Selective cyclopeptide inhibitor development toward AF9 YEATS domain and chemoproteomic study of histone lysine [beta]-hydroxybutyrylation and succinylation

Abstract

Histone posttranslational modifications (PTMs) play essential roles in regulating various chromatin-mediated processes, such as gene transcription, DNA replication and chromatin remodeling. Emerging evidence indicates that these histone markers could directly control nucleosome structure and, additionally, serve as docking platforms to recruit diverse effector proteins (so-called readers) to initiate downstream cellular events. And these effectors emerged as potential therapeutic targets for drug discovery, as dysregulation of these epigenetic regulators could be associated with many human diseases. Particularly with the widespread use of high-resolution mass spectrometry, a repertoire of novel histone PTMs has been identified in past decade. Most have been classified as lysine acylations; however, the exact regulatory mechanisms and biological functions of these novel histone PTMs remain largely elusive. Over the past four years, my research has mainly focused on the development of selective cyclopeptide inhibitors of the AF9 YEATS domain and the chemoproteomic study of the newly discovered histone lysine crotonylation (Kcr), β-hydroxybutyrylation (Kbhb) and succinylation (Ksucc). The YEATS domain is a newly identified reader of histone lysine acetylation (Kac) and crotonylation (Kcr). A unique π-π-π stacking was found in the co-crystal structure of the AF9 YEATS domain in complex with crotonylated histone peptide. Guided by this structure, our group previously developed the first-in-class peptide-based YEATS domain inhibitors with sub-micromolar activity. To further explore the YEATS domain inhibitor, I designed and synthesized cyclopeptide inhibitors based on our previous work; this process is discussed in Chapter 2. The synthetic route was optimized several times, eventually resulting in the achievement of on-bead synthesis to replace the original synthesis in solution. The best-performing cyclopeptide inhibitor, JYX-3, showed sub-micromolar inhibitory activity and 38-fold higher selectivity towards the AF9 YEATS domain than ENL. In Chapter 3, I continue exploring the molecular basis of the high selectivity underlying the AF9 YEATS-JYX-3 interaction. Through co-crystal analysis (collaborated with Prof. Haitao Li) and explorations of structure-activity relationships, I finally identified an additional Cbz-His-His unit site responsible for the high selectivity. Further cellular experiments indicated that JYX-3 can indeed selectively interact with endogenous AF9, disrupt the chromatin association of AF9, and further suppress the expression of AF9 target genes. Histone lysine β-hydroxybutyrylation and succinylation are newly identified histone PTMs. Recent studies have indicated that these two PTMs are highly involved in connecting cellular metabolism to epigenetic regulation. However, our knowledge of their regulatory mechanisms and biological significance is still limited. To fill this research gap, a chemical proteomics approach was applied to profile proteins that recognize histone Kbhb marks; this process is discussed in Chapter 4. Finally, Chapter 5 describes the design and synthesis of a photoaffinity bisubstrate probe to explore potential lysine succinyltransferases via chemoproteomic profiling. After much effort, no specific binder of Kbhb or novel succinyltransferase was identified. However, this study nevertheless shows the promise and ability of a chemoproteomic approach for interrogating PTM-mediated interactions.