A Modular Access to (±)-Tubocurine and (±)-Curine - Formal Total Synthesis of Tubocurarine.

Two consecutive Cu-catalyzed Ullmann-type C-O couplings permitted the first successful entry toward the curare alkaloids (±)-tubocurine and (±)-curine. Starting from vanillin, the synthetic sequence comprises 15 linear steps and includes a total of 24 transformations. In addition, the total synthesis of tubocurine represents a formal total synthesis of the famous arrow poison alkaloid tubocurarine.

Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication.

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand-protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (Kd=4.4µM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.

Syntheses of strychnine, norfluorocurarine, dehydrodesacetylretuline, and valparicine enabled by intramolecular cycloadditions of Zincke aldehydes.

A full account of the development of the base-mediated intramolecular Diels-Alder cycloadditions of tryptamine-derived Zincke aldehydes is described. This important complexity-generating transformation provides the tetracyclic core of many indole monoterpene alkaloids in only three steps from commercially available starting materials and played a key role in short syntheses of norfluorocurarine (five steps), dehydrodesacetylretuline (six steps), valparicine (seven steps), and strychnine (six steps). Reasonable mechanistic possibilities for this reaction, a surprisingly facile dimerization of the products, and an unexpected cycloreversion to regenerate Zincke aldehydes under specific conditions are also discussed.

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT(3)R.

Design and expression of human alpha7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties.

In order to facilitate structural studies of the extracellular domain (ECD) of human alpha7 nicotinic acetylcholine receptor (nAChR), we designed several mutants, since the wild-type-ECD forms large oligomers and microaggregates, and expressed them in the yeast Pichia pastoris. Mutant design was based on a 3D model of human alpha7-nAChR-ECD, constructed using as templates the X-ray crystal structure of the homologous acetylcholine-binding protein (AChBP) and the electron microscopy structure of the Torpedo alpha-nAChR-ECD. At least one mutant, mut10, carrying six single-point mutations (Phe3Tyr, Val69Thr, Cys116Ser, Ile165Thr, Val177Thr, Phe187Tyr) and the replacement of its Cys-loop with the corresponding and more hydrophilic AChBP Cys-loop, was expressed with a 4-fold higher expression yield (1.2 mg/L) than the wild-type alpha7-ECD, existing exclusively as a soluble oligomeric, probably pentameric, form, at concentrations up to at least 10 mg/mL, as judged by gel filtration and dynamic light scattering. This mutant displayed a significantly improved (125)I-alpha-bungarotoxin-binding affinity (K(d)=24 nM) compared to the wild-type-ECD (K(d)=70 nM), the binding being inhibited by unlabelled alpha-bungarotoxin, d-tubocurarine or nicotine (K(i) of 21.5 nM, 127 microM and 17.5 mM, respectively). Circular dichroism studies of mut10 revealed (a) a similar secondary structure composition ( approximately 5% alpha-helix, approximately 45% beta-sheet) to that of the AChBP, Torpedo alpha-nAChR-ECD, and mouse alpha1-nAChR-ECD, (b) a well-defined tertiary structure and (c) binding of small cholinergic ligands at micromolar concentrations. Furthermore, electron microscopy showed well-assembled, probably pentameric, particles of mut10. Finally, since deglycosylation did not alter its solubility or ligand-binding properties, mut10, in either its glycosylated or deglycosylated form, is a promising alpha7-ECD mutant for structural studies, useful for the rational drug design to treat alpha7-nAChR-related diseases.

Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein.

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.

Mapping residues in the ligand-binding domain of the 5-HT(3) receptor onto d-tubocurarine structure.

The serotonin 5-HT(3) receptor (5-HT(3)R) is a member of the cys-loop ligand-gated ion channel family. We have used the combination of site-directed mutagenesis, homology modeling of the 5-HT(3)R extracellular domain, and ligand docking simulations as a way to map the architecture of the 5-HT(3)R ligand binding domain. Mutation of Phe226 in loop C of the binding site to tyrosine (F226Y) has no effect on the apparent affinity of the competitive antagonist d-tubocurarine (dTC) for the receptor. On the other hand, replacement of Asn128 in loop A of the binding site with alanine (N128A) increases the apparent affinity of dTC by approximately 10-fold. Double-mutant cycle analysis employing a panel of dTC analogs with substitutions at various positions to identify specific points of interactions between the dTC analogs and Asn128 suggests that Asn128 makes a direct interaction with the 2'N of dTC. Molecular modeling of the 5-HT(3)R extracellular domain using the antagonist-bound conformation of the Aplysia californica acetylcholine binding protein as a template followed by ligand docking simulations produces two classes of structures of the 5-HT(3)R/dTC complex; only one of these has the 2'N of dTC positioned at Asn128 and thus is consistent with the data from this study and previously published data. The use of the rigid dTC analogs as "molecular rulers" in conjunction with double-mutant cycle analysis of mutant receptors can allow the spatial mapping of the position of various residues in the ligand-binding site.

