Targeting GLP-1 receptor trafficking to improve therapies for type 2 diabetes

Lead Research Organisation: Imperial College London
Department Name: Metabolism, Digestion and Reproduction

Abstract

Type 2 diabetes (T2D) kills around 4 million people worldwide every year and causes ill health in many more. A major underlying problem in T2D is that specialised cells in the pancreas known as beta cells start to fail and do not produce or secrete enough insulin, which is needed to control blood sugar. Beta cells can be stimulated to release more insulin by activating a surface protein known as glucagon-like peptide-1 receptor (GLP-1R). GLP-1R treatments, known as GLP-1R agonists, are already used in T2D. We have found that a process known as "receptor trafficking" limits the effectiveness of GLP-1R agonists. When the agonist activates the receptor, it also "internalises" it into the cell where it is hidden and can no longer be activated by agonists outside the cell. We showed this using a modified version of a GLP-1R agonist currently used in T2D (exendin-4, also known as "Exenatide"), which we engineered to reduce the internalisation process. This new agonist called "Ex-phe1" was much more effective than unmodified exendin-4 in mice with T2D, as it allowed the receptor to remain longer at the cell surface and be continually reactivated, leading to more insulin release. Also, despite being more effective in beta cells, Ex-phe1 did not cause more nausea, a well-known side effect of GLP-1R agonists.
This project has two goals. The first is to understand how changes in GLP-1R trafficking modify insulin release from beta cells. The second, to be performed in parallel, is to optimise the compounds we generated in our preliminary work to produce new, better, treatments for T2D. Our approach includes experiments in beta cells and pancreatic tissue ("islets"), which allow us to understand events in molecular detail, and in mice with T2D, which tell us what happens at the whole body level. We place a lot of emphasis on using human islets (provided for research when surplus to requirements for organ donation), as we believe this is essential to understand and treat human disease.
We highlight below some key experiments:
1) We will use a combination of computer modelling and genetic modification of the GLP-1R to identify specific interactions between the receptor and Ex-phe1 or other biased GLP-1R agonists which underpin their different trafficking and therapeutic effects. This knowledge will be critical for the design of even more effective drugs in the future.
2) We will genetically modify human islets to reduce levels of "beta-arrestin-2", a protein in beta cells that we believe is a key link between GLP-1R activation, trafficking, and beneficial insulin release. We will also breed genetically modified mice lacking beta-arrestin-2 in beta cells. We will use these systems to look at complex patterns of protein and gene activation within beta cells in response to GLP-1R agonist treatment. From this we hope to identify molecules that could in the future be targeted as new T2D treatments.
3) We will study the long-term effects of our new, improved GLP-1R agonist Ex-phe1 on human islets transplanted into diabetic mice. Importantly, we will use a new approach in which the islets are actually transplanted in the eye, where changes to islet shape, behaviour and overall health can be directly and repeatedly viewed in a microscope. The mice will be treated with a long-lasting formulation of Ex-phe1 or exendin-4 for several months, and we will monitor changes to blood sugar, allowing us to compare the treatments for their effects on overall islet function. Due to well-known differences between mouse and human islets, we believe that using this "humanised" mice is critical to understanding long-term treatment effects.
In summary, we have identified an exciting new approach based on GLP-1R trafficking for improving the treatment of T2D. In this project we will establish the molecular details of why this approach works, identify new potential drug targets and demonstrate the long-term effects of this new treatment in unique humanised mice.

