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

Abstract

The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu(5)) lacks a readily available radio-labeled tracer fordetailed structure-activity studies. This communication describes a selective mGlu(5) compound, 7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one (PBPyl) that binds with high affinity to human mGlu(5) and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC(50) of 87 nM with an 89% effect in transfected HEK293 cells and an EC(50) of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5-fold higher functional selectivity for mGlu(5) 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 [(3)H]MPEP, the mGlu(5) receptor negative allosteric modulator (NAM), PBPyl exhibited a k(i) value of 34 nM. PBPyl also displaced [(3)H]ABP688, a mGluR(5) 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 mGlu(5b) membranes, [(3)H] PBPyl exhibited saturable binding with a K(d) value of 18.6 nM. In competition binding experiments, [(3)H] PBPyl was displaced by high affinity mGlu(5) 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 mGlu(5) receptor PAM, this study offers value toward the development of novel PET imaging agents for this important therapeutic target.

Activation of the mGlu5 Receptor for the Treatment of Schizophrenia and Cognitive-Deficit-Associated Disorders

“…11). In a FLIPR assay, it had similar activity on human recombinant mGlu 5 (EC 50 ¼ 87 nM, Glu max ¼ 89%) and rat cortical neurons (EC 50 ¼ 81 nM, E max ¼ 42%), so that activity in the overexpressing human cell line might translate to a native preparation [97]. It should be noticed that only mGlu 2 showed selectivity for PBPyl (EC 50 ¼ 430 nM).…”

Neuroligin-mediated neurodevelopmental defects are induced by mitochondrial dysfunction and prevented by lutein inC. elegans

“…These results suggest that these animals up-regulate stress response defensive mechanisms. Indeed, we observed that nuo-5 and lpd-5 RNAi increased the expression of genes involved in mitochondrial quality control pathways [22,36,37]: the antioxidant response gene glutathione S-transferase gst-4, the mitochondrial unfolded protein response gene hsp-6, and the mitophagy regulator Bnip3 homolog dct-1 (Figure 2i-k; Figure S4a). Disturbing C. elegans mitochondrial function also induces autophagy, lipid remodeling [38] as well as drug detoxification and pathogenresponse genes [39].…”