LATEST! Danish Study Shows That Mutations E166V and L50F+E166V On SARS-CoV-2 Variants Weakens Nirmatrelvir (A Component Of Paxlovid) Mpro Binding!

COVID-19 Drugs: A new study by researchers from the Copenhagen University Hospital-Denmark, the Department of Immunology and Microbiology at the University of Copenhagen-Denmark and the Technical University of Denmark has found that the mutations E166V and L50F+E166V that are found on various emerging SARS-CoV-2 variants, weakens nirmatrelvir-Mpro binding. Nirmatrelvir, an oral protease inhibitor is an important component of the drug Paxlovid, of which those controlling the COVID-19 narratives are currently trying to promote extensively despite emerging data showing that it is yet another literally ineffective overpriced drug with long term toxic effects, similar the U.S. NIH and U.S. FDA promoted remdesivir and molnupiravir!


 
In order to facilitate monitoring of potentially emerging resistance, the study team studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir. Resistant variants selected in cell culture harbored different combinations of substitutions in the SARS-CoV-2 main protease (Mpro).
 
Alarmingly, reverse genetic studies in a homologous infectious cell culture system revealed up to 80-fold resistance conferred by the combination of substitutions L50F and E166V.
 
Resistant variants had high fitness increasing the likelihood of occurrence and spread of resistance. Molecular dynamics simulations revealed that E166V and L50F+E166V weakened nirmatrelvir-Mpro binding.
 
Sadly, the COVID-19 Drugs research team (must have been discreetly funded by the makers of remdesivir who are actively trying to find some use for that drug!) claimed that the SARS-CoV-2 polymerase inhibitor remdesivir retained activity against nirmatrelvir resistant variants and combination of remdesivir and nirmatrelvir enhanced treatment efficacy compared to individual compounds. (Wonder if they had done any toxicity studies on this combination of drugs!)
 
The study findings have implications for monitoring and ensuring treatment programs with high efficacy against SARS-CoV-2 and potentially emerging coronaviruses.
 
The study findings were published on a preprint server and are currently being peer reviewed.
https://www.biorxiv.org/content/10.1101/2022.06.06.494921v1

The study team assessed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Alpha, Delta, and Omicron variants’ resistance to nirmatrelvir (NIR), a protease inhibitor of SARS-CoV-2.
 
Nirmatrelvir (NIR) and Paxlovid have recently received authorization for first-line therapy for SARS-CoV-2-positive individuals with high risks of severe coronavirus disease 2019 (COVID-19) and is also under consideration for long-COVID treatment.
 
It is however known that the clinical efficacy of Nirmatrelvir(NIR) may reduce by the development of SARS-CoV-2 resistance, as observed previously for small-molecule inhibitor drugs against hepatitis B, C virus, human immunodeficiency virus (HIV), and influenza virus.
 
The study team had previously determined the efficacy of another protease inhibitor boceprevir (BOC), which is structurally similar to Nirmatrelvir (NIR), ag ainst SARS-CoV-2 in cell culture experiments. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8370243/
 
In the current research the study team extended their previous analysis by identifying amino acid substitutions (mutations) associated with resistance to BOC and NIR and evaluated the fitness of NIR-resistant SARS-CoV-2 variants.
 
The study team also assessed the sensitivity of SARS-CoV-2 Alpha, Delta, and Omicron variants compared to the original (Wuhan-Hu-1) strain to the parental drug of the same class (protease inhibitor), i.e., remdesivir (RDV) and evaluated the prospects of NIR-RDV combinations.
 
For the study,the African green monkey kidney VeroE6 cells and human lung epithelial A549 cells expressing human angiotensin-converting enzyme 2 (hACE2) were cultured, and SARS-CoV-2 stocks were generated in the cultured cells, and their sequences were confirmed by next-generation sequencing (NGS). To identify BOC- and NIR-associated mutations, concentration-response treatments were performed by infecting the cultured cells with BOC escape viruses. BOC-EV1,2 and NIR escape viruses viz NIR-EV1,2, respectively, and subsequently examining the cells by SARS-CoV-2 spike (S) protein immunostaining.

The SARS-CoV-2 escape from NIR was assessed by performing escape experiments with primary escape cultures (passage 0) and five passage cultures (passages 1 to 5). The viruses identified from the global initiative on sharing all influenza data (GISAID) database with specific mutations at specific SARS-CoV-2 main protease (Mpro) sites associated with NIR resistance were identified. Further, reverse genetics analysis and molecular dynamic (MD) simulations of Mpro were performed.
 
