Project

Discipline

Organic Chemistry; Neuroscience; Ligand-gated Ion Channels; Serotonin 5-HT3 Receptor; Fluorescence Spectroscopy; Electrophysiology; Synthetic Chemistry; Binding Assay; Biophysical Tools; Post-photoaffinity Modifications; Antagonists

Lead
Lay summary
Our brain and spinal cord consists of a vast number of nerve cells which are connected with each other in an incredibly complex fashion. Such connections at nerve endings are called synapses and there is a physical gap between a nerve cell and the next one which the nerve signal has to 'leap over'. In reality, the electrical nerve signal is 'translated' into a chemical one, which in turn produces a new electrical signal in the neighbouring nerve cell that can now travel down the nerve axon to the next synapse and so on. This chemical signal is carried by small organic molecules, neurotransmitters, which facilitate the rapid communication between the nerve cells. More specifically, the arrival of a nerve signal at the synapse will trigger the release of neurotransmitters into the synaptic cleft, which will diffuse across to the neighbouring nerve cell and bind to protein complexes on its surface. These so-called ligand-gated ion channels (LGICs) are fascinating biological macro-machines and binding of neurotransmitters to a certain site in LGICs results in a conformational transition from a non-conducting "closed" state to a conducting "open" state. High ion flux across the biological membrane in the open state triggers further events in the post-synaptic nerve cell and ultimately leads to the generation of a new nerve signal and hence transmission of the nerve impulse.LGICs are also found at the surface of cells other than neurons where they play an important role in controlling events inside the cell in response to messengers (small molecules) from outside the cell. The crucial physiological importance of LGICs becomes apparent when their function is impaired. In fact, numerous mutations in genes which encode LGICs are known to cause neurological and other diseases. Moreover, LGICs are very important drug targets from a therapy point of view. Small molecules (i.e. drugs) which either block or activate channel function (e.g. valium) can be used to control psychiatric disorders such as anxiety, drug-dependence, depression, schizophrenia and cognitive dysfunction.It is far from understood how small organic molecules (e.g. neurotransmitters, drugs) are able to activate or block these huge multi-subunit proteins. Although there have been considerable achievements in the past ten years to solve the exact three-dimensional structure of these large protein complexes the available structures are not accurate or complete enough to allow us to understand the function of these proteins and to design better drugs. LGICs are sitting in the biological membrane and such proteins are notoriously difficult to yield high-resolution structures. Thus, due to the lack of available functional and structural information a drug discovery programme is pretty much hit-and-miss and huge numbers of drug candidates need to be screened efficiently for their potency against these pharmacologically important targets. Such screening efforts are time consuming and tedious. We believe that the lack of understanding of LGIC function significantly slows down the process of finding better therapies and drugs for neurological disorders.The aim of our research is to design and synthesize molecular tools (i.e. small organic compounds) which should enable us to study the structure and function of LGICs but also allow their site-specific chemical modification. These molecular tools should complement existing biological methods and we believe will provide a new angle to study LGICs. The chemically modified LGICs will be used to (a) investigate their structure and function and (b) to develop rapid binding assays for small molecules targeting LGICs.Small organic fluorophores covalently attached to biological macromolecules provide an efficient and very sensitive way to probe molecular processes in their environment. A fluorophore is a compound that emits light of a certain wavelength when irradiated by a light source and they are very sensitive to changes in their environment, i.e. the fluorescence will change to a different wavelength.Our research programme envisages the (i) design and synthesis of compounds which are based on known high-affinity LGIC binders and that will react with LGICs using a light-induced reaction in order to attach a fluorophore near their neurotransmitter binding site. The resulting fluorescent LGICs will then be used to study the binding of known antagonists (channel blockers) and agonists (channel activators) by fluorescence spectroscopy. Due to the location of the fluorophore, the binding events will alter the fluorescence and we expect that such studies will reveal crucial structural information about the binding site and help us to understand how the binding event is structurally linked to the channel opening event. (ii) We will also explore how such fluorescent LGICs might be used in binding assays amenable for high-throughput screening and are planning to develop a fluorescence-based binding assay which would rapidly reveal if a compound of interest has the potential to be a blocker of the LGIC. In addition, we will also synthesize fluorescent or fluorophore-labelled LGIC-binders and use such tools to monitor the binding or unbinding to the LGIC. High-affinity fluorescent binders would also be very interesting for imaging applications of LGIC in living cells. (iii) Another focus of the proposed research is the synthesis of a structurally divers library of compounds which fit the common molecular signature of LGIC blockers or activators but which have more natural product-like structures. Such libraries would be screened with the newly developed binding assays and further analyzed for their influence on LGIC function using electrophysiology experiments. Such studies will allow us to understand how a molecule must be shaped in order to either activate or block the receptor.The proposed novel methodologies will be developed on the serotonin 5-HT3 receptor which is one of the simplest LGIC, and then expanded to other more complex ion channels and cell surface receptors.

