Exploration of the molecular architecture of the orthosteric binding site in the α4ß2 nicotinic acetylcholine receptor with analogs of 3-(dimethylamino)butyl dimethylcarbamate (DMABC) and 1-(pyridin-3-yl)-1,4-diazepane.

X-ray crystal structures of acetylcholine binding proteins (AChBPs) have revealed two different possible extensions to the classical ligand binding pocket known to accommodate various nicotinic agonists. One of the pockets is limited in size while the other is of considerable dimensions and protrudes along the interfacial cleft between subunits. To probe these putative extensions in functional nicotinic acetylcholine receptors (nAChRs), elongated analogs of 3-(dimethylamino)butyl dimethylcarbamate (DMABC) and 1-(pyridine-3-yl)-1,4-diazepane were prepared and characterized pharmacologically at neuronal heteromeric nAChRs. Although the new analogs, relative to parent compounds, displayed lower binding affinities, functional characterization of selected compounds revealed that they had retained partial α4ß2 nAChR agonist activity. The structure-activity relationship data did not indicate an upper limit to the size of substituents as would have been expected if the ligand was bound in the smaller pocket. The data were better in agreement with a binding mode in which substituents protrude along the interfacial cleft of the receptor. This was further supported by docking into a homology model of the α4-ß2 nAChR interface and by surface plasmon resonance biosensor analysis of binding of the compounds to acetylcholine-binding proteins, where they exhibit preference for Lymnaea stagnalis ACh binding protein (Ls-AChBP) over the Aplysia california ACh binding protein (Ac-AChBP). These results suggest new opportunities for expanding chemical space in the development of partial agonist and may be of interest in relation to development of novel smoking cessation aids.

Structure-activity relationship studies of argiotoxins: selective and potent inhibitors of ionotropic glutamate receptors.

Argiotoxin-636 (ArgTX-636), a natural product from the spider Argiope lobata, is a potent but nonselective open-channel blocker of ionotropic glutamate (iGlu) receptors. Here, three series of analogues were designed to exploit selectivity among iGlu receptors, taking advantage of a recently developed solid-phase synthetic methodology for the synthesis of ArgTX-636 and analogues. Initially, the importance of secondary amino groups in the polyamine chain was studied by the synthesis of systematically modified ArgTX-636 analogues, which were evaluated for pharmacological activity at NMDA and AMPA receptors. This led to the identification of two compounds with preference for NMDA and AMPA receptors, respectively. These were further elaborated by systematically changing the aromatic headgroup and linker amino acid leading to compounds with increased potency and selectivity for NMDA and AMPA receptors, respectively. Thus, the first structure-activity relationship study of ArgTX-636 has been carried out and has provided lead compounds for probing the ion channel region of iGlu receptors.

General synthesis of ß-alanine-containing spider polyamine toxins and discovery of nephila polyamine toxins 1 and 8 as highly potent inhibitors of ionotropic glutamate receptors.

Certain spiders contain large pools of polyamine toxins, which are putative pharmacological tools awaiting further discovery. Here we present a general synthesis strategy for this class of toxins and prepare five structurally varied polyamine toxins. Electrophysiological testing at three ionotropic glutamate receptor subtypes reveals that two of these, Nephila polyamine toxins 1 (NPTX-1) and 8 (NPTX-8), comprise intriguing pharmacological activities by having subnanomolar IC(50) values at kainate receptors.

Solid-phase synthesis and biological evaluation of Joro spider toxin-4 from Nephila clavata.

Polyamine toxins from orb weaver spiders are attractive pharmacological tools particularly for studies of ionotropic glutamate (iGlu) receptors in the brain. These polyamine toxins are biosynthesized in a combinatorial manner, providing a plethora of related, but structurally complex toxins to be exploited in biological studies. Here, we have used solid-phase synthetic methodology for the efficient synthesis of Joro spider toxin-4 (JSTX-4) (1) from Nephila clavata, providing sufficient amounts of the toxin for biological evaluation at iGlu receptor subtypes using electrophysiology. Biological evaluation revealed that JSTX-4 inhibits iGlu receptors only in high µM concentrations, thereby being substantially less potent than structurally related polyamine toxins.