title: Endogenous 2-oxoglutarate levels impact potencies of competitive HIF prolyl hydroxylase inhibitors.
abstract: The stability and transcriptional activity of the hypoxia-inducible factors (HIFs) are regulated by oxygen-dependent hydroxylation that is catalyzed by three HIF prolyl 4-hydroxylases (HPHs). Use of HPH inhibition as a mean for HIF-upregulation has recently gained interest as a potential treatment paradigm against neurodegenerative diseases like ischemia and Parkinson's disease. In the present investigation we report the development of a new and robust assay to measure HPH activity. The assay is based on capture of hydroxylated peptide product by the von Hippel-Lindau protein which is directly measured in a scintillation proximity assay. In addition we describe the determination of HPH subtype potencies of HPH inhibitors which either directly or indirectly inhibit the HPH enzyme. The potencies of the HPH inhibitors displayed almost identical IC(50) values toward the HPH1 and HPH2 subtype while the potency against the HPH3 subtype was increased for several of the compounds. For the most potent compound, a hydroxyl thiazole derivative, the potency against HPH2 and HPH3 was 7nM and 0.49nM, respectively corresponding to a 14-fold difference. These results suggest that HPH subtype-selective compounds may be developed. In addition we determined the 2-oxoglutarate concentration in brain tissue and neuronal cell lines as 2-oxoglutarate is an important co-factor used by the HPH enzyme during the hydroxylation reaction. The high intracellular 2-oxoglutarate concentration provides an explanation for the diminished cellular HIF activating potency of a competitive HPH inhibitor compared to its orders of magnitude higher HPH inhibiting potency. The present reported data suggest that in the development of specific Hif prolyl hydroxylase inhibitors the high 2-oxoglutarate tissue level should be taken into account as this might affect the cellular potency. Thus to specifically inhibit the intracellular HPH enzymatic reaction a competitive inhibitor with a low Ki should be developed.
authors.affiliation: Dept. of Neurodegeneration, H. Lundbeck A/S, Valby, Denmark. keth@lundbeck.com
authors.forename: Kenneth
authors.initials: K
authors.lastname: Thirstrup
authors.forename: Søren
authors.initials: S
authors.lastname: Christensen
authors.forename: Henriette Aaris
authors.initials: HA
authors.lastname: Møller
authors.forename: Andreas
authors.initials: A
authors.lastname: Ritzén
authors.forename: Ann-Louise
authors.initials: AL
authors.lastname: Bergström
authors.forename: Thomas Nikolaj
authors.initials: TN
authors.lastname: Sager
authors.forename: Henrik Sindal
authors.initials: HS
authors.lastname: Jensen
mesh_terms: D002460:Cell Line; D049308:Dioxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007656:Ketoglutaric Acids / Q000378:metabolism*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007656:Ketoglutaric Acids; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D049308:Dioxygenases; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C447780:EGLN2 protein, human; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases