INHIBITORS of the MAJOR CYSTEINYL PROTEINASE (GP57/51) IMPAIR HOST-CELL INVASION and ARREST the INTRACELLULAR DEVELOPMENT of TRYPANOSOMA-CRUZI INVITRO

Peptidyl diazomethane (PDAM) derivatives, a class of irreversible inhibitors for cysteine proteinase, were screened for the ability to impair Trypanosoma cruzi invasion and intracellular development in primary cultures of heart muscle cells (HMC). T. cruzi GP57/51, a purified cysteinyl proteinase, and the substrate Z-Phe-Arg-NHMec were used to determine inhibition rate constants (k'+2) by continuous kinetic assays. the k'+2 values ranged from 25 400 to 2 800. the best inhibitors of GP57/51 had bulky hydrophobic residues in the P1 position (in addition to P2), the S1 sub-site specificity of the enzyme being thus similar to mammalian cathepsin L. the effects of these PDAM on parasite infectivity were then investigated. the ability to invade HMC was markedly impaired when trypomastigotes were briefly exposed to 10-mu-M of Z-(S-Bzl)Cys-Phe-CHN2. Striking effects were observed when PDAM were added to HMC cultures that had been previously infected with trypomastigotes: Z-(S-Bzl)Cys-Phe-CHN, with an IC50 of 0.4-mu-M, and less markedly Z-Phe-Phe-CHN, and Z-Tyr-Phe-CHN2 (or Z-Phe-Tyr-CHN2) blocked amastigote replication as well as their transformation into trypomastigotes, thereby arresting intracellular development. Bz-Phe-Gly-CHN2, in contrast, failed to display antiparasite activity. Direct characterization of the target cysteinyl proteinase was sought, by incubating viable amastigotes or infected HMC with Z-[I-125]Tyr-Phe-CHN2. Affinity labeling implicated GP57/51 as the major cysteinyl proteinase target for this probe. We propose that T. cruzi intracellular development is critically dependent on GP57/51 (cruzipain). Selective inhibitors for this cysteinyl proteinase may have therapeutic potential.UNIV FED RIO de JANEIRO,INST BIOFIS CARLOS CHAGAS FILHO,CCS CID UNIV,BR-21944 RIO de JANEIRO,BRAZILINST OSWALDO CRUZ,DEPT ULTRAESTRUT & BIOL CELULAR,RIO de JANEIRO,BRAZILINST BUTANTAN,SERV FARMACOL,São Paulo,BRAZILESCOLA PAULISTA MED SCH,INST BIOFIS,BR-04023 São Paulo,BRAZILESCOLA PAULISTA MED SCH,INST BIOFIS,BR-04023 São Paulo,BRAZILWeb of Scienc

Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8

Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity. The data analysis revealed that the presence of heparin affects all steps of the enzyme reaction: (i) it decreases 3.5-fold the k1 and 4.0-fold the k−1, (ii) it affects the acyl-enzyme accumulation with pronounced decrease in k2 (2.7-fold), and also decrease in k3 (3.5-fold). The large values of ΔG = 12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys25)-S−/(His163)-Im+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme. Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface