Two approaches to discovering and developing new drugs for Chagas disease.

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A 'proof of concept' molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Two approaches to discovering and developing new drugs for Chagas disease

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A "proof of concept" molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Potent second generation vinyl sulfonamide inhibitors of the trypanosomal cysteine protease cruzain.

A new family of potent N-alkoxyvinylsulfonamide inhibitors of cruzain have been developed. Inhibitor 13 has a second order inactivation rate constant of 6,480,000s(-1)M(-1) versus cruzain, and is also highly effective against Trypanosoma cruzi trypomastigotes in a tissue culture assay.

Secretory and endocytic pathways converge in a dynamic endosomal system in a primitive protozoan.

Leishmania are a group of primitive eukaryotic trypanosomatid protozoa that are apically polarized with a flagellum at their anterior end. Surrounding the base of the flagellum is the flagellar reservoir that constitutes the site for endocytosis and exocytosis in these organisms. In the present study, we define a novel multivesicular tubular compartment involved in the intracellular trafficking of macromolecules in Leishmania. This dynamic structure appears to subtend the flagellar reservoir and extends towards the posterior end of the cell. Functional domains of several surface-expressed proteins, such as the gp63 glycosyl phosphatidyl inositol anchor and the 3'nucleotidase/nuclease transmembrane domain were fused to green fluorescent protein. These chimeric proteins were found to traffic through the secretory pathway and, while reaching their intended destinations, also accumulated within the intracellular tubular compartment. Using various compounds that are efficient fluid-phase markers used to track endocytosis in higher eukaryotes, we showed that this tubular compartment constitutes an important station in the endocytic pathway of these cells. Based on our functional observations of its role in the trafficking of expressed proteins and endocytosed markers, this compartment appears to have properties similar to endosomes of higher eukaryotes.

Characterization of the Trypanosoma cruzi Cdc2p-related protein kinase 1 and identification of three novel associating cyclins.

Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.

Aryl ureas represent a new class of anti-trypanosomal agents.

BACKGROUND: The trypanosomal diseases including Chagas' disease, African sleeping sickness and Nagana have a substantial impact on human and animal health worldwide. Classes of effective therapeutics are needed owing to the emergence of drug resistance as well as the toxicity of existing agents. The cysteine proteases of two trypanosomes, Trypanosoma cruzi (cruzain) and Trypanosoma brucei (rhodesain), have been targeted for a structure-based drug design program as mechanistic inhibitors that target these enzymes are effective in cell-based and animal models of trypanosomal infection. RESULTS: We have used computational methods to identify new lead scaffolds for non-covalent inhibitors of cruzain and rhodesain, have demonstrated the efficacy of these compounds in cell-based and animal assays, and have synthesized analogs to explore structure activity relationships. Nine compounds with varied scaffolds identified by DOCK4.0.1 were found to be active at concentrations below 10 microM against cruzain and rhodesain in enzymatic studies. All hits were calculated to have substantial hydrophobic interactions with cruzain. Two of the scaffolds, the urea scaffold and the aroyl thiourea scaffold, exhibited activity against T. cruzi in vivo and both enzymes in vitro. They also have predicted pharmacokinetic properties that meet Lipinski's 'rule of 5'. These scaffolds are synthetically tractable and lend themselves to combinatorial chemistry efforts. One of the compounds, 5'(1-methyl-3-trifluoromethylpyrazol-5-yl)-thiophene 3'-trifluoromethylphenyl urea (D16) showed a 3.1 microM IC(50) against cruzain and a 3 microM IC(50) against rhodesain. Infected cells treated with D16 survived 22 days in culture compared with 6 days for their untreated counterparts. The mechanism of the inhibitors of these two scaffolds is confirmed to be competitive and reversible. CONCLUSIONS: The urea scaffold and the thiourea scaffold are promising leads for the development of new effective chemotherapy for trypanosomal diseases. Libraries of compounds of both scaffolds need to be synthesized and screened against a series of homologous parasitic cysteine proteases to optimize the potency of the initial leads.

Upregulation of the secretory pathway in cysteine protease inhibitor-resistant Trypanosoma cruzi.

