key: cord-0886163-nok48al6
authors: Cho, Chia-Chuan D.; Li, Shuhua G.; Yang, Kai S.; Lalonde, Tyler J.; Yu, Ge; Qiao, Yuchen; Xu, Shiqing; Liu, Wenshe Ray
title: Drug Repurposing for the SARS-CoV-2 Papain-Like Protease
date: 2021-06-06
journal: bioRxiv
DOI: 10.1101/2021.06.04.447160
sha: 1a8e7285e62d0bfcbc3e3ce12b04d8666dfe70ab
doc_id: 886163
cord_uid: nok48al6

As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen’s two large polypeptide translates ORF1a and ORF1ab in human host cells to form 15 functionally important, mature nonstructural proteins. One of the two enzymes, papain-like protease or PLpro, also possesses deubiquitination and deISGylation activities that suppresses host innate immune responses toward SARS-CoV-2 infection. Therefore, PLpro is a potential COVID-19 drug target. To repurpose drugs for PLpro, we experimentally screened 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PLpro. Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit potent inhibition of PLpro at 200 μM. More comprehensive characterizations revealed 7 inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC50 value below 60 μM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC50 value below 10 μM. Among four inhibitors with an IC50 value below 10 μM, SJB2-043 is the most unique in that it doesn’t fully inhibit PLpro but has an outstanding IC50 value of 0.56 μM. SJB2-043 likely binds to an allosteric site of PLpro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PLpro holds promises but in-depth analysis of repurposed drugs is necessary to avoid omitting allosteric inhibitors.

The current prevailing pandemic of COVID-19 (coronavirus disease 2019) has caused the global health and economic consequences that is often compared to that of 1918 influenza pandemic. 1, 2 As of June 1 st , 2021, the total number of confirmed global COVID-19 cases has exceeded 170 million, of which more than 3.5 million have succumbed to death (WHO data). Encouragingly, three COVID-19 vaccines these new strains will be a concern. 3 Vaccines are also preventative making them not an option for the treatment of COVID-19 patients. For quick access to effective antivirals for COVID-19, repurposing of existing drugs has been broadly conducted. This effort has led to the identifications of a number of potential antivirals for SARS-CoV-2. 4 So far, remdesivir is the only antiviral that has been approved for the treatment of COVID-19. Remdesivir is a nucleoside analog that inhibits RNAdependent RNA polymerase (RdRp) of SARS-CoV-2. It shows high potency in inhibiting SARS-CoV-2 in vitro but appears to be only modestly effective in treating COVID-19 patients. 5 In view of the ongoing pandemic in its colossal scale and apocalyptic impact, there is a dire need of more effective antivirals with novel mechanisms of action to save lives of COVID-19 patients.

As the pathogen of COVID-19, SARS-CoV-2 has a positive-sensed genomic RNA. [6] [7] [8] It encodes 10 open reading frames (ORFs). As the largest ORF, ORF1ab comprises more than two thirds of the whole genome. In an infected host cell, ORF1ab is translated to two large polypeptide translates, ORF1a (~500 kDa) and

