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

The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLpro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PLpro inhibitors with IC50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50 values ranging from 0.56 to 0.90 μM. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PLpro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLpro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50 values of 8.89 and 8.32 μM, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 μM). The X-ray crystal structures of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PLpro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLpro assay might be a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.


INTRODUCTION
The COVID-19 pandemic has led to 120,105,958 confirmed cases and 2,657,629 deaths as of March 15, 2021, rendering it the worst pandemic since the 1918 Spanish flu. The etiological agent of COVID-19 is SARS-CoV-2, a single-stranded positive sense RNA virus that belongs to the beta coronavirus family. Two additional coronaviruses within the same family, the SARS-CoV, and MERS-CoV, have caused epidemics in humans with mortality rates of 9.6% and 34.3%, respectively. Although SARS-CoV-2 has a lower mortality rate of 2.2% compared to SARS-CoV and MERS-CoV, it has led to far greater death tolls due to its higher transmission. SARS-CoV-2 differs from SARS-CoV and MERS-CoV in that it has a long incubation time after the initial infection (~ 2 weeks), and a large percentage of infected patients continue to shed the virus while being asymptomatic, presenting a daunting task for surveillance and containment. among humans since the beginning of the pandemic. 2 Therefore, there is a dire need of additional antivirals with a novel mechanism of action. Antivirals are not substituents of vaccines, but rather an important complement to help combat the pandemic. Among the viral proteins that have been actively pursued as SARS-CoV-2 antiviral drug targets, the main protease (M pro ) and papain-like protease (PL pro ) are the most promising ones. 3,4 Both M pro and PL pro are involved in the proteolytic digestion of the viral polyproteins . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted  https://doi.org/10.1101/2021.03.15.435551 doi: bioRxiv preprint pp1a and pp1ab, yielding individual functional viral proteins for the replication complex formation. PL pro cleaves at three sites with the recognition sequence "LXGG XX". 5 PL pro has been shown to play additional roles in dysregulating host immune response and impairing the host type I interferon antiviral effect through its deubiquitinating and deISG15ylating (interferon-induced gene 15) activities, respectively. 6-8 SARS-CoV-2 PL pro cleaves ISG15 and polyubiquitin modifications from cellular proteins, and inhibition of PL pro led to the accumulation of ISG15-conjugates and poly-ubiquitin-conjugates. 9 While SARS-CoV PL pro prefers ubiquitinated substrates, the SARS-CoV-2 PL pro prefers the ISGlyated proteins as substrates. [6][7][8] PL pro is part of a membrane anchored multidomain protein named non-structural protein 3 (nsp-3), an essential component of the replicase-transcriptase complex. The multi-functional roles of SARS-CoV-2 PL pro render it a prominent antiviral drug target. A substantial morbidity and mortality associated with COVID-19 infection is caused by cytokine storm, 10 and suppressing host immune response using dexamethasone and baricitinib has been shown to provide therapeutic benefits in the treatment of severe infections.
A significant progress has been made in developing SARS-CoV-2 M pro inhibitors 3,4,[11][12][13] and the Pfizer compound PF-07304814 currently in phase 1 clinical trial. 14 In comparison, PL pro represents a more challenging drug target, and GRL0617 remains one of the most potent PL pro inhibitors reported to date despite several highthroughput screening and medicinal chemistry optimization campaigns. 8,15 GRL0617 was originally developed against SARS-CoV PL pro . 16 As SARS-CoV-2 and SARS-CoV PL pro share a sequence identity of 83% and similarity of 90%, GRL0617 was also repurposed for SARS-CoV-2 PL pro and it was reported to inhibit SARS-CoV-2 PL pro with . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  IC 50 values of around 2 µM and the SARS-CoV-2 viral replication with EC 50 values   around 20 µM from multiple studies. 8,9,15,17 In this study, we report our progress in developing novel SARS-CoV-2 PL pro inhibitors. Using the FRET-based enzymatic assay, we conducted a high-throughput screening against the Enamine 50K diversity compound library and identified two hits Jun9-13-7 and Jun9-13-9 with single-digit micromolar IC 50 values. Upon validating the hits using differential scanning fluorimetry (DSF) and native mass spectrometry binding assays, a focused library of structural analogs were designed and tested to dissect the structure-activity relationship (SAR) studies. Several compounds have been identified to inhibit SARS-CoV-2 PL pro with submicromolar potency. To prioritize lead compounds for the antiviral assay against SARS-CoV-2, we developed the FlipGFP assay that is suitable for quantifying the intracellular activity of PL pro inhibitors in the BSL2 setting.
