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Novel Scaffolds of Cell-Active Histone Demethylase Inhibitors Identified from High-Throughput Screening.

ABSTRACT: Jumonji C domain-containing histone demethylases (JHDMs) are epigenetic proteins capable of demethylating methylated lysine residues on histones proteins and for which high-quality chemical probes and eventual therapeutic leads are highly desirable. To expand the extent of known scaffolds targeting JHDMs, we initiated an unbiased high-throughput screening approach using a fluorescence polarization (FP)-based competitive binding assay we recently reported for JHDM1A (aka KDM2A). In total, 14,400 compounds in the HitFinder collection v.11 were screened, which represent all the distinct skeletons of the Maybridge Library. An eventual three compounds with two new scaffolds were discovered and further validated, which not only show in vitro binding for two different JHDMs, JHDM1A and JMJD2A (aka KDM4A), but also induce hypermethylation of their substrate in cells. These represent novel scaffolds as JHDM inhibitors and provide a basis for future optimization of affinity and selectivity.

SUBMITTER: Wang W

PROVIDER: S-EPMC4475453 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Novel Scaffolds of Cell-Active Histone Demethylase Inhibitors Identified from High-Throughput Screening.

Wang Wei W Marholz Laura J LJ Wang Xiang X

Journal of biomolecular screening 20150416 6

Jumonji C domain-containing histone demethylases (JHDMs) are epigenetic proteins capable of demethylating methylated lysine residues on histones proteins and for which high-quality chemical probes and eventual therapeutic leads are highly desirable. To expand the extent of known scaffolds targeting JHDMs, we initiated an unbiased high-throughput screening approach using a fluorescence polarization (FP)-based competitive binding assay we recently reported for JHDM1A (aka KDM2A). In total, 14,400 ...[more]

PMID: 25883088

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Project description:BACKGROUND: Previous studies from our laboratory and others have demonstrated that in addition to altering chromatin acetylation and conformation, histone deacetylase inhibitors (HDACi) disrupt the acetylation status of numerous transcription factors and other proteins. A whole genome yeast deletion library screen was used to identify components of the transcriptional apparatus that modulate the sensitivity to the hydroxamic acid-based HDACi, CG-1521. RESULTS: Screening 4852 haploid Saccharomyces cerevisiae deletion strains for sensitivity to CG-1521 identifies 407 sensitive and 80 resistant strains. Gene ontology (GO) enrichment analysis shows that strains sensitive to CG-1521 are highly enriched in processes regulating chromatin remodeling and transcription as well as other ontologies, including vacuolar acidification and vesicle-mediated transport. CG-1521-resistant strains include those deficient in the regulation of transcription and tRNA modification. Components of the SAGA histone acetyltransferase (HAT) complex are overrepresented in the sensitive strains, including the catalytic subunit, Gcn5. Cell cycle analysis indicates that both the wild-type and gcn5? strains show a G1 delay after CG-1521 treatment, however the gcn5? strain displays increased sensitivity to CG-1521-induced cell death compared to the wild-type strain. To test whether the enzymatic activity of Gcn5 is necessary in the response to CG-1521, growth assays with a yeast strain expressing a catalytically inactive variant of the Gcn5 protein were performed and the results show that this strain is less sensitive to CG-1521 than the gcn5? strain. CONCLUSION: Genome-wide deletion mutant screening identifies biological processes that affect the sensitivity to the HDAC inhibitor CG-1521, including transcription and chromatin remodeling. This study illuminates the pathways involved in the response to CG-1521 in yeast and provides incentives to understand the mechanisms of HDAC inhibitors in cancer cells. The data presented here demonstrate that components of the SAGA complex are involved in mediating the response to CG-1521. Additional experiments suggest that functions other than the acetyltransferase activity of Gcn5 may be sufficient to attenuate the effects of CG-1521 on cell growth.

Dataset Information

Novel Scaffolds of Cell-Active Histone Demethylase Inhibitors Identified from High-Throughput Screening.

Publications

Novel Scaffolds of Cell-Active Histone Demethylase Inhibitors Identified from High-Throughput Screening.

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