Current status of drug receptor nomenclature: receptor closure? The role of NC-IUPHAR

Pharmacology is at a crucial point, because we now have access to sequences, by homology, for almost all of the receptors in the human genome. The International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) has set up > 50 subcommittees to define the receptors, and their recommendations, when approved, are posted on a website freely available to all scientists. A major new effort is to functionally define relevant receptor polymorphisms. This initiative is open to all, and works only because of the freely given voluntary effort of scientists.     

Combined β-adrenergic and corticosteroid receptor activation regulates AMPA receptor function in hippocampal neurons

Shortly after stress, limbic neurons are exposed to high levels of noradrenaline and corticosterone. These hormones are necessary for optimal behavioural adaptation. Behavioural effects critically depend on noradrenaline acting via β-adrenergic receptors, but these effects are strongly modulated by corticosterone, indicating putative interactions between the two hormones. Since both noradrenaline and corticosterone are known to quickly affect properties of AMPA-type glutamate receptors (AMPAR), we here examined - in hippocampal neurons - three parameters which give insight in the functionality of AMPARs: phosphorylation, surface expression and spontaneous synaptic transmission. In homogenates of adult hippocampal slices, application of corticosterone (30 nM for 15 min) by itself did not affect phosphorylation of the AMPAR GluA1 subunit at S845 or S831. Co-application of the β-adrenergic receptor agonist isoproterenol (10 μM) largely increased S845 (but not S831) phosphorylation. Corticosterone also did not change GluA1 and GluA2 surface expression in hippocampal primary cultures. However, combined administration of corticosterone and 1 μM isoproterenol - which by itself was ineffective - enhanced surface expression. Interestingly, 10 μM isoproterenol alone enhanced GluA1 surface expression, but this was decreased by corticosterone. Finally, in hippocampal primary cultures, the inter-event interval of miniature excitatory postsynaptic currents (mEPSCs) was decreased by the combination of 1 μM isoproterenol and corticosterone (which were ineffective by themselves) while the same combination did not affect the amplitude. We conclude that AMPAR phosphorylation, surface expression and mEPSC inter-event interval respond most strongly to a combination of corticosterone and β-adrenergic receptors. These combined hormonal effects on glutamate transmission might contribute to their memory-enhancing effects.     

Discovery of aminobenzyloxyarylamides as κ opioid receptor selective antagonists: application to preclinical development of a κ opioid receptor antagonist receptor occupancy tracer

Arylphenylpyrrolidinylmethylphenoxybenzamides were found to have high affinity and selectivity for κ opioid receptors. On the basis of receptor binding assays in Chinese hamster ovary (CHO) cells expressing cloned human opioid receptors, (S)-3-fluoro-4-(4-((2-(3-fluorophenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide (25) had a K(i) = 0.565 nM for κ opioid receptor binding while having a K(i) = 35.8 nM for μ opioid receptors and a K(i) = 211 nM for δ opioid receptor binding. Compound 25 was also a potent antagonist of κ opioid receptors when tested in vitro using a [(35)S]-guanosine 5'O-[3-thiotriphosphate] ([(35)S]GTP-γ-S) functional assay in CHO cells expressing cloned human opioid receptors. Compounds were also evaluated for potential use as receptor occupancy tracers. Tracer evaluation was done in vivo, using liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods, precluding the need for radiolabeling. (S)-3-Chloro-4-(4-((2-(pyridine-3-yl)pyrrolidin-1-yl)methyl)phenoxy)benzamide (18) was found to have favorable properties for a tracer for receptor occupancy, including good specific versus nonspecific binding and good brain uptake.     

Pyrazolo-triazolo-pyrimidine derivatives as adenosine receptor antagonists: a possible template for adenosine receptor subtypes?

Adenosine, a widely distributed modulator, regulates many physiological functions through specific cell membrane G-protein-coupled receptors classified as A(1), A(2A), A(2B) and A(3). An intense medicinal chemistry effort made over the last 20 years has led to a variety of selective adenosine receptor agonists and antagonists. In particular, the pyrazolo-triazolo-pyrimidine nucleus has been strongly investigated in the last years by our group. All the modifications performed and a tentative of structure-activity-relationship is reported. In fact, the combination of different substitutions at the N(7), N(8) and N(5) positions afford compounds which showed good affinity and selectivity for the different adenosine receptor subtypes. The data herein summarized, permit to speculate on the use of this nucleus as possible template for the adenosine receptor subtypes.     

The cannabinoid receptor inverse agonist AM251 regulates the expression of the EGF receptor and its ligands via destabilization of oestrogen-related receptor α protein

BACKGROUND AND PURPOSE

AM251 is an inverse agonist of the cannabinoid 1 receptor (CB₁R) that can exert ‘off-target’ effects in vitro and in CB₁R knock-out mice. AM251 is also potent at modulating tumour cell growth, suggesting that growth factor-mediated oncogenic signalling could be regulated by AM251. Since dysregulation of the EGF receptor has been associated with carcinogenesis, we examined AM251 regulation of EGF receptor (EGFR) expression and function.

EXPERIMENTAL APPROACH

The various biological functions of AM251 were measured in CB₁R-negative human cancer cells. Pharmacological and genetic approaches were used to validate the data.

KEY RESULTS

The mRNA levels for EGFR and its associated ligands, including HB-EGF, were induced several fold in PANC-1 and HCT116 cells in response to AM251. This event was associated with enhanced expression of EGFR on the cell surface with concomitant increase in EGF-induced cellular responses in AM251-treated cells. Exposure to XCT790, a synthetic inverse agonist of the orphan nuclear oestrogen-related receptor α (ERRα), also induced EGFR and HB-EGF expression to the same extent as AM251, whereas pretreatment with the ERRα-selective agonist, biochanin A, blunted AM251 actions. AM251 promoted the degradation of ERRα protein without loss of the corresponding mRNA. Knock-down of ERRα by siRNA-based approach led to constitutive induction of EGFR and HB-EGF levels, and eliminated the biological responses of AM251 and XCT790. Finally, AM251 displaced diethylstilbestrol prebound to the ligand-binding domain of ERRα.

CONCLUSIONS AND IMPLICATIONS

AM251 up-regulates EGFR expression and signalling via a novel non-CB₁R-mediated pathway involving destabilization of ERRα protein in selected cancer cell lines.     

Anti-nociception mediated by a κ opioid receptor agonist is blocked by a δ receptor agonist

BACKGROUND AND PURPOSE

The opioid receptor family comprises four structurally homologous but functionally distinct sub-groups, the μ (MOP), δ (DOP), κ (KOP) and nociceptin (NOP) receptors. As most opioid agonists are selective but not specific, a broad spectrum of behaviours due to activation of different opioid receptors is expected. In this study, we examine whether other opioid receptor systems influenced KOP-mediated antinociception.

EXPERIMENTAL APPROACH

We used a tail withdrawal assay in C57Bl/6 mice to assay the antinociceptive effect of systemically administered opioid agonists with varying selectivity at KOP receptors. Pharmacological and genetic approaches were used to analyse the interactions of the other opioid receptors in modulating KOP-mediated antinociception.

KEY RESULTS

Etorphine, a potent agonist at all four opioid receptors, was not anti-nociceptive in MOP knockout (KO) mice, although etorphine is an efficacious KOP receptor agonist and specific KOP receptor agonists remain analgesic in MOP KO mice. As KOP receptor agonists are aversive, we considered KOP-mediated antinociception might be a form of stress-induced analgesia that is blocked by the anxiolytic effects of DOP receptor agonists. In support of this hypothesis, pretreatment with the DOP antagonist, naltrindole (10 mg·kg⁻¹), unmasked etorphine (3 mg·kg⁻¹) antinociception in MOP KO mice. Further, in wild-type mice, KOP-mediated antinociception by systemic U50,488H (10 mg·kg⁻¹) was blocked by pretreatment with the DOP agonist SNC80 (5 mg·kg⁻¹) and diazepam (1 mg·kg⁻¹).

