6,6-Spiroimine analogs of (-)-gymnodimine A: synthesis and biological evaluation on nicotinic acetylcholine receptors

Simple models of the spiroimine core of (-)-gymnodimine A have been synthesized in racemic and optically active forms. The quaternary carbon of the racemic spiroimines was created by Michael addition of a β-ketoester to acrolein, whereas the asymmetric allylic alkylation of the same β-ketoester was used to access the spiroimines in an enantioselective fashion. Both racemic and enantio-enriched mixtures were tested for their biological activities on Xenopus oocytes either expressing (human α4β2) or having incorporated (Torpedoα1(2)βγδ) nicotinic acetylcholine receptors (nAChRs). These spiroimine analogs of (-)-gymnodimine A inhibited acetylcholine-evoked nicotinic currents, but were less active than the phycotoxin. Our results reveal that the 6,6-spiroimine moiety is important for the blockade of nAChRs and support the hypothesis that it is one of the pharmacophores of this group of toxins.     

Nicotinic Acetylcholine Receptor Agonists: A Milestone for Modern Crop Protection

The destruction of crops by invertebrate pests is a major threat against a background of a continuously rising demand in food supply for a growing world population. Therefore, efficient crop protection measures in a vast range of agricultural settings are of utmost importance to guarantee sustainable yields. The discovery of synthetic agonists selectively addressing the nicotinic acetylcholine receptors (nAChRs), located in the central nervous system of insects, for use as insecticides was a major milestone in applied crop protection research. These compounds, as a result of their high target specificity and versatility in application methods, opened a new innovative era in the control of some of the world′s most devastating insect pests. These insecticides also contributed massively to extending our knowledge of the biochemistry of insect nicotinic acetylcholine receptors. The global economic success of synthetic nAChR agonists as insecticides renders the nicotinic acetylcholine receptor still one of the most attractive target sites for exploration in insecticide discovery.     

Synthesis of Hexahydro‐2‐pyrindine (=Hexahydrocyclopenta[c]pyridine) Derivatives as Conformationally Restricted Analogs of the Nicotinic Ligands Arecolone and Isoarecolone

Two hexahydropyrindine derivatives, 1,2,3,4,6,7‐hexahydro‐2‐methyl‐5H‐cyclopenta[c]pyridin‐5‐one ( 1 ) and 1,2,3,4,5,6‐hexahydro‐2‐methyl‐7H‐cyclopenta[c]pyridin‐7‐one ( 2 ), and their methiodides 14 and 26 , respectively, were synthesized. They can be considered rigid analogues of the known nicotinic agonists arecolone (=1‐(1,2,5,6‐tetrahydro‐1‐methylpyridin‐3‐yl)ethanone) and isoarecolone (=1‐(1,2,3,6‐tetrahydro‐1‐methylpyridin‐4‐yl)ethanone). The affinity for the central nicotinic receptor were measured on rat cerebral cortex. Although only the methiodide 14 , among the four conformationally restricted compounds, shows an appreciable affinity, the results obtained provide useful information on the molecular requirements at the interaction site of the central nicotinic receptors.     

Potentials of mean force for acetylcholine unbinding from the alpha7 nicotinic acetylcholine receptor ligand-binding domain

The nicotinic acetylcholine receptor is a prototype ligand-gated ion channel that mediates signal transduction in the neuromuscular junctions and other cholinergic synapses. The molecular basis for the energetics of ligand binding and unbinding is critical to our understanding of the pharmacology of this class of receptors. Here, we used steered molecular dynamics to investigate the unbinding of acetylcholine from the ligand-binding domain of human alpha7 nicotinic acetylcholine receptor along four different predetermined pathways. Pulling forces were found to correlate well with interactions between acetylcholine and residues in the binding site during the unbinding process. From multiple trajectories along these unbinding pathways, we calculated the potentials of mean force for acetylcholine unbinding. Four available methods based on Jarzynski's equality were used and compared for their efficiencies. The most probable pathway was identified to be along a direction approximately parallel to the membrane. The derived binding energy for acetylcholine was in good agreement with that derived from the experimental binding constant for acetylcholine binding protein, but significantly higher than that for the complete human alpha7 nicotinic acetylcholine receptor. In addition, it is likely that several intermediate states exist along the unbinding pathways.     

