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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • G—PHYSICS
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  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• • G—PHYSICS
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the present invention relates to the screening of binding partners of G-protein coupled receptors (GPCRs) and particularly to conformation specific binding partners of GPCRs.
• GPCRs G-protein coupled receptors
• GPCRs constitute a very large family of proteins that control many physiological processes and are the targets of many effective drugs. Thus, they are of considerable pharmacological importance.
• a list of GPCRs is given in Foord et al (2005) Pharmacol Rev. 57, 279-288, which is incorporated herein by reference. GPCRs are generally unstable when isolated, and despite considerable efforts, it has oniy been possible to crystallise bovine rhodopsin, which naturally is exceptionally stable and the beta 2 adrenergic receptor which was crystallised as a fusion protein or in complex with an antibody fragment.
• GPCRs are druggable targets, and reference is made particularly to Overington et al (2006) Nature Rev. Drug Discovery 5, 993-996 which indicates that over a quarter of present drugs have a GPCR as a target.
• GPCRs are thought to exist in multiple distinct conformations which are associated with different pharmacological classes of ligand such as agonists and antagonists, and to cycle between these conformations in order to function (Kenakin T. (1997) Ann N Y Acad Sci 812, 116-125). Switching between conformations contributes to the difficulty in obtaining crystal structures of receptors.
• GPCRs generally have poor stability when removed from their native membrane environment that severely restricts the range of conditions that can be explored without their immediate denaturation or precipitation.
• the inability to produce purified GPCRs in their native conformation prevents their use in a wide range of screening paradigms which depend on the use of purified receptors.
• GPCR screening has traditionally depended on assays in which the receptor is present in cell membranes or whole cells.
• GPCRs represent important therapeutic targets which could be exploited by biotherapeutics such as antibodies.
• the generation of therapeutic antibodies for GPCRs has been extremely difficult due in part to the lack of a suitable immunogen.
• the usual route is to take small peptide fragments of the receptor for immunization however these do not retain their native conformation and often result in binding partners that can bind to and label the receptor but have no functional agonist or antagonist activity.
• biotherapeutics such as antibodies recognise combinations of polypeptide 'loops', features that are lost when peptide fragments are used in isolation.
• Such receptors have a number of advantages as immunogens and/or selection and screening reagents for the generation of binding partners. In particular, they are useful for the generation of conformation- specific binding partners, which will frequently have functional properties, and have previously proven very difficult to generate.
• the stability of native, correctly folded receptor throughout the expression, solubilisation and purification steps facilitates a high yield of purified GPCR (milligram quantities from lab-scale cell culture).
• the stability of folded protein in a range of detergents and solubilisation buffers and additives, without distortion of function enables optimisation of conditions for immunization, for immobilization on solid surfaces without denaturation (e.g. plastic plates, resins, beads or slides, directly or via affinity tags such as poly-Histidine tags), for the purpose of antibody production and screening or for library screening (such as affibody, antibody, phage or small molecule libraries).
• a first aspect of the invention provides a method of producing a binding partner of a GPCR, the method comprising: a) providing a mutant GPCR of a parent GPCR, wherein the mutant GPCR has increased stability in a particular conformation relative to the parent GPCR; b) providing one or more test compounds; c) determining whether the or each test compound binds to the mutant
• binding partner we mean a molecule that binds to a particular GPCR.
• the molecule binds selectively to the GPCR.
• the binding partner has a K ⁇ value (dissociation constant) which is at least five or ten times lower (i.e. higher affinity) than for at least one other GPCR, and preferably more than 100 or 500 times lower.
• the binding partner of a GPCR has a K d value more than 1000 or 5000 times lower than for at least one other GPCR.
• the limits will vary dependent upon the nature of the binding partner.
• the binding partner typically has a K d value which is at least 50 times or 100 times lower than for at least one other GPCR.
• the binding partner typically has a K d value which is at least 500 or 1000 times lower than for at least one other GPCR.
• the use of stabilised GPCRs trapped in particular conformations will increase the likelihood of producing conformation-specific binding partners. Accordingly, it is appreciated that the method may be used to produce a conformation-specific binding partner of a GPCR.
• the method may also be used to identify binding partners that have functional activity such as agonists or antagonists (or other pharmacological categories) which will be determined by the conformation in which the GPCR was stabilised.
• conformation specific we mean that the binding partner of the GPCR binds selectively to a particular conformation of the GPCR and thus has a K d value for that conformation which is lower than for other conformations of the same GPCR.
• a conformation specific binding partner will bind to one conformation of a GPCR (and may cause the GPCR to adopt this conformation), but does not bind as strongly to another conformation that the GPCR may be able to adopt. It will be appreciated therefore that, while the difference in affinity between two conformations and the conformation specific binding partner may be small, typically it will be sufficient to alter the equilibrium between conformational states and encourage the GPCR to adopt a particular conformation.
• a conformational specific binding partner may be considered to be one which traps a GPCR in a conformation of biological relevance (e.g. ligand bound state).
• a conformation specific binding partner has a K d value (dissociation constant) which is at least five or ten times lower (i.e. higher affinity) than for at least one other conformation of the GPCR conformation, and preferably between 100-10000 times lower.
• the conformational specific binding partner binds to the GPCR with a K d of from mM to pM or from mM to fM, such as in the range from ⁇ M to nM or in the range from nM to pM.
• Kd values can be determined readily using methods well known in the art and as described, for example, below.
• the concentration of free ligand and bound ligand at equilibrium must be known. Typically, this can be done by using a radio-labelled or fluorescently labelled ligand which is incubated with the receptor (present in whole cells or homogenised membranes) until equilibrium is reached. The amount of free ligand vs bound ligand must then be determined by separating the signal from bound vs free ligand. In the case of a radioligand this can be done by centrifugation or filtration to separate bound ligand present on whole cells or membranes from free ligand in solution. Alternatively a scintillation proximity assay is used. In this assay the receptor (in membranes) is bound to a bead containing scintillant and a signal is only detected by the proximity of the radioligand bound to the receptor immobilised on the bead.
• the affinity constant may also be determined in a functional assay (K B ).
• K B a functional assay
• the receptor in a whole cell or membrane is activated by an agonist ligand and a response measured (e.g. mobilisation of intracellular calcium, G protein activation, increase or decrease in adenylate cyclise or cAMP, activation of a signal transduction pathway such as a MAP-kinase pathway or activation of gene transcription).
• a signal transduction pathway such as a MAP-kinase pathway or activation of gene transcription.
• the ability of an antagonist to inhibit agonist activity can be measured and for a competitive antagonist is equal to the affinity constant.
• the stability of the mutant GPCRs in a range of detergents, surfactants and solubilisation buffers enables their purification outside of their normal membrane environment. Therefore, the GPCR can be provided in an isolated form removed from non-desired antigens such as non-target GPCRs, membrane associated proteins and other membrane components such as lipoproteins, apolipoproteins, lipis, phosphoinositol lipids and liposaccharides.
• non-desired antigens such as non-target GPCRs, membrane associated proteins and other membrane components such as lipoproteins, apolipoproteins, lipis, phosphoinositol lipids and liposaccharides.
• the method of the invention allows for the selection of a binding partner of a GPCR in the absence of such non-desired antigens.
• the invention produces binding partners that have enhanced selectivity for a GPCR over other membrane components.
• Mutations of the parent GPCR that confer stability in a particular conformation are not expected to affect the binding of the parent GPCR residing in a particular conformation to a particular binding partner. However, it is appreciated that once a binding partner has been isolated by assessing binding to a mutant GPCR residing in a particular conformation, binding of that binding partner to the parent GPCR residing in the same particular conformation may also be assessed.
• the method further comprises:
• the selected binding partner binds to the mutant GPCR when residing in a particular conformation with a similar potency to its binding to the parent GPCR when residing in the same particular conformation.
• the K d values for the particular binding partner binding the mutant GPCR and the parent GPCR are within 5-10 fold of each other, such as within 2-3 fold.
• the binding of the binding partner to the mutant GPCR compared to the parent GPCR would not be more than 5 times weaker and not more than 10 times stronger.
• the selected binding partner binds to the mutant GPCR with approximately equal affinity (that is to say typically within 2-3 fold) or greater affinity than does the parent receptor, when residing in the same conformation.
• the mutants typically bind the agonist- conformation specific binding partners with the same or higher affinity than the parent GPCR and typically bind antagonist-conformation specific binding partners with the same or lower affinity than the parent GPCR.
• the mutants typically bind the antagonist- conformation specific binding partners with the same or higher affinity than the parent GPCR and typically bind agonist-conformation specific binding partners with the same or lower affinity than the parent GPCR.
• K d S Selectivity of binding partners for particular conformations of GPCR or for particular GPCRs and calculation of K d S can be determined using binding assays well known in the art and as described, for example, below.
• K d values are calculated using conventional GPCR assays in membranes wherein the binding affinity at different compound concentrations is measured. Examples of suitable assays include surface plasmon resonance assays and competition assays which are well known in the art and are described below.
• mutant GPCR used in the present aspect is selected and prepared using any of the methods as described below.
• a mutant GPCR with increased stability relative to its parent GPCR can be provided by the methods described below, and by any of the methods disclosed in PCT applications WO 2008/114020 and PCT/GB2008/004032.
• a method for selecting a mutant G-protein coupled receptor (GPCR) with increased stability comprises
• the method of selection may be considered to be a method for selecting mutants of a GPCR which have increased stability of a particular conformation, for example they may have increased conformational thermostability.
• the method may be used to create stable, conformationally locked GPCRs by mutagenesis.
• the selected mutant GPCRs are effectively purer forms of the parent molecules in that a much higher proportion of them occupies a particular conformational state.
• the deliberate selection of a chosen receptor conformation resolved from other conformations by use of a ligand (or ligands) that bind preferentially to this conformation is therefore an important feature of the selection method.
• the method may also be considered to be a method for selecting mutant GPCRs which are more tractable to crystallisation. This is because it is well known that decreased homogeneity or increased pleiotropy within a population of molecules does not favour crystallisation, and further, that an increased number of conformations of a particular molecule does not favour crystallisation.
• a further method for selecting a mutant G-protein coupled receptor (GPCR) with increased stability comprises
• Suitable GPCRs for use in the practice of the invention include, but are not limited to ⁇ -adrenergic receptor, adenosine receptor, in particular adenosine A 2a receptor, and neurotensin receptor (NTR).
• Other suitable GPCRs are well known in the art and include those listed in Hopkins & Groom supra.
• the International Union of Pharmacology produce a list of GPCRs (Foord et al (2005) Pharmacol. Rev. 57, 279-288, incorporated herein by reference and this list is periodically updated at http://www.iuphar- db.org/GPCR/ReceptorFamiliesForward). It will be noted that GPCRs are divided into different classes, principally based on their amino acid sequence similarities. They are also divided into families by reference to the natural ligands to which they bind. All GPCRs are included in the scope of the invention.
• amino acid sequences (and the nucleotide sequences of the cDNAs which encode them) of many GPCRs are readily available, for example by reference to GenBank.
• Foord et al supra gives the human gene symbols and human, mouse and rat gene IDs from Entrez Gene (http://www.ncbi.nlm.nih.gov/entrez).
• Entrez Gene http://www.ncbi.nlm.nih.gov/entrez).
• the GPCR may be derived from any source, it is particularly preferred if it is from a eukaryotic source. It is particularly preferred if it is derived from a vertebrate source such as a mammal or a bird. It is particularly preferred if the GPCR is derived from rat, mouse, rabbit or dog or non-human primate or man, or from chicken or turkey. For the avoidance of doubt, we include within the meaning of "derived from” that a cDNA or gene was originally obtained using genetic material from the source, but that the protein may be expressed in any host celi subsequently.
• a eukaryotic GPCR (such as an avian or mammalian GPCR) may be expressed in a prokaryotic host cell, such as E. coli, but be considered to be avian- or mammalian-derived, as the case may be.
• the GPCR may be composed of more than one different subunit.
• the calcitonin gene-related peptide receptor requires the binding of a single transmembrane helix protein (RAMP1 ) to acquire its physiological ligand binding characteristics.
• RAMP1 transmembrane helix protein
• Effector, accessory, auxiliary or GPCR-interacting proteins which combine with the GPCR to form or modulate a functional complex are well known in the art and include, for example, receptor kinases, G-proteins and arrestins (Bockaert et al (2004) Curr Opinion Drug Discov and Dev 7, 649-657).
• the mutants of the parent GPCR may be produced in any suitable way and provided in any suitable form.
• a series of specific mutants of the parent protein may be made in which each amino acid residue in all or a part of the parent protein is independently changed to another amino acid residue.
• the three-dimensional structure of rhodopsin is known (Li et al (2004) J MoI Biol 343, 1409-1438; Palczewski et al (2000) Science 289, 739-745), and it is possible to model certain GPCRs using this structure.
• parts of the GPCR to mutate may be based on modelling.
• each selected amino acid is replaced by Ala (ie Ala-scanning mutagenesis), although it may be replaced by any other amino acid. If the selected amino acid is Ala, it may conveniently be replaced by Leu. Alternatively, the amino acid may be replaced by GIy (ie Gly-scanning mutagenesis), which may allow a closer packing of neighbouring helices that may lock the protein in a particular conformation. If the selected amino acid is GIy 1 it may conveniently be replaced by Ala.
• amino acid used to replace the given amino acid at a particular position is typically a naturally occurring amino acid, typically an "encodeable” amino acid, it may be a non-natural amino acid (in which case the protein is typically made by chemical synthesis or by use of non-natural amino-acyl tRNAs).
• An "encodeable” amino acid is one which is incorporated into a polypeptide by translation of mRNA. It is also possible to create non-natural amino acids or introduce non-peptide linkages at a given position by covalent chemical modification, for example by post-translational treatment of the protein or semisynthesis. These post-translational modifications may be natural, such as phosphorylation, glycosylation or palmitoylation, or synthetic or biosynthetic.
• the mutants may be produced by a random mutagenesis procedure, which may be of the whole protein or of a selected portion thereof. Random mutagenesis procedures are well known in the art.
• the mutant GPCR has one replaced amino acid compared to the parent protein (ie it is mutated at one amino acid position). In this way, the contribution to stability of a single amino acid replacement may be assessed. However, the mutant GPCR assayed for stability may have more than one replaced amino acid compared to the parent protein, such as 2 or 3 or 4 or 5 or 6 replacements.
• combinations of mutations may be made based on the results of the selection method. It has been found that in some specific cases combining mutations in a single mutant protein leads to a further increase in stability. Thus, it will be appreciated that the method of selection can be used in an iterative way by, for example, carrying it out to identify single mutations which increase stability, combining those mutations in a single mutant GPCRs which is the GPCR then provided in part (a) of the method. Thus, multiply-mutated mutant proteins can be selected using the method.
• the parent GPCR need not be the naturally occurring protein. Conveniently, it may be an engineered version which is capable of expression in a suitable host organism, such as Escherichia coli.
• a convenient engineered version of the turkey ⁇ -adrenergic receptor is one which is truncated and lacks residues 1-33 of the amino acid sequence (ie PAR 34-424 ).
• the parent GPCR may be a truncated form of the naturally occurring protein (truncated at either or both ends), or it may be a fusion, either to the naturally occurring protein or to a fragment thereof.
• the parent GPCR compared to a naturally-occurring GPCR, may be modified in order to improve, for example, solubility, proteolytic stability (eg by truncation, deletion of loops, mutation of glycosylation sites or mutation of reactive amino acid side chains such as cysteine).
• the parent GPCR is a protein that is able to bind to the selected ligand which ligand is one which is known to bind the naturally occurring GPCR.
• the parent GPCR is one which, on addition of an appropriate ligand, can affect any one or more of the downstream activities which are commonly known to be affected by G-protein activation.
• the stability of the mutant is to be compared to a parent in order to be able to assess an increase in stability.
• a ligand is selected, the ligand being one which binds to the parent GPCR when residing in a particular conformation.
• the ligand will bind to one conformation of the parent GPCR (and may cause the GPCR to adopt this conformation), but does not bind as strongly to another conformation that the GPCR may be able to adopt.
• the presence of the ligand may be considered to encourage the GPCR to adopt the particular conformation.
• the method may be considered to be a way of selecting mutant GPCRs which are trapped in a conformation of biological relevance (eg ligand bound state), and which are more stable with respect to that conformation.
• step (c) corresponds to the class of ligand selected in step (b).
• the selected ligand is from the agonist class of iigands and the particular conformation is an agonist conformation, or the selected ligand is from the antagonist class of ligands and the particular conformation is an antagonist conformation.
• the selected ligand is from the agonist class of ligands and the particular conformation in which the GPCR resides in step (c) is the agonist conformation.
• the selected ligand binding affinity for the mutant receptor should be equal to or greater than that for the wild type receptor; mutants that exhibit significantly reduced binding to the selected ligand are typically rejected.
• ligand we include any molecule which binds to the GPCR and which causes the GPCR to reside in a particular conformation.
• the ligand preferably is one which causes more than half of the GPCR molecules overall to be in a particular conformation.
• the ligand is a full agonist and is able to bind to the GPCR and is capable of eliciting a full (100%) biological response, measured for example by
• the biological response is GDP/GTP exchange in a G-protein, followed by stimulation of the linked effector pathway.
• the measurement typically, is GDP/GTP exchange or a change in the level of the end product of the pathway (eg cAMP, cGMP or inositol phosphates).
• the ligand may also be a partial agonist and is able to bind to the GPCR and is capable of eliciting a partial ( ⁇ 100%) biological response.
• the ligand may also be an inverse agonist, which is a molecule which binds to a receptor and reduces its basal (ie unstimulated by agonist) activity sometimes even to zero.
• the ligand may also be an antagonist, which is a molecule which binds to a receptor and blocks binding of an agonist, so preventing a biological response. Inverse agonists and partial agonists may under certain assay conditions be antagonists.
• the above ligands may be orthosteric, by which we include the meaning that they combine with the same site as the endogenous agonist; or they may be aliosteric or allotopic, by which we include the meaning that they combine with a site distinct from the orthosteric site.
• the above ligands may be syntopic, by which we include the meaning that they interact with other ligand(s) at the same or an overlapping site. They may be reversible or irreversible.
• antagonists In relation to antagonists, they may be surmountable, by which we include the meaning that the maximum effect of agonist is not reduced by either pre- treatment or simultaneous treatment with antagonist; or they may be insurmountable, by which we include the meaning that the maximum effect of agonist is reduced by either pre-treatment or simultaneous treatment with antagonist; or they may be neutral, by which we include the meaning the antagonist is one without inverse agonist or partial agonist activity.
• Antagonists typically are also inverse agonists.
• Ligands for use in the selection method may also be aliosteric modulators such as positive aliosteric modulators, potentiators, negative aliosteric modulators and inhibitors. They may have activity as agonists or inverse agonists in their own right or they may only have activity in the presence of an agonist or inverse agonist in which case they are used in combination with such molecules in order to bind to the GPCR.
• aliosteric modulators such as positive aliosteric modulators, potentiators, negative aliosteric modulators and inhibitors. They may have activity as agonists or inverse agonists in their own right or they may only have activity in the presence of an agonist or inverse agonist in which case they are used in combination with such molecules in order to bind to the GPCR.
• the above-mentioned ligands are small organic or inorganic moieties, but they may be peptides or polypeptides.
• the ligand when it is a small organic or organic moiety, it has a M r of from 50 to 2000, such as from 100 to 1000, for example from 100 to 500.
• the ligand binds to the GPCR with a K d of from mM to pM, such as in the range of from ⁇ M (micromoiar) to nM.
• K d small organic molecule iigands
• Other small molecule Iigands include 5HT which is a full agonist at the 5HT1A receptor; eltoprazine which is a partial agonist at the 5HT1A receptor (see Newman-Tancredi et al (1997) Neurophamacology 36, 451- 459); (+)-butaclamo!
• spiperone dopamine D2 receptor inverse agonists (see Roberts & Strange (2005) Br. J. Pharmacol, 145, 34-42); and WIN55212-3 is a neutral antagonist of CB2 (Savinainen et al (2005) Br. J. Pharmacol. 145, 636-645).
• the ligand may be a peptidomimetic, a nucleic acid, a peptide nucleic acid (PNA) or an aptamer. It may be an ion such as Na + or Zn 2+ , a lipid such as oleamide, or a carbohydrate such as heparin.
• the ligand may be a polypeptide which binds to the GPCR.
