The binding domain of the GPCR:G protein complex and its derived applications.

Abstract

This invention relates to the field of structural biology and signal transduction of G protein-coupled receptors (GPCRs). In particular, this invention relates to binding domains that target and / or specifically bind to GPCR:G protein complexes. Nucleic acid sequences encoding such binding domains are also provided, along with cells that express or are capable of expressing such binding domains. The binding domains of this invention can be used as a universal tool for the structural and functional characterization of G protein-coupled receptors that complex with downstream heterotrimeric G proteins and bind to a variety of natural or synthetic ligands, for studying the kinetics of G protein activation, and for screening and drug discovery using GPCR:G protein complexes.

Description

[0001] This application is a divisional application of Chinese Patent Application 201280040585.9 (PCT / EP2012 / 062036), filed on June 21, 2012, entitled “Binding domain of GPCR:G protein complex and use therefrom”. Invention Field

[0002] This invention relates to the field of structural biology and signal transduction of G protein-coupled receptors (GPCRs). In particular, this invention relates to binding domains that target and / or specifically bind to GPCR:G protein complexes. Nucleic acid sequences encoding such binding domains are also provided, as well as cells that express or are capable of expressing such binding domains. The binding domains of this invention can be used as a general tool for the structural and functional characterization of G protein-coupled receptors that complex with downstream heterotrimeric G proteins and bind to various natural or synthetic ligands. This tool is used to study the kinetics of G protein activation and for screening and drug discovery using GPCR:G protein complexes. background

[0003] Seven-transmembrane receptors (7TMRs), also known as G protein-coupled receptors (GPCRs), are the largest class of receptors in the human genome and the most common type of target proteins used in medical therapy. Over the past 60 years, substantial progress has been made in understanding the different GPCRs, from pharmacology to in vivo functional characterization. High-resolution structural studies have now provided in-depth insights into the molecular mechanisms of GPCR activation and compositional activity (e.g., Rasmussen et al., 2011). However, molecular details of how GPCRs interact with their downstream targets and regulate their activity are still lacking. The structures of GPCRs complexed with their downstream proteins are of great interest, not only because such interactions are pharmacologically relevant, but also because an atomic-level understanding of intermolecular interactions is key to unlocking the secrets of functional selectivity and the ability of different agonists to produce different downstream effects from a single receptor. Existing structural data support the view that, even in their small size, GPCRs are complex allosteric structures with multiple signal transduction outputs.

[0004] Once activated, GPCRs transmit signals in a GTP-dependent manner via a complex of three proteins known as the heterotrimeric G protein, or Gαβγ. The binding of extracellular ligands to GPCRs regulates their ability to catalyze GDP–GTP exchange in Gαβγ, thereby modulating the intracellular level of the second messenger. The inactivated Gαβγ heterotrimer comprises two main components: Gα·GDP and the Gβγ heterodimer. Gβγ isolates the switch II element on Gα, preventing it from interacting with second messenger systems, such as those involving cAMP, diacylglycerol, and calcium. Activated GPCRs catalyze the release of GDP from Gα, allowing GTP binding and the release of the activated Gα-GTP subunit. In this state, switch II forms a helix stabilized by the γ-phosphate of GTP, enabling it to interact with effectors such as adenylate cyclases. While significant progress has been made in understanding how the Gα subunit interacts with its downstream targets and regulates their activity, it remains unclear how activated GPCRs initiate this process by catalyzing nucleotide exchanges on Gαβγ.

[0005] Drug development efforts generally focus on small molecule ligands that competitively bind to specific catalytic or active sites, using static models of the target as a starting point. This approach has identified and validated several currently available viable active site therapeutics. However, the high failure rate of new drug compounds (only about 8% of Phase I clinical therapeutics ultimately receive FDA approval, with a conservative cost of $800 million per drug) reflects the fact that much of the work is unsuccessful, often abandoned once the target is deemed undrugable (Lee & Craik, 2009). A significant portion of these failures are due to the fact that the dominant conformation of the target in question does not correspond to a druggable conformation, which is the conformation that the drug must bind to be effective for the therapeutic indication. For example, attempts to obtain the active state of a GPCR binding agonist have proven extremely difficult because this state is inherently unstable in the absence of a G protein. Currently, conformationally stable nanobodies or... The structure of the active GPCR can be obtained initially, the nanobody or It mimics G proteins and increases the affinity of agonists at orthosteric sites (Rasmussen et al., 2011). This confirms... The ability to lock the structure of the most challenging drug targets into therapeutically relevant conformations (Steyaert & Kobilka, 2011) and its use in targeted drug development, which allows for the specific screening of potential drugs with high sensitivity and selectivity to specific targets (WO2012007593). One limitation of this approach is the need to identify specific stabilized nanobodies for each GPCR target, which is not only time-consuming and expensive but also implies the use of different tools, such as biological materials used for immunization and selection purposes.

[0006] Therefore, there is a need to develop new, direct tools for the structural and pharmacological analysis of GPCR drug targets. Invention Overview

[0007] Elucidating the structure and functional characteristics of GPCRs that complex with downstream heterotrimeric G proteins and bind to a variety of natural and synthetic ligands is valuable for understanding the mechanisms of GPCR signal transduction and for drug development. For example, the structure of an active ternary complex (containing an agonist, GPCR, and G protein) has not yet been successfully obtained, and due to a lack of understanding of its biology, obtaining this structure is crucial for the structural biology of signal transduction. Crystallization of this complex has proven extremely difficult because one partner component (the receptor) requires detergent to dissolve, and the G protein is unstable in detergent. Furthermore, the nucleotides required for complex formation also cause the complex to dissociate transiently.

[0008] Therefore, and quite surprisingly, the inventors have identified tools for stabilizing GPCR and G protein complexes, capable of capturing and purifying such complexes, ultimately leading to their crystallization. This facilitates the identification of ligands or drug compounds through, for example, structure-based screening based on viruses or designed structures, high-throughput screening, or fragment-based drug discovery (see, for example, Example 10). More specifically, the inventors have identified binding domains suitable for structural and functional analysis of complexes (comprising agonists, GPCRs, and G proteins) in their active state, particularly immunoglobulin monovariable domains (see, for example, Examples 4-7). Interestingly, some of the aforementioned GPCR:G protein complex-selective binding domains have been confirmed to be specifically targeting G proteins, not GPCRs. For example, binding domains binding to Gs located at the Gαs and Gβγ interfaces have been identified without contact with β-adrenergic receptors (see, for example, Example 3), and thus these binding domains can be used to capture and stabilize other Gs-coupled receptors, as demonstrated with arginine angiotensin receptor 2 (V2R) (see, for example, Example 9). Therefore, it is particularly advantageous to have binding domains targeting G proteins in GPCR:G protein complexes, as such binding domains can be used as a general tool for stabilizing and capturing active state complexes of GPCRs that interact with specific G proteins.

[0009] Therefore, according to a first aspect, the present invention relates to binding domains for and / or specifically binding to complexes comprising GPCRs and G proteins. More specifically, the binding domains described herein bind to GPCR:G protein complexes with higher affinity than binding to G proteins and / or GPCRs individually. Furthermore, the binding domains described herein enhance the affinity of G proteins for GPCRs. Thus, the present invention provides binding domains for and / or specifically binding to conformational epitopes of complexes comprising GPCRs and G proteins, said binding domains stabilizing or locking the complex in a specific conformational state, more specifically, an active conformational state.

[0010] Generally, the binding domains described herein can bind any conformational epitope that is available or accessible to the complex of a GPCR and a G protein. These conformational epitopes may be presented by a single protein contained in the complex, and / or only upon formation of the complex. Furthermore, these conformational epitopes may or may not be presented by a single protein alone. According to a particular embodiment, the binding domains described herein specifically bind to the G protein contained in the complex, without binding to the GPCR.

[0011] Typically, G proteins are essentially in nucleotide-binding form. More specifically, G proteins (or at least their α subunits) bind GTP or GDP, depending on the activation state of a particular GPCR, as described elsewhere herein. Agonists binding GPCRs promote interactions with the GDP-bound Gαβγ heterotrimer, resulting in the exchange of GDP on Gα for GTP and the functional dissociation of the G protein into Gα-GTP and Gβγ subunits, which is required for further extracellular signaling. In a particular embodiment, the binding domain described herein specifically binds to and stabilizes the GPCR:G protein complex under nucleotide-deficient conditions; more specifically, the binding domain binds to and stabilizes such a GPCR:G protein complex in which the G protein is in a nucleotide-free form. In a particular embodiment, the binding domain described herein specifically binds to a conformational epitope located at the interface between the α and β-γ subunits of the G protein, thereby blocking the GDP / GTP binding site and interfering with GDP / GTP binding. Surprisingly, the binding domain described herein prevents or inhibits the dissociation of the GPCR:G protein complex in the presence of nucleotides (especially guanine nucleotides or their analogues, such as GTPγS). The binding domain described herein also prevents or inhibits the binding of nucleotides to the G protein.

[0012] Preferably, the binding domains described herein target and / or specifically bind to complexes comprising a GPCR, a G protein, and one or more receptor ligands. Typically, the receptor ligand is an agonist, or a normal allosteric modulator, or a combination thereof.

[0013] According to another preferred embodiment, the binding domain described herein targets and / or specifically binds to a complex comprising a Gs protein-coupled receptor and a Gs protein; or a complex comprising a Gi protein-coupled receptor and a Gi protein; or a complex comprising a Gt protein-coupled receptor and a Gt protein; or a complex comprising a Ggust protein-coupled receptor and a Ggust protein; or a complex comprising a Gz protein-coupled receptor and a Gz protein; or a complex comprising a Golf protein-coupled receptor and a Golf protein; or a complex comprising a Gq protein-coupled receptor and a Gq protein; or a complex comprising a G12 receptor and a G12 protein; or a complex comprising a G13 receptor and a G13 protein. The GPCR and / or G protein contained in the complex may be from the same or different species, particularly from mammalian species. Preferably, the GPCR is a human protein.

[0014] Generally, the binding domain of the present invention can be any non-naturally occurring molecule or portion thereof capable of specifically binding to the GPCR:G protein complex. In particular, the binding domain described herein is an immunoglobulin monovariable domain comprising an amino acid sequence containing four frame regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3) according to the following general formula (1):

[0015] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1).

[0016] Preferably, the binding domain described herein is a single variable domain of an immunoglobulin derived from camel species, particularly nanobodies or V. H H.

[0017] According to a specific implementation, the binding domain described herein is an immunoglobulin monovariable domain comprising an amino acid sequence containing four frame regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3) according to the following general formula (1):

[0018] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1),

[0019] And CDR1 is selected from:

[0020] a)SEQ ID NO:13-18,

[0021] b) A polypeptide having at least 80% amino acid identity with SEQ ID NO:13-18,

[0022] c) A polypeptide that differs from SEQ ID NO:13-18 by 3, 2, or 1 amino acid.

[0023] And CDR2 is selected from:

[0024] a)SEQ ID NO:25-30,

[0025] b) A polypeptide having at least 80% amino acid identity with SEQ ID NO:25-30,

[0026] c) A polypeptide that differs from SEQ ID NO:25-30 by 3, 2, or 1 amino acid.

[0027] And CDR3 is selected from:

[0028] a)SEQ ID NO:37-42,

[0029] b) A polypeptide having at least 80% amino acid identity with SEQ ID NO:37-42,

[0030] c) A polypeptide that differs from SEQ ID NO:37-42 by 3, 2 or 1 amino acid.

[0031] In a particularly preferred embodiment, the present invention provides an immunoglobulin single variable domain comprising an amino acid sequence comprising four frame regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3) according to the following general formula (1):

[0032] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1);

[0033] CDR1 is SEQ ID NO:13; CDR2 is SEQ ID NO:25; and CDR3 is SEQ ID NO:37.

[0034] According to a very specific implementation scheme, the binding domain, particularly the immunoglobulin monovariable domain, has an amino acid sequence selected from SEQ ID NO:1 to 6.

[0035] Furthermore, the binding domains described herein can also be contained within peptides. Additionally, the binding domains can be immobilized on solid supports.

[0036] In one particular aspect, the present invention relates to binding domains that target and / or specifically bind to G proteins.

[0037] Another aspect of the invention contemplates complexes comprising the binding domains described herein. These complexes particularly comprise GPCRs, G proteins, and optionally receptor ligands. In some applications, the complexes described herein are crystals.

[0038] The present invention also provides nucleic acid sequences, particularly nucleic acid sequences encoding any amino acid sequence of any binding domain described herein, and recombinant vectors comprising any nucleic acid sequence described herein. A particularly preferred aspect of the invention is cells comprising any vector or nucleic acid described herein, said cells being able to express or express GPCRs and / or G proteins. Cell cultures of cells according to the invention and membrane products derived therefrom also fall within the scope of the invention.

[0039] The binding domains, complexes, and cells described herein can be used in a variety of contexts and applications. Therefore, one aspect of the invention relates to the use of the binding domains described herein for stabilizing complexes comprising a GPCR and a G protein, and optionally a receptor ligand, in a functional conformational state, more specifically in an active conformational state. In a particular embodiment, the binding domains described herein can be used to prevent the complex from dissociating in the presence of nucleotides, particularly guanine nucleotides or analogues such as GTPγS. Thus, binding domains for stabilizing GPCR:G protein complexes and blocking the functional conformational state (preferably the active conformational state) of GPCRs are highly effective in a range of applications, as summarized below.

[0040] The objective of this invention is to enable crystallization and / or resolution of complex structures using the binding domains described herein, wherein the complex comprises a GPCR and a G protein and optionally a receptor ligand.

[0041] It is also believed that compounds that use the binding domains described herein, or the cells or models from which they are derived, to screen for the signal transduction activity of GPCRs fall within the scope of this invention.

[0042] Furthermore, the binding domains described herein can be used to capture one or more interacting proteins, particularly proteins that interact with G proteins and / or GPCRs.

[0043] According to a specific embodiment, the present invention provides a method for capturing and / or purifying complexes containing GPCRs and G proteins, the method comprising the steps of:

[0044] a) Provide the combined structural domains described herein, and

[0045] b) Allows the binding domain to bind to a complex containing a GPCR and a G protein, and optionally a receptor ligand, and

[0046] c) The complex formed in the optional separation step b).

[0047] In another specific embodiment, the present invention relates to a method for determining the crystal structure of a complex comprising a GPCR and a G protein, the method comprising the steps of:

[0048] a) Provide the combined structural domains described herein, and

[0049] b) Allows the binding domain to bind to a complex containing a GPCR and a G protein, and optionally a receptor ligand, and

[0050] c) Crystallize the complex formed in step b).

[0051] Some of the binding domains described herein may have therapeutic applications. Therefore, the objective of this invention remains to regulate GPCR receptor signaling, particularly G protein-mediated GPCR receptor signaling, using the binding domains described herein.

[0052] This invention also covers a method for producing a binding domain that targets and / or specifically binds to a complex comprising a GPCR and a G protein, the method comprising the steps of:

[0053] a) Express the nucleic acids described herein in a suitable cell expression system, and optionally...

[0054] b) Isolate and / or purify the binding domain.

[0055] Another aspect of the present invention relates to a method for screening binding domains that target and / or specifically bind to complexes comprising GPCRs and G proteins, the method comprising the steps of:

[0056] a) Provides multiple combined structural domains, and

[0057] b) Among the plurality of binding domains, screen for binding domains that bind to a complex containing GPCR and G protein, and

[0058] c) Separate the binding domains that bind to the complex.

[0059] Other objectives of the invention will also be apparent from the other descriptions herein. Brief description of the attached diagram

[0060] Figure 1 G protein cycling of the β2AR:Gs complex. a) Extracellular agonists binding to β2AR cause conformational rearrangement of the cytoplasmic ends of transmembrane segments that enable Gs heterotrimers (α, β, and γ) to bind to receptors (R, R*). GDP is released from the α subunit during the formation of the R:G complex. GTP binds to the nucleotide-free α subunit, leading to dissociation of the α and βγ subunits from the receptor. The subunits regulate their corresponding effector proteins—adenylate cyclase (AC) and Ca2+. 2+ Channel. After GTP in the α subunit is hydrolyzed to GDP, it reassembles from the α and βγ subunits into a Gs heterotrimer. b, Purified, nucleotide-free β2AR:Gs protein complex maintained in detergent microparticles. The Gs α subunit consists of two domains: a Ras domain (αRas) and an α-helical domain (αAH). Both domains are involved in nucleotide binding. In the nucleotide-free state, the αAH domain has a variable position relative to the αRas domain.

[0061] Figure 2The formation of a stable β2AR:Gs complex is achieved through a combination of the following effects: 1) the binding of a high-affinity agonist to the receptor at a very slow dissociation rate (as described by Rasmussen et al., 2011); 2) the formation of a nucleotide-free complex in the presence of adenosine triphosphate diphosphatase, which hydrolyzes the released GDP, preventing its rebinding and leading to a less stable R:G interaction; and 3) the exchange of DDM detergent for the stable complex of MNG-3, thus achieving a stable β2AR:Gs complex.

[0062] Figure 3: Effects of nucleotide analogs, pH, and nanobodies on the stability of the β2AR:Gs complex. a) Analytical gel filtration showed that nucleotide GDP and GTPγS (0.1 mM) caused dissociation of the β2AR-365:Gs complex. b) Phosphates mimicking the phosphate groups of the nucleotides—pyrophosphate and trisodium phosphate formate (used at 5 mM)—did not cause complex deconstruction. As additives, they improved crystal growth of two T4L-β2AR:Gs complexes (without nanobodies), T4L-β2AR:Gs:Nb37 and T4L-β2AR:Gs:Nb35. c) Determination of pH limits to guide the preparation of crystal screening. For the same purpose, the effect of ionic strength was determined using various concentrations of NaCl (data not shown). The complex was stable at 20, 100, and 500 mM, but dissociated in 2.5 M NaCl. d) Nanobody 35 (Nb35, red dashed line) binds to the T4L-β2AR:Gs:BI167107 ternary complex (blue solid line) to form an R:G:Nb35 complex (red solid line). Unlike the treated R:G complex (green dashed line), the R:G:Nb35 complex is insensitive to GTPγS treatment (green solid line). Nb35 and Nb37 bind to independent epitopes located on the Gs heterotrimer to form an R:G:Nb35:Nb37 complex (purple solid line). e) Nanobody 36 (Nb36, red dashed line) binds to the R:G complex (black solid line) to form an R:G:Nb36 complex (red solid line), which is less sensitive to GTPγS treatment (green solid line). Nb36 and Nb37 bind to independent epitopes located on the Gs heterotrimer to form an R:G:Nb36:Nb37 complex (purple solid line). f) Nanobody 37 (Nb37, green line) binds to the R:G complex (black solid line) to form the R:G:Nb37 complex (red solid line). g) The R:G:Nb37 complex is less sensitive to GTPγS treatment (blue solid line), unlike the treated R:G complex (blue dashed line).

[0063] Figure 4The stabilizing effect of MNG-3 on R:G complexes. a) Analytical gel filtration of β2AR-365:Gs complexes purified in DDM (black), MNG-3 (blue), or two MNG-3 analogues (red and green) after incubation at 4°C for 48 hours. Unlike DDM, the R:G complexes were stable in MNG detergent. b) The effect of diluted, unbound purified β2AR in DDM or MNG-3 below the detergent's critical cluster concentration (CMC), as determined by 3H-dihydroaprolol (3H-DHA) saturation binding. After 20 seconds, β2AR diluted to 1000 times below the CMC in DDM resulted in loss of 3H-DHA binding (black data point). Conversely, after 24 hours, β2AR diluted to 1000 times below the CMC in MNG-3 retained full radioligand binding capacity.

[0064] Figure 5 Purity and homogeneity of the R:G complex. a) Analytical SDS-PAGE / Coomassie blue staining of samples obtained at various stages of T4L-β2AR:G purification. BI167107 agonist-bound, dephosphorylated, and deglycosylated receptors in excess heterotrimers are used for optimal coupling efficiency with the functional portion of the G-protein. Functional purification of Gs is achieved through interaction with receptors immobilized on M1 resin, while washing away non-functional / unbound Gs. b) Representative elution curves from one of four consecutive gel filtrations, with fractions indicated in red. Fractions containing the R:G complex were combined within the short dashes shown, concentrated by rotation, and analyzed for purity and homogeneity by SDS-PAGE / Coomassie blue (a, second to last lane to the right), in c) gel filtration, and by d) anion exchange chromatography. The top figure shows the elution curve of the analytical IEC of the β2AR-365:Gs complex treated with λPP enzyme before complex formation, yielding a heterogeneous product compared to the undephosphorylated complex (bottom figure). Under conditions in the presence of multiple chemicals ( Figure 1 (Example in b) The less heterogeneous material in the fraction outside the dashed line shown in b) was used to perform gel filtration experiments to analyze the R:G complex.

[0065] Figure 6 Flowchart of the purification process for preparing the R:G complex with Nb35.

[0066] Figure 7Purification of Nb35 and determination of the R:G:Nb mixing ratio. a) Preliminary ion exchange chromatography after purification of nanobody 35 (Nb35) by nickel affinity chromatography. The nanobody (shown as red) eluted as a small peak and a homogeneous large peak in both groups was collected, concentrated by rotation, and used for crystallographic analysis after determining the appropriate mixing ratio with the R:G complex as shown in (b). b) Based on protein concentration, the agonist-bound T4L-β2AR:Gs complex was mixed with a slight excess of Nb35 (R:G complex to Nb35 molar ratio of 1-1.2) and the mixture was verified by analytical coagulation filtration.

[0067] Figure 8 T4L-β2AR:Gs:Nb35 complex crystals in the sponge-like mesophase.

[0068] Figure 9: Overall structure of the β2AR:Gs complex. a) The lattice stacking of the complex shows alternating layers of receptors and G proteins within the crystal. Abundant contacts are formed between the proteins in the aqueous layer. b) The overall structure of the asymmetric unit contents shows β2AR (green) binding to the agonist (yellow spheres) and participating in extensive interactions with Gsα (orange). Gαs, along with Gβ (cyan) and Gγ (purple), constitute the heterotrimeric G protein Gs. A Gs-binding nanobody (Nb35, red) binds the G protein between the α and β subunits. The nanobody (Nb35) promotes crystallization, as does T4 lysozyme (purple-red) fused to the N-terminus of β2AR. c) Biological complexes, omitting crystallization aids, show its position and orientation within the cell membrane.

[0069] Figure 10 The interaction between Nb35 and Gs in the T4L-β2AR:Gs:Nb35 complex of BI-167107. a) Two representative diagrams of the interaction between CDR1 (filled representation) of Nb35 (red) and Gβ (filled representation, cyan). b) Two representative diagrams of the interaction between CDR3 (filled representation) of Nb35 (red) and GαS (filled representation, orange) and Gβ (filled representation, cyan). Through interaction with GαS and Gβ, Nb35 can reduce the conformational flexibility of the complex. c) Two representative diagrams of the interaction between Nb35 (filled representation, red) and the framework region of GαS (orange).

[0070] Figure 11 Crystal contacts between Nb35 and the GαS subunit of the adjacent complex. Crystal contacts of Nb35 (red, filled in) and GαS (orange) in the related complexes (a) and (b) involving -x,y-1 / 2,-z+1 symmetry.

[0071] Figure 12: Comparison of active and inactivated β2AR structures. a) Side view and cytoplasmic view of the β2AR:Gs structure (green) compared to the inactivated carazolol-bound β2AR structure (blue; Rosenbaum et al., 2007). Significant structural changes are visible for the intracellular domains of TM5 and TM6. As measured by the α-carbon of Glu268 (yellow arrow) in both structures, TM5 extends two helical turns, while TM6 shifts them outward. b) β2AR:Gs compared to the β2AR:Nb80 structure of the nanobody in a stable active state (orange, Rasmussen et al., 2011). c) Residue positions in the E / DRY and NPxxY motifs observed from the cytoplasmic side, and other key residue positions in the β2AR:Gs and β2AR:Nb80 structures. Except for Arg131, which interacts with the nanobody in the β2AR:Nb80 structure, all residues occupy very similar positions.

[0072] Figure 13: Electron density map of residues at the R:G interface. a) D / ERY motif at the cytoplasmic terminal of TM3. b) Stacking interaction between Arg131 of the E / DRY motif and Tyr391 of the C-terminal GαS. c) NpxxY at the cytoplasmic terminal of TM7. d) Interactions of Thr68 and Tyr141 with Asp130 of the E / DRY motif. Phe139 of IL2 is embedded in a hydrophobic pocket of GαS. e) The β1-α1 loop (P loop) of GαS participates in nucleotide binding. The electron density map is a 2Fo-Fc map outlined with 1 sigma.

[0073] Figure 14 Receptor-G protein interaction. a, b) By opening transmembrane helices 5 and 6, the α5-helix of GαS enters the cavity formed on the intracellular side of the receptor. a) In the transmembrane core, the interaction is primarily nonpolar. An exception involves the α5-helix Tyr391 stacked on Arg131, which is based on the conserved DRY sequence of TM3 (see also Figure 13). Arg131 is also based on the Tyr stacked on the conserved NPxxY sequence of TM7. b) Due to the presence of the α5 helix, the receptor forms a network of polar interactions with TM5 and TM3. c) Receptor residues Thr68 and Asp130 interact with the IL2 helix of β2AR via Tyr141, positioning the helix so that Phe139 of the receptor enters (docks) the hydrophobic pocket on the surface of the G protein, thereby structurally linking the receptor-G protein interaction with the highly conserved DRY motif of β2AR.

[0074] Figure 15Conformational changes of Gαs. a) Comparison of Gαs (orange) in the β2AR:Gs complex with GTPγS-bound Gαs (gray) (PDB ID: 1AZT; Sunahara et al., 1997). GTPγS appears spherical. The helical domain (GαsAH) of Gαs exhibits dramatic displacement relative to its position in the GTPγS-bound state. b) Rotating the α5 helix of Gαs and replacing it with β2AR perturbs the β6-α5 ring, which additionally forms part of the GTPγS-binding pocket. c) The β1-α1 ring (P ring) and β6-α5 ring of Gαs interact with the phosphate and purine rings of GTPγS in the GTPγS-Gαs structure, respectively. d) In the nucleotide-free β2AR:Gs structure, the β1-α1 and β6-α5 rings are rearranged.

[0075] Figure 16 Nb37 inhibits the binding of GTPγS to Gαs. Real-time fluorescence increases were observed when Bodipy-GTPγS (100 nM) was incubated with 1 μM purified Gαs in the presence of increasing concentrations of Nb37.

[0076] Figure 17 Nb35 does not affect the binding of GTPγS to Gαs. Real-time fluorescence increases were observed when Bodipy-GTPγS (100 nM) was incubated with 1 μM purified Gαs in the presence of increasing concentrations of Nb35.

[0077] Figure 18 Nb35 inhibits the binding of GTPγS to Gsαβγ heterotrimer. Real-time fluorescence increases were observed when Bodipy‐GTPγS (100 nM) was incubated with 1 μM purified Gsαβγ heterotrimer in the presence of increasing concentrations of Nb35.

[0078] Figure 19 Purification of stable AVP:NT4LV2R:Gs complexes. a) Schematic diagram of AVP:NT4LV2R:Gs complex purification using Ni-NTA after FLAG-labeled affinity purification. b) SEC chromatogram of affinity-purified AVP:NT4LV2R:Gs. c) SDS-page monitoring of the purification protocol. Lane 1: Flux through the FLAG-labeled affinity column; Lane 2: Mixture of AVP, NT4LV2R, and Gs before purification; Lane 3: Molecular markers; Lane 4: AVP:NT4LV2R:Gs complex eluted by SEC; Lane 5: AVP:NT4LV2R:Gs complex after Ni-NTA purification following FLAG-labeled affinity purification.

[0079] Figure 20Stability of the AVP:NT4LV2R:Gs complex under SEC monitoring. Stranded line: SEC chromatogram of the AVP:NT4LV2R:Gs complex after 24 hours of incubation on ice. Blue line: SEC chromatogram of the AVP:NT4LV2R:Gs complex after 48 hours of incubation on ice. Red line: SEC chromatogram of the AVP:NT4LV2R:Gs complex after incubation with 10 μM antagonist SR121463. Invention Details

[0080] definition

[0081] This invention will be described with reference to certain embodiments and the accompanying drawings, but is not limited thereto, but only by the claims. Any references in the claims should not be construed as limiting the scope. The accompanying drawings described herein are illustrative only and not restrictive. In the drawings, the dimensions of some elements may be exaggerated for illustrative purposes and are not drawn to scale. When the term “comprising / including” is used in this specification and claims, it does not exclude other elements or steps. Where indefinite or definite articles are used, when referring to singular names such as “a / an” or “the,” this covers the plural form of the noun unless specifically stated otherwise. Furthermore, the terms first, second, third, etc., in the specification and claims are used to distinguish similar elements and are not necessarily used to describe order or chronological sequence. It should be understood that such terms are interchangeable in suitable contexts, and embodiments of the invention described herein can operate in other orders than those described or illustrated herein.

[0082] Unless otherwise defined herein, the scientific and technical terms and vocabulary used in this invention shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein, and the techniques of molecular and cell biology, genetics, protein and nucleic acid chemistry, and hybridization described herein, are generally known and conventionally used in the art. Unless otherwise indicated, the methods and techniques of this invention are generally practiced in accordance with conventional methods generally known in the art and as described in the various common and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and supplements 2002).

[0083] The term "binding domain" or "protein-binding domain" generally refers to any non-naturally occurring molecule, or a portion thereof, capable of binding to a protein or peptide using specific intermolecular interactions. A variety of molecules can function as protein-binding domains, including but not limited to protein molecules (proteins, peptides, protein-like or protein-containing molecules), nucleic acid molecules (nucleic acids, nucleic acid-like or nucleic acid-containing molecules), and carbohydrate molecules (carbohydrates, carbohydrate-like or carbohydrate-containing molecules). A more detailed description can be found in the specification.

[0084] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein to refer to a polymeric form of amino acids of any length, including encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a similar peptide backbone.

[0085] As used herein, the term "protein complex" or simply "complex" refers to a class of two or more related polypeptide chains. The proteins in a protein complex are linked by non-covalent protein-protein interactions. The "quaternary structure" is the structural arrangement of the related folded proteins in a protein complex. It is understood that a complex can be a polymeric complex containing 2, 3, 4, 5, 6, or more polypeptides. Complexes may also additionally contain non-protein molecules.

[0086] As used herein, the terms “nucleic acid molecule,” “polynucleotide,” “polynucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-restricted examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0087] As used herein, the term "ligand" or "receptor ligand" refers to a molecule that specifically binds to a GPCR, either intracellularly or extracellularly. Unless for limiting purposes, a ligand can be a protein, (poly)peptide, lipid, small molecule, protein scaffold, antibody, antibody fragment, nucleic acid, or carbohydrate. Ligands can be synthetic or naturally occurring. The term "ligand" includes "natural ligand," which is an endogenous, naturally occurring ligand of a native GPCR. In most cases, a ligand is a "regulator" that increases or decreases the intracellular response upon contact (e.g., binding) with a cell-expressed GPCR. Examples of ligands that act as regulators include agonists, partial agonists, inverse agonists, and antagonists, which can be described in more detail in the specification.