Synthesis of azabicyclo[2.2.n]alkane systems as analogues of 3-[1-methyl-2-(S)-pyrrolidinyl- methoxy]pyridine (A-84543)

This work is connected with the epibatidine field and describes the synthesis of several analogues of compounds that present affinity for nicotinic acetylcholine receptors, such as 3-[1-methyl-2-(S)-pyrrolidinylmethoxy]pyridine (A-84543). These analogues bear a 3-pyridyl ether substituent at the bridgehead carbon of the azabicyclo[2.2.n]alkane system. Particularly, in the case of the 1-substituted 2-azabicyclo[2.2.2]octane system, a new synthetic route has been developed, which involves the synthesis of a novel rigid sulfamidate that allows the straightforward introduction of nucleophiles.     

Novel approach to the design of potential bioactive alkaloid anabasine conjugates using click chemistry methodology

Anabasine‐containing azides and acetylenes were used as building blocks in copper(I)‐catalyzed 1,3‐dipolar cycloaddition reactions with a series of acetylenes and azides via click methodology to provide a range of anabasine conjugates being potential ligands for neuronal nicotinic acetylcholine receptors.     

Synthesis of bridgehead-substituted azabicyclo[2.2.1]heptane and -[3.3.1]nonane derivatives for the elaboration of α7 nicotinic ligands

Azabicyclo[2.2.1]heptane and -[3.3.1]nonane scaffolds (X = Cl, Br) containing a pyridinyl substituent at the bridgehead position were prepared via two complementary chemical pathways, either by the transformation of a methoxy group into a synthetically valuable chlorine atom at the C-6 position of the pyridine moiety or by means of a regioselective C-6 deprotonation/halogenation process of the pyridine moiety exemplified by chlorination or bromination. These newly generated scaffolds were then engaged in Suzuki-Miyaura coupling reactions to provide α7 nicotinic ligands. Both chemical series were evaluated in vitro for their affinity at α7 nicotinic receptors, revealing nanomolar potency with significant selectivity over the α4β2 nicotinic subtype. These approaches offer a general access to these α7 nicotinic scaffolds and ligands.     

Binding enhancements of nicotinic acid to water-soluble zinc porphyrins based on triple attractions of coordination, Coulomb, and CH-pi interactions

The binding of amines to artificial zinc-porphyrin receptors 1-4 was examined in basic aqueous solutions. For nicotinic acid and 3,5-dicarboxypyridine, substantial binding enhancements were observed compared to other amines with no pi system or carboxyl group. This observation suggested that interligand attractions of Coulomb and CH-pi interactions in addition to N-atom coordination can act effectively as recognition factors. The differences in the Coulomb interaction between carboxylate and sulfonate anions were also discussed.     

Total synthesis of neosurugatoxin

Total synthesis of neosurugatoxin, having a strong affinity for nicotinic receptors, is described.     

Cell‐Derived Vesicles for Single‐Molecule Imaging of Membrane Proteins

A new approach is presented for the application of single‐molecule imaging to membrane receptors through the use of vesicles derived from cells expressing fluorescently labeled receptors. During the isolation of vesicles, receptors remain embedded in the membrane of the resultant vesicles, thus allowing these vesicles to serve as nanocontainers for single‐molecule measurements. Cell‐derived vesicles maintain the structural integrity of transmembrane receptors by keeping them in their physiological membrane. It was demonstrated that receptors isolated in these vesicles can be studied with solution‐based fluorescence correlation spectroscopy (FCS) and can be isolated on a solid substrate for single‐molecule studies. This technique was applied to determine the stoichiometry of α3β4 nicotinic receptors. The method provides the capability to extend single‐molecule studies to previously inaccessible classes of receptors.     

Synthesis of new bridgehead substituted azabicyclo-[2.2.1]heptane and -[3.3.1]nonane derivatives as potent and selective α7 nicotinic ligands

New azabicyclo[2.2.1]heptane and -[3.3.1]nonane derivatives containing a pyridinyl substituent at the bridgehead position have been synthesized via an efficient ten chemical steps pathway. Both chemical series were then evaluated in vitro for their affinity at α7 nicotinic receptors revealing nanomolar potency with notably excellent selectivity over the α4β2 nicotinic subtype.     