Two compounds selected from the FlipGFP PL pro assay, Jun9-53-2 and Jun9-72-2, showed 3-fold improvement than GRL0617 in the cellular antiviral assay against SARS-CoV-2. X-ray crystal structures showed that binding of GRL0617 to the wild-type (WT) SARS-CoV-2 PL pro induced a conformational change in the BL2 loop to the more closed conformation. Overall, the SARS-CoV-2 PL pro reported herein represent promising hits for further development as SARS-CoV-2 antivirals, and FlipGFP PL pro assay might be useful in testing the cellular activity of PL pro inhibitors in the BSL-2 setting.

RESULTS AND DISCUSSION
Expression and characterization of SARS-CoV-2 PL pro .
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Two constructs of SARS-CoV-2 PL pro were expressed in E. Coli, one with Hig-tag (PL pro -His) and another without the tag (PL pro ). To profile the proteolytic activity of PL pro in cleaving the viral polyprotein, we developed a FRET based enzymatic assay with the peptide substrate 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl)-FTLRGG/APTKV-5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (Edans), which corresponds to the nsp2-nsp3 junction from the SARS-CoV-2 polyprotein. The enzymatic activity k cat /K m of PL pro -His and PL pro were 340 M -1 S -1 and 255 M -1 S -1 (Table   S1), respectively, which were similar to previous reports, 8,17 suggesting both constructs were enzymatically active. The SARS-CoV-2 PL pro was also reported to have deubiquitinating and deISGylating activities. Accordingly, we characterized the deubiquitinating and deISGylating activities of SARS-CoV-2 PL pro using the Ub-AMC and ISG-AMC substrates, respectively, in the enzymatic assay. It was found that the SARS-CoV-2 PL pro is more efficient in cleaving the ubiquitin (Ub) and ISG15 (ISG) modifications than the viral polyprotein, with k cat /K m values of 1,070 and 1.67x10 5 M -1 S -1 (Table S2), respectively. This substrate preference is in agreement with results reported by Gal et al, 17 and SARS-CoV PL pro was also reported to have similar substrate preference. 18 Significantly, the deISGylating activity is 156-fold higher than the deubiquitinating activity, which is consistent with previous reports that the SARS-CoV-2 PL pro prefers ISG15 over ubiquitin as a substrate. [5][6][7][8][9] High-throughput screening of the Enamine 50K diversity library against the SARS-CoV-2 PL pro and hit validation.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The HTS assay was optimized in 384-well plates using the FRET substrate, which gave a Z' factor of 0.668 with a signal to noise ratio (S/B) of 11.2, indicating that this was a robust assay (Fig. 1). We then performed the HTS against the enamine library, which consists of 50,240 structurally diverse compounds. GRL0617 was included as a positive control. Fig. 1. 384-well high-throughput screening assay with SARS CoV-2 PL pro . In this representative control plate (column 1 to 20 with PL pro protein, column 21 to 24 with buffer only; 1 µl DMSO was added to column 1 to 10 and 21 to 24; 1 µl GRL0617 was added to column 11 to 20), the signal to base ratio (S/B) is 11.2, and the calculated Z' factor is 0.688.
