Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19

Abstract

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

References 65

1. 1.
   A pneumonia outbreak associated with a new coronavirus of probable bat origin · 2020

   No notice
2. 2.

   Sahin, A. R. 2019 Novel coronavirus (COVID-19) outbreak: a review of the current literature. Eurasian J. Med. Oncol.10.14744/ejmo.2020.12220 (2020).

   Not in our library

3. 3.

   Coronavirus (Covid-19) update: FDA issues emergency use authorization for potential Covid-19 treatment. https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-issues-emergency-use-authorization-potential-covid-19-treatment (2020).

   Not in our library

4. 4.

   Beigel, J. H. et al. Remdesivir for the treatment of Covid-19—preliminary report. N. Engl. J. Med.10.1056/NEJMoa2007764 (2020).

   Not in our library10.1056/nejmc2022236

5. 5.

   Commissioner, O. of the FDA approves first treatment for COVID-19. FDA https://www.fda.gov/news-events/press-announcements/fda-approves-first-treatment-covid-19 (2020).

   Not in our library

6. 6.

   Commissioner, O. of the coronavirus (COVID-19) update: FDA authorizes monoclonal antibody for treatment of COVID-19. FDA https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-monoclonal-antibody-treatment-covid-19 (2020).

   Not in our library

7. 7.

   Kim JC, Spence RA, Currier PF, Lu X, Denison MR. Coronavirus protein processing and RNA synthesis is inhibited by the cysteine proteinase inhibitor E64d. Virology. 1995;208:1-8.

   Not in our library10.1006/viro.1995.1123

8. 8.

   Stobart CC, Lee AS, Lu X, Denison MR. Temperature-sensitive mutants and revertants in the coronavirus nonstructural protein 5 protease (3CLpro) define residues involved in long-distance communication and regulation of protease activity. J. Virol. 2012;86:4801-4810.

   Not in our library10.1128/jvi.06754-11

9. 9.

   Hegyi A, Ziebuhr J. Conservation of substrate specificities among coronavirus main proteases. J. Gen. Virol. 2002;83:595-599.

   Not in our library10.1099/0022-1317-83-3-595

10. 10.

    Anand K, Ziebuhr J, Wadhwani P, Mesters JR, Hilgenfeld R. Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs. Science. 2003;300:1763-1767.

    Not in our library10.1126/science.1085658

11. 11.

    Bacon BR, et al. Boceprevir for previously treated chronic HCV genotype 1 infection. N. Engl. J. Med. 2011;364:1207-1217.

    Not in our library10.1056/nejmoa1009482

12. 12.

    Chary A, Holodniy M. Recent advances in hepatitis C virus treatment: review of HCV protease inhibitor clinical trials. Rev. Recent Clin. Trials. 2010;5:158-173.

    Not in our library10.2174/157488710792007293

13. 13.

    Hoetelmans RMW, et al. Clinical pharmacology of HIV protease inhibitors: focus on saquinavir, indinavir, and ritonavir. Pharm. World Sci. 1997;19:159-175.

    Not in our library10.1023/a:1008629608556

14. 14.

    Pillaiyar T, Manickam M, Namasivayam V, Hayashi Y, Jung S-H. An overview of severe acute respiratory syndrome–coronavirus (SARS-CoV) 3CL protease inhibitors: peptidomimetics and small molecule chemotherapy. J. Med. Chem. 2016;59:6595-6628.

    Not in our library10.1021/acs.jmedchem.5b01461

15. 15.

    Hoffman RL, et al. Discovery of ketone-based covalent inhibitors of coronavirus 3CL proteases for the potential therapeutic treatment of COVID-19. J. Med. Chem. 2020;63:12725-12747.

    Not in our library10.1021/acs.jmedchem.0c01063

16. 16.

    Rota PA, et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science. 2003;300:1394-1399.

    Not in our library10.1126/science.1085952

17. 17.

