TY - JOUR A1 - Meyners, Christian A1 - Mertens, Monique A1 - Wessig, Pablo A1 - Meyer-Almes, Franz-Josef T1 - A Fluorescence-Lifetime-Based Binding Assay for Class IIa Histone Deacetylases JF - Chemistry - a European journal N2 - Class IIa histone deacetylases (HDACs) show extremely low enzymatic activity and no commonly accepted endogenous substrate is known today. Increasing evidence suggests that these enzymes exert their effect rather through molecular recognition of acetylated proteins and recruiting other proteins like HDAC3 to the desired target location. Accordingly, class IIa HDACs like bromodomains have been suggested to act as “Readers” of acetyl marks, whereas enzymatically active HDACs of class I or IIb are called “Erasers” to highlight their capability to remove acetyl groups from acetylated histones or other proteins. Small-molecule ligands of class IIa histone deacetylases (HDACs) have gained tremendous attention during the last decade and have been suggested as pharmaceutical targets in several indication areas such as cancer, Huntington's disease and muscular atrophy. Up to now, only enzyme activity assays with artificial chemically activated trifluoroacetylated substrates are in use for the identification and characterization of new active compounds against class IIa HDACs. Here, we describe the first binding assay for this class of HDAC enzymes that involves a simple mix-and-measure procedure and an extraordinarily robust fluorescence lifetime readout based on [1,3]dioxolo[4,5-f]benzodioxole-based ligand probes. The principle of the assay is generic and can also be transferred to class I HDAC8. KW - drug discovery KW - enzymes KW - fluorescent probes KW - high-throughput screening KW - hydrolases Y1 - 2017 U6 - https://doi.org/10.1002/chem.201605140 SN - 0947-6539 SN - 1521-3765 VL - 23 IS - 13 SP - 3107 EP - 3116 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Meyners, Christian A1 - Wawrzinek, Robert A1 - Kraemer, Andreas A1 - Hinz, Steffen A1 - Wessig, Pablo A1 - Meyer-Almes, Franz-Josef T1 - A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe JF - Analytical & bioanalytical chemistry N2 - High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5-f][1,3]benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N'-phenyloctandiamide. KW - Histone deacetylases KW - Acetylpolyamine amidohydrolases KW - Fluorescence life time KW - Binding assay KW - Pseudomonas aeruginosa Y1 - 2014 U6 - https://doi.org/10.1007/s00216-014-7886-5 SN - 1618-2642 SN - 1618-2650 VL - 406 IS - 20 SP - 4889 EP - 4897 PB - Springer CY - Heidelberg ER -