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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...

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric (containing α and β subunits) transcription factor, is involved in hypoxia response pathway that regulates the expression of many tumorrelated genes. The stabilized HIF-1 heterodimer couples to the general co-activators p300/CBP (CREB binding protein), forming an active transcription factor to initiate hypoxic responses. Inhibiting the transcription factor-coactivator HIF-1α-p300/CBP interaction represents an attractive approach for blocking hypoxia pathway in tumors. Recently, diverse HIF-1α-p300/CBP inhibitors have been designed and their anti-tumor activities have been evaluated. The developments of inhibitors of HIF-1α- p300/CBP are discussed in this review. An outline of structures and biological activities of these inhibitors can be traced, along with the approaches for inhibitors discovery. The challenges in identifying novel and selective potent inhibitors of HIF-1α-p300/CBP are also put forward.

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The design and discovery of a new series of (5-alkynyl-3-hydroxypicolinoyl)­glycine inhibitors of prolyl hydroxylase (PHD) are described. These compounds showed potent in vitro inhibitory activity toward PHD2 in a fluorescence polarization-based assay. Remarkably, oral administration of 17, with an IC50 of 64.2 nM toward PHD2, was found to stabilize HIF-α, elevate erythropoietin (EPO), and alleviate anemia in a cisplatin-induced anemia mouse model with an oral dose of 25 mg/kg. Rat and dog studies showed that 17 has good pharmacokinetic properties, with oral bioavailabilities of 55.7 and 54.0%, respectively, and shows excellent safety profiles even at a high dose of 200 mg/kg in these animals. Based on these results, 17 is currently being evaluated in a phase I clinical trial for anemia.