title: 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.
authors.affiliation: AstraZeneca Pharmaceuticals, Department of Neuroscience, CNS R&D, Wilmington, Delaware, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Zysk
authors.forename: Nathan
authors.initials: N
authors.lastname: Spear
authors.forename: William
authors.initials: W
authors.lastname: Fieles
authors.forename: Mark M
authors.initials: MM
authors.lastname: Stein
authors.forename: Linda S
authors.initials: LS
authors.lastname: Sygowski
authors.forename: Megan M
authors.initials: MM
authors.lastname: King
authors.forename: Valerie
authors.initials: V
authors.lastname: Hoesch
authors.forename: Richard
authors.initials: R
authors.lastname: Hastings
authors.forename: Becky
authors.initials: B
authors.lastname: Brockel
authors.forename: Mylinh
authors.initials: M
authors.lastname: Do
authors.forename: Peter
authors.initials: P
authors.lastname: Ström
authors.forename: Reto
authors.initials: R
authors.lastname: Gadient
authors.forename: Vijay
authors.initials: V
authors.lastname: Chhajlani
authors.forename: Charles S
authors.initials: CS
authors.lastname: Elmore
authors.forename: Donna L
authors.initials: DL
authors.lastname: Maier
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D054833:Isoindoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D010091:Oximes / Q000494:pharmacology; D011485:Protein Binding; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011849:Radioactive Tracers; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C510950:3-(6-methylpyridin-2-ylethynyl)cyclohex-2-enone-O-methyloxime; C581949:7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one; D018691:Excitatory Amino Acid Antagonists; D054833:Isoindoles; D010091:Oximes; D011725:Pyridines; D011849:Radioactive Tracers; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; C121465:6-methyl-2-(phenylethynyl)pyridine