Reversible sequential-binding probe receptor-ligand interactions in single cells.

With the reversible sequential (ReSeq) binding assay,we present a novel approach for the ultrasensitive profiling of receptor function in single living cells. This assay is based on the repetitive application of fluorescent ligands that have fast association-dissociation kinetics. We chose the nicotinic-acetylcholine receptor (nAChR) as a prototypical example and performed ReSeq equilibrium, kinetic, and competition-binding assays using fluorescent derivatives of the antagonist alpha-conotoxin GI (alpha-CnTx). Thereby, we determined the binding constants of unlabeled alpha-CnTx and d-tubocurarine. The high selectivity of alpha-CnTx for muscle-type nAChR made it possible to observe specific binding even in the presence of other nAChR subtypes. Imaging of individual nAChRs and ligand-binding cycles to single cells in microfluidic devices demonstrated the ultimate miniaturization and accuracy of ReSeq-binding assays even at low receptor-expression levels. We expect our approach to be of generic importance for functional screening of compounds or membrane receptors, and for the detailed characterization of rare primary cells.

Taste cell responses in the frog are modulated by parasympathetic efferent nerve fibers.

We studied the anatomical properties of parasympathetic postganglionic neurons in the frog tongue and their modulatory effects on taste cell responses. Most of the parasympathetic ganglion cell bodies in the tongue were found in extremely small nerve bundles running near the fungiform papillae, which originate from the lingual branches of the glossopharyngeal (GP) nerve. The density of parasympathetic postganglionic neurons in the tongue was 8000-11,000/mm(3) of the extremely small nerve bundle. The mean major axis of parasympathetic ganglion cell bodies was 21 microm, and the mean length of parasympathetic postganglionic neurons was 1.45 mm. Electrical stimulation at 30 Hz of either the GP nerve or the papillary nerve produced slow hyperpolarizing potentials (HPs) in taste cells. After nicotinic acetyl choline receptors on the parasympathetic ganglion cells in the tongue had been blocked by intravenous (i.v.) injection of D-tubocurarine (1 mg/kg), stimulation of the GP nerve did not induce any slow HPs in taste cells but that of the papillary nerve did. A further i.v. injection of a substance P NK-1 antagonist, L-703,606, blocked the slow HPs induced by the papillary nerve stimulation. This suggests that the parasympathetic postganglionic efferent fibers innervate taste cells and are related to a generation of the slow HPs and that substance P is released from the parasympathetic postganglionic axon terminals. When the resting membrane potential of a taste cell was hyperpolarized by a prolonged slow HP, the gustatory receptor potentials for NaCl and sugar stimuli were enhanced in amplitude, but those for quinine-HCl and acetic acid stimuli remained unchanged. It is concluded that frog taste cell responses are modulated by activities of parasympathetic postganglionic efferent fibers innervating these cells.

Ligand-induced conformational change in the alpha7 nicotinic receptor ligand binding domain.

Molecular dynamics simulations of a homology model of the ligand binding domain of the alpha7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca(2+), to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca(2+) appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change.

Laser spray: electric field-assisted matrix-assisted laser desorption/ionization.

A new liquid chromatography/mass spectrometry interface, the laser spray, has been developed. Explosive vaporization and mist formation occur when an aqueous solution effusing out from the tip of the stainless-steel capillary is irradiated from the opposite side of the capillary by a 10.6 microm infrared laser. Weak ion signals could be detected when the plume was sampled through the ion sampling orifice. When a high voltage (3-4 kV) was applied to the stainless-steel capillary, strong ion signals appeared. The ion abundances were found to be orders of magnitude greater than those obtained by conventional electrospray ionization in the case of aqueous solutions. The present method is regarded as an electric-field assisted form of matrix-assisted laser desorption/ionization in which the liquid chromatographic solvent (water, etc.) acts as a liquid matrix. Laser spray ionization is expected to become a versatile method for biological mass spectrometry because this method is compatible with the natural solvent, water.

