key: cord-0915621-mumgdy70
authors: Ma, Chunlong; Tan, Haozhou; Choza, Juliana; Wang, Yuying; Wang, Jun
title: Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays
date: 2021-11-01
journal: Acta Pharm Sin B
DOI: 10.1016/j.apsb.2021.10.026
sha: 06d2b9d21d5c51416ce77352c4efa4efbc96f238
doc_id: 915621
cord_uid: mumgdy70

SARS-CoV-2 main protease (M(pro)) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as M(pro) inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed M(pro) inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the M(pro) inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to M(pro), while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit M(pro) in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.

cleavage of viral polyproteins pp1a and pp1ab during viral replication [2] [3] [4] . M pro cleaves at more than 11 sites at the viral polyproteins and has a high substrate preference for glutamine at the P1 site 5 . In addition, the M pro is highly conserved among coronaviruses that infect human including SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-229E, and HCoV-HKU1. For these reasons, M pro becomes a high-profile drug target for the development of broadspectrum antivirals. Structurally disparate compounds including FDA-approved drugs and bioactive compounds have been reported as M pro inhibitors [6] [7] [8] [9] [10] , several of which also have in vivo antiviral efficacy against SARS-CoV-2 [11] [12] [13] [14] [15] .

FRET assay is the gold standard assay for protease and is typically used as a primary assay for the screening of M pro inhibitors. However, the FRET assay conditions used by different groups vary significantly in terms of the protein and substrate concentrations, pH, reducing reagent, and detergent. Reducing reagent is typically added in the assay buffer to prevent the non-specific oxidation or alkylation of the catalytic C145 in M pro . Nonetheless, many studies do not include reducing reagents in the FRET assay buffer, leading to debatable results 16 .

Regardless of the assay condition, FRET assay is a cell free biochemical assay, which does not mimic the cellular environment; therefore, the results cannot be used to accurately predict the cellular activity of the M pro inhibitor or the antiviral activity. Moreover, one limiting factor for M pro inhibitor development is that the cellular activity has to be tested against infectious SARS-CoV-2 in BSL-3 facility, which is inaccessible to many researchers. For these reasons, there is a pressing need of secondary M pro target-specific assays that can closely mimic the cellular environment and be used to rule out false positives.

In this study, we report our findings of validating or invalidating the literature reported M pro inhibitors using the cell lysate Protease-Glo luciferase assay and the cell-based FlipGFP assay, in conjunction to the cell-free FRET assay and thermal shift-binding assay. The purpose is to elucidate their target specificity and cellular target engagement. The Protease-Glo luciferase assay was developed in this study, and the FlipGFP assay was recently developed by us and results presented herein highlight the pressing need of stringent hit validation at the early stage of drug discovery to minimize the catastrophic failure in the following translational development.

The tag-free SARS CoV-2 M pro protein with native N-and C-termini was expressed in pSUMO construct as described previously 3 .

The FRET-based protease was performed as described previously 2 

Direct binding of testing compounds to SARS CoV-2 M pro protein was evaluated by differential scanning fluorimetry (DSF) using a Thermal Fisher QuantStudio 5 Real-Time PCR System as previously described 2 . Briefly, SARS CoV-2 M pro protein was diluted into reaction buffer to a final concentration of 3 μmol/L and incubated with 40 µmol/L of testing compounds at 30 Three hours after transfection, 1 μL of testing compound was directly added to each well without medium change. Two days after transfection, images were taken with Cytation 5 imaging reader (Biotek) using GFP and mCherry channels via 10× objective lens and were analyzed with Gen5 3.10 software (Biotek).

The mCherry signal alone in the presence of testing compounds was utilized to evaluate the compound cytotoxicity. 

The advantages and disadvantages of the cell lysate Protease-Glo luciferase assay and the cell-based

FlipGFP assay compared to the cell free FRET assay are listed in Table 1 . To minimize the bias from a particular assay, we apply all these three functional assays together with the thermal shift-binding assay for the hit validation.