[0088] The term "conformation" or "conformatory state" of a protein generally refers to the range of structures a protein may adopt at any given moment. Those skilled in the art will recognize that the determinants of conformation or conformational state include the protein's primary structure, reflected in its amino acid sequence (including modified amino acids), and the environment surrounding the protein. Protein conformation or conformational state also involves structural features such as secondary structure (e.g., α-helices, β-sheets, etc.), tertiary structure (e.g., three-dimensional folding of polypeptide chains), and quaternary structure (e.g., interactions of polypeptide chains with other protein subunits). Post-translational modifications and other modifications to the polypeptide chain (e.g., ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, etc.) can affect protein conformation. Furthermore, environmental factors (e.g., pH, salt concentration, ionic strength, and molality of the surrounding solution, interactions with other proteins and cofactors, etc.) can influence protein conformation. The conformational state of a protein can be determined by functional assays targeting activity or binding to another molecule, or by physical methods (e.g., X-ray crystallography, NMR, or spin labeling, etc.), and other methods. For a general discussion of protein conformations and conformational states, see Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993. A “specific conformation” or “specific conformational state” is any subset of the range of conformations or conformational states that a protein can adopt.

[0089] As used herein, “functional conformation” or “functional conformational state” refers to the fact that a protein has different conformational states with a dynamic range of activity, specifically from inactive to maximally active. Examples of functional conformational states include active and inactive conformations. It should be understood that “functional conformational state” is intended to cover any conformational state of a GPCR that has any activity, including inactivity; and not to cover the denatured state of the protein. A specific class of functional conformations considered herein is “conformations available for drug development,” generally referring to unique, treatment-relevant conformational states of target proteins. As an example, the active conformation of the β2-adrenergic receptor corresponds to a conformation available for drug development for the treatment of asthma. Therefore, it is understood that the ability to be used for drug development is limited by the specific conformation depending on the indication for treatment.

[0090] As used herein, the terms “locking,” “trapping,” “fixing,” or “freezing,” when referring to the functional conformational state of a GPCR (as defined herein), mean that the GPCR is held or retained within a subset of possible conformations in which it can otherwise be presented, due to the effect of the interaction between the GPCR:G protein complex and the binding domain according to the invention. Correspondingly, as used herein, “conformation-trapping,” “conformation-fixed,” “conformation-locked,” or “conformation-frozen” proteins are proteins held within a subset of possible conformations in which they can otherwise be presented due to the effect of the interaction between the GPCR:G protein complex and the binding domain according to the invention. In this context, a binding domain that specifically or selectively binds to a particular conformation or conformational state of a protein refers to a binding domain that has a higher affinity for binding to proteins within a subset of conformations or conformational states than for binding to other conformations or conformational states that the protein can present. Those skilled in the art will recognize that binding domains that specifically or selectively bind to a particular conformation or conformational state of a protein will stabilize that particular conformation or conformational state.

[0091] As used in this article, the term "complementarity-determining region" or "CDR" in the context of antibodies refers to the variable region of the H (heavy) or L (light) chain (also abbreviated as V, respectively). H and V LThese regions contain amino acid sequences that specifically bind to antigenic targets. These CDR regions are responsible for the basic specificity of antibodies against specific antigenic determinant structures. These regions are also known as "hypervariable regions." CDRs represent discontinuous amino acid segments within the variable region, and regardless of species, the positions of these key amino acid sequences within the heavy and light chain variable regions are found to be similar in the amino acid sequence of the variable chain. All regular antibodies have three CDR regions in both the variable heavy and light chains, which are discontinuous with respect to each light chain (L) and heavy chain (H) (referred to as L1, L2, L3, H1, H2, H3). In particular, immunoglobulin single variable domains (such as nanobodies (as defined elsewhere here)) generally consist of a single amino acid chain containing four "frame sequences or frame regions" or FRs (referred to as FR1, FR2, FR3, FR4) and three "complementary determinant regions" or CDRs (referred to as CDR1, CDR2, CDR3), which are discontinuous with each other. The description of CDR sequences (and FR sequences) is based on the IMGT-specific numbering system for V-domains and V-like domains (Lefranc et al., 2003).

[0092] As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope may comprise three amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more commonly, at least 8, 9, or 10 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art, including, for example, X-ray crystallography and multidimensional nuclear magnetic resonance.

[0093] As used herein, a "conformational epitope" refers to an epitope containing amino acids in a spatial conformation unique to the folded 3D conformation of a polypeptide. Generally, a conformational epitope consists of discontinuous amino acids in a linear sequence that aggregate within the folded structure of a protein. However, a conformational epitope can also consist of a linear sequence of amino acids employing a conformation specific to the folded 3D conformation of a polypeptide (and not present in the denatured state). In a protein complex, a conformational epitope consists of discontinuous amino acids in the linear sequence of one or more polypeptides that aggregate as the polypeptide folds and links together in a specific quaternary structure. Similarly, a conformational epitope can also consist herein of a linear sequence of one or more polypeptides that aggregate within a conformation specific to the quaternary structure.

[0094] As used herein, the term "specific" refers to the ability of a binding domain, particularly an immunoglobulin (such as an antibody) or immunoglobulin fragment (such as a nanobody), to preferentially bind to one antigen relative to different antigens, without necessarily implying high affinity (as defined elsewhere herein). Binding domains that can specifically bind to a particular antigen or antigenic determinant (e.g., an epitope) and / or have affinity for a particular antigen or antigenic determinant (e.g., an epitope), particularly immunoglobulins (such as antibodies) or immunoglobulin fragments (such as nanobodies), are referred to as "anti" or "target" of said antigen or antigenic determinant. If a binding domain according to the invention is specific to two different antigens or antigenic determinants, it is referred to as "cross-reactive" with both different antigens or antigenic determinants.

[0095] As used herein, the term "affinity" refers to the degree to which a binding domain, particularly an immunoglobulin (such as an antibody) or immunoglobulin fragment (such as a nanobody), binds to an antigen, such that the equilibrium between the antigen and the binding domain shifts toward the presence of a complex formed through their binding. Thus, for example, when antigen and antibody (fragment) are combined at relatively equal concentrations, the antibody (fragment) with higher affinity will bind to the available antigen, shifting the equilibrium toward a higher concentration of the resulting complex. Dissociation constant (K) d This term is commonly used to describe the affinity between a protein-binding domain and an antigen target. Typically, the dissociation constant is less than 10. -5 M. Preferably, the dissociation constant is less than 10. -6 M, more preferably less than 10 -7 M. The optimal choice has a dissociation constant less than 10. -8 M.

[0096] As used herein, the terms "specific binding" or "specific binding" generally refer to the ability of a binding domain, particularly an immunoglobulin (such as an antibody) or immunoglobulin fragment (such as a nanobody), to preferentially bind to a specific antigen present in a homogeneous mixture of different antigens. In some embodiments, the specific binding interaction will distinguish between desired and unwanted antigens in the sample, and in some embodiments, this interaction is greater than about 10 to 100 times or more (e.g., greater than about 1,000 or 10,000 times).

[0097] The terms “specific binding,” “selective binding,” “preferential binding,” and their grammatical equivalents are used interchangeably in this document. The terms “conformation specificity” or “conformation selectivity” are also used interchangeably in this document.

[0098] A “deletion” is defined herein as a change in the amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues are missing compared to the amino acid or nucleotide sequence of the parent polypeptide or nucleic acid. In the context of a protein or fragment thereof, a deletion may involve the loss of about 2, about 5, about 10, up to about 20, up to about 30, or up to about 50 or more amino acids. A protein or fragment thereof may contain more than one deletion. In the context of GPCRs, a deletion may be particularly a loop deletion, or an N- and / or C-terminal deletion.

[0099] An "insertion" or "addition" is a change in the amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues are added, compared to the amino acid or nucleotide sequence of the parent protein. An "insertion" generally refers to the addition of one or more amino acid residues to the amino acid sequence of a polypeptide, while an "addition" can be an insertion or the addition of amino acid residues at the N- or C-terminus, or at both ends. In the context of a protein or fragment thereof, insertions or additions are typically about 1, about 3, about 5, about 10, up to about 20, up to about 30, or up to about 50 or more amino acids. A protein or fragment thereof may contain more than one insertion.

[0100] As used herein, “substitution” is the result of replacing one or more amino acids or nucleotides with different amino acids or nucleotides compared to the amino acid or nucleotide sequence of a parent protein or fragment thereof. It is understood that proteins or fragments thereof may have conserved amino acid substitutions that have little effect on protein activity. Conserved substitutions are intentional combinations, such as gly, ala; val, ile, leu, met; asp, glu; asn, gln; ser, thr; lys, arg; cys, met; and phe, tyr, trp.

[0101] The term "sequence identity" is used herein to refer to the degree of similarity between sequences based on nucleotide-nucleotide or amino acid-amino acid pairs within a comparison window. Therefore, the "sequence identity percentage" is calculated as follows: by comparing two best-aligned sequences within a comparison window, the number of identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) present in the two sequences is determined, yielding the number of matching positions. This number is then divided by the total number of positions within the comparison window (i.e., the window size), and the result is multiplied by 100 to generate the sequence identity percentage. The sequence identity percentage can be determined manually or using computer programs available in the art.

[0102] As used herein, "crystal" or "crystalline structure" refers to a solid material whose constituent atoms, molecules, or ions are arranged in an ordered, repeating pattern extending through all three dimensions of space. The process of forming a crystalline structure from a liquid or a substance dissolved in a liquid is commonly referred to as "crystallization" or "crystallization." Protein crystals grow almost exclusively in solution. The most common method is to gradually reduce the solubility of its constituent molecules. Crystal growth in solution is characterized by two steps: the nucleation of microscale crystallites (possibly containing only 100 molecules), followed by the growth of said crystallites, ideally resulting in diffraction-quality crystals.

[0103] As used herein, "X-ray crystallography" is a method for determining the atomic arrangement within a crystal, in which a beam of X-rays bombards the crystal and diffracts in multiple specific directions. Based on the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional image of the electron density within the crystal. From this electron density, the average positions of the atoms within the crystal, their chemical bonds, their disorder, and a variety of other information, as known to those skilled in the art, can be determined.

[0104] As used herein, the term "atomic coordinates" refers to a set of three-dimensional coordinates of atoms within a molecular structure. In one embodiment, atomic coordinates are obtained using X-ray crystallography, according to methods generally known to those skilled in the art of biophysics. Briefly, X-ray diffraction patterns are obtained by diffracting X-rays from a crystal. The diffraction data is used to calculate an electron density map of the unit cell containing the crystal; this map is used to establish the atomic positions (i.e., atomic coordinates) within the unit cell. Those skilled in the art will understand that the set of structural coordinates determined by X-ray crystallography contains a standard deviation. In other embodiments, atomic coordinates can be obtained using other experimental biophysical structure determination methods, including electron diffraction (also known as electron crystallography) and nuclear magnetic resonance (NMR) methods. In other embodiments, atomic coordinates can be obtained using molecular modeling tools, which may be based on one or more de novo protein folding algorithms, energy minimization, and homology-based modeling. These techniques are generally known to those skilled in the art of biophysics and bioinformatics.

[0105] In this article, "structural analysis" is used to refer to determining the atomic arrangement or atomic coordinates of a protein, usually through biophysical methods such as X-ray crystallography.

[0106] The terms “compound,” “test compound,” “candidate compound,” or “drug candidate compound” are used herein to describe any naturally occurring or synthetic molecule tested in an assay, such as a screening assay or a drug discovery assay. Similarly, this class of compounds includes both organic and inorganic compounds. Compounds include polynucleotides, lipids, or hormone analogs characterized by low molecular weight. Other biopolymerized organic test compounds include small peptides or peptide-like molecules (peptide mimics) containing about 2 to about 40 amino acids, and larger polypeptides containing about 40 to about 500 amino acids, such as antibodies, antibody fragments, or antibody conjugates. Test compounds can also be protein scaffolds. For high-throughput purposes, test compound libraries, such as combinatorial libraries or randomized libraries that provide sufficient diversity, may be used. Examples include, but are not limited to, natural compound libraries, allosteric compound libraries, peptide libraries, antibody fragment libraries, synthetic compound libraries, fragment-based libraries, phage display libraries, etc. More detailed descriptions can be found in other parts of the specification.

[0107] As used herein, the terms “determine,” “measure,” “evaluate,” “monitor,” and “determine” are used interchangeably, including both quantitative and qualitative determinations.

[0108] When the term “biological activity” is used in relation to GPCRs, it refers to GPCRs that have the biochemical functions of naturally occurring GPCRs (e.g., binding function, signal transduction function, or the ability to change conformation due to ligand binding).

[0109] As used in this article, the terms “therapeutic effective amount,” “therapeutic effective dose,” and “effective amount” refer to the amount required to achieve the desired outcome.

[0110] As used herein, the term "medicinal" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual together with a compound without causing any undesirable biological effects or interacting in a harmful manner with any other component in a pharmaceutical composition containing it. Invention Details

[0112] Although the ligands that can activate GPCRs are extremely diverse, they interact with only a relatively small number of intracellular proteins, inducing profound physiological changes. The ability of heterotrimeric G proteins, β-repressor proteins, and GPCR kinases to specifically recognize GPCRs in their activated states is generally known, but our understanding of them from a structural and functional perspective is limited. Therefore, it is surprising and advantageous to identify binding domains that specifically bind to GPCR:G protein complexes and can stabilize or lock the complexes into functional conformational states (especially the activated conformational state). Furthermore, binding domains are a general tool for stabilizing and capturing desired GPCRs in their G protein-binding state, which are generally considered to represent the active state of GPCRs (as defined herein).

[0113] Therefore, a first aspect of the present invention relates to a binding domain that targets and / or specifically binds to a complex comprising a GPCR and a G protein.

[0114] The binding domains of this invention can be any non-naturally occurring molecule or portion thereof capable of specifically binding to a complex comprising GPCRs and G proteins (as defined above). According to a preferred embodiment, the binding domains described herein are protein scaffolds. The term "protein scaffold" generally refers to the folding units that form a structure, particularly a protein or peptide structure, which includes a framework for binding another molecule, such as a protein (see, for example, Skerra (2000), review). The binding domains can be derived from naturally occurring molecules, such as components of the innate or adaptive immune system, or can be entirely artificially designed. The binding domains can be immunoglobulin-based or can be based on domains present in proteins, including but not limited to microbial proteins, protease inhibitors, toxins, fibronectin, lipocalin, single-stranded antiparallel coiled helical proteins, or repeating motif proteins. Examples of binding domains known in the art include, but are not limited to: antibodies, heavy chain antibodies (hcAbs), single-domain antibodies (sdAbs), minibody, variable domains derived from camel heavy chain antibodies (VHHs or nanobodies), variable domains of neoantigen receptors derived from shark antibodies (VNARs), antigens, protein A, protein G, designed ankyrin repeat domains (DARPin), type III fibronectin repeats, anticalin, knottin, modified CH2 domains (nanobodies), peptides and proteins, lipopeptides (e.g., pepducin), DNA and RNA (see, for example, Gebauer & Skerra, 2009; Skerra, 2000; Starovasnik et al., 1997; Binz et al., 2004; Koide et al., 1998; Dimitrov, 2009; Nygren et al., 2008; WO2010066740). Typically, when using selection methods to generate a specific type of binding domain, a combinatorial library containing a common sequence or framework sequence is used to screen for binding to a target molecule (such as a protein), the common sequence or framework sequence containing random, potentially interacting residues.

[0115] The binding domains of this invention can target and / or specifically bind to any selected GPCR:G protein complex. Preferred target complexes are naturally occurring GPCR and G protein complexes, or alternatively, in the case of non-natural variants of GPCR and G protein (discussed elsewhere herein), complexes in which the GPCR and G protein will associate under appropriate physiological conditions. Those skilled in the art will understand that the structural relationship between the GPCR and G protein determines whether a particular GPCR:G protein complex can be formed, which will be further detailed below regarding members of the G protein family and the GPCR family.

[0116] "G proteins" refers to a family of guanine nucleotide-binding proteins that are involved in transmitting chemical signals extracellularly and leading to intracellular alterations. G proteins are key molecular components of intracellular signal transduction after ligands bind to the extracellular domains of GPCRs. They are also known as "heterotrimeric G proteins" or "large G proteins." G proteins consist of three subunits: α, β, and γ, and their classification is largely based on the identity of different α subunits and the nature of subsequent transduction events. Other classifications of G proteins are derived from cDNA sequence homology analysis. G proteins bind to guanosine diphosphate (GDP) or guanosine triphosphate (GTP), possessing highly homologous guanine nucleotide-binding domains and distinct domains for interaction with receptors and effectors. Different Gα protein subclasses (such as Gαs, Gαi, Gαq, and Gα12) transmit signals through different pathways involving second messenger molecules such as cAMP, inositol triphosphate (IP3), diacylglycerol, and intracellular calcium. 2+ And RhoA GTPase. For further example, the α subunit (39-46 kDa) contains a guanine nucleotide binding site and has GTPase activity; the β (37 kDa) and γ (8 kDa) subunits are closely linked and function as βγ heterodimers. Currently described, there are 23 types of α subunits (including some splicing isoforms), 6 β subunits, and 11 γ subunits. The types of G proteins and subunits are indicated by subscripts: thus, for example, the α subunit of the Gs protein (which activates adenylate cyclase) is Gsα; other G proteins include Gi, which is structurally different from Gs (a different α subunit type) and inhibits adenylate cyclase. Table 1 provides other examples.

[0117] Typically, G proteins are essentially nucleotide-binding types. More specifically, depending on the activation state of a particular GPCR, a G protein (at least the α subunit) binds GTP or GDP. Agonists binding GPCRs promote the interaction of the GDP-bound Gαβγ heterotrimer, resulting in the exchange of GDP on Gα for GTP, and the functional dissociation of the G protein into Gα-GTP and the Gβγ subunit. The dissociated Gα-GTP and Gβγ subunits can independently or in parallel regulate downstream cellular effectors (channels, kinases, or other enzymes, see Table 1). The intrinsic GTPase activity of Gγ leads to the hydrolysis of GTP to GDP, and the re-association of Gα-GDP with the Gβγ subunit, and the termination of signal transduction. Thus, G proteins function as regulatory molecular switches, capable of triggering bifurcating signals through the effects of the α and βγ subunits. The switch is activated by the receptor and then self-activated within seconds, a time sufficient to significantly amplify signal transduction.

[0118] As used herein, a “G protein-coupled receptor” or “GPCR” is a polypeptide sharing a common structural motif, having seven regions between 22 and 24 hydrophobic amino acids that form seven α-helices, each spanning the cell membrane. Each span is identified by numbering, namely, transmembrane-1 (TM1), transmembrane-2 (TM2), etc. Transmembrane helices are also linked by amino acid regions between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the extracellular or “extracellular” side of the cell membrane; these regions are referred to as “extracellular” regions 1, 2, and 3 (EC1, EC2, and EC3), respectively. Transmembrane helices are also linked by amino acid regions between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the intracellular or “intracellular” side of the cell membrane; these regions are referred to as “intracellular” regions 1, 2, and 3 (IC1, IC2, and IC3), respectively. The receptor's carboxyl ("C") terminus is located in the intracellular space, while its amino ("N") terminus is located in the extracellular space. Both of these regions can be easily identified by analyzing the primary amino acid sequence of the GPCR.

[0119] The structure and classification of GPCRs are well known in the field, and other discussions on GPCRs can be found in Probst et al., 1992; Marchese et al., 1994. & 2008; Rosenbaum et al., 2009; and the following books: Jurgen Wess (ed.), Structure-Function Analysis of G Protein-Coupled Receptors (1st edition; October 15, 1999), Wiley-Liss; Kevin R. Lynch (ed.), Identification and Expression of G Protein-Coupled Receptors (March 1998), John Wiley & Sons; Tatsuya Haga (ed.), G Protein-Coupled Receptors (September 24, 1999), CRC Press; and Steve Watson (ed.), G-Protein Linked Receptor Factsbook (1st edition; 1994), Academic Press. GPCRs can be classified into several different families based on sequence homology. Although all GPCRs share a similar 7-transmembrane α-helix structure, different families within this receptor type show no sequence homology, suggesting that the similarity in their transmembrane domain structures may define common functional requirements. A comprehensive understanding of the GPCR reservoir may be possible when the first draft of the human genome becomes available. Fredriksson et al. classified 802 human GPCRs based on phylogenetic rules. This showed that the vast majority of human GPCRs are found in five major families, known as rhodopsin, adhesion, secretin, glutamate, and Frizzled / Taste 2 (Fredriksson et al., 2003).

[0120] Members of the rhodopsin family (corresponding to class A (Kolakowski, 1994) or class 1 in the original classification system (Foord et al. (2005)) possess only a small extracellular loop, and their ligands interact with residues in the transmembrane cleft. This is the largest class to date (accounting for >90% of GPCRs), containing receptors for odorants, small molecules (such as catecholamines and amines), (neuropeptides), and glycoprotein hormones. Rhodopsin is the representative of this family and was the first GPCR whose structure was resolved (Palczewski et al., 2000). The first receptor whose structure was resolved to interact with a diffuse ligand—β2-AR (Rosenbaum)—was also resolved. (Foord et al., 2007) also belong to this family. Based on phylogenetic analysis, class B GPCRs or class 2 (Foord et al., 2005) receptors have recently been subdivided into two families: adhesins and secretins (Fredriksson et al., 2003). Adhesins and secretins are characterized by relatively long N-terminal extracellular domains involved in ligand binding. Little is known about the orientation of the transmembrane domain, but it is likely to be quite different from that of rhodopsin. The ligands for these GPCRs are hormones such as glucagon, secretins, gonadotropin-releasing hormone, and parathyroid hormone. Glutamate family receptors (class C or class 3 receptors) also have large extracellular domains and function similarly to the "Venus flytrap" because they open and close the bound agonist inside. Family members include metabolites of glutamate, Ca2+, and glutamate. 2+ - Sensitive and γ-aminobutyric acid (GABA)-B receptors.

[0121] GPCRs include, but are not limited to, serotonin olfactory receptors, glycoprotein hormone receptors, chemokine receptors, adenosine receptors, biogenic amine receptors, melanocortin receptors, neuropeptide receptors, chemokine receptors, somatostatin receptors, opioid receptors, melatonin receptors, calcitonin receptors, PTH / PTHrP receptors, glucagon receptors, secretin receptors, black widow spider venom receptors, metabolite glutamate receptors, calcium receptors, GABA-B receptors, pheromone receptors, protease-activated receptors, rhodopsin, and other G protein-coupled 7-transmembrane receptors. GPCRs also include GPCR receptors that are associated with each other as homodimers, heterodimers, or higher-order oligomers. The amino acid sequences of GPCRs (and the nucleotide sequences of their encoded cDNAs) are readily available, for example, from GenBank. http: / / www.ncbi.nlm.nih.gov / entrez ).

[0122] Therefore, according to specific embodiments, the present invention provides binding domains for and / or specifically binding to GPCR:G protein complexes, wherein the G protein is selected from Gs, Gi, Go, Gt, Ggust, Gz, Golf, Gq, G12, and G13. In one preferred embodiment, the G protein is Gs. In another preferred embodiment, the G protein is Gi. In yet another preferred embodiment, the G protein is Gt, more specifically, transducin. Correspondingly, the GPCR contained in the complex is selected from Gs-coupled receptors, Gi-coupled receptors, Go-coupled receptors, Gt-coupled receptors, Ggust-coupled receptors, Golf-coupled receptors, Gq-coupled receptors, G12-coupled receptors, and G13-coupled receptors. In one preferred embodiment, the GPCR is a Gs-coupled receptor. In another preferred embodiment, the GPCR is a Gi-coupled receptor. In yet another preferred embodiment, the GPCR is a Gt-coupled receptor, more specifically, rhodopsin. Table 1 provides specific non-limiting examples.

[0123] Table 1. Non-limiting examples of the relationship between G protein-coupled receptors and signal transduction pathways.

[0124]

[0125]

[0126]

[0127] Generally, it will be apparent to those skilled in the art that the binding domains of the present invention bind at least those GPCR:G protein complex types most relevant from a biological and / or therapeutic point of view. Therefore, it is understood that, depending on the purpose and application, the GPCRs and G proteins contained in the target complex can be naturally occurring or non-naturally occurring (i.e., artificially altered). As used herein, the term "naturally occurring" means a naturally produced GPCR or G protein. In particular, wild-type polymorphs and isotypes of GPCRs and G proteins, as well as orthologs of different species, are examples of naturally occurring proteins, found, for example, but not limited to, mammals, more specifically in humans, or viruses, or plants, or insects, etc. Thus, such GPCRs or G proteins are found in nature. As used herein, the term "non-naturally occurring" means a GPCR or G protein that is not naturally occurring. In certain settings, it is advantageous that the GPCRs and / or G proteins contained in the complex are non-naturally occurring proteins. For example, for illustrative purposes only, to increase the likelihood of obtaining crystals of GPCR:G protein complexes stable by the binding domains of the present invention, it is desirable to perform some protein modification that does not affect or only minimally affects ligand binding affinity. Alternatively or additionally, to increase the cellular expression level of the GPCR and / or G protein, or to increase stability, it may be considered to introduce certain mutations into the target GPCR and / or G protein. Non-limiting examples of non-naturally occurring GPCRs include, but are not limited to, GPCRs constitutively activated by mutation relative to their amino acid or nucleotide sequence, GPCRs with loop deletions, GPCRs with N- and / or C-terminal deletions, GPCRs with substitutions, insertions, or additions relative to their amino acid or nucleotide sequence, or any combination thereof, or other variants of naturally occurring GPCRs. Similarly, non-limiting examples of non-naturally occurring G proteins include, but are not limited to, G proteins with N- and / or C-terminal deletions, G proteins with substitutions, insertions, or additions relative to their amino acid or nucleotide sequence, or any combination thereof, or other variants of naturally occurring G proteins. The scope of this invention also includes target GPCR:G protein complexes, which comprise chimeric or hybrid GPCRs, such as chimeric GPCRs having an N- and / or C-terminus of one GPCR and a loop of a second GPCR, or GPCRs fused to a portion (such as T4 lysozyme) (see also the Examples section).

[0128] According to specific embodiments within the scope of the invention, the non-naturally occurring GPCR or G protein contained in the GPCR:G protein complex may have an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identical to that of a naturally occurring GPCR or G protein. As a further example, the β2-adrenergic receptor is used as a specific, non-limiting example of a GPCR within the scope of the invention. It should be apparent from the foregoing that, in addition to the human β2-adrenergic receptor (e.g., the sequence described in GenBank accession number NP_000015), mouse β2-adrenergic receptors (e.g., the sequence described in GenBank accession number NM 007420) or β2-adrenergic receptors from other mammals are also included. Wild-type polymorphic variants of β2-adrenergic receptors from specific species and certain other active variants are also contemplated. For example, the “human β2-adrenergic receptor” has an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the naturally occurring “human β2-adrenergic receptor” in GenBank accession number NP_000015. Similarly, and with Gαs, Gαi, and Gαt as specific, non-limiting examples of G protein subunits within the scope of this invention, it should be apparent from the foregoing that, in addition to human Gαs, Gαi, or Gαt, mouse Gαs, Gαi, or Gαt proteins, or Gαs, Gαi, or Gαt proteins from other mammals are also covered. Wild-type polymorphs of Gαs, Gαi, or Gαt from specific species and certain other active variants are also considered. For example, “human Gαs”, “human Gαi”, or “human Gαt” have amino acid sequences that are at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identical to those of naturally occurring “human Gαs”, “human Gαi”, or “human Gαt”, respectively, according to Genbank accession numbers P63092, P63096, and P11488. In addition, there are many isotypes of G protein subunits, including, for example, isotypes of Gs and Gi proteins (Gαs: GNAS; Gα0: GNAO1; Gαi: GNAI1 or GNAI2 or GNAI3; Gβ: GNB1 or GNB2 or GNB3 or GNB4 or GNB5 or GNB1L or GNB2L; Gγ: GNGT1 or GNGT2 or GNG2 or GNG3 or GNG4 or GNG5 or GNG7 or GNG8 or GNG10 or GNG11 or GNG12 or GNG13; named according to the HGNC normalized nomenclature of the human genome; accession numbers for different isotypes in different organisms are available from www.uniprot.org). Table 5 provides some specific examples of isotypes of G protein subunits.Those skilled in the art will understand that the amino acid sequences of different G protein subunits are almost 100% identical across different species and organisms, or, if not 100%, are conserved. Notably, sequence alignment of the amino acid sequences of the human, bovine, rat, and mouse β subunits of the G protein reveals that the amino acid sequences are 100% conserved across these organisms. Similarly, the amino acid sequences of the human, mouse, and bovine γ subunits of the G protein are 100% identical. The amino acid sequences of Gαs in rats and mice are also 100% identical, while human and bovine Gαs differ by only one or two amino acids, respectively. Therefore, it is anticipated that the binding domains targeting and / or specifically binding the GPCR:G protein complex, particularly binding the G protein contained within the complex, are cross-reactive. It is also apparent that the GPCR and G protein contained in the target complex can originate from the same or different species. Preferably, the GPCR and / or G protein are mammalian proteins, or plant proteins, or microbial proteins, or viral proteins, or insect proteins. More preferably, the GPCR is a human protein.

[0129] It is also anticipated that the binding domain of the present invention can generally bind to GPCR:G protein complexes containing all naturally occurring or synthetic analogs, variants, mutants, alleles, portions, fragments, and isotypes of a particular GPCR and / or G protein contained in the complex; or at least bind to those analogs, variants, mutants, alleles, portions, fragments, and isotypes of a particular GPCR and / or G protein contained in the complex that contain one or more such antigenic determinants or epitopes, said antigenic determinants or epitopes being substantially identical to the antigenic determinants or epitopes that the binding domain of the present invention binds to for the specific GPCR and / or G protein contained in the complex.

[0130] Various methods can be used to determine the specific binding between the binding domain and the target GPCR:G protein complex (as defined above), including, for example, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance assay, phage display, etc., which are routine practices in the field and are discussed, for example, in Sambrook et al., (2001), Molecular Cloning, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and further illustrated in the Examples section. For this purpose, it will be understood that unique labels or tags, such as peptide labels, nucleic acid labels, chemical labels, fluorescent labels, or radiofrequency labels, will generally be used, as further described herein.

[0131] According to specific embodiments, the binding domain of the GPCR:G protein complex binds to the complex with a higher affinity than binding only to G proteins and / or only to GPCRs, respectively. In one embodiment, the binding domain of the GPCR:G protein complex specifically binds to the GPCR contained in the complex without binding to the G protein. Preferably, in another embodiment, the binding domain of the GPCR:G protein complex specifically binds to the G protein contained in the complex without binding to the GPCR. More specifically, the binding domain of the GPCR:G protein complex specifically binds to the G protein, which is the Gs protein, contained in the complex of the Gs-coupled receptor and the Gs protein.

[0132] It is generally known that GPCRs are structurally complex membrane proteins that exhibit a range of functional behaviors in response to natural and synthetic ligands. In nature, ligand-bound GPCRs can associate with G proteins to form complexes that exhibit specific functional conformational states, more specifically, active conformational states, resulting in specific biological activities. The present invention offers the particular advantage that the binding domains described herein can stabilize various such active conformations of GPCRs complexed with G proteins and bound to a variety of natural or synthetic ligands. Those skilled in the art will recognize that binding domains that specifically bind ligand:GPCR:G protein complexes can stabilize specific conformations of the GPCR contained within the complex. In a preferred embodiment, the binding domain can stabilize or increase the stability of specific functional conformational states of the GPCR:G protein complex, preferably wherein the GPCR is in an active conformational state. Generally, the functional conformational state of the GPCR can be a basal conformational state, an active conformational state, or an inactive conformational state. Preferably, the binding domain of the present invention enables the GPCR to stabilize in its active conformation and / or locks the GPCR in its active conformation when bound to the GPCR:G protein complex, regardless of the presence of a receptor ligand.

