Addressing the architecture, dynamics and activation of the CGRP receptor

Lead Research Organisation: University of Essex
Department Name: Life Sciences

Abstract

G protein coupled receptors (GPCRs) are the largest family of proteins in the human genome and also the largest target for therapeutic drugs; thus they are of enormous scientific and practical interest. They are divided into a number of families. Of these, family-A is the best understood, but family-B includes receptors which are likely to be important in many disease states and so it is important to understand how these function, both to further our knowledge of fundamental biology and also for the design of new drugs.

Calcitonin gene-related peptide (CGRP) is found throughout the nervous system and is particularly important in regulating both the cardiovascular system (the heart and blood vessels) and also the immune system and inflammation. The receptor for CGRP is of special scientific interest as it involves a GPCR called CLR and also a second protein called RAMP1. RAMP1 is a member of a protein family that modulates a number of GPCRs of which the best characterised is CLR. CGRP is also likely to be important both in cardiovascular disorders and any disease that involves inflammation. The peptide is a major cause of migraine and drugs which block CGRP receptors have shown great promise in clinical trials; however, so far it has not been possible to use these clinically because of toxicity problems. Thus, there is an urgent need to develop new drugs that could act on CGRP receptors.

The CGRP receptor is made up of two parts. A portion called the transmembrane domain is found in the membranes of cells. This is connected to the extracellular domain, which is on the outside of cells. CGRP interacts with both parts of this structure and causes the transmembrane domain to change shape. This causes the receptor to interact with other proteins, leading to cell activation. We have a crystal structure of the part of the CGRP receptor that is on the outside of cells. Unfortunately, we do not know how CGRP binds to this, nor do we know how it binds to the transmembrane domain. This severely limits our understanding of the receptor and our ability to design drugs that could target it.

We have previously used experimental data from a technique known as site-directed mutagenesis to construct a computer model of the transmembrane domain of the CGRP receptor. This transmembrane domain is very similar to the transmembrane domains of two family-B GPCRs which were crystallised after our computer model was produced. This gives us confidence that our approach of combining experimental and computational methods is valuable. In this project, we intend to extend the approach to study how CGRP binds to both domains of the receptor and how this causes the receptor to become activated. We will use mutagenesis and also methods where we physically cross-link CGRP to the receptor to identify contact points. We will then use these to construct computational models, which we can refine through further experimentation. Using a computer, we can predict how the receptor shape will change when CGRP binds to it, so identifying the mechanism for receptor activation. This knowledge will be benefitial in the design of new drugs which can either block the receptor or promote its activation.

Technical Summary

The CGRP receptor is a particularly interesting family B G-protein coupled receptor (GPCR) having an absolute requirement for an auxiliary protein known as Receptor activity modifying protein 1 (RAMP1). Class B GPCRs consist of a large extracellular domain (ECD) and a transmembrane domain (TMD). They frequently associate with accessory proteins belonging to the family of RAMPs. They act as receptors for a number of peptide hormones and neurotransmitters. They are attractive therapeutic targets but it has proved very difficult to obtain drugs that target them. Several crystal structures exist for the ECDs and there are crystal structures for two class B GPCRs (glucagon and CRF), but neither have bound peptides and the orientation between the TMD and ECD for any receptor remains speculative, as does the mechanism whereby agonists activate the receptors.

We have recently used a combination of site-directed mutagenesis and molecular modelling to propose a structure for CGRP bound to the TMD of CLR. This shows excellent agreement with the crystal structures, which were published after our modelled structures were deposited. Thus we propose that our methodology is robust. Furthermore, the presence of the RAMP provides additional constraints on the orientation of the ECD relative to the TMD, making the CGRP receptor especially amenable to modelling by greatly reducing the number of ways in which it could be modelled incorrectly.

We propose a strategy of photoaffinity cross-linking, disulphide trapping and point mutagenesis to provide experimental information on the architecture of the receptor when bound to CGRP and as a test for the modelling. This information will then be used to produce a model of the complex. We will use molecular dynamics and other modelling techniques to plan the experiments, to interpret the results and hence to determine the conformational changes caused by CGRP binding and so establish how the receptor is activated by its native agonist.

