Search

Human transmembrane adenylyl cyclase 9 (AC9) is not regulated by heterotrimeric G proteins. Key to the resistance to stimulation by Gs-coupled receptors (GsR) is auto-inhibition by the COOH-terminal domain (C2b). The present study investigated the role of the C2b domain in the regulation of cyclic AMP production by AC9 in HEK293FT cells expressing the Glosensor22F cyclic AMP-reporter protein. Surprisingly, we found C2b to be essential for sustaining the basal output of cyclic AMP by AC9. A human mutation (E326D) in the parallel coiled-coil formed by the signalling helices of AC9 dramatically increased basal activity, which was also dependent on the C2b domain. Intriguingly, the same mutation enabled stimulation of AC9 by GsRs. In summary, auto-regulation by the C2b domain of AC9 sustains its basal activity and quenches activation by GsR. Thus AC9 appears to be tailored to support constitutive activation of cyclic AMP effector systems. A switch from this paradigm to stimulation by GsRs may be occasioned by conformation changes at the coiled-coil or removal of the C2b domain.

Human transmembrane adenylyl cyclase 9 (AC9) is not regulated by heterotrimeric G proteins. Key to the resistance to stimulation by Gs-coupled receptors (GsRs) is auto-inhibition by the COOH-terminal domain (C2b). The present study investigated the role of the C2b domain in the regulation of cyclic AMP production by AC9 in HEK293FT cells expressing the GloSensor22F cyclic AMP-reporter protein. Surprisingly, we found C2b to be essential for sustaining the basal output of cyclic AMP by AC9. A human mutation (E326D) in the parallel coiled-coil formed by the signalling helices of AC9 dramatically increased basal activity, which was also dependent on the C2b domain. Intriguingly, the same mutation enabled stimulation of AC9 by GsRs. In summary, auto-regulation by the C2b domain of AC9 sustains its basal activity and quenches activation by GsR. Thus, AC9 appears to be tailored to support constitutive activation of cyclic AMP effector systems. A switch from this paradigm to stimulation by GsRs may be occasioned by conformational changes at the coiled-coil or removal of the C2b domain.

Activation of hypothalamic–pituitary–adrenocortical (HPA) system is an essential component of the body's response to stressful challenges. The HPA system features powerful negative feedback inhibition by adrenal corticosteroids exerted both at the hypothalamus and the pituitary gland. Magnocellular neurons of the hypothalamus produce vasopressin also known as the antidiuretic hormone. The role of magnocellular vasopressin in the HPA response to stress has been controversial for a long time. The present paper summarizes the evidence indicating that magnocellular vasopressin contributes to HPA regulation in a very specific manner by inducing an escape of from feedback inhibition by corticosteroids at the level of the pituitary gland. Furthermore, it is argued that the former effect of vasopressin is important for the function of vasopressinergic magnocellular neurons as stress responsive cells that support metabolic- and immune-system homeostasis in addition to their well-recognized role in salt–water balance.

In mammals, nine genes encode trans-membrane adenylyl cyclase (tmAC) isoforms that synthesize the intracellular messenger compound cAMP from ATP. As cAMP is produced in virtually all types of cell, isoform-selective modulators of tmAC would have major research and therapeutic potential. This study investigated the effects of fungicide imidazoles previously shown to suppress cAMP production in various tissues on the activities of tmAC isoforms AC1, 2, or 9 stably expressed in human embryonic kidney 293 cells. Intact cells, as well as crude membranes, were exposed to various imidazoles or known stimulators of tmAC and the ensuing changes in the production of cAMP analyzed. In crude membranes, the activity of AC9 in the presence of GDP-β-S was enhanced by miconazole with an EC50 of ~ 8 μM, while AC1 and AC2 were inhibited with an IC50 of ~ 20 μM. Clotrimazole (10–100 μM) was an inhibitor of all the ACs tested. Substrate saturation analysis indicated that miconazole increased the Vmax of AC9 by 3-fold while having no effect on the Km. In intact cells, the effect of miconazole on cAMP production through AC9 was additive with that of isoproterenol. The stimulation of cAMP production by miconazole was inhibited by Ca2+, and this could be prevented by the calcineurin blocker FK506. In sum, activation of AC9 by miconazole is through a mechanism distinct from that of forskolin, activated G proteins, or the COOH-terminal mediated autoinhibition. However, it is subject to the AC9 isoform-specific inhibition by Ca2+/calcineurin. Differential modulation of mammalian tmAC paralogs appears to be achievable by an imidazole with phenylated side chains. Optimization of the lead compound and exploration of the underlying mechanism(s) of action in more detail could exploit this further.

