Repurposing the McoTI-II Rigid Molecular Scaffold in to Inhibitor of ‘Papain Superfamily’ Cysteine Proteases

Clan C1A or ‘papain superfamily’ cysteine proteases are key players in many important physiological processes and diseases in most living systems. Novel approaches towards the development of their inhibitors can open new avenues in translational medicine. Here, we report a novel design of a re-engineered chimera inhibitor Mco-cysteine protease inhibitor (CPI) to inhibit the activity of C1A cysteine proteases. This was accomplished by grafting the cystatin first hairpin loop conserved motif (QVVAG) onto loop 1 of the ultrastable cyclic peptide scaffold McoTI-II. The recombinantly expressed Mco-CPI protein was able to bind with micromolar affinity to papain and showed remarkable thermostability owing to the formation of multi-disulphide bonds. Using an in silico approach based on homology modelling, protein–protein docking, the calculation of the free-energy of binding, the mechanism of inhibition of Mco-CPI against representative C1A cysteine proteases (papain and cathepsin L) was validated. Furthermore, molecular dynamics simulation of the Mco-CPI–papain complex validated the interaction as stable. To conclude, in this McoTI-II analogue, the specificity had been successfully redirected towards C1A cysteine proteases while retaining the moderate affinity. The outcomes of this study pave the way for further modifications of the Mco-CPI design for realizing its full potential in therapeutics. This study also demonstrates the relevance of ultrastable peptide-based scaffolds for the development of novel inhibitors via grafting.

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Presence of Anti-cystatin C–positive Dendritic Cells or Macrophages and Localization of Cysteine Proteases in the Apical Bud of the Enamel Organ in the Rat Incisor

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.4a6533.2005 ◽

2005 ◽

Vol 53 (5) ◽

pp. 643-651 ◽

Cited By ~ 1

Author(s):

Sumio Nishikawa

Keyword(s):

Dendritic Cells ◽

Cystatin C ◽

Cysteine Protease ◽

Cathepsin L ◽

Cysteine Proteases ◽

Cysteine Protease Inhibitor ◽

Enamel Organ ◽

Apical Bud ◽

Apical Buds ◽

Proliferation And Differentiation

Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C–positive. Anti-cystatin C–labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure. Basement membrane at the periphery of apical bud was also labeled by anti-cystatin C antibody. The apical buds included a few apoptotic fragments and weak reactivity with antibody to cathepsin L, a cysteine protease. Reactivity to anti-cystatin C and anti-cathepsin ∗∗∗L antibodies was also detected in the apical bud of newborn rat incisors. These results suggest that the cystatin C–positive macrophages or dendritic cells are involved in normal incisor formation. They may be related to the clearance of apoptotic cells or protection from putative cysteine protease activity.

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Proteases as Therapeutic Targets Against the Parasitic Cnidarian Ceratonova shasta: Characterization of Molecules Key to Parasite Virulence In Salmonid Hosts

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.804864 ◽

2022 ◽

Vol 11 ◽

Author(s):

Gema Alama-Bermejo ◽

Pavla Bartošová-Sojková ◽

Stephen D. Atkinson ◽

Astrid S. Holzer ◽

Jerri L. Bartholomew

Keyword(s):

