Functional analysis of insect neuropeptide G protein-coupled receptors.

To stay alive, organisms must continuously tune their various functions according to their internal and external environment. In multicellular organisms, the complex task of regulating the different tissues (lung, heart, liver, etc) is handled by chemical messengers (acetylcholine, insulin, etc) that bind to particular receptor proteins on, or in, the appropriate targets cells for such messages. In humans, the design of drugs that affect these receptors (GPCRs) - and thus alter the tuning of our bodies - is a huge area for the pharmaceutical industry, and major research programmes have given us some of the world's most widely used drugs for a range of disease (like high blood pressure or depression).
Such GPCRs are no less important in insects, and as many of the world's most dangerous diseases (like malaria, dengue and Zika) are vectored by insects. With as many species as all other forms of life combined, it is inevitable that arthropods - and particularly insects - impinge on our lives in both positive and negative ways. They impact food security, both as pollinators and as destroyers of the world's crops; and also animal health, as vectors of animal and human disease. Climate change moves these threats ever closer to the UK, as evidenced by recent UK cases of blue tongue virus and the presence of alien insect crop pest species.
Although there is a remarkable range of pesticides in use today to control insect pests, significantly, resistance to all classes of pesticides by insects is a serious problem, and few new insecticides have been brought to market in recent years. Furthermore, ever tighter regulatory controls require removal of useful compounds from our insect control repertoire. There is thus a need for a better understanding of insect physiology and environmental stress tolerance, so that we can identify new, more selective (and thus 'greener') ways of controlling them.
As such, drugs impacting insect GPCRs have the potential to be new, more selective insect control agents. Here, we bring together a range of world-leading expertise and emerging technologies in this cross-disciplinary project between Life Sciences and Chemistry to address this issue. We will provide a deeper underdertanding of how these GPCRs signal in insects and also how they enable insects to withstand environmental stress for survival by developing new tools for use in an organismal context, based on the fruit fly, Drosophila melanogaster. We will also develop new small protein 'mimetics' which act on GPCRs (similar in function to the endogenous small proteins which activate GPCRs) that may be developed towards a new class of insect control agents which do not engender insect resistance, and which do not threaten the environment.

This programme aims to achieve a step-change in understanding insect GPCR signalling using Bioluminescence Resonance Energy Transfer (BRET), and by bringing novel SPASM technology (developed for use in cell lines) to the living animal, using the genetic model Drosophila melanogaster. These approaches have been developed from our published data (Halberg et al., Nature Comms, 2015; Christiansen E, et al. J Med Chem. 2016; Kuil et al., Bioorganic and Medicinal Chemistry, 2008).
Using the SPASM sensors, we will assess functional activation and conformation of two insect GPCRs (capaR and kininR) in vivo, in response to binding by fluorescently labeled neuropeptide ligands (capa -1, capa-2, kinin). We will also assess capaR and kininR conformation in the absence of endogenous ligands using SPASM sensors in capa and kinin mutants. We aim to couple these insights with assessment of novel capa and kinin peptide analogues against SPASM sensors in Drosophila S2 cells and Drosophila tissues by calcium and BRET assays, and to also assess how environmental stress (desiccation, cold, starvation) impact on GPCR conformation and function in Drosophila.
For rapid generation of capa and kinin peptide analogues, we will utilize approaches including fluorescent optimization and labeling, peptide library synthesis, peptide modification and optimisation, conformational restriction such as cyclisation, introduction of peptidomimic sequences, and reduced degradation.
We will also develop novel, rapid and robust BRET-based screening technologies using luminescent capaR and kininR, together with fluorescent labeled capa and kinin and peptide analogues. This BRET screening will be used towards novel insect control agents by assessment of capaR and kininR from three insect pest species (D. suzukii, fruit pest; M. persicae, agricultural pest; A. aegypti, insect vector of dengue and Zika viruses). Optimum analogues will then be screened against pest insect stress tolerance and survival.

This work will benefit the UK/international academic community, the wider public, inform policy makers and in the longer-term, fulfil economic impact.

Academic community: in addition to that described in 'Academic Beneficiaries', our work will be disseminated via meetings, publications and collaborations (Pathways to Impact). In 2015, Shireen Davies (SD)/Julian Dow (JATD) groups fulfilled >75 requests for transgenic flies and antibodies; and received many requests for information/discussion, so we are of real benefit to the Drosophila/insect community. The investigators - SD, JATD, Graeme Milligan (GM), Robert Liskamp (RJL) - are all world-leading experts in their respective fields and have collaborations with key UK and international groups in their fields (e.g. SD, JATD with neuroendocrinology researchers, e.g., nEUROSTRESSPEP consortium; environmental stress tolerance researchers e.g. Overgaard; GM with GPCR researchers e.g. Tobin and Lohse) and so are well positioned to develop avenues of investigation of mutual interest during the course of the grant. We will produce highly trained researchers for the academic or industrial market. This extends beyond the researchers directly employed on the project, to other members of the lab, including PhD, Masters and undergraduate project students.

Industry/Economic impact: The 4 investigators have recent and/or current collaborations and partnerships with industry including BASF and Zoetis (SD, JATD) and Astra Zeneca (GM). GM's spin-out, Caldan Therapeutics, with U. Southern Denmark, has benefited from a £4.45 million 'Series A' investment led by Epidarex Capital. Also, RJL has a significant track with patents and industry collaborations for novel peptides. Altogether, we have a excellent track record of utility and benefit to the major pharma and AgChem areas. It is possible that this work will deliver IP (e.g., new molecules), and if so, we already have industrial partners in place who can be approached for exploitation. At Glasgow, Knowledge Transfer/commercialisation is now managed at University level (Research Strategy and Innovations Office), and UoG has prioritised development of economic impact of its research, encouraging exploitation of our findings where possible.

Public engagement: All the PIs have had good engagement with the public via the media and BBSRC Business, International Innovations, Public Science Review, internal communications. Our work is also the subject of BBSRC and REF2020 impact case studies. Dow/Davies's BBSRC-funded research is also exhibited at the Glasgow Science Centre (Images on the Clyde) and this, and research of GM, featured in the recent BBSRC Excellence with Impact competition, in which UoG was a finalist. We also hold events at the Glasgow Science Festival and many other public engagement events.

Project Management: All the investigators play active roles in project management, essential to achieve measurable output/progress for all aspects of funded research. We utilise BaseCamp, a platform for project management which allows project-specific data display, discussion and planning, to which group members (and companies, as necessary) have secure access. We also have regular project meetings with team members, with additional weekly Drosophila group meetings (SD/JATD) as a forum for group discussion and presentation. We will also have regular monthly interdisciplinary meetings between the SD, JATD, GM and RJL groups, and attend relevant seminars at the College of Medical, Veterinary and Life Sciences as well as College of Science and Engineering (Chemistry), as appropriate.

All the investigators have excellent, relevant track records in output; collaborations and exploitation; and communication and engagement - so can achieve the maximum outputs and impact from funded projects.