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1.
J Med Chem ; 65(17): 11840-11853, 2022 09 08.
Article in English | MEDLINE | ID: covidwho-2016520

ABSTRACT

Site-selective lysine modification of peptides and proteins in aqueous solutions or in living cells is still a big challenge today. Here, we report a novel strategy to selectively quinolylate lysine residues of peptides and proteins under native conditions without any catalysts using our newly developed water-soluble zoliniums. The zoliniums could site-selectively quinolylate K350 of bovine serum albumin and inactivate SARS-CoV-2 3CLpro via covalently modifying two highly conserved lysine residues (K5 and K61). In living HepG2 cells, it was demonstrated that the simple zoliniums (5b and 5B) could quinolylate protein lysine residues mainly in the nucleus, cytosol, and cytoplasm, while the zolinium-fluorophore hybrid (8) showed specific lysosome-imaging ability. The specific chemoselectivity of the zoliniums for lysine was validated by a mixture of eight different amino acids, different peptides bearing potential reactive residues, and quantum chemistry calculations. This study offers a new way to design and develop lysine-targeted covalent ligands for specific application.


Subject(s)
Lysine , Peptides , Coronavirus 3C Proteases/chemistry , Lysine/chemistry , Peptides/chemistry , SARS-CoV-2/enzymology , Serum Albumin, Bovine/chemistry , Water/chemistry
2.
Eur J Med Chem ; 238: 114508, 2022 Aug 05.
Article in English | MEDLINE | ID: covidwho-1982957

ABSTRACT

The COVID-19 posed a serious threat to human life and health, and SARS-CoV-2 Mpro has been considered as an attractive drug target for the treatment of COVID-19. Herein, we report 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel inhibitors of SARS-CoV-2 Mpro developed by in-house library screening and biological evaluation. Similarity search led to the identification of compound F8-S43 with the enzymatic IC50 value of 10.76 µM. Further structure-based drug design and synthetic optimization uncovered compounds F8-B6 and F8-B22 as novel non-peptidomimetic inhibitors of Mpro with IC50 values of 1.57 µM and 1.55 µM, respectively. Moreover, enzymatic kinetic assay and mass spectrometry demonstrated that F8-B6 was a reversible covalent inhibitor of Mpro. Besides, F8-B6 showed low cytotoxicity with CC50 values of more than 100 µM in Vero and MDCK cells. Overall, these novel SARS-CoV-2 Mpro non-peptidomimetic inhibitors provide a useful starting point for further structural optimization.


Subject(s)
COVID-19 , Coronavirus 3C Proteases , Furans , SARS-CoV-2 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Drug Discovery/methods , Furans/chemistry , Furans/pharmacology , Humans , Hydrazines/pharmacology , Molecular Docking Simulation , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology
3.
J Mol Biol ; 434(17): 167748, 2022 09 15.
Article in English | MEDLINE | ID: covidwho-1936840

ABSTRACT

Inhibiting the main protease of SARS-CoV-2 is of great interest in tackling the COVID-19 pandemic caused by the virus. Most efforts have been centred on inhibiting the binding site of the enzyme. However, considering allosteric sites, distant from the active or orthosteric site, broadens the search space for drug candidates and confers the advantages of allosteric drug targeting. Here, we report the allosteric communication pathways in the main protease dimer by using two novel fully atomistic graph-theoretical methods: Bond-to-bond propensity, which has been previously successful in identifying allosteric sites in extensive benchmark data sets without a priori knowledge, and Markov transient analysis, which has previously aided in finding novel drug targets in catalytic protein families. Using statistical bootstrapping, we score the highest ranking sites against random sites at similar distances, and we identify four statistically significant putative allosteric sites as good candidates for alternative drug targeting.


