Affinity of beta-carbolines on rat brain benzodiazepine and opiate binding sites

The affinity of beta-carbolines, which may be formed in the body, to benzodiazepine and opiate receptors was studied by measuring their ability to inhibit the binding of [3H]-flunitrazepam and [3H]-dihydromorphine on rat brain synaptosomal membranes. All "aromatized" beta-carbolines studied (norharmane, harmane and 6-methoxyharmane) inhibited the specific binding of [3H]-flunitrazepam in micromolar concentrations, dihydro-beta-carbolines (6-methoxyharmalan, harmalol) were less potent, while all tetrahydro-beta-carbolines showed very low affinity. 6-Hydroxytetrahydroharmane, which is formed by condensation 5HT with acetaldehyde, inhibited [3H]-dihydromorphine binding in micromolar concentration, while norharmane and tetrahydro-beta-carbolines without OH-group showed little affinity. beta-Carbolines are the most potent known natural benzodiazepine receptor ligands. Because they are formed after alcohol drinking, their effects on benzodiazepine and opiate receptors may be connected with alcohol dependence although some beta-carbolines may inhibit 5HT uptake in still lower concentrations.     

Autoradiographical distribution of imidazoline binding sites in monoamine oxidase A deficient mice

This study has used receptor autoradiography to characterize imidazoline binding sites (I-BS) in monoamine oxidase (MAO) A knockout and wild-type mice. A comparison between MAO-A and MAO-B, binding of the endogenous beta-carboline [(3)H]harmane, and I-BS, has been made using sections from brain and kidney. The loss of binding to MAO-A in the knockout animals was confirmed using the selective radioligand [(3)H]Ro41-1049, with labelling reduced to background levels. The binding of [(3)H]Ro19-6327 to MAO-B was unaffected, indicating no change in this isoform in response to the loss of MAO-A. A reduction in binding to the I(2)-BS, as labelled by both [(3)H]idazoxan and [(3)H]2-BFI (2-(2-benzofuranyl)-2-imidazoline), was seen in the MAO-A knockout animals in both brain and kidney sections, whereas binding to the I(1)-BS in kidney sections remained unchanged. The loss of I(2) binding was found to be regionally dependent and was positively correlated with the relative expression of MAO-A in specific regions in the wild-type animals. Using the MAO-A knockout mice it was also possible to demonstrate a non-MAO-A population of binding sites labelled by the putative I-BS endogenous ligand, harmane.     

Sulfonamides containing curcumin scaffold: Synthesis,characterization, carbonic anhydrase inhibition and molecular docking studies

Curcumin is a multi-functional pharmacologically safe natural agent with proven cytoprotective effects to healthy human cells. In this study, a new series of sulfonamides with curcumin scaffold were synthesized, characterized and investigated for their carbonic anhydrase isoenzyme I (human) and II (bovine) isoforms. The structures of newly synthesized compounds were described by IR, ¹H NMR and ¹³C NMR spectral data. Compound 14 showed the K_i value of 0.99 µM with highest inhibitory activity among all other synthesized compounds against hCA-I enzyme. Similarly enzyme kinetic studies of compound 14, 16 and 30 against bCAII enzyme showed Ki values of 0.71, 0.67 and 0.71 µM respectively. Our biological assays results showed that most of active compounds have similar inhibitory activities compared to standard acetazolamide drug. The molecular docking predicted binding modes showed that these compounds bind with hCA-1 enzyme in similar fashion.     

Interaction of heparin with a synthetic pentadecapeptide from the C-terminal heparin-binding domain of fibronectin

