Zinc2+ ion inhibits SARS-CoV-2 main protease and viral replication in vitro

The coronaviruses plaguing humanity in the 21st century share much in common: a spontaneous route of origin from wild animals, a propensity to take human life, and, importantly, a highly conserved set of biological machinery necessary for viral replication. Most recently, the SARS-CoV-2 is decimating economies around the world and has claimed over two million human lives, reminding the world of a need for an effective drug against present and future coronaviruses. To date, attempts to repurpose clinically approved antiviral medications show minimal promise, highlighting the need for development of new antiviral drugs. Nucleotide analog inhibitors are a promising therapeutic candidate, but early data from clinical studies suggests these compounds have limited efficacy. However, novel compounds targeting the main protease responsible for critical steps in viral assembly are gaining considerable interest because they offer the potential for broad-spectrum coronavirus therapy. Here, we review the literature regarding potential inhibitors for the main protease of coronaviruses, especially SARS-CoV-2, analyze receptor-drug interactions, and draw conclusions about candidate inhibitors for future outbreaks. Promising candidates for development of a broad-spectrum coronavirus protease inhibitor include the neuraminidase inhibitor 3K, the peptidomimetic inhibitor 11a and 11b, the α-ketoamide inhibitor 13b, the aldehyde prodrug, and the phosphate prodrug developed by Pfizer. In silico and in vitro analyses have shown that these inhibitors strongly interact with the active site of the main protease, and to varying degrees, prevent viral replication via interactions with the largely conserved active site pockets. KEYWORDS: Severe Acute Respiratory Syndrome Coronavirus; Middle East Respiratory Syndrome Coronavirus; Severe Acute Respiratory Syndrome Coronavirus 2; Replicase Polypeptide; Protease; Neuraminidase Inhibitor; Peptidomimetic Inhibitor; α-Ketoamide Inhibitor; Molecular Docking

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In Silico Identification and Docking-Based Drug Repurposing Against the Main Protease of SARS-CoV-2, Causative Agent of COVID-19

10.26434/chemrxiv.12049590.v1 ◽

2020 ◽

Cited By ~ 3

Author(s):

Yogesh Kumar ◽

Harvijay Singh

Keyword(s):

Virtual Screening ◽

Active Site ◽

Viral Infections ◽

Drug Repurposing ◽

Docking Study ◽

Docking Studies ◽

Main Protease ◽

Novel Coronavirus ◽

Approved Drugs

<div>The rapidly enlarging COVID-19 pandemic caused by novel SARS-coronavirus 2 is a global</div><div>public health emergency of unprecedented level. Therefore the need of a drug or vaccine that</div><div>counter SARS-CoV-2 is an utmost requirement at this time. Upon infection the ssRNA genome</div><div>of SARS-CoV-2 is translated into large polyprotein which further processed into different</div><div>nonstructural proteins to form viral replication complex by virtue of virus specific proteases:</div><div>main protease (3-CL protease) and papain protease. This indispensable function of main protease</div><div>in virus replication makes this enzyme a promising target for the development of inhibitors and</div><div>potential treatment therapy for novel coronavirus infection. The recently concluded α-ketoamide</div><div>ligand bound X-ray crystal structure of SARS-CoV-2 Mpro (PDB ID: 6Y2F) from Zhang et al.</div><div>has revealed the potential inhibitor binding mechanism and the determinants responsible for</div><div>involved molecular interactions. Here, we have carried out a virtual screening and molecular</div><div>docking study of FDA approved drugs primarily targeted for other viral infections, to investigate</div><div>their binding affinity in Mpro active site. Virtual screening has identified a number of antiviral</div><div>drugs, top ten of which on the basis of their bending energy score are further examined through </div><div>molecular docking with Mpro. Docking studies revealed that drug Lopinavir-Ritonavir, Tipranavir</div><div>and Raltegravir among others binds in the active site of the protease with similar or higher</div><div>affinity than the crystal bound inhibitor α-ketoamide. However, the in-vitro efficacies of the drug</div><div>molecules tested in this study, further needs to be corroborated by carrying out biochemical and</div><div>structural investigation. Moreover, this study advances the potential use of existing drugs to be</div><div>investigated and used to contain the rapidly expanding SARS-CoV-2 infection.</div>

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Repurposing inhibitors for SARS-CoV2 main protease

10.21203/rs.3.rs-41342/v1 ◽

2020 ◽

Author(s):

Kumar Sharp

Keyword(s):

