Parasite Proteases Research Articles

Molecular Targets Implicated in the Antiparasitic and Anti-Inflammatory Activity of the Phytochemical Curcumin in Trichomoniasis.

Trichomoniasis, is the most prevalent non-viral sexually transmitted disease worldwide. Although metronidazole (MDZ) is the recommended treatment, several strains of the parasite are resistant to MDZ, and new treatments are required. Curcumin (CUR) is a polyphenol with anti-inflammatory, antioxidant and antiparasitic properties. In this study, we evaluated the effects of CUR on two biochemical targets: on proteolytic activity and hydrogenosomal metabolism in Trichomonas vaginalis. We also investigated the role of CUR on pro-inflammatory responses induced in RAW 264.7 phagocytic cells by parasite proteinases on pro-inflammatory mediators such as the nitric oxide (NO), tumor necrosis factor α (TNFα), interleukin-1beta (IL-1β), chaperone heat shock protein 70 (Hsp70) and glucocorticoid receptor (mGR). CUR inhibited the growth of T. vaginalis trophozoites, with an IC50 value between 117 ± 7 μM and 173 ± 15 μM, depending on the culture phase. CUR increased pyruvate:ferredoxin oxidoreductase (PfoD), hydrogenosomal enzyme expression and inhibited the proteolytic activity of parasite proteinases. CUR also inhibited NO production and decreased the expression of pro-inflammatory mediators in macrophages. The findings demonstrate the potential usefulness of CUR as an antiparasitic and anti-inflammatory treatment for trichomoniasis. It could be used to control the disease and mitigate the associated immunopathogenic effects.

The cysteine protease ATG4B of Trichinella spiralis promotes larval invasion into the intestine of the host

The cysteine proteases of parasites are vital contributors that induce parasite migration to and invasion of host tissue. In this study, we analysed the cysteine protease ATG4B of Trichinella spiralis (TsATG4B) isolated from the soluble proteins of Trichinella spiralis (T. spiralis) adult worms to ascertain its biochemical properties and functions during invasion into the intestine of the host. The 43 kDa recombinant cysteine protease ATG4B protein (rTsATG4B) consists of a conserved peptidase_C54 domain and was expressed in Escherichia coli. Gelatine zymography showed that rTsATG4B could hydrolyse gelatine and that the hydrolytic activity was prevented by the cysteine protease inhibitor E-64 (pH 5.2). Immunofluorescence assays showed that TsATG4B is expressed at different stages and is localized at the cuticles and stichosomes of worms. Far-Western blotting and confocal microscopy revealed that rTsATG4B interacts with intestinal epithelial cells (IECs) and that it was subcellularly localized to the membrane and cytoplasm in IECs. Real‑time quantitative PCR (qPCR) results indicated that the transcription level of the TsATG4B gene was the higher in 6-day-old adult worms (6 days AW) than in any other stage. An in vitro larval invasion assay verified that rTsATG4B promoted larval invasion and that invasion was inhibited when rTsATG4B was pre-incubated with E-64, whereas anti-rTsATG4B serum inhibited larval invasion in a dose-dependent manner. Collectively, these results suggested that the enzymatic activity of TsATG4B significantly influences the hydrolysis process, which is necessary for larval invasion of the host intestinal epithelium.

Cathepsin F of Teladorsagia circumcincta is a recently evolved cysteine protease.

Parasitic cysteine proteases are involved in parasite stage transition, invasion of host tissues, nutrient uptake, and immune evasion. The cysteine protease cathepsin F is the most abundant protein produced by fourth-stage larvae (L4) of the nematode Teladorsagia circumcincta, while its transcript is only detectable in L4 and adults. T. circumcincta cathepsin F is a recently evolved cysteine protease that does not fall clearly into either of the cathepsin L or F subfamilies. This protein exhibits characteristics of both cathepsins F and L, and its phylogenetic relationship to its closest homologs is distant, including proteins of closely related nematodes of the same subfamily.

Trypanosoma cruzi cleaves galectin-3 N-terminal domain to suppress its innate microbicidal activity.

