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

Abstract

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.

Keywords: 3C-like protease; BRET; SARS-CoV-2; inhibitor; self-cleaving biosensor.

FIG 1

(A) The domain organization of SARS-CoV-2 polyproteins (pp1a or pp1ab) (top) and the BRET-based self-cleaving biosensor (bottom). (B) An illustration of the BRET-based self-cleaving biosensor, showing the relative positions of GFP2, 3Clpro, and RLuc8. (C) The BRET ratio of HEK 293T cells 24 h post-transfection of the plasmid carrying biosensor pBRET-1. The 3CLpro inhibitor GC376, at the indicated working concentrations, was added right after transfection. The biosensor with a C145A mutation in 3CLpro (pBRETmut-1) was used as a noncleavable control. The data represent the mean ± standard deviation of three independent measurements. (D and E) The self-cleavage of pBRET-1 in the presence of the indicated concentrations of GC376 was detected by Western blotting using an anti-HA antibody (D) or an anti-FLAG antibody (E).

FIG 2

(A and B) The BRET ratio of HEK 293T cells 24 h post-transfection of the different biosensor plasmids (pBRET-1 to pBRET-10). The 3CLpro inhibitor GC376 was diluted into cell culture media at the indicated working concentrations right after transfection. The data represent the mean ± standard deviation of three independent measurements. (C) The amino acid sequences of the 3CLpro cleavage sites at the N- and C-terminus of the 10 biosensors, as well as the EC₅₀ of GC376 measured using these biosensors. (D) The EC₅₀ values of GC376 measured in HEK 293T cells transfected with different amounts of pBRET-10 plasmid DNA. The data represent the mean ± standard deviation of five independent measurements.