    Marra MA, et al. The genome sequence of the SARS-associated coronavirus. Science. 2003;300:1399-1404.

    Not in our library10.1126/science.1085953

18. 18.

    Vries, M. de et al. Comparative study of a 3CLpro inhibitor and remdesivir against both major SARS-CoV-2 clades in human airway models. bioRxiv10.1101/2020.08.28.272880 (2020).

    Not in our library

19. 19.
    Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors · 2020

    No notice
20. 20.

    Kalgutkar AS, et al. N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine (CP-100,356) as a ‘chemical knock-out equivalent’ to assess the impact of efflux transporters on oral drug absorption in the rat. J. Pharm. Sci. 2009;98:4914-4927.

    Not in our library10.1002/jps.21756

21. 21.

    Shen L, et al. Dose-response curve slope sets class-specific limits on inhibitory potential of anti-HIV drugs. Nat. Med. 2008;14:762-766.

    Not in our library10.1038/nm1777

22. 22.

    Erb P, Battegay M, Zimmerli W, Rickenbach M, Egger M. Effect of antiretroviral therapy on viral load, CD4 cell count, and progression to acquired immunodeficiency syndrome in a community human immunodeficiency virus–infected cohort. Arch. Intern. Med. 2000;160:1134-1140.

    Not in our library10.1001/archinte.160.8.1134

23. 23.

    Bakowski, M. A. et al. Oral drug repositioning candidates and synergistic remdesivir combinations for the prophylaxis and treatment of COVID-19. bioRxiv10.1101/2020.06.16.153403 (2020).

    Not in our library

24. 24.

    Garcia, G. et al. Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication. Cell Rep.35, 108940 (2021).

    Not in our library10.1016/j.celrep.2021.108940

25. 25.

    Koizumi Y, et al. Quantifying antiviral activity optimizes drug combinations against hepatitis C virus infection. Proc. Natl Acad. Sci. USA. 2017;114:1922-1927.

    Not in our library10.1073/pnas.1610197114

26. 26.

    Roberts A, et al. A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice. PLoS Pathog. 2007;3:e5.

    Not in our library10.1371/journal.ppat.0030005

27. 27.
    Comparison of transgenic and adenovirus hACE2 mouse models for SARS-CoV-2 infection · 2020

    No notice
28. 28.

    Frieman M, et al. Molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease. J. Virol. 2012;86:884-897.

    Not in our library10.1128/jvi.05957-11

29. 29.

    Zhuang, Z. et al. Mapping and role of T cell response in SARS-CoV-2–infected mice. J. Exp. Med.218, e20202187 (2021).

    Not in our library10.1084/jem.20202187

30. 30.

    Sun J, et al. Generation of a broadly useful model for COVID-19 pathogenesis, vaccination, and treatment. Cell. 2020;182:734-743.e5.

    Not in our library10.1016/j.cell.2020.06.010

31. 31.

    Di L, et al. Development of a new permeability assay using low-efflux MDCKII cells. J. Pharm. Sci. 2011;100:4974-4985.

    Not in our library10.1002/jps.22674

32. 32.

    Varma MV, et al. pH-Dependent solubility and permeability criteria for provisional biopharmaceutics classification (BCS and BDDCS) in early drug discovery. Mol. Pharm. 2012;9:1199-1212.

    Not in our library10.1021/mp2004912

33. 33.

    Reddy MB, et al. Pharmacokinetic/pharmacodynamic predictors of clinical potency for hepatitis C virus nonnucleoside polymerase and protease inhibitors. Antimicrob. Agents Chemother. 2012;56:3144-3156.

    Not in our library10.1128/aac.06283-11

34. 34.

    Sobue S, Tan K, Layton G, Eve M, Sanderson JB. Pharmacokinetics of fosfluconazole and fluconazole following multiple intravenous administration of fosfluconazole in healthy male volunteers. Br. J. Clin. Pharmacol. 2004;58:20-25.

