Screening methods and assays for transmembrane proteins, in particular gpcr

Abstract

The invention provides a method and a device for screening membrane proteins. The device comprises a first fusion protein comprising a membrane protein present in a boundary layer (e.g., the wall of a cell, liposome, or vesicle) and fused to one member of a binding pair, and a second fusion protein comprising an intracellular ligand fused to the membrane protein of the other member of the binding pair, wherein the combination pair is capable of generating a detectable signal.

Description

[0001] This application is a divisional application of the application filed on April 28, 2020, with international application number PCT / EP2020 / 061803, entering the Chinese national phase with application number 202080046276.7, entitled "Method and determination for screening transmembrane proteins, especially GPCRs".

[0002] This invention relates to methods and tools that can be used for determination, screening, and drug discovery and development.

[0003] In particular, the present invention relates to methods and tools for use in screening and assay techniques and for working to discover, generate, optimize and / or develop therapeutic, preventive and diagnostic reagents targeting membrane proteins (i.e., those specific to membrane proteins), the screening and assay techniques involving the use of membrane proteins (i.e., as targets to be screened, for example, to discover candidate compounds targeting said targets). The invention further relates to methods for preparing tools that can be used in screening and assay techniques.

[0004] Advantageously, the methods and tools of the present invention can be used for screening and assay techniques, as well as for the discovery, generation, optimization, and / or development of therapeutic, preventative, and diagnostic reagents targeting such membrane proteins, the screening and assay techniques involving the use of membrane proteins that can present / exist in multiple conformations (e.g., but not limited to active and inactive conformations). Such membrane proteins include, but are not limited to, transmembrane proteins, such as GPCRs and other cell surface receptors.

[0005] In a particularly preferred but non-limiting aspect, the methods and tools of the present invention can be used for screening and assay techniques, as well as for the discovery, generation, optimization, and / or development of therapeutic, preventative, and diagnostic reagents targeting such membrane proteins. These screening and assay techniques involve using membrane proteins capable of conformational changes (again, for example, but not limited to, from an inactive conformation to an active conformation) in response to a ligand binding to said protein. Similarly, such membrane proteins can be cell surface receptors, such as GPCRs.

[0006] This invention generally provides methods for performing assays (i.e., for a given compound or ligand) or for screening purposes (i.e., for screening groups, series, or libraries of compounds or ligands to identify “hit” against a target). This invention also provides apparatus that can be used in said methods, i.e., as a system or setup for performing said assays or screenings. The apparatus includes the elements described herein. These elements may also be provided or constructed as a kit of parts, and such kits of parts form another aspect of this invention. This invention also provides components for identifying and creating such apparatus, as well as methods for assembling such apparatus.

[0007] The methods and apparatus described herein can generally be used to test one or more properties of (known) compounds or ligands (i.e., those properties that can be determined using the methods described herein) and / or to identify compounds or ligands having one or more such desired properties (i.e., from groups, series, or libraries of compounds or ligands). These compounds or ligands can be any desired and / or suitable compounds or ligands, including but not limited to small molecules, small peptides, biomolecules, or other chemical entities, and examples of such compounds will be apparent to those skilled in the art based on the further disclosure herein. Furthermore, compounds identified using the methods of the present invention (i.e., “hit” from such screenings) can serve as a starting point for further drug discovery and development work (e.g., using well-known techniques of so-called “hits-to-leads” chemistry), and such further work may also involve the use of the methods of the present invention (e.g., as a functional assay or an assay for quality control purposes).

[0008] Compounds identified using the methods and techniques of this invention (i.e., “hit”), and any compounds generated or developed using such hits as a starting point, are also collectively referred to herein as “compounds of this invention” and form a further aspect of this invention. Those skilled in the art will appreciate that such compounds can be, for example, so-called “hit,” “lead,” “development candidate,” “preclinical compound,” “clinical candidate,” or commercial compounds or products, depending on their stage of development and the specific terminology used by the company or entity developing and / or commercializing them.

[0009] Advantageously, compared to conventional radioligand assays or functional assays, the methods and assays of the present invention do not require the use of labeled antagonists (e.g., fluorescently labeled or radiolabeled), and therefore can also be applied to membrane proteins where no antagonist is available or known. Furthermore, as further described herein, the methods and assays of the present invention can allow for the identification and / or characterization of allosteric agonists (positive and negative), antagonists, and / or inverse agonists (depending on the specific target and assay used).

[0010] Other features, aspects, embodiments, uses, and advantages of the invention will become clear from the further description herein.

[0011] Membrane proteins (such as cell surface receptors, including GPCRs) and their assays and screening techniques are well known in the art. It is estimated that over half of modern drugs target membrane proteins, and approximately one-third target GPCRs. Refer to standards manuals and other existing techniques cited herein.