Hits showing more than 50% inhibition were repurchased from Enamine and titrated in the FRET-based enzymatic assay to determine the IC 50 values ( Fig. 2A and Table   S3). In parallel, differential scanning fluorimetry (DSF) assay was performed as a secondary assay to characterize the binding of the hits with SARS-CoV-2 PL pro (Fig. 2B d . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Table S3). The most potent two hits Jun9-13-7 and Jun9-13-9 ( Fig. 2C) had IC 50 values of 7.29 ± 1.03 and 6.67 ± 0.05 µM, respectively. Compounds Jun9-13-7 and Jun9-13-9 also increased the thermal stability of SARS-CoV-2 PL pro by 2.98 ± 0.09 and 2.18 ± 0.29 o C (Table S3), which is consistent with their enzymatic inhibition. In comparison, GRL0617 had an IC 50 value of 2.05 ± 0.12 µM, and increased the protein stability by 3.52 ± 0.27 o C in the DSF assay (Table S3). The potency of GRL0617 in inhibiting SARS-CoV-2 PL pro from our study is consistent with recent reports. [6][7][8][9] The rest of the hits had weak enzymatic inhibition (IC 50 > 10 µM) and showed marginal binding to PL pro , therefore they were not further pursued (Table S3). Both compound Jun9-13-7 and Jun9-13-9 also inhibit the deubiquitinating and deISGylating activities with IC 50 values ranging from 4.93 to 12.5 µM ( Fig. 2D and Table S4). In contrast, neither of these two compounds inhibited the SARS-CoV-2 M pro at up to 200 µM ( fig. S2), suggesting the inhibition of SARS-CoV-2 PL pro by these two compounds are specific.
The binding of Jun9-13-7 and Jun9-13-9 to SARS-CoV-2 PL pro was further characterized using the native mass spectrometry (Fig. 2E). It was shown that both . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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Development of FlipGFP assay for testing the cellular activity of SARS-CoV-2 PL pro inhibitors.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made One of the challenges in SARS-CoV-2 antiviral drug discovery is that SARS-CoV-2 is a biosafety level 3 (BSL3) pathogen, which limits the number of drug candidates that can be screened. To help prioritize lead compounds for the antiviral assay with infectious SARS-CoV-2, we developed a cell-based FlipGFP assay for SARS-CoV-2 PL pro that is suitable for testing the intracellular activity of PL pro inhibitors in the BSL2 setting. The advantages of cell-based PL pro assay over the FRET-based enzymatic assay include but not limited to: 1) it can eliminate compounds that either cytotoxic or membrane impermeable; 2) In contrast to the standard FRET-based assay, PL pro cleaves the substrate in the cell cytoplasm in the FlipGFP assay, which is a close mimetic of the viral polyprotein cleavage by PL pro in virus-infected cell. It is known that cysteine proteases are susceptible to re-dox active compounds as well as non-specific alkylating chemicals such as ebselen. 19,20 The FlipGFP PL pro assay is expected to rule out such promiscuous compounds as the substrate is cleaved under the reducing intracellular environment.
In the assay design, the 10th and 11th β -strands from the GFP protein were separated from the rest of the GFP β -barrel (β-strands 1-9) (Fig. 4A). 21,22 The 10 th and 11 th β -strands were linked through the PL pro cleavage site and a heterodimerized coiled coils E5/K5. In the absence of the PL pro , the 10 th and 11 th β -strands are conformationally restrained and unable to associate with the GFP β -barrel 1-9. When the cleavage site is digested by the PL pro , the 11 th β -strand can then flip its orientation and associate with GFP β -barrel 1-9 together with the 10 th β -strand, leading to restoration of the green fluorescence signal (Fig. 4A). A red fluorescent protein mCherry was included within the construct via a "self-cleaving" 2A peptide to act as the transfection control (Fig. 4B), and . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made With the established assay condition, we then screened nine most potent PL pro inhibitors with IC 50 values less than 1 µM from the FRET-based enzymatic assay (Fig.   3). GRL0617 and GC376 were included as positive and negative controls, respectively.
