Screening methods and assays for use with transmembrane proteins, particularly GPCRs

Abstract

To provide a method and an arrangement for screening a membrane protein.SOLUTION: An arrangement includes: a first fused protein containing a membrane protein fused to one member of a binding pair, and existing in a boundary layer such as a wall of a cell, a liposome or a vesicle; and a second fused protein containing an intracellular ligand of the membrane protein fused to the other member of the binding pair. The binding pair can generate a detectable signal.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods and tools that can be used for assays, screening, and drug discovery and development efforts.

[0002] In particular, the present invention relates to methods and tools that can be used in screening and assay techniques involving the use of membrane proteins (i.e., as targets to be screened, e.g., to discover candidate compounds that act against said targets), and in attempts to discover, generate, optimize and / or develop therapeutic, prophylactic and diagnostic agents directed against (i.e., having specificity for) membrane proteins. The present invention further relates to methods of making tools that can be used in screening and assay techniques.

[0003] Advantageously, the methods and tools of the present invention can be used in screening and assay techniques involving the use of membrane proteins that can adopt / exist in multiple conformations (such as, but not limited to, active and inactive conformations), and in efforts to discover, generate, optimize, and / or develop therapeutic, prophylactic, and diagnostic agents directed against such membrane proteins, including, but not limited to, transmembrane proteins such as GPCRs and other cell surface receptors.

[0004] In one particularly preferred, but non-limiting aspect, the methods and tools of the present invention can be used in screening and assay techniques involving the use of membrane proteins that can undergo a conformational change (again, for example, but not limited to, a conformational change from an inactive to an active conformation) in response to binding of a ligand to said protein, and in attempts to discover, generate, optimize and / or develop therapeutic, prophylactic and diagnostic agents directed against such membrane proteins. Again, such membrane proteins may be cell surface receptors, such as GPCRs.

[0005] The present invention generally provides methods that can be used to perform an assay (i.e., for a given compound or ligand) or for screening purposes (i.e., to screen a group, series or library of compounds or ligands to identify a "hit" against a target). The present invention also provides arrangements that can be used in said methods, i.e., as systems or set-ups for performing said assays or screens. Said arrangements comprise the components described herein. Said components can also be provided or established as kits of parts, with such kits of parts forming a further aspect of the invention. The present invention also provides methods for identifying and making elements of such arrangements, as well as methods for assembling such arrangements.

[0006] The methods and arrangements described herein can generally be used to test (known) compounds or ligands for one or more properties thereof (i.e., for those properties that can be determined using the methods described herein) and / or to identify compounds or ligands (i.e., from a group, series, or library of compounds or ligands) that have such desired properties. These compounds or ligands can be any desired and / or suitable compounds or ligands, such as, but not limited to, small molecules, small peptides, biomolecules, or other chemical entities; examples of such compounds will be apparent to those of skill in the art based on the further disclosure herein. Compounds identified using the methods of the invention (i.e., "hits" from such screens) can also be used as starting points for further drug discovery and development efforts (e.g., using well-known techniques of so-called "hits-to-leads" chemistry), which may involve the use of the methods of the invention (e.g., as functional assays or assays used for quality control purposes).

[0007] Compounds identified using the methods and techniques of the invention (i.e., "hits"), and any compounds generated or developed using such hits as a starting point, are collectively referred to herein as "compounds of the invention" and form further aspects of the invention. It will be apparent to those skilled in the art that such compounds may be so-called "hits," "leads," "development candidates," "preclinical compounds," "clinical candidates," or commercial compounds or products, depending, for example, on their stage of development and on the particular terminology used by the company or entity developing and / or commercializing them.

[0008] Advantageously, compared to traditional radioligand or functional assays, the methods and assays of the present invention do not require the use of labeled antagonists (e.g., fluorescently or radioactively labeled antagonists) and can therefore be applied to membrane proteins for which antagonists are not available or known. Also, as further described herein, the methods and assays of the present invention may enable the identification and / or characterization of allosteric agonists (both positive and negative), antagonists, and / or inverse agonists (depending on the particular target and assay used).

[0009] Other features, aspects, embodiments, uses and advantages of the present invention will become apparent from the further description herein. [Background technology]

[0010] Membrane proteins (such as cell surface receptors, including GPCRs) and assay and screening techniques for membrane proteins are well known in the art. It is estimated that more than half of all modern pharmaceuticals target membrane proteins, and approximately one-third of all modern pharmaceuticals target GPCRs. Please refer to standard handbooks and further prior art cited herein.

[0011] As is well known from the field of protein dynamics, most proteins are not static entities whose function is determined solely by their primary, secondary, tertiary, and, if the protein comprises two or more polypeptide chains, quaternary structures, but are often flexible structures that can transition between different conformational states (also called "conformational changes") such that the protein may exist in equilibrium between these different states. Some of these states may be functional and / or active, while other states may be basal states (which may or may not exhibit some level of constitutive activity), essentially inactive states, and / or states with reduced activity relative to a relatively more functional or active state. The geometries of different epitopes, binding sites (including ligand binding sites), and / or catalytic sites that may be present in or on a protein may also differ between these different conformations; for example, in some of the conformational states, the binding site may not be available / accessible for ligand binding and / or the affinity for interaction between the binding site and the associated ligand(s) is reduced compared to the more active conformational states.

[0012] It is also known that for some protein / ligand combinations, binding of a ligand to a protein can change its conformation (e.g., from an inactive / less active conformation to an active / more active conformation) and / or shift the equilibrium from an inactive / less active conformation to an active / more active conformation. Binding of a ligand to one binding site of a protein can make another binding site on the protein more accessible to its associated ligand(s) and / or can result in an increase in the affinity of the other binding site for said ligand(s) and / or shift the equilibrium from a conformation in which the other binding site has a lower affinity for said ligand(s) to a conformation in which the other binding site has a higher affinity for said ligand(s). For example, for some transmembrane proteins, such as GPCRs, binding of an extracellular ligand to the protein's extracellular binding site can increase the affinity of the intracellular binding site for the intracellular ligand (e.g., affinity for the interaction between a G protein and the G protein binding site of the GPCR), and vice versa. This change in binding affinity for the intracellular ligand following binding of the extracellular ligand, and subsequent binding of the intracellular ligand to the intracellular binding site, is part of the means by which the protein transduces an extracellular signal.

[0013] Generally, for receptor proteins that can undergo conformational changes, as further described herein, an "agonist" of the receptor can be said to shift the conformational equilibrium from an inactive state (or one or more less active states) to an active state (or one or more more active states), while an "inverse agonist" of the receptor can be said to do the opposite.

[0014] A protein may form a complex with two ligands that bind to two different binding sites on the protein, and the interaction between each of the protein and the ligands may be stabilized by the binding of the other ligand (in other words, the complex may be stabilized by the binding of both ligands). Again, in this case, binding of one or both of the ligands may shift the conformational equilibrium of the protein toward (the formation and / or stabilization of) this complex. See, for example, WO 2012 / 007593, cited below.

[0015] Considering that the perceived "global" state of such a protein is largely governed by the (statistical) distribution of the protein over its various possible conformations, and therefore by the equilibrium that exists between these conformational states, when, in this specification or in the claims, a protein is said to undergo a conformational change to a particular conformation (i.e., from one or more other conformations), this should be understood to include mechanisms or circumstances by which the conformational equilibrium of the protein shifts toward said conformation (i.e., under the particular conditions used, e.g., those used for screening or related assays). Similarly, when a ligand is said to induce a conformational change to a particular conformation of a protein (i.e., from one or more other conformations), this includes mechanisms or circumstances by which binding of the ligand shifts the conformational equilibrium of the protein toward said conformation (i.e., under the particular conditions used, e.g., those used for screening or related assays).

[0016] However, it should also be noted that while any one of the mechanisms described herein (or any combination thereof) may be involved in the practice of the present invention at any given time (e.g., depending on the particular protein and / or ligand(s) to which the present invention is applied), the present invention in its broadest sense is not limited to any particular mechanism, explanation, or hypothesis, so long as application of the present invention to a particular target or protein results in the technical effect(s) outlined herein.

[0017] One challenge for screening compounds directed at membrane proteins that exist in multiple conformations is that the correct conformation of the protein may be lost when the protein is expressed or used in isolation from its native environment (even when it is feasible or possible to express the protein and ensure proper folding outside of its cellular environment). It may also be difficult to ensure that the protein is in its desired conformation (often a functional conformation, such as its active conformation) under the conditions used for screening. There may also be a need or advantage in achieving a shift in the protein's conformational equilibrium toward a conformational state that is more suitable for screening or assay purposes (such as an active state, or a state in which the relevant binding site is more accessible and / or has a better geometry for assay or screening purposes). As described further herein, such conformations are also referred to as "druggable" conformations, and according to preferred aspects of the present invention, measures are applied (as described further herein) to ensure that the protein is in such a druggable conformation and / or to ensure that the conformational equilibrium of the protein is shifted towards a more druggable conformation when the methods of the present invention are carried out.

[0018] For example, WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 175643, WO 2014 / 118297, WO 2014 / 122183, and WO 2014 / 118297 are directed to protein binding domains that can be used to stabilize specific conformational states of GPCRs for the purposes of determining their structure and for drug screening and discovery. In these references, VHH domains are used that can stabilize GPCRs in desired, particularly more druggable, conformations, e.g., functional and / or active states, e.g., conformations that arise when an activating ligand (agonist) binds to the extracellular side of the GPCR so that the GPCR can activate heterotrimeric G proteins. See, e.g., Pardon et al., Angew Chem Int Ed Engl. 2018, 57(19):5292-5295; Che et al., Cell. 2018, 172(1-2):55-67; ​​Manglik et al., Annu Rev Pharmacol Toxicol. 2017; 57:19-37; Pardon et al., Nat Protoc. 2014, 674-93; Kruse et al., Nature, 2013, 504(7478); Steyaert and Kobilka, Curr Opin Struct Biol., 2011, 567-72; and Rasmussen et al., Nature, 2011, 469(7329):175-180, and further references cited therein. VHH domains that can be used to stabilize a desired conformation of membrane proteins such as GPCRs are also referred to herein as Confobodies (Confobody™ is a registered trademark of Confo Therapeutics, Ghent, Belgium).

[0019] Some non-limiting examples of conformobodies that can bind to intracellular epitopes of GPCRs and can be used to stabilize GPCRs in a desired conformation (and can be used in the present invention) include CA2764, CA3431, CA3413, CA2780, CA2765, CA2761, CA3475, CA2770, CA3472, CA3420, CA3433, CA343 VHHs designated CA3484, CA2760, CA2773, CA3477, CA2774, CA2768, CA3424, CA2767, CA2786, CA3422, CA2763, CA2772, CA2771, CA2769, CA2782, CA2783, and CA2784 (see, e.g., WO 2012 / 007593, Tables 1 and 2, and SEQ ID NOs: 1-29); CA5 VHHs designated as Nb9-1, Nb9-8, XA8633, and CA4910 (see, e.g., Tables 1 and 2 and SEQ ID NOs: 15, 16, 17, 19, and 20 of WO 2014 / 118297); Nb9-11, Nb9-7, Nb9-7, Nb9-22, Nb9-17, Nb9-24, Nb9-9, Nb9-14, Nb9-2, Nb9-20, Nb_C3, NbH-4, Nb-E The VHHs designated Nb_1, Nb_A2, Nb_B4, Nb_D3, Nb_D1 and Nb_H1 (see, for example, Tables 1 and 2 and SEQ ID NOs: 1-19 in WO 2014 / 122183), and the VHHs designated XA8639, XA8635, XA8727 and XA9644 (see, for example, WO 2015 / 121092, Tables 2 and 3 and SEQ ID NOs: 2-6 and 74).

[0020] Some non-limiting examples of VHHs capable of binding to G proteins are CA4435, CA4433, CA4436, CA4437, CA4440 and CA4441 (see, for example, Tables 2 and 3 and SEQ ID NOs: 1-6 of WO 2012 / 175643007593). Summary of the Invention

[0021] As further described herein, the present invention generally provides improved screening methods and assay techniques that can be used to discover and develop (e.g., identify, generate, test, and optimize) compounds directed against membrane proteins (i.e., that have specificity for and / or are intended to target one or more membrane proteins, e.g., for therapeutic, prophylactic, and / or diagnostic purposes). Preferably, such compounds are specific for (i.e., selective for) one particular membrane protein compared to other (closely related) membrane proteins.

[0022] Compounds identified and / or developed using the methods of the invention can be used to modulate (as defined herein) membrane proteins, their signal transduction, and / or biological functions, pathways, and / or mechanisms in which said membrane proteins or their signal transduction participate. For example, the invention can be used to discover and develop compounds that are agonists, antagonists, inverse agonists, inhibitors, or modulators (including both positive and negative allosteric modulators) of said membrane proteins and / or signal transduction, pathways, and / or physiological and / or biological mechanisms in which said membrane proteins participate.

[0023] The present invention can be used to discover and develop compounds directed against membrane proteins, either integral or peripheral membrane proteins, in their natural environment. The present invention can be used, in particular, to discover and develop compounds directed against transmembrane proteins, as further described herein. In one specific, but non-limiting, embodiment, the compounds discovered and / or developed using the present invention are directed against receptors, particularly cell surface receptors. As further described herein, the transmembrane protein may be, in particular, a membrane protein having multiple passes through the membrane, such as a 7TM or GPCR. [In this regard, it should be noted that, in general, in the art, the terms "7TM receptor" and "7TM" are often used interchangeably with "GPCR." However, according to the IUPHAR database, there are some 7TM receptors that do not signal through G proteins. For purposes of this specification and claims, the terms "GPCR" and "7TM" are used interchangeably herein and include all transmembrane proteins, particularly transmembrane receptors, that have seven transmembrane domains, regardless of their intracellular signaling cascade or signaling mechanism. However, it should be understood that throughout this specification and claims, 7TMs that signal through G proteins are a preferred embodiment of the present invention.]

[0024] Typically, compounds discovered and / or developed using the present invention are directed to membrane proteins that are expressed and / or exposed on the surface of cells in their natural environment, and in particular membrane proteins expressed by or in cells present in the body of a subject to be treated with a compound discovered or developed using the methods and techniques of the present invention.

[0025] The present invention can be used to discover and / or develop any type of compound suitable for its intended use, which is often as a therapeutic, diagnostic, or prophylactic agent. Thus, these compounds can be small molecules, peptides, biomolecules, or other chemical entities. Examples of suitable biomolecules include, for example, antibodies and antibody fragments (such as Fab, VH, VL, and VHH domains) and compounds based on antibody fragments (such as ScFvs and diabodies, as well as other compounds or constructs comprising one or more VH, VL, and / or VHH domains), Alphabodies™ and avimer-based scaffolds, compounds based on other protein scaffolds such as PDZ domains, Protein A domains (such as Affibodies™), ankyrin repeats (such as DARPins™), fibronectins (such as Adnectins™), and lipocalins (such as Anticalins™), as well as DNA- or RNA-based binding moieties, including, but not limited to, DNA or RNA aptamers. See further description herein and, for example, Simeon and Chen, Protein Cell 2018, 9(1):3-14; Binz et al., Nat. Biotech 2005, 23:1257; and Ulrich et al., Comb Chem High Throughput Screen 2006, 9(8):619-32.

[0026] The methods and techniques of the present invention can be used, for example, to screen libraries of such compounds to identify one or more "hits" that are specific for a membrane protein (in particular, specific for a desired conformation of the membrane protein and / or capable of inducing a desired conformation of the membrane protein, e.g., a ligand-binding conformation, and in particular an agonist-binding conformation), and / or as part of a strategy to improve the affinity and / or potency of compounds directed against membrane proteins, and / or as assays used to improve the pharmacological and / or other properties of such compounds (e.g., in the case of small molecules, as part of a "hit-to-lead" campaign).

[0027] The methods and techniques of the present invention can also be used for the purpose of so-called "fragment-based drug discovery" or "FBDD" (also known as "fragment-based lead discovery" or "FBLD"). See, for example, basic books such as Lamoree and Hubbard, Essays in Biochemistry (2017) 61, 453-464; Jahnke and Erlanson, "Fragment-based approaches in drug discovery", 2006; Zartler and Shapiro, "Fragment-based drug discovery: a practical approach", 2008; and Kuo, "Fragment-based drug design: tools, practical approaches, and examples", 2011. [Brief explanation of the drawings]

[0028] [Figure 1]1 shows a schematic representation of a first arrangement of the invention, in which a second ligand (shown as (4) in FIG. 1 ) forms part of a second fusion protein (in the embodiment shown in FIG. 1 , formed by the second ligand (4), a linker (11) and the second member (7) of the binding pair (6 / 7)) that is directly (as defined herein) bound to the layer-spanning protein (2). In the setup shown in FIG. 1 : the boundary layer is designated as (1); the first environment is designated as [A]; the second environment is designated as [B]; the layer-spanning protein is designated as (2); the first ligand is designated as (3); the first binding site of the layer-spanning protein (2) exposed to the first environment [A] and to which the first ligand (3) can bind is designated as (8); the second ligand is designated as (4); the second binding site of the layer-spanning protein (2) exposed to the second environment [B] and to which the second ligand (4) can bind is designated as (9); the binding pair capable of generating a detectable signal is designated as (6 / 7) and consists of a first binding member (6) linked (directly or via a linker or spacer (10)) to the layer-spanning protein (2) and a second binding member (7) linked (directly or via a linker or spacer (11)) to the second ligand (4); The first fusion protein comprises a layer-spanning protein (2) fused directly or via a linker (10) to a first binding member (6); the second fusion protein comprises a second ligand (4) fused directly or via a linker (11) to a second binding member (7); the first and second fusion proteins are positioned relative to each other and the boundary layer (1) in such a manner that when the second ligand (4) binds to the layer-spanning protein (2) (i.e., directly via the binding site (9)), the first binding member (6) and the second binding member (7) can contact or come into close proximity with each other (or otherwise appropriately associate) to generate a detectable signal (shown by a flash symbol in Figure 1). [Figure 2]2 shows a schematic representation of a second arrangement of the invention, in which the second ligand (shown as (4) in FIG. 2 ) is separate from the second fusion protein (which in the embodiment shown in FIG. 2 is formed by the binding domain (5), the linker (11) and the second member (7) of the binding pair (6 / 7)), and the binding domain (5) present in the second fusion protein binds indirectly (as defined herein, in the case of FIG. 2 , via the second ligand (4)) to the layer-spanning protein (2).In the setup shown in Figure 2: the boundary layer is designated as (1); the first environment is designated as [A]; the second environment is designated as [B]; the layer-spanning protein is designated as (2); the first ligand is designated as (3); the first binding site of the layer-spanning protein (2) that is exposed to the first environment [A] and to which the first ligand (3) can bind is designated as (8); the second ligand is designated as (4); the second binding site of the layer-spanning protein (2) that is exposed to the second environment [B] and to which the second ligand (4) can bind is designated as (9); the binding domain or binding unit that can bind the second ligand (4) is designated as (5); The binding pair capable of producing a detectable signal is shown as (6 / 7) and consists of a first binding member (6) linked (directly or via a linker or spacer (10)) to a layer-spanning protein (2) and a second binding member (7) linked (directly or via a linker or spacer (11)) to a binding domain or binding unit (5); the first fusion protein comprises a layer-spanning protein (2) fused directly or via a linker (10) to the first binding member (6); the second fusion protein comprises a binding domain (5) fused directly or via a linker (11) to the second binding member (7); The first and second fusion proteins are positioned relative to each other and to the boundary layer (1) in such a manner that when the binding domain (5) binds to the layer-spanning protein (2) (i.e., indirectly by binding to a second ligand (4) which in turn binds to the layer-spanning protein (2) via binding site (9)), the first binding member (6) and the second binding member (7) can contact or come into close proximity to each other (or otherwise appropriately associate) to generate a detectable signal (shown by a flash symbol in Figure 2). [Figure 3]Schematically illustrates a third arrangement of the present invention, in which the second ligand (shown as (4) in FIG. 3 ) is separated from the second fusion protein and forms part of a protein complex (12) formed by the second ligand (4) and one or more additional proteins (in the case of FIG. 3 , for illustrative purposes, the complex is illustrated as comprising the second ligand (4) and two additional proteins (4a) and (4b); see also the insert of FIG. 3 ). In the embodiment shown in FIG. 3 , the second ligand (4) is also separated from the second fusion protein (formed in the embodiment shown in FIG. 3 by the binding domain (5), the linker (11), and the second member (7) of the binding pair (6 / 7)), and the binding domain (5) present in the second fusion protein binds indirectly (as defined herein, in the case of FIG. 3 , via the protein complex (12)) to the layer-spanning protein (2). In the setup shown in Figure 3: the boundary layer is designated as (1); the first environment is designated as [A]; the second environment is designated as [B]; the layer-spanning protein is designated as (2); the first ligand is designated as (3); the first binding site of the layer-spanning protein (2), exposed to the first environment [A] and to which the first ligand (3) can bind, is designated as (8); the second ligand is designated as (4), forming a complex (12) with one or more other proteins (in Figure 3, for purposes of illustration, complex (12) is represented as a complex containing three proteins / subunits, namely, a second ligand (4) and two further subunits (4a) and (4b). See also the inset of Figure 3).); the second binding site of the layer-spanning protein (2), exposed to a second environment [B] and capable of binding complex (12) (4), is designated as (9); the binding domain or binding unit capable of binding complex (12) is designated as (5); the binding pair capable of generating a detectable signal is designated as (6 / 7) and consists of a first binding member (6) linked (directly or via a linker or spacer (10)) to the layer-spanning protein (2) and a second binding member (7) linked (directly or via a linker or spacer (11)) to the binding domain or binding unit (5); the first fusion protein comprises the layer-spanning protein (2) fused directly or via a linker (10) to the first binding member (6); the second fusion protein comprises a binding domain (5) fused directly or via a linker (11) to the second binding member (7); The first and second fusion proteins are positioned relative to each other and to the boundary layer (1) in such a manner that when the binding domain (5) binds to the layer-spanning protein (2) (i.e., indirectly by binding to a complex (12) which in turn binds to the layer-spanning protein (2) via the binding site (9)), the first binding member (6) and the second binding member (7) can contact or be in close proximity to each other (or otherwise suitably associated) to generate a detectable signal (shown by a flash symbol in Figure 3). [Figure 4] 1 is a graph showing a dose-response curve for NDP-alpha-MSH obtained using the MC4R screening assay with CA4437 described in Example 1. [Figure 5A] 1 is a graph depicting dose-response curves for the indicated compounds obtained using the GLP-1R screening assay with CA4437 described in Example 2. [Figure 5B] 1 is a graph depicting dose-response curves for the indicated compounds obtained using the GLP-1R screening assay with CA4437 described in Example 2. [Figure 5C]1 is a graph depicting dose-response curves for the indicated compounds obtained using the GLP-1R screening assay with CA4437 described in Example 2. [Figure 6] 1 is a graph showing the dose-response curve for GLP-1(7-36)amide obtained using the GLP-1R screening assay with CA4435 described in Example 2. [Figure 7A] 1 is a graph showing assay results obtained using the beta-2-AR screening assay with CA4437 described in Example 3. [Figure 7B] 1 is a graph showing assay results obtained using the beta-2-AR screening assay with CA4437 described in Example 3. [Figure 8A] 1 is a graph showing assay results obtained using the beta-2-AR screening assay with CA4435 described in Example 3. [Figure 8B] 1 is a graph showing assay results obtained using the beta-2-AR screening assay with CA4435 described in Example 3. [Figure 8C] 1 is a graph showing assay results obtained using the beta-2-AR screening assay with CA4437 described in Example 3. [Figure 8D] 1 is a graph showing assay results obtained using the beta-2-AR screening assay with CA4435 described in Example 3. [Figure 8E] 1 is a graph showing assay results obtained using the beta-2-AR screening assay with the CA4435-35GS-CA4437 fusion described in Example 3. [Figure 9] 1 is a graph showing dose-response curves for the indicated compounds obtained using the MOR screening assay described in Example 4. [Figure 10] 1 is a graph showing dose-response curves for the indicated compounds obtained using the MOR screening assay described in Example 4. [Figure 11]1 is a graph showing assay results obtained using the M2R screening assay described in Example 5. [Figure 12] 1 is a graph showing assay results obtained using the beta-2AR screening assay described in Example 6. [Figure 13] 1 is a graph showing dose-response curves for the indicated compounds obtained using the AT1R screening assay described in Example 7. [Figure 14] 1 is a graph showing assay results obtained using the AT1R screening assay described in Example 7. [Figure 15] 1 shows the results of a compound library screen carried out in Example 8. [Figure 16] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant MC4R screening assay described in Example 9. [Figure 17] 10 is a graph showing assay results obtained using the recombinant MC4R screening assay described in Example 9. [Figure 18] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 10. [Figure 19] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 10. [Figure 20] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 10. [Figure 21] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 10. [Figure 22] 1 is a graph showing dose-response curves for the indicated compounds obtained using the recombinant OX2R screening assay described in Example 10. [Figure 23A]23A-23C are graphs showing assay results obtained using two recombinant APJ receptor screening assays described in Example 11. Figure 23A shows results obtained with a recombinant apelin receptor bearing the ICL of the mu-opioid receptor (MOR). [Figure 23B] Figure 23B shows the assay results obtained using two recombinant APJ receptor screening assays described in Example 11. Figure 23B shows the results obtained with a recombinant apelin receptor bearing an ICL from the beta-2AR receptor. [Figure 24] 1 shows the results of the compound library screening carried out in Example 12. [Figure 25] 1 shows the results of the compound library screening carried out in Example 12. [Figure 26] 1 shows the results of the compound library screening carried out in Example 12. [Figure 27] 1 shows the results of the compound library screening carried out in Example 12. [Figure 28] 28 is a plot of the screening results obtained in Example 14 for a collection of 78 compound fragments when tested using two assays of the invention (both using beta-2AR-LgBiT fusions, but one assay using a CA2780-SmBiT fusion and one assay using a CA4435-35GS-CA4437-LgBiT fusion). In Figure 28, the x-axis represents the results obtained in the assay using the CA4435-35GS-CA4437-LgBiT fusion, the y-axis represents the results obtained in the assay using the CA2780-SmBiT fusion, and each dot represents the result obtained in both assays for one of the 78 compounds tested. [Figure 29A]29A is a plot of the screening results obtained in Example 15 for a collection of compound fragments when tested using a radioligand assay and a corresponding assay of the invention. In Figure 29A, the x-axis represents the results obtained using an assay of the invention, the y-axis represents the results obtained in the radioligand assay in the assay, and each dot represents the result obtained for one of the compounds when tested in both the radioligand assay and the assay of the invention. [Figure 29B] 29B is a plot of the screening results obtained in Example 15 for a collection of compound fragments when tested using a radioligand assay and a corresponding assay of the invention. In Figure 29B, the x-axis represents the results obtained using an assay of the invention, the y-axis represents the results obtained in the radioligand assay in the assay, and each dot represents the result obtained for one of the compounds when tested in both the radioligand assay and the assay of the invention. [Figure 30A] 1 is a graph showing the results of testing the compounds referred to in Example 16 and Table 3 (at 100 μM and 200 μM, respectively) in the GloSensor cAMP assay for beta-2AR. [Figure 30B] 1 is a graph showing the results of testing the compounds referred to in Example 16 and Table 3 (at 100 μM and 200 μM, respectively) in the GloSensor cAMP assay for beta-2AR. [Figure 31A] 1 is a graph showing a comparison of the results obtained in Example 17 when a cell-based assay of the invention is compared to an equivalent membrane-based assay of the invention. [Figure 31B] 1 is a graph showing a comparison of the results obtained in Example 17 when a cell-based assay of the invention is compared to an equivalent membrane-based assay of the invention. [Figure 32A] Dose-response curves to apelin obtained with different VHHs are shown (Example 18). [Figure 32B] Dose-response curves to apelin obtained with different VHHs are shown (Example 18). [Figure 32C] Dose-response curves to apelin obtained with different VHHs are shown (Example 18). [Figure 33] 1 shows the dose response curves generated in Example 19 for iperoxo on the M2 receptor using the assay of the present invention, with and without LY2119620 (an allosteric modulator of the M2 receptor). [Figure 34] 2 is a plot obtained in Example 20 comparing the results of an OX2 assay of the present invention (using recombinant OX2 fusions) and an OX2 IP-One assay, where the x-axis represents data obtained in the assay of the present invention and the y-axis represents data obtained in the IP-One assay, and each dot represents the result of a single compound. [Figure 35A] 35A shows plots obtained when a large compound library was screened against recombinant OX2 receptors using the assay of the present invention in Example 21. Figure 35A shows the results obtained when compounds were tested at 30 μM, the x-axis represents the ratio of the signal obtained by the tested compound ("sample") to the signal obtained by the carrier solvent ("blank"), and each dot represents the result obtained with a single compound. [Figure 35B] 35B shows the plot obtained when a large compound library was screened against recombinant OX2 receptors using the assay of the present invention in Example 21. Figure 35B shows the results obtained when compounds were tested at 200 μM, with the x-axis representing the ratio of the signal obtained by the tested compound ("sample") to the signal obtained by the carrier solvent ("blank"), and each dot representing the result obtained with a single compound. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is described herein with reference to particular embodiments and with reference to certain non-limiting examples and drawings. Any reference signs in the claims should not be construed as limiting the scope. The drawings described are schematic only and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. When the term "comprising" is used in the specification and claims, it does not exclude other elements or steps. When an indefinite or definite article is used when referring to a singular noun (e.g., "a," "an," or "the"), it also includes the plural of that noun unless something specifically stated otherwise. Furthermore, the terms first, second, third, etc. in the specification and claims are used to distinguish between similar elements and are not necessarily used to describe an order or chronology. It should be understood that terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein may operate in orders other than those described or illustrated herein.

[0030] Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with molecular and cellular biology, structural biology, biophysics, pharmacology, genetics, and protein and nucleic acid chemistry described herein is that which is well known and commonly used in the art. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991), provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and supplements through 2002); up, Biomolecular crystallography: principles, practice and applications to structural biology, 1st ed., Garland Science, Taylor & Francis Group, LLC, an information business, NY (2009); Limbird, Cell Surface Receptors, 3rd ed., Springer (2004).

[0031] As used herein, the terms "polypeptide," "protein," and "peptide" are used interchangeably herein and refer to polymeric forms of amino acids of any length, and can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. Standard single-letter amino acid abbreviations are used throughout the application. Typically, the term "amino acid" refers to "proteinogenic amino acids," i.e., amino acids that occur naturally in proteins. In particular, amino acids are in the L-isomer form, although D-amino acids are also contemplated.

[0032] As used herein, the terms "nucleic acid molecule," "polynucleotide," "polynucleic acid," and "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Non-limiting 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.

[0033] Any of the peptides, polypeptides, nucleic acids, compounds, etc. disclosed herein may be "isolated" or "purified." "Isolated" is used herein to indicate that the referenced material (i) has been separated from one or more materials with which it naturally occurs (e.g., separated from at least some cellular material, separated from other polypeptides, or separated from its natural sequence context), and / or (ii) has been produced by a process involving the hand of man, such as recombinant DNA technology, protein engineering, chemical synthesis, etc., and / or (iii) has a sequence, structure, or chemical composition not found in nature. "Isolated" is intended to include compounds within a sample that have been substantially enriched in the compound of interest and / or from which the compound of interest has been partially or substantially purified. As used herein, "purified" indicates that the referenced material has been removed from its natural environment and is at least 60% free, at least 75% free, or at least 90% free from other components with which it is naturally associated, also referred to as "substantially pure."

[0034] As used herein, the term "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison.

[0035] Thus, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which 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, Li, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Determining the percentage of sequence identity can be performed manually or by using computer programs available in the art. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989)), BLAST and BLAST 2.0 (Altschul et al., J. Mol. Biol., 215:403 (1990)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0036] "Similarity" refers to the percentage of amino acids that are identical or that constitute conservative substitutions. Similarity can be determined using sequence comparison programs such as GAP (Deveraux et al., 1984). In this method, sequences of similar or substantially different lengths to those cited herein can be compared by inserting gaps into the alignment, and such gaps are determined, for example, by the comparison algorithm used by GAP. As used herein, a "conservative substitution" refers to the substitution of an amino acid with another amino acid whose side chain has similar biochemical properties (e.g., aliphatic, aromatic, or positively charged), and is well known to those skilled in the art. A non-conservative substitution refers to the substitution of an amino acid with another amino acid whose side chain does not have similar biochemical properties (e.g., substitution of a hydrophobic residue with a polar residue). Typically, a conservative substitution results in a sequence that is no longer identical but is still very similar. By conservative substitutions, combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp are intended.

[0037] A "deletion" is defined herein as a change in either the amino acid sequence or the nucleotide sequence in which one or more amino acid residues or nucleotide residues are absent compared to the amino acid sequence or nucleotide sequence of a parent polypeptide or nucleic acid, respectively. In the context of a protein, a deletion can involve the deletion 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 two or more deletions. In the context of a GPCR, a deletion may be a loop deletion, or an N-terminal and / or C-terminal deletion. As will be apparent to one of skill in the art, an N-terminal and / or C-terminal deletion of a GPCR is also referred to as a truncation of the amino acid sequence of the GPCR or a truncated GPCR.

[0038] An "insertion" or "addition" is a change in an amino acid sequence or nucleotide sequence in which one or more amino acids or nucleotide residues have been added, respectively, compared to the amino acid sequence or nucleotide sequence of a parent protein. An "insertion" generally refers to the addition of one or more amino acid residues within the amino acid sequence of a polypeptide, and an "addition" can refer to the insertion or addition of amino acid residues at the N-terminus, C-terminus, or both termini. In the context of a protein or fragment thereof, an insertion or addition is typically an insertion or addition of 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.

[0039] "Substitution," as used herein, results from the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid sequence or nucleotide sequence of a parent protein or fragment thereof. It is understood that a protein or fragment thereof may have conservative amino acid substitutions that do not substantially affect the activity of the protein. By conservative substitutions, combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp are intended.

[0040] The term "amino acid difference" refers to the total number of amino acid residues in a sequence that are changed (i.e., by substitution, insertion, and / or deletion) compared to a starting or reference sequence. The number of amino acid differences between a sequence and a reference sequence can usually be determined by aligning and comparing the sequences, for example.

[0041] The term "ortholog," when used in reference to an amino acid or nucleotide / nucleic acid sequence from a given species, refers to an identical amino acid or nucleotide / nucleic acid sequence from a different species. Two sequences should be understood to be orthologs of one another if they are derived from a common ancestral sequence through direct lineage and / or are otherwise closely related in terms of both their sequence and their biological function. Orthologs typically have a high degree of sequence identity, but do not necessarily (and often do not) share 100% sequence identity.

[0042] The term "recombinant" when used with reference to a cell, nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a nucleic acid or polypeptide that is not found within the native (non-recombinant) form of the cell, or expresses a native gene that is otherwise aberrantly expressed, repressibly expressed, over-expressed, or not expressed at all.

[0043] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. This process includes both transcription and translation.

[0044] As used herein, the term "operably linked" refers to a linkage in which a regulatory sequence is contiguous with a gene of interest to regulate the gene of interest, and a linkage in which the regulatory sequence acts in trans or at a distance to regulate the gene of interest. For example, a DNA sequence is operably linked to a promoter when it is ligated downstream of the promoter with respect to the transcription initiation site of the promoter, allowing transcription elongation to proceed through the DNA sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide when it is expressed as a preprotein involved in the transport of the polypeptide. Linking of a DNA sequence to a regulatory sequence is typically achieved by ligation at appropriate restriction sites using restriction endonucleases known to those skilled in the art, or by inserting adapters or linkers instead.

[0045] As used herein, the term "regulatory sequence," also referred to as "control sequence," refers to a polynucleotide sequence necessary to affect the expression of a coding sequence to which it is operably linked. Regulatory sequences are sequences that control the transcription, post-transcriptional events, and translation of a nucleic acid sequence. Regulatory sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (e.g., ribosome binding sites); sequences that improve protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. The term "regulatory sequence" is intended to include at least all components whose presence is essential for expression, and may include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0046] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. A vector can be any suitable type of vector, including, but not limited to, a phage, virus, plasmid, phagemid, cosmid, bacmid, or even an artificial chromosome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of a specific gene of interest. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Suitable vectors contain regulatory sequences, such as promoter, enhancer, and terminator sequences, as desired and depending on the particular host organism (e.g., bacterial cells, yeast cells). Typically, a recombinant vector according to the present invention comprises at least one "chimeric gene" or "expression cassette." An expression cassette is generally a DNA construct, preferably comprising (5' to 3' in the direction of transcription) a promoter region, a polynucleotide sequence of the present invention or a homolog, variant, or fragment thereof operably linked to a transcription initiation region, and a termination sequence comprising an RNA polymerase termination signal and a polyadenylation signal. It is understood that all of these regions should be operable in the living cell, such as a prokaryotic or eukaryotic cell, to be transformed. The promoter region, including the transcription initiation region, which preferably comprises an RNA polymerase binding site, and the polyadenylation signal, may be native to the living cell to be transformed, or the regions may be derived from another source that is functional in the living cell.

[0047] As used herein, the term "host cell" is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A host cell may be an isolated cell or a cell line grown in culture, or may be a cell present in a living tissue or organism. In particular, host cells are cells of bacterial or fungal origin, but may also be cells of plant or mammalian origin. The terms "host cell," "recombinant host cell," "expression host cell," "expression host system," and "expression system" are intended to have the same meaning and are used interchangeably herein.

[0048] "G-protein coupled receptors" or "GPCRs" are polypeptides that share a common structural motif: an extracellular amino terminus (N-terminus), an intracellular carboxy terminus (C-terminus), and seven hydrophobic transmembrane regions of 22-24 hydrophobic amino acids each forming seven alpha helices that span the membrane. Each span is identified by a number, i.e., transmembrane-1 (TM1), transmembrane-2 (TM2), etc. The transmembrane helices are connected by regions of amino acids between transmembrane-2 and transmembrane-3, between transmembrane-4 and transmembrane-5, and between transmembrane-6 and transmembrane-7, designated "extracellular" regions 1, 2, and 3 (EC1, EC2, and EC3), respectively, on the outer, or "extracellular," side of the cell membrane. The transmembrane helices are also connected by stretches of amino acids between transmembrane-1 and transmembrane-2, between transmembrane-3 and transmembrane-4, and between transmembrane-5 and transmembrane-6, referred to as "intracellular" regions 1, 2, and 3 (IC1, IC2, and IC3), respectively, on the inside, or "intracellular," side of the cell membrane. The "carboxy" ("C") terminus of the receptor is in the intracellular space within the cell, and the "amino" ("N") terminus of the receptor is in the extracellular space outside the cell.The structure and classification of GPCRs are generally well known in the art, and further discussion of GPCRs can be found in Cvicek et al., PLoS Comput Biol. 2016 Mar 30;12(3):e1004805.doi:10.1371 / journal.pcbi.1004805; Ventakakrishnan, Current Opinion in Structural Biology, 2014, 27:129-137; Isberg, Trends Pharmacol. Sci., 2015 Jan 22-13; Probst, DNA Cell Biol. 1992, 11:1-20; Marchese et al., Genomics 23:609-618, 1994; and the following books: Jurgen Wess (ed.) Structure-Function Analysis of G Protein-Coupled Receptors, Wiley Liss (1st ed., October 15, 1999); Kevin R. Lynch (ed.), Identification and Expression of G Protein-Coupled Receptors, published by John Wiley & Sons (March 1998); and Tatsuya Haga (ed.), G Protein-Coupled Receptors, published by CRC Press (September 24, 1999); and Steve Watson (ed.), G-Protein Linked Receptor Factsbook, published by Academic Press (1st ed., 1994).

[0049] The International Union of Basic and Clinical Pharmacology (IUPHAR) maintains a database of receptors (including GPCRs) and their known endogenous ligands and signaling mechanisms (http: / / www.guidetopharmacology.org / targets.jsp). According to this database, as of January 2019, approximately 800 GPCRs have been identified in humans, of which approximately half have sensory functions (e.g., olfaction, taste, light perception, and pheromone signaling), and approximately half mediate signaling associated with ligands ranging in size from small molecules to peptides and large proteins. As of January 2019, the IUPHAR database describes two systems for classifying GPCRs, one of which is based on six classes of GPCRs: class A (rhodopsin-like), class B (secretin receptor family), class C (metabotropic glutamate), class D (fungal mating pheromone receptors, not found in vertebrates), class E (cyclic AMP receptors, also not found in vertebrates), and class F (frizzled / smoothened).The IUPHAR database also refers to an alternative classification scheme known as "GRAFS," which divides vertebrate GPCRs into five classes (overlapping with the AF nomenclature): the glutamate family (overlapping with "Class C" above), which includes, among others, metabotropic glutamate receptors, calcium-sensing receptors, and GABAB receptors; the rhodopsin family (overlapping with "Class A" above), which includes receptors for a wide variety of small molecules, neurotransmitters, peptides, and hormones, as well as olfactory receptors, visual pigments, taste type 2 receptors, and five pheromone receptors (V1 receptors); and the adhesion family GPCRs (Class B receptors). the Frizzled family, which consists of 10 Frizzled proteins (FZD(1-10)) and Smoothened (SMO); and the secretin family, which are receptors for peptide ligands / hormones with 27-141 amino acid residues, including glucagon, glucagon-like peptides (GLP-1, GLP-2), glucose-dependent insulinotropic polypeptide (GIP), secretin, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and growth hormone-releasing hormone (GHRH). In this specification and the appended claims, the classification of types A-F will be used unless expressly stated otherwise. See also Cvicek et al., cited herein.

[0050] The term "biologically active" with respect to a GPCR refers to a GPCR that has the biochemical function of a naturally occurring GPCR (e.g., a binding function, a signaling function, or the ability to change conformation as a result of ligand binding).

[0051] Generally, the term "naturally occurring" with respect to a GPCR refers to a GPCR that is naturally produced (e.g., by a wild-type mammal such as a human). Such GPCRs are found in nature. "Non-naturally occurring" with respect to a GPCR refers to a GPCR that is not naturally occurring. Naturally occurring GPCRs that are constitutively activated by mutation and variants of naturally occurring transmembrane receptors, such as epitope-tagged GPCRs and GPCRs lacking their natural N-termini, are examples of non-naturally occurring GPCRs. Non-naturally occurring variants of naturally occurring GPCRs are often activated by the same ligand as the naturally occurring GPCR. Non-limiting examples of naturally occurring or non-naturally occurring GPCRs in the context of the present invention are further provided herein.

[0052] As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope can comprise three amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least four, five, six, or seven such amino acids, and more usually, at least eight, nine, or ten such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. As used herein, a "conformational epitope" refers to an epitope comprising amino acids in a spatial conformation unique to the folded three-dimensional conformation of a polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in a linear sequence and that are clustered in the folded structure of a protein. However, a conformational epitope may also be comprised of a linear sequence of amino acids that adopts a conformation unique to the folded three-dimensional conformation of a polypeptide (and that does not exist in the denatured state).

[0053] The term "conformation" or "conformational state" of a protein generally refers to the spatial arrangement, structure, or range of structures that a protein can adopt at any given time. Those skilled in the art will recognize that determinants of conformation or conformational state include the primary structure of a protein as reflected in the amino acid sequence of the protein (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as protein secondary structure (e.g., α-helix, β-sheet, among others), tertiary structure (e.g., the three-dimensional folding of the polypeptide chain), and quaternary structure (e.g., interactions of the polypeptide chain with other protein subunits). Post-translational and other modifications of the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, among others, can affect the conformation of a protein. Additionally, environmental factors such as the pH, salt concentration, ionic strength, and osmolality of the surrounding solution, as well as interactions with other proteins and cofactors, among others, can affect the conformation of a protein. The conformational state of a protein can be determined by functional assays for activity or binding to another molecule, or by physical methods such as X-ray crystallography, NMR, or spin labeling, among others. For a general discussion of protein conformation 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.

[0054] As used herein, "functional conformation" or "functional conformational state" refers to the fact that a protein has various conformational states with a dynamic range of activity, particularly ranging from inactive to maximally active. It will be apparent that "functional conformational state" is intended to include any conformational state of a protein with any activity, including inactive, and is not intended to include a denatured state of a protein. Non-limiting examples of functional conformations include an active conformation, an inactive conformation, or a basal conformation (further defined herein). As noted above, a particular class of functional conformations is defined as a "druggable conformation," which generally refers to a therapeutically relevant conformational state(s) of a protein. See, e.g., Johnson and Karanicolas, PLoS Comput Biol 9(3):e1002951.doi:10.1371 / journal.pcbi.1002951. Also see, for example, WO 2014 / 122183, which describes that the agonist-binding conformation of the muscarinic acetylcholine receptor M2 corresponds to the druggable conformation of this receptor associated with pain and glioblastoma, and describes VHHs that can stabilize the druggable conformation for assay and screening purposes. Therefore, it will be understood that druggability is limited to certain conformations depending on the therapeutic indication. Further details are provided further herein.

[0055] As used herein with respect to proteins that are receptors, the term "active conformation" refers more specifically to a conformation or set of receptor conformations that permits signal transduction to intracellular effector systems, such as G protein-dependent and / or G protein-independent signaling (e.g., β-arrestin signaling). Thus, "active conformation" encompasses a set of ligand-specific conformations, including agonist-specific, partial agonist-specific, or biased agonist-specific active state conformations, that result in cooperative binding of intracellular effector proteins.

[0056] In addition to the above, the terms "active conformation" and "active form" as used herein with respect to GPCRs refer to a GPCR that is folded in a manner that is (functionally) active. GPCRs can be placed into an active conformation using an activating ligand (agonist) for the receptor; such a conformational change generally allows the receptor to activate a heterotrimeric G protein. For example, a GPCR in its active conformation binds to a heterotrimeric G protein and catalyzes nucleotide exchange in the G protein, activating downstream signaling pathways. Activated GPCRs bind to inactive, GDP-bound heterotrimeric G proteins, causing the G protein to release GDP so that GTP can bind. This process creates a transient "nucleotide-free" state that allows GTP to bind. Upon GTP binding, the receptor and G protein dissociate, allowing the GTP-bound G protein to activate downstream signaling pathways, such as adenylyl cyclase, ion channels, and RAS / MAPK. The terms "inactive conformation" and "inactive form" refer to a GPCR that is folded in such a manner that it is inactive. A GPCR can be placed in an inactive conformation using an inverse agonist of the receptor. For example, a GPCR in its inactive conformation does not bind with high affinity to a heterotrimeric G protein. The terms "active conformation" and "inactive conformation" are further explained herein. As used herein, the term "basal conformation" refers to a GPCR that is folded in such a manner that it exhibits activity toward a specific signaling pathway even in the absence of an agonist (also referred to as basal activity or constitutive activity). An inverse agonist can inhibit this basal activity. Thus, the basal conformation of a GPCR corresponds to a stable conformation or distinct structural species in the absence of a ligand or accessory protein.

[0057] Similarly, the term "inactive conformation," as used herein with respect to proteins that are receptors, refers to a range of receptor conformations that do not permit or block signal transmission to intracellular effector systems. Thus, "inactive conformations" encompass a range of ligand-specific conformations, including inverse agonist-specific inactive state conformations, thereby preventing cooperative binding of intracellular effector proteins. It will be understood that the binding site of the ligand is not critical for obtaining an active or inactive conformation. Thus, orthosteric ligands as well as allosteric modulators can similarly stabilize receptors in active or inactive conformations.

[0058] The term "binding agent," as used herein, refers to all or part of a proteinaceous (protein, protein-like, or protein-containing) molecule capable of binding to a membrane protein using specific intermolecular interactions. In certain embodiments, the term "binding agent" is not intended to include naturally occurring binding partners of the relevant membrane protein, such as G proteins, arrestins, endogenous ligands, or variants or derivatives (including fragments) thereof. More specifically, the term "binding agent" refers to a polypeptide, particularly a protein domain. Suitable protein domains are self-stable, fold independently of the rest of the protein chain, and are often referred to as "binding domains." Such binding domains vary in length from about 25 amino acids up to 500 and more amino acids. Many binding domains can be classified into folds, which are recognizable and distinguishable three-dimensional structures. Some folds are common to many different proteins and therefore have been given special names. Non-limiting examples are binding domains selected from three- or four-helix bundles, armadillo repeat domains, leucine-rich repeat domains, PDZ domains, SUMO or SUMO-like domains, cadherin domains, immunoglobulin-like domains, phosphotyrosine-binding domains, pleckstrin homology domains, src homology 2 domains, among others. Thus, binding domains may be derived from naturally occurring molecules, e.g., components of the innate or adaptive immune system, or may be entirely artificially designed.

[0059] In general, binding domains may be based on immunoglobulins or may be based on domains present in proteins such as, but not limited to, microbial proteins, protease inhibitors, toxins, fibronectin, lipocalins, single-stranded antiparallel coiled-coil proteins, or repeat motif proteins. Specific examples of binding domains known in the art include, but are not limited to, antibodies, heavy chain antibodies (hcAbs), single domain antibodies (sdAbs), minibodies, variable domains derived from camelid heavy chain antibodies (VHHs or nanobodies), variable domains of novel antigen receptors derived from shark antibodies (VNAs), alphabodies, protein A, protein G, designed ankyrin repeat domains (DARPins), fibronectin type III repeats, anticalins, knottins, engineered CH2 domains (nanobodies), engineered SH3 domains, affibodies, peptides, and proteins, lipopes, and the like. Examples of suitable binding domains include peptides (e.g., pepducins) (see, e.g., Gebauer & Skerra, 2009; Skerra, 2000; Starovasnik et al., 1997; Binz et al., 2004; Koide et al., 1998; Dimitrov, 2009; Nygren et al., 2008; WO 2010066740). When selection methods are used to generate specific types of binding domains, combinatorial libraries containing consensus or framework sequences with randomized potential interacting residues are often used to screen for binding to a molecule of interest, e.g., a protein.

[0060] According to a preferred embodiment, it is specifically envisaged that the binding agent of the present invention is derived from the innate or adaptive immune system. Preferably, the binding agent is derived from an immunoglobulin. Preferably, the binding agent according to the present invention is derived from an antibody or an antibody fragment. The term "antibody" (Ab) generally refers to a polypeptide encoded by an immunoglobulin gene or a functional fragment thereof that specifically binds to and recognizes an antigen, and is known to those skilled in the art. Antibody is intended to include conventional four-chain immunoglobulins, which comprise a pair of two identical polypeptide chains, each having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50 kDa). Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen-binding site. The term "antibody" is intended to include whole antibodies, including single-chain whole antibodies, and antigen-binding fragments. In some embodiments, the antigen-binding fragment may be, but is not limited to, Fab, Fab', and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv), and fragments comprising or consisting of either a VL or VH domain, and any combination thereof, or any other functional portion of an immunoglobulin peptide capable of binding to a target antigen. The term "antibody" is also intended to include heavy-chain antibodies or fragments thereof comprising an immunoglobulin single variable domain, as further defined herein.

[0061] The term "immunoglobulin single variable domain" or "ISVD" defines a molecule in which the antigen-binding site is present in and formed by a single immunoglobulin domain (this differs from conventional immunoglobulins or fragments thereof in which typically two immunoglobulin variable domains interact to form the antigen-binding site). However, it will be clear that the term "immunoglobulin single variable domain" includes fragments of conventional immunoglobulins in which the antigen-binding site is formed by a single variable domain. Preferably, binding agents within the scope of the present invention are immunoglobulin single variable domains.

