key: cord-0729723-je46j0oc
authors: Zhang, Yuan; Li, Chunjie; Ke, Xianliang; Luo, Dan; Liu, Yan; Chen, Quanjiao; Wang, Hanzhong; Song, Xiaohui; Zheng, Zhenhua
title: Development of a biosensor assessing SARS-CoV-2 main protease proteolytic activity in living cells for antiviral drugs screening
date: 2022-05-02
journal: Virol Sin
DOI: 10.1016/j.virs.2022.04.002
sha: 9f8ba7a58323936a1d0392051636751738c0ce8b
doc_id: 729723
cord_uid: je46j0oc

nan

The biosensor reported in our study can monitor SARS-CoV-2 M pro activity in living cells instead of in vitro 28

The biosensor reported in our study is sensitive and easy to operate. 30 It is suitable for high-throughput screening. 31

It has the potential to be used in small animal models. 32 J o u r n a l P r e -p r o o f polyproteins, pp1a and pp1ab (Wu et al. 2020b; Zhou et al. 2020) . The functional polypeptides 36 are released from those two polyproteins by extensive proteolytic events, mostly mediated by 37 main protease (M pro , also known as 3CL pro ). It cleaves the polyproteins at no less than 11 38 conserved sites to produce non-structure proteins (Nsps) (Wu et al. 2020) , and no human 39 protease has been found to share similar cleavage specificity (Wu et al. 2020) . Since these 40 cleavage events play an essential role in viral replication and pathogenesis, M pro is considered 41 as one of the most attractive targets for drugs development to combat the ongoing COVID-19 42 pandemic (Pillaiyar et al. 2016). 43 Gaussia Luciferase (GLuc) is a naturally secreted luciferase (Luc) from the deep sea 44 copepod Gaussia prineps (Bowlby and Case 1991) . It is an ideal genetic reporter because it 45 offers greatly increased bioluminescence than other commonly used Lucs (Bartok et al. 2013). 46 It was reported that the fusion of mouse pro-interleukin (IL)-1β on the N-terminal of GLuc can 47 inhibit its catalytic activity because pro-IL-1β can lead protein aggregates (Bartok et al. 2013). 48 To date, no M pro inhibitor has been approved for SARS-CoV-2. In this study, we present a 49 transgene-encoded biosensor that monitors the cleavage activity of SARS-CoV-2 M pro in living 50 cells. As shown in Fig. 1A , a fusion protein constructed of mouse pro-IL-1β and GLuc lacking 51 secretion signal (i-GLuc) was generated as the negative control. A canonical M pro cleavage site 52 AVLQ↓SGFR was inserted into i-GLuc at different positions to generate three biosensors to 53 monitor M pro activity. Theoretically, pro-IL-1β renders GLuc enzyme inactive in these 54 biosensors, but M pro can cleave the biosensors, leading to protein monomerization and GLuc 55 activation. 56

Western blot assay was performed in HEK293T cell to test the cleavage of biosensors by 57 SARS-CoV-2 M pro . GC376 and walrycin B obviously decreased the bioluminescence of the 72 M pro -active biosensor (Fig. 1D ). Further, we tested the effects of these two drugs on the negative 73 control iGLuc. GC376 had no effect on the signal of iGLuc, but the iGLuc signal decreased as 74 the concentration of walrycin B increased ( Fig. 1E and 1F) . Figure 1H shows a good correlation between viral titer and GLuc signal. Cells infected with 85 virus at an MOI of 0.1 showed significant GLuc signal increase and evident cytopathic effects 86 (CPE), but no cell detachment. Thus, we used this MOI in the drug inhibition experiments. 87 GC376 was used to validate the cell line-based antiviral drugs screening system. We 88 detected the inhibitory effects of GC376 at different concentrations on SARS-CoV-2 in reporter 89 cell line via luciferase assay. As expected, the GLuc signal decreased as drug concentration 90 increased from 0 μmol/L to 20 μmol/L (Fig. 1I) , indicating the inhibition of virus by this drug. 91

Thus, this reporter cell line could be applied as an antiviral drugs screening tool. 92

In this study, we presented a transgene-encoded GLuc biosensor and a cell-based antiviral 93 drugs screening system that allowed the monitoring of SARS-CoV-2 M pro activity in living cells 94 instead of in vitro solutions. Except for biosensor based on GLuc, some other fluorescent 95 reporters to detect SARS-CoV-2 M pro activity were developed (Froggatt et al. 2020; Rawson et 96 al. 2021 ). All of these systems are suitable for high-throughput assay. EGFP is visible and 97 luciferase is more sensitive and quantifiable (Supplementary Table S1 ). Compared with other 98 enzymatic activity assays, GLuc assay is highly sensitive, quantifiable, and easy to operate, 99 suitable for high-throughput screening. However, the effect of drugs on negative control iGLuc 100 should be tested, and internal control should be involved to avoid uncertain results caused by 101 some interference factors, such as drug cytotoxicity. In addition, our system has the potential 102 to be applied in animal models because GLuc can be used in small animals as a marker.

Laboratory, Wuhan, Chinese Academy of Sciences, and we thank Tao Du, Jin Xiong and Lun 110

Wang from the laboratory for their critical support. The authors declare that they have no 111 conflict of interest. This article does not contain any studies with human or animal subjects 112 performed by any of the authors. 113

iGLuc: a luciferase-based inflammasome and protease activity 117 reporter

Flash Kinetics and Spatial Patterns of Bioluminescence in the 119

Development of a Fluorescence-Based

SARS-CoV-2 3CL(pro) Reporter Assay

Structure of M(pro) from SARS-CoV-2 and discovery of its inhibitors

Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease Inhibitors: 129 Peptidomimetics and Small Molecule Chemotherapy

Development 131 efficacy of SARS-CoV-2 M pro on i-GLuc and biosensors detected by Western blot (WB)

HEK293T cells were transfected with pHA-M pro and p-i-GLuc or biosensors expression 151 plasmids. Cells were lysed and subjected to WB at 24 hours post-transfection. C Cleavage 152 efficacy of SARS-CoV-2 M pro on i-GLuc and biosensors detected by luciferase assay

HEK293T cells were co-transfected with pHA-M pro /empty vector (EV) and p-i-GLuc or 154 biosensors expression plasmids. Cells were lysed and subjected to luciferase assay at 24 hours 155 post-transfection. D The inhibition of five potential inhibitors against M

At 4 hours 157 post-transfection, cells were treated with DMSO, anacardic acid, disulfiram, GC376, GW5074 158 and walrycin B respectively, drug concentration = 20 μmol/L. E-F The effect of walrycin B and 159 GC376 on negative control iGLuc

At 4 hours post-transfection, cells were treated with walrycin B or GC376 at 161 different concentration. G The cleavage effect of SARS-CoV-2 M

cells detected by luciferase assays. Cells were transfected with empty vector (EV) or pHA-M pro

At 24 hours post-transfection, cells were lysed and subjected to luciferase assay. H The 164 correlation between the SARS-CoV-2 viral titer and luciferase activity in VERO

Cells were infected with SARS-CoV-2 at MOI of 0-0.1. At 24 hours post-infection, 166 cells were lysed and subjected to luciferase assay. I The dose-dependent anti-viral effect of 167 GC376 in VERO-E6-i-MS-GLuc2 cells. Cells were infected with SARS-CoV-2 at an MOI of 168 0.1. At one hour post-infection

Luciferase activity was tested at 24 hours post-infection

Error bars show the standard deviation of three 171 experiments. Student's t test; ns, non-significant