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 Zn2+-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 provide insights into 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.

Low-cost synthesis of peptide libraries and their use for binding studies via temperature-related intensity change

“…To assess the peptide purity after synthesis, which may vary depending on peptide length and sequence, we recommend conducting liquid chromatography coupled to mass spectrometry (LC-MS) measurements of control peptides ( Schulte et al., 2020 , Moreno-Yruela et al., 2020 , Sereikaite et al., 2019 ) (see 18). Alternatively, matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) mass spectrometry can be performed to determine peptide purity.…”

“…Affinity data obtained with peptides of largely differing length can be expected to be biased by variations in amounts and purities. In this line, we recommend an upper limit of 30 aas for affinity determination, since a commonly observed coupling efficiency of 93%–98% corresponds to a peptide purity of 11%–55% ( Schulte et al., 2020 , Moreno-Yruela et al., 2020 , Sereikaite et al., 2019 ) . Generally, representative quality controls should always be included in any peptide library to assess the peptide purity (see Quality Control by LC-MS).…”

Diversity in the Interaction of Amino Acid- and Peptide-Based Hydroxamic Acids with Some Platinum Group Metals in Solution

“…Most of the results are summarized in excellent reviews, some of which are referred here [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The previous results clearly demonstrate the significance of hydroxamatebased compounds in many fields, for example, they are known to play a crucial role in microbial iron uptake [3,5,6,11] and are capable of inhibiting a variety of enzymes, including ureases, peroxidases, and matrix metalloproteinases [8][9][10]14,16,[18][19][20][21][22][23][24][25]. In correlation with these biological roles/effects, hydroxamate-based compounds are frequently used in the design of therapeutics targeting different diseases, like iron overload, cancers, microbial infections, inflammations, etc.…”

“…Given that a number of enzymes require metal ions for their catalytic activity, metal chelation-based metalloenzyme inhibition has become one of the strategies for the treatment of diseases. Therefore, metalloenzyme inhibitory effects of hydroxamate-based chelators have also induced accelerated interest in the past several decades [8,12,18,19,[22][23][24][25]29,56]. Excellent reviews demonstrate that in many works, numerous hydroxamate-based compounds as potential therapeutical agents, especially for cancer therapy, have been developed [8,9,18,19,33,57].…”

Automated Laser‐Transfer Synthesis of High‐Density Microarrays for Infectious Disease Screening

“…This high-throughput synthesis concept was introduced in the 1980s [5] and since continues to evolve. [6][7][8][9][10][11][12][13][14][15][16] The SPOT, [6] photolithographic, [7] and particle-based [10] syntheses are now commercialized [18][19][20] and commonly used to prepare peptides for binding studies. [21][22][23] However, each approach has at least one major drawback in regards to the peptide spot density, the possible peptide length, or the resulting peptide spot morphology (Table 1).…”