WO2016030675A1 - Gpcr receptor binding domain and uses thereof - Google Patents

Abstract

The present invention relates to the three-dimensional ligand binding domain of the Glucagon receptor (GCGR) and its use in the identification and development of compounds for drug discovery and therapy. The invention further relates to methods of designing compounds.

Description

GPCR Receptor Binding Domain and Uses Thereof

The present invention relates generally to the field of protein structure and drug screening. Specifically the invention relates to the three-dimensional binding domain of the Glucagon receptor (GCGR) and its use in the identification and development of compounds for drug discovery and therapy.

Background to the Invention The Glucagon receptor (GCGR) is a G Protein Coupled Receptor (GPCR) and a member of the secretin-like (class B) family of GPCRs. Like other GPCRs, the GCGR is composed of seven trans-membrane helices, and, being a family B GPCR, it is further characterised by the presence of a large domain at the N-terminus of the protein, located on the extra-cellular side of the cell membrane. Class B GPCRs include receptors for peptides such as secretin, glucagon, glucagon-like peptide, calcitonin and parathyroid peptide hormone and have been studied as drug targets in the treatment of various diseases, including diabetes,

osteoporosis, depression and anxiety (1). In common with other class B GPCR members, the peptide ligand binds to both the N-terminal domain and to a cavity created by the bundle of seven helices (2).

The GCGR is expressed in the external membrane of several cell types, particularly those of the liver and kidney, and plays a critical role in the regulation of blood glucose levels (3). Activation by binding of the hormonal peptide glucagon initiates an intracellular G protein signalling cascade, leading to activation of adenylate cyclase, which increases levels of cAMP, stimulating glycogenolysis and gluconeogenesis, resulting in the release of glucose from the liver. Glucagon thus counteracts the role of insulin in maintaining blood glucose homeostasis.

The role of the GCGR in glucose homeostasis has been established through observations such as the physiological effects of mutations in the GCGR gene. Excessive activation of the receptor has been associated with insulin resistance and the development of diabetes. Thus, antagonists of GCGR have been the subject of drug discovery efforts to identify therapeutic agents for diabetes and related metabolic diseases, and blocking antibodies and antagonists of GCGR have been effective in animal models of diabetes (4). A GCGR antagonist has also been reported to block glucagon-induced hyperglycemia in humans (5).

Although a number of small molecule antagonists of GCGR have been identified, none have been accompanied by clear evidence as to how they interact with GCGR, and optimisation of these compounds has relied on empirical medicinal chemistry approaches, which without the understanding of the functional and structural properties of the binding site can be time consuming and inefficient. Efforts to discover drugs targeting GPCRs have been enhanced in recent years by the determination of the three-dimensional (3D) structures of several members of this family of proteins (6). In particular the 3D structures of complexes between GPCRs and modulating compounds can be highly informative in understanding how compounds bind to the receptor, and how optimised compounds suitable as drug candidates can be obtained. A 3D structure of a truncated form of the GCGR, containing the seven trans-membrane helix domain, but lacking the N-terminal domain has been reported (7) but, despite being crystallised in the presence of an antagonist, the antagonist could not be observed in the structure and was predicted to be in the main cavity of the seven transmembrane helix core.

There is a need to characterise further the GCGR binding site to understand its structural and functional features with respect to ligand binding.

The inventors have crystallised the GCGR complexed with the small molecule inhibitor compound MK-0893 (8) and identified the binding site of GCGR for this ligand. This has enabled the development of a pharmacophore model enabling more efficient drug selection, isolation and optimisation of candidate molecules. Knowledge of the precise three-dimensional features of the binding site, including the key amino acids involved in binding allows candidate molecules to be synthesised by design and thereby improves the drug discovery process and potentially the ultimate therapeutic efficacy of drug candidates.

In addition the inventors have found that the position of the binding site is novel compared to binding sites previously identified for class A and B GPCRs and so represents a surprising discovery that could not have been predicted from the prior art. CRF1 is a class B GPCR receptor and has structural homology to GCGR, however the binding site of GCGR is located towards the outer regions of the receptor helices and not in the same internal location as the reported CRF1 binding site.

The novel binding site of the GCGR provides insight into the modulation of other class B GPCRs and has use in the screening and development of drug candidates for use in the treatment of diabetes.

Summary of the Invention According to a first aspect of the invention there is provided a polypeptide comprising a binding domain of a GPCR wherein the binding domain comprises the amino acids Arg346 (6.37), Lys349 (6.40) and Ser350 (6.41) and Asn404 (7.61), wherein the structure

coordinates are optionally varied within an root mean square deviation (rmsd) of residue backbone atoms of not more than 2.5 A. According to a further aspect of the invention there is provided a GPCR comprising a binding domain as defined herein.

According to a further aspect of the invention there is provided a method of selecting a binding partner capable of binding to a binding domain of a polypeptide as defined herein, comprising the steps of; a) providing a polypeptide or a GPCR comprising a binding domain;

b) contacting the polypeptide or GPCR with one or more candidate compounds;

c) determining whether one or more candidate compounds bind to the polypeptide or GPCR;

d) isolating one or more candidate compounds that bind to the polypeptide or GPCR;

e) optionally synthesising one or more candidate compounds. According to a further aspect of the invention there is provided a method of designing a compound that is capable of binding to a binding domain of a polypeptide as defined herein comprising the steps of; a) comparing the three-dimensional structure of a candidate compound with the three- dimensional structure of the binding domain according to the structure coordinates in Table 1, optionally varied by a root mean square deviation of residue backbone atoms of not more than 2.5 A,

b) selecting one or more compounds that are predicted to bind to the functional binding domain by molecular modelling means,

c) optionally modifying one or more selected compounds to improve binding to the functional binding domain,

d) optionally synthesising one or more selected compounds.

According to a further aspect of the invention there is provided a compound obtained by the methods as defined herein.

According to a further aspect of the invention there is provided an antibody specifically binding to the binding domain as defined herein. According to a further aspect of the invention there is provided a compound for use in the treatment of conditions associated with class B GPCRs. Brief Description of the Figures.

Figure 1

A schematic representation of compound MK-0893 in the Glucagon binding domain and the associated interactions between the compound and amino acids forming the binding domain.

Figure 2

(a) A cartoon showing the position of the Glucagon binding domain (a) in the Glucagon receptor with respect to the position of other known ligands for family A GPCRs and CRFl. It can be seen that the Glucagon receptor ligand occupies a novel site which is located on the outer regions of the receptor helices, in contrast to the CRFl ligand which is buried within the molecule.

(b) A cartoon showing an enhanced view of the Glucagon binding domain with the compound MK-0893.

Detailed Description of the Invention According to a first aspect of the invention there is provided a polypeptide comprising a binding domain of a GPCR wherein the binding domain comprises the amino acids Arg346 (6.37), Lys349 (6.40) and Ser350 (6.41) and Asn404 (7.61), wherein the structure

coordinates are optionally varied within an root mean square deviation (rmsd) of residue backbone atoms of not more than 2.5 A.

The number following each amino acid refers to its position in the sequence of the full- length unprocessed receptor and the number in brackets refers to the helix location of the residue as defined in Wootten et al. Proc Natl Acad Sci USA 110, 5211-5216, (2013) and used by Siu et al, Nature, 499; 444-451 (2013) to describe the glucagon structure. Residues in the transmembrane helices are assigned a number of the form X.YZ where X refers to which of the seven transmembrane helices it belongs, and YZ refers to its position along that helix relative to the residue most conserved across class B receptors at position 50. For example, Arg345 (6.37) refers to an arginine amino acid at position 345 in the polypeptide sequence of the GCGR, located on the 6^th trans-membrane helix and being 13 residues before the residue in the helix most conserved across family B receptors.

