The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation-interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation-binding abilities and (ii) EC 50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues-tryptophan-149 of the ␣ subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of ␣ tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position ␣149 produces a constitutively active receptor.

A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.

Apelin receptor (APJR) is a key regulator of human cardiovascular function and is activated by two different endogenous peptide ligands, apelin and Elabela, each with different isoforms diversified by length and amino acid sequence. Here we report the 2.6-Å resolution crystal structure of human APJR in complex with a designed 17-amino-acid apelin mimetic peptide agonist. The structure reveals that the peptide agonist adopts a lactam constrained curved two-site ligand binding mode. Combined with mutation analysis and molecular dynamics simulations with apelin-13 binding to the wild-type APJR, this structure provides a mechanistic understanding of apelin recognition and binding specificity. Comparison of this structure with that of other peptide receptors suggests that endogenous peptide ligands with a high degree of conformational flexibility may bind and modulate the receptors via a similar two-site binding mechanism.

The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor. Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the alpha subunit. Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein. At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding.

Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing (1)H-(15)N heteronuclear single quantum coherence (HSQC) as well as one-dimensional (1)H and (19)F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of (19)F detection in FBS has been only recently introduced (Vulpetti et al. J. Am. Chem. Soc.2009, 131 (36), 12949-12959) with the aim of targeting "fluorophilic" sites in proteins. Here, we demonstrate a more general use of (19)F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure-activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability.

No abstract

The first total synthesis of the potent antitumor agent fostriecin (CI-920) is described, confirming the relative and absolute stereochemistry assignments. Fostriecin is a unique phosphate monoester which exhibits weak topoisomerase II inhibition (IC(50) = 40 microM) and more potent and selective protein phosphatase 2A and 4 (PP2A and PP4) inhibition (IC(50) = 40-3 nM and 1.5 nM), resulting in mitotic entry checkpoint inhibition. Phase I clinical trials with fostriecin, which were the first to explore the potential of this novel mechanism of action, were halted even before therapeutic concentrations were reached or dose-limiting toxicity established due to problems of drug stability observed during storage of naturally derived material. The synthesis of fostriecin detailed herein is the first stage of efforts that may serve to address these limitations to the clinical examination of this or related promising new antitumor agents.

A nonsense suppression method was employed to incorporate a total of four natural and six unnatural residues at the 9' position of the M2 region in the beta, gamma, and delta subunits of muscle nicotinic receptors. In 33 pairwise comparisons of functional properties as influenced by structural features including side chain length, branching, and substitution of oxygen for methylene carbons, it is concluded that increased polarity in the side chains at the 9' position consistently increases the sensitivity to acetylcholine. In addition, the stereochemistry of the side chain can have marked influences on the EC50, primarily because of changes in the single-channel open time. For the case of isoleucine versus allo-isoleucine in the delta subunit, these changes are themselves modified by mutations at the 9' position in other subunits. The data suggest an especially strong interaction between the beta and delta subunits in the pore region, leading in turn to a suggested arrangement of subunits within the pentamer.