SERCA pump as a novel therapeutic target for treating neurodegenerative disorders.

The neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) and Spinocerebellar ataxias (SCAs), present an enormous medical, social, financial and scientific problem. Despite intense research into the causes of these disorders, only marginal progress has been made in the clinic and no cures exist for any of them. Most of the scientific effort has been focused on identification of the major causes of these diseases and on developing ways to target them, such as targeting amyloid accumulation for AD or targeting expression of mutant Huntingtin for HD. Calcium (Ca2+) signaling has long been proposed to play an important role in the pathogenesis of neurodegenerative disorders, but blockers of Ca2+ channels and Ca2+ signaling proteins have not been translated to clinic primarily due to side effects related to the important roles of target molecules for these compounds at the peripheral tissues. In this review article, we would like to discuss an idea that recently identified positive allosteric modulators (PAMs) of the sarco-endoplasmic reticulum calcium (SERCA) pump may provide a promising approach to develop therapeutic compounds for treatment of these disorders. This hypothesis is supported by the preclinical data obtained with animal models of AD and PD. The first critical test of this idea will be an imminent phase I study that will offer an opportunity to evaluate potential side effects of this class of compounds in humans.

Anti-GluA3 autoantibodies define a new sub-population of frontotemporal lobar degeneration patients with distinct neuropathological features.

Autoantibodies directed against the GluA3 subunit (anti-GluA3 hIgGs) of AMPA receptors have been identified in 20%-25% of patients with frontotemporal lobar degeneration (FTLD). Data from patients and in vitro/ex vivo pre-clinical studies indicate that anti-GluA3 hIgGs negatively affect glutamatergic neurotransmission. However, whether and how the chronic presence of anti-GluA3 hIgGs triggers synaptic dysfunctions and the appearance of FTLD-related neuropathological and behavioural signature has not been clarified yet. To address this question, we developed and characterized a pre-clinical mouse model of passive immunization with anti-GluA3 hIgGs purified from patients. In parallel, we clinically compared FTLD patients who were positive for anti-GluA3 hIgGs to negative ones. Clinical data showed that the presence of anti-GluA3 hIgGs defined a subgroup of patients with distinct clinical features. In the preclinical model, anti-GluA3 hIgGs administration led to accumulation of phospho-tau in the postsynaptic fraction and dendritic spine loss in the prefrontal cortex. Remarkably, the preclinical model exhibited behavioural disturbances that mostly reflected the deficits proper of patients positive for anti-GluA3 hIgGs. Of note, anti-GluA3 hIgGs-mediated alterations were rescued in the animal model by enhancing glutamatergic neurotransmission with a positive allosteric modulator of AMPA receptors. Overall, our study clarified the contribution of anti-GluA3 autoantibodies to central nervous system symptoms and pathology and identified a specific subgroup of FTLD patients. Our findings will be instrumental in the development of a therapeutic personalised medicine strategy for patients positive for anti-GluA3 hIgGs.

Synthesis and biological studies of 2-aminothiophene derivatives as positive allosteric modulators of glucagon-like peptide 1 receptor.

As a step toward the development of novel small-molecule positive allosteric modulators (PAMs) of glucagon-like peptide 1 receptor (GLP-1R) for the treatment of type 2 diabetes, obesity, and heart diseases, we discovered a novel 2-amino-thiophene (2-AT) based lead compound bearing an ethyl 3-carboxylate appendage. In this work, we report the syntheses and biological studies of more than forty 2-AT analogs, that have revealed a 2-aminothiophene-3-arylketone analogue 7 (MW 299) showing approximately a 2-fold increase in insulin secretion at 5 µM when combined with the GLP-1 peptide at 10 nM. In vivo studies using CD1 mice at a dose of 10 mg/kg, clearly demonstrated that the blood plasma glucose level was lowered by 50% after 60 min. Co-treatment of 7 with sitagliptin, an inhibitor of GLP-1 degrading enzyme Dipeptidyl Peptidase IV, further confirmed 7 to be an effective PAM of GLP-1R. The small molecular weight and demonstrated allosteric modulating properties of these compound series, show the potential of these scaffolds for future drug development.

