Novel and potent inhibitors targeting DHODH are broad-spectrum antivirals against RNA viruses including newly-emerged coronavirus SARS-CoV-2

Abstract

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broadspectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC₅₀ of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy invivoand low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.

Keywords

de novo pyrimidine biosynthesis / DHODH inhibitors / SARS-CoV-2 / influenza viruses / virus replication / mmuno-regulation

References

[1] Adalja A, Inglesby T (2019) Broad-spectrum antiviral agents: a crucial pandemic tool. Expert Rev Anti Infect Ther 17:467–470

[2] Adcock RS, Chu YK, Golden JE, Chung DH (2017) Evaluation of anti-Zika virus activities of broad-spectrum antivirals and NIH clinical collection compounds using a cell-based, high-throughput screen assay. Antiviral Res 138:47–56

[3] Carroll T, Lo M, Lanteri M, Dutra J, Zarbock K, Silveira P, Rourke T, Ma Z-M, Fritts L, O’Connor S (2017) Zika virus preferentially replicates in the female reproductive tract after vaginal inoculation of rhesus macaques. PLoS Pathog 13:e1006537

[4] Chen SF, Perrella FW, Behrens DL, Papp LM (1992) Inhibition of dihydroorotate dehydrogenase activity by brequinar sodium. Cancer Res 52:3521–3527

[5] Chen S, Ding S, Yin Y, Xu L, Li P, Peppelenbosch MP, Pan Q, Wang W (2019) Suppression of pyrimidine biosynthesis by targeting DHODH enzyme robustly inhibits rotavirus replication. Antiviral Res 167:35–44

[6] Chen L, Liu W, Zhang Q, Xu K, Ye G, Wu W, Sun Z, Liu F, Wu K, Zhong B (2020) RNA based mNGS approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 Wuhan outbreak. Emerg Microbes Infect 9:313–319

[7] Cheung NN, Lai KK, Dai J, Kok KH, Chen H, Chan K-H, Yuen K-Y, Kao RYT (2017) Broad-spectrum inhibition of common respiratory RNA viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response. J Gen Virol 98:946–954

[8] Chien JY, Hsueh PR, Cheng WC, Yu CJ, Yang PC (2006) Temporal changes in cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory syndrome. Respirology 11 (6):715–722

[9] Chung D-H, Golden JE, Adcock RS, Schroeder CE, Chu Y-K, Sotsky JB, Cramer DE, Chilton PM, Song C, Anantpadma M (2016) Discovery of a broad-spectrum antiviral compound that inhibits pyrimidine biosynthesis and establishes a type 1 interferonindependent antiviral state. Antimicrob Agents Chemother 60:4552–4562

[10] Coelho FC, Durovni B, Saraceni V, Lemos C, Codeco CT, Camargo S, De Carvalho LM, Bastos L, Arduini D, Villela DA (2016) Higher incidence of Zika in adult women than adult men in Rio de Janeiro suggests a significant contribution of sexual transmission from men to women. Int J Infect Dis 51:128–132

[11] Collaborative CP (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr Sect D 50:760

[12] Das P, Deng X, Zhang L, Roth MG, Fontoura BM, Phillips MA, De Brabander JK (2013) SAR-based optimization of a 4-quinoline carboxylic acid analogue with potent antiviral activity. ACS Med Chem Lett 4:517–521

[13] Davies B, Morris T (1993) Physiological parameters in laboratory animals and humans. Pharm Res 10:1093–1095

[14] Debing Y, Neyts J, Delang L (2015) The future of antivirals: broadspectrum inhibitors. Curr Opin Infect Dis 28:596–602

[15] Deng C, Liu S, Zhang Q, Xu M, Zhang H, Gu D, Shi L, He J, Xiao G, Zhang B (2016) Isolation and characterization of Zika virus imported to China using C6/36 mosquito cells. Virol Sin 31:176–179

[16] Diao YY, Lu WQ, Jin HT, Zhu JS, Han L, Xu MH, Gao R, Shen X, Zhao ZJ, Liu XF (2012) Discovery of diverse human dihydroorotate dehydrogenase inhibitors as immunosuppressive agents by structure-based virtual screening. J Med Chem 55:8341–8349

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

[18] Evans P (2006) Scaling and assessment of data quality. Acta Crystallogr Sect D 62:72–82

[19] Fox RI, Herrmann ML, Frangou CG, Wahl GM, Morris RE, Strand V, Kirschbaum BJ (1999) Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol 93:198–208

[20] Gao GF (2018) From “A”IV to “Z”IKV: attacks from emerging and reemerging pathogens. Cell 172:1157–1159

[21] Gao HN, Lu HZ, Cao B, Du B, Shang H, Gan JH, Lu SH, Yang YD, Fang Q, Shen YZ (2013) Clinical findings in 111 cases of influenza A (H7N9) virus infection. N Engl J Med 368:2277–2285

