key: cord-0778644-4qf9lriz
authors: Shan, Hengyue; Liu, Jianping; Shen, Jiali; Dai, Jialin; Xu, Gang; Lu, Kuankuan; Han, Chao; Wang, Yaru; Xu, Xiaolong; Tong, Yilun; Xiang, Huaijiang; Ai, Zhiyuan; Zhuang, Guanglei; Hu, Junhao; Zhang, Zheng; Li, Ying; Pan, Lifeng; Tan, Li
title: Development of potent and selective inhibitors targeting the papain-like protease of SARS-CoV-2
date: 2021-04-27
journal: Cell Chem Biol
DOI: 10.1016/j.chembiol.2021.04.020
sha: 9f653a804f36ba8e1a5dc014c07543809c4eb900
doc_id: 778644
cord_uid: 4qf9lriz

The COVID-19 pandemic has been disastrous to society and effective drugs are urgent needed. The papain-like protease domain (PLpro) of SARS-CoV-2 (SCoV2) is indispensable for viral replication and represents a putative target for pharmacological intervention. In this work, we describe the development of a potent and selective SCoV2 PLpro inhibitor, 19. The inhibitor not only effectively blocks substrate cleavage and immunosuppressive function imparted by PLpro, but also markedly mitigates SCoV2 replication in human cells with a submicromolar IC50. We further present a convenient and sensitive activity probe, 7, and complementary assays to readily evaluate SCoV2 PLpro inhibitors in vitro or in cells. Additionally, we disclose the co-crystal structure of SCoV2 PLpro in complex with a prototype inhibitor, which illuminates their detailed binding mode. Overall, these findings provide promising leads and important tools for drug discovery aiming to target SCoV2 PLpro.

The COVID-19 pandemic has been disastrous to society and effective drugs are 24 urgent needed. The papain-like protease domain (PLpro) of SARS-CoV-2 (SCoV2) is 25 indispensable for viral replication and represents a putative target for pharmacological 26 intervention. In this work, we describe the development of a potent and selective 27 SCoV2 PLpro inhibitor, 19. The inhibitor not only effectively blocks substrate 28 cleavage and immunosuppressive function imparted by PLpro, but also markedly 29 mitigates SCoV2 replication in human cells with a submicromolar IC 50 . We further 30 present a convenient and sensitive activity probe, 7, and complementary assays to 31 readily evaluate SCoV2 PLpro inhibitors in vitro or in cells. Additionally, we disclose 32 the co-crystal structure of SCoV2 PLpro in complex with a prototype inhibitor, which 33 illuminates their detailed binding mode. Overall, these findings provide promising 34 leads and important tools for drug discovery aiming to target SCoV2 PLpro. 35

The pandemic of Coronavirus Disease-2019 (COVID-19) has been ravaging for over 41 a year, causing mounting infections and millions of deaths, as well as incalculable 42 devastation on the global economy (Morens and Fauci, 2020) . To overcome 43 COVID-19, researchers worldwide are racing to discover effective drugs. Up-to-now, 44 several vaccines have been approved, and US FDA has granted Remdesivir, an 45 antiviral agent targeting RNA polymerase (RdRP), for treatment of However, the agents currently applied in clinic will most likely face anticipated drug 47 its significantly improved potency ( Figure 4C) . 227 228 To precisely characterize the affinity of 19 for SCoV2 PLpro, we performed surface 229 plasma resonance (SPR) binding analysis of 19 in comparison with GRL0617. Based 230 on the SPR results, 19 bound to SCoV2 PLpro with a K d of 2.6 µM, while that of 231 GRL0617 was 10.8 µM ( Figure 4D ). To evaluate its selectivity, 19 was profiled 232 against 10 DUBs or DUB-like proteases, including USP14 and OTUB1, which 233 reportedly favor K48-linked Ub chains as substrates. Compared to SCoV2 PLpro, 234 none of these 10 proteases were significantly inhibited by 19 at 10 μM ( Figure 4E) . The docking study of 19 was performed based on coordinates of SCoV2 PLpro-12 562 (Chain A, PDB 7E35) and Schrödinger Glide software (Friesner et al., 2006) . 563

Meanwhile, 12 as a control was docked back with same parameters. GlideScores 564 simulate binding free energies, more negative values represent tighter binders, and 565 scores of -10 or lower usually represent very good binding. In this study, the 566 GlideScore for the top-ranking predicted binding mode of 19 was -11.814, in 567 comparison, that for for 12 was -10.851. Kinetic assay was performed in assay buffer (20 mM Tris-HCl, 100 mM NaCl, 5 mM 578 2-hydroxy-1-ethanethiol, pH 7.8) with 5 mM 2-hydroxy-1-ethanethiol as the reducing 579 agent to eliminate cysteine-reactive compounds. 15 min was selected as the initial 580 condition to compare these probes with different λ ex and λ em (probe 1: λ ex = 485 nm; 581 λ em = 535nm; probe 2: λ ex =345 nm; λ em = 445nm; probe 3: λ ex =330 nm; λ em = 582 500nm; probe 5/6/7: λ ex = 405 nm; λ em = 450nm). PLpro C112S was used to determine 583 whether signal generation was dependent on enzyme activity. Enzyme concentration 584 (PLpro 1 μM) and incubation time with substrate were optimized to yield a linear 585 response in a 60-mintime frame (probe 5, 2.5μM), and a 30-mintime frame (probe 7, 586 2.5 μM) for HTS Screening. The enzyme activity was monitored using the Enspire 

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followed by the addition of acetic anhydride (0.58 mL, 6 mmol) in one portion. The 778 resulting mixture was stirred for 2 h at room temperature, then concentrated in vacuo. 779The residue was purified by silica gel column chromatography to furnish compound 780 14a in 92% yield (white solid). 1 was concentrated under reduced pressure. The residue was purified by silica gel 816 column chromatography to furnish compound 17c in 98% yield (slightly yellow oil). 817

To a solution of dimethylglycine (500 mg, 3.67 mmol), and HATU (1.67 g, 4.4 mmol) 819in dry DCM (20 mL) were added a solution of DIEA (3.2 mL, 18.3 mmol) and 820 3-amino-5-fluorobenzonitrile (454 mg, 4.4 mmol) in dry CH 2 Cl 2 (17 ml) at 0℃ under 821 argon atmosphere, and it was allowed to stir for 16 h at room temperature. The 822 reaction mixture was quenched with water and extracted with CH 2 Cl 2 . The organic 823 layers were dried over Na 2 SO 4 and concentrated under reduced pressure. The residue 824 was purified by silica gel column chromatography to furnish compound 18a in 37% 825 yield (white solid). 826To a solution of 18a (110 mg, 0.5 mmol) in saturated ammonia in MeOH, Raney-Ni 827 (11 mg, 10%) was added, and the mixture was stirred under H 2 atmosphere at room 828 temperature for 16 h. The reaction was filtered through a celite pad and the filtrate 829 was concentrated under reduced pressure. The residue was purified by silica gel 830 column chromatography to furnish compound 18c in 98% yield (slightly yellow oil). and it was allowed to stir for 16 h at room temperature. The reaction mixture was 851 quenched with water and extracted with CH 2 Cl 2 . The organic layers were dried over 852 Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by silica 853 gel column chromatography to furnish compound. 854

(11) 856The title compound was obtained as described in the general procedure in 53% yield 857 N-(3-acetamidobenzyl)-1-(1-(naphthalen-1-yl) ethyl) 864 piperidine-4-carboxamide (12) 865The title compound was obtained as described in the general procedure in 52% yield 866 

The title compound was obtained as described in the general procedure in 66% yield 875 N-(3-acetamido-5-fluorobenzyl)-1-(1-(naphthalen-1-yl) ethyl) 883

The title compound was obtained as described in the general procedure in 41% yield 885 N-((1-methylpiperidin-4-yl) methyl) -1-(1-(naphthalen-1-yl) ethyl) 892 piperidine-4-carboxamide (15) 893The title compound was obtained as described in the general procedure in 58% yield 894 (1-(1-(naphthalen-1-yl) 

The title compound was obtained as described in the general procedure in 43% yield 911