References to "binding domain" herein refer to the region within the three-dimensional structure of a protein or polypeptide involved in binding to a ligand or compound. The binding domain may be characterised by the position of particular amino acids on the polypeptide chain which form interactions with a ligand or compound. Alternatively the binding domain may be characterised by a computer generated three-dimensional structural representation which provides the atomic coordinates of the amino acids interacting with a ligand. Coordinates are derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centres) of the protein in a crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are then used to establish the individual atoms of the GPCR binding domain. It is understood that a set of structure coordinates for a polypeptide is a relative set of points that define a shape in three dimensions. Computer representations can be generated or displayed by commercially available software programs. Examples of software programs include but are not limited to QUANTA (Accelrys COPYRIGHT, 2001, 2002), O (Jones et al., Acta Crystallogr. A47, pp. 110-119 (1991)), RIBBONS (Carson, J. Appl. Crystallogr., 24, pp. 9589-961 (1991)) and PyMol (The PyMol Molecular Graphics System, Schrodinger, LLC), which are incorporated herein by reference. Examples of representations include any of a wire-frame model, a chicken-wire model, a ball-and-stick model, a space-filling model, a stick model, a ribbon model, a snake model, an arrow and cylinder model, an electron density map or a molecular surface model. Certain software programs may also imbue these three-dimensional representations with physico-chemical attributes which are known from the chemical composition of the molecule, such as residue charge, hydrophobicity, torsional and rotational degrees of freedom for the residue or segment, etc.

It is appreciated that the coordinates of the Glucagon binding domain used in the invention may be optionally varied. Such variation may be necessary in various aspects of the invention, for example in the fitting of various binding partners to the Glucagon binding domain structure. Slight variations in the coordinates of individual amino acids will have little effect on overall shape of the binding domain. For the purpose of the invention, any three-dimensional structure that has a root mean square deviation of conserved residue backbone atoms between 0.00 A and 2.50 A and preferably between 0.00 A and 2.00 A, 0.00 A and 1.50 A, 0.00 A and 1.00 A or 0.00 A and 0.50 A when superimposed on the relevant backbone atoms described by the structure coordinates of the Glucagon binding domain (Table 1) are considered identical or the same. For the purpose of the invention, structure coordinates are considered identical or the same even if slight variations are present in the individual coordinates if these do not affect the overall shape defined by the structure coordinates.

Protein structure variability and similarity is routinely expressed and measured by the root mean square deviation (rmsd), which measures the difference in positioning in space between two sets of atoms. The rmsd measures distance between equivalent atoms after their optimal superposition. The rmsd can be calculated over any sets of selected atoms including all atoms, over residue backbone atoms (i.e. the nitrogen-carbon-carbon backbone atoms of the protein amino acid residues), main chain atoms only (i.e. the nitrogen-carbon- oxygen-carbon backbone atoms of the protein amino acid residues), side chain atoms only or over C-ct atoms only. The least-squares algorithms used to calculate rmsd are well known in the art and include those described by Rossman and Argos (J Biol Chem, (1975) 250:7525), Kabsch (Acta Cryst (1976) A92:922; Acta Cryst (1978) A34:827-828), Hendrickson (Acta Cryst (1979) A35: 158), McLachan {J Mol Biol (1979) 128:49) and Kearsley (Acta Cryst (1989) A45:208). Both algorithms based on iteration in which one molecule is moved relative to the other, such as that described by Ferro and Hermans (Acta Cryst (1977) A33:345-347), and algorithms which locate the best fit directly (e.g. Kabsch's methods) may be used. Methods of comparing proteins structures are also discussed in Methods of Enzymology, vol 115: 397-420.

Typically, rmsd values are calculated using coordinate fitting computer programs and any suitable computer program known in the art may be used, for example MNYFIT (part of a collection of programs called COMPOSER, Sutcliffe et al (1987) Protein Eng 1:377-384). Other programs also include LSQMAN (Kleywegt & Jones (1994) A super position, CCP4/ESF- EACBM, Newsletter on Protein Crystallography, 31: 9-14), LSQKAB (Collaborative

Computational Project 4. The CCP4 Suite: Programs for Protein Crystallography, Acta Cryst (1994) D50:760-763), QUANTA (Jones et al, Acta Cryst (1991) A47:110-119 and commercially available from Accelrys, San Diego, CA), Insight (Commercially available from Accelrys, San Diego, CA), Sybyl^® (commercially available from Tripos, Inc., St Louis) and O (Jones et al., Acta Cryst (1991) A47:110-119). The program Sequoia (Bruns et al (1999) J Mol Biol

288(3):427-439) performs the alignment of homologous protein sequences, and the superposition of homologous protein atomic coordinates. Once aligned, the rmsd can be calculated using programs detailed above. When the sequences are identical or highly similar, the structural alignment of proteins can be done manually or automatically as outlined above. Another approach would be to generate a superposition of protein atomic coordinates without considering the sequence.

In another embodiment the binding domain comprises the comprises the amino acids Arg346 (6.37), Lys349 (6.40), Ser350 (6.41), Asn404 (7.61),Tyr343 (6.34) and Leu399 (7.56) wherein the structure coordinates are optionally varied within a root mean square deviation (rmsd) of residue backbone atoms of not more than 2.5 A

In another embodiment the binding domain comprises the amino acids Arg346 (6.37), Lys349 (6.40), Ser350 (6.41), Asn404 (7.61), Leu329 (5.61) and Leu352 (6.43) wherein the structure coordinates are optionally varied within a root mean square deviation (rmsd) of residue backbone atoms of not more than 2.5 A In another embodiment the binding domain comprises the amino acids Tyr343 (6.34) Arg346 (6.37), Lys349 (6.40), Ser350 (6.41), Leu399 (7.56), Asn404 (7.61), Leu329 (5.61), Phe345 (6.36), Ala348 (6.39), Leu352 (6.43), Thr353 (6.44) and Leu403 (7.60) wherein the structure coordinates are optionally varied within a root mean square deviation (rmsd) of residue backbone atoms of not more than 2.5 A

In a further embodiment the structure coordinates may be optionally varied within an rmsd of residue backbone atoms (i.e. the nitrogen-carbon-carbon backbone atoms of the protein) of not more than 2.5 A. In one example the structure coordinates are varied within a rmsd of residue backbone atoms of not more than 2.5 A, 2.4 A, 2.3 A, 2.2 A, 2.1 A, 2.0 A, 1.9 A, 1.8 A, 1.7 A, 1.6 A, 1.5 A, 1.4 A, 1.3 A, 1.2 A, 1.1 A, 1.0 A, 0.9 A, 0.8 A, 0.7 A, 0.6 A, 0.5 A, 0.4 A, 0.3 A, 0.2 A or 0.1 A.

Table 1 lists the atomic structure coordinates of human Glucagon binding domain, specifically amino acid residues 329, 343, 345, 346, 348, 349, 350, 352, 353, 399, 403 and 404 in the presence of compound MK-0893, a known antagonist of the Glucagon receptor (8). The characterisation of a binding domain in the presence of an antagonist provides valuable insight into the function of the binding domain and the residues crucial in forming an antagonist conformation. Likewise it is appreciated that it can also be deduced from the model produced by the atomic coordinates, those amino acid residues that would be important in forming an agonist conformation (Figures 2(a) and 2(b)).