M1/M4 receptors as potential therapeutic treatments for schizophrenia: A comprehensive study.

Muscarinic acetylcholine receptors (mAChRs) play a significant role in the pathophysiology of schizophrenia. Although activating mAChRs holds potential in addressing the full range of schizophrenia symptoms, clinical application of many non-selective mAChR agonists in cognitive deficits, positive and negative symptoms is hindered by peripheral side effects (gastrointestinal disturbances and cardiovascular effects) and dosage restrictions. Ligands binding to the allosteric sites of mAChRs, particularly the M1 and M4 subtypes, demonstrate activity in improving cognitive function and amelioration of positive and negative symptoms associated with schizophrenia, enhancing our understanding of schizophrenia. The article aims to critically examine current design concepts and clinical advancements in synthesizing and designing small molecules targeting M1/M4, providing theoretical insights and empirical support for future research in this field.

Positive allosteric modulators of the GABAB receptor: a new class of ligands with therapeutic potential for alcohol use disorder.

BACKGROUND: Positive allosteric modulators (PAMs) of the GABAB receptor constitute a new class of GABAB-receptor ligands. GABAB PAMs reproduce several pharmacological effects of the orthosteric GABAB receptor agonist, baclofen, although displaying a better safety profile. AIMS: This paper reviews the reducing or, frequently, even suppressing effects of all GABAB PAMs tested to date on multiple alcohol-related behaviours in laboratory rodents exposed to validated experimental models of human alcohol use disorder. RESULTS: Acute or repeated treatment with CGP7930, GS39783, BHF177, rac-BHFF, ADX71441, CMPPE, COR659, ASP8062, KK-92A, and ORM-27669 reduced excessive alcohol drinking, relapse- and binge-like drinking, operant alcohol self-administration, reinstatement of alcohol seeking, and alcohol-induced conditioned place preference in rats and mice. CONCLUSIONS: These effects closely mirrored those of baclofen; notably, they were associated to remarkably lower levels of tolerance and toxicity. The recent transition of ASP8062 to clinical testing will soon prove whether these highly consistent preclinical data translate to AUD patients.

Assessing potentiation of the (α4)3(ß2)2 nicotinic acetylcholine receptor by the allosteric agonist CMPI.

The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two ß2 subunits ((α4)3(ß2)2 nAChR) harbors two high-affinity "canonical" acetylcholine (ACh)-binding sites located in the two α4:ß2 intersubunit interfaces and a low-affinity "noncanonical" ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:ß2 interfaces), TC-2559 (which binds at the α4:ß2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:ß2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(ß2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod-Wyman-Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:ß2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(ß2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.

Positive Allosteric Modulators of SERCA Pump Restore Dendritic Spines and Rescue Long-Term Potentiation Defects in Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects memory formation and storage processes. Dysregulated neuronal calcium (Ca2+) has been identified as one of the key pathogenic events in AD, and it has been suggested that pharmacological agents that stabilize Ca2+ neuronal signaling can act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+-stabilizing agents and exhibit neuroprotective properties. In the present study, we evaluated effects of a set of novel SERCA PAM agents on the rate of Ca2+ extraction from the cytoplasm of the HEK293T cell line, on morphometric parameters of dendritic spines of primary hippocampal neurons in normal conditions and in conditions of amyloid toxicity, and on long-term potentiation in slices derived from 5xFAD transgenic mice modeling AD. Several SERCA PAM compounds demonstrated neuroprotective properties, and the compound NDC-9009 showed the best results. The findings in this study support the hypothesis that the SERCA pump is a potential therapeutic target for AD treatment and that NDC-9009 is a promising lead molecule to be used in the development of disease-modifying agents for AD.

Structural basis for positive allosteric modulation of AMPA and kainate receptors.

