The Use Of Novel Polymer-Lipid Nanoparticles To Study G-Protein-Coupled Receptor Activation And Dynamics.

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

Most chemical messengers in the body, work through proteins on the cell surface known as receptors. The biggest class of receptors is the G-protein-coupled receptor (GPCRs) family, which are important for cell signalling and as drug targets. GPCRs are thus of enormous interest, both in terms of basic biology and also for commercial exploitation by the pharmaceutical industry. Understanding how GPCRs work at the molecular level is fundamentally important and is one of the 'big questions' in biology today. For a few receptors we have a crystal structure showing their 3-dimensional shape when bound to a 'blocking drug'. This has deepened our understanding of receptor architecture which should aid future drug discovery. However, it is clear that GPCRs are very flexible proteins that change their shape on binding hormones and drugs, whereas the crystal structures provide snap-shots of a single conformation, usually of the non-signalling receptor. To fully understand GPCRs, we need to understand how they change their shape during signalling. This is also important for 'allosteric' agents that bind to a different part of the receptor to the natural hormone to modify receptor activity. This opens the way to a whole new class of drugs. In this application we describe a new approach to understanding GPCRs. We have found a novel way to make large quantities of natural GPCRs 'solubilised' within very small particles of their membrane environment without the need for detergent. Previously, there has been an absolute requirement for detergent to solubilise receptors. This formed a major barrier to progress in this field as its presence disrupted the normal properties and shape of the receptor, de-stabilising the protein. For the first time anywhere, we can now circumvent the detergent barrier to progress. This important advance opens up the prospect of applying powerful biophysical techniques to provide detailed information on how GPCRs dynamically change their shape in response to drugs of different classes. The project brings together four scientists from the Universities of Birmingham and Aston, with complementary skills to exploit our new technologies. We will study the adenosine A2a receptor (A2aR) as an example of the GPCR family. This is a well-characterised receptor with a large literature, defined drugs, useful tools and importantly it is one of the few receptors for which there is an atomic level crystal structure. In addition, our industrial collaborators Heptares Therapeutics will provide A2aRs constrained into functionally-defined shapes (conformations) which will be a great help to us. Finally we have a formal collaboration with an experienced computational chemist at Essex University who will construct molecular models and dynamic simulations of different functional states of the receptor based on our experimental data. We are ahead of the field and our pilot data show that we have the capability at this very moment to produce very large amounts of A2aR by growing them in a yeast and to rapidly 'solubilise' active receptors in their natural state in a styrene maleic acid lipid particle (SMALP). We will engineer A2aR so that 'reporter groups', including fluorescent groups, can be introduced at defined locations in the receptor architecture. We can then study A2aR-SMALP by a range of techniques. For example, fluorescence can tell us about the changes in the environment around important parts of the receptor when drugs bind. A related technique can also be used to provide a molecular ruler that allows us to calculate the distance between two parts of the receptor to determine how it changes on binding drugs of different signalling capability or in conformationally-constrained A2aRs provided by Heptares Therapeutics. Overall, this project will provide insight into the changes of shape that underlie GPCR activation and may aid rational drug design in the future.

Technical Summary

G-protein-coupled receptors (GPCRs) are the largest class of receptors and the largest target for prescription drugs. The processes underlying GPCR activation at the molecular level remain poorly understood. Recent crystal structures have provided static snap-shots of a few of these highly flexible proteins. A full understanding of GPCR signalling requires knowledge of the various states adopted by the receptor in response to ligands of differing efficacy (full agonist, inverse agonist) and the dynamics of the inter-conversions of these states. A formidable barrier to progress in this field is that the detergents required to solubilise GPCRs de-stabilise the receptors and disrupt conformational states. We have developed novel technologies to 'solubilise' GPCRs in a styrene maleic acid lipid particle (SMALP), without detergent, preserving native conformations and enabling powerful biophysical analyses - a world first. We aim to exploit this advantage to study the adenosine A2a receptor (A2aR) a GPCR. Conformationally stabilised receptors (StaRs) provided by Heptares Therapeutics will be an asset and the published antagonist-A2aR crystal structure provides a defined structure for modelling A2aR conformational changes. We will: 1) Enable A2aR for site-specific incorporation of reporters to allow downstream biophysical analyses. 2) Characterise constructs in HEK293T cells, which will then be over-expressed in P. pastoris, solubilised by spontaneous embedding into SMALPs and purified. 3) Use a battery of biophysical techniques to: i) Define the changes in A2aR conformation underlying activation and characterise the dynamics of inter-conversions. ii) Identify differences in conformation when the receptor is occupied by ligands of different efficacy (full agonist, partial agonist, inverse agonist). iii) Define A2aR conformation modifications induced by allosteric ligands. iv) Use constraints from biophysical data to build unified structural models of GPCR activation.

