Universal strategy to develop fluorogenic probes for kysine deacylase/demethylase activity and application in discriminating demethylation states

Abstract

Dynamically controlling the post-translational modification of the ε-amino groups of lysine residues is critical for regulating many cellular events. Increasing studies have revealed that many important diseases, including cancer and neurological disorders, are associated with the malfunction of lysine deacylases and demethylases. Developing fluorescent probes that are capable of detecting lysine deacylase and demethylase activity is highly useful for interrogating their roles in epigenetic regulation and diseases. Due to the distinct substrate recognition of these epigenetic eraser enzymes, designing a universal strategy for detecting their activity poses substantial difficulty. Moreover, designing activity-based probes for differentiating their demethylation states is even more challenging and still remains largely unexplored. Herein, we report a universal strategy to construct probes that can detect the enzymatic activity of epigenetic “erasers” through NBD-based long-distance intramolecular reactions. The probes can be easily prepared by installing the O-NBD group at the C-terminal residue of specific peptide substrates by click chemistry. Based on this strategy, detecting the activity of lysine deacetylase, desuccinylase, or demethylase with superior sensitivity and selectivity has been successfully achieved through single-step probe development. Furthermore, the demethylase probe based on this strategy is capable of distinguishing different demethylation states by both absorption and fluorescence lifetime readout. We envision that these newly developed probes will provide powerful tools to facilitate drug discovery in epigenetics in the future.

Ke Cheng, PhD

I am a chemical biologist with expertise in organic chemistry, bioconjugation, protein biology, and oncology. My research focuses on developing small molecule probes and drugs, antibody drug conjugates, and radioligand agents for targeted cancer therapy, including the design of cleavable ADC linkers and next generation payloads, as well as tumor activatable probes and chemoproteomics driven target discovery.