This paper summarizes the present knowledge on how positive allosteric modulators (PAMs) interact with the ligand-binding domain (LBD) of AMPA and kainate receptors, based on structure determinations. AMPA and kainate receptors belong to the family of ionotropic glutamate receptors that are responsible for mediating the majority of fast excitatory neurotransmission. These receptors have been related to brain disorders, e.g. Alzheimer's disease and attention deficit hyperactivity disorder. PAMs are small molecules that potentiate AMPA and kainate receptor currents by interfering with receptor desensitization. Therefore, PAMs are considered to be of interest for the development of pharmacological tools. Whereas PAMs for AMPA receptors have been known for several years, only recently have PAMs for kainate receptors been reported. Today, >80 structures are available for AMPA receptors with PAMs. These PAMs bind at the interface between two LBD subunits in the vicinity of residue 775, which is important for functional differences between flip and flop isoforms of AMPA receptors. PAMs can be divided into five classes based on their binding mode. The most potent PAM reported to date belongs to class 3, which comprises dimerized PAMs. Three structures of the kainate receptor GluK1 were determined with PAMs belonging to class 2. One PAM enhances kainate receptor currents 5- to 59-fold but shows 100-fold lower potency compared to AMPA receptors. Selective PAMs for kainate receptors will be of great use as pharmacological tools for functional investigations in vivo and might potentially prove useful as drugs in controlling the activity of neuronal networks.

Positive Allosteric Modulation of α5-GABAA Receptors Reverses Stress-Induced Alterations in Dopamine System Function and Prepulse Inhibition of Startle.

BACKGROUND: Up to 64% of patients diagnosed with posttraumatic stress disorder (PTSD) experience psychosis, likely attributable to aberrant dopamine neuron activity. We have previously demonstrated that positive allosteric modulators of α5-GABAARs can selectively decrease hippocampal activity and reverse psychosis-like physiological and behavioral alterations in a rodent model used to study schizophrenia; however, whether this approach translates to a PTSD model remains to be elucidated. METHODS: We utilized a 2-day inescapable foot shock (IS) procedure to induce stress-related pathophysiology in male Sprague-Dawley rats. We evaluated the effects of intra-ventral hippocampus (vHipp) administration GL-II-73, an α5-GABAAR, or viral overexpression of the α5 subunit, using in vivo electrophysiology and behavioral measures in control and IS-treated rats. RESULTS: IS significantly increased ventral tegmental area dopamine neuron population activity, or the number of dopamine neurons firing spontaneously (n = 6; P = .016), consistent with observation in multiple rodent models used to study psychosis. IS also induced deficits in sensorimotor gating, as measured by reduced prepulse inhibition of startle (n = 12; P = .039). Interestingly, intra-vHipp administration of GL-II-73 completely reversed IS-induced increases in dopamine neuron population activity (n = 6; P = .024) and deficits in prepulse inhibition (n = 8; P = .025), whereas viral overexpression of the α5 subunit in the vHipp was not effective. CONCLUSIONS: Our results demonstrate that pharmacological intervention augmenting α5-GABAAR function, but not α5 overexpression in itself, can reverse stress-induced deficits related to PTSD in a rodent model, providing a potential site of therapeutic intervention to treat comorbid psychosis in PTSD.

6-Aryl-quinazolines as novel GABAB receptor positive allosteric modulators.

The systemic use of GABAB orthosteric agonist baclofen might be limited due to its detrimental properties: sedation and motor impairment. In contrast, GABAB positive allosteric modulators produce less adverse effects. Using BHF-177 as a starting point, we found a new active scaffold: the 6-aryl-quinazoline scaffold. Further elaborating the scaffold, we identified several in vitro and in vivo active compounds.

Synthesis and evaluation of avermectin-imidazo[1,2-a]pyridine hybrids as potent GABAA receptor modulators.

