Functional analysis of insect neuropeptide G protein-coupled receptors.

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description We have to date
1. generated >100 peptide analogues (rational design and synthesis) which have been screened and tested as potential new mode of action pest insect biocides.
2. generated in vivo tools (constructs, cell lines, transgenic D. melanogaster lines) for GPCR function in vivo
3. established a new rapid screening platform for insect GPCR/ligands towards rapid development of insect biocides.
Exploitation Route Many of the findings will be used commercially - development of a spinout is currently underway.
Sectors Agriculture, Food and Drink,Chemicals,Environment

 
Description The development of peptide analogues as active insect biocides will form part of a spinout portfolio (Davies, Dow and Liskamp). Currently seeking investment for spinout.
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Chemicals,Environment
Impact Types Economic

 
Description Member of Animal Health, Aquaculture, Agriculture Life Sciences Scotland Industry Leadership Group (LSS ILG)
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description BBSRC
Amount £16,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2020 
End 02/2021
 
Description BBSRC IAA
Amount £17,692 (GBP)
Funding ID BBSRC IAA 
Organisation University of Glasgow 
Sector Academic/University
Country United Kingdom
Start 01/2020 
End 06/2020
 
Description BBSRC RSE Enterprise Fellowship
Amount £94,732 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2019 
End 03/2021
 
Description Scottish Enterprise High Growth Spinout Scheme Biopesticide Discovery and Development Venture
Amount £144,000 (GBP)
Funding ID PS730570C 
Organisation Scottish Enterprise 
Sector Public
Country United Kingdom
Start 04/2019 
End 08/2020
 
Title Constructs for GPCRs 
Description 29 constructs have been generated Cell lines We have generated DES constructs for expression in S2 cells with GPCR receptors of different species: (x9 +1 being generated) DES-CapaR for D.melanogaster,D. Suzukii, Aedes Aegypti, Myzus persicae and Bombus terrestris. DES-KininR for D.melanogaster,D. Suzukii, Aedes Aegypti, Myzus persicae. Generation of DES-KininR from Bombus terrestris is in progress. (1 construct) We have also generated constructs for use in BRET experiments. (x6+ 4 being generated) DES-Nluc-CapaR for D.melanogaster,D. Suzukii, Myzus persicae DES-Nluc-KiniR for D.melanogaster,D. Suzukii, Myzus persicae Genreration of Nluc construct for Aedes and Bombus is in progress (4 constructs) SPASM constructs for expression in mammalian cells have also been generated.(x4) pcDNA5-capaR-SPASM-Gq pcDNA5-capaR-SPASM-no-pep (Control) pcDNA5-KininR-SPASM-Gq pcDNA5-KininR-SPASM-no- pep (control) 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact Enable high throughout cell assays for GPCRs and to probe GPCR function. 
 
Title Development of new BRET based insect GPCR platform 
Description We have successfully developed a prototypic rapid screening system for insect GPCRs based on BRET, using Drosophila melanogaster GPCRs and neuropeptides. The platform has also been developed for insect pest GPCRs, GPCRs from beneficial species and peptide analogues, to develop rapid screening for GPCR/ligands. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact This development is likely to be of commercial interest and will contribute to spinout portfolio. 
 
Title Development of peptide analogues as novel pest insect biocides 
Description Peptide analogues have been generated by rational design and chemical synthesis based on two families of insect neuropeptides and tested as insect biocides. Chemical synthesis approaches to generate multiple distinct forms of analogues have been used/developed. The assays used for assessments of analogue activity are: GPCR activity assays in cell lines (calcium assays) and insect lethality assays. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact We have developed >100 peptide analogues of capa peptide and tested these in structure activity assays.These include biostable, bioavailable analogues. Modifications to native peptide are as follows: Truncated peptides and Ala-scan Modification of the N-terminus D-amino scan - incorporation D- amino acids - Retro-inverso peptide Cyclic peptides Control peptides N-methylated peptides 
URL https://shop.bdspublishing.com/checkout/Store/bds/Detail/WorkGroup/3-190-82952
 
Title Systematic Protein Affinity Strength Modulation sensors for insect GPCRs 
Description SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions and linking these changes to downstream signaling. Here, we have modified SPASM GPCR sensors for insect capa and kinin receptors. Although these sensors can be utilised in cell lines, we have now generated the first in vivo models using SPASM sensors in transgenic flies. These will be used to probe how GPCRs behave in vivo, for example in absence of ligand and under stress conditions for the organism. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact This should hopefully lead to a very high impact publication. 
 
