Cell-Based High-Throughput Screening Protocol for Discovering Antiviral Inhibitors Against SARS-COV-2 Main Protease (3CLpro)

Abstract

The global public health has been compromised since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019. There are no specific antiviral drugs available to combat SARS-CoV-2 infection. Besides the rapid dissemination of SARS-CoV-2, several variants have been identified with a potential epidemiologic and pathogenic variation. This fact has forced antiviral drug development strategies to stay innovative, including new drug discovery protocols, combining drugs, and establishing new drug classes. Thus, developing novel screening methods and direct-targeting viral enzymes could be an attractive strategy to combat SARS-CoV-2 infection. In this study, we designed, optimized, and validated a cell-based assay protocol for high-throughput screening (HTS) antiviral drug inhibitors against main viral protease (3CLpro). We applied the split-GFP complementation to develop GFP-split-3CLpro HTS system. The system consists of GFP-based reporters that become fluorescent upon cleavage by SARS-CoV-2 protease 3CLpro. We generated a stable GFP-split-3CLpro HTS system valid to screen large drug libraries for inhibitors to SARS-CoV-2 main protease in the bio-safety level 2 laboratory, providing real-time antiviral activity of the tested compounds. Using this assay, we identified a new class of viral protease inhibitors derived from quinazoline compounds that worth further in vitro and in vivo validation.

Keywords: Drug libraries; GFP complementation; High-throughput screening; Protease; SARS-CoV-2.

Fig. 1

GFP-split complementation method. This assay was developed as previously described [23, 24]. Split GFP into β1–9 and β10–11 resulted in losing its fluorescent capacity. β10–11 has a high affinity to bind to the β1–9 and rapidly develop green fluorescence [25]. a GFP gains the green fluorescence when β10 and β11 in anti-parallel position bind to β1–9. b E5/K5 heterodimer was used to flip β10 and β11 in parallel form prevents self-assembly of the split GFP. c Upon protease cleavage, β11 flips back, forming an anti-parallel structure with β10, which enables self-assembly with β1–9 and gains green fluorescence. The 3CLpro cleaves between E5/K5 heterodimer and β11 allowing β11 to form the anti-parallel structure with β10 (Color figure online)

Fig. 2

The expression cassettes of GFP-split-3CLpro assay. The HEK293T cells were co-transfected with the recombinant pcDNA3.1 plasmids, and the GFP fluorescence intensities were measured at 24, 48, and 72 h. a The expression cassette one consists of two plasmids, the first plasmid harbors GFP construct, and the second plasmid harbors SARS-CoV-2 main protease (3CLpro). b GFP fluorescence noticeably increased at 48 and 72 h, leading to an increase in the signal-to-noise ratio. c The expression cassette two consists of two plasmids, the first plasmid expresses the GFP β-strand 1–9, and the second plasmid harbors a construct of β-strand 10–11 that was joined to the C-terminus of 3CLpro by another 3CLpro cleavage site to facilitate auto-cleavage of the β-strand 10–11. dA stable increase in the GFP fluorescent activity initiated when the β-strand 10–11 released from 3CLpro by enzyme auto-cleavage activity and binds to β-strand 1–9

Fig. 3

GFP signal intensities of the expression cassette one and two of GFP-split-3CLpro assay. GFP fluorescence intensities were measured using Tecan F200 Pro multimode microplate reader at excitation 488 nm and emission 525 nm at different time points for 30 h post-transfection

Fig. 4

Validation of GFP-split-3CLpro screening protocol using a small molecule in-house library. a GFP-split-3CLpro protocol was evaluated by screening an in-house library of 50 small molecule compounds at 10 µM concentration. b Boceprevir showed an 80% reduction in GFP fluorescent activity at 40 µM, suggesting potent inhibition against SARS-CoV-2 main protease. Quinazoline derivatives: c QZ1; d QZ2; e QZ3; g QZ5 showed 20–50% inhibition to viral protease while f QZ4 showed approximately 80% inhibition at 40 µM (n = 5 replicates)

Fig. 5

The inhibition potential of quinazoline derivatives QZ4 to SARS-CoV-2 main protease 3CLpro. a QZ4 compound, [3-(5-methoxy-2-hydroxy benzylidene amino)-2(5-methoxy-2-hydroxyphenyl)-2,3-dihydro quinazoline-4(1H)-one]. b The increasing concentrations of QZ4 showed considerable reduction in the GFP fluorescence intensity over the time c The reduction in GFP intensities indicates that the EC₅₀ of QZ4 is approximately 6.5 µM suggesting potential inhibition against 3CLpro activity with 50% cytotoxic concentration (CC₅₀) more than 100 µM at 48 h. d EC₅₀ of boceprevir is approximately 5.2 µM at 48 h (n = 3 replicates)

Fig. 6

Molecular docking conformations of QZ4 compound to SARS-CoV-2 main protease 3CLpro. The X-ray crystal structure of 3CLpro (PDB ID: 6w63.pdb) was downloaded from Protein Databank, and the 3D structure of QZ4 ligand was downloaded from PubChem. The molecular docking between the optimized QZ4 and the 3CLpro receptor was performed using AutoDock Vina Version 2.0. Yellow: 3CLpro; CPK: ligand; magenta: reported binding site. The compound binds to the 3CLpro active site with docking energy − 10.8 kcal/mol, by generating three hydrogen bonds with Gln 189, Gln192, and Arg 188 (Color figure online)