Table 2 lists amino acids at defined structure coordinates or helix locations for the Glucagon receptor and the corresponding amino acids at the same coordinate position for other members of Family B GPCRs. The data show that when the three-dimensional structural representation of various class B members is compared to that of the Glucagon receptor there is a degree of amino acid conservation at certain three-dimensional positions suggesting that the binding domain for class B GPCRs is conserved. The data suggest that other class B GPCRs are likely to have a binding domain in the same location of the GPCR.

In another embodiment the binding domain is defined according to the coordinates as listed in Table 1.

According to a further embodiment of the invention the binding domain comprises the amino acids Arg346 (6.37), Lys349 (6.40), Ser350 (6.41) and Asn404(7.61) , wherein the structure coordinates are optionally varied within a root mean square deviation (rmsd) of residue backbone atoms of not more than 2.5 A and wherein the one or more amino acids Arg346 (6.37), Lys349 (6.40) and Ser350 (6.41), may be optionally substituted with one or more of Lys, Arg and Ala, respectively at the corresponding position in a different GPCR. Reference to "corresponding position" herein refers to the equivalent position in the amino acid sequence of a GPCR when the sequence of the GPCR is compared with that of the Glucagon receptor by alignment using, for example, MacVector and the Clustal W program. Such alignment techniques are known in the art and appreciated by the skilled person. For example, Table 2 shows the corresponding amino acid positions of other members of class B GPCRs with reference to the amino acids forming the binding domain of the Glucagon receptor. The corresponding amino acid positions occupy an equivalent helical position as shown on the first row of Table 2. For example, K311(Lys311) in CRF1 is the corresponding amino acid to R346 (Arg346) in GCGR at helical position 6.37 as shown in Table 2.

For the avoidance of doubt, Arg346 (6.37) may be substituted with Lys, Lys349 (6.40) may be substituted with Arg and Ser350 (6.41) may be substituted with Ala, provided that each substitution is in accordance with the residues listed in Table 2 for each GPCR at the corresponding amino acid position.

In a further embodiment the binding domain comprises the amino acids Arg346 (6.37), Lys349 (6.40), Ser350 (6.41), Asn404(7.61), Leu329 (5.61) and Leu352 (6.43) wherein the one or more amino acids Arg346 (6.37), Lys349 (6.40), Ser350 (6.41), Leu329 (5.61) and Leu352 (6.43) may be optionally substituted with one or more of Lys, Arg, Ala, Val and Met respectively at the corresponding position in a different GPCR.

In a further embodiment the binding domain comprises the amino acids Tyr343 (6.34), Arg346 (6.37), Lys349 (6.40), Ser350 (6.41), Leu399 (7.56), Asn404 (7.61), Leu329 (5.61), Phe345 (6.36), Ala348 (6.39), Leu352 (6.43), Thr353 (6.44) and Leu403 (7.60) wherein one or more amino acids, except Asn404 (7.61), may be optionally substituted with any of the amino acids defined in Table 2 at the corresponding position in a different GPCR.

In a further embodiment the binding domain is as defined according to any of the class B GPCRs in Table 2.

In certain embodiments one or more amino acid residues may be deleted or substituted. In particular, one or more amino acids may be substituted with a different natural amino acid, an amino acid derivative or a non-native amino acid. Sequences may differ from those specified by one or more conservative amino acid substitutions, which typically have minimal influence on the secondary structure and hydrophobic nature of the polypeptide fragment. Sequences may also differ by one or more non-conservative amino acid substitutions, deletions or insertions which do not significantly alter the function of the binding domain. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics such as substitutions within the following groups: valine - glycine; glycine - alanine; valine - isoleucine; aspartic acid - glutamic acid; asparagine - glutamine; serine - threonine; lysine - arginine; and phenylalanine - tyrosine. In certain embodiments one or more amino acids may be added or inserted to the binding domain. Suitable GPCRs are well known in the art and include those listed in Foord et al (2005) Pharmacol Rev. 57, 279-288, incorporated herein by reference, (which is periodically updated at httpi//www.iuphar-db.org/DATABASE/ReceptorFamiliesForward?type=GPCR).

It will be noted that GPCRs are divided into different classes, principally based on their amino acid sequence similarities.

Class B GPCRs include but are not limited to a Class B GPCR in the secretin class such as any of glucagon-like peptide 1 receptor (GLP1R), glucagon-like peptide 2 receptor (GLP2R), calcitonin receptor (CT), amylin/CGRP receptor (ΑΜΥια), amylin receptor (AMY₂a), amylin/CGRP receptor (AMY₃a), CGRP/adrenomedullin receptor (CGRPia),

adrenomedullin/CGRP receptor (ΑΜια), adrenomedullin/CGRP receptor (AM₂a receptor), corticotropin releasing factor receptor (CRFi), urocortins receptor (CRF₂), growth hormone releasing hormone receptor (GHRH), gastric inhibitory polypeptide receptor (GIP), glucagon receptor, secretin receptor, TIP-39 receptor (PTH2), parathyroid hormone receptor (PTH1), VIP/PACAP receptor (VPACi), PACAP receptor (PAC₂), and VIP/PACAP receptor (VPAC₂).

Alternatively, the target protein is a Class B GPCR in the adhesion class such as any of BAI1, BAI2, BAI3, CD97, CELSR1, CELSR2, CELSR3, ELTD1, EMR1, EMR2, EMR4P, GPR56, GPR64, GPR97, GPR98, GPRllO-116, GPR123-126, GPR128, GPR133, GPR144, GPR157 and LPHNl-3. In one embodiment the GPCR is a class B GPCR.

In another embodiment the GPCR is selected from one or more of GCGR, CRF2, CALCR, CALRL, GIPR, GLP1R, GLP2R, PACR, VIPR1, VIPR2, SCTR, GHRHR, PTH1R and PTH2R as listed in Table 2.

In a further embodiment the GPCR is the Glucagon Receptor (GCGR).

The GPCR may be truncated (i.e. comprises fewer amino acid residues than its full length sequence) at the N and/or C terminal. The GPCR may be expressed as a fusion protein, for example a C-terminal green-fluorescent protein (GFP) fusion or a fusion with T4-lysozyme (T4L), or E.coli apocytochrome b₅₆₂RIL (BRIL).

In a further embodiment the GPCR may be a mutant of a parent GPCR. Reference to "mutant GPCR" herein refers to a GPCR with a different genotype to the wild type (or parent) GPCR protein. Such a mutant may also result in a phenotypic difference. Reference to "parent GPCR" herein refers to a GPCR protein which retains an activity characteristic of the naturally occurring protein. The activity may be for example, ligand binding and/or the transport of ions, small molecules or macromolecules across the membrane. The parent GPCR protein may be more or less conformationally stable than the mutant membrane protein.

Mutants of the GPCR protein may be produced by any suitable method where each amino acid of the parent GPCR protein is independently changed to a different amino acid residue. Molecular biological techniques for cloning and engineering genes and cDNAs, for mutating DNA, and for expressing polypeptides from polynucleotides in host cells are well known in the art as exemplified "Molecular cloning, a laboratory manual", third edition, Sambrook, J. & Russell, D.W. (eds), Cold Spring Harbor Laboratory press, Cold Spring Harbor, NY, incorporated herein by reference.