Viral Replication ◽

Active Sites ◽

Drug Repurposing ◽

Chemotherapeutic Drugs ◽

Short Term ◽

Main Protease ◽

Potential Binding ◽

Approved Drugs ◽

Fda Approved Drugs

Abstract SARS-CoV2 main protease is important for viral replication and one of the most potential targets for drug development in this current pandemic. Drug repurposing is a promising field to provide potential short-term acceptable therapy for management of coronavirus till a specific anti-viral for coronavirus is developed. In-silico drug repurposing screening is the current fastest way to repurpose drugs by targeting active sites in fraction of seconds. In this study, SARS-CoV2 main protease is being targeted by 1050 FDA-approved drugs to inhibit its activity thereby interfering with viral replication. Chemotherapeutic drugs and anti-retroviral drugs have shown potential binding as inhibitor. In-vitro and clinical trials required to establish final fact.

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Antibiotics Used in Patients after Surgery and Effects of Human Serum Paraoxonase-I (PON1) Enzyme Activity

Protein and Peptide Letters ◽

10.2174/0929866526666190124144622 ◽

2019 ◽

Vol 26 (3) ◽

pp. 215-220 ◽

Cited By ~ 1

Author(s):

Aycan Yılmaz ◽

Esra Dilek

Keyword(s):

Enzyme Activity ◽

Human Serum ◽

Active Site ◽

Active Agent ◽

Cefuroxime Axetil ◽

Inhibition Mechanism ◽

Paraoxonase Activity ◽

Activity Measurements ◽

Active Substances

Background: Paraoxonase (PON; arilesterase, [EC 3.1.8.1]) is an enzyme from the group arilesterases (ARE). This enzyme is capable of hydrolyzing paraoxone which is the active metabolite of parathion, an organic phosphorus insecticide. PON activity was found to be low in individuals prone to development of atherosclerosis such as diabetes, familial hypercholesterolemia and kidney disorders. It was noted that PON enzyme activity decreases in relation to age increase in adults. PON enzyme activity is approximately half of that in newborns and premature babies. Approximately one year after birth, it reaches the adult level. It can be said that PON1 has significant role on living organisms. For this reason, many studies on interactions of PON-drugs are needed. </P><P> Objective: In this article, our aim is to investigate in vitro effects of four pharmaceutically active agents (fosfomycin, cefuroxime axetil, cefaclor monohydrate, and cefixime) which are often used in patients after surgery on human serum paraoxanase-I (PON1) enzyme activity. Methods: In this article, we purify paraoxonase-I enzyme from human serum by using ammonium sulfate precipitation (in the range of 60-80%), ion exchange and gel filtration chromatography. We use electrophoresis to check the purity of the enzyme. We investigate the paraoxonase activity of the enzyme at 412 nm the inhibition effects of the active substances. Paraoxone is used as the substrate. Activity measurements arw made at different inhibitor concentrations related to inhibitor studies and % Activity- [I] graphs are drawn for drug active substances. Lineweaver-Burk graphics are used to determine the Ki constants. Finally, to determine the types of inhibition we interpret these graphs. Results: The active agents used after surgery decreased the PON1 enzyme activity. They showed different inhibition mechanism. The inhibition mechanism of fosfomycin and cefaclor monohydrate was noncompetitive, cefixime was uncompetitive and cefuroxime axetil was a competitive inhibitor. The IC50 values for fosfomycin, cefuroxime axetil, cefaclor monohydrate, and cefixime were calculated to be 31.5 mM, 1.03 mM, 4.18 mM and 0.781 mM, respectively, and the Ki constants were determined to be 27.98 ± 12.25 mM, 2.20 ± 0.22 mM, 4.81 ± 2.25 mM and 1.12 ± 0.32 mM, respectively. The IC50 and Ki values showed that cefixime active agent has the maximum inhibition. Conclusion: In this study, we have detected that cefuroxime axetil inhibited competitively in vitro paraoxonase activity of this enzyme. According to this information, we thought that cefuroxime axetil linked to the active site of the enzyme. Fosfomycin and cefaclor monohydrate can be attached with amino acids out of the active site of the enzyme because they inhibit enzyme noncompetitively. Cefixime can be attached only to the enzyme-substrate complex because it inhibits enzyme uncompetitively.

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Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

Journal of Virology ◽

10.1128/jvi.02306-06 ◽

2007 ◽

Vol 81 (7) ◽

pp. 3583-3596 ◽

Cited By ~ 83

Author(s):

Laura L. Marcotte ◽

Amanda B. Wass ◽

David W. Gohara ◽

Harsh B. Pathak ◽

Jamie J. Arnold ◽

...