    Not in our library10.1111/j.1365-2125.2004.02107.x

35. 35.

    Stella VJ. A case for prodrugs: fosphenytoin. Adv. Drug Deliv. Rev. 1996;19:311-330.

    Not in our library10.1016/0169-409x(95)00112-k

36. 36.

    Uhlén, M. et al. Tissue-based map of the human proteome. Science347, 1260419 (2015).

    Not in our library10.1126/science.1260419

37. 37.

    Aoyama T, et al. Pharmacokinetics of fluconazole and fosfluconazole after intraperitoneal administration to peritoneal dialysis rats. Drug Metab. Pharmacokinet. 2005;20:485-490.

    Not in our library10.2133/dmpk.20.485

38. 38.

    Broad-spectrum inhibition of coronavirus main and papain-like proteases by HCV drugs. 10.21203/rs.3.rs-26344/v1 (2020).

    Not in our library

39. 39.

    Ghosh AK, et al. Design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors. J. Med. Chem. 2005;48:6767-6771.

    Not in our library10.1021/jm050548m

40. 40.

    Grum-Tokars V, Ratia K, Begaye A, Baker SC, Mesecar AD. Evaluating the 3C-like protease activity of SARS-Coronavirus: Recommendations for standardized assays for drug discovery. Virus Res. 2008;133:63-73.

    Not in our library10.1016/j.virusres.2007.02.015

41. 41.
    Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro) · 2015

    No notice
42. 42.

    St John SE, Tomar S, Stauffer SR, Mesecar AD. Targeting zoonotic viruses: Structure-based inhibition of the 3C-like protease from bat coronavirus HKU4–The likely reservoir host to the human coronavirus that causes Middle East Respiratory Syndrome (MERS). Bioorg. Med. Chem. 2015;23:6036-6048.

    Not in our library10.1016/j.bmc.2015.06.039

43. 43.

    Agnihothram S, et al. A mouse model for Betacoronavirus subgroup 2c using a bat coronavirus strain HKU5 variant. mBio. 2014;5:e00047-00014.

    Not in our library10.1128/mbio.00047-14

44. 44.

    St John SE, et al. X-ray structure and inhibition of the feline infectious peritonitis virus 3C-like protease: structural implications for drug design. Bioorg. Med. Chem. Lett. 2015;25:5072-5077.

    Not in our library10.1016/j.bmcl.2015.10.023

45. 45.

    St John SE, Anson BJ, Mesecar AD. X-ray structure and Inhibition of 3C-like protease from porcine epidemic diarrhea virus. Sci. Rep. 2016;6:25961.

    Not in our library10.1038/srep25961

46. 46.

    Deng X, et al. Coronaviruses resistant to a 3C-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance. J. Virol. 2014;88:11886-11898.

    Not in our library10.1128/jvi.01528-14

47. 47.

    Tomar, S. Understanding the determinants for substrate recognition, regulation of enzymatic activity and the development of broad-spectrum inhibitors of coronavirus 3-chymotrypsin-like proteases. ProQuest Dissertations, Purdue University https://www.proquest.com/openview/5998473fecf33d1f648754ab8715e95d/1?cbl=18750&pq-origsite=gscholar (2015).

    Not in our library

48. 48.

    Yen Y-C, et al. Development of an efficient enzyme production and structure-based discovery platform for BACE1 inhibitors. Biochemistry. 2019;58:4424-4435.

    Not in our library10.1021/acs.biochem.9b00714

49. 49.

    Ianevski A, He L, Aittokallio T, Tang J. SynergyFinder: a web application for analyzing drug combination dose-response matrix data. Bioinform. Oxf. Engl. 2017;33:2413-2415.

    Not in our library10.1093/bioinformatics/btx162

50. 50.

    Coughlan L, et al. Ad5:Ad48 hexon hypervariable region substitutions lead to toxicity and increased inflammatory responses following intravenous delivery. Mol. Ther. J. Am. Soc. Gene Ther. 2012;20:2268-2281.