Technical Summary

The glucagon-like peptide-1 receptor (GLP-1R) is an important target in type 2 diabetes (T2D) as GLP-1R agonists stimulate beta cell insulin release and promote weight loss. The GLP-1R undergoes rapid agonist-mediated endocytosis, leading us to question whether desensitisation limits GLP-1R agonist treatment effectiveness. We have tested this hypothesis by generating modified versions of the GLP-1R agonist exendin-4. One of these, Ex-phe1, displays a sharply reduced propensity for receptor internalisation, is a more potent insulin secretagogue, and provides enhanced glycaemic control in diabetic mice without increasing nausea, a common side effect of GLP-1R agonists. Ex-phe1 exhibits a distinct pharmacological profile to the parent compound, including fast receptor dissociation kinetics and biased signalling, with beta-arrestin-2 (barr2) recruitment selectively reduced. Here, we will elucidate the molecular mechanisms linking biased GLP-1R activation and trafficking to insulin secretion and examine the therapeutic benefits of biased GLP-1R agonism in a humanised T2D model. Specifically, we will: 1) identify the structural basis for biased GLP-1R activation and trafficking using molecular modelling, reciprocal receptor mutagenesis and a GLP-1R conformational biosensor; 2) determine biased agonist-specific kinomic and transcriptomic responses and changes to interactions with endocytic trafficking regulators in beta cells; 3) confirm the role of barr2 in beta cell responses to GLP-1R agonism both in beta cell-specific barr2 knockout mice and by silencing barr2 in human islets; and 4) establish the long-term effects of biased GLP-1R agonism in a humanised mouse model with human islets implanted into diabetic mice eyes, an approach that uniquely allows serial imaging of islet morphology and function. These studies will provide critical molecular insights into the control of GLP-1R trafficking by biased agonists and will potentially identify new targets for T2D treatment.

Planned Impact

We identify the following groups as likely beneficiaries from work conducted during this project:

1) Patients with T2D and associated metabolic conditions:

In this project, we will develop new molecules that are expected to display improved therapeutic efficacy and tolerability compared to existing treatments. As well as patients with T2D, patients with non-alcoholic fatty liver disease are another key group who are expected to benefit from treatment with biased GLP-1R agonists. GLP-1R agonism is also an investigational strategy for obesity, cardiovascular disease, and neurological diseases. Full clinical development of new agents is expected to take 5-8 years.

2) The NHS:

The S.R.B. group has considerable experience of "first in man" and phase 1 clinical trials. Molecules developed during this project will in the future be taken forward to human studies. The NHS benefits from these studies, for example through Imperial Academic Health Sciences Centre funding. Pre-clinical studies are likely to begin following successful completion of this project.

3) Imperial College London:

We have filed an International (PCT) Patent Application (PCT/GB2017/051045) for biased GLP-1R agonists developed during pilot work for this project. Via Imperial Innovations, a subsidiary of Imperial College London, we will engage with the pharmaceutical industry regarding acquisition of intellectual property. We have a proven track record in this area, for example with the spin-off company Thiakis, which developed the peptide hormone oxyntomodulin as an anti-obesity agent and was subsequently acquired by Wyeth for $150 million.

4) The UK pharmaceutical industry and UK economy:

An estimated 422 million people have diabetes worldwide. Obesity-related comorbidities, such as non-alcoholic fatty liver disease, are at least as prevalent. There is therefore a potentially enormous market for GLP-1R agonists with improved therapeutic efficacy and tolerability, with clear economic benefits for the UK pharmaceutical industry and associated organisations. G.A.R. enjoys extensive interactions with the UK and European pharmaceutical industry as a Work Package leader in the former EU-EFPIA co-funded Innovative Medicines Initiative in Diabetes (IMIDIA) consortium, and presently in the "Rhapsody" consortium under IMI2. The latter involves close collaborations with Servier, Novo Nordsik, Janssen/J&J and Sanofi Aventis, particularly as part of an initiative to identify new circulating biomarkers for T2D development and progression.

5) The wider scientific community:

During this project we will explore a new technological approach to imaging of ligand binding and receptor trafficking in living islets implanted into the anterior chamber of the eye. This would for the first time allow these cellular events to be monitored at cellular resolution in an in vivo environment. Whilst we will focus on the GLP-1R, if this technique is successful, it is likely to be of significant interest to researchers working within the wider GPCR field. Results will be disseminated at scientific conferences and by publication in peer-reviewed journals.

6) The general public:

Our departments have a strong track record of public engagement. We welcome pre-university students into our laboratory for work experience as part of a drive to widen participation. We are involved in the Imperial College Outreach scheme, which organises science education events for school students and their teachers, introducing them to the science of T2D and obesity. We regularly participate in the Imperial Festival, a public engagement event that allows the public to explore and understand our latest research. We also recently contributed to the BBC Horizon programme, "Why Are We Getting So Fat?".