ORF1ab (~800 kDa), 9 by the host protein translation system. ORF1ab is formed due to a frameshift during protein translation. Both ORF1a and ORF1ab polypeptides need to undergo proteolytic cleavage to form 15 mature proteins. These mature proteins are nonstructural proteins (Nsps) essential for the virus in its reproduction and pathogenesis. The proteolytic cleavage of ORF1a and ORF1ab is an autocatalytic process. Two internal polypeptide regions, nsp3 and nsp5, possess cysteine protease activity that cleaves themselves and all other nsps in ORF1a and ORF1ab. Nsp3 is commonly referred to as papain-like protease (PLpro) and nsp5 as main protease (Mpro). 10 Both PLpro and Mpro are essential to SARS-CoV-2. Of the two enzymes, PLpro recognizes the tetrapeptide LXGG motif that is in-between viral proteins nsp1 and nsp2, nsp2 and nsp3, and nsp3 and nsp4. Its cleavage of peptide bonds leads to the release of nsp1, nsp2, and nsp3 which are essential for host modulation and viral replication. 11 In addition, recent studies have shown that PLpro can proteolytically remove K48-crosslinked ubiquitin (Ub) and interferon-stimulated gene 15 product (ISG15), both of which play important roles in the regulation of host innate immune responses to viral infection. 12, 13 PLpro is also conserved across various coronoviruses. 14 For these reasons, PLpro is an attractive COVID-19 drug target for the development of antivirals that may be potentially used as broad-spectrum inhibitors for other coronaviruses as well. [15] [16] [17] [18] [19] In view of the urgent need of antivirals for effective COVID-19 treatments, repurposing approved drugs and late-stage clinical drug candidates against SARS-CoV-2 is an efficient strategy. Significant progress has been made in drug repurposing for Mpro. The first orally administered SARS-CoV-2 Mpro inhibitor PF-07321332 that was developed by Pfizer is currently undergoing evaluation in a Phase 1b multi-dose study in hospitalized COVID-19 participants. In contrast, inhibitors for PLpro remain relatively underdeveloped because PLpro is comparably a more challenging drug target. It has a flatter active site pocket than Mpro. Since PLpro is both a cysteine protease and a deubiquitinase, we reasoned that screening of cysteine protease inhibitors and deubiquitinase inhibitors would provide fast identification of PLpro inhibitors as initial hits for further optimization. In this study, we report our progress in identifying PLpro inhibitors by screening 33 deubiquitinase inhibitors and 37 cysteine protease inhibitors. Among these molecules, we identified seven drug candidates that can potently inhibit PLpro with an IC 50 value below 60 µM. Four molecules have an IC 50 value below 10 ߤ M. One molecule SBJ2-043 can only partially inhibit PLpro but has a remarkable low IC 50 value of 0.56

PLpro is a cysteine protease with a classical Cys-His-Asp catalytic triad (Cys111, His272 and Asp286). It is responsible for processing three cleavage sites in the viral polypeptides ORF1a and ORF1ab to release nsp1, nsp2, and nsp3. Apart from proteolytic processing, PLpro has also deubiquitinase and deISGylase activities to help SARS-CoV-2 evade host immune response. 12, 13, 20-22 Thus, to identify new PLpro inhibitors, we screened 33 deubiquitinase inhibitors and 37 cysteine protease inhibitors on their inhibition of PLpro.

To express PLpro for experimental characterizations of selected small molecules, we constructed an PLpro vector for expression in E. coli ( Figure 1A ). In this construct, PLpro was fused to the C terminus of superfolder green fluorescent protein (sfGFP) that is known to stabilize a fused partner. 23 A 6×His tag was fused to N terminus of sfGFP for affinity purification using Ni-NTA resins. Between sfGFP and PLpro was a TEV cleavage site for the proteolytic removal of PLpro from the fusion protein by TEV protease. During the purification and treatment of the sfGFP-PLpro fusion, we noticed that the cleaved PLpro quickly aggregates. Therefore, for long term storage of PLpro, we have chosen to save and use sfGFP-PLpro instead of PLpro for all our assays. In order to test catalytic activities of PLpro, we synthesized a fluorogenic protein substrate Ub-AMC (AMC: 7-amino-4-methylcoumarin) using our recently developed activated cysteine-directed protein ligation technique and purchased a fluorogenic peptide substrate Z-LRGG-AMC ( Figure 1B ). 24 The hydrolysis of Ub-AMC and Z-LRGG-AMC releases AMC whose strong blue fluorescence can be traced using a fluorometer or a fluorescent plate reader. To obtain an optimal assay condition for inhibitor characterization, both Ub-AMC and Z-LRGG-AMC are titrated at 20 nM PLpro ( Figure 1C and 1D). Based on the product formation curves, we deemed that 5 μM Ub-AMC and 50 μM Z-LRGG-AMC provide easily detectable linear production formation within 1000 s and therefore used these two conditions in our following inhibitor characterization assays. We have also characterized the influence of DMSO to the PLpro catalysis since most commercial small molecules are provided as 10 mM DMSO stocks. The stability of PLpro in the presence of DMSO restricts the highest drug concentration we can test. We titrated DMSO from 0.1% to 20% ( Figure 1E ). It showed that PLpro had slightly reduced activity at 2% DMSO and this inhibition trend by DMSO increased when the DMSO concentration improves. Considering most of commercial small molecules provided as 10 mM DMSO stocks, it will be 200 µM in an assay when it is finally diluted by 50 folds as in 2% DMSO. At this condition, the PLpro remains almost 100% activity and is good for quantifying drug inhibition effects.

