Identification and classification of papain-like cysteine proteinases.

Papain-like cysteine peptidases form a big and highly diverse superfamily of proteins involved in many important biological functions, such as protein turnover, deubiquitination, tissue remodeling, blood clotting, virulence, defense, and cell wall remodeling. High sequence and structure diversity observed within these proteins hinders their comprehensive classification as well as the identification of new representatives. Moreover, in general protein databases, many families already classified as papain like lack details regarding their mechanism of action or biological function. Here, we use transitive remote homology searches and 3D modeling to newly classify 21 families to the papain-like cysteine peptidase superfamily. We attempt to predict their biological function and provide structural characterization of 89 protein clusters defined based on sequence similarity altogether spanning 106 papain-like families. Moreover, we systematically discuss observed diversity in sequences, structures, and catalytic sites. Eventually, we expand the list of human papain-related proteins by seven representatives, including dopamine receptor-interacting protein 1 as potential deubiquitinase, and centriole duplication regulating CEP76 as retaining catalytically active peptidase-like domain. The presented results not only provide structure-based rationales to already existing peptidase databases but also may inspire further experimental research focused on peptidase-related biological processes.

Self-inhibited State of Venezuelan Equine Encephalitis Virus (VEEV) nsP2 Cysteine Protease: A Crystallographic and Molecular Dynamics Analysis.

The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 Å) crystal structure of a truncated nsP2pro (residues 463-785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.

Computational Study of the Inhibition of RgpB Gingipain, a Promising Target for the Treatment of Alzheimer's Disease.

Alzheimer's disease represents one of the most ambitious challenges for biomedical sciences due to the growing number of cases worldwide in the elderly population and the lack of efficient treatments. One of the recent attempts to develop a treatment points to the cysteine protease RgpB as a promising drug target. In this attempt, several small-molecule covalent inhibitors of this enzyme have been proposed. Here, we report a computational study at the atomic level of the inhibition mechanism of the most promising reported compounds. Molecular dynamics simulations were performed on six of them, and their binding energies in the active site of the protein were computed. Contact maps and interaction energies were decomposed by residues to disclose those key interactions with the enzyme. Finally, quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations were performed to evaluate the reaction mechanism by which these drug candidates lead to covalently bound complexes, inhibiting the RgpB protease. The results provide a guide for future re-design of prospective and efficient inhibitors for the treatment of Alzheimer's disease.

Legionella effector LegA15/AnkH contains an unrecognized cysteine protease-like domain and displays structural similarity to LegA3/AnkD, but differs in host cell localization.

Legionella pneumophila is a human pathogen that causes Legionnaires' disease, a severe form of pneumonia. It can be found in various aquatic environments ranging from cooling towers to ponds. In addition to causing disease in humans, it can also infect free-living amoebae commonly found in various aquatic environments. Once inside a human lung macrophage, it creates a niche called the Legionella-containing vacuole where it can evade phagolysosomal degradation and replicate. During infection, normal cellular functions are hijacked by proteins that are secreted by the pathogen, called bacterial effectors. Here, the structural characterization of the effector LegA15/AnkD is reported. The protein contains an ankyrin-repeat domain followed by a cysteine protease-like (CPL) domain with a putative catalytic triad consisting of His268-Asn290-Cys361. The CPL domain shows similarity to the CE clan in the MEROPS database, which contains ubiquitin-like hydrolases. The C-terminal segment of LegA15, including the CPL domain, shows structural similarity to another effector, LegA3/AnkH, while they share only 12% sequence identity. When expressed in mammalian cells, LegA15 is localized within the cytoplasm, in contrast to LegA3, which localizes to the nucleus.

The Papain-like Cysteine Protease HpXBCP3 from Haematococcus pluvialis Involved in the Regulation of Growth, Salt Stress Tolerance and Chlorophyll Synthesis in Microalgae.

