A new melatonin receptor ligand with mt1-agonist and MT2-antagonist properties

It has been difficult, so far, to obtain melatonin analogs possessing high selectivity for the respective melatonin receptors, mt1 and MT2. In the present work, we report the synthesis and pharmacological characterization of a new compound N‐{2‐[5‐(2‐hydroxyethoxy)‐1H‐indol‐3‐yl)] ethyl} acetamide or 5‐hydroxyethoxy‐N‐acetyltryptamine (5‐HEAT). To assess the activity of the compound, the following tests were performed: affinity determination for the high‐ and low‐affinity receptor states (2‐[ I]iodomelatonin binding), potency and intrinsic activity in inducing G protein activation ([ S]GTPγS binding assay). 5‐HEAT showed little selectivity for the mt1 receptor, with pKi values of 7.77 for mt1 and 7.12 for the MT2 receptors, respectively. 5‐HEAT was able to differentiate between the high‐ and the low‐affinity receptor states in the mt1 but not in the MT2 receptor. 5‐HEAT induced a high level of G protein activation when acting through the mt1 receptor, with a relative intrinsic activity of 0.92. On the contrary, it elicited only minimal MT2 receptor‐mediated G protein activation, with a relative intrinsic activity of 0.16, and was also able to inhibit the melatonin‐induced MT2 receptor‐mediated G protein activation, with a pKB value of 7.4. In conclusion, it appears that 5‐HEAT possesses very different efficacies at the two melatonin receptors, behaving as a full melatonin receptor agonist at the mt1 and as an antagonist/weak partial agonist at the MT2 receptor. Therefore, it is a promising ligand for use in functional studies aimed at distinguishing between the effects mediated by the different melatonin receptors in the human.     

云南省登革4型病毒全基因组序列特征研究

目的阐明云南省2015年5株登革4型病毒(DENV-4)流行株的全基因组序列特征及分子流行病学特点。方法采用C6/36细胞培养法分离病毒,用RT-PCR法扩增新分离DENV-4的全基因组序列,采用ClastalX1.83和MEGA6等生物信息学软件进行核苷酸和推导氨基酸序列同源性及系统进化分析。结果从2015年云南省瑞丽市登革热患者血清中分离到5株DENV-4(本地病例2株,来自缅甸腊戍和南坎市输入性病例3株)。经RT-PCR和序列测定,获得这5株DENV-4的全基因组序列(10 661nt),其开放读码框(103-10 264)编码3 386个氨基酸。全基因组或结构蛋白和非结构蛋白基因序列的系统进化和同源性分析表明,云南分离株间高度同源并聚集为一个进化支,并与泰国不同年代DENV-4基因I型(G-I)流行株具有较近的进化关系和较高的同源性,同属G-I。云南株和泰国株均与同基因型的DENV-4原型株(H241,1956年菲律宾)和中国广州1990年B5株亲缘性和同源性都较低。云南株与H241株在结构蛋白或非结构蛋白中分别存在21和45个氨基酸位点差异。结论首次获得云南省DENV-4分离株的全基因组序列并发现它们与近期东南亚DENV-4G-I流行株亲缘关系较近。首次证实云南省存在DENV-4本地流行,传播来源为相邻缅甸北部边境地区。云南分离株某些氨基酸位点的改变是否与其抗原性和毒力有关尚需进一步研究。     

云南中缅边境登革1型病毒全基因组序列特征研究

目的阐明云南省中缅边境地区2013-2015年14株登革1型病毒(DENV-1)全基因组序列特征。方法采用C6/36细胞培养法分离病毒,用RT-PCR法扩增新分离DENV-1的全基因组序列,采用ClastalX1.83和MEGA6等生物信息学软件进行核苷酸和推导氨基酸序列同源性及系统进化分析。结果从登革热患者血清中分离到14株DENV-1,其中瑞丽市9株,临沧市3株,昆明市2株。经RT-PCR和序列测定,获得这14株DENV-1的全基因组序列(10 735nt),其开放读码框(95-10 271)编码3 392个氨基酸。系统进化和同源性分析表明,13株为基因I型(G-I),其中瑞丽和临沧本地病例7株,缅甸输入性病例6株;1株为G-III(昆明的印度输入性病例)。云南13株G-I可分为2个进化群,但均与缅甸、泰国等东南亚流行株具有较近的亲缘关系。云南13株G-I的E基因的核苷酸和氨基酸同源性分别为97.02%-100%和98.78%-100%,它们与6株东南亚G-I参考株的核苷酸和氨基酸同源性分别为96.53%-99.53%和97.33%-100%,与DENV-1原型株US_Hawaii核苷酸和氨基酸同源性分别为93.76%-94.45%和95.86%-96.91%。所有云南株和东南亚参考株与US_Hawaii株在结构蛋白或非结构蛋白的氨基酸位点分别存在44和150个位点差异。结论云南中缅边境地区2013-2015年流行的DENV-1均为G-I,并具有基因多样性特点但均为来自缅甸的多个传播来源。     