Galectin-3 is the best-characterized member of galectins, an evolutionary conserved family of galactoside-binding proteins that play central roles in infection and immunity, regulating inflammation, cell migration and cell apoptosis. Differentially expressed by cells and tissues with immune privilege, they bind not only to host ligands, but also to glycans expressed by pathogens. In this regard, we have previously shown that human galectin-3 recognizes several genetic lineages of the protozoan parasite Trypanosoma cruzi, the causal agent of Chagas' disease or American trypanosomiasis. Herein we describe a molecular mechanism developed by T. cruzi to proteolytically process galectin-3 that generates a truncated form of the protein lacking its N-terminal domain-required for protein oligomerization-but still conserves a functional carbohydrate recognition domain (CRD). Such processing relies on specific T. cruzi proteases, including Zn-metalloproteases and collagenases, and ultimately conveys profound changes in galectin-3-dependent effects, as chemical inhibition of parasite proteases allows galectin-3 to induce parasite death in vitro. Thus, T. cruzi might have established distinct mechanisms to counteract galectin-3-mediated immunity and microbicide properties. Interestingly, non-pathogenic T. rangeli lacked the ability to cleave galectin-3, suggesting that during evolution two genetically similar organisms have developed different molecular mechanisms that, in the case of T. cruzi, favoured its pathogenicity, highlighting the importance of T. cruzi proteases to avoid immune mechanisms triggered by galectin-3 upon infection. This study provides the first evidence of a novel strategy developed by T. cruzi to abrogate signalling mechanisms associated with galectin-3-dependent innate immunity.

Host Invasion by Pathogenic Amoebae: Epithelial Disruption by Parasite Proteins.

The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. Entamoeba histolytica, Naegleria fowleri and Acanthamoeba spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.

Molecular Targets for Malarial Chemotherapy: A Review.

The malaria parasite resistance to the existing drugs is a serious problem to the currently used antimalarials and, thus, highlights the urgent need to develop new and effective anti-malarial molecules. This could be achieved either by the identification of the new drugs for the validated targets or by further refining/improving the existing antimalarials; or by combining previously effective agents with new/existing drugs to have a synergistic effect that counters parasite resistance; or by identifying novel targets for the malarial chemotherapy. In this review article, a comprehensive collection of some of the novel molecular targets has been enlisted for the antimalarial drugs. The targets which could be deliberated for developing new anti-malarial drugs could be: membrane biosynthesis, mitochondrial system, apicoplasts, parasite transporters, shikimate pathway, hematin crystals, parasite proteases, glycolysis, isoprenoid synthesis, cell cycle control/cycline dependent kinase, redox system, nucleic acid metabolism, methionine cycle and the polyamines, folate metabolism, the helicases, erythrocyte G-protein, and farnesyl transferases. Modern genomic tools approaches such as structural biology and combinatorial chemistry, novel targets could be identified followed by drug development for drug resistant strains providing wide ranges of novel targets in the development of new therapy. The new approaches and targets mentioned in the manuscript provide a basis for the development of new unique strategies for antimalarial therapy with limited off-target effects in the near future.

Discovery and characterization of trypanocidal cysteine protease inhibitors from the ‘malaria box’

Chagas disease, Human African Trypanosomiasis, and schistosomiasis are neglected parasitic diseases for which new treatments are urgently needed. To identify new chemical leads, we screened the 400 compounds of the Open Access Malaria Box against the cysteine proteases, cruzain (Trypanosoma cruzi), rhodesain (Trypanosoma brucei) and SmCB1 (Schistosoma mansoni), which are therapeutic targets for these diseases. Whereas just three hits were observed for SmCB1, 70 compounds inhibited cruzain or rhodesain by at least 50% at 5 μM. Among those, 15 commercially available compounds were selected for confirmatory assays, given their potency, time-dependent inhibition profile and reported activity against parasites. Additional assays led to the confirmation of four novel classes of cruzain and rhodesain inhibitors, with potency in the low-to mid-micromolar range against enzymes and T. cruzi. Assays against mammalian cathepsins S and B revealed inhibitor selectivity for parasitic proteases. For the two competitive inhibitors identified (compounds 7 and 12), their binding mode was predicted by docking, providing a basis for structure-based optimization efforts. Compound 12 also acted directly against the trypomastigote and the intracellular amastigote forms of T. cruzi at 3 μM. Therefore, through a combination of experimental and computational approaches, we report promising hits for optimization in the development of new trypanocidal drugs.

Ipomoea batatas: papain propeptide inhibits cysteine protease in main plant parasites and enhances resistance of transgenic tomato to parasites.