The papain-like cysteine proteases (PLCPs), the most important group of cysteine proteases, have been reported to participate in the regulation of growth, senescence, and abiotic stresses in plants. However, the functions of PLCPs and their roles in stress response in microalgae was rarely reported. The responses to different abiotic stresses in Haematococcus pluvialis were often observed, including growth regulation and astaxanthin accumulation. In this study, the cDNA of HpXBCP3 containing 1515 bp open reading frame (ORF) was firstly cloned from H. pluvialis by RT-PCR. The analysis of protein domains and molecular evolution showed that HpXBCP3 was closely related to AtXBCP3 from Arabidopsis. The expression pattern analysis revealed that it significantly responds to NaCl stress in H. pluvialis. Subsequently, transformants expressing HpXBCP3 in Chlamydomonas reinhardtii were obtained and subjected to transcriptomic analysis. Results showed that HpXBCP3 might affect the cell cycle regulation and DNA replication in transgenic Chlamydomonas, resulting in abnormal growth of transformants. Moreover, the expression of HpXBCP3 might increase the sensitivity to NaCl stress by regulating ubiquitin and the expression of WD40 proteins in microalgae. Furthermore, the expression of HpXBCP3 might improve chlorophyll content by up-regulating the expression of NADH-dependent glutamate synthases in C. reinhardtii. This study indicated for the first time that HpXBCP3 was involved in the regulation of cell growth, salt stress response, and chlorophyll synthesis in microalgae. Results in this study might enrich the understanding of PLCPs in microalgae and provide a novel perspective for studying the mechanism of environmental stress responses in H. pluvialis.

Characterization of a novel cysteine protease in Trichinella spiralis and its role in larval intrusion, development and fecundity.

The aim of this study was to investigate the biological properties of a novel gut-specific cysteine protease in Trichinella spiralis (TsGSCP) and its role in larval intrusion, development and fecundity. TsGSCP has a functional C1 peptidase domain; C1 peptidase belongs to cathepsin B family. The TsGSCP gene cloned and expressed in Escherichia coli BL21 showed intensive immunogenicity. qPCR and Western blotting revealed that TsGSCP mRNA and protein were expressed at various T. spiralis stages, but their expression levels in intestinal infectious larvae (IIL) were clearly higher than those in muscle larvae (ML), adult worms (AWs) and new-born larvae (NBL). Indirect immunofluorescence (IIF) analysis showed that TsGSCP was primarily located at the outer cuticle and the intrauterine embryos of this parasite. rTsGSCP showed the ability to specifically bind with IECs, and the binding site is within the IEC cytoplasm. rTsGSCP accelerated larval intrusion into host intestinal epithelial cells (IECs), whereas anti-rTsGSCP antibodies suppressed larval intrusion; the acceleration and suppression was induced by rTsGSCP and anti-rTsGSCP antibodies, respectively, in a dose-dependent manner. When ML were transfected with TsGSCP-specific dsRNA, TsGSCP expression and enzymatic activity were reduced by 46.82 and 37.39%, respectively, and the capacity of the larvae to intrude into IECs was also obviously impeded. Intestinal AW burden and adult female length and fecundity were significantly decreased in the group of mice infected with dsRNA-transfected ML compared to the control dsRNA and PBS groups. The results showed that TsGSCP plays a principal role in gut intrusion, worm development and fecundity in the T. spiralis lifecycle and might be a candidate target for vaccine development against Trichinella intrusion and infection.

Inhibition of Cysteine Proteases by 6,6'-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea.

The specificity of inhibition by 6,6'-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 µM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 µM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.

The Role of Cysteine Peptidases in Hematopoietic Stem Cell Differentiation and Modulation of Immune System Function.

Cysteine cathepsins are primarily involved in the degradation and recycling of proteins in endo-lysosomal compartments but are also gaining recognition as pivotal proteolytic contributors to various immune functions. Through their extracellular proteolytic activities within the hematopoietic stem cell niche, they are involved in progenitor cell mobilization and differentiation. Cysteine cathepsins, such as cathepsins L and S contribute to antigen-induced adaptive immunity through major histocompatibility complex class II antigen presentation whereas cathepsin X regulates T-cell migration. By regulating toll-like receptor signaling and cytokine secretion cysteine cathepsins activate innate immune cells and affect their functional differentiation. Cathepsins C and H are expressed in cytotoxic T lymphocytes and natural killer cells and are involved in processing of pro-granzymes into proteolytically active forms. Cytoplasmic activities of cathepsins B and L contribute to the maintenance of homeostasis of the adaptive immune response by regulating cell death of T and B lymphocytes. The expression pattern, localization, and activity of cysteine cathepsins is tightly connected to their function in immune cells. Furthermore, cysteine cathepsins together with their endogenous inhibitors, serve as mediators in the interplay between cancer and immune cells that results in immune cell anergy. The aim of the present article is to review the mechanisms of dysregulation of cysteine cathepsins and their inhibitors in relation to immune dysfunction to address new possibilities for regulation of their function.

The ß-link motif in protein architecture.

