Glucocorticoids rapidly activate cAMP production via Gαs to initiate non-genomic signaling that contributes to one-third of their canonical genomic effects.

Glucocorticoids are widely used for the suppression of inflammation, but evidence is growing that they can have rapid, non-genomic actions that have been unappreciated. Diverse cell signaling effects have been reported for glucocorticoids, leading us to hypothesize that glucocorticoids alone can swiftly increase the 3',5'-cyclic adenosine monophosphate (cAMP) production. We found that prednisone, fluticasone, budesonide, and progesterone each increased cAMP levels within 3 minutes without phosphodiesterase inhibitors by measuring real-time cAMP dynamics using the cAMP difference detector in situ assay in a variety of immortalized cell lines and primary human airway smooth muscle (HASM) cells. A membrane- impermeable glucocorticoid showed similarly rapid stimulation of cAMP, implying that responses are initiated at the cell surface. siRNA knockdown of Gαs virtually eliminated glucocorticoid-stimulated cAMP responses, suggesting that these drugs activate the cAMP production via a G protein-coupled receptor. Estradiol had small effects on cAMP levels but G protein estrogen receptor antagonists had little effect on responses to any of the glucocorticoids tested. The genomic and non-genomic actions of budesonide were analyzed by RNA-Seq analysis of 24 hours treated HASM, with and without knockdown of Gαs . A 140-gene budesonide signature was identified, of which 48 genes represent a non-genomic signature that requires Gαs signaling. Collectively, this non-genomic cAMP signaling modality contributes to one-third of the gene expression changes induced by glucocorticoid treatment and shifts the view of how this important class of drugs exerts its effects.

Budesonide enhances agonist-induced bronchodilation in human small airways by increasing cAMP production in airway smooth muscle.

The nongenomic mechanisms by which glucocorticoids modulate ß2 agonist-induced-bronchodilation remain elusive. Our studies aimed to elucidate mechanisms mediating the beneficial effects of glucocorticoids on agonist-induced bronchodilation. Utilizing human precision-cut lung slices (hPCLS), we measured bronchodilation to formoterol, prostaglandin E2 (PGE2), cholera toxin (CTX), or forskolin in the presence and absence of budesonide. Using cultured human airway smooth muscle (HASM), intracellular cAMP was measured in live cells following exposure to formoterol, PGE2, or forskolin in the presence or absence of budesonide. We showed that simultaneous budesonide administration amplified formoterol-induced bronchodilation and attenuated agonist-induced phosphorylation of myosin light chain, a necessary signaling event mediating force generation. In parallel studies, cAMP levels were augmented by simultaneous exposure of HASM cells to formoterol and budesonide. Budesonide, fluticasone, and prednisone alone rapidly increased cAMP levels, but steroids alone had little effect on bronchodilation in hPCLS. Bronchodilation induced by PGE2, CTX, or forskolin was also augmented by simultaneous exposure to budesonide in hPCLS. Furthermore, HASM cells expressed membrane-bound glucocorticoid receptors that failed to translocate with glucocorticoid stimulation and that potentially mediated the rapid effects of steroids on ß2 agonist-induced bronchodilation. Knockdown of glucocorticoid receptor-α had little effect on budesonide-induced and steroid-dependent augmentation of formoterol-induced cAMP generation in HASM. Collectively, these studies suggest that glucocorticoids amplify cAMP-dependent bronchodilation by directly increasing cAMP levels. These studies identify a molecular mechanism by which the combination of glucocorticoids and ß2 agonists may augment bronchodilation in diseases such as asthma or chronic obstructive pulmonary disease.

PDE8 Is Expressed in Human Airway Smooth Muscle and Selectively Regulates cAMP Signaling by ß2-Adrenergic Receptors and Adenylyl Cyclase 6.

Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing ß2-adrenergic receptor AC6 and another containing E prostanoid receptor AC2. We hypothesized that different PDE isozymes selectively regulate cAMP signaling in each compartment. According to RNA-sequencing data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age- and sex-matched donors with and without asthma. PDE8A was the third most abundant of the cAMP-degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked twofold greater cAMP responses to 1 µM forskolin in the presence of 3-isobutyl-1-methylxanthine. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescence sensor. PF-04957325, a PDE8-selective inhibitor, increased basal cAMP concentrations by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF-04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but it had no effect on cAMP concentrations or cell proliferation regulated by prostaglandin E2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin-1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells, where it specifically regulates ß2-adrenergic receptor AC6 signaling without effects on signaling by the E prostanoid receptors 2/4-AC2 complex. In airway diseases such as asthma and chronic obstructive pulmonary disease, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.

cAMP Signaling Compartmentation: Adenylyl Cyclases as Anchors of Dynamic Signaling Complexes.

