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Structural and Mechanistic Insights into the Main Protease (Mpro) Dimer Interface Destabilization Inhibitor: Unveiling New Therapeutic Avenues against SARS-CoV-2
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    Structural and Mechanistic Insights into the Main Protease (Mpro) Dimer Interface Destabilization Inhibitor: Unveiling New Therapeutic Avenues against SARS-CoV-2
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    • Ankur Singh
      Ankur Singh
      Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India
      More by Ankur Singh
    • Kuldeep Jangid
      Kuldeep Jangid
      Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India
    • Sanketkumar Nehul
      Sanketkumar Nehul
      Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India
    • Preeti Dhaka
      Preeti Dhaka
      Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India
      More by Preeti Dhaka
    • Ruchi Rani
      Ruchi Rani
      Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India
      More by Ruchi Rani
    • Akshay Pareek
      Akshay Pareek
      Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India
    • Gaurav Kumar Sharma*
      Gaurav Kumar Sharma
      Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India
      *Email: [email protected]
    • Pravindra Kumar*
      Pravindra Kumar
      Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India
      *Email: [email protected]
    • Shailly Tomar*
      Shailly Tomar
      Department of Biosciences and Bioengineering, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India
      *Email: [email protected]
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    Biochemistry

    Cite this: Biochemistry 2025, XXXX, XXX, XXX-XXX
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    https://doi.org/10.1021/acs.biochem.4c00535
    Published January 30, 2025
    © 2025 American Chemical Society

    Abstract

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    SARS-CoV-2 variant recurrence has emphasized the imperative prerequisite for effective antivirals. The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication, making it one of the prime and promising antiviral targets. Mpro features several druggable sites, including active sites and allosteric sites near the dimerization interface, that regulate its catalytic activity. This study identified six highly efficacious antiviral SARS-CoV-2 compounds (WIN-62577, KT185, bexarotene, ledipasvir, diacerein, and simepervir) using structure-based virtual screening of compound libraries against Mpro. Using SPR and ITC, the binding of selected inhibitory compounds to the target Mpro was validated. The FRET-based protease assay demonstrated that the identified molecules effectively inhibit Mpro with IC50 values in the range from 0.64 to 11.98 μM. Additionally, in vitro cell-based antiviral assays showed high efficacy with EC50 values in the range of 1.51 to 18.92 μM. The crystal structure of the Mpro-minocycline complex detailed the possible inhibition mechanism of minocycline, an FDA-approved antibiotic. Minocycline binds to an allosteric site, revealing residues critical for the loss of protease activity due to destabilization of molecular interactions at the dimeric interface, which are crucial for the proteolytic activity of Mpro. The study suggests that the binding of minocycline to the allosteric site may play a role in Mpro dimer destabilization and direct the rational design of minocycline derivatives as antiviral drugs.

    © 2025 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.4c00535.

    • Multiple sequence alignment (MSA) of cleavage site between different nonstructural proteins (Figure S1); schematic representation of SDS-PAGE profiling of purified proteins and FRET analysis of Mpro native and mutants (Figure S2); two-dimensional schematic representations of the interactions between screened drug molecules against substrate-binding pocket (Figures S3 and S4); MD simulation study of Mpro and Mpro-inhibitor complexes (Figure S5); antiviral activity of compounds (Figure S7); multiple sequence alignment (MSA) of the binding pocket of Mpro of different SARS-CoV-2 and SARS-CoV strains (Figure S8); binding energy, inhibition constant (ki), H-bond interactions, and hydrophobic interactions obtained from docking of Pep1 and Pep2 octapeptides into the catalytic site of Mpro (PDB ID: 6LU7) protein (Table S1); binding energy, H-bond interactions, and hydrophobic interactions obtained from docking of the top 16 compounds against the catalytic site and dimeric interface of Mpro (PDB ID: 6LU7) protein (Tables S2 and S3); qRT-PCR data of antiviral molecules are presented in Table S4 (a–f); and identified potential drug molecules with their known functions (Table S5) (PDF)

    Accession Codes

    SARS CoV-2 nsP5 [Main protease (Mpro)], UniProtKB P0DTD1 (R1AB_SARS2).

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    Biochemistry

    Cite this: Biochemistry 2025, XXXX, XXX, XXX-XXX
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.biochem.4c00535
    Published January 30, 2025
    © 2025 American Chemical Society

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