Marvin K. Schulte

Pharmacological Characterization of the Allosteric Modulator Desformylflustrabromine and Its Interaction with α4β2 Neuronal Nicotinic Acetylcholine Receptor Orthosteric Ligands

Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. nAChRs are involved in modulating nicotinic-based signal transmission in the central nervous system and are implicated in a range of disorders. Desformylflustrabromine (dFBr) is a positive allosteric modulator that potentiates α4β2 nAChRs. It has been reported that dFBr is selective for the α4β2 receptor relative to other common nAChR subtypes (Neurosci Lett 373:144–149, 2005). Coapplication of dFBr with acetylcholine (ACh) produces a bell-shaped dose–response curve with a peak potentiation of more than 265% (Bioorg Med Chem Lett 17:4855–4860, 2007) at dFBr concentrations <10 μM and inhibition of responses at concentrations >10 μM. The potentiation and inhibition components of dFBr-modulated responses were examined by using two-electrode voltage clamp and human α4β2 nAChRs expressed in Xenopus laevis oocytes. Currents to both partial and full agonists were potentiated by dFBr. Responses to low-efficacy agonists were potentiated significantly more than responses to high-efficacy agonists. Antagonist pIC50 values were unaffected by coapplication of dFBr. In addition to its potentiating effects, dFBr was able to induce current spikes when applied to desensitized receptors, suggestive of a shift in equilibrium from the desensitized to open conformation. In contrast to potentiation, inhibition of ACh responses by dFBr depends on membrane potential and is probably the result of open-channel block by dFBr and ACh. Our data indicate distinct mechanisms for the potentiation and inhibition components of dFBr action. dFBr could prove useful for therapeutic enhancement of responses at α4β2-containing synapses.

Identification of critical residues in loop E in the 5-HT3ASR binding site

BackgroundThe serotonin type 3 receptor (5-HT3R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family.ResultsThirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147) to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR.ConclusionOur data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, m CPBG, d-tc and lerisetron. 5-HT and m CPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.

A vertical flow chamber for Xenopus oocyte electrophysiology and automated drug screening

Xenopus laevis oocytes are used extensively in the study of ion channel coupled receptors. Efficient use of oocytes for ion channel characterization requires a system that is inherently stable, reproducible, minimizes drug volumes, and maximizes oocyte longevity. We have constructed a vertical flow oocyte recording chamber to address the aforesaid issues, where the oocyte is placed in a funnel-shaped chamber and perfused from the bottom of the funnel. The vertical rather than horizontal flow of perfusate results in an unusually stable environment for oocyte recording. Two-electrode voltage clamp recordings from a single oocyte are acquired easily and routinely over several hours while maintaining stable baseline currents and reproducible response profiles. Chamber characteristics were tested using a serotonin ligand-gated ion channel receptor (5-HT3R). Data obtained from this system corresponds well with published data. To further test the stability and reliability of this perfusion chamber, we constructed an automated oocyte perfusion system utilizing a commonly available HPLC autosampler. We were able to obtain dose-response curves for various 5-HT3AR ligands using the automated perfusion system with minimal user intervention. Such a system can easily satisfy need for automated oocyte electrophysiology in academic settings, especially small to medium sized laboratories.

Functional group interactions of a 5-HT3R antagonist

BackgroundLerisetron, a competitive serotonin type 3 receptor (5-HT3R) antagonist, contains five functional groups capable of interacting with amino acids in the 5-HT3R binding site. Site directed mutagenesis studies of the 5-HT3AR have revealed several amino acids that are thought to form part of the binding domain of this receptor. The specific functional groups on the ligand that interact with these amino acids are, however, unknown. Using synthetic analogs of lerisetron as molecular probes in combination with site directed mutagenesis, we have identified some of these interactions and have proposed a model of the lerisetron binding site.ResultsTwo analogs of lerisetron were synthesized to probe 5-HT3R functional group interactions with this compound. Analog 1 lacks the N1 benzyl group of lerisetron and analog 2 contains oxygen in place of the distal piperazine nitrogen. Both analogs show significantly decreased binding affinity to wildtype 5-HT3ASRs. Mutations at W89, R91, Y142 and Y152 produced significant decreases in binding compared to wildtype receptors. Binding affinities of analogs 1 and 2 were altered only by mutations at W89, and Y152.ConclusionsBased on the data obtained for lerisetron and analogs 1 and 2, we have proposed a tentative model of the lerisetron binding pocket of the 5-HT3ASR. According to this model, The N-benzyl group interacts in a weak interaction with R91 while the benzimidazole group interacts with W89. Our data support an interaction of the distal amino nitrogen with Y142 and Y152.

Deconstruction of the α4β2 nicotinic acetylcholine receptor positive allosteric modulator desformylflustrabromine

Desformylflustrabromine (dFBr; 1), perhaps the first selective positive allosteric modulator of α4β2 neuronal nicotinic acetylcholine (nACh) receptors, was deconstructed to determine which structural features contribute to its actions on receptors expressed in Xenopus ooycytes using two-electrode voltage clamp techniques. Although the intact structure of 1 was found to be optimal, several deconstructed analogs retained activity. Neither the 6-bromo substituent nor the entire 2-position chain is required for activity. In particular, reduction of the olefinic side chain of 1, as seen with 6, not only resulted in retention of activity/potency but in enhanced selectivity for α4β2 versus α7 nACh receptors. Pharmacophoric features for the allosteric modulation of α4β2 nACh receptors by 1 were identified.

5-HT3R Binding of lerisetron: an interdisciplinary approach to drug–Receptor interactions

The design, synthesis, and use of lerisetron-based molecular probes to investigate the 5-HT(3)R binding site are described. A SAR study, which involved distance and electronic parameter modifications of lerisetrons N-benzyl group, resulted in the discovery of a partial agonist.

Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES.

A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimers and Parkinsons disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5-10mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2- interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors.

ARCTIC PEOPLES AND BEYOND: RESEARCH OPPORTUNITIES IN NEUROSCIENCE AND BEHAVIOUR

Objectives. Arctic and northern peoples are spread across Alaska, Canada, Russia and the Scandinavian countries. Inhabiting a variety of ecosystems, these 4 million residents include Indigenous populations who total about 10% of the population. Although Arctic peoples have very diverse cultural and social systems, they have health issues related to environmental impacts and knowledge/treatment disparities that are common to other minority and Indigenous peoples around the world. Research that explores the neuroscience and behavioural aspects of these health disparities offers challenges and significant opportunities. As the next generation of neuroscientists enter the field, it is imperative that they view their contributions in terms of translational medicine to address health disparities. Study Design. A workshop was designed to bring neuroscientists together to report on the current directions of neuroscience research and how it could impact health disparities in the North. This workshop produced research recommendations for the growth of neuroscience in the North. Methods. On May 31, 2006 the National Institute of Neurological Disorders and Stroke, the Burroughs Wellcome Foundation, the Arctic Division of AAAS and the University of Alaska co-sponsored a workshop entitled “Arctic Peoples and Beyond: Decreasing Health Disparities through Basic and Clinical Research.” Also, the role and goals of the International Union for Circumpolar Health (IUCH) were presented at the meeting. Results. A set of recommendations related to research opportunities in neuroscience and behaviour research and ways to facilitate national and international partnerships were developed. Conclusions. These recommendations should help guide the development of future health research in circumpolar neuroscience and behaviour. They provide ideas about research support and informational exchange that will address health challenges.