CONCLUSIONS AND IMPLICATIONS

Systemic DOP receptor agonists blocked systemic KOP antinociception, and these results identify DOP receptor agonists as potential agents for reversing stress-driven addictive and depressive behaviours mediated through KOP receptor activation.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

Protein kinase C-mediated phosphorylation of the μ-opioid receptor and its effects on receptor signaling

Phosphorylation of the μ opioid receptor (MOPr), mediated by several protein kinases, is a critical process in the regulation of MOPr signaling. Although G protein-coupled receptor kinases are known to play an essential role in the agonist-induced phosphorylation and desensitization of MOPr, evidence suggests that other protein kinases, especially protein kinase C (PKC), also participate in the regulation of MOPr signaling. In this study, we investigated the biochemical nature and downstream effects of PKC-mediated MOPr phosphorylation. We observed in vitro phosphorylation of the MOPr C terminus by purified PKC. Protein mass spectrometry and site-directed mutagenesis implicated Ser363 of MOPr as the primary substrate for PKC, and this was confirmed in Chinese hamster ovary cells stably expressing full-length MOPr using an antibody that specifically recognizes phosphorylated Ser363. Alanine mutation of Ser363 did not affect the affinity of MOPr-ligand binding and the efficiency of receptor G-protein coupling. However, the S363A mutation attenuated the desensitization of receptor G-protein coupling induced by phorbol 12-myristate. Our research thus has identified a specific PKC phosphorylation site in MOPr and demonstrated that PKC-mediated phosphorylation of MOPr induces receptor desensitization at the G protein coupling level.     

Protective effects of glycyrrhizin against β₂-adrenergic receptor agonist-induced receptor internalization and cell apoptosis

It has been reported that treatment with β? adrenergic receptor (β?AR) agonist bronchodilators may result in airway β?ARs internalization and cardiac muscle cells apoptosis. This could lead to the loss of pharmacological effect of β?AR agonists and increase adverse cardiovascular events in asthma patients receiving β?AR agonist therapy. Glycyrrhizin, the major bioactive component of licorice root extract, has been reported to exhibit protective effect on respiratory system. Here, we investigate the effects of glycyrrhizin against β?AR agonist salbutamol-induced receptor internalization and cell apoptosis. In our study, the live cell confocal imaging and fixed-cell enzyme-linked immunosorbent assay (ELISA) assay revealed that glycyrrhizin significantly inhibited salbutamol-induced surface β?AR internalization. The underlying mechanisms were then identified to be that glycyrrhizin could reduce the association of β?ARs with β-arrestins and clathrin heavy chain as well as the level of G protein-coupled receptor kinase (GRK) mediated phosphorylation of β?ARs. The inhibition of receptor internalization by glycyrrhizin further lead to stabilization of the β?AR mRNA and protein expression, thus amplified the transmembrane signaling via the β?ARs. We also proved that glycyrrhizin could profoundly attenuate salbutamol-induced early cellular apoptosis by regulating the expressions of B-cell lymphoma 2 (Bcl-2) family genes. Taken together, our results suggest that glycyrrhizin exhibits protective effects against β?AR agonist-induced receptor internalization and cell apoptosis. These findings might have practical implications for future strategies of combined application of glycyrrhizin with β?AR receptor agonists to improve the efficacy of bronchodilators in patients with asthma and chronic obstructive pulmonary disease (COPD).     

Pharmacology and toxicology of fibrates as hypolipidemic drugs mediated by nuclear receptor peroxisome proliferator—activated receptor

PPAR（peroxisome proliferator-activated receptor) is a family of nuclear receptor.In recent years,it has been focused for the discovery and development of new drugs which are mediated by PPARs.Fibrate hypolipidemic drugs are the specific and potent ligands to PPAR alpha and have been widely used for the treatment of hyperlipidemia.But these drugs induce hepatocarcinogenesis in rodent animals after the long-term administration.However,there are species differences on these phenomena which are not seen in mammals ioncluding human.To clarify the mechanism of carcinogenesis by these drugs in important for the evaluation of safety of these drugs in human.     

