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

Abstract

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.

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

“…TLS inhibition has recently emerged as a strategy to increase chemotherapeutic sensitivity and avert resistance associated with first‐line platinating and alkylating drugs [9–11] . Rev1′s scaffolding function is essential for TLS [17,21,22] and its inhibition provides a promising avenue for maintaining genotoxic chemotherapy sensitivity in cancer cells [9,29–33] . The RIR interface of Rev1‐CT is an important binding site for the “inserter” TLS polymerases Polι, Polκ, Polη, and the “extender” polymerase Polζ (via its accessory subunit, PolD3) [23–25,28] .…”

“…Disruption of the Rev1‐CT PPIs with RIR motifs is expected to impair assembly of the multiprotein TLS complex and polymerase switching, limiting the TLS pathway‘s ability to bypass DNA damage induced by chemotherapeutics and introduce further mutations to cancer cells. Previously, we identified several small‐molecule scaffolds that inhibit essential Rev1‐CT/RIR PPIs [29–32] . To guide structure‐based design of the improved analogues, here we developed a co‐crystallization protocol for small molecules with the RIR‐site on Rev1‐CT in the context of the triple Rev1‐CT/Rev7 R124A /Rev3‐RBM1 complex.…”

“…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.…”

“…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.…”

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine

“…Though this damage tolerance pathway benefits the cell by relieving stalled replication, unregulated TLS activity can detrimentally contribute to mutagenesis and resistance to DNAdamaging cancer therapies. Recent advances have addressed these drawbacks through identification of small molecule inhibitors which target Pol-Pol interactions and/or disrupt mutagenic TLS activity, supplementing the effectiveness of current chemotherapeutic drugs (62)(63)(64)(65)(66)(67)(68). These studies provide a promising new approach for mitigating the role of TLS in chemoresistance and provide an exciting incentive for furthering our understanding of the kinetic and structural foundations of TLS.…”