Neurogranin： an integrated approach to study a key player in the molecular basis of learning and memory

Neurogranin （Ng） is a 78 - amino acid, neuron - specific protein kinase C （PKC） substrate and a calmodulin （CaM） - binding protein through an IQ motif, i.e. the Lys - Ile - Gin - Ala - Ser - Phe （KIQASF） motif, when free Ca^2＋ concentration is low. Recent biomedical researches indicate that Ng is involved in postsynaptic signal transduction, long -term potentiation （LTP, an electrophysiological model of learning and memory）, and the behaviour- learning paradigm of the Morris Water Maze. The molecular mechanisms on how Ng is linked to these biological systems are remained obscure.     

Understanding molecular recognition by G protein βγ subunits on the path to pharmacological targeting

Heterotrimeric G proteins, composed of Gα and Gβγ subunits, transduce extracellular signals via G-protein-coupled receptors to modulate many important intracellular responses. The Gβγ subunits hold a central position in this signaling system and have been implicated in multiple aspects of physiology and the pathophysiology of disease. The Gβ subunit belongs to a large family of WD40 repeat proteins with a circular β-bladed propeller structure. This structure allows Gβγ to interact with a broad range of proteins to play diverse roles. How Gβγ interacts with and regulates such a wide variety of partners yet maintains specificity is an interesting problem in protein-protein molecular recognition in signal transduction, where signal transfer by proteins is often driven by modular conserved recognition motifs. Evidence has accumulated that one mechanism for Gβγ multitarget recognition is through an intrinsically flexible protein surface or "hot spot" that accommodates multiple modes of binding. Because each target has a unique recognition mode for Gβγ subunits, it suggests that these interactions could be selectively manipulated with small molecules, which could have significant therapeutic potential.     

Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the ��2-adrenoceptors

BACKGROUND AND PURPOSE

Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs.

EXPERIMENTAL APPROACH

In binding experiments with ³H-rauwolscine, we studied the interactions of green mamba venom fractions with α₂-adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α₂-adrenoceptors expressed in mammalian cells.

KEY RESULTS

ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of ³H-rauwolscine binding to the three α₂-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α_2A-adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA₂ values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively.

CONCLUSIONS AND IMPLICATIONS

ρ-Da1b is the first toxin identified to specifically interact with α₂-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α₂-adrenoceptor subtypes.     

Influence of the molecular structure of N6-(ω-aminoalkyl)adenosines on adenosine receptor affinity and intrinsic activity

The affinities of a series of N⁶-(ω-aminoalkyl)adenosines as probes for A₁ and A₂ adenosine receptors were determined in various radioligand binding assays and the intrinsic activities were measured in adenylate cyclase assays. Clear species differences were noticed for A₁ receptor affinity of these adenosine receptor agonists, the compounds being more active in calf than in rat brain tissue. The affinity profile within the series was, however, rather similar in both membrane preparations, with N⁶-9-aminononyladenosine displaying highest affinity. The A₂ receptor affinities were comparable to values measured for the A₁ receptor in its low affinity state, as assessed with a radiolabelled antagonist in the presence of 1 mM GTP. Calculation of the intrinsic activities of the adenosine analogues from their modulating action on adenylate cyclase showed almost all the compounds to be equally effective to (−)-N⁶-(R-phenylisopropyl) adenosine, on either A₁ or A₂ adenosine receptors. N⁶-3-Aminopropyl- and N⁶-12-aminododecyladenosine, however, proved to be partial agonists, the first on A₁ and the second on A₂ adenosine receptors. The data are used as the basis for a discussion of adenosine receptor subtype selectivity and intrinsic activity in general.     

The influence of molecular structure on the affinity and efficacy of some β-adrenoceptor agonists