The ß-link is a composite protein motif consisting of a G1ß ß-bulge and a type II ß-turn, and is generally found at the end of two adjacent strands of antiparallel ß-sheet. The 1,2-positions of the ß-bulge are also the 3,4-positions of the ß-turn, with the result that the N-terminal portion of the polypeptide chain is orientated at right angles to the ß-sheet. Here, it is reported that the ß-link is frequently found in certain protein folds of the SCOPe structural classification at specific locations where it connects a ß-sheet to another area of a protein. It is found at locations where it connects one ß-sheet to another in the ß-sandwich and related structures, and in small (four-, five- or six-stranded) ß-barrels, where it connects two ß-strands through the polypeptide chain that crosses an open end of the barrel. It is not found in larger (eight-stranded or more) ß-barrels that are straightforward ß-meanders. In some cases it initiates a connection between a single ß-sheet and an α-helix. The ß-link also provides a framework for catalysis in serine proteases, where the catalytic serine is part of a conserved ß-link, and in cysteine proteases, including Mpro of human SARS-CoV-2, in which two residues of the active site are located in a conserved ß-link.

Mechanisms of Proteolytic Enzymes and Their Inhibition in QM/MM Studies.

Experimental evidence for enzymatic mechanisms is often scarce, and in many cases inadvertently biased by the employed methods. Thus, apparently contradictory model mechanisms can result in decade long discussions about the correct interpretation of data and the true theory behind it. However, often such opposing views turn out to be special cases of a more comprehensive and superior concept. Molecular dynamics (MD) and the more advanced molecular mechanical and quantum mechanical approach (QM/MM) provide a relatively consistent framework to treat enzymatic mechanisms, in particular, the activity of proteolytic enzymes. In line with this, computational chemistry based on experimental structures came up with studies on all major protease classes in recent years; examples of aspartic, metallo-, cysteine, serine, and threonine protease mechanisms are well founded on corresponding standards. In addition, experimental evidence from enzyme kinetics, structural research, and various other methods supports the described calculated mechanisms. One step beyond is the application of this information to the design of new and powerful inhibitors of disease-related enzymes, such as the HIV protease. In this overview, a few examples demonstrate the high potential of the QM/MM approach for sophisticated pharmaceutical compound design and supporting functions in the analysis of biomolecular structures.

Exploring the Versatility of the Covalent Thiol-Alkyne Reaction with Substituted Propargyl Warheads: A Deciding Role for the Cysteine Protease.

Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol-alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CDWT and catalytically inactive mutant USP16CDC205A. Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol-alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.

Structural leitmotif and functional variations of the structural catalytic core in (chymo)trypsin-like serine/cysteine fold proteinases.

Proteinases with the (chymo)trypsin-like serine/cysteine fold comprise a large superfamily performing their function through the Acid - Base - Nucleophile catalytic triad. In our previous work (Denesyuk AI, Johnson MS, Salo-Ahen OMH, Uversky VN, Denessiouk K. Int J Biol Macromol. 2020;153:399-411), we described a universal three-dimensional (3D) structural motif, NBCZone, that contains eleven amino acids: dipeptide 42 T-43 T, pentapeptide 54 T-55 T-56 T-57 T(base)-58 T, tripeptide 195 T(nucleophile)-196 T-197 T and residue 213 T (T - numeration of amino acids in trypsin). The comparison of the NBCZones among the members of the (chymo)trypsin-like protease family suggested the existence of 15 distinct groups. Within each group, the NBCZones incorporate an identical set of conserved interactions and bonds. In the present work, the structural environment of the catalytic acid at the position 102 T and the fourth member of the "catalytic tetrad" at the position 214 T was analyzed in 169 3D structures of proteinases with the (chymo)trypsin-like serine/cysteine fold. We have identified a complete Structural Catalytic Core (SCC) consisting of two classes and four groups. The proteinases belonging to different classes and groups differ from each other by the nature of the interaction between their N- and C-terminal ß-barrels. Comparative analysis of the 3CLpro(s) from SARS-CoV-2 and SARS-CoV, used as an example, showed that the amino acids at positions 103 T and 179 T affect the nature of the interaction of the "catalytic acid" core (102 T-Core, N-terminal ß-barrel) with the "supplementary" core (S-Core, C-terminal ß-barrel), which ultimately results in the modulation of the enzymatic activity. The reported analysis represents an important standalone contribution to the analysis and systematization of the 3D structures of (chymo)trypsin-like serine/cysteine fold proteinases. The use of the developed approach for the comparison of 3D structures will allow, in the event of the appearance of new representatives of a given fold in the PDB, to quickly determine their structural homologues with the identification of possible differences.

