Monkeypox virus (MPXV) is a member of the Orthopoxvirus genus and the Poxviridae family, which instigated a rising epidemic called monkeypox disease. Proteinases are majorly engaged in viral propagation by catalyzing the cleavage of precursor polyproteins. Therefore, proteinase is essential for monkeypox and a critical drug target. In this study, high-throughput virtual screening (HTVS) and molecular dynamics simulation were applied to detect the potential natural compounds against the proteinase of the monkeypox virus. Here, 32,552 natural products were screened, and the top five compounds were selected after implementing the HTVS and molecular docking protocols in series. Gallicynoic Acid F showed the minimum binding score of -10.56 kcal/mole in the extra precision scoring method, which reflected the highest binding with the protein. The top five compounds showed binding scores ≤-8.98 kcal/mole. These compound complexes were tested under 100 ns molecular dynamics simulation, and Vaccinol M showed the most stable and consistent RMSD trend in the range of 2 Å to 3 Å. Later, MM/GBSA binding free energy and principal component analysis were performed on the top five compounds to validate the stability of selected compound complexes. Moreover, the ligands Gallicynoic Acid F and H2-Erythro-Neopterin showed the lowest binding free energies of -61.42 kcal/mol and -61.09 kcal/mol, respectively. Compared to the native ligand TTP-6171 (ΔGBind = -53.86 kcal/mol), these two compounds showed preferable binding free energy, suggesting inhibitory application against MPXV proteinase. This study proposed natural molecules as a therapeutic solution to control monkeypox disease.

Fasciola hepatica is a global parasite of humans and their livestock. Regulation of parasite-secreted cathepsin L-like cysteine proteases associated with virulence is important to fine-tune parasite-host interaction. We uncovered a family of seven Kunitz-type (FhKT) inhibitors dispersed into five phylogenetic groups. The most highly expressed FhKT genes (group FhKT1) are secreted by the newly excysted juveniles (NEJs), the stage responsible for host infection. The FhKT1 inhibitors do not inhibit serine proteases but are potent inhibitors of parasite cathepsins L and host lysosomal cathepsin L, S and K cysteine proteases (inhibition constants < 10 nM). Their unusual inhibitory properties are due to (a) Leu15 in the reactive site loop P1 position that sits at the water-exposed interface of the S1 and S1' subsites of the cathepsin protease, and (b) Arg19 which forms cation-π interactions with Trp291 of the S1' subsite and electrostatic interactions with Asp125 of the S2' subsite. FhKT1.3 is exceptional, however, as it also inhibits the serine protease trypsin due to replacement of the P1 Leu15 in the reactive loop with Arg15. The atypical Kunitz-type inhibitor family likely regulate parasite cathepsin L proteases and/or impairs host immune cell activation by blocking lysosomal cathepsin proteases involved in antigen processing and presentation.

Piroplasmid parasites comprising of Babesia, Theileria, and Cytauxzoon are transmitted by ticks to farm and pet animals and have a significant impact on livestock industries and animal health in tropical and subtropical regions worldwide. In addition, diverse Babesia spp. infect humans as opportunistic hosts. Molecular phylogeny has demonstrated at least six piroplasmid lineages exemplified by B. microti, B. duncani, C. felis, T. equi, Theileria sensu stricto (T. annulata, T. parva, and T. orientalis) and Babesia sensu stricto (B. bovis, B. bigemina, and B. ovis). C1A cysteine-proteinases (C1A-Cp) are papain-like enzymes implicated in pathogenic and vital steps of the parasite life cycle such as nutrition and host cell egress. An expansion of C1A-Cp of T. annulata and T. parva with respect to B. bovis and B. ovis was previously described. In the present work, C1A-Cp paralogs were identified in available genomes of species pertaining to each piroplasmid lineage. Phylogenetic analysis revealed eight C1A-Cp groups. The profile of C1A-Cp paralogs across these groups corroborates and defines the existence of six piroplasmid lineages. C. felis, T. equi and Theileria s.s. each showed characteristic expansions into extensive families of C1A-Cp paralogs in two of the eight groups. Underlying gene duplications have occurred as independent unique evolutionary events that allow distinguishing these three piroplasmid lineages. We hypothesize that C1A-Cp paralog families may be associated with the advent of the schizont stage. Differences in the invertebrate tick host specificity and/or mode of transmission in piroplasmid lineages might also be associated with the observed C1A-Cp paralog profiles.