Association of vitamin D receptor and estrogen receptor-α gene polymorphismwith peak bone mass and bone size in Chinese women

AIM: To investigate if vitamin D receptor (VDR) gene Apa I polymorphism and estrogen receptor-alpha (ER-alpha) gene Pvu II, Xba I polymorphisms are related to bone mineral density (BMD), bone mineral content (BMC) and bone size in premenopausal Chinese women. METHODS: The VDR Apa I genotype and ER-alpha Pvu II, Xba I genotype were determined by PCR-restriction fragment length polymorphism (RFLP) in 493 unrelated healthy women aged 20-40 years of Han nationality in Shanghai city. BMD (g/cm(2)), BMC (g), and bone areal size (BAS, cm(2) ) at lumbar spine 1-4 (L(1-4)) and proximal femur (femoral neck, trochanter and Ward's triangle) were measured by duel-energy X-ray absorptionmetry. RESULTS: All allele frequencies did not deviate from Hardy-Weinberg equilibrium. After phenotypes were adjusted for age, height, and weight, a significant association was found between VDR Apa I genotype and BMC variation at L(1-4) and Ward's triangle (P<0.05), but not in BMD or BAS at lumbar spine and proximal femur. ER-a Pvu II, Xba I genotype was not related to BMC, BMD, and BAS at all sites. CONCLUSION: The study suggested that Apa I polymorphism in VDR gene may influence on attainment and maintenance of peak bone mass in premenopausal Chinese women.     

Association of estrogen receptor-alpha and vitamin D receptor genotypes with therapeutic response to calcium in postmenopausal Chinese women

AIM:Toinvestigatethecorrelationbetweencalciumtreatmentinpostmenopausalwomenandestrogenreceptor-alpha(ER-alpha)XbaIandPvuIIgenotypeandvitaminDreceptor(VDR)ApaIgenotype.METHODS:OnehundredfifteenpostmenopausalChinesewomenofHanpopulationwereenrolledandtreatedwithcalcichew-D3(1000mgcalciumand400UvitaminD3)dailyfor1year.Atentryandafter1yeartreatment,thebonemineraldensity(BMD),serumandurinaryboneturnoverbiochemicalmarkerswereevaluated.ER-alphaandVDRgeno-typewereanalyzedusingPCR-restric…     

Vitamin D receptor gene polymorphism as an important modifier of positive family history related breast cancer risk

The association between vitamin D receptor (VDR) gene polymorphisms and diseases such as breast cancer, prostate cancer and osteoporosis has been extensively investigated during recent years. To date, several polymorphisms have been found in the VDR gene. In this Finnish case-control study, comprising 483 breast cancer patients and 482 healthy population controls, we investigated the association between altered breast cancer risk and two polymorphisms in the 3' end of the gene detectable with ApaI and TaqI restriction enzymes. A statistically significant difference was observed in the ApaI genotype distribution between cases and controls. Women with the VDR variant a allele containing genotypes showed a decreased risk for breast cancer [odds ratio (OR) 0.73, 95% confidence interval (CI) 0.54-0.98] compared to women with the AA genotype. This association was especially strong among women with a positive family history of breast cancer (OR 0.14, 95% CI 0.03-0.76). Moreover, there was a trend (P for trend = 0.0007) for decreased risk with increasing number of variant alleles. The lowest risk of breast cancer was seen for the women with the aa genotype (OR 0.03, 95% CI 0.003-0.31) compared to women with the AA genotype. A tendency of decreased risk of breast cancer was also observed for the TaqI T allele containing genotypes (Tt and TT) (OR 0.68, 95% CI 0.41-1.12), but because the distribution of Taql alleles in the controls missed the Hardy-Weinberg equilibrium (P = 0.01), we were unable to properly assess the potential impact of the TaqI polymorphism in breast cancer susceptibility. In conclusion, our results suggest that the VDR ApaI genotype may be an important modifier of individual breast cancer risk among Finnish women, especially if they have a positive family history of breast cancer.     