It is widely accepted that cAMP signaling is compartmentalized within cells. However, our knowledge of how receptors, cAMP signaling enzymes, effectors, and other key proteins form specific signaling complexes to regulate specific cell responses is limited. The multicomponent nature of these systems and the spatiotemporal dynamics involved as proteins interact and move within a cell make cAMP responses highly complex. Adenylyl cyclases, the enzymatic source of cAMP production, are key starting points for understanding cAMP compartments and defining the functional signaling complexes. Three basic elements are required to form a signaling compartment. First, a localized signal is generated by a G protein-coupled receptor paired to one or more of the nine different transmembrane adenylyl cyclase isoforms that generate the cAMP signal in the cytosol. The diffusion of cAMP is subsequently limited by several factors, including expression of any number of phosphodiesterases (of which there are 24 genes plus spice variants). Finally, signal response elements are differentially localized to respond to cAMP produced within each locale. A-kinase-anchoring proteins, of which there are 43 different isoforms, facilitate this by targeting protein kinase A to specific substrates. Thousands of potential combinations of these three elements are possible in any given cell type, making the characterization of cAMP signaling compartments daunting. This review will focus on what is known about how cells organize cAMP signaling components as well as identify the unknowns. We make an argument for adenylyl cyclases being central to the formation and maintenance of these signaling complexes.

Role of ß2/3-specific GABA-A receptor isoforms in the development of hippocampus kindling epileptogenesis.

OBJECTIVE: Subunit-specific positive allosteric modulators (PAMs) of gamma-aminobutyric acid-A (GABA-A) receptors are commonly used to uncover the role of GABA-A receptor isoforms in brain function. Recently, we have designed novel PAMs selective for ß2/3-subunit containing GABA-A receptors (ß2/3-selective PAMs) that are nonbenzodiazepine site-mediated and do not show an α-subunit isoform selectivity, yet exhibit anxiolytic efficacy with reduced potential for sedation, cognitive impairment, and tolerance. In this study, we used three novel ß2/3-selective PAMs (2-261, 2-262, and 10029) with differential ß2/3-subunit potency to identify the role of ß2/3-selective receptor isoforms in limbic epileptogenesis. METHODS: Experimental epileptogenesis was induced in mice by daily hippocampus stimulations until each mouse showed generalized (stage 5) seizures. Patch-clamp electrophysiology was used to record GABA-gated currents. Brain levels of ß2/3-selective PAMs were determined for mechanistic correlations. RESULTS: Treatment with the ß2/3-selective PAMs 2-261 (30mg/kg), 2-262 (10mg/kg), and 10029 (30mg/kg), 30min prior to stimulations, significantly suppressed the rate of development of kindled seizure activity without affecting the afterdischarge (AD) signal, indicating their disease-modifying activity. The ß2/3-selective agents suppressed chemical epileptogenesis in the pentylenetetrazol model. Test doses of these agents were devoid of acute antiseizure activity in the kindling model. CONCLUSION: These findings demonstrate that ß2/3-selective PAMs can moderately retard experimental epileptogenesis, indicating the protective role of ß2/3-subunit GABA-A receptor isoforms in the development of epilepsy.

Allosteric modulation of related ligand-gated ion channels synergistically induces long-term potentiation in the hippocampus and enhances cognition.

α5 Subunit-containing GABA(A) receptors (GABA(A)Rs) and α7 neuronal nicotinic-acetylcholine receptors (nAChRs) are members of the Cys-loop family of ligand-gated ion channels (LGICs) that mediate cognitive and attentional processes in the hippocampus. α5 GABA(A)Rs alter network activity by tonic inhibition of CA1/CA3 pyramidal cells of the hippocampus. Postsynaptic α7 nAChRs in the hippocampus regulate inhibitory GABAergic interneuron activity required for synchronization of pyramidal neurons in the CA1, whereas presynaptic α7 nAChRs regulate glutamate release. Can simultaneous allosteric modulation of these LGICs produce synergistic effects on cognition? We show that combined transient application of two allosteric modulators that individually 1) inhibit α5 GABA(A)Rs and 2) enhance α7 nAChRs causes long-term potentiation (LTP) of mossy fiber stimulation-induced excitatory postsynaptic currents (EPSC) from CA1 pyramidal neurons of rat hippocampal slices. The LTP effect evoked by two compounds is replicated by 3-(2,5-difluorophenyl)-6-(N-ethylindol-5-yl)-1,2,4-triazolo[4,3-b]pyridazine (522-054), a compound we designed to simultaneously inhibit α5 GABA(A)Rs and enhance α7 nAChRs. Selective antagonists for either receptor block sustained EPSC potentiation produced by 522-054. In vivo, 522-054 enhances performance in the radial arm maze and facilitates attentional states in the five-choice serial reaction time trial with similar receptor antagonist sensitivity. These observations may translate into therapeutic utility of dual action compounds in diseases of hippocampal-based cognitive impairment.

