I24-Microfocus Macromolecular Crystallography
|
Open Access
Abstract: Here we report an efficient method to generate multiple co-structures of the A2A G protein-coupled receptor (GPCR) with small-molecules from a single preparation of a thermostabilised receptor crystallised in Lipidic Cubic Phase (LCP). Receptor crystallisation is achieved following purification using a low affinity “carrier” ligand (theophylline) and crystals are then soaked in solutions containing the desired (higher affinity) compounds. Complete datasets to high resolution can then be collected from single crystals and seven structures are reported here of which three are novel. The method significantly improves structural throughput for ligand screening using stabilised GPCRs, thereby actively driving Structure-Based Drug Discovery (SBDD).
|
Jan 2018
|
|
I24-Microfocus Macromolecular Crystallography
|
Abstract: The structural analysis of class B G protein-coupled receptors (GPCR), cell surface proteins responding to peptide hormones, has until recently been restricted to the extracellular domain (ECD). Corticotropin-releasing factor receptor type 1 (CRF1R) is a class B receptor mediating stress response and also considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of human CRF1R in complex with the small-molecule antagonist CP-376395 in a hexagonal setting with translational non-crystallographic symmetry. Molecular dynamics and metadynamics simulations on this novel structure and the existing TMD structure for CRF1R provides insight as to how the small molecule ligand gains access to the induced-fit allosteric binding site with implications for the observed selectivity against CRF2R. Furthermore, molecular dynamics simulations performed using a full-length receptor model point to key interactions between the ECD and extracellular loop 3 of the TMD providing insight into the full inactive state of multidomain class B GPCRs.
|
Oct 2017
|
|
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[12425]
Abstract: The adenosine A1 and A2A receptors belong to the purinergic family of G protein-coupled receptors, and regulate diverse functions of the cardiovascular, respiratory, renal, inflammation, and CNS. Xanthines such as caffeine and theophylline are weak, non-selective antagonists of adenosine receptors. Here we report the structure of a thermostabilized human A1 receptor at 3.3 Å resolution with PSB36, an A1-selective xanthine-based antagonist. This is compared with structures of the A2A receptor with PSB36 (2.8 Å resolution), caffeine (2.1 Å), and theophylline (2.0 Å) to highlight features of ligand recognition which are common across xanthines. The structures of A1R and A2AR were analyzed to identify the differences that are important selectivity determinants for xanthine ligands, and the role of T2707.35 in A1R (M2707.35 in A2AR) in conferring selectivity was confirmed by mutagenesis. The structural differences confirmed to lead to selectivity can be utilized in the design of new subtype-selective A1R or A2AR antagonists.
|
Jul 2017
|
|
I24-Microfocus Macromolecular Crystallography
|
Ali
Jazayeri
,
Mathieu
Rappas
,
Alastair J. H.
Brown
,
James
Kean
,
James C.
Errey
,
Nathan J.
Robertson
,
Cédric
Fiez-Vandal
,
Stephen P.
Andrews
,
Miles
Congreve
,
Andrea
Bortolato
,
Jonathan S.
Mason
,
Asma H.
Baig
,
Iryna
Teobald
,
Andrew S.
Dore
,
Malcolm
Weir
,
Robert M.
Cooke
,
Fiona H.
Marshall
Diamond Proposal Number(s):
[12425]
Abstract: Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.
|
May 2017
|
|
I24-Microfocus Macromolecular Crystallography
|
Robert K. Y.
Cheng
,
Cédric
Fiez-Vandal
,
Oliver
Schlenker
,
Karl
Edman
,
Birte
Aggeler
,
Dean G.
Brown
,
Giles A.
Brown
,
Robert M.
Cooke
,
Christoph E.
Dumelin
,
Andrew S.
Dore
,
Stefan
Geschwindner
,
Christoph
Grebner
,
Nils-Olov
Hermansson
,
Ali
Jazayeri
,
Patrik
Johansson
,
Louis
Leong
,
Rudi
Prihandoko
,
Mathieu
Rappas
,
Holly
Soutter
,
Arjan
Snijder
,
Linda
Sundström
,
Benjamin
Tehan
,
Peter
Thornton
,
Dawn
Troast
,
Giselle
Wiggin
,
Andrei
Zhukov
,
Fiona H.
Marshall
,
Niek
Dekker
Diamond Proposal Number(s):
[12425, 5065]
Abstract: Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation1, 2, 3. PARs have been the subject of major pharmaceutical research efforts3 but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar4, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously5. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist6. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.
|
Apr 2017
|
|
I24-Microfocus Macromolecular Crystallography
|
Christine
Oswald
,
Mathieu
Rappas
,
James
Kean
,
Andrew
Dore
,
James C.
Errey
,
Kirstie
Bennett
,
Francesca
Deflorian
,
John A.
Christopher
,
Ali
Jazayeri
,
Jonathan S.