[0012] As is well known in the field of protein dynamics, most proteins are not static entities whose function is determined solely by their primary, secondary, tertiary, and (when a protein contains two or more polypeptide chains) quaternary structures. Instead, they are typically flexible structures capable of transitioning between different conformational states (also known as “conformational changes”), allowing proteins to exist in equilibrium between these states. Some of these states may be functional and / or active, while others may be basal states (which may or may not exhibit a certain level of constitutive activity), essentially inactive states, and / or states with lower activity compared to more functional or active states. Furthermore, the geometry of different epitopes, binding sites (including ligand-binding sites), and / or catalytic sites that may be present in or on the protein may differ between these different conformations, for example, making the binding site unavailable for ligand binding / inaccessible for ligand binding in some conformational states, and / or causing a lower affinity between the binding site and the associated ligand compared to more active conformational states.

[0013] It is also known that for some protein / ligand combinations, ligand binding to a protein may alter 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 on a protein may also make another binding site on the protein more accessible to its associated ligand, and / or may lead to an increase in the affinity of said other binding site for said ligand, and / or shift the equilibrium from a conformation in which said other binding site has a lower affinity for said ligand to a conformation in which said other binding site has a better affinity for said ligand. For example, for some transmembrane proteins such as GPCRs, binding of an extracellular ligand to an extracellular binding site on the protein may increase the affinity of the intracellular binding site for the intracellular ligand (e.g., increase the affinity of the interaction between the G-protein and G-protein binding sites on the GPCR), and vice versa. This change in the binding affinity of extracellular ligands to intercellular ligands after binding, and the subsequent binding of intracellular ligands to intracellular binding sites, may be part of the way proteins transduce extracellular signals.

[0014] Typically, as further described herein, it can be said that for receptor proteins that can undergo conformational changes, the receptor's "agonist" shifts 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 the receptor's "reverse agonist" does the opposite.

[0015] It is also possible for a protein to form a complex with two ligands that bind to two different binding sites on the protein, and the interaction between the protein and each ligand is stabilized by the binding of the other ligand (in other words, the complex is stabilized by the binding of both ligands). Similarly, in this case, the binding of one or both ligands may also cause a conformational shift of the protein to that complex (formation and / or stabilization). See, for example, WO2012 / 007593 cited below.

[0016] Given that the perceived “global” state of a protein is largely controlled by the (statistical) distribution of the protein across its various possible conformations, and therefore by the balance that exists between these conformational states, it should be understood that in this specification or claims, when referring to a protein undergoing a conformational change to a certain conformation (i.e., from one or more other conformations), this includes a mechanism or situation in which the conformational balance of the protein shifts towards said conformation (i.e., under the specific conditions used, such as those for screening or related assays). Similarly, when referring to a ligand inducing a conformational change of a protein to a certain conformation (i.e., from one or more other conformations), this includes a mechanism or situation in which the binding of the ligand causes the conformational balance of the protein to shift towards said conformation (i.e., under the specific conditions used, such as those for screening or related assays).

[0017] However, it should also be noted that although any mechanism (or any combination thereof) described herein may be involved in the practice of the invention at any given time (and also depending on, for example, the specific protein and / or ligand applied to the invention), the invention is in its broadest sense and is not limited to any particular mechanism, explanation or hypothesis, as long as applying the invention to a particular target or protein produces the technical effects outlined herein.

[0018] One of the challenges of screening compounds for membrane proteins that exist in multiple conformations is that the correct conformation of a protein may be lost if it is expressed or used in isolation from its native environment (if expressing the protein and ensuring its correct folding outside the cellular environment is feasible or possible). Furthermore, ensuring that a protein is in its desired conformation (typically a functional conformation, such as its active conformation) under the conditions used for screening can be challenging. A shift in the conformational equilibrium of a protein toward a conformational state more suitable for the screening or assay purpose (e.g., the active state or a state in which the relevant binding site is more accessible and / or has a geometry more favorable for the assay or screening purpose) may also be required or facilitated. As further described herein, such a conformation is also referred to as a “drug-producible” conformation, and according to a preferred aspect of the invention, means (as further described herein) are applied to ensure that the protein is in such a drug-producible conformation and / or to ensure a shift in the conformational equilibrium of the protein toward a more drug-producible conformation during the conduct of the methods of the invention.

[0019] For example, WO2012 / 007593, WO2012 / 007594, WO2012 / 175643, WO2014 / 118297, WO2014 / 122183, and WO2014 / 118297 relate to protein-binding domains that can be used to stabilize specific conformational states of GPCRs, for determining their structure, and for drug screening and discovery purposes. In these references, the use of VHH domains allows GPCRs to be stabilized in desired conformations, particularly (more)drug-like conformations, such as functional and / or active states, such as the conformation that occurs when an activating ligand (agonist) binds to the extracellular side of the GPCR, thereby activating the heterotrimeric G protein. For example, references were also made to 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, as well as other references cited therein. The VHH domain, which can be used to stabilize the desired conformation of membrane proteins such as GPCRs, is also referred to as the Confobody in this paper. TM It is a registered trademark of Confo Therapeutics, Ghent, Belgium.