Publications

10 25 50
 
Description Academy of Medical Sciences "Studying the impact of biased GLP-1 receptor signalling in humans"
Amount £30,000 (GBP)
Funding ID SGL019\1078 
Organisation Academy of Medical Sciences (AMS) 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2018 
End 08/2019
 
Description DUK PhD Studentship: "Spatiotemporal Control of Signalling of the GLP-1R Variant Ala316Thr in Pancreatic Beta Cells"
Amount £199,079 (GBP)
Funding ID 19/0006094 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2020 
End 09/2023
 
Description Functional Studies on Novel GLP-1R Agonists
Amount £232,800 (GBP)
Organisation Sun Pharmaceuticals 
Sector Private
Country India
Start 02/2019 
End 01/2021
 
Description Society for Endocrinology: "Proof of concept for personalisation of GLP-1 receptor agonist treatment using biased ligands"
Amount £10,000 (GBP)
Organisation Society for Endocrinology 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2018 
End 08/2019
 
Description Understanding the tissue-specific effects of biased GLP-1 receptor agonism in type 2 diabetes
Amount € 99,000 (EUR)
Organisation European Association for the Study of Diabetes (EASD) 
Sector Charity/Non Profit
Country Germany
Start 01/2020 
End 12/2021
 
Title Biased GLP-1R agonists with improved therapeutic potential 
Description Lipid conjugated exendin-4, exendin-phe1, exendin-asp3 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact None yet 
 
Title Fluorescent conjugates of biased GLP-1R agonists for cellular and tissue imaging 
Description Fluorescent agonists for cellular and tissue imaging 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? No  
Impact Analysis of brain entry and single molecule localisation microscopy 
 
Title Further GLP-1R-active peptides for evaluation of modes of receptor binding 
Description GLP-1/exendin-4 chimeric compounds 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact None yet 
 
Title INS SNAP-GLP-1R C438A and INS SNAP-GLP-1R A316T 
Description Beta cell lines expressing mutant SNAP-GLP-1Rs on a GLP-1R -/- background 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? No  
Impact Investigation of the effects of point mutations in GLP-1R trafficking and signalling 
 
Title Lentiviral CRISPR constructs for zDHHC family of S-acyltransferases 
Description Lentiviral CRISPR constructs for generation of KO cell lines for zDHHC5, 20 and 21 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? No  
Impact Generation of zDHHC5, 20 and 21 -/- mouse beta cell lines 
 
Title SNAP-GLP-1R A316T 
Description SNAP-GLP-1R with A316T single nucleotide polymorphism 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact Investigation of the effect of SNPs on GLP-1R trafficking and signalling 
 
Title SNAP-receptor-PK plasmids 
Description SNAP-GLP-1R/GIPR/GCGR tagged with Prolink (PK) 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact Analysis of beta arrestin recruitment by enzyme fragment complementation assay 
 
Title SNAP-tagged receptor beta cell lines 
Description SNAP-GLP-1R / SNAP-GIPR-expressing beta cell lines in GLP-1R/GIPR -/- background 
Type Of Material Cell line 
Year Produced 2019 
Provided To Others? No  
Impact Use of these cell lines to investigate the effect of receptor SNPs and dual GLP-1R/GIPR agonists 
 
Title Targeted cAMP biosensors 
Description T-Epac-VV cAMP biosensor tagged with lipid raft, endosomal and TGN localisation signals 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? No  
Impact Measurement of cAMP from subcellular compartments 
 
Title Gene expression dataset 
Description Transcriptomic data of gene expression levels following GLP-1R biased agonist stimulation of beta cells and hypothalamic neuronal cells 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact None yet 
 
Title Kinase activation dataset 
Description Kinomic data of kinase activity levels following GLP-1R biased agonist stimulation of beta cells 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact None yet 
 
Description Analysis of GLP-1R agonist binding cooperativity and receptor oligomerisation 
Organisation Imperial College London
Department Institute of Reproductive and Developmental Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We are developing a number of assays to analyse differences in binding cooperativity between GLP-1 receptor agonists
Collaborator Contribution Expertise on BRET assays and oligomerisation analysis
Impact Multidisciplinary: pharmacology/cell biology/endocrinology
Start Year 2017
 
