In vitro binding of a radio-labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5

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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 akivalue 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 aKdvalue 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.

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