Modifying quinolone antibiotics yields new anxiolytics.

Patients taking fluoroquinolone antibiotics such as norfloxacin exhibit a low incidence of convulsions and anxiety. These side effects probably result from antagonism of the neurotransmitter gamma-aminobutyric acid (GABA) at the brain GABA(A) receptor complex (GRC). Modification of norfloxacin yields molecules such as compound 4 that potentiate GABA action with alpha(2) subunit selectivity. Compound 4 is anxiolytic but does not cause sedation, and may represent a new class of ligands that have anxiolytic activity without sedative liability.

Limiting activity at beta1-subunit-containing GABAA receptor subtypes reduces ataxia.

GABA(A) receptor (R) positive allosteric modulators that selectively modulate GABA(A)Rs containing beta(2)- and/or beta(3)- over beta(1)-subunits have been reported across diverse chemotypes. Examples include loreclezole, mefenamic acid, tracazolate, and etifoxine. In general,"beta(2/3)-selective" GABA(A)R positive allosteric modulators are nonbenzodiazepines (nonBZs), do not show alpha-subunit isoform selectivity, yet have anxiolytic efficacy with reduced ataxic/sedative effects in animal models and humans. Here, we report on an enantiomeric pair of nonBZ GABA(A)R positive allosteric modulators that demonstrate differential beta-subunit isoform selectivity. We have tested this enantiomeric pair along with a series of other beta(2/3)-subunit selective, alpha-subunit isoform-selective, BZ and nonBZ GABA(A) positive allosteric modulators using electrophysiological, pharmacokinetic, and behavioral assays to test the hypothesis that ataxia may be correlated with the extent of modulation at beta(1)-subunit-containing GABA(A)Rs. Our findings provide an alternative strategy for designing anxioselective allosteric modulators of the GABA(A)R with BZ-like anxiolytic efficacy by reducing or eliminating activity at beta(1)-subunit-containing GABA(A)Rs.

Positive allosteric modulators of nonbenzodiazepine γ-aminobutyric acidA receptor subtypes for the treatment of chronic pain.

Chronic neuropathic pain may be caused, in part, by loss of inhibition in spinal pain processing pathways due to attenuation of local GABAergic tone. Nociception and nocifensive behaviors are reduced after enhancement of tonically activated extrasynaptic GABAAR-mediated currents by agonist ligands for δ subunit-containing GABAARs. However, typical ligands that target δ subunit-containing GABAARs are limited due to sedative effects at higher doses. We used the spinal nerve ligation (SNL) and gp120 models of experimental neuropathic pain to evaluate compound 2-261, a nonbenzodiazepine site positive allosteric modulator of α4ß3δ GABAARs optimized to be nonsedative by selective activation of ß2/3-subunit-containing GABAARs over receptor subtypes incorporating ß1 subunits. Similar levels of 2-261 were detected in the brain and plasma after intraperitoneal administration. Although systemic 2-261 did not alter sensory thresholds in sham-operated animals, it significantly reversed SNL-induced thermal and tactile hypersensitivity in a GABAAR-dependent fashion. Intrathecal 2-261 produced conditioned place preference and elevated dopamine levels in the nucleus accumbens of nerve-injured, but not sham-operated, rats. In addition, systemic pretreatment with 2-261 blocked conditioned place preference from spinal clonidine in SNL rats. Moreover, 2-261 reversed thermal hyperalgesia and partially reversed tactile allodynia in the gp120 model of HIV-related neuropathic pain. The effects of 2-261 likely required interaction with the α4ß3δ GABAAR because 2-301, a close structural analog of 2-261 with limited extrasynaptic receptor efficacy, was not active. Thus, 2-261 may produce pain relief with diminished side effects through selective modulation of ß2/3-subunit-containing extrasynaptic GABAARs.

Enaminone Modulators of Extrasynaptic α4ß3δ γ-Aminobutyric AcidA Receptors Reverse Electrographic Status Epilepticus in the Rat After Acute Organophosphorus Poisoning.

