The front cover picture shows the modulation of hGluA2 by Selurampanel (central structure), a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist. hGluA2 is a member of the AMPA receptor family (lower right). This receptor mediates fast excitatory neurotransmission and is linked with a variety of neurodegenerative and psychiatric diseases. Overstimulation of these receptors plays a role in triggering seizures associated with epilepsy (represented by the green epileptiform discharge signal). AMPA receptor antagonists are currently in clinical development as anti-epileptic drugs (AEDs) and there is evidence from various sources that this mechanism of action is of therapeutic use in epilepsy. More information can be found in the Communication by Henri Mattes et al. on page 197 in Issue 3, 2017 (DOI: 10.1002/cmdc.201600467). (Graphics by Alan Abrams.)

Allosteric modulators are molecules that interact with a site on a receptor which is distinct from the orthosteric recognition site for the endogenous ligand. By modifying the receptor conformation, they change the affinity and/or efficacy of agonists, but often have no intrinsic activity on their own. Because of this use-dependent mechanism, they are expected to have a much better side-effect profile than agonist drugs. The first positive GABA type B (GABA(B)) receptor modulators, CGP7930 and GS39783, have been described more than 10 years ago. They were discovered in a high-throughput screen using GTP(gamma)S-35 assays, in which they enhanced both the affinity and the maximal effect of gamma-aminobutyric acid (GABA), without having any agonist activity of their own. This positive modulation was subsequently confirmed in a number of different radioligand binding, biochemical and electrophysiological assay systems. The recombinant expression of engineered receptor constructs allowed to locate the site of action of these positive modulators to the seven-transmembrane domain of the GABA(B2) subunit, through which they could to some extent directly activate the receptor in sufficiently sensitive assay systems. These early findings have fostered the search for other molecules acting in a similar way, and a number of positive GABA(B) receptor modulators, and also the first negative modulators, have been described in recent years. In vivo microdialysis experiments have demonstrated at the biochemical level that the mechanism of positive allosteric GABA(B) receptor modulation also applies in living animals. Behavioural experiments have confirmed that positive GABA(B) receptor modulators have a better side-effect profile than the therapeutically used agonist drug baclofen. Numerous studies have shown that these compounds show promising activity in animal models for anxiety, drug and alcohol abuse, pain, gastrointestinal indications and possibly more.

A series of potent quinazolinedione sulfonamide antagonists of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor were designed and synthesized. The structure-activity relationships (SAR) and in vivo activity of the series were investigated. In particular, compound 1 S (selurampanel; N-[7-isopropyl-6-(2-methylpyrazol-3-yl)-2,4-dioxo-1H-quinazolin-3-yl]methanesulfonamide) has shown excellent oral potency against maximal electroshock seizure (MES)-induced generalized tonic-clonic seizures in rodents as well as significant activity in patients suffering from various forms of epilepsy. The X-ray crystal structure of selurampanel bound to the AMPA receptor hGluA was also obtained.

Quinazoline-2,4-diones with a sulfonamide group attached to the N(3) ring atom constitute a novel class of competitive AMPA receptor antagonists. One of the synthesized compounds, 28, shows nanomolar receptor affinity, whereas other examples of the series display oral anticonvulsant activity in animal models.

A new set of quinazolinedione sulfonamide derivatives as competitive AMPA receptor antagonist with improved properties compared to 1 is disclosed. By modulating physico-chemical properties, compound 29 was identified with a low ED50 of 5.5 mg/kg in an animal model of anticonvulsant activity after oral dosage.

Allosteric receptor modulation is an attractive concept in drug targeting because it offers important potential advantages over conventional orthosteric agonism or antagonism. Allosteric ligands modulate receptor function by binding to a site distinct from the recognition site for the endogenous agonist. They often have no effect on their own and therefore act only in conjunction with physiological receptor activation. This article reviews the current status of allosteric modulation at family C G-protein coupled receptors in the light of their specific structural features on the one hand and current concepts in receptor theory on the other hand. Family C G-protein-coupled receptors are characterized by a large extracellular domain containing the orthosteric agonist binding site known as the "venus flytrap module" because of its bilobal structure and the dynamics of its activation mechanism. Mutational analysis and chimeric constructs have revealed that allosteric modulators of the calcium-sensing, metabotropic glutamate and GABA(B) receptors bind to the seven transmembrane domain, through which they modify signal transduction after receptor activation. This is in contrast to taste-enhancing molecules, which bind to different parts of sweet and umami receptors. The complexity of interactions between orthosteric and allosteric ligands is revealed by a number of adequate biochemical and electrophysiological assay systems. Many allosteric family C GPCR modulators show in vivo efficacy in behavioral models for a variety of clinical indications. The positive allosteric calcium sensing receptor modulator cinacalcet is the first drug of this type to enter the market and therefore provides proof of principle in humans.

