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Gallinamide A, originally isolated with a modest antimalarial activity, was subsequently reisolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs. Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (10, Ki = 0.0937 ± 0.01 nM and kinact/Ki = 8 730 000). From a protein structure and substrate preference perspective, cruzain, an essential Trypanosoma cruzi cysteine protease, is highly homologous. Our investigations revealed that gallinamide and its analogs potently inhibit cruzain and are exquisitely toxic toward T. cruzi in the intracellular amastigote stage. The most active compound, 5, had an IC50 = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect stage) and the host cell, and thus represents a new candidate for the treatment of Chagas disease.

The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.

Chagas' Disease, caused by the protozoan parasite Trypanosoma cruzi, is responsible for up to 41% of the heart failures in endemic areas in South America and is an emerging infection in regions of North America, Europe, and Asia. Treatment is suboptimal due to two factors. First, the lack of an adequate biomarker to predict disease severity and response to therapy; and second, up to 120-days treatment course coupled with a significant incidence of adverse effects from the drug currently used. Because the disease can manifest itself clinically a few years to decades after infection, controversy remains concerning the suitability of current drug treatment (benznidazole), and the efficacy of alternative drugs (e.g. posaconazole). We therefore followed the clinical course, and PCR detection of parasite burden, in a mouse model of infection for a full year following treatment with benznidazole or posaconazole. Efficacy of the two drugs depended on whether the treatment was performed during the acute model or the chronic model of infection. Posaconazole was clearly superior in treatment of acute disease whereas only benznidazole had efficacy in the chronic model. These results have important implications for the design and analysis of human clinical trials, and the use of specific drugs in specific clinical settings., Author summary Chagas disease is a parasitic infection that can be incapacitating, leading to heart failure with risk of sudden death. Currently, only one drug treatment is available, Benznidazole, but it demands a long period of treatment, has variable efficacy and leads to serious side effects that can lead to discontinuation of the treatment. An alternative therapy, the anti-fungal drug, Posaconazole, was repurposed for treatment of Chagas disease, but its use has been controversial with conflicting results in studies from different groups. In our approach, we followed the parasitic infection, disease symptoms and persistence of the pathogen in mice for a full year after treatment with Benznidazole or Posaconazole. Posaconazole treatment was more effective in the early infection (acute disease) whereas only Benznidazole had efficacy in the chronic model. This information can have important implications for the design and analysis of human clinical trials, and the use of specific drugs in specific clinical settings.

Although toxoplasmosis is one of the most common parasitic infections worldwide, therapeutic options remain limited. Cathepsins, proteases that play key roles in the pathogenesis of toxoplasmosis and many other protozoan infections, are important potential therapeutic targets. Because both TgCPB and TgCPL play a role in T. gondii invasion, we evaluated the efficacy of the potent, irreversible vinyl sulfone inhibitor, K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl). The inhibitor's toxicity and pharmacokinetic profile have been well-studied because of its in vitro and in vivo activity against a number of parasites. We found that it inhibited both TgCPB (EC50 = 114 nM) and TgCPL (EC50 = 71 nM) in vitro. K11777 also inhibited invasion of human fibroblasts by RH tachyzoites by 71% (p = 0.003) and intracellular replication by >99% (p

Background Chagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice. Methodology/Principal findings Both acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart. Conclusions/Significance The positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life., Author summary Chagas disease is a parasitic disease caused by the Trypanosoma cruzi. The infection may result in gastrointestinal manifestations and cardiomyopathy. Benznidazole, the current treatment, has limited efficacy and often leads to serious side effects. Aiming to develop new treatments, our group has identified new inhibitors that block the synthesis of parasitic lipids, resulting in parasite death. In this work, we evaluated the safety and efficacy of two of these compounds, 9 and 12, in mouse models of T. cruzi infection. Both compounds were well-tolerated by animals throughout the 28-day administration. In acutely infected mice, the compounds drastically reduced bloodstream parasites and increased survival. When treatment was initiated during the chronic phase, parasitemia dropped to background levels and remained undetectable for 4 weeks after drug withdrawal; parasites were re-activated by chemically-induced immunosuppression. Thus, the experimental compounds tested in these studies had an acceptable safety profile, achieved a marked reduction in parasite load and prevented heart injury due to inflammation, even in the absence of sterile cure. We conclude that the development of non-toxic medications capable of slowing the progression of cardiomyopathy is a valuable treatment option for Chagas disease patients because it could enhance the quality of life.

