Synthesis and in Vitro Receptor Binding Studies of Fluorotamoxifen Analogues

We describe the synthesis of new fluorotamoxifen analogues with the fluorine atom positioned on the end of the aliphatic chain of tamoxifen. The binding of fluorotamoxifens to cytosol estrogen receptors of rat uteri was determined with [³H]estradiol (5 nM). The fluorotamoxifens had similar or superior binding affinities compared with tamoxifen. The IC₅₀ value was as follows: tamoxifen, 5 × 10^–7 M; fluorotamoxifen (VII), 5 × 10^–7 M; N,N-diethylfluorotamoxifen (IV)—cis, 1 × 10^–6 M, and trans, 2 × 10^–7 M; and (cis) fluoromethyl-N,N-diethyltamoxifen (VI) 1 × 10^–7 M. Therefore, the fluorinated tamoxifens have potential use in imaging estrogen receptors by PET.     

Sedative-Hypnotic and Receptor Binding Studies of Fermented Marine Organisms

This study was performed to investigate the sedative-hypnotic activity of γ-aminobutyric acid (GABA)-enriched fermented marine organisms (FMO), including sea tangle (FST) and oyster (FO) by Lactobacillus brevis BJ20 (L. brevis BJ20). FST and FO were tested for their binding activity of the GABA_A-benzodiazepine and 5-HT_2C receptors, which are well-known molecular targets for sleep aids. We also measured the sleep latency and sleep duration during pentobarbital-induced sleep in mice after oral administration of FST and FO. In GABA_A and 5-HT_2C receptor binding assays, FST displayed an effective concentration-dependent binding affinity to GABA_A receptor, similar to the binding affinity to 5-HT_2C receptor. FO exhibited higher affinity to 5-HT_2C receptor, compared with the GABA_A receptor. The oral administration of FST and FO produced a dose-dependent decrease in sleep latency and increase in sleep duration in pentobarbital-induced hypnosis. The data demonstrate that FST and FO possess sedative-hypnotic activity possibly by modulating GABA_A and 5-HT_2C receptors. We propose that FST and FO might be effective agents for treatment of insomnia.     

Synthesis and Receptor Binding Studies of (±)l-Iodo-MK-801

The glutamate analogue N-methyl-D-aspartate (NMDA) binds to a subset of glutamate receptors that are coupled to a voltage-sensitive cation channel. This NMDA-linked channel is the likely binding locus of the potent anticonvulsant MK-801. To develop single-photon emission computed tomography (SPECT) probes of this brain channel, we synthesized (±)l-iodo-MK-801 and (±)l-[¹²⁵I]iodo-MK-801. The effect of (±)l-iodo-MK-801 on ligand binding to the NMDA-linked glutamate receptor site was assessed using a rat brain homogenate assay. (±)l-Iodo-MK-801 displaced the dissociative anesthetic ligand [³H]N-[l-(2-thienyl)cyclohexyl]piperidine ([³H]TCP) binding with an IC₅₀ of 1 µM, which is a 10-fold lower binding affinity than that of (±)MK-801. In in vivo autoradiographic studies, (±)MK-801 failed to block selective uptake of (±)l-iodo-MK-801 in rat brain. These results suggest that (±)l-iodo-MK-801 may not be a suitable ligand for mapping NMDA-linked glutamate receptor channels.     

Differences between Binding Affinities of some Antimuscarinic Drugs in the Parotid Gland and those in the Urinary Bladder and Ileum

Abstract: Possible differences between the muscarinic receptors in the guinea pig urinary bladder and those in the ileum and the parotid gland were investigated, using a receptor binding technique. The affinities of 18 antimuscarinic drugs were indirectly derived from the ability to inhibit the receptor-specific binding of the radioligand (–)³H-QNB. The Hill coefficients were close to unity which indicated that the drugs were bound to apparently uniform populations of receptors within each tissue. In contrast to traditional muscarinic antagonists, four drugs – namely, oxybutynine, dicyclomine, benzhexol and pirenzepine – bound with a significantly higher affinity in the parotid gland than in the urinary bladder and ileum. A tendency towards reversed selectivity was found for secoverine. Thus, the present results further support the hypothesis that differences in muscarinic receptors between tissues exist, e.g. smooth muscle compared with parotid gland, which can be detected only by certain antimuscarinic drugs.     

