Partial Inverse Agonist Research Articles

Revealing the graded activation mechanism of neurotensin receptor 1

Graded activation contributes to the precise regulation of GPCR activity, presenting new opportunities for drug design. In this work, a total of 10 μs enhanced-sampling simulations are performed to provide molecular insights into the binding dynamics differences of the neurotensin receptor 1 (NTSR1) to the full agonist SRI-9829, partial agonist RTI-3a and inverse agonist SR48692. The possible graded activation mechanism of NTSR1 is revealed by an integrated analysis utilizing the reweighted potential of mean force (PMF), deep learning (DL) and transfer entropy (TE). Specifically, the orthosteric pocket is observed to undergo expansion and contraction, with the G-protein-binding site experiencing interconversions among the inactive, intermediate and active-like states. Detailed structural comparisons capture subtle conformational differences arising from ligand binding in allosteric signaling, which can well explain the graded activation. Critical microswitches that contribute to graded activation are efficiently identified with the DL model. TE calculations enable the visualization of allosteric communication networks within the receptor, elucidating the driver-responder relationships associated with signal transduction. Fortunately, the dissociation of the full agonist from the orthosteric pocket is observed. The current findings systematically reveal the mechanism of NTSR1 graded activation, and also provide implications for structure-based drug design.

β-Carboline (FG-7142) modulates fear but not anxiety-like behaviour in zebrafish

The β-Carboline FG-7142 is a partial inverse agonist at the benzodiazepine allosteric site on the GABA-A receptor that induces anxiogenic, proconvulsant, and appetite-reducing effects in many species, including humans. Seizure-kindling effects have been well studied, however anxiogenic properties are relatively unexplored. This study aimed to investigate concentration-dependent effects of FG-7142 on anxiety-like behaviour and fear responses in zebrafish (Danio rerio) using the open-field test (OF) and novel object approach test (NOA). A U-shaped distribution was found with maximal responses in increased immobility and reduced distance moved at 10 µM in the NOA but not the OF. Follow up experiments demonstrated a lack of effect in repeated OF testing and no changes in opercular movements. Furthermore, the effect of FG-7142 was reversed with ethanol treatment. These results suggest that FG-7142 elicits a ‘freezing’ response in zebrafish via the introduction of novelty, suggesting fear-induction. These findings indicate that FG-7142 may act as an agent to promote acute fear responses in zebrafish.

GPCR molecular dynamics forecasting using recurrent neural networks

G protein-coupled receptors (GPCRs) are a large superfamily of cell membrane proteins that play an important physiological role as transmitters of extracellular signals. Signal transmission through the cell membrane depends on conformational changes in the transmembrane region of the receptor, which makes the investigation of the dynamics in these regions particularly relevant. Molecular dynamics (MD) simulations provide a wealth of data about the structure, dynamics, and physiological function of biological macromolecules by modelling the interactions between their atomic constituents. In this study, a Recurrent and Convolutional Neural Network (RNN) model, namely Long Short-Term Memory (LSTM), is used to predict the dynamics of two GPCR states and three specific simulations of each one, through their activation path and focussing on specific receptor regions. Active and inactive states of the GPCRs are analysed in six scenarios involving APO, Full Agonist (BI 167107) and Partial Inverse Agonist (carazolol) of the receptor. Four Machine Learning models with increasing complexity in terms of neural network architecture are evaluated, and their results discussed. The best method achieves an overall RMSD lower than 0.139 Å and the transmembrane helices are the regions showing the minimum prediction errors and minimum relative movements of the protein.

Computational study of the structural ensemble of CC chemokine receptor type 5 (CCR5) and its interactions with different ligands.

CC Chemokine receptor 5 (CCR5), a member of the Superfamily of G Protein-Coupled Receptors (GPCRs), is an important effector in multiple physiopathological processes such as inflammatory and infectious entities, including central nervous system neuroinflammatory diseases such as Alzheimer’s disease, recovery from nervous injuries, and in the HIV-AIDS infective processes. Thus, CCR5 is an attractive target for pharmacological modulation. Since maraviroc was described as a CCR5 ligand that modifies the HIV-AIDS progression, multiple efforts have been developed to describe the functionality of the receptor. In this work, we characterized key structural features of the CCR5 receptor employing extensive atomistic molecular dynamics (MD) in its apo form and in complex with an endogenous agonist, the chemokine CCL5/RANTES, an HIV entry inhibitor, the partial inverse agonist maraviroc, and the experimental antagonists Compound 21 and 34, aiming to elucidate the structural features and mechanistic processes that constitute its functional states, contributing with structural details and a general understanding of this relevant system.