Vitamin D receptor polymorphisms and renal cancer risk in Central and Eastern Europe

Previous studies investigated the role of vitamin D intake and cancer risk. The kidney is a major organ for vitamin D metabolism, activity, and calcium homeostasis; therefore, it was hypothesized that dietary vitamin D intake and polymorphisms in the vitamin D receptor (VDR) gene may modify renal cell carcinoma (RCC) risk. Three common VDR gene polymorphisms (BsmI, FokI, TaqI) were evaluated among 925 RCC cases and 1192 controls enrolled in a hospital-based case-control study conducted in Central and Eastern Europe. Overall associations with RCC risk were not observed; however, subgroup analyses revealed associations after stratification by median age of diagnosis and family history of cancer. Among subjects over 60 yr, reduced risks were observed among carriers of the f alleles in the FokI single-nucleotide polymorphism (SNP) (odds ratio [OR] = 0.61 for Ff and OR = 0.74 for ff genotypes) compared to subjects with the FF genotype (P trend = 0.04; P interaction = 0.004). Subjects with the BB BsmI genotype and a positive family history of cancer had lower risk compared to subjects with the bb allele (OR = 0.60; 95% CI: 0.33-1.1; P trend = 0.05). Genotype associations with these subgroups were not modified when dietary sources of vitamin D or calcium were considered. Additional studies of genetic variation in the VDR gene are warranted.     

Vitamin D receptor genotypes influence the success of calcitriol therapy for recurrent vertebral fracture in osteoporosis

Osteoporosis is a complex multi-factorial disease where environment, diet and genetics play a role in determining susceptibility. Patients with existing vertebral fracture have a heightened risk of further recurrent vertebral fracture. The efficacy of new osteoporosis therapies is often compared to calcium supplementation. 1,25-dihydroxyvitamin D3 (calcitriol) acts through the vitamin D receptor (VDR) and is effective at reducing recurrent vertebral fracture risk. Because the VDR controls calcium metabolism, we hypothesized that genetic variation at the VDR locus may influence response to both calcium and calcitriol therapy. Postmenopausal women with osteoporosis from a 3-year study comparing calcitriol versus calcium for prevention of vertebral fractures were genotyped for VDR alleles detected by FokI, BsmI, ApaI and TaqI. Data were analysed by hierarchical log-linear analysis and robust analysis of variance for relationships to fracture outcomes. Significant differences in the vertebral fracture rate in response to calcium therapy were observed between VDR genotypes (P<0.001). Calcium appeared to be equally effective as calcitriol in particular genotypes. The response to calcitriol therapy was most pronounced in patients carrying the TaqI t allele in combination with the FokI f initiation codon variant: f+t+ carriers were 11.3-fold less likely to sustain recurrent vertebral fracture in the last 2 years of the trial while on calcitriol therapy compared to calcium (P=1.4x10(-5)). Response to both calcium and calcitriol therapy is dependent on genetic variation at the VDR locus and two loci in the VDR gene may contribute to this effect.     

维生素D受体基因多态性对肾移植患者术后早期他克莫司浓度的影响

目的 研究维生素D受体(vitamin D receptor,VDR)基因多态性对肾移植术后早期他克莫司浓度的影响。方法 以360例使用他克莫司联合霉酚酸类药物以及糖皮质激素三联抗排异方案的肾移植患者为研究对象,检测患者CYP3A5 (rs776746)、VDR(VDR ApaI rs7975232,VDR BsmI rs1544410,VDR FokI rs2228570和VDR TaqI rs731236)基因型,比较分析不同基因型患者肾移植术后7 d时他克莫司血药浓度(concentration,C)、剂量(dose,D)以及浓度剂量比(ratio of concentration to dose,C/D)的差异。结果 CYP3A5非表达组(GG型)患者他克莫司的C和C/D均显著高于CYP3A5表达组(AA和AG型)(P<0.05)。以CYP3A5基因型进行分层后,在CYP3A5非表达组中VDR ApaI rs7975232 AA型患者C/D显著低于AC和CC型患者(P<0.05),然而在CYP3A5表达组中VDR ApaI rs7975232各基因型的C和C/D差异无统计学意义。此外,无论在CYP3A5表达组还是非表达组中,VDR BsmI rs1544410,VDR TaqI rs731236及VDR FokI rs2228570各基因型之间他克莫司的C、D和C/D差异均无统计学意义。结论 VDR ApaI rs7975232基因多态性与CYP3A5非表达组肾移植患者早期他克莫司血药浓度具有相关性,该基因型检测可能有助于指导肾移植患者他克莫司个体化用药。     