[0020] Some specific, but non-limiting, examples of confobodies capable of binding to intracellular epitopes of GPCRs and used to stabilize GPCRs in a desired conformation (and also applicable to this invention) are VHHs named CA2764, CA3431, CA3413, CA2780, CA2765, CA2761, CA3475, CA2770, CA3472, CA3420, CA3433, CA3434, CA3484, CA2760, CA2773, CA3477, CA2774, CA2768, CA3424, CA2767, CA2786, CA3422, CA2763, CA2772, CA2771, CA2769, CA2782, CA2783, and CA2784 (see, for example, WO2012 / 007593, Tables 1 and 2 and SEQ ID). NO:1 to 29); VHHs named CA5669, Nb9-1, Nb9-8, XA8633 and CA4910 (see, for example, WO2014 / 118297, Tables 1 and 2 and SEQ ID NO:15, 16, 17, 19 and 20); VHHs named 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-E1, Nb_A2, Nb_B4, Nb_D3, Nb_D1 and Nb_H1 (see, for example, WO2014 / 122183, Tables 1 and 2 and SEQ ID NO:1-19); and VHHs named XA8639, VHH of XA8635, XA8727 and XA9644 (see, for example, WO2015 / 121092, Tables 2 and 3 and SEQ ID NO:2 to 6 and 74).

[0021] Some specific, but non-limiting, examples of VHHs that can bind to G proteins are CA4435, CA4433, CA4436, CA4437, CA4440, and CA4441 (see, for example, WO2012 / 175643007593, Tables 2 and 3, and SEQ ID NO: 1 to 6).

[0022] 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 that target membrane proteins (i.e., compounds that are specific to one or more membrane proteins and / or designed to target one or more membrane proteins, for example, for therapeutic, preventative, and / or diagnostic purposes). Preferably, such compounds are specific to a particular membrane protein (i.e., selective for a particular membrane protein) compared to other (closely related) membrane proteins.

[0023] Compounds identified and / or developed using the methods of this invention can be used to regulate (as defined herein) membrane proteins, their signal transduction, and / or the biological functions, pathways, and / or mechanisms involved in the signal transduction of said membrane proteins or their signal transduction. For example, this invention can be used to discover and develop compounds that serve as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., positive and negative allosteric modulators) of said membrane proteins and / or the signal transduction, pathways, and / or physiological and / or biological mechanisms involved in said membrane proteins.

[0024] This invention can be used to discover and develop compounds targeting membrane proteins, which are intact or peripheral membrane proteins in their natural environment. This invention can be particularly used to discover and develop compounds targeting transmembrane proteins, as further described herein. In one specific, but not limiting, aspect, compounds discovered and / or developed using this invention will target receptors, particularly cell surface receptors.

[0025] As further described herein, transmembrane proteins can be, in particular, membrane proteins that cross the membrane multiple times, such as 7TM or GPCRs. [In this regard, it should be noted that, generally in the art, the terms “7TM receptor” and “7TM” are often used interchangeably with “GPCR”, although according to the IUPHAR database, there are some 7TM receptors that do not signal via G proteins. For the purposes of this specification and claims, the terms “GPCR” and “7TM” are used interchangeably herein to include all transmembrane proteins (especially transmembrane receptors) having seven transmembrane domains, regardless of their intracellular signaling cascades or signal transduction mechanisms, although it should be understood throughout the specification and claims that 7TMs that signal via G proteins are a preferred aspect of the invention.]

[0026] Typically, the compounds discovered and / or developed using this invention will target membrane proteins that are expressed on and / or exposed on the cell surface when in their natural environment, particularly those membrane proteins expressed by or on cells present in the body of a subject, wherein the patient will be treated with compounds discovered or developed using the methods and techniques of this invention.

[0027] This invention can be used to discover and / or develop any kind of compound suitable for its intended use, typically as a therapeutic, diagnostic, or preventative agent. Therefore, these compounds can be small molecules, peptides, biomolecules, or other chemical entities. Examples of suitable biomolecules may include, for example, antibodies and antibody fragments (e.g., Fab, VH, VL, and VHH domains) and antibody fragment-based compounds (e.g., ScFv and biantibodies, as well as other compounds or constructs containing one or more VH, VL, and / or VHH domains), compounds based on other protein scaffolds, such as Alphabodies™ and avimer-based scaffolds, PDZ domains, protein A domains (e.g., Affibodies™), ankyrin repeat sequences (e.g., DARPins™), fibronectins (e.g., Adnectins™), and lipocalin proteins (e.g., Anticalins™), as well as DNA or RNA-based binding moieties, including but not limited to DNA or RNA aptamers. For further description in this article, and for example Simeon and Chen, Protein Cell 2018, 9(1): 3–14, Binz et al., Nat. Biotech 2005, Vol 23:1257, and Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):619-32.