Mason
,
Miles
Congreve
,
Robert M.
Cooke
,
Fiona H.
Marshall
Diamond Proposal Number(s):
[14637]
Abstract: Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization4. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease6. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn’s disease but efficacy was limited, with the need for very high doses to block receptor activation6. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.
|
Dec 2016
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Elena
Segala
,
Dong
Guo
,
Robert K. Y.
Cheng
,
Andrea
Bortolato
,
Francesca
Deflorian
,
Andrew S.
Doré
,
James C.
Errey
,
Laura H.
Heitman
,
Adriaan P.
Ijzerman
,
Fiona H.
Marshall
,
Robert M.
Cooke
Abstract: The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.
|
Jul 2016
|
|
I04-Macromolecular Crystallography
|
Ali
Jazayeri
,
Andrew S.
Doré
,
Daniel
Lamb
,
Harini
Krishnamurthy
,
Stacey M.
Southall
,
Asma H.
Baig
,
Andrea
Bortolato
,
Markus
Koglin
,
Nathan J.
Robertson
,
James C.
Errey
,
Stephen P.
Andrews
,
Iryna
Teobald
,
Alastair J. H.
Brown
,
Robert M.
Cooke
,
Malcolm
Weir
,
Fiona H.
Marshall
Abstract: Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis1. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations2. Although an X-ray structure of the transmembrane domain of the GCGR3 has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined—for the corticotropin-releasing hormone receptor 1 (CRF1R)—which was located deep within the 7TM bundle5. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.
|
May 2016
|
|
I24-Microfocus Macromolecular Crystallography
|
John A.
Christopher
,
Sarah J.
Aves
,
Kirstie A.
Bennett
,
Andrew S.
Doré
,
James C.
Errey
,
Ali
Jazayeri
,
Fiona H.
Marshall
,
Krzysztof
Okrasa
,
Maria J.
Serrano-Vega
,
Benjamin G.
Tehan
,
Giselle R.
Wiggin
,
Miles
Congreve
Abstract: Fragment screening of a thermostabilized mGlu5 receptor using a high-concentration radioligand binding assay enabled the identification of moderate affinity, high ligand efficiency (LE) pyrimidine hit 5. Subsequent optimization using structure-based drug discovery methods led to the selection of 25, HTL14242, as an advanced lead compound for further development. Structures of the stabilized mGlu5 receptor complexed with 25 and another molecule in the series, 14, were determined at resolutions of 2.6 and 3.1 Å, respectively.
|
Aug 2015
|
|
I24-Microfocus Macromolecular Crystallography
|
Alexander
Heifetz
,
Gebhard F. X.
Schertler
,
Roland
Seifert
,
Christopher G.
Tate
,
Patrick M.
Sexton
,
Vsevolod V.
Gurevich
,
Daniel
Fourmy
,
Vadim
Cherezov
,
Fiona H.
Marshall
,
R. Ian
Storer
,
Isabel
Moraes
,
Irina G.
Tikhonova
,
Christofer S.
Tautermann
,
Peter
Hunt
,
Tom
Ceska
,
Simon
Hodgson
,
Mike J.
Bodkin
,
Shweta
Singh
,
Richard J.
Law
,
Philip C.
Biggin
Diamond Proposal Number(s):
[5953, 11386]
Open Access
Abstract: G-protein coupled receptors (GPCRs) are the targets of over half of all prescribed drugs today. The UniProt database has records for about 800 proteins classified as GPCRs, but drugs have only been developed against 50 of these. Thus, there is huge potential in terms of the number of targets for new therapies to be designed. Several breakthroughs in GPCRs biased pharmacology, structural biology, modelling and scoring have resulted in a resurgence of interest in GPCRs as drug targets. Therefore, an international conference, sponsored by the Royal Society, with world-renowned researchers from industry and academia was recently held to discuss recent progress and highlight key areas of future research needed to accelerate GPCR drug discovery. Several key points emerged. Firstly, structures for all three major classes of GPCRs have now been solved and there is increasing coverage across the GPCR phylogenetic tree. This is likely to be substantially enhanced with data from x-ray free electron sources as they move beyond proof of concept. Secondly, the concept of biased signalling or functional selectivity is likely to be prevalent in many GPCRs, and this presents exciting new opportunities for selectivity and the control of side effects, especially when combined with increasing data regarding allosteric modulation. Thirdly, there will almost certainly be some GPCRs that will remain difficult targets because they exhibit complex ligand dependencies and have many metastable states rendering them difficult to resolve by crystallographic methods. Subtle effects within the packing of the transmembrane helices are likely to mask and contribute to this aspect, which may play a role in species dependent behaviour. This is particularly important because it has ramifications for how we interpret pre-clinical data. In summary, collaborative efforts between industry and academia have delivered significant progress in terms of structure and understanding of GPCRs and will be essential for resolving problems associated with the more difficult targets in the future.
|
Mar 2015
|
|