• polypeptides by which we include oligopeptides
• the polypeptide may be a naturally occurring GPCR-interacting protein or other protein which interacts with the GPCR, or a derivative or fragment thereof, provided that it binds selectively to the GPCR in a particular conformation.
• GPCR-interacting proteins include those associated with signalling and those associated with trafficking, which often act via PDZ domains in the C terminal portion of the GPCR.
• Polypeptides which are known to bind certain GPCRs include any of a G protein, an arrestin, a RGS protein, G protein receptor kinase, a RAMP, a 14-3-3 protein, a NSF, a periplakin, a spinophilin, a GPCR kinase, a receptor tyrosine kinase, an ion channel or subunit thereof, an ankyrin and a Shanks or Homer protein.
• Other polypeptides include NMDA receptor subunits NR1 or NR2a, calcyon, or a fibronectin domain framework.
• the polypeptide may be one which binds to an extracellular domain of a GPCR, such as fibulin-1.
• the polypeptide may be another GPCR, which binds to the selected GPCR in a hetero-oligomer.
• GPCRs A review of protein-protein interactions at GPCRs is found in Milligan & White (2001 ) Trends Pharmacol. ScL 22, 513-518, or in Bockaert et al (2004) Curr. Opinion Drug Discov. Dev. 7, 649-657 incorporated herein by reference.
• the polypeptide ligand may conveniently be an antibody which binds to the GPCR.
• antibody we include an immunoglobulin whether natural or partly or wholley synthetically produced. Examples include immunoglobuiin isotypes and their isotypic subclasses, and monoclonal antibodies and fragments thereof comprising an antigen binding domains such as Fab, F(ab')2, single chain Fv (scFv), Fv, domain antibodies (dAbs) and diabodies. Mention is also made of camelid antibodies and engineered camelid antibodies. Such molecules which bind GPCRs are known in the art and in any event can be made using well known technology. Suitable antibodies include ones presently used in radioimmunoassay (RIAs) for GPCRs since they tend to recognise conformational epitopes.
• RIAs radioimmunoassay
• the polypeptide may also be a binding protein based on a modular framework, such as ankyrin repeat proteins, armadillo repeat proteins, leucine rich proteins, tetratriopeptide repeat proteins or Designed Ankyrin Repeat Proteins (DARPins) or proteins based on lipocalin or fibronectin domains or Affilin scaffolds based on either human gamma crystalline or human ubiquitin.
• a modular framework such as ankyrin repeat proteins, armadillo repeat proteins, leucine rich proteins, tetratriopeptide repeat proteins or Designed Ankyrin Repeat Proteins (DARPins) or proteins based on lipocalin or fibronectin domains or Affilin scaffolds based on either human gamma crystalline or human ubiquitin.
• the ligand is covalently joined to the GPCR, such as a G-protein or arrestin fusion protein.
• GPCRs for example thrombin receptor
• Some GPCRs are cleaved N-terminally by a protease and the new N-terminus binds to the agonist site.
• GPCRs are natural GPCR- ligand fusions.
• antibodies or other "universal" binding polypeptides (such as G-proteins which are known to couple with many different GPCRs) may be particularly advantageous in the use of the method on "orphan" GPCRs for which the natural ligand, and small molecule ligands, are not known.
• this step (c) is one in which it is determined whether the or each mutant GPCR has an increased stability (compared to its parent) for the particular conformation which is determined by the selected ligand.
• the mutant GPCR has increased stability with respect to binding the selected ligand as measured by ligand binding or whilst binding the selected ligand. As is discussed below, it is particularly preferred if the increased stability is assessed whilst binding the selected ligand.
• the increased stability is conveniently measured by an extended lifetime of the mutant under the imposed conditions which may lead to instability (such as heat, harsh detergent conditions, chaotropic agents and so on).
• Destabilisation under the imposed condition is typically determined by measuring denaturation or loss of structure. As is discussed below, this may manifest itself by loss of ligand binding ability or loss of secondary or tertiary structure indicators.
• the mutant GPCR may be brought into contact with a ligand before being subjected to a procedure in which the stability of the mutant is determined (the mutant GPCR and ligand remaining in contact during the test period).
• the receptor is contacted with the ligand before being heated, and then the amount of ligand bound to the receptor following heating may be used to express thermostability compared to the parent receptor. This provides a measure of the amount of the GPCR which retains ligand binding capacity following exposure to the denaturing conditions (eg heat), which in turn is an indicator of stability.
• the mutant GPCR is subjected to a procedure in which the stability of the mutant is determined before being contacted with the ligand.
• the receptor is heated first, before being contacted with the ligand, and then the amount of ligand bound to the receptor may be used to express thermostability. Again, this provides a measure of the amount of the GPCR which retains ligand binding capacity following exposure to the denaturing conditions.
• the comparison of stability of the mutant is made by reference to the parent molecule under the same conditions.
• mutants that are selected are ones which have increased stability when residing in the particular conformation compared to the parent protein.
• the preferred route may be dependent upon the specific GPCR, and will be dependent upon the number of conformations accessible to the protein in the absence of ligand. In the embodiment described in Figure 12, it is preferred if the ligand is present during the heating step because this increases the probability that the desired conformation is selected.
• the selection method includes a method for selecting a mutant GPCR with increased thermostability, the method comprising (a) providing one or more mutants of a parent GPCR, (b) selecting an antagonist or an agonist which binds the parent GPCR, (c) determining whether the or each mutant has increased thermostability when in the presence of the said antagonist or agonist by measuring the ability of the mutant GPCR to bind the selected said antagonist or agonist at a particular temperature and after a particular time compared to the parent GPCR and (d) selecting those mutant GPCRs that bind more of the selected said antagonist or agonist at the particular temperature and after the particular time than the parent GPCR under the same conditions.
• step (c) a fixed period of time at the particular temperature is typically used in measuring the ability of the mutant GPCR to bind the selected said antagonist or agonist.
• step (c) typically a temperature and a time is chosen at which binding of the selected said antagonist or agonist by the parent GPCR is reduced by 50% during the fixed period of time at that temperature (which is indicative that 50% of the receptor is inactivated; "quasi" Tm).
• the ligand when the ligand is used to assay the GPCR (ie used to determine if it is in a non-denatured state), the ligand is detectably labelled, eg radiolabeled or fluorescently labelled.
• ligand binding can be assessed by measuring the amount of unbound ligand using a secondary detection system, for example an antibody or other high affinity binding partner covalently linked to a detectable moiety, for example an enzyme which may be used in a colorimetric assay (such as alkaiine phosphatase or horseradish peroxidase). FRET methodology may also be used. It will be appreciated that the ligand used to assay the mutant GPCR in determining its stability need not be the same ligand as selected in step (b) of the method.
• Proteolytic stability may also be used to measure unfolding by loss of signals associated with secondary or tertiary structure.
• all these methods require the protein to be purified in reasonable quantities before they can be used (eg high pmol/nmol quantities), whereas the method described in the Examples makes use of pmol amounts of essentially unpurified GPCR.
• step (b) two or more ligands of the same class are selected, the presence of each causing the GPCR to reside in the same particular conformation.
• one or more ligands (whether natural or non-natural) of the same class (eg full agonist or partial agonist or antagonist or inverse agonist) may be used.
• the following steps may be used to mitigate this risk:
• ligands in a common pharmacological class as evidenced by for example a binding or functional or spectroscopic assay.
• ligands should be thought to bind to a common spatial region of the receptor, as evidenced for example by competitive binding studies using wild type and/or mutated receptors, and/or by molecular modelling, although they will not necessarily express a common pharmacophore.
• ligands of the same pharmacological class should be used to profile the receptor. These should typically show similar shifts in affinity (mutant versus parent, e.g. wild type) in spite of having different molecular recognition properties. Binding experiments should preferably be done using labelled ligand within the same pharmacological class.
• conformational substrates may exist that are specific to chemical classes of ligand within the same pharmacological class, and these may be specifically stabilised in the procedure depending on the chemical class of the selected iigand.
• the selected ligand binds to the mutant GPCR with a similar potency to its binding to the parent GPCR.
• the K d values for the particular ligand binding the mutant GPCR and the parent GPCR are within 5-10 fold of each other, such as within 2-3 fold.
• the binding of the ligand to the mutant GPCR compared to the parent GPCR would be not more than 5 times weaker and not more than 10 times stronger.
• mutant receptors which have been stabilised in the selected conformation should bind the selected ligand with approximately equal affinity (that is to say typically within 2-3 fold) or greater affinity than does the parent receptor.
• the mutants typically bind the agonists with the same or higher affinity than the parent GPCR and typically bind antagonists with the same or lower affinity than the parent GPCR.
• the mutants typically bind the antagonists with the same or higher affinity than the parent GPCR and typically bind agonists with the same or lower affinity than the parent GPCR.
• Mutants that exhibit a significant reduction (typically greater than 2-3 fold) in affinity for the selecting ligand are typically rejected.
• the rank order of binding of a set of ligands of the same class are comparable, although there may be one or two reversals in the order, or there may be an out-lier from the set.
• two or more ligands that bind simultaneously to the receptor in the same conformation may be used, for example an allosteric modulator and orthosteric agonist.
• mutant GPCRs which, while still being able to bind the selected ligand, are not able to bind, or bind less strongly than the parent GPCR, a second selected ligand which is in a different class to the first ligand.
• the mutant GPCR may be one that is selected on the basis that it has increased stability with respect to binding a selected antagonist, but the mutant GPCR so selected is further tested to determine whether it binds to a full agonist (or binds less strongly to a full agonist than its parent GPCR). Mutants are selected which do not bind (or have reduced binding of) the full agonist. In this way, further selection is made of a GPCR which is locked into one particular conformation.
• the selected ligand (with respect to part (b) of the method) and the further (second) ligand as discussed above, may be any pair of ligand classes, for example: antagonist and full agonist; full agonist and antagonist; antagonist and inverse agonist; inverse agonist and antagonist; inverse agonist and full agonist; full agonist and inverse agonist; and so on.
• the mutant receptor binds the further (second) ligand with an affinity which is less than 50% of the affinity the parent receptor has for the same further (second) ligand, more preferably less than 10% and still more preferably less than 1 % or 0.1 % or 0.01 % of affinity for the parent receptor.
• the K d for the interaction of the second ligand with mutant receptor is higher than for the parent receptor.
• the mutant ⁇ -adrenergic receptor ⁇ AR-m23 (which was selected by the selection method using an antagonist) binds an agonist 3 orders of magnitude more weakly than its parent (ie K d is 1000 x higher).
• the mutant adenosine A2a receptor Rant21 binds agonist 2-4 orders of magnitude more weakly than its parent.
• This type of counter selection is useful because it can be used to direct the mutagenesis procedure more specifically (and therefore more rapidly and more efficiently) along a pathway towards a pure conformation as defined by the ligand.
• the mutant GPCR is provided in a suitable solubilised form in which it maintains structural integrity and is in a functional form (eg is able to bind ligand).
• An appropriate solubilising system such as a suitable detergent (or other amphipathic agent) and buffer system is used, which may be chosen by the person skilled in the art to be effective for the particular protein.
• Typical detergents which may be used include, for example, dodecylmaltoside (DDM) or CHAPS or octylglucoside (OG) or many others. It may be convenient to include other compounds such as cholesterol hemisuccinate or cholesterol itself or heptane-1,2,3-triol. The presence of glycerol or proline or betaine may be useful.
• GPCR once solubilised from the membrane in which it resides, must be sufficiently stable to be assayed.
• DDM will be sufficient, but glycerol or other polyols may be added to increase stability for assay purposes, if desired. Further stability for assay purposes may be achieved, for example, by solubilising in a mixture of DDM, CHAPS and cholesterol hemisuccinate, optionally in the presence of glycerol.
• Nanodiscs may also be used for solubilising extremely unstable membrane proteins in a functional form.
• the mutant GPCR is provided in a crude extract (eg of the membrane fraction from the host cell in which it has been expressed, such as E. coli). It may be provided in a form in which the mutant protein typically comprises at least
• the mutant GPCR is usually associated with detergent molecules and/or lipid molecules.
• a mutant GPCR may be selected which has increased stability to any denaturant or denaturing condition such as to any one or more of heat, a detergent, a chaotropic agent or an extreme of pH.
• thermostability In relation to an increased stability to heat (ie thermostability), this can readily be determined by measuring ligand binding or by using spectroscopic methods such as fluorescence, CD or light scattering at a particular temperature.
• spectroscopic methods such as fluorescence, CD or light scattering at a particular temperature.
• the ability of the GPCR to bind that ligand at a particular temperature may be used to determine thermostability of the mutant. It may be convenient to determine a "quasi T m " ie the temperature at which 50% of the receptor is inactivated under stated conditions after incubation for a given period of time (eg 30 minutes). Mutant GPCRs of higher thermostability have an increased quasi Tm compared to their parents.
• the GPCR is incubated for a defined time in the presence of a test detergent or a test chaotropic agent and the stability is determined using, for example, ligand binding or a spectroscopic method as discussed above.
• a typical test pH would be chosen (eg in the range 4.5 to 5.5 (low pH) or in the range 8.5 to 9.5 (high pH).
• the mutant GPCR is stable in the presence of such detergents.
• the order of "harshness" of certain detergents is DDM, Cu ⁇ Ci 0 ⁇ C 9 ⁇ C 8 maltoside or giucoside, lauryldimethylamine oxide (LDAO) and SDS. It is particularly preferred if the mutant GPCR is more stable to any of C 9 maltoside or giucoside, Cs maltoside or giucoside, LDAO and SDS, and so it is preferred that these detergents are used for stability testing.
• thermostability is determined, and those mutants which have an increased thermostability compared to the parent protein with respect to the selected condition are chosen. It will be appreciated that heat is acting as the denaturant, and this can readily be removed by cooling the sample, for example by placing on ice. It is believed that thermostability may also be a guide to the stability to other denaturants or denaturing conditions. Thus, increased thermostability is likely to translate into stability in denaturing detergents, especially those that are more denaturing than DDM, eg those detergents with a smaller head group and a shorter alkyl chain and/or with a charged head group. We have found that a thermostable GPCR is also more stable towards harsh detergents.
• the denaturing condition When an extreme of pH is used as the denaturing condition, it will be appreciated that this can be removed quickly by adding a neutralising agent. Similarly, when a chaotrope is used as a denaturant, the denaturing effect can be removed by diluting the sample below the concentration in which the chaotrope exerts its chaotropic effect.
• the GPCR is ⁇ -adrenergic receptor (for example from turkey) and the ligand is dihydroalprenolol (DHA), an antagonist.
• DHA dihydroalprenolol
• the GPCR is the adenosine A 2a receptor (A 2a R) (for example, from man) and the ligand is ZM 241385 (4-[2-[[7-amino-2-(2-fury!) [1 ,2,4]-triazolo[2,3- ⁇ ][1 ,3,5]triazin-5- yl]amino]ethyl]phenol), an antagonist or NECA (5'-N-ethylcarboxamido adenosine), an agonist.
• a 2a R adenosine A 2a receptor
• the GPCR is the neurotensin receptor (NTR) (for example, from rat) and the ligand is neurotensin, an agonist.
• NTR neurotensin receptor
• a method for preparing a mutant GPCR selected as above comprises:
• changes to a single amino acid within the GPCR may increase the stability of the protein compared to the parent protein with respect to a particular condition in which the protein resides in a particular conformation.
• a single amino acid residue of the parent protein is changed in the mutant protein.
• the amino acid residue is changed to the amino acid residue found in the mutant tested in the method of the first aspect of the invention.
• it may be replaced by any other amino acid residue, such as any naturally-occurring amino acid residue (in particular, a "codeable" amino acid residue) or a non-natural amino acid.
• the amino acid residue is replaced with one of the 19 other codeable amino acids.
• it is the replaced amino acid residue which is present in the mutant selected in the selection method described above.
• each of the amino acids replaced is one which has been identified using the selection method described above.
• each amino acid identified is replaced by the amino acid present in the mutant protein although, as noted above, it may be replaced with any other amino acid.
• the mutant GPCR contains, compared to the parent protein, from 1 to 10 replaced amino acids, preferably from 1 to 8, typically from 2 to 6 such as 2, 3, 4, 5 or 6 replaced amino acids.
• the multiple mutants may be subject to the selection method.
• multiple mutants may be provided in step (a) of the selection.
• multiply mutagenised GPCRs may be made, whose conformation has been selected to create a very stable multiple point mutant protein.
• the mutant GPCRs may be prepared by any suitable method.
• the mutant protein is encoded by a suitable nucleic acid molecule and expressed in a suitable host cell.
• Suitable nucleic acid molecules encoding the mutant GPCR may be made using standard cloning techniques, site-directed mutagenesis and PCR as is well known in the art.
• Suitable expression systems include constitutive or inducible expression systems in bacteria or yeasts, virus expression systems such as baculovirus, semliki forest virus and lentiviruses, or transient transfection in insect or mammalian cells.
• Suitable host cells include E.
• Suitable animal host cells include HEK 293, COS, S2, CHO, NSO, DT40 and so on. It is known that some GPCRs require specific lipids (eg cholesterol) to function. In that case, it is desirable to select a host cell which contains the lipid. Additionally or alternatively the lipid may be added during isolation and purification of the mutant protein. It will be appreciated that these expression systems and host cells may also be used in the provision of the mutant GPCR in part (a) of the selection method.
• the selected or prepared mutant GPCR may be determined whether the selected or prepared mutant GPCR is able to couple to a G protein. It is also preferred if it is determined whether the selected or prepared mutant GPCR is able to bind a plurality of ligands of the same class as the selecting ligand with a comparable spread and/or rank order of affinity as the parent GPCR.
• thermostabilising mutations are scattered widely throughout the sequences of the turkey betai adrenergic receptor, human adenosine receptor, rat neurotensin receptor and human muscarinic receptor.
• Figure 17 provides an alignment of these sequences with the sequence of the human beta-2AR such that when the thermostabilising mutations are positioned onto the sequences then, in 11 instances out of a total of 70, two sequences contain mutations at the same position (denoted in Figure 17 with a star).
• a further GPCR with increased stability can be generated by aligning the amino acid sequences of the GPCRs and making the same one or more mutations at the corresponding position or positions.
• a further method for producing a mutant GPCR with increased stability relative to its parent GPCR comprises
• the one or more mutants of a first parent GPCR may be selected or prepared according to the selection or preparation methods described above. Hence, this method may also be used to create stable, conformationally locked GPCRs by mutagenesis. For example, following the selection of mutant GPCRs which have increased stability in a particular conformation, the stabilising mutation can be identified and the amino acid at a corresponding position in a second GPCR replaced to produce a mutant GPCR with increased stability in a particular conformation relative to a second parent GPCR.
• the first parent GPCR may be a GPCR which has a naturally-occurring sequence, or it may be a truncated form or it may be a fusion, either to the naturally-occurring protein or to a fragment thereof, or it may contain mutations compared to the naturally-occurring sequence, providing that it retains ligand-binding ability.
• identifying the position or positions at which the one or more mutants have at least one different amino acid residue compared to the first parent GPCR involves aligning their amino acid sequences with that of the parent GPCR, for example using the Clustal W program (Thompson et a/., 1994).
• corresponding position or positions we include the meaning of the position in the amino acid sequence of a second GPCR which aligns to the position in the amino acid sequence of the first GPCR, when the first and second GPCRs are compared by alignment, for example by using MacVector and Clustal W.
• the six stabilising mutations in turkey ⁇ 1-m23, R68S, M90V, Y227A, A282L, F327A and F338M are at positions which correspond to residues K60, M82, Y219, C265, L310 and F321 respectively in the human ⁇ 2 receptor.
• the amino acids at those positions are replaced with another amino acid.
• the amino acids are replaced with the same amino acids which replaced the amino acids at the corresponding positions in the mutant of the first parent GPCR (unless they are already that residue).
• an arginine residue was replaced with a serine residue. Therefore, at the corresponding position in the human ⁇ 2 receptor, position 60 (K60), the lysine residue is preferably replaced with a serine residue.
• Mutations can be made in an amino acid sequence, for example, as described above and using techniques well-established in the art.
• the second GPCR may be any other GPCR.
• stabilising mutations in a GPCR from one species may be transferred to a second GPCR from another species.
• stabilising mutations in one particular GPCR isoform may be transferred to a second GPCR which is a different isoform.
• the second parent GPCR is of the same GPCR class or family as the first parent GPCR.
• Phylogenetic analyses have divided GPCRs into three main classes based on protein sequence similarity, i.e., classes 1 , 2, and 3 whose prototypes are rhodopsin, the secretin receptor, and the metabotropic glutamate receptors, respectively (Foord et al (2005) Pharmacol. Rev. 57, 279-288).