[0028] The methods and techniques of the present invention can, for example, be used to screen libraries of such compounds to identify one or more “hit” specific to membrane proteins (especially desired conformations of membrane proteins, and / or conformations capable of inducing desired conformations of membrane proteins, such as ligand binding, particularly agonist binding), and / or as assays as part of a strategy to improve the affinity and / or potency of compounds against membrane proteins and / or otherwise 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” activity).

[0029] The methods and techniques of this invention can also be used for so-called "fragment-based drug discovery" or "FBDD" (also known as "fragment-based lead discovery" or "FBLD"). For example, see Lamore and Hubbard, Essays in Biochemistry (2017) 61, 453–464, and standard manuals such as 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.

[0030] This invention will be described herein with reference to specific embodiments and certain non-limiting examples and drawings. No reference numerals in the claims should be construed as limiting the scope. The described drawings are merely illustrative and non-limiting. In the drawings, for illustrative purposes, some elements may be enlarged and not drawn to scale. When the term "comprising" is used in this specification and claims, it does not exclude other elements or steps. If an indefinite or definite article, such as "a" or "an," or "the," is used when referring to a singular noun, the plural form of that noun is included unless explicitly stated otherwise. Furthermore, the terms first, second, third, etc., in the specification and claims are used to distinguish similar elements and are not necessarily used to describe an order or chronological sequence. It should be understood that the terms thus used are interchangeable where appropriate, and embodiments of the invention described herein can operate in orders other than those described or illustrated herein.

[0031] Unless otherwise defined herein, scientific and technical terms and phrases used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Generally, terms and techniques relating to molecular and cell biology, structural biology, biophysics, pharmacology, genetics, and protein and nucleic acid chemistry as described herein are well-known and commonly used in the art. Singleton, et al., *Dictionary of Microbiology and Molecular Biology*, 2DED., John Wiley and Sons, New York (1994), and Hale & Marham, *The Harper Collins Dictionary of Biology*, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many terms used in this disclosure. Unless otherwise stated, the methods and techniques of this invention are generally carried out 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. See, for example, 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 to 2002); up, Biomolecular crystallography: principles, Practice and Applications to Structural Biology, 1st ed., GarlandScience, Taylor & Francis Group, LLC, an informa Business, NY (2009); Limbird, Cell Surface Receptors, 3rd ed., Springer (2004).

[0032] As used herein, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably and refer to a polymer of amino acids of any length, which may include both coding and non-coding amino acids (chemically or biochemically modified or derived amino acids), as well as polypeptides having a modified peptide backbone. Throughout this application, the standard single-letter symbol for amino acids will be used. Generally, the term “amino acid” will refer to “protein amino acid,” that is, those amino acids that are naturally present in proteins. Most specifically, amino acids are in the L-isomer form, but D-amino acids are also contemplated.

[0033] As used herein, the terms “nucleic acid molecule,” “polynucleotide,” “polynucleic acid,” and “nucleic acid” are used interchangeably and refer to a polymer of nucleotides (deoxyribonucleotides or ribonucleotides, or analogs thereof) of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. 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.

[0034] Any peptides, polypeptides, nucleic acids, compounds, etc., disclosed herein may be “isolated” or “purified.” “Isolated” herein is used to mean that the material referred to is (i) isolated from one or more substances that coexist with it in nature (e.g., isolated from at least some cellular material, isolated from other polypeptides, isolated from its native sequence context), and / or (ii) produced by artificial processes, 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 are substantially rich in the target compound and / or wherein the target compound is partially or substantially purified. As used herein, “purified” means that the material referred to has been removed from its natural environment and is free from at least 60%, at least 75%, or at least 90% of its naturally associated other components, also referred to as “substantially pure.”

[0035] As used in this article, 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 within a comparison window.

[0036] Therefore, the "sequence identity percentage" is calculated as follows: Two best-aligned sequences are compared within a comparison window. The number of positions in both sequences where 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, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) appear is determined to produce the number of matching positions. This number of matching positions is then divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to produce the sequence identity percentage. The determination of the sequence identity percentage can be done manually or using a computer program 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 from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0037] “Similarity” refers to the percentage of identical or conserved substitutions of amino acids. Similarity can be determined using sequence comparison procedures such as GAP (Deveraux et al., 1984). In this way, sequences of similar or substantially different lengths to those cited herein can be compared by inserting vacancies into the alignment, for example, by the comparison algorithm used in GAP. As used herein, a “conservative substitution” is the substitution of an amino acid with another amino acid whose side chain has similar biochemical properties (e.g., aliphatic, aromatic, positively charged, etc.) and is well known to those skilled in the art. A non-conservative substitution is the substitution of an amino acid with another amino acid whose side chain does not have similar biochemical properties (e.g., replacing a hydrophobic residue with a polar residue). Conservative substitutions generally produce sequences that are no longer identical but are still highly similar. Conservative substitutions are meant by combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.