Cholesterol as a modulator of cannabinoid receptor CB2 signaling

Signaling through integral membrane G protein-coupled receptors (GPCRs) is influenced by lipid composition of cell membranes. By using novel high affinity ligands of human cannabinoid receptor CB2, we demonstrate that cholesterol increases basal activation levels of the receptor and alters the pharmacological categorization of these ligands. Our results revealed that (2-(6-chloro-2-((2,2,3,3-tetramethylcyclopropane-1-carbonyl)imino)benzo[d]thiazol-3(2H)-yl)ethyl acetate ligand (MRI-2646) acts as a partial agonist of CB2 in membranes devoid of cholesterol and as a neutral antagonist or a partial inverse agonist in cholesterol-containing membranes. The differential effects of a specific ligand on activation of CB2 in different types of membranes may have implications for screening of drug candidates in a search of modulators of GPCR activity. MD simulation suggests that cholesterol exerts an allosteric effect on the intracellular regions of the receptor that interact with the G-protein complex thereby altering the recruitment of G protein.

Development of Novel δ Opioid Receptor Inverse Agonists without a Basic Nitrogen Atom and Their Antitussive Effects in Mice.

Our previous results showed that naltrindole (NTI) derivatives with certain types of electron-withdrawing groups as an N-substituent showed δ opioid receptor (DOR) inverse agonistic activities. We therefore synthesized N-acylated NTI derivatives 3a-e and observed that N-benzoyl and N-cyclopropanecarbonyl derivatives SYK-736 (3b) and SYK-623 (3c) were DOR full inverse agonists and the N-acryloyl derivative 3d was a DOR partial inverse agonist. SKY-623 was over 110-fold more potent than the reference compound ICI-174,864. Both naltriben (NTB) and 7-benzylidenenaltrexone (BNTX) derivatives with N-benzoyl and N-cyclopropanecarbonyl groups were also DOR full inverse agonists. These N-acylated inverse agonists are interesting compounds because they have no basic nitrogen atom, which has been demonstrated to be an important pharmacophore. NTI and BNTX-type DOR inverse agonists SYK-623 and SYK-723 (12c) showed dose-dependent antitussive effects in a mouse cough model induced by citric acid exposure. The antitussive effects by SYK-623 and SYK-723 were significantly attenuated by pretreatment with DOR agonist SNC80.

The tarantula toxin GxTx detains K+ channel gating charges in their resting conformation.

Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K+ channel's voltage sensor allosterically modulates opening of its K+-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K+ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K+-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K+-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K+ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors.

Differential effects of functionally different histamine H4 receptor ligands on acute irritant dermatitis in mice.

The anti-inflammatory effects of histamine H4 receptor (H4R) antagonists opened new therapeutic options for the treatment of inflammatory/allergic diseases, but the role of H4R in inflammation is far from being solved. Aim of the present study was to investigate the role of structurally related H4R ligands of the aminopyrimidine class with different efficacies and functionalities (neutral antagonist ST-994, partial agonist ST-1006, inverse agonist ST-1012, and partial inverse agonist ST-1124) on croton oil-induced ear edema and pruritus in mice. The H4R ligands were administered subcutaneously before topical application of croton oil. While ST-1006 and ST-1124 were ineffective at any dose tested (10-100 mg/kg), both ST-994 and ST-1012 (30 and 100 mg/kg) significantly reduced croton oil-induced ear edema. Moreover, ST-994, ST-1006, and ST-1124, but not ST-1012, significantly inhibited croton oil-induced ear pruritus at 30 mg/kg. In accordance with results obtained with the reference H4R antagonist JNJ7777120 (100 mg/kg), histological examination of inflamed ear tissue indicated that treatment with ST-994 (30 mg/kg) led to a significant reduction in the inflammatory severity score and in the number of eosinophils infiltrating the tissue, while the number of degranulated mast cells in inflamed tissues was increased in comparison with the number of intact mast cells. These data indicate that croton oil-induced ear inflammation and pruritus seem to be clearly, but variably, affected by the H4R ligands tested. The potential advantage of dual effect of the H4R neutral antagonist ST-994 has to be carefully considered as a new therapeutic approach to the treatment of inflammatory diseases.

