High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors

Abstract

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARSCoV-2 papain-like protease (PLpro). Together with main protease (Mpro), PLpro is responsible for processing the viral replicase polyprotein into functional units. Therefore, it is an attractive target for antiviral drug development. Here we discovered four compounds, YM155, cryptotanshinone, tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 μmol/L. These compounds also exhibit strong antiviral activities in cell-based assays. YM155, an anticancer drug candidate in clinical trials, has the most potent antiviral activity with an EC50 value of 170 nmol/L. In addition, we have determined the crystal structures of this enzyme and its complex with YM155, revealing a unique binding mode. YM155 simultaneously targets three “hot” spots on PLpro, including the substratebinding pocket, the interferon stimulating gene product 15 (ISG15) binding site and zinc finger motif. Our results demonstrate the efficacy of this screening and repurposing strategy, which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.

Keywords

SARS-CoV-2 / papain-like protease / YM155 / interferon stimulating gene product 15 / drug repurposing

References

[1] Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68:352–367

[2] Bailey-Elkin BA, Knaap RC, Johnson GG, Dalebout TJ, Ninaber DK, van Kasteren PB, Bredenbeek PJ, Snijder EJ, Kikkert M, Mark BL (2014) Crystal structure of the Middle East respiratory syndrome coronavirus (MERS-CoV) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression. J Biol Chem 289:34667–34682

[3] Barretto N, Jukneliene D, Ratia K, Chen Z, Mesecar AD, Baker SC (2005a) The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J Virol 79:15189–15198

[4] Barretto N, Jukneliene D, Ratia K, Chen Z, Mesecar AD, Baker SC (2005b) The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J Virol 79:15189–15198

[5] Beigel JH, Tomashek KM, Dodd LE, Mehta AK, Zingman BS, Kalil AC, Hohmann E, Chu HY, Luetkemeyer A, Kline S (2020) Remdesivir for the treatment of Covid-19—preliminary report. N Engl J Med. 383:1813–1826

[6] Daczkowski CM, Dzimianski JV, Clasman JR, Goodwin O, Mesecar AD, Pegan SD (2017) Structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product 15 from different species. J Mol Biol 429:1661–1683

[7] de Wit E, van Doremalen N, Falzarano D, Munster VJ (2016) SARS and MERS: recent insights into emerging coronaviruses. Nat Rev Microbiol 14:523–534

[8] Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501

[9] Feng BY, Shoichet BK (2006) A detergent-based assay for the detection of promiscuous inhibitors. Nat Protoc 1:550–553

[10] Gelman MA, Glenn JS (2011) Mixing the right hepatitis C inhibitor cocktail. Trends Mol Med 17:34–46

[11] Goldman JD, Lye DC, Hui DS, Marks KM, Bruno R, Montejano R, Spinner CD, Galli M, Ahn M-Y, Nahass RG (2020) Remdesivir for 5 or 10 days in patients with severe Covid-19. N Engl J Med 383:1827–1837

[12] Guo K, Huang P, Xu N, Xu P, Kaku H, Zheng S, Xu A, Matsuura E, Liu C, Kumon H (2015) A combination of YM-155, a small molecule survivin inhibitor, and IL-2 potently suppresses renal cell carcinoma in murine model. Oncotarget 6:21137

[13] Herold J, Siddell SG, Gorbalenya AE (1999) A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold. J Biol Chem 274:14918–14925

[14] Hinton, D.M. (2020). Veklury (remdesivir) EUA Letter of Approval.

[15] Hu M, Li P, Song L, Jeffrey PD, Chenova TA, Wilkinson KD, Cohen RE, Shi Y (2005) Structure and mechanisms of the proteasomeassociated deubiquitinating enzyme USP14. EMBO J 24:3747–3756

[16] Iwasa T, Okamoto I, Suzuki M, Nakahara T, Yamanaka K, Hatashita E, Yamada Y, Fukuoka M, Ono K, Nakagawa K (2008) Radiosensitizing effect of YM155, a novel small-molecule survivin suppressant, in non-small cell lung cancer cell lines. Clin Cancer Res 14:6496–6504

[17] Jiang Z, Gao W, Huang L (2019) Tanshinones, critical pharmacological components in Salvia miltiorrhiza. Front Pharmacol 10:202

[18] Jin Z, Du X, Xu Y, Deng Y, Liu M, Zhao Y, Zhang B, Li X, Zhang L, Peng C (2020a) Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature. 582:289–293

[19] Jin Z, Zhao Y, Sun Y, Zhang B, Wang H, Wu Y, Zhu Y, Zhu C, Hu T, Du X (2020b) Structural basis for the inhibition of SARSCoV-2 main protease by antineoplastic drug carmofur. Nat Struct Mol Biol 27:529–532

[20] Kabsch W (2010) XDS. Acta Crystallogr D Biol Crystallogr 66:125–132

[21] Keaten J, Marchione M (2020) WHO study finds remdesivir didn’t help COVID-19 patients. Associated Press, New York