[0062] In general, an immunoglobulin single variable domain comprises four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3), and preferably comprises an amino acid sequence according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(1), or any suitable fragment thereof (usually comprising at least a portion of the amino acid residues forming at least one of the complementarity-determining regions). ISVDs comprising four FRs and three CDRs are known to those skilled in the art, and are described, by way of non-limiting example, in Wesolowski et al., 2009. Typical, but non-limiting, examples of immunoglobulin single variable domains include light chain variable domain sequences (e.g., VL domain sequences) or suitable fragments thereof, or heavy chain variable domain sequences (e.g., VH domain sequences or VHH domain sequences) or suitable fragments thereof, as long as they are capable of forming a single antigen-binding unit. Thus, according to a preferred embodiment, the binding agent is an immunoglobulin single variable domain which is a light chain variable domain sequence (e.g. a VL domain sequence) or a heavy chain variable domain sequence (e.g. a VH domain sequence), more particularly the immunoglobulin single variable domain is a heavy chain variable domain sequence derived from a traditional four chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. The immunoglobulin single variable domain can be a domain antibody, or a single domain antibody, or a "dAB" or dAb, or a nanobody (as defined herein), or another immunoglobulin single variable domain, or any suitable fragment of any one thereof. For a general description of single domain antibodies see the following book: "Single domain antibodies", Methods in Molecular Biology, Eds. Saerens and Muyldermans, 2012, Vol. 911. An immunoglobulin single variable domain generally comprises a single chain of amino acids which can be considered to comprise four "framework sequences" or FRs and three "complementarity determining regions" or CDRs (as defined herein above).It is evident that the framework regions of immunoglobulin single variable domains can also contribute to their antigen binding (Desmyter et al., 2002; Korotkov et al., 2009).

[0063] As further described herein, the total number of amino acid residues in a VHH, Nanobody or Confobody may be in the region of 110-120, preferably 112-115, and most preferably 113. It should be noted, however, that parts, fragments, analogs or derivatives of a VHH or Nanobody (as further described herein) are not particularly limited with respect to their length and / or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are suitable for the purposes described herein.

[0064] In the present application, the amino acid residues / positions within the immunoglobulin heavy chain variable domain are determined by the V sequences derived from camel in the literature as described by Riechmann and Muyldermans, J. Immunol. Methods, 2000 Jun. 23; 240(1-2):185-195. HH As applied to domains (see, e.g., Figure 2 therein), they are shown using the Kabat numbering system ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). See also, e.g., Figure 1 of International Application WO 2108 / 134235, which provides a table listing some of the amino acid positions in VHHs and their numbering according to several alternative numbering systems (e.g., Aho and IMGT; it should be noted that, unless expressly indicated otherwise, for the purposes of this specification and claims, the Kabat numbering system is definitive for amino acid residues / positions in VHHs, nanobodies or confobodies, and other numbering systems are given for reference only).

[0065] Regarding CDRs, as is well known in the art, there are several rules for defining and describing CDRs of VH or VHH fragments, such as the Kabat definition (based on sequence variability and is the most commonly used) and the Chothia definition (based on the location of structural loop regions). See, for example, the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and claims, although Kabat CDRs may also be referred to, it is most preferred that CDRs be defined based on the Abm definition (based on Oxford Molecular's AbM antibody modeling software), as this Abm definition is considered to be the best compromise between the Kabat and Chothia definitions. Again, see the website http: / / www.bioinf.org.uk / abs / .

[0066] Therefore, in this specification and claims, all CDRs or VHHs, nanobodies or confobodies are defined according to the Abm rules unless expressly stated otherwise herein.

[0067] It should be noted that immunoglobulin single variable domains as binding agents in the broadest sense are not limited to a particular biological source or a particular preparation method. The term "immunoglobulin single variable domain" or "ISVD" encompasses variable domains of various origins, including mouse, rat, rabbit, donkey, human, shark, and camelid variable domains. According to a particular embodiment, the immunoglobulin single variable domain is derived from a shark antibody (so-called immunoglobulin novel antigen receptor or IgNAR), more particularly from a naturally occurring heavy-chain shark antibody without a light chain, known as a VNAR domain sequence. Preferably, the immunoglobulin single variable domain is derived from a camelid antibody. More preferably, the immunoglobulin single variable domain is derived from a naturally occurring heavy-chain camelid antibody without a light chain, known as a VHH domain sequence or nanobody.

[0068] According to a particularly preferred embodiment, the binding agents of the invention are immunoglobulin single variable domains that are nanobodies (as further defined herein and including, but not limited to, VHHs). As used herein, the term "nanobody" (Nb) refers to a single domain antigen-binding fragment. It particularly refers to a single variable domain derived from a naturally occurring heavy chain antibody and is known to those skilled in the art. Nanobodies are typically derived from heavy chain-only antibodies (without light chains) found in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996) and are consequently often referred to as VHH antibodies or VHH sequences. The Camelidae family consists of Old World Camelidae (Camelus bactrianus and Camelus dromedarius) and New World Camelidae (e.g., Lama paccos, Lama glama, Lama guanicoe, and Lama vicugna). Nanobody® and Nanobodies® are registered trademarks of Ablynx NV (Belgium).For a further description of VHHs or nanobodies, see the book "Single domain antibodies", Methods in Molecular Biology, Eds. Saerens and Muyldermans, 2012, volume 911, in particular the chapter by Vincke and Muyldermans (2012), as well as the non-limiting list of patent applications mentioned as general background art, including: Vrije Universiteit Brussels WO 94 / 04678, WO 95 / 04079, WO 96 / 34103; Unilever WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231, and WO 02 / 48193; Vlaams Instituut voor WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016, and WO 03 / 055527 by Biotechnologie (VIB); WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787, and WO 06 / 122528 by Ablynx NV; see further published patent applications by NV. As known to those skilled in the art, Nanobodies are particularly characterized by the presence of one or more Camelidae "hallmark residues" in one or more framework sequences (according to Kabat numbering), e.g., as described in Table A-3 on page 75 of WO 08 / 020079, which is incorporated herein by reference. It should be noted that the Nanobodies of the invention in their broadest sense are not limited to a particular biological source or a particular method of preparation.(iii) by "humanizing" a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (iv) by "camelizing" a naturally occurring VH domain from any animal species, in particular a mammalian species such as human, or by expressing a nucleic acid encoding such a camelized VH domain; (v) by "camelizing" a "domain antibody" or "Dab" as described in the art or by expressing a nucleic acid encoding such a camelized VH domain; (vi) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (vii) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expressing the nucleic acid so obtained; and / or (8) by any combination of one or more of the foregoing. Further description of Nanobodies, including humanization and / or camelization of Nanobodies, can be found, for example, in WO 08 / 101985 and WO 08 / 142164, and further description herein. A particular class of Nanobody-binding conformational epitopes of natural targets are called Xaperones and are specifically contemplated herein. Xaperone™ is a trademark of VIB and VUB (Belgium). Xaperone™ is a camelid single domain antibody that constrains drug targets into unique, disease-relevant, druggable conformations.

[0069] Within the scope of the present invention, the term "immunoglobulin single variable domain" also encompasses variable domains that have been "humanized" or "camelized", in particular Nanobodies that have been "humanized" or "camelized". For example, both "humanization" and "camelization" can be carried out by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then changing one or more codons of said nucleotide sequence in a manner known per se in such a way that the new nucleotide sequence encodes a "humanized" or "camelized" immunoglobulin single variable domain of the invention, respectively. This nucleic acid can then be expressed in a manner known per se to provide the desired immunoglobulin single variable domain of the invention. Alternatively, the amino acid sequence of the desired humanized or camelized immunoglobulin single variable domain of the invention can be designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesized de novo using techniques for peptide synthesis known per se. It is also possible to design a nucleotide sequence encoding the desired humanized or camelized immunoglobulin single variable domain of the invention based on the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesize it de novo using techniques for nucleic acid synthesis known per se, and then express the nucleic acid so obtained in a manner known per se to provide the desired immunoglobulin single variable domain of the invention. Other suitable methods and techniques for obtaining the immunoglobulin single variable domains of the invention and / or nucleic acids encoding them, starting from naturally occurring VH sequences, or preferably VHH sequences, will be clear to the skilled person and may, for example, involve combining one or more parts (such as one or more FR sequences and / or CDR sequences) of one or more naturally occurring VHH sequences, one or more parts (such as one or more FR sequences or CDR sequences) of one or more naturally occurring VHH sequences, and / or one or more synthetic or semi-synthetic sequences in a suitable manner to provide a Nanobody of the invention or a nucleotide sequence or nucleic acid encoding it.

[0070] According to certain embodiments of the present invention, a binding agent capable of stabilizing a receptor may bind at an orthosteric or allosteric site. In other specific embodiments, a binding agent capable of stabilizing a receptor may be an active conformation-selective binding agent or a non-active conformation-selective binding agent, either by binding at an orthosteric site or by binding at an allosteric site. Generally, a conformation-selective binding agent that stabilizes the active conformation of a receptor will increase or enhance the affinity of the receptor for an active conformation-selective ligand, such as an agonist, more particularly a full agonist, partial agonist, or biased agonist, compared to the receptor in the absence of the binding agent (or in the presence of a mock binding agent, also referred to as a control binding agent or irrelevant binding agent that is not directed against and / or does not specifically bind to the receptor). Additionally, a binding agent that stabilizes the active conformation of the receptor reduces the affinity of the receptor for non-active conformation-selective ligands, such as inverse agonists, compared to the receptor in the absence of the binding agent (or in the presence of a mock binding agent). In contrast, a binding agent that stabilizes the non-active conformation of the receptor increases the affinity of the receptor for inverse agonists and reduces the affinity of the receptor for agonists, particularly full agonists, partial agonists, or biased agonists, compared to the receptor in the absence of the binding agent (or in the presence of a mock binding agent). The increase or decrease in affinity for the ligand may be measured directly and / or calculated from a decrease or increase, respectively, in EC, IC, K, K, or any other measure of affinity or potency known to those skilled in the art. It is particularly preferred that binding agents that stabilize a particular conformation of the receptor are capable of increasing or decreasing the affinity for the conformation-selective ligand by at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, and more preferably at least 100-fold, even more preferably at least 1000-fold or even more, upon binding to the receptor.It will be appreciated that affinity measurements for conformationally selective ligands that induce / inhibit specific signaling pathways can be performed using any type of ligand, including natural ligands, small molecules, and biological substances; orthosteric ligands and allosteric modulators; single compounds and compound libraries; lead compounds or fragments, etc.

[0071] As used herein, the term "affinity" refers to the degree to which a ligand binds to a target protein such that the equilibrium between the target protein and its bound ligand (as further defined herein) is shifted toward the presence of a complex formed by their binding. Thus, for example, when a GPCR and a ligand are combined at relatively equal concentrations, a high-affinity ligand will bind to the available antigen of the GPCR, shifting the equilibrium toward a higher concentration of the resulting complex. Dissociation constants are commonly used to describe the affinity between a ligand and a target protein. Typically, dissociation constants are between 10 and 10. -5 Preferably, the dissociation constant is less than 10 -6 Less than M, more preferably 10 -7 Most preferably, the dissociation constant is less than 10 -8The affinity between a ligand (including a small molecule ligand) and its target protein is less than M. Another way to describe the affinity between a ligand (including a small molecule ligand) and its target protein is to indirectly assess the potency of the ligand by measuring the association constant (Ka), the inhibition constant (Ki), or the half maximal inhibitory concentration (IC50) or half maximal effective concentration (EC50). Within the scope of the present invention, the ligand may be a binding agent that binds to a conformational epitope of a GPCR, preferably an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as a VHH or nanobody. It will be understood that within the scope of the present invention, the term "affinity" is used in the context of a binding agent, particularly an immunoglobulin or immunoglobulin fragment, such as a VHH or nanobody, that binds to a conformational epitope of a target GPCR, as well as in the context of a test compound (as further defined herein) that binds to a target GPCR, particularly an orthosteric or allosteric site of the target GPCR.

[0072] As used herein, the term "specificity" refers to the ability of a protein or other binding agent, in particular an immunoglobulin or immunoglobulin fragment such as a VHH or nanobody, to bind preferentially to one antigen over a different antigen, and does not necessarily imply high affinity.

[0073] The terms "specifically bind" and "specific binding," as used herein, generally refer to the ability of a binding agent, particularly an immunoglobulin such as an antibody or an immunoglobulin fragment such as a VHH or nanobody, to preferentially bind to a particular antigen present in a homogenous mixture of different antigens. In certain embodiments, a specific binding interaction can distinguish between desired and undesired antigens in a sample by a factor of more than about 10-fold, and in some embodiments, up to 100-fold or more (e.g., about 1000-fold or greater than 10,000-fold). In the context of a range of conformational states of a GPCR, the terms "specifically bind" and "specific binding" particularly refer to the ability of a binding agent (as defined herein) to preferentially recognize and / or bind a particular conformational state of a GPCR compared to alternative conformational states.

[0074] Also, in this specification and the appended claims, when a protein, ligand, compound, binding domain, binding unit, or other chemical entity is said to "bind" to another protein, ligand, compound, binding domain, binding unit, or other chemical entity, or to an epitope or binding site, it is to be understood that such binding is preferably "specific" binding as defined herein. Also, preferably, such binding is "selective binding" as defined herein.

[0075] As used herein, in the context of the present invention, a "conformation-selective binding agent" refers to a binding agent that binds to a target protein in a conformation-selective manner. A binding agent that selectively binds to a particular conformation or conformational state of a protein refers to a binding agent that binds to a protein in a subset of conformations or conformational states with higher affinity than to other possible conformations or conformational states of the protein. Those skilled in the art will recognize that a binding agent that selectively binds to a particular conformation or conformational state of a protein stabilizes or maintains the protein in this particular conformation or conformational state. For example, an active conformation-selective binding agent preferentially binds to GPCRs in an active conformational state and does not bind or binds to a lower extent to GPCRs in an inactive conformational state, and therefore has a higher affinity for the active conformational state. And vice versa. The terms "specifically bind," "selectively bind," "preferentially bind," and their grammatical equivalents are used interchangeably herein. The terms "conformation-specific" or "conformation-selective" are also used interchangeably herein.

[0076] As used herein, the term "stabilize," or grammatically equivalent terms, refers to an increase in the stability of a protein (described herein) or receptor (described herein) in terms of structure (e.g., conformational state) and / or a particular biological activity (e.g., intracellular signaling activity, ligand binding affinity, etc.), as described above. With respect to increased stability to structure and / or biological activity, stabilization can be readily determined by either functional assays for activity (e.g., Ca release, cAMP production or transcriptional activity, β-arrestin recruitment) or ligand binding, or by physical methods such as X-ray crystallography, NMR, or spin labeling, among others. The term "stabilize" also includes increased thermal stability of a receptor under non-physiological conditions induced by denaturing agents or conditions. As used herein, the terms "thermostabilize," "thermostabilization," and "increase the thermal stability of" refer to functional properties of a receptor, not thermodynamic properties, and refer to the resistance of a protein to irreversible denaturation induced by thermal and / or chemical approaches, including, but not limited to, heating, cooling, freezing, chemical denaturants, pH, detergents, salts, additives, proteases, or temperature. Irreversible denaturation results in irreversible unfolding of the protein's functional conformation, loss of biological activity, and aggregation of the denatured protein. With respect to increased stability to heat, stability can be readily determined by measuring ligand binding or by using spectroscopic methods, such as fluorescence, CD, or light scattering, that are sensitive to unfolding at elevated temperatures. Preferably, the binding agent is capable of increasing stability as measured by an increase in the thermal stability of the protein or receptor in its functional conformational state by at least 2°C, at least 5°C, at least 8°C, and more preferably by at least 10°C, 15°C, or 20°C. With respect to increased stability to detergents or chaotropes, typically the protein or receptor is incubated in the presence of a test detergent or chaotropic agent for a predetermined period of time, and stability is determined using, for example, ligand binding or spectroscopy, optionally at elevated temperatures as described above.Alternatively, the binding agent may increase the stability of the functional conformational state of a protein or receptor against pH extremes. With respect to pH extremes, typical test pHs are selected, for example, in the range of 6-8, 5.5-8.5, 5-9, or 4.5-9.5, more particularly, in the range of 4.5-5.5 (low pH) or 8.5-9.5 (high pH). As used herein, the terms "thermostabilize," "thermostabilize," and "increase the thermal stability of" apply to proteins or receptors embedded in lipid particles or lipid layers (e.g., lipid monolayers, lipid bilayers, etc.), as well as proteins or receptors dissolved in detergent.

[0077] Further to the above, the term "stabilization" or "stabilized," with respect to the functional conformational state of a GPCR, refers to maintaining or retaining the GPCR protein in a subset of the possible conformations it might otherwise assume, due to the effect of the interaction of the GPCR with a binding agent according to the present invention. In this context, a binding agent that selectively binds to a particular conformation or conformational state of a protein refers to a binding agent that binds with higher affinity to the protein in a certain conformation or subset of conformational states than to other possible conformations or conformational states of the protein. Those skilled in the art will recognize that a binding agent that specifically or selectively binds to a particular conformation or conformational state of a protein stabilizes this particular conformation or conformational state and its associated activity. Further details are provided further herein.

[0078] As used herein, the terms "compound" or "test compound" or "candidate compound" or "drug candidate compound" describe any naturally occurring or synthetic molecule to be tested in an assay, such as a screening assay or drug discovery assay. Thus, these compounds include organic or inorganic compounds. Compounds include polynucleotides, lipids, or hormone analogs characterized by low molecular weight. Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptidomimetics) 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. The test compound may also be protein-based. For high-throughput purposes, test compound libraries, such as combinatorial 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, and phage display libraries. Further detailed descriptions can be found further herein.

[0079] As used herein, the term "ligand" refers to a molecule that specifically binds to a protein referred to herein, such as a GPCR. A ligand can be, but is not limited to, a polypeptide, lipid, small molecule, antibody, antibody fragment, nucleic acid, or carbohydrate. A ligand can be a synthetic or naturally occurring ligand. Ligands also include "natural ligands," which are endogenous natural ligands for native GPCRs. In the context of the present invention, when a protein is a transmembrane protein such as a GPCR, a ligand can bind to the protein at a ligand-binding site that is exposed to the intracellular environment when the protein is in its native cellular environment (i.e., the ligand can be an "intracellular ligand"), or can bind to the protein at a ligand-binding site that is exposed to the extracellular environment when the protein is in its native cellular environment (i.e., the ligand can be an "extracellular ligand"). A ligand can be an agonist, partial agonist, inverse agonist, antagonist, or allosteric modulator, and can bind at either an orthosteric site or an allosteric site. In certain embodiments, a ligand may be a "conformation-selective ligand" or "conformation-specific ligand," meaning that such a ligand binds to a protein or GPCR in a conformation-selective manner. As further described herein, a conformation-selective ligand binds with higher affinity to a particular conformation of a protein than to other conformations that the protein may adopt. By way of example, an agonist is an example of an active, conformation-selective ligand, and an inverse agonist is an example of a non-active, conformation-selective ligand. For clarity, a neutral antagonist is not considered a conformation-selective ligand because it does not distinguish between different conformations of a GPCR.

[0080] As used herein, "orthosteric ligand" refers to ligands (both natural and synthetic) that bind to the active site of GPCRs and are further classified according to their potency, i.e., their effect on signal transduction through a specific pathway. As used herein, "agonist" refers to a ligand that increases the signal transduction activity of a receptor by binding to the receptor protein. A full agonist allows maximal protein stimulation. A partial agonist cannot elicit full activity even at saturating concentrations. A partial agonist can also function as a "blocker" by preventing the binding of more potent agonists. An "antagonist," also known as a "neutral antagonist," refers to a ligand that binds to the receptor without stimulating any activity. An "antagonist" is also known as a "blocker" because it can prevent the binding of other ligands and thus block agonist-induced activity. Furthermore, an "inverse agonist" refers to an antagonist that, in addition to blocking the agonist effect, reduces the basal or constitutive activity of the receptor below that of the unliganded protein.

[0081] As used herein, a ligand may be a "biased ligand" that has the ability to selectively stimulate a subset of receptor signaling activities, e.g., in the case of GPCRs, selective activation of G-protein or β-arrestin function. Such ligands are known as "biased ligands," "biased agonists," or "functionally selective agonists." More specifically, ligand bias may be incomplete bias (non-absolute selectivity), characterized by ligand stimulation of multiple receptor activities with different relative potencies for different signals, or complete bias, characterized by ligand stimulation of one receptor protein activity without any stimulation of another known receptor protein activity.

[0082] Another type of ligand is known as an allosteric regulator. "Allosteric regulator," or "allosteric modulator," "allosteric ligand," or "effector molecule," as used herein, refers to a ligand that binds to an allosteric site of a GPCR (i.e., a regulatory site that is physically distinct from the active site of the protein). In contrast to orthosteric ligands, allosteric modulators bind to the receptor protein at a different site and are non-competitive, modifying its function even when the endogenous ligand is bound. Allosteric modulators that enhance the activity of a protein are referred to herein as "allosteric activators" or "positive allosteric modulators" (PAMs), while those that decrease the activity of a protein are referred to herein as "allosteric inhibitors" or "negative allosteric modulators" (NAMs).

[0083] As used herein, the terms "determining," "measuring," "evaluating," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

[0084] The term "antibody" is intended to mean an immunoglobulin or any fragment thereof capable of binding to an antigen. The term "antibody" also refers to single-chain antibodies and antibodies with only one binding domain.

[0085] As used herein, the term "complementarity-determining region" or "CDR" in the context of an antibody refers to the variable region of either the heavy (H) or light (L) chain (also abbreviated as VH and VL, respectively) and contains amino acid sequences capable of specifically binding to an antigen target. These CDR regions are responsible for the antibody's basic specificity for a particular antigenic determinant structure. Such regions are also called "hypervariable regions." Although CDRs represent noncontiguous stretches of amino acids within the variable region, the locations of these key amino acid sequences within the variable heavy and light chain regions are found to have similar positions within the amino acid sequences of the variable chains, regardless of species. The variable heavy and light chains of all classical antibodies each have three CDR regions (termed LI, L2, L3, H1, H2, and H3) that are noncontiguous with each other for each light (L) and heavy (H) chain. Immunoglobulin single variable domains, and in particular nanobodies, generally comprise a single amino acid chain that can be considered to comprise four "framework sequences or regions" or FRs and three "complementarity-determining regions" or CDRs. Nanobodies have three CDR regions (termed CDR1, CDR2, CDR3), each of which is non-contiguous with the others. As referred to herein, amino acid positions / residues in VHHs, nanobodies or confobodies CDRs are indicated according to the Kabat numbering system, and frameworks and CDRs are defined according to the Abm definition (unless explicitly stated otherwise).

[0086] In general, for purposes of the disclosure herein and in the appended claims, a compound of the invention is considered a "modulator" of a target (and / or signal transduction, pathway(s), mechanism of action, and / or biological, physiological, and / or pharmacological function(s) involving the target) or "modulates" said target when the compound, when present in an appropriate assay or model (i.e., in an appropriate amount or concentration, e.g., a biologically active amount or concentration), alters the appropriate or intended readout of said assay or model (i.e., at least one appropriate value or parameter that can be determined using said assay or model) by at least 0.1%, e.g., at least 1%, at least 10%, and up to 50% or more, compared to the same value or parameter when measured using the same assay or model under essentially the same conditions but in the absence of said compound. Again, said modulation may result in an increase or decrease (i.e., by the percentage given in the preceding sentence) of said value or parameter. The compounds of the present invention are also preferably such compounds that can modulate said target, signal transduction, pathway(s), mechanism of action and / or said biological, physiological and / or pharmacological function in a dose-dependent manner, i.e., within or over at least one concentration range of the compound used in the assay or model.

[0087] The methods of the present invention generally comprise at least the following components (all further defined herein): · A boundary layer separating the first environment from the second environment; · Translaminar proteins; a first ligand for a transmembrane protein present in the first environment (as defined herein); a second ligand for the transmembrane protein present in the second environment (as defined herein); and a binding pair capable of generating a detectable signal, the binding pair consisting of at least a first binding member and a second binding member; An arrangement comprising: the components are disposed relative to one another (and, where applicable, operably linked to one another and / or associated with one another) in a manner as further described herein; In the arrangement, it is carried out.

[0088] In particular, in the arrangements of the present invention, and as further described herein, the first binding member of the binding pair may be part of a "first fusion protein" (as further described herein), and the second member of the binding pair may be part of a "second fusion protein" (as further described herein and different from the first fusion protein). Such first fusion proteins, such second fusion proteins (in various formats as described herein), nucleotide sequences and / or nucleic acids encoding them, and cells, cell lines or other host cells or host organisms expressing (particularly as suitable as described herein) or (suitably) capable of expressing) either the first protein and / or the second protein also form further aspects of the present invention.

[0089] In particular, an arrangement for carrying out the method of the present invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a transmembrane protein suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair (i.e., to form a first fusion protein); a first ligand for a transmembrane protein present in the first environment; and · a second ligand for the transmembrane protein present in the second environment; wherein the components are arranged (and, where applicable, operably linked and / or associated with) each other in a manner further described herein. In particular, the second member of the binding pair may be part of a second fusion protein (different from the first fusion protein comprising the layer-spanning protein and the first binding member of the binding pair), where the second fusion protein is a second fusion protein further described herein.

[0090] More particularly, the arrangement for carrying out the method of the present invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., comprising said member of the binding pair such that it is present in the second environment); a second fusion protein present in a second environment, comprising a protein capable of binding directly or indirectly to the transmembrane protein and the other binding member of said binding pair; and a first ligand for a transmembrane protein present in the first environment; wherein the components are disposed (and, where applicable, operably linked and / or associated with each other) in a manner as further described herein.

[0091] It should be noted that, in the specification and claims, when a ligand, binding domain, binding unit, or other compound or protein is said to be "capable of binding" to another protein or compound, such binding is most preferably a "specific binding," as further defined herein. Also, as further described herein, when a fusion protein is described as "comprising" a first protein, ligand, binding domain, binding member or binding unit, and a second protein, ligand, binding domain, binding member or binding unit (and optionally one or more further proteins, ligands, binding domains, binding members or binding units), it should be understood that in such a fusion protein, such proteins, ligands, binding domains, binding members or binding units are suitably linked to each other, either directly or via a suitable spacer or linker.

[0092] For purposes of this specification and the claims, a protein (such as a binding domain, binding unit, or ligand) is said to bind "directly or indirectly" to a layer-spanning protein if (i) the protein itself binds (and / or is capable of binding) to the layer-spanning protein (e.g., to an epitope or binding site of the layer-spanning protein, as further described herein); or (ii) the protein binds (and / or is capable of binding) to a ligand or protein that binds (and / or is capable of binding) to the layer-spanning protein; or (iii) the protein binds (and / or is capable of binding) to a protein complex comprising a ligand or protein that binds (and / or is capable of binding) to the layer-spanning protein. In case (i), the protein is said to bind "directly" to the layer-spanning protein, and in cases (ii) and (iii), the protein is said to bind "indirectly" to the layer-spanning protein. Also, when a protein binds to a protein complex that includes a ligand or protein that binds to a transmembrane protein, the protein may bind to the ligand or protein, or any other part, epitope, or binding site of the complex.

