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Leishmania mexicana mutants deficient in the multicopy CPB gene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specific CPB2.8 or metacyclic stage-specific CPB2 gene into a CPB-deficient mutant L. mexicana failed to restore either a Th2 response or sustained virulence. However, reexpression of multiple CPB genes from a cosmid significantly restored virulence. This was characterized by increased lesion and parasite growth and the acquisition of a Th2 response, as determined by measuring interleukin-4 production and immunoglobulin G1 (IgG1) and IgE levels. These studies confirm that L. mexicana cysteine proteases are important virulence factors and provide an explanation for the presence in L. mexicana of a multicopy tandem array of CPB genes.

Cruzain is the major cysteine protease of T. cruzi, which is the causative agent of Chagas’ disease and is a promising target for the development of new chemotherapy. With the goal of developing potent nonpeptidic inhibitors of cruzain, the Substrate Activity Screening (SAS) method was used to screen a library of protease substrates initially designed to target the homologous human protease cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor 38 was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored β-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent irreversible acyl- and aryl-oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely depend on leaving group pKa, with 2,3,5,6-tetrafluorophenoxymethyl ketone 54 identified as one of the most potent inhibitors with a second order inactivation constant of 147,000 s−1M−1. This inhibitor completely eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead to new chemotherapeutics for Chagas’ disease.

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.

Proteases constitute a major class of drug targets. Endosomal compartments harbor several protease families whose attenuation may be beneficial to a number of biological processes, including inflammation, cancer metastasis, antigen presentation, and parasite clearance. As a step toward the goal of generalized but targeted protease inhibition in the endocytic pathway, we describe here the synthesis, characterization, and cellular application of a novel multifunctional protease inhibitor. We show that pepstatin A, a potent but virtually insoluble inhibitor of cathepsins D and E, can be conjugated to a single site on cystatin C, a potent inhibitor of the papain-like cysteine proteases (PLCP) and of asparagine endopeptidease (AEP), to create a highly soluble compound capable of suppressing the activity of all 3 principal protease families found in endosomes and lysosomes. We demonstrate that this cystatin–pepstatin inhibitor (CPI) can be taken up by cells to modulate protease activity and affect biological responses.

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 appear to be required for hemoglobin hydrolysis by intraerythrocytic malaria parasites. Previous studies showed that peptidyl vinyl sulfone inhibitors of falcipain-2 blocked the development of P. falciparum in culture and exerted antimalarial effects in vivo. We now report the structure-activity relationships for inhibition of falcipain-2, falcipain-3, and parasite development by 39 new vinyl sulfone, vinyl sulfonate ester, and vinyl sulfonamide cysteine protease inhibitors. Levels of inhibition of falcipain-2 and falcipain-3 were generally similar, and many potent compounds were identified. Optimal antimalarial compounds, which inhibited P. falciparum development at low nanomolar concentrations, were phenyl vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides with P2 leucine moieties. Our results identify independent structural correlates of falcipain inhibition and antiparasitic activity and suggest that peptidyl vinyl sulfones have promise as antimalarial agents.

Chemotherapy of leishmaniasis is mainly based on antimonials. However, they are extremely toxic and cause serious side effects, and there is a worldwide increasing frequency of chemoresistance to antimonials. These issues emphasize the urgent need for affordable alternative drugs against leishmaniasis. Leishmania cysteine proteases are essential for parasite growth, differentiation, pathogenicity, and virulence and are thus attractive targets for combating leishmaniasis. Herein we demonstrate that the cysteine protease inhibitors aziridine-2,3-dicarboxylates 13b and 13e impaired promastigote growth at mid-micromolar concentrations and decreased the infection rate of peritoneal macrophages at concentrations 8- to 13-fold lower than those needed to inhibit parasite replication. Simultaneous treatment of infected cells with compound 13b and gamma interferon resulted in an even further reduction of the concentration needed for a significant decrease in macrophage infection rate. Notably, treatment with the compounds alone modulated the cytokine secretion of infected macrophages, with increased levels of interleukin-12 and tumor necrosis factor alpha. Furthermore, the decreased infection rate in the presence of compound 13b correlated with increased nitric oxide production by macrophages. Importantly, at the concentrations used herein, compounds 13b and 13e were not toxic against fibroblasts, macrophages, or dendritic cells. Together, these results suggest that the aziridine-2,3-dicarboxylates 13b and 13e are potential antileishmanial lead compounds with low toxicity against host cells and selective antiparasitic effects.