Planned Impact

The most immediate beneficiaries would be those companies with research programmes directed towards the development of CGRP antagonists for migraine, where there is clinical evidence of the effectiveness of these agents. Migraine alone is estimated to cost the UK economy £2.25 billion per annum (Steiner TJ., Lecture to the All Party Parliamentary Group on Primary Headache Disorders., 19 November 2008) and CGRP antagonists have been shown to be effective against migraine in clinical trials. There have been 66 new patent applications filed worldwide for CGRP antagonists since January 2010. Thus the development of new agents to target the CGRP receptor would be of considerable benefit both to the UK pharmaceutical industry and also the health and well-being of the UK population. The mode of binding of CGRP and the way it activates its receptor is likely to be shared by other peptides in this family such as amylin (implicated in the control of eating) and calcitonin (well-established for the treatment of osteoporosis), further adding to the value of the project. The spectrum of disorders covered by the CGRP family of peptides include many which are common amongst elderly populations (e.g. heart failure, osteoporosis) and so this project is relevant to the BBSRC's initiative on lifelong health and well-being. More broadly, the challenges resulting from CLR modelling have serendipitously resulted in the generation of a helix alignment program that can work below the twilight zone (Vohra et al., J. Roy. Soc. 2013, Taddese et al., Plant Phys 2014) and we expect other methodologies to result from this challenging problem. Here, the way in which the modelling is closely integrated with experiment be applicable to a wide range of proteins of pharmaceutical or other interest. These include G-protein coupled receptors but extend far beyond those. In this respect, the project also addresses the BBSRC initiative on Technology development for the biosciences.

Publications

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Bower R (2018) Molecular Signature for Receptor Engagement in the Metabolic Peptide Hormone Amylin in ACS Pharmacology & Translational Science

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Dal Maso E (2019) The Molecular Control of Calcitonin Receptor Signaling in ACS Pharmacology & Translational Science

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Rujan RM (2019) Calcitonin Gene-Related Peptide Antagonists and Therapeutic Antibodies. in Handbook of experimental pharmacology

 
Description 1. A homology model of the GLP1 receptor (Wotten et al., Mol Pharm, 2016)
2. Additional understanding on biased signalling (Wotten et al., Mol Pharm, 2016)
3. Additional understanding on the action of RAMPs on class B GPCs, namely glucagon. (Weston et al., JBC, 2015).
4. The cryo-EM structure of the CGRP receptor
5. The cryo-EM structure of the calcitonin receptor
Exploitation Route The model of the GLP1 receptor, a class B GPCR similar to our target receptor, the CGRP receptor, is available as supporting information to the Mol Pharm 2016 article; this may be used in target-based drug design.
The cryo-EM structures of the CGRP receptor is available from the protein databank (www.rcsb.org), pdb code 6e3y.
The cryo-EM structures of the calcitonin receptor is available from the protein databank (www.rcsb.org), pdb code 6niy (the 3.3 A structure replaces an early 4.1 A structure).
Both of the related cryo-EM structures can be used in structure-based drug design for a number of important diseases, e.g. migraine, heart disease, obesity.
Sectors Agriculture, Food and Drink,Chemicals,Pharmaceuticals and Medical Biotechnology

URL https://www.essex.ac.uk/news/2018/09/13/key-villain-in-migraine-brought-to-life
 
Description Several newspaper articles reported our work on Biased signalling, (Wootten et al., Cell 2016) under a diabetes-related theme of designing better drugs. CAR has joined Heptares Therapeutics as a Royal Society Industry Fellow as a result of earlier publications (e.g. Cell 2016) to develop improved GPCR models.
First Year Of Impact 2016
Sector Education
Impact Types Societal

 
Description Pancreatic cancer UK
Amount £74,285 (GBP)
Funding ID RPG-2017-255 
Organisation Pancreatic Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2017 
End 02/2018
 
Description Royal Society Industrial Fellowships
Amount £151,861 (GBP)
Funding ID IF160090 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 09/2017 
 
Description Royal Society Summer Studentship
Amount £2,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 08/2017 
End 09/2017
 
Description Summer studentship for Royal Society Industrial Fellowships
Amount £2,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 07/2019 
End 08/2019
 
Description Summer studentship for Royal Society Industry Fellows
Amount £2,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 06/2018 
End 07/2018
 
Description Tier 2 computer time
Amount £30,000 (GBP)
Funding ID EPSRC tier 2 award of computer time (3M core hours) at Cambridge; the cost above is an estimate of the value based on 1p per core hour 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 11/2018 
End 08/2019
 
