Recombinant Receptor Subtypes Research Articles

Preclinical characterization of zuranolone (SAGE-217), a selective neuroactive steroid GABAA receptor positive allosteric modulator

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α1β2γ2, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram β-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.

Pseudoreceptor models and 3D-QSAR for imidazobenzodiazepines at GABA A/BzR subtypes alphaxbeta3gamma2 [x = 1-3, 5, and 6] via flexible atom receptor model.

Since benzodiazepines have been used widely in the treatment of anxiety, sleeplessness, and epilepsy, the receptor sites for the benzodiazepine are of prime importance. Quantitative structure-activity relationship (QSAR) studies and receptor modeling via Flexible Atom Receptor Model (FLARM) for the binding affinities of a series of imidazobenzodiazepines at five recombinant receptor subtypes were carried out successfully. The 3D-QSAR models for all five receptor subtypes were examined by a set of test set and demonstrated their high predictability for affinities of imidazobenzodiazepines at five receptor subtypes. The pseudoreceptors yielded by FLARM were compared to the united pharmacophore/receptor model. The result shows that two hydrogen bonds and other regions in the united pharmacophore/receptor model are presented in the pseudoreceptors, which demonstrates the receptor modeling capability of FLARM. The models and pseudoreceptors can help design high affinity ligands on the GABA(A)/BZ receptor and understand the GABA(A) receptor.

3D-QSAR Study of Benzodiazepines at Five Recombinant GABA<sub>A</sub>/Benzodiazepine Receptor Subtypes

GABA(A) receptor is the major neurotransmitter system in the central nervous system(CNS) and elicits a wide range of neuronal physiological activities. Since anxiolytic/anticonvulsant agents have been employed widely in clinic, the receptor sites for the benzodiazepine are of prime importance. Studies on quantitative structure-activity relationship with CoMFA for the binding affinities of a series of imidazobenzodiazepines at five recombinant receptor subtypes were carried out successfully, and a good crossvalidated correlation was obtained for each receptor subtype. Then a set of non-cross-validated PLS models was built and permitted demonstration of high predictability for the affinities of the six ligands in the test set selected in random at all five receptorsubtypes. The modals can help design high affinitiy ligands on the GABA(A)/BZ receptor and understand the GABAA receptor modal.

Magnesium potentiation of the function of native and recombinant GABA(A) receptors.

Mg2+ decreased basal and GABA-inhibited t-butylbicyclophosphoro[35S]thionate binding to GABAA receptor ion channels in rat brain sections up to 1 mM, but increased the binding at 10 mM. The Mg2+-effect was detectable in the presence of a specific GABA site competitive antagonist. Two-electrode voltage clamp recordings of recombinant alpha1beta2gamma2S, alpha1beta2, alpha2beta2gamma2S and alpha2beta2 GABAA receptors revealed a potentiation by 0.1-1 mM Mg2+ of EC20 GABA-evoked ion currents. At 10 mM, Mg2+ decreased the currents. In the absence of GABA, Mg2+ did not evoke any currents. The results show that physiologically relevant Mg2+ concentrations affect the GABA responses on GABAA receptors in native and the main recombinant receptor subtypes, suggesting putative Mg2+ binding sites on the receptor complex.

Life on the edg

The identification of cloned receptors that mediate lysolipid phosphate signalling has greatly advanced the study of these important mediators. The amino acid sequence similarity that exists between these receptors reflects the structural similarity of LPA and S1P, and suggests that an underlying commonality exists between these two fields that have developed somewhat in parallel. Lipid phosphate mediators, particularly LPA, have been implicated in a variety of pathophysiological conditions including blood clotting27xSchumacher, K.A., Classen, H.G., and Spth, M. Thromb. Haemost. 1979; 42: 631–640PubMedSee all References27, corneal wounding28xLiliom, K. et al. Am. J. Physiol. 1998; 274: C1065–C1074PubMedSee all References28, subarachinoid haemorrhage29xTigyi, G. et al. Am. J. Physiol. 1995; 268: H2048–H2055PubMedSee all References29, inflammation30xFourcade, O. et al. Cell. 1995; 80: 919–927Abstract | Full Text PDF | PubMed | Scopus (423)See all References30 and colitis31xSturm, A., Sudermann, T., Schulte, K.M., Goebell, H., and Dignass, A.U. Gastroenterology. 1999; 117: 368–377Abstract | Full Text | Full Text PDF | PubMed | Scopus (78)See all References31, and have been shown to accumulate in malignant ascites32xXu, Y. et al. Clin. Cancer Res. 1995; 1: 1223–1232PubMedSee all References32. Furthermore, LPA is reported to be an anti-apoptotic factor in several cell types including kidney proximal tubule cells33xLevine, J.S., Koh, J.S., Triaca, V., and Lieberthal, W. Am. J. Physiol. 1997; 273: F575–F585PubMedSee all References33 and Schwann cells34xWeiner, J.A. and Chun, J. Proc. Natl. Acad. Sci. U. S. A. 1998; 96: 5233–5238Crossref | Scopus (193)See all References34, and it also promotes skin thickening35xPiazza, G.A., Ritter, J.L., and Baracka, C.A. Exp. Cell Res. 1995; 216: 51–64Crossref | PubMed | Scopus (68)See all References35. The availability of recombinant receptor subtypes will help to identify selective compounds to explore the biology of these fascinating molecules.

Pharmacology of neuronal nicotinic acetylcholine recceptors: effects of acute and chronic nicotine.

