Antiviral Methods and Protocols pp 59-79

Part of the Methods in Molecular Biology book series (MIMB, volume 1030)

In Vitro Kinetic Profiling of Hepatitis C Virus NS3 Protease Inhibitors by Progress Curve Analysis

  • Rumin Zhang
  • William T. Windsor


Kinetic profiling of drug binding to its target reveals important mechanistic parameters including drug–target residence time. In this chapter, we focus on global progress curve analysis as a convenient method for kinetic profiling. Detailed guidelines with pros and cons for various experimental designs and data analysis are provided. Kinetic profiling of Boceprevir and Telaprevir is illustrated.

Key words

HCV NS3 protease Binding kinetics Kinetic profiling Drug–target residence time Global progress curve analysis Boceprevir Telaprevir 


  1. 1.
    Zhang R, Monsma F (2010) Binding kinetics and mechanism of action: toward the discovery and development of better and best in class drugs. Expert Opin Drug Discov 5:1023–1029PubMedCrossRefGoogle Scholar
  2. 2.
    Copeland RA (2010) The dynamics of drug-target interactions: drug-target residence time and its impact on efficacy and safety. Expert Opin Drug Discov 5:305–310PubMedCrossRefGoogle Scholar
  3. 3.
    Lu H, Tonge PJ (2010) Drug-target residence time: critical information for lead optimization. Curr Opin Chem Biol 14:467–474PubMedCrossRefGoogle Scholar
  4. 4.
    Swinney DC (2009) The role of binding kinetics in therapeutically useful drug action. Curr Opin Drug Discov Dev 12:31–39Google Scholar
  5. 5.
    Vauquelin G (2010) Rebinding: or why drugs may act longer in vivo than expected from their in vitro target residence time. Expert Opin Drug Discov 5:927–941PubMedCrossRefGoogle Scholar
  6. 6.
    Copeland RA, Pompliano DL, Meek TD (2006) Drug-target residence time and its implications for lead optimization. Nat Rev Drug Discov 5:730–739PubMedCrossRefGoogle Scholar
  7. 7.
    Tummino PJ, Copeland RA (2008) Residence time of receptor-ligand complexes and its effect on biological function. Biochemistry 47:5481–5492PubMedCrossRefGoogle Scholar
  8. 8.
    Zhang R, Monsma F (2009) The importance of drug-target residence time. Curr Opin Drug Discov Dev 12:488–496Google Scholar
  9. 9.
    Fang Y (2012) Ligand-receptor interaction platforms and their applications for drug discovery. Expert Opin Drug Discov 7:969–988PubMedCrossRefGoogle Scholar
  10. 10.
    Vauquelin G (2012) Determination of drug-receptor residence time by radioligand binding and functional assays: experimental strategies and physiological relevance. Med Chem Commun 3:645–651CrossRefGoogle Scholar
  11. 11.
    Andersson K, Karlsson R, Lofas S et al (2006) Label-free kinetic binding data as a decisive element in drug discovery. Expert Opin Drug Discov 1:439–446PubMedCrossRefGoogle Scholar
  12. 12.
    Copeland RA (2005) Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists. Wiley., Hoboken, NJ, pp 141–213Google Scholar
  13. 13.
    Kwong AD, Kauffman RS, Hurter P, Mueller P (2011) Discovery and development of telaprevir: an NS3-4A protease inhibitor for treating genotype 1 chronic hepatitis C virus. Nat Biotechnol 29:993–1003PubMedCrossRefGoogle Scholar
  14. 14.
    Njoroge FG, Chen KX, Shih NY, Piwinski JJ (2008) Challenges in modern drug discovery: a case study of boceprevir, an HCV protease inhibitor for the treatment of hepatitis C virus infection. Acc Chem Res 41:50–59PubMedCrossRefGoogle Scholar
  15. 15.
    Taremi SS, Beyer B, Maher M et al (1998) Construction, expression, and characterization of a novel fully activated recombinant single-chain hepatitis C virus protease. Protein Sci 7:2143–2149PubMedCrossRefGoogle Scholar
  16. 16.
    Zhang R, Beyer BM, Durkin J et al (1999) A continuous spectrophotometric assay for the hepatitis C virus serine protease. Anal Biochem 270:268–275PubMedCrossRefGoogle Scholar
  17. 17.
    Cha S (1976) Tight-binding inhibitors-III. A new approach for the determination of competition between tight-binding inhibitors and substrates-inhibition of adenosine deaminase by coformycin. Biochem Pharmacol 25:2695–2702PubMedCrossRefGoogle Scholar
  18. 18.
    Morrison JF, Walsh CT (1988) The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 61:201–301PubMedGoogle Scholar
  19. 19.
    Sculley MJ, Morrison JF, Cleland WW (1996) Slow-binding inhibition: the general case. Biochim Biophys Acta 1298:78–86PubMedCrossRefGoogle Scholar
  20. 20.
    Szedlacsek SE, Duggleby RG (1995) Kinetics of slow and tight-binding inhibitors. Methods Enzymol 249:144–180PubMedCrossRefGoogle Scholar
  21. 21.
    Williams JW, Morrison JF, Duggleby RG (1979) Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase. Biochemistry 18:2567–2573PubMedCrossRefGoogle Scholar
  22. 22.
    Murphy DJ (2004) Determination of accurate KI values for tight-binding enzyme inhibitors: an in silico study of experimental error and assay design. Anal Biochem 327:61–67PubMedCrossRefGoogle Scholar
  23. 23.
    Copeland RA, Basavapathruni A, Moyer M, Scott MP (2011) Impact of enzyme concentration and residence time on apparent activity recovery in jump dilution analysis. Anal Biochem 416:206–210PubMedCrossRefGoogle Scholar
  24. 24.
    Kuzmic P (2008) A steady state mathematical model for stepwise “slow-binding” reversible enzyme inhibition. Anal Biochem 380:5–12PubMedCrossRefGoogle Scholar
  25. 25.
    Kuzmic P, Elrod KC, Cregar LM et al (2000) High-throughput screening of enzyme inhibitors: simultaneous determination of tight-binding inhibition constants and enzyme concentration. Anal Biochem 286:45–50PubMedCrossRefGoogle Scholar
  26. 26.
    Plesner IW, Bulow A, Bols M (2001) Accurate determination of rate constants of very slow, tight-binding competitive inhibitors by numerical solution of differential equations, independently of precise knowledge of the enzyme concentration. Anal Biochem 295:186–193PubMedCrossRefGoogle Scholar
  27. 27.
    Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108PubMedCrossRefGoogle Scholar
  28. 28.
    Yang J, Copeland RA, Lai Z (2009) Defining balanced conditions for inhibitor screening assays that target bisubstrate enzymes. J Biomol Screen 14:111–120PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Rumin Zhang
    • 1
  • William T. Windsor
    • 1
  1. 1.In Vitro Pharmacology, Merck Research LaboratoriesKenilworthUSA

Personalised recommendations