A novel GABA(A) receptor pharmacology: drugs interacting with the α(+) β(-) interface

GABA(A) receptors are ligand-gated chloride channels composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to a multiplicity of GABA(A) receptor subtypes with distinct subunit composition; regional, cellular and subcellular distribution; and pharmacology. Most of these receptors are composed of two α, two β and one γ2 subunits. GABA(A) receptors are the site of action of a variety of pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, anaesthetics and convulsants. Whereas GABA acts at the two extracellular β(+) α(-) interfaces of GABA(A) receptors, the allosteric modulatory benzodiazepines interact with the extracellular α(+) γ2(-) interface. In contrast, barbiturates, neuroactive steroids and anaesthetics seem to interact with solvent accessible pockets in the transmembrane domain. Several benzodiazepine site ligands have been identified that selectively interact with GABA(A) receptor subtypes containing α2βγ2, α3βγ2 or α5βγ2 subunits. This indicates that the different α subunit types present in these receptors convey sufficient structural differences to the benzodiazepine binding site to allow specific interaction with certain benzodiazepine site ligands. Recently, a novel drug binding site was identified at the α(+) β(-) interface. This binding site is homologous to the benzodiazepine binding site at the α(+) γ2(-) interface and is thus also strongly influenced by the type of α subunit present in the receptor. Drugs interacting with this binding site cannot directly activate but only allosterically modulate GABA(A) receptors. The possible importance of such drugs addressing a spectrum of receptor subtypes completely different from that of benzodiazepines is discussed.     

Overview of nomenclature of nuclear receptors

Nuclear receptor pharmacology has, to a certain extent, led the way, compared with other receptor systems, in the appreciation that ligands may exert very diverse pharmacology, based on their individual chemical structure and the allosteric changes induced in the receptor/accessory protein complex. This can lead to very selective pharmacological effects, which may not necessarily be predicted from the experience with other agonists/partial agonists/antagonists. If this is the case, then drug discovery may be back to drug-specific pharmacology (where each drug may have an original profile), rather than specific-drug pharmacology (where agents specific for a receptor have a distinct profile). As functional selectivity is indeed a crucial mechanism to be considered when going through the drug discovery development process, then initial screens using reconstituted systems may not show the appropriate pharmacology, simply because the required stoichiometry of corepressors and coactivators may not be present to select the best compounds; therefore, multiple effector systems are necessary to screen for differential activation, and, even then, screening with in vivo pathophysiological models may ultimately be required for the selection process-a massive but necessary task for pharmacologists. Thus, the characterization of nuclear receptors and their associated proteins and the ligands that interact with them will remain a challenge to pharmacologists.     

The importance of the adenosine A(2A) receptor-dopamine D(2) receptor interaction in drug addiction

Drug addiction is a serious brain disorder with somatic, psychological, psychiatric, socio-economic and legal implications in the developed world. Illegal (e.g., psychostimulants, opioids, cannabinoids) and legal (alcohol, nicotine) drugs of abuse create a complex behavioral pattern composed of drug intake, withdrawal, seeking and relapse. One of the hallmarks of drugs that are abused by humans is that they have different mechanisms of action to increase dopamine (DA) neurotransmission within the mesolimbic circuitry of the brain and indirectly activate DA receptors. Among the DA receptors, D(2) receptors are linked to drug abuse and addiction because their function has been proven to be correlated with drug reinforcement and relapses. The recognition that D(2) receptors exist not only as homomers but also can form heteromers, such as with the adenosine (A)(2A) receptor, that are pharmacologically and functionally distinct from their constituent receptors, has significantly expanded the range of potential drug targets and provided new avenues for drug design in the search for novel drug addiction therapies. The aim of this review is to bring current focus on A(2A) receptors, their physiology and pharmacology in the central nervous system, and to discuss the therapeutic relevance of these receptors to drug addiction. We concentrate on the contribution of A(2A) receptors to the effects of different classes of drugs of abuse examined in preclinical behavioral experiments carried out with pharmacological and genetic tools. The consequences of chronic drug treatment on A(2A) receptor-assigned functions in preclinical studies are also presented. Finally, the neurochemical mechanism of the interaction between A(2A) receptors and drugs of abuse in the context of the heteromeric A(2A)-D(2) receptor complex is discussed. Taken together, a significant amount of experimental analyses provide evidence that targeting A(2A) receptors may offer innovative translational strategies for combating drug addiction.     