The synthesis and evaluation of a refined series of α-ketoheterocycles based on the oxazole 2 (OL-135) incorporating systematic changes in the central heterocycle bearing a key set of added substituents are described. The nature of the central heterocycle, even within the systematic and minor perturbations explored herein, significantly influenced the inhibitor activity: 1,3,4-oxadiazoles and 1,2,4-oxadiazoles 9 > tetrazoles, the isomeric 1,2,4-oxadiazoles 10, 1,3,4-thiadiazoles > oxazoles including 2 > 1,2-diazines > thiazoles > 1,3,4-triazoles. Most evident in these trends is the observation that introduction of an additional heteroatom at position 4 (oxazole numbering, N > O > CH) substantially increases activity that may be attributed to a reduced destabilizing steric interaction at the FAAH active site. Added heterocycle substituents displaying well defined trends may be utilized to enhance the inhibitor potency and, more significantly, to enhance the inhibitor selectivity. These trends, exemplified herein, emerge from both enhancements in the FAAH activity and simultaneous disruption of binding affinity for competitive off-target enzymes.

Background

Leishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB) is an important virulence factor in parasites from the Leishmania (Leishmania) mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania) amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant.

Results

Predicted epitopes, obtained by in silico mapping, displayed the ability to induce cell proliferation and expression of cytokines related to Th1 and Th2 responses. Furthermore, we applied in silico simulations to investigate how the MHC/epitopes interactions could be related to the immunomodulatory effects on cytokines, finding evidence that specific interaction patterns can be related to in vitro activities.

Conclusions

Based on our results, we consider that some peptides from the CPB COOH-terminal extension may influence host immune responses in the murine infection, thus helping Leishmania survival.

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas’ disease and African sleeping sickness, respectively. Both parasites rely on essential cysteine proteases for survival, cruzain for T. cruzi and TbCatB/rhodesain for T. brucei. A recent quantitative high-throughput screen of cruzain identified triazine nitriles, which are known inhibitors of other cysteine proteases, as reversible inhibitors of the enzyme. Structural modifications detailed herein, including core scaffold modification from triazine to purine, improved the in vitro potency against both cruzain and rhodesain by 350-fold, while also gaining activity against T. brucei parasites. Selected compounds were screened against a panel of human cysteine and serine proteases to determine selectivity, and a co-crystal was obtained of our most potent analog bound to Cruzain.

Background

Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei.

Methods and Findings

We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002.

Conclusions

The mature domain of our TbCat•CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat•CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain•K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.

Author Summary

Proteases are ubiquitous in all forms of life and catalyze the enzymatic degradation of proteins. These enzymes regulate and coordinate a vast number of cellular processes and are therefore essential to many organisms. While serine proteases dominate in mammals, parasitic organisms commonly rely on cysteine proteases of the Clan CA family throughout their lifecycle. Clan CA cysteine proteases are therefore regarded as promising targets for the selective design of drugs to treat parasitic diseases, such as Human African Trypanosomiasis caused by Trypanosoma brucei. The genomes of kinetoplastids such as Trypanosoma spp. and Leishmania spp. encode two Clan CA C1 family cysteine proteases and in T. brucei these are represented by rhodesain and TbCatB. We have determined three-dimensional structures of these two enzymes as part of our ongoing efforts to synthesize more effective anti-trypanosomal drugs.

Fatty acid amide hydrolase (FAAH) degrades neuromodulating fatty acid amides including anandamide (endogenous cannabinoid agonist) and oleamide (sleep-inducing lipid) at their sites of action and is intimately involved in their regulation. Herein we report the discovery of a potent, selective, and efficacious class of reversible FAAH inhibitors that produce analgesia in animal models validating a new therapeutic target for pain intervention. Key to the useful inhibitor discovery was the routine implementation of a proteomics-wide selectivity screen against all serine hydrolases ensuring selectivity for FAAH coupled with systematic in vivo examinations of candidate inhibitors.

