Stephen D. Hess

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

Molecular and functional characterization of recombinant human metabotropic glutamate receptor subtype 5.

We have isolated and characterized overlapping cDNAs that encode two isoforms of the human metabotropic glutamate receptor subtype 5 (hmGluR5). The deduced amino acid sequences of human and rat mGluR5a are 94.5% identical. However, a region in the putative cytoplasmic domain (SER926-ALA1121) displays significant sequence divergence. Genomic analysis of this region showed that the sequence divergence results from species-specific differences in the genomic sequences, not from alternative splicing. The distribution of mGluR5 mRNA in human brain was most strongly detected throughout the hippocampus, with moderate levels in the caudate-putamen, cerebral cortex, thalamus, and deep cerebellar nuclei, and at low levels in the cerebellar cortex. Activation of both hmGluR5a and hmGluR5b transiently expressed in Xenopus oocytes and HEK293 cells was coupled to inositol phosphate (InsP) formation and elevation of the intracellular free calcium ([Ca2+]i). The agonist rank order of potency for activating recombinant hmGluR5a receptors in either system was quisqualate > L-glutamate > 1S,3R-ACPD. Both the quisqualate stimulated InsP and [Ca2+]i were inhibited by (+)-MCPG. Recombinant human mGluR5a was also stably expressed in mouse fibroblast Ltk- cells, in which the efficacy and potency of quisqualate were unchanged for more than 30 cell passages.

Functional Characterization of Human N‐Methyl‐d‐Aspartate Subtype 1A/2D Receptors

Abstract: The human NMDAR2D subunit was cloned, and the pharmacological properties of receptors resulting from injection of transcripts encoding human NMDAR1A and NMDAR2D subunits in Xenopus oocytes were characterized by profiling NMDA receptor agonists and antagonists. We found that glutamate, NMDA, glycine, and d‐serine were significantly more potent on hNMDAR1A/2D than on hNMDAR1A/2A or hNMDAR1A/2B. Also, the potencies of NMDA and glycine were higher for hNMDAR1A/2D than for hNMDAR1A/2C. Ifenprodil was more potent at hNMDAR1A/2B than at hNMDAR1A/2D, whereas 5,7‐dichlorokynurenate was more potent at hNMDAR1A/2A than at hNMDAR1A/2D. As measured in transiently transfected human embryonic kidney 293 cells, the maximal inward current in the presence of external Mg2+ occurred at −40 mV, and full block was not observed at negative potentials. Kinetic measurements revealed that the higher affinity of hNMDAR1A/2D for both glutamate and glycine relative to hNMDAR1A/2A and hNMDA1A/2B can be explained by slower dissociation of each agonist from hNMDAR1A/2D. The hNMDAR1A/2D combination represents a pharmacologically and functionally distinct receptor subtype and may constitute a potentially important target for therapeutic agents active in the human CNS.

Development of the Predictor hERG Fluorescence Polarization Assay Using a Membrane Protein Enrichment Approach

The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.

Fluorescence techniques for measuring ion channel activity.

Publisher Summary Changes in intracellular free calcium concentration ([Ca 2+ ] i ) play a crucial role in cellular physiology. A number of cell surface receptors and channels are known to regulate [Ca 2+ ] i through different molecular mechanisms. Therefore, the functional and pharmacologic properties of many of these cell surface receptors and ion channels can be studied effectively by measuring changes in [Ca 2+ ] i in intact cells. For drug discovery efforts, several ion channel and receptor systems have been targeted that play different roles in neuronal physiology and pathophysiology. These molecular targets include voltage- and ligand-gated ion channels: the human neuronal voltage-gated calcium channels (VGCCs), ligand-gated nicotinic acetylcholine receptor channels (NAChRs), ionotropic N -methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainate-type excitatory amino acid receptor (EAA) channels. All of these channels mediate elevation of [Ca 2+ ] i via Ca 2+ influx from the extracellular medium upon depolarization or activation by agonist. This chapter describes the experimental methods with particular emphasis on the validation of the assay for human VGCCs, NAChRs, and NMDA receptors.

The human N-methyl-D-aspartate receptor 2C subunit: genomic analysis, distribution in human brain, and functional expression.

Abstract: cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C‐1, ‐2, ‐3, and ‐4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C‐1 is 89.0%. The sequences of the rat and human carboxyl termini (Gly925‐Val1,236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist‐induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C‐1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7‐dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/2C‐3 or hNR1A/2C‐4 did not display agonist responses. In contrast, we observed an agonist‐induced elevation of intracellular free calcium and whole‐cell currents in cells expressing hNR1A/2C‐1 or hNR1A/2C‐2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C‐1 or hNR1A/2C‐2.

Recent Advances in the Modulation of Voltage-Gated Ion Channels for the Treatment of Epilepsy

Regardless of the voltage-gated ion channel that is targeted in a drug discovery effort for the treatment of epilepsy, two routes have been followed historically: 1). a compound initially, and often surreptitiously, discovered due to activity in animal seizure models is further optimized by medicinal chemistry, or 2). a molecular target is identified based on the phenotype of transgenic animals, or linkage studies from humans with the disease, and compounds are then investigated within a mechanistic framework. Antagonists of voltage-gated sodium channels have been pursued utilizing primarily the first approach; many of these compounds also have significant activity at other ion channels. Both approaches have been utilized to discover voltage-gated calcium channel antagonists, although most efforts to date have used the first approach. Several spontaneous mutant mice and transgenic animals have been utilized to probe the role of the numerous voltage-gated calcium channel subunits and their isoforms as potential molecular targets. Compounds that open or prolong the opening of voltage-gated potassium channels have been discovered using the first approach, with a detailed understanding of the molecular target and mechanism of action coming to light several years later. Genetic evidence from humans is limited to relatively rare forms of epilepsy, and transgenic animals with interesting phenotypes do not always translate into good molecular targets in humans. No clinically-useful antiepileptic drug (AED) has been developed to date that specifically interacts with one, or even one class, of ion channels to produce a therapeutic effect. The tools now exist to search for potent, selective, and safe ion channel modulators for the treatment of epilepsy. This review seeks to summarize the most recent pre-clinical and clinical efforts focused on voltage-gated ion-channels for the development of AEDs.