Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu3 selectivity Assay
Selectivity of mGlu5 SAMs was assessed by investigating the ability of 10uM to modulate orthosteric agonist activity at rat mGlu3 (1, 2, 3) co-expressed with Galpha15 to faciliate coupling to Ca mobilization. ..more
Assay Provider: P. Jeffrey Conn
Assay Provider Affiliation: Vanderbilt University
Selectivity of mGlu5 SAMs was assessed by investigating the ability of 10uM to modulate orthosteric agonist activity at rat mGlu3 (1, 2, 3) co-expressed with Galpha15 to faciliate coupling to Ca mobilization.
1. Noetzel, M.J., Rook, J.M., Vinson, P.N., Cho, H., Days, E., Zhou, Y., Rodriguez, A.L., Lavreysen, H., Stauffer, S.R., Niswender, C.M., Xiang, Z., Daniels, J.S., Lindsley, C.W., Weaver, C.D. and Conn, P.J., 2011. Functional Impact of Allosteric Agonist Activity of Selective Positive Allosteric Modulators of mGlu5 in Regulating CNS Function. Molecular Pharmacology, in press.
2. Hammond, A. S., Rodriguez, A. L., Townsend, S. D., Niswender, C. M., Gregory, K. J., Lindsley, C. W., Conn, P. J. (2010) Discovery of a Novel Chemical Class of mGlu(5) Allosteric Ligands with Distinct Modes of Pharmacology ACS Chem. Neurosci. 10:702-716
3. Mueller, R., Dawson, E. S., Meiler, J., Rodriguez, A. L., Chauder, B. A., Bates, B. S., Felts, A. S., Lamb, J. P., Menon, U. N.Jadhav, S. B., Kane, A. S., Jones, C. K., Gregory, K. J., Niswender, C. M., Conn, P. J., Olsen, C. M., Winder, D. G., Emmitte, K. A., Lindsley, C. W. (2012)Discovery of 2-(2-benzoxazoyl amino)-4-aryl-5-cyanopyrimidine as negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGlu(5)): from an artificial neural network virtual screen to an in vivo tool compound. ChemMedChem 7:406-414.
rmGlu3+Galpha15/TRex293 cells were cultured in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% tetracycline-free fetal bovine serum (FBS), 20 mM HEPES, 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine, 100ug/mL hygromycin, 5ug/mL blasticidin S and 500ug/mL G418.
rmGlu3+Galpha15/TRex293 cells were plated the day before the experiment at a density of 40,000 cells/well in Poly-D-Lysine coated 96-well Microplates (black walled/clear bottom, Costar) in assay medium (DMEM, 20 mM HEPES, 10% dialyzed FBS, 1 mM sodium pyruvate) and treated with 50ng of tetracyline to induce expression of mGlu3. On the day of the experiment, the media was removed and the cells washed with assay buffer (HBSS, 20 mM HEPES, 2.5 mM probenecid, pH 7.4) using an ELX405 cell plate washer. A Ca2+-sensitive fluorescent dye (Fluo4-AM, Invitrogen Corp) was added at a final concentration of 1 uM. Note: the presence of probenecid, an anion exchange inhibitor, minimizes the dye being pumped out of the intracellular environment allowing it to be available to bind intracellular Ca+2. The cells were allowed to take up the dye during an incubation period of 60 min at 37 degrees C/5% CO2. The cells were then washed with assay buffer and, after an incubation period of 3 minutes at room temperature, the assay was started.
A Flexstation2 or Flexstation3 (Molecular Devices) was utilized to evaluate changes in fluorescence intensity of Fluo4. Test compounds were provided as 10 mM stocks in DMSO. Final concentration of DMSO in the assay was 1%.
The concentration of glutamate ranged from 10nM to 1mM (final concentrations) in a 9-point curve and were made 10X in duplicate. Two additions were made within the Flexstation. First, compound alone (made at 3X; 10uM final) was added to each well at 20 seconds to detect any agonist activity. Next, different concentrations of glutamate was added at 80 seconds to detect any modulation of the glutamate response by the test compound.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)