Resistance to nirmatrelvir due to mutations in the Mpro in the subvariants of SARS-CoV-2 Omicron: Another concern?

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The world has faced 3 years of the COVID-19 pandemic with billions of infections, millions of deaths, and a massive economic crisis. Since the pandemic began, scientists have started to fight against SARS-CoV-2. One significant scientific effort was searching for successful therapeutic molecules against the virus. The repurposing of the drug candidates was initiated in the initial stage of the pandemic.^1,2,3 Along with drug repurposing, new molecule discovery was started by exploring the drug targets. The most significant drug targets are RdRp, Mpro/3CLpro, and others.^4,5 The possible repurposed and newly discovered therapeutic molecules have been tested in vivo, in vitro, and through clinical trials. They found several effective therapeutic molecules against this virus, such as nirmatrelvir, ritonavir, remdesivir, molnupiravir, etc. One recent effective drug against the SARS-CoV-2 Omicron variant and its subvariant is Paxlovid, which combines two therapeutic molecules (nirmatrelvir and ritonavir). Paxlovid shows high effectiveness against SARS-CoV-2 emerging variants and subvariants.^6,7,8,9

During the past 3 years, several variants have been created in nature with significant mutations in the S protein and other proteins like RdRp, Mpro/3CLpro, etc.^10,11 These mutations have caused drug resistance, which has been reported from time to time.^12,13 Several significant mutations have been observed in the SARS-CoV-2 Omicron variant and its subvariants that cause therapeutic escape.^14,15,16,17 The phenomenon of therapeutic escape causes immense concern to scientists. Recently, several mutations have been noted in Mpro/3CLpro, which might be responsible for the nirmatrelvir resistance (Figure 1).

Figure 1.

Nirmatrelvir is shown attached to the Mpro complex of SARS-CoV-2

The significant mutations in Mpro that cause nirmatrelvir resistance are also shown. (A) A ribbon-like model shows nirmatrelvir attached to the Mpro complex of SARS-CoV-2. The model was generated from a macromolecular crystal structure (PDB: 7SI9). (B) A ribbon-like model demonstrates the specific portion of attachment of nirmatrelvir in the Mpro complex of SARS-CoV-2. (C) A space-filling model shows the attachment of nirmatrelvir in the Mpro complex. The model was generated from a macromolecular crystal structure (PDB: 7SI9). (D) The model demonstrates significant mutations in Mpro that cause nirmatrelvir or other drug resistance. The reported significant mutations in Mpro are T21I, L50F, N142L, E166 M/V, Q189E/I, Q192T, P252L, and T304I.

SARS-CoV-2 has acquired a wide range of mutations in its non-structural proteins during its variant generation. They have allowed it to adjust quickly to the environment, increase its infectivity rate, and eventually develop drug resistance due to several mutations in the hotspot residues. This is actually for its survival. The FDA has approved the nirmatrelvir molecule (an antiviral component of Paxlovid) for treating mild-to-moderate infections to combat the developing variations predominantly observed in Omicron and its subvariants.¹⁸ It has been proven that the oral antiviral nirmatrelvir, which targets the 3CL protease of SARS-CoV-2, is clinically effective against the COVID-19 pandemic.¹⁹ This drug interacts with the Cys145 residue of the SARS-CoV-2 nsp5 by establishing a covalent bond.²⁰ New SARS-CoV-2 variants that potently evade the currently available therapeutics continue to arise due to the intense selective pressure against the spike glycoprotein. Many sublineages from Omicron have been reported so far.^21,22,23,24 Antiviral treatments are crucial for treating those who are not immunized or in cases of illness that develop suddenly. Because of its vital role in coronavirus replication, the nsp5 protease found in SARS-CoV-2 and other coronaviruses makes it an extremely promising therapeutic target.²¹ During the SARS pandemic in 2002 and the more recent SARS-CoV-2 pandemic, the major protease (Mpro/3CLpro) of the coronavirus (CoV) family was initially investigated as a target for designing various therapeutics.^25,26,27 The successful results obtained by constructing protease inhibitors for the hepatitis C virus (HCV) and HIV-1 virus instigated the development of a similar therapeutic to treat SARS-CoV-2 virus infection.^28,29

