posted on 2019-11-14, 15:44authored byMatthes Zessin, Zsófia Kutil, Marat Meleshin, Zora Nováková, Ehab Ghazy, Diana Kalbas, Martin Marek, Christophe Romier, Wolfgang Sippl, Cyril Bařinka, Mike Schutkowski
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 kcat 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 IC50 value of
1 μM for an N-methylated, N-myristoylated peptide derivative
and human HDAC11.