Mutations may be made in any part of the GPCR protein, for example in the part which spans the membrane.

In one embodiment the mutant GPCR protein is produced by alanine scanning mutagenesis, a technique well known in the art. Here each selected amino acid is replaced in turn with alanine to produce a series of mutants suitable for screening. If the selected amino acid is Ala it is replaced with Leu or Gly.

In another embodiment the mutant membrane protein is produced by random mutagenesis, which may be in the whole of the protein or in a selection part. Such techniques are well known in the art (Asubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York 2000).

In one embodiment the mutant GPCR protein comprises one or more replaced amino acids compared to the parent GPCR protein. In another embodiment the mutant membrane protein comprises two, three, four, five, six or seven replaced amino acids compared to the parent GPCR protein. In a further embodiment the GPCR membrane protein comprises between two and four replaced amino acids compared to the parent membrane protein. In a further embodiment the mutant GPCR protein comprises three or four replaced amino acids compared to the parent GPCR protein.

The stability of the mutant GPCR protein is compared to the parent or wild type GPCR protein to establish if the presence of the one or more mutations results in an increase in conformational stability. Reference to "conformational stability" herein refers to the conformation adopted by a GPCR protein that results in an improved stability with respect to any one of biological activity of the GPCR protein such as binding activity, a signalling pathway modulation activity, a transmembrane transporting activity or an enzyme activity.

Increased conformational stability may be observed when the mutant GPCR protein is bound to a ligand. A ligand may function as an inhibitor, an agonist or an antagonist. Ligand binding may cause the mutant GPCR protein to reside in a particular conformation, for example with respect to GPCRs, an agonist or antagonist conformation depending on whether the ligand functions as an agonist or antagonist. Thus the presence of the ligand may be considered to encourage the GPCR to adopt a particular conformation.

Increased stability to heat (i.e. thermostability) can be readily determined by measuring ligand binding or by using spectroscopic techniques such as fluorescence, CD or light scattering at a particular temperature. In one embodiment the thermostability of a mutant GPCR protein is determined by measuring the T_m (the temperature at which 50% of the mutant GPCR protein is inactivated under certain conditions for a given period of time, e.g. 30 minutes). Mutant GPCR proteins having a higher thermostability have higher T_m values when compared to the parent GPCR protein.

To determine increased stability to a detergent or a chaotrope, the mutant GPCR protein is incubated for a defined time in the presence of a test detergent or a test chaotropic agent and the stability is measured using for example ligand binding or a spectroscopic method as discussed above.

Suitable detergents for solubilisation of the membrane protein and/or mutant GPCR protein and for measuring conformational stability are known to the skilled person in the art and include for example, dodecylmaltoside (DDM), CHAPS, octylglucoside (OG) and many others. To determine increased stability to an extreme of pH, a typical pH test would be chosen, for example, in the range 4.5 to 5.5 (low pH) or in the range 8.5 to 9.5 (high pH).

In another embodiment the mutant GPCR has increased conformational thermostability when compared to the parent GPCR protein. In another embodiment the mutant GPCR has increased conformational thermostability when compared to the parent GPCR protein by at least 1°C.

In a further embodiment the mutant GPCR is a class B GPCR.

In a further embodiment the mutant GPCR is the Glucagon receptor. In one embodiment the polypeptide comprises a functional binding domain. By "functional" we mean that the binding domain possesses the structural three-dimensional properties to form an agonist or antagonist conformation when in contact with a ligand. The human amino acid sequence of the Glucagon receptor is provided in SEQ ID No. 1 and comprises amino acids 343 to 404 of the binding domain of the Glucagon receptor.

In a further embodiment the binding domain comprises the amino acid sequence according amino acids 343 to 404 of SEQ ID No. 1, or a sequence which has at least 85%, 88%, 90%, 95%, 98% or 99% amino acid sequence identity thereof.

Percent (%) sequence identity can be determined by methods known in the art. For example mathematical algorithms may be employed to compare amino acid sequence similarity between aligned sequences (Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264- 2268). Various other programs and software packages may be used including the ALIGN program and the FASTA algorithm (Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988: 85: 2444-2448). The BLAST, BLASTN, gapped BLAST, PSI-BLAST, BLAST 2 and WU-BLAST programs provided by the National Center for Biotechnology Information are also widely used and suitable for the purposes of the present invention.

According to a further aspect of the invention there is provided a GPCR comprising a binding domain wherein the binding domain and GPCR are described in accordance with the previous aspects. The provision of the three-dimensional structural characteristics and location of the Glucagon receptor binding domain described herein is valuable for the identification of potential compounds for therapeutic use. In view of the conservation of certain amino acids of the binding domain across class B GPCRs, the binding domain may be employed to screen for compounds that also bind to other class B GPCRs and so target a wide range of therapeutic applications.

According to a further aspect of the invention there is provided a method of selecting a binding partner capable of binding to a binding domain of a polypeptide as defined herein comprising the steps of; a) providing a polypeptide or a GPCR comprising a binding domain of a GPCR,

b) contacting the polypeptide or GPCR with one or more candidate compounds;

c) determining whether one or more candidate compounds bind to the polypeptide or GPCR;

d) isolating one or more candidate compounds that bind to the polypeptide or GPCR;

e) optionally synthesising one or more candidate compounds. The method may further comprise the step of assaying the synthesised compounds to determine in vivo or in vitro binding to a GPCR and / or to determine whether the

compound functions as an agonist, antagonist, partial agonist or inverse agonist. Such techniques are known in the art.

It is appreciated that methods of screening to identify candidate compounds are known in the art, for example described in WO 2008/004223 and WO 2009/081136 herein

incorporated by reference.

The compound or binding partner may be any of a polypeptide a protein, a peptide, a peptidomimetic, a nucleic acid, a carbohydrate, a lipid, an oligopeptide, an aptamer or a small molecule compound that binds to the GPCR. In certain embodiments the binding member is an antibody or an antibody binding fragment.

Typically, the compound is a small molecule chemical compound.

In one embodiment the compound is a library of compounds, for example a library of small molecules, a library of peptides or antibodies.

The compound may an agonist, antagonist, inverse agonist or a partial agonist.

According to a further aspect of the invention there is provided a method of designing a compound that is capable of binding to a binding domain of a polypeptide as defined herein comprising the steps of; a) comparing the three-dimensional structure of a candidate compound with the three- dimensional structure of the binding domain according to the structure coordinates in Table 1, optionally varied by a root mean square deviation of residue backbone atoms of not more than 2.5 A,

b) selecting one or more compounds that are predicted to bind to the functional binding domain by molecular modelling means,

c) optionally modifying one or more selected compounds to improve binding to the functional binding domain,

d) optionally synthesising one or more selected compounds.

Comparison of the structure of a candidate compound with the three-dimensional structure of the binding domain may be carried out by computational molecular modelling techniques using commercially available software designed for this purpose. For example N. C. Cohen et al., "Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990); see also, M. A. Navia and M. A. Murcko, "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202-210 (1992); L. M. Balbes et al., "A Perspective of Modern Methods in Computer-Aided Drug Design", in Reviews in Computational Chemistry, Vol. 5, K. B. Lipkowitz and D. B. Boyd, Eds., VCH, New York, pp. 337-380 (1994); see also, W. C. Guida, "Software For Structure-Based Drug

Design", Curr. Opin. Struct. Biology, 4, pp. 777-781 (1994) and Ballesteros, J. A. & Weinstein, H. "Integrated methods for the construction of three-dimensional models and

computational probing of structure-function relations in G protein-coupled receptors" Methods Neurosci. 25, 366-428 (1995). Candidate compounds that are predicted to bind based on molecular modelling methods must have properties which permit the required non-covalent interactions at the binding site, for example hydrogen bonding, van der Waals interactions, hydrophobic interactions and electrostatic interactions. In addition candidate compounds require the necessary three-dimensional structural properties to allow an optimal "fit" in the binding domain or pocket.