This study examined the potential of the selective extra-synaptic α5-GABAA receptor inhibitor S44819 (Egis-13529) to improve cognitive performance in preclinical models of vascular cognitive impairment (VCI). Chronic hypoperfusion of the brain in mice was induced by permanent occlusion of the right common carotid artery (rUCO). rUCO induced impairments of cognitive function in the object recognition test (OR) and the rewarded T-maze (RTM). In both tests, a single oral treatment with S44819 (OR – 0.1–3 mg/kg, RTM – 1–3 mg/kg p.o.) significantly reduced the effect of rUCO. Long-term treatment with S44819 (1–10 mg/kg twice daily p.o. for 14 days), that was initiated 24 h after surgery and was followed by a 10- or 13-day wash-out period, fully prevented the decline of cognitive performance of rUCO mice. In rats, occlusion of the middle cerebral artery (MCA) for 30 min caused a significantly diminished performance in the OR. This was prevented by S44819 given p.o. 15 mg/kg twice daily for 8 days, starting 7 days after surgery and tested following a 7-day wash-out period. Taken together, S44819 markedly and stably improved reference and working memory impaired by rUCO in mice. In rats, the compound effectively suppressed the development of cognitive impairment after mild stroke. In conclusion, as longer-term administration led to a persistent reversal of the cognitive deficits, it appears that S44819 may have symptomatic, as well as disease-modifying effects in models of VCI. Proof of concept is therefore provided for testing S44819 in the therapy of VCI and post-stroke dementia in humans.

Gamma-amino butyric acid (GABA) is an abundant neurotransmitter in the CNS. GABAergic interneurons orchestrate pyramidal neurons in the cerebral cortex, and thus control learning and memory. Ionotropic receptors for GABA (GABAAR) are heteropentameric complexes of α, β and γ integral membrane-protein subunits forming Cl- -channels operated by GABA, which are vital for brain function and are important drug targets. However, knowledge on how GABAAR bind GABA is controversial. Structural biology versus functional modelling combined with site-directed mutagenesis suggest markedly different roles for loop F of the extracellular domain of the α-subunit when complexed with GABA. Here, we report that contrary to the results of structural studies, loop F of the α-subunit controls the potency of GABA on GABAAR. We examined the effect of replacing a short, variable segment of loop F of the GABAA α5-subunit with the corresponding segment of the α2-subunit (GABAA5_LF2) and vice versa (GABAA2-LF5). When compared with their respective wild-type counterparts, GABAA5_LF2 receptors displayed enhanced sensitivity towards GABA, whilst in GABAA2-LF5 sensitivity was diminished. Mice homozygous for the genetic knock-in of the GABAA5_LF2 subunit showed a marked deficit in long- but not short-term object recognition memory. Working memory in place learning, spontaneous alternation and the rewarded T-maze were all normal. The deficit in long-term recognition memory was reversed by an α5-GABAA negative allosteric modulator compound. The data show that loop F governs GABA potency in a receptor isoform-specific manner in vitro. Moreover, this mechanism of ligand recognition appears to be operative in vivo and impacts cognitive performance.