Subject(s)
Coronavirus 3C Proteases , Allosteric Site , Coronavirus 3C Proteases/chemistry , Molecular Docking Simulation , Protein Conformation
4.
PLoS One ; 17(2): e0263251, 2022.
Article in English | MEDLINE | ID: covidwho-1938414

ABSTRACT

The main protease (3CLpro) is one of the essential components of the SARS-CoVs viral life cycle, which makes it an interesting target for overpowering these viruses. Although many covalent and noncovalent inhibitors have been designed to inhibit this molecular target, none have gained FDA approval as a drug. Because of the high rate of COVID-19 pandemic development, in addition to laboratory research, we require in silico methods to accelerate rational drug design. The unbinding pathways of two SARS-CoV and SARS-CoV-2 3CLpro noncovalent inhibitors with the PDB IDs: 3V3M, 4MDS, 6W63, 5RF7 were explored from a comparative perspective using unbiased molecular dynamics (UMD) simulations. We uncovered common weak points for selected inhibitors that could not interact significantly with a binding pocket at specific residues by all their fragments. So water molecules entered the free binding S regions and weakened protein-inhibitor fundamental interactions gradually. N142, G143, and H163 are the essential residues, which cause key protein-ligand interactions in the binding pocket. We believe that these results will help design new potent inhibitors against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , COVID-19/virology , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Drug Design , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism
5.
J Inorg Biochem ; 234: 111886, 2022 09.
Article in English | MEDLINE | ID: covidwho-1930970

ABSTRACT

The SARS-CoV-2 main protease (Mpro) is responsible for cleaving twelve nonstructural proteins from the viral polyprotein. Mpro, a cysteine protease, is characterized by a large number of noncatalytic cysteine (Cys) residues, none involved in disulfide bonds. In the absence of a tertiary-structure stabilizing role for these residues, a possible alternative is that they are involved in redox processes. We report experimental work in support of a proposal that surface cysteines on Mpro can protect the active-site Cys145 from oxidation by reactive oxygen species (ROS). In investigations of enzyme kinetics, we found that mutating three surface cysteines to serines did not greatly affect activity, which in turn indicates that these cysteines could protect Cys145 from oxidative damage.


Subject(s)
Coronavirus 3C Proteases , Cysteine , Oxidative Stress , SARS-CoV-2 , Coronavirus 3C Proteases/chemistry , Cysteine/chemistry , Protease Inhibitors , SARS-CoV-2/enzymology
6.
J Mol Biol ; 434(17): 167696, 2022 09 15.
Article in English | MEDLINE | ID: covidwho-1926683

ABSTRACT

The family of coarse-grained models for protein dynamics known as Elastic Network Models (ENMs) require careful choice of parameters to represent well experimental measurements or fully-atomistic simulations. The most basic ENM that represents each protein residue by a node at the position of its C-alpha atom, all connected by springs of equal stiffness, up to a cut-off in distance. Even at this level a choice is required of the optimum cut-off distance and the upper limit of elastic normal modes taken in any sum for physical properties, such as dynamic correlation or allosteric effects on binding. Additionally, backbone-enhanced ENM (BENM) may improve the model by allocating a higher stiffness to springs that connect along the protein backbone. This work reports on the effect of varying these three parameters (distance and mode cutoffs, backbone stiffness) on the dynamical structure of three proteins, Catabolite Activator Protein (CAP), Glutathione S-transferase (GST), and the SARS-CoV-2 Main Protease (M pro ). Our main results are: (1) balancing B-factor and dispersion-relation predictions, a near-universal optimal value of 8.5 Å is advisable for ENMs; (2) inhomogeneity in elasticity brings the first mode containing spatial structure not well-resolved by the ENM typically within the first 20; (3) the BENM only affects modes in the upper third of the distribution, and, additionally to the ENM, is only able to model the dispersion curve better in this vicinity; (4) BENM does not typically affect fluctuation-allostery, which also requires careful treatment of the effector binding to the host protein to capture.


Subject(s)
Coronavirus 3C Proteases , Cyclic AMP Receptor Protein , Glutathione Transferase , Allosteric Regulation , Coronavirus 3C Proteases/chemistry , Cyclic AMP Receptor Protein/chemistry , Elasticity , Glutathione Transferase/chemistry , Humans , Molecular Dynamics Simulation , Protein Conformation
7.
J Mol Biol ; 434(16): 167706, 2022 08 30.
Article in English | MEDLINE | ID: covidwho-1914637