Novel non-sulfonamide 5-HT6 receptor partial inverse agonist in a group of imidazo[4,5-b]pyridines with cognition enhancing properties

A small library of novel 3H-imidazo[4,5-b]pyridine and 1H-imidazo[4,5-c]pyridine derivatives was designed and synthesized as non-sulfonamide 5-HT6 receptor ligands. In vitro evaluation allowed to identify compound 17 (2-ethyl-3-(3-fluorobenzyl)-7-(piperazin-1-yl)-3H-imidazo[4,5-b]pyridine) as potent 5-HT6 receptor partial inverse agonist in Gs signaling (Ki = 6 nM, IC50 = 17.6 nM). Compound 17 displayed high metabolic stability, favorable cytochrome P450 isoenzyme (2D6, 3A4) profile, did not affect PgP-protein binding, without evoking mutagenic effects. It was orally bioavailable and brain penetrant. In contrast to intepirdine (SB-742457), which prevented 5-HT6R–elicited neurite growth and behaved as an inverse agonist of cyclin-dependent kinase 5 (Cdk5), compound 17 has no influence on neuronal differentiation. Compound 17 exerted significant pro-cognitive properties in novel object recognition (NOR) task in rats reversing both phencyclidine- and scopolamine-induced memory deficits (MED = 1 and 0.3 mg/kg, p.o, respectively). These effects were similar to those produced by intepirdine. Additionally, combination of inactive doses of compound 17 (0.1 mg/kg) and donepezil (0.3 mg/kg) produced synergistic effect to reverse scopolamine-induced memory deficits. Accordingly, investigating putative divergence between inverse agonists and neutral antagonists as cognitive enhancers in neurodegenerative and psychiatric disorders is certainly of utmost interest.

Graphic representation of pharmacology: Development of an alternative model.

Introduction:Providing clinicians with an easy to grasp and understandable representation of pharmacology is important to allow optimal clinical decisions to be made. Two of the most clinically relevant dimensions are receptor binding affinity and functional activity. The binding affinity for an agonist is described by the dissociation constant (KA), and an antagonist by the inhibition constant (Ki). Functionally, medications can act as superagonists, agonists, partial agonists, antagonists, partial inverse agonists, or inverse agonists at several receptor sites, transporters, or ion channels. Comprehending the differences between agents is complicated by the number and types of binding sites.Methods:Binding and functional data are obtained from primary literature, product labels, human cloned receptor binding, and other sources. Binding affinities are converted into ratios relative to the putative primary receptor for that category of agent. Antipsychotic binding is referenced to dopamine type 2 long (D2L) receptor binding. Binding affinity ratios (BARs) generate a 6-spoked diagram, with D2L as the hub. The most avidly bound sites are the spokes, and the disk diameter represents the BAR. Where functional data are available, they are shown as a pie chart shading the binding site's disk.Results:Binding and function diagrams are shown for the antipsychotics where binding data are available and are compared to previous methods of pharmacologic comparisons of antipsychotics.Discussion:Use of graphic models of psychotropic pharmacology improves clinician comprehension and may serve as an aid to improve rational therapeutics and patient outcomes.

Evidence for the involvement of the GABA-ergic pathway in the anticonvulsant activity of the roots bark aqueous extract of Anthocleista djalonensis A. Chev. (Loganiaceae).