[0093] Thus, in one aspect of the invention, the protein that binds to the layer-spanning protein is selected from (i) a binding domain, binding unit, or other protein that binds (and / or is capable of binding) to an epitope or binding site of the layer-spanning protein, (ii) a binding domain, binding unit, or other protein that binds (and / or is capable of binding) to a ligand or protein that binds (and / or is capable of binding) to said layer-spanning protein, and (iii) a binding domain, binding unit, or other protein that binds (and / or is capable of binding) to a protein complex comprising a ligand or protein that binds (and / or is capable of binding) to said layer-spanning protein. In each such case, such a binding domain, binding unit, or other protein is preferably a binding domain, binding unit, or other protein as further described herein.

[0094] In particular, a protein that directly or indirectly binds to a spanning protein may be selected from (i) an ISVD that binds (and / or is capable of binding) to an epitope or binding site of a spanning protein, (ii) an ISVD that binds (and / or is capable of binding) to a ligand or protein that binds (and / or is capable of binding) to said spanning protein, and (iii) an ISVD that binds (and / or is capable of binding) to a protein complex comprising a ligand or protein that binds (and / or is capable of binding) to said spanning protein. Again, in each such case, such an ISVD is preferably an ISVD as further described herein.

[0095] In a further aspect of the invention, an arrangement for carrying out the method of the invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., comprising said member of the binding pair such that said member is present in the second environment); a second fusion protein present in a second environment, the second fusion protein comprising a transmembrane protein and a protein capable of directly binding (as defined herein) to the other binding member of the binding pair; and a first ligand for a transmembrane protein present in the first environment; wherein the components are arranged (and, where applicable, operably linked and / or associated with each other) in a manner as further described herein. In this aspect of the invention, the protein that is capable of directly binding (as defined herein) to the layer-spanning protein and that is present in the second fusion protein is preferably a binding domain or binding unit, more preferably an immunoglobulin single variable domain. It should also be understood that in this aspect of the invention, the protein that is capable of directly binding (as defined herein) to the layer-spanning protein and that is present in the second fusion protein acts as a second ligand.

[0096] In another aspect of the invention, an arrangement for carrying out the method of the invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., comprising said member of the binding pair such that said member is present in the second environment); a first ligand for a transmembrane protein present in the first environment; a second ligand for the transmembrane protein, which may optionally be part of a protein complex; a second fusion protein present in a second environment, comprising a transmembrane protein and a protein capable of indirectly binding (as defined herein) to the other binding member of said binding pair; wherein the components are arranged (and, where applicable, operably linked and / or associated with each other) in a manner as further described herein. In this aspect of the invention, the second ligand may be any suitable ligand (as further described herein) and is capable of indirectly (as defined herein) binding to the layer-spanning protein, and the protein present in the second fusion protein is preferably a binding domain or binding unit, more preferably an immunoglobulin single variable domain. It will also be clear that in this aspect of the invention, the second ligand does not form part of the second fusion protein.

[0097] As described further herein, in practicing the present invention, a first ligand is often added to a further component of an already formed / established arrangement of the present invention as described herein, and it should be noted that, as a result, an arrangement of the present invention in which the first ligand is not present (i.e. before the first ligand is added) forms a further aspect of the present invention (as do methods in which a first ligand is added to an arrangement of the present invention in which said first ligand is not present or not yet present).

[0098] In this specification and claims, the term "second ligand" is used to denote a ligand, binding domain, binding unit or other chemical entity that binds directly to or is capable of binding directly to a translayer protein (or that forms part of a protein complex that binds directly to or is capable of binding directly to a translayer protein) in the methods and arrangements described herein.

[0099] As will be apparent from the further description herein, the second ligand can be part of the second fusion protein or can be separate from the second fusion protein. In either case (i.e., whether the second ligand is part of the second fusion protein or not), the second ligand is preferably one that is capable of binding to a conformational epitope of the transmembrane protein (or that is capable of binding directly to the transmembrane protein or forming part of a protein complex that is capable of binding directly to the transmembrane protein). More preferably, the second ligand (and / or the protein complex comprising the second ligand) is preferably a second ligand that specifically binds to one or more functional, active and / or druggable conformations of the layer-spanning protein, induces the formation of and / or stabilizes one or more functional, active and / or druggable conformations of the layer-spanning protein (and / or shifts the conformational equilibrium of the layer-spanning protein towards one or more such conformations), and / or induces the formation of and / or stabilizes a complex between the layer-spanning protein, the first ligand and the second ligand.

[0100] When the second ligand is part of a second fusion protein, it can be any ligand, binding domain, binding unit, peptide, protein, or other chemical entity that can be directly bound to the layer-spanning protein and that can be suitably included in the second fusion protein. Preferably, when part of a second fusion protein, the second ligand is a suitable binding domain or binding unit, in particular an immunoglobulin single variable domain, as further described herein.

[0101] When the second ligand is separate from the second fusion protein, the second ligand can be any ligand or protein capable of directly binding to the layer-spanning protein and / or forming part of a protein complex capable of binding to the layer-spanning protein. For example, as further described herein, such a second ligand can be a naturally occurring ligand of the layer-spanning protein (e.g., a naturally occurring G protein, e.g., a G protein naturally occurring in the cell or cell line used), a semisynthetic or synthetic analog or derivative of such a naturally occurring ligand or an ortholog of such a naturally occurring ligand (e.g., a "chimeric" G protein described herein). Also, when the second ligand is not part of the second fusion protein, the second fusion protein comprises a binding domain or binding unit capable of indirectly binding (as defined herein) to the layer-spanning protein, i.e., a binding domain or binding unit capable of binding to the second ligand and / or a protein complex containing the second ligand. Again, as further described herein, such a binding domain or binding unit can, in particular, be an immunoglobulin single variable domain, e.g., a camelid-derived ISVD. (or may comprise one or more such immunoglobulin single variable domains, which may be the same or different, for example two or three such immunoglobulin single variable domains, as further described herein).

[0102] As further described herein, in one aspect of the present invention, the arrangement of the present invention may be present in a suitable cell or cell line and / or the method of the present invention may be carried out using a suitable cell or cell line suitably comprising an (operable) arrangement of the present invention.

[0103] Thus, as further described herein, the present invention also relates to cells or cell lines that suitably comprise an arrangement of the present invention and / or that suitably express or are capable of suitably expressing (as defined herein) components of an arrangement of the present invention so as to provide an arrangement of the present invention (particularly an arrangement of the present invention operable in a cell or cell line). The present invention also relates to cells or cell lines that comprise and / or suitably express or are capable of suitably expressing (as defined herein) a first fusion protein as described herein. The present invention also relates to cells or cell lines that comprise and / or suitably express or are capable of suitably expressing a second fusion protein as described herein. In yet another aspect, the present invention relates to cells or cell lines that comprise and / or suitably express or are capable of suitably expressing both a first fusion protein as described herein and a second fusion protein as described herein. In aspects and embodiments in which the second ligand does not form part of the second fusion protein, such cells or cell lines may also comprise or suitably express a suitable second ligand.

[0104] Also as described herein, in one aspect of the invention, the arrangements of the invention may be present in suitable liposomes or vesicles and / or the methods of the invention may be carried out using liposomes or vesicles suitably comprising the (operable) arrangements of the invention.

[0105] Thus, as further described herein, the present invention also relates to liposomes or vesicles suitably comprising (components of) the arrangements of the present invention, particularly to provide arrangements of the present invention operable in liposomes or vesicles. The present invention also relates to liposomes or vesicles comprising a first fusion protein described herein. The present invention also relates to liposomes or vesicles comprising a second fusion protein described herein. In yet another aspect, the present invention relates to liposomes or vesicles comprising both a first fusion protein described herein and a second fusion protein described herein. In aspects and embodiments in which the second ligand does not form part of the second fusion protein, such liposomes or vesicles may also contain a suitable second ligand.

[0106] Thus, as further described herein and as illustrated by the accompanying non-limiting figures, the present invention contemplates at least three preferred embodiments of the methods and arrangements of the present invention, depending on whether the second ligand is part of a second fusion protein or not.

[0107] In a first such preferred embodiment (schematically shown in Figure 1), the second binding member of the binding pair is suitably fused or linked (directly or via a suitable linker or spacer) to a second ligand. According to this preferred embodiment, an arrangement for carrying out the method of the invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a transmembrane protein suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair; a first ligand for a transmembrane protein present in the first environment; and a second ligand for the layer-spanning protein present in a second environment and suitably fused or linked (directly or via a suitable linker or spacer) to the other binding member of said binding pair; wherein the components are disposed (and, where applicable, operably linked and / or associated with each other) in a manner as further described herein.

[0108] In particular, as further described herein, such an arrangement may comprise the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., such that said member of the binding pair is present in the second environment); a first ligand for a transmembrane protein present in the first environment; and a second fusion protein comprising a second ligand for the transmembrane protein and the other binding member of said binding pair, the second fusion protein being present in a second environment; wherein the components are disposed (and, where applicable, operably linked and / or associated with each other) in a manner as further described herein.

[0109] It will be clear to those skilled in the art that in this first embodiment, the "second ligand" is a binding domain, binding unit or other protein that binds (and / or is capable of binding) directly to an epitope or binding site of a spanning protein. Again, said binding domain or binding unit is preferably an immunoglobulin single variable domain as further described herein.

[0110] In a second such preferred embodiment (schematically shown in Figure 2), the second binding member of the binding pair does not bind directly to the layer-spanning protein, but instead is suitably fused or linked (directly or via a suitable linker or spacer) to a binding domain or binding unit that binds to a second ligand (which can then bind to the layer-spanning protein). According to this preferred embodiment, an arrangement for carrying out the method of the invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., such that said member of the binding pair is present in the second environment); a first ligand for a transmembrane protein present in the first environment; a second ligand for the transmembrane protein present in the second environment; and a second fusion protein comprising a binding domain or binding unit present in a second environment and capable of binding to a second ligand, the binding domain or binding unit being suitably fused or linked (directly or via a suitable linker or spacer) to the other binding member of said binding pair; wherein the components are disposed (and, where applicable, operably linked and / or associated with each other) in a manner as further described herein.

[0111] It will be clear to those skilled in the art that in this second embodiment, the binding domain or binding unit present in the second fusion protein binds to the layer-spanning protein "indirectly", i.e., by binding to a second ligand that binds to the layer-spanning protein. Moreover, said binding domain or binding unit is preferably (and / or preferably consists essentially of) an immunoglobulin single variable domain, as further described herein. A binding domain or binding unit can also comprise or essentially consist of two or more immunoglobulin single variable domains (e.g., two or three immunoglobulin single variable domains), each of which is capable of (specifically) binding to a second ligand (i.e., the same epitope or binding site of the second ligand, or different epitopes / binding sites of the second ligand), which may be identical or different (as further described herein), and which are suitably linked or fused (optionally via suitable linkers or spacers) to each other and to the other binding member of said binding pair to form a second fusion protein suitable for use in the present invention. For example, but not limited to, such a binding domain or binding unit may comprise two or three copies of the conformobody CA4437 (SEQ ID NO: 4 in WO 2012 / 75643 and SEQ ID NO: 2 herein), suitably linked or fused to each other and to the other binding member of said binding pair (optionally via a suitable linker or spacer) to form a second fusion protein suitable for use in the present invention. Also, in this embodiment, the second ligand may be any suitable ligand of a layer-spanning protein as further described herein. Also, again, such a "multivalent" binding domain comprising two or more ISVDs should most preferably be such that binding to the second ligand does not essentially interfere with the ability of the second ligand to bind to the layer-spanning protein and / or to form (or facilitate the formation of) a complex between the second ligand, the layer-spanning protein and the first ligand.

[0112] In a third preferred embodiment (schematically shown in Figure 3), the second binding member of the binding pair is suitably fused or linked (directly or via a suitable linker or spacer) to a binding domain or binding unit that does not bind directly to the layer-spanning protein, but instead binds to a protein complex that comprises at least a second ligand for the layer-spanning protein (which protein complex may bind to or be bound by the layer-spanning protein and / or may comprise the layer-spanning protein). According to this preferred embodiment, an arrangement for carrying out the method of the invention comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., such that said member of the binding pair is present in the second environment); a first ligand for a transmembrane protein present in the first environment; a protein complex comprising a second ligand for at least the transmembrane protein, the protein complex being present in a second environment; and a second fusion protein comprising a binding domain or binding unit present in a second environment and capable of binding to the protein complex, said binding domain or binding unit being suitably fused or linked (directly or via a suitable linker or spacer) to the other binding member of said binding pair; wherein the components are disposed (and, where applicable, operably linked and / or associated with each other) in a manner as further described herein.

[0113] It will be clear to those skilled in the art that in this third embodiment, the binding domain or binding unit present in the second fusion protein binds to the layer-spanning protein "indirectly", i.e., by binding to a protein complex comprising a second ligand, said binding domain or binding unit being preferably an immunoglobulin single variable domain as further described herein, and the second ligand being any suitable ligand for the layer-spanning protein, which may be part of a protein complex as further described herein.

[0114] The binding domain or binding unit in the second fusion protein may also comprise two or more immunoglobulin single variable domains, each capable of binding to a different epitope, portion, domain or subunit of the protein complex, e.g., capable of binding to two different epitopes of a G protein complex. For example, but not limited to, if the protein complex is a heterotrimeric G protein, the binding domain or binding unit may comprise two or three different ISVDs, each ISVD being capable of (specifically) binding to a different subunit of the G protein (preferably, at least one of the ISVDs is capable of specifically binding to the G alpha subunit present in the heterotrimeric G protein). Specific, but non-limiting examples of such binding domains or binding units may include, for example, compobody CA4435 (SEQ ID NO: 1 in WO 2012 / 75643 and SEQ ID NO: 1 herein) and compobody CA4437 (SEQ ID NO: 4 in WO 2012 / 75643 and SEQ ID NO: 2 herein), suitably linked or fused to each other and to the other binding member of said binding pair (optionally via a suitable linker or spacer) to form a second fusion protein suitable for use in the present invention.

[0115] The use of such "multivalent" (i.e., comprising two or more ISVDs) binding domains or binding units in the second fusion protein may result in improved sensitivity in the assays described herein compared to the use of the corresponding ISVD(s) in a monovalent format (i.e., comprising only one of said ISVDs).

[0116] More generally, arrangements for carrying out the methods of the invention in their various aspects and embodiments typically and preferably include at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair capable of producing a detectable signal, comprising at least a first binding member and a second binding member; a first fusion protein comprising a layer-spanning protein and one of the binding members of said binding pair (i.e., such that said member of the binding pair is present in the second environment); a first ligand for a transmembrane protein present in the first environment; a second ligand for the transmembrane protein present in the second environment; and a second fusion protein comprising the other binding member of said binding pair (i.e., such that said other member of the binding pair is also present in the second environment); At least The components are disposed relative to one another (and, where applicable, operably linked to one another and / or associated with one another) in a manner as further described herein. In particular, In the first preferred embodiment described herein, the second fusion protein comprises the other binding member of said binding pair and a second ligand; In a second preferred embodiment described herein, the second fusion protein comprises a binding domain or binding unit capable of binding to the other binding member of said binding pair and to a second ligand; and In a third preferred embodiment described herein, the second fusion protein comprises a binding domain or binding unit capable of binding to a protein complex comprising the other binding member of said binding pair and at least a second ligand.

[0117] The invention will now be illustrated by the further description herein, the experimental section below, and the accompanying non-limiting figures.

[0118] It will be apparent to those skilled in the art from the drawings and further description herein that several components of the inventive arrangements (e.g., boundary layers, layer-spanning proteins, binding pairs, optional linkers, and first ligands) are present in the various aspects and embodiments of the invention discussed herein. Thus, when a detailed description of any such component (including any preferences for any such component) is given herein, it should be understood that such description applies to all aspects and embodiments of the invention in which such component is present or used, unless expressly stated otherwise herein.

[0119] In the methods and arrangements of the present invention, the boundary layer (1) may be any layer (e.g. a wall or membrane) suitable to separate a first environment [A] from a second environment [B] (in a suitable in vitro system or in a suitable in vivo system).

[0120] For example, in a preferred embodiment of the present invention, in which the method of the present invention is carried out in a suitable cell or cell line (as further described herein), the boundary layer (1) is the cell membrane or cell wall of the cell or cell line used in the method of the present invention. In this embodiment, environment [A] is preferably the extracellular environment, and environment [B] is preferably the intracellular environment. Also in this embodiment, the first ligand (3) is preferably present in the extracellular environment, and the second ligand (4) is preferably present in the intracellular environment. Also, the first and second binding members (6) and (7) and the second fusion protein are preferably present in the intracellular environment.

[0121] In another preferred embodiment of the present invention, in which the method of the present invention is carried out in a suitable vesicle or liposome (as further described herein), the boundary layer (1) is the membrane or wall of the vesicle or liposome. In this embodiment, environment [A] is preferably the external environment of the vesicle or liposome, and environment [B] is preferably the internal environment of the vesicle or liposome. Also in this embodiment, the first ligand (3) is preferably present in the external environment of the vesicle or liposome, and the second ligand (4) is preferably present in the internal environment of the vesicle or liposome. The first and second binding members (6) and (7) and the second fusion protein are also preferably present in the internal environment of the vesicle or liposome.

[0122] However, while the present invention in some preferred embodiments is practiced using cells, liposomes or other suitable vesicles, it should be understood that the present invention in its broadest sense is not limited to the use of cells or vesicles, but may be practiced in any other suitable arrangement in which a boundary layer (1) is used to appropriately separate a first environment [A] from a second environment [B]. For example, the boundary layer may be a part or fragment of the cell wall or cell membrane present in a membrane extract, e.g., a membrane extract obtained from whole cells by techniques known per se, such as suitable osmotic and / or mechanical techniques known per se.

[0123] Thus, boundary layer (1) can be any suitable layer, wall, or membrane, particularly a biological wall or membrane (e.g., a cell wall or membrane, or a part or fragment thereof), or the wall or membrane of a liposome or other suitable vesicle. In particular, boundary layer (1) can be a suitable lipid bilayer, such as a phospholipid bilayer. When boundary layer (1) is the wall or membrane of a vesicle or liposome, boundary layer (1) can be unilamellar or multilamellar. Also, as further described herein, when boundary layer (1) is a cell membrane or cell wall, boundary layer (1) is preferably the wall or membrane of a cell or cell line that suitably expresses layer-spanning protein (2) (as defined herein), in particular suitably expressing a (first) fusion protein described herein comprising layer-spanning protein (2).

[0124] As shown diagrammatically by non-limiting Figures 1, 2 and 3, the boundary layer (1) contains a translaminar protein (2), which is the first binding site (8) for the first ligand (3) projects (as defined herein) into the first environment [A] (i.e., such that when the first ligand (3) is present in the first environment [A], the first binding site (8) is accessible for binding by said first ligand); Also, the second binding site (9) for the second ligand (4) projects (as defined herein) into the second environment [B] (i.e., such that when the second ligand (4) is present in the second environment [B], the second binding site (9) is accessible for binding by said second ligand); Thus, it penetrates the boundary layer (1).

[0125] In the present specification and claims, the term "layer-spanning protein" is used to refer to a protein used (e.g., screened) in the methods and arrangements of the present invention. In the methods and arrangements of the present invention, the layer-spanning protein (2) penetrates the boundary layer (1) (and / or is prepared and / or arranged in such a manner relative to the boundary layer) such that at least one portion of the amino acid sequence of the layer-spanning protein (2) protrudes (as defined herein) from the boundary layer (1) into a first environment [A], and at least one other portion of the amino acid sequence of the layer-spanning protein (2) protrudes (as defined herein) from the boundary layer (1) into a second environment [B]. In this context, when a portion of the amino acid sequence of the layer-spanning protein (2) is said to "protrude" from the boundary layer (1) into an environment (i.e., into the first environment [A] or the second environment [B]), this should generally be understood to mean that the portion of the sequence is exposed to the environment and / or is accessible for binding by a ligand, compound, or other chemical present in the environment. Thus, in the methods and arrangements of the present invention, at least one portion of the amino acid sequence of the layer-spanning protein (e.g., an epitope or binding site) should be accessible for binding by a ligand, compound or other chemical entity present in a first environment (in particular, binding by a first ligand (3)), and at least one other portion of the amino acid sequence of the layer-spanning protein (e.g., another epitope or binding site) should be accessible for binding by a ligand, compound or other chemical entity present in a second environment (in particular, binding by a second ligand (4)). In this respect, it should also be noted that the phrase "accessible for binding" should generally be taken to mean that a ligand, compound or other chemical present in the relevant environment can bind to a binding pocket or binding site on or within the layer-spanning protein, even if the actual binding site or binding pocket is deeper (or deeper) in the structure of the layer-spanning protein (even if the actual binding site or binding pocket is located in a part of the layer-spanning protein that does not itself physically protrude beyond the boundary layer).See, for example, the paper by Chevillard (cited herein) which shows that the binding site of a GPCR for the fragments used in the FBDD screening technique is deep within the GPCR structure (see, e.g., Figure 2 on page 1120), not on the surface of the GPCR, but nevertheless accessible for fragment binding. Theories of GPCR structure, GPCR signaling mechanisms, and GPCR ligand binding sites are also referenced from several other scientific papers cited herein.

[0126] Also, in this specification and claims, when any binding domain, binding unit, epitope, binding site, ligand, protein, or other compound or chemical or other structural entity (e.g., a protein complex) is said to be "present" in an environment (i.e., a first environment [A] or a second environment [B]), this should generally be understood to mean that the binding domain, binding unit, epitope, binding site, ligand, protein, or other compound or chemical or structural entity is exposed to the environment and / or is accessible for binding by another domain, ligand, protein, or compound present in the environment. Thus, for example, a compound or ligand present in the environment may be "free-floating" in the environment (i.e., not bound or immobilized to any other protein or structure), immobilized in the boundary layer, or fused to another protein (which may be immobilized in the boundary layer). Similarly, a binding domain or binding unit present in an environment may be part of a larger protein or structure (e.g., a fusion protein), which may be free-floating in the environment or fixed to a boundary layer or other structure, so long as the binding domain or binding unit is accessible for binding by another domain, ligand, protein, or compound present in the environment. Also, an epitope or binding site present in the environment may be part of a larger protein or structure, which may be free-floating in the environment or fixed to a boundary layer or other structure, so long as the epitope or binding site is accessible for binding by another domain, ligand, protein, or compound present in the environment.

[0127] The one or more portions of the layer-spanning protein (2) that protrude into the first environment [A] may be any loop, epitope (linear or conformational), binding site or other portion(s) of the amino acid sequence of the layer-spanning protein, and similarly, the one or more portions of the layer-spanning protein that protrude into the second environment [B] may be any loop, epitope (linear or conformational), binding site or other portion(s) of the amino acid sequence of the layer-spanning protein (provided that they are different from the portion(s) that protrude into the first environment).

[0128] In a preferred embodiment of the present invention, the layer-spanning protein (2) comprises at least two different / separate ligand binding sites, of which at least a first binding site projects (as defined herein) into a first environment [A] (particularly so as to be accessible for binding by a first ligand (3)) and at least a second binding site projects (as defined herein) into a second environment [B] (particularly so as to be accessible for binding by a second ligand (4)).

[0129] Generally, the layer-spanning protein (2) is bound to and / or anchored in the boundary layer (1) in a manner known per se for membrane (spanning) proteins that are typically anchored in the cell wall or membrane in their natural environment, for example. As further described herein, this can be achieved, for example, by appropriately expressing a nucleotide sequence or nucleic acid (as defined herein) that expresses the first fusion protein in a suitable host cell, so that the layer-spanning protein (2) is appropriately anchored in the wall or membrane of the cell. When the method of the present invention is carried out using liposomes or vesicles, this can be achieved by appropriately forming the liposomes or vesicles in the presence of the first fusion protein, so that the layer-spanning protein (2) is appropriately anchored in the wall or membrane of the liposomes or vesicles.

[0130] The transmembrane protein (2) may comprise one or more domains (in particular one or more transmembrane domains) and is usually preferably a transmembrane protein, such as a (transmembrane) receptor.

[0131] If the transmembrane protein (2) is a transmembrane protein, the transmembrane protein (2) can be a bitopic membrane protein (i.e., a transmembrane protein with one passage through the membrane) or a polytopic membrane protein (i.e., a transmembrane protein with two or more passages through the membrane). Thus, the transmembrane protein (2) can be any known or newly discovered transmembrane protein (or a synthetic or recombinant analog thereof) with known or unknown biological function and known or unknown ligand (e.g., the transmembrane protein (2) can be a so-called "orphan" GPCR).

[0132] The membrane-spanning protein (2) may be an alpha-helical protein or a beta-barrel protein, and may be a type I, II, III, or IV membrane-spanning protein, depending on the location of the protein's N- and C-termini relative to the boundary layer. Preferably, the membrane-spanning protein is a protein that, in its natural cellular environment, has its amino terminus on the outside of the cell and its carboxy terminus on the inside of the cell, although the invention in its broadest sense is not limited thereto.

[0133] Also, when the method of the invention is carried out in a cell, the arrangement of the N- and C-termini of the protein relative to the wall or membrane of the cell used is preferably the same as the arrangement of said termini in the protein's natural cellular environment.

[0134] When the methods of the present invention are practiced with liposomes or vesicles, the liposomes or vesicles can be a mixture of liposomes / vesicles in which the protein is arranged in a manner that is essentially the same as the way the protein is arranged relative to the cell wall or membrane in its natural environment (i.e., with the N-terminus and extracellular loop(s) protruding outside the vesicle and the C-terminus and intracellular loop(s) protruding inside the vesicle), and vesicles / liposomes in which the protein is arranged in the opposite manner. Typically, this will not affect the operation of the systems or mechanisms described herein.