• the second GPCR may be a GPCR which is of the same GPCR class as the first parent GPCR.
• GPCRs have been divided into families by reference to natural ligands such as glutamate and GABA.
• the second GPCR may be of the same GPCR family as the first parent GPCR.
• a list of GPCR classes and families has been produced by the International Union of Pharmacology (Foord et al (2005) Pharmacol. Rev. 57, 279-288) and this list is periodically updated at http://www.iuphar- db.org/GPCR/ReceptorFamiliesForward.
• the second parent GPCR must be able to be aligned with the first parent GPCR such that the corresponding positions of the mutations in the first GPCR can be determined in the second GPCR.
• the second parent GPCR has at least 20% sequence identity to the first parent GPCR and more preferably at least 30%, 40%, 50%, 60%, 70%, 80% or 90% sequence identity to the first parent GPCR.
• some GPCRs have low sequence identity (e.g. family B and C GPCRs) and at the same time are very similar in structure. Thus the 20% sequence identity threshold is not absolute.
• Method 4 The inventors have reasoned that the identification of structural motifs in which the one or more mutations in a mutant GPCR with increased stability reside, will be useful in producing further mutant GPCRs with increased stability.
• a further method for producing a mutant G-protein coupled receptor (GPCR) with increased stability relative to its parent GPCR comprises:
• Mapping stabilising mutations onto one or more known structural models can be used to identify particular structural motifs in which such stabilising mutations reside.
• Table (vi) lists the turkey ⁇ 1 -adrenergic receptor mutations which we have mapped onto the human ⁇ 2-adrenergic receptor and describes the corresponding structural motifs in which they reside.
• mapping of the Y227A mutation (equivalent to Y219 in the human ⁇ 2 receptor) onto the human ⁇ 2 -adrenergic receptor reveals its position at the interface between helices such that the mutation may improve packing at the helical interface (see Figures 15, 16 and 23).
• mapping of the M90V mutation (equivalent to M82 in the human ⁇ 2 receptor) onto the human ⁇ 2 -adrenergic receptor reveals it to be in helix 2 at a point where the helix is kinked (see Figures 15, 16 and 20).
• Such structural motifs are important in determining protein stability. Therefore, targeting mutations to these motifs will facilitate the generation of stabilised mutant GPCRs. Indeed, there were several instances where more than one mutation mapped to the same structural motif. For example, the Y227A, V230A and A234L mutations in the turkey ⁇ 1 adrenergic receptor mapped to the same helical interface, the V89L and M90V mutations mapped to the same helical kink and the F327A and A334L mutations mapped to the same helical surface pointing towards the lipid bilayer (Table (vi)). Thus, when one stabilising mutation has been identified, the determination of the structural motif in which that mutation is located will enable the identification of further stabilising mutations.
• the one or more mutants of a first parent GPCR may be selected or prepared according to any of the methods described above. Hence, this method may also be used to create stable, conformationally locked GPCRs by mutagenesis. For example, following the selection of mutant GPCRs which have increased stability in a particular conformation, the structural motifs in which such stabilising mutations reside can be identified. Making one or more mutations in the amino acid sequence that defines the corresponding structural motif in another GPCR can then be used to produce a mutant GPCR with increased stability in a particular conformation relative to its parent GPCR.
• thermostabilising mutations identified see Examples 1-3
• Figure 17 shows that, when the thermostabilising mutations identified (see Examples 1-3) are positioned on the sequences then, in 11 instances out of a total of 70, two sequences contain mutations at the same position (denoted in Figure 17 with a star).
• thermostabilising mutations at these positions should be of enhanced transferability for mapping onto a structural membrane protein model.
• the mutant of the first parent GPCR is a mutant human beta-2AR, rat NTR1 , turkey beta-1 AR, human Adenosine A2aR or human muscarinic M1 receptor which, when compared to its corresponding parent receptor, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the human beta2 AR as set out in Figure 17: Ala 59, VaI 81 , Ser 143, Lys 147, VaI 152, GIu 180, VaI 222, Ala 226, Ala 271 , Leu 275 and VaI 317.
• the stabilising mutations are mapped onto a known structure of a membrane protein.
• membrane protein we mean a protein that is attached to or associated with a membrane of a cell or organelle.
• the membrane protein is an integral membrane protein that is permanently integrated into the membrane and can only be removed using detergents, non-polar solvents or denaturing agents that physically disrupt the lipid bilayer.
• the structural model of a membrane protein may be any suitable structural model.
• the model may be a known crystal structure.
• GPCR crystal structures include bovine rhodopsin (Palczewski, K. et al., Science 289, 739-745. (2000)) and human ⁇ 2 adrenergic receptor (Rasmussen et al, Nature 450, 383-7 (2007); Cherezov et al (2007) Science 318:1258-65; Rosenbaum et al (2007) Science 318:1266-1273).
• the coordinates for the human ⁇ 2 adrenergic receptor structure can be found in the RCSB Protein Data Bank under accession codes: 2rh1 , 2r4r and 2r4s.
• the structural model may be a computer generated model based upon homology or using die novo structure prediction methods (Qian et al Nature (2007) 450: 259-64).
• stabilising mutations of a given mutant GPCR can be mapped onto a structural model of any membrane protein which has sufficient structural similarity to the GPCR.
• the domain of the membrane protein must have sufficient structural similarity to the GPCR domain in which the stabilising mutation resides, for a given mutation to be transferable.
• a protein domain is typically defined as a discretely folded assembly of secondary structure elements which may stand alone as a single protein or be part of a larger protein in combination with other domains. It is commonly a functional evolutionary unit.
• GPCRs are essentially single domain proteins excluding those with large N- terminal domains. Therefore, typically, the structural model is of a membrane protein which comprises at least one domain that has sufficient structural similarity to the GPCR.
• Structural similarity can be determined indirectly by the analysis of sequence identity, or directly by comparison of structures.
• sequence identity the amino acid sequence encoding the GPCR domain in which the mutant has at least one different amino acid residue compared to the first parent GPCR, is aligned with an amino acid sequence encoding a domain of a membrane protein for which a structural model is available. It will be appreciated that one or more of these sequences may contain an inserted sequence or N-terminal or C-terminal extensions which are additional to the core conserved domain. For optimal alignment, such sequences are removed so as not to skew the analysis. Membrane proteins with sufficient sequence identity across the domain in question may then be used as the structural model for mapping mutations.
• the structural membrane protein model is a model of a membrane protein which contains a domain that shares at least 20% sequence identity with the mutant GPCR domain containing the at least one different amino acid residue compared to the first parent GPCR, and more preferably at least 30%, 40%, 50%, 60%, 70%, 80% or 90% sequence identity, and yet more preferably at least 95% or 99% sequence identity.
• Sequence identity may be measured by the use of algorithms such as BLAST or
• PSI-BLAST Altschul et al, NAR (1997), 25, 3389-3402) or methods based on Hidden Markov Models (Eddy S et al, J Comput Biol (1995) Spring 2 (1 ) 9-23).
• the percent sequence identity between two polypeptides may be determined using any suitable computer program, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
• the alignment may alternatively be carried out using the Clustal W program (Thompson et a/., 1994).
• the parameters used may be as follows: Fast pairwise alignment parameters: K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.
• structural similarity can be determined directly by comparison of structural models.
• Structural models may be used to detect regions of structural similarity not evident from sequence analysis alone, and which may or may not be contiguous in the sequence. For example, family B and C GPCRs are thought to share similar structures; however, their sequence identity is very low.
• the water transporting aquaporins spinach SoPip2, E. coli AqpZ , Methanococcus AqpM, rat Aqp4, human Aqp1 and sheep AqpO share low sequence identity but all have similar structures.
• Structural models of high fidelity may be constructed for proteins of unknown structure using standard software packages such as MODELLER (SaIi A and Biundell T, J MoI Biol (1993) 234(3) 779-815), wherein the structure is modelled on a known structure of a homologous protein.
• Such modelling improves with increasing sequence identity.
• sequence identity between the sequence of unknown structure and a sequence of known 3D structure is more than 30% (Ginalski.K. Curr Op Struc Biol (2006) 16, 172-177).
• de novo structure prediction methods based on sequence alone may be used to model proteins of unknown structure (Qian et al, (2007) Nature 450:259-64).
• regions of structural similarity may be detected by direct comparison of two or more 3D structures. They may, for example, comprise secondary structure elements of a particular architecture and topology which can be detected by the use of software such as DALI (Holm, L and Sander, C (1996) Science 273, 595- 603). They may comprise local arrangements of amino acid side chains and the polypeptide backbone, or specific sets of atoms or groups of atoms in a particular spatial arrangement, which may for example also be detected by the use of graph theoretical representations (Artymiuk.P et al, (2005) J Amer Soc Info Sci Tech 56 (5) 518-528).
• the atoms or groups of atoms within the proteins or regions of proteins to be compared are typically represented as the nodes of a graph, with the edges of the graph describing the angles and distances between the nodes. Common patterns in these graphs indicate common structural motifs.
• This approach may be extended to include any descriptor of atoms or groups of atoms, such as hydrogen bond donor or acceptor, hydrophobicity, shape, charge or aromaticity; for example proteins may be spatially mapped according to such descriptors using GRID and this representation used as a basis for similarity searching (Baroni et al (2007) J Chem lnf Mod 47, 279-294). Descriptions of the methods, availability of software, and guidelines for user-defined selection of parameters, thresholds and tolerances are described in the references given above.
• the structural membrane protein model is a structural GPCR model.
• the structural model of a GPCR may be a model of the first parent GPCR.
• stabilising mutations within a mutant GPCR having increased stability can be directly mapped onto the first parent GPCR structure and the structural motifs in which such mutations are located, identified.
• structural models of other GPCRs may be used.
• stabilising mutations in a GPCR from one species may be mapped onto a known structural model of the same GPCR from another species.
• stabilising mutations in one particular GPCR isoform may be mapped onto a known structural model of another GPCR isoform.
• stabilising mutations from one GPCR may be mapped onto a GPCR of the same class or family.
• a list of GPCR classes and families has been produced by the International Union of Pharmacology (Foord et al (2005) Pharmacol. Rev. 57, 279-288) and this list is periodically updated at http://www.iuphar-db.org/GPCR/ReceptorFamiliesForward.
• the structural model may be of any GPCR provided it has sufficient structural similarity across the domain in which the mutant GPCR has at least one different amino acid compared to the first parent GPCR.
• the GPCR shares at least 20% sequence identity with the mutant of the first parent GPCR across the protein domain containing the at least one different amino acid residue compared to the first parent GPCR, and more preferably at least 30%, 40%, 50%, 60%, 70%, 80% or 90% sequence identity, and yet more preferably at least 95% or 99% sequence identity.
• the 20% sequence identity threshold is not absolute.
• GPCRs with less than 20% sequence identity to the first parent GPCR may also serve as a structural model to which stabilising mutations are transferred, wherein the low sequence identity is counterbalanced by other similarities, including, for example, the presence of the same sequence motifs, binding to the same G-protein or having the same function, or having substantially the same hydropathy plots compared to the first parent GPCR.
• Mapping of stabilising mutations onto the structural model can be done using any suitable method known in the art. For example, typically, the amino acid sequence of the GPCR for which the structural model is available is aligned with the amino acid sequence of the mutant of the first parent GPCR. The position or positions of the at least one different amino acid residue in the mutant GPCR relative to the first parent GPCR can then be located in the amino acid sequence of the GPCR for which a structural model is available.
• 'structural motif we include the meaning of a three dimensional description of the location in a GPCR structural model of a thermostabilising mutation.
• the structural motif may be any secondary or tertiary structural motif within the GPCR.
• 'tertiary structural motif we include any descriptor of atoms or groups of atoms, such as hydrogen bond donor or acceptor, hydrophobicity, shape, charge or aromaticity.
• proteins may be spatially mapped according to such descriptors using GRID and this representation used as a basis for defining a structural motif (Baroni et al (2007) J Chem lnf Mod 47, 279-
• Table (vi) lists the structural motifs in which the turkey ⁇ 1 adrenergic receptor stabilising mutations were found to reside. As seen from the table, the mutations are positioned in a number of distinct localities. Three mutations are in loop regions that are predicted to be accessible to aqueous solvent. Eight mutations are in the transmembrane ⁇ -helices and point into the lipid bilayer; three of these mutations are near the end of the helices and may be considered to be at the hydrophobic-hydrophilic boundary layer.
• the structural motif is any of a helical interface, a helix kink, a helix opposite a helix kink, a helix surface pointing into the lipid bilayer, a helix surface pointing into the lipid bilayer at the hydrophobic- hydrophilic boundary layer, a loop region or a protein binding pocket.
• Identifying a structural motif in which a stabilising mutation resides suggests the importance of that motif in protein stability. Therefore, making one or more mutations in the amino acid sequence that defines a corresponding structural motif or motifs in a second parent GPCR, should provide one or more mutants of a second parent GPCR with increased stability relative to the second parent GPCR.
• the amino acid sequence which defines a structural motif is the primary amino acid sequence of the amino acid residues which combine in the secondary or tertiary structure of the protein to form the structural motif. It will be appreciated that such a primary amino acid sequence may comprise contiguous or noncontiguous amino acid residues. Thus, identifying the amino acid sequence which defines the structural motif will involve determining the residues involved and subsequently defining the sequence. Mutations can be made in an amino acid sequence, for example as described above and using techniques well- established in the art.
• corresponding structural motif or motifs we mean the analogous structural motif or motifs identified in the structural model which are present in the second parent GPCR. For example, if a helical interface was identified, the corresponding helical interface in the second parent GPCR would be the interface between the helices which are analogous to the helices present in the structural model. If a helical kink was identified, the corresponding helical kink would be the kink in the helix which is analogous to the kinked helix present in the structural model.
• An analogous structural motif or motifs in the second parent GPCR can be identified by searching for similar amino acid sequences in the sequence of the second parent GPCR which define the motif or motifs in the structural model, for example, by sequence alignment.
• an analogous structural motif can readily be identified. It will be appreciated that if a structural model of the second parent GPCR is available, the analogous structural motif or motifs can be directly mapped onto the structure of the protein.
• the amino acid sequence defining the analogous structural motif has at least 20% sequence identity with the sequence defining the motif in the structural model, more preferably at least 30%, 40%, 50%, 60%, 70%, 80% and 90% sequence identity and yet more preferably 95% and 99% sequence identity.
• the second parent GPCR is the first parent GPCR.
• the second parent GPCR may have the naturally-occurring sequence of the first parent GPCR, or it may be a truncated form or it may be a fusion, either to the naturally occurring protein or to a fragment thereof, or it may contain mutations compared to the naturally-occurring sequence, providing that it retains ligand-binding.
• the second parent GPCR is not the first parent GPCR.
• a mutant of a first parent GPCR may have been identified that has increased stability but it is desired to generate a mutant of a different GPCR with increased stability.
• the second parent GPCR is of the same GPCR class or family as the first parent GPCR as described above.
• the second parent GPCR may be any known GPCR provided that it shares sufficient structural similarity with the first parent GPCR, such that it contains a corresponding structural motif in which the stabilising mutation of the mutant of the first parent GPCR resides.
• the second parent GPCR has at least 20% sequence identity to the first parent GPCR and more preferably at least 30%, 40%, 50%, 60%, 70%, 80% or 90% sequence identity.
• GPCRs have low sequence identity (e.g. family B and C GPCRs) but are similar in structure.
• the 20% sequence identity threshold is not absolute. Since there are potentially thousands of mutations that can be screened in a GPCR for increased stability, it is advantageous to target particular mutations which are known to be important in conferring stability. Therefore, it will be appreciated that the methods 3 and 4 may be used in a method of selecting mutant GPCRs with increased stability. In particular, carrying out methods 3 and 4 can be used to target mutations to particular amino acid residues or to amino acid sequences which define structural motifs important in determining stability.
• methods 3 and 4 further comprise:
• steps (I), (II) and (III) correspond to steps (b), (c) and (d) of method 1 described above. Accordingly, preferences for the ligand and methods of assessing stability are as defined above with respect to method 1.
• mutant GPCR with increased stability relative to its parent GPCR for example those provided by any of methods 1-4 described above, may be used in the present invention.
• mutant GPCRs with increased stability compared to their parent GPCRs particularly those with increased thermostability may be used.
• Particular examples of mutant GPCRs suitable for use in the present invention are provided below.
• the mutant GPCR is a mutant GPCR which has, compared to its parent receptor, at least one different amino acid at a position which corresponds to any one or more of the following positions: (i) according to the numbering of the turkey ⁇ -adrenergic receptor as set out in Figure 9: lie 55, GIy 67, Arg 68, VaI 89, Met 90, GIy 67, Ala 184, Arg 199, Ala 203, Leu 208, GIn 210, Ser 213, GIu 219, Arg 220, Ser 223, Thr 224, GIn 226, Lys 227, His 230, Leu 241 , Pro 260, Ser 263, Leu 267, Leu 272, Thr 279, Asn 284, GIn 311 , Pro 313, Lys 315, (iii) according to the numbering of the rat neurotensin receptor as set out in Figure 11 : Ala 69, Leu 72, Ala 73, Ala 86, Ala
• Alignment of the turkey ⁇ 1 AR, human adenosine receptor, rat neurotensin receptor and human muscarinic receptor amino acid sequences in Figure 17, shows that in 11 instances out of 70, two sequences contain mutations art the same position, namely at the following positions according to the numbering of the human beta2 AR as set out in Figure 17: Ala 59, VaI 81 , Ser 143, Lys 147, VaI 152, GIu 180, VaI 222, Ala 226, Ala 271 , Leu 275 and VaI 317. Therefore, in a further embodiment, the mutant GPCR is one which has, compared to its parent receptor, a different amino acid at any one of these positions.
• ⁇ -adrenergic receptors are well known in the art. They share sequence homology to each other and bind to adrenalin.
• the mutant GPCR is a mutant ⁇ -adrenergic receptor which, when compared to the corresponding wild-type ⁇ -adrenergic receptor, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the turkey ⁇ -adrenergic receptor as set out in Figure 9: He 55, GIy 67, Arg 68, VaI 89, Met 90, GIy 98, lie 129, Ser 151 , VaI 160, GIn 194, GIy 197, Leu 221 , Tyr 227, Arg 229, VaI 230, Ala 234, Ala 282, Asp 322, Phe 327, Ala 334, Phe 338.
• the mutant ⁇ -adrenergic receptor may be a mutant of any ⁇ -adrenergic receptor provided that it is mutated at one or more of the amino acid positions as stated by reference to the given turkey ⁇ -adrenergic receptor amino acid sequence.
• the mutant GPCR is one which has at least 20% amino acid sequence identity when compared to the given turkey ⁇ -adrenergic receptor sequence, as determined using MacVector and CLUSTALW (Thompson et al (1994) Nucl. Acids Res. 22, 4673-4680). More preferably, the mutant receptor has at least 30% or at least 40% or at least 50% amino acid sequence identity. There is generally a higher degree of amino acid sequence identity which is conserved around the orthosteric ("active") site to which the natural ligand binds.
• thermostability lie 55, GIy 67, Arg 68, VaI 89, Met 90, GIy 98, He 129, Ser 151 , Va1 160, GIn 194, GIy 197, Leu 221 , Tyr 227, Arg 229, VaI 230, Ala 234, Ala 282, Asp 322, Phe 327, Ala 334, Phe 338.
• a mutant turkey ⁇ -adrenergic receptor in which, compared to its parent, one or more of these amino acid residues have been replaced by another amino acid residue may be used.
• Mutant ⁇ -adrenergic receptors from other sources in which one or more corresponding amino acids in the parent receptor are replaced by another amino acid residue may also be used.
• the mutant GPCR is a mutant ⁇ -adrenergic receptor which has has at least one different amino acid residue in a structural motif in which the mutant receptor compared to its parent receptor has a different amino acid at a position which corresponds to any of the following positions according to the numbering of the turkey ⁇ -adrenergic receptor as set out in Figure 9: lie 55, GIy 67, Arg 68, VaI 89, Met 90, Giy 98, lie 129, Ser 151 , VaI 160, GIn 194, GIy 197, Leu 221 , Tyr 227, Arg 229, VaI 230, Ala 234, Ala 282, Asp 322, Phe 327, Ala 334, Phe 338.
