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Abstract In recent years, helicases have moved to the forefront of research as novel drug targets for a variety of human diseases, including cancer. As critical components of the cell cycle and DNA repair, these enzymes offer an attractive point of therapeutic intervention. Recent bioinformatic endeavours such as the Cancer DepMap have further highlighted the essentiality of these proteins in the progression of numerous cancers. Inhibitors of Werner helicase (WRN) entered Phase I evaluation in 2023, after studies highlighted its role in tumours with microsatellite instability (MSI) and impaired mismatch repair (MMR). Building on these advances, helicases now represent a significant drug target class and are the subject of intense research. However, these targets present significant complexities in their prosecution. Hit finding and robust validation of these putative active compounds remains challenging, with high attrition rates and significant sensitivity to false positives. Resultant hits require thorough biological characterisation via both biochemical and biophysical methods, alongside determination of selectivity against other closely related family members, to increase confidence in their provenance before commencing a detailed drug discovery project. To exemplify these approaches, we have profiled three commercially available WRN inhibitors from two chemical series through a selection of biochemical and biophysical assays. The three compounds exhibit excellent selectivity against 6 distinct helicase/translocase targets. In Michaelis-Menten mechanism of Inhibition studies, we defined contrasting mechanisms of inhibition for the two series and were also able to identify time dependent inhibition with one compound series. Analysing binding interactions of the compounds against protein and protein:substrate complexes, via a fluorescent thermal shift assay (FTSA) and microscale thermophoresis (MST), enabled us to further understand and interrogate the mechanism of inhibition. Herein, we describe some of our experiences, learnings and best practices when prosecuting these compelling, but challenging, targets. Citation Format: Yael Mamane, Zoe Caple, Katie Chapman, Ian Henderson, Allan Jordan, Vanessa Lyne, Cynthia Okoye, Laurent Rigoreau, Ganesh Kadamur, Stuart Thomson, Chris Tomlinson, Jana Wolf. Unwinding the complexities of helicases as compelling drug targets in oncology abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A003.
Mamane et al. (Mon,) studied this question.