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An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition

Gabriela Dias Noske1, Yun Song2, Rafaela Sachetto Fernandes1

  • 1Sao Carlos Institute of Physics, University of Sao Paulo, Av. Joao Dagnone, 1100 - Jardim Santa Angelina, Sao Carlos, 13563-120, Brazil.

Nature Communications
|March 21, 2023
PubMed

Insights

SARS-CoV-2 main protease (Mpro) dimerization is crucial for its activity. N-terminal processing is not essential for Mpro dimerization, but covalent inhibitors like nirmatrelvir induce dimer formation.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Virology

Background:

  • The SARS-CoV-2 main protease (Mpro) is essential for viral replication, mediating polyprotein cleavage.
  • Mpro self-processing (maturation) is critical for its dimerization and enzymatic activity.
  • Understanding Mpro's structural dynamics and dimerization mechanisms is key for antiviral drug development.

Purpose of the Study:

  • To investigate the role of N-terminal cleavage in SARS-CoV-2 Mpro dimerization and activity.
  • To elucidate the structural basis of Mpro oligomerization in solution.
  • To analyze the impact of different inhibitor classes on Mpro oligomeric states.

Main Methods:

  • Native mass spectrometry was employed to analyze Mpro oligomeric states in solution.
  • Cryo-electron microscopy (cryo-EM) provided high-resolution structural insights into N-terminal cleavage.
  • Inhibitor binding assays were performed to assess their effects on Mpro dimerization.

Main Results:

  • Native mass spectrometry revealed mixed oligomeric states of cleaved and uncleaved Mpro, indicating N-terminal processing is not critical for dimerization.
  • A 3.5 Å cryo-EM structure detailed Mpro N-terminal cleavage in a solution-based context.
  • Non-covalent inhibitors (e.g., MAT-POS-e194df51-1) inhibited dimerization, while covalent inhibitors (e.g., nirmatrelvir) promoted monomer-to-dimer conversion.

Conclusions:

  • SARS-CoV-2 Mpro dimerization is primarily driven by induced fit mechanisms upon covalent substrate processing, not solely by N-terminal self-cleavage.
  • Covalent inhibitors can stabilize the active dimeric form of Mpro.
  • These findings offer insights into Mpro's mechanism of action and potential therapeutic strategies targeting its dimerization.

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