[0133] In a particularly preferred embodiment of the invention, the binding domain is contemplated for targeting and / or specifically binding to a complex comprising a GPCR, a G protein, and a receptor ligand (as defined herein). More preferably, the binding domain targets and / or specifically binds to a complex consisting of a GPCR, a G protein, and a receptor ligand. The receptor ligand may be a small compound, peptide, antibody, or antibody fragment, etc., which triggers a response upon binding. The receptor ligand, or simply ligand, as defined herein, may be an orthosteric ligand (natural and synthetic) that binds to the receptor's active site and is classified according to its potency, or in other words, according to its effect on receptor signaling via a specific pathway. As used herein, "agonist" refers to a ligand that increases the receptor's signaling activity by binding to the receptor. A full agonist can maximally stimulate the receptor; a partial agonist cannot elicit full activity even at saturation concentrations. Partial agonists may also function as "blockers" by preventing the binding of more potent agonists. "Antagonist" refers to a ligand that binds to the receptor without stimulating any activity. "Antagonist" is also known as "blocker" because of its ability to prevent the binding of other ligands and thus block agonist-induced activity. Furthermore, "inverse agonist" refers to an antagonist that, in addition to blocking the agonist effect, reduces the basal or constitutive activity of the receptor to below that of an unbound receptor. Preferably, the binding domain of the present invention targets and / or specifically binds to a complex comprising a GPCR, a G protein, and a receptor ligand, wherein the receptor ligand is an agonist. More specifically, the agonist binds to the receptor at an orthosteric site.

[0134] The signal transduction activity (and conformational behavior) of GPCRs can also be affected by binding to another ligand called an allosteric regulator. An allosteric regulator, or modulator, or effector molecule, binds to the allosteric site of the GPCR (i.e., a regulatory site physically distinct from the protein's active site). Unlike orthosteric ligands, allosteric regulators are non-competitive because they bind to receptors at different sites and can modify receptor function even when they also bind to endogenous ligands. Therefore, allosteric regulators are not limited to simply turning receptors on or off as most drugs do. Instead, they function more like dimer switches, providing control over the strength of activation or deactivation while allowing the body to retain its natural control over triggering receptor activation by altering the affinity of the receptor for its (endogenous) ligand. Allosteric regulators that enhance protein activity are referred to herein as "allosteric activators" or "positive allosteric regulators," while those that reduce protein activity are referred to herein as "allosteric inhibitors" or "negative allosteric regulators." Therefore, in a particular embodiment, the binding domain of the present invention targets and / or specifically binds to a complex comprising a GPCR, a G protein, and a receptor ligand, wherein the receptor ligand is an allosteric regulator, preferably a positive allosteric regulator. More specifically, the positive allosteric regulator binds to the receptor at the allosteric site.

[0135] As described, the classic view of how GPCRs regulate cell physiology is that the binding of ligands (such as hormones, neurotransmitters, or sensory stimulants) stabilizes the active conformational state of the receptor, thereby allowing interaction with heterotrimeric G proteins. In addition to interaction with G proteins, the binding of agonist-associated GPCR kinases (GRKs) leads to receptor phosphorylation. A common result of GRK phosphorylation of GPCRs is reduced interaction between GPCRs and G proteins, and increased interaction between GPCRs and repressor proteins, which spatially restrict other G protein signaling, leading to receptor desensitization. Because β-repressor proteins shut down G protein signaling, a second set of parallel signaling cascades, such as the MAPK pathway, can be simultaneously triggered. GPCRs also interact with a variety of proteins (G proteins, GRKs, repressor proteins, and other receptors) outside the general GPCR-interacting protein family. These GPCR-selective partner proteins can mediate GPCR signaling, organize GPCR signaling via G proteins, guide GPCR transport, anchor GPCRs to particularly subcellular regions, and / or influence GPCR pharmacology (Ritter and Hall 2009). In this regard, the ligands used herein can also be “biased ligands” capable of selectively stimulating the signal transduction activity of a small class of receptors, such as selectively activating G proteins or β-repressor proteins. These ligands are also referred to as “biased ligands,” “biased agonists,” or “functionally selective agonists.” More specifically, ligand bias can be imperfect bias, characterized by the ligand stimulating multiple receptor activities, which have different relative potency (non-absolute selectivity) for different signals; or ligand bias can be perfect bias, characterized by the ligand stimulating one receptor activity while having no stimulatory effect on another known receptor activity. Therefore, in a particular embodiment, the binding domain of the present invention targets and / or specifically binds to a complex comprising a GPCR, a G protein, and a receptor ligand, wherein the receptor ligand is a biased ligand.

[0136] Furthermore, according to preferred embodiments of the invention, the binding domain of the invention, which targets and / or specifically binds to the GPCR:G protein complex, as described above, is derived from the innate or adaptive immune system. Preferably, the binding domain is derived from an immunoglobulin. Preferably, the binding domain according to the invention is an antibody or antibody fragment. The term "antibody" (Ab) generally refers to a polypeptide encoded by an immunoglobulin gene or a functional fragment thereof, which specifically binds to and recognizes antigens, and is known to those skilled in the art. Conventional immunoglobulin (antibody) structural units comprise tetramers. Each tetramer contains two identical pairs of polypeptide chains, each pair having a "light chain" (about 25 kDa) and a "heavy chain" (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. The term "antibody" is intended to include complete antibodies, including complete single-chain antibodies, and antigen-binding fragments. In some embodiments, the antigen-binding fragment may be an antibody fragment that binds to the antigen, including but not limited to Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (dsFv), and fragments containing VL or VH domains, or fragments composed of VL or VH domains, and any combination thereof, or any other functional portion of an immunoglobulin peptide capable of binding to the target antigen. The term "antibody" is also intended to include heavy-chain antibodies, or functional fragments thereof, such as single-domain antibodies, and more specifically, immunoglobulin single variable domains, such as VHH, or nanobodies, as defined elsewhere herein.

[0137] Preferably, the binding domain of the present invention is an immunoglobulin monovariable domain. More preferably, the binding domain is an immunoglobulin monovariable domain comprising an amino acid sequence containing four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3), preferably according to the following general formula (1):

[0138] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)

[0139] , or any suitable fragment thereof (usually containing at least some amino acid residues that form at least one complementarity-determining region).

[0140] Binding domains containing four FRs and three CDRs are known to those skilled in the art and are described as non-limiting examples in Wesolowski et al. (2009, Med. Microbiol. Immunol. 198:157). Typical but non-limiting examples of immunoglobulin monovariable domains include light chain variable domain sequences (e.g., V) typically derived from conventional four-chain antibodies. L Domain sequence) or heavy-chain variable domain sequence (e.g., V) H (Domain sequence). Preferably, the immunoglobulin monovariable domain is derived from camel antibodies, more preferably from heavy-chain camel antibodies that do not contain light chains, referred to as V. H H domain sequences or nanobodies (as otherwise described herein).

[0141] As used herein, the term "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain (V) derived from naturally occurring heavy chain antibodies. H H), as is known to those skilled in the art. These are derived from heavy-chain-only antibodies found in camels (Hamers-Casterman et al., 1993; Desmyter et al., 1996). Immunoglobulins lacking light-chain polypeptides have been found in the "camel" family. "Camels" include Old World camels (Bactrian camels (Camelus bactrianus) and Dromedary camels (Camelus dromedarius)) and New World camels (e.g., alpacas (Lama paccos), domestic llamas (Lama glama), guanocams (Lama guanicoe), and veal (Lama vicugna)). These single-variable-domain heavy-chain antibodies are referred to herein as nanobodies or V. H H antibody. Nanobody TM and Nanobodies TMNb is a trademark of Ablynx NV (Belgium). Its small size and unique biophysical properties make it superior to conventional antibody fragments in recognizing uncommon or hidden epitopes and in binding to holes or active sites of protein targets. Furthermore, Nb can be designed as multispecific and / or multivalent antibodies or linked to reporter molecules (Conrath et al., 2001). Nb is a stable and rigid single-domain protein that can be readily produced and survives in the gastrointestinal system. Therefore, Nb can be used for a variety of purposes, including drug discovery and therapy (Saerens et al., 2008), and also as a versatile and valuable tool for protein purification, functional studies, and crystallization (Conrath et al., 2009). A specific type of nanobody that functions as a crystallization molecular chaperone binding to conformational epitopes of natural targets is called a Xaperone and is specifically considered in this paper. Xaperone is a unique tool in structural biology. TM It is a trademark of VIB and VUB (Belgium). The main advantages of using camel antibody fragments as a crystallization aid are that Xaperone (1) binds to cryptic epitopes and locks proteins into their unique native conformations, (2) increases the stability of soluble and solubilized membrane proteins, (3) reduces the conformational complexity of soluble and solubilized membrane proteins, (4) increases polar surfaces, enabling the growth of diffractive crystals, (5) isolates aggregated or polymerized surfaces, and (6) allows affinity capture of active proteins.

[0142] Therefore, the immunoglobulin single variable domain of the present invention, and particularly the nanobodies of the present invention, generally comprises a single amino acid chain, which typically contains four "framework sequences" or FRs and three "complementarity-determining regions" or CDRs according to general formula (1):

[0143] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1).

[0144] The term "complementary determinant region" or "CDR" refers to the variable region within an immunoglobulin monovariable domain, containing an amino acid sequence capable of specifically binding to an antigen target. These CDR regions are responsible for the fundamental specificity of nanobodies for a specific antigenic determinant structure. Such regions are also referred to as "hypervariant regions." An immunoglobulin monovariable domain has three CDR regions, which are discontinuous with each other (designated CDR1, CDR2, and CDR3). It should be clarified that the frame regions of an immunoglobulin monovariable domain may also contribute to binding to its antigen (Desmyter et al., 2002; Korotkov et al., 2009). Non-limiting examples of such immunoglobulin monovariable domains according to the invention, as well as specific combinations of FRs and CDRs, are described herein (see Tables 2-3). The description of CDR sequences (and FR sequences) is based on the IMGT's specific numbering system for V-domains and V-like domains (Lefranc et al., 2003). Optionally, FR and CDR sequences can be described using the Kabat numbering system, such as the Camel V used in Riechmann and Muyldermans' (2000) paper. H The system of H domains. As is known to those skilled in the art, a characteristic of immunoglobulin single variable domains, particularly nanobodies, is especially the presence of one or more hallmark residues (according to the Kabat numbering system) in one or more framework sequences, such as those described in Table A-3 on page 75 of WO 08 / 020079 (which is incorporated herein by reference).

[0145] In a preferred embodiment, the present invention provides an immunoglobulin monovariable domain containing an amino acid sequence selected from amino acid sequences consisting essentially of four frame regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3), wherein the CDR sequence of the amino acid sequence has at least 70% amino acid identity with at least one CDR sequence of the immunoglobulin monovariable domain of SEQ ID NO: 1-6, preferably SEQ ID NO: 1 and / or 4 (see Table 3), preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% amino acid identity. It is understood that amino acid residues from frame regions are ignored when determining the degree of amino acid identity of the CDR sequences of one or more sequences of the immunoglobulin monovariable domain. Some preferred but non-limiting examples of the immunoglobulin monovariable domains of the present invention are given in SEQ ID NO: 1-6, preferably SEQ ID NO: 1 and / or SEQ ID NO: 4 (see Table 2).

[0146] It should be noted that the immunoglobulin single variable domain of the present invention, particularly nanobodies, is not limited, in its broadest sense, to a specific biological source or a specific preparation method. For example, the immunoglobulin single variable domain of the present invention, particularly nanobodies, can generally be obtained as follows: (1) isolating the V of naturally occurring heavy chain antibodies. H H domain; (2) Encoding the naturally occurring V H (3) The nucleotide sequence of the H domain; H The H domain is "humanized" or its expression encodes this type of humanized V. H (4) Camelizing a naturally occurring VH domain from any animal species (and especially from mammalian species, such as humans), or expressing a nucleic acid encoding such a camelized VH domain; (5) Camelizing a "domain antibody" or "Dab" as described in the art, or expressing a nucleic acid encoding such a camelized VH domain; (6) Preparing a protein, polypeptide, or other amino acid sequence known per se using synthetic or semi-synthetic techniques; (7) Preparing a nucleic acid encoding a nanobody by re-expressing the obtained nucleic acid using nucleic acid synthesis techniques known per se; and / or (8) Any combination of one or more of the above techniques.

[0147] One preferred class of immunoglobulins with a single variable domain corresponds to the V domain of naturally occurring heavy chain antibodies targeting GPCRs and G protein complexes. H H domain. Although the original library of immunoglobulin monovariable domains ( The library or synthetic library may contain a conformational binder targeting the complex, but a preferred embodiment of the invention includes immunizing camelidae with the target complex, exposing the animal's immune system to a conformational epitope specific to the complex. The animal can be immunized with a mixture of interacting monomers. Optionally, the complex can be stabilized by chemical cross-linking or by adding cooperating / allosteric ligands / metabolites (orthosteric agonists, allosteric activators, Ca2+, etc.). ++ The complex can be stabilized by adding ATP, etc. The complex can also be stabilized by covalently modifying (phosphorylating, etc.) one of the members of the complex. In an alternative embodiment, camels can be immunized with GPCRs and / or G proteins individually (and therefore not complexed with each other). Optionally, the complex can also be stabilized by, for example, adding cooperating / allosteric ligands / metabolites (orthosteric agonists, allosteric activators, Ca2+, etc.) that stabilize the GPCRs and / or G proteins. ++ (e.g., ATP, etc.) to stabilize GPCRs and / or G proteins.

[0148] Therefore, it is generally possible to obtain a suitable biological sample (such as a blood sample or any B cell sample) from the camel using a target complex containing GPCRs and G proteins, or using one or two of its constituent member proteins, an appropriate immunized camel species (i.e., a heavy chain antibody that enhances the immune response and / or targets the target complex), and to generate a V antibody against the target complex from the sample using any suitable technique known per se. H H sequences are used to generate or obtain this type of V. H H sequences. This type of technique is obvious to those skilled in the art. Optionally, it can be derived from Camel V... H This type of naturally occurring V was obtained from the original library of the H sequence. H The H domain, for example, allows screening of such libraries using one or more screening techniques known per se, with the target complex or at least one portion, fragment, antigenic determinant, or epitope thereof. Such libraries and techniques are described, for example, in WO9937681, WO0190190, WO03025020, and WO03035694. Optionally, libraries derived from the original V domain can be used. H Modified synthetic or semi-synthetic libraries of H libraries, such as those derived from the original V library through techniques such as random mutagenesis and / or CDR shuffling. H V obtained from H library H H library, as described in WO0043507. Used to obtain V against the target. H Another technique for H sequences involves using any suitable technique known per se, appropriate immunization of a transgenic mammal capable of expressing heavy chain antibodies (i.e., thereby enhancing the immune response against the target and / or the heavy chain antibody), obtaining a suitable biological sample (such as a blood sample, or any B cell sample) from said transgenic mammal, and then generating a V-sequence against the target starting from said sample. H H sequence. For example, mice expressing heavy chain antibodies, and other methods and techniques described in WO02085945 and WO04049794 can be used for this purpose.

[0149] The particularly preferred types of immunoglobulin monovariable domains of the present invention, especially the particularly preferred types of nanobodies of the present invention, include those having the same domain as naturally occurring V. H The amino acid sequence of the H domain corresponds to the immunoglobulin monovariable domain, but the amino acid sequence has been "humanized," that is, one or more amino acid residues at the corresponding positions of the VH domain of conventional human 4-chain antibodies have replaced the naturally occurring V H domain. HOne or more amino acid residues in the amino acid sequence of the H sequence (particularly in the framework sequence). This can be implemented in ways known per se, as will be apparent to those skilled in the art, for example, based on further descriptions herein and prior art regarding the humanization effects described herein. Furthermore, it should be noted that such humanized immunoglobulin single variable domains of the present invention can be obtained by any suitable means known per se (i.e., as shown in points (1)-(8) above), and therefore are not strictly limited to the use of naturally occurring V... H Peptides obtained using H-domain peptides as starting materials. Humanized immunoglobulin monovariable domains, especially nanobodies, can have several advantages, such as being superior to their corresponding naturally occurring V-domain counterparts. H Reduced immunogenicity due to the H domain. This type of humanization generally involves replacing naturally occurring V H domain residues with amino acid residues present at the same position in the human VH domain (such as the human VH3 domain). H One or more amino acid residues in the H sequence. Such humanization substitutions should be selected so that the resulting humanized immunoglobulin monovariable domain retains the advantageous properties of an immunoglobulin monovariable domain as defined herein. Those skilled in the art can select suitable humanization substitutions or combinations thereof that combine the advantageous properties provided by the humanization substitution with those of the naturally occurring V... H The optimal balance between the advantageous properties of the H-domain is achieved, or an ideal or suitable balance is realized.

[0150] The immunoglobulin monovariable domain of the present invention, particularly another particularly preferred type of the nanobody of the present invention, includes an immunoglobulin monovariable domain having an amino acid sequence that corresponds to the amino acid sequence of the naturally occurring VH domain, but has been "camelized," i.e., replaced with the VH domain present in heavy chain antibodies. H One or more amino acid residues at the corresponding position of the H domain replace one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain of a conventional 4-chain antibody. This “camelization” substitution is preferably inserted into amino acid positions forming the VH-VL interface and / or present in the VH-VL interface, and / or at amino acid positions of so-called camelid-like imprinted residues, as defined herein (see, for example, WO9404678). Preferably, the VH sequence used as the starting material or starting point for generating or designing camelized nanobodies is a mammalian VH sequence, more preferably a human VH sequence, such as the VH3 sequence. However, it should be noted that this type of camelized immunoglobulin single variable domain of the present invention can be obtained by any suitable means known per se (i.e., as shown in points (1)-(8) above), and is therefore not strictly limited to peptides obtained using peptides containing naturally occurring VH domains as starting materials.

[0151] For example, "humanization" and "camelization" can be implemented as follows: providing the encoding of the naturally occurring V... H The nucleotide sequence of the H domain or VH domain is taken, and then one or more codons in the nucleotide sequence are changed in a manner known per se, such that the new nucleotide sequence encodes the "humanized" or "camelized" immunoglobulin single variable domain of the present invention, respectively. The nucleic acid can then be expressed in a manner known per se, thereby providing the desired immunoglobulin single variable domain of the present invention. Optionally, based on naturally occurring VH domains, the nucleotide sequence is modified to encode the "humanized" or "camelized" immunoglobulin single variable domain of the present invention, respectively. H The amino acid sequences of the H domain or VH domain can be used to design the ideal humanized or camel-like immunoglobulin monovariable domain amino acid sequences of this invention, and then they can be synthesized de novo using peptide synthesis techniques known per se. Alternatively, they can be based on naturally occurring VH domains. H The nucleotide sequence encoding the ideal humanized or camel-like immunoglobulin single variable domain of the present invention is designed from the amino acid or nucleotide sequence of the H domain or VH domain, and then said nucleotide sequence is synthesized de novo using nucleic acid synthesis techniques known per se. The resulting nucleic acid is then expressed in a manner known per se, thereby providing the ideal immunoglobulin single variable domain of the present invention. This is for use from naturally occurring VH sequences, or preferably from VH domains. H Other suitable methods and techniques for obtaining the immunoglobulin monovariable domain and / or its encoded nucleic acid of the present invention via the H sequence will be apparent to those skilled in the art. These methods may, for example, include combining one or more portions of one or more naturally occurring VH sequences (such as one or more FR sequences and / or CDR sequences) in a suitable manner. H One or more portions of an H sequence (such as one or more FR sequences and / or CDR sequences), and / or one or more synthetic or semi-synthetic sequences, thereby providing the ideal nanobody of the present invention or its encoded nucleotide sequence or nucleic acid.

[0152] Also within the scope of this invention are natural or synthetic analogs, mutants, variants, alleles, portions or fragments (collectively referred to herein as “variants”) of the immunoglobulin monovariant domains of the invention as defined herein, particularly nanobodies, especially variants of the immunoglobulin monovariant domains of SEQ ID NO: 1-6 (see Tables 2-3). Therefore, according to one embodiment of the invention, the terms “immunoglobulin monovariant domain of the invention” or “nanobodies of the invention” also cover the aforementioned variants in their broadest sense. Generally, these variants have substituted, deleted, and / or added one or more amino acid residues compared to the immunoglobulin monovariant domains of the invention as defined herein. Such substitutions, insertions, or deletions may be made in one or more FRs and / or one or more CDRs, particularly variants of the FRs and CDRs of the immunoglobulin monovariant domains of SEQ ID NO: 1-6 (see Tables 2-3). As used herein, variants are sequences in which each or any frame region, each or any complementarity-determining region, exhibits at least 80% identity with the corresponding region of the reference sequence, preferably at least 85% identity, more preferably 90% identity, even more preferably 95% identity, or even more preferably 99% identity (i.e., FR1 variant with FR1 reference, CDR1 variant with CDR1 reference, FR2 variant with FR2 reference, CDR2 variant with CDR2 reference, FR3 variant with FR3 reference, CDR3 variant with CDR3 reference, FR4 variant with FR4 reference), as can be measured using algorithms such as PILEUP and BLAST electrons (50, 51). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI). http: / / www / ncbi.nlm.nih.gov / It is understood that, in order to determine the degree of amino acid identity of the CDR sequence of one or more sequences of an immunoglobulin monovariable domain, amino acid residues forming the frame region are ignored. Similarly, in order to determine the degree of amino acid identity of the FR sequence of one or more sequences of the immunoglobulin monovariable domain of the present invention, amino acid residues forming the complementary region are ignored. Such variants of the immunoglobulin monovariable domain can be particularly advantageous because they have improved titer / affinity.

[0153] As a non-limiting example, substitution can be, for example, a conserved substitution (as described herein) and / or an amino acid residue naturally present in another V. HA substitution of another amino acid residue at the same position in the H domain. Therefore, the scope of this invention includes any one or more such substitutions, deletions, or insertions, or any combination thereof, that improve the properties of the immunoglobulin monovariable domain of the invention, or at least does not excessively diminish the desired properties or balance or combination of desired properties of the nanobody of the invention (i.e., the immunoglobulin monovariable domain is no longer suitable for its intended use). Those skilled in the art can generally determine and select suitable substitutions, deletions, or insertions, or suitable combinations thereof, based on the disclosure herein, optionally after a limited number of routine experiments, which may involve, for example, introducing a limited number of possible substitutions and determining their effect on the properties of the obtained immunoglobulin monovariable domain.

[0154] According to a particularly preferred embodiment, the immunoglobulin single variable domain of the present invention, particularly the variant of the nanobody, may have a substitution, deletion or insertion of 1, 2 or 3 amino acids in 1, 2 or 3 CDRs, more specifically in (i) CDR1 or CDR2 or CDR3; (ii) CDR1 and CDR2, or CDR1 and CDR3, or CDR2 and CDR3; (iii) CDR1 and CDR2 and CDR3 having a substitution, deletion or insertion of 1, 2 or 3 amino acids, the amino acid sequences of which are listed in Table 3. More preferably, the immunoglobulin single variable domain of the present invention, particularly the variant of the nanobody, may have a conserved substitution of 1, 2 or 3 amino acids in 1, 2 or 3 CDRs (as defined herein), more specifically in the amino acid sequences of the CDRs listed in Table 3, that are (i) CDR1 or CDR2 or CDR3; (ii) CDR1 and CDR2, or CDR1 and CDR3, or CDR2 and CDR3; and (iii) CDR1 and CDR2 and CDR3, that are conserved substitutions of 1, 2 or 3 amino acids.

[0155] According to a specific embodiment, the invention provides an immunoglobulin single variable domain comprising an amino acid sequence comprising four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3), according to the following general formula (1):

[0156] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1);

[0157] And CDR1 is selected from:

[0158] a)SEQ ID NO:13-18,

[0159] b) A polypeptide having at least 80% amino acid identity with SEQ ID NO:13-18,

[0160] c) A polypeptide that differs from SEQ ID NO:13-18 by 3, 2, or 1 amino acid.

[0161] And CDR2 is selected from:

[0162] a)SEQ ID NO:25-30,

[0163] b) A polypeptide having at least 80% amino acid identity with SEQ ID NO:25-30,

[0164] c) A polypeptide that differs from SEQ ID NO:25-30 by 3, 2, or 1 amino acid.

[0165] And CDR3 is selected from:

[0166] a)SEQ ID NO:37-42,

[0167] b) A polypeptide having at least 80% amino acid identity with SEQ ID NO:37-42,

[0168] c) A polypeptide that differs from SEQ ID NO:37-42 by 3, 2 or 1 amino acid.

[0169] In a particularly preferred embodiment, the present invention provides an immunoglobulin single variable domain comprising an amino acid sequence comprising four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3), according to the following general formula (1):

[0170] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1):

[0171] CDR1 is SEQ ID NO: 13; CDR2 is SEQ ID NO: 25; and CDR3 is SEQ ID NO: 37.

[0172] Furthermore, depending on the host organism used to express the binding domains of the present invention, particularly the immunoglobulin monovariable domain, deletions and / or substitutions can be designed to remove one or more sites for post-translational modification (such as one or more glycosylation sites), which is within the capabilities of those skilled in the art. Alternatively, substitutions or insertions can be designed to introduce one or more sites for linking functional groups, such as sites that allow site-specific polyethylene glycol esterification (as described herein).

[0173] Examples of modifications, and examples of amino acid residues that can be modified in the binding domains of the present invention, particularly immunoglobulin monovariable domains (i.e., on the protein backbone, preferably on the side chains), methods and techniques for introducing such modifications, and the potential uses and advantages of such modifications, will be apparent to those skilled in the art. For example, such modifications may involve introducing (e.g., by covalent linking or another suitable method) one or more functional groups, residues, or portions into or onto the binding domains of the present invention, particularly immunoglobulin monovariable domains, particularly conferring one or more desired properties or functions upon the binding domains of the present invention. Examples of such functional groups and techniques for introducing them will be apparent to those skilled in the art, and generally may include all functional groups and techniques mentioned in the art, as well as functional groups and techniques known per se for modifying pharmaceutical proteins, particularly for modifying antibodies or antibody fragments (including ScFv and monodomain antibodies), for example, see Remington's Pharmaceutical Sciences, 16th edition, Mack Publishing Co., Easton, PA (1980). Such functional groups can be directly (e.g., covalently) linked to, for example, the binding domains of the present invention, particularly the immunoglobulin monovariable domains, or optionally linked via suitable linkers or spacers, as will be apparent to those skilled in the art. One of the most commonly used techniques for increasing the half-life of pharmaceutical proteins and / or reducing their immunogenicity involves linking suitable pharmaceutically acceptable polymers, such as poly(ethylene glycol) (PEG) or its derivatives (e.g., methoxylated poly(ethylene glycol) or mPEG). Generally, any suitable form of PEGylation can be used, such as that used in the art for the PEGylation of antibodies and antibody fragments (including, but not limited to, (single)domain antibodies and ScFv); see, for example, Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug. Deliv. Rev. 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and WO04060965. Various reagents for the PEGylation of proteins are also commercially available, for example from Nektar Therapeutics, USA. Site-directed PEGylation is preferred, particularly via cysteine ​​residues (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003)).For example, for this purpose, PEG can be linked to cysteine ​​residues naturally present in the immunoglobulin monovariable domain of the present invention, the immunoglobulin monovariable domain of the present invention can be modified to appropriately introduce one or more cysteine ​​residues for linking PEG, or an amino acid sequence containing one or more cysteine ​​residues for linking PEG can be fused to the N- and / or C-terminus of the nanobody of the present invention, all of which use protein modification techniques known to those skilled in the art. Preferably, for the immunoglobulin monovariable domain of the present invention, PEG with a molecular weight greater than 5,000, such as greater than 10,000 and less than 200,000, such as less than 100,000, can be used; for example, the molecular weight range is 20,000-80,000. In addition, modifications that are generally less preferred include N-linked or O-linked glycosylation, usually as part of co-translational and / or post-translational modifications, depending on the host cell used to express the binding domain of the present invention, particularly the immunoglobulin monovariable domain. Another technique for increasing the half-life of the binding domain includes modifying it into a bifunctional binding domain (e.g., an immunoglobulin monovariable domain targeting the GPCR:G protein complex and another targeting a serum protein such as albumin), or modifying it into a binding domain, particularly a fusion of an immunoglobulin monovariable domain with a peptide (e.g., a peptide targeting a serum protein such as albumin).

[0174] Another modification may include the introduction of one or more detectable tags or other signal-generating groups or portions, depending on the intended use of the labeled binding domain. Suitable tagging and techniques for attaching, using, and detecting the tags are apparent to those skilled in the art, including, but not limited to, fluorescent tags (such as fluorescein, isothiocyanates, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phenylenedialdehyde, and fluorescein, and fluorescent metals such as Eu, or other lanthanide metals), phosphorescent tags, chemiluminescent tags, or bioluminescent tags (such as luminol, isoaminobenzoic acid hydrazide, theromatic acridinium). Ester, imidazole, acridine salts, oxalates, dioxane, or GFP, and their analogues), radioisotopes, metals, metal chelates, or metal cations, or other metals or metal cations particularly suitable for in vivo, in vitro, or in situ diagnostic and imaging purposes, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, Δ-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, biotin-avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucosyl amylase, and acetylcholinesterase). Other suitable labeling will be apparent to those skilled in the art, including, for example, portions detectable by NMR or ESR spectroscopy. The binding domains of this invention, labeled in this way, can be used for, for example, in vitro, in vivo, or in situ assays (including immunoassays known per se, such as ELISA, RIA, EIA, and other “sandwich assays”) and in vivo diagnostic and imaging purposes, depending on the choice of specific label. It will be apparent to those skilled in the art that another modification may involve the introduction of a chelating group, such as chelating one or more of the aforementioned metals or metal cations. Suitable chelating groups include, for example, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Another modification may include the introduction of a functional group as part of a specific binding pair, such as a biotin-(streptomycin)antibiotin binding pair. Such functional groups can be used to link the binding domains of this invention to another protein, polypeptide, or chemical compound that binds to the other half of the binding pair, i.e., by forming a binding pair. For example, the immunoglobulin monovariable domain of this invention can be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier conjugated to avidin or streptomycin. For example, such conjugated immunoglobulin monovariable domains can be used as reporters, for instance, in diagnostic systems that conjugate a detectable signal generator to avidin or streptoavidin. These binding pairs can also be used, for example, to bind the immunoglobulin monovariable domains of the present invention to a carrier, including carriers suitable for pharmaceutical purposes.A non-limiting example is the liposomal formulation described by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs can also be used to connect therapeutically active agents to the binding domains of this invention.

[0175] The scope of this invention also includes the binding domains of the invention in a "multivalent" form, particularly the immunoglobulin monovariable domains of the invention, which are formed by bonding two or more monovalent immunoglobulin monovariable domains together using chemical or recombinant DNA techniques. Non-limiting examples of multivalent constructs include "bivalent," "trivalent," and "tetravalent" constructs. The immunoglobulin monovariable domains contained in the multivalent construct can be the same or different. In another specific embodiment, the immunoglobulin monovariable domains of the invention are in a "multispecific" form, formed by bonding two or more immunoglobulin monovariable domains together, at least one of which has a different specificity. Non-limiting examples of multispecific constructs include "bispecific," "trispecific," and "tetraspecific" constructs. For further example, any multivalent or multispecific (as defined herein) immunoglobulin monovariable domain of the present invention may suitably target two or more different epitopes on the same antigen, for example, two or more different portions of the G protein contained in a GPCR:G protein complex; or may target two or more different antigens, for example, an epitope of the GPCR and an epitope of the G protein. In particular, the monovalent immunoglobulin monovariable domain of the present invention is such that it binds to the target GPCR:G protein complex (as described herein) with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The multivalent or multispecific immunoglobulin monovariable domain of the present invention may also have (or be modified and / or selected to) increased affinity and / or improved selectivity for the desired GPCR:G protein complex, and / or any other desirable properties or combinations of desirable properties obtainable by using such multivalent or multispecific immunoglobulin monovariable domains.

