Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Development of phenotypic assays for identifying novel blockers of L-type calcium channels in neurons.

L-type calcium channels (LTCCs) are highly expressed in the heart and brain and are critical for cardiac and neuronal functions. LTCC-blocking drugs have a long and successful record in the clinic for treating cardiovascular disorders. In contrast, establishment of their efficacy for indications of the central nervous system remains challenging given the tendency of existing LTCC drugs being functionally and mechanistically more selective for peripheral tissues. LTCCs in vivo are large macromolecular complexes consisting of a pore-forming subunit and other modulatory proteins, some of which may be neuro-specific and potentially harbor mechanisms for neuronal selectivity. To exploit the possibility of identifying mechanistically novel and/or neuro-selective blockers, we developed two phenotypic assays-a calcium flux-based primary screening assay and a patch clamp secondary assay, using rat primary cortical cultures. We screened a library comprised of 1278 known bioactive agents and successfully identified a majority of the potent LTCC-blocking drugs in the library. Significantly, we identified a previously unrecognized LTCC blocker with a novel mechanism, which was corroborated by patch clamp and binding studies. As such, these phenotypic assays are robust and represent an important step towards identifying mechanistically novel and neuro-selective LTCC blockers.