High-throughput screening for identification of small molecule inhibitors of histone acetyltransferases using scintillating microplates (FlashPlate)

The role of histone acetyltransferases (HATs) in the regulation of crucial cellular functions, e.g., gene transcription, differentiation, and prolifer ation, has recently been documented and there is increasing evidence that a berrant expression of these enzymes may have a role to play in the developm ent of the malignant phenotype. The availability of potent and selective sm all molecule inhibitors of HATs would provide useful proof of principle pro bes for further validation of these enzymes as drug discovery targets and m ay also provide lead molecules for clinical drug development. We have devel oped a microplate assay for HAT activity suitable for high-throughput scree ning. In the assay, following incubation of histone H3, [H-3]acetyl-CoA, an d enzyme (recombinant p300/CBP-associated factor expressed as a glutathione S-transferase fusion protein), radiolabeled histone was captured onto the walls of a scintillating microplate (FlashPlate) generating a scintillation signal. The assay was reproducible, amenable to automation, and generated a wide signal to noise ratio. Although antiacetylated histone antibodies we re initially used to capture the radiolabeled product, it was subsequently shown that a signal was effectively produced by histone passively binding t o the walls of the FlashPlate. This resulted in a simple "mix and measure" assay that is currently being used for the identification of HAT inhibitors . (C) 2001 Academic Press.