• the parent may be a ⁇ -adrenergic receptor which has a naturally-occurring sequence, or it may be a truncated form or it may be a fusion, either to the naturally occurring protein or to a fragment thereof, or it may contain mutations compared to the naturally-occurring sequenced provided that it retains ligand-binding ability.
• corresponding amino acid residue we include the meaning of the amino acid residue in another ⁇ -adrenergic receptor which aligns to the given amino acid residue in turkey ⁇ -adrenergic receptor when the turkey ⁇ -adrenergic receptor and the other ⁇ -adrenergic receptor are compared using MacVector and CLUSTALW.
• Figure 9 shows an alignment between turkey ⁇ -adrenergic receptor and human ⁇ 1 , ⁇ 2 and ⁇ 3 ⁇ -adrenergic receptors.
• lie 72 of human ⁇ 1 corresponds to lie 55 of turkey ⁇ -adrenergic receptor
• lie 47 of human ⁇ 2 corresponds to Ne 55 of turkey ⁇ -adrenergic receptor
• Thr51 of human ⁇ 3 corresponds to He 55 of turkey ⁇ -adrenergic receptor.
• Other corresponding amino acid residues in human ⁇ 1 , ⁇ 2 and ⁇ 3 can readily be identified by reference to Figure 9.
• the particular amino acid is replaced with an Ala.
• the particular amino acid residue is an Ala, it is preferred that it is replaced with a Leu (for example, see turkey ⁇ -adrenergic Ala 234, Ala 282 and Ala 334 in Figure 1 ).
• mutant ⁇ -adrenergic receptor has a different amino acid compared to its parent at more than one amino acid position since this is likely to give greater stability.
• Particularly preferred human ⁇ 1 receptor mutants are those in which one or more of the following amino acid residues are replaced with another amino acid residue: K85, M 107, Y244, A316, F361 and F372.
• the given amino acid residue is replaced with Ala or VaI or Met or Leu or lie (unless they are already that residue).
• Mutant human ⁇ 1 receptors which have combinations of 3 or 4 or 5 or 6 mutations as described above are preferred.
• Particularly preferred human ⁇ 2 receptor mutants are those in which one or more of the following amino acids are replaced with another amino acid residue: K60, M82, Y219, C265, L310 and F321.
• the given amino acid residue is replaced with Ala or VaI or Met or Leu or lie (unless they are already that residue).
• Mutant human ⁇ 2 receptors which have combinations of 3 or 4 or 5 or 6 mutations as described above are preferred.
• Figure 26 shows the effect on thermostability when six thermostabilising mutations in ⁇ 1-m23 (R68S, M90V, Y227A, A282L, F327A, F338M) were transferred directly to the human ⁇ 2 receptor (equivalent mutations K60S, M82V, Y219A, C265L, L310A, F321 M), making human ⁇ 2-m23.
• the Tms for human ⁇ 2 and ⁇ 2-m23 were 29 0 C and 41 0 C respectively, thus exemplifying the transferability of thermostabilising mutations from one receptor to another receptor.
• a particularly preferred human ⁇ 2 receptor mutant is one which comprises the mutations K60S, M82V, Y219A, C265L, L310A, F321 M.
• Particularly preferred human ⁇ 3 receptor mutants are those in which one or more of the following amino acids are replaced with another amino acid residue: W64, M86, Y224, P284, A330 and F341.
• the given amino acid residue is replaced with Ala or VaI or Met or Leu or lie (unless they are already that residue).
• Mutant human ⁇ 3 receptors which have combinations of 3 or 4 or 5 or 6 mutations as described above are preferred.
• suitable mutants include the mutant turkey ⁇ - adrenergic receptors, and also include mutant ⁇ -adrenergic receptors where amino acids in corresponding position have been replaced by another amino acid, typically the same amino acid as indicated in Tables 1 and 2 in Example 1.
• Particularly preferred mutants are those which contain mutations in the amino acids which correspond to the given amino acid residge by reference to turkey ⁇ - adrenergic receptor: (R68S, Y227A, A282L, A334L) (see m6-10 in Table 2 below); (M90V, Y227A, F338M) (see m7-7 in Table 2 below); (R68S, M90V, V230A, F327A, A334L) (see m10-8 in Table 2 below); and (R68S, M90V, Y227A, A282L, F327A, F338M) (see m23 in Table 2 below).
• Adenosine receptors are well known in the art. They share sequence homology to each other and bind to adenosine.
• the mutant GPCR is a mutant adenosine receptor which, when compared to the corresponding wild-type adenosine, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the human adenosine A 2a receptor as set out in Figure 10: GIy 114, GIy 118, Leu 167, Ala 184, Arg 199, Ala 203, Leu 208, GIn 210, Ser 213, GIu 219, Arg 220, Ser 223, Thr 224, GIn 226, Lys 227, His 230, Leu 241 , Pro 260, Ser 263, Leu 267, Leu 272, Thr 279, Asn 284, GIn 311 , Pro 313, Lys 315, Ala 54, VaI 57, His 75, Thr 88, GIy 114, GIy 118, Thr 119, Lys 122, GIy 123, Pro 149
• the mutant adenosine receptor may be a mutant of any adenosine receptor provided that it is mutated at one or more of the amino acid positions as stated by reference to the given human adenosine A 2a receptor amino acid sequence.
• the mutant GPCR is one which has at least 20% amino acid sequence identity when compared to the given human adenosine A 2a receptor sequence, as determined using MacVector and CLUSTALW.
• the mutant GPCR has at least 30% or at least 40% or at least 50% or at least 60% sequence identity.
• individual replacement of the following amino acid residues in the human adenosine A 2a receptor sequence (as shown in Figure 10) lead to an increase in thermostability when measured with the agonist 5'-N-ethylcarboxamidoadenosine (NECA):
• mutant human adenosine A 2a receptor in which, compared to its parent, one or more of these amino acid residues have been replaced by another amino acid residue may be used.
• Mutant adenosine receptors from other sources in which one or more corresponding amino acids in the parent receptor are replaced by another amino acid residue may also be used.
• the mutant GPCR is a mutant adenosine receptor which has at least one different amino acid residue in a structural motif in which the mutant receptor compared to its parent receptor has a different amino acid at a position which corresponds to any of the following positions according to the numbering of the human adenosine A 2a receptor as set out in Figure 10: GIy 114, GIy 118, Leu 167, Ala 184, Arg 199, Ala 203, Leu 208, GIn 210, Ser 213, GIu 219, Arg 220, Ser 223, Thr 224, GIn 226, Lys 227, His 230, Leu 241 , Pro 260, Ser 263, Leu 267, Leu 272, Thr 279, Asn 284, GIn 311 , Pro 313, Lys 315.
• the parent may be an adenosine receptor which has a naturally-occurring sequence, or it may be a truncated form or it may be a fusion, either to the naturally-occurring protein or to a fragment thereof, or it may contain mutations compared to the naturally-occurring sequence, provided that it retains iigand-binding ability.
• corresponding amino acid residue we include the meaning of the amino acid residue in another adenosine receptor which aligns to the given amino acid residue in human adenosine A 2a receptor when the human adenosine A 2a receptor and the other adenosine receptor are compared using MacVector and CLUSTALW.
• Figure 10 shows an alignment between human adenosine A 2a receptor and three other human adenosine receptors (A2b, A3 and A1 ).
• Ser 115 in the A 2b receptor corresponds to GIy 114 in the A 2a receptor.
• Ala 60 in the A 3 receptor corresponds to Ala 54 in the A 2a receptor, and so on.
• Other corresponding amino acid residues in human adenosine receptors A 2b , A 3 and A 1 can readily be identified by reference to
• the mutant adenosine receptor has a different amino acid compared to its parent at more than one amino acid position.
• Particularly preferred human adenosine A2b receptors are those in which one or more of the following amino acid residues are replaced with another amino acid residue: A55, T89, R123, L236 and V240.
• the given amino acid residue is replaced with Ala or VaI or Met or Leu or lie (unless they are already that residue).
• Mutant human adenosine A2b receptors which have combinations of 3 or 4 or 5 mutations as described above are preferred.
• Particularly preferred human adenosine A3 receptors are those in which one or more of the following amino acid residues are replaced with another amino acid residue: A60, T94, W128, L232 and L236.
• the given amino acid residue is replaced with Ala or VaI or Met or Leu or lie (unless they are already that residue).
• Mutant human adenosine A3 receptors which have combinations of 3 or 4 or 5 mutations as described above are preferred.
• Particular preferred human adenosine A1 receptors are those in which one or more of the following residues are replaced: A57, T91 , A125, L236, and L24Q.
• the given amino acid residue is replaced with Ala or VaI or Met or Leu or lie (unless they are already that residue).
• Suitable mutants include these mutant human adenosine A 2a receptors, and also include other mutant adenosine receptors where amino acids in corresponding positions have been replaced by another amino acid, typically the same amino acid as indicated in Example 2.
• Particularly preferred adenosine receptor mutants are those which contain mutations in the amino acids which correspond to the given amino residue by reference to human adenosine A2a receptor: (A54L, K122A, L235A) (Rant 17); (A54L, T88A, V239A, A204L) (Rant 19); and (A54L, T88A, V239A, K122A) (Rant 21 ).
• Neurotensin receptors are known in the art. They share sequence homology and bind neurotensin.
• the mutant GPCR is a mutant neurotensin receptor which, when compared to the corresponding wild-type neurotensin receptor, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the rat neurotensin receptor as set out in Figure 11 : Ala 69, Leu 72, Ala 73, Ala 86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111 , Asp 113, lie 116, Ala 120, Asp 139, Phe 147, Ala 155, VaI 165, GIu 166, Lys 176, Ala 177, Thr 179, Met 181 , Ser 182, Arg 183, Phe 189, Leu 205, Thr 207, GIy 209, GIy 215, VaI 229, Met 250, lie 253, Leu 256, lie 260, Asn 262, VaI 268, Asn 270, Thr 279,
• the mutant GPCR is one which has at least 20% amino acid sequence identity when compared to the given rat neurotensin receptor sequence, as determined using MacVector and CLUSTALW.
• the mutant GPCR has at least 30% or at least 40% or at least 50% amino acid sequence identity.
• the mutant neurotensin receptor may be a mutant of any neurotensin receptor provided that it is mutated at one or more of the amino acid positions as stated by reference to the given rat neurotensin receptor amino acid sequence.
• Figures 11 and 28 lead to an increase in thermostability when considered with respect to the presence of neurotensin.
• a mutant rat neurotensin receptor in which, compared to its parent, one or more of these amino acid residues have been replaced by another amino acid residue may be used.
• Mutant neurotensin receptors from other sources in which one or more corresponding amino acids in the parent receptor are replaced by another amino acid residue may also be used.
• the mutant GPCR is a mutant neurotensin receptor which has at least one different amino acid residue in a structural motif in which the mutant receptor compared to its parent receptor has a different amino acid at a position which corresponds to any of the following positions according to the numbering of the rat neurotensin receptor as set out in Figure 11 : Ala 69, Leu 72, Ala 73, Ala 86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111 , Asp 113, lie 116, Ala 120, Asp 139, Phe 147, Ala 155, VaI 165, GIu 166, Lys 176, Ala 177, Thr 179, Met 181 , Ser 182, Arg 183, Phe 189, Leu 205, Thr 207, GIy 209, GIy 215, VaI 229, Met 250, He 253, Leu 256, lie 260, Asn 262, VaI 268, Asn 270, Thr
• the parent may be a neurotensin receptor which has a naturally-occurring sequence, or it may be a truncated form or it may be a fusion, either to the naturally-occurring protein or to a fragment thereof, or it may contain mutations compared to the naturally-occurring sequence, providing that it retains ligand-binding ability.
• corresponding amino acid residue we include the meaning of the amino acid residue in another neurotensin receptor which aligns to the given amino acid residue in rat neurotensin receptor when the rat neurotensin receptor and the other neurotensin receptor are compared using MacVector and CLUSTALW.
• Figure 11 shows an alignment between rat neurotensin receptor and two human neurotensin receptors 1 and 2. It can be seen, for example, that Ala 85 of the human neurotensin receptor 1 corresponds to Ala 86 of the rat neurotensin receptor, that Phe 353 of the human neurotensin receptor 1 corresponds to Phe 358 of the rat neurotensin receptor, and so on. Other corresponding amino acid residue in the human neurotensin receptors 1 and 2 can readily be identified by reference to Figure 11.
• the particular amino acid in the parent is replaced with an Ala.
• the particular amino acid residue in the parent is an Ala, it is preferred that it is replaced with a Leu.
• the mutant neurotensin receptor has a different amino acid compared to its parent at more than one amino acid position.
• Particularly preferred human neurotensin receptors are those in which one or more of the following amino acid residues are replaced with another amino acid residue: Ala 85, His 102, lie 259, Phe 337 and Phe 353.
• the given amino acid residues is replaced with Ala or VaI or Met or Leu or lie (unless they are already that residue).
• NTR1 human neurotensin receptors
• NTR2 human neurotensin receptors
• V54, R69, T229, P331 and F347 amino acid residues
• the given amino acid residue is replaced with Ala or VaI or Met or Leu or He (unless they are already that residue).
• Mutant human neurotensin receptors (NTR2) which have combinations of 3 or 4 or 5 mutations as described above are preferred.
• Suitable mutants include these mutant rat neurotensin receptors, and also include other mutant neurotensin receptors where amino acids in corresponding positions have been replaced by another amino acid, typically the same amino acid as indicated in Example 3.
• Particularly preferred neurotensin receptor mutants are those which contain mutations in the amino acid residues which correspond to the given amino acid residue by reference to the rat neurotensin receptor: (F358A, A86L, I260A, F342A) (Nag7m); (F358A, H103A, I260A, F342A) (Nag7n). Mutant muscarinic receptor
• Muscarinic receptors are known in the art. They share sequence homology and bind muscarine.
• the mutant GPCR is a mutant muscarinic receptor which, when compared to the corresponding wild-type muscarinic receptor, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the human muscarinic receptor M1 as set out in Figure 17: Leu 65, Met 145, Leu 399, lie 383 and Met 384.
• the mutant GPCR is one which has at least 20% amino acid sequence identity when compared to the given human muscarinic receptor sequence, as determined using MacVector and CLUSTALW.
• the mutant GPCR has at least 30% or at least 40% or at least 50% amino acid sequence identity.
• the mutant muscarinic receptor may be a mutant of any muscarinic receptor provided that it is mutated at one or more of the amino acid positions as stated by reference to the given muscarinic receptor amino acid sequence.
• mutant human muscarinic receptor in which, compared to its parent, one or more of these amino acid residues have been replaced by another amino acid residue may be used.
• Mutant muscarinic receptors from other sources in which one or more corresponding amino acids in the parent receptor are replaced by another amino acid residue may also be used.
• the parent may be a muscarinic receptor which has a naturally-occurring sequence, or it may be a truncated form or it may be a fusion, either to the naturally-occurring protein or to a fragment thereof, or it may contain mutations compared to the naturally-occurring sequence, providing that it retains ligand-binding ability.
• the mutant GPCR is a mutant muscarinic receptor.
• the mutant muscarinic receptor may have at least one different amino acid residue in a structural motif in which the mutant receptor compared to its parent receptor has a different amino acid at a position which corresponds to any of the following positions according to the numbering of the human muscarinic receptor as set out in Figure 17: Leu 65, Met 145, Leu 399, lie 383 and Met 384.
• corresponding amino acid residue we include the meaning of the amino acid residue in another muscarinic receptor which aligns to the given amino acid residue in human muscarinic receptor when the human muscarinic receptor and the other muscarinic receptor are compared using MacVector and CLUSTALW.
• the particular amino acid is replaced with an Ala.
• the particular amino acid residue is an Ala, it is preferred that it is replaced with a Leu.
• mutant GPCRs used in the invention have increased stability to any one of heat, a detergent, a chaotropic agent and an extreme of pH.
• mutant GPCRs used in the invention have increased thermostability.
• the mutant GPCRs used in the invention including the mutant ⁇ -adrenergic, adenosine and neurotensin receptors, have an increased thermostability compared to its parent when in the presence or absence of a ligand thereto.
• the ligand is an antagonist, a full agonist, a partial agonist or an inverse agonist, whether orthosteric or allosteric.
• the ligand may be a polypeptide, such as an antibody.
• the mutant GPCRs used in the invention for example a mutant ⁇ -adrenergic receptor or a mutant adenosine receptor or a mutant neurotensin receptor or a mutant muscarinic receptor is at least 2 0 C more stable than its parent preferably at least 5°C more stable, more preferably at least 8 0 C more stable and even more preferably at least 10 0 C or 15 0 C or 20 0 C more stable than its parent.
• thermostability of the parent and mutant receptors are measured under the same conditions.
• thermostability is assayed under a condition in which the GPCR resides in a particular conformation.
• this selected condition is the presence of a ligand which binds the GPCR.
• mutant GPCRs used in the invention when solubilised and purified in a suitable detergent has a similar thermostability to bovine rhodopsin purified in dodecyl maltoside. It is particularly preferred that the mutant GPCR retains at least 50% of its ligand binding activity after heating at 40 0 C for 30 minutes. It is further preferred that the mutant GPCR retains at least 50% of its ligand binding activity after heating at 55°C for 30 minutes.
• the mutant GPCR provided in step (a) of the method of the first aspect of the invention may be extended or truncated, contain internal deletions or insertions or otherwise altered beyond introduction of stabilising mutations; for example by introduction or deletion of sites for posttranslational modification e.g. glycosylation or phosphorylation or fatty acylation. It may also be chemically modified synthetically, for example by peptide semisynthesis or crosslinking or alkylation. In any event, the mutant GPCR provided has increased stability in a particular conformation relative to its parent GPCR.
• Membrane proteins are often detergent-solubilised and they are purified as protein-detergent complexes.
• the type and concentration of detergent is crucial to keep the protein in its native conformation. Some detergents may prevent proteins from binding to plastic and polystyrene surfaces used as common ELISA supports.
• adsorption to the solid phase can cause partial denaturation of the protein.
• mutant GPCRs confers several advantages when the GPCRs are used as immunogens or selection reagents for screening, enabling them to be used as such in a variety of contexts.
• the mutant GPCR may be in a whole cell preparation, a cell membrane fragment, solubilised in detergent or it may be incorporated into a lipid monolayer, a lipid bilayer, a bead-linked lipid particle, another solid-supported lipid layer or a proteoliposome.
• the lipid layers may be supported directly as a layer on the surface of the solid support or may be tethered as a layer or vesicle as described in Cooper M. A. J MoI Recognit. 2004 Jul-Aug;17(4):286-315.
• membrane proteins are deposited on a surface together with lipid in the form of proteoliposomes.
• the most commonly employed methods for the formation of proteoliposomes start with either prokaryote or eukaryote cells.
• Membrane proteins can be isolated either in a mixed micelle with detergent, dissolved in an organic solvent or aggregated as membrane fragments by sonication in buffer.
• the membrane proteins can be reconstituted into vesicles by: a) organic solvent- mediated reconstitutions (for example reverse-phase evaporation, rehydration of lipid-protein films), b) mechanical means (for example sonication, French press, freeze-thaw), or c) detergent-mediated means (for example dialysis, dilution, or direct incorporation into pre-formed vesicles or into bicelies)
• organic solvent- mediated reconstitutions for example reverse-phase evaporation, rehydration of lipid-protein films
• mechanical means for example sonication, French press, freeze-thaw
• detergent-mediated means for example dialysis, dilution, or direct incorporation into pre-formed vesicles or into bicelies
• the detergent soiubilised form of the mutant GPCR may be a partly pure or highly pure preparation. Purification, enabled by the improved stability and optimisation of solubilisation conditions, confers the advantage of removal of extraneous "sticky" antigens and lipids and other cell surface material such as carbohydrate to which, for example, phage may stick to. This is particularly beneficial when the 'extraneous' material is charged or hydrophobic since phage particles, for example, are known to stick to hydrophobic or charged surfaces non-specifically. Moreover, certain phage antibodies may themselves bind promiscuously to antigens. A normal level of purity, as assessed by SDS-PAGE, is 80-95%.
• the detergent soiubilised form of the mutant GPCR is at least 80% pure, or at least 85% pure, and still more preferably at least 90%, or at least 93%, or at least 95%, pure of other proteins.
• higher levels of purity e.g. at least 99%, can be achieved using additional purification techniques.
• Solubilised receptor preparations are typically made in a buffer of 2OmM Tris (pH 7.0), 0.1 M (NhU) 2 SO 4 , 10% glycerol, 0.07% CHS, 0.33% DOM, 0.33% Chaps, 0.33mM DOPC/DOPS (7:3), and 1 protease inhibitor tablet per 50 ml buffer.