SARS-CoV-2 viruses now in circulation have Mpro mutations that make them resistant to nirmatrelvir. For instance, the M49I mutation was discovered in 1,883 genomes in May 2022, with a minor increase in late 2021.³⁰ Sasi et al. highlighted that the potency of nirmatrelvir is decreased by five mutations, namely Q189E, Q192T, N142L, Q189I, and E166M. The IC₅₀ of nirmatrelvir was reduced by a factor of 24 against the E166M mutation.³¹ According to phylogenetic analyses, nirmatrelvir-resistant variants are transmissible and appear to have existed before nirmatrelvir was introduced into the human population.³² In addition, Lan et al.²⁰ designed specific mutations to hinder nirmatrelvir’s ability to attach to its substrate to study nirmatrelvir resistance. They used 12 wild-type SARS-CoV-2 replicons and one subvariant of the Omicron (BA.1), followed by specific enzymatic assays and cell-based complementation. The result demonstrated that E166V conferred strong nirmatrelvir resistance, approximately 55-fold, with a significant drop in wild-type replicon fitness (nearly 20-fold), but not in the BA.1 subvariant (2-fold), in both cases. However, L50F improved the fitness of wild-type replicons. Due to these variations, Omicron possibly may have a lower resistance barrier than the wild-type variant.²⁰ Despite the prevalent mutation in the Mpro site of the Omicron variant, namely P132H, nirmatrelvir has maintained its in vitro efficacy against the sublineages of Omicron, namely BQ.1.1,³³ XBB,³³ BA.1,³⁴ BA.1.1,³⁵ BA.5,³⁵ BA.4,³⁵ BA.2,³⁵ BA.2.12.1,³⁵ BA.2,³⁵ BA.2.12.1,³⁵ and BA.2.75.³⁶

The outcomes of the clinical trials testing nirmatrelvir are encouraging, but there are still many challenges in their widespread application. First, the trial participants received treatment when the infection was still in its early stages. In practice, it can be challenging to administer antivirals to patients as soon as they are diagnosed as positive. Second, despite careful patient selection, nirmatrelvir monotherapy produces suboptimal clinical results.^37,38 Nirmatrelvir has been shown to reduce viral infection in hamsters’ respiratory organs after they are infected by the BA.2 variant.³⁹ The transmission of the virus was suppressed entirely in a group of animals given a human-equivalent dose of molnupiravir. Still, a pharmacokinetic human-equivalent dose of nirmatrelvir did not significantly lower the SARS-CoV-2 titers in ferrets. It also did not prevent viral transmission to untreated ferrets in direct contact. While the prophylactic nirmatrelvir treatment resulted in an infection in all contacts, prophylactic molnupiravir treatment of uninfected ferrets close to the infected group prevented SARS-CoV-2 transmission.⁴⁰ An analysis by Iketani et al.¹⁹ of 13 recombinant SARS-CoV-2 clones revealed that higher levels of resistance needed the accumulation of further mutations, because three noteworthy mutations, namely T21I, P252L, and T304I, caused only low-level resistance. The most significant resistance was acquired by the E166V mutation (nearly 100-fold). However, this mutation also reduced the ability of the virus to replicate, which was later recovered by compensatory modifications like T21I and L50F. The results also show that the viral resistance to nirmatrelvir can quickly develop in vitro through a variety of pathways, and the unique mutations found here provide a solid framework to investigate the mechanism of resistance in more detail and guide the development of potent protease inhibitors in future generations.¹⁹ According to molecular dynamics simulations, a combination of L50F with E166M, and E166V alone, reduced the binding efficacy between nirmatrelvir and Mpro. The polymerase inhibitor remdesivir and bebtelovimab (a monoclonal antibody) maintained their anti-nirmatrelvir activity against the emerging resistant lineages. However, a combination of these compounds with nirmatrelvir has shown to be more effective compared with the administration of the individual components. These discoveries affect how therapies for the SARS-CoV-2 virus are monitored and ensured to be effective.⁴¹

The quick and aggressive preclinical development of nirmatrelvir helped reduce the health care burden due to the COVID-19 pandemic in 2022. This drug has shown to be exceptionally efficient in combating the Omicron wave, with preserved in vitro and clinical efficacy. In the first quarter of 2022, nirmatrelvir generated $1.5 billion in sales, making it one of the most common antivirals prescribed throughout the world.⁴² When this drug is taken, the immune system actively destroys the virus, including any potentially resistant versions that may have developed. It makes sense to concentrate efforts on surveillance of immunocompromised patients receiving nirmatrelvir treatment to detect the emergence of a drug-resistant virus. If medication resistance could be chosen in vitro, as has happened previously with other viral infections, it will undoubtedly produce similar results in vivo. Although most of the existing COVID-19 treatments have been given as monotherapies, it is feasible that future treatments can be beneficial by employing a drug cocktail to reduce the possibility of SARS-CoV-2 escape.¹⁹ Therefore, scientists need to work on other drug cocktails and discover more antiviral molecules to increase the therapeutic options for treating emerging variants and subvariants of SARS-CoV-2. Simultaneously, it would hinder drug resistance by the SARS-CoV-2 variants and subvariants and help to reduce the health care burden due to the ongoing COVID-19 pandemic.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.