ABSTRACT

New variants of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) emerged and spread rapidly all over the world, which strongly supports the need for pharmacological options to complement vaccine strategies. Main protease (Mpro or 3CLpro) is a critical enzyme in the life cycle of SARS-CoV-2 and appears to be highly conserved among different genera of coronaviruses, making it an ideal target for the development of drugs with broad-spectrum property. PF-07304814 developed by Pfizer is an intravenously administered inhibitor targeting SARS-CoV-2 Mpro. Here we showed that PF-07304814 displays broad-spectrum inhibitory activity against Mpros from multiple coronaviruses. Crystal structures of Mpros of SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor PF-07304814 revealed a conserved ligand-binding site, providing new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures complemented by comprehensive comparison defined the key structural determinants essential for inhibition and illustrated the binding mode of action of Mpros from different coronaviruses. In view of the importance of Mpro for the medications of SARS-CoV-2 infection, insights derived from the present study should accelerate the design of pan-coronaviral main protease inhibitors that are safer and more effective.


Subject(s)
Coronavirus 3C Proteases , Coronavirus Protease Inhibitors , Indoles , Leucine , Pyrrolidinones , SARS-CoV-2 , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus Protease Inhibitors/chemistry , Coronavirus Protease Inhibitors/pharmacology , Drug Design , Humans , Indoles/chemistry , Indoles/pharmacology , Leucine/chemistry , Leucine/pharmacology , Ligands , Protein Binding , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology
8.
Biophys Chem ; 287: 106829, 2022 08.
Article in English | MEDLINE | ID: covidwho-1850725

ABSTRACT

The viral main protease (Mpro) from a novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a key enzyme essential for viral replication and has become an attractive target for antiviral drug development. The Mpro forms a functional dimer and exhibits a pH-dependent enzyme activity and dimerization. Here, we report a molecular dynamics (MD) investigation to gain insights into the structural stability of the enzyme dimer at neutral and acidic pH. Our data shows larger changes in structure of the protein with the acidic pH than that with the neutral pH. Structural analysis of MD trajectories reveals a substantial increase in intersubunit separation, the loss of domain contacts, binding free energy and interaction energy of the dimer which implies the protein instability and tendency of dimer dissociation at acidic pH. The loss in the interaction energy is mainly driven by electrostatic interactions. We have identified the intersubunit hydrogen-bonding residues involved in the decreased dimer stability. These findings may be helpful for rational drug design and target evaluation against COVID-19.


Subject(s)
COVID-19 , Coronavirus 3C Proteases , SARS-CoV-2 , COVID-19/metabolism , COVID-19/virology , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Humans , Hydrogen-Ion Concentration , Molecular Docking Simulation , Molecular Dynamics Simulation , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism
9.
Proc Natl Acad Sci U S A ; 119(16): e2117142119, 2022 04 19.
Article in English | MEDLINE | ID: covidwho-1774040

ABSTRACT

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.


Subject(s)
Coronavirus 3C Proteases , Coronavirus RNA-Dependent RNA Polymerase , SARS-CoV-2 , Antiviral Agents/chemistry , Coronavirus 3C Proteases/chemistry , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Polyproteins/chemistry , Protein Conformation , Proteolysis , SARS-CoV-2/enzymology , Substrate Specificity/genetics
10.
Acta Crystallogr D Struct Biol ; 78(Pt 3): 363-378, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1758984

ABSTRACT

The SARS-CoV-2 main protease (Mpro) has a pivotal role in mediating viral genome replication and transcription of the coronavirus, making it a promising target for drugs against the COVID-19 pandemic. Here, a crystal structure is presented in which Mpro adopts an inactive state that has never been observed before, called new-inactive. It is shown that the oxyanion loop, which is involved in substrate recognition and enzymatic activity, adopts a new catalytically incompetent conformation and that many of the key interactions of the active conformation of the enzyme around the active site are lost. Solvation/desolvation energetic contributions play an important role in the transition from the inactive to the active state, with Phe140 moving from an exposed to a buried environment and Asn142 moving from a buried environment to an exposed environment. In new-inactive Mpro a new cavity is present near the S2' subsite, and the N-terminal and C-terminal tails, as well as the dimeric interface, are perturbed, with partial destabilization of the dimeric assembly. This novel conformation is relevant both for comprehension of the mechanism of action of Mpro within the catalytic cycle and for the successful structure-based drug design of antiviral drugs.


Subject(s)
COVID-19/virology , Coronavirus 3C Proteases/chemistry , SARS-CoV-2/chemistry , Catalytic Domain , Crystallography, X-Ray , Humans , Models, Molecular , Protein Conformation , Protein Multimerization
11.
Proc Natl Acad Sci U S A ; 119(15): e2120913119, 2022 04 12.
Article in English | MEDLINE | ID: covidwho-1758464

ABSTRACT

SignificanceThe coronavirus main protease (Mpro) is required for viral replication. Here, we obtained the extended conformation of the native monomer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro by trapping it with nanobodies and found that the catalytic domain and the helix domain dissociate, revealing allosteric targets. Another monomeric state is termed compact conformation and is similar to one protomer of the dimeric form. We designed a Nanoluc Binary Techonology (NanoBiT)-based high-throughput allosteric inhibitor assay based on structural conformational change. Our results provide insight into the maturation, dimerization, and catalysis of the coronavirus Mpro and pave a way to develop an anticoronaviral drug through targeting the maturation process to inhibit the autocleavage of Mpro.


Subject(s)
Antiviral Agents , COVID-19 , Coronavirus 3C Proteases , Protease Inhibitors , SARS-CoV-2 , Allosteric Regulation/drug effects , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/enzymology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Humans , Luciferases , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Conformation , Protein Multimerization
12.
Commun Biol ; 5(1): 160, 2022 03 01.
Article in English | MEDLINE | ID: covidwho-1721596

ABSTRACT

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Rates of MProWT and MProM catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MProWT, to MProM leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.


Subject(s)
Coronavirus 3C Proteases/metabolism , Catalysis , Catalytic Domain , Circular Dichroism , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Models, Molecular , Mutation , Pyrrolidines/chemistry , Sulfonic Acids/chemistry , Thermodynamics
13.
Int J Mol Sci ; 23(4)2022 Feb 21.
Article in English | MEDLINE | ID: covidwho-1715403

ABSTRACT

As the etiological agent for the coronavirus disease 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenges the ongoing efforts of vaccine development and drug design. Due to the accumulating cases of breakthrough infections, there are urgent needs for broad-spectrum antiviral medicines. Here, we designed and examined five new tetrapeptidomimetic anti-SARS-CoV-2 inhibitors targeting the 3C-Like protease (3CLPro), which is highly conserved among coronaviruses and essential for viral replications. We significantly improved the efficacy of a ketoamide lead compound based on high-resolution co-crystal structures, all-atom simulations, and binding energy calculations. The inhibitors successfully engaged the catalytic dyad histidine residue (H41) of 3CLPro as designed, and they exhibited nanomolar inhibitory capacity as well as mitigated the viral loads of SARS-CoV-2 in cellular assays. As a widely applicable design principle, our results revealed that the potencies of 3CLPro-specific drug candidates were determined by the interplay between 3CLPro H41 residue and the peptidomimetic inhibitors.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Peptidomimetics/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemistry , COVID-19/drug therapy , Catalytic Domain , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Drug Design , Fluorescence Resonance Energy Transfer , Histidine/chemistry , Ligands , Molecular Dynamics Simulation , Peptidomimetics/chemistry , Protease Inhibitors/chemistry , Structure-Activity Relationship , Vero Cells
14.
J Biol Chem ; 298(4): 101739, 2022 04.
Article in English | MEDLINE | ID: covidwho-1693313

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (Mpro, also called 3CLpro) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here, we report the biophysical and enzymatic characterization of native Mpro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high-throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate, which is better suited for high-throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus Mpro enzyme assays to facilitate the discovery and development of therapies targeting Mpro.


