Characterization of Inhibitor Binding Through Multiple Inhibitor Analysis: A Novel Local Fitting Method

  • Thomas V. RieraEmail author
  • Tim J. Wigle
  • Robert A. Copeland
Part of the Methods in Molecular Biology book series (MIMB, volume 1439)


Understanding inhibitor binding modes is a key aspect of drug development. Early in a drug discovery effort these considerations often impact hit finding strategies and hit prioritization. Multiple inhibitor experiments, where enzyme inhibition is measured in the presence of two simultaneously varied inhibitors, can provide valuable information about inhibitor binding. These experiments utilize the inhibitor concentration dependence of the observed combined inhibition to determine the relationship between two compounds. In this way, it can be determined whether two inhibitors bind exclusively, independently, synergistically, or antagonistically. Novel inhibitors can be tested against each other or reference compounds to assist hit classification and characterization of inhibitor binding. In this chapter, we discuss the utility and design of multiple inhibitor experiments and present a new local curve fitting method for analyzing these data utilizing IC50 replots. The IC50 replot method is analogous to that used for determining mechanisms of inhibition with respect to substrate, as originally proposed by Cheng and Prusoff (Cheng and Prusoff Biochem Pharmacol 22: 3099–3108, 1973). The IC50 replot generated by this method reveals distinct patterns that are diagnostic of the nature of the interaction between two inhibitors. Multiple inhibition of the histone methyltransferase EZH2 by EPZ-5687 and the reaction product S-adenosylhomocysteine is presented as an example of the method.

Key words

Enzyme inhibition Multiple inhibitor Binding site Yonetani-Theorell IC50 replot Local curve fitting 



We acknowledge William Janzen for helpful discussions in the preparation of this chapter and Suzanne Jacques for providing data for test fitting.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Thomas V. Riera
    • 1
    Email author
  • Tim J. Wigle
    • 2
  • Robert A. Copeland
    • 1
  1. 1.Epizyme, Inc.CambridgeUSA
  2. 2.Ribon TherapeuticsLexingtonUSA

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