Description Analysis of GLP-1R agonist binding cooperativity and receptor oligomerisation 
Organisation Imperial College London
Department School of Public Health
Country United Kingdom 
Sector Academic/University 
PI Contribution We are developing a number of assays to analyse differences in binding cooperativity between GLP-1 receptor agonists
Collaborator Contribution Expertise on BRET assays and oligomerisation analysis
Impact Multidisciplinary: pharmacology/cell biology/endocrinology
Start Year 2017
 
Description Analysis of GLP-1R trafficking differences in response to a panel of exendin-4 analogues with single amino acid substitutions 
Organisation Imperial College London
Department Section of Endocrinology and Investigative Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Analysis of GLP-1R trafficking in response to a panel of biased exendin-4 analogues
Collaborator Contribution Panel of exendin-4 analogues exhibiting varying degrees of GLP-1R biased agonism
Impact Multi-disciplinary: Pharmacology/Cell Biology On-going partnership that might provide a novel therapeutic approach to T2DM Paper submitted and under review Biased agonists now patented (ICL)
Start Year 2015
 
Description Analysis of biased agonist GLP-1R responses in the choroid plexus 
Organisation University of Birmingham
Department Institute of Metabolism and Systems Research (IMSR)
Country United Kingdom 
Sector Academic/University 
PI Contribution Plasmids, lentiviruses, modified agonists
Collaborator Contribution Choroid samples and ICV/choroid plexus techniques
Impact No outputs yet
Start Year 2019
 
Description Analysis of biased agonist brain responses 
Organisation University College London
Department Division of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Brain samples from biased agonist treated mice
Collaborator Contribution Analysis of neuronal activity
Impact No outputs yet
Start Year 2019
 
Description Analysis of chemically-modified GLP-1 analogues 
Organisation ImmuPharma
Department UREKA
Country United Kingdom 
Sector Private 
PI Contribution Analysis of GLP-1R trafficking and signalling bias from chemically-modified GLP-1 analogues
Collaborator Contribution Chemically-modified GLP-1 analogues
Impact No outputs yet
Start Year 2018
 
Description Analysis of chemically-modified GLP-1 analogues 
Organisation University of Bordeaux
Country France 
Sector Academic/University 
PI Contribution Analysis of GLP-1R trafficking and signalling bias from chemically-modified GLP-1 analogues
Collaborator Contribution Chemically-modified GLP-1 analogues
Impact No outputs yet
Start Year 2018
 
Description Analysis of incretin receptor expression and function in zebrafish 
Organisation University of Geneva
Department Department of Biochemistry
Country Switzerland 
Sector Academic/University 
PI Contribution Fluorescent GLP-1R agonists and SNAP-tagged expression vectors
Collaborator Contribution expertise in the generation of transgenic zebrafish lines, labelling and localisation of dual GLP-1R/GCGR in zebrafish embryos
Impact No outputs to date
Start Year 2019
 
Description Development of novel tools to study GPCR palmitoylation 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Genetically modified beta cell lines (CRISPR/Cas9)
Collaborator Contribution Plasmids and tools to engineer cell lines to screen for specific palmitoyltransferase partners in beta cells
Impact N/A
Start Year 2019
 
Description Generation of fluorescently labelled GLP-1R agonists 
Organisation Imperial College London
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Testing fluorescently labelled GLP-1R agonists for a variety of applications Modified agonists and fluorophores
Collaborator Contribution Chemical sysnthesis of fluorescently labelled GLP-1R agonists
Impact No outputs yet
Start Year 2018
 
Description Investigation of in vivo effects of biased agonists in eye-implanted pancreatic islets 
Organisation Imperial College London
Department Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Biased agonists and expertise in in vivo evaluation of incretin signalling
Collaborator Contribution mouse model of islets implantation and imaging from anterior chamber of the eye
Impact None yet
Start Year 2019
 
Description Lipidomic analysis of GLP-1R biased agonist signalling 
Organisation University of Geneva
Department Department of Biochemistry
Country Switzerland 
Sector Academic/University 
PI Contribution Pancreatic beta cell and neuronal samples stimulated with biased GLP-1R agonists
Collaborator Contribution Analysis of sample lipid species composition
Impact Data on lipid composition downstream of GLP-1R signalling
Start Year 2019
 