Importance of the field: The NMDA receptor is a complex ligand gated, voltage-dependent ion channel. It has been a drug target for > 25 years for neurological and psychiatric indications. Whereas the initial optimism to turn preclinically active compounds rapidly into drugs for human use was dampened, new insights into cellular receptor localization, role of subunits and receptor operation have kept the interest alive to modulate this receptor for therapeutic intervention.Areas covered in this review: The article describes the NMDA receptor antagonists patented since 2006. Also included are novel NMDA receptor ligands potentially useful for positron emission tomography imaging.What the reader will gain: The first section summarizes the current status of NMDA receptor pharmacology. This serves as a base for the next sections discussing the patented compounds with respect to their mode of action, potency and, in some cases, drugability.Take home message: The most important recent strategies aiming for inhibition of NMDA receptor-mediated neurotransmission avoid for safety reasons full receptor blockade but allow a low degree of normal receptor function. Approaches pursued by the latest patents comprise blocking the channel with compounds of low affinity, antagonizing receptor activity by highly potent NR2B ligands, partial agonism at the glutamate or glycine-binding site and improvement of pharmacokinetic properties of well established, safe antagonists by deuteration.

Docking of randomly selected compounds from the chemical universe database GDB-11, which contains all organic molecules up to 11 atoms of C, N, O, F possible under consideration of simple chemical stability and synthetic feasibility rules, into the NMDA receptor glycine site (1pb7.pdb) lead to the identification of 3-(aminomethyl)piperazine-2,5-dione 3 and its close analog 5-(aminomethyl)piperazine-2,3-dione 4 as possible new ligands for this drug target, which is implicated in synaptic plasticity, neuronal development, learning and memory. Synthesis of these compounds in 4 and 6 steps, respectively, and testing by radioligand displacement assays and electrophysiological measurements in Xenopus oocytes show that while 4 is inactive, 3 is indeed an inhibitor of glycine, with an estimated KD of 50μM.

Retaining agonistic activity at the glycine coagonist site of the NMDA receptor in molecules derived from glycine or d-serine has proven to be difficult because in the vicinity of the alpha-amino acid group little substitution is tolerated. We have solved this problem by replacing the hydroxy group of d-serine with an amido group, thus keeping the hydrogen donor function and allowing for further substitution and exploration of the adjacent space. Heterocyclic substitutions resulted in a series of 3-acylamino-2-aminopropionic acid derivatives, with high affinities in a binding assay for the glycine site. In a functional assay assessing the activation of the glycine site, these compounds displayed a wide range of intrinsic efficacies, from antagonism to a high degree of partial agonism. Structure-activity relationships reveal that lipophilic substituents, presumably filling an additional hydrophobic pocket, are accepted by the glycine site, provided that they are separated from the alpha-amino acid group by a short linker.

Gamma-Aminobutyric acid B (GABA(B)) receptors are heterodimers composed of two subunits GABA(B(1)) and GABA(B(2)), the former existing in two isoforms GABA(B(1a)) and GABA(B(1b)). The contributions of individual receptor subunits and isoforms to GABA(B) auto-and heteroreceptor functions were investigated, using release experiments in cortical slice preparations from corresponding knockout mice. Presynaptic GABA(B) autoreceptors are located on GABAergic terminals and inhibit GABA release, whereas presynaptic GABA(B) heteroreceptors control the release of other neurotransmitters (e.g. glutamate). Neither baclofen nor the selective antagonist CGP55845 at maximally active concentrations affected [(3)H]GABA release in slices from GABA(B(1))-/- mice. The amount of [(3)H]GABA released per pulse was unaffected by the stimulation frequency in slices from GABA(B(1))-/- and GABA(B(2))-/- demonstrating a loss of GABA(B) autoreceptor function in these knockout animals. The GABA(B) receptor agonist baclofen was ineffective in modulating glutamate release in cortical slices from GABA(B(2))-/- mice, showing that heteroreceptor function was abolished as well. Next we investigated knockout mice for the two predominant GABA(B(1)) isoforms expressed in brain, GABA(B(1a)) and GABA(B(1b)). In cortical, hippocampal and striatal slices from both GABA(B(1a))-/- and GABA(B(1b))-/- mice, the frequency dependence of [(3)H]GABA released per pulse was maintained, suggesting that both isoforms participate or can substitute for each other in GABA(B) autoreceptor function. By contrast, the efficacy of baclofen to inhibit glutamate release was substantially reduced in GABA(B(1a))-/-, but essentially unaltered in GABA(B(1b))-/- mice. Our data suggest that functional GABA(B) heteroreceptors regulating glutamate release are predominantly, but not exclusively composed of GABA(B(1a)) and GABA(B(2)) subunits.

