Inhibition Mechanism of SARS-CoV-2 Main Protease with Ketone-Based Inhibitors Unveiled by Multiscale Simulations: Insights for Improved Designs*.
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ABSTRACT:
SUBMITTER: Ramos-Guzman CA
PROVIDER: S-EPMC8653175 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We present a detailed theoretical analysis of the reaction mechanism of proteolysis catalyzed by the main protease of SARS-CoV-2. Using multiscale simulation methods, we have characterized the interactions established by a peptidic substrate in the active site, and then we have explored the free energy landscape associated with the acylation and deacylation steps of the proteolysis reaction, characterizing the transition states of the process. Our mechanistic proposals can explain most of the experimental observations made on the highly similar ortholog protease of SARS-CoV. We point to some key interactions that may facilitate the acylation process and thus can be crucial in the design of more specific and efficient inhibitors of the main protease activity. In particular, from our results, the P1' residue can be a key factor to improve the thermodynamics and kinetics of the inhibition process.
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Project description:A critical step for SARS-CoV-2 assembly and maturation involves the autoactivation of the main protease (MProWT) from precursor polyproteins. Upon expression, a model precursor of MProWT mediates its own release at its termini rapidly to yield a mature dimer. A construct with an E290A mutation within MPro exhibits time dependent autoprocessing of the accumulated precursor at the N-terminal nsp4/nsp5 site followed by the C-terminal nsp5/nsp6 cleavage. In contrast, a precursor containing E290A and R298A mutations (MProM) displays cleavage only at the nsp4/nsp5 site to yield an intermediate monomeric product, which is cleaved at the nsp5/nsp6 site only by MProWT. MProM and the catalytic domain (MPro1-199) fused to the truncated nsp4 region also show time-dependent conversion in vitro to produce MProM and MPro1-199, respectively. The reactions follow first-order kinetics indicating that the nsp4/nsp5 cleavage occurs via an intramolecular mechanism. These results support a mechanism involving an N-terminal intramolecular cleavage leading to an increase in the dimer population and followed by an intermolecular cleavage at the C-terminus. Thus, targeting the predominantly monomeric MPro precursor for inhibition may lead to the identification of potent drugs for treatment.
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Project description:Inhibition of the main protease (Mpro) of SARS-CoV-2 has been suggested to be vital in shutting down viral replication in a host. Most efforts aimed at inhibiting MPro activity have been channeled into competitive inhibition at the active site, but this strategy will require a high inhibitor concentration and impressive inhibitor-MPro binding affinity. Allosteric inhibition can potentially serve as an effective strategy for alleviating these limitations. In this study, the ability of antiviral natural products to inhibit MPro in an allosteric fashion was explored with in silico techniques. Molecular docking revealed a strong interaction between casticin, an antiviral flavonoid, and Mpro at a site distant from the active site. This site, characterized as a distal site, has been shown to have an interdependent dynamic effect with the active site region. Mpro only, Mpro-peptide (binary) and Mpro-peptide-casticin (ternary) complexes were subjected to molecular dynamics simulations for 50 ns to investigate the modulatory activity of casticin binding on Mpro. Molecular dynamic simulations revealed that binding of casticin at the distal site interferes with the proper orientation of the peptide substrate in the oxyanion hole of the active site, and this could lead to a halt or decrease in catalytic activity. This study therefore highlights casticin as a potential allosteric modulator of the SARS-CoV-2 main protease, which could be optimized and developed into a potential lead compound for anti-SARS-CoV-2 chemotherapy. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s42250-022-00411-7.
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Project description:Integrins are large cell-surface adhesion receptors that can be activated to a high affinity state by the formation of an intracellular complex between the integrin β-subunit tail, the membrane, and talin. The F2 and F3 subdomains of the talin head play a key role in formation of this complex. Here, activation of the integrin αIIb/β3 dimer by the talin head domain was probed using multiscale molecular dynamics simulations. A number of novel insights emerge from these studies, including (i) the importance of the integrin αIIb subunit F992 and F993 residues in stabilizing the "off" state of the αIIb/β3 dimer, (ii) a crucial role for negatively charged groups in the F2-F3/membrane interaction, (iii) binding of the talin F2-F3 domain to negatively charged lipid headgroups in the membrane induces a reorientation of the β transmembrane (TM) domain, (iv) an increase in the tilt angle of the β TM domain relative to the bilayer normal helps to destabilize the α/β TM interaction and promote a scissor-like movement of the integrin TM helices. These results, combined with various published experimental observations, suggest a model for the mechanism of inside-out activation of integrins by talin.