Evidence indicates that cysteine proteases play essential role in malaria parasites; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria. Studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis. The best characterized Plasmodium cysteine proteases are falcipains, which are papain family enzymes. Falcipain-2 and falcipain-3 are major hemoglobinases of P. falciparum. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. Overall, the complexes of falcipain-2 and falcipain-3 with small and macromolecular inhibitors provide structural insight to facilitate the design or modification of effective drug treatment against malaria. Drug development targeting falcipains should be aided by a strong foundation of biochemical and structural studies.

The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas’ disease, affects millions of individuals and continues to be an important global health concern. The poor efficacy and unfavorable side effects of current treatments necessitate novel therapeutics. Cruzain, the major cysteine protease of T. cruzi, is one potential novel target. Recent advances in a class of vinyl-sulfone inhibitors are encouraging; however, as most potential therapeutics fail in clinical trials and both disease progression and resistance call for combination therapy with several drugs, the identification of additional classes of inhibitory molecules is essential. Using an exhaustive virtual-screening and experimental-validation approach, we identify several additional small-molecule cruzain inhibitors. Further optimization of these chemical scaffolds could lead to the development of novel drugs useful in the treatment of Chagas’ disease.

A series of α-ketooxazoles containing conformational constraints in the C2 acyl side chain of 2 (OL-135) were examined as inhibitors of fatty acid amide hydrolase (FAAH). Only one of the two possible enantiomers displayed potent FAAH inhibition (S vs R enantiomer), and their potency is comparable or improved relative to 2, indicating that the conformational restriction in the C2 acyl side chain is achievable. A cocrystal X-ray structure of the -ketoheterocycle 12 bound to a humanized variant of rat FAAH revealed its binding details, confirmed that the (S)-enantiomer is the bound active inhibitor, shed light on the origin of the enantiomeric selectivity, and confirmed that the catalytic Ser241 is covalently bound to the electrophilic carbonyl as a deprotonated hemiketal. Preliminary in vivo characterization of the inhibitors 12 and 14 is reported demonstrating that they raise brain anandamide levels following either intraperitoneal (i.p.) or oral (p.o.) administration indicative of effective in vivo FAAH inhibition. Significantly, the oral administration of 12 caused dramatic accumulation of anandamide in the brain, with peak levels achieved between 1.5–3 h and these elevations were maintained over 9 h. Additional studies of these two representative members of the series (12 and 14) in models of thermal hyperalgesia and neuropathic pain are reported, including the demonstration that 12 administered orally significantly attenuated mechanical (>6 h) and cold (>9 h) allodynia for sustained periods consistent with its long acting effects in raising the endogenous concentration of anandamide.

The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ∼2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.

Author Summary

Diseases like malaria and sleeping sickness are caused by tropical parasites and represent a major cause of mortality and morbidity in the developing world. A pragmatic approach to discover new drugs for these diseases is to search for drug leads among existing small molecule collections generated in the for-profit pharmaceutical industry. In this study, we searched for new drug leads among a collection of small molecules donated by Celera Genomics. This collection of molecules was originally developed to inhibit a class of human enzymes (cathepsins) implicated in diseases like osteoporosis and psoriasis. Similar enzymes are also present in most tropical parasites, making this collection a logical place to search for new drug leads. The end result of this effort saw the identification of compounds that inhibit the growth of one or more tropical parasites and that will serve as good starting points for the development of new drugs for tropical parasitic diseases.

Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.

Synopsis

Malaria causes hundreds of millions of illnesses and more than a million deaths each year. Illness is caused by infection of red blood cells, with repeated rounds of red cell invasion, parasite development, and red cell rupture. Among enzymes with important roles in malaria parasites are proteases, which break down other proteins. Functions of proteases include the breakdown of red cell hemoglobin, the release of parasites from red cells, and the invasion of red cells by free parasites. This work concerns the identification and characterization of a protease inhibitor of malaria parasites termed falstatin. Falstatin inhibits one class of proteases, cysteine proteases, from both malaria parasites and humans. It is produced from soon before until soon after the processes of red cell rupture and invasion. Incubation of malaria parasites with an antibody that prevents the effects of falstatin markedly inhibited red cell invasion. Thus, falstatin appears to facilitate red cell invasion, presumably by preventing the action of proteases that hinder this process. Falstatin may therefore be a potential new target for vaccines or drugs to control malaria.