The synthetic pentadecapeptide FN-C/H II (KNNQKSEPLIGRKKT-NH(2)) has the sequence of the carboxy-terminal heparin-binding domain of module III(14) of fibronectin. Interaction of FN-C/H II with bovine lung heparin has been studied by (1)H and (23)Na NMR spectroscopy and by heparin affinity chromatography. FN-C/H II binds to heparin from pD <2 up to pD approximately 10; at higher pD, the binding decreases as the lysine side-chain ammonium groups are titrated. Na(+) counterions are displaced from the counterion condensation volume that surrounds sodium heparinate by FN-C/H II, which provides direct evidence that the binding involves electrostatic interactions. The pK(A) values for each of the five ammonium groups of FN-C/H II increase upon binding to heparin which, together with chemical shift data, indicates that the binding involves both delocalized and direct electrostatic interactions between ammonium groups of FN-C/H II and carboxylate and/or sulfate groups of heparin. NMR data also provide evidence for the direct interaction of the guanidinium group of the arginine side chain with anionic sites on heparin. The affinity of heparin for FN-C/H II and for 13 analogue peptides in which lysine and arginine residues were systematically substituted with alanine increases as the number of basic residues increases. The relative contribution of each lysine and arginine to the affinity of heparin for FN-C/H II is R(12) > K(13) > K(14) > K(1) > K(5). Nuclear Overhauser enhancement (NOE) data indicate that, while FN-C/H II is largely unstructured in aqueous solution, the bound peptide interconverts among overlapping, turn-like conformations over the L(9) - T(15) segment of the peptide. NOE data for the interaction of FN-C/H II with a heparin-derived hexasaccharide, together with the number of Na(+) ions displaced from heparin by FN-C/H II as determined by (23)Na NMR, indicates that the peptide binds to a hexasaccharide segment of heparin. Identical NMR and heparin affinity chromatography results were obtained for the interaction of FN-C/H II and its D-amino acid analogue peptide with heparin, which is of interest for the potential use of peptides as therapeutic agents for diseases in which cell adhesion plays a critical role.     

Molecular determinants of β-carboline inhibition of the glycine receptor

β-Carbolines are potent modulators of GABA type A receptors and they have recently been shown to inhibit glycine receptors in a subunit-specific manner. The present study screened four structurally similar β-carbolines, 1,2,3,4-tetrahydronorharmane, norharmane, harmane and 6-methoxyharmalan, at recombinantly expressed α1, α1β, α2 and α3 glycine receptors with the aims of identifying structural elements of both the receptor and the compounds that are important for binding and subunit specificity. The four compounds exhibited only weak subunit specificity, rendering them unsuitable as pharmacological probes. Because they displayed competitive antagonist activity, we investigated the roles of known glycine binding residues in coordinating the four compounds. The structural similarity of the compounds, coupled with the differential effects of C-loop mutations (T204A, F207Y) on compound potency, implied direct interactions between variable β-carboline groups and mutated residues. Mutant cycle analysis employing harmane and norharmane revealed a strong pairwise interaction between the harmane methyl group and the C-loop in the region T204 and F207. These results which define the orientation of the bound β-carbolines were supported by molecular docking simulations. The information may also be relevant to understanding the mechanism β-carboline of binding to GABA type A receptors where they are potent pharmacological probes.     

High affinity (3H)tryptamine binding sites in various organs of the rat

(3H)Tryptamine binds with high affinity (KD values around 4 nmol/l) to membranes from different organs of the rat. The number of binding sites was high in the liver, intermediate in the heart and cerebral cortex and low in other organs including ileum, kidney, spleen, stomach and lung. Binding to kidney membranes was investigated in more detail. It was rapid and reversible (t1/2 7 min.). The structure-activity profile was characterized by high affinity of some beta-carbolines (IC50 = 20 - 200 nmol/l), medium affinity of serotonin (IC50 = 858 nmol/l) and low of methysergide (IC50 greater than 20 mumol/l). The findings correlate very well with binding characteristics to cerebral cortex membranes. Therefore, rat brain cortex and other organs contain the same type of (3H)tryptamine binding site and may be equally appropriate for further analysis.     

Synthesis,spectral and antimicrobial evaluation of some novel 1-methyl-3-alkyl-2,6-diphenylpiperidin-4-one oxime carbonates

Synthesis of some novel biologically active piperidin-4-one oxime carbonates from 1-methyl-3alkyl-2,6-diphenylpiperidin-4-one oximes and substituted chloroformates was carried out in the presence of potassium carbonate as base and tetrabutylammonium bromide (TBAB) as catalyst. The newly synthesized compounds were characterized by IR, ¹H, ¹³C NMR and LC–mass spectra. Based on the ¹H NMR analysis, all the compounds were found to adopt normal chair conformation with equatorial orientation of all the substituents. For all the synthesized compounds (5a–5l) antimicrobial activity has been tested against bacterial and fungal strains using Streptomycin and Amphotericin B as standards.     