Seizures induced by organophosphorus nerve agent exposure become refractory to treatment with benzodiazepines because these drugs engage synaptic γ-aminobutyric acid-A receptors (GABAARs) that rapidly internalize during status epilepticus (SE). Extrasynaptic GABAARs, such as those containing α4ß3δ subunits, are a putative pharmacological target to comprehensively manage nerve agent-induced seizures since they do not internalize during SE and are continuously available for activation. Neurosteroids related to allopregnanolone have been tested as a possible replacement for benzodiazepines because they target both synaptic and extrasynaptic GABAARs receptors. A longer effective treatment window, extended treatment efficacy, and enhanced neuroprotection represent significant advantages of neurosteroids over benzodiazepines. However, neurosteroid use is limited by poor physicochemical properties arising from the intrinsic requirement of the pregnane steroid core structure for efficacy rendering drug formulation problematic. We tested a non-steroidal enaminone GABAAR modulator that interacts with both synaptic and extrasynaptic GABAARs on a binding site distinct from neurosteroids or benzodiazepines for efficacy to control electrographic SE induced by diisopropyl fluorophosphate or soman intoxication in rats. Animals were treated with standard antidotes, and experimental therapeutic treatment was given following 1 h (diisopropyl fluorophosphate model) or 20 min (soman model) after SE onset. We found that the enaminone 2-261 had an extended duration of seizure termination (>10 h) in the diisopropyl fluorophosphate intoxication model in the presence or absence of midazolam (MDZ). 2-261 also moderately potentiated MDZ in the soman-induced seizure model but had limited efficacy as a stand-alone anticonvulsant treatment due to slow onset of action. 2-261 significantly reduced neuronal death in brain areas associated with either diisopropyl fluorophosphate- or soman-induced SE. 2-261 represents an alternate chemical template from neurosteroids for enhancing extrasynaptic α4ß3δ GABAAR activity to reverse SE from organophosphorous intoxication.

Enaminone amides as novel orally active GABAA receptor modulators.

A series of enaminone esters and amides have been developed as potent allosteric modulators of gamma-aminobutyric acidA (GABAA) receptors. The compounds bind to a novel modulatory site that is independent of the benzodiazepine (BZ), isosteric GABA, and neuroactive steroid binding sites. Structure-activity relationship (SAR) studies resulted in the synthesis of the c-Bu amide 16h with an in vitro potency of 7 nM based on inhibition of [35S]TBPS binding. The activity of the enaminones as positive allosteric modulators was confirmed with electrophysiological measurements in oocytes expressing alpha1beta2gamma2L GABAA receptors. The i-Pr, s-Bu, c-Pr, and c-Bu amides (16e-h) were orally active in mice with profound central nervous system depressant effects. The i-Pr amide 16e was an orally active anxiolytic in the mouse light-dark paradigm.

Reversible lipidization for the oral delivery of leu-enkephalin.

The endogenous opioid peptide leu-enkephalin (ENK) was chemically modified by a method known as reversible aqueous lipidization (REAL) with a novel amine-reacting lipophilic dimethylmaleic anhydride analog, 3,4-bis(decylthiomethyl)-2,5-furandione. The binding affinity of the product, REAL-ENK, to opioid receptors was greatly reduced. This prodrug was stable in neutral and basic phosphate buffers but underwent rapid hydrolysis under acidic conditions in the presence of 50% acetonitrile. It also showed increased stability toward enzymatic degradations in various tissue preparations. The half-lives of REAL-ENK in mouse small intestinal mucosal homogenate and liver homogenate were 12 and 80 min, representing a 12- and 32-fold increase over those of ENK itself. In contrast to ENK (t(1/2) 6.7 min), REAL-ENK was stable in mouse plasma. More importantly, REAL-ENK produced significant and sustained antinociception mediated by peripheral opioid receptors in a rodent inflammatory pain model. Pharmacokinetic studies employing a radioimmunoassay (RIA) demonstrated that significantly higher and sustained plasma peptide levels were detected up to 24 h following the oral administration of REAL-ENK in normal mice. The peak concentration and area under the curve of oral REAL-ENK were 4.4 and 21 times higher than that of oral ENK. Our results indicate that like its disulfide-based counterpart, amine-based REAL may be an enabling technology which can be applied to enhance metabolic stability, increase oral absorption, and preserve and possibly prolong the pharmacological activity of peptide drugs.

Limited central side effects of a ß-subunit subtype-selective GABAA receptor allosteric modulator.