Title Method for generating GPCR models 
Description We have published a method for generating class B G protein-coupled receptor (GPCR) structures from X-ray, NMR or homology modelled sub-structures that is particularly suited to the 2 domain extracellular domain (ECD)/transmembrane (TM) structure of class B GPCRs. The essential feature is to generate partially overlapping fragments, which can be achieved through carefully docking of the full peptide ligand to both the ECD and the TM domain. The method also involves a two-step approach to handing photoaffinity labelling by first generating the model containing the labels in the presence of constraints and then translating the constraints into equivalent constraints for the wild-type receptor. The method is described in a series of 2016 publications, including: Wootten, D.; Reynolds, C. A.; Smith, K. J.; Mobarec, J. C.; Koole, C.; Savage, E. E.; Pabreja, K.; Simms, J.; Sridhar, R.; Furness, S. G.; Liu, M.; Thompson, P. E.; Miller, L. J.; Christopoulos, A.; Sexton, P. M. The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism. Cell 2016, 165, 1632-43. Weston, C.; Winfield, I.; Harris, M.; Hodgson, R.; Shah, A.; Dowell, S. J.; Mobarec, J. C.; Woodlock, D. A.; Reynolds, C. A.; Poyner, D. R.; Watkins, H. A.; Ladds, G. Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors. J. Biol. Chem. 2016, 291, 21925-21944. 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? Yes  
Impact The method was taken up by Heptares Therapeutics and was used to model a particular receptor (by Dr Conor Scully) as part of a drug design program. This enabled the programme to get off to a good start and the programme is progressing well. As a result, a REF impact case is being prepared. 
 
Title Supervised molecular dynamics method for studying th ebinding of class B GPCR peptides 
Description Supervised molecular dynamics, SuMD, is an adaptive sampling method for computationally modelling the binding of a ligand from outside of an enzyme/receptor to the receptor binding site. The method had been published previously, but we adapted the method for the binding of class B peptides to their receptor. Without such a method the simulations could take months. 
Type Of Material Improvements to research infrastructure 
Year Produced 2018 
Provided To Others? Yes  
Impact The article was spotted by Miles Congreve at Sosei Heptares and circulated internally; the company are interested in drugs for class B G protein coupled receptors 
 
Title CGRP 
Description Computer models of the CGRP receptor have been deposited in the Essex Research Repository and are given a DOI from the relevant publications 
Type Of Material Computer model/algorithm 
Year Produced 2017 
Provided To Others? Yes  
Impact Deeper understanding into the structure and function of the CGRP receptor that may be relevant to drug design and heart disease/migraine. 
URL http://repository.essex.ac.uk
 
Title CTR/AMY 
Description Molecular models of the calcitonin receptor (CTR) and the Amylin Receptor (AMY1R, i.e. CTR in complex with a receptor activity modifying protein). These models are stored in the Essex Research Repository and are referenced from associated publications / articles submitted. 
Type Of Material Computer model/algorithm 
Year Produced 2018 
Provided To Others? No  
Impact Deeper understanding into the structure and dynamics of these calcitonin-based receptor models that are related to various diseases including osteoporosis, migraine and diabetes. 
URL http://repository.essex.ac.uk/
 
Title GLP-1R 
Description Computer models have been generated of the GLP-1 receptor, the adrenomedullin receptor and the PTH2 receptor; these have been validated by collaborative experimental studies. The models are available from ftp.essex.ac.uk/pub/oyster/ 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact The models have had a significant impact on explaining pharmacological data on important drug targets, as indicated in the following publications: 1. Wotten, D., Reynolds, C. A., Smith, K. J., Mobarec, J. C., Koole, C., Savage, E. E., Pabreja, K., Simms, J., Sridhar, R., and Furness, S. G., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2016) The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism. Cell 165,1632-1643. doi.org/10.1016/j.cell.2016.05.023 2. Wootten, D., Reynolds, C. A., Smith, K. J., Mobarec, J. C., Furness, S. G., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2016) Key interactions by conserved polar amino acids located at the transmembrane helical boundaries in Class B GPCRs modulate activation, effector specificity and biased signalling in the glucagon-like peptide-1 receptor. Biochem. Pharmacol. 118,68-87. doi.org/10.1016/j.bcp.2016.08.015 3. Wootten, D., Reynolds, C. A., Koole, C., Smith, K. J., Mobarec, J. C., Simms, J., Quon, T., Coudrat, T., Furness, S. G., and Miller, L. J. (2016) A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism: lessons from class B crystal structures. Mol. Pharmacol. 89,335-347. doi.org/10.1124/mol.115.101246 4. Weston, C., Winfield, I., Harris, M., Hodgson, R., Shah, A., Dowell, S. J., Mobarec, J. C., Woodlock, D. A., Reynolds, C. A., and Poyner, D. R. (2016) Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors. J. Biol. Chem. 291,21925-21944. doi.org/10.1074/jbc.M116.751362 5. Weaver, R. E., Mobarec, J. C., Wigglesworth, M. J., Reynolds, C. A., and Donnelly, D. (2016) High affinity binding of the peptide agonist TIP-39 to the parathyroid hormone 2 (PTH 2) receptor requires the hydroxyl group of Tyr-318 on transmembrane helix 5. Biochem. Pharmacol. doi.org/10.1016/j.bcp.2016.12.013 6. Watkins, H. A., Chakravarthy, M., Abhayawardana, R. S., Gingell, J. J., Garelja, M., Pardamwar, M., McElhinney, J. M., Lathbridge, A., Constantine, A., and Harris, P. W. (2016) Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties. J. Biol. Chem. 291,11657-11675. doi.org/10.1074/jbc.M115.688218 
URL http://ftp.essex.ac.uk/pub/oyster/
 