• solubilization buffer 0.5ml of this solubilization buffer is added to approximately 2x10 6 cells, and these cell suspensions are sonicated using a probe sonicator (6x1 -s pulses) and placed on a rocker at 4 0 C. After 2 h, the solutions are centrifuged at 4 0 C for 20min at 14,000rpm using a tabletop centrifuge. The supematants containing solubilised receptor are then transferred to new tubes and kept frozen at -80 0 C until analysis.
• the mutant GPCR may be engineered to include a molecular tag at the C terminus or N-terminus as is well known in the art.
• the tag may be any of a FLAG tag, a His tag, a c-Myc tag, a DDDDK tag, an HSV tag, a Halo tag or a biotin tag.
• Such tags can be used to facilitate phage-based selection protocols in solution and may also be used to confer binding to a solid support.
• mutant GPCRs will facilitate selection and enrichment of mutant GPCRs from impure preparations using affinity columns, affinity filters, magnetic beads and other examples of selective solid supported reagents, filtration, centrifugation, size- exclusion chromatography and dialysis amongst other methods
• affinity columns affinity filters
• magnetic beads magnetic beads
• the increased stability of mutant GPCRs in a range of detergents and solubilisation buffers and additives lends them particularly well to being immobilised onto solid surfaces.
• the mutant GPCR is immobilised onto a solid support.
• Various supports are known in the art and include, for example, beads, columns, slides, chips or plates. Immobilisation may be via covalent or non-covalent interaction.
• the support may be coated with any of avidin, streptavidin, a metal ion, an antibody to the parent GPCR or an antibody to a molecular tag attached to the mutant GPCR.
• the tag may be one recognised by an antibody such as a FLAG tag, or may be a poly-histidine tag enabling binding to a metal ion such as nickel or cobalt, as described for example in Venturi et a/, Biochemica et Biophysica Acta 1610 (2003) 46-50.
• the mutant GPCR may be chemically modified for example with a biotin tag which can be bound to a surface coated with avidin or streptavidin.
• a mutant GPCR may be immobilised via an antibody raised to the native receptor sequence.
• the support may be coated with a polymeric support such as carboxylated dextran.
• the mutant GPCR may be covalently immobilised onto a surface coated with a carboxylated polymer via amine coupling.
• water-soluble carbodiimide mediated activation of a carboxymethylated support such as dextran or hyaluronic acid allows for direct covalent capture of a mutant GPCR via available amino moieties of the protein to form a stable amide linkage.
• GPCRs can be engineered or further derivatised with sulfydryl-reactive reagents (e.g.
• PDEA pyridinyldithioethanamine
• SPDP 3-(2-pyridinyldithio)propioic acid N- hydroxysuccinimide ester)
• stable thioether bonds may be formed using maleimide coupling reagents such as sulfosuccinimidyI-4- ( ⁇ /-maleimidomethyl)cyclohexanecarboxylate (Sulfo-SMCC) and N-( ⁇ - maleimidobutyrloxy)sulfosuccinimide ester (GMBS).
• a solid support may also be derivatised with cystamine to effect coupling with disulfide-activated GPCRs.
• treatment with hydrazine followed by a reductive amination enables coupling with aldheydes.
• the aldehyde groups may be native to the receptor or formed by mild oxidation of any c/s-diols present in the solid support (present in sugar residues of carbohydrates such as dextran, agarose, sepharose, hyaluronic acid and polyaginate).
• amino-presenting surfaces can be treated with commercially available bifunctional linking reagents to effect coupling with free amino or sulfhydryl groups on the receptor as described by (Ernst, O. P. et al.. Meth. Enzymol. 2000, 375, 471-489 and Nunomura, W.e et al. J. Biol. Chem. 2000, 275, 24540-24546).
• the orientation of the mutant GPCR will depend on the desired outcome of the binding partner identification.
• the mutant GPCR will typically be immobilised via the C-terminus or other intracellular domain to enable the extracellular domains to be outward facing in the assay.
• the mutant GPCR is typically immobilised by the N-terminus or extracellular domain. Immobilisation by the N-terminus or extracellular domain may also identify therapeutic molecules that bind to the intracellular domain.
• the mutant GPCR is not immobilised and is, for example, solubilised in detergent or present in a whole cell preparation.
• the test compound for example, a library of test compounds
• the test compound may be immobilised on a solid support, such as a chip surface.
• suitable techniques include photolithography (Affymetrix, Santa Clara, Calif.), mechanical microspotting (Schena et al., Science (1995) vol. 270, p.
• test compound/GPCR mutant pair on the chip is used to reveal the identity of the test compound.
• Other suitable methods are described, for example, in Fang, Y. et al. Drug Discovery Today 2003, 8, 755-761 , and references Alves, I. D.et al. Curr. Prot. & Peptide Sci. 2005, 6, 293-312.; Barry, R.et al. Proteomics 2004, 4, 3717-3726.; Besenicar, M.ef al. Chem. Phys.
• neither the GPCR nor the test compound are immobilised, for example in phage-display selection protocols in solution.
• the method of the invention maybe used to identify ligands of 'orphan' GPCRs where the natural ligand is unknown.
• Ligands of orphan GPCRs may be identified from biological samples such as blood or tissue extracts or from libraries of ligands.
• ligands of mutant GPCRs may be identified where the ligands were interacting proteins such as accessory proteins.
• peptide or protein therapeutics may be identified by the methods of the invention as could conformation-specific antibodies. For example, antibodies prepared as described below could be assessed for conformational-specific GPCR binding.
• antibodies may be identified from supernatants obtained from B- cells taken from immunised animals, from hybridomas obtained following immortalisation of B-cells from the immunised animal or from recombinant antibody libraries which may be expressed on phage particles or through an in vitro expression system such as ribosome display.
• the methods of the invention may also be used to determine the mechanism of action of compounds where the mechanism of action has not previously been known. For example, a mixture of GPCRs representing the 'GPCRome' or a subset thereof could be screened against compounds to identify if their mechanism of action was via binding to a GPCR.
• the invention may be used as a biochemical affinity purification system wherein particular compounds are purified from a mixture of compounds.
• the test compound may be provided as a biological sample.
• the sample could be any suitable sample taken from an individual.
• the sample may be a fluid sample such as blood, serum, plasma or spinal fluid.
• the sample could be a tissue or cell extract.
• the one or more test compounds is a polypeptide.
• the test compound may be a particular type of polypeptide which is known to bind to certain GPCRs but where the identification of a conformation- specific polypeptide is desired.
• the polypeptide may be a candidate therapeutic molecule, for example an anticalin (Skerra J Biotechnol (2001) 74(4):257-75).
• the one or more test compounds is a peptide.
• the one or more test compounds is an affibody, a peptidomimetic, a nucleic acid, a peptide nucleic acid (PNA) or an aptamer, or a lipid or a carbohydrate.
• PNA peptide nucleic acid
• the one or more test compounds is a binding protein based on a modular framework, such as ankyrin repeat proteins, armadillo repeat proteins, leucine rich proteins, tetrariopeptide repeat proteins or Designed Ankyrin Repeat Proteins (DARPins) or proteins based on lipocalin or fibronectin domains or Affilin scaffolds based on either human gamma crystalline or human ubiquitin.
• a binding protein based on a modular framework such as ankyrin repeat proteins, armadillo repeat proteins, leucine rich proteins, tetrariopeptide repeat proteins or Designed Ankyrin Repeat Proteins (DARPins) or proteins based on lipocalin or fibronectin domains or Affilin scaffolds based on either human gamma crystalline or human ubiquitin.
• DARPins Designed Ankyrin Repeat Proteins
• the one or more test compounds is an antibody.
• the test compound may be an antibody that has been raised against a mutant GPCR of a parent GPCR, wherein the mutant GPCR has increased stability in a particular conformation relative to the parent GPCR.
• the mutant GPCR is the same mutant GPCR as was provided in step (a).
• antibody includes but is not limited to polyclonal, monoclonal, chimaeric, single chain, Fab fragments and fragments produced by a Fab expression library.
• fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as genetically engineering derivatives of antibodies such as single chain antibodies (scFv), fusion proteins, domain antibodies (dAbs) and diabodies.
• scFv single chain antibodies
• dAbs domain antibodies
• diabodies diabodies.
• recombinant DNA technology may be used to produce further antibodies or chimeric molecules which retain the binding specificity of an original antibody.
• Such technology may involve fusing the DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions of a different immunoglobulin, as described, for example, in E P-A- 184187, GB 2188638A or EP-A-239400.
• a hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes which may or may not alter the binding specificity of antibodies ⁇ produced.
• the term "antibody" is to be construed as covering any specific binding member or substance having a binding domain with the required specificity.
• antibodies and fragments thereof may be human or humanised antibodies, as is well known in the art. Various procedures known within the art may be used to raise antibodies against a mutant GPCR, or against fragments or fusions thereof.
• an antibody to a mutant GPCR having increased stability relative to a parent GPCR may be produced by immunising a lymphocyte with an immunogen of the mutant GPCR, screening the antibodies so produced for an antibody which binds to the GPCR, and isolating the antibody.
• immunising a lymphocyte' we include both in vivo immunisation, where a whole animal is immunised and in vitro immunisation, where lymphocytes are immunised in vitro, for example as described in US 5,290,681.
• the immunogen of a mutant GPCR is generally all of the mutant GPCR but may also be a part of the mutant GPCR, for example a fragment of the mutant GPCR.
• the production of a stabilised GPCR may facilitate identification of a part of the GPCR not previously obvious from the native conformation that would benefit from a targeted immunisation approach.
• the fragment of the mutant GPCR may be any part of the GPCR which is able to elicit an immune response such as an antibody response. It is known that peptides having as few as 5 amino acids may elicit an antibody response, although typically larger peptides are used.
• the fragment of the immunogen may have at least 5 amino acids, typically from 5 to 1000 amino acids, such as 5 to 500, 5 to 200, 5 to 100, 5 to 50, 5 to 40, 5 to 30, 5 to 20, for example 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.
• the immunogen may be a fusion of the mutant GPCR, wherein the GPCR is fused to a second protein known to be immunogenic to lymphocytes.
• immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
• the immunogen may also further include an adjuvant to enhance the immunological response to a mutant GPCR in a mammal in vivo.
• adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface-active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
• An additional example of an adjuvant that can be employed includes MPL-TDM adjuvant (monophosphoryl Lipid A 1 synthetic trehalose dicorynomycolate).
• Stabilised mutant GPCRs may aiso be linked together on a backbone to produce polyvalent molecules to further increase antigenicity.
• the immunogen of the mutant GPCR may be a variant of the mutant GPCR, provided that it is able to elicit an immune response to the mutant GPCR and does not significantly reduce the stability of the mutant GPCR.
• variants include polypeptides which have one or more amino acid substitutions compared to the mutant GPCR, and as many as 5% substitutions.
• the substitutions are conservative substitutions where, for example, a "variant" refers to a protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein that is still able to elicit an immune response against the mutant GPCR and does not significantly reduce the stability of the mutant GPCR.
• substitutions is intended combinations such as GIy, Ala; VaI, lie, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
• Such variants of a mutant GPCR can be made using standard methods of protein engineering and site-directed mutagenesis.
• the immunogen may also be a polynucleotide encoding the stabilised GPCR or fragment thereof.
• the polynucleotide may be taken up into cells in vivo and expressed on the cell surface where it will stimulate an immune response.
• the immunogen of a mutant GPCR may be provided as a whole cell preparation, in a cell membrane fragment, solubilised in detergent, in a lipid monolayer, in a lipid bilayer, in a bead-linked lipid particle, in a solid-supported lipid layer or in a proteoliposome, as described above.
• polyclonal antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, chicken, mouse or other mammal) may be immunized by one or more injections with the immunogen.
• the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the serum or egg yolk) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum
• Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
• a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
• peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
• the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
• Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
• the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
• the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT- deficient cells.
• Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et ai, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
• monoclonal antibodies can be generated using the 'Selected Lymphocyte Antibody Method' (SLAM) as is well known in the art and described, for example, in Babcook et a/. (1996) Proc. Natl. Acad. Sci. 93:7843-7848. Briefly, a single lymphocyte that is producing an antibody with a desired specificity or function within a large population of lymphoid cells is identified.
• SLAM 'Selected Lymphocyte Antibody Method'
• lymphoid cells derived from an in vivo immunization are screened for those that produce antibodies which bind to selected antigens using an adapted haemolytic plaque assay (Jerne & Nordin, 1963, Science, 140:405) and the genetic information that encodes the specificity of the antibody subsequently retrieved from that lymphocyte.
• an adapted haemolytic plaque assay Jerne & Nordin, 1963, Science, 140:405
• Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
• DNA encoding monoclonal antibodies raised against mutant GPCRs with increased stability can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
• the hybridoma cells described above serve as a preferred source of such DNA.
• the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
• host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
• the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
• non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
• Antibody fragments that contain the idiotypes to the mutant GPCR may also be produced by techniques known in the art including, but not limited to: (i) an F (ab . )2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F a _, fragment generated by reducing the disulfide bridges of an F ⁇ a v )2 fragment; (iii) an F ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v fragments.
• Bispecific antibodies may also be produced.
• Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a mutant GPCR.
• the second binding target is any other antigen, and advantageously is a cell-surface protein orrecept ⁇ r, including another GPCR or receptor subunit.
• bispecific antibodies would be useful for pairs of GPCRs which can form heterodimers (Milligan. Biochim Biophys Acta. 2007 Apr;1768(4):825-35.). In this case, the bispecific antibody may selectively target specific heterodimeric receptors.
• Bispecific antibodies may also be useful for viral entry co-receptors where one of the co-receptors is a GPCR, e.g CD4 and the GPCRs CCR5 or CXCR4 (Alkhatib G, Berger EA. Eur J Med Res. 2007 Oct 15;12(9):375-84).
• GPCR e.g CD4 and the GPCRs CCR5 or CXCR4
• Methods for making bispecific antibodies are known in the art.
• the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy- chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).
• test compound may be a library of test compounds.
• the library may be a peptide or protein library produced, for example, by ribosome display or an antibody library prepared either in vivo, ex vivo or in vitro. Methodologies for preparing and screening such libraries are known in the art.
• the test compound may be an antibody library.
• another method for generating antibodies specific to a GPCR involves screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria, yeast, filamentous phages, ribosomes or ribosomal subunits or other display systems. In this method, large libraries of antibody sequences or antibody fragment sequences are obtained from diverse sources such healthy donors, patients or animals (healthy or not). These sequences are cloned and expressed in an appropriate system and antibodies typically selected by binding to a GPCR with increased stability immobilised on a solid surface.
• an antibody library is a recombinant combinatorial antibody library, for example a scFv or Fab phage display library, prepared using human VL and VH cDNAs prepared from mRNA derived from human lymphocytes (McCafferty et al., Nature 348:552-553 (1990)).
• antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M 13 or fd, and displayed as functional antibody fragments on the surface of the phage particle.
• filamentous particle contains a single-stranded DNA copy of the phage genome
• selections based on the ability of the antibody to bind to the GPCR with increased stability also result in selection of the gene encoding the antibody exhibiting those properties.
• Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-57 1 (1993).
• examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No.
• the methods may also be used to identify a polynucleotide capable of expressing a polypeptide binding partner of a GPCR, for example as described above in relation to phage display methods. Aliquots of an expression library in a suitable vector may be tested for the ability to give the required result. It will be appreciated that several cycles of identifying pools of polynucleotides comprising a polynucleotide having the required property and then rescreening those polynucleotides may be required in order to identify a single species of polynucleotide with the required property.
• the invention includes screening methods to identify drugs or lead compounds of use in treating a disease or condition. It is appreciated that screening assays which are capable of high throughput operation are particularly preferred.
• test compound may be a drug-like compound or lead compound for the development of a drug-like compound.
• a drug-like compound is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament.
• a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water- soluble.
• a drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes or the blood:brain barrier, but it will be appreciated that these features are not essential.
• lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
• the method further comprises modifying a test compound which has been shown to bind to the mutant GPCR when residing in a particular conformation, and determining whether the modified test compound binds to the mutant GPCR when residing in the particular conformation. It will be appreciated that it may be further determined whether the modified test compound also binds to the parent GPCR when residing in the particular conformation.
• Various methods may be used to determine binding between a GPCR and a test compound including, for example, enzyme linked immunosorbent assays
• ELISA ELISA
• surface plasmon resonance assays chip-based assays
• immunocytofluorescence yeast two-hybrid technology
• phage display which are common practice in the art and are described, for example, in Plant et al
• Fluorescence Energy Resonance Transfer methods, for example, well known to those skilled in the art, may be used, in which binding of two fluorescent labelled entities may be measured by measuring the interaction of the fluorescent labels when in close proximity to each other.
• mutant GPCR is provided as a whole cell preparation, a membrane protoplast or a proteoliposome
• biophysical techniques such as patch clamping, magic angle spinning NMR, fluorescence correlation spectroscopy, fluorescence resonance energy transfer and analytical ultracentrifugation may be used to analyse binding of the mutant GPCR to the test compound (as described in New, R. C, Liposomes: a practical approach. 1st ed.; Oxford University Press: Oxford, 1990, and Graham, J. M.; Higgins, J. A., Membrane Analysis.
• Methods which allow quantitative, non-invasive determination of both the affinity and kinetics of such interactions include direct assays that allow continuous monitoring of membrane- protein binding, or in some cases by ultra-rapid separation of bound and free interactants followed by quantification of the membrane-bound and membrane- free analyte.
• a bound test compound can be detected using a unique label or tag associated with the compound such as a peptide label, a nucleic acid label (Kerr et al., JACS (1993) vol. 115, p. 2529-2531 ; and Brenner & Lerner, Proc. Natl. Acad. Sci. USA (1992) vol. 89, p. 5381-5383), a chemical label (Ohlmeyer et al., Proc. Natl. Acad. Sci. USA (1993) vol. 90, p. 109222-10926; and Maclean et al., Proc. Natl. Acad. Sci. USA (1997) vol. 94, p.
• a unique label or tag associated with the compound such as a peptide label, a nucleic acid label (Kerr et al., JACS (1993) vol. 115, p. 2529-2531 ; and Brenner & Lerner, Proc. Natl. Acad.
• binding is preferably assayed using immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
• the stabilised GPCR is immobilised on a microtitre plate or other solid surface.
• the plates are first incubated with BSA or other similar protein to block non-specific binding sites.
• Samples containing the antibody such as serum or egg yolk or hybridoma cell culture supernatant
• Bound antibodies are detected by the addition of a second detection antibody which binds to the first antibody and allows detection via its conjugation to an enzyme which catalyzes a reaction which can be subsequently detected.
• the binding affinity of antibodies can also be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem, 107:220 (1980).
• antibodies having a high degree of specificity and a high binding affinity for the target GPCR are isolated.
• the hybridoma clones identified as expressing specific antibody can subsequently be subcloned by limiting dilution procedures and grown using standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
• the monoclonal antibodies secreted by the subclones can then be isolated or further purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
• binding to a stabilised GPCR may be assessed as follows.
• the mutant GPCR with increased stability is coated onto wells of a microtiter plate overnight at 4 0 C.
• the wells are washed in PBS and blocked for 1 hour at 37 °C in MPBS (3% milk powder in PBS).
• Purified phage from a phagemid library e.g. expressing a repertoire of human scFv (10 transducing units (tu)
• the blocked phage are added to the blocked GPCR wells and incubated for 1 hour.
• PBST PBS containing 0.1 % v/v Tween 20
• the bound phage particles are then eluted and used to infect 10 ml exponentially growing E. coli TG 1.
• the infected cells are grown in 2TY broth for 1 hour at 37 0 C, then spread onto 2TYAG plates and incubated overnight at 30 0 C. Cultures from this first round of panning selection are superinfected with helper phage and rescued to give, for example, scFv antibody-expressing phage particles for the second round of panning.
• An alternative binding assay for phage display antibodies includes the use of soluble selections using biotinylated mutant GPCR protein at a final concentration typically of 100 nM.
• purified scFv phage (1012 tu) from a scFv phagemid library (as described above) is suspended in 1 ml 3% MPBS and blocked for 30 minutes.
• the biotinylated GPCR is then added and incubated at room temperature for 1 hour.
• the phage/antigen complexes are subsequently added to 250 ⁇ l of Dynal M280 Streptavidin magnetic beads that have been blocked for 1 hour at 37 0 C in 1 ml of 3% MPBS, and incubated with the beads for a further 15 minutes at room temperature.
• the beads are captured using a magnetic rack and washed 4 times in 1 ml of 3% MPBS/0.1% (v/v) Tween 20 followed by 3 washes in PBS. After the last PBS wash, beads are resuspended in 100 ⁇ l PBS and used to infect 5 ml exponentially growing E.coli. Again, cultures from this first round of soluble selection would be superinfected with helper phage and rescued to give scFv antibody-expressing phage particles for a second round of soluble selection.
• screening assays which are capable of high throughput operation are particularly preferred to determine binding to a mutant GPCR, for example chip-based assays.
• Stabilised mutant GPCRs are particularly suited to such assays unlike their parent GPCRs which are not stable enough when purified to be used in these formats.
• technology called VLSIPSTM has enabled the production of extremely small chips that contain hundreds of thousands or more of different molecular probes, i.e. the test compounds. These biological chips have probes arranged in arrays, each probe assigned a specific location. Biological chips have been produced in which each location has a scale of, for example, ten microns. The chips can be used to determine whether target molecules interact with any of the probes on the chip. After exposing the array to target molecules under selected test conditions, scanning devices can examine each location in the array and determine whether a target molecule has interacted with the probe at that location.
• a test compound to a mutant GPCR on a chip surface may be detected by scanning the chip surface for radioactivity or fluorescence.
• the address of the interacting pair on the chip reveals the identity of the test compound or where there is an array of mutant GPCRs on the chip, the identity of the receptor (see, for example, Kuimelis et al., Addressable Protein Arrays, U.S. Ser. No.
• the array of mutant GPCRs may be used as a method for obtaining selectivity data on compounds either for various conformations of the same GPCR or for various GPCRs.
• SPA surface plasmon resonance assays
• the mutant GPCR immobilised on a SPA bead, may be incubated with a single ligand which is labelled for example with a fluorescent group or the ligand may be a radioligand.
• the ability of a test compound to bind to the mutant GPCR may then be determined via its ability to displace the fluorescent ligand or the radioligand.
• the mutant GPCR is immobilised on a chip surface and binding of test compounds is detected by surface plasmon resonance and related techniques employing evanescent waves. Changes in refractive index can be used to determine the amount of bound compound, the affinity of interaction and the association and dissociation kinetics.
• An example of this approach has been described for rhodopsin which was immobilised on carboxylated dextran surfaces modified with long alkyl groups. Following amine coupling of the detergent- solubilised receptor, lipid/detergent-mixed micelles were adhered over the immobilized surface. The detergent was eluted in the subsequent buffer flow and the remaining lipid formed a bilayer on the chip surface. (Karlsson OP, Lofas S.. Anal Biochem. 2002 Jan 15;300(2): 132-8.
• test compound is a peptide or protein
• a ligand for an orphan receptor or an interacting protein the bound ligand may be eluted and then identified by mass spectrometry such as matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) or electrospray ionization mass spectrometry (ESI-MS) (Williams C. Addona TA.
• mass spectrometry such as matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) or electrospray ionization mass spectrometry (ESI-MS) (Williams C. Addona TA.
• MALDI-TOF matrix assisted laser desorption/ionization time of flight mass spectrometry
• ESI-MS electrospray ionization mass spectrometry
• the mutant GPCR may be immobilised on a column or bead, or expressed in tagged form in a cell and co-purified with the ligand from such complex mixtures using reagents directed to the tag or directly to the GPCR
• the method further comprises determining if the binding partner affects the function of the GPCR to which it binds and isolating a test compound that affects the function of the GPCR.
• the binding partner alters the binding of the GPCR to its ligand.
• ligand we include any molecule which binds to the GPCR and which causes the GPCR to reside in a particular conformation as described above.
• the ligand is the natural ligand of that GPCR or an analogue thereof. Binding of a GPCR to its ligand can be assayed using standard ligand binding methods known in the art and, for example, as described above.
• the ligand may be radiolabeled or fluorescently labelled. The binding assay can be performed using the stabilised mutant GPCR or the parent GPCR.
• the stabilised GPCR is purified or expressed in a cell such a mammalian, bacterial or insect cell.
• the parent receptor is expressed in a cell such as a mammalian, bacterial or insect cell.
• the assay may be carried out on whole cells or on membranes obtained from the cells.
• the binding partner will be characterised by its ability to alter the binding of the labelled ligand.
• the binding partner decreases binding between the GPCR and its ligand.
• the binding partner may decrease binding by a factor of at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 15 fold, 20, fold, 50 fold, 100 fold, 250 fold, 500 fold or 1000 fold.
• the binding partner decreases binding by a factor of between 100-1000 fold, such as between 10-100 fold.
• the binding partner increases binding between the GPCR and its ligand.
• the binding partner may increase binding by a factor of at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 15 fold, 20, fold, 50 fold, 100 fold, 250 fold, 500 fold or 1000 fold.
• the binding partner increases binding by a factor of between 100-1000 fold, such as between 10-100 fold.
• the parent GPCR or stabilised mutant GPCR is expressed in vivo, for example, in mammalian or insect cells where the GPCR is allowed to couple to well know GPCR signal transduction pathways (Eglen R.M. Functional G protein-coupled receptor assays for primary and secondary screening. Comb Chem High Throughput Screen. 2005 Jun;8(4):311-8).
• Such assays include: calcium mobilisation (Gonzalez JE, Maher MP. Cellular fluorescent indicators and voltage/ion probe reader (VIPR) tools for ion channel and receptor drug discovery. Receptors Channels. 2002;8(5-6):283-95, Dupriez VJ, Maes K, Le Poul E, Burgeon E, Detheux M.
• Binding partners are typically selected which modulate the activation of the receptor.
• the binding partner will typically mimic the activity of the natural ligand of the receptor and produce an increase in receptor activation, G protein activation or signal transduction. This will occur in the absence of an additional agonist.
• An agonist binding partner may increase receptor activation by a factor of at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 15 fold, 20, fold, 50 fold, 100 fold, 250 fold, 500 fold, 1000 fold, or 10000 fold.
• a binding partner may increase receptor activation.
• the binding partner may act as a direct agonist, in which case receptor activation is typically increased by between 2-1000 fold.
• the binding partner may act to amplify the activity of an agonist.
• the binding partner may increase the potency of the agonist, in which case receptor activation is typically increased by between 2-1000 fold, such as between 10-100 fold, or the binding partner may increase the maximal response produced by the agonist, in which case receptor activation is typically increased by between 2-10 fold.
• activating/amplifying the activity of receptors that are already switched on by endogenous ligand may be preferable to turning on all available receptors, since it is more physiologically specific and may mitigate issues such as desensitisation and undesirable side-effects (Christopoulos A (2002) Nat Rev Drug Discov 1 :198-210).
• the binding partner will typically act to block the activity of the receptor or its activation by an agonist.
• the antagonist binding partner may do this by blocking the binding of the agonist or by locking the receptor in an inactive form such that it is unable to couple to G proteins.
• An antagonist binding partner may decrease receptor activation by a factor of at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 15 fold, 20, fold, 50 fold, 100 fold, 250 fold, 500 fold, 1000 fold or 10000 fold.
• an antagonist binding partner would reduce receptor activation to a non-detectable level.
• test compound may be desirable to produce a test compound or combination of test compounds that bind to more than one GPCR.
• the test compound may bind to more than one GPCR of the same class of family.
• a list of GPCR classes and families has been produced by the International Union of Pharmacology (Foord et al (2005) Pharmacol. Rev. 57, 279-288) and this list is periodically updated at http://www.iuphar- db.org/GPCR/ReceptorFamiliesForward.
• the test compound may bind to one target GPCR and at least one further GPCR.
• the at least one further GPCR may be a GPCR that has been implicated in a disease pathway, or a GPCR that is involved in the same signalling pathway as the target GPCR, for example a signalling pathway that regulates or modulates a disease pathway.
• the at least one further GPCR may be one which enhances or suppresses the action of a test compound on the target GPCR. Such enhancement or suppression of action can be determined using methods well known in the art including binding assays and functional assays as described, for example, above.
• more than one mutant GPCR is provided in step (a).
• at least 2, 3, 4 or 5 mutant GPCRs of a different parent GPCR may be provided in step (a).
• test compounds are selected for which bind to more than one GPCR.
• the compound may be a cross-reactive compound including, for example, a small molecule, affibody, antibody or diaboody. It will be appreciated that such an approach may lead to improvements in a binding partner's efficacy or potency.
• a test compound may be selected which binds to a first and second GPCR, where the first and second GPCRs may be any pair of GPCRs.
• the method can be used to select for a bivalent partner that binds to GPCRs which form a heterodimer, in which case the bivalent partner could bind to both receptors at the same time.
• GPCR heterodimers include dopamine D1 and adenosine A1 , opioid receptor heterodimers, cannabinoid CB1 and orexin receptors (Marshall FH.Heterodimerization of G- protein-coupled receptors in the CNS. Curr Opin Pharmacol.
• a binding partner could be selected for which binds to and modulates the activity of two independent receptors for the purpose of enhanced therapeutic activity.
• CCK1 /opioid receptor peptides bind to both CCK1 and the opoid receptor (Garcfa-L ⁇ pez MT, Gonzalez-Muniz R, Martin-Martinez M, Herranz R.Strategies for design of non peptide CCK1 R agonist/antagonist ligands. Curr Top Med Chem. 2007;7(12):1180-94).
• Other appropriate examples include a combined beta 2 agonist and muscarinic antagonist; a dopamine D2 antagonist and 5HT2 antagonist; a D2 antagonist and 5HT6 antagonist; and a M1 agonist and 5HT6 antagonist.
• the more than one GPCR provided in step (a) may or may not reside in the same conformation.
• a diabody or similar bivalent binding partner may agonise at one end of the molecule and antagonise at another end, in which case the GPCRs would not have to reside in the same conformation.
• test compound typically binds to more than one GPCR, the test compound binds to each GPCR with a similar potency.
• the K d values for the particular binding partner binding to each of the respective GPCRs are within 5-10 fold of each other, such as within 2-3 fold.
• the methods of the invention allow for combinations of test compounds that bind to one or more GPCRs to be isolated, either by repeating the method with single test compounds, providing multiple test compounds in one cycle of the method or by using a library of test compounds in the method.
• test compounds which, while still able to bind to a first GPCR, are not able to bind, or bind less strongly than to the first GPCR, to at least one other GPCR, for example a second GPCR.
• the first and second GPCRs may be any pair of GPCRs.
• the test compound may be one that is selected on the basis that it binds to a first GPCR, but the test compound so selected is further tested to determine whether it binds a second GPCR (or binds less strongly to a second GPCR than the first GPCR). Test compounds are selected which do not bind (or have reduced binding to) the second GPCR. Where the binding partner is therapeutic molecule, such an approach may help to reduce the binding partner's toxicity.
• the test compound binds the further (second) GPCR with an affinity which is less than 50% of the affinity the compound has for first GPCR, more preferably less than 10% and still more preferably less than 1% or 0.1 % or 0.01% of the affinity the compound has for the first GPCR.
• the K d for the interaction of the test compound with the first GPCR is higher than for the second GPCR.
• a second aspect of the invention provides a method for producing a binding partner of a GPCR, the method comprising synthesising a binding partner identifiable by carrying out the method according to the first aspect of the invention.
• binding partners can be synthesised by any suitable method known in the art including the techniques of organic chemistry, molecular biology or biochemistry.
• the binding partner may be made by expressing the nucleic acid molecule encoding the binding partner in a suitable host cell as is known in the art.
• Antibodies may be synthesised using any of the methods described above including, for example, recombinant DNA technology.
• a third aspect of the invention provides a binding partner obtained by any of the methods of the first aspect of the invention.
• the binding partner is a conformation-specific binding partner, as described above.
• a fourth aspect of the invention provides a binding partner, for example a conformation-specific binding partner, obtainable by any of the methods of the first aspect of the invention.
• the binding partner may be any of a polypeptide; an anticalin; a peptide; an antibody; a chimeric antibody; a single chain antibody; an aptamer; a darpin; a
• Fab, F(ab') 2l Fv, ScFv or dAb antibody fragment a small molecule; a natural product; an affibody; a peptidomimetic; a nucleic acid; a peptide nucleic acid molecule; a lipid; a carbohydrate; a protein based on a modular framework including ankyrin repeat proteins, armadillo repeat proteins, leucine rich proteins, tetrariopeptide repeat proteins or Designed Ankyrin Repeat Proteins (DARPins); or proteins based on lipocalin or fibronectin domains or Affilin scaffolds based on either human gamma crystalline or human ubiquitin.
• DARPins Designed Ankyrin Repeat Proteins
• the binding partner is an antibody.
• the antibody may be specific to a non-contiguous epitope in the GPCR or may be specific to a contiguous epitope in the GPCR.
• the relevant epitopes in the parent and mutant GPCR recognised by the antibody are similar, whether they be contiguous or non-contiguous.
• extracellular epitopes such as C- or N-terminii or polypeptide loops of the parent and mutant GPCR are preferably similar.
• the binding partner to the mutant GPCR with a similar potency to its binding to the parent GPCR.
• the K d values for the particular binding partner binding the mutant GPCR and the parent GPCR are within 5-10 fold of each other, such as within 2-3 fold.
• the binding of the binding partner to the mutant GPCR compared to the parent GPCR would be not more than 5 times weaker and not more than 10 times stronger.
• mutant receptors which have been stabilised in the selected conformation should bind the binding partner with approximately equal affinity (that is to say typically within 2-3 fold) or greater affinity than does the parent receptor.
• the mutants typically bind the agonists with the same or higher affinity than the parent GPCR and typically bind antagonists with the same or lower affinity than the parent GPCR.
• the mutants typically bind the antagonists with the same or higher affinity than the parent GPCR and typically bind agonists with the same or lower affinity than the parent GPCR. It is appreciated that the methods of the invention may be used as a biosensor to detect target substances such as molecules, especially biomolecules.
• the biosensor may be used to detect biomarkers of disease or drug treatment which may be used as a diagnostic or prognostic.
• the mutant GPCR may be immobilised on a sensor surface and binding of compounds detected, for example, by surface plasmon resonance as described above.
• compound binding to the receptor can be detected by changes in intrinsic tryptophan fluorescence or using fluorescence resonance energy transfer between an intrinsic tryptophan resident donor and a fluorescent acceptor (Lakowicz JR 1999. Principles of fluorescence spectroscopy, Plenum New York, Martin DD, Budamagunta MS, Ryan RO, Voss JC, Oda MNJ Biol Chem. 2006 JuI 21 ;281(29):20418-26).
• mutant GPCRs may be used in acoustic biosensors, wherein the mutant GPCRs are immobilised on quartz crystal resonator sensors (QCRS) and the acoustic sensor response used to detect compound - mutant GPCR binding interactions (Cooper MA 1 Drug Discov Today. 2006 Dec; 11 (23-24): 1068-74. Epub 2006 Oct 20).
• QCRS quartz crystal resonator sensors
• a fifth aspect of the invention provides a biosensor comprising a mutant GPCR of a parent GPCR wherein the mutant GPCR has increased stability in a particular conformation relative to the parent GPCR, and wherein when a target substance binds to said mutant GPCR, a detectable signal is produced.
• Preferences for the mutant GPCRs and their methods of production are as defined above with respect to the first aspect of the invention.
• the biosensor is in a chip form or a bead supported form, where the mutant GPCRs are immobilised on a chip or bead and used to detect target substances.
• the mutant GPCRs may be provided in soluble form, in which case the biosensor would comprise a solution.
• mutant GPCRs are reconstituted onto chip surfaces suitable for direct biosensor analysis via flow-mediated surface reconstitution (Karlsson et al., Analytical Biochemistry 300, 132-138 (2002)).
• flow-mediated surface reconstitution Karlsson et al., Analytical Biochemistry 300, 132-138 (2002).
• rapid immobilization and reconstitution of GPCRs on carboxylated dextran surfaces modified with long alkyl groups can be achieved following amine coupling of a detergent-solubilized receptor; lipid/detergent-mixed micelles are adhered as they are injected over the immobilized surface, taking advantage of integrated flow cells present in many biosensor systems.
• the detergent can then be eluted in the subsequent buffer flow leaving functional, intact mutant GPCRs for subsequent screening and analysis.
• mutant GPCR preparations containing detergent are ideally suited for use in a flow-based biosensor, such as quartz crystal microbaiance biosensor, an evanescent wave biosensor, a planar wave guide biosensor, a surface Raman sensor, or a surface plasmon resonance biosensor.
• solubilized receptors can be captured on a GE Healthcare (Biacore) CM4 or CM5 dextran sensor chip.
• the dextran matrix of the sensor chip is activated by 35 uL of 50 mM /V-hydroxysuccinimide and 200 mM ⁇ /-ethyl- ⁇ /- [(dimethylamino)propyl]carbodiimide at a flow rate of 5 uL/min, followed by a 7- min injection of 0.1 mg/mL detergent-solubilised GPCR receptor (for example with 25 mM CHAPS in 10 mM MOPS, pH 7.5). Any remaining reactive carboxy groups are deactivated using a 7-min pulse of 1 M ethanolamine hydrochloride, pH 8.5.
• the biosensor chip is washed at high flow rate with the surface plasmon resonance running solution until a stable baseline is restored (ca. 30 min).
• This washing step works like a flow dialysis procedure and ensures the removal of the detergent from the sensor chip surface; however, it will be appreciated that hydrophobic parts of the GPCR may still be attached to some lipid or detergent molecules in order to maintain functional integrity.
• the target substance may be any of a molecule, a biomolecule, a peptide, a protein, a carbohydrate, a lipid, a GPCR ligand, a synthetic molecule, a drug, a drug metabolite or a disease biomarker.
• the detectable signal is any of a change in colour; fluorescence; evanescence; surface plasmon resonance; electrical conductance or charge separation; ultraviolet, visible or infrared absorption; luminescence; chemiluminescence; electrochemiluminescence; fluorescence anisotropy; fluorescence intensity; fluorescence lifetime; fluorescence polarisation; fluorescence energy transfer; molecular mass; electron spin resonance; nuclear magnetic resonance; hydrodynamic volume or radius; specific gravity; scintillation; field effect resistance; electrical impedance; acoustic impedance; quantum evanescence; resonant scattering; fluorescent quenching; fluorescence correlation spectroscopy; acoustic load; acoustic shear wave velocity; binding force; or interfacial stress.
• Figure 1 Amino acid changes in ⁇ AR that lead to thermostability. Stability quotient indicates the % remaining binding activity of the mutants after heating the sample for 30 min at 32 0 C. All values are normalized to PAR 34-424 (50%, showed as a discontinuous line) to remove any experimental variability between assays. Bars show the stability for each mutant. The letters on the x-axis indicate the amino acid present in the mutant. The original amino acid and its position in PAR 34-424 is indicated below. Bars corresponding to the same amino acid in PAR 34-424 are in the same colour with arrows indicating the best mutations. Errors were calculated from duplicate measurements; the best mutants were subsequently re-assayed to determine the Tm for each individual mutation and to give an accurate rank order of stability for each mutant (see Example 1).
• FIG. 2 Side chains in rhodopsin that are at equivalent positions to the thermostable mutations in PAR 34-424 .
• the equivalent amino acid residues in rhodopsin to the amino acid residues mutated in PAR 34-424 were located in the rhodopsin structure, based upon an alignment among rhodopsin, pi adrenergic receptor, neurotensin receptor, and adenosine A 2a receptor (data not shown).
• Side chains in the same transmembrane helix are shown as space filling models in the same colour.
• the name and position of the amino acid residues are those in rhodopsin.
• detergent-solubilised receptors were incubated for 30 minutes at the temperatures indicated before carrying out the binding assay.
• detergent-solubilised receptors were pre-incubated with [ 3 H]-DHA, followed by incubation at the temperatures indicated.
• ⁇ AR-m23 circles
• PAR 34-424 squares
• FIG. 5 Competition binding of agonists to ⁇ AR-m23 and ⁇ AR 34-424 . Binding assays were performed on receptors partially purified in DDM; ⁇ AR-m23 (triangles) and ⁇ AR 34-424 (squares). [ 3 H]-DHA was used at a concentration three times greater than the K 0 of partially purified receptor (see Methods). [ 3 H]-DHA binding was competed with increasing concentrations of the agonists, norepinephrine (a) and isoprenaline (b), or with an antagonist, alprenolol (c).