选择性毒蕈碱M3受体阻断剂进展与治疗膀胱过度活动症的评价

目的:膀胱过度活动症是发生在男、女性常见的慢性下泌尿道综合征,严重影响人群的生活质量,本文介绍其治疗药毒蕈碱受体阻断剂进展的侧面。方法:采用国内、外文献综述方法。结果与结论:选择性M3受体阻断剂作为膀胱过度活动症治疗药的一支奇葩,进展十分迅猛,对毒蕈碱受体有高度的选择性,标志一个新的里程开始,在改善膀胱过度活动症各项指标上,包括尿失禁、尿急迫、尿逼迫,在治疗膀胱过度活动症上展现了良好的前景。     

Muscarinic Receptor Density in the Rat Urinary Bladder after Denervation,Hypertrophy and Urinary Diversion *

Abstract: Parasympathetic denervation of the urinary bladder results in supersensitivity to muscarinic agonists and in bladder hypertrophy. In the present study, the effects of denervation on the muscarinic receptors in the rat bladder were investigated, using a receptor binding technique with (-)³H-QNB as radioligand. The density of muscarinic receptors was increased in denervated, hypertrophied bladders but it was decreased, below that in control bladders, when the development of hypertrophy was prevented by urinary diversion. A decreased receptor density was also found in innervated bladders after urinary diversion, whereas the receptor density was unaffected by hypertrophy alone. Competition experiments with methacholine revealed no changes in the agonist binding properties of the receptors. When the present data are combined with those in previous functional studies, it seems unlikely that the muscarinic receptors in the bladder are involved in the development of supersensitivity. It is suggested that the density of muscarinic receptors in the bladder may be related to the bladder function.     

The Binding Mode Prediction and Similar Ligand Potency in the Active Site of Vitamin D Receptor with QM/MM Interaction,MESP, and MD Simulation

Non‐secosteroidal ligands are well‐known vitamin D receptor (VDR) agonists. In this study, we described a combined QM/MM to define the protein–ligand interaction energy a strong positive correlation in both QM–MM interaction energy and binding free energy against the biological activity. The molecular dynamics simulation study was performed, and specific interactions were extensively studied. The molecular docking results and surface analysis shed light on steric and electrostatic complementarities of these non‐secosteroidal ligands to VDR. Finally, the drug likeness properties were also calculated and found within the acceptable range. The results show that bulky group substitutions in side chain decrease the VDR activity, whereas a small substitution increased it. Functional analyses of H393A and H301A mutations substantiate their roles in the VDR agonistic and antagonistic activities. Apart from the His393 and His301, two other amino acids in the hinge region viz. Ser233 and Arg270 acted as an electron donor/acceptor specific to the agonist in the distinct ligand potency. The results from this study disclose the binding mechanism of VDR agonists and structural modifications required to improve the selectivity.     

Long-Term Efficacy and Safety of Repeated Intravescial OnabotulinumtoxinA Injections Plus Hydrodistention in the Treatment of Interstitial Cystitis/Bladder Pain Syndrome

Intravesical onabotulinumtoxinA (BoNT-A) injection can relieve symptoms of interstitial cystitis/bladder pain syndrome (IC/BPS), but lacks sustainability. Repeated injections have been shown to provide a superior outcome to a single injection, but data on long-term efficacy and safety is limited. In this prospective study, we enrolled patients with refractory IC/BPS, and treated them with 100 U of BoNT-A injection plus hydrodistention followed by repeated injections every six months for up to two years or until the patient wished to discontinue. A “top-up” dose was offered after the fourth injection. Of these 104 participants, 56.7% completed four BoNT-A injections and 34% voluntarily received the fifth injection due to exacerbated IC symptoms. With a follow-up period of up to 79 months, O’Leary-Sant symptom and problem indexes (ICSI, ICPI, OSS), pain visual analogue scale (VAS) functional bladder capacity, frequency episodes, and global response assessment (GRA) all showed significant improvement (p < 0.0001). Those who received repeated injections had a better success rate during the long-term follow-up period. The incidence of adverse events did not rise with the increasing number of BoNT-A injections. A higher pre-treatment ICSI and ICPI score was predictive for successful response to repeated intravesical BoNT-A injections plus hydrodistention.     