Keyword(s):

Crystal Structure ◽

Viral Replication ◽

Active Site ◽

Polymerase Activity ◽

Rna Dependent Rna Polymerase ◽

Multifunctional Protein ◽

Biologically Relevant ◽

N Terminus ◽

Key Residues

ABSTRACT Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3Cpro and the viral polymerase 3Dpol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-Å resolution and the G64S fidelity mutant of 3Dpol at a 3.0-Å resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3Dpol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3Dpol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.

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In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells

Biological Chemistry ◽

10.1515/bc.2008.122 ◽

2008 ◽

Vol 389 (8) ◽

Cited By ~ 31

Author(s):

Sinisa Urban ◽

Rosanna P. Baker

Keyword(s):

Enzyme Activity ◽

Active Site ◽

Membrane Lipids ◽

Fold Increase ◽

Living Cells ◽

Transmembrane Segment ◽

Intramembrane Proteases ◽

Gating Mechanism

Abstract Intramembrane proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid intramembrane proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in protease activity in living cells. Conversely, mutations in other parts of the protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.

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SARS-CoV-2 Main Protease Active Site Ligands in the Human Metabolome

Molecules ◽

10.3390/molecules26051409 ◽

2021 ◽

Vol 26 (5) ◽

pp. 1409

Author(s):

Anna Maria Sardanelli ◽

Camilla Isgrò ◽

Luigi Leonardo Palese

Keyword(s):

Active Site ◽

Therapeutic Strategy ◽

Silybum Marianum ◽

Antiviral Therapies ◽

Global Pandemic ◽

Main Protease ◽

Toxicological Profile ◽

Starting Point ◽

Antiviral Activities

In late 2019, a global pandemic occurred. The causative agent was identified as a member of the Coronaviridae family, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we present an analysis on the substances identified in the human metabolome capable of binding the active site of the SARS-CoV-2 main protease (Mpro). The substances present in the human metabolome have both endogenous and exogenous origins. The aim of this research was to find molecules whose biochemical and toxicological profile was known that could be the starting point for the development of antiviral therapies. Our analysis revealed numerous metabolites—including xenobiotics—that bind this protease, which are essential to the lifecycle of the virus. Among these substances, silybin, a flavolignan compound and the main active component of silymarin, is particularly noteworthy. Silymarin is a standardized extract of milk thistle, Silybum marianum, and has been shown to exhibit antioxidant, hepatoprotective, antineoplastic, and antiviral activities. Our results—obtained in silico and in vitro—prove that silybin and silymarin, respectively, are able to inhibit Mpro, representing a possible food-derived natural compound that is useful as a therapeutic strategy against COVID-19.

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In Silico Identification and Docking-Based Drug Repurposing Against the Main Protease of SARS-CoV-2, Causative Agent of COVID-19

10.26434/chemrxiv.12049590 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yogesh Kumar ◽

Harvijay Singh

Keyword(s):

Virtual Screening ◽

Active Site ◽

Viral Infections ◽

Drug Repurposing ◽

Docking Study ◽

Docking Studies ◽

Main Protease ◽

Novel Coronavirus ◽

Approved Drugs

<div>The rapidly enlarging COVID-19 pandemic caused by novel SARS-coronavirus 2 is a global</div><div>public health emergency of unprecedented level. Therefore the need of a drug or vaccine that</div><div>counter SARS-CoV-2 is an utmost requirement at this time. Upon infection the ssRNA genome</div><div>of SARS-CoV-2 is translated into large polyprotein which further processed into different</div><div>nonstructural proteins to form viral replication complex by virtue of virus specific proteases:</div><div>main protease (3-CL protease) and papain protease. This indispensable function of main protease</div><div>in virus replication makes this enzyme a promising target for the development of inhibitors and</div><div>potential treatment therapy for novel coronavirus infection. The recently concluded α-ketoamide</div><div>ligand bound X-ray crystal structure of SARS-CoV-2 Mpro (PDB ID: 6Y2F) from Zhang et al.</div><div>has revealed the potential inhibitor binding mechanism and the determinants responsible for</div><div>involved molecular interactions. Here, we have carried out a virtual screening and molecular</div><div>docking study of FDA approved drugs primarily targeted for other viral infections, to investigate</div><div>their binding affinity in Mpro active site. Virtual screening has identified a number of antiviral</div><div>drugs, top ten of which on the basis of their bending energy score are further examined through </div><div>molecular docking with Mpro. Docking studies revealed that drug Lopinavir-Ritonavir, Tipranavir</div><div>and Raltegravir among others binds in the active site of the protease with similar or higher</div><div>affinity than the crystal bound inhibitor α-ketoamide. However, the in-vitro efficacies of the drug</div><div>molecules tested in this study, further needs to be corroborated by carrying out biochemical and</div><div>structural investigation. Moreover, this study advances the potential use of existing drugs to be</div><div>investigated and used to contain the rapidly expanding SARS-CoV-2 infection.</div>

