For the first time the V carinata
For the first time, the V. carinata transcriptome was analyzed and its transcript coding sequences comprehensively characterized. The non-redundant contigs encoding putative CysPs were identified and their Triclosan patterns in different tissues were investigated. Our study show that four proteases; pyroglutamyl peptidase, metacaspase and the papain-like MF770496 and MF0497, are present in higher levels in the leaf base. It suggests their relevance in the region of bromeliad tanks with a potential impact in protein turnover and degradation as well as in nitrogen assimilation from microorganism present in this environment. The well documented molecular functions of the proteases, and the transcriptome and expression dataset provided here will extend the mechanism underlying the processes of protein degradation and nitrogen metabolism in Bromeliads.
Introduction Cysteine proteases play essential roles in the life cycles of various protozoan organisms making them attractive targets for new antiparasitic drugs. Especially for the neglected diseases HAT (Human African Trypanosomiasis) and the various forms of leishmaniasis, but also for malaria and Chagas disease, new drugs are urgently needed. The parasites causing these diseases express numerous related cysteine proteases that belong to the papain family; according to the MEROPS database system [1,2] they are classified as clan CA, family C1 proteases and share structural similarity to human cathepsins. In detail, these are the cathepsin L-like proteases rhodesain from Trypanosoma brucei rhodesiense, falcipains 2 and 3 from Plasmodium falciparum, cruzain from Trypanosoma cruzi, and various cathepsin L-like proteases from Leishmania species, e.g. LmCPB2.8 from Leishmania mexicana. There are also some cathepsin B-like proteases expressed by the parasites, e.g. TbCatB from T. b. rhodesiense or the Leishmania CPCs. Some of these proteases have already been shown to be suitable targets for new antiparasitic drugs with the vinyl sulfone K11777 which is in preclinical studies against Chagas disease as one of the most promising compounds [, , ]. In previous studies we identified aziridine-based peptides as inhibitors selective for cathepsin L-like cysteine proteases [, , , ]. These consist of a trans-configured dibenzyl aziridine-2,3-dicarboxylate fragment Azi(OBn)2 (i.e. (S,S) or (R,R) configuration) and the dipeptide Boc-Leu-Pro attached to the aziridine nitrogen. Among the various stereoisomers of the peptide Boc-Leu-Pro-Azi(OBn)2 some displayed selectivity for parasite proteases over human cathepsin L (Table 1). Some compounds concomitantly showed antileishmanial activity against L. major promastigotes and amastigotes [, , ], namely compounds I, II, V (Table 1). Among these derivatives the most promising is compound V which is selective for the leishmanial cathepsin L-like enzyme over the mammalian cathepsins, and which displays good antileishmanial activity. In an ongoing study derivatives with various dipeptides attached to the aziridine nitrogen were synthesized in which the (S)-Leu residue of compound V was exchanged against another hydrophobic (S)-configured amino acid (Ala, Val, Ile, Nva, Nle, Chg, Cha, Phg, Phe, hPhe, Trp) . Most of these derivatives showed inhibition of the Leishmania enzyme in the same range as compound V, but only the inhibitors with Val and Ile also displayed antileishmanial activity against L. major promastigotes and amastigotes: (EC50 [μM] amastigotes/promastigotes: Val: 34.8/2.2 ± 0.6; Ile: 9.8/2.0 ± 0.6: toxicity against J774.4 macrophages > 250 μM for both). Compound V, in contrast to its Val and Ile analogs, inhibits both the cathepsin L-like CPB2.8 and the L. major cathepsin B-like enzyme CPC (Ki = 18.2 μM) . Also in L. major lysates, inhibition of both cathepsin L- and cathepsin B-like Leishmania proteases had been observed with this compound  in contrast to compounds I and II. Thus, we decided to extend our studies towards Leishmania protease inhibitors based on this most promising inhibitor V. Previous studies had already shown that dibenzyl esters are superior to diethyl esters in terms of enzyme inhibition and antiparasitic activity . On the other hand, a free acid group at the aziridine ring has several advantages over the diesters. First, the water solubility is enhanced in comparison to diesters. Second, a free acid improves affinity to cysteine proteases by forming an ionic interaction with the histidinium residue of the active site [7,11]. Furthermore, the free acids show fast covalent inhibition of cysteine proteases since this ionic interaction leads to a positioning of the electrophilic aziridine ring, which is favorable for the nucleophilic ring opening by the active site cysteine residue . The new target compounds thus should consist of the dipeptide sequence Boc-(S)-Leu-(R)-Pro attached to the aziridine nitrogen, and either of two hydrophobic ester groups or of a free acid and a hydrophobic ester group at the aziridine ring (Fig. 1).