[22] Grandin C, Hourani M-L, Janin YL, Dauzonne D, Munier-Lehmann H, Paturet A, Taborik F, Vabret A, Contamin H, Tangy F (2016) Respiratory syncytial virus infection in macaques is not suppressed by intranasal sprays of pyrimidine biosynthesis inhibitors. Antiviral Res 125:58–62

[23] Han Q, Chang C, Li L, Klenk C, Cheng J, Chen Y, Xia N, Shu Y, Chen Z, Gabriel G (2014) Sumoylation of influenza A virus nucleoprotein is essential for intracellular trafficking and virus growth. J Virol 88:9379–9390

[24] Harvey R, Brown K, Zhang Q, Gartland M, Walton L, Talarico C, Lawrence W, Selleseth D, Coffield N, Leary J (2009) GSK983: a novel compound with broad-spectrum antiviral activity. Antiviral Res 82:1–11

[25] Hoffmann H-H, Kunz A, Simon VA, Palese P, Shaw ML (2011) Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis. Proc Natl Acad Sci 108:5777–5782

[26] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X (2020) Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet 395:497–506

[27] Hung I, To K, Lee C, Lee K, Yan W, Chan K, Ngai C, Law K, Chow F, Liu R (2013) Hyperimmune intravenous immunoglobulin treatment: a multicentre double-blind randomized controlled trial for patients with severe A(H1N1)pdm09 infection. Chest 144:464–473

[28] Ianevski A, Andersen PI, Merits A, Bjørås M, Kainov D (2019) Expanding the activity spectrum of antiviral agents. Drug Discov Today 24:1224–1228

[29] Jasenosky LD, Neumann G, Kawaoka Y (2010) Minigenome-based reporter system suitable for high-throughput screening of compounds able to inhibit ebolavirus replication and/or transcription. Antimicrob Agents Chemother 54:3007

[30] Jordan PC, Stevens SK, Deval J (2018) Nucleosides for the treatment of respiratory RNA virus infections. Antiviral Chem Chemother 26:1–19

[31] Jordheim LP, Durantel D, Zoulim F, Dumontet C (2013) Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nat Rev Drug Discov 12:447–464

[32] Kottkamp AC, De Jesus E, Grande R, Brown JA, Jacobs AR, Lim JK, Stapleford KA (2019) Atovaquone inhibits arbovirus replication through the depletion of intracellular nucleotides. J Virol 93: e00389–00319

[33] Li S, Luan G, Ren X, Song W, Xu L, Xu M, Zhu J, Dong D, Diao Y, Liu X (2015) Rational design of benzylidenehydrazinylsubstituted thiazole derivatives as potent inhibitors of human dihydroorotate dehydrogenase with in vivo anti-arthritic activity. Sci Rep 5:14836

[34] Liu Z, Guo Z, Wang G, Zhang D, He H, Li G, Liu Y, Higgins D, Walsh A, Shanahan-Prendergast L (2009) Evaluation of the efficacy and safety of a statin/caffeine combination against H5N1, H3N2 and H1N1 virus infection in BALB/c mice. Eur J Pharm Sci 38:215–223

[35] Lucas-Hourani M, Dauzonne D, Jorda P, Cousin G, Lupan A, Helynck O, Caignard G, Janvier G, André-Leroux G, Khiar S (2013) Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity. PLoS Pathog 9:e1003678

[36] Lucas-Hourani M, Dauzonne D, Munier-Lehmann H, Khiar S, Nisole S, Dairou J, Helynck O, Afonso PV, Tangy F, Vidalain P-O (2017) Original chemical series of pyrimidine biosynthesis inhibitors that boost the antiviral interferon response. Antimicrob Agents Chemother 61:e00383–00317

[37] Luke TC, Kilbane EM, Jackson JL, Hoffman SL (2006) Metaanalysis: convalescent blood products for spanish influenza pneumonia: a future H5N1 Treatment? Ann Intern Med 145:599–609

[38] Luthra P, Naidoo J, Pietzsch CA, De S, Khadka S, Anantpadma M, Williams CG, Edwards MR, Davey RA, Bukreyev A (2018) Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses. Antiviral Res 158:288–302

[39] Marschall M, Niemann I, Kosulin K, Bootz A, Wagner S, Dobner T, Herz T, Kramer B, Leban J, Vitt D (2013) Assessment of drug candidates for broad-spectrum antiviral therapy targeting cellular pyrimidine biosynthesis. Antiviral Res 100:640–648

[40] Matrosovich M, Matrosovich T, Garten W, Klenk H-D (2006) New low-viscosity overlay medium for viral plaque assays. Virol J 3:63

[41] Matsuoka Y, Lamirande EW, Subbarao K (2009) The mouse model for influenza. Curr Protoc Microbiol 13:15G–3

[42] McNicholl IR, McNicholl JJ (2001) Neuraminidase inhibitors: zanamivir and oseltamivir. Ann Pharmacother 35:57–70