Toward atomic-scale understanding of ligand recognition in the muscle nicotinic receptor.

The nicotinic receptor at the motor endplate has served as a prototype for understanding structure, function and ligand recognition in the superfamily of pentameric ligand-gated ion channels. Yet despite this advanced state of knowledge, atomic-scale understanding of such elementary processes as ligand recognition has remained elusive owing to the lack of a high-resolution x-ray structure. However, the field has recently entered a state of rapid advancement following the discovery and atomic structural determination of the water-soluble acetylcholine binding protein (AChBP), a homolog of the receptor ligand binding domain. The AChBP structure provides the theoretical foundation for generating homology models of the corresponding receptor ligand binding domains within this structural family of receptors. Experimental assignment of residue equivalence between AChBP and receptor subunits subsequently yielded homology models ready for experimental testing. One such test is computational determination of ligand docking orientation in conjunction with mutagenesis of predicted contact residues and measurements of ligand binding affinity. Applied to different analogs of the competitive antagonist curare, docking computations that incorporate intrinsic protein flexibility reveal fundamentally distinct orientations of each analog bound to AChBP. The different contact residues predicted for each analog were tested and confirmed by mutagenesis of AChBP followed by measurements of ligand binding. By applying the same computational and experimental approaches to the adult human muscle AChR, we find that the two curare analogs also dock in distinctly different orientations. Thus subtle structural changes in the ligand, and by extension, structural differences in non-conserved residues among receptor subtypes and species, can dramatically alter the orientation of the bound ligand. The results have important implications for design of drugs targeting nicotinic receptors and members of the superfamily of pentameric ligand-gated ion channels.

Curariform antagonists bind in different orientations to acetylcholine-binding protein.

Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to approximately 170 degrees rotation and approximately 30 degrees degree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.

Orientation of d-tubocurarine in the muscle nicotinic acetylcholine receptor-binding site.

Ligand modification and receptor site-directed mutagenesis were used to examine binding of the competitive antagonist, d-tubocurarine (dTC), to the muscle-type nicotinic acetylcholine receptor (AChR). By using various dTC analogs, we measured the interactions of specific dTC functional groups with amino acid positions in the AChR gamma-subunit. Because data for mutations at residue gammaTyr(117) were the most consistent with direct interaction with dTC, we focused on that residue. Double mutant thermodynamic cycle analysis showed apparent interactions of gammaTyr(117) with both the 2-N and the 13'-positions of dTC. Examination of a dTC analog with a negative charge at the 13'-position failed to reveal electrostatic interaction with charged side-chain substitutions at gamma117, but the effects of side-chain substitutions remained consistent with proximity of Tyr(117) to the cationic 2-N of dTC. The apparent interaction of gammaTyr(117) with the 13'-position of dTC was likely mediated by allosteric changes in either dTC or the receptor. The data also show that cation-pi electron stabilization of the 2-N position is not required for high affinity binding. Molecular modeling of dTC within the binding pocket of the acetylcholine-binding protein places the 2-N in proximity to the residue homologous to gammaTyr(117). This model provides a plausible structural basis for binding of dTC within the acetylcholine-binding site of the AChR family that appears consistent with findings from photoaffinity labeling studies and with site-directed mutagenesis studies of the AChR.

Structure of the agonist-binding sites of the Torpedo nicotinic acetylcholine receptor: affinity-labeling and mutational analyses identify gamma Tyr-111/delta Arg-113 as antagonist affinity determinants.

Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the gamma-subunit which, along with the analogous residue in delta-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and alpha-conotoxin (alpha Ctx) MI. nAChR gamma-subunit, isolated from nAChR-rich membranes photolabeled with [3H]dTC, was digested with Staphylococcus aureus V8 protease, and a 3H-labeled fragment was purified by reversed-phase high-performance liquid chromatography. Amino-terminal sequence analysis of this fragment identified 3H incorporation in gamma Tyr-111 and gamma Tyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at gamma Trp-55, the primary site of [3H]dTC photoincorporation within gamma-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940-32950]. The Torpedo nAChR delta-subunit residue corresponding to gamma Tyr-111 (delta Arg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the alpha-delta agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]alpha-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and alpha Ctx MI with nAChRs containing gamma Y111R or delta R113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the gamma Y111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of alpha Ctx MI, with delta R113Y enhancing and gamma Y111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of gamma- (or delta-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.

