Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His6-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.

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Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

10.1101/2020.12.16.422677 ◽

2020 ◽

Author(s):

Maria Kuzikov ◽

Elisa Costanzi ◽

Jeanette Reinshagen ◽

Francesca Esposito ◽

Laura Vangeel ◽

...

Keyword(s):

Enzymatic Activity ◽

Small Molecules ◽

Drug Target ◽

Treatment Options ◽

Cleavage Sites ◽

X Ray ◽

Binding Characteristics ◽

Main Protease ◽

Ic50 Values

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 uM and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Angs., showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

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Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets for drug repurposing

10.1101/2020.09.16.297945 ◽

2020 ◽

Cited By ~ 1

Author(s):

Bjoern Meyer ◽

Jeanne Chiaravalli ◽

Philip Brownridge ◽

Dominic P. Bryne ◽

Leonard A. Daly ◽

...

Keyword(s):

Protease Activity ◽

Drug Repurposing ◽

Cellular Proteins ◽

Cleavage Sites ◽

Antibody Testing ◽

Cellular Targets ◽

Main Protease ◽

Antigenic Proteins ◽

Culture Models ◽

Basic Biology

AbstractSARS-CoV-2 is the causative agent behind the COVID-19 pandemic, and responsible for tens of millions of infections, and hundreds of thousands of deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication, and inhibitors targeting proteases have already shown success at inhibiting SARS-CoV-2 in cell culture models. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify multiple previously unknown cleavage sites in multiple viral proteins, including major antigenic proteins S and N, which are the main targets for vaccine and antibody testing efforts. We discovered significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We showed that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, showed a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19 disease.

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Differential cleavage of the norovirus polyprotein precursor by two active forms of the viral protease

Journal of General Virology ◽

10.1099/vir.0.82797-0 ◽

2007 ◽

Vol 88 (7) ◽

pp. 2013-2018 ◽

Cited By ~ 18

Author(s):

Ulrike Scheffler ◽

Wolfram Rudolph ◽

Julia Gebhardt ◽

Jacques Rohayem

Keyword(s):

Molecular Mechanisms ◽

Protein Translation ◽

Cleavage Sites ◽

Viral Protease ◽

Catalytic Activities ◽

Cleavage Activity ◽

Catalytic Cleavage ◽

Scissile Bond ◽

Polyprotein Precursor

Protein translation in noroviruses requires translational processing of a polyprotein precursor by the viral protease. So far, the molecular mechanisms of catalytic cleavage by the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the viral protease were examined in vitro by using synthetic peptides (11–15 residues) corresponding to the cleavage sites of the norovirus polyprotein. Both predicted forms of the viral protease, the 3C-like protease (3Cpro) and the 3CD-like protease polymerase protein (3CDpropol), displayed a specific trans cleavage activity of peptides bearing Gln–Gly at the scissile bond. In contrast, peptides bearing Glu–Gly at the scissile bond (p20/VPg and 3Cpro/3Dpol junctions) were resistant to trans-cleavage by 3Cpro and 3CDpropol. Interestingly, the VPg/3Cpro scissile bond (Glu–Ala) was cleaved only by 3CDpropol, and examination of relative cleavage efficiencies revealed significant differences in processing of peptides, indicating differential cleavage patterns for 3Cpro and 3CDpropol.

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Predicted Coronavirus Nsp5 Protease Cleavage Sites in the Human Proteome: A Resource for SARS-CoV-2 Research

10.1101/2021.06.08.447224 ◽

2021 ◽

Author(s):

Benjamin M Scott ◽

Vincent Lacasse ◽

Ditte G Blom ◽

Peter D Tonner ◽

Nikolaj S Blom

Keyword(s):

Protein Interactions ◽

Cleavage Site ◽

Nonstructural Protein ◽

Data Bank ◽

Molecular Pathways ◽

Cleavage Sites ◽

Host Proteins ◽

Structure Information ◽

Human Proteins

Background: The coronavirus nonstructural protein 5 (Nsp5) is a cysteine protease required for processing the viral polyprotein and is therefore crucial for viral replication. Nsp5 from several coronaviruses have also been found to cleave host proteins, disrupting molecular pathways involved in innate immunity. Nsp5 from the recently emerged SARS-CoV-2 virus interacts with and can cleave human proteins, which may be relevant to the pathogenesis of COVID-19. Based on the continuing global pandemic, and emerging understanding of coronavirus Nsp5-human protein interactions, we set out to predict what human proteins are cleaved by the coronavirus Nsp5 protease using a bioinformatics approach. Results: Using a previously developed neural network trained on coronavirus Nsp5 cleavage sites (NetCorona), we made predictions of Nsp5 cleavage sites in all human proteins. Structures of human proteins in the Protein Data Bank containing a predicted Nsp5 cleavage site were then examined, generating a list of 92 human proteins with a highly predicted and accessible cleavage site. Of those, 48 are expected to be found in the same cellular compartment as Nsp5. Analysis of this targeted list of proteins revealed molecular pathways susceptible to Nsp5 cleavage and therefore relevant to coronavirus infection, including pathways involved in mRNA processing, cytokine response, cytoskeleton organization, and apoptosis. Conclusions: This study combines predictions of Nsp5 cleavage sites in human proteins with protein structure information and protein network analysis. We predicted cleavage sites in proteins recently shown to be cleaved in vitro by SARS-CoV-2 Nsp5, and we discuss how other potentially cleaved proteins may be relevant to coronavirus mediated immune dysregulation. The data presented here will assist in the design of more targeted experiments, to determine the role of coronavirus Nsp5 cleavage of host proteins, which is relevant to understanding the molecular pathology of SARS-CoV-2 infection.

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Amoxicillin Haptenation of α-Enolase is Modulated by Active Site Occupancy and Acetylation

Frontiers in Pharmacology ◽

10.3389/fphar.2021.807742 ◽

2022 ◽

Vol 12 ◽

Author(s):

Juan M. González-Morena ◽

Francisco J. Sánchez-Gómez ◽

Yolanda Vida ◽

Ezequiel Pérez-Inestrosa ◽

María Salas ◽

...

Keyword(s):

Posttranslational Modifications ◽

Covalent Binding ◽

Site Occupancy ◽

Cellular Protein ◽

Glycolytic Enzyme ◽

Diagnostic Tools ◽

Target Sequence ◽

Allergic Reactions ◽

Extracellular Milieu

Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.

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NHBA is processed by kallikrein from human saliva

10.1101/395988 ◽

2018 ◽

Cited By ~ 1

Author(s):

Elisa Pantano ◽

Sara Marchi ◽

Massimiliano Biagini ◽

Martina Di Fede ◽

Vincenzo Nardi-Dei ◽

...

Keyword(s):

Protein A ◽

Human Saliva ◽

Body Fluids ◽

Meningococcal Infection ◽

Heparin Binding ◽

Rich Region ◽

Main Protease ◽

Human Proteins

AbstractNeisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein and a component of the Bexsero vaccine. NHBA is characterized by the presence of a highly conserved Arg-rich region involved in binding to heparin and heparin sulphate proteoglycans present on the surface of host epithelial cells, suggesting a possible role of NHBA duringN. meningitidiscolonization. NHBA has been shown to be cleaved by the bacterial NalP protein, a meningococcal protease and by human lactoferrin (hLF), a host protease present in different body fluids (saliva, breast milk and serum). Cleavage occurs upstream or downstream the Arg-rich region. Since the human nasopharynx is the only known reservoir of infection, we further investigated the susceptibility of NHBA to human proteases present in the saliva to assess whether proteolytic cleavage could happen during the initial steps of colonization. Here we show that human saliva proteolytically cleaves NHBA; and identified human kallikrein 1 (KLK1) as the main protease responsible for this cleavage. Kallikrein is an important enzyme present in blood plasma, lymph, urine, saliva, pancreatic juices, and other body fluids that catalyze the proteolysis of several human proteins. We report thein vitrocharacterization of NHBA cleavage by kallikrein; the identification of the cleavage in the recombinant NHBA protein and, on the native protein, when expressed on live bacteria. Overall, this findings provide new insights on NHBA as target of host proteases, highlights a potential role of NHBA in theNeisseria meningitidisnasopharyngeal colonization, and of kallikrein as a defensive agent against meningococcal infection.

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Anti-Influenza Activity of the Ribonuclease Binase: Cellular Targets Detected by Quantitative Proteomics

International Journal of Molecular Sciences ◽

10.3390/ijms21218294 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8294 ◽

Cited By ~ 1

Author(s):

Vera Ulyanova ◽

Raihan Shah Mahmud ◽

Alexander Laikov ◽

Elena Dudkina ◽

Maria Markelova ◽

...