Research Letter: Structural Combination of Established 5-HT2A Receptor Ligands: New Aspects of the Binding Mode

MH.MZ, MDL 100907, and altanserin are structurally similar 4-benzoyl-piperidine derivatives and are well accommodated to receptor interaction models. We combined structural elements of different high-affinity and selective 5-HT_2A antagonists, as MH.MZ, altanserin, and SR 46349B, to improve the binding properties of new compounds. Three new derivatives were synthesized with a 4-benzoyl-piperidine moiety as the lead structure. The in vitro affinity of the novel compounds was determined by a [³H]MDL 100907 competition binding assay. The combination of MH.MZ and SR 46349B resulted in a compound ( 8 ) with a moderate affinity toward the 5-HT_2A receptor (K_i = 57 nm ). The remarkably reduced affinity of other compounds ( 4a ), ( 4b ), and (4c) (K_i = 411, 360 and 356 nm respectively) indicates that MH.MZ can only bind to the 5-HT_2A receptor with the p-fluorophenylethyl residue in a sterically restricted hydrophobic binding pocket.     

Interaction of Follicle‐Stimulating Hormone (FSH) Receptor Binding Inhibitor‐8: A Novel FSH‐Binding Inhibitor,with FSH and its Receptor

Follicle‐stimulating hormone (FSH) receptor binding inhibitor (FRBI‐8) is a novel octapeptide purified from human ovarian follicular fluid. In vitro, it inhibits the binding of FSH to granulosa cells and in vivo, it induces atresia in developing follicles in rodents. This peptide, when administered to marmosets and bonnet monkeys, altered the circulating progesterone levels. This study was carried out to elucidate structure of the FRBI‐8 and understand its mechanism of inhibiting interaction of FSH to its receptors. Homology modeling predicted that the FRBI‐8 adopts a turn and random coil. This is further confirmed by circular dichroism and NMR. Docking studies of the FRBI‐8 with reported FSH–FSHR hormone binding (FSHR_HB) domain complex using zdock algorithm revealed that the FRBI‐8 binds to FSHβL2–FSHR_HB binding interface which is otherwise known to be crucial for activation of signal transduction cascade. FRBI‐8 analogs were designed by replacing the acidic amino acid residues at positions 2, 5 and 6 with Ala, individually. Docking studies revealed that D6A mutant (FRBI‐8^D6A) had a higher binding affinity than the native FRBI‐8. In vitro radioreceptor assay with FRBI‐8^D6A showed 50% lower IC₅₀ compared with the FRBI‐8, confirming the in silico observations. Thus, the study reveals that both FRBI‐8 and FRBI‐8^D6A interfered with the binding of FSH to its receptor.     

MUC15 promotes growth and invasion of glioma cells by activating Raf/MEK/ERK pathway

MUC15 is a novel mucin associated with the cell membrane that is overexpressed in human gliomas. Its function in glioma is unclear. In this study, high MUC15 levels were detected in glioma tissues and cells. We found that transfection with MUC15 siRNA in U251 and T98G cells reduced MUC15 expression and decreased cell proliferation, invasion, and migration (P < .05). After transfecting U251 and T98G cells with pcDNA3.1-myc-His-MUC15 plasmid to overexpress MUC15, MUC15 expression was significantly upregulated and cell proliferation, invasion, and migration were increased (P < .05). MUC15 activated the Raf/MEK/ERK signalling pathway and the ERK inhibitor PD98059 partly reversed MUC15-enhanced proliferation, invasion, and migration of glioma cells (P < .05). The results indicate that MUC15 plays a part in glioma tumorigenesis, and the Raf/MEK/ERK signalling is involved in the regulation of MUC15 on glioma cell activity.     