The role of proline residues in the structure and function of human MT2 melatonin receptor

Melatonin functions as an essential regulator of various physiological processes in all vertebrate species. In mammals, two G protein-coupled melatonin receptors (GPCR) mediate some melatonin's actions: MT1 and MT2. Transmembrane domains (TM) of most GPCRs contain a set of highly conserved proline residues that presumably play important structural and functional roles. As TM segments of MT2 receptor display several interesting differences in expression of specific proline residues compared to other rhodopsin-like receptors (rGPCRs), we investigated the role of proline residues in the structure and function of this receptor. All prolines in TM segments of MT2 receptor were individually replaced with alanine and/or glycine. In addition, the unusual NAxxY motif located in TM7 was mutated to generate highly conserved NPxxY motif found in the majority of rGPCR proteins. Following transient expression in CHO-K1 cells, binding properties of the mutant receptors and their ability to transduce signals were analyzed using (125)I-mel- and [(35)S]GTPgammaS-binding assays, respectively. The impact of the performed mutations on the receptor structure was assessed by molecular dynamic simulations of MT2 receptors embedded in the fully hydrated phospholipid bilayer. Our results indicate that residues P174, P212 and P266 are important for the ligand binding and/or signaling of the human MT2 receptor. We also show that changes within the unusual NAxxY sequence in the TM7 (mutations A305P and A305V) produce defective MT2 receptors indicating an important role of this motif in the function of melatonin receptors.     

Placental melatonin production and melatonin receptor expression are altered in preeclampsia: new insights into the role of this hormone in pregnancy

Abstract: The melatonin system in preeclamptic pregnancies has been largely overlooked, especially in the placenta. We have previously documented melatonin production and expression of its receptors in normal human placentas. In addition, we and others have shown a beneficial role of melatonin in placental and fetal functions. In line with this, decreased maternal blood levels of melatonin are found in preeclamptic compared with normotensive pregnancies. However, melatonin production and expression of its receptors in preeclamptic compared with normotensive pregnancy placentas has never been examined. This study compares (i) melatonin‐synthesizing enzyme expression and activity, (ii) melatonin and serotonin, melatonin’s immediate precursor, levels and (iii) expression of MT1 and MT2 melatonin receptors in placentas from preeclamptic and normotensive pregnancies. Protein and mRNA expression of aralkylamine N‐acetyltransferase (AANAT) and hydroxyindole O‐methyltransferase (HIOMT), the melatonin‐synthesizing enzymes, as well as MT1 and MT2 receptors were determined by RT‐qPCR and Western blot, respectively. The activities of melatonin‐synthesizing enzymes were assessed by radiometric assays while melatonin levels were determined by LC‐MS/MS. There is a significant inhibition of AANAT, melatonin’s rate‐limiting enzyme, expression and activity in preeclamptic placentas, correlating with decreased melatonin levels. Likewise, MT1 and MT2 expression is significantly reduced in preeclamptic compared with normotensive pregnancy placentas. We propose that reduced maternal plasma melatonin levels may be an early diagnostic tool to identify pregnancies complicated by preeclampsia. This study indicates a clinical utility of melatonin as a potential treatment for preeclampsia in women where reduced maternal plasma levels have been identified.     