Candidate compounds identified in steps b) may be modified to optimize the non-covalent interactions such that a candidate compound is predicted to bind with a high affinity or induce a conformational change in the GPCR which may be required for functionality. Such techniques may be performed using computer software, for example as described in WO2008/068534 herein incorporated by reference.

Step c) may comprise a further step where modified compounds are tested according to step b).

Synthesis of selected compounds according to step d) may be carried out by organic synthetic techniques known in the art, or other synthetic methods including molecular biology techniques depending on the nature of the compound. According to a further aspect of the invention there is provided a compound obtained by the methods of the previous aspects.

The compound may be for use in medicine or more specifically in the treatment of conditions associated with class B GPCRs.

Conditions associated with class B GPCRs including but not limited to diabetes, anxiety, depression, stress and disorders of the hyperthallamic pituitary axis, obesity, hypertension, osteoporosis, gastrointestinal disorders, inflammatory disorders, migraine. Antagonists of the Glucagon receptor have use in the treatment of Type II diabetes and hyperglycemia which is characterised by the presence of excess glucose. According to a fu rther aspect of the invention there is provided an antibody that specifically binds to the polypeptide or GPCR of the previous aspects. It is appreciated that an antibody is an immunoglobulin, whether natural or partly or wholly synthetically produced . The term also covers any polypeptide, protein or peptide having a binding domain that is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically prod uced. Examples of antibodies are the immunoglobulin isotypes and their isotypic su bclasses and fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd, and a bi-specific antibody. In further embodiments, the antibody may be a camelid antibody, in pa rticular a camelid heavy chain antibody. Fu rther the antibody fragment may be a domain antibody or a nanobody derived from a camelid heavy chain antibody. I n a fu rther embodiment the antibody may be a shark antibody or a shark derived antibody. As antibodies can be modified in a nu mber of ways, the term "antibody" should be construed as covering any binding member or substance having a binding domain with the required specificity. The antibody of the invention may be a monoclonal antibody, a humanised antibody or a fragment, derivative, fu nctiona l equivalent or homologue thereof. The term includes any polypeptide comprising an immunoglobu lin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobu lin binding domain, or equivalent, fused to another polypeptide are therefore included .

The invention is described further by way of the following non-limiting examples. Examples

Example 1. Generation of a stabilised Receptor (StaR)

Using the full-length human GCGR with an intact intracellu lar loop 2 (ICL2) with no T4L (T4 lysozyme) insertion, as the template, a conformationally thermostabilized receptor was generated using a previously published mutagenesis approach (Robertson N. et al. The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery. Neuropharmacology 60, 36-44 (2011)). M utants were analyzed for

thermostability in the presence of the radioligand [³H] M K-0893. The GCGR StaR is the full- length receptor with 12 thermostabilizing mutations.

Example 2. Truncations and T4 Lysozyme fusions

A panel of N- and C-terminal tru ncations of the GCGR StaR receptor was designed and the truncated receptors were expressed in transiently transfected human embryonic kidney (HEK 293) cells as C-terminal fusions with green-fluorescent protein (GFP). Receptors were solubilized in 50 mM Tris-HCI pH 8.0, 150 mM NaCI, and 2 % (w/v) n-decyl-β-ο- maltopyranoside (DM, Affymetrix) and their expression levels and stability was assayed by fluorescence-detection size exclusion chromatography (FSEC, Kawate & Gouaux, 2006). The most suitable construct emerging from this screen comprised residues 135-417. In parallel, a panel of T4 lysozyme (T4L) insertions at various points into intracellular loop (ICL) 2 was analysed in a similar fashion, identifying the insertion between residues 255 and 259 in ICL2 as the most promising fusion (construct GCGR_3728).

Example 3. Expression and purification of a GCGR fusion protein

GCGR with a C-terminal deca-histidine tag was expressed in Spodoptera frugiperda 21 (Sf21) cells in ESF 921 medium (Expression Systems) supplemented with 10 % (v/v) fetal bovine serum (Sigma-Aldrich) and 1 % (v/v) Penicillin/Streptomycin (PAA Laboratories). Cells were infected at a density of 2 x 10⁶ cells/ml with 20 ml of baculovirus per liter of culture, corresponding to an approximate multiplicity of infection (moi) of 2. Cultures were grown at 27^°C with constant shaking and harvested 48 hours post infection. Cells were pelleted and washed with 250 ml PBS and stored at -80^°C.

All purification steps were carried out at 4°C unless indicated otherwise. To prepare membranes, cells from 12L of culture were thawed at room temperature and resuspended in 400 ml ice-cold 50 mM Tris-HCI pH 7.4, 150 mM NaCI, 2 μΜ MK-0893 supplemented with EDTA-free protease inhibitors (Roche). Cells were disrupted using a microfluidizer (M-110L Pneumatic, Microfluidics) and membranes were collected by ultracentrifugation at 140.000 x g. Membranes were resuspended in 180ml of the same lysis buffer, divided into 6 aliquots, and stored at -80 °C until further use. For purification, a single aliquot of membranes was thawed at room temperature and solubilized with 2 % (w/v) DM for 1.5 hours. Insoluble material was removed by ultra-centrifugation and the receptors were immobilized by batch binding to NiNTA metal-affinity resin (Qiagen) for 2 hours, in the presence of 10 mM imidazole. The resin was packed into a XK-16 column (GE Healthcare) and washed with steps of 10 and 30 mM imidazole in 50 mM Tris-HCI pH 7.4, 150 mM NaCI, 0.15 % (w/v) DM, and 2 μΜ MK-0893 for a total of 15 column volumes before bound material was eluted with 250 mM imidazole. The protein was then concentrated using an Amicon Ultra-15 centrifugal filter unit (Millipore), MWCO 100 kDa, and subjected to preparative gel filtration in 20 mM Tris-HCI pH 7.4, 150 mM NaCI, 0.15 % (w/v) DM, and 2 μΜ MK-0893 on a Superdex 200 10/300 GL gel filtration column (GE Healthcare). Receptor purity was analyzed using SDS- PAGE. Protein concentration was determined with a NanoDrop spectrophotometer using the receptor's calculated extinction coefficient at 280 nm (e₂so, ca\c = 1-65 (mg/ml x cm) ).

Example 4. Crystallisation of a GCGR fusion protein with MK-0893 GCGR_3728 was crystallized in lipidic cubic phase (LCP) at 22.5 °C. The protein was concentrated to 20-30 mg/ml by ultrafiltration and mixed with monoolein (Nu-Check) su pplemented with 10 % (w/w) cholesterol (Sigma) and 10 μΜ M K-0893 using the twin- syringe method (Caffrey & Cherezov, 2009). The final protein :lipid ratio was 1:1.5 (w/w). With the help of a dispensing robot (Mosquito LCP, TTP Labtech), 60 nl boli were dispensed on 96-well Laminex Glass Bases (Molecular Dimensions), overlaid with 0.75 μΙ precipitant solution and sealed off with Laminex Film Covers (Molecular Dimensions). GCGR_3728 crystals, -20-30 μιη in size, grew in 100 mM N-(2-acetamido)iminodiacetic acid (ADA) pH 6.0, 200-240 mM Na/K tartrate, 28- 32 % (v/v) polyethylene glycol 400, and 2 μΜ M K-0893.