Planned Impact

The research described in this proposal will increase understanding of the molecular processes underlying GPCR activation and how these processes are initiated or blocked by the receptor binding different classes of ligand (agonist/antagonist/inverse agonist/allosteric modulator). This will have a significant impact in the field of cell signalling in academia and also within the Pharmaceutical Industry. The insights provided may lead to rational drug design in the future producing drugs which are better able to activate or block discrete aspects of receptor signalling. This would lead to improved medicines which would increase the quality of life. Our research is directly relevant to the Pharmaceutical Industry in the UK as is evident from the 'Letter of Support and Collaboration' from our industrial collaborator Heptares Therapeutics Ltd. (attached). Any advance we make would be exploited by Pharma companies. Our technologies may be applicable to studying other membrane proteins thereby extending the impact of our research beyond cell signalling. Two PDRAs will be trained in a wide range of multidisiplinary skills which will be an asset to their career development and to subsequent employers.

Publications

10 25 50

publication icon
Dörr JM (2014) Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs. in Proceedings of the National Academy of Sciences of the United States of America

publication icon
Routledge S (2014) Antifoams: the overlooked additive? in Pharmaceutical Bioprocessing

 
Description We have now used our technology to isolate 2 different GPCRS from yeast, bacteria and mammalian cells. These receptors have been purified to homogeneity. We have further characterised one of these biophysically, to understand how drugs change the shape of the protein; this knowledge will be essential in designing improved drugs for the future.



We have used unnatural amino acid mutagenesis to introduce fluorescent reporters into our GPCRs. These are tools to measure the shape of the receptor. We have been able to use these to detect changes to the GPCRs when we add drugs.
Exploitation Route The research can be used to design new classes of drugs. This is of interest to the pharmaceutical industry
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The findings have so far been reported in the magazine International Innovations, which is widely read by EU policy makers. Our work also needs to be seen in the wider context of the growth of use of SMALPs as agents to study commercially important membrane proteins; by demonstrating that they can be used to isolate G-protein coupled receptors (the largest target for therapeutic drugs), our results will be important for more efficient and effective drug discovery. To this end, we are presenting our results to the wider scientific community via papers and posters, as described elsewhere.
First Year Of Impact 2013
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description BBRSRC Responsive mode grant
Amount £27,991 (GBP)
Funding ID BB/R016755/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 08/2021
 
Description Biomarkers of the ageing tear film
Amount £125,520 (GBP)
Funding ID BB/K013319/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2017
 
Description Engineering yeast for GPCR expression and functional studies
Amount £125,520 (GBP)
Funding ID BB/L502194/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2017
 
Description Investigating GPCR:RAMP interactions using nanobodies
Amount £404,022 (GBP)
Funding ID BB/R016615/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2018 
End 06/2021
 
Description MRC-CiC
Amount £97,623 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 08/2016 
End 06/2017
 
Description Wellcome Trust Vacation Studentship
Amount £1,500 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Vacation Scholarship
Sector Charity/Non Profit
Country United Kingdom
Start 07/2016 
End 08/2016
 
Title Use of SMALPs 
Description Our work has shown how it is possible to purify membrane proteins without detergents, by means of SMALPs 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact This has resulted in colleagues at both Aston and Birmingham universities publishing papers using the technology 
 
Description Collaboration with Dr Alice Rothnie 
Organisation Aston University
Country United Kingdom 
Sector Academic/University 
PI Contribution Providing SMALP-based technology knowledge.
Collaborator Contribution Applying the SMALP technology to ABC transporters
Impact Paper published in Biocemical Journal.
Start Year 2013
 
Description Collaboration with Dr Ed Hulme 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Public 
PI Contribution Collaboration on muscarinic receptor with NIMR, London
Collaborator Contribution Dr Hulme supplied the expression plasmid for the muscarinic receptor
Impact Evaluation of purification strategies of the SMALPed muscarinic receptor
Start Year 2013
 