Systemic sclerosis (SSc) is a connective tissue disease that is characterized by fibrosis in the skin and internal organs, such as the lungs. Activated differentiation of progenitor cells, which are mainly resident fibroblasts, into myofibroblasts is considered a key mechanism underlying the overproduction of extracellular matrix and the resultant tissue fibrosis in SSc. Calpains are members of the Ca2+-dependent cysteine protease family, whose enzymatic activities participate in signal transduction and tissue remodeling, potentially contributing to fibrosis in various organs. However, the roles of calpain in the pathogenesis of SSc remain unknown. This study aimed to examine the anti-fibrotic properties of N-acetyl-Leu-Leu-norleucinal (ALLN), one of the cysteine proteinase inhibitors that primarily inhibit calpain, in vitro and in vivo, to optimally translate into the therapeutic utility in human SSc.

Plant cysteine proteinase inhibitors (cystatins) play important roles in plant defense mechanisms. Some proteins that interact with cystatins may defend against abiotic stresses. Here, we showed that AtCaN2, a Ca(2+)-dependent nuclease in Arabidopsis, is transcribed in senescent leaves and stems and interacts with an Arabidopsis cystatin (AtCYSb) in a yeast two-hybrid screen. The interaction between AtCYSb and AtCaN2 was confirmed by in vitro pull-down assay and bimolecular fluorescence complementation. Agarose gel electrophoresis showed that the nuclease activity of AtCaN2 against λDNA was inhibited by AtCYSb, which suggests that AtCYSb regulates nucleic acid degradation in cells.

Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. The proteinase inhibitors function as modulators for controlling the extent of deleterious proteinase activity. The Kazal-type proteinase inhibitors (KPIs) in family I1 are among the well-known families of proteinase inhibitors, widely found in mammals, avian and a variety of invertebrates. Like those classical KPIs, the invertebrate KPIs can be single or multiple domain proteins containing one or more Kazal inhibitory domains linked together by peptide spacers of variable length. All invertebrate Kazal domains of about 40-60 amino acids in length share a common structure which is dictated by six conserved cysteine residues forming three intra-domain disulfide cross-links despite the variability of amino acid sequences between the half-cystines. Invertebrate KPIs are strong inhibitors as shown by their extremely high association constant of 10(7)-10(13)M(-1). The inhibitory specificity of a Kazal domain varies widely with a different reactive P(1) amino acid. Different invertebrate KPI domains may arise from gene duplication but several KPI proteins can also be derived from alternative splicing. The invertebrate KPIs function as anticoagulants in blood-sucking animals such as leech, mosquitoes and ticks. Several KPIs are likely involved in protecting host from microbial proteinases while some from the parasitic protozoa help protecting the parasites from the host digestive proteinase enzymes. Silk moths produce KPIs to protect their cocoon from predators and microbial destruction.

Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution.

Theileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs) are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP) is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells.

Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18 kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.

Current chemotherapy of human African trypanosomiasis or sleeping sickness relies on drugs developed decades ago, some of which show toxic side effects. One promising line of research towards the development of novel anti-trypanosomal drugs are small-molecule inhibitors of Trypanosoma brucei cysteine proteinases.

Tamoxifen is one of the most commonly employed endocrine therapies for patients with estrogen receptor α (ERα)-positive breast cancer. Unfortunately the clinical benefit is limited due to intrinsic and acquired drug resistance. We previously reported a genome-wide association study that identified common SNPs near the CTSO gene and in ZNF423 associated with development of breast cancer during tamoxifen therapy in the NSABP P-1 and P-2 breast cancer prevention trials. Here, we have investigated their roles in ERα-positive breast cancer growth and tamoxifen response, focusing on the mechanism of CTSO. We performed in vitro studies including luciferase assays, cell proliferation, and mass spectrometry-based assays using ERα-positive breast cancer cells and a panel of genomic data-rich lymphoblastoid cell lines. We report that CTSO reduces the protein levels of BRCA1 and ZNF423 through cysteine proteinase-mediated degradation. We also have identified a series of transcription factors of BRCA1 that are regulated by CTSO at the protein level. Importantly, the variant CTSO SNP genotypes are associated with increased CTSO and decreased BRCA1 protein levels that confer resistance to tamoxifen. Characterization of the effect of both CTSO SNPs and ZNF423 SNPs on tamoxifen response revealed that cells with different combinations of CTSO and ZNF423 genotypes respond differently to Tamoxifen, PARP inhibitors or the combination of the two drugs due to SNP dependent differential regulation of BRCA1 levels. Therefore, these genotypes might be biomarkers for selection of individual drug to achieve the best efficacy.

