Identification of HCV Inhibitors from a Cell-Based Sub-Genomic Replicon Screen

Abstract

A high throughput screen of the Pfizer compound collection was carried out using a hepatitis C virus (HCV) genotype 1b subgenomic replicon cell line. Those confirmed hits that demonstrated broad spectrum activity without overt cytotoxicity were further evaluated, leading to the identification of a series of pyrrolopyridines with excellent antiviral activity in a fully infectious HCV cell-based assay and pharmacokinetic properties.

Share and Cite:

D. Pryde, T. Tran, M. Gardner, C. Pickford, S. Shaw, M. Westby, T. Parkinson, C. Smith-Burchnell, R. Webster and S. Dayal, "Identification of HCV Inhibitors from a Cell-Based Sub-Genomic Replicon Screen," Open Journal of Medicinal Chemistry, Vol. 3 No. 1, 2013, pp. 16-25. doi: 10.4236/ojmc.2013.31003.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] D. Lavanchy, “The Global Burden of Hepatitis C,” Liver International, Vol. 29, Suppl. 1, 2009, pp. 74-81. doi:10.1111/j.1478-3231.2008.01934.x
[2] R. S. Brown, “Hepatitis C and Liver Transplantation,” Nature, Vol. 436, No. 7035, 2005, pp. 973-978. doi:10.1038/nature04083
[3] J. H. Hoofnagle and L. B. Seeff, “Targeting the NonStructural Proteins of Hepatitis C Virus: Beyond Hepatitis C Virus Protease and Polymerase,” The New England Journal of Medicine, Vol. 355, 2006, pp. 2444-2451. doi:10.1056/NEJMct061675
[4] S. J. Hadziyannis, H. Sette, T. R. Morgan, V. Balan, M. Diago, P. Marcellin, G. Ramadori, H. Bodenheimer, D. Bernstein, M. Rizzetto, S. Zeuzem, P. J. Pockros, A. Lin and A. M. Ackrill, “Peginterferon-α2a and Ribavirin Combination Therapy in Chronic Hepatitis C: A Randomized Study of Treatment Duration and Ribavirin Dose,” Annals of Internal Medicine, Vol. 140, No. 5, 2004, pp. 346-355.
[5] S. M. Lemon, J. A. McKeating, T. Pietschmann, T. L. Tellinghuisen, D. N. Frick, J. S. Glenn, J. Symons and P. A. Furman, “Development of Novel Therapies for Hepatitis C,” Antiviral Research, Vol. 86, No. 1, 2010, pp. 79-92. doi:10.1016/j.antiviral.2010.02.003
[6] L. S. Smith, M. Nelson, S. Naik and J. Woten, “Telaprevir: An NS3/4A Protease Inhibitor for the Treatment of Chronic Hepatitis C,” The Annals of Pharmacotherapy, Vol. 45, No. 5, 2011, pp. 639-648. doi:10.1345/aph.1P430
[7] K. X. Chen and G. F. Njoroge, “1—The Journey to the Discovery of Boceprevir: An NS3-NS4 HCV Protease Inhibitor for the Treatment of Chronic Hepatitis C,” Progress in Medicinal Chemistry, Vol. 49, 2010, pp. 1-36. doi:10.1016/S0079-6468(10)49001-3
[8] P. L. Beaulieu, “Filibuvir, a Non-Nucleoside NS5B Polymerase Inhibitor for the Potential Oral Treatment of Chronic HCV Infection,” IDrugs, Vol. 13, No. 12, 2010, pp. 938-948.
[9] R. A. Fridell, D. Qiu, L. Valera, C. Wang, R. E. Rose and M. Gao, “Distinct Functions of NS5A in Hepatitis C Virus RNA Replication Uncovered by Studies with the NS5A Inhibitor BMS-790052,” Journal of Virology, Vol. 85, No. 14, 2011, pp. 7312-7320. doi:10.1128/JVI.00253-11
[10] T. P. Holler, T. Parkinson and D. C. Pryde, “Targeting the Non-Structural Proteins of Hepatitis C Virus: Beyond Hepatitis C Virus Protease and Polymerase,” Expert Opinion on Drug Discovery, Vol. 4, No. 3, 2009, pp. 293-314. doi:10.1517/17460440902762802
[11] Genotype 1b (Con1, Licensed from Reblikon GmbH) HCV Replicon Cells Were Resuspended to a Concentration of 1.4 × 105 Cells/ml by Addition of Pre-Warmed Medium (DMEM + 10% FCS). 45 μl of This Suspension Was Added to Each Well a 384-Well Assay Plate (Lumitrac, Greiner) Already Containing 0.5 l of Test Compound. All Plates Were Covered with gas Permeable Seals and Incubated at 37oC, 5% CO2 for 48 Hours. After 48 Hours, the Plate Was Removed from the Incubator and Left to Cool to Room Temperature for 15 -30 mins. An Equal Volume of Reconstituted Lyophilised Britelite plus Substrate (PerkinElmer) to Medium Was Added to Each Well. Luminescence Was Read Immediately on an EnVision (Perkin Elmer) Plate Reader.
[12] 1[2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(2,4-Disulfophenyl)-2H-Tetrazolium], CAS Registry Number, 150849-52-8.