江苏汉族青少年维生素D受体基因多态性分析

目的 检测江苏地区青少年维生素D受体(VDR)基因型分布情况,建立正常青少年VDR基因多态性分布谱.方法 应用聚合酶链反应-限制性片断长度多态性技术(PCR-RFLP)对139名江苏地区汉族健康青少年VDR基因型进行检测分析.结果 139名青少年中,VDR基因AA、Aa和aa基因型分别为8(5.76%)名、55(39.57%)名和76(54.67%)名,BB、Bb和bb基因型分别为7(5.04%)、34(24.46%)和98(70.50%).结论 江苏地区汉族健康青少年VDR基因型分布男女性别之间趋于一致,与文献报道的国人、日本人没有显著性差异,与韩国人、欧洲人差异明显.     

Polymorphism of alpha-estrogen receptor and aryl hydrocarbon receptor genes in dementia patients in Shanghai suburb

AIM: To explore the possible association of different polymorphic forms of human alpha-estrogen receptor (ER-alpha) and aryl hydrocarbon receptor gene (Ahr) with the risk to senile dementia in farmers in Shanghai suburb. METHODS: Senile dementia patients (n=52) were examined for ER-alpha and Ahr gene polymorphism genotyping. Healthy individuals (n=125) in the same area were selected as a community control group. Two polymorphic loci, Pvu II locus and Xba I locus, of human ER- a gene were investigated by a PCR-RFLP-based procedure. The population frequencies of two polymorphic loci in exon 10 of Ahr gene, G1721A (R554K) and G1768A (V570I) were compared between patients and healthy controls using an allele-specific PCR (AS-PCR) procedure. RESULTS: The mutant allele frequencies of ER-alpha gene in the AD group were significantly higher than those in the control group (P=0.023, OR=2.94, 95 % CI 1.13-7.71 for Pvu II locus; P=0.046, OR=2.28, 95 % CI 1.003-5.17 for Xba I locus). The mutant allele frequencies among female AD patients were higher than those in the female controls (P=0.016, OR=3.68, 95 % CI 1.22-11.08 for Pvu II locus, P=0.029, OR=2.95, 95 % CI 1.10-7.94 for Xba I locus). The mutant form, neither in the homozygous, nor in the heterozygous form was detected at the locus of Ahr G1768A in a normal local population. No significant difference of Ahr genotype frequency at locus G1721A was noticed between the patients and healthy individuals. CONCLUSION: The distribution of ER polymorphisms was significantly different between Chinese and some other ethnic populations. The results suggested that ER-alpha gene polymorphisms might be related to the individual susceptibility to AD, especially in the females. However, it did not support the association of Ahr gene polymorphism with higher risk of senile dementia.     

VDR基因启动子单核苷酸多态性与前列腺癌的关系

目的了解维生素D受体（vitamin D receptor,VDR）基因启动子上存在的单核苷酸多态性（single nucleotide polymorphism,SNP）与前列腺癌的关系,进而探讨导致前列腺发病的遗传因素。方法提取60例前列腺癌及50例良性前列腺增生患者外周血标本中基因组DNA,应用聚合酶链反应-限制性片段长度多态性（polymerase chain reaction—restriction fragment length polyinorphisin,PCR—RFLP）检测并分析了VDR基因起始密码SNP在两组中的分布。常规病理方法分析前列腺癌的Gleason评分。对比分析VDR基因上存在的SNP与前列腺癌发生发展的相关性。结果前列腺癌组和前列腺增生组Fok Ⅰ等位基因多态性分布筹异有统计学意义（P〈0．05）,高危组和低危组前列腺癌中VDR基因SNP分布的的差异有统计学意义（P〈0．05）。结论VDR基因起始密码SNP不同类型可能与前列腺癌的发生有相关性。不同SNP类型可能成为前列腺癌发展的危险因素。