Gut extracts from cereal aphids (Sitobion avenae) showed significant levels of proteolytic activity, which was inhibited by reagents specific for cysteine proteases and chymotrypsin-like proteases. Gut tissue contained cDNAs encoding cathepsin B-like cysteine proteinases, similar to those identified in the closely related pea aphid (Acyrthosiphon pisum). Analysis of honeydew (liquid excreta) from cereal aphids fed on diet containing ovalbumin showed that digestion of ingested proteins occurred in vivo. Protein could partially substitute for free amino acids in diet, although it could not support complete development. Recombinant wheat proteinase inhibitors (PIs) fed in diet were antimetabolic to cereal aphids, even when normal levels of free amino acids were present. PIs inhibited proteolysis by aphid gut extracts in vitro, and digestion of protein fed to aphids in vivo. Wheat subtilisin/chymotrypsin inhibitor, which was found to inhibit serine and cysteine proteinases, was more effective in both inhibitory and antimetabolic activity than wheat cystatin, which inhibited cysteine proteases only. Digestion of ingested protein is unlikely to contribute significantly to nutritional requirements when aphids are feeding on phloem, and the antimetabolic activity of dietary proteinase inhibitors is suggested to result from effects on proteinases involved in degradation of endogenous proteins.

The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of beta-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC50 values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.

Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases.

We have solved the crystal and molecular structures of hepatitis A viral (HAV) 3C proteinase, a cysteine peptidase having a chymotrypsin-like protein fold, in complex with each of three tetrapeptidyl-based methyl ketone inhibitors to resolutions beyond 1.4 A, the highest resolution to date for a 3C or a 3C-Like (e.g. SARS viral main proteinase) peptidase. The residues of the beta-hairpin motif (residues 138-158), an extension of two beta-strands of the C-terminal beta-barrel of HAV 3C are critical for the interactions between the enzyme and the tetrapeptide portion of these inhibitors that are analogous to the residues at the P4 to P1 positions in the natural substrates of picornaviral 3C proteinases. Unexpectedly, the Sgamma of Cys172 forms two covalent bonds with each inhibitor, yielding an unusual episulfide cation (thiiranium ring) stabilized by a nearby oxyanion. This result suggests a mechanism of inactivation of 3C peptidases by methyl ketone inhibitors that is distinct from that occurring in the structurally related serine proteinases or in the papain-like cysteine peptidases. It also provides insight into the mechanisms underlying both the inactivation of HAV 3C by these inhibitors and on the proteolysis of natural substrates by this viral cysteine peptidase.

We have investigated whether varied assay conditions account for the conflicting reports on measured elastolytic activity of alveolar macrophages (AM) cultured in direct contact with the 3H-elastin substrate coated onto 16-mm wells in serum-containing media. The data indicate that measured elastolytic activity in this assay system was dependent on the amount of 3H-elastin/culture well. 3H-elastin > 350 micrograms/well, in contrast to the < or = 200 micrograms/well commonly used in this assay system, resulted in optimal measurement of elastolytic activity that was linear with respect to culture time (up to 72 h examined) and was directly proportional to number of AM/well (up to 1.0 x 10(6) examined). The sensitivity of measured elastolytic activity to tissue inhibitor of metalloproteinases (TIMP) and to Z-phe-phe (a specific cysteine proteinase inhibitor) was not affected by amount of 3H-elastin/well, but appears to be dependent on the time period of AM culture. TIMP (at 5 microM, maximal dose examined) inhibited the measured elastolytic activity by 25% in 24-h cultures compared to 69% in 72-h cultures; Z-phe-phe (at 10 microM, dose at which maximal effect was obtained) inhibited the elastolytic activity by 45% in the 24-h cultures compared to 34% in the 72-h cultures. These findings indicate that differences in substrate levels and in culture time have a significant effect on the results obtained in measurement of AM-mediated elastolytic activity in culture, which may account for the conflicting reports in the literature. Thus standard optimal assay condition are required for valid interpretation of results of AM-mediated elastolytic activity measurements.

