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. 2024 Dec;39(1):2387417.
doi: 10.1080/14756366.2024.2387417. Epub 2024 Aug 20.

In-cell bioluminescence resonance energy transfer (BRET)-based assay uncovers ceritinib and CA-074 as SARS-CoV-2 papain-like protease inhibitors

Mei Li  1   2 , Zhu-Chun Bei  2 , Yongtian Yuan  1   2 , Baogang Wang  2 , Dongna Zhang  2 , Likun Xu  2 , Liangliang Zhao  2 , Qin Xu  1 , Yabin Song  2
Affiliations

In-cell bioluminescence resonance energy transfer (BRET)-based assay uncovers ceritinib and CA-074 as SARS-CoV-2 papain-like protease inhibitors

Mei Li et al. J Enzyme Inhib Med Chem. 2024 Dec.

Abstract

Papain-like protease (PLpro) is an attractive anti-coronavirus target. The development of PLpro inhibitors, however, is hampered by the limitations of the existing PLpro assay and the scarcity of validated active compounds. We developed a novel in-cell PLpro assay based on BRET and used it to evaluate and discover SARS-CoV-2 PLpro inhibitors. The developed assay demonstrated remarkable sensitivity for detecting the reduction of intracellular PLpro activity while presenting high reliability and performance for inhibitor evaluation and high-throughput screening. Using this assay, three protease inhibitors were identified as novel PLpro inhibitors that are structurally disparate from those previously known. Subsequent enzymatic assays and ligand-protein interaction analysis based on molecular docking revealed that ceritinib directly inhibited PLpro, showing high geometric complementarity with the substrate-binding pocket in PLpro, whereas CA-074 methyl ester underwent intracellular hydrolysis, exposing a free carboxyhydroxyl group essential for hydrogen bonding with G266 in the BL2 groove, resulting in PLpro inhibition.

Keywords: SARS-CoV-2; bioluminescence resonance energy transfer (BRET); high-throughput screening (HTS); papain-like protease (PLpro); protease inhibitors.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

