key: cord-297787-t9neub6d
authors: Fu, Ziyang; Huang, Bin; Tang, Jinle; Liu, Shuyan; Liu, Ming; Ye, Yuxin; Liu, Zhihong; Xiong, Yuxian; Cao, Dan; Li, Jihui; Niu, Xiaogang; Zhou, Huan; Zhao, Yong Juan; Zhang, Guoliang; Huang, Hao
title: Structural basis for the inhibition of the papain-like protease of SARS-CoV-2 by small molecules
date: 2020-07-18
journal: bioRxiv
DOI: 10.1101/2020.07.17.208959
sha: 
doc_id: 297787
cord_uid: t9neub6d

SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease has been implicated in virus maturation, dysregulation of host inflammation and antiviral immune responses. We showed that PLpro preferably cleaves the K48-ubiquitin linkage while also being capable of cleaving ISG15 modification. The multiple functions of PLpro render it a promising drug target. Therefore, we screened an FDA-approved drug library and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising IC50 of 2.1 μM. The co-crystal structure of SARS-CoV-2 PLpro-C111S in complex with GRL0617 suggests that GRL0617 is a non-covalent inhibitor. NMR data indicate that GRL0617 blocks the binding of ISG15 to PLpro. The antiviral activity of GRL0617 reveal that PLpro is a promising drug target for therapeutically treating COVID-19. One Sentence Summary Co-crystal structure of PLpro in complex with GRL0617 reveals the druggability of PLpro for SARS-CoV-2 treatment.

druggability of PLpro for SARS-CoV-2 treatment.

The COVID-19 pandemic has caused devastating damage to the world and it has resulted in 21 over 12 million confirmed cases and over half a million death as of July 14, 2020 (1). The novel 22 SARS-CoV-2 coronavirus is the etiological agent responsible for the pandemic, and it belongs to 23 the beta coronavirus family (2-4). Similar to the other two known beta coronaviruses, i.e. SARS 24 and MERS, SARS-CoV-2 also causes severe acute respiratory syndromes (5, 6). Unexpectedly, 25 SARS-CoV-2 has been reported to have more mild symptoms but much higher transmission rate 26 (7, 8) , therefore it has caused the biggest catastrophe to the world healthcare since the Spanish flu 27 in 1918-1920 (9). There are currently no FDA-approved drugs for SARS, MERS or SARS-CoV-2. 28 Remdesivir, an anti-HIV drug developed by Gilead, was given Emergency Use Authorization (EUA) 29 permission by the U.S. Food and Drug Administration (FDA) for COVID-19 treatment (10). 30 Meanwhile, the discovery of antibodies or vaccines for COVID-19 is still in progress. Therefore, 31

anti-SARS-CoV-2 drugs are urgently needed. Besides Mpro, PLpro has been considered another promising target for drug discovery to treat 47 length SARS-COV-2 PLpro protein was expressed in E. coli and subsequently purified. A 75 commercially available fluorogenic peptide substrate RLRGG-AMC, representing the C-terminal 76 residues of ubiquitin, was used to report the enzymatic activity of PLpro. The 1 st round of screening 77 provided ~30 compounds with over 50% inhibition at 100 μM. Hits from the 1 st round of screening 78 went into the 2 nd round of validation using the same enzymatic assay. After removing compounds 79 with poor solubility, strong reactivity and low intrinsic fluorescence, seven relatively potent 80 compounds were measured for IC50 (n=3). These 7 drugs showed modest IC50 values ranging from 81 29 to 91 μM (Fig. S3 ). Although these compounds can potentially provide a starting point for further 82 optimization, their low potency imply a need of large amounts of resources and time input. 83 Therefore, we cherry-picked GRL0617 from promising SARS-Cov PLpro inhibitors based on the 84 structural similarity between the SARS-CoV and SARS-CoV-2 PLpro proteins (25). The in-vitro 85 IC50 of GRL0617 against SARS-COV-2 PLpro was 2.1 ± 0.2 μM, suggesting a promising lead 86 compound and therefore it was subjected to further structural and mechanistic studies ( Fig. 2A) . A co-crystal structure would be crucial to understand the mechanism of inhibition of SARS-105 COV-2 by GRL0617, therefore we determined the X-ray co-crystal structure of SARS-COV-2 106 PLpro C111S at 3.2 Å (Fig. 3A -E and Table S1 ). The crystal of PLpro/GRL0617 belongs to the 107 space group I4122 with just one protein molecule in each asymmetric unit. GRL0617 resides in the 108 S3 and S4 subsites of PLpro and is apart from the catalytical triad with a minimum distance of 10.5 109 Å to S111 in the C111S structure. Based on the binding site of GRL0617, it inhibits SARS-COV-2 110

