virology blog
About viruses and viral disease
Richard joins TWiV to discuss the SARS-CoV-2 antiviral drug Molnupiravir , including how it was discovered, its mechanism of action, whether it is a mutagen for cells, and the future of drugs for treatment of COVID-19.
Hosts: Vincent Racaniello, Alan Dove, and Brianne Barker
Guest: Richard Plemper
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Show notes at microbe.tv/twiv
Multiple vaccines have been developed that have made substantial contributions to controlling the COVID-19 pandemic, but where are the antivirals? Only repurposed drugs have been used and not with much success. That situation seems about to change with the authorization of drugs that target the RNA polymerase (Molnupiravir) and a viral protease (Paxlovid).
Molnupiravir is an orally available pro-drug of the nucleoside analog N4-hydroxycytidine (NHC). The latter is a nucleoside analogue which is incorporated into RNA by the viral RNA-dependent RNA polymerase. Once incorporated into RNA, NHC is recognized as either C or U by the RNA polymerase. As a consequence, many mutations are introduced into the viral genome, causing lethal mutagenesis and inhibition of infectivity. NHC has been shown to block SARS-CoV-2 transmission in ferrets.
Interim results in 775 patients of a phase 3 clinical trial with molnupiravir show that the drug reduced hospitalization or death about 50% compared with placebo in patients with mild to moderate COVID-19: 7.3% of patients who received molnupiravir were either hospitalized or died through Day 29 (28/385), compared with 14.1% of placebo-treated patients (53/377). No deaths were reported in patients who received molnupiravir, as compared to 8 deaths in patients who received placebo, through day 29. The trial required that all patients have laboratory-confirmed COVID-19 with symptom onset within 5 days of assignment to control or drug group. Patients were recruited from multiple countries and included those with risk factors for poor disease. Merck expects to produce 10 million doses of the drug in 2021 and has submitted an application for emergency use authorization (EUA).
Paxlovid is an inhibitor of the 3CL or main protease of SARS-CoV-2, an essential viral enzyme that is required to process precursor proteins into functional products. The drug works by binding to the active site of the protease. Such inhibitors have been successfully developed and are approved for the treatment of AIDS and hepatitis C. Paxlovid inhibits viral reproduction in cell culture and in virus-infected mice when given orally. In a phase I trial in 4 participants, the drug was safe and well tolerated and reached levels greater than needed to inhibit reproduction in cell culture.
Interim analysis of a phase 2/3 randomized, placebo-controlled study of Paxlovid showed that the drug reduced the risk of hospitalization or death by 89%. This study enrolled non-hospitalized patients with COVID-19 who were at risk for severe illness. The patients were treated with Paxlovid within 3 days of symptom onset. Of the patients who received the drug, 3/389 were hospitalized through day 28 (0.8%) compared with 27/385 in the placebo group hospitalized and 7 deaths (7%). Similar results were obtained in a group of patients treated within 5 days of symptom onset. Based on these results, Pfizer plans to submit an application for EUA.
The only antiviral repurposed for SARS-CoV-2 that had any efficacy is remdesivir, whose widespread adoption is limited by the need to administer the drug intravenously. Because they are taken orally, Molnupiravir and Paxlovid should have a far greater impact on the pandemic, especially for people who refuse to be vaccinated. If these drugs had been available before the pandemic – which was certainly possible – it might have been largely prevented.
Molnupiravir might be the first highly effective antiviral drug given emergency use authorization for treatment of COVID-19. Should we be concerned about the results of a recent study which show that the drug is mutagenic in cells?
Molnupiravir is an orally available pro-drug of the nucleoside analog N4-hydroxycytidine (NHC). The latter is a nucleoside analogue which is incorporated into RNA by the viral RNA-dependent RNA polymerase (pictured above). Once incorporated into RNA, NHC is recognized as either C or U by the RNA polymerase. As a consequence, many mutations are introduced into the viral genome, causing lethal mutagenesis and inhibition of infectivity. NHC has been previously shown to have broad-spectrum anti-RNA virus activity and blocks transmission of influenza virus in a guinea pig model of infection. It has been shown to block SARS-CoV-2 transmission in ferrets and results of a phase 2/3 clinical trial look promising, leading to a request for emergency use authorization.
N4-hydroxycytidine could be metabolized by the host to produce the 2’-deoxyribonucleotide form, which could be incorporated into cellular DNA and lead to mutagenesis. To test this hypothesis, a mutagenesis assay was used in Chinese hamster ovary cells (CHO-K1). These cells have one copy of the gene encoding the enzyme hypoxanthine phosphoribosyltransferase (HPRT), which makes the cells sensitive to the base analog 6-thioguanine (6-TG). If NHC were mutagenic, changes in the HPRT gene would allow cells to survive in the presence of 6-TG.
Cells were exposed to NHC for 32 days and assayed for sensitivity to 6-TG. The drug conferred 6-TG resistance in a dose-dependent manner. Two other antivirals that are base analogs, ribavirin and favipiravir, displayed either no or modest mutagenic activity in this assay. Sequence analysis of HPRT mRNA revealed the presence of base changes.
Molnupiravir is a far more active coronavirus antiviral than favipiravir and ribavirin, yet NHC has the distinct ability of causing mutations in cell DNA. The concern is that such mutations could lead to cancer or birth defects in a developing fetus. Whether or not Molnupiravir might cause cancer in humans is not known. However Merck, the developer of Molnupiravir, is required to carry out a series of gene toxicity studies before phase I testing of the compound in humans. Included is the Ames test, which uses bacteria to assess mutagenic activity of a compound. Bacteria do have the enzyme which can convert NHC to the DNA form. The results of these safety studies will not be published until after the drug receives EUA, but presumably nothing was observed that would preclude clinical trials.
Consequently until we have further information about preclinical studies on NHC, we should be cautious in our interpretation of the results of mutagenesis assays in CHO cells.