Since PLpro is a cysteine protease that is also a deubiquitinase, we initiated our search of its inhibitors using 31 deubiquitinase inhibitors ( sequence identity and have a very high level of sequence and structural similarity in their substrate binding pockets. 13, 19, 27, 28 In line with other recent studies, our results

show that GRL0617 is also effective at inhibiting PLpro from SARS-CoV-2 (IC 50 =1.4 µM). 12, 27, 29 Given its strong potency and relatively small size, structure-activity relationship studies of GRL0617 will likely lead to more potent PLpro inhibitors with high antiviral activities. TCID is an inhibitor for ubiquitin C-terminal hydrolase L3 with a reported IC 50 of 0.6 µ M. 30 TCID has two ketones conjugated to a highly electron withdrawing tetrachlorobenzene. Both ketones are highly prone to hydrate or react with a nucleophilic residue in PLpro. It will be interesting to study how this molecule interacts with PLpro covalently or noncovalently. A structural investigation of its complex with PLpro will serve as a starting point for the development of more potent inhibitors for PLpro. PR619 is a thiocyanate reagent that broadly inhibits a series of deubiquitinases. 31 The propensity of exchanging the cyano group with a protein thiolate makes PR619 likely to target cysteine proteases broadly. How exactly PR619 inactivates PLpro needs to be investigated further though a possible mechanism is the covalent transfer of the cyano group to the active site cysteine. S130

is an inhibitor that was originally discovered as an ATG4B inhibitor. 32 ATG4B functions similar to a deubiquitinase and hydrolyzes the preprotein of ATG8. Since mutating the catalytic cysteine in ATG4B does not significantly affect the binding of S130 to ATG4B, S130 likely interacts with ATG4B noncovalently. A similar mechanism to inhibit PLpro is also expected. S130 has a large aromatic moiety in which four rings are conjugated. It will be interesting to see how this extended large aromatic moiety interacts with PLpro to exert strong binding. SJB2-043, SJB3-019a and DUB-IN-3 are three molecules that inhibit PLpro only partially at the highest tested concentration of 200 μM. SJB2-043 and SJB3-019a are two structurally similar compounds that inhibit USP1. 33 Both have two quinone oxygens that potentially interact with nucleophilic residues in PLpro. Since both molecules did not inhibit PLpro completely but displayed clearly measurable IC 50 values, they likely bind to an allosteric site of PLpro. Further investigations of these two molecules in their mechanisms of inhibiting PLpro will likely reveal novel targeting sites for the development of PLpro inhibitors. SJB2-043 has the lowest IC 50 value among all tested small molecules, indicating very strong binding to PLpro. A potential application of this strong binding is to use it to develop a proteasome targeting chimera for PLpro. This is a direction we are actively exploring. DUB-IN-3 is a USP8 inhibitor that has two nitrile groups conjugated to a diazine. 34 The electron deficient nature of the diazine makes the two nitriles reactive toward nucleophilic residues in PLpro. It is possible that one of these two nitriles will hit on the catalytic cysteine in PLpro to form a reversible covalent complex. The low Hill coefficient of the DUB-IN-3 inhibition curve also indicates a complicated inhibition mechanism.

For six deubiquitinase inhibitors that showed high potency in inhibiting PLpro catalyzed AMC release from Z-LRGG-AMC, we have also characterized their inhibition of PLpro in its hydrolysis of Ub-AMC. Ub-AMC is much larger than Z-LRGG-AMC and involves a much bigger interface than Z-LRGG-AMC to interact with PLpro. For inhibitors that do not directly target the active site, they may display different inhibition characteristics when Ub-AMC instead of Z-LRGG-AMC is used as a substrate. We tested all six inhibitors in the presence of 20 nM PLpro and 5 μM Ub-AMC ( Figure 6 ). When 5 μM Ub-AMC was used as a substrate, IC 50 values of GRL0617, TCID and PR619 were determined as 1.80, 10.5 and 12.9 μM, respectively.