Summary The affinity and efficacy of a number of sympathomimetic amines structurally related to prenalterol and the selective ₁-adrenoceptor agonist RO 363 were determined using a combination of radioligand binding and organ bath techniques. Affinity of the molecules (pK _D) was calculated from their ability to displace the radioligand [¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP) from -adrenoceptor sites in left atrial (₁) and uterine (₂) membrane homogenates. These pK _D values were used to calculate efficacy from the positive inotropic and uterine relaxant responses elicited by the drugs in organ bath experiments. The drugs studied were either arylethanolamines i.e., (–)-isoprenaline (ISO), p-hydroxyisoprenaline (pOH-ISO), compounds XIV and XVI or aryloxypropanolamine-derivatives, i.e., oxymethylene-isoprenaline (OM-ISO), prenalterol and Compound XI which possessed ap-phenol or catechol ring and an isopropyl or a homoveratryl amine substituent. Only ISO, OM-ISO, pOH-ISO and Compound XVI were active as agonists in both tissue preparations. These drugs were partial agonists which exhibited a wide range of pD₂ values and did not display any marked selectivity for either -adrenoceptor subtype. Compound XI and prenalterol were inactive as agonists and together with the partial agonists behaved as competitive antagonists to ISO in the two preparations. All drugs tested displaced [¹²⁵I]CYP from -adrenoceptor sites, however, there was also a wide range of potency amongst the drugs.Analysis of the structure-affinity and structure-efficacy relationships indicated that removal of the 3-hydroxyl group from the catechol ring reduces both affinity and efficacy without altering the selectivity of the drug for either -adrenoceptor subtype. While aryloxypropanolamine derivatives have generally higher affinities than arylethanol-amines, especially at -adrenoceptor sites, their efficacies are generally reduced at both -adrenoceptors. The presence of a homoveratryl group in aryloxypropanolamines enhances slightly the affinity for ₁- and reduces affinity for ₂-adrenoceptors. With this amine group, efficacy is markedly reduced at ₂- as opposed to ₂-adrenoceptor sites.Thus for prenalterol, the small degree of cardioselectivity can be attributed to the oxymethylene group whilst its lack of agonist activity (i.e., efficacy) reflects a combined action of this group and the absence of the 3-hydroxyl group on the phenyl ring. In RO363 it can be deduced that the oxymethylene group, together with the homoveratryl substituent are responsible for the observed selective affinity of the drug for ₁- as opposed to ₂-adrenoceptors.     

A combined modelling and experimental study of the surface energetics of α-lactose monohydrate

The surface energy of α-lactose monohydrate measured by inverse gas chromatography (IGC) is reported along with a dynamic molecular modelling study of the interaction of the various molecular probes with different surfaces of α-lactose monohydrate. The IGC results show that α-lactose monohydrate is acidic in nature. Using quantitative calculations of the energy of adsorption, the acidic nature of the surface is confirmed and the calculated values agree closely with the experimentally measured values. Along with the acidic nature, dynamic molecular modelling also reveals that the presence of a channel and water molecules on a surface affects the surface energetics of that face. The presence of water on the surface can decrease or increase the surface energy by either blocking or attracting a probe molecule, respectively. This property of water depends on its position and association with other functional groups present on the surface. The effect of a channel or cavity on the surface energy is shown to depend on its size, which determines whether the functional groups in the channel are assessable by probe molecules or not. Overall molecular modelling explains, at the molecular level, the effect of different factors affecting the surface energy of individual faces of the crystal. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:741–752, 2010     

Structure-based ensemble-QSAR model： a novel approach to the study of the EGFR tyrosine kinase and its inhibitors

Aim： To develop a novel 3D-QSAR approach for study of the epidermal growth factor receptor tyrosine kinase （EGFR TK） and its inhibi- tors. Methods： One hundred thirty nine EGFR TK inhibitors were classified into 3 clusters. Ensemble docking of these inhibitors with 19 EGFR TK crystal structures was performed. Three protein structures that showed the best recognition of each cluster were selected based on the docking results. Then, a novel QSAR （ensemble-QSAR） building method was developed based on the ligand conforma- tions determined by the corresponding protein structures. Results： Compared with the 3D-QSAR model, in which the ligand conformations were determined by a single protein structure, ensemble-QSAR exhibited higher R2 （0.87） and Q2 （0.78） values and thus appeared to be a more reliable and better predictive model. Ensemble-QSAR was also able to more accurately describe the interactions between the target and the ligands. Conclusion： The novel ensemble-QSAR model built in this study outperforms the traditional 3D-QSAR model in rationality, and provides a good example of selecting suitable protein structures for docking prediction and for building structure-based QSAR using available protein structures.     

Bioactivity and pharmacological properties of α-mangostin from the mangosteen fruit: a review

Introduction: α-Mangostin (α-MG) is the most representative xanthone isolated from the pericarp of mangosteen, possessing extensive biological activities and pharmacological properties, considered as an antineoplastic agent, antioxidant, anti-proliferation and induces apoptosis.

Areas covered: The bioactivity and pharmacological properties of α-MG are being actively investigated by various industrial and academic institutions. The bioactivities of α-MG have been summarized in several previous reviews, which were worthy of high compliment. However, recently, many new literatures about the bioactivities of α-MG have been further reported from 2016 to 2017. Herein, the activities of α-MG are supplemented and summarized in this text.