Characterization and evaluation of d-(+)-tubocurarine chloride as a chiral selector for capillary electrophoretic enantioseparations.

A new macrocyclic of the bis(benzylisoquinoline) alkaloid family, d-(+)-tubocurarine chloride (DTC), has been evaluated as a chiral selector for the separation of optical isomers of organic carboxylates using capillary electrophoresis (CE). The pertinent physicochemical properties, such as absorption spectrum, isoionic point, and solution conformation, of DTC were determined. The effects of varying such experimental parameters as DTC concentration, pH, and methanol content in the running buffer were assessed. CE separation of the enantiomers of 18 different compounds was achieved using DTC as the chiral selector under optimized background electrolytic conditions.

Interaction of d-tubocurarine analogs with the mouse nicotinic acetylcholine receptor. Ligand orientation at the binding site.

The binding of d-tubocurarine and several of its analogs to the mouse nicotinic acetylcholine receptor (AChR) was measured by competition against the initial rate 125I-alpha-bungarotoxin binding to BC3H-1 cells. The changes in affinity due to methylation or halogenation at various functional groups on d-tubocurarine was measured to both the high affinity (alphagamma-site) and the low affinity site (alphadelta-site). We show that quaternization by methylation of the 2'-N ammonium group enhances the affinity for both the acetylcholine binding sites of mouse AChR, whereas this change does not affect affinity for the Torpedo AChR sites. The effect of N-methylation suggests the presence of interactions with the ammonium moiety that cannot be readily attributed to the known conserved residues thought to stabilize this functional group. Methylation of both the 7'- and 12'-phenols produced net affinity changes at both sites. The changes resulted from contributions at both the 7'- and the 12'-positions; however, these effects were dependent on whether the ammoniums were also methylated. Substitution of bromine or iodine at the 13'-position decreased the affinity considerably to the high affinity alphagamma-site of mouse AChR, whereas the affinity for the Torpedo alphagamma-site was slightly increased. Furthermore, binding to the mouse AChR was unaffected by the conformational state, whereas these ligands strongly preferred the desensitized conformation of the Torpedo AChR. Comparison of binding changes upon 13'-halogenation to the changes in amino acid residues at the ACh binding sites of the mouse and Torpedo AChR shows mouse residue Ile-gamma116 as likely to be involved in interacting with the 13'-position of d-tubocurarine. It is predicted that this residue is involved in the conformational equilibrium between the resting and desensitized conformations.

Molecular recognition by natural macrocycles. I. d-tubocurarine as a host molecule for organic anions.

The binding of 8-anilino-1-naphthalenesulfonate and 15 anions of substituted benzoic, aliphatic dicarboxylic, and N-acetyl-alpha-amino acids to a macrocyclic alkaloid d-tubocurarine in aqueous solution has been studied by fluorometry, conductometry, and 1H NMR. The binding constants vary from ca. 50 to 3300 M-1 depending on the guest structure, charge and hydrophobicity. The results of fluorescence and NMR studies show that the host-guest complexation of the anions of aromatic acids involves the formation of a salt bridge between the quaternary nitrogen of the alkaloid and the anionic group of the guest as well as hydrophobic/Van der Waals interactions between the guest and host aromatic moieties. The binding of dianions of aliphatic dicarboxylic acids most probably is purely electrostatic. In general, d-tubocurarine possesses binding ability comparable to that of synthetic cyclophanes. It binds enantiospecifically anions of N-acetyl-alpha-amino acids and discriminates between positional isomers of anions of hydroxy and carboxy substituted benzoic acids.

Nuclear magnetic resonance (NMR) analysis of ligand receptor interactions: the cholinergic system--a model.

Elucidation of the molecular mechanisms that govern ligand-receptor recognition is essential to the rational design of specific pharmacological reagents. Whereas often the receptor and its binding site are the target of investigation, study of the ligand in its free and bound state can also reveal important information regarding this recognition process. Nuclear magnetic resonance (NMR) spectroscopy can be extremely useful for such studies. In this review, we discuss the attributes of NMR in the study of ligand receptor interactions. The cholinergic receptor and its binding to the neurotransmitter, acetylcholine, and cholinergic antagonists serve as a model system, illustrating the power of ligand analysis by NMR. The results discussed prove that the region of residues alpha 180-205 of the nicotinic acetylcholine receptor are an essential component of the cholinergic binding site and that ligand binding involves a positively charged hydrophobic motif.