Pyrithione Zn selectively inhibits hypoxia-inducible factor prolyl hydroxylase PHD3.

Increasing evidence emphasizes the role of the hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) isoforms in regulating non-HIF substrates, but isoform selective PHD inhibitors under physiological conditions have not yet been reported. Here we have identified pyrithione Zn (PZ) as a potent, isoform-selective PHD3 inhibitor. The IC50 value of PZ was determined as 0.98 µM for PHD3, while it did not show any inhibitory activity toward full length and truncated PHD2 up to 1 mM. The selective efficacy of PZ was further demonstrated at the cellular level by observing inhibition of the PHD3-dependent DNA damage response pathway without stabilization of HIF-1α.

Recent Advances in Developing Inhibitors for Hypoxia-Inducible Factor Prolyl Hydroxylases and Their Therapeutic Implications.

Hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs) are members of the 2-oxoglutarate dependent non-heme iron dioxygenases. Due to their physiological roles in regulation of HIF-1α stability, many efforts have been focused on searching for selective PHD inhibitors to control HIF-1α levels for therapeutic applications. In this review, we first describe the structure of PHD2 as a molecular basis for structure-based drug design (SBDD) and various experimental methods developed for measuring PHD activity. We further discuss the current status of the development of PHD inhibitors enabled by combining SBDD approaches with high-throughput screening. Finally, we highlight the clinical implications of small molecule PHD inhibitors.

Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay.

Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and it coordinates cholesterol and lipid metabolism. Because targeting the FXR-CDCA interaction might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently labeled CDCA (CDCA-F), we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR-LBD) among nuclear receptors. The assay was then used for screening inhibitors against the FXR-CDCA interaction, thereby discovering four relatively potent inhibitors. The selected inhibitors were further studied for changes in intrinsic tryptophan fluorescence of FXR-LBD to gain structural insights into the interaction. Furthermore, transactivation effects of the inhibitors on the human bile salt excretory pump (BSEP) promoter were examined to reveal their cellular activities in the FXR-mediated pathway. Therefore, we demonstrated that the developed assay would offer an efficient primary screening tool for identifying FXR modulators.

Actinomycin D identified as an inhibitor of discoidin domain receptor 2 interaction with collagen through an insect cell based screening of a drug compound library.

Discoidin domain receptors belong to the cell surface receptor tyrosine kinase family and recognize collagens for their activating ligands. They have been implicated for cell growth and migration and their elevated expressions were observed in various human cancers. When we expressed human Discoidin domain receptor 2 (DDR2) in insect cells, the protein was targeted properly into the cell membrane and this could enforce the cells to adhere on culture plate coated with type I collagen. By taking advantage of this, we established a novel insect cell based screening protocol to identify chemicals which inhibit the interaction between DDR2 and collagen. We screened a drug-compound library to select an anti-cancer drug, actinomycin D, as the inhibitory compound. Actinomycin D prevented the activation of DDR2 by type I collagen in human embryonic kidney 293 cells with an IC(50) value of 9 microM, while it did not interfere with the activation of other receptor tyrosine kinases by their ligands. In conclusion we identified a new biological function of actinomycin D and the insect cell based method provides a useful protocol for screening inhibitors against the association of DDR2 with collagen.