Insert Table 1 In the cell-based FlipGFP assay, the cells were transfected with two plasmids, one expresses the SARS-CoV-2 M pro , and another expresses the GFP reporter 22 . The GFP reporter plasmid expresses three proteins including the GFP β10-β11 fragment flanked by the K5/E5 coiled coil, the GFP β1-9 template, and the mCherry (Fig. 1A) . mCherry serves as an internal control for the normalization of the expression level or the quantification of compound toxicity. In the assay design, β10 and β11 were conformationally constrained in the parallel position by the heterodimerizing K5/E5 coiled coil with a M pro cleavage sequence (AVLQ↓SGFR). Upon cleavage of the linker by M pro , β10 and β11 become antiparallel and can associate with the β1-9 template, resulting in the restoration of the GFP signal. In principle, the ratio of GFP/mCherry fluorescence is proportional to the enzymatic activity of M pro . The FlipGFP M pro assay has been used by several groups to characterize the cellular activity of M pro inhibitors 17, 19, 20 .

In the cell lysate Protease-Glo luciferase assay, the cells were transfected with pGloSensor-30F luciferase reporter (Fig. 1B) 23 . The pGloSensor-30F luciferase reporter plasmid expresses two proteins, the inactive, circularly permuted firefly luciferase (FFluc) and the active Renilla luciferase (Rluc). Renilla luciferase was included as an internal control to normalize the protein expression level. The firefly luciferase was split into two fragments, the Table 2 ). The discrepancy indicates that the mechanism of action of rupintrivir might be independent of M pro inhibition.

Overall, the FlipGFP and Protease-Glo luciferase assays are validated as target-specific assays for SARS-CoV-2 M pro .

Insert Fig. 1 and Table 2 3

The HCV protease inhibitors have been proven a rich source of SARS-CoV-2 M pro inhibitors 2,28,29 . From screening a focused protease library using the FRET assay, we discovered simeprevir, boceprevir, and In the current study, we found that boceprevir showed moderate inhibition in the cellular FlipGFP M pro assay with an IC 50 of 18.33 µmol/L ( Fig. 2A and B) , a more than 4-fold increase compared to the IC 50 in the FRET assay (4.13 µmol/L). The IC 50 value of boceprevir in the cell lysate Protease-Glo luciferase assay was 4.49 µmol/L (Fig. 2E ). In comparison, telaprevir and narlaprevir showed weaker inhibition than boceprevir in both the FlipGFP and Protease-Glo luciferase assays ( Fig. 2A , C, D, F, and G), which is consistent with their weaker potency in the FRET assay (Table 2) . Overall, boceprevir, telaprevir, and narlaprevir have been validated as SARS-CoV-2 M pro inhibitors in both the cellular FlipGFP assay and the cell lysate Protease-Glo luciferase assay. Therefore, the antiviral activity of these three compounds against SARS-CoV-2 are likely due to M pro inhibition. Although the inhibition of M pro by boceprevir is relatively weak compared to GC-376, several highly potent M pro inhibitors were subsequently designed as hybrids of boceprevir and GC-376 11,17,31 including the Pfizer oral drug candidate PF-07321332, which contain the dimethylcyclopropylproline at the P2 substitution.

Insert Fig. 2 

HIV protease inhibitors, especially Kaletra, have been hotly pursued as potential COVID-19 treatment at the beginning of the pandemic. Kaletra was first tested in clinical trial during the SARS-CoV outbreak in 2003 and showed somewhat promising results based on the limited data 49 . However, a double-blinded, randomized trial concluded that Kaletra was not effective in treating severe COVID-19 50, 51 . In SARS-CoV-2 infection ferret models, Kaletra showed marginal effect in reducing clinical symptoms, while had no effect on virus titers 52 .

Keletra is a combination of lopinavir and ritonavir. Lopinavir is a HIV protease inhibitor, and ritonavir is used as a booster. Ritonavir does not inhibit the HIV protease and it is a cytochrome P450-3A4 inhibitor 53 . When used in combination, ritonavir can enhance other protease inhibitors by preventing or slowing down the metabolism. In cell culture, lopinavir was reported to inhibit the nanoluciferase SARS-CoV-2 reporter virus with an EC 50 of 9 µmol/L 24 . In two other studies, lopinavir showed moderate antiviral activity against SARS-CoV-2 activity with EC 50 values of 19 ± 8 µmol/L 34 and 25 µmol/L 35 . As such, it was assumed that lopinavir inhibited SARS-CoV-2 through inhibiting the M pro . However, lopinavir showed no activity against SARS-CoV-2 M pro in the FRET assay from our previous study (IC 50 > 60 µmol/L) 2 . Wu et al. 54 also showed that lopinavir was a weak inhibitor against SARS-CoV M pro with an IC 50 of 50 µmol/L. In the current study, we further confirmed the lack of binding of lopinavir to SARS-CoV-2 M pro in the thermal shift assay (ΔT m = -0.60 ºC, Table 2 ). The result from the FlipGFP assay was J o u r n a l P r e -p r o o f not conclusive as lopinavir was cytotoxic. Lopinavir was not active in the Protease-Glo luciferase assay.