Example 5. Diffraction data collection and processing

X-ray diffraction data were measured on a Pilatus 6M hybrid-pixel detector at Diamond Light Sou rce beamline 124 using a 10 μιη diameter microbeam. A complete dataset to 2.6 A was obtained by combining datasets from 2 crystals. The crystals belonged to the primitive orthorhombic space grou p P2i2i2i and harbor a single receptor-T4L fusion protein per asymmetric unit giving a Matthews coefficient of 2.44 A³/Da (49.7% solvent content). Data merging and scaling was carried out with AIMLESS (Evans & M urshudov, 2012; Collaborative Computational Project, Nu mber 4, 1994).

Example 6. Structure solution and refinement

The structu re was solved by molecular replacement using the program PHASER (McCoy et al. 2007; Collaborative Computational Project, Number 4, 1994) by sequentially searching for a truncated version (no loops) of the glucagon receptor (pdbcode: 4L6R) and the T4 lysozyme. Several high resolution structures of T4 lysozyme were explored; the one from chain B of the dopamine receptor structure (pdbcode: 3PBL) produced the highest Z-score. Initial refinement of the molecular replacement solution was performed with REFMAC using jelly-body restraints (M urshudov et al., 2011; Collaborative Computational Project, N umber 4, 2007). Thereafter, the model was iteratively built using C007^~ (Emsley et al., 2010) into 2F₀-F_C maps, cross-validated using PHENIX prime-and-switch maps and simulated annealing composite omit maps and refined with PHENIX using positional and individual atomic displacement parameters (Adams et al., 2010). This iterative model building and refinement procedure yielded a structu re with good crystallographic and stereochemical statistics as judged using MolProbity (Chen et al., 2010) , embedded within PHENIX. Regions of weak or no electron density were excluded from the model such that the final structu re consists of GCGR resid ues 138-199, 211-255, 259-373, and 378-417 (Figu res 1 and 2).

Table 2 below shows the position of the amino acid resid ues that interact with the compound M K-0893 (first row) and the corresponding amino acids for other known Family B GPCRs in the same position when compared by alignment. There is a high degree of amino acid identity at positions 7.61 (Asparagine), 5.61 (Leucine), 6.41 (Serine), 6.37 (Arginine), 6.40 (Lysine) and 6.43 (Leucine) indicating conservation across family B GPCRs. Table 1: Coordinates of amino acid residues forming the Glucagon binding domain.

This is extracted from a Protein Data Bank format file, in which each row corresponds to an individual C, N or 0 atom, the fourth column refers to the amino acid type, the sixth column refers to the sequence position of the amino acid, and columns seven, eight, nine refer to the x, y, z co-ordinates (in A ) of the atom relative to a common origin for all atoms.

ATOM 2687 N LEU A 329 2. .961 -8. .837 -63. .761 1. .00 41. .54 A N

ATOM 2688 CA LEU A 329 4. .216 -8. .123 -63. .956 1. .00 42. .55 A C

ATOM 2689 CB LEU A 329 4. .741 -7. .575 -62. .630 1. .00 44. .16 A C

ATOM 2690 CG LEU A 329 4. .104 -6. .272 -62. .155 1. .00 44. .67 A C

ATOM 2691 CD1 LEU A 329 4. .865 -5. .717 -60. .969 1. .00 50. .19 A C

ATOM 2692 CD2 LEU A 329 4. .055 -5. .267 -63. .283 1. .00 36. .61 A C

ATOM 2693 C LEU A 329 5. .273 -9. .011 -64. .600 1. .00 40. .66 A C

ATOM 2694 O LEU A 329 5. .861 -8. .645 -65. .618 1. .00 42. .69 A O

ATOM 2803 N TYR A 343 5. .432 3. .239 -67. .396 1. .00 43. .44 A N

ATOM 2804 CA TYR A 343 4. .694 3. .851 -66. .299 1. .00 38. .57 A C

ATOM 2805 CB TYR A 343 3. .597 4. .770 -66. .838 1. .00 37. .37 A C

ATOM 2806 CG TYR A 343 2. .744 5. .371 -65. .748 1. .00 40. .28 A C

ATOM 2807 CD1 TYR A 343 3. .218 6. .416 -64. .972 1. .00 41. .00 A C

ATOM 2808 CE1 TYR A 343 2. .449 6. .965 -63. .969 1. .00 45. .98 A C

ATOM 2809 CZ TYR A 343 1. .187 6. .469 -63. .728 1. .00 54. .50 A C

ATOM 2810 OH TYR A 343 0. .421 7. .019 -62. .725 1. .00 61. .87 A O

ATOM 2811 CE2 TYR A 343 0. .693 5. .427 -64. .484 1. .00 47. .01 A C

ATOM 2812 CD2 TYR A 343 1. .471 4. .884 -65. .486 1. .00 42. .80 A C

ATOM 2813 C TYR A 343 4. .077 2. .822 -65. .361 1. .00 39. .22 A C

ATOM 2814 O TYR A 343 4. .088 3. .000 -64. .144 1. .00 37. .17 A O

ATOM 2820 N PHE A 345 5. .120 -0. .118 -64. .706 1. .00 40. .07 A N

ATOM 2821 CA PHE A 345 6. .178 -0. .693 -63. .892 1. .00 43. .31 A C

ATOM 2822 CB PHE A 345 7. .403 -1. .023 -64. .745 1. .00 41. .09 A C

ATOM 2823 CG PHE A 345 8. .508 -1. .685 -63. .977 1. .00 42. .09 A C

ATOM 2824 CD1 PHE A 345 8. .581 -3. .064 -63. .900 1. .00 47. .20 A C

ATOM 2825 CE1 PHE A 345 9. .593 -3. .681 -63. .192 1. .00 43. .11 A C

ATOM 2826 CZ PHE A 345 10. .540 -2. .919 -62. .547 1. .00 40. .72 A C

ATOM 2827 CE2 PHE A 345 10. .475 -1. .540 -62. .609 1. .00 37. .58 A C

ATOM 2828 CD2 PHE A 345 9. .466 -0. .931 -63. .321 1. .00 38. .56 A C

ATOM 2829 C PHE A 345 6. .570 0. .257 -62. .765 1. .00 44. .29 A C

ATOM 2830 O PHE A 345 6. .747 -0. .159 -61. .620 1. .00 28. .29 A O

ATOM 2831 N ARG A 346 6. .712 1. .534 -63. .101 1. .00 40. .17 A N

ATOM 2832 CA ARG A 346 7. .137 2. .529 -62. .127 1. .00 45. .63 A C

ATOM 2833 CB ARG A 346 7. .490 3. .847 -62. .831 1. .00 44. .09 A C

ATOM 2834 CG ARG A 346 8. .784 3. .760 -63. .645 1. .00 34. .42 A C

ATOM 2835 CD ARG A 346 9. .186 5. .088 -64. .267 1. .00 33. .64 A C

ATOM 2836 NE ARG A 346 8. .302 5. .472 -65. .362 1. .00 38. .49 A N

ATOM 2837 CZ ARG A 346 7. .384 6. .431 -65. .282 1. .00 43. .58 A C

ATOM 2838 NH1 ARG A 346 7. .233 7. .115 -64. .156 1. .00 42. .96 A N

ATOM 2839 NH2 ARG A 346 6. .621 6. .709 -66. .330 1. .00 43. .67 A N

ATOM 2840 C ARG A 346 6. .071 2. .743 -61. .048 1. .00 38. .62 A C

ATOM 2841 O ARG A 346 6. .399 2. .990 -59. .890 1. .00 31. .92 A O

ATOM 2850 N ALA A 348 4. .071 0. .339 -60. .061 1. .00 31. .84 A N

ATOM 2851 CA ALA A 348 4. .144 -0. .887 -59. .274 1. .00 26. .14 A C

ATOM 2852 CB ALA A 348 4. .354 -2. .090 -60. .182 1. .00 26. .25 A C

ATOM 2853 C ALA A 348 5. .252 -0. .807 -58. .231 1. .00 27. .06 A C ATOM 2854 O ALA A 348 5..183 -1..461 -57..196 1..00 28..64 A O

