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Updated: Jul 5, 2025
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
A miniaturized mode-of-action profiling platform enables high throughput characterization of the molecular and
Lilia Falkenstern1,2, Victoria Georgi1,3, Stefanie Bunse1,3
1Bayer AG, Müllerstrasse 178, 13353, Berlin, Germany.
Abstract:
The market approval of Tazemetostat (TAZVERIK) for the treatment of follicular lymphoma and epithelioid sarcoma has established "enhancer of zeste homolog 2" (EZH2) as therapeutic target in oncology. Despite their structural similarities and common mode of inhibition, Tazemetostat and other EZH2 inhibitors display differentiated pharmacological profiles based on their target residence time. Here we established high throughput screening methods based on time-resolved fluorescence energy transfer, scintillation proximity and high content analysis microscopy to quantify the biochemical and cellular binding of a chemically diverse collection of EZH2 inhibitors. These assays allowed to further characterize the interplay between EZH2 allosteric modulation by methylated histone tails (H3K27me3) and inhibitor binding, and to evaluate the impact of EZH2's clinically relevant mutant Y641N on drug target residence times. While all compounds in this study exhibited slower off-rates, those with clinical candidate status display significantly slower target residence times in wild type EZH2 and disease-related mutants. These inhibitors interact in a more entropy-driven fashion and show the most persistent effects in cellular washout and antiproliferative efficacy experiments. Our work provides mechanistic insights for the largest cohort of EZH2 inhibitors reported to date, demonstrating that-among several other binding parameters-target residence time is the best predictor of cellular efficacy.
Insights
EZH2 inhibitors, including Tazemetostat, show varying target residence times impacting efficacy. Longer residence times in EZH2 inhibitors predict better cellular effectiveness in cancer treatment.
Area of Science:
- Oncology
- Pharmacology
- Biochemistry
Background:
- Tazemetostat's approval highlights enhancer of zeste homolog 2 (EZH2) as a key oncology target.
- EZH2 inhibitors, despite structural similarities, exhibit distinct pharmacological profiles due to target residence time.
Purpose of the Study:
- To establish high-throughput screening methods for quantifying EZH2 inhibitor binding.
- To characterize the interaction between EZH2 allosteric modulation and inhibitor binding.
- To evaluate the impact of EZH2 mutations on drug target residence times.
Main Methods:
- Utilized time-resolved fluorescence energy transfer, scintillation proximity, and high-content analysis microscopy.
- Quantified biochemical and cellular binding of diverse EZH2 inhibitors.
- Assessed EZH2 allosteric modulation by H3K27me3 and the Y641N mutant's effect on residence times.
Main Results:
- All tested compounds showed slower off-rates from EZH2.
- Clinical candidate EZH2 inhibitors demonstrated significantly longer target residence times against wild-type and mutant EZH2.
- Inhibitors with longer residence times exhibited more persistent cellular effects and antiproliferative efficacy.
Conclusions:
- Target residence time is a critical determinant of EZH2 inhibitor cellular efficacy.
- Entropy-driven interactions characterize potent EZH2 inhibitors.
- This study provides mechanistic insights into the largest cohort of EZH2 inhibitors to date.
