Abstract 5490: Biochemical tools to characterize specific Werner syndrome helicase inhibitors and accelerate drug discovery

Abstract WRN has various roles in the maintenance of genome stability and is commonly found upregulated in cancers. Therefore, new WRN inhibitors have emerged for selective cancer therapy. Eurofins Discovery researchers have developed assays to accelerate helicase drug discovery focusing on WRN. Relaying on three different biochemical assays: Binding, ATPase and unwinding, these tools can be used to screen, validate, and characterize inhibitors mode of action (MOA). Using clinical-stage WRN helicase inhibitor HRO761 and VVD214, we present a complete MOA study demonstrating that the assays generated by Eurofins Discovery teams are well suited for WRN drug discovery project. The full length protein was used in the three biochemical assays. First protein stability was tested to qualify the protein used in all assays. ATPase activity was performed using DNA double strand substrate with ADP Hunter Plus assay kit (Eurofins DiscoverX). Unwinding activity was performed using the same substrate labelled in 5' end by the fluorophore TAMRA and at the 3' end by the quencher BHQ2. Compounds IC50 were validated in the two assays. MOAs were characterized by testing the reversibility of the interaction, substrate competition and ATP cooperativity. Binding analysis was assessed using UHPLC/MS/MS. Methods were first determined and then Kd, Bmax, kon, koff were analyzed. Stability assay demonstrated that our enzyme is stable with cycle of freezing/thawing. IC50s for ATPase activity correlate well with available publication on the HRO761 and VVD214 with 220 nM and 3.5µM respectively. The panel of selectivity testing, developed by the Eurofins Discovery team, showed high selectivity of the two compounds for WRN. ATPase activity assay pharmacologies of the two compounds were validated in unwinding activity assay . IC50s were 29nM and 6.4µM respectively. Reversibility testing showed that HRO761, is a reversible inhibitor and that VVD214 is an irreversible inhibitor. WRN mass spectrometry binding assay was successfully developed using HRO761, as reference compound. The binding assay using ATP as nonspecific ligand demonstrates a decrease in HRO761 binding to its site suggesting a competition or a negative cooperativity. Enzymatic assay showed that HR0761 is a mixed ATP allosteric inhibitor. Further experiment demonstrates that ATP prevent HR0761 binding when it binds first to the helicase. In this project, we have performed a complete characterization of two different compounds MOA. We were capable of validating HRO761 as an allosteric inhibitor with ATP negative cooperativity and VVD2014 as covalent inhibitor. This demonstration provides strong evidence that the developed assays are well suited for helicase drug candidate discovery, development and characterization. These will help providing innovative solution to fight against cancer an especially the one involving WRN over expression. Citation Format: Thibault Annaval, Jeremy Lhomedet, Amelie Moreau, Chantal Rein, Alastair King, Sophie Raynal, Vanessa Jahnke. Biochemical tools to characterize specific Werner syndrome helicase inhibitors and accelerate drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5490.