Characterization of Inhibitor Binding Through Multiple Inhibitor Analysis: A Novel Local Fitting Method
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 wordsEnzyme 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.
- 2.Segel IH (1975) Enzyme kinetics : behavior and analysis of rapid equilibrium and steady state enzyme systems. Wiley, New YorkGoogle Scholar
- 5.Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus CR, Sacks JD, Raimondi A, Majer CR, Song J, Scott MP, Jin L, Smith JJ, Olhava EJ, Chesworth R, Moyer MP, Richon VM, Copeland RA, Keilhack H, Pollock RM, Kuntz KW (2012) A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nat Chem Biol 8(11):890–896. doi: 10.1038/nchembio.1084, nchembio.1084 [pii]Google Scholar
- 8.Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, Porter Scott M, Chesworth R, Moyer MP, Copeland RA, Richon VM, Pollock RM, Kuntz KW, Keilhack H (2013) Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc Natl Acad Sci U S A 110(19):7922–7927. doi: 10.1073/pnas.1303800110, 1303800110 [pii]CrossRefGoogle Scholar
- 9.Majer CR, Jin L, Scott MP, Knutson SK, Kuntz KW, Keilhack H, Smith JJ, Moyer MP, Richon VM, Copeland RA, Wigle TJ (2012) A687V EZH2 is a gain-of-function mutation found in lymphoma patients. FEBS Lett 586 (19):3448–3451. doi: 10.1016/j.febslet.2012.07.066, S0014-5793(12)00634-5 [pii]
- 10.Sneeringer CJ, Scott MP, Kuntz KW, Knutson SK, Pollock RM, Richon VM, Copeland RA (2010) Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas. Proc Natl Acad Sci U S A 107(49):20980–20985. doi: 10.1073/pnas.10125251071012525107 [pii]
- 11.Wigle TJ, Knutson SK, Jin L, Kuntz KW, Pollock RM, Richon VM, Copeland RA, Scott MP (2011) The Y641C mutation of EZH2 alters substrate specificity for histone H3 lysine 27 methylation states. FEBS Lett 585(19):3011–3014. doi: 10.1016/j.febslet.2011.08.018, S0014-5793(11)00608-9 [pii]
- 12.McCabe MT, Graves AP, Ganji G, Diaz E, Halsey WS, Jiang Y, Smitheman KN, Ott HM, Pappalardi MB, Allen KE, Chen SB, Della Pietra A, 3rd, Dul E, Hughes AM, Gilbert SA, Thrall SH, Tummino PJ, Kruger RG, Brandt M, Schwartz B, Creasy CL (2012) Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27). Proc Natl Acad Sci U S A 109(8):2989–2994. doi: 10.1073/pnas.11164181091116418109 [pii]
- 13.Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R, Paul JE, Boyle M, Woolcock BW, Kuchenbauer F, Yap D, Humphries RK, Griffith OL, Shah S, Zhu H, Kimbara M, Shashkin P, Charlot JF, Tcherpakov M, Corbett R, Tam A, Varhol R, Smailus D, Moksa M, Zhao Y, Delaney A, Qian H, Birol I, Schein J, Moore R, Holt R, Horsman DE, Connors JM, Jones S, Aparicio S, Hirst M, Gascoyne RD, Marra MA (2010) Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet 42(2):181–185. doi: 10.1038/ng.518ng.518 [pii]
- 14.Morin RD, Mendez-Lago M, Mungall AJ, Goya R, Mungall KL, Corbett RD, Johnson NA, Severson TM, Chiu R, Field M, Jackman S, Krzywinski M, Scott DW, Trinh DL, Tamura-Wells J, Li S, Firme MR, Rogic S, Griffith M, Chan S, Yakovenko O, Meyer IM, Zhao EY, Smailus D, Moksa M, Chittaranjan S, Rimsza L, Brooks-Wilson A, Spinelli JJ, Ben-Neriah S, Meissner B, Woolcock B, Boyle M, McDonald H, Tam A, Zhao Y, Delaney A, Zeng T, Tse K, Butterfield Y, Birol I, Holt R, Schein J, Horsman DE, Moore R, Jones SJ, Connors JM, Hirst M, Gascoyne RD, Marra MA (2011) Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature 476(7360):298–303. doi: 10.1038/nature10351nature10351 [pii]
- 15.Ryan RJ, Nitta M, Borger D, Zukerberg LR, Ferry JA, Harris NL, Iafrate AJ, Bernstein BE, Sohani AR, Le LP (2011) EZH2 codon 641 mutations are common in BCL2-rearranged germinal center B cell lymphomas. PLoS One 6(12):e28585. doi: 10.1371/journal.pone.0028585, PONE-D-11-17903 [pii]
- 16.Yap DB, Chu J, Berg T, Schapira M, Cheng SW, Moradian A, Morin RD, Mungall AJ, Meissner B, Boyle M, Marquez VE, Marra MA, Gascoyne RD, Humphries RK, Arrowsmith CH, Morin GB, Aparicio SA (2011) Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation. Blood 117(8):2451–2459. doi:10.1182/blood-2010-11-321208blood-2010-11-321208 [pii]Google Scholar