Allosteric agonists of 7TM receptors: expanding the pharmacological toolbox

Approximately 1% of the genome of higher organisms encodes seven-transmembrane (7TM) G-protein-coupled receptors, which control an extensive range of physiological processes and represent drug targets for nearly half of all drugs that are prescribed currently. To date, most drugs that target 7TM receptors interact via the same domain as the endogenous agonist, called the orthosteric site. However, the advent of functional screening assays has greatly increased the number of allosteric ligands identified. Such ligands bind to topographically distinct sites on 7TM receptors. In addition to modulating the affinity of orthosteric ligands, allosteric ligands can also alter the efficacy of orthosteric ligands and activate 7TM receptors in their own right. In this article, we briefly review the current status of putative allosteric agonists of 7TM receptors, and discuss the promises and challenges that this class of ligand might pose for pharmacologists and the drug-discovery industry.     

Ligands for the benzodiazepine binding site--a survey

Gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian Central Nervous System (CNS). GABA participates in the regulation of neuronal excitability through interaction with specific membrane proteins (the GABAA receptors). The binding of GABA to these postsynaptic receptors, results in an opening of a chloride channel integrated in the receptor which allows the entry of Cl- and consequently leads to hyperpolarization of the recipient cell. The action of GABA is allosterically modulated by a wide variety of chemical entities which interact with distinct binding sites at the GABAA receptor complex. One of the most thoroughly investigated modulatory site is the benzodiazepine binding site. The benzodiazepines constitute a well-known class of therapeutics displaying hypnotic, anxiolytic and anticonvulsant effects. Their usefulness, however, is limited by a broad range of side effects comprising sedation, ataxia, amnesia, alcohol and barbiturate potentiation, tolerance development and abuse potential. Consequently, there has been an intensive search for modulatory agents with an improved profile, and a diversity of chemical entities distinct from the benzodiazepines, but with GABA modulatory effects have been identified. The existence of endogenous ligands for the GABAA receptor complex beside GABA has often been described, but their role in the regulation of GABA action is still a matter of controversy. The progress of molecular biology during the last decade has contributed enormously to the understanding of benzodiazepine receptor pharmacology. A total of 14 GABAA receptor subunits have been cloned from mammalian brain and have been expressed/co-expressed in stable cell lines. These transfected cells constitute an important tool in the characterization of subtype selective ligands. In spite of the rapidly expanding knowledge of the molecular and pharmacological mechanisms involved in GABA/benzodiazepine related CNS disorders, the identification of clinically selective acting drugs is still to come.     

G protein-coupled receptor hetero-dimerization: contribution to pharmacology and function

The concept that G protein-coupled receptors (GPCRs) can form hetero-dimers or hetero-oligomers continues to gain experimental support. However, with the exception of the GABA_B receptor and the sweet and umami taste receptors few reported examples meet all of the criteria suggested in a recent International Union of Basic and Clinical Pharmacology sponsored review (Pin et al., 2007) that should be required to define distinct and physiologically relevant receptor species. Despite this, there are many examples in which pairs of co-expressed GPCRs reciprocally modulate their function, trafficking and/or ligand pharmacology. Such data are at least consistent with physical interactions between the receptor pairs. In recent times, it has been suggested that specific GPCR hetero-dimer or hetero-oligomer pairs may represent key molecular targets of certain clinically effective, small molecule drugs and there is growing interest in efforts to identify ligands that may modulate hetero-dimer function selectively. The current review summarizes key recent developments in these topics.     

Particle Size Limits to Pass the Acceptance Value (AV)-Based USP Content Uniformity Test for Tablets

The papers published by Yalkowsky et al. and Rohrs et al. offer a method to correlate the probability of passing USP content uniformity (CU) test with maximum allowed particle size and distribution. Their work also provides a guidance for setting the particle size specification especially for a low dose drug. However, their methods are developed based on the previous USP CU test and the up-to-date version of USP/NF (e.g., USP42/NF37) has adopted a new Acceptance Value (AV)-based CU criterion. In this study, using the same assumptions and conditions that are utilized in their papers, we develop a new method to calculate the upper limit of particle size that ensures the pass of the AV-based CU test at a given probability (e.g., 0.99). It turns out that, compared to the previous CU test (e.g., the CU test in USP28/NF23), to pass the AV-based CU test, similar but slightly smaller particle size is needed.     

Histaminergic pharmacology of homo-oligomeric β3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology

A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric β3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to β3 GABA(A) receptors with micromolar affinity (K(D)<300 μM), thus extending previous evidence that β3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with β3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric β3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for β3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether β3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site.     

Schizophrenia - advances in drug therapy

It has become increasingly clear that classical drug treatments for schizophrenia, while valuable for many patients, are far from optimal. Not only do they induce unpleasant side-effects, but they are also limited in their antipsychotic efficacy in a substantial proportion of patients. Using the uniquely effective but pharmacologically complex drug, clozapine, as a model, a variety of receptors in addition to the dopamine D2 site have been identified as targets for drug development; the 5-HT2A site has received the greatest interest in this respect. Several new drugs in development as antipsychotics have pharmacological effects at 5-HT2 and other clozapine-sensitive receptors; the receptor mechanisms underlying both the antipsychotic efficacy and limitations of these compounds are reviewed here, as are some other potential drug therapies that do not share clozapine's receptor pharmacology.     

