Developing Chemical Biology Probes for Lysine Deacylase/Demethylase Fluorescence Detection and Proteomic Profiling

Abstract

Dynamically controlling the post-translational modification (PTM) of the ε-amino groups of lysine residues is critical for regulating many cellular events. Increasing studies have revealed that many important diseases, including cancers and neurological disorders, are associated with the malfunction of lysine deacylases (HDACs) and lysine demethylases (KDMs). Developing chemical biology probes that are capable of detecting HDACs and KDMs is highly useful for interrogating their roles in epigenetic regulation and diseases. The main objective of this thesis is to develop novel chemical biology probes for detecting HDACs/KDMs. Chapter 1 provides a brief introduction to PTMs, with a focus on lysine acylations and methylations. Next, a series of fluorescent probes for HDACs and KDMs activity detection are presented. Additionally, photoreactive affinity-based probes (AƒBPs) to identify the readers and erasers of lysine PTMs are introduced.

Due to the distinct substrate recognition of the 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. In Chapter 2, we reported 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 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.

Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor associated with limited treatment options and high drug resistance, presenting significant challenges in the pursuit of effective treatment strategies. Epigenetic modifications have emerged as promising diagnostic biomarkers and therapeutic targets for GBM. For instance, histone deacetylase 6 (HDAC6) has been identified as a potential pharmacological target for GBM. Furthermore, the overexpression of monoamine oxidase A (MAO A) in glioma has been linked to tumor progression, making it an attractive target for therapy. In Chapter 3, we successfully engineered HDAC-MB, an activatable multifunctional small molecule probe, with the goal of efficiently detecting and killing glioma cells. HDAC-MB can be selectively activated by HDAC6, leading to the “turn on” of near-infrared fluorescence and demonstrating effective inhibition of MAO A, along with potent photodynamic therapy (PDT) effects. Consequently, HDAC-MB not only enables the imaging of HDAC6 in live glioma cells but also exhibits the synergistic effect of MAO A inhibition and PDT, effectively inhibiting glioma invasion and inducing cellular apoptosis. The distinctive combination of features displayed by HDAC-MB positions it as a versatile and highly effective tool for the accurate diagnosis and treatment of glioma cells.

As a newly identified PTM, the biological roles and regulatory factors of non-histone lactylation remain limited and undiscovered. In Chapter 4, we designed and synthesized four lactylated peptides whose sequences were selected from non-histone proteins. Through a series of systematic screenings with different HDACs, the results showed that sirtuin 1 (Sirt1) was the robust eraser of the lactylation modification in vitro. Furthermore, a single-step fluorogenic probe Plac-NBD was developed to detect the delactylation activity of Sirt1 in a continuous manner. To investigate Klac-interacting proteins under native cellular environments, we constructed two affinity-based probes PL1 and PL2, in which the photoactive alkyl diazirine was conjugated to the lactylated peptide substrates. They were shown to retain the strong binding affinity of their parental peptides while possessing the photo-activatable cross-linking ability to capture its potentially recognizing proteins. Using these chemical probes, we have successfully established the relationship between Sirt1 and lysine lactylation of non-histone proteins. We envision that such chemical probes will serve as useful tools for delineating the roles of lysine lactylation in biological functions and diseases.

Finally, Chapter 5 provides a summary of the designs and applications of the above chemical biology probes, and suggests directions for future works. We envision that these newly developed probes will help to further elucidate the roles of HDACs and KDMs in epigenetic control and regulation, thereby contributing to epigenetic drug discovery.

Date of Award	21 Aug 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Hongyan SUN (Supervisor)