A novel chemotherapy in development for Chagas' disease targets cruzain, the major cysteine protease of Trypanosoma cruzi. Peptidomimetic inhibitors disrupt the intracellular cycle of the parasite and rescue animals from a lethal infection. Inhibitor killing of parasites results from interruption of autocatalytic cruzain processing and transport to lysosomes, and massive accumulation of precursor protein in the Golgi complex. To further understand the mechanisms of protease processing and transport in this primitive eukaryote, and uncover potential mechanisms for resistance to these drugs, we generated cysteine-protease inhibitor (CPI)-resistant epimastigotes in vitro and investigated the mechanisms involved at the biochemical and structural levels. Resistance to 20-fold the lethal CPI concentration, achieved after a year of gradual drug increase, was accompanied by a modest decrease in growth rate. A marked increase in the number of vesicles trafficking from the Golgi complex to the flagellar pocket occurs in resistant cells. No mature protease reaches lysosomes though accumulation of endocytosed gold particles in lysosomes appears to be normal. Higher molecular mass cruzain species, consistent with complexes of cruzain precursors and inhibitor, are secreted by CPI-resistant parasites into the culture supernatant. Release of these cruzain precursors may be facilitated by an enhanced acidification of trans-Golgi cisternae in resistant parasites. The pH within Golgi cisternae is higher in control epimastigotes and most mature cruzain is lysosomal. Cruzain activity is negligible in CPI-resistant epimastigote extracts compared to the parental clone. Activity is restored following withdrawal of the inhibitor. No cross-resistance to the therapeutic drugs nifurtimox and benznidazole occurred and, conversely, parasites resistant to these drugs were sensitive to CPI. Protease inhibitors are thus potential therapeutical alternatives in cases of nifurtimox/benznidazole resistance. Cumulatively, these results suggest that CPI-resistance induces upregulation of Golgi complex function and post-Golgi secretory pathway, and release of precursors before the enzyme reaches its site of biologic activity.

Efficient identification of inhibitors targeting the closed active site conformation of the HPRT from Trypanosoma cruzi.

BACKGROUND: Currently, only two drugs are recommended for treatment of infection with Trypanosoma cruzi, the etiologic agent of Chagas' disease. These compounds kill the trypomastigote forms of the parasite circulating in the bloodstream, but are relatively ineffective against the intracellular stage of the parasite life cycle. Neither drug is approved by the FDA for use in the US. The hypoxanthine phosphoribosyltransferase (HPRT) from T. cruzi is a possible new target for antiparasite chemotherapy. The crystal structure of the HPRT in a conformation approximating the transition state reveals a closed active site that provides a well-defined target for computational structure-based drug discovery. RESULTS: A flexible ligand docking program incorporating a desolvation correction was used to screen the Available Chemicals Directory for inhibitors targeted to the closed conformation of the trypanosomal HPRT. Of 22 potential inhibitors identified, acquired and tested, 16 yielded K(i)'s between 0.5 and 17 microM versus the substrate phosphoribosylpyrophosphate. Surprisingly, three of eight compounds tested were effective in inhibiting the growth of parasites in infected mammalian cells. CONCLUSIONS: This structure-based docking method provided a remarkably efficient path for the identification of inhibitors targeting the closed conformation of the trypanosomal HPRT. The inhibition constants of the lead inhibitors identified are unusually favorable, and the trypanostatic activity of three of the compounds in cell culture suggests that they may provide useful starting points for drug design for the treatment of Chagas' disease.

Cysteine protease inhibitors as chemotherapy: lessons from a parasite target.

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

Cysteine protease inhibitors as chemotherapy for parasitic infections.

