key: cord-0930560-vx4069zq
authors: Ma, Chunlong; Hu, Yanmei; Wang, Yuyin; Choza, Juliana; Wang, Jun
title: Drug-Repurposing Screening Identified Tropifexor as a SARS-CoV-2 Papain-like Protease Inhibitor
date: 2022-04-11
journal: ACS Infect Dis
DOI: 10.1021/acsinfecdis.1c00629
sha: eec1e3beca0bc5e0a0edf6ee088e150ab9dc7b9e
doc_id: 930560
cord_uid: vx4069zq

[Image: see text] The global COVID-19 pandemic underscores the dire need for effective antivirals. Encouraging progress has been made in developing small-molecule inhibitors targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (M(pro)). However, the development of papain-like protease (PL(pro)) inhibitors faces several obstacles. Nevertheless, PL(pro) represents a high-profile drug target given its multifaceted roles in viral replication. PL(pro) is involved in not only the cleavage of viral polyprotein but also the modulation of host immune response. In this study, we conducted a drug-repurposing screening of PL(pro) against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PL(pro) inhibitors with IC(50) values ranging from 3.39 to 8.28 μM. The three hits showed dose-dependent binding to PL(pro) in the thermal shift assay. In addition, tropifexor inhibited the cellular PL(pro) activity in the FlipGFP assay with an IC(50) of 10.6 μM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells at noncytotoxic concentrations. Overall, tropifexor represents a novel PL(pro) inhibitor that can be further developed as SARS-CoV-2 antivirals.

proteins. 9 Therefore, inhibiting PL pro is a two-pronged approach to protecting host cells from viral infection.

PL pro is a 35 kDa domain withinNsp3, which is a 215 kDa multidomain protein that is a key component of the viral replication complex. 10 Compared to PL pro from SARS-CoV, SARS-CoV-2 PL pro displays decreased deubiquitination activity and enhanced deISGlyation activity. 9, 11 In contrast to M pro , PL pro is a more challenging drug target mainly for two reasons. First, the protein substrate of PL pro consists of LXGG. 12 Accordingly, there is a lack of drug binding pockets in the S1 and S2 subsites. As such, a majority of reported PL pro inhibitors are noncovalent inhibitors that bind to the S3 and S4 subsites that are located more than 10 Å away from the catalytic cysteine C111. 13−15 Second, PL pro cleaves the same substrate sequence LXGG as the human deubiquitinase, 16 which presents a challenge in developing selective PL pro inhibitors. Despite extensive high-throughput screening and lead optimization, 11, [13] [14] [15] 17, 18 GRL0617 and its analogues remain the most potent PL pro inhibitors reported so far. To identify structurally novel PL pro inhibitors, we conducted a drug-repurposing screening and identified EACC, KY-226, and tropifexor as potent PL pro inhibitors with IC 50 values ranging from 3.39 to 8.28 μM. EACC is a reversible autophagy inhibitor. 19 KY-226 is a potent, selectivity, and orally bioavailable allosteric protein tyrosine phosphatase 1B (PTP1B) with an IC 50 of 0.25 μM. 20 Tropifexor is a highly potent agonist of the farnesoid X receptor and is currently undergoing phase II clinical trial for nonalcoholic steatohepatitis (NASH) and liver fibrosis. 21 Their antiviral mechanism of action was further characterized in the thermal shift assay and the FlipGFP protease assay. Gratifyingly, tropifexor also had potent antiviral activity against SARS-CoV-2 in Calu-3 cells with an EC 50 of 4.03 μM. Overall, tropifexor represents a potent PL pro inhibitor with a novel scaffold that can be further developed as SARS-CoV-2 antivirals.