Pacific Northwest ◽

Cathepsin D ◽

Cell Metabolism ◽

Cathepsin L ◽

Cysteine Proteases ◽

Aminopeptidase N ◽

Cysteine Protease Inhibitor ◽

Parasite Development ◽

Future Research ◽

3D Protein Structure

Proteases and their inhibitors play critical roles in host-parasite interactions and in the outcomes of infections. Ceratonova shasta is a myxozoan pathogen that causes enteronecrosis in economically important salmonids from the Pacific Northwest of North America. This cnidarian parasite has host-specific genotypes with varying virulence, making it a powerful system to decipher virulence mechanisms in myxozoans. Using C. shasta genome and transcriptome, we identified four proteases of different catalytic types: cathepsin D (aspartic), cathepsin L and Z-like (cysteine) and aminopeptidase-N (metallo); and a stefin (cysteine protease inhibitor), which implied involvement in virulence and hence represent target molecules for the development of therapeutic strategies. We characterized, annotated and modelled their 3D protein structure using bioinformatics and computational tools. We quantified their expression in C. shasta genotype 0 (low virulence, no mortality) and IIR (high virulence and mortality) in rainbow trout Oncorhynchus mykiss, to demonstrate that there are major differences between the genotypes during infection and parasite development. High proliferation of genotype IIR was associated with high expression of the cathepsin D and the stefin, likely correlated with high nutrient demands and to regulate cell metabolism, with upregulation preceding massive proliferation and systemic dispersion. In contrast, upregulation of the cathepsin L and Z-like cysteine proteases may have roles in host immune evasion in genotype 0 infections, which are associated with low proliferation, low inflammation and non-destructive development. In contrast to the other proteases, C. shasta aminopeptidase-N appears to have a prominent role in nematocyst formation in both genotypes, but only during sporogenesis. Homology searches of C. shasta proteases against other myxozoan transcriptomes revealed a high abundance of cathepsin L and aminopeptidase homologs suggesting common gene requirements across species. Our study identified molecules of potential therapeutic significance for aquaculture and serves as a baseline for future research aimed at functional characterisation of these targets.

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A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells

10.1101/2020.10.23.347534 ◽

2020 ◽

Cited By ~ 1

Author(s):

Drake M. Mellott ◽

Chien-Te Tseng ◽

Aleksandra Drelich ◽

Pavla Fajtová ◽

Bala C. Chenna ◽

...

Keyword(s):

Protease Inhibitor ◽

Host Cell ◽

Cysteine Protease ◽

Cleavage Site ◽

Cathepsin L ◽

Cysteine Proteases ◽

Protein Processing ◽

Spike Protein ◽

Cysteine Protease Inhibitor ◽

Viral Infectivity

ABSTRACTK777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.SIGNIFICANCEThe virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.

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Cysteine Protease Inhibitor (AcStefin) Is Required for Complete Cyst Formation of Acanthamoeba

Eukaryotic Cell ◽

10.1128/ec.00308-12 ◽

2013 ◽

Vol 12 (4) ◽

pp. 567-574 ◽

Cited By ~ 17

Author(s):

Jung-Yub Lee ◽

Su-Min Song ◽

Eun-Kyung Moon ◽

Yu-Ran Lee ◽

Bijay Kumar Jha ◽

...

Keyword(s):

Protease Inhibitor ◽

Cysteine Protease ◽

Protease Activity ◽

Cathepsin L ◽

Cyst Formation ◽

Cysteine Proteases ◽

Cysteine Protease Inhibitor ◽

Content Type ◽

Human Cathepsin ◽

Interfering Rna

ABSTRACTThe encystation ofAcanthamoebaleads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protectAcanthamoebafrom intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established inAcanthamoeba. In the present study, we identified and characterizedAcanthamoebacysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation ofAcanthamoeba.

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A mouse testis serine protease, TESP1, as the potential SPINK3 receptor protein on mouse sperm acrosome

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaab059 ◽

2021 ◽

Vol 27 (10) ◽

Author(s):

Shiyam Sundar Ramachandran ◽

Rubhadevi Balu ◽

Ravikumar Vilwanathan ◽

Jeyakanthan Jeyaraman ◽

Sudhakar Gandhi Paramasivam

Keyword(s):

