In silico validation of novel inhibitors of malarial aspartyl protease, plasmepsin V and antimalarial efficacy prediction.

Plasmepsin V (Plm V) is an essential aspartic protease required for survival of the malaria parasite, Plasmodium falciparum (Pf). Plm V is required for cleaving the PEXEL motifs of many Pf proteins and its inhibition leads to a knockout effect, indicating its suitability as potential drug target. To decipher new inhibitors of PfPlm V, molecular docking of four HIV-1 protease inhibitors active against PfPlmV was performed on Glide module of Schrödinger suite that supported saquinavir as a lead drug, and therefore, selected as a control. Saquinavir contains an important hydroxyethylamine (HEA) pharmacophore, which was utilized as backbone coupled with piperazine scaffold to build new library of compounds. Newly designed HEA compounds were screened virtually against Plm V. Molecular docking led to a few hits (1 and 3) with higher docking score over the control drug. Notably, compound 1 showed the highest docking score (-11.90 kcal/mol) and XP Gscore (-11.948 kcal/mol). The Prime MMGBSA binding free energy for compound 1 (-60.88 kcal/mol) and 3 (-50.96 kcal/mol) was higher than saquinavir (-37.51 kcal/mol). The binding free energy for the last frame of molecular dynamic simulation supported compound 1 (-92.88 kcal/mol) as potent inhibitor of PfPlm V over saquinavir (-72.77 kcal/mol), and thus, deserves experimental validations in culture and subsequently in animal models.Communicated by Ramaswamy H. Sarma.

Rupatadine protects the intestinal mucosa from injury by 5-flurouracil via modulation of inflammation, apoptosis and intestinal permeability.

Fluorouracil (5-FU) is a widely used chemotherapeutic agent in various malignant tumors. However, intestinal toxicity is considered the irritant unavoidable adverse effect during the course therapy. The aim of the current study was to screen the effect of a new selective histamine receptor 1 blocker and platelet-activating factor (PAF) blocker on 5-FU induced intestinal toxicity. Five groups (6 rats each) of adult male rats (Wistar) were arranged as follows: (1) control group that was treated with carboxymethylcellulose, (2) a group that received rupatadine (higher dose) only, (3) a group that received 5-FU and (4) and (5) groups that received 5-FU plus lower or higher dose rupatadine, respectively. At end of the experiment, we determined intestinal malondialdehyde (MDA), glutathione reduced (GSH), nitric oxide (NO), tumor necrosis factor (TNF-α), interleukin 1ß, 6, 10 (IL-1ß, IL-6, IL-10), PAF, histamine, myeloperoxidase, cysteine-aspartic acid protease-3 (caspase-3), and nuclear factor kappa B (NF-κB) as well as the histological analysis. 5-FU injection caused marked elevation of MDA, NO, TNF-α, IL-1ß, IL-6, PAF, histamine, myeloperoxidase, caspase-3, and NF-κB expressions. The intoxicated animals showed deficient GSH and IL-10 along with significant loss of villi, disorganized crypts, and inflammatory cell infiltration. Rupatadine pretreatment reduced the previously mentioned parameters, preserved a nearly normal intestinal mucosa picture with replenished GSH and elevated IL-10. In conclusion, rupatadine is a dual histamine receptor 1, and a PAF blocker could reduce 5-FU-induced oxidative damage, inflammation, apoptosis, and ulceration of the intestinal epithelium. Rupatadine may be a valuable modality to decrease 5-FU induced intestinal mucositis.

In vivo tumor growth inhibition by Solanum tuberosum aspartic protease 3 (StAP3) treatment.

Solanum tuberosum aspartic Proteases (StAPs) show selective plasma membrane permeabilization, inducing cytotoxicity of cancer cells versus normal cells in vitro. Herein, we aimed to evaluate both StAP3 systemic toxicity and antitumoral activity against human melanoma in vivo. The toxicity of a single high dose of StAP3 (10 µg/g body weight, intraperitoneally) was assessed in a Balb/c mice model. Subcutaneous A375 human melanoma xenografts in athymic nude (nu/nu) mice were induced. Once tumors developed (mean larger dimension = 3.8 ± 0.09 mm), mice were StAP3-treated (6 µg/g body weight, subcutaneously under the tumor at a single dose). For both models, controls were treated with physiologic saline solution. StAP3-treated mice showed a significant inhibition of tumor growth (p < 0.05) compared with controls. No signs of toxicity were detected in StAP3-treated mice in both models. These results suggest the potential of these plant proteases as anticancer agents.

Akkermansia muciniphila Aspartic Protease Amuc_1434* Inhibits Human Colorectal Cancer LS174T Cell Viability via TRAIL-Mediated Apoptosis Pathway.

