Enzymatic reaction catalyzed by CtBP. b. Crystal structure of CtBP1 with MTOB (1) and NAD⁺ (PBDID: 4LCE).

Figure 2

Analogues of 9 designed to explore the structure-activity relationship.

Figure 3

Plot of IC₅₀ with recombinant enzyme (from NADH assay) vs. GI₅₀ from cell growth/viability assay for hydroxyimine analogues. The most potent compounds (14g and 14h) are highlighted by the red box. Analogues 14c, 14f, 14i, and 14j have been omitted for clarity.

Figure 4

Change in Bik promoter expression in HCT-116 p53^−/− cells upon treatment with CtBP inhibitors. * P<0.05 compared to 1, 9, 14f, and 14h. ** P<0.05 compared to 1, 9, 14f, and 14g.

Figure 5

Crystal structure of CtBP1 with hydroxyimine 9 and NADH (PBDID: 4U6Q).

Figure 6

Docked poses of 9 (white), 14a (cyan), 14g (magenta) and 14j (yellow) within CtBP active site.

Scheme 1

Design of CtBP Inhibitors from MTOB (1).

Scheme 2

Design of Deconstruction Analogues of Phenylpyruvic Acid (3).

Scheme 3

Synthesis of analogues 14a-b, d-g, and i-m. Reagents and Conditions: (a) Hydantoin, Na₂CO₃ (sat aq), ethanolamine, EtOH/H₂O (1:1), 120 °C, 5-10 h. (b) i. 20% NaOH (aq), 100 °C, 3h; 12N HCl, 54-88% for 2 steps. (c) NH₂OH·HCl, NaOH, H₂O, rt, 12h, 54-91%.

Scheme 4

Synthesis of analogues 14cand 14h. Reagents and Conditions: (a) 1,4-diacetylpiperazine-2,5-dione, NEt₃, DMF, rt, 12 h. (b) 6N HCl, reflux, 4 h; 58% (17c), 56% (17h). (c) NH₂OH·HCl, NaOH, H₂O, rt, 12h; 92% (17c), 66% (17h).