Drug development faces the ever-increasing challenge of discovering new bioactive small molecules that have not already been investigated in the more than 150-year history of medicinal chemistry. [1] This problem is particularly critical for drug targets that require very small organic ligands, for which the range of potential structures is limited. One possible way to overcome this difficulty would be to search through the entire chemical space of these small molecules using virtual screening tools and to identify promising ligands for synthesis and testing. Herein we report the first example of such an approach for ligands of the N-methyl-d-aspartic acid (NMDA) receptor glycine site. This receptor is an important drug target implicated in synaptic plasticity, neuronal development, learning, and memory. Inhibiting the NMDA receptor may help prevent neuronal cell death caused by glutamate excitotoxicity in acute and chronic neurodegenerative disorders such as stroke, epilepsy, Huntington’s, and Alzheimer’s disease. [2] Starting with our recently reported chemical universe database (GDB) that lists all compounds of C, N, O, and F up to 11 atoms obeying simple stability and synthetic feasibility rules, [3] we show that virtual screening followed by synthesis and testing leads to several new NMDA glycine site ligands. Both ligand binding assays and functional investigations reveal that the identified ligands interact with the glycine binding site of the NDMA receptors, inhibiting receptor function by direct competition with glycine. The crystal structure of the NMDA receptor glycine site was recently reported. [4] Glycine is bound through a series of hydrogen bonds at the bottom of a narrow channel, leaving limited free space. The known NMDA glycine site ligands are indeed very small analogues of glycine, such as d-alanine and dserine, [5] d-cycloserine, [6] and small cyclic amino acids, [7] sug

Allosteric modulators act more physiologically than orthosteric ligands, targeting only endogenously activated receptors and not their whole population, which is why they are expected to produce less side effects and tolerance. To inspect the role of the positive allosteric modulator GS39783 in GABAB receptor desensitization, we examined receptor function and cell surface expression in a recombinant GABAB cell line and in primary neuronal cultures upon persistent treatments with GABAB agonists, and combinations of agonists and GS39783. The potency of GABA to inhibit 7beta-forskolin-induced cAMP formation in recombinant cells decreased after the exposure to a saturating GABA concentration, but not after a combination of a low GABA concentration and GS39783, that activated the receptor to the same extent. Concordantly, a significant decrease of cell surface receptors was found after GABA-induced desensitization, unlike after the combined treatment with GABA and GS39783. Similar observations regarding receptor function were found in primary neurons for baclofen-induced inhibition of spontaneous Ca2+ oscillations. However, the cell surface receptor density remained unaffected upon baclofen-induced desensitization in the primary neurons, possibly due to different mechanisms of desensitization in the neurons and the recombinant cell line. These findings indicate that the degree of occupancy of the orthosteric site determines desensitization rather than the degree of receptor activation. In summary, our results conform to predictions that positive allosteric modulators have less propensity for the development of tolerance due to receptor desensitization than classical agonists.

It has been estimated that only 15% of the compounds classified as silent G protein-coupled receptor antagonists are indeed devoid of either positive or negative intrinsic efficacy. Considering that 40% of all drugs on the market target G protein-coupled receptors mainly as orthosteric ligands, elucidating their intrinsic properties is becoming increasingly important. While agonism can be demonstrated using appropriately sensitive experimental setups, the detection of inverse agonism can be limited by a low degree of constitutive activity in many assay systems. In this study, changes in ligand behavior upon a lasting pretreatment with gamma-aminobutyric acid (GABA), that induced receptor desensitization, were observed, measuring the second messenger cyclic AMP (cAMP) in a GABA(B) receptor-expressing recombinant cell line. The GABA(B) receptor partial agonist 2-OH-saclofen lost its ability to inhibit 7beta-forskolin-induced cAMP production upon GABA-pretreatment. The "silent" receptor antagonists CGP62349, CGP52432, CGP56999 and SCH50911, on the other hand, stimulated 7beta-forskolin-induced cAMP production under these conditions. The inverse agonism of CGP56999 was inhibited by the efficacy-deficient 2-OH-saclofen, proving it was truly mediated through the orthosteric site of the GABA(B) receptor. Finally, the positive allosteric modulator GS39783, which previously only marginally inhibited cAMP production, suppressed it by 60% both alone and in the presence of the competitive receptor antagonist 2-OH-saclofen, thus GS39783 became an allosteric receptor agonist at desensitized GABA(B) receptors. These changes likely reflect adaptations in the mechanisms of GABA(B) receptor function following desensitization and may be important in the elucidation of intrinsic ligand efficacies as well as for the consequences of continuous drug treatment.