The root bark of Anthocleista djalonensis A. Chev. (Loganiaceae) is widely used in traditional medicine in Northern Cameroon to treat epilepsy and related conditions, such as migraine, insomnia, dementia, anxiety, and mood disorders. To investigate the anticonvulsant effects and the possible mechanisms of this plant, an aqueous extract of Anthocleista djalonensis (AEAD) was evaluated by using animal models of bicuculline-, picrotoxin-, pilocarpine-, and pentylenetetrazole-induced convulsions. Their effects on brain γ-aminobutyric acid (GABA) concentration and GABA-T activity were also determined. This extract significantly protected mice against bicuculline-induced motor seizures. It provided 80% protection against picrotoxin-induced tonic-clonic seizures, and strongly antagonized convulsions induced by pilocarpine. AEAD also protected 100% of mice against pentylenetetrazole-induced seizures. Flumazenil, a central benzodiazepine receptor antagonist and FG7142, a partial inverse agonist in the benzodiazepine site of the GABAA receptor complex, were found to have an inhibitory effect on the anticonvulsant action of AEAD in pentylenetetrazole test. Finally, the brain GABA concentration was significantly increased and GABA-T activity was inhibited by AEAD. The effects of Anthocleista djalonensis suggested the presence of anticonvulsant properties that might involve an action on benzodiazepine and/or GABA sites in the GABAA receptor complex or by modulating GABA concentration in the central nervous system (CNS).

The Tarantula Toxin Guangxitoxin-1E Traps K+ Channel Voltage Sensor in a Fully Resting Conformation

Allosteric ligands modulate protein activity by altering the energy landscape of conformational change in ligand-protein complexes. Here, we investigate how specific steps in voltage-activation path of an ion channel are altered by a potent allosteric inhibitor. The K+ channel peptide inhibitor guangxitoxin-1E (GxTX) is partial inverse agonist of the voltage gated potassium channel subtype Kv2.1 from rat. At saturating concentrations of GxTX, Kv2.1 requires higher voltages to activate. The GxTX modulated conductance requires more voltage to activate. It has more sigmoidal activation kinetics, activates slower, and deactivates more rapidly. With GxTX bound, all gating charge requires more voltage to translocate, indicating that GxTX stabilizes resting voltage sensors by 20,000-fold, or 9 kBT. Remarkably, with GxTX bound, the rate of fast outward gating charge movement lost its positive voltage dependence. This suggests that GxTX can bind to channels in all conformations, but when gating charge is most intracellular, the toxin-channel complex adopts a stable conformation from which escape is time-dependent, but not voltage-sensitive. Channel kinetics were analyzed with a model that included a single conducting state for the channel, preceded by two voltage-dependent gating steps in each subunit, one independent and one concerted between subunits. When fit to the sigmoidicity and activation kinetics of Kv2.1 ionic currents, this model was predictive of the measured gating currents. However, with GxTX bound, this model was no longer able to predict gating current kinetics, indicating that a new conformational transition was rate limiting toxin-channel activation. We conclude that GxTX modulates Kv2.1 ion channels by “trapping” each voltage sensor in a conformation which is insensitive to voltage, yet where all gating charge remains on the intracellular side of the transmembrane electric field. Following escape from the GxTX-“trapped” state, gating charges can translocate outward, allowing channels to open with GxTX bound.

New ligands of the ghrelin receptor based on the 1,2,4-triazole scaffold by introduction of a second chiral center

Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the ‘C-terminal part’ of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog.

AC-3933, a benzodiazepine partial inverse agonist, improves memory performance in MK-801-induced amnesia mouse model

AC-3933, a novel benzodiazepine receptor partial inverse agonist, is a drug candidate for cognitive disorders including Alzheimer's disease. We have previously reported that AC-3933 enhances acetylcholine release in the rat hippocampus and ameliorates scopolamine-induced memory impairment and age-related cognitive decline in both rats and mice. In this study, we further evaluated the procognitive effect of AC-3933 on memory impairment induced by MK-801, an N-methyl-d-aspartate receptor antagonist, in mice. Unlike the acetylcholinesterase inhibitor donepezil and the benzodiazepine receptor inverse agonist FG-7142, oral administration of AC-3933 significantly ameliorated MK-801-induced memory impairment in the Y-maze test and in the object location test. Interestingly, the procognitive effects of AC-3933 on MK-801-induced memory impairment were not affected by the benzodiazepine receptor antagonist flumazenil, although this was not the case for the beneficial effects of AC-3933 on scopolamine-induced memory deficit. Moreover, the onset of AC-3933 ameliorating effect on scopolamine- or MK-801-induced memory impairment was different in the Y-maze test. Taken together, these results indicate that AC-3933 improves memory deficits caused by both cholinergic and glutamatergic hypofunction and suggest that the ameliorating effect of AC-3933 on MK-801-induced memory impairment is mediated by a mechanism other than inverse activation of the benzodiazepine receptor.