The initial hit Jun9-13-9 was also included. Compounds were added 3 h post transfection, and GFP and mCherry fluorescence signals were measured at 48 h post transfection using the Cytation 5 imager reader. A dose dependent decrease of the GFP signal was observed with increasing concentrations of GRL0617 (Fig. 4D), and quantification of the normalized GFP/mCherry ratio gave an EC 50 value of 9.29 ± 3.45 µM. In contrast, GC376 had no effect on the intensity of green fluorescence signal . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (Fig. 4E), suggesting the FlipGFP assay is suitable for the screening of PL pro inhibitors. Among the ten compounds tested in the cell-based FlipGFP assay, . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Cellular antiviral activity of PL pro inhibitors against SARS-CoV-2
Based on the FlipGFP-PL pro assay results, we selected two compounds Jun9-53-2 and Jun9-72-2 and tested their cellular antiviral activity against the infectious SARS-CoV-2 in two cell lines, the Caco-2 hACE2, and the Vero E6. The Caco-2 hACE2 cell line expresses endogenous TMPRSS2 and was transfected to overexpress hACE2, rending it a physiological relevant cell line model that is close to the human airway epithelial cells. GRL0617 was included as a positive control. It was found that all three compounds had more potent inhibition in Caco-2 hACE2 cells than the Vero E6 cells (Fig. 5). Consistent with the enzymatic inhibition results, Jun9-53-2 and Jun9-72-2 had more potent cellular antiviral activity than GRL0617 in the two cell lines tested. The . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

X-ray crystal structures of SARS-CoV-2 PL pro in complex with GRL0617.
The complex structure of SARS-CoV-2 PL pro with GRL0617 was determined at 2.50 Å resolution, providing insight to its mechanism of inhibition. There are two monomers per asymmetric unit in the P2 1 space group. Unambiguous electron density reveals GRL0617 binds to the S3-S4 subpockets of the PL pro active site in a noncovalent manner (Fig. 6A)  One of the unique aspects of GRL0167 is that it does not interact with the catalytic core, but instead binds to a distal portion of the active site. Other research groups have determined complex structures of PL pro with GRL0617 with its catalytic cysteine, Cys 111, mutated to a serine, presumably to increase its propensity to crystallize (PDB IDs: 7JIR (2.1 Å) and 7CJM (3.2 Å)). 24,25 When the three structures are compared, the GRL0167 adopts a nearly identical pose. Minor differences in the side chain conformations of Glu167 and Gln269 are observed. However, there is a significant difference in the pose of Leu162 between the WT and the C111S mutants (Fig. 6D). In our WT structure, Leu162 inserts into the core of the protein, where it maintains an interatomic distance of 3.4 Å with the catalytic cysteine. In contrast, Leu162 of both C111S structures flip outwards, towards the solvent. In the higher resolution structure (PDB ID 7JIR), an acetate from the crystallization condition is modelled in the active site.
When superimposed with our WT structure, this acetate clashes with Cys111 (closest distance 2.5 Å) and Leu162 (3.0 Å). In the lower-resolution C111S mutant complexed . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made with GRL0617 (PDB ID 7CJM), no acetate is modelled, but Leu162 adopts the same conformation as the higher-resolution C111S structure (PDB ID 7JIR). Further inspection of the 2F o -F c map of 7JCM reveals there is unmodeled density corresponding to the acetate from PDB ID 7JIR. Interestingly, this experiment did not use acetate in their crystallization condition. Therefore, the density in the catalytic core of both C111S structures likely corresponds to a species of unknown identity that preferentially interacts with a serine residue.

CONCLUSION
Given the devastating impact of the COVID-19 pandemic, the SARS-CoV outbreak in 2003 was a dire warning that was gravely overlooked in retrospect. Looking forward, it is imperative that therapeutics are developed that are not only effective against SARS-CoV-2, but against future strains of similar coronaviruses. PL pro is a high profile drug target, partly because it is highly conserved between SARS-CoV and SARS-CoV-2, sharing 83% sequence similarity. Furthermore, inhibitors like GRL0617 are equally effective against both viruses, with a Ki of 0.49 µM and 0.57 µM, against SARS-CoV PL pro and SARS-CoV-2 PL pro . 26 Likewise, all critical active site residues that interact with GRL0617 are conserved, consequently, the binding poses are nearly identical ( fig.   S4). 26 These similarities would indicate that PL pro inhibitors might retain their activity against future strains of SARS coronaviruses.