[0038] "Deletion" is defined herein as a change in the amino acid or nucleotide sequence of one or more amino acid or nucleotide residues that are absent compared to the amino acid or nucleotide sequence of the parent polypeptide or nucleic acid. In the context of proteins, deletions may involve the loss of about 2, about 5, about 10, up to about 20, up to about 30, or up to about 50 or more amino acids. A protein or a fragment thereof may contain more than one deletion. In the context of GPCRs, deletions may also be loop deletions or N- and / or C-terminal deletions. As will be apparent to those skilled in the art, N- and / or C-terminal deletions of GPCRs are also referred to as truncation of the GPCR amino acid sequence or truncated GPCRs.

[0039] An "insertion" or "addition" is a change in the amino acid or nucleotide sequence that results in the addition of one or more amino acid or nucleotide residues compared to the amino acid or nucleotide sequence of the parent protein. An "insertion" typically refers to the addition of one or more amino acid residues within the amino acid sequence of a polypeptide, while an "addition" can refer to an insertion or the addition of amino acid residues at the N- or C-terminus or both ends. In the context of a protein or fragment thereof, an insertion or addition is typically about 1, about 3, about 5, about 10, up to about 20, up to about 30, or up to about 50 or more amino acids. A protein or fragment thereof may contain more than one insertion.

[0040] As used herein, a “substitution” is generated by replacing one or more amino acids or nucleotides with different amino acids or nucleotides compared to the amino acid or nucleotide sequence of a parent protein or fragment thereof. It should be understood that proteins or fragments thereof may have conserved amino acid substitutions that have substantially no effect on protein activity. Conserved substitutions refer to combinations such as gly, ala; val, ile, leu, met; asp, glu; asn, gin; ser, thr; lys, arg; cys, met; and phe, tyr, trp.

[0041] The term "amino acid difference" refers to the total number of amino acid residues in a sequence that have changed (i.e., through 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 comparing these sequences, for example, through alignment.

[0042] The term "orthologous homolog," when used to refer to amino acid or nucleotide / nucleic acid sequences from a given species, refers to the same amino acid or nucleotide / nucleic acid sequence from different species. It should be understood that two sequences are orthologous homologs of each other when they are derived linearly (descent) from a common ancestral sequence and / or are closely related in terms of their sequence and their biological function. Orthologous homologs typically have a high degree of sequence identity but may not (and usually do not) share 100% sequence identity.

[0043] The term "recombinant" when used to refer to cells, nucleic acids, proteins, or vectors indicates that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells that have been so modified. Thus, for example, recombinant cells express nucleic acids or polypeptides not found in the natural (non-recombinant) form of the cells, or express native genes that are otherwise abnormally expressed, underexpressed, overexpressed, or not expressed at all.

[0044] As used in this article, the term "expression" refers to the process by which a gene's nucleic acid sequence produces a polypeptide. This process includes transcription and translation.

[0045] As used herein, the term "operably ligated" refers to ligation in which the regulatory sequence is adjacent to the target gene to control the target gene, and trans- or distantly acting to control the regulatory sequence of the target gene. For example, when a DNA sequence is ligated downstream of the transcription start site of a promoter, the DNA sequence is operably ligated to the promoter, allowing transcriptional elongation to proceed through the DNA sequence. If the DNA of the signal sequence is expressed as a preprotein involved in polypeptide transport, the DNA of the signal sequence is operably ligated to the DNA encoding the polypeptide. Ligation of the DNA sequence to the regulatory sequence is typically achieved by using a restriction endonuclease known to those skilled in the art at a suitable restriction site or adapter, or alternatively, a linker.

[0046] As used herein, the term "regulatory sequence," also known as "control sequence," refers to a polynucleotide sequence essential to the expression of a coding sequence to which it is operationally linked. Regulatory sequences are sequences that control transcription, post-transcriptional events, and translation of nucleic acid sequences. 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 enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and sequences that enhance protein secretion when needed. The nature of such control sequences depends on the host organism. The term "regulatory sequence" is intended to include at least all components whose presence is essential for expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion coupler sequences.

[0047] 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 is linked. Vectors can be of any suitable type, including but not limited to bacteriophages, viruses, plasmids, phage particles, granules, rod particles, or even artificial chromosomes. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can integrate into the host cell's genome after introduction and thus replicate along with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of certain target genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, etc., as needed and depending on the specific host organism (e.g., bacterial cells, yeast cells). Typically, recombinant vectors according to the invention contain at least one "chimeric gene" or "expression cassette". The expression cassette is typically a DNA construct, preferably comprising (5' to 3' in the transcriptional direction): a promoter region, a polynucleotide sequence of the invention operatively linked to the transcription start region, a homolog, a variant, or a fragment thereof, and a termination sequence comprising a termination signal for RNA polymerase and a polyadenylation signal. It should be understood that all these regions should be functional in the biological cells to be transformed, such as prokaryotic or eukaryotic cells. The promoter region containing the transcription start region (which preferably includes an RNA polymerase binding site) and the polyadenylation signal can be native to the biological cells to be transformed or can be derived from alternative sources, wherein the region is functional in the biological cells.

