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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 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.

Kinetics of inhibition have been determined for the interaction of human leukocyte elastase (HLE) with two series of peptide trifluoromethyl ketones (TFMKs): X-Val-CF3,X-Pro-Val-CF3,X-Val-Pro-Val-CF3, and X-Lys(Z)-Val-Pro-Val-CF3, where X is MeOSuc or Z. These compounds are "slow-binding" inhibitors of HLE and, thus, allow the determination of Ki, the dissociation constant for the stable complex of inhibitor and enzyme, as well as kon and koff, the rate constants for formation and decomposition of this complex. Maximal potency is reached with Z-Lys(Z)-Val-Pro-Val-CF3, which displays a Ki less than 0.1 nM. Upon binding to HLE, these compounds undergo addition by the hydroxyl of the active site serine to form a hemiketal. The evidence supporting a hemiketal intermediate includes Ki values of 1.6 and 80,000 nM for Z-Val-Pro-Val-CF3 and its alcohol analogue, linear free energy correlations between inhibitory potency and catalytic efficiency for structurally related TFMKs and substrates, and the pH dependence of kon for the inhibition of HLE by Z-Val-Pro-Val-CF3, which is sigmoidal and displays a pKa of 6.9. Hemiketal formation is probably not rate limiting, however. Kinetic solvent isotope effects of unity suggest that kon cannot be rate limited by a reaction step, like hemiketal formation, that is subject to protolytic catalysis. A general mechanism that is consistent with these results is one in which formation of the hemiketal is rapid and is followed or preceded by a slow step that rate limits kon.(ABSTRACT TRUNCATED AT 250 WORDS)