The ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the urgent need for effective antiviral treatments. The Coronaviridae family, encompassing SARS-CoV-2, poses a persistent threat due to its endemic nature and frequent zoonotic events.
Structural Characteristics of Coronaviruses
Coronaviruses are enveloped viruses with a positive-sense, single-stranded RNA genome. They encode four structural proteins (S, E, M, N) and three key enzymes (RdRp, Mpro, PLpro) (V’Kovski et al., 2021). Notable outbreaks, such as those caused by SARS-CoV and MERS-CoV, emphasize the necessity for broad-spectrum antivirals.
Drug Discovery Efforts and Treatment Landscape
A substantial effort in drug discovery has identified potential antiviral treatments for SARS-CoV-2, many repurposed from other indications (Edwards et al., 2022; Taibe et al., 2022). However, the emergence of variants has prompted investigations into the efficacy of these treatments. Monoclonal antibodies targeting the S protein exhibit reduced efficacy against Omicron variants (Cox et al., 2023).
In contrast, drugs targeting viral enzymes, including Mpro inhibitor nirmatrelvir and RdRp inhibitors remdesivir and molnupiravir, show robust cross-variant efficacy against various human coronaviruses (de Wit et al., 2020; Sheahan et al., 2017, 2020; Brown et al., 2019; Li et al., 2022, 2023; Liu et al., 2023).
Clinical Challenges and Approved Treatments
Despite these advancements, clinically approved options remain limited. Nucleoside analogues remdesivir and molnupiravir, while promising, show inconsistent clinical benefits in randomized trials (Butler et al., 2023; Consortium, 2022). The Mpro inhibitor nirmatrelvir, when co-administered with ritonavir (Paxlovid), is currently the most widely approved antiviral treatment for non-hospitalized COVID-19 patients, but concerns exist regarding its efficacy against Omicron subvariants and reported viral rebound (Arbel et al., 2022; Pandit et al., 2023).
Repositioning of Ritonavir and Cobicistat
Ritonavir, initially developed as an antiretroviral drug for HIV, has been repurposed as a pharmacoenhancer in Paxlovid. Its derivative, cobicistat, serves as a booster for antiretroviral drugs, lacking anti-HIV activity but retaining CYP3A inhibition capability (Xu et al., 2010). A previous study demonstrated unexpected antiviral effects of cobicistat against SARS-CoV-2, prompting further investigation (Shytaj et al., 2022).
High-Throughput Screening of Cobicistat and Ritonavir
In this study, automated image analysis enabled a high-throughput parallel screening of the in vitro antiviral effects of cobicistat and ritonavir. Eight SARS-CoV-2 variants, including Omicron, and other human coronaviruses (HCoV-229E and MERS-CoV) were examined. The data revealed that both drugs exhibited broad-spectrum anti-coronavirus activity at low micromolar concentrations.
Comparative Analysis of Cobicistat and Ritonavir
Despite both drugs showing efficacy, cobicistat consistently demonstrated greater potency. This was evidenced by more pronounced maximal inhibition of viral replication, lower EC50 values, and higher combination sensitivity scores when co-treated with nirmatrelvir.
Implications for Future Treatments
Considering the historical experience with CYP3A inhibitors, the study suggests that dose adjustments to standard administration protocols could enhance the utilization of CYP3A inhibitors, particularly cobicistat, as first-line monotherapy or in combination against human coronaviruses. This finding may open new avenues for developing more potent antiviral strategies against SARS-CoV-2 and related coronaviruses.
The exploration of clinically approved compounds for repurposing in the context of antiviral drug discovery is a valuable strategy, providing a shortcut for the development of effective treatments. This study builds on previous research demonstrating the antiviral activity of cobicistat against early SARS-CoV-2 isolates, extending the investigation to encompass multiple SARS-CoV-2 variants and other coronaviruses. The findings indicate that cobicistat’s antiviral effects are conserved across a broad spectrum of coronaviruses, with the parent drug, ritonavir, exhibiting similar but consistently lower potency in vitro.
Consistency in Antiviral Concentrations
The observed antiviral concentrations for both cobicistat and ritonavir against a diverse panel of SARS-CoV-2 variants and other human coronaviruses align with previous studies. Comparable EC50 values were noted for cobicistat in the early SARS-CoV-2 isolate Ger/BavPat1/2020, while ritonavir demonstrated effectiveness against the Wuhan SARS-CoV-2 variant and a recombinant MERS-CoV/luciferase construct. This consistency reinforces the broad-spectrum nature of the antiviral activities of these drugs across various coronaviruses.
Mechanism of Action and Unexpected Potency Discrepancy
The exact mechanism underlying the direct antiviral activity of cobicistat and ritonavir remains unclear. Surprisingly, ritonavir, despite structural similarities with cobicistat and its original design as a CYP3A inhibitor, exhibited consistently lower potency. The presence of morpholine moieties in cobicistat, differing from ritonavir, may contribute to enhanced antiviral effects, as shown in previous studies on various viruses. Notably, the calculated binding entropy for cobicistat with the main protease (Mpro) suggests an unstable association, and previous experiments have ruled out Mpro inhibition by cobicistat.
Potential Intracellular Mechanism and Lipid Alterations
Previous research indicated that cobicistat can block SARS-CoV-2 fusion in cells overexpressing ACE-2 and the SARS-CoV-2 S protein. The current study expands on this, revealing that the antiviral effects of cobicistat and ritonavir are independent of ACE-2, suggesting a potential inhibition of viral entry through a conserved effect on the S protein. Noteworthy is ritonavir’s known ability to induce lipid metabolism alterations. However, the lack of enhanced antiviral effects with pre-treatment of cells implies that any lipid alterations induced by these drugs may act intracellularly, possibly following the first cycle of viral entry. Future studies are required to elucidate this hypothesis.
Synergistic Effects with Nirmatrelvir and Therapeutic Implications
The observed synergistic effects when combining cobicistat or ritonavir with the Mpro inhibitor nirmatrelvir align with its metabolism through CYP3A. However, the significant synergy scores at higher concentrations of cobicistat and ritonavir suggest a potential contribution of their direct antiviral activity. The calculated combination sensitivity scores (CSS) indicate that the cobicistat/nirmatrelvir combination is more potent in suppressing SARS-CoV-2 replication, suggesting potential therapeutic advantages. Clinical dose escalation studies will be crucial to assess the safety and feasibility of implementing higher dosages for this purpose.
Implications for Future Antiviral Strategies
Despite the study’s limitations, such as the lack of a mechanistic explanation for the differing potencies of cobicistat and ritonavir, the findings suggest that these CYP3A inhibitors can be repurposed as broad-spectrum anti-coronavirus agents. Adjusting dosing regimens could potentially achieve concentrations necessary for effective viral suppression, opening avenues for the development of more potent antiviral strategies against SARS-CoV-2 and related coronaviruses. Further in vivo studies and clinical trials are warranted to validate these in vitro findings and explore the potential of cobicistat and ritonavir as promising candidates for COVID-19 treatment.
reference link : https://www.sciencedirect.com/science/article/pii/S0166354223002449#sec4