Role of GABA Deficit in Sensitivity to the Psychotomimetic Effects of Amphetamine.

Some schizophrenia patients are more sensitive to amphetamine (AMPH)-induced exacerbations in psychosis-an effect that correlates with higher striatal dopamine release. This enhanced vulnerability may be related to gamma-aminobutyric acid (GABA) deficits observed in schizophrenia. We hypothesized that a pharmacologically induced GABA deficit would create vulnerability to the psychotomimetic effects to the 'subthreshold' dose of AMPH in healthy subjects, which by itself would not induce clinically significant increase in positive symptoms. To test this hypothesis, a GABA deficit was induced by intravenous infusion of iomazenil (IOM; 3.7 μg/kg), an antagonist and partial inverse agonist of benzodiazepine receptor. A subthreshold dose of AMPH (0.1 mg/kg) was administered by intravenous infusion. Healthy subjects received placebo IOM followed by placebo AMPH, active IOM followed by placebo AMPH, placebo IOM followed by active AMPH, and active IOM followed by active AMPH in a randomized, double-blind crossover design over 4 test days. Twelve healthy subjects who had a subclinical response to active AMPH alone were included in the analysis. Psychotomimetic effects (Positive and Negative Syndrome Scale (PANSS)), perceptual alterations (Clinician Administered Dissociative Symptoms Scale (CADSS)), and subjective effects (visual analog scale) were captured before and after the administration of drugs. IOM significantly augmented AMPH-induced peak changes in PANSS positive symptom subscale and both subjective and objective CADSS scores. There were no pharmacokinetic interactions. In conclusion, GABA deficits increased vulnerability to amphetamine-induced psychosis-relevant effects in healthy subjects, suggesting that pre-existing GABA deficits may explain why a subgroup of schizophrenia patients are vulnerable to AMPH.

Effect of GABAA Partial Inverse Agonists on Drug‐facilitated ICSS Reward

We examined the ability of gamma-aminobutyric acid A receptor (GABAA) partial inverse agonists Ro15-4513 and FG-7142 and the antagonist flumazenil to block the reward-facilitating effects of methamphetamine on intracranial self-stimulation (ICSS) using a rate-frequency procedure. A total of 13 adult male C57BL6/J mice implanted with chronic bipolar stimulating electrodes directed toward the medial forebrain bundle and served as subjects. Following surgery, mice were trained to respond on an operant lever for electrical stimulation in 10 one min trials descending each subsequent min. A dose of 3 mg/kg methamphetamine robustly facilitated operant responding for ICSS (Top Figure). When administered alone, doses of 1 and 3 mg/kg flumazenil; 0.3 and 1 mg/kg RO15-4513; 0.1, 0.3 and 1 mg/kg FG-7142 did not significantly affect ICSS compared to the vehicle control. Higher doses of RO15-4513 and FG-7142 significantly decreased responding for ICSS relative to the vehicle control. Preadministration of flumazenil had no effect on methamphetamine facilitated ICSS. However, doses of 0.3 and 1 mg/kg RO15-4513, which themselves did not significantly attenuate ICSS, decreased the ICSS facilitation produced by 3 mg/kg methamphetamine (Bottom Figure). Similarly, 0.3 and 1 mg/kg FG-7142 dose-dependently decreased methamphetamine-facilitated ICSS. These results suggest an important role of GABAA receptors in modulating methamphetamine's reward-facilitating effects as well as the possibility that negative GABAA receptor modulation may have therapeutic value for the treatment of substance abuse disorders.