Cohnella 1759 cysteine protease shows significant long term half-life and impressive increased activity in presence of some chemical reagents.

Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme Km and Vmax values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme's co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.

Targeting SARS-CoV-2 M3CLpro by HCV NS3/4a Inhibitors: In Silico Modeling and In Vitro Screening.

Currently the entire human population is in the midst of a global pandemic caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2). This highly pathogenic virus has to date caused >71 million infections and >1.6 million deaths in >180 countries. Several vaccines and drugs are being studied as possible treatments or prophylactics of this viral infection. M3CLpro (coronavirus main cysteine protease) is a promising drug target as it has a significant role in viral replication. Here we use the X-ray crystal structure of M3CLpro in complex with boceprevir to study the dynamic changes of the protease upon ligand binding. The binding free energy was calculated for water molecules at different locations of the binding site, and molecular dynamics (MD) simulations were carried out for the M3CLpro/boceprevir complex, to thoroughly understand the chemical environment of the binding site. Several HCV NS3/4a protease inhibitors were tested in vitro against M3CLpro. Specifically, asunaprevir, narlaprevir, paritaprevir, simeprevir, and telaprevir all showed inhibitory effects on M3CLpro. Molecular docking and MD simulations were then performed to investigate the effects of these ligands on M3CLpro and to provide insights into the chemical environment of the ligand binding site. Our findings and observations are offered to help guide the design of possible potent protease inhibitors and aid in coping with the COVID-19 pandemic.

Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases.

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the "lock and key" mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.

Complexos imínicos de cobre com atividade antiparasitária frente à doença de chagas / Imine copper complexes with antiparasitic activity against Chagas disease

Neste trabalho foram sintetizados e caracterizados três complexos de cobre com ligantes imínicos, com o objetivo de avaliar sua atividade tripanocida. Esses complexos foram caracterizados por diversas técnicas espectroscópicas, como UV-Vis, Infravermelho e EPR, além de análise elementar e espectrometria de massa. Juntamente com outros complexos similares previamente sintetizados pelo nosso grupo, tiveram suas atividades avaliadas frente à forma tripomastigota do parasita T. cruzi, responsável pela fase aguda da doença de Chagas, por ensaios de viabilidade celular, com determinação do valor de seus IC50, concentração em que observamos a morte de 50% da cultura celular, pela metodologia denominada MTT. Todos os complexos mostraram-se eficientes frente a tripomastigotas, apresentando valores de IC50 abaixo de 10 µM, com quatro deles obtendo índice de seletividade maior que 10, fator importante para definir agentes promissores antichagásicos. Complexos selecionados também tiveram sua atividade verificada frente à forma amastigota do parasita, responsável pela fase crônica da doença, utilizando método de imageamento por microscópio de fluorescência e contagem celular. Estudos de inibição da cruzaína, uma cisteíno-protease importante para o metabolismo do parasita foram conduzidos em colaboração com o laboratório do Prof. Wagner Alves de Souza Júdice, da Universidade de Mogi das Cruzes. Quatro dos compostos testados apresentaram atividade inibitória frente a cruzaína, sendo dois de cobre, um de zinco e um ligante livre. Os estudos também permitiram diferenciar os mecanismos de inibição dos compostos, com os complexos de cobre apresentando um mecanismo de inibição clássico e o composto de zinco e o ligante livre apresentando o mecanismo de inibição competitiva parabólica com cooperatividade

In this work, three copper complexes with iminic ligands were synthesized and characterized, with the objective of evaluating their trypanocidal activity. These complexes were characterized by several spectroscopic techniques, such as UV-Vis, Infrared and EPR, in addition to elementary analysis and mass spectrometry. Together with other similar complexes previously synthesized by our group, their activities were evaluated against the trypomastigote form of the parasite T. cruzi, responsible for the acute phase of Chagas disease, by cell viability tests, with determination of the value of their IC50, concentration in that we observed the death of 50% of the cell culture, by the methodology called MTT, all presenting IC50 values below 10 µM, with four of them obtaining a selectivity index greater than 10, important factor for defining promising antichagasic agents. Selected complexes also had their activity verified against the amastigote form of the parasite, responsible for the chronic phase of the disease, using a fluorescence microscope and cell counting imaging method. Inhibition studies of cruzain, a cysteine protease important for the metabolism of the parasite, were conducted in collaboration with the laboratory of Professor Wagner Alves de Souza Júdice at the University of Mogi das Cruzes. Four of the tested compounds showed inhibitory activity against cruzain, two of copper, one of zinc and a free ligand. The studies also allowed to differentiate the mechanisms of inhibition of the compounds, with the copper complexes presenting a classic inhibition mechanism and the zinc compound and the free ligand presenting the competitive parabolic inhibition mechanism with cooperativity