Previous attempts to discover SARS-CoV-2 PL pro inhibitors through HTS by others have failed to identify hits with improved enzymatic inhibition and cellular antiviral activity. 8,9 Structural analogs of GRL0617 were also designed and synthesized, . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 16, 2021. ; https://doi.org/10.1101/2021.03.15.435551 doi: bioRxiv preprint however, none of them showed improved enzymatic inhibition. 15 Part of the reason for the difficulty in targeting SARS-CoV-2 PL pro is the lack of S1 and S2 pockets, which leaves only S3 and S4 pockets for inhibitor binding. Among the reported SARS-CoV or SARS-CoV-2 PL pro inhibitors, GRL0617 is one of the most potent compound. However, it had weak antiviral activity (EC 50 > 20 µM). In this study, we attempted to identify more potent SARS-CoV-2 PL pro inhibitors through a HTS. Based on two promising hits Jun9-13-7 and Jun9-13-9, a library of analogs was designed and synthesized, among which several compounds had submicromolar IC 50 values in the FRET-based enzymatic assay.
To alleviate the burden of relying on BSL-3 facility to test the antiviral activity of PL pro inhibitors, we developed the cell-based FlipGFP PL pro assay, which can be used to quantify the intracellular enzymatic inhibition of PL pro in a BSL-2 lab. The FlipGFP PL pro assay is a close mimetic of the virus-infected cell in which PL pro cleaves its substrate in the native intracellular reducing environment. The advantage of the FlipGFP PL pro assay overt the standard FRET-based enzymatic assay is that it can rule out compounds that are either cytotoxic or membrane impermeable or non-specifically modify the catalytic cysteine through oxidation or alkylation. GRL0617 showed an EC 50 of 9.29 µM in the FlipGFP PL pro assay, while the SARS-CoV-2 M pro inhibitor GC376 was not active (EC 50 > 60 µM), suggesting this assay was suitable for testing PL pro inhibitors. Among the ten PL pro inhibitors tested in the FlipGFP PL pro assay, five compounds Jun9-53-2, Jun9-72-2, Jun9-75-4, Jun9-85-1, and Jun9-87-1 had EC 50 values less than 10 µM. Next, two prioritized hits Jun9-53-2 and Jun9-72-2 were advanced to the cellular antiviral assay against SARS-CoV-2. Both compounds showed improved antiviral activity than . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 16, 2021. ; https://doi.org/10.1101/2021.03.15.435551 doi: bioRxiv preprint GRL0617 and the EC 50 values were consistent between the FlipGFP PL pro assay and the cellular antiviral assay.
We also solved the X-ray crystal structure of the wild-type SARS-CoV-2 PL pro in complex with GRL0617. Binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. In contrast, a larger inhibitor VIR251 stabilizes the BL2 loop in the open conformation. 5 The intrinsic flexibility of the BL2 loop implies that structurally diverse inhibitors might be able to fit in the S3-S4 pockets.
In conclusion, the SARS-CoV-2 PL pro inhibitors developed in this study represent promising hits for further development as SARS-CoV-2 antivirals, and the FlipGFP PL pro assay is a suitable surrogate for testing the cellular activity of PL pro inhibitors in the BSL-2 setting.

MATERIALS AND METHODS
Cell lines and viruses. VERO E6 cells (ATCC, CRL-1586) were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 5% heat inactivated FBS in a 37 o C incubator with 5% CO 2 .
SARS-CoV-2, isolate USA-WA1/2020 (NR-52281), was obtained through BEI Resources and propagated once on VERO E6 cells before it was used for this study.
Studies involving the SARS-CoV-2 were performed at the UTHSCSA biosafety level-3 laboratory by personnel wearing powered air purifying respirators.
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Protein expression and purification.
Detailed expression and purification of C-terminal His tagged SARS-CoV-2 PL Pro (PL pro -His) were described in our previous publication. 4  For the IC 50 measurement with FRET Peptide-Edans substrate: the reaction was carried out in 96-well format with 200 nM PL pro protein as described previously 3,4 . For the IC 50 measurements with Ub-AMC or ISG15-AMC substrate, the reaction was carried out in 384-well format. The final PL pro protein concentration is 50 nM and substrate concentration is 2.5 µM when Ub-AMC is applied; The final PL pro protein concentration is 2 nM and substrate concentration is 0.5 µM when ISG15-AMC is applied.