Expert opinion: As previously said, α-MG possesses good bioactivities pharmacological properties. More recently, it found that α-MG has the effect of maintaining cardiovascular system and gastrointestinal health and controlling free radical oxidation. Furthermore, α-MG has more applications in cosmetics, with the effects of anti-aging, anti-wrinkle, acne treatment, maintenance of skin lubrication. The application of α-MG in treating rheumatoid arthritis has been disclosed and the MG-loaded self-micro emulsion (MG-SME) was designed to improve its pharmacokinetic deficiencies. As mentioned above, α-MG can be a promising drug, also worthy of developing, and further research is crucial for the future application of α-MG.     

Knowledge and pharmacological management of Alzheimer’s disease by managing community pharmacists: a nationwide study

Background Managing community pharmacists can play a leading role in supporting community dwelling individuals with Alzheimer’s disease and their caregivers. Objective The main purpose of this study was to assess knowledge of managing community pharmacists towards Alzheimer’s disease and its pharmacological management. Setting Community pharmacies in the Maltese islands. Method A nationwide survey was conducted with full-time managing community pharmacists in possession of a tertiary education degree in pharmacy studies. The level of knowledge was investigated using the Alzheimer’s Disease Knowledge Scale and the Alzheimer’s Disease Pharmacotherapy Measure. Participants were also asked to rate a number of statements related to disease management. Results Maltese managing community pharmacists (57 % response rate) had inadequate knowledge on risk factors, caregiving issues and pharmacological management of Alzheimer’s disease. Age and number of years working in a community pharmacy setting were found to be negatively correlated with increased knowledge. Conclusion The findings highlight the need of providing training and continued educational support to managing community pharmacists in order to provide quality advice to individuals with dementia and their caregivers in the community.     

Metabolomics study on the synergistic interaction between Salvia miltiorrhiza and Lignum dalbergiae odoriferae used as ��Jun-Shi�� herbs in a S. miltiorrhiza recipe

A metabolomic method was established to investigate the plasma metabolic difference between healthy rabbits and rabbits with Qi-stagnancy and blood stasis. All rabbits were administrated with an extraction of Salvia miltiorrhiza and S. miltiorrhiza coupled with Lignum dalbergiae odoriferae. The main compounds in plasma samples were detected by high-performance liquid chromatography/diode array detector/electrospray-mass spectrometry (HPLC/DAD/ESI-MS). The data were analyzed by principal component analysis (PCA). The results showed that Qi-stagnancy and blood stasis had a close relationship with dopamine. In addition, the results also indicated that the major plasma metabolic difference between rabbits administrated with S. miltiorrhiza and S. miltiorrhiza coupled with Lignum dalbergiae odoriferae were 4-methylbenzamide, Danshensu and β-(3,4-dihydroxybenzene)-α-hydroxyl propanoic acid isopropyl ester. The above results could provide experimental evidence for the theory of traditional Chinese medicine.     

Special issue on molecular targets, biomarkers and animal models for anti-cancer pharmacological research： potentials and challenges from chemoprevention to chemotherapeutic treatment

With the current advances in the field of cancer research particularly in cancer chemoprevention, we have much to celebrate but we also have a lot of challenges before us. Some of the challenges include finding appropriate animal models for predicting efficacy of chemopreventive compounds for human cancers, of which are usually highly complex and involved multiple genes in nature and translating our basic research into clinical practice. One of the other challenges will be to disseminate our ideas into the larger biomedical and pharmaceutical community. With this in mind, I have been asked by the Editorial Team of Acta Pharmacologica Sinica to organize a special issue to discuss the current advances in cancer chemoprevention from prevention to treatment. The distinction and the potential overlapping nature of cancer chemoprevention versus chemotherapeutic treatment has become somewhat blurred, since prevention of carcinogenesis encompasses preventing any or all of the three stages of carcinogenesis namely initiation, promotion and progression. Oftentimes, many cancer chemopreventive compounds can possess all three biological properties that culminate in the overall protection against cancer. To be most effective, cancer chemoprevention of earlier lesions will be the ultimate goal if we are to eradicate this dreadful disease, since advanced metastasized cancers are almost lethal and unresponsive to radiation and chemotherapy. The reviews covered in this special issue, which are based on strong preclinical studies, will hopefully provide the impetus to translate this information to clinical practice and improved patient care.     