Fragment-Based Discovery of Irreversible Covalent Inhibitors of Cysteine Proteases Using Chlorofluoroacetamide Library.

Fragment-based approach combined with electrophilic reactive compounds is a powerful strategy to discover novel covalent ligands for protein target. However, the promiscuous reactivity often interferes with identification of the fragments possessing specific binding affinity to the targeted protein. In our study, we report the fragment-based covalent drug discovery using the chemically tuned weak reactivity of chlorofluoroacetamide (CFA). We constructed a small fragment library composed of 30 CFA-appended compounds and applied it to the covalent ligand screening for cysteine protease papain as a model protein target. Using the fluorescence enzymatic assay, we identified CFA-benzothiazole 30 as a papain inhibitor, which was found to irreversibly inactivate papain upon enzyme kinetic analysis. The formation of the covalent papain-30 adduct was confirmed using electrospray ionization mass spectrometry analysis. The activity-based protein profiling (ABPP) experiment using an alkynylated analog of 30 (i.e., 30-yne) revealed that 30-yne covalently labeled papain with high selectivity. These data demonstrate potential utility of the CFA-fragment library for de novo discovery of target selective covalent inhibitors.

Drupin, a cysteine protease from Ficus drupacea latex accelerates excision wound healing in mice.

Wound healing is a tightly regulated physiological process that restores tissue integrity after injury. Plant latex proteases (PLPs) are considered an integral part in herbal wound care as it interferes at different phases of the wound healing process. Although many studies have reported the involvement of PLPs in healing process, an in-depth investigation is required to understand the molecular mechanism. Hence, the effect of PLPs with fibrinolytic activity on wound healing was investigated systematically using mouse excision wound model. Among 29 latices from Ficus genus tested, Ficus drupacea exhibited potent fibrinolytic activity. Cysteine protease responsible for fibrinolysis was purified from the F. drupacea latex named it as drupin, tested for its wound healing efficacy. The accelerated wound healing was mediated by downregulation of matrix metalloprotease (MMP)-9 without altering MMP-8 expression. Besides, drupin enhanced the rate of collagen synthesis at the wound site by increasing arginase 1 activity. And also, drupin increased the expression of arginase 1 in macrophages and involved in cell proliferation, and migration via MAP kinase and PI3K/Akt pathways. Overall, the present study highlights the interference of drupin in wound healing by increased arginase 1 activity and collagen synthesis, and cell proliferation and migration.

ZCPG, a cysteine protease from Zingiber montanum rhizome exhibits enhanced anti-inflammatory and acetylcholinesterase inhibition potential.

A 48 kDa Zingiber montanum cysteine protease glycoprotein (ZCPG) purified previously was studied for anti-inflammatory and acetylcholinesterase inhibitory activity. The lipoxygenase inhibition by ZCPG was linear, with an IC50 value of 2.25 µM. MTT, LDH, and cell cycle analysis in THP-1 derived macrophages corroborate no significant cytotoxicity at a lower concentration. ZCPG inhibited the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in lipopolysaccharide-stimulated THP-1 macrophages. In contrast, an increase in the production of interleukin-10, an anti-inflammatory cytokine, was observed. A reverse-transcription polymerase chain reaction study further confirmed that ZCPG inhibited the expression of IL-1ß, inducible nitric oxide synthase, and TNF-α by suppressing their mRNA transcription and expression in LPS stimulated THP-1 macrophages. Furthermore, the nature of acetylcholinesterase (AChE) inhibition by ZCPG is dose-dependent, competitive, and reversible. The AChE inhibitory activity was stable in a broad range of temperatures and pH. In vitro data were further validated by molecular interaction studies with a detailed inspection of the ZCPG probable binding modes in the active sites of AChE that provides the lead to deliver the structural determinants necessary for the activity towards AChE.

Kinetic characterization of a novel cysteine peptidase from the protozoan Babesia bovis, a potential target for drug design.

C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.