High-Throughput Screening of SARS-CoV-2 PL pro Inhibitors. Using the previously optimized FRET assay condition, 15 we performed a high-throughput screening of SARS-CoV-2 PL pro against the MedChemExpress bioactive compound library, which consists of 9,791 compounds including FDA-approved drugs, clinical candidates, and natural products. The assay was performed in a 384-well plate with a Z′ of 0.688, and GRL0617 was included as the positive control. All compounds were originally screened at 40 μM, and hits showing more than 50% inhibition were further titrated to determine the IC 50 values. GRL0617 was included as a positive control. In total, three compounds, EACC, KY-226, and tropifexor ( Figure 1A ), were identified as positive hits with IC 50 values of 8.28, 3.39, and 5.11 μM, respectively ( Figure  1B ). In comparison, the IC 50 value for the positive control GRL0617 was 1.66 μM ( Figure 1B) . Next, the broad-spectrum activity of the three hits was tested against SARS-CoV PL pro ( Figure 1C ) and MERS-CoV PL pro ( Figure 1D ). It was found that EACC, KY-226, and tropifexor retained potent inhibition against SARS-CoV PL pro with IC 50 values of 6.28, 3.53, and 5.54 μM, respectively ( Figure 1C ). In contrast, EACC and KY-226 were weak inhibitors of MERS-CoV PL pro with IC 50 values of 27.8 and 30.6 μM, while GRL0617 was inactive (IC 50 > 60 μM) ( Figure 1D ). Nevertheless, tropifexor showed higher potency against MERS-CoV PL pro with an IC 50 of 2.32 μM ( Figure 1D ). The hits were further counterscreened against the SARS-CoV-2 M pro to rule out promiscuous cysteine protease inhibitors. 22−25 It was found that EACC and KY-226 were not active (IC 50 ≥ 60 μM), while tropifexor had weak inhibition with an IC 50 of 43.65 μM, which corresponds to a selectivity index (SI) of 8.5 ( Figure 1E ). These results suggest that the inhibition of SARS-CoV-2 PL pro by tropifexor is specific. The inhibition of PL pro 's deubiquitination and deISGlyation activities was characterized using the Ub-AMC and ISG15-AMC substrates, respectively. 14,15 While EACC and KY-226 were inactive in inhibiting the deubiquitinase activity of PL pro (IC 50 > 100 μM), tropifexor showed moderate activity with an IC 50 of 18.85 μM ( Figure 1F ). Similarly, EACC and KY-226 were not active in inhibiting the deISGlyation activity of PL pro (IC 50 > 80 μM), tropifexor showed does-dependent inhibition with an IC 50 of 27.22 μM ( Figure 1G ). Tropifexor is a hydrophobic compound with a C log P of 5.69. To rule out the possibility that the observed PL pro inhibition was due to nonspecific binding, we repeated the FRET assay against SARS-CoV-2 PL pro in the presence of 0.01% BSA, and it was found that tropifexor retained potent inhibition with an IC 50 of 10.36 μM ( Figure 1H ), suggesting that the inhibition of PL pro by tropifexor is unlikely due to nonspecific hydrophobic interactions. Tropifexor had similar IC 50 values against SARS-CoV-2 PL pro with and without a 30 min preincubation ( Figure  1I ), suggesting a reversible binding. The mechanism of inhibition of tropifexor was further studied in an enzymatic kinetic experiment, and GRL0617 was included as a control. The Lineweaver−Burk plots showed that both GRL0617 and tropifexor are competitive inhibitors of SARS-CoV-2 PL pro ( Figure 1J ,K).

Overall, tropifexor appears to be the most promising hit with consistent inhibition against SARS-CoV-2, SARS-CoV, and MERS-CoV PL pro s. In addition, tropifexor also inhibited the deubiquitination and deISGlyation activities of SARS-CoV-2 PL pro , albeit at lower potency.

Pharmacological Characterization of the Hits in the Thermal Shift Assay and the Cell-Based FlipGFP PL pro Assay. The mechanism of action of EACC, KY-226, and tropifexor in inhibiting SARS-CoV-2 PL pro was further characterized by the thermal shift assay and the cell-based FlipGFP PL pro assay. 15, 22, 23, 26 Thermal shift assay measures the direct binding between the compound and the protein; therefore, it can rule out hits that might bind to the FRET substrate in the enzymatic assay. Similar to the positive control GRL0617, all three hits displayed dose-dependent binding to PL pro , as revealed by the enhanced melting temperatures with increasing drug concentrations ( Figure 2 ).

Next, we tested the three hits in the FlipGFP PL pro assay. 15, 22, 23 The FlipGFP PL pro was recently developed by us as a surrogate assay to quantify the cellular activity of PL pro inhibitors in the biological safety level 2 facility, and we have shown that there is a positive correlation between the FlipGFP IC 50 values with the SARS-CoV-2 antiviral EC 50 values. 15 The FlipGFP assay is a virus-free cell-based protease assay in which the 293T cells were transfected with PL pro and the GFP reporter. The GFP reporter consists of two fragments, 27,28 the β1−9 template and the β10-11 strands that are constrained in the parallel inactive conformation through a PL pro substrate linker. Upon cleavage of the substrate linker, the β10 and β11 strands become parallel and can associate with the β1−9 template, leading to increased GFP signal. mCherry is included as an internal control to normalize transfection efficacy and compound cytotoxicity. In principle, the normalized GFP/ mCherry ratio is proportional to the enzymatic activity of PL pro . The advantage of the FlipGFP assay over the FRET assay is that it can rule out compounds that are cytotoxic, membrane-impermeable, and having off-target effects that prevent cellular on-target engagement. 22, 23 In the FlipGFP assay, the positive control GRL0617 showed dose-dependent inhibition with an IC 50 of 14.67 μM, while the negative control GC376 was not active (IC 50 > 60 μM) ( Figure 3A ,B). The results from EACC and KY-226 were not conclusive due to the cell cytotoxicity of the compounds. Tropifexor had an IC 50 of 10.60 μM but a low selectivity index (CC 50 = 29.77 μM, SI = 2.8) ( Figure 3A ,B). Given the low selectivity, the results from the FlipGFP are not stringently conclusive. Nevertheless, tropifexor reduced the GFP/ mCherry ratio by 50% at 10 μM, which was not cytotoxic. In summary, the FlipGFP assay results suggest that tropifexor might have antiviral activity against SARS-CoV-2.