[0176] Furthermore, the binding domains of the present invention, particularly the immunoglobulin monovariable domains of the present invention, can generally target or specifically bind any conformational epitope presented by, contacted by, or part of a complex comprising a GPCR and a G protein. Binding domains that specifically bind to “three-dimensional” or “conformational” epitopes are binding domains that specifically bind to the tertiary or quaternary structures of folded proteins or protein complexes. These binding domains bind linear (i.e., unfolded, denatured) polypeptide chains with significantly reduced (i.e., reduced to at least 1 / 2, 5, 10, 50, or 100th) affinity. The structures bound by such binding domains typically contain amino acids that are discontinuous in the linear sequence of the protein (complex). In other words, the binding of such binding domains to polypeptides depends on the polypeptide folding into a specific three-dimensional conformation. It should be clearly understood that the conformational epitopes selectively recognized by the binding domain of the present invention can be GPCR-specific epitopes, or G protein-specific epitopes, or GPCR:G protein complex-specific epitopes, which are formed only when associated with the component proteins and thus by combining the amino acid residues of the GPCR and the G protein. In one embodiment, the binding domain of the present invention, particularly the immunoglobulin monovariable domain, specifically binds to any conformational epitope of any desired G protein or a portion thereof. In another embodiment, the conformational epitope can be a portion of the intracellular or extracellular region of any desired GPCR, or an intramembrane region, or a domain or loop structure. It is evident that in non-associated GPCRs and / or G proteins, some of these conformational epitopes are accessible, while others are accessible only when forming a complex. According to a particular embodiment, the binding domain of the present invention, particularly the immunoglobulin monovariable domain, specifically binds to conformational epitopes located at the interface between the α and β subunits of a G protein, as described elsewhere in this document as non-limiting examples (see the Examples section).

[0177] According to other specific embodiments, the binding domain binds to GPCR:G protein complexes in which the G protein is in its nucleotide-free form. According to other specific embodiments, the binding domain of the present invention, particularly the immunoglobulin monovariable domain, inhibits or prevents the dissociation of the GPCR:G protein complex in the presence of nucleotides (particularly guanine nucleotides (such as GDP or GTP), or analogs thereof, such as non-hydrolyzable GTP analogs (such as GTPγS)), or GDP in combination with aluminum fluoride or beryllium fluoride, or in the presence of minimal nucleotide analogs (such as pyrophosphate or forcarnet). In the absence of the binding domain of the present invention, the GPCR:G protein complex typically dissociates in the presence of these nucleotides.

[0178] According to one particular aspect, the present invention also provides binding domains that target or specifically bind to G proteins (i.e., G proteins only, not complexed with GPCRs). In a particular embodiment, the binding domains described herein target and / or specifically bind to Gs proteins. According to a particular embodiment of this aspect, the binding domains of the present invention prevent or inhibit the binding of nucleotides (particularly guanine nucleotides or analogs (as described above)) to G proteins. Alternatively, the binding domains of the present invention may also substitute for guanine nucleotides or analogs in G proteins. Non-limiting examples of assays for determining the degree of inhibition of guanine nucleotide binding to proteins or the degree of substitution of guanine nucleotides for proteins are provided in the Example section, such as Examples 3 and 8. Furthermore, it is understood that all specific embodiments involving the binding domains of the present invention (as described above) are also applicable to this particular aspect of the invention.

[0179] The functional diversity of GPCRs is intrinsically linked to the flexibility of these proteins that contribute to their conformational profiles. The conformational energy landscape is intrinsically related to factors such as the presence of binding ligands (effecton molecules, agonists, antagonists, inverse agonists, etc.), lipid environments, or binding interactions with proteins. Therefore, in one embodiment, the binding domain of the present invention, particularly the immunoglobulin monovariable domain, increases the stability of the complex containing the GPCR and G protein when binding the binding domain. In other embodiments, the binding domain of the present invention binds any conformational epitope that induces the formation of a functional conformational state of the GPCR, particularly the active conformational state of the GPCR, which is presented by, is accessible to, or is part of the GPCR:G protein complex. More specifically, the binding domain of the present invention can stabilize the active state of the GPCR contained in the GPCR:G protein complex by increasing the affinity of the G protein for the receptor. Similarly, the binding domain of the present invention can stabilize the GPCR:G protein complex bound to an agonist and / or enhance the affinity of the agonist for the GPCR:G protein complex. Preferably, via K... d The reduction in the measured binding domain can increase the affinity of the G protein for the GPCR, and / or the affinity of the agonist for the GPCR:G protein complex by at least 2-fold, at least 5-fold, more preferably at least 10-fold. Optionally, in any assay setting comparing the strength of receptor-G protein interaction under conditions of presence of a stable binding domain versus absence of the binding domain, or in any other affinity or titer measurement known to those skilled in the art, the binding domain can induce EC. 50 or IC 50 The change is at least 2 times, at least 5 times, and more preferably at least 10 times.

[0180] As used herein, the term "functional conformational state" refers to the fact that proteins, particularly membrane proteins (such as GPCRs), possess a variety of conformational states with a dynamic range of activity, specifically from inactive to maximally active (see the review by Kobilka and Deupi, 2007). It should be clarified that "functional conformational state" is not intended to cover the denatured state of a protein. For example, the basal conformational state can be defined as the low-energy state of the receptor in the absence of a ligand. The probability of a protein undergoing a transition to another conformational state is a function of the energy difference between the two states and the height of the energy barrier between them. In the case of receptor proteins (such as GPCRs), the energy of ligand binding can be used to alter the energy barrier between the two states, or to alter the relative energy levels between the two states, or both. Changes in the energy barrier affect the rate of transition between the two states, while changes in energy levels affect the equilibrium distribution of the receptor between the two states. Binding an agonist or partial agonist can lower the energy barrier and / or lower the energy of the more active conformational state relative to the inactive conformational state. Reverse agonists can increase the energy barrier and / or decrease the energy of the inactive conformation state relative to the active conformation state. Coupled receptors and their G proteins can further alter the energy landscape. The cooperative interaction between β2AR and Gs observed in ligand binding assays forms the basis of a ternary complex model of GPCR activation (Delean et al., 1980). In this ternary complex consisting of an agonist, receptor, and G protein, the receptor exhibits enhanced affinity for the agonist, and the G protein shows a specific alteration in its affinity for guanine nucleotides, favoring GTP over GDP.

[0181] It should be noted that the activity of integrated membrane proteins (including GPCRs) is also influenced by the structure of the lipid molecules surrounding them in the membrane. Membrane proteins are not rigid entities but deformable to ensure a good hydrophobic match with the surrounding lipid bilayer. An important parameter is the hydrophobic thickness of the lipid bilayer, defined by the length of the fatty acyl chains of the lipids. Furthermore, the structure of the lipid head group regions can also be important in defining the structure of those portions of the membrane protein located in the lipid head group regions. Among other lipids, palmitoylation and GPCR binding of cholesterol also play a structural role in monomeric receptors and contribute to the formation / stabilization of receptor oligomers (Lee 2004; Chini and Parenti 2009).

[0182] Another aspect of the invention relates to complexes comprising the binding domain of the invention. More specifically, complexes comprising the binding domain of the invention, a GPCR, a G protein, and optionally a receptor ligand are provided. As a non-limiting example, stable complexes can be purified by gel filtration, as is done for quaternary complexes containing nanobodies, GPCRs, G proteins, and receptor ligands (see the Examples section). In certain embodiments, the complex may be crystalline. Accordingly, crystals of the complex and methods for preparing said crystals are also provided, described in more detail elsewhere herein.

[0183] In another aspect, nucleic acid sequences encoding the amino acid sequences of any binding domain of the present invention, particularly immunoglobulin monovariable domains, are also part of the present invention, with non-limiting examples provided in Table 4. According to a preferred embodiment, the present invention relates to nucleic acid sequences of the binding domains of the present invention, particularly immunoglobulin monovariable domains, wherein the sequence has greater than 80%, preferably greater than 90%, more preferably greater than 95%, such as 99% or higher sequence identity (as defined herein) with at least one nucleic acid sequence of the binding domain of SEQ ID NO:49-54 (see Table 4). To calculate the percentage sequence identity, the nucleic acid sequence of the tag (e.g., the His tag or the EPEA tag) should be ignored. Furthermore, the nucleic acid sequence may include the nucleic acid sequences described herein.

[0184] Furthermore, the present invention also considers expression vectors comprising nucleic acid sequences encoding any binding domain of the present invention, particularly immunoglobulin monovariable domains, and host cells expressing such expression vectors. Suitable expression systems include constitutive and inducible expression systems of bacteria or yeast, viral expression systems (such as baculoviruses, Semliki Forest viruses, and lentiviruses), or transient transfection in insect or mammalian cells. The binding domains of the present invention, particularly immunoglobulin monovariable domains, can be cloned, expressed, and / or purified according to techniques known to those skilled in the art.

[0185] Therefore, this invention encompasses cells or cell cultures expressing the binding domains of the invention, particularly immunoglobulin monovariable domains, said domains targeting and / or capable of specifically binding to complexes comprising GPCRs and G proteins. Cells according to the invention can be any prokaryotic or eukaryotic organism. Preferably, the cells are eukaryotic cells endogenously or recombinantly expressing the target GPCR and / or G protein, such as yeast or insect cells, or cultured cell lines, such as mammalian cell lines, preferably human cell lines. The nature of the cells used generally depends on the ease and cost of producing the native protein, the desired glycosylation characteristics, the source of the target protein, the intended use, or any combination thereof. Eukaryotic cells or cell lines used for protein production are generally known in the art, including cell lines with modified glycosylation pathways, and non-limiting examples are provided below.

[0186] Animal or mammalian host cells suitable for accommodating, expressing, and producing proteins for subsequent isolation and / or purification include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; and Kolkekar et al., 1997, Biochemistry, 36:10901-10909), CHO-K1Tet-On cell line (Clontech), CHO (CAMR, Salisbury, Wiltshire, UK) designated ECACC 85050302, CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1 / SF (CAMR, Salisbury, Wiltshire, UK) designated ECACC 93061607, and CHO (ECACC 93061607). RR-CHOK1 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase-negative CHO cells (CHO / -DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (US Patent No. 5,721,121): monkey kidney CV1 cells transformed with SV40 (COS cells, COS-7, ATCC CRL-1651): human embryonic kidney cells (e.g., 293 cells or 293T cells, or subclonal 293 cells for suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59, or GnTI KO HEK293S cells, Reeves et al., 2002, PNAS, 99:13419): juvenile hamster kidney cells (BHK, ATCC). CCL-10): Monkey kidney cells (CV1, ATCC CCL-70): African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81): Mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod.)The following cells were selected for further analysis: human cervical cancer cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatocellular carcinoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT060562, ATCC CCL-51); buffalo rat hepatocytes (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad. Sci., 383:44-68); MCR 5 cells; FS4 cells. According to a specific implementation scheme, the cells are mammalian cells selected from Hek293 cells or COS cells.

[0187] Exemplary non-mammalian cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems (e.g., review Jarvis, Virology, Vol. 310, No. 1, May 25, 2003, pp. 1-7), plant cells such as tobacco cells, tomato cells, maize cells, algal cells, or yeast, such as species of the genera *Saccharomyces*, *Schizosaccharomyces*, *Hansenula*, *Yarrowia*, or *Pichia*. According to a specific implementation, the eukaryotic cell is a yeast cell derived from a species of the genus *Saccharomyces cerevisiae*, a species of the genus *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), a species of the genus *Hansenula* (e.g., *Hansenula polymorpha*), a species of the genus *Yarrowia* (e.g., *Yarrowia lipolytica*), a species of the genus *Kluyveromyces* (e.g., *Kluyveromyces lactis*), a species of the genus *Pichia* (e.g., *Pichia pastoris*), or a species of *Komagataella* (e.g., *Komagataella pastoris*). According to a specific implementation, the eukaryotic cell is a *Pichia* cell, and in the most specific implementation, it is a *Pichia pastoris* cell.

[0188] Target cells (e.g., mammalian cells) can be transfected according to the principles summarized by Sambrook and Russell (Molecular Cloning, A Laboratory Manual, 3rd Edition, Volume 3, Chapter 16, Sections 16.1–16.54). Alternatively, viral transduction can be performed using reagents such as adenovirus vectors. Selecting an appropriate viral vector system, regulatory region, and host cell is common knowledge to those skilled in the art. According to standard practice, the transfected cells are kept in culture and frozen for later use.

[0189] Accordingly, another aspect of the invention relates to a method for producing a bonding domain according to the invention, the method comprising at least the steps of:

[0190] a) Expressing the nucleic acid according to the invention in a suitable cell expression system (as defined above), and optionally...

[0191] b) Isolate and / or purify the binding domain.

[0192] The binding domains, complexes, cells, or cell lines described herein can be used in a variety of environments and applications, such as, but not limited to, for capturing and / or purifying GPCR:G protein complexes, and for crystallization studies and high-resolution structural analysis of GPCR:G protein complexes. Therefore, one object of the present invention is to utilize the binding domains according to the invention, particularly immunoglobulin monovariable domains (such as nanobodies), as tools for stabilizing GPCR:G protein complexes, and further relates to utilizing these binding domains as aids in the co-crystallization of GPCRs complexed with G proteins, or in other words, to promote the crystallization of GPCR:G protein complexes. Additional and / or optional binding domains and preferred cell systems expressing the binding domains (as described herein) can be used for other purposes, such as ligand screening, drug discovery, and immunology, which will be fully described elsewhere below.

[0193] Stabilizing GPCRs: G protein complexes and locking GPCRs in a G protein-bound state.

[0194] Therefore, according to one aspect, the present invention relates to the use of the aforementioned binding domain for stabilizing complexes comprising GPCRs and G proteins. According to a preferred embodiment, the stabilized complex further comprises a receptor ligand, more specifically an agonist. As used herein, the term “stabilize” or “increase stability” refers to increasing the stability of a GPCR:G protein complex in terms of the structure (conformational state) and / or specific biological activity (intracellular signaling activity) of one or both constituent proteins (particularly GPCRs and / or G proteins). In a particularly preferred embodiment, the binding domain of the present invention can be used to stabilize a GPCR:G protein complex, causing the GPCR to be locked or immobilized in an active or G protein-binding state. A GPCR in such an active or G protein-binding state will perform its native biological activity. Methods for determining (increased) stability of GPCR:G protein complexes have been described above, and further examples will be given in the Examples section.

[0195] It is understood that increased stability in terms of the structure and / or specific biological activity of GPCRs includes stability against other denaturing agents or denaturing conditions, including heat, detergents, dissociation agents, and extreme pH. Accordingly, in other embodiments, the binding domain according to the invention can increase the stability of the GPCR:G protein complex under non-physiological conditions induced by dilution, concentration, buffer composition, heating, cooling, freezing, detergents, dissociation agents, pH, etc. Accordingly, the terms "thermal stability" and "increased thermal stability" refer to the functional properties of the GPCR:G protein complex rather than its thermodynamic properties, and refer to the resistance of the constituent protein to irreversible denaturation induced by heat and / or chemical means, including but not limited to heating, cooling, freezing, chemical denaturants, pH, detergents, salts, additives, proteases, or temperature. Irreversible denaturation leads to irreversible unfolding of the protein's functional conformation, loss of biological activity, and aggregation of denatured proteins. As used herein, the terms “(thermal) stable” and “increased (thermal) stability” apply to GPCR:G protein complexes embedded in lipid particles or lipid layers (e.g., lipid monolayers, lipid bilayers, etc.) and to GPCR:G protein complexes dissolved in detergents.

[0196] Increased thermal stability can be conveniently determined by measuring ligand binding or using spectroscopic methods (such as fluorescence, CD, or light scattering sensitive to unfolding at elevated temperatures). Preferably, the binding domain increases stability, as measured by an increase in thermal stability of the GPCR:G protein complex of at least 2°C, at least 5°C, at least 8°C, and more preferably at least 10°C, 15°C, or 20°C. According to another preferred embodiment, the binding domain increases the thermal stability of GPCR:G protein complexes containing receptor ligands (more specifically, agonists or orthoallosteric regulators of GPCR-dependent signaling pathways). According to another preferred embodiment, the binding domain of the present invention increases the stability of the GPCR:G protein complex in the presence of detergents or liquid release agents. Preferably, the binding domain increases the stability of the GPCR:G protein complex to denaturation induced by heat or chemical methods. For increased stability to heat, detergents, or clinker, the GPCR:G protein is typically incubated for a defined time in the presence of a test detergent or clinker, and stability is determined using, for example, ligand binding or spectroscopic methods, optionally at increased temperatures, as discussed above. According to yet another preferred embodiment, the binding domain according to the invention is capable of increasing the stability of the functional conformational state of the GPCR to extreme pH values. Preferably, the binding domain is capable of increasing the stability of the GPCR:G protein complex to extreme pH values. For extreme pH values, typical test pH values ​​can be selected, for example, in the ranges of 6 to 8, 5.5 to 8.5, 5 to 9, 4.5 to 9.5, more specifically in the range of 4.5 to 5.5 (low pH) or 8.5 to 9.5 (high pH).

[0197] In a particularly preferred embodiment, the binding domain according to the invention can be used to prevent the complex from dissociating in the presence of nucleotides, particularly guanine nucleotides or analogues thereof. More specifically, guanine nucleotides include GDP and GTP, and analogues of guanine nucleotides include, but are not limited to, combinations of GTPγS or GDP with aluminum fluoride or beryllium fluoride, or nucleotide fragments (such as pyrophosphate or phosphonoformate).

[0198] Capture and / or purify GPCR:G protein complex

[0199] Understandably, the ability to form stable GPCR:G protein complexes is particularly effective for capturing and / or purifying GPCR:G protein complexes, allowing for subsequent crystallization, ligand characterization and compound screening, immunization, etc. Furthermore, a particular advantage is that the binding domains of this invention can be a universally effective tool applicable to a range of GPCR:G protein complexes.

[0200] Accordingly, the present invention also considers methods for capturing and / or purifying complexes containing GPCRs and G proteins, the methods comprising the steps of:

[0201] a) Provides a combined structural domain according to the invention, and

[0202] b) Allows the binding domain to bind to a complex containing GPCRs and G proteins, and

[0203] c) The complex formed in the optional separation step b).

[0204] In a particular embodiment, the present invention provides a method for capturing a complex comprising GPCRs and G proteins, the method comprising the steps of:

[0205] a) A solution containing multiple GPCRs and G proteins is used on a solid support having immobilized binding domains according to the invention, and

[0206] b) Formation of a complex of binding domains, GPCRs, and G proteins, and

[0207] c) Remove weakly bound or unbound molecules.

[0208] The present invention also considers a method for purifying a complex containing GPCRs and G proteins, the method comprising the steps of:

[0209] a) Contact a solution containing GPCRs and G proteins with the binding domains according to the invention, and

[0210] b) Formation of a complex containing a binding domain, GPCR, and G protein, and

[0211] c) Separate the complex from step b).

[0212] The complex of GPCR and G protein was basically purified.

[0213] According to a specific implementation, the binding domain described herein can also be used to capture target GPCR:G protein complexes that further contain receptor ligands and / or one or more other interacting proteins.

[0214] The methods described above for capturing / purifying target GPCR:G protein complexes include, but are not limited to, affinity-based methods such as affinity chromatography, affinity purification, immunoprecipitation, protein detection, immunohistochemistry, and surface display, which are commonly known in the art.

[0215] Crystallization and analysis of the structure of GPCR:G protein complex

[0216] Crystallization of membrane proteins, including GPCRs, remains a formidable challenge. While methods have emerged that allow for the production of milligram-scale expression and purification, achieving stability in these molecules is perhaps the most difficult obstacle to overcome. Firstly, the binding domains according to the invention can increase the stability of detergent-soluble GPCR:G protein complexes, protecting them from degradation and / or aggregation by proteolytic hydrolysis, and facilitating the purification and concentration of homogeneous samples of properly folded proteins. Those skilled in the art will recognize that such samples are a preferred starting point for the generation of diffraction crystals.

[0217] Crystallization is another major bottleneck in methods for determining the structure of macromolecules using X-ray crystallography. Successful crystallization requires nucleation and subsequent growth into crystals of suitable size. Crystal growth generally occurs spontaneously in supersaturated solutions, resulting from homogeneous nucleation. In classic sparse matrix screening experiments, proteins can be crystallized, where extensive sampling of the concentrations of precipitant, additives, and protein allows for the identification of suitable supersaturation conditions for nucleation and crystal growth for a specific protein. Related to sparse matrix screening methods is the generation of structural differences within the protein itself, for example, by adding protein-binding ligands, or by generating different mutations (preferably in the surface residues of the target protein), or by attempting to crystallize different types of orthogonal homologs of the target protein (Chang 1998). An unexpected finding of this invention is the effectiveness of specifically binding the binding domain of the GPCR:G protein complex to introduce a degree of structural alteration upon binding while preserving the overall folding of the complex.

[0218] Because crystallization involves the unfavorable loss of conformational entropy of molecules to be assembled in the crystal lattice, methods to reduce the conformational entropy of the target still in solution should enhance the likelihood of crystallization by reducing the net entropic penalty of lattice formation. The "surface entropy reduction" method has proven to be very effective (Derewenda 2004). Similarly, binding partners (such as ions, small molecule ligands, and peptides) can reduce conformational heterogeneity by binding and stabilizing subgroups of protein conformational states. While such binding partners are effective, not all proteins have known binding partners, and even when binding partners are known, their affinity, solubility, and chemical stability may be incompatible with crystallization experiments. Therefore, it is surprising to find that the binding domains of the present invention can be used as tools to minimize the conformational heterogeneity of the target GPCR:G protein complex by binding to a specific conformation of the G protein, increasing the likelihood of obtaining well-ordered crystals.

[0219] Crystallization of GPCRs for high-resolution structural studies is particularly challenging due to the amphiphilic surfaces of these membrane proteins. Embedded in the membrane bilayer, the contact sites between the protein and the acyl chains of the phospholipids are hydrophobic, while the polar surfaces are exposed to the polar head groups of the lipids and the aqueous phase. To obtain well-ordered three-dimensional crystals—a prerequisite for high-resolution X-ray structural analysis—GPCRs are dissolved with the aid of detergents and purified as protein-detergent complexes. Detergent flocs cover the hydrophobic surfaces of membrane proteins in a belt-like manner (Hunte and Michel 2002; Ostermeier et al., 1995). The GPCR-detergent complexes form three-dimensional crystals in which contacts between adjacent protein molecules are generated through the polar surfaces of the proteins protruding from the detergent flocs (Day et al., 2007). It is evident that detergent flocs require space within a crystal lattice. While attractive interactions between microclusters can stabilize crystal stacking (Rasmussen et al., 2007; Dunn et al., 1997), these interactions do not result in rigid crystal contacts. Since many membrane proteins, including GPCRs, contain relatively small or highly flexible hydrophilic domains, one strategy to increase the likelihood of obtaining well-ordered crystals is to expand the polar surface of the protein and / or reduce its flexibility. The most physiological approach is to use natural signal transduction partners, such as G proteins or repressor proteins. Unfortunately, the interaction of GPCRs with G proteins or repressor proteins is highly lipid-dependent, making it difficult to form complexes with sufficient stability for crystallography. Therefore, the binding domain of this invention, by binding to a G protein, can be used to expand the polar surface of the GPCR, supplementing the amount of protein surface that facilitates primary contact between the molecules of the lattice and the polar surfaces of G proteins and nanobodies. The binding domain of this invention can also reduce the flexibility of its extracellular region to grow well-ordered crystals. Immunoglobulin monovariable domains, including nanobodies, are particularly well-suited for this purpose because they bind conformational epitopes and contain a rigid globular domain, unlike conventional antibodies or fragments derived from them (such as Fab), which lack flexible linker regions.

[0220] Therefore, according to a preferred embodiment, the present invention provides a binding domain that can be used as a tool for crystallizing complexes containing GPCRs and G proteins, ultimately resolving their structures. More preferably, the complex crystallized using the binding domain of the present invention further includes a receptor ligand, more specifically an agonist. In a particularly preferred embodiment, the GPCR contained in the complex is in an active state or conformation.

[0221] Therefore, a variety of crystallization methods specifically designed for membrane proteins can be used to crystallize the binding domains of the GPCR:G protein complex and optionally the receptor ligand complex, many of which are reviewed in Caffrey (2003 & 2009). Generally, the methods are lipid-based, involving the addition of lipids to the complex prior to crystallization. Such methods have previously been used to crystallize other membrane proteins. Many of these methods, including lipid cubic phase crystallization and bicelle crystallization, utilize the spontaneous self-assembly properties of lipids and detergents as vesicles (vesicle fusion method), discoidal microclusters (bicelle method), and lipid crystals or intermediate phases (in the intermediate or cubic phase method). Lipid cubic phase crystallization methods are described, for example, in Landau et al., 1996; Gouaux 1998; Rummel et al., 1998; Nollert et al., 2004; Rasmussen et al., 2011, these publications are incorporated by reference to the published contents of these methods. The Bicelle crystallization method is described in, for example, Faham et al., 2005; Faham et al., 2002, and these publications are incorporated by reference to the public information of these methods.

[0222] According to another embodiment, the present invention relates to the use of the binding domain described herein for resolving the structure of a target complex comprising a GPCR and a G protein, the complex optionally further comprising a receptor ligand. As used herein, “resolving structure” refers to determining the atomic or atomic coordinate arrangement of a protein, typically by biophysical methods such as X-ray crystallography.

[0223] In X-ray crystallography, correctly combined diffraction data yields the 3D Fourier transform amplitude of the electron density of a molecule per unit cell. If the phase is known, the electron density can be easily obtained via Fourier synthesis. For protein complexes, when the proportion of proteins with known structures (the study model) is low (less than 50% amino acid content), and / or when the crystal exhibits limited diffraction quality, the successful inference of phase information from molecular substitution (MR) alone is questionable. While combinations of multiple isomorphic substitutions (MIR) and MR phasing have been successfully demonstrated for protein complexes (e.g., Ostermeier et al., 1995; Li et al., 1997; Hunter et al., 2000), the need to produce good heavy-atom derivatives is almost always challenging. In the last two decades, the application of anomalous dispersive data has generally surpassed classical MIR methods, primarily using selenomethionine (SeMet) incorporation (MAD or SAD) (Hendrickson 1991). In practice, anomalous experimental data using Se-edge energies generally provide better and less biased phase information than MIR or model-based MR phasing data. Accordingly, a particular embodiment relates to the use of the binding domain according to the invention for phasing GPCR:G complexes via MR or MAD. In particular, immunoglobulin monovariable domains, including nanobodies, are generally highly expressed and suitable for SeMet incorporation. For further illustration and not limitation, phasing complexes containing GPCRs, G proteins, and nanobodies by introducing all SeMet sites only into the nanobodies circumvents the requirement of incorporating SeMet sites into the GPCRs or G proteins.

[0224] In many cases, obtaining diffraction-grade crystals is a major obstacle to resolving atomic resolution structures. Therefore, according to specific embodiments, the binding structural domains described herein can be used to improve the diffraction quality of crystals, thereby enabling the resolution of the crystal structure of target complexes.

[0225] Furthermore, obtaining structural information about GPCR targets is highly desirable, for example, to help guide GPCR drug discovery. In addition to crystallizing more GPCRs, methods for obtaining receptor structures that bind to different types of ligands, including agonists, antagonists, allosteric modulators, and / or G proteins, are particularly needed. This invention specifically provides a general tool for obtaining crystals of GPCR:G protein complexes. In particular, agonist-bound GPCR:G protein complex crystals provide a three-dimensional representation of the active state of the GPCR. These structures will help clarify conformational changes associated with ligand binding and G protein interaction sites, leading to more precise mechanistic hypotheses and ultimately new therapeutic agents. Such states are difficult to stabilize given the inherent conformational flexibility of ligand-activated GPCRs and the greater heterogeneity exhibited by agonist-bound receptors. Therefore, work in this area can benefit from the stabilizing effect of agonist-bound receptor conformations bound to their heterotrimeric G protein complexes, achieved by adding such complex-specific binding domains. Particularly suitable are binding domains that bind to G proteins and form part of such complexes, because such binding domains can be used as a universal tool to stabilize all GPCRs that transmit signals via the same G proteins (e.g., Gs-coupled receptors, Gi-coupled receptors, etc.).

[0226] According to an alternative embodiment, the present invention covers a method for determining the crystal structure of a complex comprising GPCRs and G proteins, the method comprising the steps of:

[0227] a) Provides a combined structural domain according to the invention, and

[0228] b) Allows the binding domain to bind to a complex containing GPCRs and G proteins, and

[0229] c) Crystallize the complex formed in step b).

[0230] In a specific embodiment of the method for determining the crystal structure described above, the target complex containing GPCRs and G proteins further includes a receptor ligand that binds to the GPCRs, or more specifically, an agonist.

[0231] The crystal structure can be determined using biophysical methods, such as X-ray crystallography. The method may also include the step of obtaining the atomic coordinates of the crystal (as defined above).

[0232] Compounds for identifying GPCR:G protein complex

[0233] In the processes of compound screening, drug discovery, and lead optimization, there is a need for faster, more efficient, lower-cost, and especially more informative screening assays that provide simultaneous information on the characteristics of various compounds and their effects on various cellular pathways (i.e., efficacy, specificity, toxicity, and drug metabolism). Therefore, there is a need for rapid and inexpensive screening of large numbers of compounds to identify novel specific ligands for target GPCRs, preferably conformation-specific ligands, which may be potential drug candidates. This invention solves this problem by providing a binding domain that stabilizes the GPCR:G protein complex in a functional conformational state, which can then be used as an immunogen or selector for screening in various conditions. A key advantage of the binding domain according to the invention is that the GPCR contained in the GPCR:G protein complex can remain in a stable functional conformation, particularly the active conformation. For example, library compounds that selectively bind to the active conformation of this receptor have a greater tendency to act as agonists because the orthosteric or allosteric stability of the active conformation of the GPCR elicits a biological response. Another advantage is that the binding domain increases the thermal stability of the active conformation of the GPCR contained in the complex, thus protecting the GPCR from irreversible denaturation or thermal degradation caused by unnatural conditions used in compound screening and drug discovery, without relying on mutant GPCRs with increased stability. Another key advantage of the conformationally selective binding domain according to the invention is that it allows for rapid and reliable screening and differentiation of GPCRs and receptor agonists, inverse agonists, antagonists, and / or regulators of GPCR-dependent pathways, as well as inhibitors, thereby increasing the likelihood of identifying ligands with desirable pharmacological properties.