Ligand-binding assays for cyanobacterial neurotoxins targeting cholinergic receptors

Toxic cyanobacterial blooms are a threat to public health because of the capacity of some cyanobacterial species to produce potent hepatotoxins and neurotoxins. Cyanobacterial neurotoxins are involved in the rapid death of wild and domestic animals by targeting voltage gated sodium channels and cholinergic synapses, including the neuromuscular junction. Anatoxin-a and its methylene homologue homoanatoxin-a are potent agonists of nicotinic acetylcholine receptors. Since the structural determination of anatoxin-a, several mass spectrometry-based methods have been developed for detection of anatoxin-a and, later, homoanatoxin-a. Mass spectrometry-based techniques provide accuracy, precision, selectivity, sensitivity, reproducibility, adequate limit of detection, and structural and quantitative information for analyses of cyanobacterial anatoxins from cultured and environmental cyanobacterial samples. However, these physicochemical techniques will only detect known toxins for which toxin standards are commercially available, and they require highly specialized laboratory personnel and expensive equipment. Receptor-based assays are functional methods that are based on the mechanism of action of a class of toxins and are thus, suitable tools for survey of freshwater reservoirs for cyanobacterial anatoxins. The competition between cyanobacterial anatoxins and a labelled ligand for binding to nicotinic acetylcholine receptors is measured radioactively or non-radioactively providing high-throughput screening formats for routine detection of this class of neurotoxins. The mouse bioassay is the method of choice for marine toxin monitoring, but has to be replaced by fully validated functional methods. In this paper we review the ligand-binding assays developed for detection of cyanobacterial and algal neurotoxins targeting the nicotinic acetylcholine receptors and for high-throughput screening of novel nicotinic agents.     

Preparation of Nicotinic Acid from Oxidation of 3-Picoline with Oxygen Under Catalysis of T(0-CI)PPMn

The oxidation of 3-picoline to nicotinic acid took place efficiently in an ethanol solution with O2 as the oxidant under the catalysis of T(o-C1)PPMn at 40-150℃ and 0.5-3.0 MPa oxygen pressure.The influences of temperature,oxygen pressure,reaction time,concentration of 3-picoline,concentration of sodium hydroxide,and concentration of T(0-C1)PPMn catalyst,etc.on the production of nicotinic acid were investigated.The results show that T(0-C1)PPMn presented excellent catalytic activity in the oxidation of 3-picoline to nicotinic acid and the yield of nicotinic acid varied greatly with the reaction temperature,oxygen pressure,T(0-C1)PPMn concentration,etc.     

A model for short alpha-neurotoxin bound to nicotinic acetylcholine receptor from Torpedo californica: comparison with long-chain alpha-neurotoxins and alpha-conotoxins

Short-chain alpha-neurotoxins from snakes are highly selective antagonists of the muscle-type nicotinic acetylcholine receptors (nAChR). Although their spatial structures are known and abundant information on topology of binding to nAChR is obtained by labeling and mutagenesis studies, the accurate structure of the complex is not yet known. Here, we present a model for a short alpha-neurotoxin, neurotoxin II from Naja oxiana (NTII), bound to Torpedo californica nAChR. It was built by comparative modeling, docking and molecular dynamics using 1H NMR structure of NTII, cross-linking and mutagenesis data, cryoelectron microscopy structure of Torpedo marmorata nAChR [Unwin, N., 2005. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J. Mol. Biol. 346, 967-989] and X-ray structures of acetylcholine-binding protein (AChBP) with agonists [Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., Sixma, T.K., 2004. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41 (6), 907-914] and antagonists: alpha-cobratoxin, a long-chain alpha-neurotoxin [Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., Marchot, P., 2005. Crystal structure of Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J. 24 (8), 1512-1522] and alpha-conotoxin [Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., Smit, A.B., 2005. Crystal structure of nicotinic acetylcholine receptor homolog AChBP in complex with an alpha-conotoxin PnIA variant. Nat. Struct. Mol. Biol. 12 (7), 582-588]. In complex with the receptor, NTII was located at about 30 A from the membrane surface, the tip of its loop II plunges into the ligand-binding pocket between the alpha/gamma or alpha/delta nAChR subunits, while the loops I and III contact nAChR by their tips only in a 'surface-touch' manner. The toxin structure undergoes some changes during the final complex formation (for 1.45 rmsd in 15-25 ps according to AMBER'99 molecular dynamics simulation), which correlates with NMR data. The data on the mobility and accessibility of spin- and fluorescence labels in free and bound NTII were used in MD simulations. The binding process is dependent on spontaneous outward movement of the C-loop earlier found in the AChBP complexes with alpha-cobratoxin and alpha-conotoxin. Among common features in binding of short- and long alpha-neurotoxins is the rearrangement of aromatic residues in the binding pocket not observed for alpha-conotoxin binding. Being in general very similar, the binding modes of short- and long alpha-neurotoxins differ in the ways of loop II entry into nAChR.     

