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P2020
HIF-Prolyl Hydroxylase; Preparation of Multiple Reagents to Develop TR-FRET Assay for HTS
Presenter John Martin, GlaxoSmithKline, USA
Additional Authors: Huizhen Zhao, Thau Ho, Melissa Pappalardi, Hong Zhang, Matthew Burns, Gilbert Scott, John Kerrigan, Patricia McCormick, Sharon Sweitzer, Robert Kirkpatrick, Lusong Luo, Kyung Johanson, Christopher Jones, Benjamin Schwartz
Hypoxia Inducible Factors (HIF 1, 2 and 3) are heterodimeric transcription factors which help regulate cellular responses to changing oxygen levels. Under normoxia, prolines of the a subunit of HIFs are hydroxylated by prolyl-4-hydroxylases (knowns as PHDs or EGLNs). This modification serves as a recognition element for a specific E3 ligase (VBC complex), which ubiquitinates the HIFs, thus targeting them for proteosomal degradation. Under hypoxic conditions, the a subunit remains unmodified, accumulates, dimerizes with its b sub-unit, and translocates to nucleus where it binds its co-factors (p300/CREB) and initiates transcription of proteins for the cell to compensate for low oxygen levels. One such response is the increase in production of erythropoietin (EPO). EPO in turn stimulates the production of oxygen carrying red blood cells. Recombinant forms of EPO are currently administered to treat chemotherapy induced anemia. Thus, an inhibitor of EGLNs would afford an orally bioavailable alternative to stimulate EPO production.
For in-house drug discovery efforts, a biochemical HTS assay was constructed to monitor inhibition of EGLN activity. To enable this TR-FRET assay, multiple complex protein reagents were produced. The CODD domain of HIF2a was expressed, purified, and tagged with Cy5. The VBC complex was prepared from its individual components (VHL, Elongin A and Elongin B) by re-folding, and subsequently biotinylated to allow for capture of a Eu-streptavidin chelate. Lastly EGLN3 was produced, and when mixed with the other components catalyzes the hydroxylation of the Cy5-HIF2a CODD. The hydroxylated product is specifically recognized by the Eu-labelled VBC complex, resulting in energy transfer.