In vitro binding of a radio-labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5
Article first published online: 6 DEC 2012
Copyright © 2012 Wiley Periodicals, Inc.
Volume 67, Issue 3, pages 135–144, March 2013
How to Cite
Zysk, J. R., Spear, N., Fieles, W., Stein, M. M., Sygowski, L. S., King, M. M., Hoesch, V., Hastings, R., Brockel, B., Do, M., Ström, P., Gadient, R., Chhajlani, V., Elmore, C. S. and Maier, D. L. (2013), In vitro binding of a radio-labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5. Synapse, 67: 135–144. doi: 10.1002/syn.21625
- Issue published online: 17 JAN 2013
- Article first published online: 6 DEC 2012
- Accepted manuscript online: 14 NOV 2012 04:59AM EST
- Manuscript Accepted: 6 NOV 2012
- Manuscript Received: 30 JUN 2012
- metabotropic glutamate receptor subtype 5;
- positive allosteric modulator;
The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu5) lacks a readily available radio-labeled tracer fordetailed structure-activity studies. This communication describes a selective mGlu5 compound, 7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one (PBPyl) that binds with high affinity to human mGlu5 and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC50 of 87 nM with an 89% effect in transfected HEK293 cells and an EC50 of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5-fold higher functional selectivity for mGlu5 in a full mGlu receptor panel. Unlabeled PBPyl was tested for specific binding using a liquid chromatography mass spectrometry (LC/MS/MS)-based filtration binding assay and exhibited 40% specific binding in recombinant membranes, a value higher than any candidate compound tested. In competition binding studies with [3H]MPEP, the mGlu5 receptor negative allosteric modulator (NAM), PBPyl exhibited a k i value of 34 nM. PBPyl also displaced [3H]ABP688, a mGluR5 receptor NAM, in tissue sections from mouse and rat brain using autoradiography. Areas of specific binding included the frontal cortex, striatum and nucleus accumbens. PBPyl was radiolabeled to a specific activity of 15 Ci/mmol and tested for specific binding in a filter plate format. In recombinant mGlu5b membranes, [3H] PBPyl exhibited saturable binding with a Kd value of 18.6 nM. In competition binding experiments, [3H] PBPyl was displaced by high affinity mGlu5 positive and negative modulators. Further tests showed that PBPyl displays less than optimal characteristics as an in vivo tool, including a high volume of distribution and ClogP, making it more suitable as an in vitro compound. However, as a first report of direct binding of an mGlu5 receptor PAM, this study offers value toward the development of novel PET imaging agents for this important therapeutic target. Synapse, 2013. © 2012 Wiley Periodicals, Inc.