Structural analysis of three sulfated oligosaccharides isolated from human milk

The structures of two sulfated octasaccharides and one sulfated nonasaccharide isolated from human milk have been investigated. Using 13C and 1H NMR spectroscopy and ESMS, the following structures 1-3 were established: [formula: see text].     

Solubilization of adrenal medullary opioid receptors

The opioid-binding activity of digitonin extract of bovine adrenal medullary membranes was studied. [3H]Diprenorphine binding to the solubilized material was rapid and saturable. The dissociation constant of [3H]diprenorphine binding was 0.76 nM. Several opioids displaced the [3H]diprenorphine binding. The complex of [3H]diprenorphine and the solubilized binding sites was eluted as a single peak on a Sepharose 6B column and showed an apparent molecular weight of 200,000. These results indicate that active opioid receptors are solubilized with digitonin from bovine adrenal medullary membranes.     

Structural variation in the antithrombin III binding site region and its occurrence in heparin from different sources

A tetrasaccharide possessing a biosynthetically permissible structural variability in and adjacent to the antithrombin III (ATIII) binding site has been isolated from heparin lyase depolymerized bovine lung heparin by using strong anion-exchange high-pressure liquid chromatography (SAX-HPLC). On the basis of two-dimensional 500-MHz 1H NMR experiments, including phase-sensitive correlated spectroscopy (COSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), and fast-atom bombardment mass spectrometry (FAB-MS), the primary structure of this tetrasaccharide was unambiguously established as delta UAp2S (1----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D-GlcNp2S3S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid). The 1H NMR ROESY experiment proved to be particularly valuable in offering sequence information. Heparins from a variety of species and tissue sources were examined by oligosaccharide mapping using SAX-HPLC and gradient polyacrylamide gel electrophoresis. Two of these heparins are used as anticoagulants; they are porcine intestinal mucosal heparin and bovine lung heparin. The predominant ATIII-binding site in porcine heparin contained an N-acetylated glucosamine residue. We now report the structure of the predominant ATIII-binding site in bovine heparin as----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D- GlcNp2S3S6S(1----4)-alpha-L-IdoAp2S(1----4)-alpha-D-GlcNp 2S6S(1----. This study shows the presence of one or both types of ATIII-binding-site variants in all of the heparins that were examined.     

Binding of an imidazopyridoindole at imidazoline I2 receptors

3,5,6,11-Tetrahydro-2H-imidazo[1',2':1,2]pyrido[3,4-b]indole (10) might be viewed as a fusion structure of two classes of I(2) imidazoline receptor ligands: 2-(2-benzofuranyl)-2-imidazolines and beta-carbolines. Its high affinity (K(i)=7.3 nM) provides insight to how the two classes of agents might bind relative to one another at I(2) receptors.     

Plasma protein binding and endothelial enzyme interactions in the lung

The influence of plasma albumin binding of the synthetic angiotensin-converting enzyme (ACE) substrate [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP) on BPAP hydrolysis by pulmonary endothelial ACE was studied in isolated rabbit lungs perfused with a salt solution containing either 5% bovine serum albumin (BSA) or 5% dextran. The single-pass indicator-dilution method was used to measure the fraction (M) of [3H]BPAP hydrolyzed. Lung M was greater with albumin-free perfusate than when BSA was present. M decreased as the time (ti) that the BPAP was in contact with the BSA before reaching the lung was increased, suggesting that some BSA binding sites for BPAP were not in equilibrium during bolus transit through the lungs. The M vs. ti data were correlated using a model incorporating both rapid and slow binding kinetics of BPAP and BSA. For the slow BPAP-BSA interaction, the dissociation rate constant was approximately 0.015 s-1, and the fraction of the BPAP bound to these slowly equilibrating sites at equilibrium was approximately 22%. The results indicate that transient plasma protein binding kinetics can affect lung BPAP hydrolysis.     

Identification of a residue in the gamma-aminobutyric acid type A receptor alpha subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding

GABAA receptors that contain either the alpha4- or alpha6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including beta-carbolines, bind to these diazepam-insensitive receptor subtypes. Some beta-carbolines [e.g. ethyl beta-carboline-3-carboxylate (beta-CCE) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)] display a higher affinity for alpha4- compared to alpha6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric alpha6/alpha4 subunits and co-expressed these with wild-type rat beta2 and gamma2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (alpha6N204/alpha4I203). Substitutions at alpha6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive alpha1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the beta-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the alpha1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by approximately 19-fold and approximately 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the alpha subunit in diazepam-sensitive and -insensitive receptors.     