Title Transgenic Drosophila lines 
Description Transgenic lines for SPASM GPCRs; and neuropeptide nulls based on CRISPR/Cas9. Total of 12 lines. SPASM Lines (x4) pUAST-capaR-SPASM-Gq pUAST-capaR-SPASM-no-pep (Control) pUAST-KininR-SPASM-Gq pUAST-KininR-SPASM-no- pep (control) Nluc Lines (x2) pUAST-Nluc-CapaR pUAST-Nluc-KininR Crispr lines (x4+2 being generated) Capa: Capa1-Null( Homologous recombination) Capa2-Null Capa 1-2-PK1 Null.(Lethal/semilethal). Capa2+PK1 null Capa1+Capa2 Null (testing lines) Kinin: Kinin Null (testing lines) 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact These transgenic lines will provide mechanistic insights into GPCR function in vivo. They also represent the first CRISPR neuropeptide lines. 
 
Description Peptide analogues 
Organisation U.S. Department of Agriculture USDA
Country United States 
Sector Public 
PI Contribution Devised physiological and functional assays for neuropeptide analogues developed by USDA.
Collaborator Contribution Provision of neuropeptide analogues.
Impact Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2882-7. doi: 10.1073/pnas.1501518112. Epub 2015 Feb 17. Insect capa neuropeptides impact desiccation and cold tolerance. Terhzaz S1, Teets NM2, Cabrero P3, Henderson L3, Ritchie MG4, Nachman RJ5, Dow JA3, Denlinger DL6, Davies SA1.
Start Year 2013
 
Company Name SOLASTA BIO LIMITED 
Description SOLASTA Bio (solastabio.com) is a bioinsecticides discovery startup company with a unique proposition: insect control products that are nature-inspired rather than selected from a synthetic chemistry library. SOLASTA Bio designs "Insect Water Taps" (IWTs) that are specific, efficacious and safe for non-target organisms and the environment. Our market creating innovation, the multi-technology IWT platform, is developed from the award-winning H2020 nEUROSTRESSPEP and UK Research Innovation projects. SOLASTA Bio is currently supported by Scottish Enterprise (Scotland's economic development agency) and the founders are engaging with investors and SME grant funders to secure the finance to launch the business. The company's management team has world-leading technological expertise; and global commercial and organisational experience having attracted a former Agrochemical Corporate (Ag Corp) executive. SOLASTA Bio candidates are aimed at the €28B global insecticides market and will be licenced to Ag Corp, who are best placed to complete large-scale field trials and have global sales channels. The company is incubated at University of Glasgow, with excellent laboratory space, and facilities for genome analysis, fast screening, insect rearing and testing facilities. SOLASTA Bio's IWT platform, portfolio of lead bioinsecticides and the founding team are the core assets of the business. The company's strategy is to focus on developing the existing lead products against key insect pests to greenhouse/ small field proof of concept studies. This data package will be used to secure agreements with Ag Corp with a view to entering co-development and licensing deals, providing upfront payments, milestones and product royalties. The IWT platform represents a profound change for how insect control agents are discovered, being rooted in insect physiology rather than traditional synthetic chemistry. This provides the company with a disruptive competitive advantage, balancing risk with high reward. Achieving deals with Ag Corp will validate the IWT platform and the company's business model sufficiently to secure a major Series A investment from VC's that will enable SOLASTA Bio to expand and develop its operations. This will signpost IWT platform potential to the global AG industry modern biopesticides that meet grower and consumer demands. 
Year Established 2020 
Impact Capital raised to date : Innovate UK grant, £650k (50% matching equity from investment below) Investment £350k 3 Management positions - CEO, CSO, COO 3 full time R&D staff (from April 2020) Planned recruitment for 3 R&D staff (June 2020)
Website http://solastabio.com
 
Description Finalist for Scottish Knowledge Exchange Awards 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Finalist for Scottish Knowledge Exchange Awards 2020. Video presented and exhibition mounted.
Year(s) Of Engagement Activity 2020
URL https://interface-online.org.uk/scottish-knowledge-exchange-awards-2020
 
Description Industry workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact - Industry workshop on new biopesticides, BASF, USA, 2017.
Year(s) Of Engagement Activity 2017
 
Description Innovation Workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact BBSRC Idea to Investment, part of BBSRC commercialisation and KE activities. Provided an opportunity to engage directly with the wider network of BBSRC (from investors, specialist consultants, businesses and CEOs).
Year(s) Of Engagement Activity 2019