Description Collaboration with Dr Nick Holliday, Nottingham University 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Measurement of beta-arrestin interactions
Collaborator Contribution Dr Holliday supplied constructs to allow the use of the above assay
Impact Evaluation of the assay
Start Year 2013
 
Description Collaboration with Professor Chris Reynolds, University of Essex 
Organisation University of Essex
Country United Kingdom 
Sector Academic/University 
PI Contribution A collaborator at Essex University is assisting us with molecular modelling of the A2a and M1 receptors
Start Year 2012
 
Description Collaboration with UCB 
Organisation UCB Pharma
Department UCB Celltech
Country United Kingdom 
Sector Private 
PI Contribution We have supplied SMALP-solubilised GPCRs for antibody production; we are currently expanding this to RAMP-receptor complexes
Collaborator Contribution UCB are currently using our material to produce antibodies
Impact This lead to an application for a new project grant to the BBSRC and also resulted in the award of a university awarded PhD studentship.
Start Year 2015
 
Description FCS to study SMALPed proteins 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We have supplied SMALP-purified adenosine 2a receptor for analysis by fluorescence correlation spectroscopy (FCS) by Dr Steve Briddon at Nottingham University
Collaborator Contribution Dr Briddon has performed and interpreted the FCS
Impact We have shown that it is possible to identify single molecules of the adenosine A2a receptor by FCS
Start Year 2017
 
Description Interaction with Schultz Group, USA 
Organisation Scripps Research Institute
Country United States 
Sector Charity/Non Profit 
PI Contribution Interaction with group and transfer of materials for unnatural amino acid incorporation
Collaborator Contribution We introduced unnatural amino acids into the A2a receptor expressed in yeast
Impact This opened the way to us using unnatural amino acid mutagenesis in eukaryotic expression systems
Start Year 2012
 
Description Mass spectroscopy of GPCRs 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We have supplied purified GPCRs
Collaborator Contribution The partners are analysing the GPCRs by mass spectroscopy
Impact This is an academic collaboration; we hope it will lead to new papers
Start Year 2015
 
Description SMALPing of calcitonin receptor 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution We have supplied reagents and provided training for a research student to purify the calcitonin receptor using SMALPs
Collaborator Contribution Provision of a cell line expressing the calcitonin receptor and a student to do the experimental work
Impact Production of the purified calcitonin receptor for structural studies
Start Year 2015
 
Description Study of rhodopsin by SMALPs 
Organisation University of Essex
Country United Kingdom 
Sector Academic/University 
PI Contribution We helped Dr Reeves from Essex University to SMALP bovine rhodopsin
Collaborator Contribution Dr Reeves analysed the activation of the SMALPed rhodopsin
Impact Oral communication at Pharmacology 2018
Start Year 2017
 
Description Using SMALPs for mass-spectroscopy analysis of GPCRs 
Organisation University of Auckland
Department School of Biological Sciences
Country New Zealand 
Sector Academic/University 
PI Contribution We SMALPed the adenosine 2a and calcitonin receptors to allow them to be analysed by mass-spectroscopy
Collaborator Contribution Analysis of receptor by mass-spec
Impact Grant applications submitted as follows: Royal Society of New Zealand Marsden Fund, Hay, Robinson and Wheatley, 15-UOA-248, $870,000 NZD (unfunded) University of Auckland Faculty Research Development Fund $160,000 (awarded) Human Frontiers of Science Programme, Hay, Robinson and Wheatley, RGP0022/2016, $900,000 USD (application under assessment)
Start Year 2015
 
Description Using SMALPs for mass-spectroscopy analysis of GPCRs 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We SMALPed the adenosine 2a and calcitonin receptors to allow them to be analysed by mass-spectroscopy
Collaborator Contribution Analysis of receptor by mass-spec
Impact Grant applications submitted as follows: Royal Society of New Zealand Marsden Fund, Hay, Robinson and Wheatley, 15-UOA-248, $870,000 NZD (unfunded) University of Auckland Faculty Research Development Fund $160,000 (awarded) Human Frontiers of Science Programme, Hay, Robinson and Wheatley, RGP0022/2016, $900,000 USD (application under assessment)
Start Year 2015
 
Description XFMS of SMALPed proteins 
Organisation University of Essex
Country United Kingdom 
Sector Academic/University 
PI Contribution We have supplied SMALP purified Adenosine 2a receptor for analysis by XFMS by Dr Vassily Bavro
Collaborator Contribution Dr Bavro has run the XFMS at a synchrotron in the USA and interpreted the results
Impact We have established the XFMS can be used to label the Adenosine 2a receptor after SMALP solubilisation
Start Year 2017
 