[0048] As used herein, the term "host cell" is intended to refer to a cell into which the recombinant vector has been introduced. It should be understood that these terms refer not only to the specific test cell but also to the progeny of such cells. Because certain modifications may occur in the progeny due to mutations or environmental influences, such progeny may actually differ from the parent cell but are still included within the scope of the term "host cell" as used herein. Host cells can be isolated cells or cell lines grown in a culture, or they can be cells present in living tissue or organisms. In particular, host cells are of bacterial or fungal origin, but can also be 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.

[0049] G-protein coupled receptors (GPCRs) are polypeptides sharing a common structural motif, possessing an extracellular N-terminus, an intracellular C-terminus, and seven hydrophobic transmembrane regions (regions of 22 to 24 hydrophobic amino acids forming seven α-helices), each spanning the cell membrane. Each span is numbered, namely transmembrane-1 (TM1), transmembrane-2 (TM2), etc. The transmembrane helices are linked by amino acid regions between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the extracellular or "extracellular" side of the cell membrane, referred to as "extracellular" regions 1, 2, and 3 (EC1, EC2, and EC3), respectively. The transmembrane helices are also linked by amino acid regions between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the intracellular or "intracellular" side of the cell membrane, referred to as "intracellular" regions 1, 2, and 3 (IC1, IC2, and IC3), respectively. The receptor’s “carboxyl” (“C”) terminus is located in the intracellular space within the cell, and the receptor’s “amino” (“N”) terminus is located in the extracellular space outside the cell.The structure and classification of GPCRs are generally well known in the field, 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 book: Jurgen Wess (Ed) Structure-Function Analysis of G Protein-Coupled Receptors, by Wiley Liss (1st edition; October 15, 1999); Kevin R. Lynch (Ed) Identification and Expression of G Protein-Coupled Receptors, published by John Wiley & Sons (March 1998); 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 edition; 1994).

[0050] The International Union of Basic and Clinical Pharmacology (IUPHAR) maintains a database of receptors (including GPCRs) and their known endogenous ligands and signal transduction mechanisms (http: / / www.guidetopharmacology.org / targets.jsp). According to this database, as of January 2019, approximately 800 GPCRs have been identified in humans, about half of which have sensory functions (e.g., olfaction, taste, light perception, and pheromone signaling), and about half mediate signal transduction associated with ligands (ranging in size from small molecules to peptides to large proteins). The IUPHAR database as of January 2019 describes two systems for classifying GPCRs, one based on six classes as follows: Class A (rhodopsin-like receptors), Class B (secretin receptor family), Class C (metabolite glutamate), Class D (fungal mating pheromone receptors, not found in vertebrates), Class E (cyclic AMP receptors, also not found in vertebrates), and Class F (curled / smooth). The IUPHAR database also mentions an alternative classification scheme called "GRAFS," which divides vertebrate GPCRs into five classes (overlapping with the AF nomenclature), as follows: Glutamate family (overlapping with the "C class" above), which specifically includes metabolized glutamate receptors, calcium-sensitive receptors, and GABAB receptors; Rhodopsin family (overlapping with the "A class" above), which includes receptors for various small molecules, neurotransmitters, peptides, and hormones, as well as olfactory receptors, visual pigments, type 2 gustatory receptors, and five pheromone receptors (V1 receptors); Adhesion family G... PCR (phylogenetically associated with class B receptors); the coiled family, consisting of 10 coiled proteins (FZD(1-10)) and smooth (SMO); and the secretin family, which are peptide ligand / hormone receptors with 27-141 amino acid residues, including glucagon, glucagon-like peptides (GLP-1, GLP-2), glucose-dependent insulinotropic peptide (GIP), secretin, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), and growth hormone-releasing hormone (GHRH). In this specification and the appended claims, unless otherwise expressly stated, type A to F will be used. Further reference is made to Cvicek et al., cited herein.

[0051] In the context of GPCRs, the term "bioactivity" refers to a GPCR that possesses the biochemical functions of a naturally occurring GPCR (such as binding function, signal transduction function, or the ability to change conformation due to ligand binding).

[0052] Generally, the term "naturally occurring" in relation to GPCRs 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. The term "non-naturally occurring" in relation to GPCRs refers to a GPCR that is not naturally occurring. Examples of non-naturally occurring GPCRs include naturally occurring GPCRs that have constitutive activity through mutation and variants of naturally occurring transmembrane receptors (e.g., epitope-tagged GPCRs and GPCRs lacking their natural N-terminus). Non-naturally occurring versions of naturally occurring GPCRs are typically activated by the same ligands as the naturally occurring GPCRs. This document further provides non-limiting examples of naturally occurring or non-naturally occurring GPCRs in the context of this invention.