• LogEC 5 o and corresponding EC 50 values for the different ligands were calculated by nonlinear regression using GraphPad Prism software and the error for logEC 50 s were lower than 10%.
• the EC 50 S for ligand binding to ⁇ AR 34-424 and ⁇ AR-m23 are: norepinephrine, ⁇ AR 34-424 1.5 ⁇ M, ⁇ AR-m23 3.7 mM; isoprenaline, ⁇ AR 34-424 330 nM, ⁇ AR-m23 20 ⁇ M; alprenolol, ⁇ AR 78 nM, ⁇ AR-m23 112 nM.
• FIG. 6 Stability of ⁇ AR-m23 and ⁇ AR 34-424 in five different detergents.
• Samples of ⁇ AR 34-424 (a), and ⁇ AR-m23 (b) solubilized in DDM were partially purified on Ni-NTA agarose columns allowing the exchange into various different detergents: DDM (squares), DM (triangles), OG (inverted triangles), LDAO (diamonds) and NG (circles).
• ⁇ AR is so unstable in OG, NG and LDAO that it was not possible to measure any activity after purification at 6°C.
• Assays were carried out as described in the Methods and the Tm is shown at the intersection between the curves and the discontinuous line. Results are from duplicate measurements in a representative experiment performed in parallel,
• FIG. 7 Curve of thermostability of ⁇ AR 34-424 (Tm). Binding assays were performed using [ 3 H]-dihydroalprenolol (DHA) as radioligand as described under "Methods”. Samples were heated for 30 minutes at different temperatures before the assay. Tm represents the temperature at which the binding decreased to the 50%, value showed as a discontinuous line. Data points are from duplicates of one single experiment. This experiment has been repeated several times with similar results.
• DHA dihydroalprenolol
• FIG. 8 Saturation binding assays of membranes of ⁇ AR 34-424 and ⁇ AR-m23. Binding assays were performed as described in "Methods" using
• Figure 9 Alignment of the turkey ⁇ -adrenergic receptor with human ⁇ 1 , ⁇ 2 and ⁇ 3 receptors.
• Figure 12 Flow chart showing the two different assay formats of ligand (+) and ligand (-) used to determine receptor thermostablity.
• FIG. 13 Pharmacological profile of thermostable mutant adenosine A2a receptor, Rant21. Saturation binding of (A) antagonist and (B) agonist to solubilised receptors. (C-F) Inhibition of [ 3 H]ZM241385 binding by increasing concentrations of antagonists (C) XAC and (D) Theophylline, and agonists (E) NECA and (F) R-PIA; binding of [ 3 H]ZM241385 (10 nM) in the absence of unlabelled ligand was set to 100%.
• Figure 14 Thermostable mutants show a decreased dependence on lipids (A) and an increased survival at higher concentration of DDM (B) upon heating compared to the wild-type receptor. Receptors were solubilised in 1% DDM
• FIG. 15 Mapping of the M90V, Y227A, A282L and F338M m23 mutations in turkey betai adrenergic receptor onto homologous residues (M82, Y219, C265 and A321 respectively) in the human beta2 adrenergic receptor structure (Rasmussen et al (2007) Nature 15;383-387; pdb accession codes 2R4R and 2R4S) reveals their position at a helical interface and helical kink respectively. Amino acid residues in equivalent positions to the thermostabilising mutations in the turkey ⁇ 1 adrenergic receptor are shown as labelled space filling models.
• Figure 16 Mapping of m23 mutations in turkey betai adrenergic receptor onto homologous residues in the human beta2 adrenergic receptor structure (Cherezov et al (2007) Science, 318:1258-65; pdb accession code 2RH1 ).
• the Ca trace of the ⁇ 2AR is shown with the fusion moiety (T4 lysozyme) removed.
• the six mutations in ⁇ AR-m23 (R68S, M90V, Y227A, A282L, F327A, F338M) are equivalent to amino acid residues K60, M82, Y219, C265, L310, F321 in the human ⁇ 2AR.
• Lys ⁇ O is on the intracellular end of Helix 1 and points into the lipid- water interface.
• Met82 is near the middle of Helix 2 and points into the ligand binding pocket; the nearest distance between the substrate carazolol and the Met side chain is 5.7 A.
• Tyr219 is towards the intracellular end of helix 5 and is at the helix ⁇ -helix 6 interface.
• Cys265 is at the end of the loop region between helices 5 and 6 and points away from the transmembrane regions.
• Leu310 and Phe321 are both in helix 7 and both point out into the lipid bilayer.
• Figure 17 Multiple sequence alignment of human beta-2AR, rat NTR1 , turkey beta-1 AR, human Adenosine A2aR and human muscarinic M1 receptors. In each sequence, thermostabilising mutations are marked with a box. Mutations occurring in two or more sequences are denoted with a star.
• FIG. 18 Mapping of turkey betalAR mutation I55A (human beta2AR 147) onto human beta2AR structure (pdb accession code 2RH1). Mutation is at the interface between 3 helices (H1 , H2 kink, H7 kink). Left: side view; right: top view.
• Figure 19 Mapping of turkey betalAR V89L mutation (human beta2AR V81 ) onto human beta2AR structure (pdb accession code 2RH1 ). Mutation is in the kink in helix 2. The helices are numbered and the bound antagonist is shown as a space filling model. Amino acid residues in equivalent positions to the thermostabiiising mutations in the turkey ⁇ 1 adrenergic receptor are shown as space filling models and are arrowed for clarity. Left: side view; right: top view.
• FIG. 20 Mapping of turkey betalAR M90V mutation (human beta2AR M82) onto human beta2AR structure (pdb accession code 2RH1 ). Mutation is in kink in helix 2 oriented towards the binding pocket. The helices are numbered and the bound antagonist is shown as a space filling model. Amino acid residues in equivalent positions to the thermostabiiising mutations in the turkey ⁇ 1 adrenergic receptor are shown as space filling models and are arrowed for clarity. Left: side view; right: top view.
• FIG. 21 Mapping of turkey betalAR 1129V mutation (human beta2AR 1121 ) onto human beta2AR structure (pdb accession code 2RH1 ). Mutation is opposite a kink in helix 5. The helices are numbered and the bound antagonist is shown as a space filling model. Amino acid residues in equivalent positions to the thermostabiiising mutations in the turkey ⁇ 1 adrenergic receptor are shown as space filling models and are arrowed for clarity. Left: side view; right: bottom view.
• FIG. 22 Mapping of turkey betalAR F338M mutation (human beta2AR F321 ) onto human beta2AR structure (pdb accession code 2RH1 ). Mutation is in kink in helix 7. The helices are numbered and the bound antagonist is shown as a space filling model. Amino acid residues in equivalent positions to the thermostabiiising mutations in the turkey ⁇ 1 adrenergic receptor are shown as space filling models and are arrowed for clarity. Left: side view; right: top view.
• FIG. 23 Mapping of turkey betalAR Y227A mutation (human beta2AR Y219) onto human beta2AR structure (pdb accession code 2RH1 ). Mutation is at helix- helix interface. The helices are numbered and the bound antagonist is shown as a space filling model. Amino acid residues in equivalent positions to the thermostabiiising mutations in the turkey ⁇ 1 adrenergic receptor are shown as space filling models and are arrowed for clarity. Left: side view; right: bottom view.
• FIG. 24 Mapping of turkey beta 1AR A282L mutation (human beta2AR C265) onto human beta2AR structure (pdb accession code 2RH1 ). Mutation is in loop region. The helices are numbered and the bound antagonist is shown as a space filling model. Amino acid residues in equivalent positions to the thermostabilising mutations in the turkey ⁇ 1 adrenergic receptor are shown as space filling models and are arrowed for clarity. Left: side view; right: top view.
• FIG. 25 Mapping of turkey betalAR R68S mutation (human beta2AR K60) onto human beta2AR structure (pdb accession code 2RH1 ). Mutation is at the lipid-water boundary, pointing into the solvent. The helices are numbered and the bound antagonist is shown as a space filling model. Amino acid residues in equivalent positions to the thermostabilising mutations in the turkey ⁇ 1 adrenergic receptor are shown as space filling models and are arrowed for clarity. Left: side view; right: angled top view.
• thermostabilities of three ⁇ adrenergic receptors (turkey ⁇ 1 ( ⁇ ), human ⁇ 1 (T) and human ⁇ 2 (•)) and two thermostabilised receptors (turkey ⁇ 1-m23 (A) and human ⁇ 2-m23 ( ⁇ )).
• the six thermostabilising mutations in ⁇ 1-m23 (R68S, M90V, Y227A, A282L, F327A, F338M) were all transferred directly to the human ⁇ 2 receptor (K60S, M82V, Y219A, C265L, L310A, F321 M) making ⁇ 2-m23, based upon the alignment in Figure 9.
• the resulting mutants were transiently expressed in mammalian cells, solubilised in 0.1 % dodecylmaltoside and assayed for thermostability in the minus-ligand format (heating the apo-state, quenching on ice, adding 3H-DHA).
• the apparent Tms for turkey ⁇ 1 and ⁇ 2-m23 were 23 0 C and 45 0 C respectively, giving a ⁇ Tm of 22°C as seen previously in E.coli expressed receptor.
• the Tms for human ⁇ 2 and ⁇ 2-m23 were 29°C and 41 0 C respectively, showing that the apo receptor was stabilised by 12°C.
• thermostabilising mutations from one receptor to another receptor, which in this case are 59% identical.
• the human ⁇ 1 receptor (Tm ⁇ 12°C) is much less stable than the turkey ⁇ 1 receptor.
• Figure 27 Percentage identity of the turkey ⁇ 1 adrenergic receptor, human adenosine receptor and rat neurotensin receptor to human ⁇ adrenergic receptors, human adenosine receptors and human neurotensin receptors, respectively.
• Figure 29 Schematic representation of typical lead isolation process for the identification of inhibitory scFv binders.
• FIG 31 Specificity phage ELISA using ⁇ -andregenic receptor and 3 unrelated control proteins (CD86-CD4, Notch1-Fc and the NRR region of Notchi ). Proteins coated on two ELISA plates, amino-plate (A) and His-plate (B) are shown. Beta- AR phage clones (hashed bars) from left to right (C1 , E2, A3, G3, C4, D4, F4, H4, D5, F5, G5, C6, D6, C7, F7, B8 and C8) names originates from the screen shown in Figure 30. In the graph, sticky anti ⁇ -AR clones are indicated by their clone names over the corresponding bar. Also shown are binding of control phage populations specific to the control proteins, anti-CD86 (grey bars), anti-N1 EGF (white bars) and anti-N1 NRR (dotted bars).
• Figure 33 Two capture stages of the biotinylated ⁇ 1AR onto a streptavidin- coated flow cell. A -1200 RU captured; B -4000 RU captured.
• FIG. 34 Biacore responses for alprenolol. The highest concentration is 666 nM and each concentration was tested three times in a three-fold dilution series. The responses are concentration dependent and fairly reproducible. The lines depict the fit of a simple 1 :1 interaction model and the parameters determined from this fit are listed in the inset (the number in parentheses is the error in the last digit).
• Figure 35 Normalised responses to alprenolol. The responses are normalized with respect to the Rmax determined (from the fitting) for each curve.
• Figure 37 Normalised responses to propranol. The responses are normalized with respect to the Rmax determined (from the fitting) for each curve.
• Figure 39 Capture of ⁇ 1 AR to a density of 8000 RU.
• FIG 41 Biacore responses to salmeterol using 2 different surfaces. Salmeterol was tested using a highest concentration of 1.67 uM, with each concentration tested two or three times. A. 4200 RU surface; B. 8000 RU surface. Larger responses are observed with the 8000 RU surface.
• Figure 44 Biacore responses for carvedilol. A. 5200 RU surface; B. 8000 RU surface.
• GPCRs G protein-coupled receptors
• ⁇ AR-m23 The most stable mutant receptor, ⁇ AR-m23, contained 6 point mutations that led to a Tm 21 0 C higher than the native protein and, in the presence of bound antagonist, ⁇ ARm23 was as stable as bovine rhodopsin. In addition, ⁇ AR-m23 was significantly more stable in a wide range of detergents ideal for crystallisation and was preferentially in an antagonist conformation in the absence of ligand.
• ⁇ AR from turkey erythrocytes is an ideal target for structural studies because it is well characterised and is expressed at high-levels in insect cells using the baculovirus expression system[10,11].
• the best overexpression of ⁇ AR is obtained using a truncated version of the receptor containing residues 34-424 ( ⁇ AR 34- 42 4 ) [9] and this was used as the starting point for this work.
• Alanine scanning mutagenesis was used to define amino residues in ⁇ AR 34-424 that, when mutated, altered the thermostability of the receptor; if an alanine was present in the sequence it was mutated to a leucine residue.
• a total of 318 mutations were made to amino acid residues 37-369, a region that encompasses all seven transmembrane domains and 23 amino acid residues at the C terminus; mutations at 15 amino residues were not obtained due to strong secondary structure in the DNA template.
• the receptors were functionally expressed in E. coli and assayed for stability.
• the assay for thermostability was performed on unpurified detergent-solubilised receptors by heating the receptors at 32°C for 30 minutes, quenching the reaction on ice and then performing a radioligand binding assay, using the antagonist [ 3 H]-dihydroalprenolol, to determine the number of remaining functional ⁇ AR 34-424 molecules compared to the unheated control. Heating the unmutated ⁇ AR 34-424 at 32°C for 30 min before the assay reduced binding to approximately 50% of the unheated control (Fig. 7); all the data for the mutants were normalised by including the unmutated ⁇ AR 34 . 424 as a control in every assay performed.
• each of the 18 residues was mutated to 2-5 alternative amino acid residues of varying size or charge (Fig. 1). Out of these 18 mutants, 12 were not improved by further changes, 5 had better thermostability if another amino acid was present and one mutation from the first screen turned out to be a false positive.
• residues would be expected to interact with each other in the ⁇ AR structure, such as the consecutive amino acids G67 and R68 (V63 and Q64 in rhodopsin), or the amino acids within the cluster Y227, R229, V230 and A234 in helix 5 (Y223, Q225, L226 and V230 in rhodopsin).
• Other amino acid residues that could interact in ⁇ AR were Q194A in external loop 2 and D322A in external loop 3 (G182 and P285 in rhodopsin, respectively).
• Tm in this context is the temperature that gave a 50% decrease in functional binding after heating the receptor for 30 minutes.
• Each mutation increased the Tm of ⁇ AR 34-424 by 1 -3°C, with the exception of M90A and Y227A that increased the Tm by 8°C.
• thermostability PCR reactions were performed using various mixes of primers to combine up to 5 different mutations in a random manner and then tested for thermostability (Table 1 ). The best of these combinations increased the Tm more than 10 0 C compared to the Tm of ⁇ AR 34-424 . In some cases, there was a clear additive effect upon the Tm with the sequential incorporation of individual mutations. This is seen in a series of 3 mutants, m4-1 , m4-7 and m4-2, with the addition of V230A to m4-1 increasing the Tm by 2 0 C and the additional mutation D332A in m4-7 increasing the Tm a further 3 0 C.
• Table 1 Combinations of mutations by PCR. 10 PCR reactions were performed combining different pairs of primers that contained the selected mutations. Successful PCR reactions are shown in the table. The stability of these new mutants was assayed as described in Figure 7 and the Tm calculated. The results are shown as the mean ⁇ S. E. from duplicates.
• thermostable mutants obtained which were still expressed at high levels in E. coli, were m6-10, m7-7 and m10-8. These mutants contained collectively a total of 10 different mutations, with 8 mutations occurring in at least two of the mutants.
• a second round of mutagenesis was performed using m10-8 as the template and adding or replacing mutations present in m6-10 and m7-7 (Fig. 3); some of these mutations were very close in the primary amino acid sequence of ⁇ AR and therefore were not additive as noted above, but many mutations improved the Tm further (Table 2).
• thermostability assays used to develop PAR 34-424 mutants were performed by heating the receptor in the absence of the antagonist, but it is well known that bound ligand stabilises receptors. Therefore, stability assays for ⁇ AR 34-424 and ⁇ AR-m23 were repeated with antagonist bound to the receptors during the heating step (Fig. 4). As expected, the Tm of the receptor that contained bound antagonist during the incubation was higher than that for the receptor without antagonist. For ⁇ AR 34 .
• the Tm was 6°C higher with bound antagonist and for ⁇ AR-m23 the Tm increased 2 0 C to 55°C; the smaller increase in thermostability observed for ⁇ AR-m23 when antagonist binds suggests that the receptor is already in a more stable conformation similar to the antagonist bound state than ⁇ AR 34-424 (see also below).
• the Tm of ⁇ AR-m23 with antagonist bound is very similar to the Tm of dark-state rhodopsin in dodecylmaltoside (DDM)[12], whose structure has been solved by two independent laboratories[13,14]. This suggested that ⁇ AR-m23 is sufficiently stable for crystallisation.
• thermostability assays used to derive ⁇ AR-m23 were performed on receptors soiubilised in DDM.
• the aim of the thermostabilisation process was to produce a receptor that is ideal for crystallography, which means being stable in a variety of different detergents and not just DDM.
• coli expressing ⁇ AR-m23 or ⁇ AR 34-424 were soiubilised in DDM, bound to Ni-NTA agarose then washed with either DDM, decylmaltoside (DM), octylglucoside (OG), lauryldimethylamine oxide (LDAO) or nonylglucoside (NG). Stability assays were performed on the receptors in each of the different detergents (Fig 6). ⁇ AR 34-424 was only stable in DDM and DM, with no active receptors eluting from the resin washed with OG, NG or LDAO. In contrast, functional ⁇ AR-m23 was still present in all detergents and the Tm could be determined.
• Tm 52°C the smaller detergents were considerably more denaturing than either DDM (Tm 52°C) or DM (Tm 48°C), with T m s of 25 0 C (NG), 23°C (LDAO) and 17°C (OG).
• Tm 48°C T m s of 25 0 C
• LDAO 23°C
• OG 17°C
• the difference in Tm between ⁇ AR-m23 and ⁇ AR 34-424 is about 20 c C, irrespective of whether the receptors were soiubilised in either DDM or DM; it is therefore not surprising that no active ⁇ AR 34-424 could be found in even NG, because the predicted Tm would be about 5°C, thus resulting in rapid inactivation of the receptor under the conditions used for purification.
• thermostability The selection strategy used for the generation of ⁇ AR-m23 was chosen deliberately to be based upon thermostability, because it is far simpler to apply than selecting for stability in detergents of increasing harshness. However, it is clear that increasing the thermostability of ⁇ AR 34-424 also resulted in increasing tolerance to small detergents ideal for crystallising integral membrane proteins.
• the crystals that have been most studied so far were obtained using the purified beta-36 construct (amino acid residues 34-367 of the turkey beta receptor containing the following changes: point mutations C116L and C358A; the 6 thermostabilising point mutations in m23; replacement of amino acid residues 244-278 with the sequence ASKRK; a C terminal His6 tag) expressed in insect cells using the baculovirus expression system, after transferring the receptor into the detergent octyl-thioglucoside.
• the precipitant used was PEG600 or PEG1000 and the crystals obtained are elongated plates.
• a GPCR may be subjected to a variety of well-known techniques for structure determination.
• the most common technique for crystallising membrane proteins is by vapour diffusion (20, 21 ), usually using initially a few thousand crystallisation conditions set up using commercial robotic devices (22). However, sometimes the crystals formed by vapour diffusion are small and disordered, so additional techniques may then be employed.
• One technique involves the co-crystallisation (by vapour diffusion) of the membrane protein with antibodies that bind specifically to conformational epitopes on the proteins' surface (23, 24); this increases the hydrophilic surface of the protein and can form strong crystal contacts.
• a second alternative is to use a different crystallisation matrix that is commonly called either lipidic cubic phase or lipidic mesophase (25, 26), which has also been developed into a robotic platform (27). This has proven very successful for producing high quality crystals of proteins with only small hydrophilic surfaces e.g. bacteriorhodopsin (28). Membrane protein structures can also be determined to high-resolution by electron crystallography (29).
• ⁇ AR-m23 The evolution of ⁇ AR-m23 from PAR 34-424 by a combination of alanine scanning mutagenesis and the selection of thermostable mutants has resulted in a GPCR that is ideal for crystallography.
• the Tm for ⁇ AR-m23 is 21 0 C higher than for ⁇ AR 34-424 and, in the presence of antagonist, ⁇ AR-m23 has a similar stability to rhodopsin.