[0135] As further described herein, generally and preferably, the layer-spanning protein (2) is a protein that exists in (i.e., can adopt) two or more conformations (e.g., a basal state / conformation, an active state / conformation, and / or an inactive state / conformation, and / or a ligand-bound or ligand-free conformation) and / or can undergo a conformational change (e.g., a functional conformational change). In particular, the layer-spanning protein (2) can exhibit at least one functional conformation and at least one non-functional conformation (e.g., a basal conformation) and / or can undergo a conformational change from a non-functional conformation to a functional conformation; more particularly, it can exhibit an active (or more active) conformation and an inactive (or less active) conformation and / or can undergo a conformational change from an inactive (or less active) conformation to an active (or more active) conformation. The layer-spanning protein (2) can also be a protein capable of adopting at least one ligand-bound (particularly agonist-bound) conformation and at least one ligand-free conformation. In particular, the layer-spanning protein (2) can be a protein capable of adopting at least one ligand-bound (particularly agonist-bound) conformation, which is an active or functional conformation.

[0136] As described herein, certain classes of functional conformations of (transmembrane) proteins (e.g., certain GPCRs) are called / defined as "druggable conformations." Thus, in one particular aspect, the membrane-spanning protein (2) may be a protein that can assume at least one such druggable conformation (often an active conformation, although the invention is not limited to the use of druggable conformations that are active conformations) and at least one conformation that is not a druggable conformation (often an inactive conformation) and / or may be a membrane-spanning protein that can undergo a conformational change from a non-druggable conformation to a druggable conformation.

[0137] In particular, the layer-spanning protein (2) can be a protein that undergoes a conformational change upon binding of a ligand (especially an agonist) to the protein. This conformational change upon ligand binding can be, for example, from an active to an inactive conformation or from a functional to a non-functional conformation, but is preferably from a non-functional to a functional conformation and / or from an inactive to an active conformation. In a particular embodiment, it is a change from a non-druggable conformation to a druggable conformation.

[0138] For example, if the layer-spanning protein (2) is a receptor, such as a cell surface receptor (or a synthetic analog thereof), the layer-spanning protein (2) can be a protein that undergoes a conformational change when the receptor's natural or synthetic (extracellular) ligand binds to the receptor.

[0139] When the layer-spanning protein (2) is a GPCR, in a preferred, but non-limiting embodiment, the conformational change can be from a conformation that is essentially incapable of binding a G protein to a conformation that binds a G protein (or is capable of being bound by a G protein).

[0140] As referred to herein, a ligand capable of inducing a conformational change of the transmembrane protein (2) from a non-functional state to a functional state (e.g., from an inactive state, such as a basal state, to an active state) is also referred to herein as an "agonist" of the transmembrane protein (2). In particular, when the transmembrane protein (2) is a GPCR, the "agonist" may be capable of inducing a conformational change from a conformation that is essentially incapable of binding a G protein to a conformation that binds a G protein.

[0141] In a preferred embodiment of the present invention, the interlayer protein (2) is a protein that undergoes (or is capable of undergoing) a conformational change (as described herein) when a first ligand (3) binds to the interlayer protein (2), and conversely, the first ligand (3) is a first ligand that can induce a conformational change in the interlayer protein (2) when the first ligand (3) binds to the interlayer protein (2) (and / or the present invention is used to identify such a first ligand). In a more preferred embodiment, the conformational change is from an inactive or less active state to a functional or(more) active state, and the first ligand (3) used is capable of inducing a conformational change in the interlayer protein from an inactive or less active state to a functional or(more) active state when the first ligand binds to the interlayer protein (2). Furthermore, when the layer-spanning protein (2) is a GPCR, the conformational change upon ligand binding can be, in a preferred but non-limiting embodiment, a change from a conformation that is essentially incapable of binding a G protein to a conformation that binds a G protein.

[0142] As further described herein, the transmembrane protein may be a protein capable of forming a complex with a first ligand and a second ligand. In particular, the transmembrane protein may be a protein capable of forming a complex with an intracellular ligand and an extracellular ligand in its natural environment. For example, from the references cited herein, it is known that most GPCRs form complexes with extracellular ligands and G proteins (which are the most common natural intracellular ligands of GPCRs), and that such complexes are stabilized by the binding of the G proteins to an intracellular conformational epitope of the GPCR. Similarly, in the present invention, the second ligand preferably stabilizes the (formation of) a complex of the transmembrane protein, the first ligand, and the second ligand. For example, for this purpose, and as further described herein, if the layer-spanning protein (2) is a GPCR, the second ligand can be a G protein that associates with the GPCR in its natural environment (i.e., transduces signals via the GPCR), another naturally occurring G protein that can bind to the GPCR and stabilize the formation of said complex, or a synthetic or semi-synthetic analog or derivative of a GPCR that can bind to the GPCR and stabilize the formation of said complex. As also mentioned herein, the second ligand can be a confobody, i.e., an immunoglobulin single variable domain (e.g., a VHH or nanobody) designed / produced to stabilize the formation of a complex between the confobody, the layer-spanning protein, and the first ligand.

[0143] In a preferred, but non-limiting embodiment of the present invention, the transmembrane protein (2) is a "seven-transmembrane protein," particularly a 7TM that is a receptor (such as a cell surface receptor). In a particularly preferred embodiment, the transmembrane protein (2) may be a 7TM that signals via a G protein. Such a 7TM is also known in the art as a GPCR. [As noted above, the terms "GPCR" and "7TM" are used interchangeably herein and include all transmembrane proteins having seven transmembrane domains, regardless of the intracellular signaling cascade or signaling mechanism. However, it should be understood that throughout this specification and claims, a 7TM that signals via a G protein is a preferred embodiment of the present invention.]

[0144] The membrane-spanning protein (2) can be a naturally occurring protein or receptor, or a synthetic or semi-synthetic analog of a naturally occurring protein or receptor (again, obtained through protein chemistry or recombinant DNA techniques, as generally described herein). Such a synthetic analog can be, for example, an analog of a naturally occurring membrane-spanning protein in which, compared to the sequence of the naturally occurring protein, one or more amino acid residues or stretches of amino acid residues (including one or more loops or portions thereof and / or one or more domains and / or portions thereof) have been inserted, deleted, and / or replaced by other amino acid residues or stretches of amino acid residues (e.g., by essentially corresponding stretches or loops of amino acids from another (preferably structurally related) membrane protein) (in other words, the naturally occurring sequence contains one or more "amino acid differences," as defined herein, compared to the naturally occurring sequence). Often, the native sequence of the naturally occurring protein used is obtained from the species to be treated with the compounds of the invention or from an animal (preferably a mammal) used for animal model purposes to test the compounds of the invention.

[0145] As will be apparent to those skilled in the art, such synthetic analogs can be obtained using standard techniques of protein chemistry and / or recombinant DNA techniques known per se. For example, when the invention is carried out in a cell as described herein, a synthetic analog can be obtained by appropriately expressing in said cell a DNA sequence (or other suitable nucleotide sequence) encoding said synthetic analog.

[0146] Also, as is well known in the art, 7TM and other transmembrane proteins typically contain one or more intracellular loops and one or more extracellular loops. Similarly, a transmembrane protein used in an arrangement of the invention may contain one or more loops that protrude into a first environment (as defined herein) and one or more loops that protrude into a second environment (as defined herein). For example, when a method of the invention is performed in a cell, a transmembrane protein used in an arrangement of the invention may contain one or more loops that protrude into an intracellular environment and one or more loops that protrude into an extracellular environment. Similarly, when a method of the invention is performed in a vesicle or liposome, a transmembrane protein used in an arrangement of the invention may contain one or more loops that protrude into the internal environment of the liposome or vesicle and one or more loops that protrude into the external environment of the liposome or vesicle. In each case, the loops projecting into the first environment are most preferably loops that are capable of (and are positioned to) form a functional ligand binding site (particularly a functional binding site for a first ligand) and / or are capable of (and are positioned to) form a functional ligand binding site when the layer-spanning protein adopts a suitable conformation, and the loops projecting into the second environment are most preferably loops that are capable of (and are positioned to) form a functional ligand binding site (particularly a functional binding site for a second ligand) and / or are capable of (and are positioned to) form a functional ligand binding site when the layer-spanning protein adopts a suitable conformation (e.g., upon binding of a first ligand to the layer-spanning protein).

[0147] In one specific, but non-limiting, aspect, the loops of the spanning protein that project into one environment essentially correspond to the extracellular loops of the spanning protein, and the loops of the spanning protein that project into the other environment essentially correspond to the intracellular loops (again, in each case, preferably, the extracellular loops form a functional ligand-binding site and the intracellular loops form another functional ligand-binding site). Preferably, the loops of the spanning protein that project into the first environment [A] essentially correspond to the extracellular loops of the spanning protein, and the loops of the spanning protein that project into the second environment [B] essentially correspond to the intracellular loops of the spanning protein, particularly when the second environment [B] is the environment inside a cell or liposome (again, preferably, the extracellular loops form a functional ligand-binding site that projects into the first environment and the intracellular loops form a different functional ligand-binding site that projects into the second environment).

[0148] For example, when the transmembrane protein is a transmembrane protein (such as a 7TM), the transmembrane protein may comprise one or more extracellular loops of the transmembrane protein (particularly one or more extracellular loops of the 7TM) and one or more intracellular loops of the transmembrane protein (particularly one or more intracellular loops of the 7TM), particularly such a transmembrane protein in which the extracellular loops form or are capable of forming a functional ligand binding site and the intracellular loops form or are capable of forming a different functional ligand binding site. Again, the ligand binding site formed by the extracellular loops preferably protrudes into one environment (as defined herein), and the ligand binding site formed by the intracellular loops preferably protrudes into the other environment (as defined herein). In particular, when the method of the present invention is carried out in a cell or liposome, the extracellular loops protrude into the environment outside the cell or liposome, and the intracellular loops protrude into the environment inside the cell or liposome. Also, preferably, the intracellular loop is such that it forms or is capable of forming (and is arranged to form) a functional ligand-binding site for a second ligand (in other words, in the present invention, the ligand-binding site of the second ligand is preferably constituted by and / or comprises one or more intracellular loops of a transmembrane protein. It is also possible that the ligand-binding site of the first ligand is constituted by and / or comprises one or more extracellular loops, although, as further described herein, the actual binding / docking site of the first ligand may be located deep within the structure of the transmembrane protein).

[0149] For example, if the transmembrane protein is a 7TM, the transmembrane protein may comprise three intracellular loops (i.e., three intracellular loops from the 7TM) and three extracellular loops (i.e., three extracellular loops from the 7TM), where the three intracellular loops form or can form a functional ligand binding site, and the three extracellular loops form or can form a different functional ligand binding site. Again, preferably, the functional ligand binding site formed by the three intracellular loops projects into one environment (preferably, the second environment [B]), and the functional ligand binding site formed by the three extracellular loops projects into another environment (preferably, the second environment [A]). Also, the three intracellular loops preferably form a functional binding site for a second ligand (and the three extracellular loops may form a functional binding site for a first ligand, or the binding site may be located deep within the structure of the 7TM). Most preferably, the three intracellular loops form a binding site for a second ligand that protrudes into the second environment [B] (i.e., the environment inside a cell or liposome, when the method of the invention is carried out in a cell or liposome, respectively), and the three extracellular loops protrude into the first environment [A] (and may form a functional binding site for a first ligand, or said binding site may be located deep within the structure of the 7TM).

[0150] In one aspect of the invention, the intracellular and extracellular loops of the transmembrane protein are derived from or essentially derived from the same transmembrane protein (i.e., are the same as or essentially the same as the intracellular and extracellular loops present in the native transmembrane protein). In this aspect of the invention, the transmembrane protein may have the same or essentially the same amino acid sequence as the native transmembrane protein used as a target in the screening or assay methods of the invention.

[0151] In another embodiment of the present invention, the intracellular and extracellular loops of the transmembrane protein may be derived from different transmembrane proteins. In particular, in this embodiment of the present invention, the intracellular and extracellular loops may be derived from different but related transmembrane proteins, for example, from two different but related 7TMs, such as two GPCRs. In particular, in this embodiment of the present invention, the intracellular loop may be derived from a first 7TM or GPCR, and the extracellular loop may be derived from a second 7TM or GPCR that is different from the first 7TM or GPCR. The transmembrane domains of such chimeric proteins may be derived from the first or second 7TM or GPCR, preferably all from essentially the same GPCR, and more preferably from the same GPCR as the extracellular loops (although they may contain some amino acid residues from the GPCR from which the intracellular loop is derived, depending on the position selected for recombinantly deleting the native intracellular loop and inserting the replacement intracellular loop).

[0152] In this aspect of the invention, the resulting chimeric interlayer protein is still most preferably a interlayer protein that can be suitably used in the methods and arrangements of the invention. Again, in the case of 7TM, the interlayer protein comprises three intracellular loops and three extracellular loops, the three intracellular loops forming a functional ligand-binding site for a second ligand (which is then selected to be capable of binding to the ligand-binding site (9) formed by the intracellular loops). Again, the binding site formed by the three intracellular loops preferably extends into the second environment [B] (i.e., the environment inside the cell or liposome, if the method of the invention is carried out in a cell or liposome, respectively), and the three extracellular loops preferably extend into the first environment [A] (and may form a functional binding site for the first ligand, or the binding site may be located deep within the structure of the 7TM).

[0153] Thus, in a further aspect, the present invention relates to an arrangement as further described herein, wherein the transmembrane protein is a 7TM protein comprising seven transmembrane domains, three intracellular loops, and three extracellular loops (which are linked together in the order known per se for 7TM proteins, i.e., [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-[C-terminal sequence]), wherein the intracellular loops are derived from a first 7TM protein and the extracellular loops are derived from a second 7TM protein different from the first 7TM protein, and the intracellular loops form functional ligand-binding sites. Preferably, the TM domains from the transmembrane protein are essentially derived from the same 7TM protein as the extracellular loops.

[0154] The intracellular loops and 7TM are also such that, taken together, they form a functional ligand-binding site, in particular a functional ligand-binding site to which a (suitable) second ligand (as defined herein) can bind, which again preferably extends into the second environment [B].

[0155] In one specific embodiment, such a chimeric layer-spanning protein comprises an intracellular loop derived from the beta-2-adrenergic receptor. In another specific embodiment, such a chimeric layer-spanning protein comprises an intracellular loop derived from the mu-opioid receptor. For some non-limiting examples of such chimeric receptors, see also the assignee's co-pending PCT application entitled "Chimeric proteins and methods to screen for compounds and ligands binding to GPCRs," which has the same international filing date as the present application and incorporates the same priority application.

[0156] The present invention particularly relates to arrangements comprising such chimeric 7TM and a second ligand capable of binding to the ligand binding site formed by said intracellular loop.

[0157] For the remainder, if the second ligand is appropriately selected to be capable of binding to the ligand binding site (9) of the chimeric layer-spanning protein to provide an operable arrangement of the invention (and if the chimeric layer-spanning protein is itself operable in the arrangement of the invention), such arrangement of the invention in which the chimeric layer-spanning protein is used can essentially be the arrangement further described herein.

[0158] In addition, such chimeric layer-spanning proteins, the nucleotide sequences and nucleic acids encoding them, and cells, cell lines or host organisms comprising such nucleotide sequences or nucleic acids and / or capable of expressing such chimeric layer-spanning proteins, as well as further uses of such chimeric layer-spanning proteins, nucleotide sequences, nucleic acids, cells, cell lines and host organisms form further aspects of the present invention.

[0159] Another aspect of the invention is a composition or kit of parts comprising at least said chimeric transmembrane protein and a ligand capable of binding to an intracellular loop present in said GPCR, said ligand being preferably a protein, more preferably a protein comprising or consisting essentially of an immunoglobulin single variable domain (e.g. a VHH domain), and in particular may be a conformobody (as described herein).

[0160] As described above, the chimeric layer-spanning protein is preferably a 7TM / GPCR. Also, in one specific embodiment, the chimeric layer-spanning protein comprises an intracellular loop derived from the beta-2-adrenergic receptor. In another specific embodiment, the chimeric layer-spanning protein comprises an intracellular loop derived from the mu-opioid receptor.

[0161] As further described herein and shown diagrammatically in Figures 1-3, in the arrangements of the present invention, the layer-spanning protein (2) is typically and preferably fused or linked to a first member (6) of a binding pair (6 / 7), either directly or via a suitable spacer or linker (10), to form a first fusion protein. The second binding member (7) of the binding pair (6 / 7) is typically and preferably part of a second fusion protein distinct from the first fusion protein, which second fusion protein is also further described herein. The first fusion protein, the second fusion protein (in its various formats as described herein), nucleotide sequences and / or nucleic acids encoding the first or second fusion protein, and cells, cell lines, or other host cells or organisms expressing (particularly suitably expressed as described herein) or capable of expressing (suitably) the first and / or second fusion protein (and preferably both), as well as their various uses as further described herein, form further aspects of the present invention.

[0162] The binding pair (6 / 7) used in the arrangements of the present invention generally comprises at least two separate binding members (6) and (7), also referred to herein as the "first binding member" and the "second binding member," respectively. The binding pair (6 / 7) and its respective members (6) and (7) should be such that the binding pair (6 / 7) is capable of generating a detectable signal when members (6) and (7) contact or are in close proximity to one another. Such a detectable signal may be, for example, a luminescent, fluorescent, or chemiluminescent signal, may be based on a reporter gene, or may be based on DNA ligation. Some specific, but non-limiting examples of technologies (including binding pairs and their associated detectable signals) include protein complementation-based technologies, such as the NanoBit™ system, the NanoLuc™ system, the hGLuc system (Remy and Michnick, Nature Methods, 2006, 977), BiFC (Bimolecular Fluorescence Complementation), and DHFR-PCA (Dihydrofolate Reductase Protein-Fragment Complementation Assay); direct interaction-based technologies, such as BRET (Bioluminescence Resonance Energy Transfer), FRET (Fluorescence / Förster Resonance Energy Transfer), and BioID (Proximity-Dependent Biotin Identification); reporter gene-based systems (e.g., KISS / Kinase Substrate Sensor) or proximity ligation assays (Weilbrecht et al., Expert Review of Proteomics, 7:3, 401-409). Generally, technologies based on protein complementation and luminescent, fluorescent, or chemiluminescent signals (such as NanoLuc™ or NanoBit™) are preferred.

[0163] In a particularly preferred embodiment, when the method of the invention is carried out in a suitable cell, the first member (6) and the second member (7) of the binding pair (6 / 7) are preferably both polypeptides, proteins, amino acid sequences or other chemical entities obtainable by suitable expression of nucleic acid or nucleotide sequences encoding same, preferably in the cell used in the method of the invention.

[0164] The first and second binding members may be part of a suitable reporter assay, or may be any other pair of domains or units that are capable of generating a detectable signal when they contact or are in close proximity to each other, such as an enzyme and substrate combination, or binding pairs commonly used in experimental studies of protein-protein interactions. As noted, in order to reduce the level of baseline / background signal, it is preferred that the two members of the binding pair by themselves do not have substantial binding affinity for each other.

[0165] Some preferred, but non-limiting examples of suitable binding pairs are pGFP and Promega's NanoBiT® system, the latter being particularly preferred because the large and small BiTs that make up the NanoBiT® system have, by themselves, low affinity for each other.

[0166] The first binding member (6) can be fused to the layer-spanning protein (2) in any suitable manner, so long as the resulting first fusion protein allows the first member (6) to contact (or otherwise come into suitable close proximity with) the second member (7) of the binding pair (6 / 7) when the second fusion protein formed by the second ligand (4) and the second member (7) binds to the layer-spanning protein (2) via the second binding site (9). Also, preferably, the first binding member (6) is fused or linked to the layer-spanning protein (2) in a manner that does not substantially affect the conformation and / or conformational changes that the layer-spanning protein (2) may undergo under the conditions used to practice the methods of the present invention.

[0167] Thus, although the present invention does not generally preclude the first binding member (6) from being directly fused or linked to the layer-spanning protein (2), it is generally preferred that the first binding member (6) be fused or linked to the layer-spanning protein (2) via a suitable linker (10). For example, the use of a flexible linker having a total length of between 5 and 50 amino acids, preferably between 10 and 30 amino acids, e.g., about 15-20 amino acids, is generally preferred. Suitable linkers will be apparent to those skilled in the art and include GlySer linkers (e.g., 15GS linkers).

[0168] In the present invention, the first and second binding members of binding pair (6 / 7) will be present in the same environment (as defined herein) relative to boundary layer (1) such that they can contact or be in close proximity to each other (as further described herein) and thereby generate a detectable signal. In particular, as shown schematically in Figures 1, 2, and 3, the first and second binding members of binding pair (6 / 7) will be present in the same environment (as defined herein) as second binding site (9) of layer-spanning protein (2) (also relative to boundary layer (1)) such that the first and second binding members of binding pair (6 / 7) can contact when a second fusion protein binds to said binding site directly (as shown in Figure 1) or indirectly (as shown in Figures 2 and 3). To this end, first binding member (6) will generally be attached, either directly or via a linker (10), to an amino acid residue / position within / on layer-spanning protein (2) that is exposed to the same environment as second binding site (9). As further described herein, the environment (shown as environment [B] in Figures 1-3) can be, for example, an intracellular environment (when the methods of the invention are performed in a cell) or the environment inside a vesicle or liposome.

[0169] In preferred embodiments of the invention, the first binding member (6) will be fused, either directly or via a linker (10), to one end of the primary amino acid sequence of the layer-spanning protein (2). This may be the N-terminus or C-terminus of the layer-spanning protein (2), so long as in the final arrangement of the invention the first binding member (6) is on the same side of the boundary layer (1) as the second binding site (9). Thus, in embodiments of the invention performed in cells, as further described herein, in which the second binding site (9) is exposed to the intracellular environment, the first member (6) may be fused to the end of the primary amino acid sequence that terminates in the intracellular environment (which in the case of 7TM would typically be the C-terminus).

[0170] The first fusion protein may be prepared and produced using suitable techniques of protein chemistry and / or recombinant DNA technology known per se. Such techniques will be clear to the skilled person based on the further disclosure herein and the standard manuals and other scientific literature referred to herein. When the method of the present invention is carried out in a cell (as further described herein), the first fusion protein is preferably provided by suitably expressing in said cell a nucleotide sequence and / or nucleic acid encoding the first fusion protein. This can also be carried out using suitable techniques of recombinant DNA technology known per se, and cells which suitably express (or are capable of suitably expressing) the first fusion protein form a further aspect of the present invention.

[0171] As further described herein, in the arrangements of the present invention, the second member (7) of the binding pair (6 / 7) will usually and preferably also form part of a fusion protein, which generally comprises said second binding unit fused or linked, either directly or via a suitable spacer or linker (11), to another ligand, protein, binding domain or binding unit, which ligand, protein, binding domain or binding unit can bind directly (as defined herein) or indirectly (as defined herein) to the layer-spanning protein (2). For this purpose, as further described herein, said ligand, protein, binding domain or binding unit may be, for example, a second ligand (wherein (4) is a second ligand, resulting in an arrangement of the invention of the type shown schematically in Figure 1), a binding domain or binding unit capable of binding to a second ligand (wherein (4) is a second ligand and (5) is a binding domain or binding unit that binds to the second ligand, resulting in an arrangement of the invention of the type shown schematically in Figure 2), or a binding domain or binding unit capable of binding to a protein complex that is capable of binding to a transmembrane protein (wherein (4) is a second ligand, (12) is said protein complex comprising a second ligand, and (5) is a binding domain or binding unit that binds to the protein complex, as shown schematically in Figure 3).

[0172] In the second fusion protein, the second binding member (7) is most preferably linked to the other ligand, protein, binding domain, or binding unit in a suitable manner that allows the second binding member (7) to contact (or otherwise be in suitable close proximity to) the first member (6) of the binding pair (6 / 7) when the second fusion protein binds directly or indirectly to the second binding site (9) of the layer-spanning protein (2). To this end, the second binding member (7) may be fused or linked directly to the other ligand, protein, binding domain, or binding unit, but preferably they are linked via a suitable linker (11), preferably a flexible linker having, for example, a total of between 5 and 50 amino acids, preferably between 10 and 30 amino acids, e.g., about 15-20 amino acids, is generally preferred. Suitable linkers will be apparent to those skilled in the art and include GlySer linkers (e.g., 15GS linkers).

[0173] As described herein, the second ligand can be any ligand, protein, binding domain or binding unit that can bind to the layer-spanning protein, i.e., via the binding site (9) (if the second ligand is part of a second fusion protein, the second ligand should most preferably also be one that can be suitably included in the second fusion protein).

[0174] Generally, in the present invention (regardless of whether said binding site is bound directly or indirectly by the second fusion protein used in the arrangement of the present invention), binding site (9) can be a conformational epitope of the layer-spanning protein (2). More specifically, said binding site (9) can be a conformational epitope of the layer-spanning protein (2) that changes its "shape" (i.e., the spatial arrangement of the domains, loops, and / or amino acid residues that form the epitope) when the layer-spanning protein (2) undergoes a conformational change, e.g., from an inactive or less active state to an active, more active, and / or functional state, and / or a conformational change that occurs when a first ligand binds to the layer-spanning protein.

[0175] Preferably, when the binding site 9 changes shape due to the interlayer protein 2 undergoing a conformational change, the binding site 9 and the second ligand may increase the affinity of the interaction between the binding site 9 and the second ligand 4. In particular, when the interlayer protein 2 undergoes a conformational change from an inactive or less active state to an active, more active, functional, and / or druggable state, and / or when a first ligand 3 (particularly a first ligand 3 that acts as an agonist for the interlayer protein) binds to the interlayer protein 2, the binding site 9 and the second ligand may increase the affinity of the interaction between the binding site 9 and the second ligand 4.

