Development of biotin-tagged affinity ligands and fluorophore-conjugated probes for the study of native kainate receptors

The social burden and cost of brain disorders is enormous, and no serious progress can be expected in the treatment or the prevention of these disorders without better understanding of the way neuronal activity is regulated in the central nervous system. Neurones communicate via proteins known as neurotransmitter receptors. Nearly all of the excitatory neurotransmitter receptors in the brain are activated by the amino acid glutamate. Glutamate is released from neurones at synapses in a highly regulated and activity dependent manner. There are several different classes of receptors which respond to glutamate. The least well characterised of these are the kainate receptors (KARs).

KARs regulate the activity of neuronal networks, which is essential for normal brain function. They are also involved in neuronal development and neurological conditions such as epilepsy, chronic pain, migraine, schizophrenia and neurodegeneration and they represent a potential target for therapeutic drug design. Drug discovery efforts aimed at identifying new and potentially therapeutic compounds rely on detailed knowledge of the molecular composition, structure and pharmacological properties of the neurotransmitter receptors to which new compounds are targeted. Over the last few years, it has become clear that receptor proteins operate within a complex web of interactions with other proteins. Many interactions serve to modulate the transport, localisation and mobility of receptor proteins, while others affect their acute functioning and pharmacological properties. Many proteins have been found to interact with glutamate receptors. This gives rise to complex protein-protein interactions that can have a profound effect on synaptic function. However, the general principles that govern the modulation of the function and pharmacological properties of native KARs by their interaction partners in different types of neurones in various brain regions are not clear. It is important to gain more information about KAR associated proteins, because increased understanding of the mechanisms that regulate KARs will allow their involvement in normal brain function and neurological disorders to be better defined.

In this research project we aim to define the molecular organisation, distribution, protein interactions, pharmacological properties and regulation of KARs in the central nervous system by using newly developed compounds into affinity probes. Based on molecular modelling of KAR interactions with various drugs we will design, synthesise and characterise new pharmacological reagents, which specifically bind to KARs with high affinity and can be used for the selective identification, isolation and analysis of the native receptors and protein interaction partners directly in brain samples. These new pharmacological tools will be used to isolate KARs and associated proteins. The systematic analysis of these isolated protein complexes will provide basic information about the molecular composition, interactions and regulation of KARs in different functionally important regions of the brain and subcellular compartments of neurones. It is very likely that some of the interaction partners (called auxiliary subunits) significantly influence the pharmacological properties of KARs and this may have important implications for drug development. Therefore, we will establish the effects of auxiliary subunits on the pharmacological properties of KARs.

The results of the proposed studies may help the future development of therapeutic agents for the treatment of epilepsy, chronic pain, migraine, schizophrenia and neurodegenerative diseases. Many of these disorders currently have no effective treatments and are extremely deleterious to the health and wealth of the nation.

Kainate receptors (KARs) are ligand gated ion channels that are activated by the major excitatory neurotransmitter glutamate and they are key players in the modulation of neuronal-network activity throughout the central nervous system (CNS). Furthermore, KARs play an important role in neuronal differentiation, synaptic plasticity, epileptogenesis, chronic pain, migraine, schizophrenia and neurodegeneration. Recently developed drugs and transgenic mice in combination with electrophysiological studies highlighted the functional significance of KARs in several brain regions, but studies often produced conflicting results. Discrepancies between the functional and pharmacological characteristics of recombinant versus native receptors suggest the existence of important modulatory components in neurones that can influence essential properties of KARs. While several studies identified individual interaction partners for KARs, the molecular composition of the native KARs in neurones are poorly understood. Some of these interaction partners (e.g. auxiliary subunits) are likely to have major effects on the pharmacological properties of KARs that are likely to explain conflicting functional observations. We will use molecular modelling to develop new high affinity ligands for the isolation and localisation of native KARs and their interaction partners. We will apply these affinity ligands in combination with proteomics and immunochemical analysis of isolated KAR complexes to establish regional, cell-type and subcellular compartment specific differences in KAR-interacting proteins. We will also study the effects of different auxiliary subunits on the pharmacological properties of KARs. Better understanding of the KAR associated functional protein network and molecular interactions will ultimately provide better understanding of these receptors as pharmacological targets which could be considered for future treatment strategies.

Understanding how the brain works by regulating neuronal activity is a topic of great public and academic interest. The somewhat elusive kainate receptors (KARs) play a key role in the regulation of neuronal activity. The recent discovery of the structure of KAR ligand binding sites created exciting new opportunities for rational drug design and the development of new, more potent and selective pharmacological reagents for the analysis of endogenous KARs and their complex interactions in their native environment in neurones. This project is thus timely and should have major impact at both academic and social levels. To deliver that impact, it is important that the research is disseminated as effectively as possible. The target audiences are academics, pharmaceutical industry, health professionals, schools and the wider public.

ACADEMICS: The various ways neuroscientists, neuropharmacologists, postdoctoral scientists employed on the project and the very wide range of undergraduate and postgraduate student benefit from this project are described under 'Academic beneficiaries'.

PHARMACEUTICAL INDUSTRY: Mutually beneficial collaborations with industry is an integral part of the way that we achieve our aims. DEJ has worked as a consultant for Eli Lilly's project to develop KAR antagonists for the treatment of a range of neurological disorders and has been a member of Lilly's Centre for Cognitive Neuroscience since its inception in 2008. DEJ has close links with biotechnology companies (Ascent Scientific and Tocris Biosciences). These established collaborations will be used to commercialise and distribute new research tools developed during this project (high affinity GluK1 selective antagonists, biotinylated affinity ligands and fluorophore-conjugated probes) to the wider research community. Furthermore, there is significant interest in KARs by industry as potential drug targets for epilepsy, schizophrenia, neurodegenerative disorders, migraine and chronic pain therefore our results are likely to have an impact in this area.

HEALTH PROFESSIONALS: Because KARs have been linked to a range of neurological disorders where effective treatment is currently unavailable, they offer inviting possibilities for therapeutic exploitation. However, a much better understanding of KARs will be essential for the development of new therapies. Therefore, the medical community will also benefit from any new developments in the field.

SCHOOLS: The future of science depends on enthusiastic young scientists. Both applicants are involved in University open days, making explanation of science accessible to prospective students and parents.

THE WIDER PUBLIC: Both applicants are committed to engage public interest and to shape public perception about the benefits of scientific discovery. We will take every opportunity to directly engage the public and schools through initiatives coordinated by the Bristol University Centre for Public Engagement, Bristol Neuroscience and by local attractions (e.g. the children's science activity centre '@Bristol'). Our published findings will be promoted to the public through Bristol Neuroscience and the University Press Office.