• the increased Tm of ⁇ AR-m23 has resulted in an increased stability in a variety of small detergents that inactivate ⁇ AR 34-424 .
• the selection strategy employed resulted in a receptor that is preferentially in the antagonist- bound conformation, which will also improve the chances of obtaining crystals, because the population of receptor conformations will be more homogeneous than for wild type ⁇ AR 34-424 .
• the selection strategy employed resulted in a receptor that is preferentially in the antagonist- bound conformation, which will also improve the chances of obtaining crystals, because the population of receptor conformations will be more homogeneous than for wild type ⁇ AR 34-424 .
• thermostabilisation of the receptor It is not at all clear why the particular mutations we have introduced lead to the thermostabilisation of the receptor. Equivalent positions in rhodopsin suggest that the amino acid residues mutated could be pointing into the lipid bilayer, into the centre of the receptor or at the interfaces between these two environments. Given the difficulties in trying to understand the complexities of the thermostabilisation of soluble proteins[15], it seems unlikely that membrane proteins will be any easier to comprehend; we found that there was no particular pattern in the amino acid residues in ⁇ AR that, when mutated, led to thermostability. However, since nearly 5% of the mutants produced were more stable than the native receptor, alanine scanning mutagenesis represents an efficient strategy to rapidly identify thermostable mutants.
• ⁇ AR-m23 The procedure we have used to generate ⁇ AR-m23 is equally applicable to any membrane protein that has a convenient assay for detecting activity in the detergent solubilized form. While we have selected for stability as a function of temperature as the most convenient primary parameter, the procedure can easily be extended to test primarily for stability, for example, in a harsh detergent, an extreme of pH or in the presence of chaotropic salts. Conformational stabilisation of a variety of human receptors, channels and transporters will make them far more amenable to crystallography and will also allow the improvement in resolution of membrane proteins that have already been crystallised. It is to be hoped that conformational stabilisation will allow membrane protein crystallisation to become a far more tractable problem with a greater probability of rapid success than is currently the case. This should allow routine crystallisation of human membrane proteins in the pharmaceutical industry, resulting in valuable structural insights into drug development.
• ⁇ AR Mutagenesis of ⁇ AR.
• the ⁇ AR cDNA was ligated into pRGIII to allow the functional expression of ⁇ AR in E. coli as a MaIE fusion protein[16].
• Mutants were generated by PCR using the expression plasmid as template using the QuikChange Il methodology (Stratagene). PCR reactions were transformed into XLIO-GoId ultracompetent cells (Stratagene) and individual clones were fully sequenced to check that only the desired mutation was present.
• Different mutations were combined randomly by PCR by including all the pairs of primers that introduced the following mutations: Mut4, G67A, G068A, V230A, D322A and F327A; Mut6, R068S, Y227A, A234L, A282L and A334L; Mut7, M90V, 1129V, Y227A, A282L and F338M; Mut10, R68S, M90V, V230A, F327A and A334L.
• the PCR mixes were transformed and the clones sequenced to determine exactly which mutations were introduced.
• cells were broken by freeze-thaw (five cycles), resuspended in 500 ⁇ l of buffer [2OmM Tris pH 8, 0.4 M NaCI, " 1 mM EDTA and protease inhibitors (CompleteTM, Roche)]. After an incubation for 1h at 4°C with 100 ⁇ g/ml lysozyme and DNase I (Sigma), samples were solubilized with 2% DDM on ice for 30 minutes. Insoluble material was removed by centrifugation (15,000xg, 2 min, 4 0 C) and the supernatant was used directly in radioligand binding assays.
• the supernatant (membranes) was centrifuged at 200,000 xg for 30 min at 4 0 C; the membrane pellet was resuspended in 15 ml of 20 mM Tris pH 7.5 and stored in 1 ml aliquots at -80 0 C after flash-freezing in liquid nitrogen.
• the protein concentration was determined by the amido black method[17]. These samples were used in radioligand binding assays after thawing and being solubilized in 2% DDM as above.
• DDM-solubilized ⁇ AR was partially purified with Ni-NTA agarose (Qiagen). 200 ⁇ l of Ni-NTA agarose was added to 2 ml of solubilized samples (10 mg/ml of membrane protein) in 2OmM Tris pH 8, 0.4 M NaCI, 20 mM imidazole pH 8 and incubated for
• Receptors were eluted in 2 x 100 ⁇ l of buffer (0.4 M NaCI, 1mM EDTA, 250 mM imidazole pH 8, plus the relevant detergent).
• the K D for [ 3 H]-DHA binding to semipurified PAR 34-424 and ⁇ AR-m23 was, respectively 3.7 nM and 12.5 nM and the final concentration of [ 3 H]-DHA used in the competition assays was 3 times the K D /e 12 nM for ⁇ AR 34-424 and 40 nM for ⁇ AR-m23.
• Radioligand binding and thermostability assays Single point binding assays contained 2OmM Tris pH 8, 0.4 M NaCI, 1 mM EDTA, 0.1 % DDM (or corresponding detergent) with 50 nM [ 3 H]-DHA and 20-100 ⁇ g membrane protein in a final volume of 120 ⁇ !; equilibration was for 1 h at 4 0 C. Thermostability was assessed by incubating the binding assay mix, with or without [ 3 H]-DHA at the specified temperature for 30 minutes; reactions were placed on ice and [ 3 H]-DHA added as necessary and equilibrated for a further hour. Receptor-bound and free radioligand were separated by gel filtration as described previously[18].
• Nonspecific binding was determined in the presence of 1 ⁇ M of s-propranolol. Saturation curves were obtained using a range of [ 3 H]-DHA concentration from 0.4 nM to 100 nM. Competition assays were performed using a concentration of [ 3 H]-DHA of 12 nM for ⁇ AR 34 . 424 and 40 nM for ⁇ AR-m23 (ie three times the K D ) and various concentrations of unlabeled ligands (0-100 mM). Radioactivity was counted on a Beckman LS6000 liquid scintillation counter and data were analyzed by nonlinear regression using Prism software (GraphPad).
• thermostable mutations in rhodopsin structure The pdb file for the rhodopsin structure, accession code 1 GZM[14], was downloaded from the Protein Data Bank website (www.pdb.org) and displayed in the program PyMOLXH Hybrid (DeLano Scientific).
• the equivalent amino acid residues in rhodopsin for the thermostable mutations in ⁇ AR were located in the rhodopsin structure based upon an alignment among the four GPCRs with which we are most familiar, namely rhodopsin, ⁇ 1 adrenergic receptor, neurotensin receptor and adenosine A 2a receptor[19].
• Example 2 Mutants of the adenosine Ag 5 receptor (A 22 R) with increased thermostability
• Wildtype A 2a R has a Tm of 31 0 C with ZM241385 bound. a. Rant17 A54L+K122A+L235A Tm 48 0 C (ZM241385 bound) b. Rant19 A54L,T88A,V239A+A204L Tm 47°C (ZM241385 bound) c. Rant21 A54L,T88A,V239A+K122A Tm 49°C (ZM241385 bound)
• Rant 21 did not bind NECA sufficiently for an accurate Kj determination (hence indicated as >1 x 10 '1 ).
• the affinity of Rant21 for agonist binding is weakened 232 fold for R-PIA and at least by 1900 fold for NECA.
• Table (v) Summary of stability of wild-type and mutant receptors in different detergents. Solubilisation of receptors and detergent exchange was performed during the IMAC step. S. D. is ⁇ 1 0 C. It was not possible to determine the Tm for some receptor-detergent combinations, because the receptor was too unstable (t).
• thermostability A356L, H103A, D345A, A86L, A385L, Y349A, C386A, K397A, H393A, 1116A 1 F358A, S108A, M181A, R392A, D113A, G209A, L205A, L72A, A120L, P399A, Y351A, V268A, T207A, A155L, S362A, F189A, N262A, L109A, W391A, T179A, S182A, M293A, L256A, F147A, D139A, S100A, K176A, L111A, A90
• Mutants tested for thermostability by heating in the absence of the agonist were re-tested using a slightly different assay where the mutants were heated in the presence of 3 H-neurotensin (Ligand(+) format in Figure 12).
• Mutants with improved thermostability are: A69L, A73L, A86L, A90L, H103A, V165A, E166A, G215A, V229A, M250A, I253A, A177L, R183A, I260A, T279A, T294A, G306A, L308A, V309A, L310A, V313A, F342A, F358A, V360A, S362A, N370A, S373A, F380A, A385L, P389A, G390A, R395A.
• mutants that have a significantly enhanced expression level compared to the wildtype receptor and could be used to boost preceptor production levels for crystallisation: A86L, H103A, F358A, S362A, N370A, A385L, G390A. All of these also have increased thermostability.
• Wildtype NTR has a Tm of 35°C with neurotensin bound.
• Example 4 Identification of structural motifs in which stabilising GPCR mutations reside.
• the structure of the ⁇ 2 adrenergic receptor has been determined (20, 21 ), which is 59% identical to the turkey ⁇ receptor, but with a distinctly different pharmacological profile (22, 23).
• a distinctly different pharmacological profile 22, 23.
• the beta adrenergic receptors were first aligned using ClustalW in the MacVector package; thermostabilising mutations in turkey ⁇ 1 were highlighted along with the corresponding residue in the human ⁇ 2 sequence.
• the human ⁇ 2 model (pdb accession code 2RH1 ) was visualised in Pymol and the desired amino acids were shown as space filling models by standard procedures known in the art. The structural motifs in which the stabilising mutations were located, were determined by visual inspection.
• Table (vi) lists the equivalent positions in the ⁇ 2 receptor corresponding to the thermostabilising mutations in ⁇ AR-m23 and the structural motifs in which they reside.
• the mutations are positioned in a number of distinct localities. Three mutations are in loop regions that are predicted to be accessible to aqueous solvent (loop). Eight mutations are in the transmembrane ⁇ -helices and point into the lipid bilayer (lipid); three of these mutations are near the end of the helices and may be considered to be at the hydrophilic boundary layer (lipid boundary).
• Such structural motifs are important in determining protein stability. Therefore, targeting mutations to these motifs will facilitate the generation of stabilised mutant GPCRs. Indeed, there were several instances where more than one mutation mapped to the same structural motif. For example, the Y227A, V230A and A234L mutations in the turkey ⁇ 1 adrenergic receptor all mapped to the same helical interface, the V89L and M90V mutations mapped to the same helical kink and the F327A and A334L mutations mapped to the same helical surface pointing towards the lipid bilayer (Table (vi)). Thus, when one stabilising mutation has been identified, the determination of the structural motif in which that mutation is located will enable the identification of further stabilising mutations.
• mutant GPCRs having increased stability in a particular conformation relative to a parent GPCR provides a number of advantages for screening of binding partners.
• the present methods reduce the amount of material required for a screen.
• GPCRs are present in whole cells or in membranes from whole cells which are usually screened by incubation with individual compounds in isolated chambers rather than libraries of compounds. Therefore the present invention provides advantages in terms of time required to carry out a compound screen.
• the ability to lock a GPCR in a particular conformation provides advantages in that it increases the likelihood of identifying a ligand with the required pharmacological properties.
• GPCRs are able to assume a number of different conformations and binding compounds will be identified across different pharmacological types. Reagent costs can be reduced due to the ability to miniaturize the assay formats and this is facilitated by the present method.
• a mutant GPCR having increased stability in a particular conformation is immobilised on a solid surface and incubated with buffer containing an encoded library of compounds. After a suitable period of time, to allow binding between the mutant GPCR and compounds from the library which selectively bind to the mutant GPCR 1 the buffer is removed. Next there follows a number of wash steps to remove compounds which have not bound specifically to the mutant GPCR. The reading code, tag or address (such as DNA or RNA) is then used to identify the small molecule bound either whilst still bound to the mutant GPCR or following elution from the GPCR. The conformation specific binding partner is subsequently isolated. Selection of antibodies using immobilised GPCRS
• Immobilised stabilised GPCRs can be used to select antibodies to the receptor from mixtures of antibodies such as would be present in plasma from an animal immunised with the stabilised GPCR, the native receptor or a peptide from the receptor.
• Antibodies could be identified from supernatants obtained from B-cells taken from immunised animals or from hybridomas obtained following immortalization of B-cells from the immunised animal or from recombinant antibody libraries which may be expressed on phage particles or through an in vitro expression system such as ribosome display.
• the method has the advantage of selecting antibodies to particular conformations of a receptor.
• a stabilised GPCR locked in the antagonist or ground state of the receptor would increase the probability of selecting an antagonistic antibody whereas a stabilised GPCR locked in the activated or R* state would increase the probability of selecting an activating antibody.
• antibodies are often selected to GPCRs that bind to peptide epitopes of the receptor but do not have antagonist or agonist properties and therefore are not useful as therapeutic agents.
• Example 6 Antibody phage selection on ⁇ -adregenic receptor
• ⁇ -AR stabilised ⁇ -adrenergic receptor
• GPCRs G protein coupled receptors
• a typical phage display strategy comprises several stages in the process to identify inhibitory scFv clones ( Figure 29).
• the first part is the selection of phage libraries on antigen to isolate a population of phage antibody binders using various methodologies (for example, panning selection, soluble selection, etc.). This resulting population of phage antibody binders is referred to as a selection output. This process is repeated 2-4 times to enrich for specific antigen binders.
• a pool of clones representing a selection output is assessed for recognition of antigen by phage ELISA.
• Individual clones can also be assessed by monoclonal phage ELISA and for diversity by sequence analysis, however the preferred method is to subclone the selected population into a recombinant antibody expression vector (pSANG 10-3F) and then perform the assessment by monoclonal scFv ELISA and DNA sequencing. This circumvents the problem of identifying phage antibody binders that subsequently exhibit poor expression as scFv fragments. Selection outputs yielding diverse ELISA positives can then be prioritised for functional screening of larger panels of phage antibodies.
• Selection outputs are subjected to a screening campaign, employing a functional high-throughput assay, of periplasmic extracts in order to identify a population of scFv inhibitors.
• the hits from the screening campaign are profiled as scFv by further functional assays, as well as IC50 analysis which assess the efficiency of their inhibitory action.
• Immobilisation strategies were based on taking advantage of the His tagged antigen. In the first instance, immobilisation was done using control proteins rather than ⁇ AR-m23. A variety of surfaces, including Ni-NTA plates, were employed in the initial assessment. All were compared against standard passive absorption onto Nunc plates. In all cases, the outcome was evaluated using polyclonal phage ELISA after 2 rounds of selection.
• ⁇ AR was covalently immobilised on Amino plates (Nunc Catalog No: 436008). Coating and washing buffers were supplemented with 2OnM ligand and 0.1 % dec-M. The ELISA plate was coated overnight with ⁇ -AR at 24 ⁇ g/ml and 2 control proteins (CD86 and Notchi ) at 5 ⁇ g/ml. Next day the wells were washed and blocked with PBS-M. 50 ⁇ l/well of polyclonal phage from 2 rounds of selection were added (in PBS-M, at a concentration of 0.1 x relative to the initial culture volume) and incubated for 1 hour at room temperature.
• 17 of the positive phage clones from the monoclonal ELISA were tested for binding specificity to ⁇ -AR and 3 unrelated proteins.
• the other control proteins include the extracellular domain of murine CD86 and the negative regulatory region of murine Notchi expressed as a CD4 fusion (CD86 and NI(NRR) respectively) in a transient expression system as described in Chappie et al 2006 (25). In this ELISA both nickel chelate and amino-piate were used for comparison.
• scFv antibody single chain Fv
• selection A Selected antibody genes within the round 2 phage population
• pSANG10-3F vector (26) was sub-cloned into the pSANG10-3F vector (26) and transformed into BL21(DE3) cells.
• 96 colonies were picked and periplasmic expression of the scFv antibody was induced in a 96 well format using standard methods in the art.
• scFv were recovered from the periplasm and used for ELISA on an Amino plate (Nunc). Washed and blocked wells were incubated for 1h with 50 ⁇ l/well of scFv.
• unique positive antibody clones are selected for larger scale preparation (50-50OmI) using periplasmic extraction and immobilised metal affinity chromatography. This material is assessed for the ability to interfere in ligand binding to receptor using robust reporter assays (e.g. inhibition of cAMP generation or inhibition of ligand binding to transfected cells).
• Binding studies were performed at 1 O 0 C using a Biacore S51 optical biosensor equipped with a streptavidin-coated CM5 chip and equilibrated with running buffer (20 mM trisHCI, 150 mM NaCI, 1 mM EDTA, 1% DMSO, 0.1% decyl maltoside, pH 7.8).
• J31AR36-M23 was minimally biotinylated using EZ-link sulfo-NHS-LC-LC biotin (Pierce #21338): The biotin was added to the receptor preparation, (spiked with 100 uM alprenolol) and allowed to react for three hours at 4 0 C, after which free biotin was removed via column chromatography.
• Figure 33 shows the two capture stages of the biotinylated b1AR onto a streptavidin-coated flow cell. In the first stage, we captured ⁇ 1200 RU; in the second, -4000 RU.
• the biacore sensor chips coated in the stabilised beta receptor could be used to characterise the binding of drugs with activity at the beta receptor. Flow through of the compound allowed the on rate to be determined. Subsequent washing enabled determination of the off rate. These parameters could then be used to determine a kinetic affinity measurement (Kd).
• Figure 34 shows the responses for alprenolol which was tested in replicate in a three-fold dilution series for binding to the receptor surface The highest concentration is 666 nM and each concentration was tested three times. The responses are concentration dependent and are reproducible. The grey lines depict the fit of a simple 1 :1 interaction model and the parameters determined from this fit are listed in the inset (the number in parentheses is the error in the last digit).
• Figure 35 is an alternative depiction of the data set in Figure 34.
• the responses are normalized with respect to the Rmax determined (from the fitting) for each curve.
• the alprenolol data demonstrates that the biotinylated b1AR is active and this biosensor approach can be used to characterize the compound/receptor interactions.
• Figure 36 shows the responses for propranolol binding to ⁇ 1AR. 111 nM is the highest concentration and each concentration was tested two or three times. The propranolol data are plotted as normalized responses in Figure 37.
• Figure 38 shows the alprenolol binding responses with 333 nM as the highest concentration. The receptor appeared to be nearly as active as when it was first captured.
• the S51 biosensor has the ability to monitor two reaction spots at one time so we captured the receptor to a density of 8000 RU on another streptavidin-coated spot ( Figure 39).
• Salmeterol dissociated much faster and bound the receptor much more weakly than alprenolol or propranolol did. This is to be expected since salmeterol has a low affinity for the ⁇ 1-AR and in particular has a low affinity for the antagonist stabilised form of the receptor which is used here.
• we detected some complexity in the interaction as indicated by the poor fit of the model to the responses during the dissociation phase (t> 60 sec).
• Figure 42A and B shows the data plotted as normalized responses.
• Isoproterenol a non-selective ⁇ -adrenergic agonist
• isoproterenol displayed a much slower association rate than the other compounds. The compound was injected for 90 sec.
• Figure 44A and B show responses for 111uM and 333 nM carvedilol binding to the two b1AR surfaces. Carvedilol displays a much slower dissociation rate than the other compounds examined so far.
• Example 8 Use of the adenosine A2a StaR for compound screening in drug discovery
• thermostabilised adenosine A2a receptor (A2a StaR) conformationally selected in the antagonist form (refered to as Rant22) was used to screen compounds from a library in order to identify compounds with activity at the A2a receptor.
• the StaR was generated as previously described (Magnani et al, Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor.Proc Natl Acad Sci U S A. 2008 Aug 5;105 (31): 10744-9).
• HEK293T cells transfected with Rant22 A2a receptors were grown in a monolayer in T-175 flasks at 37 0 C and 5% CO 2 in Dulbeccos Modified Eagle Medium supplemented with 10% fetal bovine serum. Cells were harvested by scraping the cells from the T-175 surface and collected by centrifugation.
• Cell pellets were resuspended in 10ml 2OmM HEPES, pH 7.4 plus protease inhibitor cocktail tablets (Roche) and were homogenised for 30s at 20,500 rpm using a tissuemizer. Homogenates were centrifuged at 200 ⁇ g for 15 min at 4 0 C. The supernatant was removed and reserved on ice. This procedure was repeated a further two times and the pooled supematants were then centrifuged at 40,000xg for 45 min at 4 0 C. Membranes were resuspended in 1 ml aliquots of 2OmM HEPES, pH 7.4 plus protease inhibitor tablets. Protein concentration was determined by a BCA protein assay (Pierce).

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