One-Atom Substitution Enables Direct and Continuous Monitoring of Histone Deacylase Activity

Abstract

We developed a one-step direct assay for the determination of histone deacylase (HDAC) activity by substituting the carbonyl oxygen of the acyl moiety with sulfur, resulting in thioacylated lysine side chains. This modification is recognized by class I HDACs with different efficiencies ranging from not accepted for HDAC1 to kinetic constants similar to that of the parent oxo substrate for HDAC8. Class II HDACs can hydrolyze thioacylated substrates with approximately 5−10-fold reduced k cat values, which resembles the effect of thioamide substitution in metallo-protease substrates. Class IV HDAC11 accepts thiomyristoyl modification less efficiently with an ∼5-fold reduced specificity constant. On the basis of the unique spectroscopic properties of thioamide bonds (strong absorption in spectral range of 260−280 nm and efficient fluorescence quenching), HDAC-mediated cleavage of thioamides could be followed by ultraviolet−visible and fluorescence spectroscopy in a continuous manner. The HDAC activity assay is compatible with microtiter plate-based screening formats up to 1536-well plates with Z′ factors of >0.75 and signal-to-noise ratios of >50. Using thioacylated lysine residues in p53-derived peptides, we optimized substrates for HDAC8 with a catalytic efficiency of >250000 M −1 s −1 , which are more than 100-fold more effective than most of the known substrates. We determined inhibition constants of several inhibitors for human HDACs using thioacylated peptidic substrates and found good correlation with the values from the literature. On the other hand, we could introduce N-methylated, N-acylated lysine residues as inhibitors for HDACs with an IC 50 value of 1 μM for an N-methylated, N-myristoylated peptide derivative and human HDAC11.

Dethioacylation by Sirtuins 1–3: Considerations for Drug Design Using Mechanism-Based Sirtuin Inhibition

“…70-6-compared to SIRT1-3). 18,37 Although, expression levels of the various deacylases in the cell of course have a major impact on the degree of conversion as well. To ensure that the observed dethioacylations were not artifacts arising from inhibitor/substrate degradation or thioamide to oxoamide conversion, followed by standard sirtuin deacylation, we performed compound stability tests in assay buffer.…”

Mitochondria-targeted inhibitors of the human SIRT3 lysine deacetylase

“…2B ). Because ε- N -thioacetyllysine residues have been shown to be processed by SIRT1–3 49 and HDAC8, 52 the ε- N ′-methylthiourea functionality ( 4 ) was chosen for further investigations.…”

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

“…To probe this, we selected two H3 N-terminal peptides for modification in a combinatorial manner according to reported histone PTMs (Supplementary Table 4) [48][49] . The included modifications were O-phosphoserine (pS), O-phosphothreonine (pT), ε-N-mono-, di-, and trimethyllysine (Kme1, Kme2 and Kme3, respectively), Kac, and ε-N-thioacetyllysine (Kthioac), which is a Kac analogue with enhanced stability towards hydrolases [50][51] that is only cleaved to some extent by HDACs 6 and 8 52 and class I sirtuins 53 .…”