A series of oxyguanidine analogues of the cysteine protease inhibitor WRR-483 were synthesized and evaluated against cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi. Kinetic analyses of these analogues indicated that they have comparable potency to previously prepared vinyl sulfone cruzain inhibitors. Co-crystal structures of the oxyguanidine analogues WRR-666 (4) and WRR-669 (7) bound to cruzain demonstrated different binding interactions with the cysteine protease, depending on the aryl moiety of the P1' inhibitor subunit. Specifically, these data demonstrate that WRR-669 is bound noncovalently in the crystal structure. This represents a rare example of noncovalent inhibition of a cysteine protease by a vinyl sulfone inhibitor.

Cysteine proteinases are key virulence factors of the protozoan parasiteEntamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of theE. histolyticagenome project has revealed more than 40 genes encoding cysteine proteinases. We have focused onE. histolyticacysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasiveE. histolyticaand is highly expressed and released. Recombinant EhCP1 was expressed inEscherichia coliand refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence.

Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

Cysteine proteinases have a number of important functions in the life cycle of protozoan parasites. Based on our previous studies demonstrating the role of cysteine proteinases in invasion by Entamoeba histolytica, we evaluated the cysteine proteinases of E. invadens, a related protozoan which causes invasive disease of reptiles. E. invadens readily encysts in axenic culture and provides a model to investigate the role of cysteine proteinases in encystation. Broad bands of approximately 130-230, 55, and 35 kDa were detected on gelatin substrate gels and were inhibited with specific cysteine proteinase inhibitors. Maximal enzymatic activity was detected with peptide substrates containing arginine in the P2 position. A 567-bp fragment containing the active site of an E. invadens cysteine proteinase gene was amplified by PCR and had 37.7, 79.1, and 67.9% identity to the derived amino acid sequences of the acp 1, 2, and 3 genes, respectively, of E. histolytica. The PCR product hybridized with a single band of 1.1 kb on a Southern blot of EcoRI-restricted E. invadens genomic DNA. Long-term inhibition of cysteine proteinase activity during encystation resulted in significantly fewer cysts (P < 0.02); however, this effect appeared to be secondary to decreased trophozoite cell division. No difference in chitin synthase activity was detected between controls and encysting cells with inhibited cysteine proteinases, suggesting that these proteinases are not critical for activation of a zymogen form of chitin synthase. These studies demonstrate that cysteine proteinases may be critical for the survival of E. invadens, and specific inhibition may ultimately interrupt transmission.

The enteric protozoan parasite Entamoeba histolytica is the cause of potentially fatal amebic colitis and liver abscesses. E. histolytica trophozoites colonize the colon, where they induce inflammation, penetrate the mucosa, and disrupt the host immune system. The early establishment of E. histolytica in the colon occurs in the presence of antimicrobial human (LL-37) and murine (CRAMP [cathelin-related antimicrobial peptide]) cathelicidins, essential components of the mammalian innate defense system in the intestine. Studying this early step in the pathogenesis of amebic colitis, we demonstrate that E. histolytica trophozoites or their released proteinases, including cysteine proteinase 1 (EhCP1), induce intestinal cathelicidins in human intestinal epithelial cell lines and in a mouse model of amebic colitis. Despite induction, E. histolytica trophozoites were found to be resistant to killing by these antimicrobial peptides, and LL-37 and CRAMP were rapidly cleaved by released amebic cysteine proteases. The cathelicidin fragments however, did maintain their antimicrobial activity against bacteria. Degradation of intestinal cathelicidins is a novel function of E. histolytica cysteine proteinases in the evasion of the innate immune system in the bowel. Thus, early intestinal epithelial colonization of invasive trophozoites involves a complex interplay in which the ultimate outcome of infection depends in part on the balance between degradation of cathelicidins by amebic released cysteine proteinases and upregulation of proinflammatory mediators which trigger the inflammatory response.

Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.