Description Mass spectrometry analysis of GLP-1R biased agonist signalling 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Preparation of mass spec samples with factors pulled down with GLP-1R following stimulation with biased agonists in pancreatic beta cells
Collaborator Contribution Mass spec analysis of GLP-1R binding partners
Impact Datasets of binding partners
Start Year 2019
 
Description Mass spectrometry analysis of GLP-1R interactions downstream of biased agonist stimulation 
Organisation Imperial College London
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Pulled-down samples of SNAP-GLP-1R from pancreatic beta cells
Collaborator Contribution Mass spectrometry sample preparation and analysis
Impact No outputs yet
Start Year 2018
 
Description Mathematical modelling of human data 
Organisation The CNR Institute of Neuroscience, Padova, Italy
Country Italy 
Sector Academic/University 
PI Contribution Human physiological study data
Collaborator Contribution Study of incretin effects in healthy and type 2 diabetic subjects through the use of mathematical models
Impact No outputs yet
Start Year 2018
 
Description Molecular dynamics simulations of class B GPCR interactions with the lipid environment 
Organisation Council of Scientific and Industrial Research (CSIR)
Department Centre for Cellular and Molecular Biology (CCMB)
Country India 
Sector Academic/University 
PI Contribution Experimental validation of simulation results
Collaborator Contribution In silico analysis of GLP-1R-lipid interactions
Impact No outputs yet
Start Year 2019
 
Description Sharing of materials and knowledge for the investigation of biased GLP-1R/GIPR agonists 
Organisation MedImmune
Department MedImmune Cambridge
Country United Kingdom 
Sector Private 
PI Contribution Generation of cell lines and analysis of GLP-1R/GIPR biology
Collaborator Contribution Cell lines Antibodies
Impact Tagged receptor cell sublines
Start Year 2018
 
Description Single particle tracking of biased agonist-stimulated GLP-1Rs 
Organisation University of Birmingham
Department Institute of Metabolism and Systems Research (IMSR)
Country United Kingdom 
Sector Academic/University 
PI Contribution Cell lines, plasmids and biased agonists
Collaborator Contribution SPT tracking TIRFM and image analysis
Impact No outputs yet
Start Year 2019
 
Description Single-molecule localisation microscopy analysis of plasma membrane GPCR dynamics 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Preparation of samples and labelling
Collaborator Contribution Imaging and data analysis
Impact Joint publication: Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells. PLoS Biol. 2019 Aug 20;17(8):e3000097. doi: 10.1371/journal.pbio.3000097.
Start Year 2019
 
Description Structural analysis of GLP-1R-biased agonist complex 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution Biased agonists
Collaborator Contribution Expertise in structural biology
Impact No outputs yet
Start Year 2019
 
Description Transcriptome and kinome analysis of GLP-1R biased agonist signalling 
Organisation Imperial College London
Department Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Data on GLP-1R trafficking and signalling differences
Collaborator Contribution RNA seq and kinome analysis
Impact No output yet
Start Year 2017
 
Description Transcriptome and kinome analysis of GLP-1R biased agonist signalling 
Organisation Imperial College London
Department Department of Paediatrics
Country United Kingdom 
Sector Academic/University 
PI Contribution Data on GLP-1R trafficking and signalling differences
Collaborator Contribution RNA seq and kinome analysis
Impact No output yet
Start Year 2017
 
Title "RETRO study" 
Description "RETRO study" - a human physiological study investigating the effects of biased GLP-1R agonists in humans. Academy of Medical Sciences. "Studying the impact of biased GLP-1 receptor signalling in humans". Ben Jones (PI), £30,000. 08/2018 - 08/2019 Please note this is a physiological study rather than a Clinical Trial of an Investigational Medicinal Product 
Type Therapeutic Intervention - Drug
Current Stage Of Development Refinement. Clinical
Year Development Stage Completed 2018
Development Status Under active development/distribution
Impact Human physiological study investigating the effects of biased GLP-1R agonists in humans. 
 
Description COMPARE Workshop on Single-Molecule imaging and spectroscopy of membrane proteins 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Participation in COMPARE Workshop on Single-Molecule imaging and spectroscopy of membrane proteins
Year(s) Of Engagement Activity 2019
 
Description Session chair, Diabetes UK Professional Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Session chair, Diabetes UK Professional Conference
Year(s) Of Engagement Activity 2019