COVID-19 News: More Mpro Mutations Emerging On Newer SARS-CoV-2 Variants. Are Current Antivirals Still Effective?

COVID-19 News: The global fight against the COVID-19 pandemic has entered a new phase with the emergence of diverse SARS-CoV-2 variants, each presenting unique challenges. As we grapple with the impact of these variants, a critical aspect under scrutiny is the main protease (Mpro or 3CLpro) of the virus. Mpro plays a pivotal role in the viral life cycle by cleaving the polyprotein at multiple sites, making it an attractive target for antiviral therapies. In this COVID-19 News report, we delve into the intricate details of Mpro mutations across different Variants of Concern (VOCs) - Alpha, Beta, Gamma, Delta, and Omicron - exploring their implications for the effectiveness of current antiviral drugs.


 
Showing Mpro residues mutational status of proline in one promoter, mutated residues are represented as red dots whereas orange sticks represent proline residues which are conserved using the three-dimensional structure of Mpro (PDB ID 6Y2G).

The Genomic Landscape of SARS-CoV-2 and the Role of Mpro
SARS-CoV-2, an RNA virus with a genome size of approximately 30 Kb, encodes essential proteins such as spike, envelope, nucleocapsid, and membrane. Among these, the Mpro protease, also known as 3CLpro, stands out as a key player in the viral replication process. It cleaves the polyprotein at 11 different sites, facilitating viral replication and proliferation. Targeting Mpro has emerged as a promising therapeutic strategy due to its absence of human homologs and high conservation among coronaviruses.
 
The Mpro crystal structure reveals a homo-dimeric configuration with three domains in each monomer. Domains I and II form antiparallel barrel structures, while domain III regulates dimerization. The catalytic dyad, Cys145 and His41, resides in a cleft between domains I and II. The N-finger region and a linker region are crucial for catalytically active dimer formation. Understanding the mutational landscape of Mpro is crucial for unraveling the virus's adaptive strategies and devising effective antiviral interventions.
 
Exploring the Mutational Landscape Across Variants
To comprehensively analyze the mutational landscape, we focused on five VOCs: Alpha, Beta, Gamma, Delta, and Omicron. A dataset comprising 222,980 sequences allowed the study team to identify 51,733 mutations in the Mpro protein. The distribution of mutations varied among the variants, revealing distinct patterns.

The Alpha variant exhibited mutations at 59 positions, with K90R, M17V, and Q110H being prevalent. In the Beta variant, mutations were identified at 98 positions, with K90R, A193V, and K100R prominent. The Gamma variant displayed mutations at 175 positions, with K90R, H246Y, and T21I prevalent. The Delta variant showed mutations at 208 positions, with K90R, V296I, and N274S prominent. Notably, the Omicron variant exhibited mutations at 22 positions, with P132H, K90R, and T169S being most prevalent.
 
Insights into Amino Acid Substitutions
Amino acid substitutions can sign ificantly impact protein folding, stability, and interactions. In our analysis, positive substitutions dominated at 71%, influenced by mutations like K90R in the Beta variant and P132H in the Omicron variant. Aliphatic substitutions (18%) and aromatic substitutions (6%) suggested roles in stabilizing protein structure. Polar substitutions (5%) indicated alterations in surface properties, while negative substitutions were minimal at 0.17%.
 
Examining Specific Structural Regions
Structural regions crucial for Mpro functionality were investigated, revealing intriguing insights into mutation distribution. The N-finger region, essential for catalytic activity, showed fewer mutations. The linker region, connecting domains II and III, exhibited mutations linked to drug resistance. Domains I, II, and III displayed varying mutation counts, emphasizing the critical role of domain III in enzyme activity.
 
Mutational Analysis of Drug Binding Sites
The study extended to assess the mutational status of drug binding sites, focusing on the nirmatrelvir binding site, interdomain (ritonavir) binding site, and dimeric interface. Key residues within the nirmatrelvir binding site crucial for drug interaction remained highly conserved, suggesting potential therapeutic efficacy.

Notably, the interdomain (ritonavir) binding site exhibited significant alterations, emphasizing the importance of understanding how variations in these residues might impact ritonavir binding efficacy. The dimeric interface, essential for Mpro's functional dimeric form, displayed mutations, raising questions about potential implications for enzyme function and susceptibility to therapeutic interventions.
 
Insights into Sub-Pockets and Catalytic Site Mutations
The catalytic site of Mpro comprises five sub-pockets (S1 to S5), crucial for substrate recognition and binding. The analysis revealed that the S1 sub-pocket, deeply embedded within the protein structure, displayed the least mutation, emphasizing its importance in effective substrate recognition. In contrast, the S3 sub-pocket exhibited a higher mutation rate, suggesting potential implications for substrate recognition activity.
 
Proline Substitutions and Structural Stability
Proline residues play a vital role in providing structural rigidity to proteins. Analysis of proline positions in Mpro revealed mutations at ten out of thirteen positions, raising concerns about potential impacts on protein stability. Mutations at the dimeric interface pocket containing proline residues emphasized potential structural disabilities affecting drug-binding stability.
 
Conclusion: Implications for Antiviral Therapies
In conclusion, this extensive analysis provides a nuanced understanding of the evolving mutational landscape of SARS-CoV-2 Mpro across different variants. The distribution of mutations, amino acid substitutions, and insights into specific structural regions and drug binding sites offer valuable perspectives for therapeutic development.
 
The conservation of crucial residues within the nirmatrelvir binding site suggests potential efficacy of existing antiviral drugs. However, the observed mutations in the interdomain (ritonavir) binding site and dimeric interface raise concerns about the effectiveness of certain therapeutic interventions.
 
Understanding the dynamics of Mpro mutations is vital for adapting antiviral strategies to the evolving nature of the virus. This analysis lays the groundwork for future research aimed at developing targeted antiviral therapies resistant to mutational alterations in SARS-CoV-2 Mpro, contributing to the ongoing battle against the global pandemic.
 
The study findings were published in the preprint server and is currently being peer reviewed.
https://www.researchsquare.com/article/rs-3902142/v1
 
Thailand Medical News would like to add that these study findings not only questions the efficacy of approved antiviral drugs but also the effectiveness of many past repurposed drugs, supplements and even herbs and phytochemicals used to treat COVID-19.
 
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