Cysteine proteinases are key virulence factors of Entamoeba histolytica that are released during the process of invasion. We used a chemical mutant of E. histolytica strain HM-1:IMSS, clone L6, which is deficient in virulence, phagocytosis, and cysteine proteinase activity to help define the mechanisms of cysteine proteinase release. All cysteine proteinase genes of wild type HM-1 were present in the L6 mutant genome, but three of the major expressed proteinases, ehcp1, ehcp2, and ehcp5 were both transcribed, translated, and released at lower levels in L6. We hypothesized that a central protein such as the calcium binding protein 1, EhCaBP1, which is required for both phagocytosis and exocytosis might be deficient in this mutant. We found that both mRNA and proteinase levels of EhCaBP1 were decreased in L6. These findings provide an important link between phagocytosis, passive release of multiple cysteine proteinases, and attenuated virulence of this E. histolytica mutant.

Peroxiredoxins are an important class of antioxidant enzymes found from Archaea to humans, which reduce and thereby detoxify peroxides and peroxynitrites. The major thiol-containing surface antigen of the invasive ameba, Entamoeba histolytica, is a peroxiredoxin and is likely to be important during the transition from the anaerobic environment of the large intestine to human tissues. The closely related species, Entamoeba dispar, is incapable of invasion and more sensitive to hydrogen peroxide, yet also has a peroxiredoxin. We cloned and expressed the two active recombinant enzymes and found that their activity was similar by a fluorometric stopped-flow assay, giving a Km of

Transformation of Entamoeba histolytica has been previously reported, but the foreign genes have all been replicated episomally. Pantropic retroviral vectors based on the Moloney murine leukemia virus with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) have an extremely broad host range and can be concentrated to high titer. To investigate whether these pseudotyped, pantropic vectors can mediate gene transfer and expression in E. histolytica, we constructed a retroviral vector, in which a hygromycin phosphotransferase is expressed from the E. histolytica actin promoter. Data confirm the infection, integration, and expression of a foreign gene mediated by the provirus. To our knowledge, this is the most evolutionarily distant example of successful integration and expression of a mammalian retrovirus. Pantropic retroviral vectors may thus facilitate genetic analysis in species lacking transformation systems.

Cysteine proteinases are hypothesized to be important virulence factors of Entamoeba histolytica, the causative agent of amebic dysentery and liver abscesses. The release of a histolytic cysteine proteinase from E. histolytica correlates with the pathogenicity of both axenic strains and recent clinical isolates as determined by clinical history of invasive disease, zymodeme analysis, and cytopathic effect. We now show that pathogenic isolates have a unique cysteine proteinase gene (ACP1). Two other cysteine proteinase genes (ACP2, ACP3) are 85% identical to each other and are present in both pathogenic and nonpathogenic isolates. ACP1 is only 35 and 45% identical in sequence to the two genes found in all isolates and is present on a distinct chromosome-size DNA fragment. Presence of the ACP1 gene correlates with increased proteinase expression and activity in pathogenic isolates as well as cytopathic effect on a fibroblast monolayer, an in vitro assay of virulence. Analysis of the predicted amino acid sequence of the ACP1 proteinase gene reveals homology with cysteine proteinases released by activated macrophages and invasive cancer cells, suggesting an evolutionarily conserved mechanism of tissue invasion. The observation that a histolytic cysteine proteinase gene is present only in pathogenic isolates of E. histolytica suggests that this aspect of virulence in amebiasis is genetically predetermined.

The broad host range IncP (IncP1) plasmids of gram-negative bacteria encode DNA primases that are involved in conjugal DNA synthesis. The primase of RK2/RP4 is required for efficient DNA transfer to certain gram-negative bacteria, indicating that the enzyme primes complementary strand synthesis in the recipient. In vitro, the primase initiates synthesis of oligoribonucleotides at 3'-dGdT-5' dinucleotides on the template strand. In this report, replication-defective M13 phage are used to assay the ability of the RK2-encoded primase to initiate complementary strand synthesis in vivo on single-strand templates containing the RK2 origin of conjugal transfer (oriT) or the RK2 origin of vegetative replication (oriV). The results show that sequences from either strand of the oriT region serve as efficient substrates for the RK2 primase and can enhance the growth of the defective M13 vectors delta E101 and delta Elac to levels approaching wild-type. The primise-oriT interaction appeared specific, since neither the oriV sequence nor another RK2 region, trfB, significantly enhanced growth of the defective phage, either in the presence or in the absence of the primase. In contrast to ColEl and F, this study also shows that the oriV region of RK2 lacks sites that are recognized by the host-specified DNA priming systems. The results suggest that the oriT region contains sites on both DNA strands that are efficient substrates for the plasmid-encoded primase, facilitating initiation of complementary strand DNA synthesis in both donor and recipient during conjugation.