[(11)C]ABP688 (2) has recently been demonstrated to be a useful PET tracer for in vivo imaging of the metabotropic glutamate receptors type 5 (mGluR5) in rodents. We describe here the identification and preclinical profiling of ABP688 and its tritiated version [(3)H]ABP688, and show that its high affinity (K(d)=2nM), selectivity, and pharmacokinetic properties fulfill all requirements for development as a PET tracer for clinical imaging of the mGlu5 receptor.

c-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS). A great number of major CNS-active, investigational, recreational, or clinically used drugs exert their effects through the GABAergic system. The antiepileptic drugs tiagabine (a GABA-transport blocker) and vigabatrine (c-vinyl GABA, a GABAtransaminase inhibitor) both act by increasing GABA concentrations in the nervous tissue. However, most pharmacologically active compounds interacting with GABAergic neurotransmission are targeted at the receptors for GABA (1,2). The hallucinogenic and CNS-depressant drug muscimol is a selective agonist at the GABAA receptor, a ligand-gated ion channel, which by enabling the influx of chloride ions hyperpolarizes the postsynaptic neuronal membrane. On the other hand, the convulsant drugs bicuculline and picrotoxin inhibit this receptor. Barbiturates (such as pentobarbital) and benzodiazepines (diazepam, chlordiazepoxide) are in clinical use as anesthetic, anxiolytic, anticonvulsant, and muscle-relaxant agents. Both classes of drugs enhance the chloride conductance of the GABAA receptor, albeit through different mechanisms. Most barbiturates prolong the open time of the chloride channel also in the absence of GABA, whereas the

We studied the effects of the positive allosteric modulator GS39783 on GABA(B) receptors at a biochemical level in vivo. Changes in extracellular levels of cyclic AMP following GABA(B) receptor activation were monitored in the striatum of freely moving rats using microdialysis. Locally applied GABA(B) agonist R(-)-baclofen inhibited cyclic AMP formation stimulated by a water-soluble forskolin analogue in a concentration-dependent manner (EC50 7.3 mu M, maximal inhibition 40%). The selective GABA(B) antagonist CGP56999 reversed R(-)-baclofen-induced cyclic AMP inhibition to control levels, but not higher. Orally applied GS39783 lacked effects on its own but, together with a threshold concentration of R(-)-baclofen (1 mu M), significantly decreased cyclic AMP formation in a dose-dependent fashion. Effects of GS39783 were revoked with CGP56999, showing dependence on GABA(B) receptor activation and suggesting allosteric modulation as a mechanism of action in vivo. Administered with a maximally active dose of R(-)-baclofen, GS39783 failed to further inhibit cyclic AMP formation. The data obtained with CGP56999 and the lack of effect of GS39783 alone suggest that there is no detectable endogenous activation of GABA(B) receptors controlling cyclic AMP formation in rat striatum. To our knowledge, these results provide the first biochemical demonstration of in vivo activity of a G protein-coupled receptor-positive allosteric modulator.

We determined the effects of the allosteric γ-aminobutyric acid B receptor modulators CGP7930 and GS39783 on binding and function of orthosteric ligands with distinct intrinsic properties. In radioligand binding (saturation or displacement) experiments, the affinities of a number of competitive antagonists were decreased by the modulators, with no change in receptor number. The binding curves of the partial agonist CGP47656 comprised a high and a low affinity component; the affinity of the former was increased by the allosteric agents. The maximal stimulation of GTP[γ]35S binding via recombinant GABAB receptors by CGP47656 was increased 4-fold in the presence of 30μM CGP7930 or GS39783. Two compounds known so far as “silent” competitive GABAB receptor antagonists, CGP35348 and 2-OH-saclofen, did not stimulate GTP[γ]35S binding on their own, but became low efficacy partial agonists in the presence of the two modulators. The potency of GABA to inhibit the formation of cAMP induced by a forskolin analog in a recombinant CHO cell line expressing GABAB receptors was increased by the modulators. CGP35348 and 2-OH-saclofen, like CGP47656, were partial agonists on their own in this assay, and the allosteric modulators increased the potency as well as the efficacy of all three compounds. With CGP52432, there was a trend towards inverse agonism in the cAMP assay. These results show that the intrinsic properties of orthosteric ligands are highly dependent on the characteristics of the assay system used and that allosteric modulators are useful tools for elucidating these properties.