Fluorovinylsulfones And -Sulfonates As Potent Covalent Reversible Inhibitors Of The Trypanosomal Cysteine Protease Rhodesain: Structure-Activity Relationship, Inhibition Mechanism, Metabolism, And In Vivo Studies

Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of alpha-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, alpha-fluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (K-i = 3 nM). Furthermore, the non-fluorinated 2d-(H) shows favorable metabolism and biodistribution by accumulation in mice brain tissue after intraperitoneal and oral administration. The highest antitrypanosomal activity was observed for inhibitors with an N-terminal 2,3-dihydrobenzo[b][1,4]dioxine group and a 4-Me-Phe residue in P2 (2e/4e) with nanomolar EC50 values (0.14/0.80 mu M). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations.