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

Hilbert, Brendan J.; Morris, Benjamin L.; Ellis, Keith C.; Paulsen, Janet L.; Schiffer, Celia A.; Grossman, Steven R.; Royer, William E.

doi:10.1021/cb500820b

Cited by 25 publications

(45 citation statements)

References 42 publications

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“…For this set, we chose to focus on exploring the structure-activity relationship of the phenyl ring. Our set of analogues included electronically and sterically diverse substituents; all were evaluated computationally by docking them into the site identified in the CtBP– 9 crystal structure 25 to prioritize synthesis and evaluation. Docking scores calculated with the HINT scoring function 26 (Table 1), which has been shown to correlate with free energy of binding, identified thirteen compounds that we selected for synthesis and evaluation as inhibitors of CtBP ( 14a-m , Figure 2).…”

Section: Resultsmentioning

confidence: 99%

“…This crystal structure and associated biophysical data have been reported separately. 25 More detailed computational docking with this crystal structure model was performed to support the SAR studies. While no clear trend in docking scores vs. protein or cellular inhibition IC 50 ’s was found, the docked poses of designed analogues at the CtBP active site revealed factors important for binding and inhibition (Figure 6).…”

Section: Resultsmentioning

confidence: 99%

“…The crystal structure 25 of CtBP1/ 9 /NAD + was used as the target for docking studies using GOLD v5.2, 28 with the binding defined as all the residues within 10 Å of the bound ligand 9 . Since the ligands are analogs of 9 , it is reasonable to expect that they will adopt binding modes very similar to that of the parent compound.…”

Section: Methodsmentioning

confidence: 99%

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

Korwar

Morris

Parikh

et al. 2016

Bioorganic & Medicinal Chemistry

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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 = 0.24 μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50 = 0.18 μM) and 3-chloro- (IC50 = 0.17 μM) 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Korwar

Morris

Parikh

et al. 2016

Bioorganic & Medicinal Chemistry

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“…3). CtBP1 crystallizes with one subunit in the asymmetric unit of hexagonal space group P6 4 22 (23,43). Three mutually perpendicular intersecting crystallographic 2-fold axes generate the D 2 symmetric tetramer shown in Fig.…”

Section: Tetramer Models Based On Crystal Lattices Of Ctbp1 and Ctbp2mentioning

confidence: 99%

Assembly of human C-terminal binding protein (CtBP) into tetramers

Bellesis

Jecrois

Hayes

et al. 2018

Journal of Biological Chemistry

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C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD- and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate- and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer.

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“…Attempts have been made to identify molecules targeting CtBP-mediated transcriptional repression through multiple angles (Blevins et al, 2017). For example, phenylpyruvate analogs have been generated that indirectly inhibit CtBP-mediated transcription suppression through inhibiting the enzymatic activity of CtBP, with limited cellular potency (IC50s ranging from 0.85 to 4 mM) (Hilbert et al, 2015;Korwar et al, 2016;Sumner et al, 2017). A second approach utilizes a cyclic peptide, CP61, which prevents the dimerization and function of CtBP in MCF7 cells at 50 lM (Birts et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

CPP‐E1A fusion peptides inhibit CtBP‐mediated transcriptional repression

Blevins

Zhang

et al. 2018

Molecular Oncology

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The carboxyl‐terminal binding proteins (CtBP) are transcriptional corepressors that regulate the expression of multiple epithelial‐specific and pro‐apoptotic genes. Overexpression of CtBP occurs in many human cancers where they promote the epithelial‐to‐mesenchymal transition, stem cell‐like features, and cell survival, while knockdown of CtBP in tumor cells results in p53‐independent apoptosis. CtBPs are recruited to their target genes by binding to a conserved PXDLS peptide motif present in multiple DNA‐binding transcription factors. Disrupting the interaction between CtBP and its transcription factor partners may be a means of altering CtBP‐mediated transcriptional repression and a potential approach for cancer therapies. However, small molecules targeting protein–protein interactions have traditionally been difficult to identify. In this study, we took advantage of the fact that CtBP binds to a conserved peptide motif to explore the feasibility of using peptides containing the PXDLS motif fused to cell‐penetrating peptides (CPP) to inhibit CtBP function. We demonstrate that these peptides disrupt the ability of CtBP to interact with its protein partner, E1A, in an AlphaScreen assay. Moreover, these peptides can enter both lung carcinoma and melanoma cells, disrupt the interaction between CtBP and a transcription factor partner, and inhibit CtBP‐mediated transcriptional repression. Finally, the constitutive expression of one such peptide, Pep1‐E1A‐WT, in a melanoma cell line reverses CtBP‐mediated oncogenic phenotypes including proliferation, migration, and sphere formation and limits tumor growth in vivo. Together, our results suggest that CPP‐fused PXDLS‐containing peptides can potentially be developed into a research tool or therapeutic agent targeting CtBP‐mediated transcriptional events in various biological pathways.

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

Cited by 25 publications

References 42 publications

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

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

Assembly of human C-terminal binding protein (CtBP) into tetramers

CPP‐E1A fusion peptides inhibit CtBP‐mediated transcriptional repression

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