Mucin2 (Muc2) is the main component of the intestinal mucosal layer and is highly expressed in mucous colorectal cancer. Previous studies conducted by our lab found that the recombinant protein Amuc_1434 (expressed in Escherichia coli prokaryote cell system, hereinafter termed Amuc_1434*), derived from Akkermansia muciniphila, can degrade Muc2. Thus, the main objective of this study was to explore the effects of Amuc_1434* on LS174T in colorectal cancer cells expressing Muc2. Results from this study demonstrated that Amuc_1434* inhibited the proliferation of LS174T cells, which was related to its ability to degrade Muc2. Amuc_1434* also blocked the G0/G1 phase of the cell cycle of LS174T cells and upregulated the expression of tumor protein 53 (p53), which is a cell cycle-related protein. In addition, Amuc_1434* promoted apoptosis of LS174T cells and increased mitochondrial ROS levels in LS174T cells. The mitochondrial membrane potential of LS174T cells was also downregulated by Amuc_1434*. Amuc_1434* can activate the death receptor pathway and mitochondrial pathway of apoptosis by upregulating tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL). In conclusion, our study was the first to demonstrate that the protein Amuc_1434* derived from Akkermansia muciniphila suppresses LS174T cell viability via TRAIL-mediated apoptosis pathway.

Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits.

Plant proteases play a fundamental role in several processes like growth, development and in response to biotic and abiotic stress. In particular, aspartic proteases (AP) are expressed in different plant organs and have antimicrobial activity. Previously, we purified an AP from Salpichroa origanifolia fruits called salpichroin. The aim of this work was to determine the cytotoxic activity of this enzyme on selected plant and human pathogens. For this purpose, the growth of the selected pathogens was analysed after exposure to different concentrations of salpichroin. The results showed that the enzyme was capable of inhibiting Fusarium solani and Staphylococcus aureus in a dose-dependent manner. It was determined that 1·2 µmol l-1 of salpichroin was necessary to inhibit 50% of conidial germination, and the minimal bactericidal concentration was between 1·9 and 2·5 µmol l-1 . Using SYTOX Green dye we were able to demonstrate that salpichroin cause membrane permeabilization. Moreover, the enzyme treated with its specific inhibitor pepstatin A did not lose its antibacterial activity. This finding demonstrates that the cytotoxic activity of salpichroin is due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of the AP could represent a potential alternative for the control of pathogens that affect humans or crops of economic interest. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insights into the antimicrobial activity of an aspartic protease isolated from Salpichroa origanifolia fruits on plant and human pathogens. The proteinase inhibited Fusarium solani and Staphylococcus aureus in a dose-dependent manner due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of salpichroin suggests its potential applications as an important tool for the control of pathogenic micro-organisms affecting humans and crops of economic interest. Therefore, it would represent a new alternative to avoid the problems of environmental pollution and antimicrobial resistance.

Biocontrol activity of recombinant aspartic protease from Trichoderma harzianum against pathogenic fungi.

The use of cell wall degrading enzymes of Trichoderma is a promising alternative for improving food storage. The aspartic protease P6281 secreted by the fungus Trichoderma harzianum plays an important role in mycoparasitism on phytopathogenic fungi. In this study, recombinant P6281 (rP6281) expressed in Pichia pastoris showed high activity of 321.8 U/mL. Maximum activity was observed at pH 2.5 and 40 °C, and the enzyme was stable in the pH range of 2.5-6.0. rP6281 significantly inhibited spore germination and growth of plant and animal pathogenic fungi such as Botrytis cinerea, Mucor circinelloides, Aspergillus fumigatus, Aspergillus flavus, Rhizoctonia solani, and Candida albicans. Transmission electron microscopy revealed that rP6281 efficiently damages the cell wall of Botrytis cinerea. In addition, the protease significantly inhibited the development of grey mold that causes rotting of apple, orange, and cucumber, indicating that rP6281 may be developed as an effective anti-mold agent for fruit storage.

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.

Secreted fungal aspartic proteases: A review.

The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application.

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris.

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25-60 °C and pH 3.0-9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL(-1). In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.

A novel aspartic protease with HIV-1 reverse transcriptase inhibitory activity from fresh fruiting bodies of the wild mushroom Xylaria hypoxylon.

A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession), and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6-8 and in the temperature range 35-60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 µM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

Cholesterol and membrane phospholipid compositions modulate the leakage capacity of the swaposin domain from a potato aspartic protease (StAsp-PSI).

