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

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.

BRET-Based Self-Cleaving Biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery.

The 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments, and more inhibitors are under development. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors should be evaluated in Biosafety Level 3 (BSL-3) laboratories. Here, we design DNA-coded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After overexpression of the biosensors in human embryonic kidney (HEK) 293T cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their previously reported anti-SARS-CoV-2 activities. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery. IMPORTANCE The virus proteases 3CLpro are validated drug targets for developing antivirals to treat coronavirus diseases, such as COVID-19. However, the development of 3CLpro inhibitors relies heavily on BSL-3 laboratories. Here, we report a series of BRET-based self-cleaving biosensors that can be used to set up cell-based assays to evaluate the cell permeability and cellular activity of SARS-CoV-2 3CLpro inhibitors in BSL-2 laboratories. The cell-based assay is suitable for high-throughput screening for 3CLpro inhibitors because of the simplicity and good reproducibility of our biosensors. The design strategy can also be used to design biosensors for other viral proteases for which the activation processes involve the self-cleavage of polyproteins.