Ligand Conformational Bias Drives Enantioselective Modification of a Surface-Exposed Lysine on Hsp90

Targeted covalent modification of surface-exposed lysines is challenging due to their low intrinsic reactivity and high prevalence throughout the proteome. Strategies for optimizing the rate of covalent bond formation by a reversibly bound inhibitor (kinact) typically involve increasing the reactivity of the electrophile, which increases the risk of off-target modification. Here, we employ an alternative approach for increasing kinact of a lysine-targeted covalent Hsp90 inhibitor, independent of the reversible binding affinity (Ki) or the intrinsic electrophilicity. Starting with a noncovalent ligand, we appended a chiral, conformationally constrained linker, which orients an arylsulfonyl fluoride to react rapidly and enantioselectively with Lys58 on the surface of Hsp90. Biochemical experiments and high-resolution crystal structures of covalent and noncovalent ligand/Hsp90 complexes provide mechanistic insights into the role of ligand conformation in the observed enantioselectivity. Finally, we demonstra...