| In Vitro |
PF-592379 appears to be a full agonist (Emax=95%) when compared with the standard Pramipexole, a D2/D3 receptor agonist for the treatment of Parkinson’s disease[1]. PF-592379 is a potent and selective dopamine 3 agonist with EC50 and Ki of 21 nM and 322 nM, respectively[2]. In vitro binding assays show that PF-592379 (PF-592,379) selectively binds human D3 receptors with a high affinity (Ki=215 nM). Although PF-592379 also binds to human D4 receptors (Ki=4165 nM), it displays a 19-fold binding selectivity for human D3 over D4 receptors. PF-592379 fails to bind human D2 (Ki≥10 μM), D1 (Ki≥10 μM), or D5 (Ki≥10 μM) receptors at concentrations of up to 10 µM, and thus is at least 46-fold selective for D3 over D2, D1, and D5 receptors[3].
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| Kinase Assay |
The binding affinities of PF-592379 (PF-592,379) and 7-OH-DPAT are characterized at each the five dopamine receptor subtypes using the following radioligands: [3H]SCH23390 (D1 and D5, 70 Ci mmol, 1 nM), [3H]U-86170 (D2, 62 Ci/mmol, 2 nM), and [3H]spiperone (D3 and D4, 96 Ci/mmol, 0.2 nM). CHO cells expressing recombinant human D1, D2, D3, D4, and D5 receptors are rinsed with, and harvested in, ice-cold Ca2+/Mg2+-free phosphate-buffered saline prior to pelleting (500g, 5 min), resuspension in 25 mM Tris, 5 mM EDTA, and 5 mM EGTA, pH 7.5, and freezing the cells in liquid nitrogen. Upon thawing, the cells are homogenized and centrifuged at 1,000g to remove nuclei and unbroken cells, with the supernatant subsequently centrifuged at 47,000g. The membrane pellet is then washed with Tris, EGTA, EDTA, resuspended in 20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 1 mM EDTA, and frozen in liquid nitrogen prior to storage of membrane aliquots at -70°C. Membranes are then thawed and diluted into 20 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM MgCl2, or 1 mM EDTA, 10 mM MgSO4, with binding reactions carried out in a total volume of 0.9 ml for 1 h at room temperature, and stopped by vacuum filtration. Nonspecific binding is assessed with 3 µM SCH23390 (D1-like antagonist) or 3 µM haloperidol (D2-like antagonist). Competition binding experiments employ 11 concentrations of PF-592379 or 7-OH-DPAT run in duplicate. IC50 values are determined by fitting the data to a one-site model by nonlinear least-squares minimization, and Ki values are calculated with the Cheng-Prusoff equation[3].
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