[0176] In particular, the interaction of the second ligand (4) and the binding site (9) may be such that when the layer-spanning protein (2) is in an active, more active and / or functional state, the second ligand (4) may bind to the binding site (9) with high affinity, and / or when the first ligand (3) (particularly the first ligand (3) that acts as an agonist for the layer-spanning protein (2)) binds to the layer-spanning protein (2), the second ligand (4) may bind to the binding site (9) with high affinity. For example, the affinity of the second ligand 4 for the binding site 9 can increase by 10-fold, e.g., 100-fold or more, when the spanning protein 2 undergoes such a conformational change that its interaction with the second ligand 4 and the binding site 9 changes from, for example, an affinity in the micromolar range (i.e., greater than 1000 nM) when the spanning protein is in an inactive, less active, or ligand-free conformation to an affinity in the nanomolar range (i.e., less than 1000 nM, e.g., less than 100 nM) when the spanning protein 2 is in a functional, active, or more active and / or ligand-bound conformation. For example, in the case of GPCRs, it is known that the affinity of the interaction between a G protein and a G protein binding site increases when a ligand (especially an agonist) binds to the extracellular binding site of the GPCR. Furthermore, WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 75643, WO 2014 / 118297, WO 2014 / 122183 and WO 2014 / 118297 describe VHH domains (ConfoBodies) that have a higher affinity for a layer-spanning protein when the layer-spanning protein is in a functional, active or more active and / or ligand-binding conformation compared to when the layer-spanning protein is in an inactive, less active or ligand-free conformation (e.g. in the nanomolar range for a functional, active or ligand-binding conformation versus the micromolar range for an inactive or ligand-free conformation).

[0177] The second ligand itself may also undergo a conformational change upon binding to the layer-spanning protein (2). In embodiments in which the second fusion protein indirectly binds to the layer-spanning protein (2), this may mean that the binding domain or binding unit (5) of the second fusion protein that binds to the second ligand (4) may also be such that it has a higher affinity for the conformation that the second ligand (4) adopts when bound to the layer-spanning protein (2) compared to the conformation that the second ligand (4) adopts when not bound to the layer-spanning protein (2). For example, G proteins are known to undergo a conformational change upon binding to GPCRs, and a VHH domain present in the second fusion protein may have a higher affinity for the GPCR-bound conformation of the G protein compared to the unbound conformation of the GPCR.

[0178] In a preferred embodiment, the binding site (9) is a binding site of the transmembrane protein (2) that functions as a binding site for the transmembrane protein's natural ligand when the transmembrane protein (2) is in its natural environment. More specifically, the binding site (9) can be a binding site of the transmembrane protein (2) that functions as an intracellular binding site for the transmembrane protein's natural intracellular ligand when the transmembrane protein (2) is in its natural environment. For example, if the transmembrane protein (2) is a receptor, the binding site (9) can be a binding site of the transmembrane protein (2) that functions as an intracellular binding site for one or more intracellular ligands of the transmembrane protein (2) involved in signal transduction when the transmembrane protein (2) is in its natural environment.

[0179] In certain embodiments, when the layer-spanning protein (2) is a GPCR, the binding site (9) can be a binding site for a G protein (and / or a G protein complex). As further described herein, in such cases, the second ligand can be a natural, synthetic, or recombinant protein, or other ligand, capable of binding to the G protein binding site of the GPCR.

[0180] If the second ligand (4) is not a conformation-induced binding domain or binding unit, it will typically be a protein or proteinaceous ligand. In embodiments of the present invention carried out in suitable cells or cell lines, the second ligand (4) may be a protein native to the cells or cell line used, or a suitable (recombinant) protein expressed in the cells or cell line used. For example, if the second ligand (4) is not part of a second fusion protein, the second ligand (4) may be a ligand of the transmembrane protein (2) naturally present in the cells or cell line (e.g., if the transmembrane protein (2) is a GPCR, the second ligand (4) may be a G protein naturally expressed by the cells or cell line used). Alternatively, the second ligand may be a protein that is recombinantly expressed in the cell or cell line used, for example, if the cell or cell line does not naturally express a suitable ligand for the layer-spanning protein (2), or if it is desired to use a ligand that is different from the ligand(s) naturally expressed by the cell or cell line (e.g., if it is desired to use an analog, derivative, or ortholog of a naturally expressed ligand, in which case the native expression of the naturally expressed ligand may also be temporarily or permanently suppressed or knocked out in the cell or cell line used). If the second ligand (4) forms part of a second fusion protein, the second ligand will typically be recombinantly expressed as part of the second fusion protein.

[0181] As further described herein, the second ligand (4) can either be part of the second fusion protein or can be separate from the second fusion protein. In either case (i.e., whether the second ligand is part of the second fusion protein or not), the second ligand preferably can bind to a conformational epitope of the layer-spanning protein (or binds directly to the layer-spanning protein or is part of a protein complex that can bind directly to the layer-spanning protein). More preferably, the second ligand (and / or the protein complex comprising the second ligand) preferably specifically binds to one or more functional, active, and / or druggable conformations of the layer-spanning protein, induces and / or stabilizes the formation of one or more functional, active, and / or druggable conformations of the layer-spanning protein (and / or shifts the conformational equilibrium of the layer-spanning protein toward one or more such conformations); and / or induces and / or stabilizes the formation of a complex of the layer-spanning protein, the first ligand, and the second ligand.

[0182] When the second ligand is part of a second fusion protein, it may be any ligand, binding domain, binding unit, peptide, protein or other chemical entity that can bind directly to the layer-spanning protein and that can suitably be included in the second fusion protein. Preferably, when the second ligand is part of a second fusion protein, it will be a suitable binding domain or binding unit, in particular an immunoglobulin single variable domain, as further described herein.

[0183] When the second ligand is separated from the second fusion protein, the second ligand can be any ligand or protein capable of directly binding to the layer-spanning protein and / or forming part of a protein complex capable of binding to the layer-spanning protein. For example, as further described herein, such a second ligand can be a naturally occurring ligand of the layer-spanning protein, a semisynthetic or synthetic analog or derivative of such a naturally occurring ligand, or an ortholog of such a naturally occurring ligand. Also, when the second ligand is not part of the second fusion protein, the second fusion protein will comprise a binding domain or binding unit capable of indirectly binding (as defined herein) to the layer-spanning protein, i.e., a binding domain or binding unit capable of binding to the second ligand and / or a protein complex containing the second ligand. Also, as further described herein, such a binding domain or binding unit can be, in particular, an immunoglobulin single variable domain, such as a camelid-derived ISVD. As also mentioned herein, such a binding domain or binding unit may comprise two or more (e.g., two or three) identical or different ISVDs (suitably ISVDs fused or linked, optionally via a suitable linker or spacer), where, if the two or more ISVDs are the same, they generally bind to the same binding site or epitope of the second ligand, and where, if different, they can bind to the same or different epitopes or binding sites of the second ligand (and, where the second ligand is a protein complex such as a G protein complex, they can bind to the same or different subunits of said protein complex).

[0184] It will also be clear to those skilled in the art that if the second ligand does not form part of the second fusion protein, the binding domain or binding unit present in the second fusion protein and capable of binding to the second ligand should not essentially interfere with the binding of the second ligand to the layer-spanning protein. For example, it is preferred that the second ligand binds to a binding site or epitope of said second ligand that is different from the binding site of the second protein that binds to the layer-spanning protein (preferably sufficiently removed from the binding site of the second protein that binds to the layer-spanning protein so as to avoid any major steric hindrance).

[0185] When second ligand (4) is a naturally occurring ligand of interlaminar protein (2), the second ligand may be, for example, a ligand involved in a signal transduction pathway or signal transduction involving interlaminar protein (2). For example, when second ligand (4) is a receptor, second ligand (4) may be a naturally occurring ligand of the receptor, particularly a naturally occurring intracellular ligand of the receptor, for example, an extracellular ligand that binds to an extracellular binding site of the receptor, or an intracellular ligand that binds to an intracellular binding site of the receptor, for example, when the receptor has some constitutive activity and binds to an intracellular binding site of the receptor as part of a pathway that provides said constitutive activity. Suitable examples of such natural ligands will be apparent to those skilled in the art based on the disclosure herein and generally depend on the interlaminar protein (2) used. For example, if the layer-spanning protein (2) is a 7TM or GPCR, the second ligand (4) may (preferably) be a G protein, such as, but not limited to, a naturally occurring G protein (e.g., a G protein naturally occurring in the cell or cell line used), or a synthetic or semi-synthetic analog or derivative of a naturally occurring G protein (such as a chimeric G protein), all of which are further described herein.

[0186] As further described herein, particularly in aspects and embodiments of the invention practiced using cells or cell lines, the second ligand (4) may also be part of a complex comprising the second ligand (4) and, optionally, one or more additional proteins. For example, if the transmembrane protein is a GPCR and the second ligand is a G protein or a G protein analog or derivative, the second ligand may be part of a complex formed by the G protein and, optionally, one or more additional proteins. One preferred, but non-limiting, example of such a complex is a G protein trimer comprising a G alpha subunit, a G beta subunit, and a G gamma subunit. The complex may also include the transmembrane protein itself (e.g., a GPCR and a G protein or a GPCR and a G protein trimer). It will be apparent to those skilled in the art that, when the second ligand forms part of such a complex, it is generally preferred that the second ligand not form part of the second fusion protein. Instead, the second fusion protein will comprise a binding domain or binding unit capable of binding to the second ligand or the complex. For example, if the second ligand forms part of a G protein complex, the binding domain or binding unit of the second fusion protein can be a VHH domain that binds to the complex, e.g., a subunit within the complex or an interface between two or more of the subunits. As described herein, an example of such a VHH domain is the VHH designated "CA4435" (SEQ ID NO: 1 in WO 2012 / 75643 and SEQ ID NO: 1 herein).

[0187] The second ligand (4) may be a synthetic or semi-synthetic analog or derivative of such a naturally occurring ligand, e.g., an analog or derivative having a primary amino acid sequence that differs from that of the corresponding natural ligand by deletion, insertion, and / or substitution of a limited number of amino acid residues or stretches of amino acid residues. Such analogs or derivatives may also be provided using suitable recombinant DNA techniques known per se, and in one embodiment may involve expression in a suitable host or host cell of a nucleotide sequence or nucleic acid encoding the analog or derivative (preferably as part of an entire second fusion protein that also includes the second binding member (7) and optional linker (11), if present). For example, if the layer-spanning protein (2) is a 7TM or GPCR, the second ligand (4) may be an analog or derivative of a (preferred) G protein and may have one or more amino acid differences from the native sequence (as defined herein), so long as the analog or derivative still has sufficient affinity for the layer-spanning protein (2) to render it suitable for use in the methods of the invention.

[0188] For example, in one particular embodiment, such an analog or derivative of a naturally occurring G protein may be a naturally occurring G protein in which one or more amino acid residues (and / or one or more stretches of amino acid residues) have been replaced by one or more amino acid residues (and / or one or more stretches of amino acid residues) that are located at (essentially) the same or corresponding position(s) in another naturally occurring G protein.

[0189] Where the G-protein is a heterotrimeric protein, such substitution of one or more amino acid residues (and / or one or more stretches of amino acid residues) may be present or carried out in any one, any two, or all three of the G-alpha, G-beta, and / or G-gamma subunits, and may in particular be a substitution in the G-alpha subunit.

[0190] For example, it is well known that in humans there are multiple genes encoding different G-alpha subunits, each encoding a different G-alpha isoform, which can be grouped into different functional subfamilies (see, for example, Flock et al., Nature, 2015, 524(7564), 173-179; and Nehme et al., PLoS One, 2017, 12(4)). Such analogs or derivatives of naturally occurring G-alpha subunits that can be used in the present invention can also be obtained by replacing one or more amino acid residues (and / or one or more stretches of amino acid residues) in (the amino acid sequence of) a naturally occurring G-alpha subunit with one or more amino acid residues (and / or one or more stretches of amino acid residues) at (essentially) the same or corresponding position(s) in another naturally occurring alpha subunit (which may belong to the same subfamily as the original subunit or a different subfamily). Some specific, but non-limiting examples are those in which one or more amino acid residues and / or one or more stretches of amino acid residues are G-alpha isoforms. i a naturally occurring Gα subunit substituted with one or more amino acid residues and / or one or more stretches of amino acid residues at (essentially) the same or corresponding position(s) of the subunit; s The subunit, or one or more amino acid residues and / or one or more stretches of amino acid residues, is Gα q a naturally occurring Gα subunit substituted with one or more amino acid residues and / or one or more stretches of amino acid residues at (essentially) the same or corresponding position(s) of the subunit; s Often, but not exclusively, such replaced / substituted amino acids or stretches of amino acids are present at or adjacent to the C-terminus of the alpha subunit.

[0191] Some specific, but non-limiting, examples of such "chimeric" G proteins and their designs can also be found in the scientific literature, see, for example, Flock et al. and Nehme et al., again cited above.

[0192] The second ligand (4) may also be another type of ligand designed to bind to the binding site (9) of the layer-spanning protein (2), preferably in a manner further described herein.

[0193] In a particularly preferred embodiment, when the method herein is carried out in a suitable cell, the second ligand (4) is preferably a polypeptide, protein, amino acid sequence or other chemical entity, preferably obtainable by suitable expression of a nucleic acid or nucleotide sequence encoding it in the cell used in the method of the invention.

[0194] As referred to herein, second ligand (4), whether a transmembrane protein (2) (e.g., a naturally occurring G protein), a synthetic or semisynthetic analog or derivative of such a naturally occurring ligand (e.g., a chimeric G protein described herein), or another type of ligand (e.g., a conformobody described further herein), is generally a second ligand capable of binding, particularly specifically binding, to an epitope, particularly binding site (9), of transmembrane protein (2). In particular, second ligand (4) may be a second ligand capable of binding, particularly specifically binding, to an epitope that would be an intracellular epitope when transmembrane protein (2) is in its natural cellular environment.

[0195] As referred to herein, the epitope (i.e., binding site (9)) may be a linear epitope or a conformational epitope, preferably a conformational epitope (as described herein). For example, if the layer-spanning protein (2) is a GPCR, the epitope may include one or more amino acid residues and / or stretches of amino acid residues in at least one intracellular loop of the GPCR, and in particular may be a conformational epitope formed by and / or including one or more amino acid residues and / or stretches of amino acid residues in at least two different intracellular loops of the GPCR.

[0196] The epitope of the second ligand (4) may be (part of) an epitope of the transmembrane protein (2) that is involved in signal transduction mediated by the transmembrane protein (2). For example, the second ligand may bind to an epitope on the transmembrane protein (2) that is located within the binding site of a downstream signaling protein. For example, if the transmembrane protein (2) is a GPCR, the second ligand (4) may be a binding domain or binding unit capable of specifically binding to a conformational epitope contained in, located within, or overlapping with the G protein binding site of the GPCR.

[0197] If the layer-spanning protein (2) used is a protein that can assume / exist in two or more conformations (such as a ground state / conformation, an active state / conformation and / or an inactive state / conformation) and / or that can undergo a conformational change (especially a functional conformational change), the second ligand (4) is preferably a protein that is capable of binding, in particular specifically binding, to the functional conformational state of the layer-spanning protein (2).

[0198] In certain preferred embodiments, the second ligand (4), upon binding to the interlayer protein (2), stabilizes and / or induces a functional and / or active conformational state of the interlayer protein (2) (and / or shifts the conformational equilibrium of the interlayer protein (2) from an inactive or less active state(s) to a more active state), shifts the interlayer protein to a more druggable conformation (and / or shifts the conformational equilibrium of the interlayer protein (2) from a less druggable conformation(s) to a more druggable conformation(s)), The second ligand is capable of altering the conformation of the (associated binding pocket of) the first ligand (3) so that it is more suitable or accessible for binding of the first ligand (3), or increasing the overall affinity of the interaction with the first ligand (3) (and / or shifting the conformational equilibrium of the protein towards such a conformation), and / or inducing and / or stabilizing the formation of a complex comprising the second ligand, the layer-spanning protein and the first ligand (and / or shifting the conformational equilibrium of the layer-spanning protein towards the formation of such a complex), or any combination thereof.

[0199] Thus, when the layer-spanning protein (2) is a GPCR, the second ligand (4) can be a second ligand capable of binding to, and in particular stabilizing and / or inducing, a functional conformational state of the GPCR, more preferably an active conformational state of the GPCR, and preferably a second ligand (4) that preferentially / specifically binds to the protein or GPCR when it is bound to an agonist (e.g., by a first ligand (3) that acts as an agonist of the protein or GPCR) compared to the conformational state of the protein or GPCR when it is not bound by any first ligand (3) or when it is bound by a ligand (3) that acts as an inverse agonist, and / or that increases (i.e., by at least 2-fold, particularly at least 5-fold, and more preferably at least 10-fold) the affinity of the protein or GPCR for at least one compound or ligand that acts as an agonist of the protein or GPCR.

[0200] As mentioned, one preferred class of compounds for use in the present invention as a second ligand (particularly when the second ligand is comprised in a second fusion protein) are generally described in WO 2012 / 007593, WO 2012 / 007594, WO 2012 / 75643, WO 2014 / 118297, WO 2014 / 122183 and WO 2014 / 118297 and comprise VHH domains capable of stabilising GPCRs in a desired conformation (comphobodies).

[0201] WO 2012 / 75643 also discloses a number of VHH domains that can indirectly bind to GPCRs, i.e., by binding to a G protein or a G protein complex. Some preferred, but non-limiting, examples of these are the VHH designated "CA4435" (SEQ ID NO: 1 in WO 2012 / 75643 and SEQ ID NO: 1 herein), which can bind to a G protein complex, and the VHH designated "CA4437" (SEQ ID NO: 4 in WO 2012 / 75643 and SEQ ID NO: 2 herein), which can bind to a G protein. Such a VHH domain can be suitably included in a second fusion protein to provide a second fusion protein that can indirectly bind to a GPCR by binding to a G protein or a G protein complex.

[0202] Thus, in one preferred embodiment of the present invention, the second fusion protein comprises at least one such VHH or confobody and a second binding member (7).

[0203] Generally, in the present invention, the first binding member (6) and the second binding member (7) will be in close proximity to each other when the second fusion protein binds directly or indirectly (both as defined herein) to the layer-spanning protein (2). In particular, the first and second binding members will be in close proximity to each other when the second ligand (4) present in the second fusion protein binds directly to the layer-spanning protein (2), or when the binding domain or binding unit (5) present in the second fusion protein binds indirectly to the layer-spanning protein, i.e., when said binding domain or binding unit (5) binds to the second ligand (4) or, in the embodiment shown in Figure 3, to the protein complex (12), which then binds to or is bound by the layer-spanning protein (2). Preferably, the first and second binding members should not have high affinity for each other by themselves, so that their association (and the associated generation of a detectable signal) is primarily driven by the binding of the second ligand (directly or indirectly) to the transmembrane protein, bringing the first and second binding members into close proximity with each other, and thus essentially not, or only to a low extent, due to the affinity between the first and second ligands (the NanoBiT system from Promega is an example of such a suitable binding pair). However, it should also be noted that any such affinity between the first and second ligands generally provides a baseline for the detectable signal, which should not essentially interfere with the assay of the present invention, as the readout of this assay primarily looks at any change in the detectable signal, for example, upon addition of the first ligand to an arrangement of the present invention that does not contain the first ligand (more generally, it should also be noted that for some uses of the methods and arrangements of the present invention, it may be preferable to have some level of baseline signal, where the readout may then also include a decrease in signal compared to the baseline).

[0204] Thus, in general, in the present invention, the detectable signal (or any change in said signal) generated by the first and second binding members will be proportional to the amount of second fusion protein bound, directly or indirectly, to the layer-spanning protein (2), which will then be due to the binding interaction between the second ligand (4) and the layer-spanning protein (in particular one or more specific conformations that the second ligand and the layer-spanning protein can assume, such as functional, active and / or druggable conformations), and / or any change to said binding interaction (in particular any change to said binding interaction resulting from a conformational change in the layer-spanning protein and / or a shift in the conformational equilibrium of the layer-spanning protein, e.g., due to binding of the first ligand to the layer-spanning protein and / or formation of a complex between the first ligand, the layer-spanning protein and the second ligand).

[0205] Included herein are methods for identifying and making the various elements of the above-described arrangements and compositions, as well as methods for assembling such arrangements and compositions. Such methods can be combined with or form part of any assay or method for measuring or determining one or more properties of the first ligand.

[0206] As a non-limiting example, the methods for determining one or more properties of a first ligand described herein may include one or more steps directed to determining whether the second ligand binds to the layer-spanning protein, specifically binds to the layer-spanning protein, specifically binds to a domain of the layer-spanning protein located in a second environment, is a conformation-selective binder of the layer-spanning protein, stabilizes a conformation of the layer-spanning protein, stabilizes an inactive conformation of the layer-spanning protein, stabilizes a functional, active, and / or druggable conformation of the layer-spanning protein, and / or stabilizes a complex of the layer-spanning protein and the first ligand.

[0207] Based on this and the further disclosure of the present invention, it will be clear to one skilled in the art that the methods and arrangements of the present invention can be used to measure or determine one or more properties of a first ligand (in particular, properties of the first ligand that are related to, influence and / or determine the interaction between the first ligand and the layer-spanning protein), one or more properties of a second ligand (in particular, properties of the second ligand that are related to, influence and / or determine the interaction between the second ligand and the layer-spanning protein), and / or one or more properties of any binding domain or binding unit present in a second fusion protein (in particular, if the binding domain or binding unit directly binds to the layer-spanning protein, properties of the binding domain or binding unit that are related to, influence and / or determine the interaction between the binding domain or binding unit and the layer-spanning protein; or if the binding domain or binding unit binds to a second ligand and / or a protein complex comprising it, properties of the binding domain or binding unit that are related to, influence and / or determine the interaction between the binding domain or binding unit and the second ligand or the complex).

[0208] More particularly, with respect to a first ligand, the methods and arrangements of the present invention can be used to measure or determine the ability of the first ligand to bind to a layer-spanning protein, affect a conformational change in the layer-spanning protein, and / or affect a change in the conformational equilibrium of the layer-spanning protein. For example, as further described herein, the methods and arrangements of the present invention can measure or determine the ability of a given first ligand to act as an agonist, antagonist, inverse agonist, inhibitor, or modulator (e.g., allosteric) of the layer-spanning protein, and / or screen or identify small molecules, proteins, or other compounds or chemicals that act or can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric) of the layer-spanning protein. In this regard, it will be clear to those skilled in the art based on the disclosure herein that if the methods and arrangements of the invention are used for such purposes (i.e. for purposes relating to a first ligand), then the other components used in the arrangements of the invention (e.g. the second ligand and / or any binding domains or binding units present in the second fusion protein) will typically (and preferably) be selected such that they have known properties (i.e. their properties relevant to their use in the methods and arrangements of the invention are known and / or characterized) and / or their use in the methods and arrangements of the invention is already validated.

[0209] The assays of the present invention can also be performed in the presence of a compound with a known effect on the membrane-spanning protein (e.g., in the presence of a known agonist, antagonist, inverse agonist, inhibitor, or modulator (e.g., allosteric modulator) of the membrane-spanning protein), at a concentration at which the "known" compound is known to have its effect on the membrane-spanning protein. The known compound is typically present in the same environment as the first ligand (i.e., the ligand whose characteristics are determined using the assays of the present invention). For example, in methods of the present invention in which a first ligand is added to an arrangement of the present invention in which the first ligand is not already present, the known compound can be added essentially simultaneously with the first ligand, separately before the addition of the first ligand, or after the addition of the first ligand. The readout from the assay can vary depending on the order in which the first ligand and the known compound are added, and, if the first ligand and the known compound are not added essentially simultaneously, the time between the moment the first ligand is added and the moment the known compound is added (or vice versa). By varying the order and / or timing of addition of the first ligand and the known compound, it is also possible to determine different properties of the first ligand and / or identify different first ligands having such properties.

[0210] For example, but not limited to, the assays of the present invention can be performed in the presence of a known agonist of the interlayer protein (i.e., in the same environment as the first ligand). In this setting, the assays of the present invention can be used to determine, for example, whether and how the first ligand is able to counteract the agonist effect of a known compound, e.g., since the first ligand acts as an antagonist (thus, in this setting, the assays of the present invention can be used to identify and / or characterize potential antagonists and agonists of the interlayer protein). The setting with a known agonist can also be used to identify and / or characterize a first ligand that can be used, for example, as an allosteric modulator that increases or decreases the effect of the agonist and / or that can act as an inverse agonist of the interlayer protein. It may also be possible to perform a competition assay between the first ligand and a known compound.

[0211] The methods and arrangements of the present invention can be used to measure or determine the ability of a second ligand to bind to a layer-spanning protein, in particular its ability to bind to and / or stabilize a specific conformation (such as a functional, active, or druggable conformation) of the layer-spanning protein, and / or its ability to stabilize and / or induce the formation of a complex between a first ligand, a second ligand, and the layer-spanning protein. For example, as further described herein, the methods and arrangements of the present invention can be used to measure or determine the ability of a given VHH to act as a conformobody for a layer-spanning protein, or to identify, optimize, or validate VHHs that can act as conformobodies. For this purpose, typically, a VHH or candidate VHH is present in a second fusion protein (i.e., as a second ligand) and tested for its ability to directly bind to the layer-spanning protein or to one or more specific conformations of the layer-spanning protein. As further described herein, the methods and arrangements of the invention can also be used to measure or determine the ability of an analogue, derivative or orthologue of a natural ligand of a spanning protein to act as a ligand for the spanning protein (e.g., to test analogues, derivatives or orthologues of a naturally occurring G protein to act as a ligand for an associated GPCR). In this case, the second ligand will typically not be present in a second fusion protein (although a second fusion protein comprising said analogue, derivative or orthologue as the second ligand may be used), but instead the second fusion protein will comprise a binding domain or binding unit (such as a VHH) that is capable of binding to the second ligand (or a complex comprising it).In this regard, it will be clear to those skilled in the art based on the disclosure herein that when the methods and arrangements of the invention are used for such purposes (i.e. for purposes relating to a second ligand), the other components used in the arrangements of the invention (e.g. the first ligand and / or any binding domains or binding units present in the second fusion protein) will typically (and preferably) be selected such that they have known properties (i.e. their properties relevant to their use in the methods and arrangements of the invention are known and / or characterized) and / or such that they have already been validated for use in the methods and arrangements of the invention.

