Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.

American trypanosomiasis, or Chagas' disease, is the leading cause of heart disease in Latin America. Currently there is an urgent need to develop antitrypanosomal therapy due to the toxicity of existing agents and emerging drug resistance. A novel series of potent thio semicarbazone small-molecule inhibitors of the Trypanosoma cruzi cysteine protease cruzain have been identified. Some of these inhibitors have been shown to be trypanocidal. We initially discovered that 3'-bromopropiophenone thio semicarbazone (1i) inhibited cruzain and could cure mammalian cell cultures infected with T. cruzi. 3'-Bromopropiophenone thio semicarbazone showed no toxicity for mammalian cells at concentrations that were trypanocidal. Following this lead, more than 100 compounds were designed and synthesized. A specific structure-activity relationship (SAR) was established, and many potent analogues with IC(50) values in the low nanomolar range were identified. Eight additional analogues were trypanocidal in a cell culture assay, and this indicates that aryl thio semicarbazone is a productive scaffold for killing the parasites. Kinetic studies show that these are time-dependent inhibitors. Molecular modeling studies of the enzyme-inhibitor complex have led to a proposed mechanism of interaction as well as insight into the SAR of the thio semicarbazone series. The nonpeptide nature of this series, small size, and extremely low cost of production suggest this is a promising direction for the development of new antitrypanosome chemotherapy.

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Schistosomiasis is a global health problem caused by several species of schistosome blood flukes. The initial stage of infection is invasion of human skin by a multicellular larva, the cercaria. We identified proteins released by cercariae when they are experimentally induced to exhibit invasive behavior. Comparison of the proteome obtained from skin lipid-induced cercariae (the natural activator), a cleaner mechanical induction procedure, and an uninduced proteomic control allowed identification of protein groups contained in cercarial acetabular gland secretion versus other sources. These included a group of proteins involved in calcium binding, calcium regulation, and calcium-activated functions; two proteins (paramyosin and SPO-1) implicated in immune evasion; and protease isoforms implicated in degradation of host skin barriers. Several other protein families, traditionally found as cytosolic proteins, appeared concentrated in secretory cells. These included proteins with chaperone activity such as HSP70, -86, and -60. Comparison of the three experimental proteomes also allowed identification of protein contaminants from the environment that were identified because of the high sensitivity of the MS/MS system used. These included proteins from the intermediate host snail in which cercariae develop, the investigator, and the laboratory environment. Identification of proteins secreted by invasive larvae provides important new information for validation of models of skin invasion and immune evasion and aids in rational development of an anti-schistosome vaccine.

We have synthesized a library of thiosemicarbazones and screened them against three parasitic cysteine proteases, cruzain, falcipain-2, and rhodesain, and against the respective parasite sources of these three proteases, Trypanosoma cruzi, Plasmodium falciparum, and Trypanosoma brucei. The screens identified compounds that were effective against the enzymes and the parasites but also some compounds that were parasiticidal despite a lack of activity against the proteases. Several compounds were effective in killing all tested parasites. These promising lead compounds were tested for general toxicity in mice, and only one produced observable toxicity after 62 h. Our results suggest that thiosemicarbazones represent validated drug leads that kill several species of protozoan parasites through the inhibition of cysteine proteases as well as other novel targets.

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Cysteine proteases are an important class of enzymes involved in the degradative processing of peptides and proteins. 1,2 They are ubiquitous in nature and play vital roles in numerous physiological processes including arthritis, osteoporosis, Alzheimer's disease, cancer cell invasion, and apoptosis. 1-3 Cysteine proteases are also essential to the life cycles of many pathogenic protozoa. 4,5 One such parasite is Trypanosoma cruzi, the etiologic agent of Chagas' disease. Cruzain, 6,7 the major cysteine protease of T. cruzi, has been identified as a potential therapeutic target for treatment of Chagas' disease. [7][8][9] Several strategies have been pursued in the design of cysteine protease inhibitors. 2,10,11 Peptidyl aldehydes, 12 diamino ketones, 13 and nitriles 14 are reversible inhibitors that form hemithioacetals, peptide ketals, and thioimidates, respectively, with the thiol of the active site cysteine residue, mimicking the initial covalent enzyme adduct in normal proteolytic turnover. Epoxysuccinyl derivatives, 15 peptidyl Michael acceptors, 16-18 (acyloxy)methyl ketones, 19 and halomethyl ketones are examples of inhibitors which irreversibly inactivate cysteine proteases via alkylation of the active site cysteine residue. Several classes of nonpeptidic reversible inhibitors of cysteine proteases also have been described. [20][21][22] In connection with efforts to develop potent and selective inhibitors of cruzain, we became interested in the vinyl sulfone inhibitor series first introduced by Hanzlik 17 and further developed by Palmer et al. 18,23 Compound 1a is a potent and selective inhibitor of cruzain, with a second-order rate constant (k inact /K i ) of 203 000 s -1 M -1 . 18 Inhibitors 1a and especially 1b have also proven highly effective against T. cruzi, both in tissue culture and in vivo experiments (mouse model). 24 Although considerable effort has been devoted to the optimization of interactions of inhibitors with the cruzain S 1 and S 2 binding sites, 18,23,25 virtually nothing is known about the interactions of substrates or inhibitors with the S 1 ′ and S 2 ′ sites. The prime site region in cruzain contains a large open surface defined by Trp 177, and available X-ray structures suggest that there is considerable room for prime site inhibitor binding. 7,13,22,25 A recent X-ray structure of cathepsin K, the active site of which is homologous to that of cruzain, with covalently bound APC3328, a dipeptidyl phenyl vinyl sulfone inhibitor related to 1b, reveals that the phenyl residue of the phenyl sulfonyl unit does not make optimal interactions with prime site residues. 26 Accordingly, we decided to probe the possibility that additional selectivity and potency in the vinyl sulfonyl series could be achieved by extending the inhibitor structure into the prime site region, via modification of the sulfonyl substituent as suggested by structure 2. However, we anticipated that it might be easier to synthesize a family of vinyl sulfonamides 3 or vinyl sulfonate esters 4, using the vinyl sulfonyl chl...

The series of vinyl-sulfone-based inhibitors examined in complex with cruzain was designed to probe recognition and binding potential of an aromatic-rich region of the enzyme. Analysis of the interactions formed shows that aromatic interactions play a less significant role, whereas the strength and importance of hydrogen bonding in the conformation adopted by the inhibitor upon binding to the enzyme was highlighted. A derivative of one inhibitor examined is currently under development as a therapeutic agent against Chagas' disease.