ATOM 2855 N LYS A 349 6. .272 -0. .001 -58. .508 1. .00 33. .83 A N

ATOM 2856 CA LYS A 349 7. .363 0. .200 -57. .562 1. .00 32. .53 A C

ATOM 2857 C LYS A 349 6. .851 0. .813 -56. .267 1. .00 31. .69 A C

ATOM 2858 O LYS A 349 7. .373 0. .536 -55. .193 1. .00 37. .02 A O

ATOM 2859 CB LYS A 349 8. .447 1. .095 -58. .165 1. .00 33. .76 A C

ATOM 2860 CG LYS A 349 9. .297 0. .435 -59. .238 1. .00 33. .97 A C

ATOM 2861 CD LYS A 349 10. .242 1. .442 -59. .873 1. .00 35. .90 A C

ATOM 2862 CE LYS A 349 11. .073 2. .163 -58. .820 1. .00 41. .13 A C

ATOM 2863 NZ LYS A 349 11. .869 3. .285 -59. .395 1. .00 53. .72 A N

ATOM 2864 N SER A 350 5. .828 1. .652 -56. .373 1. .00 30. .31 A N

ATOM 2865 CA SER A 350 5. .280 2. .324 -55. .203 1. .00 32. .53 A C

ATOM 2866 CB SER A 350 4. .720 3. .693 -55. .590 1. .00 32. .44 A C

ATOM 2867 OG SER A 350 5. .739 4. .534 -56. .102 1. .00 36. .63 A O

ATOM 2868 C SER A 350 4. .198 1. .486 -54. .530 1. .00 33. .70 A C

ATOM 2869 O SER A 350 4. .229 1. .266 -53. .318 1. .00 29. .11 A O

ATOM 2877 N LEU A 352 3. .421 -1. .763 -54. .837 1. .00 25. .88 A N

ATOM 2878 CA LEU A 352 3. .842 -3. .068 -54. .339 1. .00 30. .82 A C

ATOM 2879 C LEU A 352 4. .803 -2. .994 -53. .153 1. .00 28. .43 A C

ATOM 2880 O LEU A 352 5. .187 -4. .028 -52. .614 1. .00 34. .17 A O

ATOM 2881 CB LEU A 352 4. .493 -3. .890 -55. .454 1. .00 37. .72 A C

ATOM 2882 CG LEU A 352 3. .613 -4. .349 -56. .616 1. .00 41. .26 A C

ATOM 2883 CD1 LEU A 352 4. .371 -5. .357 -57. .458 1. .00 42. .83 A C

ATOM 2884 CD2 LEU A 352 2. .296 -4. .938 -56. .119 1. .00 32. .41 A C

ATOM 2885 N THR A 353 5. .212 -1. .795 -52. .748 1. .00 23. .86 A N

ATOM 2886 CA THR A 353 5. .952 -1. .684 -51. .494 1. .00 25. .51 A C

ATOM 2887 CB THR A 353 7. .196 -0. .761 -51. .592 1. .00 36. .54 A C

ATOM 2888 OG1 THR A 353 6. .796 0. .606 -51. .742 1. .00 54. .11 A O

ATOM 2889 CG2 THR A 353 8. .071 -1. .167 -52. .750 1. .00 34. .02 A C

ATOM 2890 C THR A 353 5. .021 -1. .172 -50. .407 1. .00 30. .48 A C

ATOM 2891 O THR A 353 5. .089 -1. .619 -49. .268 1. .00 32. .10 A O

ATOM 3206 N LEU A 399 6. .725 8. .726 -52. .634 1. .00 25. .92 A N

ATOM 3207 CA LEU A 399 6. .636 7. .641 -53. .599 1. .00 33. .67 A C

ATOM 3208 CB LEU A 399 6. .159 6. .367 -52. .899 1. .00 25. .17 A C

ATOM 3209 CG LEU A 399 7. .247 5. .366 -52. .497 1. .00 33. .41 A C

ATOM 3210 CD1 LEU A 399 8. .414 6. .050 -51. .808 1. .00 29. .45 A C

ATOM 3211 CD2 LEU A 399 6. .669 4. .269 -51. .609 1. .00 33. .13 A C

ATOM 3212 C LEU A 399 5. .724 7. .960 -54. .790 1. .00 39. .01 A C

ATOM 3213 O LEU A 399 6. .080 7. .677 -55. .933 1. .00 41. .85 A O

ATOM 3243 N LEU A 403 8. .960 11. .517 -56. .352 1. .00 38. .92 A N

ATOM 3244 CA LEU A 403 9. .840 10. .597 -57. .069 1. .00 32. .02 A C

ATOM 3245 CB LEU A 403 10. .054 9. .318 -56. .257 1. .00 30. .08 A C

ATOM 3246 CG LEU A 403 10. .896 9. .454 -54. .991 1. .00 31. .43 A C

ATOM 3247 CD1 LEU A 403 11. .272 8. .082 -54. .452 1. .00 30. .16 A C

ATOM 3248 CD2 LEU A 403 12. .137 10. .294 -55. .270 1. .00 23. .84 A C

ATOM 3249 C LEU A 403 9. .306 10. .230 -58. .446 1. .00 28. .93 A C

ATOM 3250 O LEU A 403 9. .994 9. .581 -59. .229 1. .00 35. .08 A O

ATOM 3251 N ASN A 404 8. .072 10. .627 -58. .732 1. .00 44. .07 A N

ATOM 3252 CA ASN A 404 7. .441 10. .293 -60. .002 1. .00 44. .15 A C

ATOM 3253 CB ASN A 404 5. .936 10. .559 -59. .928 1. .00 41. .80 A C

ATOM 3254 CG ASN A 404 5. .263 10. .452 -61. .277 1. .00 41. .81 A C

ATOM 3255 OD1 ASN A 404 4. .984 11. .462 -61. .921 1. .00 49. .30 A O

ATOM 3256 ND2 ASN A 404 5. .009 9. .225 -61. .719 1. .00 38. .47 A N

ATOM 3257 C ASN A 404 8. .052 11. .063 -61. .169 1. .00 47. .34 A C

ATOM 3258 O ASN A 404 8. .179 12. .286 -61. .113 1. .00 45. .38 A O Table 2: Residues interacting with MK-0893

