Structure-Based Virtual Screening and Functional Validation of Potential Hit Molecules Targeting the SARS-CoV-2 Main Protease

Abstract

The recent global health emergency caused by the coronavirus disease 2019 (COVID-19) pandemic has taken a heavy toll, both in terms of lives and economies. Vaccines against the disease have been developed, but the efficiency of vaccination campaigns worldwide has been variable due to challenges regarding production, logistics, distribution and vaccine hesitancy. Furthermore, vaccines are less effective against new variants of the SARS-CoV-2 virus and vaccination-induced immunity fades over time. These challenges and the vaccines’ ineffectiveness for the infected population necessitate improved treatment options, including the inhibition of the SARS-CoV-2 main protease (Mpro). Drug repurposing to achieve inhibition could provide an immediate solution for disease management. Here, we used structure-based virtual screening (SBVS) to identify natural products (from NP-lib) and FDA-approved drugs (from e-Drug3D-lib and Drugs-lib) which bind to the Mpro active site with high-affinity and therefore could be designated as potential inhibitors. We prioritized nine candidate inhibitors (e-Drug3D-lib: Ciclesonide, Losartan and Telmisartan; Drugs-lib: Flezelastine, Hesperidin and Niceverine; NP-lib: three natural products) and predicted their half maximum inhibitory concentration using DeepPurpose, a deep learning tool for drug–target interactions. Finally, we experimentally validated Losartan and two of the natural products as in vitro Mpro inhibitors, using a bioluminescence resonance energy transfer (BRET)-based Mpro sensor. Our study suggests that existing drugs and natural products could be explored for the treatment of COVID-19.

Protegrin-2, a potential inhibitor for targeting SARS-CoV-2 main protease Mpro

“…4 A ). This sensor is based on bioluminescence resonance energy transfer (BRET), wherein the efficiency of energy transfer between a donor and acceptor largely depends on the distance between the two proteins [29] , [30] , [31] , [32] , [33] , [34] . The sensor consists of a green fluorescent protein, mNeonGreen, that serves as the resonance energy acceptor and a bioluminescent protein, NanoLuc, that serves as the energy donor sandwiching the cognate M pro N-terminal autocleavage site.…”

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

“…Further, given the slow bioluminescence activity of the R162A mutant NLuc leading to prolonged half-life of the bioluminescence activity, the R162A mutant NLuc could be utilized in assays requiring continuous monitoring of bioluminescence activity over long periods without requiring any specialized reagent such as in monitoring biochemical activity of enzymes, including proteases, that display a slow turn-over rate. 31, 32…”

“…For live cell experiments, HEK293T cells were transfected with pmNG-(EAAK)3-NLuc, pmNG- (EAAK)3-NLuc(Q32A) and pmNG-(EAAK)3-NLuc(R162A) plasmid DNA. After 24 or 48 h of transfection, fluorescence, bioluminescence, and BRET 4-7, 31-33 measurements were performed by the addition of furimazine (Promega, Wisconsin, USA) at a dilution of 1:200 using a Tecan SPARK® multimode plate reader at 37 °C. For fluorescence measurements, untransfected cells were used to determine blank values, which were subtracted from the fluorescence values of cells expressing the mNG fluorescence proteins.…”

“…The cell lysate was prepared as described earlier. 32, 74 Briefly, post 48 h of transfection, the cells were washed with chilled Dulbecco’s Phosphate-Buffered Saline (DPBS) and lysed in a buffer containing 50 mM HEPES (pH 7.5), 50 mM NaCl, 0.1% Triton-X 100, 1 mM dithiothreitol (DTT) & 1 mM ethylenediamine tetraacetic acid (EDTA) on ice, followed by centrifugation at 4 °C for 1 h at 14,000 rotations per min (RPM). The supernatant was collected and stored at -80 °C until further usage.…”

“…24-26 Additional applications of NLuc include nanoparticle conjugates utilized for in vivo imaging 27 , sensitive viral infection detection 28 , to evaluate one of metabolic intermediate in fatty acid metabolism 29 or to test antibiotic susceptibility 30 . Further, NLuc has been successfully applied in the development of various Bioluminescence Resonance Energy Transfer (BRET)-based biomolecular assays 4-7, 31-33 such as to study molecular/organelle interactions 34, 35 , for optogenetics purposes 36 , for drug descovery 37, 38 , as genetic encoded light source for a photodynamic anticancer therapy 39 , biomolecule detection 40 , monitoring molecular tension 41 , and assays to monitor SARS-CoV-2 protease activity 42, 43 . Additionally, NLuc possessing synthetic allosteric regulation has been generated through circular permutation of the protein for developing biosensors.…”