Solubilization of rat lung vasoactive intestinal peptide receptors in the active state. Characterization of the binding properties and comparison with membrane-bound receptors

We demonstrate here that rat lung membrane vasoactive intestinal peptide (VIP) receptors can be extracted in the active state using digitonin. Sepharose 4B gel filtration chromatography was utilized to demonstrate the formation of specific binding complexes between 125I-VIP and solubilized receptors. A rapid soluble receptor assay was established to separate 125I-VIP-receptor complexes from free 125I-VIP, which entailed differential precipitation of the 125I-VIP-receptor complex with polyethylene glycol and bovine gamma-globulin. Using this assay, several detergents were tested for their suitability to extract active VIP receptors, and most favorable results were obtained with digitonin, as judged by specific binding of 125I-VIP to the solubilized receptors. Time course studies indicated that the binding of 125I-VIP to digitonin extract was more rapid than to rat lung membranes. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitonin extract, as in the membrane. The values for the dissociation constants (Kd) were 200 pM for Class I and 8 nM for Class II receptors while the values for binding capacity (Bmax) were 200 and 2300 fmol/mg for Class I and II sites, respectively. Although the binding parameters of the two classes were similar to those in the membrane, the pharmacological properties were different, as evidenced by the inability of rat growth hormone releasing factor, a potent VIP agonist in the membrane, to displace specifically bound 125I-VIP from solubilized receptors. The ability to solubilize active VIP receptors represents an important step toward purification of the functional protein.     

水菖蒲杀虫活性组分菖蒲螺酮的分离与鉴定（英文）

储粮害虫的危害造成储粮严重损失,化学农药的种种弊端使开发新型环境友好型药剂非常迫切, 植物由于其自身的特点, 成为开发新型药剂的重要来源。我们的前期研究表明, 水菖蒲Acorus calamus提取物对多种储粮害虫具有明显的触杀活性,并且分离得到了其主要活性组分β-细辛醚。本研究采用硅胶柱层析法,并以对玉米象Sitophilus zeamais的触杀活性进行追踪, 对水菖蒲提取物进行分离, 得到另一活性组分。经气相色谱-质谱联用、红外吸收光谱和核磁共振波谱方法鉴定,该活性组分为单体化合物菖蒲螺酮（1-异丙基-4,8-二甲基螺[4.5]癸-2,7-二酮）。将菖蒲螺酮以314.54, 251.63, 188.72, 125.82和62.91 μg/cm²的浓度处理玉米象96 h后, 玉米象的死亡率分别为85.6%, 72.2%, 53.3%, 38.9%和15.6%。处理96 h后,菖蒲螺酮对玉米象成虫的LD₅₀为158.00 μg/cm²。这些结果说明菖蒲螺酮对玉米象具有明显的生物活性,是水菖蒲提取物中除β-细辛醚以外的又一活性组分。     

Centrally Acting Imidazolines Stimulate Vascular Alpha 1A-Adrenergic Receptors in Rat-Tail Artery

  1. Centrally acting imidazoline antihypertensive agents clonidine and moxonidine also act peripherally to contract blood vessels. While these agents act at both I₁-imidazoline and alpha 2 adrenergic receptors centrally, the receptor types by which they mediate contraction require further definition. We therefore characterized the receptor subtype by which these agents mediate contraction of proximal rat-tail artery.2. Dose–response curves were determined for phenylephrine and for several imidazoline ligands, using endothelium denuded, isolated ring segments, of tail arteries from adult male Sprague-Dawley rats. Ring segments were mounted on a force transducer with platinum wires and immersed in a tissue bath containing Krebs solution, to which drugs could be added. Signals were digitized and recorded by a computer.3. Tail artery contractions expressed as a percent of contraction to 106 mM potassium were phenylephrine (96%), moxonidine (88%), clonidine (52%), and UK14304 (30%). Neither rilmenidine nor harmane caused contraction. Contraction of tail artery to moxonidine or clonidine could be blocked by alpha 1 antagonist urapidil or prazosin, and also by alpha 1A subtype selective antagonist WB4101. Schild plots were generated and a calculated pA2 value of 9.2 for prazosin in the presence of clonidine confirms clonidine as an agonist at alpha 1A receptors in proximal segments of rat-tail artery.4. Our work suggests that clonidine and moxonidine are promiscuous compounds at micromolar concentrations and that harmane and rilmenidine are more selective compounds for in vivo imidazoline research.*This work represents a portion of a dissertation to be submitted to the School of Graduate Studies, Loma Linda University, for the degree of Doctor of Philosophy.     