These IC 50 values are higher than but comparable to IC 50 values determined when 50 μM Z-LRGG-AMC was used as a substrate, indicating that all three inhibitors are likely involved in a similar mechanism to inhibit PLpro. GRL0617 is known to bind the PLpro active site. Therefore, it is highly likely that TCID and PR619 bind to the PLpro active site as well. When Ub-AMC was used as a substrate, SJB2-043 had a determined IC 50 value of 0.091 μM. This IC 50 value is much lower than the determined IC 50 value when Z-LRGG-AMC was used as a substrate. Similar to the observed pattern when Z-LRGG-AMC was used as a substrate, SJB2-043 did not inhibit PLpro completely. It is obvious that SJB2-043 behaves very differently from GRL0617, TCID and PR619. It binds likely to an allosteric site of PLpro. This allosteric binding influences apparently the catalytic hydrolysis of Ub-AMC more than Z-LRGG-AMC. This is likely attributed to the much larger binding interface of Ub-AMC that responses more sensitively to the PLpro structural perturbation. When

Ub-AMC was used as a substrate, SBJ3-019a displayed an inhibition curve that is more complicated than that from SBJ2-043. It showed two inhibition stages with the first leading to partial inhibition and the second continuously inhibiting PLpro without reaching its inhibition plateau at the highest concentration we tested. Since it could not be fitted to a simple inhibition mechanism, we did not calculate its IC 50 value.

When Ub-AMC was used as a substrate, DUB-IN-3 displayed a simple inhibition curve that did not reach its plateau at the highest inhibitor concentration. Its estimated IC 50 value was above 10 μM and similar to the IC 50 value determined when Z-LRGG-AMC was used as a substrate. This similarity indicates that DUB-IN-3 likely binds to the PLpro active site to convene its inhibition.

To understand how some inhibitors may interact with PLpro, we conducted molecular docking using Autodock Vina. As a starting point for docking, we used the crystal structure of PLpro bound with GRL0617 (PDB ID: 7CMD). 27, 29 GRL0617 and water molecules were removed from the active site to prepare PLpro for the docking analysis. To simplify our analysis, we limited docking around the active site. A binding pocket was defined based on the known residues of the S3/S4 binding pocket site of PLpro. 3D Conformers of selected compounds were generated using OpenBabel. We validated the docking protocol by conducting re-docking of GRL0617. Our docking results suggested a very similar binding mode of GRL0617 with PLpro as the co-crystal structure ( Figure 7A ). GRL0617 formed two hydrogen bonds between its amides and Asp164 and Tyr268, one hydrogen bond between its oxygen atom and Gln269. In addition, the naphthalene group of GRL0617 involves the hydrophobic interactions with aromatic residues Tyr264 and Tyr268. Other inhibitors ML364, LDN-57444, TCID and DUB-IN-3 were also docked against PLpro.

The protein-ligand interactions and their detailed binding modes were illustrated in might likely bind to an allosteric site of PLpro to exert its inhibition effect. As a pilot study, the current work indicates that drug repurposing for COVID-19 by targeting PLpro holds promises but in-depth investigation of these inhibitors in their mechanisms of action will likely reveal unique inhibition mechanisms of PLpro for more advanced development of potent PLpro inhibitors.

The deubiquitinase inhibitor library and cysteine protease library were purchased from MedChemExpress. Two other deubiquitinase inhibitors, C527 and VLX1579, were purchased from Cayman Chemicals. The PLpro substrate, Z-LRGG-AMC, was purchased from Cayman Chemicals.

Protein Preparation: The crystal structure of the SARS-CoV-2 PLpro in complex with inhibitor GRL0617 (PDB ID : 7CMD) was obtained from the RCSB Protein Data Bank (https://www.rcsb.org/). Only chain A of the homo-tetrameric structure was used in our docking study. The cognate ligand was extracted from the structure, which would be applied in the following redocking test. In AutoDockTools-1.5.7 , water molecules were deleted and polar hydrogens were added to the structure.

Finally, the prepared protein structure was converted into a PDBQT file for further docking studies. 

PLpro gene fragment was purchased from Integrated DNA Technologies (IDT), amplified with the primer pair that has XbaI and XhoI cutting sites. The amplified gene was inserted into pBAD-sfGFP vector. In the final construct (pBAD-sfGFP-PLpro), a hexahistidine tag is located at the N-terminus of sfGFP. A TEV cutting site is placed after the C-terminus of sfGFP which is followed by PLpro. Chemically 

The Ub-G76C-6H was expressed according to the protocol described before. 24 Briefly, an overnight culture was inoculated to 2YT medium with 100 times dilution.