The γ-aminobutyric acid type A (GABAA) receptors are pentameric transmembrane protein complexes. They have attracted extensive attention from the scientific community due to their significant pharmacological potential. Here we report the first synthesis of avermectin-imidazo[1,2-a]pyridine hybrids promising as GABAA receptor positive allosteric modulators (PAMs). An efficient multi-step protocol was elaborated for the installation of the 6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridine pendant to the Avermectin B1a and Ivermectin skeletons through a linker. A variety of linkers were used in order to study the effect of disturbances in the hybrid structure on the GABAA receptor affinity. In vitro experiments showed that the lead compounds exhibited high potency (IC50 = 207 and 359 nM) for binding at the benzodiazepine site of GABAA receptors. In silico studies suggest that the hybrids are able to bind at the Ivermectin binding site of the GABAA receptor. The functional properties of the highest-affinity hybrid (compound 15e) as GABAAR PAM were evaluated by patch-clamp electrophysiological recordings of GABA-mediated currents in rat cerebellar Purkinje neurons. The results obtained suggest that the potentiating effect of hybrid compound 15e is due to its interaction both with benzodiazepine- and Ivermectin-binding sites of GABAARs. Drug-induced behavioral responses in adult zebrafish for hybrids correlate with an alternative mode of action of avermectin and imidazo[1,2-a]pyridine pharmacophores. The investigation of avermectin-imidazo[1,2-a]pyridine hybrid molecules with activity as GABAA receptor modulators is important for the discovery of safe and effective drugs for the treatment of neurological disorders and pest control agents.

In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators.

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-ß-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

Identification of novel positive allosteric modulators of GLP1R that stimulate its interaction with ligands and Gα subunits.

Glucagon-like peptide-1 (GLP-1) is a major incretin hormone that enhances the release of insulin from pancreatic ß-cells by activating the glucagon-like peptide-1 receptor (GLP1R), which belongs to secretin-like class B of G protein-coupled receptors (GPCRs). Owing to the absence of small molecule agonist drugs to GLP1R, focus has been placed on chemical modulators that bind to the allosteric site of GLP1R. In this study, we identified novel small-molecule positive allosteric modulators of GLP1R from a chemical library consisting of commercial drug compounds using an assay system that measures the direct interaction between a purified GLP1R and its ligand, exendin-4. Two newly identified compounds, benzethonium and tamoxifen, significantly enhanced the affinity of peptide ligands for GLP1R although they lacked agonist activity by themselves. In addition, benzethonium augmented the ligand-induced accumulation of cAMP in GLP1R-transfected HEK293T cells. These compounds significantly increased the affinity of GLP1R to the alpha-subunit of G proteins, suggesting that they stabilize GLP1R in a conformation with a higher affinity to peptide ligand as well as G proteins. These compounds may lead to the design of an orally active positive allosteric modulator for GLP1R.

In Silico Screening of Novel α1-GABAA Receptor PAMs towards Schizophrenia Based on Combined Modeling Studies of Imidazo [1,2-a]-Pyridines.

The ionotropic GABAA receptor (GABAAR) has been proven to be an important target of atypical antipsychotics. A novel series of imidazo [1,2-a]-pyridine derivatives, as selective positive allosteric modulators (PAMs) of α1-containing GABAARs with potent antipsychotic activities, have been reported recently. To better clarify the pharmacological essentiality of these PAMs and explore novel antipsychotics hits, three-dimensional quantitative structure-activity relationships (3D-QSAR), molecular docking, pharmacophore modeling, and molecular dynamics (MD) were performed on 33 imidazo [1,2-a]-pyridines. The constructed 3D-QSAR models exhibited good predictive abilities. The dockings results and MD simulations demonstrated that hydrogen bonds, π-π stackings, and hydrophobic interactions play essential roles in the binding of these novel PAMs in the GABAAR binding pocket. Four hit compounds (DS01-04) were then screened out by the combination of the constructed models and computations, including the pharmacophore model, Topomer Search, molecular dockings, ADME/T predictions, and MD simulations. The compounds DS03 and DS04, with higher docking scores and better predicted activities, were also found to be relatively stable in the binding pocket by MD simulations. These results might provide a significant theoretical direction or information for the rational design and development of novel α1-GABAAR PAMs with antipsychotic activities.