Potato aspartic proteases (StAPs) and their swaposin domain (StAsp-PSI) are proteins with cytotoxic activity which involves plasma membrane destabilization. The ability of these proteins to produce cell death varies with the cellular type. Therefore, StAPs and StAsp-PSI selective cytotoxicity could be attributed to the different membrane lipid compositions of target cells. In this work we investigate the possible mechanism by which StAPs and StAsp-PSI produce selective membrane destabilization. Results obtained from leakage assays show that StAsp-PSI is a potent inducer of the leakage of LUVs containing anionic phospholipids, especially those containing phosphatidylglycerol. Based in these results, we suggest that the cytotoxic activity of StAsp-PSI on pathogenic microorganisms could be mediated by the attraction between the exposed positive domains of StAsp-PSI and the negatively charged microorganism membrane. On the other hand, our circular dichroism spectroscopic measurements and analysis by size exclusion chromatography and followed by electrophoresis, indicate that hydrophobic environment is necessary to StAsp-PSI oligomerization and both StAsp-PSI disulfide bounds and membrane with negative charged phospholipids are required by StAsp-PSI to produce membrane destabilization and then induce cell death in tumors and microorganism cell targets. Additionally, we demonstrate that the presence of cholesterol into the LUV membranes strongly diminishes the capacity of StAsp-PSI to produce leakage. This result suggests that the lack of hemolytic and cytotoxic activities on human lymphocytes of StAsp-PSI/StAPs may be partly due by the presence of cholesterol in these cell membrane types.

Inhibition of BACE1 for therapeutic use in Alzheimer's disease.

Since BACE1 was reported as the beta-secretase in Alzheimer's disease (AD) over ten years ago, encouraging progress has been made toward understanding the cellular functions of BACE1. Genetic studies have further confirmed that BACE1 is essential for processing amyloid precursor protein (APP) at the beta-secretase site. Only after this cleavage can the membrane-bound APP C-terminal fragment be subsequently cleaved by gamma-secretase to release so-called AD-causing Abeta peptides. Hence, in the past decade, a wide variety of BACE1 inhibitors have been developed for AD therapy. This review will summarize the major historical events during the evolution of BACE1 inhibitors designed through different strategies of drug discovery. Although BACE1 inhibitors are expected to be safe in general, careful titration of drug dosage to avoid undesirable side effects in BACE1-directed AD therapy is also emphasized.

Heterologous expression and characterization of recombinant OsCDR1, a rice aspartic proteinase involved in disease resistance.

The Oryza sativa constitutive disease resistance 1 (OsCDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling. This apoplastic enzyme is a member of the group of 'atypical' plant aspartic proteinases. Recombinant OsCDR1 expressed in Escherichia coli exhibited protease activity against succinylated-casein substrate. Inactivating the enzyme through modification of an aspartate residue present in the deduced active site completely abolished its proteinase activity. Infiltration of the OsCDR1 fusion protein into leaves of Arabidopsis plants induced PR2 transcripts in both the infiltrated leaf (primary) and in non-treated secondary leaves while the inactive recombinant protein failed to induce either local or systemic PR2. These findings demonstrate that OsCDR1 is capable of inducing systemic defense responses in plants.

The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens.

Plant-specific insert domain (PSI) is a region of approximately 100 amino acid residues present in most plant aspartic protease (AP) precursors. PSI is not a true saposin domain; it is the exchange of the N- and C-terminal portions of the saposin like domain. Hence, PSI is called a swaposin domain. Here, we report the cloned, heterologous expression and purification of PSI from StAsp 1 (Solanum tuberosum aspartic protease 1), called StAsp-PSI. Results obtained here show that StAsp-PSI is able to kill spores of two potato pathogens in a dose-dependent manner without any deleterious effect on plant cells. As reported for StAPs (S. tuberosum aspartic proteases), the StAsp-PSI ability to kill microbial pathogens is dependent on the direct interaction of the protein with the microbial cell wall/or membrane, leading to increased permeability and lysis. Additionally, we demonstrated that, like proteins of the SAPLIP family, StAsp-PSI and StAPs are cytotoxic to Gram-negative and Gram-positive bacteria in a dose dependent manner. The amino acid residues conserved in SP_B (pulmonary surfactant protein B) and StAsp-PSI could explain the cytotoxic activity exerted by StAsp-PSI and StAPs against Gram-positive bacteria. These results and data previously reported suggest that the presence of the PSI domain in mature StAPs could be related to their antimicrobial activity.

Cytotoxic effect of potato aspartic proteases (StAPs) on Jurkat T cells.

StAPs are potato aspartic proteases with cytotoxic activity against plant pathogens and spermatozoa. StAPs cytotoxic activity is selective, since these proteins do not exert toxic effect on plant cells and erythrocytes. In this work, we investigated the capacity of StAPs to exert cytotoxicity on human leukaemia cells. Obtained results show that StAPs induce apoptosis on Jurkat T cells after a short time of incubation in a dose-dependent manner. However, no significative effect on the T lymphocytes viability was observed at all StAPs incubation times and concentrations tested. These results suggest that StAPs can be conceptually promising leads for cancer therapy.