Analysis of the evolution, localization and biologic function of papain family cysteine proteases in metazoan and protozoan parasites has provided important and often surprising insights into the biochemistry and cellular function of this diverse enzyme family. Furthermore, the relative lack of redundancy of cysteine proteases in parasites compared to their mammalian hosts makes them attractive targets for the development of new antiparasitic chemotherapy. The treatment of experimental models of parasitic diseases with cysteine protease inhibitors has provided an important 'proof of concept' for the use of cysteine protease inhibitors in vivo. Evidence has now accumulated that cysteine protease inhibitors can selectively arrest replication of a microbial pathogen without untoward toxicity to the host. Furthermore, this can be achieved with reasonable dosing schedules and oral administration of the drug. Initial studies have confirmed the efficacy of cysteine protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. Work on Trypanosoma brucei, the agent of African trypanosomiasis, is preliminary but also promising. Target validation studies have shown that biotinylated or radiolabeled irreversible inhibitors specifically bind to the cysteine protease targets thought to represent the major activity within the parasite. In the case of T. cruzi, the effect of inhibitors appears to be predominantly in blocking protease processing. Transfection studies using variant constructs have supported this model. Finally, the generation of null mutants for the multiple protease genes in Leishmania mexicana has provided the first genetic support for the key role of this enzyme family in parasite virulence. Safety studies in rodents and analysis of uptake of inhibitors by parasites and host cells suggest that the selectivity of inhibitors for the parasite targets may reside in the lack of redundancy of parasite proteases, the higher concentration of host proteases in intracellular compartments, and differential uptake of inhibitors by parasites. Attempts to elicit resistance to cysteine protease inhibitors in parasite cultures suggest that mechanisms of induced resistance are independent of resistance to the traditional antiparasitic agents. This suggests that cysteine protease inhibitors may provide an alternative to traditional therapy in drug-resistant organisms.

Protease trafficking in two primitive eukaryotes is mediated by a prodomain protein motif.

Trypanosome protozoa, an early lineage of eukaryotic cells, have proteases homologous to mammalian lysosomal cathepsins, but the precursor proteins lack mannose 6-phosphate. Utilizing green fluorescent protein as a reporter, we demonstrate that the carbohydrate-free prodomain of a trypanosome cathepsin L is necessary and sufficient for directing green fluorescent protein to the lysosome/endosome compartment. A proper prodomain/catalytic domain processing site sequence is also required to free the mature protease for delivery to the lysosome/endosome compartment. A nine-amino acid prodomain loop motif, implicated in prodomain-receptor interactions in mammalian cells, is conserved in the protozoa. Site-directed mutagenesis now confirms the importance of this loop to protease trafficking and suggests that a protein motif targeting signal for lysosomal proteases arose early in eukaryotic cell evolution.

[Trypanocidal effect of cysteine protease inhibitors in vitro and in vivo in experimental Chagas disease]. / Efecto antiparasitario de inhibidores de cisteín proteasas in vitro e in vivo en la enfermedad de Chagas experimental.

Endemic in most American countries, Chagas' disease causes high morbidity and mortality. Recent experimental and clinical evidence shows the importance of chemotherapy in both the acute and chronic phases of this disease. However, treatment is yet limited by the toxicity associated to available drugs. This review describes the design, evolution, and selection of dipeptides that interrupt the intracellular cycle of T. cruzi and cure acute experimental infections in laboratory animals. Peptido-mimetic inhibitors specifically bind cruzain, a T. cruzi cystein protease. The inhibitors cause alterations in the Golgi complex and ER, accumulation of unprocessed enzyme within Golgi cisternae, and decrease of mature cruzain within lysosomes. The most effective compound, N-Pip-F-hF-VS phi, cured an acute lethal infection in experimental animals. Myocardial lesions, lymphocyte infiltration and intracellular amastigote clusers were absent in treated animals. Preliminary toxicology and pharmacokinetic analyses suggest the lack of toxicity associated to high doses and prolonged treatment regimes. Protease inhibitors may soon become good chemotherapeutic alternatives for acute and chronic Chagas' disease.

A new member of YER057c family in Trypanosoma cruzi is adjacent to an ABC-transporter.

