Unravelling WRN helicase: Structural Insights Reveal Conformational States and Opportunities for MSI-H Cancer Drug Discovery

Werner syndrome helicase (WRN) is a RecQ-family DNA helicase essential for genome maintenance and is a synthetic lethal target in microsatellite instability-high (MSI-H) cancers. Despite its therapeutic promise, the conformational dynamics that enable WRN to unwind DNA, and how inhibitors disrupt this activity, remains poorly understood. Here, we present crystal structures of apo WRN and WRN bound to single-stranded DNA (ssDNA), capturing key conformations in the helicase catalytic cycle. These structures reveal how WRN engages DNA through conserved polar and aromatic interactions, and how domain rearrangements, including an ordering of the aromatic-rich loop (ARL), drive directional translocation. Biochemical and biophysical data demonstrate how nucleotide and inhibitor binding remodel these conformations and suggest that known clinical inhibitors (HRO761 and VVD-133214) function by locking WRN in inactive, 'off-DNA' states. Resistance emerged rapidly in vitro, through acquired point mutations as well as altered WRN expression. Together, our findings provide a structural framework for the WRN structural cycle and support the development of next-generation 'on-DNA' inhibitors to overcome resistance Overall design: Samples were collected to charactersie the changes in gene expression of cell lines after acute or chronic treatment of WRN inhibitors to identify changes. One million cells of 3 biological replicates were washed with PBS and snap-frozen in dry ice. Samples were collected from HCT116, 0.1% DMSO treated HCT116, HRO761 and VVD-133214 chronically treated HCT116 in the early (HRO761: 1uM; VVD-133214: 600nM) and late (HRO761: 13uM; VVD-133214: 3uM) stages of resistance development, and HRO761 and VVD-13321 acute treated HCT116. For acute treatment, the 0.1% DMSO HCT116 cell line was grown in 13uM HRO761 or 3uM VVD-13321 for one passage.