Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: Fold-shift Assay
Silent allosteric modulators of metabotropic glutamate receptor 5 (mGlu5) occupy allosteric sites but have no intrinsic modulator activity. Thus, such compounds have potential as crucial tools to dissect receptor function and efficacy of novel positive and negative allosteric modulators both in vitro and in vivo. Previous mGlu5 SAMs, VU0365396 and 5MPEP, have low affinity and are therefore more ..
Depositor Specified Assays
Assay Provider: P. Jeffrey Conn
Assay Provider Affiliation: Vanderbilt University
Silent allosteric modulators of metabotropic glutamate receptor 5 (mGlu5) occupy allosteric sites but have no intrinsic modulator activity. Thus, such compounds have potential as crucial tools to dissect receptor function and efficacy of novel positive and negative allosteric modulators both in vitro and in vivo. Previous mGlu5 SAMs, VU0365396 and 5MPEP, have low affinity and are therefore limited in their utility as tools (1, 2). The mGlu5 acetylene PAM series is prone to "molecular switches" within the SAR and certain PAMs had previously been shown to exhibit these "molecular switches" when key residues within the the common allosteric site were mutated (3). Thus, this series was predicted to also contain hitherto unappreciated mGlu5 SAMs.
To identify and evaluate mGlu5 SAMs, we screened previously identified inactive compounds in an acetylene PAM series at 10uM, to confirm they were inactive. Confirmed "inactives" were then screened in a 3 pt binding assay to rapidly identify compounds that retained appreciable affinity for the common allosteric site and thus categorise these compounds as "SAMs". SAMs with sub-100nM affinities were then selected for determination of binding affinity, using a more rigorous 10 pt inhibition binding curve,
1. Rodriguez, A. L., Nong, Y., Sekaran, N. K., Alagille, D., Tamagnan, G. D., Conn, P. J. (2005) A close structural analog of 2-methyl-6-(phenylethynyl)-pyridine acts as a neutral allosteric site ligand on metabotropic glutamate receptor subtype 5 and blocks the effects of multiple allosteric modulators. Mol. Pharmacol. 68:1793-1802.
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. Gregory, K. J., Nguyen, E. D., Reiff, S. D., Squire, E. F., Stauffer, S. R., Lindsley, C. W., Meiler, J., Conn, P. J. (2013) Probing the Metabotropic Glutamate Receptor 5 (mGlu5) Positive Allosteric Modulator (PAM) Binding Pocket: Discovery of Point Mutations that Engender a "Molecular Switch" in PAM Pharmacology. Mol Pharmacol (in press)
4. 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.
Creation and culture of the rat mGlu5 cell line. Monoclones of stable HEK293A cells expressing rat mGlu5 were generated, selected, and tested for functional Ca+2 response (4). The selected clone (designated as "R10A") exhibited robust Ca+2 response to addition of glutamate, demonstrated specific binding of the specific mGlu5 radioligand [3H]-mPEPy, and showed an mGlu5-postive signal in a Western blot analysis.
rmGlu5/HEK293A cells were cultured in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS), 20 mM HEPES, 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine, and 500 ug/ml G418 (Mediatech, Inc., Herndon, VA). All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
rmGlu5/HEK293A 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). 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)