Properties Influencing the Relative Binding Affinity of Pteroate Derivatives and Drug Conjugates Thereof to the Folate Receptor

Purpose  Using in vitro competition assays, determine salient chemical features of pteroates and pteroate-drug conjugates which afford high affinity to the folate receptor. Materials and Methods  Both folate binding protein-coated polystyrene plates and adherent human cell-based assays were used to evaluate the effects of assay temperature and buffer composition on pteroate/pteroate-drug conjugate binding affinity. Following assay selection and optimization, the relative binding affinities of ten vitamers and derivatives as well as seven pteroate-drug conjugates were evaluated. Results  Compared to polystyrene plates containing immobilized folate binding protein, adherent KB cells were determined to be an equally effective, more desirable source of folate receptor for such analyses. Using the latter method, we discovered that a charged group positioned in close proximity to the pteroate’s aryl moiety is critical for retaining high binding affinity. We also found that a diverse set of bioactive small molecule agents can be attached to folic acid in a manner that does not appreciably disturb this vitamin’s intrinsic high affinity for the folate receptor. However, conjugation of lipophilic, high protein-binding agents to folate was sometimes found to dramatically reduce affinity, which is a finding that best exemplifies the need for having a reliable in vitro assay for determining a compound’s RA. Conclusion  Molecules which bind best to the human folate receptor are those that contain hydrophilic regions distal to the ligand’s aryl group, and for drug conjugates, an extended hydrophilic spacer placed in-between the pteroate and drug cargo moieties.     

真核表达重组小鼠IL-15/Fc融合蛋白的制备和活性鉴定

目的 制备真核表达小鼠白细胞介素15(IL-15)和IgGγ1 Fc段融合蛋白,并探讨其对抗原特异性CD8+T细胞的作用.方法 运用RT-PCR方法,从B6小鼠脾细胞中分离小鼠IL-15和IgG γ1绞链区-CH2-CH3 Fc cDNA.在IL-15 3'端和Fc 5'端引入BamH I酶切位点,将IL-15和Fc直接连接,构建小鼠IL-15/Fe融合蛋白基因,再连接到真核表达质粒载体pcDNA3.1(a+)上,转化CHO-S细胞表达、纯化后,研究其活性.结果 融合蛋白486 bp的编码由162个氨基酸组成,其中含48个信号肽氨基酸的小鼠IL-15前体蛋白和由681 bp编码227个氨基酸的IgGγ1-CH2-CH3功能区构成;表达蛋白在IL-15信号肽引导下能高效分泌到培养液中,有二聚体和三聚体两种天然结构,单体分子量约50 kDa,能特异性的结合IL-15R并抑制抗原诱导的CD8+T细胞的增殖反应.结论 成功构建了小鼠IL-15/Fc融合蛋白真核表达载体,并在CHO-S细胞中得到高效表达.此融合蛋白具有较高的生物学活性,可有效地抑制抗原特异性CD8+细胞的增殖反应.     

MTS2/P15和Bcl-2蛋白产物在鼻咽低分化鳞癌中的表达及意义

目的观察抑癌基因ＭＴＳ２／ Ｐ１５及凋亡抑制基困Ｂｃｌ－２在低分化鼻咽癌（ＮＰＣ）中的表达,探讨其与低分化 ＮＰＣ发生、发展和预后的关系。方法用免疫组化 ＳＰ法检测 ４２例低分化 ＮＰＣ组织和门例无瘤鼻咽（ＮＰ）组织的Ｐ１５、Ｂｃｌ－２蛋白的表达情况。结果低分化ＮＰＣ中Ｐ１５、Ｒｃｌ－２蛋白的阳性表达率分别为５７．１％和８３．３％,ＮＰ组织中Ｐ１５蛋白表达率为１００％,而Ｂｃｌ－２蛋白大多在假复层纤毛柱状上皮的基底层细胞有表达,但较ＮＰＣ细胞染色弱,而在鳞状上皮多为阴性,可认为Ｂｃｌ－２蛋白在ＮＰ组织为阴性表达。Ｐ１５及Ｂｃｌ－２蛋白在ＮＰＣ与ＮＰ组中表达比较,两者差异有统计学意义（Ｐ＜０．０５）。Ｐ１５与Ｂｃｌ－２蛋白在ＮＰＣ中表达与临床分期、颈淋巴结转移、３年生存时间无关,其差异均无统计学意义（Ｐ＞０．０５）。两蛋白阳性表达之间无相关性。结论Ｐ１５蛋白在低分化ＮＰＣ中表达缺失和Ｂｃｌ－２蛋白在低分化ＮＰＣ中的过度表达对ＮＰＣ中的发生起一定作用。     

