Sara Amberntsson, Alison J Foster, Bhavik Chouhan, Stephen Wilkinson, Stephanie Harlfinger, Graham Smith, Jason G Kettle, Michael Niedbala, Stefan Kavanagh, Dominic P Williams
Toxicology Research, 2025, 14, 3, tfaf054,
https://doi.org/10.1093/toxres/tfaf054
Interest in inhibiting target proteins through covalent binding mechanisms has increased in the last decade due to the potential for beneficial pharmacological properties. However, the inherent targeted covalent inhibitor (TCI) adverse off-target reactivity risk requires a mitigation strategy early during drug discovery. The aim of this research was to design a pre-clinical hepatic safety assessment strategy for TCIs considering risk associated with electrophilic warhead reactivity and reactive metabolites formation at clinically-relevant plasma concentrations.
The mitigation strategy was applied to compound 35, a potent irreversible inhibitor to KRASG12C. Drug induced liver injury was assessed in primary human hepatocyte spheroids. GSH and ATP depletion were investigated for compound 35 and 6 other marketed TCIs containing an acrylamide warhead which binds irreversibly to cysteine-containing target proteins. None of the TCIs showed GSH depletion prior to ATP depletion after 7-days exposure, suggesting that GSH depletion was not driving cytotoxicity in the spheroids. The calculated hepatotoxicity margin towards plasma exposure of 2.5 for compound 35 was found to be in the same range as for the two KRASG12Cinhibitors adagrasib and sotorasib, with clinically reported treatment-related adverse aminotransferase elevations leading to dose modifications. The safety evaluation reported here suggests no negative discrepancy in liver toxicity for compound 35 versus similar approved TCI’s. Finally, the risk associated with detected oxidative metabolites was further mitigated as the pan-CYP450 inhibitor 1-aminobenzotriazole (ABT) had no effect on the cytotoxicity response following incubation of compound 35 in the presence and absence of ABT.