[22] Kelly RJ, Thomas A, Rajan A, Chun G, Lopez-Chavez A, Szabo E, Spencer S, Carter CA, Guha U, Khozin S (2013) A phase I/II study of sepantronium bromide (YM155, survivin suppressor) with paclitaxel and carboplatin in patients with advanced nonsmall-cell lung cancer. Ann Oncol 24:2601–2606

[23] Klemm T, Ebert G, Calleja DJ, Allison CC, Richardson LW, Bernardini JP, Lu BG, Kuchel NW, Grohmann C, Shibata Y (2020) Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. EMBO J. https://doi.org/10.15252/embj.2020106275

[24] Lei J, Hilgenfeld R (2016) Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin. Virol Sin 31:288–299

[25] Liebschner D, Afonine PV, Baker ML, Bunkóczi G, Chen VB, Croll TI, Hintze B, Hung LW, Jain S, McCoy AJ (2019) Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D Struct Biol 75:861–877

[26] Lu DY, Wu HY, Yarla NS, Xu B, Ding J, Lu TR (2018) HAART in HIV/AIDS treatments: future trends. Infect Disord Drug Targets 18:15–22

[27] McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674

[28] Mehra MR, Desai SS, Ruschitzka F, Patel AN (2020) Hydroxychloroquine or chloroquine with or without a macrolide for treatment of COVID-19: a multinational registry analysis. The Lancet. https://doi.org/10.1016/S0140-6736(20)31324-6

[29] Nakahara T, Kita A, Yamanaka K, Mori M, Amino N, Takeuchi M, Tominaga F, Hatakeyama S, Kinoyama I, Matsuhisa A (2007) YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts. Cancer Res 67:8014–8021

[30] World Health Organization (2020). Coronavirus disease 2019(COVID-19): situation report.

[31] Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, Doig A, Guilliams T, Latimer J, McNamee C (2019) Drug repurposing: progress, challenges and recommendations. Nat Rev Drug Discovery 18:41–58

[32] Ratia K, Pegan S, Takayama J, Sleeman K, Coughlin M, Baliji S, Chaudhuri R, Fu W, Prabhakar BS, Johnson ME (2008) A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication. Proc Natl Acad Sci 105:16119–16124

[33] Ratia K, Saikatendu KS, Santarsiero BD, Barretto N, Baker SC, Stevens RC, Mesecar AD (2006) Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. Proc Natl Acad Sci USA 103:5717–5722

[34] Ren J, Fu L, Nile SH, Zhang J, Kai G (2019) Salvia miltiorrhiza in treating cardiovascular diseases: a review on its pharmacological and clinical applications. Front Pharmacol 10:753

[35] Sanders JM, Monogue ML, Jodlowski TZ, Cutrell JB (2020) Pharmacologic treatments for coronavirus disease 2019 (COVID-19): a review. JAMA. https://doi.org/10.1001/jama.2020.6019

[36] Satoh T, Okamoto I, Miyazaki M, Morinaga R, Tsuya A, Hasegawa Y, Terashima M, Ueda S, Fukuoka M, Ariyoshi Y (2009) Phase I study of YM155, a novel survivin suppressant, in patients with advanced solid tumors. Clin Cancer Res 15:3872–3880

[37] Shi H-X, Yang K, Liu X, Liu X-Y, Wei B, Shan Y-F, Zhu L-H, Wang C (2010) Positive regulation of interferon regulatory factor 3 activation by Herc5 via ISG15 modification. Mol Cell Biol 30:2424–2436

[38] Shin D, Mukherjee R, Grewe D, Bojkova D, Baek K, Bhattacharya A, Schulz L, Widera M, Mehdipour AR, Tascher G (2020) Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature. 587:657–662

[39] Terwilliger TC, Adams PD, Read RJ, McCoy AJ, Moriarty NW, Grosse-Kunstleve RW, Afonine PV, Zwart PH, Hung LW (2009) Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr D Biol Crystallogr 65:582–601

[40] Tong T, Wu Y-Q, Ni W-J, Shen A-Z, Liu S (2020) The potential insights of traditional Chinese medicine on treatment of COVID-19. Chin Med 15:1–6

[41] Wang Y, Zhang D, Du G, Du R, Zhao J, Jin Y, Fu S, Gao L, Cheng Z, Lu Q (2020) Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial. Lancet 395:1569–1578

[42] Zhang J, Xie B, Hashimoto K (2020) Current status of potential therapeutic candidates for the COVID-19 crisis. Brain Behav Immunity 87:59–73

[43] Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, Xiang J, Wang Y, Song B, Gu X (2020a) Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 395:1054–1062

[44] Zhou L, Zuo Z, Chow MSS (2005) Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. J Clin Pharmacol 45:1345–1359

[45] Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL (2020b) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270–273

RIGHTS & PERMISSIONS

2021 The Author(s) 2021