The recent pandemic associated with SARS-CoV-2, a virus of the Coronaviridae family, has resulted in an unprecedented number of infected people. The highly contagious nature of this virus makes it imperative for us to identify promising inhibitors from pre-existing antiviral drugs. Two druggable targets, namely 3C-like proteinase (3CLpro) and 2'-O-ribose methyltransferase (2'-O-MTase) were selected in this study due to their indispensable nature in the viral life cycle. 3CLpro is a cysteine protease responsible for the proteolysis of replicase polyproteins resulting in the formation of various functional proteins, whereas 2'-O-MTase methylates the ribose 2'-O position of the first and second nucleotide of viral mRNA, which sequesters it from the host immune system. The selected drug target proteins were screened against an in-house library of 123 antiviral drugs. Two promising drug molecules were identified for each protein based on their estimated free energy of binding (ΔG), the orientation of drug molecules in the active site and the interacting residues. The selected protein-drug complexes were then subjected to MD simulation, which consists of various structural parameters to equivalently reflect their physiological state. From the virtual screening results, two drug molecules were selected for each drug target protein [Paritaprevir (ΔG = -9.8 kcal/mol) & Raltegravir (ΔG = -7.8 kcal/mol) for 3CLpro and Dolutegravir (ΔG = -9.4 kcal/mol) and Bictegravir (ΔG = -8.4 kcal/mol) for 2'-OMTase]. After the extensive computational analysis, we proposed that Raltegravir, Paritaprevir, Bictegravir and Dolutegravir are excellent lead candidates for these crucial proteins and they could become potential therapeutic drugs against SARS-CoV-2. Communicated by Ramaswamy H. Sarma.

Two cysteine proteinase inhibitors from cowpea, VuCys1 and VuCys2, were produced in E. coli ArcticExpress (DE3). The recombinant products strongly inhibited papain and chymopapain as well as the midgut proteases from Callosobruchus maculatus larvae, a bruchid that uses cysteine proteases as major digestive enzymes. Heat treatment at 100°C for up to 60min or incubation at various pH values caused little reduction in the papain inhibitory activity of both inhibitors. Moreover, minor conformational variations, as probed by circular dichroism spectroscopy, were observed after VuCys1 and VuCys2 were subjected to these treatments. The crystal structure of VuCys1 was determined at a resolution of 1.95Å, revealing a domain-swapped dimer in the asymmetric unit. However, the two lobes of the domain-swapped dimer are positioned closer to each other in VuCys1 in comparison to other similar cystatin structures. Moreover, some polar residues from opposite lobes recruit water molecules, forming a hydrogen bond network that mediates contacts between the lobes, thus generating an extended open interface. Due to the closer distance between the lobes, a small hydrophobic core is also formed, further stabilizing the folded domain-swapped dimer. These structural features might account for the extraordinary thermal and pH stability of VuCys1.

The main proteinase (M(pro)) of the severe acute respiratory syndrome (SARS) coronavirus is a principal target for the design of anticoronaviral compounds. Benzotriazole esters have been reported as potent nonpeptidic inhibitors of the enzyme, but their exact mechanism of action remains unclear. Here we present crystal structures of SARS-CoV M(pro), the active-site cysteine of which has been acylated by benzotriazole esters that act as suicide inhibitors. In one of the structures, the thioester product has been hydrolyzed and benzoic acid is observed to bind to the hydrophobic S2 pocket. This structure also features the enzyme with a shortened N-terminal segment ("amputated N finger"). The results further the understanding of the important role of the N finger for catalysis as well as the design of benzotriazole inhibitors with improved specificity.

Kazal-type serine proteinase inhibitors (KPIs) function in physiological and immunological processes requiring proteinase action. In the present study, the first Cherax quadricarinatus KPI gene (designated CqKPI) was identified and characterized. The open reading frame of CqKPI contains 405 nucleotides and encodes a protein of 134 amino acids. CqKPI has two Kazal domains comprising 44 amino acid residues with the conserved amino acid sequence C-X3-C-X7-C-X6-Y-X3-C-X6-C-X12-C. Each Kazal domain has six conserved cysteine residues, which can form a structural conformation of three pairs of disulfide bonds stabilizing the Kazal domain. CqKPI exhibited high similarity with previously identified KPIs from crayfish hemocytes. The results of tissue distribution showed that CqKPI had the highest expression level in hemocytes, and this was in agreement with phylogenic relationships. Recombinant CqKPI (rCqKPI) was heterologously expressed in Escherichia coli and purified for further study. The proteinase inhibition assays suggested that rCqKPI could potently inhibit elastase and weakly inhibit trypsin, subtilisin A, and proteinase K, but not α-chymotrypsin. It can firmly bind to Bacillus hwajinpoensis, Staphylococcus aureus, and Vibrio parahaemolyticus, with weak binding to Candida albicans. In addition, CqKPI inhibited bacterial secretory proteinase activity and inhibited the growth of B. hwajinpoensis and C. albicans. These data suggest that CqKPI might be involved in anti-bacterial immunity, acting as an inhibitor of the proteinase cascade in the resistance to invasion of pathogens.