It was grown in 37 °C to reach OD 600 of 0.7-0.9 and the protein was expressed overnight at 18 °C by 1 mM IPTG induction. The cells were harvested by centrifugation and resuspended in lysis buffer (50 mM NaH 2 PO 4 , 500 mM NaCl, 5 mM imidazole, 1 mM TCEP, pH 7.8) for sonication, and then the lysate was removed by centrifugation (16000 rpm, 30 min at 4 °C). The supernatant was collected and acidified with 6 M HCl dropwise until it reached pH 2 then stirred for 5-10 min to precipitate impurities. The impurities were removed by centrifugation (10000 rpm, 30 min at 4 °C), and then the supernatant was neutralized to pH 7.8 using 6M NaOH.

The supernatant was loaded onto Ni-NTA resin and washed with washing buffer (50 mM NaH 2 PO 4 , 500 mM NaCl, 25 mM imidazole, 1 mM TCEP at pH 7.8) and then eluted with elution buffer (washing buffer substituting 25 mM imidazole with 300 mM imidazo le). Finally, the elution was desalted into 50 mM ABC buffer using HiPrep Desalting column (GE Healthcare: Chicago, IL, USA) and lyophilized into 100 nmol per aliquot.

Ub-AMC was synthesized according to the Activated Cysteine-directed Protein Ligation (ACPL) technique described before. 24 to make a reaction solution with 24 mM Gly-AMC, 20 mM borate and 0.5 mM TCEP in a 60% DMSO solution. The reaction was initiated by adding 5 µL 500 mM NTCB (dissolved in DMSO) and the pH value was adjusted to 9.5 by 6 M NaOH. The reaction was performed at 37 °C for 16 h and terminated by desalting it to 50 mM ABC buffer with HiTrap Desalting column (GE Healthcare: Chicago, IL, USA). It was further purified using Ni-NTA resin and collected the flow-through to remove the side product, dehydroalanine, that we discovered during the ACPL reactions. 35 The purified Ub-AMC was analyzed by Orbitrap ESI-MS, then flash frozen and stored in -80 °C.

To have a preliminary test of inhibition ability of each drug, a screening assay was µM Z-LRGG-AMC. The fluorescence of AMC that was generated as a result of PLpro enzymatic activity was recorded with plate reader by using 380 nm as excitation wavelength and 440 nm as the emission wavelength. The initial slope of the fluorescence vs time graph for each drug was analyzed by calculating the slope of the curve between 0-10 min. The calculation was done by using linear regression analysis in GraphPad 8.0. Then, the initial slopes were normalized against reaction substitute drugs with DMSO.

The inhibition assay was performed the same way as the screening assay except varying the drug concentration with 200 µM and 100 µM serially diluted 5 for 10 times to obtain the inhibition curve. All the reactions are performed as triplicates. The further calculation was done through GraphPad 8.0. Initial slopes were calculated from the slope of first 10 min fluorescence increasement as described above. It was then normalized against the control reaction that has DMSO only. The normalized PLPro activity against drug concentration was analyzed with the inhibition curve (three parameters) analysis option in GraphPad 8.0 to determine the IC 50 values.

Responding to Covid-19 -A Once-in-a-Century Pandemic?

Escaping Pandora's Box -Another Novel Coronavirus

Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV

Bepridil is potent against SARS-CoV-2 in vitro

Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial. 1474-547X (Electronic)

Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Genetic diversity and evolution of SARS-CoV-2

Prediction of the SARS-CoV-2

structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates

Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity

The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds

SARS-CoV-2: the many pros of targeting PLpro

Broad-spectrum coronavirus antiviral drug discovery

Papain-Like Proteases as Coronaviral Drug Targets: Current Inhibitors, Opportunities, and Limitations. Pharmaceuticals (Basel) 2020

Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: A framework for anti-COVID-19 drug design

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme

Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme

Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner

Expressed Protein Ligation without Intein

A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

X-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases

The complex structure of GRL0617 and SARS-CoV-2 PLpro reveals a hot spot for antiviral drug discovery

A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

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

Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line

Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes

Discovery of a small molecule targeting autophagy via ATG4B inhibition and cell death of colorectal cancer cells in vitro and in vivo

Smallmolecule inhibitors of USP1 target ID1 degradation in leukemic cells

Synthesis and biological evaluation of 9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile analogues as potential inhibitors of deubiquitinating enzymes

A&M Presidential Impact Fellowship Fund and Texas A&M X-Grant.

The authors declare no competing financial interests.