Different parasites cause severe lose in quantity and quality of crops. Many parasites develop haustorial cells and stylets that penetrate the host using secreted enzymes and mechanical pressure. Cysteine proteases are pre-pro-enzyme produced by parasites that areessential for normal parasitism. Papain is also a kind of cysteine proteases suchthat its propeptide segment has inhibitory properties and limits the protease activity of papain. To investigate the inhibitory effects of papain propeptide on some parasite proteases, we cloned inhibitory propeptide of papain of Ipomoea batatas, and enzymatic fragments of Diabrotica virgifera cathepsin L-like protease-1, Meloidogyne incognita cathepsin L-like protease 1, Heterodera glycines cysteine protease-1, Cuscuta chinesis cysteine protease and Orobanche cernua cysteine protease. After purification of recombinant inhibitory propeptide and enzymatic fragments, the inhibition activity of propeptide on cysteine proteases was measured. Finally inhibitory propeptide was transformed into tomato and transgenic plants resistance to parasites (bioassay) were examined. We demonstrated papain-propeptide inhibits cysteine protease of mentioned parasites. In transgenic tomato plants, papain-inhibitory propeptide effectively interrupted haustoria development. Haustoria-digitate cells of dodder could not differentiate and develop into the phloem and xylem hyphae on transgenic tomatoes. Parasites grown on transgenic tomatoes showed reduction in vigor and productivity due to defective connection of haustoria. Lower ratio of female nematodes and a decrease of nematode egg mass per transgenic line indicated biocontrol of nematode. The changes in growth factors of parasite challenged transgenic lines relative to controls, indicates the efficacy of papain propeptide in control of parasitism.

Giardiaintestinalis cystatin is a potent inhibitor of papain, parasite cysteine proteases and, to a lesser extent, human cathepsin B.

Cystatins are important regulators of papain-like cysteine proteases. In the protozoan parasite Giardiaintestinalis, papain-like cysteine proteases play an essential role in the parasite's biology and pathogenicity. Here, we characterized a cysteine protease inhibitor of G.intestinalis that belongs to type-I-cystatins. The parasite cystatin is shown to be a strong inhibitor of papain (Ki ≈0.3nm) and three parasite cysteine proteases (CP14019, CP16160 and CP16779, Ki ≈0.9-5.8nm), but a weaker inhibitor of human cathepsin B (Ki ≈79.9nm). The protein localizes mainly in the cytoplasm. Together, these data suggest that cystatin of G.intestinalis plays a role in the regulation of cysteine protease activities in the parasite and, possibly, in the interaction with the host.

Structural Insights Into Key Plasmodium Proteases as Therapeutic Drug Targets.

Malaria, caused by protozoan of genus Plasmodium, remains one of the highest mortality infectious diseases. Malaria parasites have a complex life cycle, easily adapt to their host’s immune system and have evolved with an arsenal of unique proteases which play crucial roles in proliferation and survival within the host cells. Owing to the existing knowledge of enzymatic mechanisms, 3D structures and active sites of proteases, they have been proven to be opportune for target based drug development. Here, we discuss in depth the crucial roles of essential proteases in Plasmodium life cycle and particularly focus on highlighting the atypical “structural signatures” of key parasite proteases which have been exploited for drug development. These features, on one hand aid parasites pathogenicity while on the other hand could be effective in designing targeted and very specific inhibitors for counteracting them. We conclude that Plasmodium proteases are suitable as multistage targets for designing novel drugs with new modes of action to combat malaria.

Rational Lead Optimization Based on the Modeled Structure of Cysteine Protease of Leishmania donovani

In present study, the molecular modeling techniques were applied to generate a refined model of a cysteine protease of Leishmania donovani using the crystal structure of a homologous protease and used for lead optimization. The structures of a series of complexes of the protease with the designed inhibitors were predicted using a novel docking technique comprising of repeated cycles of molecular dynamics and energy minimization. Calculation of the free energies of binding of the model with the designed inhibitors suggested that three compounds can form stable complexes with dissociation constants in the nanomolar range (0.038-1.41 nM). Search in the human genome revealed that a number of proteases of the cathepsin family had high homology with the parasite protease with amino acid identity around 45 %. The X-ray structures of all these were available in the protein data bank. The structures of the complexes of the selected inhibitors with a few homologous human proteases of known 3-D structures were also predicted using the same technique of optimization. The electrostatic potentials around the binding sites of the proteases were highly negative, which served as a clue for the introduction of positively charged groups in the designed inhibitors for higher affinity. The comparison of interaction energies and hydrogen bonding patterns among these complexes and similar complexes with homologous human proteases allowed us to short-listed three molecules as effective antileishmanial cysteine protease inhibitors.