GABAergic anxiolytics have well-documented centrally mediated side effects including sedation, potentiation of ethanol, tolerance, abuse liability and memory impairment. Most research directed towards identifying an anxioselective GABAergic therapeutic has been based upon the theory that these side effects could be mitigated by avoiding α1/5-subunit GABAA receptors while specifically targeting those with the α2/3-subunit. Unfortunately, there are prominent exceptions to this theory and it has yet to be translated into clinical success. We previously demonstrated that ß2/3-subunit-selective GABAA receptor-positive allosteric modulators act as anxiolytics with reduced sedation and ethanol potentiation regardless of their activity at α1-subunit GABAA receptors. The prototypical ß2/3-subunit-selective positive allosteric modulator, 2-261, is further characterized here for additional side effects commonly associated with central GABAA receptor activation. In mice, 10 times the anxiolytic dose (10 mg/kg) of 2-261 does not induce behavioral tolerance in the elevated plus maze following a 2 week subchronic treatment. In rats, an anxiolytic dose (10 mg/kg) of 2-261 is inactive in conditioned place preference, suggesting a reduced abuse liability. In rats, 10 times the anxiolytic dose (100 mg/kg) of 2-261 does not have a significant amnestic effect in the radial arm maze, suggesting a greater therapeutic index for memory impairment. These results suggest that ß2/3-subunit subtype-selective GABAA receptor-positive allosteric modulators not only have reduced sedative liability, but also a reduction in other central side effects commonly associated with broader GABAA receptor activation. ß2/3-subunit-selective compounds may represent a novel design template for anxiolytics with benzodiazepine-like efficacy and mitigated side effects.

Pharmacological profile of a 17ß-heteroaryl-substituted neuroactive steroid.

RATIONALE: In order to improve upon the pharmacological properties of the neuroactive steroid ganaxolone, it was used as the starting point in the design of novel neurosteroids that replace the 17ß-acetyl side chain with an isoxazole bioisostere. OBJECTIVES: UCI-50027 (3-[3α-hydroxy-3ß-methyl-5α-androstan-17ß-yl]-5-(hydroxymethyl)isoxazole) was designed as an orally active neuroactive steroid specifically targeted at the gamma-aminobutyric acid(A) receptor (GABAAR). METHODS: UCI-50027 was tested in vitro in Xenopus oocytes expressing human GABAARs and in vivo as an anticonvulsant, for ataxic effects and for anxiolytic activity. RESULTS: In vitro, UCI-50027 dose-dependently enhanced the activity of GABA at human α1ß2γ2L, α2ß1γ2L, and α4ß3δ GABAARs. Consistent with its action as a positive allosteric modulator (PAM), it had no direct activity in the absence of GABA. UCI-50027 protected against acute pentylenetetrazol (PTZ)-induced convulsions with an ED50 of 6 mg/kg p.o. In the rotarod (RR) paradigm in mice, the AD50 (the ataxic dose where half of the animals fail the RR test) was found to be 38 mg/kg p.o., giving a therapeutic index (TI = RR AD50/PTZ ED50)∼6 versus 2.8 for ganaxolone. In the mouse-elevated plus maze (EPM) model for anxiety, UCI-50027 showed a minimum effective dose (MED) ≤0.3 mg/kg p.o. Thus, the TI (TI = RR AD50/EPM MED) for the compound as an anxiolytic is ≥127 versus 3.3 for ganaxolone. CONCLUSIONS: UCI-50027 is an orally active neuroactive steroid with pharmacological activity consistent with a GABAAR PAM that has an improved separation between anticonvulsant/anxiolytic and rotarod effects, potent activity as an anticonvulsant and anxiolytic when compared to ganaxolone.

Design, synthesis, and activity of a series of arylpyrid-3-ylmethanones as type I positive allosteric modulators of α7 nicotinic acetylcholine receptors.

A series of novel arylpyrid-3-ylmethanones (7a-aa) were designed as modulators of α7 nicotinic acetylcholine receptors (nAChRs). The methanones were found to be type I positive allosteric modulators (PAMs) of human α7 nAChRs expressed in Xenopus ooctyes. Structure-activity relationship (SAR) studies resulted in the identification of compound 7v as a potent and efficacious type I PAM with maximum modulation of a nicotine EC5 response of 1200% and EC50 = 0.18 µM. Compound 7z was active in reversing the effect of scopolamine in the novel object recognition (NOR) paradigm with a minimum effective ip dose of 1.0 mg/kg (2.7 µmol/kg). This effect was blocked by the selective α7 nAChR antagonist methyllycaconitine (MLA). These compounds are potent type I positive allosteric modulators of α7 nAChRs that may have therapeutic value in restoring impaired sensory gating and cognitive deficits in schizophrenia and Alzheimer's disease.

Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators.

Activation of brain alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of alpha7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective alpha7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-alpha-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at alpha7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of alpha7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.