Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting main protease and papain-like protease

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID is still under investigation. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules targeting the SARS-CoV-2 virus-specific proteins or capable of suppressing the host inflammatory responses to viral infection would provide an effective antiviral therapy against emerging variants of concern. Evolutionarily conserved papain-like protease (PLpro) and main protease (Mpro) play an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting both these viral proteases represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral and anti-inflammatory potential of natural triterpenoids: azadirachtin, withanolide_A, and isoginkgetin. These molecules inhibit the Mpro and PLpro proteolytic activities with half-maximal inhibitory concentrations (IC₅₀) values ranging from 1.42 to 32.7 μM. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to Mpro and PLpro. As expected, the two compounds, withanolide_A and azadirachtin, exhibit potent anti-SARS-CoV-2 activity in cell-based assays, with half-maximum effective concentration (EC₅₀) values of 21.73 and 31.19 μM, respectively. The anti-inflammatory roles of azadirachtin and withanolide_A when assessed using HEK293T cells, were found to significantly reduce the levels of CXCL10, TNFα, IL6, and IL8 cytokines, which are elevated in severe cases of COVID-19. Interestingly, azadirachtin and withanolide_A were also found to rescue the decreased type-I interferon response (IFN-α1). The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2-specific enzymes and also host immune pathways involved in virus-mediated inflammation.