Demonstration of α-Adrenoceptors in the Rabbit Bladder Base and Urethra with 3H-Dihydroergocryptine Ligand Binding

Abstract: The purpose of this work was to demonstrate the presence of α-adrenoceptors in a crude membrane preparation made from rabbit bladder base and urethra. This was achieved by radioligand binding studies, using ³H-dihydro-α-ergocryptine (³H-DHE) as the radioligand. The specific binding, i.e. the binding that could be inhibited by 10?⁵ M phentolamine, was saturable with 73 fmol ³H-DHE bound per mg membrane protein. Binding was at steady state after 60 min., and reversible. Rate constants for association and dissociation were 3 × 10⁷ M^?1 min.^?1, and 2× 10^?2 min.^?1, respectively. A number of compounds were tested for their abilities to compete with ³H-DHE for the binding sites. The relative affinity of some adrenoceptor agonists was: (-)-adrenaline>(-)-noradrenaline(±)-isoprenaline. Stereoselectivity was shown, since (-)-noradrenaline had 42 times higher affinity than (+)-noradrenaline. Adrenoceptor antagonists inhibited ³H-DHE binding in the following order of potency: DHE>phentolamine(±)-propranolol. The dissociation constant, K_D, for DHE to the binding sites was estimated in three different ways. The constants were derived from saturation, competition, and kinetic studies, and gave K_D values of 1.1,1.4 and 0.7 nM, respectively. The results suggest that α-adrenoceptors were labelled by ³H-DHE in the tissue homogenates.     

Effect of Intratrigonal Botulinum Toxin in Patients with Bladder Pain Syndrome/Interstitial Cystitis: A Long-Term,Single-Center Study in Real-Life Conditions

The high percentage of treatment failures seen in patients with bladder pain syndrome/interstitial cystitis (BPS/IC) managed conservatively frequently demands invasive treatment options. We aimed to evaluate the long-term efficacy and adverse events of intratrigonal botulinum toxin injection in such circumstances, as well as to determine possible predictors of response to toxin treatment. A retrospective cohort study included 47 female BPS/IC patients treated with onabotulinum toxin A (OnabotA) in a tertiary hospital between the years 2009 and 2022. All patients received 100 U of OnabotA in ten injections limited to the trigonal area. Patients were divided into three groups based on their treatment response as responders, non-responders and lost to follow-up due to non-medical reasons. The clinical and surgical records of the individuals were retrieved, including the 10-point visual analogue scale (VAS), the number of treatments, the time between injections, and the age at the first injection. A total of 25 patients (>50% of the cohort) were long-term responders, but none of the evaluated parameters was a predictor for this circumstance: age, pain intensity, or duration of improvement following the injection. The time between injections was stable (around 1 year). No severe adverse events were registered. The intratrigonal injection of botulinum toxin in patients with BPS/IC was an effective and safe long-term treatment for patients’ refractory to conservative forms of treatment. Age, basal pain intensity, and time to injection request did not predict long-term response to OnaBotA.     

Using Ro 15-1788 to investigate the benzodiazepine receptor in vivo: Studies on the anticonvulsant and sedative effect of melatonin and the convulsant effect of the benzodiazepine Ro 05-3663

Both the anticonvulsant and sedative effects of diazepam (5 mg/kg) were reversed by subsequent administration of the suggested specific benzodiazepine antagonist Ro 15-1788. In contrast neither the seizure threshold raising or sedative effect of melatonin (200 mg/kg) was reversed by Ro 15-1788. Ro 15-1788 had no effect on the convulsant action of the benzodiazepine Ro 05-3663. These data therefore argue against the suggestion that melatonin produces its sedative and anticonvulsant effects in vivo by interacting with the benzodiazepine receptor, and also strengthens the suggestion that Ro 05-3663 does not act at this site. The use of Ro 15-1788 in demonstrating whether a drug acts in vivo at the benzodiazepine site to produce a pharmacological response is discussed.