In the mammalian central nervous system (CNS) GABAA receptors (GABAARs) mediate neuronal inhibition and are important therapeutic targets. GABAARs are composed of 5 subunits, drawn from 19 proteins, underpinning expression of 20–30 GABAAR subtypes. In the CNS these isoforms are heterogeneously expressed and exhibit distinct physiological and pharmacological properties. We report the discovery of S44819, a novel tricyclic oxazolo-2,3-benzodiazepine-derivative, that selectively inhibits α5-subunit-containing GABAARs (α5-GABAARs). Current α5-GABAAR inhibitors bind to the “benzodiazepine site”. However, in HEK293 cells expressing recombinant α5-GABAARs, S44819 had no effect on 3H-flumazenil binding, but displaced the GABAAR agonist 3H-muscimol and competitively inhibited the GABA-induced responses. Importantly, we reveal that the α5-subunit selectivity is uniquely governed by amino acid residues within the α-subunit F-loop, a region associated with GABA binding. In mouse hippocampal CA1 neurons, S44819 enhanced long-term potentiation (LTP), blocked a tonic current mediated by extrasynaptic α5-GABAARs, but had no effect on synaptic GABAARs. In mouse thalamic neurons, S44819 had no effect on the tonic current mediated by δ-GABAARs, or on synaptic (α1β2γ2) GABAARs. In rats, S44819 enhanced object recognition memory and reversed scopolamine-induced impairment of working memory in the eight-arm radial maze. In conclusion, S44819 is a first in class compound that uniquely acts as a potent, competitive, selective antagonist of recombinant and native α5-GABAARs. Consequently, S44819 enhances hippocampal synaptic plasticity and exhibits pro-cognitive efficacy. Given this profile, S44819 may improve cognitive function in neurodegenerative disorders and facilitate post-stroke recovery.

Previous work has shown that S44819 is a novel GABAA receptor (GABAAR) antagonist, which is selective for extrasynaptic GABAARs incorporating the α5 subunit (α5-GABAARs). The present study reports on the preclinical neuropsychopharmacological profile of S44819. Significantly, no sedative or pro-convulsive side effects of S44819 were found at doses up to 30 mg/kg i.p. Object recognition (OR) memory in intact mice was enhanced by S44819 (0.3 mg/kg p.o.) given before the acquisition trial. Mice treated with phencyclidine for two weeks and tested six days after the cessation of treatment failed to show OR memory. This deficit was corrected by a single administration of S44819 (0.1, 0.3 or 1 mg/kg p.o.) prior to the acquisition trial. The amnestic effect of ketamine in rats tested in the eight-arm radial maze (reference and working memory versions) was blocked by S44819 (3 mg/kg p.o.). Extinction of cued fear was preserved during treatment with S44819 (3 mg/kg/diem i.p.). Administration of S44819 had no significant effect in the Vogel-conflict test, the elevated plus maze, the forced swim, the marble-burying and the tail-suspension tests. In contrast, anxiolytic/antidepressant-like effects of the compound were found in paradigms that have mnemonic components, such as social interaction, fear-potentiated startle and social avoidance induced by negative life experience. In summary, S44819 enhanced intact recognition memory and ameliorated memory deficits induced by inhibition of NMDA receptors. Anxiolytic/antidepressant efficacy was limited to paradigms involving cognitive function. In conclusion, S44819 is a novel psychoactive pro-cognitive compound with potential as a therapeutic agent in dementia.

The neurotransmitter γ-amino butyric acid (GABA) has a fundamental role in CNS function and ionotropic (GABAA) receptors that mediate many of the actions of GABA are important therapeutic targets. This study reports the mechanism of action of novel GABAA antagonists based on a tricyclic oxazolo-2,3-benzodiazepine scaffold. These compounds are orthosteric antagonists of GABA on heteropentameric GABAA receptors of αxβ2γ2 configuration expressed in HEK293 cells. In silico modelling predicted that the test compounds docked in the GABA binding-pocket and would interact with amino-acid residues in the α- and β-subunit interface that are known to be important for the binding of GABA. Intriguingly, optimal docking also required an interaction with the non-conserved amino-terminal segment of Loop-F of the α–subunit. Testing of a compound with altered regiochemistry of the oxazolone moiety supported the model with respect to the conserved GABA-interacting residues in vitro as well as in vivo. The prediction regarding loop-F was examined by replacing the amino-terminal variable segment of loop-F of the α5-subunit with the corresponding residues in the α1- and α2-subunits. When tested with the novel inhibitors, the receptors formed by the modified α5-subunits displayed the pharmacologic phenotype of the source of loop-F. In summary, these data show that the variable amino-terminal segment of loop-F of the α-subunit determines the pharmacologic selectivity of the novel tricyclic inhibitors of GABAA receptors.