The study findings showed that in the concentration-response treatments, the Wuhan-Hu-1 strain was suppressed by three-fold of 50% effective concentration (EC50) BOR and seven-fold EC50 NIR.
 
BOR escape viruses demonstrated 4.7-fold increased EC50 and conferred 6.8-fold cross-resistance to NIR. The short-term concentration-response treatments showed 80-fold resistance conferred by the L50F + E166V combination mutations, whereas the L50F+A173V and T21I+T304I (in NIR-EV1) mutations conferred negligible resistance.
 
Interestingly, in the long-term concentration-response treatments, all the mutants transmitted with 7.5-fold NIR EC50 and the L50F+E166V mutant (NIR-EV2) spread with 15-fold NIR EC50. NIR-resistant SARS-CoV-2 variants demonstrated high fitness and harbored several mutations in Mpro but did not gain more mutations in the passages of the escape experiments.
 
Molecular dynamic or MD simulations showed that the L50F+E166V and E166V mutations weakened NIR-Mpro binding, whereas the L50F mutation improved NIR-Mpro binding. Further, the L50F+E166V and E166V mutations may have shifted NIR-Mpro conformational equilibrium towards non-catalytical states, reducing NIR inhibition probability by 63% and 50%, respectively.
 
The study team said that the weakening of the NIR-Mpro binding by the E166V mutation could be due to the loss of the interactions between the NIR-E166 and S protein subunit 1 (S1) and the loss of interactions between NIR and Mpro residues 187 to 192, which leads to the opening of the SARS-CoV-2 S4 and S2 subunits. In variants with double mutations, reductions in NIR interactions with F140, H163, H172, and L141 in S1 were also observed.
 
The L50F mutation most likely enhanced NIR-Mpro binding, a gain of NIR interactions with T190 and R188 at S4.
 
Importantly the study findings highlighted the mutations associated with the escape of SARS-CoV-2 variants from NIR (or NIR-associated resistance) due to weakened NIR-Mpro binding and reduced inhibition probability of NIR.
 
Thailand Medical News would also like to highlight an important fact which is none of the existing drugs and antivirals approved in the world to treat COVID-19 is effective to curtail viral persistence which is only just emerging to be a very important factor contributing to long COVID and increased mortality in the long term effects of COVID-19!
 
For the latest on COVID-19 Drugs, keep on logging to Thailand Medical News.
 
Read Also:
 
https://www.thailandmedical.news/news/molnupiravir--what-wrong-with-america-why-is-the-american-government-and-agencies-funding-and-pushing-potentially-toxic-drugs-to-treat-covid-19
 
https://www.thailandmedical.news/news/yale-study-reveals-that-enzyme-present-in-sars-cov-2-known-as--coronavirus-3-5-exoribonuclease-exon-is-responsible-for-antiviral-drug-resistance
 
https://www.thailandmedical.news/news/covid-19-drugs-study-published-in-journal-of-the-heart-rhythm-society-warns-that-remdesivir-can-cause-dangerously-low-heart-rate-in-covid-19-patients
 
https://www.thailandmedical.news/news/covid-19-antivirals-interim-who-solidarity-trial-results-confirms-that-remdesivir,-lopinavir-and-interferon-have-no-effect-on-covid-19-mortality
 
https://www.thailandmedical.news/news/breaking-sars-cov-2-ba-2-variant-spawning-mutations-on-its-mpro-sites-that-reduce-efficacy-of-the-antiviral-paxlovid-many-such-variants-in-america-no
 
https://www.thailandmedical.news/news/warning-mutations-in-new-emerging-ba-2,-ba-4-subvariants-are-changing-the-main-coronavirus-protease-drastically,-rendering-many-previous-antivirals-ob
 
https://www.thailandmedical.news/news/covid-19-drugs-university-of-cincinnati-study-warns-against-use-of-u-s-fda-approved-remdesivir-due-to-drug-interactions-and-increased-toxicity
 
https://www.thailandmedical.news/news/urgent-studies-needed-on-paxlovid-does-it-only-help-alleviate-symptoms-and-suppress-viral-load-for-a-while-but-does-not-help-in-total-viral-clearance
 
https://www.thailandmedical.news/news/much-touted-merck-s-covid-19-drug-molnupiravir-shown-to-be-less-effective-in-new-analysis-experts-question-its-efficacy-against-the-omicron-variant