[0053] As used herein, an epitope refers to an antigenic determinant of a polypeptide. An epitope can contain three amino acids in a spatial conformation, which is unique to epitopes. Typically, epitopes consist of at least four, five, six, or seven such amino acids, and more commonly, at least eight, nine, or ten. Methods for determining the spatial conformation of amino acids are known in the art, including, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. As used herein, a conformational epitope refers to an epitope containing amino acids in a spatial conformation that is unique to the folded 3D conformation of the polypeptide. Typically, conformational epitopes consist of discontinuous amino acids in a linear sequence that aggregate within the folded structure of the protein. However, conformational epitopes can also consist of a linear amino acid sequence that adopts a conformation unique to the folded 3D conformation of the polypeptide (and is not in a denatured state).

[0054] The term "conformation" or "conformatory state" of a protein generally refers to the spatial arrangement, structure, or range of structures a protein may adopt at any given time. Those skilled in the art will recognize that the determinants of conformation or conformational state include the protein's primary structure, reflected in its amino acid sequence (including modified amino acids), and its surrounding environment. Protein conformation or conformational state also involves structural features such as secondary structure (e.g., α-helices, β-sheets, etc.), tertiary structure (e.g., three-dimensional folding of polypeptide chains), and quaternary structure (e.g., interactions between polypeptide chains and other protein subunits). Post-translational modifications and other alterations to the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, can all affect protein conformation. Furthermore, environmental factors such as pH, salt concentration, ionic strength, and osmotic pressure of the surrounding solution, as well as interactions with other proteins and cofactors, can influence protein conformation. The conformational state of a protein can be determined by functional assays of its activity or binding to another molecule, or by physical methods such as X-ray crystallography, NMR, or spin labeling. For a general discussion of protein conformation and conformational states, see Cantor and Schimmel, Biophysical Chemistry, Part 1: The Conformation of Biological Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993.

[0055] As used herein, "functional conformation" or "functional conformational state" refers to the fact that a protein possesses different conformational states with a dynamic range of activity (particularly from inactive to maximally active). It is important to clarify that "functional conformational state" refers to any conformational state of a protein that is active (including inactive), and does not include denatured states. Non-limiting examples of functional conformations include active conformations, inactive conformations, or basal conformations (as further defined herein). As noted above, certain categories of functional conformations are defined as "drugable conformations" and generally refer to therapeutically relevant conformational states of proteins. For example, refer to Johnson and Karanicolas, PLoS Comput Biol 9(3): e1002951.doi:10.1371 / journal.pcbi.1002951 and, for example, WO2014 / 122183, which describe the agonist-binding active conformation of the muscarinic acetylcholine receptor M2 corresponding to a druggable conformation of this receptor associated with pain and gliobastoma, and describe VHHs capable of stabilizing said druggable conformations for assay and screening purposes. Therefore, it should be understood that druggability is limited to a specific conformation depending on the therapeutic indication. Further details are provided in this article.

[0056] For proteins that act as receptors, as used herein, the term “active conformation” more specifically refers to the conformation or receptor conformation profile that allows signal transduction to intracellular effector systems (e.g., G protein-dependent signaling and / or G protein-independent signaling (e.g., β-repressor signaling)). Therefore, “active conformation” encompasses a range of ligand-specific conformations, including agonist-specific, partially agonist-specific, or biased agonist-specific conformations, in order to induce the co-binding of intracellular effector proteins.

[0057] In addition to the above, for GPCRs, the terms "active conformation" and "active form" as used herein refer to GPCRs folded in a manner to have (functional) activity. GPCRs can be placed in the active conformation using an activating ligand (agonist) of the receptor, and this conformational change typically enables the receptor to activate heterotrimeric G proteins. For example, a GPCR in the active conformation binds to a heterotrimeric G protein and catalyzes nucleotide exchange of the G protein to activate downstream signaling pathways. An activated GPCR binds to the inactive GDP-binding form of the heterotrimeric G protein, causing the G protein to release its GDP, thus enabling GTP binding. This process produces a transient "nucleotide-free" state, allowing GTP binding. Once GTP binds, the receptor and G protein dissociate, and the G protein that enabled GTP binding activates downstream signaling pathways, such as adenylate cyclases, ion channels, RAS / MAPK, etc. The terms "inactive conformation" and "inactive form" refer to GPCRs folded in a manner to be inactive. GPCRs can be placed in the inactive conformation using a reverse agonist of the receptor. For example, GPCRs in the inactive conformation do not bind to heterotrimeric G proteins with high affinity. The terms "active conformation" and "inactive conformation" will be further explained in this paper. As used herein, the term "basal conformation" refers to a GPCR folded in such a manner that it exhibits activity against a specific signaling pathway, even in the absence of an agonist (also known as basal activity or constitutive activity). Inverse agonists inhibit this basal activity. Thus, in the absence of ligands or accessory proteins, the basal conformation of a GPCR corresponds to a stable conformation or a prominent structural class.

[0058] Similarly, for proteins acting as receptors, the term "inactive conformation" as used herein refers to the receptor conformational profile that does not allow or blocks signal transduction to the intracellular effector system. Therefore, "inactive conformation" encompasses a range of ligand-specific conformations, including inverse agonist-specific inactive conformations, thereby preventing the co-binding of intracellular effector proteins. It should be understood that the ligand binding site is not critical for acquiring an active or inactive conformation. Therefore, orthosteric ligands and allosteric modulators can also stabilize receptors in active or inactive conformations.

