High-throughput evaluation of novel WRN inhibitors

Abstract

DNA repair is a critical component for the maintenance of genomic stability and cancer prevention. Werner syndrome helicase (WRN), a RecQ family helicase involved in DNA double-strand break (DSB) repair, has been identified as a promising therapeutic target for multiple cancer types with high microsatellite instability (MSI-H). Microsatelite unstable tumors are characterized by a vulnerability in the DNA mismatch repair mechanism and depend on WRN for survival. Internal validation confirmed that CRISPR-mediated knockout of WRN was lethal in MSI-H, but not microsatellite stable (MSS) tumor cells. Additionally, this effect was confirmed as contingent upon the helicase activity of the enzyme. The challenge in targeting WRN lies in identifying inhibitors that effectively engage the helicase without causing toxicity to normal or microsatellite stable (MSS) cells. To address this challenge, we initiated a collaborative effort combining in vitro biochemical assays with cell-based assays using a panel of MSI and MSS cells. This approach aimed to evaluate compounds derived from knowledge-based designs as well as hits identified through our internal screening efforts, including cell-based phenotypic screens, Automated Ligand Identification System (ALIS), biochemical ADP glo HTS, and DEL. The assay suite comprises biochemical ATPase and helicase assays, in addition to cell viability and two target engagement assays. The primary functional target engagement assay utilized a high-content imaging method to detect a biomarker of DNA DSBs, using histone H2AX phosphorylation (pH2AX). A cellular thermal shift assay served as an orthogonal assessment of target engagement. This work enabled a knowledge-based drug discovery approach that leveraged structural design through computational modeling capabilities, resulting in a potent and novel series of spirocyclic WRN inhibitors specifically targeting MSI-H tumor cells. Our findings underscore the potential of WRN as a drug target for treating MSI-H cancers and emphasize the significance of interdisciplinary approaches in the discovery and advancement of new therapeutic agents.