key: cord-0863485-0yn0tnfx
authors: Ma, Chunlong; Wang, Yuyin; Choza, Juliana; Wang, Jun
title: Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor
date: 2021-12-03
journal: bioRxiv
DOI: 10.1101/2021.12.02.471030
sha: 3977d9197eef53f6269f90fd526132be8199e0b2
doc_id: 863485
cord_uid: 0yn0tnfx

The global COVID-19 pandemic underscores the dire need of 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 (Mpro). However, the development of papain-like protease (PLpro) inhibitors faces several obstacles. Nevertheless, PLpro represents a high-profile drug target given its multifaceted roles in viral replication. PLpro is involved in not only the cleavage of viral polyprotein but also modulation of host immune response. In this study, we conducted a drug-repurposing screening of PLpro against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PLpro inhibitors with IC50 values ranging from 3.39 to 8.28 µM. The three hits showed dose-dependent binding to PLpro in the thermal shift assay. In addition, tropifexor inhibited the cellular PLpro activity in the FlipGFP assay with an IC50 of 10.6 µM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells with an EC50 of 4.03 µM, a 7.8-fold increase compared to GRL0617 (EC50 = 31.4 µM). Overall, tropifexor represents a novel PLpro inhibitor that can be further developed as SARS-CoV-2 antivirals.

The etiological agent of COVID-19 is SARS-CoV-2, a single-stranded, positive-sense RNA virus that belong to the β -coronavirus genera. Given the catastrophic impact of COVID-19 on public health and global economy, researchers around of the globe are working relentlessly to develop vaccines and antiviral drugs. This effort led to the approval of vaccines and antiviral drugs in record breaking speed. Two mRNA vaccines from Moderna and Pfizer, and one adenovirus vaccine from Johnson and Johnson were approved by FDA. 1 Although vaccines are the mainstay in combating the pandemic, antiviral drugs are nevertheless needed as complementary strategies. Vaccines are preventative, while antiviral drugs can be used for the treatment of COVID patients. In addition, the mRNA vaccines target the viral spike protein, which is prone to mutation as shown by the variants of concerns including the Delta variant and the most recent Omicron variant. 2 As a result, vaccines might need to be frequently updated to match the circulating strains. In comparison, small molecule antiviral drugs targeting the conserved viral proteins are expected to have broad-spectrum antiviral activity and a high genetic barrier to drug resistance. The viral RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir is the first FDA-approved COVID drug. 3 In addition, the second RdRp inhibitor molnupirivir [4] [5] [6] and the main protease (M pro ) inhibitor PF-07321332 (Paxlovid) 7 are likely to become the first oral COVID drugs.

Despite the encouraging progress, additional antiviral drugs with a novel mechanism of action are still in dire need to override the emergence of new mutations. They can be used either alone or in combination with existing RdRp inhibitors or M pro inhibitors to combat not only current COVID-19 pandemic, but also future coronavirus outbreaks. SARS-CoV-2 expresses two viral proteases, the M pro and papain-like protease (PL pro ), during viral replication. Both M pro and PL pro are cysteine proteases that mediate the cleavage of viral polyprotein during viral replication. 8 In addition, PL pro desregulates the host immune responses by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. 9 Therefore, inhibiting PL pro is a two-pronged approach in protecting host cells from viral infection.

PL pro is a 35-KDa domain of Nsp3, 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, majority of reported PL pro inhibitors are non-covalent inhibitors that bind to the S3 and S4 subsites that are located more than 10 Å away from the catalytic cysteine C111. [13] [14] [15] Second, PL pro bears structural similarities to human deubiquitinases and delSGylases, 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 analogs 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. Their mechanism of action was further characterized in the thermal shift binding 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.

Using the previously optimized FRET assay condition, 15 we performed a highthroughput 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. 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 broadspectrum 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 counter screened against the SARS-CoV-2 M pro to rule out promiscuous cysteine protease inhibitors. [19] [20] [21] [22] 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 the inhibition of SARS-CoV-2 PL pro by tropifexor is specific. The inhibition of PL pro 's deubiquitination and deISGlyation activities were 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 ). 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. Next, we tested the three hits in the FlipGFP PL pro assay. 15, [19] [20] 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 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, 24-25 the β 1-9 template, and the β 10-11 strands that are constrained in the parallel inactive conformation through a PL pro substrate linker. Upon cleavage of the substrate linker, the β 10 and β 11 strands become parallel and can associate with the β 1-9 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 advantages of FlipGFP assay compared to 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. [19] [20] 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 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) . In summary, the FlipGFP assay results suggest tropifexor might have antiviral activity against SARS-CoV-2. Values represent the average ± standard deviation of three replicates.

The antiviral activity of EACC, KY-226, and tropifexor in inhibiting SARS-CoV-2 replication in Calu-3 cells was tested using the immunofluorescence assay ( Figure 4 ).

Calu-3 is TMPRSS2-positive and is a close mimetic of the human respiratory epithelial cells, 26 

To gain insights of the binding mode of the three hits, we performed molecular docking with Schrödinger Glide using the wild-type SARS-CoV-2 PL pro structure we recently solved (PDB: 7JRN). 15 EACC, KY-226 and tropifexor all fit snuggly into the U-shape binding pocket that is covered by the BL2 loop where GRL0617 binds ( Figure 5A ). EACC forms two hydrogen bonds with PL pro , one from the EACC carbonyl with the Gln269 main chain amide NH, and another from the EACC imide NH with the Asp164 side chain carboxylate ( Figure 5B ). In addition, the nitro-thienyl ring forms a π -π interaction with the Tyr268 side chain phenol. For KY-226, the benzene ring in the biphenyl substitution similarly forms a π -π interaction with the Tyr268 side chain phenol.

The sulfone from KY-226 forms a hydrogen bond with the Arg166 side chain guanidine NH ( Figure 5C ). The carboxylate from tropifexor forms an ionic bond with the Lys157 side chain ammonium ( Figure 5D ). 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 analogs 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, 28 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). 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. 29 In addition to the inhibition of PL pro mediated cleavage of viral polyprotein substrate, tropifexor also inhibited the deubiquitination and deISGlation activities of SARS-CoV-2 PL pro .

Consistent with the enzymatic inhibition, tropifexor showed dose-dependent stabilization of SARS-CoV-2 PL pro in the thermal shift binding 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 = 6.97) 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.

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 mM NaCl, 0.4 mM EDTA, 20% glycerol, and 4 mM DTT as described previously. 30, 32 Differential Scanning Fluorimetry (DSF). The thermal shift binding assay (TSA) was carried out using a Thermo Fisher QuantStudio 5 Real-Time PCR system as described previously. 15 

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