Minisymposium on Cys-loop receptors: Nicotinic acetylcholine and GABA-A receptors
Friday, September 26, 2008 at 09:15 - 17:30 in the Benzon Auditorium
The Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen
Program
Friday, 26 September 2008
| Programme - morning | |
| 09:15-09:25 | Welcome Thomas Balle, Department of Medicinal Chemistry, PHARMA, University of Copenhagen, Denmark<br/> |
| Chairman: | Thomas Balle, University of Copenhagen |
| 09:25 - 10:00 | The Cys-loop receptors: Key effectors of synaptic neurotransmision Daniel B. Timmermann, NeuroSearch A/S, Ballerup, Denmark |
| 10:00 - 10:10 | Discussions |
| 10:10 - 10:55 | Structure and gating mechanism of the ACh receptor Nigel Unwin, MRC Laboratory of Molecular Biology, Cambridge, UK |
| 10:55 - 11.10 | Discussions |
| 11:10 - 11:30 | Coffee |
| Chairman: | Kasper Harpsøe, University of Copenhagen |
| 11:30 - 12:15 | New structural insights into ligand interactions at the AChP binding pocket Yves Bourne, Glycobiology & Structural Neurobiology, Universités Aix-Marseille I & II |
| 12:15 - 12:30 | Discussion |
| 12:30 - 13:30 | Lunch |
| Programme - afternoon | |
| Chairman: | Jette Sandholm Kastrup, University of Copenhagen |
| 13:30 - 14:15 | Structure of the extracellular domain of nAChR alpha1 Cosma D. Dellisanti, Molecular and Computational Biology, University of Southern California, Los Angeles, CA, USA |
| 14:15 - 14:30 | Discussions |
| 14:30 - 15:00 | Insight into partial agonism of a4b2 receptors - from the ligands perspective Thomas Balle, Department of Medicinal Chemistry, PHARMA, University of Copenhagen, Denmark |
| 15:00-15:10 | Discussions |
| 15:10-15:30 | Coffee |
| Chairman: | Anders A. Jensen, University of Copenhagen |
| 15:30-16:15 | From structure to function: Diverse binding and gating modes for Cys-loop receptors Dennis A. Dougherty, Divisionof Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California |
| 16:15-16:30 | Discussions |
| 16:30-16:45 | Distinct b-subunit domains determine agonist efficacy at heteromeric nAChRs Helle Hald, Department of Medicinal Chemistry, PHARMA, University of Copenhagen, Denmark and Neurosearch A/S, Ballerup, Denmark |
| 16:45-16:50 | Discussions |
| 16:50-17:20 | GABAA receptors - discovery of novel drug candidates Philip K. Ahring, NeuroSearch A/S, Ballerup, Denmark |
| 17:20-17:30 | Discussions |
| 17:30-17:35 | Concluding remarks Philip K. Ahring, Neurosearch A/S, Ballerup, Denmark |
| 17:35-18:20 | Beer and snacks |
| 19:00- | Dinner 'in town' sponsored by Neurosearch A/S |
The participation in the symposium and dinner is free of charge and is open for attendance by all interested parties.
Registration is required: Please register by sending an e-mail to tb(at)farma.ku.dk and indicate whether you will participate 'in the symposium' only or 'in both the symposium and dinner' before Friday, September 19.
Available seats at the dinner are limited to 40 persons. These will be allocated on a 'first come, first served' basis.
The symposium is organized on behalf of the Drug Research Academy, PHARMA by Thomas Balle and Jette Sandholm Kastrup, Department of Medicinal Chemistry, The Faculty of Pharmaceutical Sciences, University of Copenhagen, and Philip K. Ahring, NeuroSearch A/S. Correspondence e-mail: Thomas Balle, tb(at)farma.ku.dk.
You can find both programme and abstracts as a pdf-files here.
Abstracts - morning session
The Cys-loop receptors: Key effectors of synaptic neurotransmission
Daniel Timmermann
Neurosearch A/S
The Cys-loop family of ligand-gated ion channels include cation-permeant receptor channels gated by acetylcholine (nicotinic receptors, nAChRs) and serotonin (5-HT3R) as well as anion-permeable receptors for g-aminobutyric acid (GABAARs) and glycine (GlyRs), which all play significant roles in mediating fast excitatory, respectively inhibitory, synaptic transmission. This receptor family therefore also constitutes a major drug target class where intensive efforts are being invested in designing ligands which selectively activate or inhibit specific Cys-loop receptor subtypes. During the past five years, a number of seminal discoveries have revolutionized our understanding of the structure and function of Cys-loop receptors (i.e. high-resolution crystal and electron microscopy structures of the neuromuscular nAChR and ACh binding proteins), findings that will undoubtedly aid rational design of selective drug molecules.
This presentation will provide a general overview of the physiology, basic function and molecular biology of the Cys-loop family members.
Structure and Gating Mechanism of the ACh Receptor
Nigel Unwin
MRC Laboratory of Molecular Biology, Cambridge
Postsynaptic membranes from the (muscle-derived) electric organ of the Torpedo ray convert readily into tubular crystals, having ACh receptors and intervening lipid molecules organised like they are in vivo. These crystals are being analysed by electron microscopy to determine the structure of the ACh receptor in the closed- and open-channel forms. So far only the closed channel has been revealed in near-atomic detail. To analyse the receptor in the open-channel form, images are obtained from crystals on e.m. grids which have been sprayed with ACh and then freeze-plunged, within 10 ms, into liquid nitrogen-cooled ethane. This technique is allowing the open-channel form to be described with increasing accuracy as more images are evaluated. The activation by ACh triggers an extended conformational change in the ligand-binding domain, which is coupled to movements of the M2 helices, leading to a widening of the pore at the gate in the middle of the membrane.
