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Development of a fluorescence polarization based assay for histone deacetylase ligand discovery.

ABSTRACT: Histone deacetylases (HDACs) regulate many important physiological processes and the discovery of small molecules that modulate HDAC activity has both academic and clinical relevance. HDAC inhibitors, most notably SAHA, have been pursued as cancer chemotherapeutics but may be useful in treating psychiatric disorders, malaria, and other diseases. Herein, we describe an inexpensive and robust assay, based on fluorescence polarization, for HDAC ligand discovery. The assay is well suited for high-throughput screening and enzyme kinetic studies.

SUBMITTER: Mazitschek R

PROVIDER: S-EPMC2408937 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Development of a fluorescence polarization based assay for histone deacetylase ligand discovery.

Mazitschek Ralph R Patel Vishal V Wirth Dyann F DF Clardy Jon J

Bioorganic & medicinal chemistry letters 20080407 9

Histone deacetylases (HDACs) regulate many important physiological processes and the discovery of small molecules that modulate HDAC activity has both academic and clinical relevance. HDAC inhibitors, most notably SAHA, have been pursued as cancer chemotherapeutics but may be useful in treating psychiatric disorders, malaria, and other diseases. Herein, we describe an inexpensive and robust assay, based on fluorescence polarization, for HDAC ligand discovery. The assay is well suited for high-th ...[more]

PMID: 18430569

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Project description:Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by activating the BCL-2 family member BAX. Studies investigating molecular regulation of BAX are commonly limited to methodologies measuring endpoint phenotypes and do not assess activation of monomeric BAX. Here, we present FLAMBE, a fluorescence polarization ligand assay for monitoring BAX early activation, that measures activation-induced release of a peptide probe in real time. Using complementary parallel and tandem biochemical techniques, we validate, corroborate, and apply FLAMBE to a contemporary repertoire of BAX modulators, characterizing their contributions within the early steps of BAX activation. Additionally, we use FLAMBE to reveal that historically "dead" BAX mutants remain responsive to activation as quasi-functional monomers. We also identify data metrics for comparative analyses and demonstrate that FLAMBE data align with downstream functional observations. Collectively, FLAMBE advances our understanding of BAX activation and fills a methodological void for studying BAX with broad applications in cell biology and therapeutic development. MOTIVATION In vitro BAX activation studies are invaluable platforms for studying cellular and pharmacological modulators of apoptosis. The gold standard for studying BAX function relies on membrane permeabilization assays, which assess the pore-forming activity of oligomeric BAX. However, there are currently no rapid or kinetic assays to interrogate real-time activation of monomeric BAX in solution, thereby limiting any molecular insights that occur upstream of mitochondrial permeabilization. Furthermore, available methods to observe the activation of monomeric BAX suffer from low throughput and static observations. To address this methodological gap, we developed FLAMBE, a kinetic fluorescence polarization-based assay to measure monomeric BAX activation in solution via concomitant displacement of a labeled peptide. This approach maintains the benefits of rapid kinetic data generation in a low-cost microplate format without requiring specialized equipment or large quantities of protein. FLAMBE compliments available experimental strategies and expands the accessibility of investigators to monitor early steps within the BAX activation continuum.

Dataset Information

Development of a fluorescence polarization based assay for histone deacetylase ligand discovery.

Publications

Development of a fluorescence polarization based assay for histone deacetylase ligand discovery.

Similar Datasets

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