Synthesis and Biochemical Characterization of Peptide-based Chemical Probes for Substrate Profiling of Bromodomains and SIRT2 Deacetylase

Abstract

Dissertation gesperrt bis 05.02.2028!

Lysine Nǫ-acetylation is a widespread and dynamic post-translational protein modification that plays critical roles in numerous cellular processes such as regulating chromatin architecture and gene transcription. This modification is established by lysine acetyltransferases (KATs), removed by lysine deacetylases (KDACs) and recognized by bromodomains (Brds), which serve as molecular readers of acetylated lysines. Aberrant activity of KDACs or Brds correlates with the onset and progression of diseases, highlighting their potential for new chemical tools for investigating the readers, writers, and erasers of lysine acetylation. In the first project of this thesis, acetyl-lysine derivatives and peptide prodes were established and evaluated as binding partners of Brds. Lysine analogs with extended side chain lengths were synthesized by the Lossen rearrangement and upon incorporation into peptide probes derived from established Brd binding sequences tested for interactions with Brds BRD3(2), CBP, BAZ2B, and BRD4(1) in pulldown assays. While all Brds favored canonical acetyl- lysine, BRD3(2) and CBP exhibited moderate interaction with acetyl-homolysine. By contrast the BAZ2B Brd was recruited to peptides with shortened acetyl-ornithine. Complementary deacetylation assays with bacterial sirtuin SrtN showed highest activity for substitutes with acetyl-lysine, minimal activity for acetyl-homolysine, and none for acetyl-ornithine. The second part of this thesis focused on developing enzyme-specific peptide-based affinity probes for sirtuin KDACs, particularly SIRT2. Fragments from the SIRT2-specific inhibitor SirReal2 were attached to amino acid scaffolds and subsequently incorporated into peptides de- rived from SIRT2 substrate sites to form a bivalent SIRT2 ligands. Novel synthetic routes were developed for six probe-head fragments, which were subsequently conjugated to azido lysine us- ing Huisgen cycloaddition. Dimethylpyrimidine (DMP) and aminothiazole groups, previously identified as SIRT2 binding fragments, served as trapping moieties. After incorporating the SIRT2-trapping amino acids into peptides, pulldown assays demonstrated strong SIRT2 bind- ing to DMP-based probes LysTPy and LysTAcaPy, whereas aminothiazole-based probes showed reduced affinity. LysTPy-probes further showed a strong impact of the amino acid sequence in SIRT2 binding. Probes derived from p53K382 and NF-κBB p65K310 demonstrated supe- rior SIRT2 binding compared to probes derived from histone H3K18 or Ran binding sequences. Enrichment experiments from whole cell lysates confirmed the strong enrichment of SIRT2 for LysTPy in the p53, NF-κBB, and histone H3-derived probes, showcasing the potential of these probes for targeting SIRT2 in complex biological samples.