PLpro in a non-covalent manner. The overall GRL0617 bound PLpro structure is very similar to 111 the available apo-structure of C111S (PDB 6WRH) with a backbone RMSD of 0.76 Å, except for 112 two residues on the BL2 loop, i.e. Y268 and Q269 (Fig. 3D ). Upon binding GRL0617, the sidechain 113 of Y268 and Q269 shifted towards GRL0617 to form polar and hydrophobic interactions with the 114 compound and stabilize its binding (Fig. 3D) . Specifically, the sidechain oxygen of Y268 forms a 115 hydrogen bond with the NH2 group on the benzene ring of GRL0617, and the backbone NH of 116 Q269 with the carbonyl oxygen of GRL0617 ( GRL0617. In addition, hydrophobic integration also plays pivotal roles in GRL0617's binding to 122

PLpro. The naphthalene group of GRL0617 is buried in the pocket formed by aromatic residues 123 Y264, Y268 and the hydrophobic sidechains of P247 and P248 ( Fig. 3B and C) . that the original pocket in MERS PLpro might be too shallow to allow GRL0617 to bind with 130 extensive contacts, and the naphthalene moiety of GRL0617 would also be in steric clash with the 131 pocket of MERS PLpro ( Fig. S4C) . In contrast to SARS and SARS-CoV-2, the BL-2 loop of MERS 132 is one residue longer but it lacks the critical Y268 of SARS-COV-2 (Fig. S2) . The extra residue of 133 MERS PLpro may rearrange the hydrogen-bonding interaction network of the BL2 loop and the 134 lack of the aromatic tyrosine clearly resulted in the removal of T-shaped Pi-Pi stacking and van der 135

Waals interactions with the naphthalene group of GRL0617 ( Fig. 3E and Fig.S4 A-F) . response of peak intensity recovery was evident, which indicated that GRL0617 competes with 147 ISG15 for the binding site on PLpro, namely the S3 and S4 sites and blocked the binding of ISG15 148

to PLpro (Fig. S5A) . The superposition of the HSQC spectra of 0.1 mM ISG15 only and the 0.1 149 mM/0.15 mM PLpro/0.25 mM GRL0617 mixture are essentially identical, which indicated that 150 GRL0617 is a potent binder to PLpro and almost completely blocked the binding of ISG15 to PLpro 151 at a molar ratio of 1.67 (0.25 mM /0.15 mM) (Fig. 4C and Fig. S5B ). No peak shifting was observed 152 in the superimposed HSQC (Fig. S5B) , suggesting that GRL0617 is a bona fide PLpro binder 153 because the HSQC spectrum of 15 N-ISG15 is not disturbed at all by 2.5 excess molar ratio of 154 GRL0617. In comparison with the complex structure of SARS-COV-2 PLpro with Ub (PDB ID: 155 6XAA) (Fig. 4F ) or ISG15 (PDB ID: 6XA9) (Fig. 4E) , GRL0617 blocked the access of the C-156 terminal tail of Ub and ISG15 to the catalytically active site of SARS-COV-2 PLpro. As suggested 157 from the linkage cleaving preference experiments (Fig. 1) , only K48 linkage can be effectively 158 cleaved by SARS2-PLpro, therefore a very weak binding of monoUb to PLpro was expected. 159

Indeed, titrations of PLpro into 15 N-Ub caused very limited peak shifting even at molar ratio of 3, 160 confirming the suspected weak binding (Fig. S6) . Therefore, GRL0617 was not further titrated into 161 the 15 N-Ub/PLpro mixture. Taken together, our NMR and X-ray analysis indicates that GRL0617 162 is a potent PPI (protein-protein interaction) inhibitor for PLpro blocking the binding of ISG15. inhibitor Remdesivir (27, 28). In our study, we show that PLpro is an equally promising target but 173 more challenging because the co-crystal structure is hard to obtain and often irreproducible, like 174 other coronaviral PLpros (16). Our co-crystal structure of PLpro C111S in complex with the potent 175 inhibitor GRL0617 validated that SARS-COV-2 PLpro is a druggable target. Our structural 176 characterization paves the way for future drug discovery targeting PLpro and provids a solid 177 template for modeling and modifying potential inhibitors, including GRL0617 and its naphthalene 178 analogs. Based on the structure, GRL0617 resides in the S3/S4 site of PLpro, naturally it will also 179 inhibit the processing of viral polyproteins of SARS-CoV-2 since these viral polyproteins share the 180 same substrate cleavage sequence with Ub and ISG15. Therefore, inhibition of SARS2-CoV-2 181 PLpro can simutaneouly prevent viral matureation and attaching host antiviral immune system. Our 182 NMR study reveals that GRL0617 is a potent protein-protein interaction (PPI) inhibitor targeting 183