Keyword(s):

Influenza A ◽

Cellular Localization ◽

A549 Cells ◽

Cellular Protein ◽

High Morbidity ◽

Biological Regulation ◽

Cellular Targets ◽

Antiviral Effects

Unpredictable influenza pandemics, annual epidemics, and sporadic poultry-to-human avian influenza virus infections with high morbidity and mortality rates dictate a need to develop new antiviral approaches. Targeting cellular pathways and processes is a promising antiviral strategy shown to be effective regardless of viral subtypes or viral evolution of drug-resistant variants. Proteomics-based searches provide a tool to reveal the druggable stages of the virus life cycle and to understand the putative antiviral mode of action of the drug(s). Ribonucleases (RNases) of different origins not only demonstrate antiviral effects that are mediated by the direct RNase action on viral and cellular RNAs but can also exert their impact by signal transduction modulation. To our knowledge, studies of the RNase-affected cell proteome have not yet been performed. To reveal cellular targets and explain the mechanisms underlying the antiviral effect employed by the small extra-cellular ribonuclease of Bacillus pumilus (binase) both in vitro and in vivo, qualitative shotgun and quantitative targeted proteomic analyses of the influenza A virus (IAV) H1N1pdm09-infected A549 cells upon binase treatment were performed. We compared proteomes of mock-treated, binase-treated, virus-infected, and virus-infected binase-treated cells to determine the proteins affected by IAV and/or binase. In general, IAV demonstrated a downregulating strategy towards cellular proteins, while binase had an upregulating effect. With the help of bioinformatics approaches, coregulated cellular protein sets were defined and assigned to their biological function; a possible interconnection with the progression of viral infection was conferred. Most of the proteins downregulated by IAV (e.g., AKR1B1, AKR1C1, CCL5, PFN1, RAN, S100A4, etc.) belong to the processes of cellular metabolism, response to stimulus, biological regulation, and cellular localization. Upregulated proteins upon the binase treatment (e.g., AKR1B10, CAP1, HNRNPA2B1, PFN1, PPIA, YWHAB, etc.) are united by the processes of biological regulation, cellular localization, and immune and metabolic processes. The antiviral activity of binase against IAV was expressed by the inversion of virus-induced proteomic changes, resulting in the inhibition of virus-associated processes, including nuclear ribonucleoprotein export (NCL, NPM1, Nup205, and Bax proteins involved) and cytoskeleton remodeling (RDX, PFN1, and TUBB) induced by IAV at the middle stage of single-cycle infection in A549 cells. Modulation of the immune response could be involved as well. Overall, it seems possible that binase exerts its antiviral effects in multiple ways.

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Beauvericin and enniatin: emerging toxins and/or remedies?

World Mycotoxin Journal ◽

10.3920/wmj2010.1245 ◽

2010 ◽

Vol 3 (4) ◽

pp. 415-430 ◽

Cited By ~ 39

Author(s):

F. Tedjiotsop Feudjio ◽

R. Dornetshuber ◽

M. Lemmens ◽

O. Hoffmann ◽

R. Lemmens-Gruber ◽

...

Keyword(s):

Molecular Mechanisms ◽

Cancer Therapeutics ◽

Mitochondrial Pathway ◽

In Vitro Cytotoxicity ◽

Cellular Targets ◽

Drug Candidates ◽

Food And Feed ◽

Drug Efflux Pumps ◽

Pharmacologically Active

Beauvericin (BEA) and enniatins (ENN) are emerging Fusarium mycotoxins that are known to contaminate food and feed. BEA- and ENN-mediated cytotoxicity towards various mammalian and cancer cell lines is only partly understood yet and engages several cellular targets and molecular mechanisms. Thus, the channel forming ability of BEA and ENN selectively directs a flux of cations – particularly calcium – into the cell. The resulting increased intracellular calcium levels might be at least in part responsible for their cytotoxicity. Additionally, BEA and ENN activate programmed cell death via the internal mitochondrial pathway (release of cytochrome c, activation of pro-apoptotic proteins such as Bax and activation of caspases). Several cellular signalling pathways and regulators are influenced by these fusariotoxins including MAPK, NF-κB and p53. The in vitro cytotoxicity implicates that these compounds could be potentially used as cancer therapeutics. However, considering their high prevalence in grains destined for consumption, also potential systemic toxicity towards humans and animals has to be considered. Interestingly, the few studies that have addressed this issue in animals so far predominantly reported minor effects at least as far as acute toxicity is concerned. However, consequences especially of chronic exposure but also at pharmacologically active doses in humans/animals have not been explored in detail. Nevertheless, both compounds exhibit interesting pharmacological characteristics (as they are cytotoxic especially to cancer cells, inhibit drug efflux pumps, are non-mutagenic, inhibit bone resorption) which suggest them as potential drug candidates to fight disseminated cancer. Thus, detailed studies on the consequences of chronic and bolus BEA and ENN exposure are eagerly needed.

