Affinity-Based Fluorescence Polarization Assay for High-Throughput Screening of Prolyl Hydroxylase 2 Inhibitors

Abstract

Prolyl hydroxylase domain 2 (PHD2) enzyme, a Fe II and 2-oxoglutarate (2-OG) dependent oxygenase, mediates key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). PHD2 has been shown to have the therapeutic potentials for conditions including anemia and ischemic disease. Currently, many activity-based assays have been developed for identifying PHD2 inhibitors. Here we report an affinity-based fluorescence polarization method using FITC-labeled HIF1α (556−574) peptide as a probe for quantitative and site-specific screening of small molecule PHD2 inhibitors. KEYWORDS: PHD2, FITC-labeled probe, affinity-based, fluorescence polarization assay H ypoxia is linked to human diseases such as anemia and ischemia. 1,2 In humans, the response to hypoxia is mediated by up-regulation of the hypoxia inducible transcription factor (HIF). 3−5 HIF is a heterodimeric transcription factor composed of an oxygen-dependent α-subunit and constitutively expressed β-subunit. Under normoxic conditions, HIFα, the regulatory subunit of HIF dimer, is constitutively produced with a half-life of approximately 5 min. It is hydroxylated by prolyl hydroxylases1−3 (PHD1−3), recognized by von Hippel Lindau protein (pVHL), and then rapidly ubiquitylated and subsequently degraded by the 26S proteasome. 6 PHDs are members of the dioxygenase family that require O 2 , Fe II , and 2-oxoglutarate (2-OG) for their catalytic activity, which are responsible for the C4 trans hydroxylation of HIFα at Pro402 and Pro564 that initiates the path to protein degradation. 7 It is currently believed that PHD2 plays a dominant role in controlling the cellular HIFα levels. 8 Inhibitor of PHD2 has been pursued as a promising therapy for conditions including anemia and ischemic disease.To discover small molecules that can regulate PHD2 activity, many activity-based assays have been developed. The development of activity-based assay was based on the catalytic activity of PHD2, which utilizes 2-OG and oxygen as cosubstrates to catalyze the prolyl hydroxylations. 9−11 This property has led to the development of several generic activity-based assays, which detected the activity by measuring the ratio of HIFα peptide and its hydroxylated product, such as fluorescence-based assay using o-phenylenediamine, 12 MALDI-TOF MS, 13 AlphaScreen assay, 14 homogeneous time-resolved fluorescence assay, 15 and fluorescence polarization assay based on HIF-von Hippel− Lindau protein-Elongin B−Elongin C (VBC) interaction. 16 However, activity-based assays are not always well-suited to the initial stages of medicinal chemistry, for example, for fragmentbased screening, and are only possible when substrates are available. Recently, affinity-based assays that utilize nondenaturing electrospray ionization mass spectrometry (ESI-MS), 17 affinity selection mass spectroscopy assay (AS-MS), 15 or nuclear magnetic resonance (NMR) 18 technology have been developed for studying the binding of metal ions and small molecules with PHD2 protein. Amo...

A small-molecule probe for monitoring binding to prolyl hydroxylase domain 2 by fluorescence polarisation

“…The IC 50 values of FG-4592, AKB-6548, and 1 for inhibition of the PHD2/probe 12 interaction were 120.8 AE 3.8 nM, 215.1 AE 2.1 nM, and 21.5 AE 2.3 nM, respectively. These values are in accord with the IC 50 values determined by the HIF-1a peptide-based FP (FG-4592, 591.4 nM; AKB-6548, 608.7 nM; 1, 77.7 nM) 11 and SPE MS substrate turnover (FG-4592, 2.587 mM; 1, 0.276 mM) assays (Fig. 6).…”

“…We and others have reported various assays for measuring PHD catalysis, [7][8][9][10][11] including by monitoring 2OG consumption 7 and by monitoring substrate depletion/product formation by mass spectrometry (MS)/nuclear magnetic resonance (NMR), 8,9 or antibody based methods. 10 Whilst these assays can be useful for identifying potent PHD inhibitors, they do not provide information on binding constants, in particular to the biologically relevant PHD-Fe complexes.…”

“…employing matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) (50 mM), 8 NMR (10 mM), 9 or peptide-based FP assays (100 nM). 11 We investigated the dependency of the binding affinity on factors including pH (range 4-10), DMSO concentration, and incubation time ( Fig. S6-S9, ESI †).…”

Discovery of prolyl hydroxylase 2 inhibitors with new chemical scaffolds as in vivo active erythropoietin inducers through a combined virtual screening strategy

“…The 35 hit compounds with diverse scaffolds were commercially purchased from Topscience (Co., Ltd, China). Their inhibitory activity toward PHD2 was evaluated in vitro using a fluorescence polarization assay developed by our group previously (Lei et al, ). All these compounds were preliminarily tested for PHD2 inhibitory activity at 20 μM, and full IC 50 values were evaluated for compounds showing an inhibitory rate more than 50% at the tested concentration.…”

Discovery of Fluorescence Polarization Probe for the ELISA-Based Antagonist Screening of α1-Adrenergic Receptors

“…We also measured the Z′ factor in both the traditional assay and the ELISA-like assay ( Figure S4b), which is a statistical benchmark to judge whether the response in a particular assay is large enough to warrant further attention. 30,31 The Z′ factors calculated for our ELISA-based FP assay are 0.84, 0.77, and 0.70 for α 1A -, α 1B -, and α 1D -AR proteins, respectively ( Figure S4Bb), and in the traditional FP assay, the Z′ factors are −0.49, −0.64, and −0.56, respectively ( Figure S4Ab). A Z′ factor greater than 0.5 means the assay is considered acceptable for a good HTS, while that less than 0 confirms there is too much overlap between the positive and negative controls for the assay to be used.…”