Taken together, lopinavir is not a M pro inhibitor.

Ritonavir was not active in both the FlipGFP Mpro and the Protease-Glo luciferase assays, which is consistent with the lack of activity in the FRE assay and the thermal shift binding binding assay (Table 2) .

Therefore, ritonavir is not a M pro inhibitor.

We also tested additional HIV antivirals including atazanavir, nelfinavir, and cobicistat. Atazanavir and nelfinavir were reported as a potent SARS-CoV-2 antiviral with EC 50 values of 2.0 ± 0.12 38 Table 2 ). The results of atazanavir, nelfinavir, and cobicistat from the FlipGFP assay were not conclusive due to compound cytotoxicity.

None of the compounds showed inhibition in the Protease-Glo luciferase assay.

Collectively, our results have shown that the HIV protease inhibitors including lopinavir, ritonavir, atazanavir, nelfinavir, and cobicistat are not M pro inhibitors. Nonetheless, given the potent antiviral activity of lopinavir, atazanavir, nelfinavir, and cobicistat against SARS-CoV-2, it might worth to conduct resistance selection to elucidate their drug target(s). Fig. 3 

Several bioactive compounds have been identified as SARS-CoV-2 M pro inhibitors through either virtual screening or FRET-based HTS. We are interested in validating these hits using the FlipGFP and the Protease-Glo luciferase assays.

Manidipine was identified as a SARS-CoV-2 M pro inhibitor from a virtual screening and was subsequently shown to inhibit M pro with an IC 50 of 4.81 µmol/L in the FRET assay 40 . No antiviral data was provided. When we repeated the FRET assay, the IC 50 was 64.2 µmol/L ( Table 2) . Manidipine also did not show binding to M pro in the thermal shift assay. Furthermore, manidipine showed no activity in either the FlipGFP assay or the Protease-Glo luciferase assay (Fig. 4A, B, and F) . Therefore, our results invalidated manidipine as a SARS-CoV-2 M pro inhibitor. A recent study independently confirmed our results and suggested that manidipine might form colloidal aggregates 55 These results suggest that the FlipGFP assay can be used to faithfully predict the antiviral activity of M pro inhibitors. The lower activity of calpain inhibitors II and XII in the FlipGFP assay and the Calu-3 antiviral assay might due to the competition with host proteases, resulting in the lack of cellular target engagement with M pro .

In conclusion, calpain inhibitors II and XII are validated as M pro inhibitors but their antiviral activity against SARS-CoV-2 is cell type dependent. Accordingly, TMPRSS2 positive cell lines such as Calu-3

should be used to test the antiviral activity of calpain inhibitors II and XII analogs.

Ebselen is among one of the most frequently reported promiscuous M pro inhibitors. It was first reported by Jin et al. 16 that ebselen inhibits SARS-CoV-2 M pro with an IC 50 of 0.67 µmol/L and the SARS-CoV-2 replication with an EC 50 of 4.67 µmol/L. However, it was noted that no reducing reagent was added in the FRET assay, and we reasoned that the observed inhibition might be due to non-specific J o u r n a l P r e -p r o o f modification of the catalytic cysteine 145 by ebselen. To test this hypothesis, we repeated the FRET assay with and without reducing reagent DTT or GSH, and found that ebselen completely lost the M pro inhibition in the presence of DTT or GSH 41 . Similarly, ebselen also non-specifically inhibited several other viral cysteine proteases in the absence of DTT including SARS-CoV-2 PL pro , EV-D68 2A pro and 3C pro , and EV-A71 2A pro and 3C pro41 . The inhibition was abolished with the addition of DTT. Ebselen also had no antiviral activity against EV-A71 and EV-D68, suggesting that the FRET assay results obtained without reducing reagent cannot be used to predict the antiviral activity. In this study, we found that ebselen showed no inhibition in either the FlipGFP assay or the Protease-Glo luciferase assay (Fig. 4A , E, and I), providing further evidence for the promiscuous mechanism of action of ebselen. Another independent study by Gurard-Levin et al. 58 using mass spectrometry assay reached similar conclusion that the inhibition of M pro by ebselen is non-specific and inhibition was abolished with the addition of reducing reagent DTT or glutathione. In contrary to the potent antiviral activity reported by Jin et al. 16 , the study from Gurard-Levin et al. 58 found that ebselen was inactive against SARS-CoV-2 in Vero E6 cells (EC 50 > 100 µmol/L). Chen et al. 59 reported that ebselen and disulfiram had synergistic antiviral effect with remdesivir against SARS-CoV-2 in Vero E6 cells. It was also proposed that ebselen and disulfiram act as zinc ejectors and inhibited not only the PL pro60 , but also the nsp13 ATPase and nsp14 exoribonuclease activities 59 , further casting doubt on the detailed mechanism of action of ebselen.