Melatonin treatment restores mitochondrial function in Alzheimer's mice: a mitochondrial protective role of melatonin membrane receptor signaling

Mitochondrial dysfunction is a hallmark of Alzheimer's disease (AD) and is observed in mutant amyloid precursor protein (APP) transgenic mouse models of familial AD. Melatonin is a potent antioxidant, can prevent toxic aggregation of Alzheimer's beta-amyloid (Aβ) peptide and, when taken long term, can protect against cognitive deficits in APP transgenic mice. To study the effects of melatonin on brain mitochondrial function in an AD model, APP/PS1 transgenic mice were treated for 1 month with melatonin. Analysis of isolated brain mitochondria from mice indicated that melatonin treatment decreased mitochondrial Aβ levels by two- to fourfold in different brain regions. This was accompanied by a near complete restoration of mitochondrial respiratory rates, membrane potential, and ATP levels in isolated mitochondria from the hippocampus, cortex, or striatum. When isolated mitochondria from untreated young mice were given melatonin, a slight increase in respiratory rate was observed. No such effect was observed in mitochondria from aged mice. In APP-expressing neuroblastoma cells in culture, mitochondrial function was restored by melatonin or by the structurally related compounds indole-3-propionic acid or N(1)-acetyl-N(2)-formyl-5-methoxykynuramine. This restoration was partially blocked by melatonin receptor antagonists indicating melatonin receptor signaling is required for the full effect. Therefore, treatments that stimulate melatonin receptor signaling may be beneficial for restoring mitochondrial function in AD, and preservation of mitochondrial function may an important mechanism by which long term melatonin treatment delays cognitive dysfunction in AD mice.     

A case-control study of melatonin receptor type 1A polymorphism and acute myocardial infarction in a Spanish population

Coronary artery disease (CAD) is a complex disease with genetic and environmental determinants. Although a large number of genetic polymorphisms involved in the pathogenesis of atherosclerosis have been identified, there is still no evidence of a genetic association with CAD. As melatonin might play a role in the pathogenesis of atherosclerosis through its anti-inflammatory and antioxidant properties, we tested whether the expression of six single nucleotide polymorphisms (SNPs) of the melatonin receptor differs in acute myocardial infarction (AMI) patients with acute myocardial infarction (n = 300) compared with healthy age- and sex-matched controls (n = 250). Finally, only MEL1A receptor SNP rs28383653 was selected because of Hardy-Weinberg equilibrium (χ(2) = 0.49). The distribution of genotype frequencies for this SNP showed that the unfavourable CT genotype was significantly more frequent in patients with AMI than in controls (4.5% versus 1.3%; P = 0.006). Multivariable analysis showed a significantly higher frequency of the unfavourable CT genotype in AMI patients with peripheral arteriopathy (28% versus 10%; P = 0.01). This finding suggests a synergism effect between the unfavourable genotype (CT) of the MELIA receptor SNP and the vascular disease in this subgroup of patients. To our knowledge, this is the first study to report an association between a genetic polymorphism of the melatonin receptor 1A and CAD.     

The functional role of cysteines adjacent to the NRY motif of the human MT1 melatonin receptor

All G protein-coupled melatonin receptors have two conserved cysteines in the interphase between transmembrane helix III and the second intracellular loop, in the region assumed important in receptor/G protein coupling. The cysteines are also potential targets of receptor S-nitrosylation. The effects of site-directed mutagenesis of these cysteines in the human MT1 melatonin receptor were investigated. The cysteines were mutated into serines either individually or as a pair and stable Chinese hamster ovary cell lines expressing the wild-type and mutant MT1 receptors were created. Receptor expression level, subcellular localization, ligand binding and G protein activation of the cell lines were analyzed. Serine substitution of C127 (Cys(3.52)) did not affect the ligand binding affinity and agonist potency but had an influence on receptor trafficking and G protein activation capacity. Serine substitution of C130 (Cys(3.55)) resulted in a decrease in the potency of melatonin to activate G proteins. When both cysteines were mutated into serines, normal MT1 receptor binding and activation were abolished. Computer modeling revealed that the mutations did not change the structure of the ligand binding pocket. Cysteine S-nitrosylation had no influence on G protein activation through MT1 receptors. Taken together, these data show that the two conserved cysteines in the end of transmembrane domain III of the MT1 melatonin receptor, especially C130 (Cys(3.55)), are needed for normal G protein activation and receptor trafficking.     