[0234] To further illustrate, a very clear concept is that most GPCRs exhibit higher agonist-binding affinity when complexed with G proteins. This is attributed to the cooperative interaction between the agonist-occupied receptor and the G protein. The binding domain of the present invention, which inhibits the dissociation of the GPCR and G protein complex, thus stabilizes the active conformational state of the R:G protein complex, thereby increasing the affinity of the GPCR for the agonist and decreasing the affinity for the inverse agonist. Therefore, the binding domain that recognizes the active functional conformation of the R:G complex can, for example, be used in high-throughput screening assays to screen for agonists because it increases the affinity of the receptor for the agonist relative to the inverse agonist. The binding domain that recognizes the active functional conformation of the G:R complex can also be used in high-throughput screening assays to screen for biased agonists that have the ability to selectively stimulate the signal transduction activity of a certain class of receptors, for example, selectively activating G proteins relative to the function of β-repressor proteins. Depending on the specific implementation, binding domains of G proteins that specifically bind to GPCRs (e.g., Tables 2-3) can be used as a general tool in screening programs targeting multiple GPCRs, because specific G proteins (e.g., Gs) can bind to multiple GPCRs (e.g., Gs-coupled receptors, including 5-HT receptors 5-HT4 and 5-HT7, ACTH receptors, adenosine receptor A). 2a and A 2b Type I, arginine angiotensin receptor 2, β-adrenergic receptors β1, β2 and β3, calcitonin receptor, calcitonin gene-related peptide receptor, corticotropin-releasing hormone receptor, dopamine receptor D1-like family (D1 and D5), FSH-receptor, gastric inhibitory polypeptide receptor, glucagon receptor, histamine H2 receptor, luteinizing hormone / human chorionic gonadotropin receptor, melanocortin receptor, parathyroid hormone receptor 1, prostaglandin receptors D2 and I2, secretin receptor, thyroid-stimulating hormone receptor, etc.; see also Table 1) form complexes.

[0235] Therefore, according to another aspect of the invention, the use of binding domains, or the use of complexes, cells, membrane articles containing binding domains, as described above, is covered for screening and / or identification procedures for conformation-specific binding partners of GPCR:G protein complexes, which may ultimately lead to potential new drug candidates.

[0236] According to one embodiment, the present invention considers a method for identifying compounds capable of selectively binding to GPCR:G protein complexes, the method comprising the steps of:

[0237] (i) Provide a complex containing GPCRs and G proteins

[0238] (ii) Contacting the complex with a binding domain that targets and / or specifically binds to the complex, allowing the binding domain to bind to the complex, and

[0239] (iii) Provide test compounds, and

[0240] (iv) Evaluate whether the test compound binds to the complex, and

[0241] (v) Selectively bind compounds to the complex.

[0242] It is evident that the binding domains used in any of the above methods can stabilize the functional conformational state of the GPCR:G protein complex and prevent the complex from dissociating. Preferably, the GPCR:G protein complex is in the active conformational state (as defined above). According to a particularly preferred embodiment of the above screening method, the GPCR:G protein complex further includes a receptor ligand.

[0243] It should be noted that particularly preferred embodiments combining structural domains are as described above with respect to the preceding aspects of the invention.

[0244] Therefore, the binding domains of the present invention can be used for screening assays. Screening assays for drug discovery can be solid-phase or liquid-phase assays, such as binding assays, like radioligand binding assays. It is understood that in some cases, high-throughput screening of test compounds is preferred, and the above methods can be used as “library screening” methods, a term commonly known to those skilled in the art. Therefore, the test compound can be a library of test compounds. In particular, high-throughput screening assays for therapeutic compounds, such as agonists, antagonists, or inverse agonists and / or modulators, form part of the present invention. For high-throughput purposes, compound libraries, such as allosteric compound libraries, peptide libraries, antibody libraries, fragment-based libraries, synthetic compound libraries, natural compound libraries, phage display libraries, etc., can be used. Methods for preparing and screening such libraries are known in the art. In a preferred embodiment, the high-throughput screening method involves providing a chemical or peptide library containing a large number of combinations of potential therapeutic ligands. Such “combinatorial libraries” or “compound libraries” are then screened in one or more assays to identify library members (specific chemical species or subclasses) exhibiting desired characteristic activities, as described herein. A "compound library" is a stored collection of chemicals, typically used for high-throughput screening. A "combinatorial library" is a collection of multiple chemical compounds generated through chemical or biological synthesis by combining various chemical "building blocks" (such as reagents). The preparation and screening of combinatorial libraries are well known to those skilled in the art. The identified compounds can thus function as conventional "lead compounds" or be used themselves as potential or actual therapeutic agents. Therefore, in another embodiment, the screening method described herein further includes modifying a test compound that has shown binding to a GPCR:G protein complex exhibiting conformational activity, and determining whether the modified test compound binds to a GPCR held in a specific conformation.

[0245] When high-throughput screening of target GPCR:G protein complexes is preferred for conformation-specific binding partner objects, this can be facilitated by immobilizing the binding domains according to the invention, or GPCR:G protein complexes stabilized by the binding domains, on suitable solid surfaces or supports that can be arrayed or otherwise multiplexed. Non-limiting examples of suitable solid supports include beads, columns, glass slides, chips, or plates. More specifically, the solid support can be granular (e.g., bead-like or granular, generally used in extraction columns) or sheet-like (e.g., membranes or filter paper, glass or plastic slides, microtiter plates, test strips, capillary filling devices, etc.), and can be flat, pleated, or hollow fibers or tubes. The following matrices are given as examples, not exhaustive, including silica (porous amorphous silica), i.e., FLASH series columns containing 60A irregular silica (32-63µm or 35-70µm) supplied by Biotage (a division of Dyax Corp.); agarose or polyacrylamide supports, such as the Sepharose series supplied by Amersham Pharmacia Biotech, or Affi-Gel supports supplied by Bio-Rad. Additionally, macroporous polymers, such as the pressure-stabilized Affi-Prep support supplied by Bio-Rad, are also available. Other available supports include dextran, collagen, polystyrene, methacrylates, calcium alginate, controlled-pore glass, aluminum, titanium, and porous ceramics. Optionally, the solid surface may include a portion of a mass-dependent sensor, such as a surface plasmon resonance detector. Other examples of commercially available support are discussed, for example, in Protein Immobilisation, RF Taylor, ed., Marcel Dekker, Inc., New York, (1991).

[0246] Immobilization can be non-covalent or covalent. Specifically, non-covalent immobilization or adsorption of the binding domain according to the invention, or a GPCR:G protein complex stabilized by the binding domain, on a solid surface can be achieved by coating the surface with any antibody, streptoacidin or avidin protein, or metal ions (e.g., biotin tags, histidine tags, etc.) according to standard techniques known to those skilled in the art, using methods according to conventional coupling chemistry. Optionally, the binding domain according to the invention, or a GPCR:G protein complex stabilized by the binding domain, can be covalently crosslinked to a solid surface using conventional coupling chemistry. The solid surface may naturally include crosslinkable residues suitable for covalent linkage, or it may be coated or derived to introduce suitable crosslinkable groups according to methods generally known in the art. In a particular embodiment, the immobilized protein retains sufficient function after direct covalent coupling to an ideal matrix via a reaction portion without chemical spacers. Jung et al. (2008) discussed other examples and more detailed information regarding methods for immobilizing antibodies (fragments) on solid supports; similarly, Cooper (2004) reviewed methods for membrane receptor immobilization; both are incorporated herein by reference. It is noteworthy that, for example, mutating specific amino acids (in proteins with known or suggested structures) to lysine or cysteine ​​(or other desirable amino acids) can provide specific sites for covalent coupling. It is also possible to reprogram specific proteins, altering the distribution of surface-available amino acids involved in chemical coupling (Kallwass et al., 1993), which effectively controls the orientation of the coupled protein. Similar methods can be used for the binding domains according to the invention, as well as conformationally stabilized GPCR:G protein complexes, thereby providing a means of directional immobilization without the addition of other peptide tails or domains containing native or non-native amino acids. In the case of antibodies or antibody fragments (such as nanobodies), introducing mutations into the frame region is preferred to minimize disruption to the antigen-binding activity of the antibody (fragment).

[0247] Conventionally, immobilized proteins can be used in immunoadsorption methods, such as immunoassays (e.g., ELISA), or immunoaffinity purification methods, which involve contacting the immobilized protein according to the invention with a test sample (i.e., containing a test compound, etc.) according to standard methods conventional in the art. Optionally, particularly for high-throughput purposes, the immobilized protein can be arranged or multiplexed. Preferably, the immobilized protein according to the invention can be used to screen and select compounds that specifically bind to conformationally stable GPCR:G protein complexes, particularly GPCRs in their active conformational state.

[0248] Understandably, depending on the type of application or the type of screening required, a fixed binding domain, or a (conformally stable) GPCR:G protein complex, or its constituent proteins, can be selected. Furthermore, the choice of the GPCR:G protein stabilizing binding domain (targeting a specific conformational epitope of the GPCR:G protein complex) will determine the orientation of the protein, and therefore, the ideal outcome for compound identification is, for example, a compound that specifically binds to the extracellular, intracellular, or intracellular portion of the conformationally stable GPCR, or a compound that specifically binds to the conformationally stable G protein.

[0249] Optionally, the test compound (or a library of test compounds) may be immobilized on a solid surface, such as a chip surface, while providing binding domains and GPCR:G protein complexes in, for example, detergent solutions or membrane-like preparations (see below).

[0250] Most preferably, the binding domain, GPCR:G protein complex or its constituent protein, or the test compound are not fixed, as in, for example, phage display selection processes in solution or radioligand binding assays. In a preferred embodiment, the binding domain, GPCR:G protein complex (or the respective constituent protein) used in any of the above screening methods is provided as a whole cell or cell (organelle) extract, such as a membrane extract or fraction thereof, or may be incorporated into lipid layers or vesicles (including natural and / or synthetic lipids), high-density lipid particles, or any nanoparticles, such as nanodisks, or provided as a VLP, thereby preserving full function of the corresponding protein. GPCR products formed from membrane fragments or membrane-detergent extracts are reviewed in detail in Cooper (2004), which is incorporated herein by reference. Alternatively, the binding domain, GPCR:G protein complex, or constituent protein may also be dissolved in a detergent. Non-limiting examples of dissolved receptor products are also provided in the Examples section.

[0251] Various methods can be used to determine the binding between a stable GPCR:G protein complex and a test compound, including conventional practices in the art such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance assay, chip-based assays, immunofluorescence, yeast double hybridization, and phage display, as described, for example, in Sambrook et al., (2001), Molecular Cloning, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Other methods for detecting the binding between the test compound and the GPCR include ultrafiltration using ion spray mass spectrometry / HPLC, or other (bio)physical and analytical methods. Fluorescence energy resonance transfer (FRET) methods, such as those commonly known to those skilled in the art, can also be used. It will be understood that the binding of the test compound can be detected using unique markers or tags associated with the compound, such as peptide tags, nucleic acid tags, chemical tags, fluorescent tags, or radiofrequency tags, as described elsewhere herein.

[0252] The efficacy of compounds and / or compositions containing compounds can be tested using any suitable in vitro assays, cell-based assays, in vivo assays and / or animal models, or any combination thereof, whichever is known in itself, depending on the specific disease or condition involved.

[0253] In a particular embodiment, it is determined whether the compound alters the binding of the GPCR to the receptor ligand (as defined herein). The binding of the GPCR to its ligand can be determined using standard ligand binding methods known in the art as described herein. For example, the ligand may be radiolabeled or fluorescently labeled. The determination can be performed on intact cells, on membranes obtained from cells, or on receptors dissolved in detergent water. The compound is characterized by its ability to alter the binding of the labeled ligand (see also the Examples section). The compound may reduce or increase the binding between the GPCR and its ligand, for example, by at least 2, 3, 4, 5, 10, 20, 30, 50, or 100-fold. Therefore, according to more specific embodiments, the complex used for any of the screening methods described above also includes a receptor ligand. Preferably, the receptor ligand is selected from small molecules, peptides, antibodies or any fragments thereof, natural products, etc. More preferably, the receptor ligand is a full agonist, a partial agonist, an inverse agonist, or an antagonist, as described above.

[0254] In addition to establishing binding to the target GPCR:G protein complex in its functional conformation, it is desirable to determine the functional effects of compounds on the biological activity of the GPCR:G protein complex, particularly on the GPCR and its downstream interacting partners. Specifically, the binding domains according to the invention can be used to screen compounds that regulate (increase or decrease) the biological activity of the GPCR:G protein complex or its components (GPCR or G protein). The desired regulation of biological activity depends on the selected GPCR. Compounds can bind to the target GPCR:G protein complex, particularly binding to one or both of its components, resulting in regulation (activation or inhibition) of downstream receptor signaling. This regulation of GPCR signaling can occur orthosterically or allosterically. Compounds can bind to the target complex comprising a GPCR that binds to the G protein or its components, thereby activating or increasing receptor signaling; or optionally, thereby decreasing or inhibiting receptor signaling. Compounds can also bind to the target complex in such a way that they block the constitutive activity of the GPCR. Compounds can also bind to the target complex in such a way that they mediate allosteric regulation (e.g., binding to the GPCR or G protein at an allosteric site). In this manner, compounds can modulate receptor function by binding to different regions of GPCRs or G protein complexes (e.g., at allosteric sites). See, for example, George et al. (2002), Kenakin (2002), and Rios et al. (2001). The compounds of the present invention can also bind to target complexes in such a manner as to prolong the duration of GPCR-mediated signal transduction or to enhance receptor signal transduction by increasing receptor-ligand affinity. Furthermore, the compounds can also bind to target complexes in such a manner as to inhibit or enhance the assembly of functional homopolymers or heteropolymers of GPCRs.

[0255] Furthermore, cell-based assays are crucial for assessing the mechanisms of action of novel biological targets and the biological activity of chemical compounds. Current cell-based assays used for GPCRs include measuring pathway activation (Ca2+). 2+ (Release, cAMP generation, or transcriptional activity); protein transport by labeling GPCRs and downstream elements with GFP; and direct measurement of protein-protein interactions using Fórster resonance energy transfer (FRET), bioluminescent resonance energy transfer (BRET), or yeast two-hybrid methods. Introducing the binding domains of this invention into relevant compartments (intracellular or extracellular) within the cell using any means generally known and conventionally used in the art can lead to novel or better cell-based assays.

[0256] In particular, it is necessary to "de-orphanize" GPCRs for which no natural activating ligands have yet been identified. Using GPCRs according to the invention with their binding domains stably in a functional conformation, a screening method for identifying ligands in "orphan" GPCRs, whose natural ligands are unknown, can be implemented. For example, various methods are employed for "de-orphanization," including array screening against families of known ligands. Ligands in orphan GPCRs can be identified from biological samples. Therefore, in certain embodiments, the test compound is provided as a biological sample. In particular, the sample can be any suitable sample collected from an individual. For example, the sample can be a bodily fluid, such as blood, serum, plasma, or cerebrospinal fluid. Optionally, the sample is a tissue or cell extract.

[0257] Test compounds used in any of the screening methods described above may be selected from polypeptides, peptides, small molecules, natural products, peptide mimics, nucleic acids, lipids, lipopeptides, carbohydrates, antibodies, or any fragments thereof, such as Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv) and fragments containing VL or VH domains, heavy chain antibodies (hcAb), single-domain antibodies (sdAb), microantibodies, variable domains derived from camel heavy chain antibodies (VHH or nanobodies), variable domains derived from novel antigen receptors derived from shark antibodies (VNAR), protein scaffolds (including αbody), protein A, protein G, designed ankyrin repeat domains (DARPin), type III fibronectin repeats, anticalins, knottins, and modified CH2 domains (nanobodies), as defined above.

[0258] Test compounds may optionally be covalently or non-covalently linked to detectable labels. Suitable detectable labels and techniques for linking, using, and detecting them are apparent to those skilled in the art, including but not limited to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Effective labels include magnetic beads (e.g., dynabead), fluorescent dyes (e.g., all Alexa Fluor dyes, fluorescein isothiocyanate, Texas Red, Rhodamine, green fluorescent protein, etc.), and radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase), and colorimetric labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Methods for detecting such labels are known to those skilled in the art. Thus, for example, radioactive labels can be detected using film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect divergent light. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product produced by the enzyme's action on the substrate. Colorimetric labels are detected by simply visualizing colored labels. Other suitable detectable labels have also been described previously in the first aspect of this invention concerning binding domains.

[0259] According to a particularly preferred embodiment, the test compound is an antibody or any fragment thereof, including nanobodies as described above. For example, but not limited to, the test compound can be an antibody generated against a GPCR:G protein complex stabilized by a binding domain according to the invention (as defined most broadly herein). Methods for generating antibodies in vivo are known in the art. Preferably, animals are immunized in a manner similar to that described earlier herein. The invention also relates to a method for selecting antibodies that specifically bind to a conformationally stabilized GPCR:G protein complex, involving screening an expression library encoding an immunoglobulin gene or a portion thereof on said GPCR:G protein complex, said library being expressed in bacteria, yeast, filamentous phage, ribosomes or ribosomal subunits or other display systems.

[0260] A particular aspect of the invention relates to a solid support that anchors the bonding domains according to the invention. Thus, such solid supports (as described above) can be used in any of the screening methods described above.

[0261] Regulation of GPCR receptor signaling

[0262] The binding domain of this invention can also be used to modulate GPCR signaling, particularly G protein-mediated GPCR signaling, including the elimination of G protein-mediated GPCR signaling. The terms "modulate" and "regulated" refer to an increase or decrease in the activity of a protein or protein complex, particularly the GPCR:G protein complex. In particular, the binding domain of this invention can be an allosteric regulator or an allosteric inhibitor. The terms "allosteric regulator" or "allosteric inhibitor" in the context of this invention refer to a non-competitive regulator or inhibitor that exerts its effect by binding to sites other than the active site of the receptor, modulating receptor activity or inactivating the receptor in signal transduction. "Positive allosteric regulators (PAMs)" increase signal transduction, while "negative allosteric regulators (NAMs)" decrease signal transduction. In particular, allosteric inhibitors can also eliminate signal transduction. Assays for evaluating the regulation of GPCR signaling by the binding domain of this invention are as described above.

[0263] In this respect, according to specific embodiments, the binding domains of the present invention, particularly the immunoglobulin monovariable domain, can also be used for precursor identification and design of peptide mimics. Utilizing biologically relevant peptide or protein structures as a starting point for precursor identification represents one of the most powerful methods in modern drug discovery. Peptide mimics are compounds that spatially mimic the key element (pharmacophore) of a natural peptide or protein while retaining the ability to interact with biological targets and produce the same biological effects. Peptide mimics are designed to circumvent some of the challenges associated with natural peptides, such as stability to protein degradation (duration of activity) and low bioavailability. They can often significantly improve other properties, such as receptor selectivity or potency.

[0264] Therapeutic and diagnostic applications

[0265] Some of the aforementioned binding domains may have therapeutic uses and may be administered to a subject with a condition, thereby treating the subject's condition. The therapeutic use of the binding domain is determined by the target GPCR:G protein complex to which the binding domain binds, as signaling via the GPCR is associated with the condition. In some cases, the GPCR can be activated by conditions involving the binding ligand. In other embodiments, the GPCR can be constitutively activated, for example, by mutation. The binding domains of the present invention can be applied to treat GPCR-mediated conditions such as schizophrenia, migraine, reflux, asthma, bronchospasm, benign prostatic hyperplasia, ulcers, epilepsy, sore throat, allergies, rhinitis, cancers such as prostate cancer, glaucoma, and stroke. Other exemplary GPCR-related conditions can be found in the NCBI worldwide website under On-line Mendelian Inheritance in the MAN database. Therefore, specific embodiments of the present invention also contemplate the binding domains of the present invention, or pharmaceutical compositions comprising the binding domains, for the treatment of GPCR-related diseases or conditions. It is understood that the therapeutic use also depends on the specific conformational epitope of the GPCR:G protein complex targeted by the binding domain.

[0266] In a fixed pharmaceutical composition, the binding domain of the present invention may be mixed with another drug or may be administered separately, before, simultaneously with, or after other pharmaceutical substances. Generally, these procedures involve administering an effective amount of the binding domain to an individual suffering from a GPCR-related disease or condition that modulates the signaling activity of GPCRs in the host and treats the individual's condition.

[0267] In some implementations, when it is necessary to reduce the activity of certain GPCRs, one or more compounds that reduce GPCR activity may be applied, and when it is necessary to increase the activity of certain GPCRs, one or more compounds that increase GPCR activity may be applied.

[0268] According to the method of the invention, a variety of individuals are treatable. These individuals are generally mammals or mammals, where such terms are used broadly to describe organisms falling into the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In several embodiments, the individual is a human. The treatment method of the invention is typically implemented on individuals suffering from such conditions or on individuals who need to avoid such conditions.

[0269] In another aspect, the present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of the binding domain of the present invention and at least one pharmaceutically acceptable carrier, adjuvant, or diluent.

[0270] "Carrier" or "adjuvant," particularly "pharmaceutical-grade carrier" or "pharmaceutical-grade adjuvant," refers to any suitable excipient, diluent, carrier, and / or adjuvant that does not induce antibodies harmful to the individual receiving the composition, nor does it provide protection. Therefore, pharmaceutically-grade carriers are inherently non-toxic and non-therapeutic, as is known to those skilled in the art. Suitable carriers or adjuvants typically comprise one or more compounds included in the following non-exhaustive list: large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, and inactivated viral particles. As a non-limiting example, the carrier or adjuvant may be Ringer's solution, dextran solution, or Hank solution. Anhydrous solutions, such as non-volatile oils and ethyl oleate, may also be used. A preferred excipient is 5% dextran in physiological saline. Excipients may contain small amounts of additives, such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.

[0271] The binding domains described herein, or their pharmaceutically acceptable salts, can be administered orally, by inhalation, or parenteral administration. In certain embodiments, nanobodies are delivered via intrathecal or intraventricular administration. The active compound can be administered alone or preferably formulated into a pharmaceutical composition. The amount required to effectively treat certain diseases or conditions expressing antigens that recognize the binding domain depends on common factors such as the nature and severity of the condition to be treated and the weight of the mammal. However, unit doses are typically from 0.01 to 50 mg, for example 0.01 to 10 mg, or 0.05 to 2 mg of the binding domain or its pharmaceutically acceptable salt. Unit doses are typically administered once or more daily, for example two, three, or four times daily, more commonly one to three times daily, so that the total daily dose is typically in the range of 0.0001 to 1 mg / kg; therefore, for a 70 kg adult, a suitable total daily dose is 0.01 to 50 mg, for example 0.01 to 10 mg, or more commonly 0.05 to 10 mg. More preferably, the compound or its pharmaceutically acceptable salt is administered in the form of a unit dose composition, such as a unit dose oral, parenteral, or inhaled composition. Such compositions are prepared by mixing and are suitable for oral, inhalation, or parenteral administration, and may be in the form of tablets, capsules, oral liquid products, powders, granules, lozenges, rehydrated powders, injectable and infusionable solutions or suspensions or suppositories or aerosols. Tablets and capsules for oral administration are typically present in unit doses and contain conventional excipients such as binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavoring agents, and humectants. Tablets can be coated according to methods generally known in the art. Suitable fillers used include cellulose, mannitol, lactose, and other similar agents. Suitable disintegrants include starch, polyvinylpyrrolidone, and starch derivatives such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable humectants include sodium lauryl sulfate. These solid oral compositions can be prepared by conventional methods such as blending, filling, and tableting. Repeated blending operations can be used to distribute the active agent throughout the aforementioned composition, which has been extensively filled with filler. Such operations are, of course, conventional in the art. Oral liquid products can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or can be presented as dry powders for rehydration with water or other suitable solvents prior to use. Such liquid products may contain conventional additives, such as suspending agents, such as sorbitol, syrups, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats; emulsifiers, such as lecithin, dehydrated sorbitan monooleate, or gum arabic; water-free solvents (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters such as glycerol, propylene glycol, or ethanol; preservatives, such as methylparaben or propylparaben or sorbic acid; and conventional flavoring or coloring agents as needed.Oral formulations also include conventional sustained-release formulations, such as tablets or granules with an enteric coating. Preferably, the composition for inhalation is presented as a solution of nasal spray, aerosol, or nebulizer, alone or in combination with an inert carrier (such as lactose), or as an ultrafine powder for inhalation for administration into the respiratory tract. In the above cases, the particles of the active compound appropriately have a diameter of less than 50 micrometers, preferably less than 10 micrometers, for example, 1 to 5 micrometers, such as 2 to 5 micrometers. Advantageous inhalation doses are 0.05 to 2 mg, for example, 0.05 to 0.5 mg, 0.1 to 1 mg, or 0.5 to 2 mg. For parenteral administration, a fluid unit dosage form containing the compound of the present invention and a sterile solvent is prepared. Depending on the solvent and concentration, the active compound may be in suspension or dissolved form. Parenteral solutions are typically prepared by dissolving the compound in a solvent, filtering and sterilizing it before filling into suitable vials or ampoules, and sealing it. Advantageously, adjuvants (such as local anesthetics), preservatives, and buffers are also dissolved in the solvent. To enhance stability, the composition can be frozen after vial filling and moisture removed under vacuum. Parenteral suspensions are prepared in essentially the same manner, except that the compound is suspended rather than dissolved in the solvent, and sterilized by exposure to ethylene oxide before suspension in a sterile solvent. Advantageously, the composition includes surfactants or humectants to promote uniform distribution of the active compound. Where appropriate, small amounts of bronchodilators may be included, such as sympathomimetic amines like isoproterenol, ethyl isoproterenol, salbutamol, phenylephrine, and ephedrine; xanthine derivatives such as theophylline and aminophylline; and corticosteroids such as prednisolone and adrenal stimulants such as ACTH. As a matter of common practice, the composition is usually accompanied by handwritten or printed instructions for the relevant medical treatment.

[0272] Binding domains, particularly immunoglobulin monovariable domains, can be delivered intracellularly, as described with respect to peptides, polypeptides, and proteins. If the antigen is extracellular or an extracellular domain, the binding domain can function by binding to that domain without requiring intracellular delivery. The binding domains of the present invention described herein can target intracellular conformational epitopes of GPCRs:G proteins. To utilize these binding domains as effective and safe intracellular therapeutic agents, intracellular delivery can be enhanced using protein transduction or delivery systems known in the art. Protein transduction domains (PTDs) have attracted considerable attention in the field of drug delivery due to their ability to translocate across biological membranes. PTDs are relatively short sequences (11-35 amino acids) that confer significant translocation activity to proteins and other macromolecular loads conjugated, compounded, or fused with them (Sawant and Torchilin 2010). For example, the HIV-derived TAT peptide (YGRKKRRQRRR) has been widely used for the intracellular delivery of a range of active agents, from small molecules to proteins, peptides, various drug nanocarriers, and imaging agents. Alternatively, receptor-mediated endocytosis can be used for intracellular drug delivery. For example, the transferrin receptor-mediated internalization pathway is an efficient cellular uptake pathway and has been developed for site-specific delivery of drugs and proteins (Qian et al., 2002). This is achieved either by chemically conjugating transferrin to a therapeutic drug or protein, or genetically by fusing a therapeutic peptide or protein into the structure of transferrin. Because naturally occurring proteins (such as the iron-binding protein transferrin) are biodegradable, non-toxic, and non-immunogenic, they are highly effective in drug targeting. Furthermore, the abundance of their receptors on the cell surface allows for site-specific targeting. Other delivery systems include, but are not limited to, polymer-based and liposome-based delivery systems.

[0273] The efficacy of the binding domains of the present invention and compositions comprising said domains can be tested using any suitable in vitro assays, cell-based assays, in vivo assays and / or animal models known per se, or any combination thereof, depending on the specific disease or condition involved.

[0274] Screening, selection and production of combined structural domains

[0275] In another aspect, the present invention also covers a method for screening binding domains that target and / or specifically bind to complexes comprising GPCRs and G proteins, the method comprising the steps of:

[0276] a) Provides multiple combined structural domains, and

[0277] b) Among the plurality of binding domains, screen for binding domains that bind to a complex containing GPCR and G protein, and

[0278] c) Separate the binding domains that bind to the complex.

[0279] In a preferred embodiment of this aspect of the invention, binding domains are generated and screened for their specific binding to a complex comprising a GPCR and a G protein, and optionally a receptor ligand. Binding domains can also be generated and screened for their specific binding to a G protein. As described herein, binding domains can be generated in various ways. In the case of immunoglobulin single variable domains (such as nanobodies), animals are typically immunized with a target complex comprising a GPCR that binds to a G protein and a receptor ligand, as described above (e.g., V). H H sequences (as a non-restrictive example) and examples elsewhere in this paper.

[0280] To immunize animals with the target complex, the protein of the target complex (i.e., GPCR and G protein) can be produced and purified using conventional methods that express the recombinant form of the protein in host cells, and the protein can be purified using affinity chromatography and / or antibody-based methods. In a particular embodiment, the baculovirus / Sf-9 system can be used for expression, but other expression systems (e.g., bacterial, yeast, or mammalian cell systems) can also be used. Exemplary methods for expressing and purifying GCPR are described, for example, in Kobilka (1995), Eroglu et al. (2002), Chelikani et al. (2006), and the book “Identification and Expression of G Protein-Coupled Receptors” (Kevin R. Lynch, ed., 1998), etc. Functional GPCR:G protein complexes can also be reconstructed using purified receptors (e.g., β2-AR or MOR) (reconstructed into recombinant HDL particles with a stoichiometric excess of G protein (Gs or Gi), as described by Whorton et al., (2009) (regarding β2-AR:Gs) or Kuszak et al., (2009) (regarding MOR:Gi). GPCRs can also be reconstructed in phospholipid vesicles and loaded with a stoichiometric excess of G protein. Similarly, methods for reconstructing active GPCRs in phospholipid vesicles are known and described in: Luca et al. (2003), Mansoor et al. (2006), Niu et al., (2005), Shimada et al., (2002), and Eroglu et al., (2003), etc. In some cases, GPCRs and phospholipids can be reconstructed at high densities (e.g., 1 mg receptor per mg phospholipid). In many cases, GPCRs can be located in two orientations within phospholipid vesicles (the normal orientation and the "inverted" orientation, in which the intracellular loop is located on the outside of the vesicle). Other immunologic methods for GPCRs include, but are not limited to, using intact cells or membranes derived from expressing GPCRs and / or G proteins.

[0281] In a particular embodiment, the target complex used for cross-linking with the bifunctional cross-linking agent is immunized in animals (see also the Examples section). Chemical cross-linking can be performed using standard techniques generally known to those skilled in the art (see, for example, Hermanson, GT (2008) Bioconjugate Techniques, 2nd edition, Elsevier Inc., p. 1202).

[0282] Any suitable animal, such as a warm-blooded animal, particularly a mammal, such as a rabbit, mouse, rat, camel, sheep, cow, or pig, or a bird, such as a chicken or turkey, can be immunized using any technique generally known in the art that is suitable for generating an immune response.

[0283] Screening for binding domains of conformational epitopes that specifically bind to target complexes can be performed, for example, by screening combinations, collections, or libraries of cells (e.g., B cells obtained from appropriately immunized camels) that express binding domains on their surfaces, by screening (raw or immunized) libraries of binding domains, or by screening (raw or immunized) libraries of nucleic acid sequences encoding amino acid sequences of binding domains. These screenings can all be performed in manner known per se, and the method may optionally include one or more other suitable steps, such as, but not limited to, affinity maturation, expression of a desired amino acid sequence, screening for binding and / or activity against a desired antigen, determination of a desired amino acid or nucleotide sequence, introduction of one or more humanized substitutions, formatting in a suitable multivalent and / or multispecific pattern, screening for desired biological and / or physiological properties (i.e., using suitable assays known in the art), and / or combinations of one or more such steps in any suitable order.