[13] Genotype 1b (Con1) HCV Replicon Cells Were Resuspended to a Concentration of 1.4 × 105cells/ml by Addition of Pre-Warmed Medium (DMEM +10% FCS). 45 μl of This Suspension Was Added to Each Well of a 384-Well Assay Plate (Greiner) Already Containing 0.5 l of Test Compound. All Plates Were Covered with Gas Permeable Seals and Incubated at 37oC, 5% CO2 for 48 Hours. After 48 Hours, 5 μl of WST-1 Cell Proliferation Reagent (Roche) Was Added to Each Well and the Plate Returned to the Incubator for 1 Hour. After This Incubation Period Absorbance Was Read at 450 nm on an EnVision (Perkin Elmer) Plate Reader.
[14] Genotype 1a (H77, Licensed from Apath LLC) HCV Replicon Cells Were Resuspended to a Concentration of 1.4 × 105 Cells/ml by Addition of Pre-Warmed Assay Medium (DMEM + 10% FCS). 45 μl of This Suspension Was Added to Each Well of a 384-Well Assay Plate (Lumitrac, Greiner), Already Containing 0.5 l of Test Compound. All Plates Were Covered with Gas Permeable Seals and Incubated at 37oC, 5% CO2 for 48 Hours. After 48 Hours, the Assay Plate Was Removed from the Incubator and Left to Cool to Room Temperature for 15-30 mins. Medium Was Removed from the Wells and 5 μl Lysis Buffer (Renilla Luciferase Assay Kit, Promega) Was Added to Each Well. The Plate Was Incubated at Room Temperature on a Rocker for 15 mins then 15 μl Assay Substrate was Added to Each Well. Luminescence Was Read Immediately Using an EnVision Plate Reader.
[15] Details of the Biochemical Assays and compound Screening within Them Will Be Described Elsewhere.
[16] J. Blagg, C. Mowbray, D. C. Pryde, G. Salmon, E. Schmid, D. Fairman and K. Beaumont, “Small, NonPeptide C5a Receptor Antagonists: Part 1,” Bioorganic & Medicinal Chemistry Letters, Vol. 18, 20, 2008, pp. 5601-5604. doi:10.1016/j.bmcl.2008.08.106
[17] J. Blagg, C. Mowbray, D. Pryde, G. Salmon, D. Fairman, E. Schmid and K. Beaumont, “Small, Non-Peptide C5a Receptor Antagonists: Part 2,” Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 20, 2008, pp. 5605-5608. doi:10.1016/j.bmcl.2008.08.101
[18] M. P. Edwards and D. A. Price, “Chapter 23—Role of Physicochemical Properties and Ligand Lipophilicity Efficiency in Addressing Drug Safety Risks,” Annual Reports in Medicinal Chemistry, Vol. 45, 2010, pp. 380-391. doi:10.1016/S0065-7743(10)45023-X
[19] 5-(2-Thienyl)-N-(2-Thienylmethyl)-7-(Trifluoromethyl)-pyrAzolo[1,5-a]Pyrimidine-2-Carboxamide, CAS Registry Number 314049-21-3.
[20] For the Weak Inhibition of Myc/Max Transcription Factors by Structurally Similar Compounds See A. Kiessling, B. Sperl, A. Hollis, D. Eick and T. Berg, “Selective Inhibition of c-Myc/Max Dimerization and DNA Binding by Small Molecules,” Chemistry & Biology, Vol. 13, No. 7, 2006, pp. 749-751. doi:10.1016/j.chembiol.2006.05.011
[21] S. K. Chunduru, C. A. Benetatos, T. J. Nitz and T. R. Bailey, “Compounds, Compositions and Methods for Treatment and Prophylaxis of Hepatitis C Viral Infections and Associated Diseases,” US Patent No. US2004039533, 2005.
[22] G. Koutsoudakis, A. Kaul, E. Steinmann, S. Kallis, V. Lohmann, T. Pietschmann and R. Bartenschlager, “Characterization of the Early Steps of Hepatitis C Virus Infection by Using Luciferase Reporter Viruses,” Journal of Virology, Vol. 80, No. 11, 2006, pp. 5308-5320. doi:10.1128/JVI.02460-05
[23] B. Feng, J. B. Mills, R. E. Davidson, R. J. Mireles, J. S. Janiszewski, M. D. Troutman and S. M. de Morais, “In Vitro P-Glycoprotein Assays to Predict the in Vivo Interactions of P-Glycoprotein with Drugs in the Central Nervous System,” Drug Metabolism and Disposition, Vol. 36, No. 2, 2008, pp. 268-275. doi:10.1124/dmd.107.017434
[24] G. Allan, J. Davis, M. Dickins, I. Gardner, T. Jenkins, H. Jones, R. Webster and H. Westgate, “Pre-Clinical Pharmacokinetics of UK-453,061, a Novel Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI), and Use of in Silico Physiologically Based Prediction Tools to Predict the Oral Pharmacokinetics of UK-453,061 in Man,” Xenobiotica, Vol. 38, No. 6, 2008, pp. 620-640. doi:10.1080/00498250802069088
[25] K. Finlayson, L. Turnbull, C. T. January, J. Sharkey and J. S. Kelly, “[3H]Dofetilide Binding to HERG Transfected Membranes: A Potential High Throughput Preclinical Screen,” European Journal of Pharmacology, Vol. 430, No. 1, 2001, pp. 147-148. doi:10.1016/S0014-2999(01)01362-0

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.