Subject(s)
Coronavirus 3C Proteases , Enzyme Assays , Fluoresceins , SARS-CoV-2 , Antiviral Agents/chemistry , COVID-19/drug therapy , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/isolation & purification , Coronavirus 3C Proteases/metabolism , Enzyme Assays/methods , Fluoresceins/chemistry , Fluoresceins/metabolism , High-Throughput Screening Assays , Humans , Protease Inhibitors/chemistry , SARS-CoV-2/enzymology , SARS-CoV-2/genetics
15.
J Am Chem Soc ; 144(7): 2905-2920, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1683927

ABSTRACT

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacokinetics , Catalytic Domain , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Cysteine Proteinase Inhibitors/metabolism , Cysteine Proteinase Inhibitors/pharmacokinetics , Drug Evaluation, Preclinical , Humans , Microbial Sensitivity Tests , Microsomes, Liver/metabolism , Molecular Docking Simulation , Protein Binding , SARS-CoV-2/enzymology , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacokinetics , Vero Cells
16.
Int J Mol Sci ; 23(3)2022 Feb 06.
Article in English | MEDLINE | ID: covidwho-1674672

ABSTRACT

The inflammatory protease caspase-1 is associated with the release of cytokines. An excessive number of cytokines (a "cytokine storm") is a dangerous consequence of COVID-19 infection and has been indicated as being among the causes of death by COVID-19. The anti-inflammatory drug colchicine (which is reported in the literature to be a caspase-1 inhibitor) and the corticosteroid drugs, dexamethasone and methylprednisolone, are among the most effective active compounds for COVID-19 treatment. The SERM raloxifene has also been used as a repurposed drug in COVID-19 therapy. In this study, inhibition of caspase-1 by these four compounds was analyzed using computational methods. Our aim was to see if the inhibition of caspase-1, an important biomolecule in the inflammatory response that triggers cytokine release, could shed light on how these drugs help to alleviate excessive cytokine production. We also measured the antioxidant activities of dexamethasone and colchicine when scavenging the superoxide radical using cyclic voltammetry methods. The experimental findings are associated with caspase-1 active site affinity towards these compounds. In evaluating our computational and experimental results, we here formulate a mechanism for caspase-1 inhibition by these drugs, which involves the active site amino acid Cys285 residue and is mediated by a transfer of protons, involving His237 and Ser339. It is proposed that the molecular moiety targeted by all of these drugs is a carbonyl group which establishes a S(Cys285)-C(carbonyl) covalent bond.


Subject(s)
Anti-Inflammatory Agents/pharmacology , COVID-19/drug therapy , Caspase 1/drug effects , Caspase Inhibitors/pharmacology , Coronavirus 3C Proteases/drug effects , Anti-Inflammatory Agents/chemistry , COVID-19/metabolism , Caspase 1/chemistry , Caspase 1/metabolism , Caspase Inhibitors/chemistry , Colchicine/chemistry , Colchicine/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Dexamethasone/pharmacology , Humans , Models, Molecular , Molecular Docking Simulation , Pentacyclic Triterpenes/pharmacology , Protein Interaction Domains and Motifs , Raloxifene Hydrochloride/chemistry , Raloxifene Hydrochloride/pharmacology , Viral Protease Inhibitors/chemistry , Viral Protease Inhibitors/pharmacology
17.
Proteins ; 90(5): 1090-1101, 2022 05.
Article in English | MEDLINE | ID: covidwho-1669631

ABSTRACT

An attractive drug target to combat COVID-19 is the main protease (Mpro ) because of its key role in the viral life cycle by processing the polyproteins translated from the viral RNA. Studying the crystal structures of the protease is important to enhance our understanding of its mechanism of action at the atomic-level resolution, and consequently may provide crucial structural insights for structure-based drug discovery. In the current study, we report a comparative structural analysis of the Mpro substrate binding site for both apo and holo forms to identify key interacting residues and conserved water molecules during the ligand-binding process. It is shown that in addition to the catalytic dyad residues (His41 and Cys145), the oxyanion hole residues (Asn142-Ser144) and residues His164-Glu166 form essential parts of the substrate-binding pocket of the protease in the binding process. Furthermore, we address the issue of the substrate-binding pocket flexibility and show that two adjacent loops in the Mpro structures (residues Thr45-Met49 and Arg188-Ala191) with high flexibility can regulate the binding cavity' accessibility for different ligand sizes. Moreover, we discuss in detail the various structural and functional roles of several important conserved and mobile water molecules within and around the binding site in the proper enzymatic function of Mpro . We also present a new docking protocol in the framework of the ensemble docking strategy. The performance of the docking protocol has been evaluated in predicting ligand binding pose and affinity ranking for two popular docking programs; AutoDock4 and AutoDock Vina. Our docking results suggest that the top-ranked poses of the most populated clusters obtained by AutoDock Vina are the most important representative docking runs that show a very good performance in estimating experimental binding poses and affinity ranking.