Serine Protease ◽

Binding Affinity ◽

Trypsin Inhibitor ◽

Homology Modelling ◽

Protein Structures ◽

Protein Docking ◽

Female Reproductive Tract ◽

Dynamics Simulation ◽

Receptor Protein ◽

Sperm Acrosome

Abstract Serine protease inhibitor Kazal type 3 (SPINK3) from mouse seminal vesicles is a Kazal-type trypsin inhibitor. It has been shown to bind to the sperm acrosome and modify sperm activity by influencing the sub-cellular Ca2+ influx. Previously, SPINK3 was reported to suppress in vitro sperm capacitation. However, under natural coitus, SPINK3 is removed from the mouse acrosome in the female reproductive tract, leading to successful fertilisation. Identification of the SPINK3 binding partner becomes essential to develop a contraceptive that works by prolonging the binding of SPINK3 to the sperm acrosome. We identified the SPINK3 receptor by using recombinant SPINK3 (rSPINK3). Testicular serine protease 1 (TESP1) was identified as the receptor for SPINK3 by 2D gel electrophoresis coupled with western blot analysis. To authenticate TESP1 as the receptor for SPINK3, sperm cells were incubated with TESP1 peptide antibody followed by determining the intracellular [Ca2+]i concentration by flow cytometry using Fluo-3 AM as a calcium probe. Furthermore, the 3D structures of SPINK3 and TESP1 were predicted by homology modelling (Schrodinger suite) using the crystal structure of pancreatic secretory trypsin inhibitor (PDB ID—1TGS) and human prostasin (PDB ID—3DFJ) as templates. The modelled protein structures were validated and subjected to molecular dynamics simulation (MDS) using GROMACS v5.0.5. Protein–protein docking was performed using HDOCK and the complex was validated by MDS. The results predicted that SPINK3 and TESP1 had strong binding affinity, with a dock score of −430.70 and 14 hydrogen bonds as key active site residues. If the binding affinity between SPINK3 and TESP1 could be increased, the SPINK3-TESP1 association will be prolonged, which will be helpful in the development of a male contraceptive.

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Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S

Plants ◽

10.3390/plants9121676 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1676

Author(s):

Roberto Carlos Sallai ◽

Bruno Ramos Salu ◽

Rosemeire Aparecida Silva-Lucca ◽

Flávio Lopes Alves ◽

Thiago Henrique Napoleão ◽

...

Keyword(s):

Protease Inhibitor ◽

Animal Feed ◽

Cathepsin L ◽

Cysteine Proteases ◽

Reversed Phase ◽

Size Exclusion ◽

Cysteine Protease Inhibitor ◽

Araucaria Angustifolia ◽

Tropical Regions ◽

Defensive Role

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.

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Cryptostatin, a chagasin-family cysteine protease inhibitor of Cryptosporidium parvum

Parasitology ◽

10.1017/s0031182012000297 ◽

2012 ◽

Vol 139 (8) ◽

pp. 1029-1037 ◽

Cited By ~ 13

Author(s):

J.-M. KANG ◽

H.-L. JU ◽

J.-R. YU ◽

W.-M. SOHN ◽

B.-K. NA

Keyword(s):

Cryptosporidium Parvum ◽

Cysteine Protease ◽

Cathepsin L ◽

Sodium Dodecyl ◽

Cysteine Proteases ◽

Cysteine Protease Inhibitor ◽

Immunogold Electron Microscopy ◽

Electron Microscopy Analysis ◽

Wide Ph Range ◽

Human Cathepsin

SUMMARYCysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 β-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with Ki's in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and β-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.

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In silico comparison of SARS-CoV-2 spike protein-ACE2 binding affinities across species and implications for virus origin

Scientific Reports ◽

10.1038/s41598-021-92388-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sakshi Piplani ◽

Puneet Kumar Singh ◽

David A. Winkler ◽

Nikolai Petrovsky

Keyword(s):

