Chemical Tools for Studying Lysine Lactylation, Crotonylation and Targeted Degradation of BRD4 by Nanoprobes

Abstract

Histone post-translational modification (PTM) is a crucial part of epigenetics, providing another layer of gene expression regulatory mechanisms on top of what is encoded in our genetic sequence. Emerging evidence shows that these histone marks display diverse modes of function, from the dynamics of primarily a single type of histone PTM to the timely orchestration of several marks, to systematically regulate basal cellular activities or unique events including transcription, DNA damage repair, replication, and chromatin condensation. The dynamic nature of histone codes is maintained by fluctuating expression and activation of writer and eraser enzymes dedicated to specific modification sites and types. Meanwhile, reader modules interpret the histone code through specific interactions, commonly followed by recruiting transcription factors or chromatin-remodeling complexes and consequently driving changes in cellular phenotypes and behaviors.

The primary objective of this dissertation is to explore the post-translational modification erasers and readers of lactylation and crotonylation within the cell, and the targeted degradation of the intracellular PTM reader BRD4 (bromodomain-containing protein 4) for anti-cancer therapy. Chapter 1 provides a detailed introduction to PTMs, including the basic concepts of PTMs, and the PTM processes discovered to date, with a focus on the research progress of crotonylation and lactylation modifications. It also introduces the main role played by the BRD4 protein in the ubiquitination PTM process. In Chapter 2, an alkyne-functionalized chemical probe, p-H4K16laAlk, was developed to capture the potential readers and erasers of Kla (lysine lactylation). We employed a variety of biochemical and cellular techniques to investigate the delactylation activity of SIRT3, including NAD⁺ consumption/cycling assays, isothermal titration calorimetry (ITC), LC-MS analysis, kinetic studies, and cellular assays using siRNA knockdown. It also introduces innovative methods to verify the interaction between SIRT3 and lactylated histones. Collectively, these findings and newly developed probes provide new directions for further investigation of lysine lactylation. The other fluorescence probe p-H4K16la-NBD is designed to detect delactylation activity continuously. In Chapter 3, a chemical probe, Kpcro, was designed to validate crotonylation interaction, the Kpcro probe can be recognized by the cellular crotonylation reader or eraser. Covalent modification of specific readers or erasers can be achieved through UV excitation, and the protein regions recognizing crotonylation modifications can be screened through a click reaction. BPTF bromodomain was identified as a reader of endogenous H4K12cr, and the molecular mechanism provides important insight into the development of selective modulators targeting bromodomains. In Chapter 4, a PROTAC prodrug named MB-ARV771 was designed, ingeniously integrating the well-documented PROTAC molecule ARV-771 with methylene blue (MB). The GSH-triggered release of MB-ARV771 successfully degrades target protein BRD4 and amplifies anticancer effects. This study provides a comprehensive elucidation of the GSH-triggered release, in vitro anticancer efficacy, and the synergistic effect of photodynamic therapy and BRD4 protein degradation, thus showcasing the potential of MB-ARV771 in cancer therapeutics. This innovative approach highlights a promising direction for the development of highly effective PROTAC-based anticancer drugs.

Finally, the last part briefly summarizes this dissertation, highlighting the main directions of current PTM and anticancer therapy research.

Date of Award	23 Aug 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Hongyan SUN (Supervisor)