Fluorovinylsulfones and -Sulfonates as Potent Covalent Reversible Inhibitors of the Trypanosomal Cysteine Protease Rhodesain: Structure–Activity Relationship, Inhibition Mechanism, Metabolism, and In Vivo Studies

Abstract

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 α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, αfluorovinylsulfones/-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 nonfluorinated 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 EC 50 values (0.14/0.80 μM). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations.

Identification, Characterization, and Synthesis of Natural Parasitic Cysteine Protease Inhibitors: Pentacitidins Are More Potent Falcitidin Analogues

“…Rhodesain (Rhod), − Staphylococcus aureus sortase A (SrtA), and human matriptase-2 (TMPRSS6) were expressed and purified as published previously, cathepsins B and L (CatB and CatL; human liver, Calbiochem) and α-chymotrypsin (α-CT; Sigma-Aldrich, St. Louis, MO, United States) were purchased. For these proteases except SrtA, fluorescence increase upon cleavage of the fluorogenic substrates was monitored without incubation with a TECAN Infinite F200 Pro fluorimeter (excitation λ = 365 nm; emission λ = 460 nm) in white, flat-bottom 96-well microtiter plates (Greiner Bio-One, Kremsmünster, Austria) with a total volume of 200 μL.…”

Identification, Characterization and Synthesis of Natural Parasitic Cysteine Protease Inhibitors – More Potent Falcitidin Analogs

“…Fluorometric Assays. Rhodesain (Rhod), [48][49][50] Staphylococcus aureus sortase A (SrtA) 51 and human matriptase-2 (TMPRSS6) 52 were expressed and purified as published previously, Molecular docking. Molecular docking was performed against falcipain-2 (complex with E-64, PDB-ID 3BPF), 57 falcipain-3 (complex with Leupeptin, PDB-ID 3BPM) 57 and chymotrypsin (complex with D-Leucyl-L-phenylalanyl-p-fluorobenzylamide PDB-ID 1AFQ).…”

Development of Reduced Peptide Bond Pseudopeptide Michael Acceptors for the Treatment of Human African Trypanosomiasis

“…Rhodesain plays essential roles both in disease progression and the survival of the parasite: in fact, it is involved in the crossing of the blood-brain barrier, which promotes the neurological stage [21,22], and in the elusion of host immune response, since it is implicated in the formation of the variant surface glycoproteins (VSGs) expressed on the Trypanosoma coat [23,24]. In recent decades, several classes of potent peptide-based rhodesain inhibitors have been developed, while peptidomimetics and small molecules have also shown remarkable activity [25][26][27][28][29][30][31][32][33][34][35]. Unfortunately, although many ligands exhibited noteworthy inhibitory properties, most of them showed poor selectivity towards the target enzyme.…”