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Characterization of a Robust Enzymatic Assay for Inhibitors of 2-Oxoglutarate-Dependent Hydroxylases

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California, kkanelak{at}its.jnj.com

Heather L. Palomino

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Lina Li

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Jiejun Wu

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Wen Yan

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Mark D. Rosen

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Michele C. Rizzolio

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Meghana Trivedi

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California, University of California, San Diego, Skaggs School of Pharmacy and Pharmaceutical Sciences, La Jolla, California

Magda F. Morton

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Young Yang

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Hariharan Venkatesan

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Michael H. Rabinowitz

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Nigel P. Shankley

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

Terrance D. Barrett

Department of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development L.L.C., San Diego, California

The prolyl-4-hydroxylase proteins regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylation of proline residues targeting HIF-1 for proteasomal degradation. Using the purified catalytic domain of prolyl hydroxylase 2 (PHD2_181-417), an enzymatic assay has been developed to test inhibitors of the enzyme in vitro. Because PHD2 hydroxylates HIF-1, with succinic acid produced as an end product, radiolabeled [5-¹⁴C]-2-oxoglutaric acid was used and formation of [¹⁴C]-succinic acid was measured to quantify PHD2_181-417 enzymatic activity. Comparison of the separation of 2-oxoglutaric acid and succinic acid by either ion exchange chromatography or precipitation with phenylhydrazine showed similar results, but the quantification and throughput were vastly increased using the latter method. The PHD2 reaction was substrate and concentration dependent. The addition of iron to the enzyme reaction mix resulted in an increase in enzymatic activity. The K_m value for 2-oxoglutaric acid was determined to be 0.9 µM, and known PHD2 inhibitors were used to validate the assay. In addition, the authors demonstrate that this assay can be applied to other 2-oxoglutaric acid-dependent enzymes, including the asparaginyl hydroxylase, factor-inhibiting HIF-1 (FIH). A concentration-dependent increase in succinic acid production using recombinant FIH enzyme with a synthetic peptide substrate was observed. The authors conclude that a by-product enzyme assay measuring the conversion of 2-oxoglutaric acid to succinic acid using the catalytic domain of the human PHD2 provides a convenient method for the biochemical evaluation of inhibitors of the 2-oxoglutaric acid-dependent hydroxylases. (Journal of Biomolecular Screening 2009:627-635)

Key Words: HIF-1 • prolyl hydroxylase • 2-oxoglutaric acid • iron • FIH

This version was published on July 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 6, 627-635 (2009)
DOI: 10.1177/1087057109333976


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