[43] Mei-jiao G, Shi-fang L, Yan-yan C, Jun-jun S, Yue-feng S, Ting-ting R, Yong-guang Z, Hui-yun C (2019) Antiviral effects of selected IMPDH and DHODH inhibitors against foot and mouth disease virus. Biomed Pharmacother 118:109305

[44] Min JY, Subbarao K (2010) Cellular targets for influenza drugs. Nat Biotechnol 28:239–240

[45] Munier-Lehmann H, Vidalain PO, Tangy F, Janin YL (2013) On dihydroorotate dehydrogenases and their inhibitors and uses. J Med Chem 56:3148–3167

[46] Murshudov GN, Skubák P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr Sect D 67:355–367

[47] Qing M, Zou G, Wang Q-Y, Xu HY, Dong H, Yuan Z, Shi P-Y (2010) Characterization of dengue virus resistance to brequinar in cell culture. Antimicrob Agents Chemother 54:3686–3695

[48] Raveh A, Delekta PC, Dobry CJ, Peng W, Schultz PJ, Blakely PK, Tai AW, Matainaho T, Irani DN, Sherman DH (2013) Discovery of potent broad spectrum antivirals derived from marine actinobacteria. PLoS ONE 8:e82318

[49] Rozman B (2002) Clinical pharmacokinetics of leflunomide. Clin Pharmacokinet 41:421–430

[50] Song W, Li S, Tong Y, Wang J, Quan L, Chen Z, Zhao Z, Xu Y, Zhu L, Qian X (2016) Structure-based design of potent human dihydroorotate dehydrogenase inhibitors as anticancer agents. Med Chem Commun 7:1441–1448

[51] Tang WW, Young MP, Mamidi A, Regla-Nava JA, Kim K, Shresta S (2016) A mouse model of Zika virus sexual transmission and vaginal viral replication. Cell Rep 17:3091–3098

[52] Van Hoecke L, Job ER, Saelens X, Roose K (2017) Bronchoalveolar lavage of murine lungs to analyze inflammatory cell infiltration. JoVE 123:e55398

[53] Wan S, Yi Q, Fan S, Lv J, Zhang X, Guo L, Lang C, Xiao Q, Xiao K, Yi Z (2020) Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP). medRxiv

[54] Wang CH, Liu C-Y, Wan Y-L, Chou C-L, Huang K-H, Lin H-C, Lin S-M, Lin T-Y, Chung KF, Kuo H-P (2005) Persistence of lung inflammation and lung cytokines with high-resolution CT abnormalities during recovery from SARS. Respir Res 6:42

[55] Wang QY, Bushell S, Qing M, Xu HY, Bonavia A, Nunes S, Zhou J, Poh MK, Florez de Sessions P, Niyomrattanakit P (2011) Inhibition of dengue virus through suppression of host pyrimidine biosynthesis. J Virol 85:6548–6556

[56] Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, Shi Z, Hu Z, Zhong W, Xiao G (2020) Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 30:269–271

[57] Wong CK, Lam CWK, Wu AKL, Ip WK, Lee NLS, Chan IHS, Sung JJY (2004) Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome. Clin Exp Immunol 136:95–103

[58] Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, Hu Y, Tao ZW, Tian JH, Pei YY (2020) A new coronavirus associated with human respiratory disease in China. Nature 579:265–269

[59] Yang Y, Cao L, Gao H, Wu Y, Wang Y, Fang F, Lan T, Lou Z, Rao Y (2019) Discovery, optimization, and target identification of novel potent broad-spectrum antiviral inhibitors. J Med Chem 62:4056–4073

[60] Yokota S (2003) Influenza-associated encephalopathy–pathophysiology and disease mechanisms. Jpn J Clin Med 61:1953–1958

[61] Yuen KY, Wong SS (2005) Human infection by avian influenza A H5N1. Hong Kong Med J 11:189–199

[62] Zeng H, Waldman WJ, Yin DP, Knight DA, Shen J, Ma L, Meister GT, Chong AS, Williams JW (2005) Mechanistic study of malononitrileamide FK778 in cardiac transplantation and CMV infection in rats. Transplantation 79:17–22

[63] Zhang Y, Li J, Zhan Y, Wu L, Yu X, Zhang W, Ye L, Xu S, Sun R, Wang Y (2004) Analysis of serum cytokines in patients with severe acute respiratory syndrome. Infect Immun 72:4410–4415

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

[65] Zhu J, Han L, Diao Y, Ren X, Xu M, Xu L, Li S, Li Q, Dong D, Huang J (2015a) Design, synthesis, X-ray crystallographic analysis, and biological evaluation of thiazole derivatives as potent and selective inhibitors of human dihydroorotate dehydrogenase. J Med Chem 58:1123–1139

[66] Zhu JD, Meng W, Wang XJ, Wang HC (2015b) Broad-spectrum antiviral agents. Front Microbiol 6:517

RIGHTS & PERMISSIONS

2020 The Author(s)