Screening for inhibitors of histone deacetylase by incorporating a spraying method to micro-arrayed compound screening

• Published in: Combinatorial chemistry & high throughput screening Vol. 7; no. 2; p. 93
• Main Authors: Sabet, R S, Marcotte, P, Glaser, K, Burns, D J, Warrior, U, Groebe, D R
• Format: Journal Article
• Language: English
• Published: United Arab Emirates 01.03.2004
• Subjects: Drug Evaluation, Preclinical - methods; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Fluorescence; Histone Deacetylase Inhibitors; Histone Deacetylases - metabolism; Humans; K562 Cells; Miniaturization - methods
• ISSN: 1386-2073
• DOI: 10.2174/138620704773120784

We have developed a method of spraying assay reagents onto a target gel in the Micro-Arrayed Compound Screening ( micro ARCS) format. After application of target gels to compound sheets, subsequent reagents can be applied by spraying onto the target gel. The spraying method conserves on assay reagents by up to 10-fold, eliminates the need for casting additional agarose gels, and increases the throughput of a screen by 3-fold. To demonstrate the efficacy of applying the spraying method to micro ARCS, we screened over 600,000 compounds for inhibitors of histone deacetylase (HDAC). Commercially available HDAC substrate and reaction developer were sprayed directly onto the gel to initiate the reaction and to amplify the signal, respectively. Picks in the primary screen were retested at a density of 384 compounds per sheet in the micro ARCS format. IC(50) values for active compounds were confirmed in a 96-well plate assay. The screen identified several small molecule inhibitors of the enzyme, including members of several classes of known HDAC inhibitors. The combination of the high-density format of micro ARCS, the efficiency of the spraying method, and a timed sequence of adding assay reagents permitted a screening throughput of 200,000 tests an hour. We present the details of the screening format and the analysis of the hits from the screen.