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 (k(inact)) typically involve increasing the reactivity of the electrophile, which increases the risk of off-target modification. Here, we employ an alternative approach for increasing k(inact) of a lysine targeted covalent Hsp90 inhibitor, independent of the reversible binding affinity (K-i) 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 LysS8 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 demonstrate selective covalent targeting of cellular Hsp90, which results in a prolonged heat shock response despite concomitant degradation of the covalent ligand/Hsp90 complex. Our work highlights the potential of engineering ligand conformational constraints to dramatically accelerate covalent modification of a distal, poorly nucleophilic lysine on the surface of a protein target.