The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in millions of infections worldwide. While the search for an effective antiviral is still ongoing, experimental therapies based on repurposing of available antivirals is being attempted, of which HIV protease inhibitors (PIs) have gained considerable interest. Inhibition profiling of the PIs directly against the viral protease has never been attempted in vitro, and while few studies reported an efficacy of lopinavir and ritonavir in SARS-CoV-2 context, the mechanism of action of the drugs remains to be validated.
Methods We carried out an in-depth analysis of the efficacy of HIV PIs against the main protease of SARS-CoV-2 (Mpro) in cell culture and in vitro enzymatic assays, using a methodology that enabled us to focus solely on any potential inhibitory effects of the inhibitors against the viral protease. For cell culture experiments a dark-to-bright GFP reporter substrate system was designed.
Results Lopinavir, ritonavir, darunavir, saquinavir, and atazanavir were able to inhibit the viral protease in cell culture, albeit in concentrations much higher than their achievable plasma levels, given their current drug formulations. While inhibition by lopinavir was attributed to its cytotoxicity, ritonavir was the most effective of the panel, with IC50 of 13.7 µM. None of the inhibitors showed significant inhibition of SARS-CoV-2 Mpro in our in vitro enzymatic assays up to 100 µM concentration.
Conclusion Targeting of SARS-CoV-2 Mpro by some of the HIV PIs might be of limited clinical potential, given the high concentration of the drugs required to achieve significant inhibition. Therefore, given their weak inhibition of the viral protease, any potential beneficial effect of the PIs in COVID-19 context might perhaps be attributed to acting on other molecular target(s), rather than SARS-CoV-2 Mpro.
To our knowledge, direct determination of the inhibition efficacy of HIV PIs against SARS-CoV-2 Mpro in cell culture has not yet been published, although many in silico studies analyzing interaction between SARS-CoV-2 Mpro and potential inhibitors were published (Additional file 1: Table Table1).1). Antiviral assays using lopinavir and ritonavir in Calu-3 cells were previously carried out for MERS-CoV, and the IC50 for lopinavir, ritonavir and their combination was 11.6, 24.9, and 8.5 µM respectively [3]. In our analysis, we found that a combination of lopinavir plus ritonavir achieved the lowest IC50, this however was due to the high cytotoxicity of lopinavir, and not as a result of direct inhibition of SARS-CoV-2 Mpro. Ritonavir on the other hand was much more tolerable than lopinavir, and achieved the lowest IC50. This should be taken into consideration, given the current drug formulation of lopinavir which is in combination with ritonavir, where ritonavir is used as a pharmacokinetic enhancer due to its inhibition of the cytochrome P450 3A4 isoenzyme, thereby increasing the bioavailability of lopinavir. As a result, administration of ritonavir in combination with other PIs was found to decrease its minimum blood plasma concentration level, as compared to the generic formulation of the drug. Also, while our result regarding ritonavir was in direct contrast to what Choy et al. reported in their short communication, we believe that difference in methodologies is to blame for this discrepancy, as we examined the efficacy of ritonavir against the viral Mpro protease per see.
It is important to note that the IC50 of the inhibitors were in the micromolar range which is not considered optimal for the inhibition of the viral enzyme. Previous studies have reported that the minimum concentrations (Cmin) for lopinavir, darunavir, saquinavir, and atazanavir in patient’s serum under antiretroviral treatment was found to be 9.3, 3.3, 3.8, and < 1 µM, respectively. It would indeed be challenging to achieve such high plasma levels of the inhibitors in order to block the viral replication, moreover, the cytotoxic effects of some of the inhibitors, in addition to the side effects commonly observed with PIs questions the use of anti-HIV PIs in the context of SARS-CoV-2.
Additionally, using an in vitro enzymatic assay, we were able to directly analyze any potential inhibition of Mpro by the HIV PIs. Our results show that none of the inhibitors was able to significantly inhibit Mpro in vitro.
A drawback of this study is that we were not able to assess the interaction between the PIs and the papain-like protease of SARS-CoV-2, as our methodology only enabled us to study the viral main protease. Whether or not these exert any inhibitory effect on the papain-like protease is a subject for future studies, although, a similar methodology may be adapted for SARS-CoV-2 papain-like protease, and other proteases as well. Also, while our cell culture assays were not performed in SARS-CoV-2 target cells, our methodology enabled us to directly examine any potential inhibition of the viral Mpro by the PIs, therefore, it is unlikely that different results will be obtained in target cells.
Conclusion In conclusion, to our knowledge, thorough analysis of the efficacies of PIs against SARS-CoV-2 remains scarce, and the targets of the drugs are yet to be verified. While few studies examined the efficacy of some PIs against the replication of SARS-CoV-2, we set out to study whether or not the inhibitors exert a direct effect on the viral protease. In our experiments, even though some of the PIs developed for the treatment of HIV were able to inhibit SARS-CoV-2 Mpro, they were only able to do so at high concentrations. The combination of lopinavir plus ritonavir resulted in the lowest IC50 in cell culture, albeit at the cost of cellular viability. Although, darunavir and atazanavir required a much higher concentration to achieve the inhibition, cytotoxicity was not observed even at a concentration of 200 µM. It should be noted that there might be other molecular targets for the HIV PIs, as nelfinavir was recently shown to inhibit spike protein-mediated fusion of SARS-CoV-2.
Taking everything into consideration, the use of HIV PIs in the context of COVID-19 might be of limited clinical potential, beneficial effects of which might perhaps be attributed to acting on other molecular target(s), rather than Mpro itself. Data from clinical trials will indeed shed more light on their clinical efficacy
Reference & source information: https://www.ncbi.nlm.nih.gov/
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