Kinetic characterization and identification of a novel inhibitor of hypoxia-inducible factor prolyl hydroxylase 2 using a time-resolved fluorescence resonance energy transfer-based assay technology

Pharmacological Characterization of 1-(5-Chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic Acid (JNJ-42041935), a Potent and Selective Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor

“…This ironbinding mechanism of action has further implications for PHD inhibitors that act in this way because the potency for inhibition of PHD enzymes seems to be dependent on the affinity of iron at the active site of the enzyme. This observation is supported by our own observations (Kanelakis et al, 2009) and by the observations of McDonough et al (2006) and Dao et al (2009), which find the K M of PHD2 for iron to be ϳ1 M. The iron chelators tested in the current study had a similar potency and had steep concentration-response curves consistent with the inhibition being dependent on the depletion of active enzyme. Together, these results imply that iron dissociates from the active site of the PHD enzyme and then binds to the most potent iron chelator available (i.e., desferrioxamine and not PHD), resulting in reduced activity of PHD.…”

“…For the full-length PHD2 protein, the histidine tag was not removed because attempts to do so resulted in inactive protein. This is consistent with the experience of others (McDonough et al, 2006;Dao et al, 2009). PHD3 was cloned into baculovirus with an N-terminal histidine tag and was purified on an Ni 2ϩ HiTrap column.…”

“…Warshakoon et al (2006a,b,c,d) describe several different series of compounds that may well act via different molecular mechanisms. More recently, Tegley et al, (2008) and Dao et al (2009) described the compounds identified from a high-throughput screening campaign, which acted in a 2-OG-competitive fashion, whereas Smirnova et al (2010) described hits from a cell-based high-throughput screening campaign that probably inhibit PHD enzymes via iron chelation although this has not been demonstrated. The differences in the mechanism of action can have a dramatic affect on the translation of the pharmacology across different test systems.…”

Preliminary Recognition of c-Myc Gene Protein Using an Optical Biosensor with Gold Colloid Nanoparticles Based on Localized Surface Plasmon Resonance

“…Among many advantages of the optical biosensors, sensitivity and real-time detection of biomolecular interactions gave them wide application (Chan and Nie 1998;McFarland and Van Duyne 2003). As one knows, the optical sensor techniques are based on various sensing transduction mechanisms, such as fluorescence (Gong et al 2006Lee et al 2008;Dao et al 2009), chemiluminescence (Chan et al 2002;Ashkenazi, Abu-Rabeah, and Marks 2009), light absorption and scattering (Malinsky et al 2001;Xiang et al 2008), reflectance (Hicks et al 2005;Filik et al 2008), surface plasmon resonance (SPR) (Chen 2009;Stepanov et al 2009;Joung et al 2008), and Raman scattering (Nie and Emory 1997;Zhang et al 2005;Jiang et al 2003;Jeong et al 2009). Biosensing with surface plasmon resonance (SPR) was first established in the early 1980s and quickly became the benchmark technique for label-free detection over the following 30 years (Hoa, Kirk, and Tabrizian 2007;Fan et al 2008;Leung, Shankar, and Mutharasan 2007;Boozer et al 2006;Shankaran, Gobi, and Miura 2007).…”

Novel Approach to High Throughput Screening for Activators of Transcription Factors

“…This assay, developed by Amgen team, is the best one in terms of sensitivity and applicability for HTS. The assay, in addition to other expensive reagents, utilizes Europium (Dao et al 2009). All enzymatic assays are based on the use of PHD2 recombinant enzyme produced in either baculovirus or E.coli expression system.…”