    Not in our library10.1038/mt.2012.162

51. 51.

    Coughlan L, et al. In vivo retargeting of adenovirus type 5 to alphavbeta6 integrin results in reduced hepatotoxicity and improved tumor uptake following systemic delivery. J. Virol. 2009;83:6416-6428.

    Not in our library10.1128/jvi.00445-09

52. 52.

    Stopher D, McClean S. An improved method for the determination of distribution coefficients. J. Pharm. Pharm. 1990;42:144.

    Not in our library10.1111/j.2042-7158.1990.tb05373.x

53. 53.

    Szakacs Z, Haegele G, Tyka R. 1H/31P NMR pH indicator series to eliminate the glass electrode in NMR spectroscopic pKa determinations. Anal. Chim. Acta. 2004;522:247-258.

    Not in our library10.1016/j.aca.2004.07.005

54. 54.

    Obach RS. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: an examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab. Dispos. 1999;27:1350-1359.

    Not in our library

55. 55.

    Walker GS, et al. Biosynthesis of drug metabolites and quantitation using NMR spectroscopy for use in pharmacologic and drug metabolism studies. Drug Metab. Dispos. 2014;42:1627-1639.

    Not in our library10.1124/dmd.114.059204

56. 56.

    Yates P, Eng H, Di L, Obach RS. Statistical methods for analysis of time-dependent inhibition of cytochrome P450 enzymes. Drug Metab. Dispos. 2012;40:2289-2296.

    Not in our library10.1124/dmd.112.047233

57. 57.

    Kalvass JC, Maurer TS, Pollack GM. Use of plasma and brain unbound fractions to assess the extent of brain distribution of 34 drugs: comparison of unbound concentration ratios to in vivo P-glycoprotein efflux ratios. Drug Metab. Dispos. 2007;35:660-666.

    Not in our library10.1124/dmd.106.012294

58. 58.

    Yang, Z. In Vivo Metabolite Kinetics. In Drug Metabolism Handbook 41–64 10.1002/9780470439265.ch4 (John Wiley & Sons, Ltd, 2008).

    Not in our library

59. 59.

    Green MHL, Muriel WJ. Mutagen testing using TRP+ reversion in Escherichia coli. Mutat. Res. 1976;38:3-32.

    Not in our library10.1016/0165-1161(76)90076-5

60. 60.

    Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat. Res. Environ. Mutgen. Relat. Subj. 1983;113:173-215.

    Not in our library10.1016/0165-1161(83)90010-9

61. 61.

    OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test) (1997).

    Not in our library

62. 62.

    Redfern WS, et al. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. Cardiovasc. Res. 2003;58:32-45.

    Not in our library10.1016/s0008-6363(02)00846-5

63. 63.

    Brown AM, Rampe D. Drug-induced long QT syndrome: is HERG the root of all evil. Pharm. N. 2000;7:15-20.

    Not in our library

64. 64.

    Weirich J, Antoni H. Rate-dependence of antiarrhythmic and proarrhythmic properties of class I and class III antiarrhythmic drugs. Basic Res. Cardiol. 1998;93:125-132.

    Not in our library10.1007/s003950050236

65. 65.

    Yap YG, Camm AJ. Arrhythmogenic mechanisms of non-sedating antihistamines. Clin. Exp. Allergy. 1999;29:174-181.

    Not in our library10.1046/j.1365-2222.1999.0290s3174.x

Cited by papers we hold 5

• Naturally Occurring Mutations of SARS-CoV-2 Main Protease Confer Drug Resistance to Nirmatrelvir · 2023
  No notice
• The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir · 2023
  No notice
• Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors · 2023
  No notice
• The SARS‐CoV‐2 main protease (M pro ): Structure, function, and emerging therapies for COVID‐19 · 2022
  No notice
• Crystal structure of SARS-CoV-2 main protease in complex with protease inhibitor PF-07321332 · 2021
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.