[0284] Another aspect of the invention relates to a kit comprising a binding domain according to the invention. The kit may also include combinations of reagents such as buffers, molecular tags, vector constructs, reference sample materials, and suitable solid supports, cells, nucleic acids, etc. Such kits can be used in any application of the invention described herein. For example, the kit may include a library of test compounds for compound screening purposes.

[0285] Finally, a last aspect of the invention is the use of any binding domain according to the invention for isolating the amino acid sequence responsible for specifically binding the conformational epitope of the GPCR:G protein complex and constructing artificial binding domains based on said amino acid sequence. It will be understood that in the binding domains according to the invention, the frame region and complementarity-determining region are known, and the study of binding domain derivatives binding the same conformational epitope in the GPCR:G protein complex will allow for the acquisition of key amino acids involved in binding said conformational epitope. This knowledge can be used to construct minimal binding domains and generate their derivatives, which can be routinely performed using techniques known to those skilled in the art.

[0286] The following examples are intended to facilitate a further understanding of the invention. The invention has been described herein with reference to exemplary embodiments, but it should be understood that the invention is not limited thereto. Other modifications and embodiments within the scope of the invention will be recognized by those skilled in the art and who have received the teachings herein. Therefore, the invention is limited only by the claims appended herein. Example

[0287] Example 1: Formation and purification of a stable agonist-β2AR-Gs ternary complex

[0288] A stable complex was formed by mixing approximately 100 μM of Gs heterotrimer with an excess molar amount (approximately 130 μM) of BI-167107-bound T4L-β2AR (or β2AR-365) in 2 ml of buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% DDM, 1 mM EDTA, 3 mM MgCl2, 10 μM BI-167107) and incubating at room temperature for 3 hours (see [link to relevant documentation]). Figure 2 BI-167107, identified from screening and characterizing approximately 50 different β2AR agonists, has a dissociation half-life of approximately 30 hr, providing a higher degree of stabilization for receptors bound to active G proteins than other complete agonists (such as isoproterenol) (Rasmussen et al., 2011). To maintain the nucleotide-free, high-affinity state of the complex, adenosine triphosphate bisphosphatase (25 mU / ml, NEB) was added after 90 min to hydrolyze the remaining GDP released from Gαs during receptor binding. The GMP obtained from the hydrolysis of GDP by adenosine triphosphate bisphosphatase has a very low affinity for the G protein in the complex. Rebinding of GDP leads to the dissociation of the R:G complex ( Figure 3a ).

[0289] At 4°C, the R:G complex in DDM showed significant dissociation after 48 hours. Figure 4 a). More than 50 amphiphiles were screened and characterized (data not shown), and MNG-3 (NG-310, Affymetrix-Anatrace; Chae et al., 2011) and its closely related analogues were identified as detergents that substantially stabilize the complex. Figure 4 a, b). By adding the R:G mixture (2 ml) to 8 ml of buffer (20 mM HEPES, pH 7.5, 100 mM NaCl) containing 1% MNG-3 at room temperature. In BI-167107, after 1 hour, the complex was exchanged into MNG-3.

[0290] At this stage, the mixture contains R:G complexes, nonfunctional Gs, and excess β2AR. To separate the functional R:G complexes from the nonfunctional Gs and to complete detergent exchange, the R:G complexes were immobilized on M1Flag resin and washed in a buffer containing 0.2% MNG-3 (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 μM BI-167107, and 3 mM CaCl2). To prevent cysteine-bridged R:G complex aggregation, 100 μM TCEP was added to the eluted proteins before concentration using a 50 kDa MWCO Millipore concentrator. A final size exclusion chromatography step was performed on a Superdex 200 10 / 300 GL column (GE Healthcare) equilibrated with buffer containing 0.02% MNG-3, 10 mM HEPES pH 7.5, 100 mM NaCl, 10 μM BI-167107, and 100 μM TCEP to separate excess free acceptor from the R:G complex. Figure 5 b). Mixed peak fraction ( Figure 5 b) The fraction was concentrated to approximately 90 mg / ml using a 100 kDa MWCO Viva rotary concentrator. -1 and stained with SDS-PAGE / Coomassie Brilliant Blue ( Figure 5 a) and gel filtration ( Figure 5 c) Analysis. To verify that it was a pure, homogeneous, and dephosphorylated product, the R:G complex was routinely analyzed by ion exchange chromatography. Figure 5 d).

[0291] Example 2: Generation of nanobodies bound to the agonist:β2AR:Gs ternary complex

[0292] The α-helical domain of Gαs was observed to be flexible in negative staining EM imaging (data not shown). Targeting stabilization of this domain was achieved by generating nanobodies that bind to the agonist-β2AR-Gs ternary complex. The nanobodies are single-domain antibodies derived from heavy-chain-only antibodies from domestic llamas (Muyldermans, 2001). To identify nanobodies binding to receptor-coupled Gs-proteins (loaded with agonists), two domestic llamas (Llama glama) were immunized with a β2AR:Gs:BI167107 ternary complex cross-linked with bis(sulfosuccinicotinamide)glutarate (BS2G, Pierce). Both animals were immunized four times with 50–100 μg injections every other week. After immunization, peripheral blood lymphocytes were isolated from the immunized animals, and total RNA was extracted to prepare cDNA. Total RNA was isolated from approximately 10⁷ lymphocytes, as described by Chomczynski and Sacchi (1987). First-strand cDNA synthesis was performed using dN6 primers and superscript RT, according to the manufacturer's (Invitrogen) instructions. As previously described, the fragment encoding the VHH gene was amplified from this cDNA by PCR using specific primers (Conrad et al., 2001). Pst1 and BstEII were modified at the beginning and end of the VHH read frame, respectively, using nested PCR. VHH was cloned as a Pst1-BstEII fragment into the phage display vector pMESy4. For each alpaca, an independent phage display library was constructed, containing the corresponding nanobody library as a geneIII fusion (Domanska et al., 2011). R:G complex-specific nanobodies were enriched by two rounds of biopanning: i) β2AR:Gs:BI167107 ternary complexes embedded in ApoL biotinylated high-density lipoprotein particles (rHDL, Whoton et al., 2007) or ii) BS2G cross-linked β2AR:Gs:BI167107 ternary complexes. For the first round of biopanning, biotinylated rHDL particles containing the β2AR:Gs:BI167107 ternary complex were immobilized at 1 μg / well on Maxisorp plates (Nunc) coated with neutralvidin in 20 mM Hepes (pH 8.0), 100 mM NaCl, 1 mM EDTA, 100 μM TCEP, and 100 nM BI167107. For the second round of biopanning, the BS2G cross-linked β2AR:Gs:BI167107 ternary complex was coated onto Maxisorp plates at a solid-phase concentration of 1 μg / well. For each round of biopanning, 10 μg of the complex was added. 11Phages were used to immobilize antigens and incubated for 1 to 2 hours. Unbound phages were then removed from the antigen-containing wells by washing 14 times with 20 mM HEPES, 100 mM NaCl, and pH 8. Finally, the wells were incubated for 10 minutes with 200 μL of 20 mM HEPES (pH 8.0), 100 mM NaCl, 1 mM EDTA, 100 μM TCEP, and 100 nM BI167107 to remove non-specific phages. To elute complex-specific phages, the wells were treated with trypsin, and the phages were re-coated and used to infect logarithmically growing TG1 cells (OD600 ± 0.5). Forty-eight colonies were randomly selected from each enriched library and grown in 1 ml of 2xTY containing ampicillin and glucose. Nanobody expression was induced using IPTG-induced cultures to prepare periplasmic extracts containing partially purified nanobodies. The binding of nanobodies contained in these peritoneal extracts to the agonist:β2AR:Gs ternary complex was analyzed by ELISA.

[0293] Nanobodies enriched on biotinylated rHDL particles containing the β2AR:Gs:BI167107 ternary complex were analyzed by comparative ELISA between the following complex and blank rHDL particles immobilized on avidin-coated Maxisorb plates. Nanobodies enriched on the solid-phase-coated BS2G crosslinked β2AR:Gs:BI167107 ternary complex were analyzed by comparative ELISA between the same solid-phase-coated complex and uncoated wells. Monoclonal antibodies were prepared from colonies that scored positive by the comparative ELISA, DNA was extracted, and the sequence of the encoded nanobodies was analyzed using standard methods (amino acid sequences are shown in Tables 2-3). For Nb35, Nb36, and Nb37, binding to the β2AR:Gs:BI167107 ternary complex was further validated by analytical gel filtration. Figure 3d , 3e , 3f, 3g).

[0294] Example 3: Nb35, Nb36, and Nb37 bind to Gs and prevent complex dissociation via GTPγS.

[0295] To determine whether the nanobodies generated for the β2AR:Gs:BI167107 ternary complex (Table 2) bound to the receptor or Gs, the binding of these nanobodies to individual purified receptors was subsequently monitored in an ELISA. In a solid phase (Maxisorb, Nunc) coated with agonist-bound β2AR-356, all nanobodies in Table 2 were scored negatively, representing a high density of reconstituted phospholipid vesicles of agonist-bound β2AR-356 (Rasmussen et al., 2011). In this ELISA, the β2AR-specific nanobody—Nb80 (Rasmussen et al., 2011)—scored positively. None of the β2AR:Gs:BI167107 conjugates described in Table 2 bound to the reconstituted individual receptors, suggesting binding to epitopes contained within Gs. Size exclusion chromatography revealed that Nb35 and Nb37 bound to separate epitopes located on the Gs heterotrimer, forming an R:G:Nb35:Nb37 complex (…). Figure 3d Similarly, Nb36 and Nb37 bind to separate epitopes located on the Gs heterotrimer to form the R:G:Nb36:Nb37 complex. Figure 3e ).

[0296] GDP, GTP, and non-hydrolyzable GTP analogues disrupt the β2AR:Gs complex. Figure 3a This leads to the dissociation of the GPCR G protein complex in vitro and in vivo. The interaction between Nbs and the non-hydrolyzable GTP analog GTPγS on the integrity of the agonist:β2AR:Gs ternary complex was analyzed by analytical-grade size exclusion chromatography in the presence and absence of GTPγS. Nanobodies 35, 36, and 37 were found to protect the β2AR:Gs:BI167107 complex from dissociation by GTPγS. Figure 3d , 3e &3g).

[0297] Example 4. Crystallization of β2AR-Gs complex assisted by nanobody

[0298] G protein-coupled receptors (GPCRs) are responsible for most cellular responses to hormones and neurotransmitters, as well as vision, smell, and taste. A paradigm of GPCR signaling is the activation of heterotrimeric GTP-binding proteins (G proteins) via agonist-occupied receptors. In an attempt to understand the structural basis of GPCR signaling, the β2AR-Gs complex was crystallized, and its structure was resolved by X-ray crystallography.

[0299] One challenge in crystal formation is the preparation of stable β2AR:Gs complexes in detergent solutions. β2AR and Gs are effectively coupled in lipid bilayers, but not in detergents used to dissolve and purify these proteins (Example 1). It has been found that relatively stable β2AR:Gs complexes can be prepared by mixing purified GDP-Gs (approximately 100 μM final concentration) with a molar excess of a high-affinity agonist bound to purified β2AR (BI167107; Rasmussen et al., 2011) in a dodecyl maltodextrin solution. A nonselective purine pyrophosphatase—adenosine triphosphate diphosphatase—can be added to hydrolyze GDP released from Gs that form the complex with β2AR. The complex is then purified by sequential antibody affinity chromatography and size exclusion chromatography. The stability of the complex is enhanced by exchanging it with a newly developed maltodextrin-based detergent (NG-310, Anatrace) (Chae et al., 2010). The complex could be incubated at room temperature for 24 hours without any noticeable degradation; however, initial attempts to crystallize the complex in detergent microparticles, bicellles, and lipid cubic phases (LCP) using a sparse matrix screening method failed.

[0300] To further evaluate the quality of the complex, the protein was analyzed by single-particle electron microscopy (EM). The results confirmed that the complex was monodisperse (data not shown), but revealed other potential bottlenecks for obtaining quality crystallographic diffraction. First, the detergent used to stabilize the complex formed large microclusters, resulting in very little polar surface remaining on the extracellular side of the β2AR:Gs complex for lattice contact formation. Therefore, the unstructured amino terminus of β2AR was replaced with T4 lysozyme (T4L). T4L had previously been used to promote crystallization of inactivated β2AR by inserting it between the cytoplasmic ends of transmembrane segments (TM) 5 and 6 (Rosenbaum et al., 2007). This fusion protein (T4L-β2AR) exhibited normal ligand binding and Gs-coupled properties. In LCP, crystallization experiments were conducted using a modified monoolein (7.7 MAG, provided by Martin Caffrey), which was designed to be compatible with the large hydrophilic component of the T4L-β2AR:Gs complex (Misquitta et al., 2004). Although we were able to obtain diffraction... Small crystals, but their quality cannot be improved by using additives and other modifications.

[0301] Another potential challenge in crystal formation revealed by single-particle EM analysis is the increased variability in the placement of the α-helical component of the Gαs subunit. Gαs consists of two domains: a ras-like GTPase domain (GαsRas), which interacts with the β2AR and Gβ subunits, and an α-helical domain (GαsAH) (Sprang et al., 1997). The interface between the two Gαs subdomains forms a pocket for binding nucleotides. Figure 1 The EM 2D mean and 3D reconstruction suggest that, in the absence of guanine nucleotides, GαsAH has a variable position relative to the T4L-β2AR-GαsRAS-Gβγ complex. Figure 1 b).

[0302] In further work to promote the crystallization of the complex, we explored co-crystallization of the complex and nanobody 35. Nb35 protected the complex from dissociation by GTPγS, suggesting that the Gs:Nb interaction was stabilized. Figure 3a The T4L-β2AR:Gs complex containing BI-167107 and Nb35 were mixed in a molar ratio of 1:1.2 (see [link to original text]). Figure 6 and 7 The presence of a slight molar excess of Nb35 was verified by gel filtration analysis. Figure 7 b). Using the previously reported twin-syringe mixing method (Caffrey 2009), the mixture was incubated at room temperature for 1 hour before being mixed with 7.7 MAG (provided by Martin Caffrey) containing 10% cholesterol (C8667, Sigma) at a 1:1 protein-to-lipid ratio (w / w). The concentration of the R:G:Nb complex in the 7.7 MAG was approximately 25 mg / ml. The protein:lipid mixture was delivered to 24-well or 96-well glass sandwich plates at 40 nmol per drop using an LCP dispersion robot (Gryphon, Art Robbins Instruments), and the entire volume was covered with 0.8 μl of precipitation solution. Several crystallization precursors were preliminarily identified using internal screening with reagents from a partially StockOptions Salt kit (Hampton Research). The mixture was prepared in 18–22% PEG 400, 100 mM MES, and pH 6.5. Figure 1 c), 350 to 450 mM potassium nitrate, 10 mM phosphonoformic acid ( Figure 3b Crystals for data collection were grown in 1 mM TCEP and 10 μM BI167107. The crystals reached their actual size within 3–4 days at 20 °C. Figure 8 It was then picked out from the sponge-like mesophase and instantaneously frozen in liquid nitrogen without any additional cryoprotectant.

[0303] Example 5: Nb35 promotes crystal formation of R:G complex

[0304] BI-167107 is bound to a T4L-β2AR:Gs:Nb35 complex crystallized in space group P21, with a single complex in each asymmetric unit. Figure 9a This demonstrates the interaction of crystallographic stacking. Figure 9b The structure of the complete complex, including T4L and Nb35, is shown. Figure 9c The β2AR:Gs complex itself was shown.

[0305] The T4L-β2AR:Gs:Nb35 complex, bound to BI-167107, was arranged in the alternative aqueous and lipid layers, exhibiting lattice contacts formed almost exclusively between the soluble components of the complex, leaving receptor molecules suspended between the G protein layers, widely separated from each other on the membrane plane. This extensive lattice contact formation between all soluble proteins likely results in strong overall diffraction and very clear electron density of the G proteins.

[0306] Nb35 and T4L promote crystal formation of the T4L-β2AR:Gs:Nb35 complex bound to BI-167107. Nb35 binds to a conformational epitope on Gs, pressing on the interface between the Gβ and Gα subunits and the complementarity-determining region (CDR, as defined by IMTG designation; Lefranc, 2003) 1, which primarily interacts with Gβ. Figure 10 a), while the long CDR3 ring interacts with both the Gβ and Gα subunits simultaneously ( Figure 10 b). Some framework regions of Nb35 also interact with Gα of the same complex ( Figure 10 C). Other framework regions from one complex interact with Gα subunits from two adjacent complexes ( Figure 11 The T4L is primarily responsible for crystal contacts within the lattice. It forms a relatively sparse interaction with the amino terminus of the acceptor, but is pressed against the amino terminus of the Gβ subunit of one complex and the carboxyl terminus of the Gγ subunit of another complex.

[0307] Example 6. Structure of the active state β2AR

[0308] The β2AR:Gs structure provides the first high-resolution view of the structural basis of the transmembrane signal transduction mechanism of GPCRs and the functional properties of the ternary complex. Figure 12a The structures of the receptors binding the agonist in the β2AR:Gs complex and in β2AR bound with inactivated kalanol were compared. The greatest difference between the inactivated and active structures was the outward shift of TM6 at the Cα carbon of E268, as measured. The cytoplasmic ends of the TM5 helix extend and shift outwards less, by 7 residues. In the third intracellular loop (ICL3), a 26-amino acid segment is out of order. Another significant difference between the inactivated and active structures is the second intracellular loop (ICL2), which forms an extended loop in the inactivated β2AR structure, while forming an α-helix in the β2AR:Gs complex. This helix was also observed in the β2AR-Nb80 structure. Figure 12b However, this may not be a feature specific to the active state, as this structure has also been observed in the inactivated structure of highly homogeneous avian β1AR (Warne et al., 2008).

[0309] At the β2AR-GαsRas interface, the electron density map of β2AR has the highest quality, while the extracellular portion is much weaker, possibly due to the lack of lattice contact with the extracellular surface. Figure 9a The result was that the model of a high-affinity agonist (BI-167107) could not be definitively constructed in the ligand-binding pocket. However, the overall structure of β2AR in the T4L-β2AR:Gs complex was remarkably similar to the structure of our most recent active-state β2AR stabilized by the G protein-mimicking nanobody (Nb80). These structures largely deviated from the cytoplasmic ends of TM5 and TM6 ( Figure 12b This may be due to the presence of T4L, which replaces ICL3 in the β2AR-Nb80 structure. Regardless, the β2AR-Nb80 complex exhibits the same high affinity for the agonist isoproterenol as the β2AR:Gs complex (Rasmussen et al., 2011), consistent with the high structural homology around the ligand-binding pocket. Electron density maps of β2AR-Nb80 crystals provide a more reliable observation of amino acid conformational rearrangements around the ligand-binding pocket and between the ligand-binding pocket and the Gs coupling interface (Rasmussen et al., 2011).

[0310] Figure 12c The positions of highly conserved sequence motifs in the β2AR:Gs complex, including D / ERY and NPxxY, are shown compared to the β2AR-Nb80 complex (see also Figure 13). These conserved sequences have been suggested to be important for activating or maintaining the receptor in an active-inactive state (Hofmann et al., 2009). The positions of these amino acids are essentially the same in both structures, confirming that Nb80 is a very good substitute for G proteins. The only difference between the two structures is Arg131. In the β2AR-Nb80 structure, Arg131 interacts with Nb80, while in the β2AR:Gs structure, Arg131 presses onto Tyr391 of Gαs (Figure 13).

[0311] The active state of β2AR is stabilized through extensive interactions with (GαsRas). Figure 14 It has no direct interaction with the Gβ or Gγ subunits. The total embedding surface of the β2AR-GαsRas interface is (GαsRas is) β2AR is The interface is formed by ICL2, TM5, and TM6 of β2AR, and by the α5-helix, αN-β1 linker, top of the β3 chain, and α4-helix of GαsRas (see Table 6 for specific interactions). The β2AR sequence involved in this interaction appears to play a role in G protein coupling; however, when comparing these segments with other GPCRs, no obvious common sequences for Gs-coupling specificity are found. This may not be surprising, given that β2AR also couples to Gi, and many GPCRs couple to more than one G protein isoform. Therefore, the structural basis for G protein coupling specificity must involve more subtle secondary and tertiary structural features. However, a notable interaction involves Phe139, located at the start of the ICL2 helix, situated within a hydrophobic pocket formed by Gαs His41 at the start of the β1 chain, Val213 at the start of the β3 chain, and Phe376, Arg380, and Ile383 in the α5-helix. Figure 14 c). The β2AR mutant F139A exhibits severely impaired coupling with Gs (Moro et al., 1993). On almost all Gs-coupled receptors, the residues corresponding to Phe139 are either Phe or Leu, but the residues described are more variable in GPCRs known to couple with other G proteins. Interestingly, the interaction between the conserved DRY sequence Asp130 and Tyr141 at the center of the ICL2 helix stabilizes the ICL2 helix (…). Figure 14 c). Tyr141 is shown to be a substrate of insulin receptor tyrosine kinase (Baltensperger et al., 1996): however, the functional importance of this phosphorylation is currently unknown.

[0312] Example 7. Structure of activated Gs

[0313] The most surprising observation in the β2AR:Gs complex is the large displacement of GαsAH relative to GαsRas (a rotation of approximately 180° around the connection between the domains). Figure 15a) In the crystal structure of Gαs, a nucleotide-binding pocket is formed at the interface between GαsRas and GαsAH. The binding of guanine nucleotides stabilizes the interaction between these two domains. The stabilizing effect of losing this binding guanine nucleotide is consistent with the high flexibility of GαsAH observed in single-particle EM analysis of the detergent-dissolved complex (data not shown). This is also consistent with the increased deuterium exchange at the interface between the two domains during complex formation (data not shown). Recently, Hamm, Hubbell, and colleagues used two-electron electronic resonance (DEER) spectroscopy to confirm a significant change in the distance between nitrooxy probes located on the α-helical domains of Ras and Gi after complex formation with photoactivated rhodopsin (up to [missing information]). (Van Eps 2011). Therefore, it is perhaps not surprising that GαsAH is replaced by GαsRas; however, its position in the crystal structure likely reflects an interaction with the crystal stack rather than an effect on its physiological conformation.

[0314] The conformational association between β2AR and the nucleotide binding pocket primarily involves the helix at the amino and carboxyl ends of Gαs. Figure 14 ). Figure 15 b focused on the GαsRAS region undergoing the greatest conformational change when comparing the GαsRAS structures of the Gs-β2AR complex and the Gαs-GTPγS complex (Sunahara et al., 1997). The most significant difference observed was the α5 helix, which shifted towards the receptor. It then rotates to the carboxyl terminus and protrudes into the transmembrane core of β2AR. Related to this movement, the β6-α5 ring, which interacts with the guanine ring in the Gαs-GTPγS structure, shifts outward, deviating from the nucleotide-binding pocket. Figure 15 The movement of the α5 helix is ​​also associated with changes in the interactions between this helix and the β6 sheet, the αN-β1 ring, and the α1 helix. The β1 chain forms another link between the β2AR and the nucleotide-binding pocket. The C-terminus of this chain alters the conformation around Gly47, and other changes are made to the β1-α1 ring (P ring) of the γ-phosphate in the GTP-binding form. Figure 15 The observations of the crystal structure are consistent with the deuterium exchange experiments, in which enhanced deuterium exchange is observed in the β1 sheet and at the amino terminus of the α5 helix after the formation of the nucleotide-free β2AR:Gs complex (data not shown).

[0315] The structure of the Gs heterotrimer has not yet been determined, therefore the Gαs-Gβγ interface cannot be directly compared before and after the formation of the β2AR:Gs complex. Based on the structure of the Gi heterotrimer bound to GDP (Wall et al., 1995), no significant change in the interaction between GαsRAS and Gβγ was observed after complexing with β2AR. This is also consistent with deuterium exchange studies (data not shown). It should be noted that Nb35 binds at the interface between GαsRas and Gβ ( Figure 2 b). Therefore, the possibility that Nb35 can affect the relative orientation of the GαsRas-Gβγ interface in the crystal structure cannot be ruled out. However, single-grain EM studies provide evidence that Nb35 does not disrupt the interaction between GαsAH and GαsRas (data not shown).

[0316] Example 8: Nb35 and Nb37 bind to different epitopes on Gs and inhibit nucleotide binding.

[0317] To investigate the effects of nanobodies (Nb35 and Nb37) on Gs itself, nanobodies, bodipy-GTPγS-FL, and various Gs protein preparations were added to a final volume of 200 μL in 20 mM Tris-HCl, pH 8.0, 3 mM MgCl2, and 1 mM DTT. Samples containing heterotrimeric Gs proteins also included 0.1% DDM. Bodipy-GTPγS-FL is a stable fluorescent GTP analog. Its fluorescence intensity increases upon binding to G proteins; therefore, Bodipy-GTPγS-FL can be used for real-time measurement of nucleotide binding to G proteins (McEwen et al., 2001). Fluorescence was measured in 96-well microtiter plate mode on an M5 Molecular Precision instrument.

[0318] In the first experiment ( Figure 16 Incubation of Nb37 with 1 μM purified GαS and 100 nM Bodipy-GTPγS-FL resulted in a short-term (300 s) increase in fluorescence, minimizing the accumulation of the hydrolysis product bodipy-phosphate (Jameson et al., 2005). As previously described, the Gαs subunit of the heterotrimeric Gs protein was purified (Sunahara et al., 1997). Based on this experiment, Nb37 appears to block GTPγS binding to Gsα alone in a dose-dependent manner. These results also suggest that the binding epitope of Nb37 is limited to the Gαs subunit of the heterotrimeric Gs protein. In similar experiments ( Figure 17The increased amount of Nb35 was incubated with 1 μM purified GαS and 100 nM Bodipy-GTPγS-FL. Consistent with the observed results, Nb35 bound epitopes containing GαsRAS and Gβ elements (see Example 7), and Nb35 had no effect on the binding of GTPγS to the GαS subunit alone.

[0319] In another experiment ( Figure 18 The increased concentrations of Nb35 were incubated with 1 μM purified Gsαβγ heterotrimer and 100 nM Bodipy-GTPγS-FL. This experiment suggests that Nb35 blocks the binding of GTPγS to free Gsαβγ heterotrimer in a dose-dependent manner.

[0320] Example 9. Nb35 stabilizes other agonist-GPCR-Gs complexes

[0321] The Gsα subunit (or Gs protein) is a heterotrimeric G protein subunit that activates the cAMP-dependent pathway by activating adenylate cyclase. Gs-coupled G protein-coupled receptors include: 5-HT receptors (5-HT4 and 5-HT7 types), ACTH receptors, adenosine receptors (A2a and A2b types), arginine angiotensin receptor 2, β-adrenergic receptors (β1, β2, and β3 types), calcitonin receptors, calcitonin gene-related peptide receptors, corticotropin-releasing hormone receptors, dopamine receptors (D1-like family, D1 and D5), FSH-receptors, gastric inhibitory peptide receptors, glucagon receptors, glucagon-like peptide-1 receptors (GLP-1-R), histamine H2 receptors, luteinizing hormone / human chorionic gonadotropin receptors, melanocortin receptors, parathyroid hormone receptor 1, prostaglandin receptors (D2 and I2 types), secretin receptors, and thyroid-stimulating hormone receptors, among others.

[0322] To determine whether nanobodies binding to Gs in β2AR:Gs:BI167107 also stabilize other GPCR:Gs:agonist complexes, a complex of arginine angiotensin receptor 2 (accession number P30518; V2R_HUMAN) with Gs, Nb35, and arginine angiotensin (AVP:NT4LV2R:Gs) was prepared, and the stability of this complex in SEC was confirmed. Arginine angiotensin (AVP), also known as vasopressin, is a natural ligand that activates arginine angiotensin receptor 2.

[0323] A stable complex was formed by mixing approximately 90 μM of His-labeled Gs heterotrimer with AVP-bound NT4LV2R (90 μM) and NB35 (100 μM) in 0.1 mL buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% DDM, 1 mM EDTA, 3 mM MgCl2, 10 μM AVP) and incubating at room temperature for 2 hours. Figure 19 a). Subsequently, the AVP:NT4LV2R:Gs complex was purified in a two-step consecutive affinity purification process. The purification was monitored by SDS-PAGE. Figure 19 First, the complex was used on a Ni-NTA column after adding 300 μl of 1% MNG to a buffer of 10 mM HEPES, pH 7.5, and 100 mM NaCl. After thorough washing with buffer, the complex was eluted in 0.2% MNG, 10 mM HEPES, pH 7.5, 100 mM NaCl, 10 μM AVP, and 200 mM imidazole. Subsequently, the complex was used on a FLAG-tagged affinity column, thoroughly washed with the same buffer containing 0.01% MNG, and eluted with the FLAG peptide. The process was monitored by SDS-PAGE. Figure 19 (b) This process showed that the complex containing NT4LVTR, GαS, Gβ, Gγ, and Nb35 could be purified accordingly. The complex was further purified by SEC on a Superdex 200 column in 10 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% MNG, 1 mM EDTA, 3 mM MgCl2, and 1 μM AVP. The process was monitored by SDS-PAGE. Figure 19 c) shows that monodisperse complexes containing NT4LVTR, GαS, Gβ, Gγ and Nb35 can be purified accordingly.

[0324] To verify the stability of the AVP:NT4LV2R:Gs complex, the purified sample was incubated on ice for 24 hours and then reused in SEC to verify its monodispersity characteristics and its molecular weight (MW). Figure 20 As expected, excessive antagonist SR121463 (10 μM) disrupted the AVP:NT4LV2R:Gs complex.

[0325] Example 10. Screening for improvements in agonists or orthoallosteric regulators using nanobody-stabilized GPCR:G protein complexes.

[0326] Nanobodies of non-major conformational isomers of selectively stable GPCRs will allow for more efficient screening of ligands that selectively interact with a particular low-abundance conformational isomer. Furthermore, conformational isomer-selective nanobodies can be used to expose allosteric or hidden sites suitable for drug development, or to reverse-mask binding pockets that are undesirable for drug screening. When a particular conformational isomer is in its active state, the identified ligand has a high probability of acting as an agonist, a result supported by computer-simulated in-silico docking experiments described by Costanzi & Vilar (2011). In fact, their results suggest that activated structures are more favorable for identifying agonists than antagonists, while inactivated structures are more favorable for identifying receptor blockers than agonists.

[0327] The evidence provided is that Nb35 stabilizes the complex between activated β2AR and the G protein by binding to the interface of the Gαs and Gβγ subunits. An example of a screening assay to identify ligands that selectively interact with conformational isomers of β2AR in low-abundance active states could be a radioligand assay, which utilizes Nb35 to shift the β2AR population further toward its active state. Such radioligand assays can be performed similarly with minimal modifications, as described by Seifert et al. (1998). This paper describes an assay involving β2AR as a target of selection by replacing the target of β2AR with any other GPCR that interacts with the Gαs subunit, allowing for the performance of similar screening methods to identify agonist ligands targeting that particular GPCR. Candidate agonists can be identified by screening libraries of small molecules (compounds typically between 250 and 1000 Da) or even fragment-based libraries (compounds typically <250 Da). Stabilizing non-major conformational isomers will significantly improve the performance of fragment library screening, especially since initial hits in fragment-based drug screening often have low titer / affinity. Nb35 will selectively increase affinity for compounds that are specific to selective conformational isomers suitable for drug development, and therefore have profound implications for the identification of de novo fragments.