Tcp17 is a Trypanosoma cruzi gene located contiguous to the ABC-transporter tcpgp2. The protein contains 160 amino acid residues with a predicted molecular mass of 16.5kDa. Western blot analysis using a polyclonal antiserum against recombinant TCP17 revealed that the protein is only expressed in the epimastigote form of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. A sequence comparison of TCP17 showed a remarkable homology with a conserved family of prokaryotic and eukaryotic proteins called YER057c whose function has not yet been characterized. Here, we propose a new signature of this family considering the N-terminal: [IV]-X(4)-[AV]-[AP]-X-[AP]-X(3)-Y-X(9)-[LIVF]-X(2)-[SA]-G-[QS], and the C-terminal: [AT]-R-X(2)-[IVFY]-X-[VC]-X(2)-L-P-X(4)-[LIVM]-E-[IVM] -[DE] motifs. Immunofluorescence and immunoelectron microscopy studies suggest that the protein has a wide distribution in the cell, with a higher concentration in the external side of the plasma membrane, on the Golgi complex and on cytoplasmic vacuoles. Although the physiological function of TCP17 is unknown, its conservation in evolution suggests biological relevance in the parasite.

Cysteine protease inhibitors cure an experimental Trypanosoma cruzi infection.

Trypanosoma cruzi is the causative agent of Chagas' disease. The major protease, cruzain, is a target for the development of new chemotherapy. We report the first successful treatment of an animal model of Chagas' disease with inhibitors designed to inactivate cruzain. Treatment with fluoromethyl ketone-derivatized pseudopeptides rescued mice from lethal infection. The optimal pseudopeptide scaffold was phenylalanine-homophenylalanine. To achieve cure of infection, this pseudopeptide scaffold was incorporated in a less toxic vinyl sulfone derivative. N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl also rescued mice from a lethal infection. Six of the treated mice survived over nine months, three without further treatment. Three mice that had entered the chronic stage of infection were retreated with a 20-d regimen. At the conclusion of the experiments, five of the six mice had repeated negative hemacultures, indicative of parasitological cure. Studies of the effect of inhibitors on the intracellular amastigote form suggest that the life cycle is interrupted because of inhibitor arrest of normal autoproteolytic cruzain processing at the level of the Golgi complex. Parasites recovered from the hearts of treated mice showed the same abnormalities as those treated in vitro. No abnormalities were noted in the Golgi complex of host cells. This study provides proof of concept that cysteine protease inhibitors can be given at therapeutic doses to animals to selectively arrest a parasitic infection.

Cysteine protease inhibitors alter Golgi complex ultrastructure and function in Trypanosoma cruzi.

Cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi, is a target of rational drug design for chemotherapy of Chagas' disease. The precise biological role of cruzain in the parasite life cycle and the mechanism involved in the trypanocidal effect of cysteine protease inhibitors are still unclear. Here we report biological and ultrastructural alterations caused by cysteine protease inhibitors in T. cruzi epimastigotes. Cruzain, a glycoprotein that transits the Golgi-endosomal pathway, localized to pre-lysosomes/lysosomes in the posterior end of untreated epimastigotes by fluorescent microscopy utilizing either a biotinylated cysteine protease inhibitor to tag the active site, or a specific anti-cruzain antibody. Radiolabeled or biotinylated cysteine protease inhibitors bound exclusively to cruzain in intact epimastigotes confirming that cruzain is accessible to, and is targeted by the inhibitors. Treatment of T. cruzi epimastigotes with specific cysteine protease inhibitors arrested growth, altered the intracellular localization of cruzain, and induced major alterations in the Golgi complex. Following treatment, cruzain accumulated in peripheral dilations of Golgi cisternae. There was a concomitant 70% reduction in gold-labeled cruzain transported to lysosomes. Cisternae abnormalities in the Golgi compartment were followed by distention of ER and nuclear membranes. Brefeldin A increased the number and size of cisternae in epimastigotes. Pre-treatment of epimastigotes with cysteine protease inhibitors followed by exposure to brefeldin A induced a more rapid appearance of the cysteine protease inhibitor-induced Golgi alterations. Our results suggest that cysteine protease inhibitors prevent the normal autocatalytic processing and trafficking of cruzain within the Golgi apparatus. Accumulation of cruzain may decrease mobility of Golgi membranes and result in peripheral distention of cisternae. These major alterations of the Golgi complex parallel the death of T. cruzi epimastigotes.

A primitive enzyme for a primitive cell: the protease required for excystation of Giardia.