Antiviral Activity of Hits against SARS-CoV-2 in Calu-3 Cells. The antiviral activity of EACC, KY-226, and tropifexor in inhibiting SARS-CoV-2 replication in Calu-3 cells was tested ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article SARS-CoV-2 PL pro structure we recently solved (PDB: 7JRN). 15 The binding sites were calculated by site map, and the GRL0617 binding site was identified as the top-ranked binding site; therefore, it was selected for docking. GRL0617 was included as a positive control. The docking pose of GRL0617 was superimposable with the binding mode in the Xray crystal structure ( Figure 5A ). Tropifexor, EACC, and KY-226 all fit snuggly into the U-shaped binding pocket that is covered by the BL2 loop where GRL0617 binds ( Figure 5B − D). Among the three hits, tropifexor showed the most favorable binding pose with a Glide score of −4.085 ( Figure  5B ). The docking poses might provide a guidance for the following lead optimization.

Although PL pro is a validated antiviral drug target, the development of PL pro inhibitors falls behind M pro and RdRp inhibitors. As of date, no PL pro inhibitors have been advanced to the in vivo animal model studies yet. The naphthalene compounds such as GRL0617 and its analogues are the only class of validated PL pro inhibitors with antiviral activity against SARS-CoV-2. However, the low metabolic stability of this series of compounds might prevent its further development. 14, 31 In this study, we aimed to identify structurally novel PL pro inhibitors that can serve as starting points for further optimization. Through screening the MedChemExpress bioactive compound library, three hits EACC, KY-226, and tropifexor were identified as SARS-CoV-2 PL pro inhibitors with IC 50 values in the single-digit micromolar range. Among the three hits, tropifexor appears to be the most promising hit as it also showed potent inhibition against SARS-CoV PL pro (IC 50 = 5.54 μM) and MERS-CoV PL pro (IC 50 = 2.32 μM). In addition to the inhibition of the PL pro -mediated cleavage of the viral polyprotein substrate, tropifexor also inhibited the deubiquitination and deISGlyation activities of SARS-CoV-2 PL pro . Consistent with the enzymatic inhibition, tropifexor showed a dose-dependent stabilization of SARS-CoV-2 PL pro in the thermal shift assay. Importantly, tropifexor displayed cellular PL pro inhibitory activity in the FlipGFP assay and the antiviral activity against SARS-CoV-2 in Calu-3 cells. Although the low selectivity index (SI = 6.2) of tropifexor in the antiviral assay prevents its direct repurposing as a SARS-CoV-2 antiviral, the discovery of tropifexor as a novel PL pro inhibitor provides an additional scaffold for further medicinal chemistry optimization. Follow-up studies will focus on improving the target and cellular selectivity. Furthermore, tropifexor is a fairly large molecule (MW: 603.59); efforts will be made to reduce the size as well as the hydrophobicity of the compound to optimize ligand efficiency and druglikeness properties.

Protein Expression and Purification. Detailed expression and purification procedures untagged SARS-CoV-2 PL pro and SARS-CoV-2 M pro were described in our previous publications. 15,32 SARS-CoV papain-like protease gene (ORF 1ab 1541-1855) (accession # AEA10621.1) from strain SARS coronavirus MA15 with Escherichia coli codon optimization in the pET28b-(+) vector was ordered from GenScript. Then, the SARS-CoV PL pro gene (ORF 1ab 1541-1855) was subcloned from the pET28b-(+) to pE-SUMO vector according to the manufacturer's protocol (LifeSensors Inc., Malvern, PA). The forward primer with the Bsa I site is GCGGTCTCAAGGT-GAGGTGAAGACCATCAAAGTGTTCACCACC; the reverse primer with a Bsa I site is GCGGTCTCTCTAGAT-TATTTAATGGTGGTGGTATAGCTGGTTTCCTTGTAG. The expression and purification protocol of SARS-CoV PL pro are identical to those of SARS-CoV-2 PL pro . 15 MERS-CoV PL pro gene (ORF 1ab 1482-1803) (accession # KY581684) from strain MERS coronavirus Hu/ UAE_002_2013 with E. coli codon optimization in the pET28b-(+) vector was ordered from GenScript. Then, the MERS-CoV PL pro gene (ORF 1ab 1482-1803) was subcloned into the pE-SUMO vector with the pair primers: G C G G T C T C A A G G T C A G C T G A C C A T C -GAGGTGCTGGTTACCGTGG and GCGGTCTCTCTA-GATTAGTTGCAATCGCTGCTATATTTTTGACCCGG-GAAC. The expression and purification protocol of MERS-CoV papain-like protease are identical to those of SARS-CoV-2 PL pro . 15 FRET Substrate Synthesis. The SARS-CoV-2 PL pro FRET substrate 1 is Dabcyl-FTLRGG/APTKV(Edans); this substrate was also used as SARS-CoV PL pro and MERS-CoV PL pro substrates. SARS-CoV-2 M pro FRET substrate 2 is Dabcyl-KTSAVLQ/SGFRKME-(Edans). These FRET substrates were synthesized by solid-phase synthesis through iterative cycles of coupling and deprotection using the previously optimized procedure. 33 Ub-AMC and ISG15-AMC were purchased from BostonBiochem (catalog nos. U-550-050 and UL-553-050, respectively).

Enzymatic Assays. The high-throughput screening was carried out in 384-well format, as described previously. 15 The bioactive compound library consisting of 9,791 compounds was purchased from MedChemExpress (catalog no. HY-L001). The enzymatic reactions for SARS-CoV-2, SARS-CoV, and MERS-CoV PL pro s were carried out in a reaction buffer consisting of 50 mM HEPES pH 7.5, 5 mM DTT, and 0.01% Triton X-100. For the IC 50 measurement with the FRET peptide−Edans substrate, the reaction was carried out in 96well format with a 100 μL reaction volume. SARS-CoV-2 PL pro (200 nM), SARS-CoV PL pro (200 nM), or MERS-CoV PL pro (2 μM) was preincubated with various concentrations of testing compounds at 30°C for 30 min before the addition of the FRET peptide substrate to initiate the reaction. The reaction was monitored in a Cytation 5 image reader with filters for excitation at 360/40 nm and emission at 460/40 nm at 30°C for 1 h. The initial enzymatic reaction velocity was calculated from the initial 10 min enzymatic reaction via a linear regression function and was plotted against the substrate concentrations in Prism 8 with a four-parameter dose− response function. For the IC 50 measurements with Ub-AMC or ISG15-AMC substrate, the reaction was carried out in 384well format in a 50 μL reaction volume. In the Ub-AMC cleavage assay, the final SARS-CoV-2 PL pro concentration is 50 nM, and the substrate Ub-AMC concentration is 2.5 μM. In the ISG15-AMC assay, the final SARS-CoV-2 PL pro concentration is 2 nM, and the substrate ISG15-AMC concentration is 0.5 μM. The SARS-CoV-2 M pro enzymatic assays were carried out in the reaction buffer containing 20 mM HEPES pH 6.5, 120 mM NaCl, 0.4 mM EDTA, 20% glycerol, and 4 mM DTT, as described previously. 32, 34 To rule out that the inhibition of tropifexor on PL Pro is due to aggregation, 200 nM PL Pro was incubated with serial concentrations of tropifexor (0, 0.1, 0.3, 1, 3, 10, 30, 100 μM) in the reaction buffer in the presence or absence of 0.01% BSA (0.1 mg/mL) at 30°C for 30 min. The reaction was initiated ACS Infectious Diseases pubs.acs.org/journal/aidcbc Article by adding a 10 μM FRET substrate and monitored every 90 s for 1 h at 30°C. The initial velocity was determined in the first 15 min by linear regression. The IC 50 values were determined by fitting the curves with nonlinear regression using log (concentration of inhibitor) vs response with variable slopes in Prism 8.

To determine whether preincubation affects the IC 50 value of tropifexor, 200 nM PL Pro was mixed with serial concentrations of tropifexor (0, 0.1, 0.3, 1, 3, 10, 30, 100 μM) in the reaction buffer with or without preincubation at 30°C for 30 min, and the reaction was initiated by adding a 10 μM FRET substrate. IC 50 values were determined as previously described.