[0212] The methods and arrangements of the present invention can also be used to measure or determine the ability of a binding domain or binding unit present in a second fusion protein to bind to a given second ligand and / or a protein complex containing the second ligand. For example, as further described herein, the methods and arrangements of the present invention can be used to measure or determine the ability of a given VHH to bind to a G protein and / or to identify, optimize, or validate such a VHH that can indirectly bind to a layer-spanning protein (which can then be used, for example, as a binding domain or binding unit in an arrangement of the present invention as described herein, or for any other suitable purpose). Such methods and arrangements of the present invention can also be used to measure or determine the ability of a given VHH to bind to a protein complex containing a G protein and / or to identify, optimize, or validate such a VHH (again, such a VHH can be used as a binding domain or binding unit in an arrangement of the present invention as described herein, or for any other suitable purpose). In this regard, it will be clear to those skilled in the art based on the disclosure herein that when the methods and arrangements of the invention are used for such purposes (i.e. for purposes involving binding domains or binding units that indirectly bind to layer-spanning proteins), the other components (e.g. the first and second ligands) used in the arrangements of the invention will typically (and preferably) be selected such that they have known properties (i.e. their properties that are relevant to their use in the methods and arrangements of the invention are known and / or characterized) and / or such that they have already been validated for use in the methods and arrangements of the invention.

[0213] In the present invention, generally, the detectable signal is preferably generated in response to, and more preferably proportional to, a conformational change in the layer-spanning protein and / or a shift in the conformational equilibrium of the layer-spanning protein. As further described herein, without being limited to any particular mechanism or explanation, said conformational change and / or shift in the conformational equilibrium of the layer-spanning protein may then be caused by the binding of a first ligand to the layer-spanning protein (or otherwise causing a conformational change in the layer-spanning protein) and / or by the formation of a complex of the first ligand, the layer-spanning protein and a second ligand (which second ligand may, for example, stabilize the complex or otherwise induce or promote the formation of the complex). Thus, more generally, in the present invention, a detectable signal (or any change therein, as further described herein) will be generated in response to the presence of a first ligand in a first environment and / or in response to binding of the first ligand to the layer-spanning protein (or otherwise causing a conformational change in the layer-spanning protein and / or a shift in the conformational equilibrium of the layer-spanning protein).

[0214] Also, typically, particularly when using the methods and arrangements of the present invention to test, optimize and / or validate a first ligand and / or identify small molecules, proteins, ligands or other chemicals that can act as agonists, antagonists, inverse agonists, inhibitors or modulators (e.g., allosteric) of a layer-spanning protein, the detectable signal (or any change therein, as further described herein) will be proportional to the amount and / or concentration of the first ligand present in the first environment (and / or the environment to which the layer-spanning protein is exposed) and / or the affinity of the first ligand for the layer-spanning protein (e.g., compared to other ligands tested).

[0215] Therefore, based on the description herein, it will be apparent to one skilled in the art that, in one aspect of the present invention, the methods and arrangements described herein are used to detect the presence of a first ligand in a first environment and / or to determine the amount and / or concentration of a first ligand in a first environment. The methods and arrangements described herein may also be used to measure the amount of signal generated when different concentrations of a first ligand are present in a first environment, e.g., to establish a relationship between the amount / concentration of the first ligand in the first environment and the (level and / or change in) detectable signal. The methods and arrangements described herein may also be used to determine the affinity of a first ligand for a layer-spanning protein, e.g., by comparing the signal generated by one or more known concentrations of the first ligand in the first environment with the signal generated in the same arrangement by known concentrations of other ligands with known affinities for the layer-spanning protein.

[0216] As further described herein, the methods and arrangements of the present invention may also be used to determine whether a given (first) ligand is an agonist, antagonist, inverse agonist, inhibitor or modulator, e.g., allosteric, of a transmembrane protein.

[0217] It will also be apparent to those skilled in the art that when the methods and arrangements of the invention are used to determine one or more characteristics of a first ligand, the arrangements of the invention will typically first be set up or otherwise established in the absence of the first ligand, then the arrangement is contacted with the ligand (e.g., by adding the ligand to the first environment), and the detectable signal (or any change therein) resulting from the presence of the first ligand is then measured (optionally compared to the signal in the absence of the first ligand and / or to one or more reference values). Thus, the arrangements described herein in the absence of the first ligand (e.g., before the first ligand is added) form further aspects of the invention.

[0218] Another aspect of the present invention is a method of preparing an arrangement of the present invention as described herein, the method comprising the step of adding a first ligand to an arrangement of the present invention (as described herein) that does not (yet) comprise the first ligand, and the resulting arrangement can then be used to measure or otherwise determine at least one property of the first ligand, in particular a property of the first ligand that can be measured or otherwise determined using the arrangement of the present invention.

[0219] As will be apparent to one of skill in the art based on the disclosure herein, an arrangement of the present invention in the absence of a first ligand (i.e., an arrangement of the present invention that does not yet include a first ligand) comprises at least the following components: · A boundary layer separating the first environment from the second environment; · Translaminar proteins; a ligand for the transmembrane protein present in the second environment; and a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; wherein the components are arranged relative to one another (and, where applicable, operably linked and / or associated with one another) in a manner further described herein (i.e., essentially as described for arrangements of the invention comprising a first ligand).

[0220] In particular, an arrangement of the invention in the absence of a first ligand (i.e. an arrangement of the invention that does not yet comprise a first ligand) comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a transmembrane protein that is suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair (i.e., forming a first fusion protein); and · a second ligand for the transmembrane protein present in the second environment; wherein the components are arranged with respect to each other (and, where applicable, operably linked and / or associated with each other) in a manner as further described herein (i.e., essentially as described for inventive arrangements comprising a first ligand). In particular, the second member of the binding pair may be part of a second fusion protein (different from the first fusion protein comprising the layer-spanning protein and the first binding member of the binding pair), which second fusion protein is further described herein.

[0221] More specifically, an arrangement of the present invention in the absence of a first ligand (i.e., an arrangement of the present invention that does not yet comprise a first ligand) comprises at least the following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., said member of the binding pair is present in the second environment); a second fusion protein comprising a layer-spanning protein and a protein capable of binding directly or indirectly to the other binding member of said binding pair, the second fusion protein being present in a second environment; wherein the components are arranged relative to one another (and, where applicable, operably linked and / or associated with one another) in a manner further described herein (i.e., essentially as described for arrangements of the invention comprising a first ligand).

[0222] Other aspects, embodiments and references to arrangements of the invention that do not comprise a first ligand are other aspects, embodiments and references to arrangements of the invention that comprise a first ligand as described herein but in the absence of the first ligand.

[0223] Generally, any such inventive arrangement in which no first ligand is present becomes a corresponding inventive arrangement comprising a first ligand once the first ligand is added as part of the methods described herein. Accordingly, another aspect of the present invention is a method of preparing an inventive arrangement as described herein, which method comprises adding a first ligand to an inventive arrangement (as described herein) that does not (yet) comprise the first ligand. The resulting arrangement may then be used to measure or otherwise determine at least one property of the first ligand, particularly a property of the first ligand that can be measured or otherwise determined using the inventive arrangement.

[0224] The present invention also relates to a method of measuring or otherwise determining at least one property of a compound or ligand, the method comprising at least: adding said compound or ligand as a first ligand to an arrangement of the invention that does not already contain a first ligand; and measuring or otherwise determining at least one property of said compound or ligand, said property being a property that can be measured or otherwise determined using said arrangement; The steps include:

[0225] In this aspect of the invention, said property is preferably a property represented by the ability (eg affinity) of the compound or ligand to bind to and / or modulate the transmembrane protein.

[0226] The present invention also relates to a method of measuring or otherwise determining the ability of a compound or ligand to alter a detectable signal produced by a binding pair present in an arrangement of the invention as further described herein, the method comprising at least: adding said compound or ligand as a first ligand to an arrangement of the invention that does not already contain a first ligand; and determining whether adding said compound or ligand results in a change in a detectable signal produced by the binding pair used in said arrangement, and optionally measuring said change in said detectable signal. The steps include:

[0227] Thus, in another aspect, the present invention provides a method for producing ... medicament for a medicament comprising: a) The following components: · A boundary layer separating the first environment from the second environment; · Translaminar proteins; a ligand for the transmembrane protein present in the second environment; and a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; wherein said components are arranged (and, where applicable, operatively linked and / or associated with each other) in a manner as further described herein; and; b) adding a first ligand to the first environment; Regarding a method comprising the steps of: The method preferably: c) measuring the signal produced by the binding pair and / or measuring the change in the signal produced by the binding pair The method further includes the steps of:

[0228] In a more particular aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) The following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a transmembrane protein suitably fused or linked (directly or via a suitable linker or spacer) to one of the binding members of said binding pair (i.e., to form a first fusion protein); and · a second ligand for the transmembrane protein present in the second environment; wherein said components are arranged (and, where applicable, operatively linked and / or associated with each other) in a manner as further described herein; and; b) adding a first ligand to the first environment; Regarding a method comprising the steps of: The method preferably: c) measuring the signal produced by the binding pair and / or measuring the change in the signal produced by the binding pair The method further includes the steps of:

[0229] In another particular aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) The following components: · A boundary layer separating the first environment from the second environment; a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; a first fusion protein comprising a transmembrane protein and one of the binding members of said binding pair (i.e., said member of the binding pair is present in the second environment); a second fusion protein comprising a layer-spanning protein and a protein capable of binding directly or indirectly to the other binding member of said binding pair, the second fusion protein being present in a second environment; wherein said components are arranged (and, where applicable, operatively linked and / or associated with each other) in a manner as further described herein; and; b) adding a first ligand to the first environment; Regarding a method comprising the steps of: The method preferably: c) measuring the signal produced by the binding pair and / or measuring the change in the signal produced by the binding pair The method further includes the steps of:

[0230] As further described herein, in this aspect of the invention, the first ligand can be any desired and / or suitable compound or ligand, including, but not limited to, a small molecule, a small peptide, a biomolecule, or other chemical entity. It will be apparent to those skilled in the art that the method described in this aspect (and other methods of the invention) can be used to measure or otherwise determine at least one property of a compound or ligand added to an arrangement as a first ligand, in particular the ability of the compound or ligand to produce a change in a detectable signal generated by a binding pair, the ability of the compound or ligand to bind to a transmembrane protein, the ability of the compound or ligand to affect a conformational change in a transmembrane protein, and / or the ability of the compound or ligand to modulate (as defined herein) a transmembrane protein and / or signaling pathway(s) and / or biological mechanism(s) in which the transmembrane protein is involved. In particular, the methods may be used to determine whether such compounds or ligands are capable of acting as agonists, antagonists, inverse agonists, inhibitors or modulators (e.g. allosteric modulators) of the transmembrane protein and / or the signal transduction pathway(s) and / or biological mechanism(s) that the transmembrane protein is / are involved in. The methods and arrangements of the present invention may also be used to identify and / or screen compounds or ligands that have the ability to cause a change in the detectable signal generated by the binding partner, to bind to the transmembrane protein, to affect a conformational change in the transmembrane protein, to modulate the transmembrane protein and / or the signal transduction pathway(s) and / or biological mechanism(s) that the transmembrane protein is / are involved in, and / or to act as agonists, antagonists, inverse agonists, inhibitors and / or modulators (e.g. allosteric modulators) of the transmembrane protein, and such uses of the methods and arrangements described herein form further aspects of the present invention.

[0231] It should be noted that in a further aspect, the methods and arrangements of the invention can also be used to measure or otherwise determine at least one property of a second ligand, for example, the ability of the second ligand to bind to the layer-spanning protein, the ability of the second ligand to bind to and / or stabilize a particular conformation of the layer-spanning protein (such as an active and / or druggable conformation), and / or the ability of the second ligand to stabilize a complex of the layer-spanning protein, the first ligand, and the second ligand. Typically, in this aspect of the invention, one or more first ligands with a known ability to bind to and / or modulate the layer-spanning protein are used to determine whether an arrangement of the invention comprising a (candidate) second ligand produces a detectable signal when said first ligand is added to said arrangement (e.g., at one or more known concentrations).

[0232] For example, this aspect of the invention can be used to identify or optimize binding domains or binding units (e.g., ISVDs) that can directly bind to a layer-spanning protein (as defined herein), in particular binding domains or binding units that are specific and / or selective for the conformation of the layer-spanning protein that results when the first ligand used binds to the layer-spanning protein. Binding domains or binding units thus identified, optimized, and / or validated can be used, for example, in arrangements of the invention (i.e., as part of a second fusion protein) and / or to induce or stabilize a specific conformation of the layer-spanning protein (e.g., for screening or crystallization purposes as described in the prior art cited herein for conformation-specific ligands of GPCRs). Thus, for example, this aspect of the invention can be used to identify, optimize, and / or validate ISVDs for use as conformobodies, which can then be used for the purposes described herein and / or for uses known per se for conformation-specific ISVDs. Again, see further prior art cited herein.

[0233] Generally, in this aspect of the invention, the binding domain, binding unit, ligand or other protein to be tested or verified for (direct) binding to the layer-spanning protein is part of a second fusion protein. Thus, the invention provides a method for measuring or otherwise determining at least one property of a binding domain, binding unit, ligand or other protein, comprising at least: providing an arrangement of the invention that does not already comprise a first ligand, wherein a second fusion protein comprises said binding domain, binding unit, ligand or other protein; adding a first ligand to the arrangement; determining whether adding said first ligand results in a change in a detectable signal produced by the binding pair used in said arrangement, and optionally measuring said change in said detectable signal.

[0234] As will be clear to those skilled in the art based on the disclosure herein, the at least one property of the binding domain, binding unit, ligand or other protein will be, in particular, the ability of the binding domain, binding unit, ligand or other protein to bind to the layer-spanning protein (in particular to the predicted conformation of the layer-spanning protein when the first ligand used is bound to the layer-spanning protein), the ability of the binding domain, binding unit, ligand or other protein to stabilize the predicted conformation of the layer-spanning protein when the first ligand used is bound to the layer-spanning protein, and / or the ability of the binding domain, binding unit, ligand or other protein to promote or induce the formation of a complex between the first ligand used, the layer-spanning protein and the binding domain, binding unit, ligand or other protein, and / or the ability of the binding domain, binding unit, ligand or other protein to stabilize such a complex.

[0235] In another aspect of the invention, the arrangement described herein is again used to measure or determine at least one property of a second ligand and / or to identify, optimize, and / or validate a candidate second ligand, but in this aspect the second fusion protein does not comprise the second ligand to be tested or the candidate second ligand, but instead comprises a binding domain or binding unit known to bind to the second ligand to be tested or the candidate second ligand. In other words, in this aspect the second fusion protein comprises a binding domain or binding unit capable of indirectly binding (as defined herein) to the layer-spanning protein, i.e., via the second ligand to be tested or the candidate second ligand, or via a protein complex comprising it, provided that said second ligand or complex is capable of binding to the layer-spanning protein (particularly to the conformation of the layer-spanning protein that results when the first ligand binds to the layer-spanning protein). As with the previous embodiment, this embodiment can also be used to identify, optimize, and / or validate (candidate) ligands for a layer-spanning protein, e.g., ligands that are synthetic or semi-synthetic analogs or derivatives of a naturally occurring ligand for the layer-spanning protein. For example, if the layer-spanning protein is a GPCR, this embodiment of the invention can be used to identify, optimize, and / or validate analogs or derivatives of G proteins that are natural ligands for said GPCR, or to determine whether an orthologue of the natural G protein of the relevant GPCR is capable of binding to said GPCR, and / or to stabilize the complex between the GPCR and the first ligand used.

[0236] Accordingly, the present invention provides a method for measuring or otherwise determining at least one property of a ligand or other protein, comprising at least: providing an arrangement of the invention that does not already comprise a first ligand, wherein said ligand or another protein is present and / or is used as a second ligand, and wherein a second fusion protein comprises a binding domain or binding unit that can bind to said ligand or other protein and / or a protein complex comprising said ligand or other protein; adding a first ligand to the arrangement; determining whether adding said first ligand results in a change in a detectable signal produced by the binding pair used in said arrangement, and optionally measuring said change in said detectable signal.

[0237] As described herein, in one particular aspect of the invention, the method of the invention is carried out using a suitable cell or cell line in which all components of the arrangement of the invention are suitably present and arranged to provide a workable arrangement of the invention. Such a cell or cell line will suitably contain a layer-spanning protein (2) in its cell wall or membrane, i.e., such that the layer-spanning protein (2) is present in and penetrates the cell wall or membrane of the cell, such that at least a portion of the amino acid sequence of the layer-spanning protein protrudes into the extracellular environment (as defined herein) and at least one other portion of the amino acid sequence of the layer-spanning protein protrudes into the intracellular environment (as defined herein). Also, preferably, and as further described herein, the layer-spanning protein (2) will form part of a first fusion protein as described herein, and the arrangement will also include a second fusion protein as described herein. More preferably, the extracellular environment is the "first environment" (i.e., the environment in which the first ligand (3) is present or to which the first ligand (3) is added) and the intracellular environment is the "second environment" (i.e., the environment in which the binding partner (6 / 7) and the second fusion protein are present).

[0238] Therefore, in a further aspect, the present invention relates to a method or arrangement as described herein, wherein the boundary layer (2) is the wall or membrane of a cell.

[0239] Also as described herein, when the method of the invention is carried out in a cell or a suitable cell line, the cell or cell line used preferably contains one or more, and preferably all, of the following components of the arrangement of the invention: a first fusion protein comprising a layer-spanning protein (2) and a first binding member (6); a second fusion protein comprising a second binding member (7) and a protein capable of binding directly or indirectly (as defined herein) to the layer-spanning protein (2); and / or If the second fusion protein binds directly to the transmembrane protein (2), the second ligand (4) and / or protein constituting the protein complex (12) Appropriately express.

[0240] In the context of a cell or cell line expressing one or more components of the arrangement of the present invention, and more generally in the context of this description and claims, the term "suitably express" means that the cell or cell line expresses or is capable of expressing (i.e., under the conditions used to carry out the method of the present invention) a nucleotide sequence or nucleic acid encoding said component, such that when expressed, said component is capable of functioning as an operative part of the arrangement of the present invention. For example, with respect to the interlayer protein (2), this means that the interlayer protein is expressed as part of a first fusion protein such that the interlayer protein (2) is suitably anchored or otherwise integrated into the cell wall or membrane of the cell, such that the expressed interlayer protein (2) penetrates the cell wall or membrane, with at least a portion of the interlayer protein's amino acid sequence projecting (as defined herein) into the extracellular environment and at least one other portion of the interlayer protein's amino acid sequence projecting (as defined herein) into the intracellular environment. With respect to the first and second fusion proteins, "suitably expressed" means that the first and second fusion proteins are expressed (most preferably expressed in an intracellular environment) such that when the second fusion protein binds directly or indirectly to the layer-spanning protein (2) in a manner further described herein, the first and second binding members of the binding pair (6 / 7) can contact or be in close proximity to each other.

[0241] When cells are used to practice the methods of the present invention, any suitable expression of each component of the arrangement of the present invention can be transient or constitutive, so long as all necessary components of the arrangement of the present invention are present in a suitable and operable manner in sufficient amounts at the appropriate time.

[0242] In one aspect of the invention, for embodiments of the invention in which the second fusion protein indirectly binds to the layer-spanning protein (i.e., the second ligand (4) is not part of the second fusion protein), the cell or cell line used preferably naturally expresses the second ligand (4) and / or proteins that make up the protein complex (12). For example, and without limitation, in this aspect of the invention, the layer-spanning protein (2) may be a GPCR, the second ligand (4) may be a G protein naturally expressed by the cell or cell line used, and / or the protein complex (12) may be a G protein trimer comprising a G alpha subunit, a G beta subunit, and a G gamma subunit naturally expressed by the cell or cell line used. More generally, in these aspects of the invention, the cells or cell lines used may be cells or cell lines that naturally express one or more natural ligands (particularly intracellular ligands) of the interlayer protein (2) and / or naturally express one or more ligands that can function as secondary ligands for the interlayer protein (2), depending on the interlayer protein (2) used or screened.

[0243] The cells or cell lines can be any cells or cell lines suitable for use in the methods and arrangements of the present invention, including but not limited to mammalian cells and insect cells. Some preferred, but non-limiting examples are human cell lines such as HEK293T.

[0244] Suitable techniques for transiently or stably expressing a desired protein in such cells or cell lines so that the transmembrane protein (2) is appropriately anchored to the cell wall or membrane of said cells will be apparent to those skilled in the art and include, for example, techniques involving the use of suitable transfection reagents such as SigmaAldrich's X-tremeGENE™ or polyethyleneimine (PEI).

[0245] Where the invention is practiced using cells or cell lines that suitably express one or more components of the arrangements of the invention, the methods of the invention will generally also include the step of culturing or maintaining said cells or cell lines under conditions such that they suitably express said components.

[0246] Thus, in another aspect, the present invention relates to a cell or cell line comprising a fusion protein, wherein the fusion protein comprises a layer-spanning protein (as described herein) fused, directly or via a suitable linker, to a binding domain or binding unit that is a first binding member of a binding pair, the binding pair comprising at least the binding domain or binding unit as the first binding member and a further binding domain or binding unit as the second binding member, the first and second binding members of the binding pair being capable of generating a detectable signal when they contact each other or are in close proximity to each other. The present invention also relates to a cell or cell line that expresses or is capable of expressing (i.e. under appropriate conditions) such a fusion protein.

[0247] Such a cell or cell line may be further described herein and preferably expresses or is capable of expressing said fusion protein in such a manner that the interlayer protein is integrated into the cell wall or cell membrane of the cell or cell line and penetrates said cell wall or cell membrane, more preferably such that at least a portion of the amino acid sequence of the interlayer protein protrudes (as defined herein) into the extracellular environment and at least one other portion of the amino acid sequence of the interlayer protein protrudes (as defined herein) into the intracellular environment. More preferably, in said cell or cell line, the first binding member of the binding pair is present in the intracellular environment of the cell (as defined herein), and / or the cell or cell line expresses or is capable of expressing the fusion protein, and upon such expression, the first binding member is present in the intracellular environment of the cell (as defined herein).

[0248] Furthermore, the layer-spanning protein present in the fusion protein is preferably a layer-spanning protein as further described herein, more preferably having at least two ligand binding sites, one protruding into the extracellular environment (as defined herein) and one protruding into the intracellular environment (as defined herein). Furthermore, as described herein, the layer-spanning protein is preferably such that, upon binding of a ligand to the ligand-binding site of the layer-spanning protein, in particular, upon binding of a ligand present in the extracellular environment to the ligand-binding site of the layer-spanning protein present in the extracellular environment (as defined herein), the layer-spanning protein is capable of undergoing a conformational change from one conformation of the layer-spanning protein to another conformation (in particular, a conformational change from an essentially inactive or less active conformation to an active or more active conformation). As further described herein, the layer-spanning protein is preferably also such that it can be stabilized in a functional and / or active (or more active) conformation (particularly a druggable conformation and / or ligand-binding conformation, more particularly an agonist-binding conformation) by binding of a suitable ligand, binding domain or binding unit (e.g., a natural ligand of a conformobody or layer-spanning protein described herein, such as a natural intracellular ligand) to the intracellular binding site of the layer-spanning protein (which may be a binding site of the layer-spanning protein that is an intracellular binding site when the layer-spanning protein is in its natural environment and / or a binding site of the layer-spanning protein that is an intracellular binding site when the layer-spanning protein is present in a cell or cell line used in the invention, preferably both). In particular, as also described herein, the layer-spanning protein may be capable of forming a complex when a first ligand binds to the extracellular binding site and a second ligand binds to the intracellular binding site.More specifically, as described herein, the layer-spanning protein may be capable of forming a complex in which the layer-spanning protein is in a functional or active conformation induced by binding of a first ligand to an extracellular binding site, and said active or functional conformation is stabilized by binding of a second ligand to an intracellular binding site, and the second ligand is capable of stabilizing said functional, active or ligand-bound conformation and / or said complex. In a preferred, but non-limiting embodiment, the layer-spanning protein is a transmembrane protein, particularly a 7TM. Also, the members of the binding pair and any linkers used may be those further described herein.

[0249] In another aspect, the present invention relates to a cell or cell line comprising a fusion protein, the fusion protein comprising a protein capable of binding (directly or indirectly as described herein) to a layer-spanning protein (as described herein), which protein is fused, directly or via a suitable linker, to a binding domain or binding unit that is a binding member of a binding pair, the binding pair comprising said binding domains or binding units as at least a first binding member and a second binding member, and wherein said first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other. The present invention also relates to a cell or cell line that expresses or is capable of expressing (i.e., under appropriate conditions) such a fusion protein.

[0250] The protein present in the fusion protein and capable of binding to the layer-spanning protein is preferably a protein that can be present in a second fusion protein, as further described herein. Also, the binding pair members and any linkers used may be further described herein. Also as described herein, the protein can be directly (as described herein) or indirectly (as described herein) bound to the layer-spanning protein. Again, in this embodiment, the layer-spanning protein to which the protein can bind is preferably a layer-spanning protein, as further described herein, and may in particular be a transmembrane protein, more in particular a 7TM.

[0251] As described herein, when the protein present in the fusion protein directly binds to a spanning protein, it preferably specifically binds to one or more functional, active and / or druggable conformations of the spanning protein and induces and / or stabilizes the formation of one or more functional, active and / or druggable conformations of the spanning protein (and / or shifts the conformational equilibrium of the spanning protein towards one or more such conformations); and / or induces and / or stabilizes the formation of a complex of said protein, the spanning protein and a further ligand of the spanning protein (all as further described herein). Also, when the protein present in the fusion protein directly binds to a spanning protein, it is preferably capable of binding to an intracellular binding site of the spanning protein. The intracellular binding site of the spanning protein may be a binding site of the spanning protein that is an intracellular binding site when the spanning protein is in its natural environment and / or a binding site of the spanning protein that is an intracellular binding site when the spanning protein is present in a cell or cell line used in the present invention (and preferably both).

[0252] Also, when the protein present in the fusion protein directly binds to a transmembrane protein, it is preferably a VHH domain or a binding domain or binding unit derived from a VHH domain, in particular a ConfoBody (described herein).