Structure optimization of positive allosteric modulators of GABAB receptors led to the unexpected discovery of antagonists/potential negative allosteric modulators.

Positive allosteric modulators (PAMs) of GABAB receptor represent an interesting alternative to receptor agonists such as baclofen, as they act on the receptor in a more physiological way and thus are devoid of the side effects typically exerted by the agonists. Based on our interest in the identification of new GABAB receptor PAMs, we followed a merging approach to design new chemotypes starting from selected active compounds, such as GS39783, rac-BHFF, and BHF177, and we ended up with the synthesis of four different classes of compounds. The new compounds were tested alone or in the presence of 10 µM GABA using [35S]GTPγS binding assay to assess their functionality at the receptor. Unexpectedly, a number of them significantly inhibited GABA-stimulated GTPγS binding thus revealing a functional switch with respect to the prototype molecules. Further studies on selected compounds will clarify if they act as negative modulators of the receptor or, instead, as antagonists at the orthosteric binding site.

Discovery and structure-activity relationships study of positive allosteric modulators of the M3 muscarinic acetylcholine receptor.

The M3 muscarinic acetylcholine receptor (mAChR) is a member of the family of mAChRs, which are associated with a variety of physiological functions including the contraction of various smooth muscle tissues, stimulation of glandular secretion, and regulation of a range of cholinergic processes in the central nerve system. We report here the discovery and a comprehensive structure--activity relationships (SARs) study of novel positive allosteric modulators (PAMs) of the M3 mAChR through a high throughput screening (HTS) campaign. Compound 9 exhibited potent in vitro PAM activity towards the M3 mAChR and significant enhancement of muscle contraction in a concentration-dependent manner when applied to isolated smooth muscle strips of rat bladder. Compound 9 also showed excellent subtype selectivity over other subtypes of mAChRs including M1, M2, and M4 mAChRs, and moderate selectivity over the M5 mAChR, indicating that compound 9 is an M3-preferring M3/M5 dual PAM. Moreover, compound 9 displayed acceptable pharmacokinetics profiles after oral dosing to rats. These results suggest that compound 9 may be a promising chemical probe for the M3 mAChR for further investigation of its pharmacological function both in vitro and in vivo.

Development of 1,3-thiazole analogues of imidazopyridines as potent positive allosteric modulators of GABAA receptors.

Structure-activity relationship studies were conducted in the search for 1,3-thiazole isosteric analogs of imidazopyridine drugs (Zolpidem, Alpidem). Three series of novel γ-aminobutyric acid receptor (GABAAR) ligands belonging to imidazo[2,1-b]thiazoles, imidazo[2,1-b][1,3,4]thiadiazoles, and benzo[d]imidazo[2,1-b]thiazoles were synthesized and characterized as active agents against GABAAR benzodiazepine-binding site. In each of these series, potent compounds were discovered using a radioligand competition binding assay. The functional properties of highest-affinity compounds 28 and 37 as GABAAR positive allosteric modulators (PAMs) were determined by electrophysiological measurements. In vivo studies on zebrafish demonstrated their potential for the further development of anxiolytics. Using the OECD "Fish, Acute Toxicity Test" active compounds were found safe and non-toxic. Structural bases for activity of benzo[d]imidazo[2,1-b]thiazoles were proposed using molecular docking studies. The isosteric replacement of the pyridine nuclei by 1,3-thiazole, 1,3,4-thiadiazole, or 1,3-benzothiazole in the ring-fused imidazole class of GABAAR PAMs was shown to be promising for the development of novel hypnotics, anxiolytics, anticonvulsants, and sedatives drug-candidates.

Examining the Effects of (α4)3(ß2)2 Nicotinic Acetylcholine Receptor-Selective Positive Allosteric Modulator on Acute Thermal Nociception in Rats.