Protozoan parasites of the genus Giardia are one of the earliest lineages of eukaryotic cells. To initiate infection, trophozoites emerge from a cyst in the host. Excystation is blocked by specific cysteine protease inhibitors. Using a biotinylated inhibitor, the target protease was identified and its corresponding gene cloned. The protease was localized to vesicles that release their contents just prior to excystation. The Giardia protease is the earliest known branch of the cathepsin B family. Its phylogeny confirms that the cathepsin B lineage evolved in primitive eukaryotic cells, prior to the divergence of plant and animal kingdoms, and underscores the diversity of cellular functions that this enzyme family facilitates.

Leishmania major: comparison of the cathepsin L- and B-like cysteine protease genes with those of other trypanosomatids.

Cysteine proteases play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based strategy of drug design. As a first step toward applying this strategy to design inhibitors as antiparasitic agents for leishmaniasis, we have isolated and sequenced the full-length clones of two cysteine protease genes from Leishmania major. One of the genes is structurally similar to the cathepsin L-like family and the other is similar to the cathepsin B-like family of cysteine proteases. The L. major cathepsin L-like sequence has a proregion that shares high sequence similarity with other cathepsin L sequences but not cathepsin B sequences and has a proline/threonine-rich C-terminal extension. The cathepsin L-like gene occurs in multiple copies, whereas there may be only one copy of the cathepsin B-like gene. Northern blot analyses show that both genes are expressed in the promastigote and amastigote stages, and pulse field gel electrophoresis revealed that the cathepsin L- and B-like genes are each found on two nonhomologous chromosomes. The L. major L-like amino acid sequence is 75% identical to the L. mexicana sequence, 74% identical to the L. pifanoi sequence, 47% identical with the Trypanosoma cruzi sequence, 47% identical with the T. congolense sequence, and 45% identical with the T. brucei sequence. L. major is one of two trypanosomatid species for which a cathepsin B-like gene has been identified and sequenced; its amino acid sequence is 82% identical to the one from L. mexicana. Tree inference based on distance and parsimony methods of kinetoplastid cathepsin L proteins yielded independent support for phylogenetic hypotheses inferred from analyses of ribosomal RNA genes. Because the cathepsin L locus has a high level of phylogenetic signal with respect to trypanosomatid taxa, this locus has great potential utility for investigating the evolutionary history of trypanosomatids and related organisms.

Cellular and molecular biological analyses of nifurtimox resistance in Trypanosoma cruzi.

We induced nifurtimox resistance in both epimastigotes and tissue culture-derived trypomastigotes of several Trypanosoma cruzi strains. The magnitude of nifurtimox resistance was strain-dependent. A variety of karyotype changes occurred in the nifurtimox-resistant (NR) strains. Chromosome-specific DNA probes identified karyotype changes common to the NR strains that were moderately resistant to nifurtimox but not the NR strains that were highly resistant to nifurtimox. A marked increase in nifurtimox resistance in one NR strain was accompanied by a 100% increase in nuclear DNA mass and a 50% increase in kinetoplast DNA mass. These data suggest that nifurtimox resistance can be accompanied by a wide spectrum of DNA changes. Both trypanothione reductase and heat-shock proteins may modulate the effects of exposure of T. cruzi to nifurtimox. However, we did not detect qualitative or quantitative differences in these genes or their transcripts between the NR strains and the sensitive strains from which they were derived. An understanding of the spectrum of diversity in nifurtimox resistance at the cellular and molecular levels demonstrated in this report is critical in the development of drug therapies against Chagas' disease.

The cysteine protease of Trypanosoma cruzi as a model for antiparasite drug design.

By combining new technology in molecular biology, X-ray crystallography, computer graphics and biochemistry, structure-based drug design provides a parallel and cost-effective strategy for identification of new antiparasite chemotherapy. James McKerrow, Mary McGrath and Juan Engel here discuss an example of the amplication of this strategy is its use in targeting the major cysteine protease in Trypanosoma cruzi. Tools from molecular biology helped overcome the obstacle of limited parasite material to allow production of reagent quantities of enzyme for inhibitor screening. Computer graphics analysis and X-ray crystallography are allowing rapid identification of new inhibitors based on either leads already identified or compounds selected by computer graphics screening of chemical databases.