Despite the accumulating evidence to support the promiscuous mechanism of action of ebselen, several studies continue to explore ebselen and its analogs as SARS-CoV-2 M pro and PL pro inhibitors 42 nearly identical complex structures. It was found that selenium coordinates directly to Cys145 and forms a S-Se bond 42 . Accordingly, a mechanism involving hydrolysis of the organoselenium compounds was

proposed. Similar to their previous study, the M pro enzymatic reaction buffer (50 mmol/L Tris pH 7.3, 1 mmol/L EDTA) did not include the reducing reagent DTT. Therefore, the M pro inhibition by these ebselen analogs might be non-specific and the antiviral activity might arise from other mechanisms 42 .

Overall, it can be concluded that ebselen is not a specific M pro inhibitor, and its antiviral activity against SARS-CoV-2 might involve other drug targets such as nsp13 or nsp14.

Disulfiram is an FDA-approved drug for alcohol aversion therapy. Disulfiram has a polypharmacology and was reported to inhibit multiple enzymes including urease 63 µM. Follow up studies by us and others showed that disulfiram did not inhibit M pro in the presence of DTT or GSH 26, 41 . In this study, disulfiram had no inhibition against M pro in either the FlipGFP assay or the Protease-Glo luciferase assay (Fig. 5A, B, N) .

Similar to disulfiram, carmofur, PX-12 and tideglusib, which were previously claimed by Ma et al. 41 as M pro inhibitors, showed no inhibitory activity in either the FlipGFP or Protease-Glo luciferase assay ( Fig. 5A , C, D, E, O-Q), which is consistent with their lack of inhibition in the FRET assay in the presence of DTT.

Shikonin and baicalein are polyphenol natural products with known polypharmacology. Both compounds showed no inhibition in either the FlipGFP or the Protease-Glo luciferase assay (Fig. 5A , F, G, R, and S), suggesting they are not M pro inhibitors. These two compounds were previously reported to inhibit SARS-CoV-2 M pro in the FRET assay 16 and had antiviral activity against SARS-CoV-2 in Vero E6 cells. However, our recent study showed that shikonin had no inhibition against SARS-CoV-2 M pro in the FRET assay in the presence of DTT 41 . Studies from Gurard-Levin et al. 26 using FRET assay and mass spectrometry assay reached the same conclusion. X-ray crystal structure of SARS-CoV-2 M pro in complex with shikonin showed that shikonin binds to the active site in a non-covalent manner 44 .

In addition to the proposed mechanism of action of M pro inhibition, Zandi et al. 66 

The M pro is perhaps the most extensive exploited drug target for SARS-CoV-2. A variety of drug discovery techniques have been applied to search for M pro inhibitors. Researchers around the world are racing to share their findings with the scientific community to expedite the drug discovery process.

However, the quality of science should not be compromised by the speed. The mechanism of action of drug candidates should be thoroughly characterized in biochemical, binding, and cellular assays.

Pharmacological characterization should address both target specificity and cellular target engagement. compounds that non-specifically inhibit multiple unrelated viral or host cysteine proteases are most likely promiscuous inhibitors that should be triaged. 5) X-ray crystal structures cannot be used to justify the target specificity or cellular target engagement. Promiscuous compounds have been frequently cocrystallized with M pro including ebselen, carmofur, and shikonin (Table 2) . Overall, we hope our studies will promote the awareness of the promiscuous SARS-CoV-2 M pro inhibitors among the scientific community and call for more stringent hit validation. J o u r n a l P r e -p r o o f

