Targeting papain-like protease for broad-spectrum coronavirus inhibition

Abstract

The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics. Drugging the multi-functional papain-like protease (PLpro) domain of the viral nsp3 holds promise. However, none of the known coronavirus PLpro inhibitors has been shown to be in vivo active. Herein, we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity, including against the Sarbecoviruses (SARS-CoV-1 and SARS-CoV-2), Merbecovirus (MERS-CoV), as well as the Alphacoronavirus (hCoV-229E and hCoV-OC43). Importantly, F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice. F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage, as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity. Despite the significant difference of substrate recognition, mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue, whereas an allosteric inhibitor of MERS-PLpro interacting with its 271E position. Our proof-ofconcept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anti-coronavirus agents. The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.

Keywords

protease / inhibitor / coronavirus / Nsp3 / antiviral

References

[1] Báez-SantosYM, Barraza SJ, WilsonMW, AgiusMP, Mielech AM, DavisNM, BakerSC, LarsenSD, MesecarAD (2014) X-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases. J Med Chem 57(6):2393–2412

[2] Baez-SantosYM, Mielech AM, DengX, BakerS, Mesecar AD (2014) Catalytic function and substrate specificity of the papainlike protease domain of nsp3 from the Middle East respiratory syndrome coronavirus. J Virol 88(21):12511–12527

[3] Baez-SantosYM, St John SE, MesecarAD (2015) The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds. Antiviral Res 115:21–38

[4] BarrettoN, Jukneliene D, RatiaK, ChenZ, Mesecar AD, BakerSC (2005) The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J Virol 79 (24):15189–15198

[5] Blanco-MeloD, Nilsson-Payant BE, LiuWC, UhlS, Hoagland D, MøllerR, JordanTX, OishiK, PanisM, Sachs D et al (2020) Imbalanced host response to SARS-CoV-2 drives development of COVID-19. Cell 181(5):1036–1045.e9

[6] ChanJF, LauSK, ToKK, ChengVC, WooPC, Yuen KY (2015) Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease. Clin Microbiol Rev 28(2):465–522

[7] ChanJF, ZhangAJ, YuanS, Poon VK, ChanCC, LeeAC, ChanWM, FanZ, TsoiHW, WenL et al (2020) Simulation of the clinical and pathological manifestations of coronavirus disease 2019 (COVID-19) in a golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clin Infect Dis 71 (9):2428–2446

[8] ChuH, ShuaiH, HouY, ZhangX, WenL, HuangX, HuB, YangD, WangY, Yoon C et al (2021) Targeting highly pathogenic coronavirus-induced apoptosis reduces viral pathogenesis and disease severity. Sci Adv 7(25):eabf8577

[9] ClasmanJR, Everett RK, SrinivasanK, MesecarAD (2020) Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease. Antiviral Res 174:104661

[10] FuZ, HuangB, TangJ, Liu S, LiuM, YeY, LiuZ, XiongY, Zhu W, CaoD et al (2021) The complex structure of GRL0617 and SARS-CoV-2 PLpro reveals a hot spot for antiviral drug discovery. Nat Commun 12(1):488

[11] GaoX, MuZ, YuX, QinB, WojdylaJ, Wang M, CuiS (2018) Structural insight into conformational changes induced by ATP binding in a type III secretion-associated ATPase from Shigella flexneri. Front Microbiol 9:1468

[12] GaoX, QinB, ChenP, Zhu K, HouP, WojdylaJA, WangM, CuiS (2021) Crystal structure of SARS-CoV-2 papain-like protease. Acta Pharm Sin B 11(1):237–245

[13] GiannakopoulosNV, Arutyunova E, LaiC, LenschowDJ, HaasAL, VirginHW (2009) ISG15 Arg151 and the ISG15-conjugating enzyme UbE1L are important for innate immune control of sindbis virus. J Virol 83(4):1602–1610

[14] GuZ, EilsR, SchlesnerM (2016) Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32(18):2847–2849

[15] İnandıklıoğluN, AkkocT (2020) Immune responses to SARS-CoV, MERS-CoV and SARS-CoV-2. Adv Exp Med Biol 1288:5–12

[16] KimS, ChenJ, ChengT, Gindulyte A, HeJ, HeS, LiQ, ShoemakerBA, Thiessen PA, YuB et al (2021) PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Res 49(D1):D1388–D1395

[17] KlemmT, EbertG, CallejaDJ, Allison CC, RichardsonLW, BernardiniJP, LuBG, KuchelNW, Grohmann C, ShibataY et al (2020) Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. EMBO J 39(18):e106275

[18] KumarS, Stecher G, LiM, KnyazC, TamuraK (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35(6):1547–1549

[19] LiK, Wohlford-Lenane CL, ChannappanavarR, ParkJE, Earnest JT, BairTB, BatesAM, Brogden KA, FlahertyHA, GallagherT et al (2017) Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice. Proc Natl Acad Sci U S A 114(15):E3119–E3128

[20] OsipiukJ, AziziSA, DvorkinS, Endres M, JedrzejczakR, JonesKA, KangS, KathayatRS, Kim Y, LisnyakVG et al (2021) Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors. Nat Commun 12(1):743

[21] RatiaK, PeganS, TakayamaJ, Sleeman K, CoughlinM, BalijiS, Chaudhuri R, FuW, PrabhakarBS, Johnson ME et al (2008) A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication. Proc Natl Acad Sci U S A 105(42):16119–16124

[22] ShanH, LiuJ, ShenJ, Dai J, XuG, LuK, HanC, WangY, Xu X, TongY et al (2021) Development of potent and selective inhibitors targeting the papain-like protease of SARS-CoV-2. Cell Chem Biol 28(6):855–865.e9

[23] SharmaA, GarciaG Jr, WangY, Plummer JT, MorizonoK, ArumugaswamiV, Svendsen CN (2020) Human iPSC-derived cardiomyocytes are susceptible to SARS-CoV-2 infection. Cell Rep Med 1(4):100052

[24] ShinD, Mukherjee R, GreweD, BojkovaD, BaekK, BhattacharyaA, SchulzL, WideraM, MehdipourAR, Tascher G et al (2020) Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature 587(7835):657–662

[25] SieversF, Higgins DG (2018) Clustal Omega for making accurate alignments of many protein sequences. Protein Sci 27(1):135–145

[26] WolffG, Limpens RWAL, Zevenhoven-DobbeJC, LaugksU, ZhengS, de JongAWM, Koning RI, AgardDA, GrünewaldK, KosterAJ et al (2020) A molecular pore spans the double membrane of the coronavirus replication organelle. Science 369(6509):1395–1398

[27] WongLR, YeZW, LuiPY, Zheng X, YuanS, ZhuL, FungSY, YuenKS, Siu KL, YeungML et al (2020) Middle east respiratory syndrome coronavirus ORF8b accessory protein suppresses type I IFN expression by impeding HSP70-dependent activation of IRF3 kinase IKKϵ. J Immunol 205(6):1564–1579

[28] YuGC, LamTT, ZhuH, GuanY (2018) Two methods for mapping and visualizing associated data on phylogeny using Ggtree. Mol Biol Evol 35(12):3041–3043

[29] YuanS, ChuH, ZhangK, Ye J, SinghK, KaoRY, ChowBK, ZhouJ, Zheng BJ (2016) A novel small-molecule compound disrupts influenza A virus PB2 cap-binding and inhibits viral replication. J Antimicrob Chemoth 71(9):2489–2497

[30] YuanS, WangR, ChanJF, Zhang AJ, ChengT, ChikKK, YeZW, WangS, Lee AC, JinL et al (2020) Metallodrug ranitidine bismuth citrate suppresses SARS-CoV-2 replication and relieves virus-associated pneumonia in Syrian hamsters. Nat Microbiol 5(11):1439–1448

[31] YuanS, ChuH, HuangJ, Zhao X, YeZW, LaiPM, WenL, CaiJP, Mo Y, CaoJ et al (2020) Viruses harness YxxO motif to interact with host AP2M1 for replication: a vulnerable broad-spectrum antiviral target. Sci Adv 6(35):eaba7910

[32] YuanS, YinX, MengX, Chan JF, YeZW, RivaL, PacheL, ChanCC, Lai PM, ChanCC et al (2021) Clofazimine broadly inhibits coronaviruses including SARS-CoV-2. Nature 593(7859):418–423

[33] ZhaoY, DuX, DuanY, Pan X, SunY, YouT, HanL, JinZ, ShangW, YuJ et al (2021) High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors. Protein Cell 12(11):877–888

RIGHTS & PERMISSIONS

2022 The Author(s) 2022