Chemopreventative properties of β‐blockers

Recently carvedilol, a β‐AR GRK/β‐arrestin biased agonist, was shown to have cancer preventative effects; yet, atenolol, a β2‐AR inverse agonist, was less potent and efficacious. These disparate results lead to the hypothesis that the GRK/β‐arrestin biased agonist β‐blockers act as chemopreventive agents. JB6 P+ cells are an established chemoprevention model that is transformable by epidermal growth factor (EGF). Thus, JB6 P+ cells treated with 10 ng/mL EGF were utilized to screen partial agonist, inverse agonist, neutral antagonist, and biased agonist β‐blockers for inhibition of growth in soft agar. Additionally, parallel cytotoxicity assays were conducted for each β‐blocker to ensure that any observable inhibition is not due to cell death. EGF‐mediated colony formation is fully inhibited by carvedilol with a LogIC50 of ‐6.365 ± 0.158 and partially (47.9% ± 7.6%) inhibited by atenolol with a LogIC50 of ‐5.037 ± 0.266 at non‐toxic concentrations. Preliminary studies indicate that: 1) nebivolol acts similarly to carvedilol;2) acebutolol, ICI 118,551, metoprolol, and bucindolol have little to no effect; and 3) labetalol, carazolol, and propranolol have effects similar to atenolol. Expression of only β2‐ARs can explain the changes in potency, but the differences in efficacy suggest a unique biological effect. Thus, categorization of the β‐blockers by type paradoxically indicates that β‐blockers that can activate the EGF receptor are the most potent inhibitors of EGF‐mediated transformation of JB6 P+ cells and thus my serve as chemopreventative agents.

Ligand‐based pharmacophore modeling of 5‐HT2A receptor biased agonism

The central dogma of pharmacology posits that ligands that function as agonists of a protein receptor will activate all effectors coupled to that receptor in equal proportions. A new conceptualization of ligand-receptor interactions has emerged, showing that some receptor agonists can differentially activate distinct effector systems. A single ligand can therefore function as a full agonist of one effector while being a partial agonist, neutral antagonist, or inverse agonist of another effector. Although this new conceptualization has profound implications for drug function, this area have been slowed by our poor understanding of the structural properties of ligands that determine biased activation of distinct effector systems. In these studies, we studied biased agonism of serotonin 2A (5-HT2A) receptors because there are several known 5-HT2A receptor biased agonists, extensive work has determined the effector systems activated by 5-HT2A receptor biased agonists, the downstream signaling cascades triggered by 5-HT2A receptor biased agonists are known, and the in vivo consequences of biased 5-HT2A receptor agonism are well characterized and profound. We used ligand-based computational approaches, including molecular modeling tools for pharmacophore modeling (Phase), docking (Glide), property prediction (QikProp), and QSAR z95-8. The results of these models were tested using in vitro and in vivo approaches. Our efforts have revealed distinct molecular interactions that are important determinants of biased agonism of 5-HT2A receptors and have successfully predicted several compounds that bind to these receptors.

The extracellular loop 2 (ECL2) of the human histamine H4 receptor substantially contributes to ligand binding and constitutive activity.

In contrast to the corresponding mouse and rat orthologs, the human histamine H4 receptor (hH4R) shows extraordinarily high constitutive activity. In the extracellular loop (ECL), replacement of F169 by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH4R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gαi2 and Gβ1γ2, and the membranes were studied in [3H]histamine binding and functional [35S]GTPγS assays. The potency of various ligands at the hH4R-F168A mutant decreased compared to the wild-type hH4R, for example by 30- and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH4R was completely lost in the hH4R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH4R. By analogy, JNJ7777120 was a partial inverse agonist at the hH4R, but a partial agonist at the hH4R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH4R.

The legacy of the benzodiazepine receptor: from flumazenil to enhancing cognition in Down syndrome and social interaction in autism.

The study of the psychopharmacology of benzodiazepines continues to provide new insights into diverse brain functions related to vigilance, anxiety, mood, epileptiform activity, schizophrenia, cognitive performance, and autism-related social behavior. In this endeavor, the discovery of the benzodiazepine receptor was a key event, as it supplied the primary benzodiazepine drug-target site, provided the molecular link to the allosteric modulation of GABAA receptors and, following the recognition of GABAA receptor subtypes, furnished the platform for future, more selective drug actions. This review has two parts. In a retrospective first part, it acknowledges the contributions to the field made by my collaborators over the years, initially at Hoffmann-La Roche in Basle and later, in academia, at the University and the ETH of Zurich. In the second part, the new frontier of GABA pharmacology, targeting GABAA receptor subtypes, is reviewed with special focus on nonsedative anxiolytics, antidepressants, analgesics, as well as enhancers of cognition in Down syndrome and attenuators of symptoms of autism spectrum disorders. It is encouraging that a clinical trial has been initiated with a partial inverse agonist acting on α5 GABAA receptors in an attempt to alleviate the cognitive deficits in Down syndrome.