Methods of identifying modulators of gpr17
a modulator and modulator technology, applied in the field of methods of identifying modulators of gpr17, can solve the problems of low reported affinity of 5-ala and pbg, unsuitable for routine screening assays or even high throughput screening, and impracticality in routine handling, etc., to achieve significant superiority over published gpr17 agonists, high specificity and potency, and high affinity and selectivity
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Example (1)
hGPR17 Expressing Cell Systems And Media
[0162]The sequence for the human GPR17 (short isoform) was subcloned into the vector pLXSN (Clontech Laboratories, CA 94043, USA), which can be used for retroviral transfection of the human astrocytoma cell line 1321N1. Since it was postulated by Ciana et. al. that GPR17 is activated by uracil nucleotides such as UDP, UDP-glucose and UDP-galactose, 1321N1 astrocytoma cells were chosen as an appropriate test system, because it was known that this cell line does not express endogenous nucleotide receptors. For the retroviral transfection of 1321N1 astrocytoma cells the packaging cell line GP+envAM12 was cultivated in HXM medium (50 ml of foetal calf serum (FCS), 5 ml of penicillin G / streptomycin solution (final concentration 100 U / ml penicillin, 100 μg / ml streptomycin), 1% ultra glutamine, 0.75 ml of hypoxanthine (10 mg / ml), 12.5 ml of xanthine (10 mg / ml), 1.25 ml of mycophenolic acid (10 mg / ml) and 2 ml of hygromycin B (50 mg / ml) wer...
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(4)
Description of 35SGTPYS Binding Assay
[0168][35S]GTPγS binding experiments were carried out in 96-well plates using 5 μg of protein / well. Membranes were added to 400 μl of incubation buffer (10 mM HEPES, 10 mM MgCl2, 100 mM NaCl, pH 7.4) containing 10 μM GDP and RA-II-150 in the indicated concentrations. Reactions were started by adding 50 μl of 0.07 nM [35S]GTPγS and then incubated for 60 min at 30° C. Membrane-bound radioactivity was separated by vacuum filtration. After drying, glass fiber filter mats were melted with scintillation wax, and radioactivity was measured by liquid scintillation counting.
example 5
[0169]Arrestin recruitment was detected in BRET2 assays using HEK293 cells stably expressing human GPR17—Rluc and GFP2-β-arrestin2.
[0170]Cells were detached by trypsinization, counted and washed once in assay buffer (HBSS, 20 mM HEPES (S12), pH 7.0). After centrifugation at 800 rpm for 4 min, the pelleted cells were resuspended in an appropriate volume of assay buffer to a density of 1×106 cells per ml. In order to stabilize readings, cells were allowed to incubate at 28° C. for 30 min while slowly shaking (180 rpm), prior to experiments.
[0171]The coelenterazine 400A stock solution was freshly diluted (3:100) in PBS containing 20% of ethanol to obtain a 30 μM solution, which was always kept in the dark, due to its light-sensitivity.
[0172]As a first step, the agonist solutions were prepared and 10 μl of each 8-fold dilution was dispensed in the 384-well assay plate. DMSO concentrations were adjusted and did not exceed 0.1% in final concentrations.
[0173]Harvested and s...
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