Paxlovid provides a glimpse into the future against SARS-CoV-2

A recent study posted to the bioRxiv* preprint server reveals that an antiviral drug, nirmatrelvir, is potent and effective against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron variant.

Study: Structural Basis for Nirmatrelvir in Vitro Efficacy against the Omicron Variant of SARS-CoV-2. Image Credit: Cryptographer / Shutterstock.com

Study: Structural Basis for Nirmatrelvir in Vitro Efficacy against the Omicron Variant of SARS-CoV-2. Image Credit: Cryptographer / Shutterstock.com

Background

The coronavirus disease 2019 (COVID-19) pandemic remains a global health crisis as new variants of its etiologic agent, SARS-CoV-2, continue to emerge over time. The SARS-CoV-2 Omicron (B.1.1.529) variant is the latest variant of concern (VOC) that carries over 30 mutations, a majority of which reside within the receptor-binding domain (RBD) of its spike (S) protein.

The last two decades have witnessed the emergence of several communicable diseases like severe acute respiratory syndrome (SARS), the Middle East respiratory syndrome (MERS), Ebola virus disease (EVD), and Zika virus disease. COVID-19 is the latest infectious disease to emerge that has caused an unprecedented health emergency globally.

SARS-CoV-2 is a beta coronavirus containing a single-stranded (ss), positive-sense ribonucleic acid (RNA) genome that is around 30 kilobases (Kb0 long and encodes two polyproteins including pp 1a and pp 1ab, as well as four structural proteins. The main protease (Mpro), also known as 3CL protease, of SARS-CoV-2 cleaves the polyproteins at 11 sites to create shorter non-structural proteins that are crucial for viral replication.

About the study

In the current study, researchers evaluate the efficacy of nirmatrelvir against the SARS-CoV-2 Omicron variant. Nirmatrelvir is an Mpro inhibitor that has been previously demonstrated by the authors of the current study to be effective against SARS-CoV-2 in vitro.

The SARS-CoV-2 Omicron VOC contains a mutation (P132H) at site 132 in Mpro, where the aa proline (P) is replaced by histidine (H). Therefore, the researchers investigated the biochemical potency of this drug against the mutated 3CL protease of the B.1.1.529 variant.

The Mpro from both the SARS-CoV-2 wildtype (WT) strain and Omicron variant were expressed and purified to obtain near-homogeneous proteins. The mutation P132H was introduced by site-directed mutagenesis and the protein was expressed in BL21 (DE3) cells.

The proteases were purified by nickel (Ni)-affinity chromatography using a step gradient that was followed by C-terminal His-tag cleavage with PreScission protease. A second Ni-affinity column was used to remove non-cleaved Mpro and PreScission protease. Size exclusion chromatography was performed as the final step to yield a near-100% pure protein.

Study findings

Enzyme catalytic activity was determined by an established Mpro fluorescence resonance energy transfer (FRET)-based cleavage assay. The turnover number (kcat) for the WT Mpro was about 0.41s-1 and 0.39 s-1 for the P132H Mpro.

The authors observed comparable Michaelis constants (Km) and catalytic efficiencies (kcat/Km) for both the WT and P132H 3CL protease, thus suggesting that the enzymatic properties are similar for both the proteases.

The inhibitory activity of nirmatrelvir was evaluated and found to yield a mean inhibitory constant (Ki) of 0.93 nanomolar (nM) for WT Mpro and 0.64 nM for the P132H Mpro. Statistical analysis (t-test) confirmed no significant differences between them.

Furthermore, the crystal structure of P132H Mpro bound to nirmatrelvir was elucidated at 1.62 Å resolution and compared with that of the WT strain. This comparison revealed that the binding of the drug to mutated Mpro is the same as with the WT Mpro, thereby confirming no structural changes around P132H substitution in the Omicron Mpro. The only observed change was the conformation of the Glu240 side chain, which adopts a different rotamer to avoid a clash with the H sidechain.

Structural Characterization of nirmatrelvir bound to SARS-Cov-2-MproP132H. (a) Superposition of the x-ray crystal structures of nirmatrelvir bound to SARS-Cov-2-Mpro P132H (in magenta and green) and wildtype SARS-CoV-2-Mpro (grey). Key interactions are indicated via dashed lines. (b) Residues surrounding Proline 132 (in ball & stick representation) (c) Residual deuterium exchange plot indicates no significant differential uptake between wild-type and P132H SARS-CoV-2-Mpro.

Structural Characterization of nirmatrelvir bound to SARS-Cov-2-MproP132H. (a) Superposition of the x-ray crystal structures of nirmatrelvir bound to SARS-Cov-2-Mpro P132H (in magenta and green) and wildtype SARS-CoV-2-Mpro (grey). Key interactions are indicated via dashed lines. (b) Residues surrounding Proline 132 (in ball & stick representation) (c) Residual deuterium exchange plot indicates no significant differential uptake between wild-type and P132H SARS-CoV-2-Mpro.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) was performed on both the proteases and subsequently digested with two protease columns (Protease XIII/Pepsin and Nepenthesin-1) for LC-MS (liquid chromatography-mass spectrometry) analysis. This experiment revealed no significant differences between the backbone dynamics of P132H and WT Mpro.

Conclusions

The present in vitro study demonstrated the inhibitory activity of nirmatrelvir against the mutated 3CL protease of the SARS-CoV-2 Omicron variant and found that its potency is similar to that against the wT SARS-CoV-2 Mpro. Taken together, these findings indicate the structural basis for retaining in vitro potency against the mutated Mpro and suggest that they can be used to assess inhibitory activity against prospective newer variants harboring mutations in the Mpro of SARS-CoV-2.

Although vaccines are intended to trigger immune responses against the virus and offer long-term protection in the form of immune memory, which is vital to ending the COVID-19 pandemic, a significant proportion of the human population has yet to receive a COVID-19 vaccine. While some individuals are skeptical and hesitant to take a vaccine, others wait for vaccination due to lack of vaccine access or cannot receive vaccines due to pre-existing medical conditions. Therefore, research and development of new therapeutics should continue and advance, along with improvised vaccination strategies to mitigate the pandemic.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Tarun Sai Lomte

Written by

Tarun Sai Lomte

Tarun is a writer based in Hyderabad, India. He has a Master’s degree in Biotechnology from the University of Hyderabad and is enthusiastic about scientific research. He enjoys reading research papers and literature reviews and is passionate about writing.

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