Background/Objectives

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major health problem in Ethiopia. Parasites in disparate regions are transmitted by different vectors, and cluster in distinctive genotypes. Recently isolated strains from VL and HIV-VL co-infected patients in north and south Ethiopia were characterized as part of a longitudinal study on VL transmission.

Methodology/Principal Findings

Sixty-three L. donovani strains were examined by polymerase chain reaction (PCR) targeting three regions: internal transcribed spacer 1 (ITS1), cysteine protease B (cpb), and HASPB (k26). ITS1- and cpb - PCR identified these strains as L. donovani. Interestingly, the k26 - PCR amplicon size varied depending on the patient's geographic origin. Most strains from northwestern Ethiopia (36/40) produced a 290 bp product with a minority (4/40) giving a 410 bp amplicon. All of the latter strains were isolated from patients with HIV-VL co-infections, while the former group contained both VL and HIV-VL co-infected patients. Almost all the strains (20/23) from southwestern Ethiopia produced a 450 bp amplicon with smaller products (290 or 360 bp) only observed for three strains. Sudanese strains produced amplicons identical (290 bp) to those found in northwestern Ethiopia; while Kenyan strains gave larger PCR products (500 and 650 bp). High-resolution melt (HRM) analysis distinguished the different PCR products. Sequence analysis showed that the k26 repeat region in L. donovani is comprised of polymorphic 13 and 14 amino acid motifs. The 13 amino acid peptide motifs, prevalent in L. donovani, are rare in L. infantum. The number and order of the repeats in L. donovani varies between geographic regions.

Conclusions/Significance

HASPB repeat region (k26) shows considerable polymorphism among L. donovani strains from different regions in East Africa. This should be taken into account when designing diagnostic assays and vaccines based on this antigen.

Author Summary

HASPB belongs to a hydrophilic repeat-containing surface antigen family found in Leishmania. The L. infantum/L. donovani protein has been used for diagnosis of visceral leishmaniasis, and is a putative vaccine candidate for this disease. Visceral leishmaniasis is a fatal disease, and approximately one third of the cases are found in East Africa. The k26 – PCR, which amplifies the repeat region of HASPB, produced different amplicon sizes for recent Ethiopian L. donovani depending on the strain's geographic origin. Further analysis showed that the number and order of the peptide motifs, either 13 or 14 amino acids long, comprising the L. donovani repeats varies between endemic regions of East Africa. Polymorphism in the amino acid sequence of the peptides was also observed. In addition, the 13 amino acid peptide motifs prevalent in L. donovani are rare in L. infantum. The observed polymorphisms in the HASPB repeat region suggests that custom antigens may be needed for diagnosis or vaccination in distinct endemic foci.

Background

Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum in all Mediterranean countries. The Leishmania parasite is transmitted by the bite of a corresponding sand fly vector and primarily maintained in nature by wild and domestic reservoirs, including dogs, foxes and jackals. Infected dogs are the primary reservoir host in endemic regions and are the most significant risk disposing humans to infection. The present study aimed at assessing the prevalence of infection with Leishmania and identification of Leishmania infantum in domestic dogs in the West Bank, Palestine.

Methods

The infection rate among domestic dogs collected from seven districts in the Palestinian West Bank was investigated by examination of parasites in culture from the buffy coat using serological and molecular methods; based on ELISA, internal transcribed spacer 1 (ITS1) and cysteine protease (CPB) PCR.

Results

Out of 215 dogs examined for Leishmania, 36 (16.7%) were positive in at least one method. Twenty three animals (11.5%) were positive for Leishmania DNA, whereas, ELISA and culture revealed 16 (7.5%), and 4 (1.5%) respectively. CPB-PCR on one of three culture-positive isolates revealed Leishmania infantum as the causative agent for Leishmania infection in dogs.

Conclusions

Our study showed that canine leishmania infection is prevalent with varying degrees in all the seven studied districts in Palestine despite the absence of human VL cases in 4 of these districts. The causative agent was confirmed to be Leishmania infantum.

