Small molecule scaffolds that disrupt the Rev1-CT/RIR protein-protein interaction

Ozen, Zuleyha; Dash, Radha Charan; McCarthy, Kaitlyn R.; Chow, Samantha A; Rizzo, Alessandro A.; Korzhnev, Dmitry M.; Hadden, M. Kyle

doi:10.1016/j.bmc.2018.07.029

Cited by 11 publications

(15 citation statements)

References 21 publications

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“…One strategy to inhibit TLS for chemotherapeutic sensitivity is to disrupt essential PPIs that control assembly of the multiprotein TLS complex with small molecules [9,29–33] . Our attempts to inhibit TLS focused on blocking PPIs of Rev1‐CT with RIR motifs from Polη, Polι, Polκ, and PolD3.…”

Section: Introductionmentioning

confidence: 99%

“…The majority of contacts between Rev1‐CT and RIR are mediated by the FF pair and mutation of these residues completely abrogates the PPI [23–25] . We have used biochemical high‐throughput screening, [29–30] virtual screening, [32] and rational design approaches [31] to identify small molecules that disrupt Rev1‐CT/RIR PPIs. The identified scaffolds demonstrated concentration‐dependent displacement of FAM‐Polκ‐RIR peptide from its complex with Rev1‐CT in fluorescence polarization and/or intensity (FP/FI) assays, as well as direct binding to the RIR site on Rev1‐CT in NMR titration experiments.…”

Section: Introductionmentioning

confidence: 99%

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Structure‐Based Drug Design of Phenazopyridine Derivatives as Inhibitors of Rev1 Interactions in Translesion Synthesis

et al. 2021

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Rev1 is a protein scaffold of the translesion synthesis (TLS) pathway, which employs low‐fidelity DNA polymerases for replication of damaged DNA. The TLS pathway helps cancers tolerate DNA damage induced by genotoxic chemotherapy, and increases mutagenesis in tumors, thus accelerating the onset of chemoresistance. TLS inhibitors have emerged as potential adjuvant drugs to enhance the efficacy of first‐line chemotherapy, with the majority of reported inhibitors targeting protein‐protein interactions (PPIs) of the Rev1 C‐terminal domain (Rev1‐CT). We previously identified phenazopyridine (PAP) as a scaffold to disrupt Rev1‐CT PPIs with Rev1‐interacting regions (RIRs) of TLS polymerases. To explore the structure‐activity relationships for this scaffold, we developed a protocol for co‐crystallization of compounds that target the RIR binding site on Rev1‐CT with a triple Rev1‐CT/Rev7R124A/Rev3‐RBM1 complex, and solved an X‐ray crystal structure of Rev1‐CT bound to the most potent PAP analogue. The structure revealed an unexpected binding pose of the compound and informed changes to the scaffold to improve its affinity for Rev1‐CT. We synthesized eight additional PAP derivatives, with modifications to the scaffold driven by the structure, and evaluated their binding to Rev1‐CT by microscale thermophoresis (MST). Several second‐generation PAP derivatives showed an affinity for Rev1‐CT that was improved by over an order of magnitude, thereby validating the structure‐based assumptions that went into the compound design.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structure‐Based Drug Design of Phenazopyridine Derivatives as Inhibitors of Rev1 Interactions in Translesion Synthesis

et al. 2021

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“…[1,2,8,9] Severalo ft hese switching events are mediated by protein-protein interactions (PPIs) between the C-terminal domain of the Y-familyp olymerase Rev1 (Rev1-CT) and the Rev1 interacting regions (RIR) from the Y-familyp olymerases polh,p olk,p oli,a nd polz. [14][15][16] These compounds increase sensitivity to cisplatin and decrease cisplatin-mediated mutagenesis in human cancer cells, highlighting their potential as combination therapies for variousforms of cancer. [14][15][16] These compounds increase sensitivity to cisplatin and decrease cisplatin-mediated mutagenesis in human cancer cells, highlighting their potential as combination therapies for variousforms of cancer.…”

Section: Introductionmentioning

confidence: 99%

“…[10][11][12][13] We recently identified the first small molecules( Figure1A, 1 and 2)t hat disrupt the Rev1-CT/ RIR PPI. [14][15][16] These compounds increase sensitivity to cisplatin and decrease cisplatin-mediated mutagenesis in human cancer cells, highlighting their potential as combination therapies for variousforms of cancer.…”

Section: Introductionmentioning

confidence: 99%

Virtual Pharmacophore Screening Identifies Small‐Molecule Inhibitors of the Rev1‐CT/RIR Protein–Protein Interaction

et al. 2019

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Translesion synthesis (TLS) has emerged as a mechanism through which several forms of cancer develop acquired resistance to first‐line genotoxic chemotherapies by allowing replication to continue in the presence of damaged DNA. Small molecules that inhibit TLS hold promise as a novel class of anticancer agents that can serve to enhance the efficacy of these front‐line therapies. We previously used a structure‐based rational design approach to identify the phenazopyridine scaffold as an inhibitor of TLS that functions by disrupting the protein–protein interaction (PPI) between the C‐terminal domain of the TLS DNA polymerase Rev1 (Rev1‐CT) and the Rev1 interacting regions (RIR) of other TLS DNA polymerases. To continue the identification of small molecules that disrupt the Rev1‐CT/RIR PPI, we generated a pharmacophore model based on the phenazopyridine scaffold and used it in a structure‐based virtual screen. In vitro analysis of promising hits identified several new chemotypes with the ability to disrupt this key TLS PPI. In addition, several of these compounds were found to enhance the efficacy of cisplatin in cultured cells, highlighting their anti‐TLS potential.

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“…Previous studies demonstrate that the noncatalytic function of Rev1 relies on Rad5, that the C terminus of Rev1 physically interacts with the N terminus of Rad5, and that blocking this interaction inactivates the noncatalytic activity of Rev1, but not the transferase activity (21,22). Recent work has revealed that Rev1 acts as a scaffold protein interacting with other TLS polymerases (23,24). In S. cerevisiae and human cells, Rev1 promotes Pol recruitment through physical interaction with it when DNA is damaged (25)(26)(27), and Rev1 forms a complex with Pol to bypass thymine dimers induced by UV irradiation (28).…”

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Sml1 Inhibits the DNA Repair Activity of Rev1 in Saccharomyces cerevisiae during Oxidative Stress

Yao

Zhou

et al. 2020

Appl Environ Microbiol

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In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress. IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.

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Small molecule scaffolds that disrupt the Rev1-CT/RIR protein-protein interaction

Cited by 11 publications

References 21 publications

Structure‐Based Drug Design of Phenazopyridine Derivatives as Inhibitors of Rev1 Interactions in Translesion Synthesis

Structure‐Based Drug Design of Phenazopyridine Derivatives as Inhibitors of Rev1 Interactions in Translesion Synthesis

Virtual Pharmacophore Screening Identifies Small‐Molecule Inhibitors of the Rev1‐CT/RIR Protein–Protein Interaction

Sml1 Inhibits the DNA Repair Activity of Rev1 in Saccharomyces cerevisiae during Oxidative Stress

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