None
Graphical abstract
Figure 1.
Figure 1.
SARS-CoV-2 papain-like protease (PLpro) and its dual biological role in SARS-CoV-2 replication and innate immunity inhibition. (a) Schematic organisation of the SARS-CoV-2 genome and polyproteins. Genes encoding replicase, structural, and accessory proteins are highlighted in different colours. The locations of PLpro and 3-chymotrypsin like protease (3CLpro, also termed main protease, Mpro) in viral polyproteins are highlighted in red and blue. Red and blue arrows indicate the cleavage sites of PLpro and 3CLpro, respectively. (b) Crystal structure of SARS-CoV-2 PLpro (PDB: 6XA9). The right-handed architecture of PLpro is shown in cartoon representation. The thumb, palm, and finger domains and catalytic triad are labelled, and active site residues (C111, H272, and D286) are represented as sticks. The zinc ion is slate blue. (c) The dual role of SARS-CoV-2 PLpro. PLpro cleaves the LXGG↓XX motif in viral polyproteins to release nsp1-3, playing an indispensable role in viral replication. It also recognises and cleaves the same motif in the C-terminal tails of ubiquitin (Ub) and interferon (IFN)-stimulated gene 15 (ISG15), deubiquitinating or deISGylating cellular signalling proteins, contributing to suppressing innate immune responses. (d) Surface view of the substrate-binding pocket of PLpro. The catalytic-triad residues are shown as cyan sticks and labelled. The terminal LRGG motif of the ISG15 substrate (PDB: 6XA9) located in the binding pocket is shown as grey sticks. Residues of substrate are labelled per the occupied position in the binding site: P4 for L, P3 for R, P2 for G, and P1 for G. The surface of the subsites is coloured as indicated. The blocking loop 2 (BL2) spanning residues 267–271 in the S3 and S4 subsites is shown as a cartoon and labelled. Residue Y268 on the BL2 loop is shown as orange or grey sticks, corresponding to two different conformations. (e) SARS-CoV-2 PLpro cleavage site sequences.
Figure 2.
Figure 2.
Performance of BRET reporter with CyOFP1, mScarlet, or mNeonGreen as the acceptor. (a) Schematic representation of the BRET. (b) Nluc emission spectrum (blue solid lines) overlaid with excitation (dashed lines) and emission (solid lines) spectrum of CyOFP1 (orange), mScarlet (red), and mNeonGreen (green). The spectral separation between donor and acceptors, brightness of acceptors, and donor emission/acceptor excitation overlap (colour-shaped) are indicated. The Nluc spectrum was derived from emission spectral scans of cells expressing recombinant Nluc, and the spectrum and brightness of fluorescent proteins are obtained from previous reports (c) Emission spectral scan of cells expressing BRET reporters (sloid lines) and their T2A-linked analogues (dotted lines). The emission intensities were normalised to the peak emission intensities of Nluc measured in individual samples (n = 1 transfection). The solid and dashed vertical arrows indicate the net BRET ratios of BRET reporters and the background BRET ratios from T2A-linked reporters. (d) Quantitative correlation between BRET signal and the amount of BRET reporter plasmid DNA co-transfected into cells. The x-axis shows the percentage of T2A-linked reporter plasmid DNA replaced by the BRET reporter construct. The data is from two independent experiments with triplet samples (open squares and circles). The trend lines (solid colour lines) with 95% confidence intervals (colour shading) were fitted to the base 10 logarithm of BRET against percentage using a linear regression model. The signal-to-background ratio (S/B) and signal-to-noise ratio (S/N) were calculated using x = 100% as positive and x = 0% as negative controls. The limit of detection (LOD) was calculated as 3 times the ratio of the intercept standard error to the slope, both derived from the fitted regression.
Figure 3.
Figure 3.
In-cell assay of PLpro proteolytic cleavage activity using the BRET reporter. (a) Schematic representation of the used BRET reporters. The framed text indicates the PLpro substrate peptide sequence in linkers with yellow highlights on the LxGG motif. (b) PLpro construct co-transfection resulted in a significant BRET signal decrease in cells expressing BRET reporters harbouring substrate peptides in the linker region. The plasmid DNA ratio of the protease construct (WT or C111S) to the reporter was set at 1:1. The data was from one experiment with triplicates. Points with a range bar indicate the mean with a 95% confidence interval. The significance of the difference in the mean between WT and C111S was determined using a two-sample t test. *P < 0.05, **P < 0.01, ***P < 0.001. (c) Cleavage efficiency of PLpro on different substrate peptides. BRET signals were normalised to the mean signals of cells expressing no cleavage reporter (C111S and uncleavable reporter co-transfected cells) and then compared to complete cleavage reporter (T2A). The dataset used is the same for (b). The significance of the difference was determined by a post-hoc pairwise t test with an ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (d) Western blot analysis showing the cleavage of the nsp2/3-linked BRET reporter in cells co-transfected with PLpro. Cells were co-transfected by reporter (T2A-linked or nsp2/3-linked) and PLpro (WT or C111S) plasmids, with a 3:1 reporter-to-PLpro plasmid DNA ratio used. GRL0617 (GRL) and rac5c (Rac5c) were added at 100 μM after 6 h of transfection. Blots of protein extracts from 24 h post-transfection were probed with anti-V5 antibody. Red arrows indicate bands of the complete BRET reporter (BRET), PLpro, and cleaved C-terminal product (Nluc). (e) BRET signal in cells co-transfected with the different amount of PLpro plasmids. The nsp2/3-linked BRET reporter plasmid in co-transfection was fixed at 140 ng/well. The BRET signal was normalised to that of the C111S mutant co-transfected cells. The data is from two independent experiment with triplicates in each. The significance of the difference was determined by a post-hoc pairwise t test with an ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (blue) vs 2 ng/well; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 (green) vs 4 ng/well; p values in orange vs 6 ng/well. (f) Quantitative correlation between BRET signal and the amount of PLpro plasmid DNA co-transfected. The plasmid DNA ratio of reporter (nsp2/3 linked) to protease constructs (PLpro plus C111S) was set at 24:1. The x-axis shows the percentage of PLpro plasmid DNA replaced by the C111S construct. The data is derived from one experiment with triplicates. The data analysis and calculation were performed as described in Figure 2d.