It has been proposed that the Plasmodium falciparum cysteine protease falcipain and aspartic proteases plasmepsin I and plasmepsin II act cooperatively to hydrolyze hemoglobin as a source of amino acids for erythrocytic parasites. Inhibitors of each of these proteases have potent antimalarial effects. We have now evaluated the antimalarial effects of combinations of cysteine and aspartic protease inhibitors. When incubated with cultured P. falciparum parasites, cysteine and aspartic protease inhibitors exhibited synergistic effects in blocking parasite metabolism and development. The inhibitors also demonstrated apparent synergistic inhibition of plasmodial hemoglobin degradation both in culture and in a murine malaria model. When evaluated for the treatment of murine malaria, a combination of cysteine and aspartic protease inhibitors was much more effective than higher concentrations of either compound used alone. These results support a model whereby plasmodial cysteine and aspartic proteases participate in the degradation of hemoglobin, and they suggest that combination antimalarial therapy with inhibitors of the two classes of proteases is worthy of further study.

The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.

Attempts to crystallize a complex of papain (C. papaya) with a cysteine protease inhibitor from the parasitic pathogen T. brucei failed. However, over an extended period the mixture produced an ordered crystal of the protease carrying two peptide fragments in the active site. These correspond to dipeptides and tripeptides that are assigned as fragments of the inhibitor, which has presumably suffered proteolytic cleavage.

Attempts to cocrystallize the cysteine protease papain derived from the latex of Carica papaya with an inhibitor of cysteine proteases (ICP) from Trypanosoma brucei were unsuccessful. However, crystals of papain that diffracted to higher resolution, 1.5 Å, than other crystals of this archetypal cysteine protease were obtained, so the analysis was continued. Surprisingly, the substrate-binding cleft was occupied by two short peptide fragments which have been assigned as remnants of ICP. Comparisons reveal that these peptides bind in the active site in a manner similar to that of the human cysteine protease inhibitor stefin B when it is complexed to papain. The assignment of the fragment sequences is consistent with the specificity of the protease.

Toxoplasma gondii enters host cells via an active, self-driven process to fulfill its need for intracellular replication and survival. Successful host cell invasion is governed by sequential release of secretory proteins from three specialized organelles, including the micronemes, which contribute adhesive proteins necessary for parasite attachment and penetration. Cumulative evidence from studies of Trypanosoma species and malaria parasites has shown that cysteine protease inhibitors represent potent anti-parasitic agents capable of curing infections in vivo. In this study, we screened a series of selective cysteine protease inhibitors for their effects on T. gondii cell invasion. Two of these compounds, morpholinourea-leucyl-homophenolalaninyl-phenyl-vinyl-sulfone and N-benzoxycarbonyl-(leucyl)3-phenyl-vinyl-sulfone, impaired T. gondii invasion and gliding motility at low-micromolar concentrations. Unexpectedly, these inhibitors did not affect surface proteolysis of microneme products but instead impaired an earlier step by precluding the secretion of microneme-derived adhesins to the parasite surface. Our findings suggest that cysteine protease activity is required for microneme secretion and cell invasion by T. gondii.

Background: Malaria parasite egress from host erythrocytes requires cysteine protease activity, but the identity of key parasite proteases is unknown.

Results: SERA6 is an essential parasite cysteine protease that resides in the parasitophorous vacuole and is activated by SUB1, a parasite serine protease.

Conclusion: SERA6 may play a role in egress.

Significance: SERA6 is a potential new antimalarial drug target.

The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte.

By studying the inactivation of malaria parasite culture by cysteine protease inhibition using confocal microscopy of living cells, and electron microscopy of high-pressure frozen and freeze-substituted cells, we report the precise step in the release of malaria parasites from erythrocytes that is likely regulated by cysteine proteases: the opening of the erythrocyte membrane, liberating parasites for the next round of infection. Inhibition of cysteine proteases within the last few minutes of cycle does not affect rupture of the parasitophorus vacuole but irreversibly blocks the subsequent rupture of the host cell membrane, locking in resident parasites, which die within a few hours of captivity. This irreversible inactivation of mature parasites inside host cells makes plasmodial cysteine proteases attractive targets for anti-malarials, as parasite-specific cysteine protease inhibitors may significantly augment multi-target drug cocktails.