Utilization of an In Vivo Reporter for High Throughput Identification of Branched Small Molecule Regulators of Hypoxic Adaptation

SummarySmall molecules inhibiting hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are the focus of drug development efforts directed toward the treatment of ischemia and metabolic imbalance. A cell-based reporter produced by fusing HIF-1α oxygen degradable domain (ODD) to luciferase was shown to work as a capture assay monitoring stability of the overexpressed luciferase-labeled HIF PHD substrate under conditions more physiological than in vitro test tubes. High throughput screening identified novel catechol and oxyquinoline pharmacophores with a “branching motif” immediately adjacent to a Fe-binding motif that fits selectively into the HIF PHD active site in in silico models. In accord with their structure-activity relationship in the primary screen, the best “hits” stabilize HIF1α, upregulate known HIF target genes in a human neuronal line, and exert neuroprotective effects in established model of oxidative stress in cortical neurons.

Graphical AbstractFigure optionsDownload high-quality image (306 K)Download as PowerPoint slideHighlights
► HIF1 ODD-luciferase fusion protein designed for in vivo imaging has been used for HTS
► Mutations analysis validates the reporter as a surrogate for endogenous HIF stability
► HTS of 85,000 compounds identified novel small molecule inhibitors of HIF PHDs
► New branched HIF PHD inhibitors are neuroprotective in oxidative stress model