Inverse agonist exposure enhances ligand binding and G protein activation of the human MT1 melatonin receptor, but leads to receptor down-regulation

Melatonin binds and activates G protein-coupled melatonin receptors. The density and affinity of the endogenous melatonin receptors change throughout the 24-hr day, and the exposure of recombinant melatonin receptors to melatonin often results in desensitization of the receptors. Receptor density, G protein activation and expression level were analyzed in CHO cell lines stably expressing the human MT1 receptors after 1 or 72 hr of exposure to melatonin (agonist, 10 nm) and luzindole (antagonist/inverse agonist, 10 microm). The 72-hr exposure to luzindole significantly increased the apparent receptor density in cell lines with both high and low MT1 receptor expression levels (MT1(high) and MT1(low) cells, respectively). In the constitutively active MT1(high) cells, luzindole pretreatment also stimulated the functional response to melatonin in [(35)S]GTPgammaS binding assays, whereas melatonin pretreatment attenuated the functional response at both time points. Receptor ELISA was used to analyze the cell membrane and total expression level of the MT1 receptor in intact and permeabilized cells, respectively. Luzindole pretreatment decreased the total cellular level of MT1 receptor in the MT1(high) cells at both time points but increased the cell surface expression of MT1 receptor at 72 hr. Melatonin significantly decreased MT1 receptor cell surface expression only in MT1(high) cells after a 1-hr treatment. These results indicate that melatonin treatment desensitizes MT1 receptors, whereas luzindole increases ligand binding and G-protein activation. Luzindole also stimulates downregulation of the MT1 receptor protein, interfering with the synthesis and/or degradation of the receptor.     

Expression of the melatonin receptor (MT) 1 in benign and malignant human bone tumors

The beneficial effects of melatonin on bone homeostasis have been shown in various diseases. As this indoleamine causes dose-dependent modulation of bone-forming osteoblast and bone-resorbing osteoclast activities by receptor-independent and -dependent pathways, we investigated the expression of G-protein-coupled melatonin receptors (MTs) in malignant and non-malignant human bone lesions. By TaqMan polymerase chain reaction (PCR), we analyzed 30 specimens from osteosarcoma and 11 from benign bone tumors for MT1-mRNA expression. Furthermore, we determined mRNA expression levels of the osteoclast activity-stimulating receptor activator of nuclear factor-kappa B ligand (RANKL) and its counterpart osteoprotegerin (OPG). Although mean MT1-mRNA levels were similar (P = 0.596) in malignant (4.39 +/- 4.98-fold) and benign samples (4.64 +/- 6.81-fold), the highest MT1-mRNA levels (up to 27-fold) were observed in individual osteosarcomas, particularly, in two specimens of patients with local recurrence of the tumor. Moreover, mean RANKL- and OPG-mRNA levels were similar in malignant and benign specimens (RANKL: 7.38 +/- 9.61-fold versus 3.57 +/- 3.11-fold, P = 0.207; OPG: 23.45 +/- 32.76 versus 8.07 +/- 7.23-fold, P = 0.133). Again, highest RANKL- and OPG-mRNA levels (up to 41- and 160-fold, respectively) were observed in individual osteosarcomas. Expression of MT1-mRNA was confirmed in two human osteosarcoma cell lines (HOS, MG63). High expression levels of MT1-mRNA together with low OPG-mRNA were found in both osteosarcoma cell lines, while in normal human osteoblasts and bone marrow stromal cells, high OPG-mRNA levels were associated with low MT1-mRNA levels. These data on the abundant expression of MT1-mRNA in human bone tumors and osteosarcoma cells lines suggest an important role for MT1 in bone pathology.     

Metabolic analysis of the melatonin biosynthesis pathway using chemical labeling coupled with liquid chromatography‐mass spectrometry

Characterization of the melatonin (MLT) biosynthesis pathway in plants is still limited. Additionally, a metabolomic analysis of MLT biosynthesis in plants is still a challenge due to analyte structural and chemical diversity, low analyte abundances, and plant matrix complexities. Herein, a sensitive liquid chromatography‐mass spectrometry (LC‐MS) method enabling the simultaneous determination of seven plant MLT biosynthetic metabolites was developed. In the proposed strategy, the targeted metabolites, which included tryptophan (Trp), tryptamine (TAM), 5‐hydroxytryptophan (5HTP), serotonin (5HT), N‐acetylserotonin (NAS), 5‐methoxytryptamine (5MT), and MLT, were purified from plant extracts using a one‐step dispersive solid‐phase extraction (DSPE). The samples were then chemically labeled with dansyl chloride (DNS‐Cl), followed by analysis using LC‐MS. The limit of detection (LOD) values ranged from 0.03 to 1.36 pg/mL and presented a 22‐ to 469‐fold decrease when compared to the unlabeled metabolites. Due to the high sensitivity of the proposed method, the consumption of plant materials was reduced to 10 mg FW. Ultimately, the established method was utilized to examine the distributions of MLT and its intermediates in rice shoots and roots with or without cadmium (Cd) stress. The results suggested that under normal condition, MLT may also be generated via a Trp/TAM/5HT/5MT/MLT path (Pathway II) in addition to the previously reported Trp/TAM/5HT/NAS/MLT path (Pathway I), although Pathway I was shown to be dominant. During Cd stress, MLT was also shown to be produced through these two pathways, with Pathway II shown to be dominant in rice shoots and roots.     