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Last update: 21.02.2013

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Ligand-gated ion channels are protein complexes located at nerve terminals, also called synapses. They are responsible for the rapid transmission of nerve impulses from one nerve cell to the next one. The crucial physiological importance of ligand-gated ion channels becomes apparent when their function is impaired. In fact, numerous mutations in ligand-gated ion channel genes are known to cause neurological diseases. In addition, ligand-gated ion channels are the site of action of many therapeutic drugs.The ligand-gated ion channels work when small organic molecules (neurotransmitters) are released into the synaptic cleft from the pre-synaptic cell and bind to the ligand-gated ion channel resulting in a conformational transition from a non-conducting "closed" state to a conducting "open" state. High ion flux across the biological membrane in the open channel state triggers further events in the post-synaptic cell and finally leads to the generation of a new action potential and transmission of the nerve impulse.There is evidence that small molecules which block or activate channel function specifically may be useful for treatment of certain psychiatric disorders such as anxiety, drug-dependence, schizophrenia and cognitive dysfunction. Although there have been considerable achievements in the past ten years to solve the exact three-dimensional structure of these large proteins the structures are not accurate enough to allow rational structure-based drug design. As a result many different libraries of compounds are being synthesised and have to be tested against the ion channel receptors. Current screening methods are time consuming and some even rely on using radioactive compounds.The aim of this proposal is to develop novel synthetic biophysical tools which will aid our understanding of the function of these ion channel receptors but also allow their selective chemical modification. These proposed chemical modifications will yield semi-synthetic ion channels with added biophysical properties (e.g. fluorescence). Such fluorescent ion channel receptors would be very powerful tools in sophisticated fluorescence spectroscopy studies determining how small molecules interact with these huge multi-subunit proteins and how they activate them. Furthermore, the added biophysical properties of the modified ion channels will be exploited in a binding assay which is based on detection of fluorescence and hence could be used for screening of drugs targeting ion channels. Another goal of the proposed research is the generation of small libraries of compounds which fit the common molecular signature of ion channel activators and inhibitors but which have more natural product-like structures. Such libraries would be screened with the newly developed assay and positive hits will be further analysed for their influence on ion channel function using electrophysiology experiments. The proposed novel methodologies shall be developed on the serotonin 5-HT3 ion channel receptor which is one of the simplest ligand-gated ion channels. The concept would then be expanded to other more complex ion channels and cell-surface receptors (e.g. G-protein coupled receptors). Due to its highly interdisciplinary nature the proposed work is likely to attract interest from researchers in other fields such as biology, pharmacology and spectroscopy. The chance of success is high with the prospect of yielding a new and highly rewarding angle to look at ion channel function. Whereas in the past ion channels have purely been studied using physiological and biochemical methods the proposed research involves intelligent molecular design to develop a tool for studying the binding of small organic molecules to ion channels which also gives an element of structural and functional information. This will lead to a better understanding of ion channel function and reveal details of their structure which will ultimately lead to better and more selective drugs.