Subject(s)
COVID-19 , Coronavirus 3C Proteases/chemistry , SARS-CoV-2 , Binding Sites , COVID-19/drug therapy , Drug Discovery , Endopeptidases , Humans , Ligands , Molecular Docking Simulation , Peptide Hydrolases , Protease Inhibitors/pharmacology , Water
18.
Phys Chem Chem Phys ; 24(7): 4324-4333, 2022 Feb 16.
Article in English | MEDLINE | ID: covidwho-1671657

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 has been declared a global health crisis. The development of anti-SARS-CoV-2 drugs heavily depends on the systematic study of the critical biological processes of key proteins of coronavirus among which the main proteinase (Mpro) dimerization is a key step for virus maturation. Because inhibiting the Mpro dimerization can efficiently suppress virus maturation, the key residues that mediate dimerization can be treated as targets of drug and antibody developments. In this work, the structure and energy features of the Mpro dimer of SARS-CoV-2 and SARS-CoV were studied using molecular dynamics (MD) simulations. The free energy calculations using the Generalized Born (GB) model showed that the dimerization free energy of the SARS-CoV-2 Mpro dimer (-107.5 ± 10.89 kcal mol-1) is larger than that of the SARS-CoV Mpro dimer (-92.83 ± 9.81 kcal mol-1), indicating a more stable and possibly a quicker formation of the Mpro dimer of SARS-CoV-2. In addition, the energy decomposition of each residue revealed 11 key attractive residues. Furthermore, Thr285Ala weakens the steric hindrance between the two protomers of SARS-CoV-2 that can form more intimate interactions. It is interesting to find 11 repulsive residues which effectively inhibit the dimerization process. At the interface of the Mpro dimer, we detected three regions that are rich in interfacial water which stabilize the SARS-CoV-2 Mpro dimer by forming hydrogen bonds with two protomers. The key residues and rich water regions provide important targets for the future design of anti-SARS-CoV-2 drugs through inhibiting Mpro dimerization.


Subject(s)
Coronavirus 3C Proteases/chemistry , SARS-CoV-2/enzymology , COVID-19 , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Pandemics , Protein Multimerization
19.
ACS Appl Mater Interfaces ; 14(1): 191-200, 2022 Jan 12.
Article in English | MEDLINE | ID: covidwho-1655440

ABSTRACT

At present, the most powerful new drugs for COVID-19 are antibody proteins. In addition, there are some star small molecule drugs. However, there are few studies on nanomaterials. Here, we study the intact graphene (IG), defective graphene (DG), and graphene oxide (GO) interacting with COVID-19 protein. We find that they show progressive inhibition of COVID-19 protein. By using molecular dynamics simulations, we study the interactions between SARS-CoV-2 3CL Mpro and graphene-related materials (GRMs): IG, DG, and GO. The results show that Mpro can be absorbed onto the surfaces of investigated materials. DG and GO interacted with Mpro more intensely, causing the decisive part of Mpro to become more flexible. Further analysis shows that compared to IG and GO, DG can inactivate Mpro and inhibit its expression effectively by destroying the active pocket of Mpro. Our work not only provides detailed and reliable theoretical guidance for the application of GRMs in treating with SARS-CoV-2 but also helps in developing new graphene-based anti-COVID-19 materials.


Subject(s)
Coronavirus 3C Proteases/chemistry , Graphite/chemistry , Molecular Dynamics Simulation , SARS-CoV-2/metabolism , Adsorption , Binding Sites , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Coronavirus 3C Proteases/metabolism , Graphite/metabolism , Humans , Ligands , SARS-CoV-2/isolation & purification
20.
STAR Protoc ; 3(1): 101158, 2022 03 18.
Article in English | MEDLINE | ID: covidwho-1650422

ABSTRACT

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).


Subject(s)
Coronavirus 3C Proteases/chemistry , Models, Molecular , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/genetics , Crystallization , Crystallography, X-Ray , Humans
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