[0328] In an incubation buffer (50 mM Hepes pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 100 mM NaCl, and 0.5% w / v BSA), an appropriate amount (typically 10 μg) of homogenized human β2AR membrane extract from HEK293T cells containing membranes anchored to G protein subunits was incubated in parallel at 30 °C for 1 h with Nb35 or unrelated nanobodies (unstable active GPCR conformation). The nanobodies were exogenously provided in a large molar excess (e.g., ≥1 μM) relative to the adrenergic receptor. The nanobodies-bound membranes were then added to 96-well plates containing the library compound and 2 nM of a 3H-dihydroapranol (DHA) antagonistic radioligand. The total volume of each well was adjusted to 100 μl with incubation buffer, and the reaction mixture was further incubated at 30 °C for an additional 1 h. Subsequently, the radioligands bound to the membrane were harvested using a GF / C glass fiber 96-well filter plate (Perkin Elmer) pre-soaked in 0.3% polyethyleneimine. The filter plate was washed with ice-cold washing buffer (50 mM Tris-HCl pH 7.4) and dried at 50°C for 30 min. Then, 25 μl of MicroScintillator was added. TM Following the administration of Nb35 (PerkinElmer), radioactivity (cpm) was measured in a Wallac MicroBeta TriLux scintillation counter. Library compounds exhibiting a significant reduction in cpm in the presence of Nb35 without the application of unrelated nanobodies were considered agonist ligands. Candidate agonist hits identified through initial library screening were re-screened in a dose-response manner. The IC50 values ​​of the % radioligand substitution curves for each candidate agonist in the presence of Nb35 and unrelated nanobodies were calculated using Graphpad Prism software. To confirm the effective agonist activity of the de novo identified compounds, the dose-dependent effects of these compounds were evaluated in cellular β2AR signaling assays. One example of such assays relies on the detection of second messenger molecules, such as cAMP following Gαs-mediated signaling (e.g., HitHunter cAMP assay, DiscoverX). In addition to using membrane extracts and exogenously applied Nb35, radioligand assays can be performed on β2AR-expressing cell lines (or membrane-derived cells) co-transfected with Nb35 as an intracellular antibody, thereby shifting the β2AR population to its active state. Optionally, recombinant G proteins and β2AR can be used to stabilize the active state of β2AR via Nb35.

[0329] Materials and methods in the embodiments

[0330] Expression and purification of β2AR, Gs heterotrimer and nanobody-35

[0331] In Sf-9 insect cell cultures infected with recombinant baculovirus (BestBac, Expression Systems), the T4 lysozyme-β2AR construct (T4L-β2AR, detailed below), truncated at position 365 and fused to the N-terminus, was expressed. The construct was dissolved in dodecyl-β-D-maltose glycoside (DDM) according to the aforementioned method (Kobilka et al., 1995). Figure 6 (Abstract on purification). Most analytical and deuterium exchange assays used the truncated β2AR construct (β2AR-365; SEQ ID NO: 55) after residue 365. M1Flag affinity chromatography (Sigma) was performed as the initial purification step, followed by aprol-Sepharose chromatography to select for functional receptors. A subsequent M1Flag affinity chromatography step exchanged the receptor-bound aprol with the high-affinity agonist BI-167107. The agonist-bound receptor was eluted, dialyzed against buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.1% DDM, and 10 μM BI-167107), treated with λ phosphatase (New England Biolabs), and concentrated to approximately 50 mg / mL using a Millipore concentrator with a 50 kDa molecular weight cutoff (MWCO). -1 Between spin-concentrations, the β2AR-365 construct, instead of T4L-β2AR, was treated with PNGase F (New England Biolabs) to remove N-terminal N-linked glycosylation. The purified receptor was routinely analyzed by SDS-PAGE / Coomassie Brilliant Blue staining (see [link to analysis]). Figure 5 a).

[0332] Bovine Gα was expressed in High 5 insect cells (Invitrogen) grown in Insect Xpress serum-free medium (Lonza). s Short (Bovine Gα) s(short), rat Gβ1 and rat Gγ2 fused with the His6 tag (see Table 5). Cultures were grown to a density of 1.5 million cells / ml and then infected with three separate Autographa californica nucleopolyhedroviruses, each containing the gene for one 1:1 co-infected G protein subunit (viruses donated by Dr. Alfred Gilman). After incubation for 40–48 hours, infected cells were harvested by centrifugation and resuspended in 75 ml lysis buffer per liter of culture (50 mM HEPES pH 8.0, 65 mM NaCl, 1.1 mM MgCl2, 1 mM EDTA, 1x PTT (35 μg / ml phenylmethanesulfonyl fluoride, 32 μg / ml tosylphenylalanyl chloromethyl ketone, 32 μg / ml tosylphenylalanyl chloromethyl ketone), 1x LS (3.2 μg / ml leucopeptide and 3.2 μg / ml soybean trypsin inhibitor), 5 mM β-ME, and 10 μM GDP). The suspension was pressurized with 600 psig N2 for 40 minutes in a nitrogen cavitation bomb (Parr Instrument Company). After depressurization, the lysate was centrifuged to remove nuclei and unlysed cells, then ultracentrifuged at 180,000 x g for 40 minutes. Using a Dounce homogenizer, the precipitated membrane was resuspended in 30 ml wash buffer per liter of culture (50 mM HEPES pH 8.0, 50 mM NaCl, 100 μM MgCl2, 1 x PTT, 1 x LS, 5 mM β-ME, 10 μM GDP) and centrifuged again at 180,000 x g for 40 minutes. The washed precipitate clumps were resuspended in a small volume of wash buffer and flash-frozen with liquid nitrogen.

[0333] Thaw the frozen membrane and dilute with fresh wash buffer to a total protein concentration of 5 mg / ml. Add sodium cholate detergent to the suspension to a final concentration of 1.0%, MgCl2 to a final concentration of 5 mM, and 0.05 mg of purified protein phosphatase 5 (internally prepared) per liter of culture volume. Stir the sample on ice for 40 min, then centrifuge at 180,000 x g for 40 min to remove insoluble residue. Dilute the supernatant 5-fold with Ni-NTA loading buffer (20 mM HEPES pH 8.0, 363 mM NaCl, 1.25 mM MgCl2, 6.25 mM imidazole, 0.2% Anzergent 3-12, 1 x PTT, 1 x LS, 5 mM β-ME, 10 μM GDP), adding the buffer slowly and carefully to avoid causing the bile acid concentration to drop too quickly below its critical micelle concentration. Add 3 ml of pre-equilibrated Ni-NTA resin (Qiagen) in Ni-NTA wash buffer 1 (20 mM HEPES pH 8.0, 300 mM NaCl, 2 mM MgCl2, 5 mM imidazole, 0.2% cholic acid, 0.15% Anzergent 3-12, 1x PTT, 1x LS, 5 mM β-ME, 10 μM GDP) per liter of culture volume, and stir the sample on ice for 20 minutes. The resin was collected into a gravity column and washed with 4 column volumes of Ni-NTA wash buffer 1, Ni-NTA wash buffer 2 (20 mM HEPES pH 8.0, 50 mM NaCl, 1 mM MgCl2, 10 mM imidazole, 0.15% Anzergent 3-12, 0.1% DDM, 1x PTT, 1x LS, 5 mM β-ME, 10 μM GDP) and Ni-NTA wash buffer 3 (20 mM HEPES pH 8.0, 50 mM NaCl, 1 mM MgCl2, 5 mM imidazole, 0.1% DDM, 1x PTT, 1x LS, 5 mM β-ME, 10 μM GDP). Elute the protein with Ni-NTA elution buffer (20 mM HEPES pH 8.0, 40 mM NaCl, 1 mM MgCl2, 200 mM imidazole, 0.1% DDM, 1x PTT, 1x LS, 5 mM β-ME, 10 μM GDP). Mix the protein fraction and add MnCl2 to a final concentration of 100 μM. Add 50 μg of purified λ protein phosphatase (internally prepared) per liter of culture volume, and incubate the eluent on ice with stirring for 30 minutes.The eluent was passed through a 0.22 μm filter and directly loaded onto a MonoQ HR 16 / 10 column (GE Healthcare) equilibrated in MonoQ buffer A (20 mM HEPES pH 8.0, 50 mM NaCl, 100 μM MgCl2, 0.1% DDM, 5 mM β-ME, 1x PTT). The column was washed with 150 mL of buffer A at 5 mL / min, and the bound proteins were eluted with a linear gradient of over 350 mL of MonoQ buffer B (identical to buffer A except for 1 M NaCl) up to 28%. The fraction was collected into tubes containing sufficient GDP to bring the final concentration to 10 μM. The Gs-containing fraction was concentrated to 2 mL using a stirred ultrafiltration cell with a 10 kDa NMWL regenerated cellulose membrane (Millipore). The concentrated sample was run on a Superdex 200prep grade XK 16 / 70 column (GE Healthcare) equilibrated with S200 buffer (20 mM HEPES pH 8.0, 100 mM NaCl, 1.1 mM MgCl2, 1 mM EDTA, 0.012% DDM, 100 μM TCEP, 2 μM GDP). The fraction containing pure Gs was mixed, glycerol was added to a final concentration of 10%, and the protein was concentrated to at least 10 mg / ml using a 30 kDa MWCO regenerated cellulose Amicon centrifuge ultrafiltration device. The concentrated sample was then aliquoted, flash-frozen, and stored at -80°C. The typical yield of the final, purified Gs heterotrimer from an 8-liter cell culture volume is 6 mg.

[0334] Following the aforementioned method, nanobody-35 (Nb35) (SEQ ID NO: 1) was expressed in the periplasm of *Escherichia coli* strain WK6. The nanobody was extracted and purified by nickel affinity chromatography (Rasmussen et al., 2011), followed by ion exchange chromatography using a Mono S 10 / 100 GL column (GE Healthcare). Figure 7 a). The selected Nb35 fraction was dialyzed against buffer (10 mM HEPES, pH 7.5, 100 mM NaCl) and concentrated to approximately 65 mg / mL using a 10 kDa MWCO Millipore concentrator. -1 .

[0335] Protein modification

[0336] To increase the likelihood of obtaining R:G complex crystals, two strategies were employed to increase the polar surface area of ​​the receptor's extracellular surface. The method involved replacing the flexible and hypothetically unstructured N-terminus with the previously used globular protein T4 lysozyme (T4L), crystallizing and resolving the receptor bound to caralol (Rosenbaum et al., 2007). The construct used in this study (T4L-β2AR) contains a cleavable signal sequence followed by an M1Flag epitope (DYKDDDDA; SEQ ID NO: 70), a TEV protease recognition sequence (ENLYFQG; SEQ ID NO: 71), phage T4 lysozymes from N2 to Y161 (containing C54T and C97A mutations), and two remaining alanine linkers fused to the human β2AR sequence from D29 to G365 (T4L-β2AR fusion construct as defined in SEQ ID NO: 69). The PNGaseF-inaccessible glycosylation site of β2AR at N187 was mutated to Glu. Replacing M96 and M98 in the first extracellular loop with Thr was used to increase the low expression level of additional T4L-β2AR. Threonine mutations did not affect ligand binding. 3 The affinity for H-dihydroapranol is increased, but this results in a slight decrease in affinity for isoproterenol, approximately 2-fold (data not shown). It should be noted that the wild-type reference β2AR used in this paper is defined by SEQ ID NO: 72.

[0337] Microcrystal data collection and processing

[0338] Data collection was performed on the Advanced Photon Source beamline 23 ID-B. Hundreds of crystals were screened, and the final dataset was compiled using diffraction wedges typically 10 degrees from the 20 strongest diffraction crystals. All data preparation was performed using an HKL2000 (Otwinowski et al., 1997). Although in many cases, more than [a certain number of crystals] were visible in the initial framework... While diffraction is possible, radioactive degradation and anisotropic diffraction result in lower integrity within the high-resolution shell. Analysis of the final dataset by the UCLA Anisotropic Diffraction Service (Strong et al., 2006) suggests that diffraction along the a* reciprocal axis is better than diffraction along other directions. Based on an F / sigF cutoff of 3 along each reciprocal space axis, anisotropic truncation of the reflections before refinement limits the resolution to 2.9, 3.2, and 3.2 Å along a*, b*, and c*, respectively. Due to the lower integrity of the high-resolution shell, the overall resolution of the structure is reported to be only [missing information]. However, it should be noted that the refinement and graph calculation process includes some steps to achieve... The diffraction data.

[0339] Structural analysis and refinement

[0340] The structure was resolved by molecular substitution using Phaser (McCoy et al., 2007a,b). The order of molecular substitution studies was found to be crucial in the structural resolution. The model used was derived from the following sequence: β and γ subunits of the Gi heterotrimeric G protein (PDB ID: 1GP2), Gsαras domain (PDB ID: 1AZT), active-state β2-adrenergic receptor (PDB ID: 3P0G), β2-binding nanobody (PDB ID: 3P0G), T4 lysozyme (PDB ID: 2RH1), and the Gsα helical domain (PDB ID: 1AZT). After determining the initial structure by molecular substitution, rigid body refinement and simulated annealing were performed in Phenix (Afonine et al., 2005) and BUSTER (Blanc et al., 2004), followed by constraint refinement and manual reconstruction in Coot (Emsley et al., 2004). After iterative refinement and manual adjustments, the structure was refined in CNS using the DEN method. While the resolution of this structure exceeded the generally most effective resolution of DEN, the presence of several poorly resolved regions suggests that integrating additional information to guide the refinement could yield better results. The DEN reference model used was the one described above for molecular substitution studies, with the exception of NB35, which is highly ordered and for which higher resolution structures could not be obtained. PyMOL (The PyMOL Molecular Graphics System, version 1.3) was used. Chart by LLC. Table 7 presents the refined statistics.

[0341] Combination

[0342] As previously described, membranes expressing β2AR or β2AR-Gs peptide fusions were prepared from Sf9 cells infected with baculovirus, and the following procedures were implemented. 3 H-dihydroaprolol ( 3 H-DHA) binding (Swaminath et al., 2002). For competitive binding, before harvesting to the GF / B membrane, use... 3 H-DHA (final 1.1 nM) and increasing concentrations of (-)-isoproterenol (ISO) were incubated on the membrane for 1 hour. Competitive data were fitted with a two-site binding model, and high and low Ki and scores of ISO were calculated using GraphPad Prism.

[0343] Purification of NT4LV2R

[0344] The N-terminal fused T4LV2R construct (NT4L-V2R; SEQ ID NO: 73) was expressed in Sf9 cells using the baculovirus system (PfastBac). (4 × 10⁻⁶) 6 Cells were infected at a density of cells / ml and cultured in shaking flasks at 27°C for 48 h. After harvesting, cells were cultured in a solution containing 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 μM Tolvaptan (Sigma), and 2 mg / ml. -1 Cells were lysed by osmotic shock in a buffer containing iodoacetamide, used to block reactive cysteine. The buffer solution contained 0.5% dodecyl maltodextrin (DDM), 0.3% cholate, 0.03% cholesterol hemisuccinate (CHS), 20 mM HEPES (pH 7.5), 0.5 M NaCl, 30% v / v glycerol, and 2 mg / mL HEPES. -1 NT4L-V2R was extracted from the Sf9 membrane using a Dounce homogenizer in a dissolution buffer of iodoacetamide and 1 μM Tolvaptan. After centrifugation, nickel-NTA agarose was added to the supernatant, and the mixture was stirred for 2 h. The membrane was then washed in fractions at 100 g spin for 5 min each time with a wash buffer of 0.1% DDM, 0.03% Cholate, 0.01% CHS, 20 mM HEPES (pH 7.5), and 0.5 M NaCl. The resin was poured into a glass column, and the bound receptor was eluted in a wash buffer supplemented with 300 mM imidazole. NT4L-V2R was further purified using anti-Flag M1 affinity resin, exchanging ligands with the agonist AVP. The Ni-NTA resin eluent was loaded onto the anti-Flag M1 resin and washed thoroughly in the presence of 10 μM AVP. Then, the ligands were extracted from the anti-Flag M1 affinity resin with 0.2 mg / mL of the solution in the presence of 1 μM AVP. -1 Flag peptide and 2 mM EDTA were used to elute the receptor and then concentrated using a 100 kDa MWCO concentrator.

[0345] Table 2. List of Nanobodies

[0346]

[0347]

[0348] Table 4. Nucleic acid sequences of nanobodies

[0349]

[0350]

[0351] Table 5: Examples of isotypes of G protein subunits

[0352]

[0353]

[0354]

[0355]

[0356] Table 6. Potential intermolecular interactions within the R:G interface

[0357]

[0358] Table 7. Statistics on Data Collection and Refinement

[0359]

[0360] *The values ​​in parentheses represent the highest shell statistics. a These regions were omitted from the model due to poor electron density resolution. Unmodeled purification tags are not included within these residue ranges. b The construct omits residues 1-28 of β2AR, and T4L is fused to the amino terminus of transmembrane helix 1, which is conducive to crystallization. c The T4L residue was omitted from the construct. d As defined by MolProbity.

[0361] References

[0362] -Afonine, PV, Grosse-Kunstleve, RW, & Adams, PD (2005). A robust bulk-solvent correction and anisotropic scaling procedure. Actacrystallographica. Section D, Biological crystallography, 61, 850-5.

[0363] -Baltensperger, K. et al. The beta-adrenergic receptor is a substrate for the insulin receptor tyrosine kinase. J Biol Chem 271, 1061-1064 (1996).

[0364] -Binz et al., Nature Biotech., 22:575-582 (2004).

[0365] -Blanc, E., Roversi, P., Vonrhein, C., Flensburg, C., Lea, S. M., Bricogne, G., et al. (2004). Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta crystallographica. Section D, Biological crystallography, 60, 2210-2221.

[0366] -Caffrey (2003). Membrane protein crystallization. J Struct. Biol. 2003 142: 108-132.

[0367] -Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat Protoc 4, 706-731, (2009).

[0368] -Chae, P.S. et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat Methods 7, 1003-1008 (2010).

[0369] -Chelikani et al. Protein Sci. 2006 15: 1433-1440.

[0370] -Chini, B., & Parenti, M. (2009). G-protein-coupled receptors, cholesterol and palmitoylation: facts about fats. Journal of molecular endocrinology, 42(5), 371-379.

[0371] -Chomczynski, P. and Sacchi, N., 1987. Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal.

[0372] Biochem. 162, p. 156.

[0373] -Conrath K, Pereira AS, Martins CE, Timóteo CG, Tavares P, Spinelli S, Kinne J, Flaudrops C, Cambillau C, Muyldermans S, Moura I, Moura JJ, Tegoni M, Desmyter A. Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase. Protein Sci. 2009 Mar; 18(3): 619-28.

[0374] -Conrath K.E., M.Lauwereys, M.Galleni et al., Antimicrob Agents Chemother 45(10), 2807(2001).

[0375] -Costanzi S, Vilar S(2011). In Silico screening for agonists and blockers of the beta(2) adrenergic receptor: Implications of inactive and activated state structures. Journal of computational chemistry 33: 561-572.

[0376] -Day P.W., Rasmussen S.G., Parnot C., Fung J.J., Masood A., Kobilka T.S., Yao X.J., Choi H.J., Weis W.I., Rohrer D.K., et al., A monoclonal antibody for G protein-coupled receptor crystallography, Nat Methods 4(2007), pp. 927–929.

[0377] -Delean, A., J.M. Stadel, et al. (1980). "A TERNARY COMPLEX MODEL EXPLAINS THE AGONIST-SPECIFIC BINDING-PROPERTIES OF THE ADENYLATE CYCLASE-COUPLED BETA-ADRENERGIC-RECEPTOR." JOURNAL OF BIOLOGICAL CHEMISTRY 255(15): 7108-7117.

[0378] -Derewenda Z.S. Rational protein crystallization by mutational surface engineering, Structure(Camb) 12(2004), pp. 529–535.

[0379] -Domanska, K. et al. Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic beta2-microglobulin variant. Proc Natl Acad Sci U S A 108, 1314-1319, (2011).

[0380] -Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-2132, (2004).

[0381] -Eroglu et al. EMBO 2002 3: 491^96

[0382] -Eroglu et al., Proc. Natl. Acad. Sci. 2003 100:10219-10224

[0383] -Faham et al., Crystallization of bacteriorhodopsin from bicelle formulations at room temperature. Protein Sci. 2005 14:836-40. 2005

[0384] -Faham et al., Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J Mol Biol. 2002 Feb 8; 316(1):1-6.

[0385] -Foord, S.M., T.I. Bonner, et al. (2005). "International Union of Pharmacology. XLVI. G protein-coupled receptor list." Pharmacological reviews 57(2):279-288.

[0386] -Fredriksson, R., M.C. Lagerstrom, et al. (2003). "The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints." Molecular Pharmacology 63(6):1256-1272.

[0387] -Gebauer & Skerra (2009) Current opinion in chemical biology 13, 245-255

[0388] -George et al., Nat Rev Drug Discov 1:808-820(2002)

[0389] -Gouaux, It's not just a phase: crystallization and X-ray structure determination of bacteriorhodopsin in lipidic cubic phases. Structure. 1998 6:5-10;

[0390] -Hamers-Casterman, C., T. Atarhouch, S. Muyldermans et al., Naturally occurring antibodies devoid of light chains. Nature 363, 446-448, doi: 10.1038 / 363446a0 (1993).

[0391] -Hendrickson WA. Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science. 1991 Oct 4; 254(5028):51-8.

[0392] -Hofmann K.P., P. Scheerer, P.W. Hildebrand et al., Trends Biochem Sci 34(11), 540 (2009).

[0393] -Hunte C. and Michel H., Crystallisation of membrane proteins mediated by antibody fragments, Curr Opin Struct Biol 12(2002), pp. 503–508.

[0394] -Jameson, E.E. et al. (2005). Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues. J Biol Chem 280, 7712-7719.

[0395] -Kenakin, Trends Pharmacol Sci 25:186-192(2002)

[0396] -Kobilka et al. (2007) Trends in pharmacological sciences 28, 397-406.

[0397] -Kobilka, B. K. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal Biochem 231, 269-271 (1995).

[0398] -Koide et al., J. Mol Biol, 284: 1141-1151 (1998))

[0399] -Kolakowski, L. F. (1994). "GCRDB-A G-PROTEIN-COUPLED RECEPTOR DATABASE." Receptors & Channels 2(1): 1-7.

[0400] -Kolb, P., D. M. Rosenbaum, et al. (2009). "Structure-based discovery of beta(2)-adrenergic receptor ligands." Proceedings of the National Academy of Sciences of the United States of America 106(16): 6843-6848.

[0401] -Kuszak, A. J., S. Pitchiaya, et al. (2009). "Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2." The Journal of biological chemistry 284: 26732-26741.

[0402] - M. C. and H. B. (2008). "Structural diversity of G protein-coupled receptors and significance for drug discovery." Nature reviews. Drug discovery 7:339-357.

[0403] -Landau et al., Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. 1996 93:14532-5

[0404] -Lee, A.G. (2004). How lipids affect the activities of integral membrane proteins. Biochimica et biophysica acta, 1666(1-2), 62-87.

[0405] -Lee GM, Craik CS (2009). Trapping moving targets with small molecules. Science. Apr 10; 324(5924):213-5.

[0406] -Lefranc, M.P., C. Pommie, et al. (2003). "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains." Developmental and Comparative Immunology 27(1):55-77.

[0407] -Li H., Dunn J.J., Luft B.J. and Lawson C.L., Crystal structure of Lyme disease antigen outer surface protein A complexed with an Fab, Proc Natl Acad Sci U S A 94(1997), pp 3584–3589

[0408] -Luca et al., Proc. Natl. Acad. Sci. 2003 100:10706-11

[0409] -Lynch Kevin R. (Ed.) Identification and Expression of G Protein-Coupled Receptors published by John Wiley & Sons (March 1998)

[0410] -Mansoor et al., Proc. Natl. Acad. Sci. 2006 103:3060-3065

[0411] -Marchese et al., Genomics 23:609-618, 1994

[0412] -McCoy, A.J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr D Biol Crystallogr 63, 32-41 (2007).

[0413] -McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., Read, R.J., et al. (2007). Phaser crystallographic software. Journal of applied crystallography, 40(Pt 4), 658-674.

[0414] -McEwen, D.P., Gee, K.R., Kang, H.C. & Neubig, R.R. (2001) Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins. Anal Biochem 291, 109-117.

[0415] -Misquitta, L. V. et al., Membrane protein crystallization in lipidic mesophases with tailored bilayers. Structure 12, 2113-2124, (2004).

[0416] -Moro, O., Lameh, J., Hogger, P. & Sadee, W. Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling. J Biol Chem 268, 22273-22276 (1993).

[0417] -Niu et al., Biophys J. 2005 89: 1833-1840

[0418] -Nollert et al., Lipidic cubic phases as matrices for membrane protein crystallization Methods. 2004 34: 348-53

[0419] -Ostermeier C., Iwata S., Ludwig B. and Michel H., Fv fragment-mediated crystallization of the membrane protein bacterial cytochrome c oxidase, Nat Struct Biol 2(1995), pp. 842–846.

[0420] -Otwinowski, Z., & Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods in enzymology, 276, 307–325.

[0421] -Palczewski, K. et al., Crystal structure of rhodopsin: A G protein-coupled receptor [see comments]. Science 289, 739-745 (2000).

[0422] -Probst et al., 1992, DNA Cell Biol. 1992 1 1:1-20;

[0423] -Qian Z.M., Li H., Sun H. and Ho K (2002). Targeted drug delivery via the transferring receptor-mediated endocytosis pathway. Pharmacol Rev 54, 561-587.

[0424] -Rasmussen et al. (2011) Nature 469, 175-180.

[0425] -Rasmussen S.G., Choi H.J., Rosenbaum D.M., Kobilka T.S., Thian F.S., Edwards P.C., Burghammer M., Ratnala V.R., Sanishvili R. and Fischetti R.F. et al., Crystal structure of the human beta2 adrenergic G-protein-coupled receptor, Nature 450 (2007), pp. 383–387.

[0426] -Riechmann and Muyldermans J. Immunol. Methods 2000; 240:185-195.

[0427] -Rios et al., Pharmacol Ther 92:71-87 (2001).

[0428] -Ritter, S.L., & Hall, R.a. (2009). Fine-tuning of GPCR activity by receptor-interacting proteins. Nature reviews. Molecular cell biology, 10(12), 819-30. Nature Publishing Group. doi: 10.1038 / nrm2803.)

[0429] -Rosenbaum D.M., S.G. Rasmussen, and B.K. Kobilka, Nature 459(7245), 356(2009).

[0430] -Rosenbaum, D.M. et al. Structure and function of an irreversible agonist-beta(2) adrenoceptor complex. Nature 469, 236-240(2011).

[0431] -Rosenbaum, D.M., V. Cherezov, et al. (2007). "GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function." Science 318: 1266-1273.

[0432] -Rummel et al., Lipidic Cubic Phases: New Matrices for the Three-Dimensional Crystallization of Membrane Proteins. J. Struct. Biol. 1998 121: 82-91;

[0433] -Sawant R, Torchilin V. Intracellular transduction using cell-penetrating peptides. Mol Biosyst. 2010 Apr; 6(4): 628-40. Epub 2009 Dec 21.

[0434] -Seifert et al. (1998). Reconstitution of beta2-adrenoceptor-GTP-binding-protein interaction in Sf9 cells--high coupling efficiency in a beta2-adrenoceptor-G(s alpha) fusion protein. Eur. J. Biochem. 255: 369-382.

[0435] -Shimada et al., J. Biol. Chem. 2002 277:31774-80

[0436] -Skerra, J. Molecular Recognition, 13:167-187 (2000)

[0437] -Sprang, S. R. G protein mechanisms: insights from structural analysis. Annu Rev Biochem 66, 639-678 (1997).

[0438] -Starovasnik et al., Proc. Natl. Acad. Sci. USA, 94:10080-10085 (1997)

[0439] -Steyaert J, Kobilka BK (2011). Nanobody stabilization of G protein-coupled receptor conformational states. Curr Opin Struct Biol. Aug; 21(4):567-72.

[0440] -Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE / PPE protein complex from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 103, 8060-8065, (2006).

[0441] -Sunahara, R.K., Tesmer, J.J., Gilman, A.G. & Sprang, S.R. Crystal structure of the adenylyl cyclase activator Gsalpha [see comments]. Science 278, 1943-1947 (1997).

[0442] -Swaminath, G., Steenhuis, J., Kobilka, B. & Lee, T.W. Allosteric modulation of beta2-adrenergic receptor by Zn(2+). Mol Pharmacol 61, 65-72. (2002).

[0443] -Edited by Tatsuya Haga, G Protein-Coupled Receptors, published by CRC Press (September 24, 1999)

[0444] -Van Eps, N. et al. Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit. Proc Natl Acad Sci U S A (2011).

[0445] -Wall, M.A. et al. The structure of the G protein heterotrimer Gia1 b1 g2. Cell 83, 1047-1058 (1995).

[0446] -Warne, T. et al. Structure of a beta1-adrenergic G-protein-coupled receptor. Nature 454, 486-491, (2008).

[0447] -Edited by Watson, S., G-Protein Linked Receptor Factsbook, published by Academic Press (1st edition; 1994).

[0448] -Edited by Wess Jurgen, Structure-Function Analysis of G Protein-Coupled Receptors, published by Wiley-Liss (1st edition; October 15, 1999)

[0449] -Whorton, MR et al. A monomeric G protein-coupled receptor isolated in ahigh-density lipoprotein particle efficiently activates its G protein. ProcNatl Acad Sci USA 104, 7682-7687 (2007).

[0450] -Whorton, MR, SGFRasmussen, et al. (2009). "The effect of ligandefficacy on the formation and stability of a GPCR-G protein complex." PNAS106:1-6.

[0451] The present invention relates to, for example but not limited to, the following embodiments:

[0452] 1. Binding domains that target and / or specifically bind to complexes containing GPCRs and G proteins.

[0453] 2. According to the binding domain of Implementation Scheme 1, its affinity for binding to the complex is higher than that for binding to heterotrimeric G protein alone and / or GPCR alone.

[0454] 3. According to the binding domain of implementation scheme 1, it specifically binds to G proteins but not to GPCRs.

[0455] 4. According to the binding domain of implementation scheme 1, the complex further includes a receptor ligand.

[0456] 5. According to the binding domain of implementation scheme 4, the complex consists of GPCR, G protein and receptor ligand.

[0457] 6. The binding domain according to any one of embodiments 4 or 5, wherein the receptor ligand is an agonist.

[0458] 7. A binding domain according to any one of the implementation schemes 1 to 6, wherein the G protein is in a nucleotide-free form.

[0459] 8. The binding domain according to any one of embodiments 1 to 7, which specifically binds to a conformational epitope located at the interface between the α and β subunits of the G protein.

[0460] 9. According to any of the combined structural domains in implementation schemes 1 to 8, its

[0461] a) Preventing or inhibiting the dissociation of the complex in the presence of nucleotides, or

[0462] b) Prevent or inhibit the binding of nucleotides to G proteins, or

[0463] c) Nucleotides that can replace G proteins.

[0464] 10. A binding domain according to any one of embodiments 7 to 9, wherein the nucleotide is a guanine nucleotide, such as GDP or GTP, or an analogue thereof, such as GTPγS.

[0465] 11. A binding domain according to any one of the implementation schemes 1 to 10, wherein the GPCR is in the active conformation.