Description Biased signalling in the GLP1 receptor 
Organisation Mayo Clinic
Department Molecular Pharmacology and Experimental Therapeutics
Country United States 
Sector Hospitals 
PI Contribution homology Modelling the GLP1 receptor; molecular dynamics studies of the GLP1 receptor; docking studies on ligands binding to the receptor
Collaborator Contribution Experimental mutagenesis studies of the GLP1 receptor and an analysis of the preferred signalling pathways of the receptor in the presence of various agonists, namely GLP1 peptide, oxyntmodulin and exendin-4
Impact Wootten, D.; Reynolds, C. A.; Koole, C.; Smith, K. J.; Mobarec, J. C.; Quon, T.; Coudrat, T.; Furness, S. G. B.; Miller, L. J.; Christopoulos, A.; Sexton, P. M. A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism: lessons from class B crystal structures. Mol. Pharmacol. (resubmitted) 2016, 89, 335-347
Start Year 2015
 
Description Biased signalling in the GLP1 receptor 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution homology Modelling the GLP1 receptor; molecular dynamics studies of the GLP1 receptor; docking studies on ligands binding to the receptor
Collaborator Contribution Experimental mutagenesis studies of the GLP1 receptor and an analysis of the preferred signalling pathways of the receptor in the presence of various agonists, namely GLP1 peptide, oxyntmodulin and exendin-4
Impact Wootten, D.; Reynolds, C. A.; Koole, C.; Smith, K. J.; Mobarec, J. C.; Quon, T.; Coudrat, T.; Furness, S. G. B.; Miller, L. J.; Christopoulos, A.; Sexton, P. M. A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism: lessons from class B crystal structures. Mol. Pharmacol. (resubmitted) 2016, 89, 335-347
Start Year 2015
 
Description GLP-1 signalling and bias (with Imperial) 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Preliminary data on the structure of the GLP-1 receptor (GLP-1R)
Collaborator Contribution Preliminary data on identification of biased ligands for GLP-1R
Impact Award of an MRC grant entitled XXX on GLP-1R signalling and bias, (PI: , Co-Is: ); CAR was very pleased to supply a letter of support
Start Year 2017
 
Description Modelling remote homologues (with Johnson Matthey) 
Organisation Johnson Matthey
Country United Kingdom 
Sector Private 
PI Contribution Sequence alignment in the twilight zone in the context of modelling remote homologues for building models of enzymes required for carrying out chemical reactions
Collaborator Contribution Supplied sequences
Impact alignments
Start Year 2018
 
Description Modelling the Adenosine Receptor 
Organisation University of Cambridge
Department Department of Pharmacology
Country United Kingdom 
Sector Academic/University 
PI Contribution Molecular modelling of the adenosine A1 receptor
Collaborator Contribution Molecular pharmacology of the A1 receptor
Impact multi-disciplinary: molecular modelling and experimental pharmacology
Start Year 2017
 
Description Royal Society Industrial Fellowship: Markov State Modelling 
Organisation Heptares Therapeutics Ltd
Country United Kingdom 
Sector Private 
PI Contribution Expertise in modelling GPCRs
Collaborator Contribution Access to specialist software; specialist knowledge on GPCRs and drug design
Impact Summer student trained in bioinformatics
Start Year 2017
 
Description Drug Design Workshop for the giften and able 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 8 Gifted and able students from Bromfords School, Essex, attended a week-long drug design workshop, which greatly increased the pupils enthusiasm for science.
Year(s) Of Engagement Activity 2016