Nuclear magnetic resonance determination of metal-proton distances in a synthetic calcium binding site of rabbit skeletal troponin C

The binding of gadolinium to a synthetic peptide of 13 amino acid residues representing the calcium binding loop of site 3 of rabbit skeletal troponin C [AcSTnC(103-115)amide] has been studied by using proton nuclear magnetic resonance (1H NMR) spectroscopy. In particular, the proton line broadening and enhanced spin-lattice relaxation have been used to determine proton-metal ion distances for several assigned nuclei in the peptide-metal ion complex. These distances have been used in conjunction with other constraints and a distance algorithm procedure to demonstrate that the structure of the peptide-metal complex as shown by 1H NMR is consistent with the structure of the EF calcium binding loop in the X-ray structure of parvalbumin but that the available 1H NMR distances do not uniquely define the solution structure.     

Identification of Na+/K+-ATPase inhibitors in bovine plasma as fatty acids and hydrocarbons

A preparative purification of endogenous inhibitors of the Na+/K+-ATPase has been carried out from bovine blood. Dried plasma was deproteinized, hexane-extracted and desalted, followed by further purification through a series of reverse-phase HPLC fractionations. Fractions active in inhibiting Na+/K+-ATPase activity and displacing ouabain were collected and purified further. By comparison with ouabain, the final extract was found to have a steeper concentration-effect curve in the inhibition of Na+/K+-ATPase. In displacement of [3H]ouabain, the extract had again a steeper concentration-effect curve than does ouabain, and in addition it enhanced ouabain binding at high dilutions. These properties are indicative of nonspecific interactions with the Na+/K+-ATPase. The active fraction was identified by TLC, HPLC, NMR, GLC and GC-MS, to be a mixture of three unesterified fatty acids, mainly oleic acid (72% of the total) and three saturated hydrocarbons. The assignment of structures was corroborated by comparison with authentic samples.     

Simple compounds with high pharmacologic potential: beta-carbolines. Origins, syntheses, biological properties

We reviewed the origins, the synthetic pathways and the biological properties of beta-carbolines, the condensation products of tryptophan and indole alkylamines with aldehydes. They were found in many plants, some of which have been used as hallucinogens. They also occur as minor constituents in tobacco smoke. In mammalian body, beta-carboline derivatives occur normally in plasma, platelets and urine, moreover it seems that some are formed in human body after alcohol intake. Due to interesting biological effects described in recent years (inhibition of monoamine oxidase, binding to benzodiazepine receptors, comutagenic and carcinogenic properties, 5-hydroxy tryptamine uptake inhibition), many attempts were made to prepare beta-carbolines starting from various indole derivatives. We reviewed the published methods up to 1975 and summarized the main patents related with pharmacological properties of synthetic beta-carbolines.     

A novel piperazine linked β-amino alcohols bearing a benzosuberone scaffolds as anti-proliferative agents

A new series of 1-((9-chloro-2,3-dimethyl-6,7-dihydro-5H-benzo[7]annulen-8-yl)methoxy)-3-(4-phenylpiperzin-1-yl) propan-2-ols (6a-k) have been designed, synthesized and their structures were established by spectroscopic data (FT-IR, ¹H NMR, ¹³C NMR, HRMS) and further confirmed by X-ray analysis. The newly synthesized compounds 6a-k were evaluated for their in vitro anti-proliferative activity against four cancer cell lines such as HeLa (cervical), MDA-MB-231 (breast), A549 (lung) and MIAPACA (pancreatic). Among the compounds tested, the compound 6e displayed most potent activity against four cancer cell lines with GI₅₀ values ranging from 0.010 to 0.097 μM. The structure and anti-proliferative activity relationship was further supported by in silico molecular docking study of the active compounds against Colchicine binding site of β-tubulin.