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Project description:SARS-CoV-2 Mpro, also known as the main protease or 3C-like protease, is a key enzyme involved in the replication process of the virus that is causing the COVID-19 pandemic. It is also the most promising antiviral drug target targeting SARS-CoV-2 virus. In this work, the catalytic mechanism of Mpro was studied using the full model of the enzyme and a computational QM/MM methodology with a 69/72-atoms QM region treated at DLPNO-CCSD(T)/CBS//B3LYP/6-31G(d,p):AMBER level and including the catalytic important oxyanion-hole residues. The transition state of each step was fully characterized and described together with the related reactants and products. The rate-limiting step of the catalytic process is the hydrolysis of the thioester-enzyme adduct, and the calculated barrier closely agrees with the available kinetic data. The calculated Gibbs free energy profile, together with the full atomistic detail of the structures involved in catalysis, can now serve as valuable models for the rational drug design of transition state analogs as new inhibitors targeting the SARS-CoV-2 virus.
| S-EPMC8224256 | biostudies-literature
Project description:We here investigate the mechanism of SARS-CoV-2 3CL protease inhibition by one of the most promising families of inhibitors, those containing an aldehyde group as a warhead. These compounds are covalent inhibitors that inactivate the protease, forming a stable hemithioacetal complex. Inhibitor 11a is a potent inhibitor that has been already tested in vitro and in animals. Using a combination of classical and QM/MM simulations, we determined the binding mode of the inhibitor into the active site and the preferred rotameric state of the catalytic histidine. In the noncovalent complex, the aldehyde group is accommodated into the oxyanion hole formed by the NH main-chain groups of residues 143 to 145. In this pose, P1-P3 groups of the inhibitor mimic the interactions established by the natural peptide substrate. The reaction is initiated with the formation of the catalytic dyad ion pair after a proton transfer from Cys145 to His41. From this activated state, covalent inhibition proceeds with the nucleophilic attack of the deprotonated Sγ atom of Cys145 to the aldehyde carbon atom and a water-mediated proton transfer from the Nε atom of His41 to the aldehyde oxygen atom. Our proposed reaction transition-state structure is validated by comparison with X-ray data of recently reported inhibitors, while the activation free energy obtained from our simulations agrees with the experimentally derived value, supporting the validity of our findings. Our study stresses the interplay between the conformational dynamics of the inhibitor and the protein with the inhibition mechanism and the importance of including conformational diversity for accurate predictions about the inhibition of the main protease of SARS-CoV-2. The conclusions derived from our work can also be used to rationalize the behavior of other recently proposed inhibitor compounds, including aldehydes and ketones with high inhibitory potency.
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Project description:The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (Mpro). FRET-based screening against recombinant SARS-CoV-2 Mpro identified six compounds that inhibit proteolysis with nanomolar IC50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 Mpro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit Mpro competitively. Lead E24 inhibited viral replication with a nanomolar EC50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of Mpro inhibition that should facilitate the design of future COVID-19 treatments.
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Project description:SARS-CoV-2 main protease (Mpro) cleaves the viral polypeptide 1a and 1ab in a site-specific ((L-Q|(S, A, G)) manner and produce functional enzymes for mediating viral replication. Numerous studies have reported synthetic competitive inhibitors against this target enzyme but increase in substrate concentration often reduces the effectiveness of such inhibitors. Allosteric inhibition by natural compound can provide safe and effective treatment by alleviating this limitation. Present study deals with in silico allosteric inhibition analysis of quercetin, against SARS-CoV-2-Mpro. Molecular docking of quercetin with Mpro revealed consistent binding of quercetin at a site other than active site in multiple runs, with the highest binding energy of -?8.31 kcal/mol, forming 6 H-bonds with residues Gln127, Cys128, Lys137, Asp289 and Glu290. Molecular dynamic simulation of 50 ns revealed high stability of Mpro-quercetin complex with RMSD values ranging from 0.1 to 0.25 nm. Moreover, native-Mpro and Mpro-quercetin complex conformations extracted at different time points from simulation trajectories were subjected to active site-specific docking with modelled substrate peptide (AVLQSGFR) by ZDOCK server. Results displayed site-specific cleavage of peptide when docked with native-Mpro. While substrate peptide remained intact when docked with Mpro-quercetin complex, also the binding energy of peptide reduced from 785 to 86 from 1 to 50 ns as quercetin induced alterations in the active site cavity reducing its affinity for the substrate. Further, no interactions were noticed between peptide and active site residues of Mpro-quercetin complex conformations at 40 and 50 ns. Hence, quercetin displayed effective allosteric inhibition potential against SARS-CoV-2 Mpro, and can be developed into an efficient treatment for COVID-19.
| S-EPMC7802979 | biostudies-literature