To determine the binding mode of tropifexor, K M and V max were determined at different concentrations of GRL0617 (0, 0.3, 1, 3, 10 μM) or tropifexor (0, 1, 3, 10, 30 μM). SARS-CoV-2 PL Pro (200 nM) was mixed with the indicated concentrations of GRL0617 or tropifexor in the reaction buffer and incubated at 30°C for 30 min. The reaction was initiated by adding different concentrations of FRET peptides (5, 10, 25, 50, 100, 200 μM) . Michaelis−Menten and Lineweaver−Burk curves were plotted in Prism 8.

Differential Scanning Fluorimetry (DSF). The thermal shift assay (TSA) was carried out using a Thermo Fisher QuantStudio 5 real-time PCR system, as described previously. 15, 32 Briefly, 4 μM SARS-CoV-2 PL pro protein in the PL pro reaction buffer (50 mM HEPES pH 7.5, 5 mM DTT, and 0.01% Triton X-100) was incubated with various concentrations of testing compounds at 30°C for 30 min. A 1× SYPRO orange dye was added, and the fluorescence of each well was monitored under a temperature gradient range from 20 to 90°C with a 0.05°C/s incremental step. The melting temperature (Tm) was calculated as the mid-log of the transition phase from the native to the denatured protein using a Boltzmann model in Protein Thermal Shift Software v1.3.

Cell-Based FlipGFP PL pro Assay. Plasmid pcDNA3-PLpro-flipGFP-T2A-mCherry was constructed from pcDNA3-TEV-flipGFP-T2A-mCherry. 15 SARS-CoV-2 PL pro expression plasmid pcDNA3.1-SARS2 PL pro was ordered from Genscript (Piscataway NJ) with codon optimization. For transfection, 293T cells were seeded into a 96-well Greiner plate (catalog no. 655090) overnight with 70−90% confluency; 50 ng of pcDNA3-PLPro-flipGFP-T2A-mCherry plasmid and 50 ng of protease expression plasmid pcDNA3.1-PL pro were added to each well in the presence of a transfection reagent TransIT-293 (Mirus) according to the manufacturer's protocol. Three hours after transfection, 1 μL of the testing compound was added to each well at 100-fold dilution. Images were acquired 2 days after transfection with a Cytation 5 imaging reader (Biotek) GFP and mCherry channels and were analyzed with Gen5 3.10 software (Biotek). SARS-CoV-2 PL pro protease activity was calculated by the ratio of the GFP signal over the mCherry signal. The FlipGFP PL pro assay IC 50 value was determined by plotting the GFP/mCherry signal over the compound concentration with a four-parameter dose− response function in Prism 8. The mCherry signal alone was utilized to evaluate the transfection efficiency and compound cytotoxicity.

Antiviral Assay in Calu-3 Cells. Calu-3 cells (ATCC, HTB-55) grown in minimal Eagle's medium supplemented with 1% nonessential amino acids, 1% penicillin/streptomycin, and 10% FBS are plated in 384-well plates. The next day, 50 nL of drug suspended in DMSO is added as an 8-pt dose− response with 3-fold dilutions between test concentrations in triplicate, starting at 40 μM final concentration. The negative control (DMSO, n = 32) and positive control (10 μM remdesivir, n = 32) are included on each assay plate. Calu-3 cells are pretreated with controls and test drugs (in triplicate) for 2 h prior to infection. In BSL3 containment, SARS-CoV-2 (isolate USA-WA1/2020) diluted in a serum-free growth medium is added to plates to achieve an MOI = 0.5. Cells are incubated continuously with drugs and SARS-CoV-2 for 48 h. Molecular modeling of the binding of EACC, KY-226, and tropifexor to SARS-CoV-2 PL pro . Docking was performed using Schrodinger Glide extra precision (XP). The SARS-CoV-2 PL pro structure was downloaded from the PDB code 7JRN. The binding sites were calculated by the site map, and the GRL0617 binding site is the highest-scored binding site, and therefore, it was chosen for docking. The docking grid was centered around GRL0617 with the coordinates of X = 9.88, Y = −11.74, and Z = 32.55. GRL0617 was added as a positive control for the docking. The final docking poses were generated in PyMOL.

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Complete contact information is available at: https://pubs.acs.org/10.1021/acsinfecdis.1c00629Author Contributions § C.M. and Y.H. contributed equally. J.W., C.M., and Y.H. conceived and designed the study. C.M. performed the highthroughput screening, enzymatic assays, and thermal shift assay. Y.H. performed enzymatic kinetic study and FRET assays with and without BSA or preincubation. Y.W. and J.C. helped with the protein expression and purification and the enzymatic assays. J.W. wrote the manuscript with input from C.M.

The authors declare no competing financial interest.