Analysis of the efficacy of HIV protease inhibitors against SARS-CoV-2's main protease

Abstract

Background: 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 (M^pro) 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 M^pro in our in vitro enzymatic assays up to 100 µM concentration.

Conclusion: Targeting of SARS-CoV-2 M^pro 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 M^pro.

Keywords: HIV protease inhibitors; In vitro assay; Inhibition profiling; Protease; SARS-CoV-2.

Fig. 1

The dark-to-bright reporter system utilized to investigate M^pro activity in cell culture. a Sequences of M^pro and the reporter substrate (PR-Sub). b Schematic representation of the dark-to-bright reporter system. Homology model structure of PR-Sub is also shown

Fig. 2

Optimization of HEK-293 T cell transfection with SARS-CoV-2-M^pro and the dark-to-bright GFP substrate. a Cells transfected with PR-Sub and CoV-2 M^pro under native microscopic light. b Visualization of cells transfected with PR-Sub under fluorescent microscope. c Visualization of cells transfected with PR-Sub and CoV-2 M^pro under fluorescent microscope. Co-transfection with both plasmids resulted in 28–34% GFP fluorescence

Fig. 3

Determination of IC₅₀ in cell culture. Relative activity (%) is plotted on the left Y axis versus logarithmic transformation of the inhibitor's concentration (nM). Percentage of cytotoxicity is plotted on the right Y axis. Error bars represent SD (n = 3)

Fig. 4

Inhibition profiling using an enzymatic assay. Results show that none of the inhibitors showed significant inhibition of SARS-CoV-2 M^pro at a concentration of 100 µM in the reaction. The control reaction contained DMSO without a protease inhibitor. Error bars represent SD (n = 2)