Functional evidence for a twisted conformation of the NMDA receptor GluN2A subunit N-terminal domain

Ionotropic glutamate receptors (iGluRs) possess in their extracellular region a large N-terminal domain (NTD) that precedes the agonist-binding domain and displays a clamshell-like architecture similar to the bacterial leucine/isoleucine/valine-binding protein (LIVBP). In addition to their role in receptor assembly, in NMDA receptors (NMDARs), the NTDs of GluN2A and GluN2B subunits form a major site for subunit-specific regulation of ion channel activity, in particular through binding of allosteric modulators such as the synaptically-enriched zinc ion. A recent crystallographic study of the isolated GluN2B NTD has revealed an unexpected twisted closed-cleft conformation caused by a rotation of ∼50° in the interlobe orientation compared with all other known LIVBP-like structures (Karakas et al., 2009). By measuring currents carried by recombinant NMDARs, we now provide functional evidence, through disulfide cross-linking and the identification of a new zinc-binding residue (D283), that the GluN2A NTD of intact GluN1/GluN2A receptors adopts a similar twisted conformation in its closed-cleft state. We propose that the twisted NTD conformation is a distinct structural feature of NMDARs (at least for GluN2A and GluN2B subunits), arguing for interactions between the NTDs in the tetrameric complex that are likely to differ between NMDA and AMPA/kainate receptors.     

Keynote review: in vitro safety pharmacology profiling: an essential tool for successful drug development

Broad-scale in vitro pharmacology profiling of new chemical entities during early phases of drug discovery has recently become an essential tool to predict clinical adverse effects. Modern, relatively inexpensive assay technologies and rapidly expanding knowledge about G-protein coupled receptors, nuclear receptors, ion channels and enzymes have made it possible to implement a large number of assays addressing possible clinical liabilities. Together with other in vitro assays focusing on toxicology and bioavailability, they provide a powerful tool to aid drug development. In this article, we review the development of this tool for drug discovery, its appropriate use and predictive value.     

A critical pocket close to the glutamate binding site of mGlu receptors opens new possibilities for agonist design

A recent publication from Ogawa et al. suggested a possible allosteric chloride binding site in the extracellular domain of metabotropic glutamate receptors (mGluRs) by comparison with a similar site found in atrial natriuretic peptide receptor. We simultaneously reported about (S)-PCEP an agonist of subtype 4 mGluR that would bind to a similar pocket, adjacent to the glutamate binding site. Here we disclose LSP1-2093, a new derivative of (S)-PCEP that holds a nitrophenyl substituent. Docking experiments predict that the nitro group binds to the receptor at the putative chloride ion site. It is thus possible to take advantage of this putative chloride binding site to develop new types of mGluR agonists. This pocket is present in the structural family of Leucine Isoleucine Valine Binding Protein that includes class C GPCRs, suggesting that extended agonists may be identified at receptors bearing such a structural domain.     

Snaclecs (snake C-type lectins) that inhibit or activate platelets by binding to receptors

More than 300 known species of venomous snakes are classified into five families, Hydrophidae, Elapidae, Viperidae, Crotalidae and Colubidae. Venom proteins have also been demonstrated recently in other snakes and some lizards (Fry et al., 2006). Venom components that affect hemostasis are most generally found in Viperidae, and Crotalidae snakes but the others often contain some as well. This review concentrates on structural and functional properties of venom components of the C-type lectin related class (now named snaclecs) that inhibit or activate platelets by binding to receptors.     

Pharmacology of human memory and cognition: illustrations from the effects of benzodiazepines and cholinergic drugs

The current research methods, findings and questions that are being addressed in studies of the pharmacology of human memory and cognition are reviewed. Memory is not a unitary function. Neuropsychological studies of brain-damaged memory-impaired patients, as well as neuroimaging and drug studies in normal individuals indicate that different forms of learning and memory are subserved by different brain systems. Animal drug studies have also provided evidence that, while distinct, memory systems are not independent, but operate in close interaction with one another. Recent human studies of benzodiazepines and of cholinergic drugs demonstrate the value of the psychological models and of the experimental paradigms that are available from cognitive sciences for exploring how drugs alter cognitive and memory functions. They also show how drugs can be used as tools for analyzing the distinct neurochemical mechanisms underlying independent cognitive processes, and so find effective drugs rationally from a knowledge of the neurochemical bases of cognition. This research leads to specific recommendations concerning treatments that may improve memory functioning, for instance in Alzheimer's disease.     