Receptor 6.34 6.37 6.40 6.41 7.56 7.61 5.61 6.36 6.39 6.43 6.44 7.60

GCGR Y343 R346 K349 S350 L399 N404 L329 F345 A348 L352 T353 L403

CRF1 Q308 K311 K314 A315 F362 N367 L294 R310 V313 L317 V318 L366

CRF2 Q304 K307 K310 A311 F358 N363 L290 R306 V309 L313 V314 F362

CALCR M353 K356 K359 A360 1406 N411 L339 L355 V358 M362 1363 C410

CALRL L330 K333 R336 A337 1383 N388 L316 M332 V335 L339 1340 F387

GIPR Y335 R338 R341 S342 L391 N396 L321 L337 A340 L344 T345 1395

GLP1R 1345 R348 K351 S352 L401 N406 V331 C347 A350 L354 T355 V405

GLP2R Y379 R382 K385 S386 Q435 N440 L365 Y381 A384 L388 V389 A439

PACR 1347 R350 R353 S354 L399 N404 L331 L349 A352 L356 L357 L403

VIPR1 P335 R338 R341 S342 L387 N392 L319 S337 A340 L344 L345 L391

VIPR2 Q322 R325 K328 S329 L374 N379 L306 K324 A327 L331 L332 L378

SCTR H336 R339 R342 S343 L387 N392 L320 K338 A341 L345 L346 L391

GHRHR Q323 R326 K329 S330 L375 N380 L307 W325 S328 L332 F333 L379

PTH1R Q402 K405 K408 S409 1458 N463 L385 R404 L407 L411 V412 C462

PTH2R Q357 K360 K363 S364 1412 N417 L340 R359 A362 L366 V367 C416

Sequences

The human Glucagon Receptor sequence can be found at:

http://www.uniprot.org/uniprot/P47871

SEQ ID No.l:

M PPCQPQRPLLLLLLLLACQPQVPSAQVM DFLFEKWKLYGDQCH HN LSLLPPPTELVCN R TFDKYSCWPDTPANTTAN ISCPWYLPWH H KVQH RFVFKRCGPDGQWVRGPRGQPWRDASQ CQM DGEEI EVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHAN L FASFVLKASSVLVI DGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQYGIVANYC WLLVEGLYLH N LLGLATLPERSFFSLYLGIGWGAPM LFVVPWAVVKCLFENVQCWTSNDN MGFWWILRFPVFLAILIN FFIFVRIVQLLVAKLRARQM HHTDYKFRLAKSTLTLI PLLGV HEVVFAFVTDEHAQGTLRSAKLFFDLFLSSFQGLLVAVLYCFLN KEVQSELRRRWH RWRL GKVLWEERNTSN H RASSSPGHGPPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPF References

1. Pal et al. Acta Pharmacologica Sinica, 33; 300-311 (2012)

2. Hoare, Drug Discovery Today, 10; 414-427 (2005)

3. Jiang and Zhang, Am J Physiol Endocrinol Metab, 284: E671-678, 2003

4. Yan et al. J Pharmacol Exper Ther, 329; 102- 11 (2011)

5. Peterson and Sullivan, Diabetologia, 44; 2018-24 (2001)

6. Congreve et al. Advances Pharmacology, 62; 1-36 (2011)

7. Fai Yiu Siu et al, Nature, 499; 444-451 (2013)

8. Xiong et al, J. Med. Chem 55, 6137-6148 (2012)

9. Wootten et al. Proc Natl Acad Sci USA 110, 5211-5216, (2013).

Claims

Claims

1. A polypeptide comprising a binding domain of a GPCR wherein the binding domain comprises the amino acids Arg346 (6.37), Lys349 (6.40) and Ser350 (6.41) and Asn404

(7.61), wherein the structure coordinates are optionally varied within an root mean square deviation (rmsd) of residue backbone atoms of not more than 2.5 A.

2. A polypeptide according to claim 1 wherein the binding domain further comprises the amino acids Tyr343 (6.34) and Leu399 (7.56).

3. A polypeptide according to claim 1 or 2 wherein the binding domain further comprises the amino acids Leu329 (5.61) and Leu352 (6.43).

4. A polypeptide according to claim 2 wherein the binding domain further comprises the amino acids Leu329 (5.61), Phe345 (6.36), Ala348 (6.39), Leu352 (6.43), Thr353 (6.44) and Leu403 (7.60).

5. A polypeptide according to any preceding claim wherein the structure coordinates are varied within a rmsd of residue backbone atoms of not more than 2.4 A, 2.3 A, 2.2 A, 2.1 A,

2.0 A, 1.9 A, 1.8 A, 1.7 A, 1.6 A, 1.5 A, 1.4 A, 1.3 A, 1.2 A, 1.1 A, 1.0 A, 0.9 A, 0.8 A, 0.7 A, 0.6 A, 0.5 A, 0.4 A, 0.3 A, 0.2 A or 0.1 A.

6. A polypeptide according to any preceding claim wherein the amino acids Arg346 (6.37), Lys349 (6.40) and Ser350 (6.41) may be optionally substituted with one or more of Lys, Arg and Ala respectively at the corresponding position in a different GPCR.

7. A polypeptide according to claim 6 further comprising the amino acids, Leu329 (5.61) and Leu352 (6.43) which may be optionally substituted with one or more of Val and Met respectively at the corresponding position in a different GPCR.

8. A polypeptide according to claims 6 or 7 wherein the amino acids may be optionally substituted with any of the amino acids defined in Table 2 at a corresponding position in a different GPCR.

9. A polypeptide according to any preceding claim wherein the GPCR is a class B GPCR

10. A polypeptide according to any preceding claim wherein the GPCR is a GPCR receptor listed in Table 2.

11 . A polypeptide according to any preceding claim wherein the GPCR is the Glucagon receptor.

12. A polypeptide according to any preceding claim wherein the GPCR is a mutant GPCR of a parent GPCR.

13. A polypeptide according to claim 12 wherein the mutant GPCR is conformationally stable.

14. A polypeptide according to any preceding claim wherein the binding domain is a functional binding domain.

15. A polypeptide according to any preceding claim wherein the binding domain is defined according to amino acids 343 to 404 of SEQ ID No.l or a sequence having at least 85%, 88%, 90%, 95%, 98% or 99% amino acid sequence identity thereof.

16. A GPCR comprising a binding domain according to any preceding claim.

17. A method of selecting a binding partner capable of binding to a binding domain of a polypeptide according to claims 1-16, comprising the steps of; a) providing a polypeptide or a GPCR comprising a binding domain of a GPCR

b) contacting the polypeptide or GPCR with one or more candidate compounds;

c) determining whether one or more candidate compounds bind to the polypeptide or GPCR;

d) isolating one or more candidate compounds that bind to the polypeptide or GPCR;

e) optionally synthesising one or more candidate compounds.

18. A method according to claim 17 wherein the compound is a library of compounds.

19. A method according to claims 17 or 18 wherein the compound is an antagonist.

20. A method of designing a compound that is capable of binding to a binding domain of a polypeptide according to claims 1-16 comprising the steps of; a) comparing the structure of a candidate compound with the three-dimensional structure of the binding domain according to the structure coordinates in Table 1, optionally varied by a root mean square deviation of residue backbone atoms of not more than 2.5 A, b) selecting one or more compounds that are predicted to bind to the functional binding domain by molecular modelling means, c) optionally modifying one or more selected compounds to improve binding to the functional binding domain,

d) optionally synthesising one or more selected compounds.

21. A compound obtained by the methods of claims 17 to 19.

22. An antibody specifically binding to the binding domain of claims 1 to 16.