Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide

17 beta-Hydroxysteroid dehydrogenase type 1 (17 beta-HSD1) plays a pivotal role in the progression of estrogen-related diseases because of its involvement in the biosynthesis of estradiol (E2), constituting a valuable therapeutic target for endocrine treatment. In the present study, we successfully cocrystallized the enzyme with the reversible inhibitor 2-methoxy-16 beta-(m-carbamoylbenzyl)-E2 (2-MeO-CC-156) as well as the enzyme with the irreversible inhibitor 3-(2-bromoethyl)-16 beta-(m-carbamoylbenzy1)-17 beta-hydroxy-1,3,5(10)-estratriene (PBRM). The structures of ternary complexes of 17 beta-HSD1-2-MeO-CC-156- NADP(+) and 17 beta-HSD1-PBRM-NADP(+) comparatively show the formation of a covalent bond between His(221) and the bromoethyl side chain of the inhibitor in the PBRM structure. A dynamic process including beneficial molecular interactions that favor the specific binding of a low-reactivity inhibitor and subsequent N-alkylation event through the participation of His(221) in the enzyme catalytic site clearly demonstrates the covalent bond formation. This finding opens the door to a new design of alkyl halide-based specific covalent inhibitors as potential therapeutic agents for different enzymes, contributing to the development of highly efficient inhibitors.