[0253] Furthermore, as described herein, when the protein present in the fusion protein indirectly binds to the layer-spanning protein, it is preferably capable of binding to a ligand capable of binding to the layer-spanning protein. The ligand may be a "secondary ligand," as described herein, if the second ligand does not form part of the second fusion protein. Furthermore, the ligand preferably specifically binds to one or more functional, active, and / or druggable conformations of the layer-spanning protein, induces the formation of one or more functional, active, and / or druggable conformations of the layer-spanning protein (and / or shifts the conformational equilibrium of the layer-spanning protein toward one or more such conformations); and / or induces and / or stabilizes the formation of a complex of the ligand, the layer-spanning protein, and a further ligand of the layer-spanning protein (all as further described herein). Furthermore, the ligand is preferably capable of binding to an intracellular binding site of the layer-spanning protein. Said intracellular binding site of a transmembrane protein may be a binding site of the transmembrane protein that is an intracellular binding site when the transmembrane protein is in its natural environment and / or a binding site of the transmembrane protein that is an intracellular binding site when the transmembrane protein is present in a cell or cell line used in the invention (and preferably both). As described herein, said ligand may also be part of a protein complex that is capable of binding to the transmembrane protein (i.e. the intracellular binding site of the transmembrane protein), in which case the protein present in the fusion protein is also capable of binding to said protein complex.

[0254] Furthermore, when the protein present in the fusion protein indirectly binds to a transmembrane protein, it is preferably a VHH domain or a binding domain or binding unit derived from a VHH domain. In a preferred embodiment, when the protein present in the fusion protein indirectly binds to a transmembrane protein and the transmembrane protein is a GPCR, the ligand that binds to the GPCR is a G protein, and the protein present in the fusion protein is capable of specifically binding to the G protein or a G protein complex, such as a G protein trimer comprising a G-alpha subunit, a G-beta subunit, and a G-gamma subunit. The G protein may be native to the cell or cell line used, or may be a suitable analog or derivative of a natural G protein (as described herein and recombinantly expressed in the cell or cell line) or a suitable ortholog of a G protein native to the cell or cell line used (and recombinantly expressed in the cell or cell line used).

[0255] Regardless of whether the protein present in said fusion protein is directly or indirectly bound to the transmembrane protein, the cell or cell line preferably expresses or is capable of expressing said fusion protein in its intracellular environment. Another aspect of the invention relates to such a cell or cell line comprising such a fusion protein in its intracellular environment.

[0256] In another aspect, the present invention relates to a cell or cell line comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair.

[0257] The present invention also relates to cells or cell lines that express or are capable of expressing (ie under appropriate conditions) such first and second fusion proteins.

[0258] The present invention particularly relates to a cell or cell line comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair; When the second fusion protein binds (directly or indirectly, as described herein) to the layer-spanning protein that forms part of the first fusion protein, the first and second binding members of the binding pair can contact or be in close proximity to each other.

[0259] The present invention also relates to a cell or cell line comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair; The first and second binding members of the binding pair are present in the intracellular environment of the cell (as described herein).

[0260] The present invention further provides a cell or cell line comprising a first fusion protein and a second fusion protein, the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first binding member and the second binding member of the binding pair are capable of generating a detectable signal when they contact or are in close proximity to each other; said first fusion protein comprises a transmembrane protein (as described herein) fused, either directly or via a suitable linker, to said first binding member of a binding pair; the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, the protein being fused, either directly or via a suitable linker, to a second binding member of the binding pair; a cell or cell line capable of producing a detectable signal (in particular a detectable signal produced by the first and second binding members of a binding pair) when a second fusion protein binds (directly or indirectly as described herein) to a transmembrane protein forming part of the first fusion protein.

[0261] The present invention further relates to a cell or cell line comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair; When a ligand of the transmembrane protein present in the extracellular environment binds to the transmembrane protein, the cell or cell line produces a detectable signal and / or a change in a detectable signal (in particular a detectable signal and / or a change in such a signal generated by the first and second binding members of the binding pair).

[0262] In a particular aspect, the present invention provides a cell or cell line comprising a first fusion protein and a second fusion protein, the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact or are in close proximity to each other; said first fusion protein comprises a transmembrane protein (as described herein) fused, either directly or via a suitable linker, to said first binding member of a binding pair; the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; a cell or cell line which produces a detectable signal and / or a change in a detectable signal (in particular a detectable signal and / or a change in such a signal generated by the first and second binding members of the binding pair) when an agonist of the transmembrane protein present in the extracellular environment binds to the transmembrane protein;

[0263] Such cells or cell lines comprising or expressing such first and second fusion proteins are also further described herein, and preferably they express or are capable of expressing said first fusion protein in such a manner that the layer-spanning protein is integrated into the cell wall or cell membrane of the cell or cell line and penetrates said cell wall or cell membrane, more preferably such that at least a portion of the amino acid sequence of the layer-spanning protein protrudes into the extracellular environment (as defined herein) and at least one other portion of the amino acid sequence of the layer-spanning protein protrudes into the intracellular environment (as defined herein).

[0264] The cell or cell line preferably expresses or is capable of expressing the first and second fusion proteins, and upon such expression, the first and second binding members of the binding pair can contact or be in close proximity to each other when the second fusion protein binds (directly or indirectly, as described herein) to the membrane-spanning protein that forms part of the first fusion protein. It will be clear to those skilled in the art that this generally means that such a cell or cell line will express the first and second fusion proteins in such a manner that, upon such expression, the first and second binding members of the binding pair will be present in the same environment relative to the cell wall or membrane (as defined herein). Preferably, the cell or cell line expresses or is capable of expressing the first and second fusion proteins, and upon such expression, both the first and second binding members of the binding pair are present in the intracellular environment of the cell (as defined herein). This also generally means that the cell or cell line preferably expresses or is capable of expressing the second fusion protein in their intracellular environment.

[0265] Additionally, in aspects of the invention relating to cells or cell lines that express or are capable of expressing such first and second fusion proteins, the layer-spanning proteins, proteins that can bind directly or indirectly to the layer-spanning proteins, members of binding pairs, and any linkers used may all be as further described herein.

[0266] In a further aspect, the present invention also relates to methods, particularly assay or screening methods, that include the use of the cells or cell lines described herein. As further described herein, such assay and screening methods can be used, in particular, to identify compounds and other chemicals that bind (particularly specifically bind) to a transmembrane protein, can modulate the transmembrane protein, and / or modulate the signal transduction, signal transduction pathway, and / or biological or physiological activity(ies) involved in the transmembrane protein, its signal transduction, and / or its signal transduction pathway. As such, the cells and cell lines described herein can be used in methods to identify compounds or other chemicals that can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric) of a transmembrane protein.

[0267] The present invention also relates to the use of the cells or cell lines described herein, particularly in assay and screening methods and techniques. Such methods and uses may also be further described herein with respect to the methods and uses of the arrangements of the invention and will generally also include culturing or maintaining the cells or cell lines under conditions in which they suitably express the desired fusion protein(s).

[0268] In all these aspects, such cells, cell lines and their uses are also preferably as further described herein.

[0269] In another aspect of the invention, the method of the invention is carried out using a suitable liposome or vesicle, all components of the arrangement of the invention being suitably present and arranged to provide a workable arrangement of the invention. Such liposome or vesicle suitably comprises a transmembrane protein (2) in its wall or membrane, i.e., the transmembrane protein (2) is present in and penetrates the wall or membrane of the liposome or vesicle, with at least one portion of the transmembrane protein's amino acid sequence projecting (as defined herein) into the environment outside the liposome or vesicle, and at least one other portion of the transmembrane protein's amino acid sequence projecting (as defined herein) into the environment inside the liposome or vesicle. Also preferably, and as further described herein, in aspects of the invention embodied in liposomes or vesicles, the environment outside the liposome or vesicle is the "first environment" (i.e., the environment in which the first ligand (3) is present or to which the first ligand (3) is added), and the environment inside the liposome or vesicle is the "second environment" (i.e., the environment in which the binding pair (6 / 7) and the second fusion protein are present).

[0270] Therefore, in a further aspect, the present invention relates to a method or arrangement as described herein, wherein the boundary layer (2) is the wall or membrane of a liposome or other (suitable) vesicle.

[0271] Also as described herein, when the method of the present invention is carried out with a liposome or vesicle, the liposome or vesicle preferably comprises the following components of the arrangement of the present invention: a first fusion protein comprising a layer-spanning protein (2) and a first binding member (6); a second fusion protein comprising a second binding member (7) and a protein capable of binding directly or indirectly (as defined herein) to the layer-spanning protein (2); and / or If the second fusion protein indirectly binds to the transmembrane protein (2), the second ligand (4) and / or protein constituting the protein complex (12) suitably (i.e. in such a manner as to provide an operable arrangement of the invention).

[0272] Liposomes or vesicles containing the components can generally be provided by forming liposomes or vesicles in the presence of the relevant components of the arrangement of the present invention so that the components are properly incorporated into the liposomes or vesicles. This can generally be carried out by methods and techniques known per se for forming liposomes or vesicles, preferably in an appropriate aqueous buffer or another suitable aqueous medium. Such methods may also include the step of separating liposomes or vesicles in which the components of the desired arrangement of the present invention are properly and operably contained from vesicles or liposomes that do not contain all the necessary components of the arrangement and / or whose components do not form an operative arrangement of the present invention. The components of the arrangement to be incorporated into liposomes or vesicles can be provided in a manner known per se, for example by recombinant expression in a suitable host cell or host organism, followed by isolation and purification of the resulting expressed components.

[0273] Generally, in aspects of the invention embodied in liposomes or vesicles, the second ligand will not form part of the second fusion protein, and a sufficient amount of the second ligand will also be provided so that it is properly contained in the vesicle or liposome.

[0274] The liposomes or vesicles may be any liposomes or vesicles suitable for use in the methods and arrangements of the present invention, including, but not limited to, liposomes based on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The liposomes and vesicles may also contain and / or be based on (e.g., reconstituted from) one or more membrane fractions obtained from cells expressing the desired component(s) of the arrangements of the present invention.

[0275] Thus, in another aspect, the present invention relates to a liposome or vesicle comprising a fusion protein, the fusion protein comprising a transmembrane protein fused, directly or via a suitable linker, to a binding domain or binding unit that is a first binding member of a binding pair (as described herein), the binding pair comprising at least the binding domain or binding unit as the first binding member and a further binding domain or binding unit as the second binding member, the first and second binding members of the binding pair being capable of generating a detectable signal when they contact each other or are in close proximity to each other. The present invention also relates to a method of preparing such a liposome or vesicle, the method comprising at least the step of incorporating such a fusion protein into the liposome or vesicle and / or forming the liposome or vesicle in the presence of the fusion protein.

[0276] As further described herein, said liposomes or vesicles preferably have a membrane-spanning protein anchored to or otherwise suitably incorporated into the wall or membrane of the liposome or vesicle, spanning said wall or membrane, more preferably at least a portion of the amino acid sequence of the membrane-spanning protein protruding (as defined herein) into the environment outside the liposome or vesicle and at least one other portion of the amino acid sequence of the membrane-spanning protein protruding (as defined herein) into the interior environment of the liposome or vesicle. More preferably, the first binding member of the binding pair is present in the interior environment of the liposome or vesicle (as defined herein).

[0277] Furthermore, the interlayer protein present in the fusion protein is preferably a interlayer protein as further described herein, more preferably having at least two ligand binding sites, one protruding into the external environment (as defined herein) of the liposome or vesicle and one protruding into the internal environment (as defined herein) of the liposome or vesicle. Furthermore, as described herein, the interlayer protein is preferably such that it is capable of undergoing a conformational change from one of its conformations to another (in particular from an essentially inactive or less active conformation to an active or more active conformation) upon binding of a ligand to the ligand binding site of the interlayer protein, in particular upon binding of a ligand present in the external environment of the liposome or vesicle to the ligand binding site of the interlayer protein present in the external environment of the liposome or vesicle (as defined herein). As further described herein, the interlayer protein is preferably also one that can be stabilized in a functional and / or active (or more active) conformation (particularly a druggable conformation and / or a ligand-binding conformation, more particularly an agonist-binding conformation) by binding of a suitable ligand, binding domain or binding unit (e.g., a conformobody as described herein or a natural ligand of the interlayer protein) to the intracellular binding site of the interlayer protein (which may be a binding site of the interlayer protein that is an intracellular binding site when the interlayer protein is in its natural environment and / or a binding site of the interlayer protein that is present in the internal environment of the liposome or vesicle when the interlayer protein is present in the liposome or vesicle used in the present invention, preferably both). In particular, as also described herein, the interlayer protein may be capable of forming a complex when a first ligand binds to a binding site that is present in the external environment (as defined herein) of the liposome or vesicle and a second ligand binds to a binding site that is present in the internal environment (as defined herein) of the liposome or vesicle.More particularly, as described herein, the membrane-spanning protein may be capable of forming a complex in which the membrane-spanning protein is in a functional or active conformation induced by binding of a first ligand to a binding site (as defined herein) present in the external environment of the liposome or vesicle, and said active or functional conformation is stabilized by binding of a second ligand to a binding site (as defined herein) present in the internal environment of the liposome or vesicle, the second ligand being capable of stabilizing said functional, active or ligand-bound conformation and / or said complex. In a preferred, but non-limiting, aspect, the membrane-spanning protein is a transmembrane protein, particularly a 7TM. Also, the members of the binding pair and any linkers used may be those further described herein.

[0278] In another aspect, the present invention relates to a liposome or vesicle comprising a fusion protein, the fusion protein comprising a protein capable of binding (directly or indirectly, as described herein) to a membrane-spanning protein (as described herein), the protein fused, directly or via a suitable linker, to a binding domain or binding unit that is a binding member of a binding pair, the binding pair comprising said binding domain or binding unit as at least a first binding member and a second binding member, the first and second binding members of the binding pair being capable of generating a detectable signal when they contact each other or are in close proximity to each other. The present invention also relates to a method of preparing such a liposome or vesicle, the method comprising at least the step of incorporating such a fusion protein into the liposome or vesicle and / or the step of forming the liposome or vesicle in the presence of the fusion protein.

[0279] The protein present in the fusion protein and capable of binding to the layer-spanning protein is preferably a protein that can be present in a second fusion protein, as further described herein. Also, the binding pair members and any linkers used may be further described herein. Also as described herein, the protein may be directly (as described herein) or indirectly (as described herein) bound to the layer-spanning protein. Again, in this embodiment, the layer-spanning protein that the protein can bind to is preferably a layer-spanning protein, as further described herein, and may in particular be a transmembrane protein, more in particular a 7TM.

[0280] As described herein, when the protein present in said fusion protein binds directly to a spanning protein, it preferably binds specifically to one or more functional, active and / or druggable conformations of the spanning protein and induces and / or stabilizes the formation of one or more functional, active and / or druggable conformations of the spanning protein (and / or shifts the conformational equilibrium of the spanning protein towards one or more such conformations); and / or induces and / or stabilizes the formation of a complex of said protein, the spanning protein and a further ligand of the spanning protein (all as further described herein). Also, when the protein present in said fusion protein binds directly to a spanning protein, it is preferably such that it is capable of binding to a binding site of the spanning protein that is an intracellular binding site when the spanning protein is in its native environment and / or to a binding site of the spanning protein that is present in the internal environment (as defined herein) of a liposome or vesicle when the spanning protein is present in said liposome or vesicle when the liposome or vesicle is used in the present invention (and preferably both).

[0281] Also, when the protein present in the fusion protein directly binds to a transmembrane protein, it is preferably a VHH domain or a binding domain or binding unit derived from a VHH domain, in particular a ConfoBody (as described herein).

[0282] Also, as described herein, if the protein present in the fusion protein indirectly binds to the layer-spanning protein, it is preferably capable of binding to a ligand capable of binding to the layer-spanning protein. The ligand may be a "secondary ligand," as described herein, if the second ligand does not form part of a second fusion protein. The ligand also preferably specifically binds to one or more functional, active, and / or druggable conformations of the layer-spanning protein, and induces and / or stabilizes the formation of one or more functional, active, and / or druggable conformations of the layer-spanning protein (and / or shifts the conformational equilibrium of the layer-spanning protein towards one or more such conformations); and / or induces and / or stabilizes the formation of a complex of the ligand, the layer-spanning protein, and a further ligand of the layer-spanning protein (all as further described herein). The ligand is also preferably capable of binding to a binding site of the transmembrane protein that is an intracellular binding site when the transmembrane protein is in its natural environment and / or to a binding site of the transmembrane protein that is present in the internal environment (as defined herein) of a liposome or vesicle when the transmembrane protein is present in said liposome or vesicle when said liposome or vesicle is used in the present invention (and preferably both). As described herein, the ligand may also be part of a protein complex that is capable of binding to the transmembrane protein, in which case the protein present in the fusion protein is also capable of binding to said protein complex.

[0283] Furthermore, when the protein present in the fusion protein indirectly binds to a transmembrane protein, it is preferably a VHH domain or a binding domain or binding unit derived from a VHH domain. Also, in a preferred embodiment, when the protein present in the fusion protein indirectly binds to a transmembrane protein, the transmembrane protein is a GPCR, the ligand binding to the GPCR is a G protein, and the protein present in the fusion protein can specifically bind to the G protein or a G protein complex such as a G protein trimer comprising a G alpha subunit, a G beta subunit, and a G gamma subunit.

[0284] Regardless of whether the protein present in the fusion protein is directly or indirectly bound to the transmembrane protein, the fusion protein is preferably present in the interior environment of a liposome or vesicle (as defined herein), and if a second ligand does not form part of the fusion protein, the interior environment of the liposome or vesicle will also contain an appropriate amount of a second ligand.

[0285] In another aspect, the present invention relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair.

[0286] The present invention also relates to a method for preparing such liposomes or vesicles, which method comprises at least the steps of incorporating such a fusion protein into a liposome or vesicle and / or forming a liposome or vesicle in the presence of said fusion protein.

[0287] The present invention particularly relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein of the invention, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair; When the second fusion protein binds (directly or indirectly, as described herein) to the layer-spanning protein that forms part of the first fusion protein, the first and second binding members of the binding pair can contact or be in close proximity to each other.

[0288] The present invention also relates to a method for preparing such liposomes or vesicles, which method comprises at least the step of incorporating said fusion protein into a liposome or vesicle and / or the step of forming a liposome or vesicle in the presence of said fusion protein.

[0289] The present invention also relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair; The first and second binding members of the binding pair are present in the interior environment of the liposome or vesicle (as defined herein).

[0290] The present invention also relates to a method for preparing such liposomes or vesicles, which method comprises at least the step of incorporating said fusion protein into a liposome or vesicle and / or the step of forming a liposome or vesicle in the presence of said fusion protein.

[0291] The present invention further relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair; When the second fusion protein binds (directly or indirectly, as described herein) to the transmembrane protein forming part of the first fusion protein, the liposome or vesicle is capable of producing a detectable signal (particularly a detectable signal produced by the first and second binding members of the binding pair).

[0292] The present invention also relates to a method for preparing such liposomes or vesicles, which method comprises at least the step of incorporating said fusion protein into a liposome or vesicle and / or the step of forming a liposome or vesicle in the presence of said fusion protein.

[0293] The present invention further relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair; When a ligand for a transmembrane protein present in the environment outside the liposome or vesicle binds to the transmembrane protein, said liposome or vesicle produces a detectable signal and / or a change in a detectable signal (in particular a detectable signal and / or a change in such a signal produced by the first and second binding members of the binding pair).

[0294] The present invention also relates to a method for preparing such liposomes or vesicles, which method comprises at least the step of incorporating said fusion protein into a liposome or vesicle and / or the step of forming a liposome or vesicle in the presence of said fusion protein.

[0295] In a particular aspect, the present invention relates to a liposome or vesicle comprising a first fusion protein and a second fusion protein: the first fusion protein comprises a binding domain or binding unit that is a first binding member of a binding pair, and the second fusion protein comprises a binding domain or binding unit that is a second binding member of the binding pair, and the first and second binding members of the binding pair are capable of generating a detectable signal when they contact each other or are in close proximity to each other; said first fusion protein comprises a transmembrane protein fused to said first binding member of a binding pair, either directly or via a suitable linker (as described herein); the second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the layer-spanning protein, which protein is fused, either directly or via a suitable linker, to a second binding member of the binding pair; When an agonist for a transmembrane protein present in the environment outside the liposome or vesicle binds to the transmembrane protein, said liposome or vesicle produces a detectable signal and / or a change in a detectable signal (in particular a detectable signal and / or a change in such a signal produced by the first and second binding members of the binding pair).

[0296] The present invention also relates to a method for preparing such liposomes or vesicles, which method comprises at least the step of incorporating said fusion protein into a liposome or vesicle and / or the step of forming a liposome or vesicle in the presence of said fusion protein.

[0297] Such liposomes or vesicles comprising such first and second fusion proteins may be as further described herein, preferably they are suitably anchored to or otherwise incorporated into the wall or membrane of the liposome or vesicle and have a layer-spanning protein that spans said wall or membrane, more preferably at least a portion of the amino acid sequence of the layer-spanning protein protruding into the environment outside the liposome or vesicle (as defined herein) and at least one other portion of the amino acid sequence of the layer-spanning protein protruding into the environment inside the liposome or vesicle (as defined herein).

[0298] The liposome or vesicle also preferably allows the first and second binding members of the binding pair to contact or be in close proximity to each other when the second fusion protein binds (directly or indirectly, as described herein) to the membrane-spanning protein that forms part of the first fusion protein. It will be apparent to those skilled in the art that this generally means that the first and second binding members of the binding pair are in the same environment (as defined herein) relative to the wall or membrane of the liposome or vesicle. Preferably, the liposome or vesicle will have both the first and second binding members of the binding pair in the interior environment (as defined herein) of the liposome or vesicle.

[0299] Additionally, in aspects of the invention relating to liposomes or vesicles containing such first and second fusion proteins, the membrane-spanning proteins, proteins that can bind directly or indirectly to the membrane-spanning proteins, members of binding pairs, and any linkers used may all be further described herein.

[0300] In a further aspect, the present invention also relates to methods, particularly assay or screening methods, that include the use of the liposomes or vesicles described herein. As further described herein, such assay and screening methods can be used, in particular, to identify compounds and other chemicals that bind (particularly specifically bind) to a transmembrane protein, modulate the transmembrane protein, and / or modulate signal transduction, signal transduction pathways, and / or biological or physiological activity(ies) involving the transmembrane protein, its signal transduction, and / or its signal transduction pathway. As such, the liposomes or vesicles described herein can be used in methods to identify compounds or other chemicals that can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric) of a transmembrane protein.

[0301] The present invention also relates to the use of the liposomes or vesicles described herein, particularly in assay and screening methods and techniques, such methods and uses also as will be further described herein with respect to the methods and uses of the arrangements of the invention.

[0302] In all these aspects, such liposomes or vesicles and their uses are also preferably as further described herein.

[0303] It will be apparent to those skilled in the art that compounds discovered, developed, produced, and / or optimized using the methods and techniques described herein may be used for any suitable or desired purpose. Said purpose generally relates to the target for which the compound was screened / produced, the signaling pathway(s) and / or mechanism of action associated with the target, and / or the biological, physiological, and / or pharmacological function that includes said target, pathway(s), signaling pathway(s), and / or mechanism of action. Typically and preferably, compounds of the present invention are capable of, and / or are selected to, modulate said target, signaling pathway(s), mechanism of action, and / or said biological, physiological, and / or pharmacological function in a desired or intended manner. As described herein, this modulation can take any desired or intended form, including, but not limited to, upregulation and downregulation of the target, signaling pathway(s), mechanism of action, and / or said biological, physiological, and / or pharmacological function. As such, the compounds of the present invention can function, for example, as agonists, antagonists, inverse agonists, inhibitors, or another type of modulator (e.g., allosteric modulators) of said target and / or its signal transduction, pathway(s), mechanism of action, and / or said biological, physiological, and / or pharmacological function. All of this can be determined using suitable in vitro, cellular, and / or in vivo assays (e.g., suitable efficacy or potency assays) and / or suitable animal models, depending on the specific target, signal transduction, pathway(s), mechanism of action, and / or said biological, physiological, and / or pharmacological function involved. Suitable assays and models will be apparent to those skilled in the art.

[0304] Typically, when a compound of the present invention is an agonist (or antagonist, respectively) of a target, the compo...

Claims

1. At a minimum, the following components: a boundary layer separating the first environment from the second environment; translaminar proteins; a first ligand for said layer-spanning protein present in said first environment; a second ligand for the layer-spanning protein present in the second environment; and a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; Including, the transmembrane protein is a GPCR; the second ligand is a G protein or a G protein complex; the first binding member of the binding pair is part of a first fusion protein comprising the first binding member fused or linked to the Fc receptor, either directly or via a suitable linker or spacer; an arrangement in which the second binding member of the binding pair is part of a second fusion protein comprising the second binding member fused or linked directly or via a suitable linker or spacer to an immunoglobulin single variable domain capable of binding to the G protein or G protein complex.

2. 2. The arrangement of claim 1, wherein the boundary layer is a cell wall or membrane of a cell, the first environment is an extracellular environment, and the second environment is an intracellular environment.

3. 2. The arrangement of claim 1, wherein the boundary layer is a wall or membrane of a liposome or vesicle, the first environment is the environment outside the liposome or vesicle, and the second environment is the environment inside the liposome or vesicle.

4. At a minimum, the following components: a boundary layer separating the first environment from the second environment; translaminar proteins; a second ligand for the layer-spanning protein present in the second environment; and a binding pair consisting of at least a first binding member and a second binding member, the binding pair being capable of generating a detectable signal; Including, the transmembrane protein is a GPCR; the second ligand is a G protein or a G protein complex; the first binding member of the binding pair is part of a first fusion protein comprising the first binding member fused or linked to the Fc receptor, either directly or via a suitable linker or spacer; an arrangement in which the second binding member of the binding pair is part of a second fusion protein comprising the second binding member fused or linked directly or via a suitable linker or spacer to an immunoglobulin single variable domain capable of binding to the G protein or G protein complex.

5. 5. The arrangement of claim 4, wherein the boundary layer is a cell wall or membrane of a cell, the first environment is an extracellular environment, and the second environment is an intracellular environment.

6. 5. The arrangement of claim 4, wherein the boundary layer is a wall or membrane of a liposome or vesicle, the first environment is the environment outside the liposome or vesicle, and the second environment is the environment inside the liposome or vesicle.

7. a) providing an arrangement according to any one of claims 4 to 6; b) adding a first ligand to the first environment of the arrangement; A method comprising:

8. c) measuring the signal produced by said binding pair and / or measuring the change in the signal produced by said binding pair The method of claim 7 further comprising:

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