Functional Characterisation of insect nicotinic Acetylcholine Receptors

By 2050 it is estimated that the global population will have increased by a third. The calorific requirements to sustain this population will need to increase by 50% and, with a growing preference for meat consumption, additional provision of feedstocks will also be needed. It has been estimated that this will require a 70% increase in crop yields by 2050. Thus increasing demands will be placed on available agricultural land to deliver efficient, reliable and sustainable food production. Insecticides are important tools for ensuring efficient crop yields. New insecticides need to be created on a regular basis to overcome resistance in pest populations as well as providing more benign environmental impacts and human safety profiles. In particular, new insecticides need to exhibit low toxicity to non-target species, particularly the major pollinators such as bees, while retaining efficacy against both the 'chewing' and 'sucking' pests that can devastate many major crops. The target of the most commercially important insecticides is the neuronal acetylcholine signalling pathway and of this class of insecticides, the safest and most effective are molecules (neonicotinoids and spinosyns) that act at the nicotinic acetylcholine receptor (nAChR).

The nAChR is in fact composed of five protein subunits that form a complex. In the well established model insect, the fruit fly Drosophila melanogaster, there are ten different genes encoding nAChR subunit proteins, and these can combine to form many different classes of nAChRs, each with the potential to interact differently with insecticidal compounds. To date, very little is known about how these subunits combine, what other proteins act as accessory molecules interacting with receptor subunits, or what impact the 'parts list' of each complex has on its pharmacological properties.

The aim of this project is to characterise functionally distinct classes of nAChRs in Drosophila melanogaster, a genetically and biochemically tractable insect model system that will generate insights broadly applicable to other insect species. Knowledge of the functional classes of nAChRs in Drosophila will pave the way for more targeted future research in the pest insects that cause havoc to major crops and for which there are currently very few genetics tools. In this application we will apply cutting edge spatial proteomics methods created by Kathryn Lilley with insect gene editing and advanced genetics techniques available from Steve Russell to advance the insect receptor biochemical and pharmacological approaches established at Syngenta, one of the world's leading agrochemical companies. Together, the proposed research programme will lead to a thorough characterisation of different native nAChR classes and substantially advance our understanding of a class of insecticide targets that are crucial for protecting global agriculture. The ability to design effective new insecticides that are safe and have low environmental impact will be essential to the continuing drive to increase agricultural yields and better feed the growing global population.

We will characterise functionally distinct classes of nicotinic Acetyl Choline receptor (nAChR), the target of most commercially important insecticides. nAChRs are composed of 5 subunits, built from combinations of 10 different subunit genes, and a set of poorly characterised interacting proteins. There are several functional classes of nAChRs that differ in their pharmacological properties but studies aimed at re-constructing these classes have been largely unsuccessful due to inefficient expression of insect subunits and little information on the identity and roles of accessory proteins. The design of novel insecticides with improved potency and selectivity requires a better understanding of the composition of native nAChR classes and their differing pharmacology. We will apply CRISPR based editing to make Drosophila lines tagging individual nAChR subunits with immunological epitopes and fluorescent proteins. Fluorescent tags allow analysis of receptor composition and distribution in vivo in the adult brain and via methods that generate membrane nano-discs, we will complement affinity purification approaches to determine interacting protein partners of specific nAChRs, improving our understanding of how nAChRs operate and informing future work to reconstruct functional receptors in vivo. We will use CRISPR to create null mutants in each receptor subunit, determining their impact on whole animal phenotypes and insecticide responses. The work will be underpinned by biochemical and pharmacological characterisation using methods established at Syngenta. Together, the work will lead to a thorough characterisation of different native nAChR classes and substantially advance our understanding of a class of insecticide targets that are crucial for protecting global agriculture. We will provide a platform technology to drive novel insecticide development at Syngenta and more widely in the agrochemical industry.

The most immediate and obvious impact of the work proposed in this application will be on the insecticide development programme at Syngenta. Through a better understanding or nAChR subunit composition, distribution and ligand binding properties, the ability to engineer in vitro and in vivo systems for assaying insecticide binding will be much improved. The development of next generation insecticide compounds, with improved environmental impact and safety profiles will benefit global agriculture and contribute to the food security agenda.

Specifically the work will:
1. Enable more effective management of insecticide resistance. Understanding which are the critical proteins mediating the effects of current and future insecticides will inform resistance management practices and allow more effective monitoring for resistance in pest populations. Syngenta has active resistance monitoring programmes and will integrate this knowledge as it becomes available. Syngenta is an active member of Crop Life's Insecticide Resistance Action Committee, a body through which most insecticide companies cooperate to manage resistance.
2. Facilitate the design of novel insecticides. Syngenta already uses homology based modelling as an aid to design of novel chemical leads. This approach will benefit as the protein composition of key targets is established and will be extended to the prediction of species selectivity. Our research will also inform efforts to obtain meaningful crystal structures of ligand binding domains.
3. Enable discovery tool development. Syngenta uses biochemical tools to discover novel chemical leads. We expect the output of this work to facilitate successful heterologous expression of functional insect nACh receptors, allowing early understanding of potential selectivity.

The development of null mutants for all Drosophila nAChR subunit genes as well as endogenously tagged copies of the subunits in a uniform genetic background will impact on our basic understanding of receptor biology. The provision of Drosophila lines through stock centres will facilitate research into receptor pharmacology, the underlying biology of insecticide specificity and basic neurobiology.

The methods we develop for the analysis of nAChR complexes and associated proteins will impact on fundamental aspects of membrane protein proteomics as well as providing rich datasets for datamining.

We will deliver these impacts through the scientific collaboration with Syngenta, through the outreach and communication deliverables described in the pathways to impact as well as traditional routes of publication in peer reviewed journals.