A recent break-through paper has revealed for the first time the high-resolution, three-dimensional structure of a mammalian trans-membrane adenylyl cyclase (tmAC) obtained by cryo-electronmicroscopy (cryo-EM). Reporting the structure of adenylyl cyclase 9 (AC9) in complex with activated Gsα, the cryo-EM study revealed that AC9 has three functionally interlinked, yet structurally distinct domains. The array of the twelve transmembrane helices is connected to the cytosolic catalytic core by two helical segments that are stabilized through the formation of a parallel coiled-coil. Surprisingly, in the presence of Gsα, the isoform-specific carboxyl-terminal tail of AC9 occludes the forskolin- as well as the active substrate-sites, resulting in marked autoinhibition of the enzyme. As AC9 has the lowest primary sequence homology with the eight further mammalian tmAC paralogues, it appears to be the best candidate for selective pharmacologic targeting. This is now closer to reality as the structural insight provided by the cryo-EM study indicates that all of the three structural domains are potential targets for bioactive agents. The present paper summarizes for molecular physiologists and pharmacologists what is known about the biological role of AC9, considers the potential modes of physiologic regulation, as well as pharmacologic targeting on the basis of the high-resolution cryo-EM structure. The translational potential of AC9 is considered upon highlighting the current state of genome-wide association screens, and the corresponding experimental evidence. Overall, whilst the high- resolution structure presents unique opportunities for the full understanding of the control of AC9, the data on the biological role of the enzyme and its translational potential are far from complete, and require extensive further study.

Trans-membrane adenylyl cyclase (tmAC) isoforms show markedly distinct regulatory properties that have not been fully explored. AC9 is highly expressed in vital organs such as the heart and the brain. Here, we report that the isoform-specific carboxyl-terminal domain (C2b) of AC9 inhibits the activation of the enzyme by Gs-coupled receptors (GsCR). In human embryonic kidney cells (HEK293) stably overexpressing AC9, cAMP production by AC9 induced upon the activation of endogenous beta-adrenergic and prostanoid GsCRs was barely discernible. Cells expressing AC9 lacking the C2b domain showed a markedly enhanced cAMP response to GsCR. Subsequent studies of the response of AC9 mutants to the activation of GsCR revealed that residues 1268-1276 in the C2b domain were critical for auto-inhibition. Two main species of AC9 of 130 K and >= 170 K apparent molecular weight were observed on immunoblots of rodent and human myocardial membranes with NH2-terminally directed anti-AC9 antibodies. The lower molecular weight AC9 band did not react with antibodies directed against the C2b domain. It was the predominant species of AC9 in rodent heart tissue and some of the human samples. There is a single gene for AC9 in vertebrates, moreover, amino acids 957-1353 of the COOH-terminus are encoded by a single exon with no apparent signs of mRNA splicing or editing making it highly unlikely that COOH-terminally truncated AC9 could arise through the processing or editing of mRNA. Thus, deductive reasoning leads to the suggestion that proteolytic cleavage of the C2b auto-inhibitory domain may govern the activation of AC9 by GsCR.

This study examined the potential of the selective extra-synaptic α5-GABA

Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development.

International audience

Gamma-amino butyric acid (GABA) is an abundant neurotransmitter in the CNS. GABAergic interneurons orchestrate pyramidal neurons in the cerebral cortex, and thus control learning and memory. Ionotropic receptors for GABA (GABA

Previous work has shown that S44819 is a novel GABAA receptor (GABA

The neurotransmitter γ-amino butyric acid (GABA) has a fundamental role in CNS function and ionotropic (GABA