Characteristics of SARS-CoV-2 and COVID-19

GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M pro and cathepsin L

Crystal structure of SARS-CoV-2 papain-like protease

SARS-CoV-2 M pro inhibitors and activity-based probes for patient-sample imaging

Drug development and medicinal chemistry efforts toward SARS-coronavirus and Covid-19 therapeutics

The SARS-CoV-2 main protease as drug target

Bepridil is potent against SARS-CoV-2 in vitro

Recent advances in developing smallmolecule inhibitors against SARS-CoV-2

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19. medRxiv 2021

Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2

Efficacy of GC-376 against SARS-CoV-2 virus infection in the K18 hACE2 transgenic mouse model

Postinfection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection

The preclinical inhibitor GS441524 in combination with GC376 efficaciously inhibited the proliferation of SARS-CoV-2 in the mouse respiratory tract

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

Rational design of hybrid SARS-CoV-2 main protease inhibitors guided by the superimposed cocrystal structures with the peptidomimetic inhibitors GC-376, telaprevir, and boceprevir

Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and a FlipGFP-based reporter assay

Development of a fluorescence-based, high-throughput SARS-CoV-2 3CL pro reporter assay

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models

Designing a green fluorogenic protease reporter by flipping a beta strand of GFP for imaging apoptosis in animals

A novel bioluminescent protease assay using engineered firefly luciferase

A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-infective drugs for COVID-19

Both boceprevir and GC376 efficaciously inhibit SARS-CoV-2 by targeting its main protease

Evaluation of SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption ionization mass spectrometry

Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication

Hepatitis C virus drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to suppress viral replication in cell culture

Malleability of the SARS-CoV-2 3CL M pro active-site cavity facilitates binding of clinical antivirals

Identification of existing pharmaceuticals and herbal medicines as inhibitors of SARS-CoV-2 infection

SARS-CoV-2 M pro inhibitors with antiviral activity in a transgenic mouse model

Direct observation of protonation state modulation in SARS-CoV-2 main protease upon inhibitor binding with neutron crystallography

Structural basis for the inhibition of the SARS-CoV-2 main protease by the anti-HCV drug narlaprevir

Remdesivir, lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro

chloroquine, montelukast sodium, candesartan, oxytetracycline, and atazanavir are not SARS-CoV-2 main protease inhibitors

Identify potent SARS-CoV-2 main protease inhibitors via accelerated free energy perturbation-based virtual screening of existing drugs

Atazanavir, alone or in combination with ritonavir, inhibits SARS-CoV-2 replication and proinflammatory cytokine production

Structure-based virtual screening and biochemical validation to discover a potential inhibitor of the SARS-CoV-2 main protease

Identification of 14 known drugs as inhibitors of the main protease of SARS-CoV-2

Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are nonspecific promiscuous SARS-CoV-2 main protease inhibitors

Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives

Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug carmofur

Crystal structure of SARS-CoV-2 main protease in complex with the natural product inhibitor shikonin illuminates a unique binding mode

Cellular Activities of SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics. bioRxiv 2021

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro

Role of lopinavir/ritonavir in the treatment of SARS: initial virological and clinical findings

A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19

Lopinavir-ritonavir in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Antiviral efficacies of FDAapproved drugs against SARS-CoV-2 infection in Ferrets

Pharmacological and therapeutic properties of ritonavir-boosted protease inhibitor therapy in HIV-infected patients

Small molecules targeting severe acute respiratory syndrome human coronavirus

Colloidal aggregators in biochemical SARS-CoV-2 repurposing screens

calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Evaluation of SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption ionization mass spectrometry

Synergistic inhibition of SARS-CoV-2 replication using disulfiram/ebselen and remdesivir

Multi-targeting of functional cysteines in multiple conserved SARS-CoV-2 domains by clinically safe Zn-ejectors

Identification of ebselen and its analogues as potent covalent inhibitors of papain-like protease from SARS-CoV-2

Ebsulfur and ebselen as highly potent scaffolds for the development of potential SARS-CoV-2 antivirals

Structural basis for inactivation of Giardia lamblia carbamate kinase by disulfiram

Disulfiram is a direct and potent inhibitor of human O 6 -methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damage

Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes

Baicalein and baicalin inhibit SARS-CoV-2 RNA-dependent-RNA polymerase

Identify potent SARS-CoV-2 main protease inhibitors via accelerated free energy perturbation-based virtual screening of existing drugs

protease inhibitors by a quantitative high-throughput screening