Atazanavir Is a Competitive Inhibitor of SARS-CoV-2 Mpro, Impairing Variants Replication In Vitro and In Vivo

Abstract

Atazanavir (ATV) has already been considered as a potential repurposing drug to 2019 coronavirus disease (COVID-19); however, there are controversial reports on its mechanism of action and effectiveness as anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the pre-clinical chain of experiments: enzymatic, molecular docking, cell-based and in vivo assays, it is demonstrated here that both SARS-CoV-2 B.1 lineage and variant of concern gamma are susceptible to this antiretroviral. Enzymatic assays and molecular docking calculations showed that SARS-CoV-2 main protease (Mpro) was inhibited by ATV, with Morrison’s inhibitory constant (Ki) 1.5-fold higher than GC376 (a positive control) dependent of the catalytic water (H2Ocat) content. ATV was a competitive inhibitor, increasing the Mpro’s Michaelis–Menten (Km) more than sixfold. Cell-based assays indicated that different lineages of SARS-CoV-2 is susceptible to ATV. Using oral administration of ATV in mice to reach plasmatic exposure similar to humans, transgenic mice expression in human angiotensin converting enzyme 2 (K18-hACE2) were partially protected against lethal challenge with SARS-CoV-2 gamma. Moreover, less cell death and inflammation were observed in the lung from infected and treated mice. Our studies may contribute to a better comprehension of the Mpro/ATV interaction, which could pave the way to the development of specific inhibitors of this viral protease.

Commercially Available Flavonols Are Better SARS-CoV-2 Inhibitors than Isoflavone and Flavones

“…This is probably due to their impact on the orientation of the compounds inside the enzyme target, which was supported by the experimental evidence in the inhibitory percentage of kaempferol, quercetin, and myricetin (10 μM) to SARS-CoV-2 M pro . The EC 50 value for myricetin was comparable to that of atazanavir (ATV), a repurposed SARS-CoV-2 inhibitor that targets M pro [ 27 ], reinforcing the good prospects of flavonols as SARS-CoV-2 antivirals. The CC 50 value in most cases is about 60 times higher than the EC 50 value, which provided a selective index (SI) consistent with an adequate safety profile.…”

“…The inoculum was removed, and cells were incubated with different concentrations of genistein, apigenin, luteolin or fisetin (0.00, 0.63, 1.25, 2.50, 5.00, and 10.0 μM), kaempferol, myricetin, or quercetin (0.00, 0.10, 0.30, 1.00, 3.16, and 10.0 μM), and RDV (0.00, 0.0001, 0.001, 0.01, 0.10, 0.50, 1.0, 5.0, and 10.0 μM) in DMEM with 10% FBS. After 48 h, the virus content in the supernatant was quantified by plaque forming assays in Vero cells (2.0 × 10 4 cells/well) according to our previous publications [ 10 , 27 , 28 ]. The virus titers were calculated by scoring for plaque-forming units (PFU/mL); and non-linear regression analysis of the dose–response curves were also performed to calculate the 50% effective concentration (EC 50 ).…”

“…Vero cells (2.0 × 10 4 cells/well) were treated for 3 days with different concentrations of flavonoids or RDV (ranging from 1 to 600 μM) following the procedure described previously [ 27 ]. Absorption was read at 595 nm using a plate spectrophotometer; the 50% cytotoxic concentration (CC 50 ) was calculated using non-linear regression analysis from a dose–response curve.…”

Developing New Treatments for COVID-19 through Dual-Action Antiviral/Anti-Inflammatory Small Molecules and Physiologically Based Pharmacokinetic Modeling

“…Following molecular docking studies, which showed that atazanavir has a strong binding affinity to the main protease of SARS-CoV-2 [ 50 ] and further demonstrating high bioavailability within the respiratory tract [ 51 ], atazanavir was deemed an interesting candidate for anti-SARS-CoV-2 activity, and was used in in vivo studies of humanized murine models of SARS-CoV-2 infection [ 50 ]. In these studies, it improved survival in atazanavir-treated groups, compared with non-treated controls.…”

“…In addition, it showed sufficient anti-inflammatory activity, significantly reducing the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and keratinocyte-derived chemokines (KCs) in the lungs of treated mice. Atazanavir further protected animals from severe lung injury, which could lead to hemorrhage and shrinking of the lobe, bronchiole, and alveoli, which was prominent in untreated groups [ 50 ].…”