Fragment-based structure-activity relationship analysis of CK-571 reveals non-interacting groups drive smooth muscle...

Smooth muscle myosin-2 (SMM) is a promising target for treating asthma and COPD, but selective inhibition remains challenging due to the high conservation of myosin-2 isoforms. CK-571 is the first potent and selective SMM inhibitor, despite binding to an allosteric site conserved across isoforms. We performed a fragment-based structure-activity relationship analysis to deconstruct CK-571 and define the role of each segment. The isoquinoline-carbamate and chloro-fluorobenzyl moieties were found to be inactive when isolated. Surprisingly, the presence of a methyl group and a solvent-exposed pair of non-interacting dihydroxyl groups dramatically enhanced potency by up to 100-fold, despite no direct contact with the protein. These findings highlight the critical contribution of non-interacting, flexible groups in optimizing ligand potency and selectivity for conserved targets and establish a framework for the development of improved SMM-targeted therapeutics.