Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors.

BACKGROUND: Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε)-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε)-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. PRINCIPAL FINDINGS: High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. CONCLUSIONS: These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.

A 1,536-well-based kinetic HTS assay for inhibitors of Schistosoma mansoni thioredoxin glutathione reductase.

Abstract: Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-mul final volume assay is based on the Ellman reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate.