In agreement with earlier studies on CPs (15), most of the compounds containing ethyl ester moieties, namely, compounds s1 to s8, did not inhibit or only weakly inhibited the enzymes. the NTDs is the group of most neglected diseases, affecting the poorest, mainly rural areas, including leishmaniases, sleeping sickness (African trypanosomiasis), and Chagas’ disease (3). These three NTDs have the highest rates of death. However, the NTD drug discovery pipeline is almost empty, thus leading to a lack of efficient and safe drugs (2, 4). Because of climate warming and tourism, the occurrence of leishmaniasis is also reported in says around the Mediterranean Sea (1). Leishmaniasis is usually caused by more than 20 species of protozoan parasites belonging to the genus genome encodes a total of 65 CPs, grouped into 4 clans [CA, CD, CF, and PC(C)] and 13 families. Leishmanial CPs belonging to the CAC1 family are the lysosomal cathepsin L (CL)-like enzymes CPA and CPB, as well as the cathepsin B (CB)-like enzyme CPC (19). They share some homology with the related mammalian enzymes; however, their substrate specificities are different. While human CB accepts an Arg at the P2 position (benzyloxycarbonyl-Arg-Arg-7-amino-4-methylcoumarine [Cbz-Arg-Arg-AMC] is usually a quite good substrate), the leishmanial homolog CPC does not, preferring Phe in that position, comparable to human and leishmanial CL enzymes (20). In the case of CPs, it was shown that these enzymes are essential for parasite growth, differentiation, pathogenicity, and virulence (19, 21, 22). However, the extent to which the additional inhibition of related host EPZ020411 cathepsins may have an anti-infective effect or, in contrast, may even support the infection is not yet fully comprehended (23,C25). Therefore, it is necessary to develop inhibitors selective for cysteine proteases. In previous studies, we recognized two peptidomimetic aziridine-2,3-dicarboxylate-based inhibitors, Boc-(CPC), as documented with a biotin-tagged derivative of 13b (27). The inhibitor compound 13b induced an accumulation of undigested debris in autophagy-related lysosome-like vacuoles in experiment was carried out using the BALB/c mouse model of EPZ020411 contamination. After application of compound 13b, a poor exacerbation of the contamination was observed; this was characterized KLF10/11 antibody by a significantly increased secretion of the Th2 cell cytokine interleukin 4 by murine splenic cells. This effect was probably caused by inhibition of murine CL (data not shown). This is in accordance with studies by the Katunuma group indicating that inhibition of human CL results in the potentiation of Th2-type immune responses and thus leads to an exacerbation of inflammation (23,C25). These studies also showed that CB-specific inhibitors can switch T-cell development from Th2- to Th1-type immune responses in mice, resulting EPZ020411 in an amelioration of contamination. In summary, there is an urgent need for inhibitors which selectively inhibit the CL-like parasite CPs and do not affect the mammalian equivalents. There is no X-ray structure available for leishmanial papain-like CPs, making the development of selective inhibitors a matter of trial and error by synthesis and screening of a broad variety of related inhibitors. Therefore, we extended our study by synthesizing a series of aziridine-2,3-dicarboxylates based on compounds 13b and 13e as lead structures. This series comprises structural isomers (s11 to s14), derivatives with ethyl ester moieties (s1 to s8), a derivative with an extended peptide chain (s15), and derivatives with nonproteinogenic amino acids within the peptide sequence in order to improve hydrolytic stability (-Ala in s21, -aminoisobutyric acid [Aib] in s22, and norvaline [Nva], norleucine [Nle], cyclohexylglycine [Chg], cyclohexylalanine [Cha], and phenylglycine [Phg] in s26 to s30 and s32). The influence of the configuration of the three-membered aziridine ring (or CPC), and a recombinantly expressed CL-like protease from (promastigote lysates. This was done with the compounds alone and in combination with the standard cysteine protease inhibitors E64 and CA074 in order to evaluate the extent to which the proteolytic activity is usually further decreased by the addition of aziridine-based cysteine protease inhibitors. The most promising compounds were analyzed for the ability to inhibit the EPZ020411 growth and viability of promastigotes and amastigotes and for cytotoxicity against the macrophage cell collection J774.1. MATERIALS AND METHODS Syntheses. Synthesis of the potential inhibitors.