New structural insights into ligand interactions at the AChBP binding pocket
Yves Bourne
Universités Aix-Marseille I & II
The soluble acetylcholine-binding protein (AChBP) is a structural and functional surrogate of the extracellular ligand-binding domain (LBD) of homopentameric nicotinic acetylcholine receptors. Nicotinic agonists bind at each subunit interface of AChBP within a “core agonist” nest of conserved aromatic side chains, contributed by loops C and F on the principal and complementary sides of the interface, respectively. We have solved a set of four crystal structures of AChBP bound with selective and non-selective full and partial agonists. The structures reveal distinctive modes of binding for the full versus partial agonists within the binding pocket along with distinctive contributions of loops C and F to either trap the bound ligand or confer it residual mobility.
These structures represent new templates for the design of novel partial nicotinic agonists with exquisite subtype-selectivity.
Abstracts – Afternoon session
Structure of the extracellular domain of nAChR alpha1
Cosma Dellisanti
University of Southern California
We determined the crystal structure of the extracellular domain (ECD) of the mouse nAChR a1 subunit bound to antagonist a-bungarotoxin at 1.94Å resolution. This structure is the first view at atomic resolution of a nAChR subunit ECD, and reveals the details of receptor-specific features including the signature Cys-loop and the N-linked carbohydrate chain. Intriguingly, the structure shows an ordered water molecule and two hydrophilic residues deep in the b-sandwich core of the a1 subunit, in close vicinity to the Cys-loop disulphide bridge and the asparagine residue (Asn-141) where the glycosylation moiety is harbored. The two hydrophilic core residues are highly conserved in nAChRs, but correspond to hydrophobic residues in the non-channel homolog AChBPs. Site-directed mutagenesis and electrophysiology analyses were carried out to assess the functional role of the glycosylation and the hydrophilic core residues. Our structural and functional studies show essential features of the nAChR and provide new insights into the gating mechanism.
Insight into partial agonism of a4b2 receptors - from the ligands perspective
Thomas Balle
University of Copenhagen
A ligand based approach to understanding receptor activation procesess “Complementary 3D-QSAR modelling of binding affinity and functional potency” was succesfully applied to a series of nicotinic a4b2 agonists. The method is proposed as a tool to pinpoint the molecular features of the ligands responsible for high affinity binding vs those driving agonist behaviour and thus reveals the difference between a “good binder” and a “good agonist”. When interpreted in terms of interactions with the receptor, the results suggest two different areas in the receptor where ligand-receptor interactions can result in partial agonism (and possibly also antagonism).
From Structure to Function: Diverse Binding and Gating Modes for Cys-Loop Receptors
Dennis A. Dougherty
California Institute of Technology
The valuable structural insights from the Torpedo receptor, the ACh binding proteins, prokaryotic channels, and single receptor subunits have vastly expanded our understanding of Cys-loop receptors. In recent years, a major focus of our work has been to evaluate the degree to which these images present universal themes across the receptor family. Using the powerful tool of unnatural amino acid mutagenesis coupled with whole cell and single channel electrophysiology, we have probed the binding sites and gating mechanisms of many Cys-loop receptors. We find surprising diversity in the modes of action across the family, suggesting that nature has preserved the structural motifs of the family, but that mechanistically there is considerable variety.
Distinct b-subunit domains determine agonist efficacy at heteromeric nAChRs
Helle Hald
University of Copenhagen and Neurosearch A/S
The efficacies of two nicotinic agonists, (-)-cytisine and the novel compound NS3861 were investigated on heteromeric nAChRs, composed of pairwise combinations of a3, a4, b2 and b4 subunits or chimeric subunit constructs. Although both agonists preferentially activated wt a3b4 but not a4b2 receptors, cytisine was found to activate only receptors containing the b4-subunit, whereas NS3861 exclusively activated a3-containing nAChRs. However, the efficacy of NS3861 was much greater in b2- relative to b4-containing receptors. Moreover, studies on chimeric subunit combinations revealed that the efficacy of NS3861 was determined exclusively by the extracellular portions of a- and b-subunits, whereas cytisine efficacy required both extra- and intracellular portions of the b4-subunit. These results imply a decisive role for the b-subunit in determining agonist efficacy and suggest the existence of agonist-specific interactions with efficacy determinants at the agonist binding site of heteromeric nAChRs.
GABAA Receptors - Discovery of novel drug candidates
Philip K. Ahring
NeuroSearch A/S
GABAA receptors are the major inhibitory receptor class in the mammalian brain and a number of marketed drugs act through modulation of these receptors. Commonly known are the benzodiazepines for anxiety, Z-hypnotics and steroids/barbiturates for anaesthesia. Due to unwanted side effects (i.e. sedation, amnesia, tolerance), the clinical use of benzodiazepines have declined in recent years. Thus, designing new molecules, with the potential for becoming next generation drugs substituting for benzodiazepines, has been a goal for pharmaceutical research for some years now. At NeuroSearch we have a major drug discovery project focusing on synthesis of novel subtype selective GABAA receptor molecules that are now filtering into clinical research. In the presentation you will see an example of such a novel molecule (NS11394) and get a taste of the data seen with this compound including the more “traditional” anxiety related tests as well as more novel tests related to pain behavior. Finally, the presentation will touch upon the challenges that are met in deciphering structure activity relationships for novel compounds and why basic research in the Cys-loop receptor field is highly important for the pharmaceutical industry.
