Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases

Abstract

Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn<sup>2+</sup>-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution demonstrate their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes<br>

Synthesis of Nε‐acetyl‐L‐homolysine by the Lossen rearrangement and its application for probing deacetylases and binding modules of acetyl‐lysine

“…Synthesis strategies for the L-Asu hydroxamic acid derivative-L-AsuHd-have been reported. [29][30][31] When incorporated into peptides, L-AsuHd serves as trapping-probe for HDACs allowing the isolation of HDAC complexes from cell lysates and profiling of HDAC substrate specificity. 29,30,32 In order to synthesize Fmoc-hLys(Ac)-OH and Fmoc-hLys(Alloc)-OH from L-Asu (1), we adapted our synthesis for AsuHd to the Lossen rearrangement (Figure 2).…”

“…[29][30][31] When incorporated into peptides, L-AsuHd serves as trapping-probe for HDACs allowing the isolation of HDAC complexes from cell lysates and profiling of HDAC substrate specificity. 29,30,32 In order to synthesize Fmoc-hLys(Ac)-OH and Fmoc-hLys(Alloc)-OH from L-Asu (1), we adapted our synthesis for AsuHd to the Lossen rearrangement (Figure 2). 33 In a first step, we protected the α-carboxylate and amino group with 9-borabicyclo[3.3.1]nonane (9-BBN).…”

“…SrtN (1 μM) and peptide substrate (100 μM) in assay buffer (50 mM NaH 2 PO 4 , 100 mM NaCl, 25 μM ZnCl 2 , pH 8) were incubated at 37 C. Reactions were started by addition of NAD + (2 mM). At various time points (3,5,10,20,30,60, 90, 120, 150 min), samples (50 μl) were taken, and the deacetylation reactions were stopped by addition of TFA (50 μl, 4% in water). The reactions were analyzed by analytical HPLC, and the rates were calculated from the signal intensities recorded at 280 nm.…”

Rapid Response to Pandemic Threats: Immunogenic Epitope Detection of Pandemic Pathogens for Diagnostics and Vaccine Development Using Peptide Microarrays

“…However, discontinuous conformational epitopes are still difficult to construct. Other, more cumbersome synthesis routes have to be followed to enable such arrays. , Another challenge concerns posttranslationally modified peptides (phosphorylation, glycosylation, hydroxamic acid-modification, etc. ), which are currently difficult and expensive to be synthesized in situ in the array format.…”

Hydroxamic Acid-Modified Peptide Library Provides Insights into the Molecular Basis for the Substrate Selectivity of HDAC Corepressor Complexes

“…This correlation is a significant finding that directly addresses the outstanding question of whether the substrate turnover of peptides of this type translates well into inhibitor potency, as posed by Moreno-Yruela et al in their peptide microarray study. 29 It also provides validation of the Asu(NHOH) sidechain as an effective substitute for acetyl-lysine for developing histone tail-mimetic peptide inhibitors. We therefore decided to focus our work on inhibitor peptides.…”