Journal: Nature Communications

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

doi: 10.1038/s41467-020-20250-9

Figure Lengend Snippet: a Approved drugs and examples of candidates in clinical trials that target class I HDACs . Shared features include a cap group, a hydrophobic linker region, and zinc-binding group. b Display and probing of modified 15-mer histone peptides for capture of HDAC3 (10 nM) on microarray slides. Incorporation of a 2-aminosuberic acid hydroxamic acid derivative (Asuha) or a 2-aminosuberic acid o -aminoanilide derivative (Asuapa) but not the carboxylic acid (Asu) allow probing of HDAC binding in microarray format ( n = 2 independent experiments). Source data are provided as a Source Data file. *Saturated chemiluminescence signal.

Article Snippet: Binding intensities were evaluated using FIJI including the Microarray Profile addon (OptiNav).

Techniques: Binding Assay, Modification, Microarray

Journal: Nature Communications

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

doi: 10.1038/s41467-020-20250-9

Figure Lengend Snippet: a Fine mapping of relative HDAC1 binding affinities in microarray format (data represent mean ± SEM, n = 4 independent experiments). See Supplementary Figs. ‒ for complete data sets for HDACs 1‒3 and 8. X: Asuha. Source data are provided as a Source Data file. b Resynthesized peptide sequences including the parent peptide ( 1a ), two frame-shifted sequences ( 2a , 4a ), a C-truncated sequence ( 3a ), and a N-truncated sequence ( 5a ). c Comparison of the inhibitory curves of resynthesized peptides 1a ‒ 5a with their corresponding microarray binding curves against HDAC1 (microarray data represent mean ± SEM, n = 4 independent experiments; inhibition data represent n = 2 independent experiments). See Supplementary Fig. for data on HDACs 2, 3 and 8 and for half-maximal inhibition concentrations. Source data are provided as a Source Data file.

Article Snippet: Binding intensities were evaluated using FIJI including the Microarray Profile addon (OptiNav).

Techniques: Binding Assay, Microarray, Sequencing, Inhibition

Journal: Nature Communications

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

doi: 10.1038/s41467-020-20250-9

Figure Lengend Snippet: a Heat map depicting effects of neighboring chemical modifications on the affinity of peptides H3(1‒16)K9Asuha and H3(10‒25)K18Asuha. p: phosphorylation, ac: acetylation, thioac: thioacetylation, me1: monomethylation, me2: demethylation, me3: trimethylation. Combinations of 4, 5, and 6 modifications abolished binding to H3(1‒16)K9Asuha peptides and have not been included. The pEC 50 values represent the concentration of HDAC enzyme required for half-maximal binding signal, n = 4 independent experiments). White squares represent weak binders (pEC 50 < 7.2). Source data are provided as a Source Data file. b Resynthesized peptide sequences with Kac at the position of study, and relative deacetylation by class I HDACs. Enzyme and substrate were incubated for 1 h at 37 °C, and relative conversion was determined by LCMS (see Supplementary Fig. for sample assay traces). Microarray data represent mean ± SEM, n = 4 indepe n dent experiments; conversion data represent mean ± SD, n ≥ 2 indepe n dent experiments. *HDAC3 tested in combination with the DAD domain of NCoR2. † Matrix steric effects might contribute to the effects at these positions.

Article Snippet: Binding intensities were evaluated using FIJI including the Microarray Profile addon (OptiNav).

Techniques: Binding Assay, Concentration Assay, Incubation, Microarray

Journal: Nature Communications

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

doi: 10.1038/s41467-020-20250-9

Figure Lengend Snippet: a Dose-response HDAC inhibition curves for Asuha (X)-containing peptides ( 9a ‒ 13a ), the HDAC inhibitor SAHA and lysine-containing controls ( 9b ‒ 13b ). See Supplementary Fig. for curves corresponding to 6a , 14a ‒ 17a , and corresponding controls. b Sequence of resynthesized peptides. c Dose-response curves for peptides 11a , 13a , 14a, and 16a . d Summary of microarray screening data and the determined potency of resynthesized Asuha-containing peptides in solution. See Supplementary Fig. for visual representation. *HDAC3 tested in combination with the DAD domain of NCoR2. All microarray data represent mean ± SEM, n = 4 independent experiments; all inhibition data represent n = 2 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Binding intensities were evaluated using FIJI including the Microarray Profile addon (OptiNav).

Techniques: Inhibition, Sequencing, Microarray