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

Abstract

Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor that suppresses multiple proapoptotic and epithelial genes. CtBP is overexpressed in many human cancers, and its overexpression increases stem cell-like features, epithelial-mesenchymal transition, and cancer cell survival. Knockdown of CtBP also increases apoptosis independent of p53 in cell culture. Therefore, targeting CtBP with small molecules that disrupt its interaction with transcription factor partners may be an effective cancer therapy. To elicit its corepressing effect, CtBP binds to a conserved peptide motif in each transcription factor partner. We developed an AlphaScreen high-throughput screening assay to monitor the interaction between CtBP and E1A (which mimics the interaction between CtBP and its transcriptional partners). We screened the LOPAC library of 1280 bioactive compounds and identified NSC95397, which inhibits the CtBP-E1A interaction (IC50 = 2.9 µM). The inhibitory activity of NSC95397 was confirmed using two secondary assays and a counterscreen. NSC95397 also behaved as a weak substrate of CtBP dehydrogenase activity and did not inhibit another dehydrogenase, lactase dehydrogenase. Finally, NSC95397 was able to disrupt CtBP-mediated transcriptional repression of a target gene. These studies present a new possibility for the development of a therapeutic agent targeting tumors through disrupting the CtBP transcriptional complex.

Keywords: AlphaScreen; CtBP1; E1A; NSC95397; protein-protein interaction.

Figure 1. The CtBP1-E1A interaction can be monitored using an AlphaScreen assay

(A) Varying concentrations of E1A was added to 25 nM (■), 50 nM (▼), 100 nM (◆), 150 nM (●), 200 nM (□), and 250 nM (△) CtBP1 to generate AlphaScreen signals. (B) Competition displacement in the AlphaScreen assay with an untagged 14 amino acid E1A peptide demonstrated that the IC₅₀ value for the peptide was 20.9 ± 1.1 μM.

Figure 2. A pilot screen of 1,280 compounds from the LOPAC library using the AlphaScreen assay identified 7 primary hits

The LOPAC library was screened using the CtBP1-E1A AlphaScreen 1536-well assay protocol described in the Materials and Methods section. The screen included a total of eight assay plates, one in which plain DMSO was added, and seven in which the compounds from the LOPAC collection were added in an interplate-dose response manner (seven doses starting at 40 μM and then 5-fold dilutions). (A) Signal/background (S/B) and Z′-factor values for each assay plate from the LOPAC pilot screen. Plate parameters were calculated using median signal from the control columns; column 3 being high signal with all reagents added and column 1 being low signal with only detection reagents added (without proteins). (B) Hits from the dose response screen of the LOPAC collection were selected based on curve response class (CRC) analysis^, . Dose response curves for seven hits identified with CRC of 1.1, IC₅₀ < 5 μM, and maximal inhibition of > 80% are shown.

Figure 3. NSC95397 is confirmed in secondary and counter-screen assays

(A) The chemical structure of NSC95397. (B) NSC95397 does not inhibit the interaction between two unrelated proteins, Six1 and Eya2, in an AlphaScreen assay, demonstrating compound specificity. (C) NSC95397 (▼) inhibits the CtBP1-E1A peptide interaction with an IC₅₀ value of 0.6 ± 0.12 μM in a secondary fluorescence polarization assay using a fluorescein labeled E1A peptide (EPGQPLDLSCQRPR). Two other primary hits, fusaric acid (■) and FSCPX (▲) are false positives and are inactive in the fluorescence polarization assay. (D) NSC95397 disrupts the CtBP1-E1A interaction with an IC₅₀ value of 10.0 ± 1.3 μM in a competitive ELISA.

Figure 4. NSC95397 is a weak substrate for CtBP1 and does not inhibit other cellular dehydrogenases

(A) CtBP1 enzymatic activity (nmol NADH/min/mg protein) was determined spectrophotometrically in the presence of 150 μM NADH and the indicated concentrations of NSC95397 ( ) and MTOB (■). No activity was observed in the absence of substrate. (B) NSC95397 is unable to inhibit another NADH dependent enzyme, LDH. NSC95397 did not significantly inhibit enzymatic activity in the presence of 5 μM pyruvate (■) nor did it exhibit significant consumption of NADH without pyruvate ( ). (C) The effect of a known substrate, MTOB, on the CtBP1-E1A interaction was monitored using both the AlphaScreen and fluorescence polarization assays. NSC95397 (▼) was able to disrupt the interaction, while MTOB (■), the more efficient CtBP1 substrate, was ineffective at disrupting the interaction.

Figure 5. NSC95397 releases CtBP1-mediated transcriptional repression of the E-cadherin promoter in a luciferase assay

(A) H1299 cells expressing high endogenous levels of CtBP1 were transfected with the E-cadherin promoter-luciferase construct and luciferase activity was measured after treatment with increasing concentrations of NSC95397. (B) H1299 cells were transfected with a SIX1-specific MEF3 promoter-luciferase construct and luciferase activity was measured after treatment with increasing concentrations of NSC95397.