Mechanism of binding of the inhibitor (E)-3-(furan-2-yl)-N-hydroxyacrylamide to a histone deacetylase-like amidohydrolase.
Histone deacetylases have proven to be attractive novel targets for the treatment of cancer. The first inhibitor of histone deacetylases was approved for the treatment of cutaneous T-cell lymphoma in 2006. The identification of new lead structures with improved effectiveness and fewer side effects is necessary. This report investigates the mechanism of inhibition of a histone deacetylase-like amidohydrolase by stopped-flow and equilibrium titration techniques. The interaction between the inhibitor (E)-3-(furan-2-yl)-N-hydroxyacrylamide and the enzyme generates a fluorescence resonance energy transfer from the intrinsic tryptophan residues of the enzyme to the chromophore of the inhibitor. The apparent equilibrium binding constant was determined to be 1.9 muM. Several independent experimental results provide evidence of the existence of solely one HDAH conformer. The association kinetics showed two phases representing two unimolecular processes. Kinetic arguments and accurate investigation of the very fast time range suggest a fast pre-equilibrium, in which the inhibitor binds to the surface of the enzyme. In the next step, the first complex undergoes a conformational change that allows the inhibitor to translocate into the active site. Finally, the intermediate complex is stabilized by another conformational rearrangement. All kinetic data are in agreement with a reversible three-step mechanism and analyzed using a global fit, yielding the association constant of the pre-equilibrium (K(1) = 0.28 x 10(6) M(-1)) and the forward and reverse rate constants of the consecutive conformational changes (k(2) = 6.6 s(-1), k(-2) = 1.5 s(-1), k(3) = 0.8 s(-1), and k(-3) = 0.3 s(-1)).
Sykora
Jaromir
J
Department of Chemical Engineering and Biotechnology, University of Applied Sciences, Schnittspahnstrasse 12, 64287 Darmstadt, Germany.
Meyer-Almes
Franz-Josef
FJ
D000421:Alcaligenes / Q000201:enzymology; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001572:Benzofurans / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D001884:Bordetella / Q000201:enzymology; D003460:Crystallization; D031541:Fluorescence Resonance Energy Transfer; D056572:Histone Deacetylase Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006655:Histone Deacetylases / Q000737:chemistry* / Q000378:metabolism*; D006877:Hydroxamic Acids / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics
C523909:3-(3-(benzofuran-2-carbonyl)phenyl)-N-hydroxyacrylamide; D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D001572:Benzofurans; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D006655:Histone Deacetylases