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O-Cyanobenzaldehydes (CNBA) Irreversibly Modify both Bur-ied and Exposed Lysine Residues in Live Cells

ABSTRACT: Lysine residue represents an attractive site for covalent drug development due to high abundance (5.6%) and critical functions. However, very few lysines have been characterized to be accessible to covalent ligands and perturb the protein functions, owning to their protonation state and adjacent steric hindrance. Herein, we report a new lysine bioconjugation chemistry, O-Cyanobenzaldehydes (CNBA), that enables selective modification of the lysine ε-amine to form iso-indolinones under physiological conditions. Proteome profiling enabled the mapping of 85 endogenous kinases in live cells, highlighting its potential for modifying buried catalytic lysines within the kinome. Further protein crystallography and mass spectrometry confirmed that K271_ABL1 and K162_AURKA are covalently targetable sites in kinases. Leverag-ing a structure-based drug design (SBDD), we incorporated CNBA into the core structure of Nutlin-3 to irreversibly inhibit MDM2-p53 interaction by targeting an exposed lysine K94 on the surface of MDM2. Importantly, we have demonstrated the potential application of CNBA as a lysine-recognized bioconjugation agent for developing new Antibody-drug conju-gates (ADCs). The results collectively validate CNBA as a new selective and efficient modifying agent with broad applica-tions for both buried and exposed lysine residues in live cells.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Cell Culture

SUBMITTER: Jiangnan Zheng

LAB HEAD: Ruijun Tian

PROVIDER: PXD060708 | Pride | 2025-03-27

REPOSITORIES: Pride

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			Action	DRS
	MaxQuant_HFX_Jurkat_ABPP.zip	Other
	MaxQuant_HFX_Jurkat_Drug.zip	Other
	P059_20240613_HFX_Jurkat_E_1.raw	Raw
	P059_20240613_HFX_Jurkat_E_2.raw	Raw
	P059_20240613_HFX_Jurkat_E_3.raw	Raw

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Project description:Specific histone modifications play important roles in chromatin functions such as activation or repression of gene transcription. These participation must occur as a dynamic process, however, most of histone modification state maps reported to date only provide static pictures linking certain modification with active or silenced states. This study focused on the global histone modification variation that occurs in response to transcriptional reprogramming produced by a physiological perturbation in yeast. We have performed genome-wide chromatin immunoprecipitation analysis for eight specific histone modifications before and after of a saline stress. The most striking change is a quick deacetylation of lysines 9 and 14 of H3 and lysine 8 of H4 associated to repression of genes. Genes that are activated increase the acetylation levels at these same sites, but this acetylation process of activated genes seems minor quantitatively to that of the deacetylation of repressed genes. The observed changes in tri-methylation of lysines 4, 36 and 79 of H3 and also di-methylation of lysine 79 of H3 are much more moderate than those of acetylation. Additionally, we have produced new genome-wide maps for six histone modifications at more than five times higher resolution of previous available data and analyzed for the first time in S. cerevisiae genome wide profiles of two more, acetylation of lysine 8 of H4 and di-methylation of lysine 79 of H3. In this research we have shown that dynamic of acetylation state of histones during activation or repression of transcription is a process much quicker than methylation and therefore the changes produced in the acetylation may affect methylation but the reverse path is not possible. The experiments described in this study compare ChIP with a histone modification antibody to a control ChIP with a core histone antibody. Budding yeast samples were analyzed in exponential growing conditions (YPD) or after 10 minutes of 0.4M NaCl stress. For each experiment 1 or 2 biological replicates were performed.

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O-Cyanobenzaldehydes (CNBA) Irreversibly Modify both Bur-ied and Exposed Lysine Residues in Live Cells

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