Is the vascular system a main target for thyroid hormones? From molecular and biochemical findings to clinical perspectives

The cardiovascular system is an important target for thyroid hormones (THs). Until recently, our understanding of the biological role of THs has been largely based on a catalog of effects observed in excess or deficiency of THs. In the last decades, however, some important progress has been done in defining the molecular and biochemical basis of thyroid hormone action at the cellular and nuclear level. Most of the molecular and cellular mechanisms responsible for the effects of THs on the heart have been clarified, whereas few data are available about the mechanisms of action of THs on the vasculature. Data reported so far describe the thyroid hormone effects on the vascular system as indirect consequences of thermogenic or hemodynamic derangements. The aim of this review is to focus on the direct role of THs in the vascular system, to analyze the main factors involved in this regulatory process, to evaluate the causes of imbalance, their relationships to some pathophysiological conditions, and, finally, to hypothesize effective therapeutic approaches. Our review considers data on the molecular and biochemical properties of iodothyronine deiodinases, with particular attention to D2, the enzyme for the local conversion of the precursor thyroxine (T4) into the biologically active triiodothyronine (T3). We summarize data on the deiodinase tissue distribution, subcellular localization, topology and structure-activity relationships. We also discuss the physiological role of deiodinases and their involvement in the TH-mediated regulation of vascular function.     

Binding affinity of levomepromazine and two of its major metabolites to central dopamine and α-adrenergic receptors in the rat

N-Monodesmethyl levomepromazine and levmepromazine sulfoxide have previously been found in higher plasma concentrations than the parent drug in patients who received oral doses of levomepromazine. In the present study levomepromazine, N-monodesmethyl levomepromazine and levomepromazine sulfoxide have been assayed for their binding affinity to rat striatal dopamine receptors and to -adrenergic receptors in rat cortex, and compared with the potency of chlorpromazine and some of its metabolites in the same systems. Levomepromazine sulfoxide was relatively inactive in the dopamine receptor binding test but much more active in the -adrenergic receptor binding test, where it had a binding affinity similar to 7-hydroxy chlorpromazine. Levomepromazine and N-monodesmethyl levomepromazine were active in both systems, having a slightly higher potency than chlorpromazine in the -adrenergic binding test, and a somewhat lower potency than chlorpromazine in the dopamine receptor binding test. The results indicate that N-monodesmethyl levomepromazine may significantly contribute to the antipsychotic effects of levomepromazine while the sulfoxide metabolite lacks neuroleptic potency, and that both metabolites may contribute to the autonomic side-effects of the drug.     

What is a food and what is a medicinal product in the European Union? Use of the benchmark dose (BMD) methodology to define a threshold for “pharmacological action”

The decision criterion for the demarcation between foods and medicinal products in the EU is the significant “pharmacological action”. Based on six examples of substances with ambivalent status, the benchmark dose (BMD) method is evaluated to provide a threshold for pharmacological action. Using significant dose–response models from literature clinical trial data or epidemiology, the BMD values were 63 mg/day for caffeine, 5 g/day for alcohol, 6 mg/day for lovastatin, 769 mg/day for glucosamine sulfate, 151 mg/day for Ginkgo biloba extract, and 0.4 mg/day for melatonin. The examples for caffeine and alcohol validate the approach because intake above BMD clearly exhibits pharmacological action. Nevertheless, due to uncertainties in dose–response modelling as well as the need for additional uncertainty factors to consider differences in sensitivity within the human population, a “borderline range” on the dose–response curve remains. “Pharmacological action” has proven to be not very well suited as binary decision criterion between foods and medicinal product. The European legislator should rethink the definition of medicinal products, as the current situation based on complicated case-by-case decisions on pharmacological action leads to an unregulated market flooded with potentially illegal food supplements.     

(3R, 3’R)-zeaxanthin protects the retina from photo-oxidative damage via modulating the inflammation and visual health molecular markers

Purpose: Zeaxanthin protects the macula from ocular damage due to light or radiation by scavenging harmful reactive oxygen species. In the present study, zeaxanthin product (OmniXan®; OMX), derived from paprika pods (Capsicum annum; Family-Solanaceae), was tested for its efficacy in the rat retina against photooxidation.

Methods: Forty-two male 8-week-old Wistar rats exposed to 12L/12D, 16L/8D and 24L/0D hours of intense light conditions were orally administrated either 0 or 100?mg/kg BW of zeaxanthin concentration. Retinal morphology was analyzed by histopathology, and target gene expressions were detected with real-time polymerase chain reaction methods.

Results: OMX treatment significantly increased the serum zeaxanthin concentration (p?<?0.001) and ameliorated oxidative damage by increasing the antioxidant enzyme activities in the retina induced by light (p?<?0.001). OMX administration significantly upregulated the expression of genes, including Rhodopsin (Rho), Rod arrestin (SAG), Gα Transducin 1 (GNAT-1), neural cell adhesion molecule (NCAM), growth-associated protein 43 (GAP43), nuclear factor-(erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase (HO-1) and decreased the expression of nuclear factor-κB (NF- κB) and GFAP by OMX treatment rats. The histologic findings confirmed the antioxidant and gene expression data.