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Targeting furin activity through in silico and in vitro drug repurposing strategy for SARS-CoV-2 spike glycoprotein cleavage repression

10.21203/rs.3.rs-25856/v1 ◽

2020 ◽

Cited By ~ 2

Author(s):

Bruno O. Villoutreix ◽

John Creemers ◽

Yannick Léger ◽

Geraldine Siegfried ◽

Etienne Decroly ◽

...

Keyword(s):

Molecular Mechanisms ◽

Drug Repurposing ◽

Protein S ◽

Central China ◽

Host Cells ◽

Spike Protein ◽

Cleavage Sites ◽

Furin Cleavage ◽

High Death

Abstract In December 2019, a new coronavirus was identified in the Hubei province of central china and named SRAS-CoV-2. This new virus induces COVID-19, a severe respiratory disease with high death rate. The spike protein (S) of SARS-CoV-2 contains furin-like cleavage sites absent the other SARS-like viruses. The viral infection requires the priming or cleavage of the S protein and such processing seems essential for virus entry into the host cells. Furin is highly expressed in the lung tissue and the expression is further increased in lung cancer, suggesting the exploitation of this mechanism by the virus to mediate enhanced virulence as shown by the higher risk of COVID-19 in these patients. In this study, we used structure- based virtual screening and a collection of about 8,000 unique approved and investigational drugs suitable for docking to search for molecules that could inhibits furin activity. Sulconazole, a broad-spectrum anti-fungal agent, was found to be of potential interest. Using Western blot analysis, Sulconazole was found to inhibit the cleavage of the cell surface furin substrate MT1-MMP that contains two furin cleavage sites similar to those of the SARS- CoV-2 spike protein. Sulconazole and analogs could be interesting for repurposing studies and to probe the yet not fully understood molecular mechanisms involved in cell entry.

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Blockade of SARS-CoV-2 infection in-vitro by highly potent PI3K-α/mTOR/BRD4 inhibitor

10.1101/2021.03.02.433604 ◽

2021 ◽

Author(s):

Arpan Acharya ◽

Kabita Pandey ◽

Michellie Thurman ◽

Kishore B Challagundla ◽

Kendra R Vann ◽

...

Keyword(s):

Molecular Mechanisms ◽

Human Cells ◽

Host Cells ◽

Productive Infection ◽

Disease Evolution ◽

Cellular Targets ◽

Proteomic Approach ◽

Ic50 Value ◽

Life Threatening

Pathogenic viruses like SARS-CoV-2 and HIV hijack the host molecular machinery to establish infection and survival in infected cells. This has led the scientific community to explore the molecular mechanisms by which SARS-CoV-2 infects host cells, establishes productive infection, and causes life-threatening pathophysiology. Very few targeted therapeutics for COVID-19 currently exist, such as remdesivir. Recently, a proteomic approach explored the interactions of 26 of 29 SARS-CoV-2 proteins with cellular targets in human cells and identified 67 interactions as potential targets for drug development. Two of the critical targets, the bromodomain and extra-terminal domain proteins (BETs): BRD2/BRD4 and mTOR, are inhibited by the dual inhibitory small molecule SF2523 at nanomolar potency. SF2523 is the only known mTOR PI3K-α/(BRD2/BRD4) inhibitor with potential to block two orthogonal pathways necessary for SARS-CoV-2 pathogenesis in human cells. Our results demonstrate that SF2523 effectively blocks SARS-CoV-2 replication in lung bronchial epithelial cells in vitro, showing an IC50 value of 1.5 uM, comparable to IC50 value of remdesivir (1.1 uM). Further, we demonstrated that the combination of doses of SF2523 and remdesivir is highly synergistic: it allows for the reduction of doses of SF2523 and remdesivir by 25-fold and 4-fold, respectively, to achieve the same potency observed for a single inhibitor. Because SF2523 inhibits two SARS-CoV-2 driven pathogenesis mechanisms involving BRD2/BRD4 and mTOR signaling, our data suggest that SF2523 alone or in combination with remdesivir could be a novel and efficient therapeutic strategy to block SARS-CoV-2 infection and hence be beneficial in preventing severe COVID-19 disease evolution.

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