Cysteine Protease Inhibitors from the Methanol Extract of the Root Bark of Securidaca longepedunculata with Antimalarial Potentials in Chloroquine-Resistant P. berghei Parasite

Malaria parasite resistance against Artemisinin-based Combination Therapy (ACT) in some parts of the world necessitates the search for antimalarial compounds or plant extracts with novel mode of action and active against the chloroquine-resistant strain of the parasite to serve as alternative to ACT. Cysteine protease inhibitors' fraction of the methanol extract of the root bark of Securidaca longepedunculata (CPI) was investigated for antiplasmodial activity against chloroquine-resistant P. berghei-infected mice and its inhibitory effect on papain, P. berghei cysteine proteases and heme biocystallization were also evaluated. The methanol extract of the root bark was obtained by soxhlet extraction with 1,000 mL of 70% (v/v) methanol for 48 hours and concentrated to dryness at 45∘C. CPI was obtained using PBS (pH 7) extraction followed by cold acetone precipitation. Peter's four-day suppressive and Rane's four-day curative test was employed to assess the antimalarial potentials of CPI. Data was analysed with one way ANOVA followed by Dunnett's post hoc test, differences were considered significant at p≤0.05. The suppressive effect of CPI was significant (p≤0.05) at 34 and 23 mg/kg doses. Doses of 34, 23 and 11 mg/kg produced significant (p≤0.05) dose-dependent curative effect. CPI inhibited the proteolytic activity of papain enzyme and P. berghei cysteine proteases in vitro with IC50 values of 20.1 and 5.6 μg/mL respectively. The present study showed that cysteine protease inhibitors fraction of the methanol extract of the root bark of Securidaca longepedunculata is a potential source of novel antimalarial agents that could target malaria parasite cysteine proteases.

Single-Molecule Analyses Reveal Rhomboid Proteins Are Strict and Functional Monomers in the Membrane

Intramembrane proteases hydrolyze peptide bonds within the membrane as a regulatory paradigm that is conserved across all forms of cellular life. Many of these enzymes are thought to be oligomeric, and that their resulting quaternary interactions form the basis of their regulation. However, technical limitations have precluded directly determining the oligomeric state of intramembrane proteases in any membrane. Using single-molecule photobleaching, we determined the quaternary structure of 10 different rhomboid proteins (the largest superfamily of intramembrane proteases) and six unrelated control proteins in parallel detergent micelle, planar supported lipid bilayer, and whole-cell systems. Bacterial, parasitic, insect, and human rhomboid proteases and inactive rhomboid pseudoproteases all proved to be monomeric in all membrane conditions but dimeric in detergent micelles. These analyses establish that rhomboid proteins are, as a strict family rule, structurally and functionally monomeric by nature and that rhomboid dimers are unphysiological.

Cysteine proteases as digestive enzymes in parasitic helminths.

We briefly review cysteine proteases (orthologs of mammalian cathepsins B, L, F, and C) that are expressed in flatworm and nematode parasites. Emphasis is placed on enzyme activities that have been functionally characterized, are associated with the parasite gut, and putatively contribute to degrading host proteins to absorbable nutrients [1–4]. Often, gut proteases are expressed as multigene families, as is the case with Fasciola [5] and Haemonchus [6], presumably expanding the range of substrates that can be degraded, not least during parasite migration through host tissues [5]. The application of the free-living planarian and Caenorhabditis elegans as investigative models for parasite cysteine proteases is discussed. Finally, because of their central nutritive contribution, targeting the component gut proteases with small-molecule chemical inhibitors and understanding their utility as vaccine candidates are active areas of research [7].

Antiprotozoal and cysteine proteases inhibitory activity of dipeptidyl enoates

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k2nd (M−1s−1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC50 values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values against Leishmania donovanii.

2 H-1,2,3-Triazole-Based Dipeptidyl Nitriles: Potent, Selective, and Trypanocidal Rhodesain Inhibitors by Structure-Based Design.