Neuronal nicotinic acetylcholine receptors mediate nicotine's diverse effects on the brain, spinal cord and autonomic nervous system. These receptors are composed of alpha and beta subunits. Eight different alpha and three different beta subunits have been identified in vertebrate nervous systems, giving rise to the possibility of multiple subtypes of nicotinic receptors, defined by their constituent subunits. The pharmacological and channel conductance properties of the recombinant receptor subtypes studied in cellular expression systems differ from one another. In addition, the regulation of the receptor density and function during and after acute and chronic exposure to nicotine appears to differ among the subtypes. The predominant receptor subtypes in specific brain regions and peripheral neurons are beginning to be identified and their characteristics studied using new ligands and methods. As more is learned of the differences among the receptor subtypes, it should be possible to identify which specific subtype mediates a specific function within the nervous system and which subtypes are associated with the reinforcing and addictive actions of nicotine.

Molecular Cloning and Pharmacological Characterization of Guinea Pig 5-HT1B and 5-HT1D Receptors

Human 5-HT1B and 5-HT1D receptors have been implicated as molecular targets for the treatment of acute migraine based upon the pharmacological actions and clinical efficacy of sumatriptan, an agonist for human 5-HT1B/1D receptors. The guinea pig has served as an animal model to assess 5-HT1B/1D receptor function, most recently in evaluating 5-HT1B/1D receptor agonists as potential anti-migraine agents. Since two distinct, but closely-related receptors displaying “5-HT1D receptor pharmacology” have been cloned previously from most mammalian species, the genes encoding these receptors were isolated from a guinea pig liver genomic DNA library using oligonucleotide probes targeted to nonconserved regions of recombinant human 5-HT1B and 5-HT1D receptors. Sequence analysis indicates that guinea pig 5-HT1B and 5-HT1D receptors are comprised 390 and 378 amino acids, respectively. Comparison of the deduced amino acid sequences of guinea pig 5-HT1B and 5-HT1D receptor subtypes show that they display overall and transmembrane (TM) identities of 63% and 77%, respectively. Both clones contain a conserved threonine residue in TM7, a structural feature imparting “5-HT1D receptor pharmacology”. Guinea pig 5-HT1B and 5-HT1D receptor genes were transiently expressed in Cos-7 cells and their binding properties were evaluated using [3H]5-HT. Both cloned receptor subtypes displayed “5-HT1D receptor pharmacology” with the following rank order of binding affinities: 5-CT > 5-HT > sumatriptan > 8-OH-DPAT > (−)-pindolol. Ketanserin displayed modest (five-fold) 5-HT1D receptor selectivity, while methiothepin exhibited a similar selectivity for the 5-HT1B subtype. In particular, ketanserin exhibits profound differences in 5-HT1D receptor affinity (and selectivity) across species. High correlations were observed between the binding affinities of serotonergic ligands for 5-HT1D binding sites measured in guinea pig cortical membranes and both cloned guinea pig 5-HT1B (r2 = 0.88) and 5-HT1D (r2 = 0.80) receptors, indicating that the development of subtype selective compounds (i.e. 5-HT1B versus 5-HT1D) using native tissues may be more difficult to achieve without the advantage of using recombinant receptor subtypes. Additionally, there is a good correspondence between binding profiles of recombinant guinea pig 5-HT1B and 5-HT1D receptor subtypes and to their respective cloned human homologs. However, species differences in binding affinities of a subset of compounds are evident. These data extend previous observations that subtype selective (i.e. 5-HT1D) compounds identified in one species may not discriminate between closely related receptors (i.e. 5-HT1B and 5-HT1D) in all animal model systems. © 1997 Elsevier Science Ltd.

Chapter 28 The systems-level organization of cholinergic innervation in the human cerebral cortex and its alterations in Alzheimer's disease

The cholinergic innervation of the cerebral cortex has now become one of the most dynamic areas of research and offers exciting opportunities for exploring the chemical neuroanatomy of cognition, age-related memory impairments, and Alzheimer's disease. The cholinergic pathway from the basal forebrain is the most massive of all extra-thalamic corticofugal projections of the cerebral cortex. Developments based on electron microscopy, immunocytochemistry, antibodies to recombinant receptor subtypes, in situ hybridization, single unit recordings, and selective immunotoxic lesioning have introduced a wealth of new information on the organization and function of this pathway in various animal species, including humans. The cholinergic pathway emanating from the basal forebrain constitutes one of the most important modulatory afferents of the mammalian cortex. The initial expectation that the cholinergic deficiency would provide a unifying pathophysiological basis for Alzheimer's disease and that cholinergic therapies would cure the dementia are clearly too optimistic. Nonetheless, the cortical cholinergic denervation remains one of the earliest, most severe, and most consistent transmitter changes in this disease. The cholinergic depletion may provide an important substrate for the neuropsychological features of Alzheimer's disease and may eventually yield important clues to its pathogenesis.

Classification and nomenclature of somatostatin receptors

There is considerable controversy about the classification and nomenclature of somatostatin receptors. To date, five distinct receptor genes have been cloned and named chronologically according to their respective publication dates, but two were unfortunately given the same appellation (SSTR 4). Consensually, a nomenclature for the recombinant receptors has been agreed according to IUPHAR guidelines (sst 1, sst 2, sst 3, sst 4 and sst 5). However, a more informative classification is to be preferred for the future, employing all classification criteria in an integrated scheme. It is already apparent that the five recombinant receptors fall into two classes or groups, on the basis of not only structure but also pharmacological characteristics. One class (already referred to by some as SRIF 1) appears to comprise sst 2, sst 3 and sst 5 receptor subtypes. The other class (SRIF 2) appears to comprise the other two recombinant receptor subtypes (sst 1 and sst 4). This promising approach is discussed but it is acknowledged that much more data from endogenous receptors in whole tissues are needed before further recommendations on somatostatin receptor nomenclature can be made.