Description Medical University of Vienna, Austria 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Questions and discussions after talk

The talk increased awareness of the use of SMALPs
Year(s) Of Engagement Activity 2013
 
Description Article in community magazine describing recent research 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Some comments that the article was interesting

I was subsequently invited to speak about my work, to a community group
Year(s) Of Engagement Activity 2015
 
Description Article in community magazine on the importance of scientific evaluation 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact There were a few positive comments, but generally limited response

Difficult to say!
Year(s) Of Engagement Activity 2015
 
Description Australian Peptide Society, Penang, Malaysia 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Questions and discussion after the talk

The talk increased awareness of the use of SMALPs
Year(s) Of Engagement Activity 2013
 
Description Biomedical Research Centre, Sheffield Hallam University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Questions and discussion after the talk

The talk increased awareness of protein isolatin and purification methods amongst PhD students and undergraduates
Year(s) Of Engagement Activity 2014
 
Description German Research School for Simulation Sciences, Julich, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Questions and discussion after talk

Talk lead to discussions with molecular modellers that are continuing
Year(s) Of Engagement Activity 2013
 
Description How do drugs work 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Poster for village fete Poster

The poster generated much interest at the fete
Year(s) Of Engagement Activity 2013
 
Description Incorporation of unnatural amino acids into recombinant human adenosine-2a receptor produced in Pichia pastoris. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Poster presented at 7th Conference on Recombinant Protein Production (RPP), Germany, 6th-8th March 2013

This resulted in enquiries about placements in the laboratory
Year(s) Of Engagement Activity 2013
 
Description Moving molecules 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Article for community newsletter describing the BBSRC funded project The newsletter is circulated around the villages of Highley, Billingsley, Chelmarsh, Deuxhill and Glazeley, my home communities Article for newsletter

Around 5 people commented that they had found the article interesting
Year(s) Of Engagement Activity 2013
 
Description Moving molecules 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Article for community newsletter describing the BBSRC funded project The newsletter is circulated around the villages of Highley, Billingsley, Chelmarsh, Deuxhill and Glazeley, my home communities Article for newsletter

A number of lay people commented on the article.
Year(s) Of Engagement Activity 2013
 
Description New ways of looking at GPCRs 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Project website

This has lead to several enquiries for PhD places
Year(s) Of Engagement Activity 2013
URL http://www.aston.ac.uk/lhs/staff/az-index/poynerdr/
 
Description PEGS Europe Summit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact This was the keynote presentation to in excess of 100 delegates at a conference about optimising protein expression and purification

This raised the profile of our work on SMALPs to a international audience
Year(s) Of Engagement Activity 2014
 
Description Protein modelling masterclass 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Hands-on demonstration of protein modelling and its application in drug discovery to 6th formers
Year(s) Of Engagement Activity 2017
 
Description Receptive Collaboration 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Article in International Innovation, November 2013, 113-115 One article

We have been approached for further articles
Year(s) Of Engagement Activity 2013
 
Description Research talk at the 'Mercian GPCR Meeting' at the University of Essex 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A GPCR meeting of approximately 50 attendees including international speakers and researchers from industry, with interactive question sessions.
Year(s) Of Engagement Activity 2016
 
Description Research talk at the multinational pharmaceutical company GSK (Stevenage) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I gave a research talk of our findings to various research team members from within GSK research laboratories
Year(s) Of Engagement Activity 2016
 
Description Talk at village group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk to around 15 people at group in the village of Highley about my work as scientist
Year(s) Of Engagement Activity 2017
 
Description Talk to community group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact There was a lot of discussion following the talk

I was asked if a 6th former could spend a day in my lab, by a the friend of his parent, who attended the talk. This visit took place on 2-11-15
Year(s) Of Engagement Activity 2015
 
Description That Eureka Moment 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Article in community newsletter describing progress on the grant Follow-on from Moving Molecules

Several individuals commented on reading the article and how it had given them a better insight into the scientific process
Year(s) Of Engagement Activity 2013
 
Description University of Nottingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Questions and discussion after talk

This has helped a current collaboration with colleagues at Nottingham on the use of SMALPs
Year(s) Of Engagement Activity 2013