PLpro. In comparison, Mpro is another highly explored drug target with several potent covalent 184 inhibitors been reported (11, 12, 14) . However, no covalent inhibitors of PLpro have ever been 185 reported. Our study suggests that focusing on the discovery of non-covalent inhibitors could be an 186 effective strategy targeting PLpro. Althrough the seven FDA-approved drug obtained in our 187 screening show low potency against PLpro, we cannot rule out the potential of these drug to 188 therapeutically treat COVID-19 because they may have higher antiviral activities by inhibiting 189 other pathways in the virus lifecycle. The catalytic triad residues (S111 in place of C111, H272 and D286) are shown in yellow;

Coronavirus disease (COVID-2019) situation reports-176" 226 (2020);www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports

A pneumonia outbreak associated with a new coronavirus of probable bat 228 origin

A new coronavirus associated with human respiratory disease in China

Genomic characterisation and epidemiology of 2019 novel coronavirus: 232 implications for virus origins and receptor binding

A familial cluster of pneumonia associated with the 2019 novel 234 coronavirus indicating person-to-person transmission: a study of a family cluster

A Novel Coronavirus from Patients with Pneumonia in China

The SARS-CoV-2 outbreak: What we know

SARS-CoV-2 and COVID-19: The 241 most important research questions

Reorganize and survive-a recommendation for healthcare services 243 affected by COVID-19-the ophthalmology experience

Remdesivir in COVID-19: A critical review of 245 pharmacology, pre-clinical and clinical studies

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

Structure-based design of antiviral drug candidates targeting the SARS-CoV-249 2 main protease

Structural basis for the inhibition of SARS-CoV-2 main protease by 251 antineoplastic drug carmofur

Crystal structure of SARS-CoV-2 main protease provides a basis for design 253 of improved alpha-ketoamide inhibitors

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral 255 replication by targeting the viral main protease

The SARS-coronavirus papain-like 257 protease: structure, function and inhibition by designed antiviral compounds

Ubiquitin-Linkage Specificity and deISGylating activity of SARS-CoV papain-like 261 protease

Deubiquitinating and interferon antagonism activities of coronavirus 263 papain-like proteases

Modulation of Extracellular ISG15 Signaling by Pathogens and Viral 265 Effector Proteins

Research and Development on Therapeutic Agents and Vaccines for COVID-267 19 and Related Human Coronavirus Diseases

Therapeutic options for the 2019 novel coronavirus (2019-nCoV)

Rapid repurposing of drugs for 271 COVID-19

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-273 distributive deubiquitinating enzyme

Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities 275 of the SARS Coronavirus Papain-like Protease

A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks 277 SARS virus replication

Catalytic function 279 and substrate specificity of the papain-like protease domain of nsp3 from the Middle East 280 respiratory syndrome coronavirus

Structural basis for inhibition of the RNA-dependent RNA polymerase from 282

SARS-CoV-2 by remdesivir

Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase

Inhibitor recognition specificity of MERS-CoV papain-like protease may 286 differ from that of SARS-CoV

Y268 and Q269 are shown in marine in bound state, and in cyan in unbound state

SARS-COV2 PLpro C111S/GRL0617 (marine sticks) and MERS-CoV PLpro

Figure 4 NMR studies show that GRL0617 blocks the binding of ISG15 to SARS-COV-2

A) 1 H, 15 N-HSQC spectrum of 15 N labeled ISG15; (B) HSQC spectrum of 15 N labeled 341

Peak broadening and peak intensity loss indicates 342 binding of ISG15 to PLpro; (C) HSQC spectrum of 15 N labeled ISG15 (0.1 mM) in the 343 mixture of 0.15 mM PLpro and 0.25 mM GRL0617. Recovery of peak intensity suggests 344 that GRL0617 binds to PLpro and displaces ISG15

PDB 6XA9) was modelled on (D) by superposition, 347 showing steric clash of GRL0617 with the ISG15 C-terminal tail; (F) Ub in the complex 348 structure of UbPA/SARS-CoV-2 PLpro (PDB 6XAA) was modelled on

showing steric clash of GRL0617 with the Ub C-terminal tail