One-Atom-Substitution Enables Direct and Continuous Monitoring of Histone Deacylase Activity.
Fiche publication
Date publication
novembre 2019
Journal
Biochemistry
Auteurs
Membres identifiés du Cancéropôle Est :
Dr ROMIER Christophe
Tous les auteurs :
Zessin M, Kutil Z, Meleshin M, Novakova Z, Ghazy E, Kalbas D, Marek M, Romier C, Sippl W, Bařinka C, Schutkowski M
Lien Pubmed
Résumé
We developed a one-step direct assay for determination of histone deacylase (HDAC) activity by substituting the carbonyl oxygen of the acyl moiety by sulfur resulting in thioacylated lysine side chains. This modification is recognized by class I HDACs with different efficiency ranging from not accepted for HDAC1 to kinetic constants similar to the parent oxo substrate for HDAC8. Class II HDACs are able to hydrolyze thioacylated substrates with about 5-10-fold reduced kcat values resembling the effect of thioamide substitution in metallo-protease substrates. Class IV HDAC11 accepts thiomyristoyl modification less efficient with about 6-fold reduced specificity constant. Based on the unique spectroscopic properties of thioamide bonds (strong absorp-tion in spectra range 260-280 nm and efficient fluorescence quenching) HDAC-mediated cleavage of thioamides could be fol-lowed by UV-Vis 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 higher than 0.75 and a signal-to-noise ratios higher than 50. Using thioacylated lysine residues in p53-derived peptides we optimized substrates for HDAC8 with catalytic efficiency over 250,000 M-1s-1, which are more than 100-fold more effective than most of the known substrates. We determined inhibition con-stants of several inhibitors for human HDACs using thioacylated peptidic substrates and found good correlation with the values known 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.
Référence
Biochemistry. 2019 Nov 4;: