We propose a structure-based protocol for the development of customized covalent inhibitors. Starting from a known inhibitor, in the first and second steps appropriate substituents of the warhead are selected on the basis of quantum mechanical (QM) computations and hybrid approaches combining QM with molecular mechanics (QM/MM). In the third step the recognition unit is optimized using docking approaches for the noncovalent complex. These predictions are finally verified by QM/MM or molecular dynamic simulations. The applicability of our approach is successfully demonstrated by the design of reversible covalent vinylsulfone-based inhibitors for rhodesain. The examples show that our approach is sufficiently accurate to identify compounds with the desired properties but also to exclude nonpromising ones.

Dipeptidyl nitroalkenes are potent reversible inhibitors of cysteine proteases. Inhibitor 11 resulted to be the most potent one with K i values of 0.49 and 0.44 nM against rhodesain and cruzain, respectively. According to enzymatic dilution and dialysis experiments, as well as computational and NMR studies, dipeptidyl nitroalkenes are tightly binding covalent reversible inhibitors.KEYWORDS: Rhodesain, cruzain, inhibitors, Chagas' disease, sleeping sickness T he target proteases of the presented study, rhodesain and cruzain, are parasite proteases, which belong to the papain-family of cysteine proteases.1,2 They are related to human cathepsins.1,2 Rhodesain is expressed by the protozoa Trypanosoma brucei rhodesiense, which causes the African sleeping sickness. 3,6,7 Cruzain is expressed by T. cruzi, the parasite causing Chagas' disease occurring in South and Central America.4,5 Both proteases are essential for the life cycles of the pathogens. Consequently, their inhibition is an important strategy for the treatment of these diseases. 8,9 K11777 (Figure 1a), a dipeptidyl vinyl sulfone, irreversibly inactivates cysteine proteases 10 by conjugate addition of the thiolate of the cysteine at the active site to the double bond. The resulting carbanion is subsequently protonated driving the process thermodynamically to the more stable enzyme− inhibitor complex (Figure 1b).Irreversible inhibitors can give rise to undesired side reactions. Turning dipeptidyl vinyl sulfones into compounds that reversibly react with thiols by introducing thioethers or halogen atoms into the structure has been reported. 11 In case of the halogenated vinylsulfones this leads to covalent reversible inhibition. 12We envisioned dipeptidyl nitroalkenes as inhibitors for cysteine proteases. Based upon the inhibition mechanism of

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A potent and highly selective anhydride-based inhibitor of Leishmania mexicana cysteine protease CPB2.8 (IC50 = 3.7 μM) was investigated by inhibition assays, NMR biomimetic experiments and docking studies.

Novel rhodesain inhibitors were developed by combining an enantiomerically pure 3-bromoisoxazoline warhead with a 1,4-benzodiazepine scaffold as specific recognition moiety. All compounds were proven to inhibit rhodesain with K i values in the low-micromolar range. Their activity towards rhodesain was found to be coupled to an in vitro antitrypanosomal activity, with IC 50 values ranging from the mid-micromolar to a low-micromolar value for the most active rhodesain inhibitor (R,S,S)-3. All compounds showed a good selectivity against the target enzyme since all of them were proven to be poor inhibitors of human cathepsin L.