Challenges and opportunities of trapping ligands

Because gonadotropin-releasing hormone (GnRH) analogs constitute an important class of therapeutics for various reproductive and hormone-dependent disorders, many novel compounds have been discovered and studied. Several orally active nonpeptide GnRH antagonists have recently gained increased attention. In the study published in this issue of Molecular Pharmacology, Kohout et al. (p. 238) used small-molecule TAK-013 (sufugolix; developed previously by Takeda Chemical Industries) as a tool to elucidate the mechanism of its insurmountable antagonism. On the basis of receptor mutagenesis combined with molecular modeling, the authors hypothesized that certain amino acid sequences uniquely present in the human GnRH receptor amino terminus and extracellular loop 2 may form a "trap door" retarding dissociation of TAK-013. Such a trapping mechanism could be both ligand- and receptor species-specific. Although analogous models were previously proposed for other G protein-coupled receptors, the study by Kohout et al. (2007) provides an important advance in the GnRH antagonists field and an illustration of the fact that preclinical studies using animal models with nonhuman receptors may have very limited value in predicting drug efficacy in human disease. There are many examples showing that high-affinity protein, peptide, or nonpeptide agonists or antagonists have also enhanced clinical efficacy. However, there are also numerous studies indicating that very high receptor binding affinity is not a guarantee of drug efficacy and that other factors, including pharmacokinetic profile, ligand-induced receptor desensitization, and "trafficking," are critical in design and development of optimal drugs.     

Communication between multiple drug binding sites on P-glycoprotein

P-glycoprotein, a member of the ATP-binding cassette transporter family, is able to confer resistance on tumors against a large number of functionally and chemically distinct cytotoxic compounds. Several recent investigations suggest that P-glycoprotein contains multiple drug binding sites rather than a single site of broad substrate specificity. In the present study, radioligand-binding techniques were used to directly characterize drug interaction sites on P-glycoprotein and how these multiple sites interact. The drugs used were classified as either 1) substrates, which are known to be transported by P-glycoprotein (e.g., vinblastine) or 2) modulators, which alter P-glycoprotein function but are not themselves transported by the protein (e.g., XR9576). Drug interactions with P-glycoprotein were either competitive, at a common site, or noncompetitive, and therefore at distinct sites. Based on these data, we can assign a minimum of four drug binding sites on P-glycoprotein. These sites fall into two categories: transport, at which translocation of drug across the membrane can occur, and regulatory sites, which modify P-glycoprotein function. Intriguingly, however, some modulators interact with P-glycoprotein at a transport site rather than a regulatory site. The pharmacological data also demonstrate that both transport and regulatory sites are able to switch between high- and low-affinity conformations. The multiple sites on P-glycoprotein display complex allosteric interactions through which interaction of drug at one site switches other sites between high- or low-affinity conformations. The data are discussed in terms of a model for the mechanism of transport by P-glycoprotein.     

GPCR-GIP networks: a first step in the discovery of new therapeutic drugs?

G protein-coupled receptors (GPCRs) are transmembrane molecules that, on interaction with G proteins upon ligand binding, can associate with a large variety of transmembrane or soluble proteins, termed 'GPCR-interacting proteins' (GIPs). Some special transmembrane GIPs are themselves GPCRs that form homo- or heterodimers, while other transmembrane GIPs are ionic channels, ionotropic receptors and single transmembrane proteins that control GPCR pharmacology and trafficking. Most soluble GIPs interact with the C-termini of GPCRs and often physically link GPCRs to large protein networks, called 'receptosomes', that include ionic channels, protein kinases, small G proteins, cytoskeletal proteins and adhesion molecules. Here, we review the nature and functions of some of these networks, such as the glutamate and serotonin receptosomes, and focus on the fine-tuning of GPCR signaling by GIPs. Finally, we discuss the possibilities for developing new therapeutic drugs capable of modulating GPCR signaling by disrupting or reinforcing specific GPCR-GIP interactions.     

Development of an inhalation unit risk factor for cadmium

An inhalation unit risk factor (URF) was developed for cadmium. The URF is based on excess lung cancer mortality in a key epidemiological study of cadmium smelter workers (Park et al., 2012). The Park et al. (2012) study is an update of the Thun et al. (1985) cohort that was previously used to derive a URF in USEPA (1985). Park et al. re-analyzed the cadmium smelter worker population (near Denver, CO) using more detailed work history information, a revised cadmium exposure matrix, a detailed retrospective exposure assessment for arsenic (potential confounder), and updated mortality data (through 2002). Grouped observed and expected number of lung cancer mortalities along with cumulative cadmium exposures were used in the current study to obtain the maximum likelihood estimate and asymptotic variance of the slope (β) for the linear multiplicative relative risk model using Poisson regression modeling. Life-table analyses were used to derive the final URF for cadmium of 4.9E-04 per μg Cd/m³. The corresponding lifetime air concentration at the 1 in 100,000 no significant excess risk level is 0.020 μg Cd/m³, which can be used to protect the general public in Texas against the potential carcinogenic effects from chronic exposure to cadmium and cadmium compounds.