Melatonin as a naturally occurring co-substrate of quinone reductase-2, the putative MT3 melatonin membrane receptor: hypothesis and significance

The nature of the MT3 melatonin receptor/binding site has been a long pondered mystery for scientists. Even though it is a presumptive membrane receptor, neither its transduction cascade nor its biological consequences, after its stimulation, have been uncovered. Moreover, solid data support the idea that the MT3 melatonin binding site is an enzyme, quinone reductase 2 (QR2), rather than a membrane melatonin receptor. Based on the data available and our preliminary studies, we hypothesize that melatonin is a co-substrate of QR2. We surmise that melatonin binds to a co-substrate binding site (MT3 binding site) donating an electron to the enzyme co-factor, flavin adenine dinucleotide (FAD). FAD can be reduced to either FADH or FADH2 while melatonin is converted to N1-acetyl-N2-formyl-5-methoxykynuramine and/or cyclic 3-hydroxymelatonin. QR2 is considered to be a detoxifying and antioxidant enzyme and its behavior changes depending on available co-substrates. As a naturally occurring substance, melatonin's levels fluctuate with the light/dark cycle, with aging and with health/disease state. As a result, these alterations in melatonin production under physiological or pathological conditions would probably influence the activity of QR2.     

安徽省人感染H7N9禽流感病毒基因组特征分析

目的 分析安徽省2013年分离的5株人感染H7N9禽流感病毒全基因组特征。方法 从美国国家生物技术信息中心(NCBI)和全球禽流感基因共享数据库(GISAID)中下载具有代表性的H7N9、H7N3、H7N7和H9N2等毒株序列,运用分子生物信息学软件分析病毒全基因组特征。结果 我省流行的H7N9病毒HA基因与A/duck/Fujian/6390/2010(H7N3)相似度最高,NA基因与A/northern shoveler/Hong Kong/MPL133/2010(H2N9)株相似度最高,6个内部基因片段与中国北京、香港、湖南、江苏地区分离的H9N2毒株相似度接近。氨基酸序列比对发现NS1蛋白218~230位氨基酸缺失、M2蛋白的N31S突变、HA蛋白的G186V 、Q226L突变以及NA蛋白69~73位的删除,并且我省人感染H7N9病毒均带有PB2的E627K突变,同时PA-100A、PA-356R、PA-409N这些易感人类的特征氨基酸也在本次流行的H7N9病毒中发现;此外HA蛋白裂解位点仅有1个碱性氨基酸、糖基化位点高度保守以及未发现NA蛋白R294K突变也是我省H7N9病毒主要特征。结论 我省人感染H7N9病毒与中国其他省份流行株高度同源,该病毒获得跨种传播、毒力增强、耐药等能力均与病毒蛋白功能域有关。     

Melatonin restores the pluripotency of long-term-cultured embryonic stem cells through melatonin receptor-dependent m6A RNA regulation

N6-methyladenosine (m6A) methylation is the most common and abundant modification on mammalian messenger RNA (mRNA) and regulates the pluripotency of embryonic stem cells (ESCs). Research has shown that melatonin plays a fundamental role in DNA and histone modifications. However, the effect of melatonin on RNA modification is unknown. Here, for the first time, we investigated the effect of melatonin on m6A modifications in long-term-cultured ESCs. Pluripotency studies indicated that 10 μmol/L melatonin sufficiently maintained ESCs with stemness features over 45 passages (more than 90 days). Notably, treatment of ESCs with melatonin led to a significant decrease in the nuclear presence of m6A methyltransferase complex and decreased global m6A modification. Depletion of melatonin receptor 1 (MT1) by CRISPR/Cas9 significantly reduced the effects of melatonin on ESC pluripotency and m6A modification. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that melatonin promotes stabilization of core pluripotency factors, such as Nanog, Sox2, Klf4, and c-Myc, by preventing m6A-dependent mRNA decay. Using cell signaling pathway profiling systems, melatonin was shown to regulate m6A modification predominantly through the MT1-JAK2/STAT3-Zfp217 signal axis. This study reveals a new dimension regarding melatonin regulation of gene expression at the RNA level.