Figure 4.
Figure 4.
Evaluation of reported PLpro inhibitors using the developed in-cell BRET assay. GRL0617 (a), rac5c (b), disulfiram (c), ebselen (d), 6-thioguanine (e), and proanthocyanidins (f) were tested with serial concentrations. Red crossbars with points indicate mean values with raw data from one experiment (n = 3). The raw BRET signals were used to evaluate PLpro inhibition, while the fluorescence intensities (RFU) detected before BRET substrate adding were used to quantify cytotoxicity. The dose-response curves were fitted using the four-parameter log-logistic regression model. IC50 and CC50 were predicted from the fitted regression model and presented as estimates with 95% confidence interval. For inhibitors that showed no or insufficient inhibition in the assay, an ANOVA with post-hoc pairwise t tests was performed to confirm the existence of a significant difference compared to the vehicle controls. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 5.
Figure 5.
High-throughput screening identified SARS-CoV-2 PLpro inhibitors from the protease inhibitor library. (a) Overview of results of HTS. All compounds (285) were tested at 20 μM, with vehicle (0.2% DMSO) as the negative control. Cells co-transfected by the C111S mutant serve as positive controls. The red dashed line indicates the BRET threshold for the hit selection. Z-factor and Z’-factor were calculated as described in the Material and Methods section. (b) Chemical structures of hits. (c), (d), and (e) Results of follow-up dose-response assays. Data from one (c) or two (d and e) independent experiments (n = 3) are presented as mean values (red crossbar) along with raw data (points). For the repeated assays, the raw BRET or RFU readouts were normalised to the mean of positive controls (C111S for BRET and Vehicle for RFU) within the same assay. The dose-response analysis was performed as described in Figure 4.
Figure 6.
Figure 6.
FRET-based enzymatic assay validating the PLpro inhibition activity of ceritinib and CA-074. GRL0617 (a), ceritinib (b), and CA-074 (c) at serial concentrations were tested in the FRET-based proteolysis assay for SARS-CoV-2 PLpro. The RFU readings of each sample were normalised by subtracting readings recorded at 0 min. The data is from one experiment with triplicates and is presented as mean values with a 95% confidence interval (points with a range bar). The purple dashed lines indicate the linear trendline fitted using data from the first three time points of vehicle controls. The dose-response curves were fitted using data normalised (vehicle controls as 100%) from three time points at which the vehicle controls recorded the highest readings within the linear range. For GRL0617 (d) and ceritinib (e), data recorded at 10, 15, and 20 min were used. For CA-074 (f), data recorded at 25, 35, and 45 min were used. Points with a range bar indicate the mean with a 95% confidence interval. The dose-response analysis was performed as described in Figure 4.
Figure 7.
Figure 7.
Ligand-protein interaction analysis based on the docking model of ceritinib and CA-074. Molecular docking was performed using AutoDock Vina, with the receptor derived from the crystal structure of GRL0617-bound SARS-CoV-2 PLpro (PDB: 7JRN). Ligand-protein interactions were profiled using PLIP and LigPlot+ based on the co-crystal structure (GRL0617) or the best docking models (ceritinib and CA-074). (a) Co-crystallographic pose of GRL0617 superimposed with the best docking poses of ceritinib and CA-074 in substrate-binding pocket of SARS-CoV-2 PLpro. The receptor is presented as surface model, and compounds as stick model. For clarity, the surface areas of substrate-binding subsites S2, S3, S4, and the adjacent BL2 groove are shaded in different colours. (b) Superimposition of docking models of ceritinib (left) and CA-074 (right) with the terminal five residues of the ISG15 substrate. Cyan and gray cartoons represent SARS-CoV-2 PLpro with inhibitor (PDB 7JRN) or ISG15 substrate (PDB 6XA9) binding. Spheres depict atoms that are nearly superimposable between the inhibitor and the substrate. (c) and (d) Close-up view of ceritinib and CA-074 interacting with PLpro substrate binding sites. The receptor is presented as a cartoon model showing the surface of binding sites, with a blue highlight to indicate residues involving hydrophobic interactions. Residues responsible for interactions are shown as cyan sticks and labelled. The aromatic ring centre involved in putative π-π stacking is indicated using grey spheres. The putative hydrogen bonds, π-π stacking, halogen bond, and hydrophobic interactions are shown as coloured dashed lines as indicated.
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References

    1. Hiscott J, Alexandridi M, Muscolini M, Tassone E, Palermo E, Soultsioti M, Zevini A.. The global impact of the coronavirus pandemic. Cytokine Growth Factor Rev. 2020;53:1–9. - PMC - PubMed
    1. Naseer S, Khalid S, Parveen S, Abbass K, Song H, Achim MV.. COVID-19 outbreak: Impact on global economy. Front Public Health. 2022;10:1009393. - PMC - PubMed
    1. Penninx BWJH, Benros ME, Klein RS, Vinkers CH.. How COVID-19 shaped mental health: from infection to pandemic effects. Nat Med. 2022;28(10):2027–2037. - PMC - PubMed
    1. Kumari M, Lu RM, Li MC, Huang JL, Hsu FF, Ko SH, Ke FY, Su SC, Liang KH, Yuan JP, et al. . A critical overview of current progress for COVID-19: development of vaccines, antiviral drugs, and therapeutic antibodies. J Biomed Sci. 2022;29(1):68. - PMC - PubMed
    1. Toussi SS, Hammond JL, Gerstenberger BS, Anderson AS.. Therapeutics for COVID-19. Nat Microbiol. 2023;8(5):771–786. - PubMed

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