[0466] 12. The binding domain according to any one of the implementation schemes 1 to 11, wherein the G protein is selected from Gs, Gi, Go, Gt, Ggust, Gz, Golf, Gq, G12, and G13.

[0467] 13. The binding domain according to any one of the implementation schemes 1 to 11, wherein the GPCR is selected from Gs-coupled receptors, Gi-coupled receptors, Go-coupled receptors, Gt-coupled receptors, Ggust-coupled receptors, Golf-coupled receptors, Gq-coupled receptors, G12-coupled receptors, and G13-coupled receptors.

[0468] 14. The binding domain of any one of embodiments 1 to 13, wherein the GPCR is a human protein.

[0469] 15. A binding domain according to any one of embodiments 1 to 14, wherein the binding domain is an immunoglobulin monovariable domain comprising an amino acid sequence comprising four frame regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3) according to the following general formula (1):

[0470] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1).

[0471] 16. The binding domain according to embodiment 15, wherein the immunoglobulin monovariable domain comprises an amino acid sequence comprising four frame regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3) according to the following general formula (1):

[0472] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1),

[0473] And CDR1 is selected from:

[0474] a)SEQ ID NO:13-18,

[0475] b) A polypeptide having at least 80% amino acid identity with SEQ ID NO:13-18,

[0476] c) A polypeptide that differs from SEQ ID NO:13-18 by 3, 2, or 1 amino acid.

[0477] And CDR2 is selected from:

[0478] a)SEQ ID NO:25-30,

[0479] b) A polypeptide having at least 80% amino acid identity with SEQ ID NO:25-30,

[0480] c) A polypeptide that differs from SEQ ID NO:25-30 by 3, 2, or 1 amino acid.

[0481] And CDR3 is selected from:

[0482] a)SEQ ID NO:37-42,

[0483] b) A polypeptide having at least 80% amino acid identity with SEQ ID NO:37-42,

[0484] c) A polypeptide that differs from SEQ ID NO:37-42 by 3, 2 or 1 amino acid.

[0485] 17. According to the binding domain of implementation scheme 15, wherein the immunoglobulin monovariable domain is a nanobody (V H H).

[0486] 18. The binding domain according to embodiment 17, wherein the nanobody has an amino acid sequence selected from SEQ ID NO:1 to 6 or a variant thereof.

[0487] 19. A binding domain according to any one of embodiments 1 to 18, wherein the binding domain is contained in the polypeptide.

[0488] 20. A bonding structural domain according to any one of embodiments 1 to 19, wherein the bonding structural domain is fixed to a solid support.

[0489] 21. A complex comprising a binding domain according to any one of embodiments 1 to 20.

[0490] 22. The complex according to embodiment 21 further comprises a GPCR, a G protein, and an optional receptor ligand.

[0491] 23. The complex according to any one of embodiments 21 to 22 is crystalline.

[0492] 24. A nucleic acid sequence encoding an amino acid sequence of a binding domain according to any one of implementation schemes 1 to 20.

[0493] 25. A recombinant vector containing the nucleic acid sequence according to implementation plan 24.

[0494] 26. Cells containing a vector according to embodiment 25 or a nucleic acid sequence according to embodiment 24.

[0495] 27. Cells according to implementation scheme 26 that express or are able to express GPCRs and / or G proteins.

[0496] 28. Cell cultures of cells according to any one of implementation schemes 26 to 27.

[0497] 29. A cell or cell culture membrane product according to any one of embodiments 26 to 27 or according to embodiment 28.

[0498] 30. The use of the binding domain according to any one of embodiments 1 to 20 for stabilizing a complex, said complex comprising a GPCR and a G protein and optionally a receptor ligand in a functional conformational state.

[0499] 31. According to the use of embodiment 30, it is used to prevent the complex from dissociating in the presence of nucleotides, particularly guanine nucleotides or analogues thereof, such as GTPγS.

[0500] 32. The use of the binding domain according to any one of embodiments 1 to 20 for crystallizing and / or resolving the structure of a complex, said complex comprising a GPCR and a G protein and optionally a receptor ligand.

[0501] 33. The binding domain of any one of embodiments 1 to 20, or the cell of any one of embodiments 26 to 27, or the use of the membrane product of embodiment 29, for screening compounds that regulate the signal transduction activity of GPCRs.

[0502] 34. The use of any one of the implementation schemes 30 to 33, wherein the GPCR is in the active conformation.

[0503] 35. The use of the binding domain according to any one of embodiments 1 to 20 for capturing one or more interacting proteins.

[0504] 36. A method for capturing and / or purifying complexes containing GPCRs and G proteins, the method comprising the steps of:

[0505] a) Provide a combined structural domain according to any one of implementation schemes 1 to 20, and

[0506] b) Allows the binding domain to bind to complexes containing GPCRs and G proteins, and optionally receptor ligands, and

[0507] c) The complex formed in the optional separation step b).

[0508] 37. A method for determining the crystal structure of a complex containing GPCRs and G proteins, comprising the following steps:

[0509] a) Provide a combined structural domain according to any one of implementation schemes 1 to 20, and

[0510] b) Allows the binding domain to bind to complexes containing GPCRs and G proteins and optionally receptor ligands, and

[0511] c) Crystallize the complex formed in step b).

[0512] 38. The use of the binding domain according to any one of embodiments 1 to 20 for regulating GPCR receptor signaling, particularly G protein-mediated GPCR receptor signaling.

[0513] 39. A method for producing a binding domain that targets and / or specifically binds to a complex comprising a GPCR and a G protein, the method comprising the steps of:

[0514] a) Express the nucleic acid according to implementation scheme 24 in a suitable cell expression system, and optionally...

[0515] b) Isolate and / or purify the binding domain.

[0516] 40. A method for screening binding domains that target and / or specifically bind to complexes containing GPCRs and G proteins, the method comprising the steps of:

[0517] a) Provides multiple combined structural domains, and

[0518] b) Among the plurality of binding domains, screen for binding domains that bind to a complex containing GPCR and G protein, and

[0519] c) Separate the binding domains that bind to the complex. sequence list <110> The non-profit organization, the Institute of Biotechnology at the Flemish National University (VIB VZW). Vrije University of Brussels The Board of Trustees of the Leland Stanford Junior University The Regents of the University of Michigan <120> The binding domain of the GPCR:G protein complex and its derived applications. <130> JS / GPROT / 380 <150> US 61 / 571,159 <151> 2011-06-21 <150> EP 11181357.2 <151> 2011-09-15 <160> 73 <170> PatentIn version 3.5 <210> 1 <211> 138 <212> PRT <213> Domestic alpaca (Lama glama) <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Lys Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Ile Ser Gln Ser Gly Ala Ser Ile Ser Tyr Thr Gly Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Cys Pro Ala Pro Phe Thr Arg Asp Cys Phe Asp Val Thr Ser 100 105 110 Thr Thr Tyr Ala Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 His His His His His His Glu Pro Glu Ala 130 135 <210> 2 <211> 138 <212> PRT <213> Lama pacos <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Val Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Ile Ser Asn Gly Gly Gly Thr Thr Ser Tyr Ala Ser Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Gly Leu Lys Pro Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Cys Pro Ala Pro Phe Thr Asn Asp Cys Met Asp Ile Thr Ser 100 105 110 Thr Thr Tyr Ala Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 His His His His His His Glu Pro Glu Ala 130 135 <210> 3 <211> 129 <212> PRT <213> Lama pacos <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Thr Ile Phe Ser Val Thr 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Gly Phe Thr Asn Thr Arg Asn Thr Asn Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Ser Ala Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Arg Arg Trp Gly Gly Thr Asn Trp Asn Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu 115 120 125 Ala <210> 4 <211> 129 <212> PRT <213> Vicugna pacos <400> 4 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Phe Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Lys Asn 20 25 30 Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45 Ala Ala Ser Pro Thr Gly Gly Ser Thr Ala Tyr Lys Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Val Leu Leu 65 70 75 80 Gln Met Asn Val Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His 85 90 95 Leu Arg Gln Asn Asn Arg Gly Ser Trp Phe His Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu 115 120 125 Ala <210> 5 <211> 129 <212> PRT <213> Vicugna pacos <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Phe Asp Ile Thr 20 25 30 Pro Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Val Val 35 40 45 Ala Asp Leu Thr Ser Arg Gly Thr Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Ser Asp Asp Thr Gly Val Tyr Tyr Cys Asn 85 90 95 Val Lys Arg Trp Gly Gly Ile Gly Trp Asn Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu 115 120 125 Ala <210> 6 <211> 129 <212> PRT <213> Vicugna pacos <400> 6 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Arg Phe Ser Asn Phe 20 25 30 Pro Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Ile Gly Gly Ser Thr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Leu Gly Arg Leu Val Pro Pro Thr Thr Glu Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser His His His His His His Glu Pro Glu 115 120 125 Ala <210> 7 <211> 25 <212> PRT <213> domestic alpacas <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 8 <211> 25 <212> PRT <213> domestic alpacas <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 9 <211> 25 <212> PRT <213> domestic alpacas <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser 20 25 <210> 10 <211> 25 <212> PRT <213> domestic alpacas <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Phe Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 11 <211> 25 <212> PRT <213> domestic alpacas <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser 20 25 <210> 12 <211> 25 <212> PRT <213> domestic alpacas <400> 12 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser 20 25 <210> 13 <211> 8 <212> PRT <213> domestic alpacas <400> 13 Gly Phe Thr Phe Ser Asn Tyr Lys 1 5 <210> 14 <211> 8 <212> PRT <213> domestic alpacas <400> 14 Gly Phe Thr Phe Ser Asn Tyr Val 1 5 <210> 15 <211> 8 <212> PRT <213> domestic alpacas <400> 15 Gly Thr Ile Phe Ser Val Thr Val 1 5 <210> 16 <211> 8 <212> PRT <213> domestic alpacas <400> 16 Gly Ser Ile Phe Ser Lys Asn Thr 1 5 <210> 17 <211> 8 <212> PRT <213> domestic alpacas <400> 17 Gly Thr Ile Phe Asp Ile Thr Pro 1 5 <210> 18 <211> 8 <212> PRT <213> domestic alpacas <400> 18 Gly Phe Arg Phe Ser Asn Phe Pro 1 5 <210> 19 <211> 17 <212> PRT <213> domestic alpacas <400> 19 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Asp <210> 20 <211> 17 <212> PRT <213> domestic alpacas <400> 20 Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Asp <210> twenty one <211> 17 <212> PRT <213> domestic alpacas <400> twenty one Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 15 Gly <210> twenty two <211> 17 <212> PRT <213> domestic alpacas <400> twenty two Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala 1 5 10 15 Ala <210> twenty three <211> 17 <212> PRT <213> domestic alpacas <400> twenty three Met Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Val Val Ala 1 5 10 15 Asp <210> twenty four <211> 17 <212> PRT <213> domestic alpacas <400> twenty four Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Leu <210> 25 <211> 8 <212> PRT <213> domestic alpacas <400> 25 Ile Ser Gln Ser Gly Ala Ser Ile 1 5 <210> 26 <211> 8 <212> PRT <213> domestic alpacas <400> 26 Ile Ser Asn Gly Gly Gly Thr Thr 1 5 <210> 27 <211> 7 <212> PRT <213> domestic alpacas <400> 27 Phe Thr Asn Thr Arg Asn Thr 1 5 <210> 28 <211> 7 <212> PRT <213> domestic alpacas <400> 28 Ser Pro Thr Gly Gly Ser Thr 1 5 <210> 29 <211> 7 <212> PRT <213> domestic alpacas <400> 29 Leu Thr Ser Arg Gly Thr Thr 1 5 <210> 30 <211> 8 <212> PRT <213> domestic alpacas <400> 30 Ile Ser Ile Gly Gly Ser Thr Thr 1 5 <210> 31 <211> 38 <212> PRT <213> Lama pacos <400> 31 Ser Tyr Thr Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 32 <211> 38 <212> PRT <213> Lama pacos <400> 32 Ser Tyr Ala Ser Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Gly Leu Lys Pro Ala Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 33 <211> 38 <212> PRT <213> Lama pacos <400> 33 Asn Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Ser 1 5 10 15 Ala Lys Asn Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 34 <211> 38 <212> PRT <213> Lama pacos <400> 34 Ala Tyr Lys Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser 1 5 10 15 Ala Lys Asn Thr Val Leu Leu Gln Met Asn Val Leu Lys Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 35 <211> 38 <212> PRT <213> Lama pacos <400> 35 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Lys Met Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Asp Asp 20 25 30 Thr Gly Val Tyr Tyr Cys 35 <210> 36 <211> 38 <212> PRT <213> Lama pacos <400> 36 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 37 <211> twenty one <212> PRT <213> domestic alpacas <400> 37 Ala Arg Cys Pro Ala Pro Phe Thr Arg Asp Cys Phe Asp Val Thr Ser 1 5 10 15 Thr Thr Tyr Ala Tyr 20 <210> 38 <211> twenty one <212> PRT <213> domestic alpacas <400> 38 Ala Arg Cys Pro Ala Pro Phe Thr Asn Asp Cys Met Asp Ile Thr Ser 1 5 10 15 Thr Thr Tyr Ala Tyr 20 <210> 39 <211> 13 <212> PRT <213> domestic alpacas <400> 39 Asn Val Arg Arg Trp Gly Gly Thr Asn Trp Asn Asp Tyr 1 5 10 <210> 40 <211> 13 <212> PRT <213> domestic alpacas <400> 40 His Leu Arg Gln Asn Asn Arg Gly Ser Trp Phe His Tyr 1 5 10 <210> 41 <211> 13 <212> PRT <213> domestic alpacas <400> 41 Asn Val Lys Arg Trp Gly Gly Ile Gly Trp Asn Asp Tyr 1 5 10 <210> 42 <211> 12 <212> PRT <213> domestic alpacas <400> 42 Ala Lys Tyr Leu Gly Arg Leu Val Pro Pro Thr Thr 1 5 10 <210> 43 <211> 11 <212> PRT <213> domestic alpacas <400> 43 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 44 <211> 11 <212> PRT <213> domestic alpacas <400> 44 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 45 <211> 11 <212> PRT <213> domestic alpacas <400> 45 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 46 <211> 11 <212> PRT <213> Lama pacos <400> 46 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 47 <211> 11 <212> PRT <213> Lama pacos <400> 47 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 48 <211> 11 <212> PRT <213> Lama pacos <400> 48 Glu Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 49 <211> 417 <212> PRT <213> Lama pacos <400> 49 Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly Ala Gly Gly Ala Gly Gly Cys Thr Thr 20 25 30 Gly Gly Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Gly Gly Gly Gly 35 40 45 Thr Cys Thr Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr 50 55 60 Gly Thr Gly Cys Gly Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr 65 70 75 80 Cys Ala Cys Cys Thr Thr Cys Ala Gly Cys Ala Ala Thr Thr Ala Thr 85 90 95 Ala Ala Ala Ala Thr Gly Ala Ala Cys Thr Gly Gly Gly Thr Cys Cys 100 105 110 Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Ala Ala Ala 115 120 125 Gly Gly Gly Gly Cys Thr Cys Gly Ala Gly Thr Gly Gly Gly Thr Cys 130 135 140 Thr Cys Ala Gly Ala Thr Ala Thr Thr Thr Cys Thr Cys Ala Gly Ala 145 150 155 160 Gly Thr Gly Gly Thr Gly Cys Thr Ala Gly Cys Ala Thr Ala Ala Gly 165 170 175 Thr Thr Ala Cys Ala Cys Ala Gly Gly Cys Thr Cys Cys Gly Thr Gly 180 185 190 Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala 195 200 205 Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys Gly Cys 210 215 220 Cys Ala Ala Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr 225 230 235 240 Cys Thr Ala Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys 245 250 255 Thr Gly Ala Ala Gly Cys Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys 260 265 270 Gly Gly Cys Cys Gly Thr Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr 275 280 285 Gly Cys Cys Ala Gly Ala Thr Gly Thr Cys Cys Gly Gly Cys Cys Cys 290 295 300 Cys Ala Thr Thr Cys Ala Cys Gly Ala Gly Ala Gly Ala Thr Thr Gly 305 310 315 320 Thr Thr Thr Thr Gly Ala Cys Gly Thr Gly Ala Cys Thr Ala Gly Thr 325 330 335 Ala Cys Cys Ala Cys Gly Thr Ala Thr Gly Cys Cys Thr Ala Cys Ala 340 345 350 Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Gly Ala Cys Cys Cys Ala 355 360 365 Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala 370 375 380 Cys Ala Cys Cys Ala Cys Cys Ala Thr Cys Ala Cys Cys Ala Thr Cys 385 390 395 400 Ala Cys Gly Ala Ala Cys Cys Thr Gly Ala Ala Gly Cys Cys Thr Ala 405 410 415 Gly <210> 50 <211> 417 <212> PRT <213> Lama pacos <400> 50 Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly Ala Gly Gly Gly Gly Gly Cys Thr Thr 20 25 30 Gly Gly Thr Gly Cys Ala Gly Cys Cys Thr Gly Gly Gly Gly Gly Gly 35 40 45 Thr Cys Thr Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr 50 55 60 Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr 65 70 75 80 Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Ala Ala Cys Thr Ala Thr 85 90 95 Gly Thr Cys Ala Thr Gly Ala Ala Cys Thr Gly Gly Gly Thr Cys Cys 100 105 110 Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Ala Ala Ala 115 120 125 Gly Gly Gly Gly Cys Thr Cys Gly Ala Gly Thr Gly Gly Gly Thr Cys 130 135 140 Thr Cys Ala Gly Ala Thr Ala Thr Thr Thr Cys Thr Ala Ala Thr Gly 145 150 155 160 Gly Cys Gly Gly Thr Gly Gly Thr Ala Cys Cys Ala Cys Ala Ala Gly 165 170 175 Thr Thr Ala Thr Gly Cys Ala Ala Gly Cys Thr Cys Cys Gly Thr Gly 180 185 190 Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala 195 200 205 Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys Gly Cys 210 215 220 Cys Ala Ala Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr 225 230 235 240 Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Ala Cys Gly Gly Cys Cys 245 250 255 Thr Gly Ala Ala Gly Cys Cys Thr Gly Cys Gly Gly Ala Cys Ala Cys 260 265 270 Gly Gly Cys Cys Gly Thr Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr 275 280 285 Gly Cys Ala Ala Gly Ala Thr Gly Thr Cys Cys Gly Gly Cys Cys Cys 290 295 300 Cys Ala Thr Thr Cys Ala Cys Gly Ala Ala Cys Gly Ala Thr Thr Gly 305 310 315 320 Thr Ala Thr Gly Gly Ala Cys Ala Thr Ala Ala Cys Thr Ala Gly Thr 325 330 335 Ala Cys Cys Ala Cys Gly Thr Ala Thr Gly Cys Cys Thr Ala Cys Ala 340 345 350 Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Gly Ala Cys Cys Cys Ala 355 360 365 Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala 370 375 380 Cys Ala Cys Cys Ala Cys Cys Ala Thr Cys Ala Cys Cys Ala Thr Cys 385 390 395 400 Ala Cys Gly Ala Ala Cys Cys Thr Gly Ala Ala Gly Cys Cys Thr Ala 405 410 415 Gly <210> 51 <211> 390 <212> PRT <213> Vicugna pacos <400> 51 Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly Ala Gly Gly Ala Gly Gly Cys Thr Cys 20 25 30 Gly Gly Thr Gly Cys Ala Gly Gly Cys Thr Gly Gly Gly Gly Gly Gly 35 40 45 Thr Cys Thr Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr 50 55 60 Gly Thr Ala Cys Ala Gly Thr Cys Thr Cys Thr Gly Gly Ala Ala Cys 65 70 75 80 Cys Ala Thr Cys Thr Thr Cys Ala Gly Thr Gly Thr Cys Ala Cys Thr 85 90 95 Gly Thr Cys Ala Thr Gly Gly Gly Cys Thr Gly Gly Thr Ala Cys Cys 100 105 110 Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala 115 120 125 Gly Cys Ala Gly Cys Gly Cys Gly Ala Gly Thr Thr Gly Gly Thr Cys 130 135 140 Gly Cys Ala Gly Gly Thr Thr Thr Thr Ala Cys Thr Ala Ala Thr Ala 145 150 155 160 Cys Thr Ala Gly Ala Ala Ala Cys Ala Cys Ala Ala Ala Cys Thr Ala 165 170 175 Thr Gly Thr Ala Gly Ala Cys Thr Cys Cys Gly Thr Gly Ala Ala Gly 180 185 190 Gly Gly Cys Cys Gly Cys Thr Thr Cys Ala Cys Cys Ala Thr Cys Thr 195 200 205 Cys Cys Ala Ala Ala Gly Ala Cys Ala Gly Cys Gly Cys Cys Ala Ala 210 215 220 Gly Ala Ala Cys Ala Cys Gly Ala Thr Gly Thr Ala Thr Cys Thr Ala 225 230 235 240 Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala 245 250 255 Ala Ala Cys Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys Ala Gly Cys 260 265 270 Cys Gly Thr Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr Ala Ala Thr 275 280 285 Gly Thr Ala Cys Gly Thr Cys Gly Gly Thr Gly Gly Gly Gly Cys Gly 290 295 300 Gly Thr Ala Cys Gly Ala Ala Thr Thr Gly Gly Ala Ala Thr Gly Ala 305 310 315 320 Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Gly 325 330 335 Ala Cys Cys Cys Ala Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr 340 345 350 Cys Cys Thr Cys Ala Cys Ala Cys Cys Ala Cys Cys Ala Thr Cys Ala 355 360 365 Cys Cys Ala Thr Cys Ala Cys Gly Ala Ala Cys Cys Thr Gly Ala Ala 370 375 380 Gly Cys Cys Thr Ala Gly 385 390 <210> 52 <211> 390 <212> PRT <213> Lama pacos <400> 52 Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly Ala Gly Gly Gly Gly Gly Cys Thr Thr 20 25 30 Cys Gly Thr Gly Cys Ala Gly Gly Cys Thr Gly Gly Gly Gly Gly Gly 35 40 45 Thr Cys Thr Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr 50 55 60 Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Ala Gly 65 70 75 80 Cys Ala Thr Cys Thr Thr Cys Ala Gly Thr Ala Ala Gly Ala Ala Thr 85 90 95 Ala Cys Cys Ala Thr Gly Gly Cys Cys Thr Gly Gly Thr Thr Cys Cys 100 105 110 Gly Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala 115 120 125 Gly Gly Ala Gly Cys Gly Ala Gly Ala Gly Thr Thr Gly Gly Thr Cys 130 135 140 Gly Cys Ala Gly Cys Thr Ala Gly Thr Cys Cys Thr Ala Cys Gly Gly 145 150 155 160 Gly Thr Gly Gly Thr Ala Gly Cys Ala Cys Ala Gly Cys Gly Thr Ala 165 170 175 Thr Ala Ala Ala Gly Ala Cys Thr Cys Cys Gly Thr Gly Ala Ala Gly 180 185 190 Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala Thr Cys Thr 195 200 205 Cys Cys Ala Gly Ala Gly Ala Cys Ala Gly Cys Gly Cys Cys Ala Ala 210 215 220 Gly Ala Ala Cys Ala Cys Gly Gly Thr Gly Thr Thr Gly Cys Thr Gly 225 230 235 240 Cys Ala Ala Ala Thr Gly Ala Ala Cys Gly Thr Cys Cys Thr Gly Ala 245 250 255 Ala Ala Cys Cys Thr Gly Ala Gly Gly Ala Thr Ala Cys Thr Gly Cys 260 265 270 Cys Gly Thr Cys Thr Ala Thr Thr Ala Cys Thr Gly Thr Cys Ala Thr 275 280 285 Cys Thr Ala Cys Gly Thr Cys Ala Ala Ala Ala Thr Ala Ala Cys Cys 290 295 300 Gly Thr Gly Gly Thr Thr Cys Thr Thr Gly Gly Thr Thr Cys Cys Ala 305 310 315 320 Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Gly 325 330 335 Ala Cys Cys Cys Ala Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr 340 345 350 Cys Cys Thr Cys Ala Cys Ala Cys Cys Ala Cys Cys Ala Thr Cys Ala 355 360 365 Cys Cys Ala Thr Cys Ala Cys Gly Ala Ala Cys Cys Thr Gly Ala Ala 370 375 380 Gly Cys Cys Thr Ala Gly 385 390 <210> 53 <211> 390 <212> PRT <213> Vicugna pacos <400> 53 Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr 20 25 30 Gly Gly Thr Gly Cys Ala Gly Gly Cys Thr Gly Gly Gly Gly Gly Gly 35 40 45 Thr Cys Gly Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Thr Thr 50 55 60 Gly Thr Gly Cys Ala Gly Thr Cys Thr Cys Thr Gly Gly Thr Ala Cys 65 70 75 80 Gly Ala Thr Cys Thr Thr Cys Gly Ala Thr Ala Thr Cys Ala Cys Thr 85 90 95 Cys Cys Cys Ala Thr Gly Gly Gly Cys Thr Gly Gly Thr Ala Cys Cys 100 105 110 Gly Cys Cys Ala Gly Ala Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala 115 120 125 Gly Cys Ala Gly Cys Gly Cys Gly Ala Ala Gly Thr Gly Gly Thr Cys 130 135 140 Gly Cys Ala Gly Ala Thr Cys Thr Thr Ala Cys Thr Ala Gly Thr Cys 145 150 155 160 Gly Cys Gly Gly Thr Ala Cys Cys Ala Cys Ala Ala Ala Thr Thr Ala 165 170 175 Cys Gly Cys Ala Gly Ala Cys Thr Cys Cys Gly Thr Gly Ala Ala Gly 180 185 190 Gly Gly Cys Cys Gly Gly Thr Thr Cys Ala Cys Cys Ala Thr Cys Thr 195 200 205 Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys Gly Cys Cys Ala Ala 210 215 220 Gly Ala Ala Ala Ala Thr Gly Thr Thr Gly Thr Ala Thr Cys Thr Gly 225 230 235 240 Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Ala 245 250 255 Ala Ala Thr Cys Thr Gly Ala Cys Gly Ala Cys Ala Cys Ala Gly Gly 260 265 270 Cys Gly Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Ala Ala Cys 275 280 285 Gly Thr Gly Ala Ala Ala Cys Gly Gly Thr Gly Gly Gly Gly Ala Gly 290 295 300 Gly Thr Ala Thr Thr Gly Gly Cys Thr Gly Gly Ala Ala Cys Gly Ala 305 310 315 320 Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys Cys Ala Gly Gly Gly Gly 325 330 335 Ala Cys Cys Cys Ala Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr 340 345 350 Cys Cys Thr Cys Ala Cys Ala Cys Cys Ala Cys Cys Ala Thr Cys Ala 355 360 365 Cys Cys Ala Thr Cys Ala Cys Gly Ala Ala Cys Cys Thr Gly Ala Ala 370 375 380 Gly Cys Cys Thr Ala Gly 385 390 <210> 54 <211> 390 <212> PRT <213> Vicugna pacos <400> 54 Cys Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Gly Cys Ala Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly Ala Gly Gly Ala Gly Gly Cys Thr Thr 20 25 30 Gly Gly Thr Gly Cys Ala Gly Thr Cys Thr Gly Gly Gly Gly Gly Gly 35 40 45 Thr Cys Thr Cys Thr Gly Ala Gly Ala Cys Thr Cys Thr Cys Cys Thr 50 55 60 Gly Thr Gly Thr Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr 65 70 75 80 Cys Ala Gly Ala Thr Thr Cys Ala Gly Thr Ala Ala Cys Thr Thr Thr 85 90 95 Cys Cys Thr Ala Thr Gly Ala Thr Gly Thr Gly Gly Gly Thr Cys Cys 100 105 110 Gly Cys Cys Ala Gly Gly Cys Cys Cys Cys Ala Gly Gly Ala Ala Ala 115 120 125 Gly Gly Gly Gly Cys Thr Cys Gly Ala Gly Thr Gly Gly Gly Thr Cys 130 135 140 Thr Cys Gly Cys Thr Gly Ala Thr Thr Ala Gly Cys Ala Thr Thr Gly 145 150 155 160 Gly Thr Gly Gly Thr Ala Gly Thr Ala Cys Cys Ala Cys Gly Ala Ala 165 170 175 Thr Thr Ala Thr Gly Cys Gly Gly Ala Cys Thr Cys Cys Gly Thr Gly 180 185 190 Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala 195 200 205 Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Cys Gly Cys 210 215 220 Cys Ala Ala Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Thr Thr 225 230 235 240 Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys 245 250 255 Thr Gly Ala Ala Ala Cys Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys 260 265 270 Gly Gly Cys Cys Gly Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr 275 280 285 Gly Cys Ala Ala Ala Ala Thr Ala Thr Cys Thr Thr Gly Gly Thr Cys 290 295 300 Gly Gly Cys Thr Gly Gly Thr Cys Cys Cys Ala Cys Cys Gly Ala Cys 305 310 315 320 Thr Ala Cys Thr Gly Ala Gly Gly Gly Cys Cys Ala Gly Gly Gly Gly 325 330 335 Ala Cys Cys Cys Ala Gly Gly Thr Cys Ala Cys Cys Gly Thr Cys Thr 340 345 350 Cys Cys Thr Cys Ala Cys Ala Cys Cys Ala Cys Cys Ala Thr Cys Ala 355 360 365 Cys Cys Ala Thr Cys Ala Cys Gly Ala Ala Cys Cys Thr Gly Ala Ala 370 375 380 Gly Cys Cys Thr Ala Gly 385 390 <210> 55 <211> 1095 <212> PRT <213> Artificial <220> <223> Beta2AR construct <400> 55 Gly Gly Gly Cys Ala Ala Cys Cys Cys Gly Gly Gly Ala Ala Cys Gly 1 5 10 15 Gly Cys Ala Gly Cys Gly Cys Cys Thr Thr Cys Thr Thr Gly Cys Thr 20 25 30 Gly Gly Cys Ala Cys Cys Cys Ala Ala Thr Ala Gly Ala Ala Gly Cys 35 40 45 Cys Ala Thr Gly Cys Gly Cys Cys Gly Gly Ala Cys Cys Ala Cys Gly 50 55 60 Ala Cys Gly Thr Cys Ala Cys Gly Cys Ala Gly Cys Ala Ala Ala Gly 65 70 75 80 Gly Gly A...

Claims

1. A method for identifying compounds capable of selectively binding to complexes comprising a GPCR and a G protein and optionally a receptor ligand, the method comprising the steps of: (i) Screening for binding domains of specific binding complexes comprising a GPCR, a G protein, and optionally a receptor ligand, wherein the binding domain binds the G protein in the complex and stabilizes the complex in an active conformational state. The binding domain is an immunoglobulin monovariable domain containing an amino acid sequence comprising four frame regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3) according to the following general formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1), Where CDR1 is SEQ ID NO: 13, CDR2 is SEQ ID NO: 25, and CDR3 is SEQ ID NO: 37; or Where CDR1 is SEQ ID NO: 15, CDR2 is SEQ ID NO: 27, and CDR3 is SEQ ID NO: 39; or CDR1 is SEQ ID NO: 16, CDR2 is SEQ ID NO: 28, and CDR3 is SEQ ID NO: 40; (ii) Provide a complex; (iii) Make the complex come into contact with the binding domain, allowing the binding domain to bind to the complex; (iv) Provide test compounds; (v) Evaluate whether the test compound binds to the complex; and (vi) Screening for compounds that selectively bind to the complex; The binding domain mentioned therein is the VHH domain.

2. The method according to claim 1, wherein the test compound is evaluated for whether it regulates the signal transduction activity of GPCRs and compounds capable of regulating the signal transduction activity of GPCRs are screened.

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