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.

A novel synthetic and processing strategy for converting elemental sulfur into polymeric and nanocomposite materials is reported. We describe a facile one-pot reaction using elemental sulfur and oleylamine as comonomers to prepare high sulfur content copolymers and lead sulfide nanoparticle (PbS NP) nanocomposites. This process enables the preparation of solution processable copolymers and nanocomposites, where the loading and dispersion of PbS NP inclusions could be precisely controlled.We demonstrate the dual roles of oleylamine with sulfur for both the copolymerization of sulfur copolymers as well as the in situ synthesis of PbS NPs in a one-pot fashion.

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

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.

Electrophilic (het)arenes can undergo reactions with nucleophiles yielding πor Meisenheimer (σ-) complexes or the products of the S N Ar addition/elimination reactions. Such building blocks have only rarely been employed for the design of enzyme inhibitors. Herein, we demonstrate the combination of a peptidic recognition sequence with such electrophilic (het)arenes to generate highly active inhibitors of disease-relevant proteases. We further elucidate an unexpected mode of action for the trypanosomal protease rhodesain using NMR spectroscopy and mass spectrometry, enzyme kinetics and various types of simulations. After hydrolysis of an ester function in the recognition sequence of a weakly active prodrug inhibitor, the liberated carboxylic acid represents a highly potent inhibitor of rhodesain (K i = 4.0 nM). The simulations indicate that, after the cleavage of the ester, the carboxylic acid leaves the active site and re-binds to the enzyme in an orientation that allows the formation of a very stable π-complex between the catalytic dyad (Cys-25/His-162) of rhodesain and the electrophilic aromatic moiety. The reversible inhibition mode results because the S N Ar reaction, which is found in an alkaline solvent containing a low molecular weight thiol, is hindered within the enzyme due to the presence of the positively charged imidazolium ring of His-162. Comparisons between measured and calculated NMR shifts support this interpretation.