Attempts to Achieve Targeted Covalent Inhibition of Hsp90β

Recent advances in targeted covalent inhibition have broadened the therapeutic landscape via the development of warheads that target various nucleophilic amino acids. Although cysteine was once the primary focus of such efforts, lysine has emerged as another appealing residue for covalent modification, due to its high prevalence within the proteome, functional importance, and low mutation rate. Despite this amino acid's reduced nucleophilicity at physiological pH, rational, structure-based techniques have enabled the identification of inhibitors that exploit the unique protein microenvironment that a given lysine occupies. Strategies to inhibit Hsp90 via covalent modification of Lys58 include the use of N-acyl-N-aryl sulfonamide (ArNASA) warheads and sulfur(VI) fluoride exchange (SuFEx) chemistry, both of which have demonstrated enhanced cellular selectivity and potency. However, such compounds fail to distinguish between the > 95% identical nature of the ligand binding sites in cytosolic Hsp90α and Hsp90β. In this study, the incorporation of electrophilic warheads onto Hsp90β-selective inhibitors resulted in compounds that demonstrate favorable binding to Hsp90β, yet subsequent analyses to confirm covalent modification of Lys58 were inconclusive. Nevertheless, the kinetics of inhibitor binding to the Hsp90 N-terminal ATP-binding pocket were obtained, whereby some compounds exhibited their highest affinity towards Hsp90β after a 2 h incubation.