Binding Affinity ◽

In Silico ◽

Sequence Similarity ◽

Homology Modelling ◽

Protein Docking ◽

Dynamics Simulation ◽

Spike Protein ◽

High Sequence Similarity ◽

Virus Origin ◽

Species Specific

AbstractThe devastating impact of the COVID-19 pandemic caused by SARS–coronavirus 2 (SARS-CoV-2) has raised important questions about its origins and the mechanism of its transfer to humans. A further question was whether companion or commercial animals could act as SARS-CoV-2 vectors, with early data suggesting susceptibility is species specific. To better understand SARS-CoV-2 species susceptibility, we undertook an in silico structural homology modelling, protein–protein docking, and molecular dynamics simulation study of SARS-CoV-2 spike protein’s ability to bind angiotensin converting enzyme 2 (ACE2) from relevant species. Spike protein exhibited the highest binding to human (h)ACE2 of all the species tested, forming the highest number of hydrogen bonds with hACE2. Interestingly, pangolin ACE2 showed the next highest binding affinity despite having a relatively low sequence homology, whereas the affinity of monkey ACE2 was much lower despite its high sequence similarity to hACE2. These differences highlight the power of a structural versus a sequence-based approach to cross-species analyses. ACE2 species in the upper half of the predicted affinity range (monkey, hamster, dog, ferret, cat) have been shown to be permissive to SARS-CoV-2 infection, supporting a correlation between binding affinity and infection susceptibility. These findings show that the earliest known SARS-CoV-2 isolates were surprisingly well adapted to bind strongly to human ACE2, helping explain its efficient human to human respiratory transmission. This study highlights how in silico structural modelling methods can be used to rapidly generate information on novel viruses to help predict their behaviour and aid in countermeasure development.

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Structure prediction of SPAK C-terminal domain and analysis of its binding to RFXV/I motifs by homology modelling, docking, and molecular dynamics simulation studies

Current Computer - Aided Drug Design ◽

10.2174/1573409916666200712140941 ◽

2020 ◽

Vol 16 ◽

Author(s):

Mubarak A. Alamri ◽

Ahmed D. Alafnan ◽

Obaid Afzal ◽

Alhumaidi B. Alabbas ◽

Safar M. Alqahtani

Keyword(s):

Molecular Dynamic ◽

Dynamic Stability ◽

Md Simulation ◽

Homology Modelling ◽

Antihypertensive Agents ◽

Structure And Function ◽

Dynamics Simulation ◽

Dimensional Structure ◽

Terminal Domain ◽

And Function

Background: The STE20/SPS1-related proline/alanine-rich kinase (SPAK) is a component of WNKSPAK/OSR1 signaling pathway that plays an essential role in blood pressure regulation. The function of SPAK is mediated by its highly conserved C-terminal domain (CTD) that interacts with RFXV/I motifs of upstream activators, WNK kinases, and downstream substrate, cation-chloride cotransporters. Objective: To determine and validate the three-dimensional structure of the CTD of SPAK and to study and analyze its interaction with the RFXV/I motifs. Methods: A homology model of SPAK CTD was generated and validated through multiple approaches. The model was based on utilizing the OSR1 protein kinase as a template. This model was subjected to 100 ns molecular dynamic (MD) simulation to evaluate its dynamic stability. The final equilibrated model was used to dock the RFQV-peptide derived from WNK4 into the primary pocket that was determined based on the homology sequence between human SPAK and OSR1 CTDs. The mechanism of interaction, conformational rearrangement and dynamic stability of the binding of RFQV-peptide to SPAK CTD were characterized by molecular docking and molecular dynamic simulation. Results: The MD simulation suggested that the binding of RFQV induces a large conformational change due to the distribution of salt bridge within the loop regions. These results may help in understanding the relation between the structure and function of SPAK CTD and to support drug design of potential SPAK kinase inhibitors as antihypertensive agents. Conclusion: This study provides deep insight into SPAK CTD structure and function relationship.

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A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts

Pathogens ◽

10.3390/pathogens10040388 ◽

2021 ◽

Vol 10 (4) ◽

pp. 388

Author(s):

Hương Giang Lê ◽

A-Jeong Ham ◽

Jung-Mi Kang ◽

Tuấn Cường Võ ◽

Haung Naw ◽

...

Keyword(s):

Protease Inhibitor ◽

Cysteine Protease ◽

Expression Profiles ◽

Critical Role ◽

Cyst Formation ◽

Cysteine Proteases ◽

Cysteine Protease Inhibitor ◽

Naegleria Fowleri ◽

Nægleria Fowleri ◽

Cystatin Family

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

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