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

Abstract

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.

REV7 in Cancer Biology and Management

“…These findings, together with the results from other similar attempts targeting the REV1-CTD, indicate that targeting the REV1/Polζ-associated TLS with small-molecule inhibitors is a promising strategy for cancer therapy [ 109 , 110 , 111 , 112 , 113 ].…”

Targeting protein–protein interactions in the DNA damage response pathways for cancer chemotherapy

“…In a series of works, researchers at the University of Connecticut identified multiple small molecule TLS inhibitors targeting Rev1-CT/RIR PPIs. [328][329][330][331][332] Two initial scaffolds, thiophene and piperazine, identified in an FP-based HTS assay disrupted the Rev1-CT PPI with a fluorescently tagged FAM-Polκ-RIR peptide (Figure 9C). 328 The thiophene scaffold compound was shown to bind the RIR-interface of Rev1-CT by NMR, sensitized fibrosarcoma cells to cisplatin, and reduced cisplatin-induced mutagenesis.…”

“…Recently, a 2.5 Å resolution X-ray crystal structure has been determined for the most potent phenazopyridine compound bound to Rev1-CT (in the context of Rev1-CT/Rev7/Rev3-RBM1 complex; PDB: 6WS5), which guided the design of second-generation PAP derivatives exhibiting low μM binding affinities to Rev1-CT that are improved by an order of magnitude relative to the first-generation compounds. 332 The second PPI interface of Rev1-CT binds the Rev7 subunit of Polζ, an interaction integral to the Polζ recruitment to DNA. 315,316 Researchers at Duke University and Massachusetts Institute of Technology utilized an ELISA assay HTS and identified, a 1,4-dihydroquinolin-4-one derivative, JH-RE-06, as a disruptor of Rev7/Rev1-CT PPI (Figure 9D).…”

“…Recently, a 2.5 Å resolution X-ray crystal structure has been determined for the most potent phenazopyridine compound bound to Rev1-CT (in the context of Rev1-CT/Rev7/Rev3-RBM1 complex; PDB: ), which guided the design of second-generation PAP derivatives exhibiting low μM binding affinities to Rev1-CT that are improved by an order of magnitude relative to the first-generation compounds. 332 …”

Protein–Protein Interactions in Translesion Synthesis

“…More detailed information about the identification and development of these compounds has been reviewed elsewhere [60]. Structures of the following compounds complexed to their TLS protein target are available in the RCSB database: compound 1, PDB ID 3WGW [61]; compound 4, PDB ID 6WS0 [62]; compound 6, PDB ID 6C8C [63].…”