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Phenothiazines as dual inhibitors of SARS-CoV-2 main protease and COVID-19 inflammation

Canadian Journal of Chemistry ◽

10.1139/cjc-2021-0139 ◽

2021 ◽

Author(s):

Katrina L Forrestall ◽

Darcy E Burley ◽

Meghan Kirsten Cash ◽

Ian Pottie ◽

Sultan Darvesh

Keyword(s):

Viral Replication ◽

Active Site ◽

Acute Infection ◽

Binding Energies ◽

Neurotransmitter Receptors ◽

Main Protease ◽

Dual Action ◽

Inhibition Constants ◽

Anti Inflammatory ◽

Essential Enzyme

COVID-19, caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2) currently has no treatment for acute infection. The main protease (Mpro) of SARS-CoV-2 is an essential enzyme for viral replication and an attractive target for disease intervention. The phenothiazine moiety has demonstrated drug versatility for biological systems, including inhibition of butyrylcholinesterase, a property important in the cholinesterase anti-inflammatory cascade. Nineteen phenothiazine drugs were investigated using in silico modelling techniques to predict binding energies and inhibition constants (Ki values) with SARS-CoV-2 Mpro. Since most side-effects of phenothiazines are due to interactions with various neurotransmitter receptors and transporters, phenothiazines with few such interactions were also investigated. All compounds were found to bind to the active site of SARS-CoV-2 Mpro and showed Ki values ranging from 1.30 to 52.4 µM. Nine phenothiazines showed inhibition constants <10 µM. The compounds with limited interactions with neurotransmitter receptors and transporters showed micromolar (µM) Ki values. Docking results were compared with remdesivir and showed similar interactions with key residues Glu-166 and Gln-189 in the active site. This work has identified several phenothiazines with limited neurotransmitter receptor and transporter interactions and that may provide the dual action of inhibiting SARS-CoV-2 Mpro to prevent viral replication and promote the release of anti-inflammatory cytokines to curb viral-induced inflammation. These compounds are promising candidates for further investigation against SARS-CoV-2.

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In Silico Screening of Naturally Occurring Coumarin Derivatives for the Inhibition of the Main Protease of SARS-CoV-2

10.26434/chemrxiv.12234728 ◽

2020 ◽

Author(s):

Sona Lyndem ◽

Sharat Sarmah ◽

Sourav Das ◽

Atanu Singha Roy

Keyword(s):

Active Site ◽

Preliminary Screening ◽

Naturally Occurring ◽

Main Protease ◽

Coumarin Compounds ◽

Potential Inhibitors ◽

Novel Drug ◽

Catalytic Dyad

<p>The dissemination of a novel corona virus, SARS-CoV-2, through rapid human to human transmission has led to a global health emergency. The lack of a vaccine or medication for effective treatment of this disease has made it imperative for developing novel drug discovery approaches. Repurposing of drugs is one such method currently being used to tackle the viral infection. The genome of SARS-CoV-2 replicates due to the functioning of a main protease called M<sup>pro</sup>. By targeting the active site of M<sup>pro</sup> with potential inhibitors, this could prevent viral replication from taking place. Blind docking technique was used to investigate the interactions between 29 naturally occurring coumarin compounds and SARS-CoV-2 main protease, M<sup>pro</sup>, out of which 17 coumarin compounds were seen to bind to the active site through the interaction with the catalytic dyad, His41 and Cys145, along with other neighbouring residues. On comparing the ΔG values of the coumarins bound to the active site of M<sup>pro</sup>, corymbocoumarin belonging to the class pyranocoumarins, methylgalbanate belonging to the class simple coumarins and heraclenol belonging to the class furanocoumarins, displayed best binding efficiency and could be considered as potential M<sup>pro</sup> protease inhibitors. Preliminary screening of these naturally occurring coumarin compounds as potential SARS-CoV-2 replication inhibitors acts as a stepping stone for further <i>in vitro</i> and <i>in vivo</i> experimental investigation and analytical validation. </p>

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