Macrocyclic inhibitors of rhodesain (RD), a parasitic cysteine protease and drug target for the treatment of human African trypanosomiasis, have shown low metabolic stability at the macrocyclic ether bridge. A series of acyclic dipeptidyl nitriles was developed using structure-based design (PDB ID: 6EX8 ). The selectivity against the closely related cysteine protease human cathepsin L (hCatL) was substantially improved, up to 507-fold. In the S2 pocket, 3,4-dichlorophenylalanine residues provided high trypanocidal activities. In the S3 pocket, aromatic residues provided enhanced selectivity against hCatL. RD inhibition ( Ki values) and in vitro cell-growth of Trypanosoma brucei rhodesiense (IC50 values) were measured in the nanomolar range. Triazole-based ligands, obtained by a safe, gram-scale flow production of ethyl 1 H-1,2,3-triazole-4-carboxylate, showed excellent metabolic stability in human liver microsomes and in vivo half-lives of up to 1.53 h in mice. When orally administered to infected mice, parasitaemia was reduced but without complete removal of the parasites.

Characterization of a novel, papain-inducible murine model of eosinophilic rhinosinusitis.

Eosinophilic chronic rhinosinusitis (ECRS) is a disease characterized by eosinophilic inflammatory infiltrate and a local type 2 cytokine milieu. Current animal models fail to recapitulate many of the innate and adaptive immunologic hallmarks of the disease, thus hindering the development of effective therapeutics. In the present study, mice were exposed intranasally to the cysteine protease papain, which shares functional similarities with parasitic proteases and aeroallergens, to generate a rapidly inducible murine model of eosinophilic rhinosinusitis. C57BL/6 mice were intranasally instilled with 20 μg papain or heat-inactivated papain (HP) on days 0-2 and days 7-10, and then euthanized on day 11. Nasal lavage fluid (NALF) was analyzed to quantify eosinophils and inflammatory cytokine secretion. Sinonasal tissue was sectioned and stained for goblet cells or homogenized to analyze cytokine levels. Serum samples were assayed for immunoglobulin E (IgE) by enzyme-linked immunoassay. Sinonasal mucosal tissue was dissociated and analyzed by flow cytometry. Compared with HP treatment, papain induced significant eosinophilia in NALF, goblet cell hyperplasia, innate and adaptive immune cell infiltration, type 2 cytokine production, and IgE responses. Flow cytometric analysis of sinonasal tissues revealed significant inflammatory cell infiltration and interleukin-13-producing cell populations. In this study, we demonstrated that the cysteine protease papain induces allergic sinonasal eosinophilic rhinosinusitis and resembles T-helper 2 cell inflammation and innate immune characteristics of ECRS. This model permits further study into the molecular mechanisms underlying ECRS pathology and provides a model system for the evaluation of potential pharmacologic interventions.

Papers of note in Science 358 (6362)

This week’s articles describe how bacteria sense surfaces; the lysine acylation of host proteins mediated by a Vibrio cholerae toxin; and druggable proteases of malaria parasites.

A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.

Regulated exocytosis by secretory organelles is important for malaria parasite invasion and egress. Many parasite effector proteins, including perforins, adhesins, and proteases, are extensively proteolytically processed both pre- and postexocytosis. Here we report the multistage antiplasmodial activity of the aspartic protease inhibitor hydroxyl-ethyl-amine-based scaffold compound 49c. This scaffold inhibits the preexocytosis processing of several secreted rhoptry and microneme proteins by targeting the corresponding maturases plasmepsins IX (PMIX) and X (PMX), respectively. Conditional excision of PMIX revealed its crucial role in invasion, and recombinantly active PMIX and PMX cleave egress and invasion factors in a 49c-sensitive manner.

Plasmodiumparasite entrance and exit

Malaria Sweats and fever are the hallmarks of malaria. Red blood cells are the replication factories for malaria parasites. Fever occurs when the parasites' merozoite stages burst en masse from red blood cells into the circulation. Nasamu et al. and Pino et al. discovered that two parasite proteases, plasmepsin IX and X, are essential for mass cell exit (see the Perspective by Boddey). Plasmepsin X is also used by the merozoites to enter a fresh red blood cell to continue the replicative cycle. These two plasmepsins act by regulating the maturation of enzymes required to disrupt host cell membranes. Because these functions are essential for the parasite, the authors used protease inhibitors to show that plasmepsins provide potential drug targets. Science , this issue p. [518][1], p. [522][2]; see also p. [445][3] [1]: /lookup/doi/10.1126/science.aan1478 [2]: /lookup/doi/10.1126/science.aaf8675 [3]: /lookup/doi/10.1126/science.aaq0002