Conclusions: This study suggests that OMX is a potent substance that can be used to protect photoreceptor cell degeneration in the retina exposed to intense light.     

Prospects of multitarget drug designing strategies by linking molecular docking and molecular dynamics to explore the protein–ligand recognition process

The designing of drugs that can simultaneously affect different protein targets is one novel and promising way to treat complex diseases. Multitarget drugs act on multiple protein receptors each implicated in the same disease state, and may be considered to be more beneficial than conventional drug therapies. For example, these drugs can have improved therapeutic potency due to synergistic effects on multiple targets, as well as improved safety and resistance profiles due to the combined regulation of potential primary therapeutic targets and compensatory elements and lower dosage typically required. This review analyzes in-silico methods that facilitate multitarget drug design that facilitate the discovery and development of novel therapeutic agents. Here presented is a summary of the progress in structure-based drug discovery techniques that study the process of molecular recognition of targets and ligands, moving from static molecular docking to improved molecular dynamics approaches in multitarget drug design, and the advantages and limitations of each.     

Age and gender dependent bioavailability of R- and R,S-α-lipoic acid: a pilot study

Lipoic acid (LA) shows promise as a beneficial micronutrient toward improving elder health. Studies using old rats show that (R)-α-LA (R-LA) significantly increases low molecular weight antioxidants that otherwise decline with age. Despite this rationale for benefiting human health, little is known about age-associated alterations in absorption characteristics of LA, or whether the commercially available racemic mixture of LA (R,S-LA) is equally as bioavailable as the naturally occurring R-enantiomer. To address these discrepancies, a pilot study was performed to establish which form of LA is most effectively absorbed in older subjects relative to young volunteers. Young adults (average age=32 years) and older adults (average age=79 years) each received 500 mg of either R- or R,S-LA. Blood samples were collected for 3h after supplementation. After a washout period they were given the other chiral form of LA not originally ingested. Results showed that 2 out of 6 elder males exhibited greater maximal plasma LA and area under the curve for the R-form of LA versus the racemic mixture. The elder subjects also demonstrated a reduced time to reach maximal plasma LA concentration following R-LA supplementation than for the racemic mixture. In contrast, young males had a tendency for increased bioavailability of R,S-LA. Overall, bioavailability for either LA isoform was much more variable between older subjects compared to young adults. Plasma glutathione levels were not altered during the sampling period. Thus subject age, and potential for varied response, should be considered when determining an LA supplementation regimen.     

Are women more susceptible than men to drug-induced QT prolongation? Concentration-QTc modelling in a phase 1 study with oral rac-sotalol

Aim

To study the differences in QT_c interval on ECG in response to a single oral dose of rac-sotalol in men and women.

Methods

Continuous 12-lead ECGs were recorded in 28 men and 11 women on a separate baseline day and following a single oral dose of 160 mg rac-sotalol on the following day. ECGs were extracted at prespecified time points and upsampled to 1000 Hz and analyzed manually in a central ECG laboratory on the superimposed median beat. Concentration–QTc analyses were performed using a linear mixed effects model.

Results

Rac-sotalol produced a significant reduction in heart rate in men and in women. An individual correction method (QT_cI) most effectively removed the heart rate dependency of the QT_c interval. Mean QT_cI was 10 to 15 ms longer in women at all time points on the baseline day. Rac-sotalol significantly prolonged QT_cI in both genders. The largest mean change in QT_cI (ΔQT_cI) was greater in females (68 ms (95% confidence interval (CI) 59, 76 ms) vs. 27 ms (95% CI 22, 32 ms) in males). Peak rac-sotalol plasma concentration was higher in women than in men (mean C_max 1.8 μg ml⁻¹ (range 1.1–2.8) vs. 1.4 μg ml⁻¹ (range 0.9–1.9), P = 0.0009). The slope of the concentration–ΔQT_cI relationship was steeper in women (30 ms per μg ml⁻¹ vs. 23 ms per μg ml⁻¹ in men; P = 0.0135).

Conclusions

The study provides evidence for a greater intrinsic sensitivity to rac-sotalol in women than in men for drug-induced delay in cardiac repolarization.     

Partial Agonists of the ��3��4* Neuronal Nicotinic Acetylcholine Receptor Reduce Ethanol Consumption and Seeking in Rats

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3–CHRNA5–CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2^* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.