Synthesis of two fluoro analogues of the nicotinic acetylcholine receptor agonist UB-165

Two racemic fluoropyridine analogues 4 and 5 of the potent nicotinic agonist UB-165 have been synthesized. Halogenated pyridines 7 and 12 provided the organometallic reagents needed for the Negishi and Suzuki coupling reactions used for the preparation of 4 and 5, and the N-vinyloxycarbonyl protecting group of 8 and 15 was cleaved using a novel trifluoroacetic acid-mediated deprotection protocol. Analogue 4 retained high binding affinity at rat brain alpha4beta2 and alpha7 nicotinic receptors.     

Mechanisms of imidacloprid resistance in Nilaparvata lugens by molecular modeling

<正>Homology models of the ligand binding domain of the wild-type and Y151S mutant brown planthopper {Nilaparvata lugens)α1 and rat(Rattus norvegicus)β2 nicotinic acetylcholine receptor(nAChR) subunits were generated based on the crystal structure of acetylcholine binding protein of Lymnaea stagnalis.Neonicotinoid insecticide imidacloprid was docked into the putative binding site of wild-type and mutantα1β2 dimeric receptors by Surflex-docking,and the calculated docking energies were in agreement with experimental results.The resistance mechanisms and corresponding binding modes of imidacloprid on nAChRs containing the Y151S target-site mutation were discussed.     

Barriers to ion translocation in cationic and anionic receptors from the Cys-loop family

Understanding the mechanisms of gating and ion permeation in biological channels and receptors has been a long-standing challenge in biophysics. Recent advances in structural biology have revealed the architecture of a number of transmembrane channels and allowed detailed, molecular-level insight into these systems. Herein, we have examined the barriers to ion conductance and origins of ion selectivity in models of the cationic human alpha7 nicotinic acetylcholine receptor (nAChR) and the anionic alpha1 glycine receptor (GlyR), based on the structure of Torpedo nAChR. Molecular dynamics simulations were used to determine water density profiles along the channel length, and they established that both receptor pores were fully hydrated. The very low water density in the middle of the nAChR pore indicated the existence of a hydrophobic constriction. By contrast, the pore of GlyR was lined with hydrophilic residues and remained well-hydrated throughout. Adaptive biasing force simulations allowed us to reconstruct potentials of mean force (PMFs) for chloride and sodium ions in the two receptors. For the nicotinic receptor we observed barriers to ion translocation associated with rings of hydrophobic residues-Val13' and Leu9'-in the middle of the transmembrane domain. This finding further substantiates the hydrophobic gating hypothesis for nAChR. The PMF revealed no significant hydrophobic barrier for chloride translocation in GlyR. For both receptors nonpermeant ions displayed considerable barriers. Thus, the overall electrostatics and the presence of rings of charged residues at the entrance and exit of the channels were sufficient to explain the experimentally observed anion and cation selectivity.     

α-芋螺毒素构效关系与分子设计

α-芋螺毒素(α-conotoxins)是从芋螺毒液中提取到的一类活性多肽. 与其它家族的芋螺毒素相比, 它们含二硫键少, 结构相对简单, 由于作用于神经肌肉接头的N-乙酰胆碱受体(nAChRs)的不同亚型, 拮抗乙酰胆碱, 可作为鉴定nAChRs亚型及其亚基的有效工具, 已成为芋螺毒素结构改造的最佳先导化合物. 利用HyperChem软件包的量子化学半经验方法AM1对8个具有代表性的α-芋螺毒素进行了量子化学计算, 研究了它们的电子结构及构效关系. 结果表明, 空间结构的相似性使它们作用于同一受体, 局部结构差异而导致的电子结构的较大差别是它们能作用于不同受体亚型的重要原因. 在此基础上, 以α-芋螺毒素GI为模型设计了7个类似物并进行了量子化学计算, 比较了类似物与GI在空间结构及电子结构方面的特征.     

Efficient N-arylation catalyzed by a copper(I) pyrazolyl-nicotinic acid system

Catalyst 6-(1H-pyrazol-1-yl)nicotinic acid L-CuCl behaves as a very active promoter of the N-arylation reactions, as it has been demonstrated with varieties of substrates under mild reaction conditions. A Cu(I) complex based on L of precatalyst has been isolated by a hydrothermal method and structurally characterized.     

A cation-pi binding interaction with a tyrosine in the binding site of the GABAC receptor

GABA(C) (rho) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. As in other members of this family, the agonist binding site of GABA(C) receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-pi interaction to a tryptophan, the GABA(C) binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-pi binding ability of the side chain and EC(50) for receptor activation, thus demonstrating a cation-pi interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-pi binding energies that reflect the nature of the cationic center of the agonist.