For the Lineweaver-Burk plots of GRL0617, Jun9-13-7 and Jun9-13-9, assay was carried as follows: 50 µl of 400 nM PL pro protein was added to 50 µl reaction buffer containing testing compound and various concentrations of FRET Peptide-Edans substrate to initiate the enzyme reaction. The initial velocity of the enzymatic reaction with and without testing compounds was calculated by linear regression for the first 10 min of the kinetic progress curve, the plotted against substrate concentrations in Prism 8 with Michaelis-Menten equation and linear regression of double reciprocal plot.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The main protease (M pro ) enzymatic assays were carried out in M pro 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. 3,4,19,20 Cell-based FlipGFP PL pro assay Plasmid pcDNA3-TEV-flipGFP-T2A-mCherry was ordered from Addgene (Cat # 124429). SARS CoV-2 PL pro cleavage site LRGGAPTK or SARS CoV-2 M pro cleavage site AVLQSGFR was introduced into pcDNA3-flipGFP-T2A-mCherry via overlapping PCRs to generate a fragment with SacI and HindIII sites at the ends. SARS CoV-2 M pro and PL pro expression plasmids pcDNA3.1 SARS2 M pro and pcDNA3.1 SARS2 PL pro was ordered from Genscript (Piscataway NJ) with codon optimization.
For transfection, 96-well Greiner plate (Cat # 655090) was seeded with 293T cells to overnight 70-90% confluency. 50 ng pcDNA3-flipGFP-T2A-mCherry plasmid and 50 ng protease expression plasmid pcDNA3.1 were used each well in the presence of transfection reagent TransIT-293 (Mirus). 3 hrs after transfection, 1 µl testing compound was added to each well at 100-fold dilution. Images were acquired 2 days after transfection with 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 ration of GFP signal sum intensity over mCherry signal sum intensity. FlipGFP-PLP assay IC 50 value was calculated by plotting GFP/mCherry signal over the applied compound concentration with a 4 parameters dose-response function in prism 8. The mCherry signal alone was utilized to determine the compound cytotoxicity.
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Differential scanning fluorimetry (DSF).
The thermal shift binding assay (TSA) was carried out using a Thermal Fisher QuantStudio TM 5 Real-Time PCR System as described previously. 3,4,19,20 Briefly, 4 µM SARS-CoV-2 PL pro protein (PL pro ) in PL pro reaction buffer was incubated with 40 µM of compounds at 30 °C for 30 min. 1X SYPRO orange dye was added and fluorescence of the well was monitored under a temperature gradient range from 20 °C to 90 °C with 0.05 °C/s incremental step. Measured T m was plotted against compound concentration with one-site binding function in Prism 8.

Native Mass Spectrometry.
Before MS analysis, the protein was buffer exchanged using two Micro Bio-Spin columns (Bio-Rad) and diluted into 0.2M ammonium acetate to a concentration of 10 µM. Each drug was diluted with 100% ethanol to concentrations of 100, 200, and 400 Native mass spectrometry (MS) was performed using a Q-Exactive HF quadrupole-Orbitrap mass spectrometer with the Ultra-High Mass Range research modifications (Thermo Fisher Scientific) as described in our previous publications 3,4 .
Immunofluorescence assay. Antiviral immunofluorescence assay was carried out as previously described. 4,28 Briefly, Vero E6 cells or Caca2-hACE2 cells in 96-well plates . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 16, 2021. ; https://doi.org/10.1101/2021.03.15.435551 doi: bioRxiv preprint (Corning) were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at a MOI of 0.1 in DMEM supplemented with 1% FBS. Immediately before the viral inoculation, the tested compounds in a three-fold dilution concentration series were also added to the wells in X-ray diffraction data for SARS-CoV-2 PL pro + GRL0617 was collected on the SBC 19-BM beamline at the Advanced Photon Source (APS) in Argonne, IL, and processed with the HKL2000 software suite. 29 The CCP4 versions of MOLREP was used for molecular replacement using a previously solved apo SARS-CoV-2 PL pro structure, PDB ID: 6WZU as a reference model. 30,31 Rigid and restrained refinements were performed using . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made