Neuronal nicotinic acetylcholine receptor (nAChR)-based therapeutics are sought as a potential alternative strategy to opioids for pain management. In this study, we examine the antinociceptive effects of 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrazol-4-yl)isoxazole (CMPI), a novel positive allosteric modulator (PAM), with preferential selectivity to the low agonist sensitivity (α4)3(ß2)2 nAChR and desformylflustrabromine (dFBr), a PAM for α4-containing nAChRs. We used hot plate and tail flick tests to measure the effect of dFBr and CMPI on the latency to acute thermal nociceptive responses in rats. Intraperitoneal injection of dFBr, but not CMPI, dose-dependently increased latency in the hot plate test. In the tail flick test, the effect achieved at the highest dFBr or CMPI dose tested was only <20% of the maximum possible effects reported for nicotine and other nicotinic agonists. Moreover, the coadministration of dFBr did not enhance the antinociceptive effect of a low dose of nicotine. Our results show that the direct acute effect of dFBr is superior to that for CMPI, indicating that selectivity to (α4)3(ß2)2 nAChR is not advantageous in alleviating responses to acute thermal nociceptive stimulus. However, further studies are necessary to test the suitability of (α4)3(ß2)2 nAChR-selective PAMs in chronic pain models.

Chemical conversion of nicotinamide into type I positive allosteric modulator of α7 nAChRs.

Structural modifications of nicotinamide, a form of vitamin B3, gave rise to a series of compounds (8aa-8ce) that exhibit activities as type I positive allosteric modulators (PAMs) of human α7 nAChR expressed in Xenopus oocytes in two-electrode voltage clamp assay. The compound 8ai was a potent and efficacious PAM with an EC50 = 3.34 ±â€¯1.13 µM and the maximum activation effect of α7 current over 1474 ±â€¯246% in the presence of acetylcholine (100 µM). It is highly specific to α7 nAChR over other subtypes of nAChR and 5-HT3A receptors. The structure-activity relationship analysis identified a key skeleton of nicotinamide nucleus critical for biological activity. Taken together, the 8ai as a type I PAM of α7 nAChR may be beneficial for improvement of cognitive deficit.

GABAB receptor positive allosteric modulators with different efficacies affect neuroadaptation to and self-administration of alcohol and cocaine.

Drugs of abuse induce widespread synaptic adaptations in the mesolimbic dopamine (DA) neurons. Such drug-induced neuroadaptations may constitute an initial cellular mechanism eventually leading to compulsive drug-seeking behavior. To evaluate the impact of GABAB receptors on addiction-related persistent neuroplasticity, we tested the ability of orthosteric agonist baclofen and two positive allosteric modulators (PAMs) of GABAB receptors to suppress neuroadaptations in the ventral tegmental area (VTA) and reward-related behaviors induced by ethanol and cocaine. A novel compound (S)-1-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-4-methyl-6,7,8,9-tetrahydro-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (ORM-27669) was found to be a GABAB PAM of low efficacy as agonist, whereas the reference compound (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) had a different allosteric profile being a more potent PAM in the calcium-based assay and an agonist, coupled with potent PAM activity, in the [35 S] GTPγS binding assay in rat and human recombinant receptors. Using autoradiography, the high-efficacy rac-BHFF and the low-efficacy ORM-27669 potentiated the effects of baclofen on [35 S] GTPγS binding with identical brain regional distribution. Treatment of mice with baclofen, rac-BHFF, or ORM-27669 failed to induce glutamate receptor neuroplasticity in the VTA DA neurons. Pretreatment with rac-BHFF at non-sedative doses effectively reversed both ethanol- and cocaine-induced plasticity and attenuated cocaine i.v. self-administration and ethanol drinking. Pretreatment with ORM-27669 only reversed ethanol-induced neuroplasticity and attenuated ethanol drinking but had no effects on cocaine-induced neuroplasticity or self-administration. These findings encourage further investigation of GABAB receptor PAMs with different efficacies in addiction models to develop novel treatment strategies for drug addiction.