[0059] As used herein, the term "binding agent" 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 a particular embodiment, the term "binding agent" is not intended to include naturally occurring binding partners of the relevant membrane protein, such as G proteins, repressor proteins, endogenous ligands; or variants or derivatives thereof (including fragments). More specifically, the term "binding agent" refers to polypeptides, and more specifically, protein domains. A suitable protein domain is an element of the overall protein structure; it is self-stabilizing and folds independently of the rest of the protein chain and is generally referred to as a "binding domain." The length of such binding domains varies from about 25 amino acids to 500 amino acids and more. Many binding domains can be classified as folds and are identifiable, identifiable, 3-D structures. Some folds are so common in many different proteins that they have been given specific names. Non-limiting examples are selected from the following binding domains: 3- or 4-helical 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, etc. Therefore, the binding domain can be derived from naturally occurring molecules, for example, components of the innate or adaptive immune system, or it can be entirely artificially designed.

[0060] Typically, the binding domain can be based on immunoglobulins or on domains present in proteins, including but not limited to microbial proteins, protease inhibitors, toxins, fibronectin, lipocalin, single-stranded antiparallel coiled helical proteins, or repeating motif proteins. Specific examples of binding domains known in the art include, but are not limited to: antibodies, heavy chain antibodies (hcAbs), single-domain antibodies (sdAbs), microantibodies, variable domains (VHHs or nanobodies) derived from camel heavy chain antibodies, variable domains (VNAs) of neoantigen receptors derived from shark antibodies, antigen bodies, protein A, protein G, designed ankyrin repeat domains (DARPin), fibronectin type III repeat sequences, anticalin, knottin, engineered CH2 domains (nanobodies), engineered SH3 domains, affibody, peptides and proteins, lipopeptides (e.g., pepducin) (see, for example, Gebauer & Skerra, 2009; Skerra, 2000; Starovasnik et al., 1997; Binz et al., 2004; Koide et al., 1998; Dimitrov, 2009; Nygren et al., 2008; WO2010066740). Typically, when using selection methods to generate specific types of binding domains, combinatorial libraries containing shared sequences or framework sequences with random potential interacting residues are used to screen for binding to the target molecule (e.g., a protein).

[0061] According to preferred embodiments, the binder of the present invention is particularly envisioned to originate from the innate or adaptive immune system. Preferably, the binder is derived from immunoglobulins. Preferably, the binder according to the present invention is derived from antibodies or antibody fragments. The term "antibody" (Ab) generally refers to a polypeptide or functional fragment thereof encoded by an immunoglobulin gene that specifically binds to and recognizes an antigen, and is known to those skilled in the art. Antibodies are intended to include conventional four-stranded immunoglobulins, which comprise two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50 kDa). Typically, in conventional immunoglobulins, the variable domain (VH) of the heavy chain and the variable domain (VL) of the light chain interact to form an antigen-binding site. The term "antibody" is intended to include intact antibodies, including single-stranded intact antibodies and antigen-binding fragments. In some embodiments, the antigen-binding fragment may be an antigen-binding antibody fragment, including but not limited to Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (dsFv), and fragments containing or composed of VL or VH domains, as well as any combination of any other functional portions of immunoglobulin peptides that bind to the target antigen. The term "antibody" is also intended to include heavy-chain antibodies or fragments thereof, including immunoglobulin monovariable domains, as further defined herein.

[0062] The term "immunoglobulin single variable domain" or "ISVD" defines a molecule in which an antigen-binding site is present on and formed by a single immunoglobulin domain (unlike conventional immunoglobulins or fragments thereof, where typically two immunoglobulin variable domains interact to form an antigen-binding site). However, it should be clear that the term "immunoglobulin single variable domain" does indeed include fragments of conventional immunoglobulins in which the antigen-binding site is formed by a single variable domain. Preferably, the binding agent within the scope of this invention is an immunoglobulin single variable domain.

[0063] Typically, an immunoglobulin monovariable domain (ISVD) is an amino acid sequence comprising four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3), preferably according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1), or any suitable fragment thereof (which typically contains at least some amino acid residues forming at least one complementarity-determining region). ISVDs comprising four FRs and three CDRs are known to those skilled in the art and are described as non-limiting examples in Wesolowski et al. 2009. Typical but non-limiting examples of immunoglobulin ISVDs 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, provided that they can form a single antigen-binding unit. Therefore, according to a preferred embodiment, the binder 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 specifically, the immunoglobulin single variable domain is a heavy chain variable domain sequence derived from a conventional 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 “dAB” or dAb, or a nanobody (as defined herein), or another immunoglobulin single variable domain, or any suitable fragment of either. For a general description of single-domain antibodies, refer to “Single domain antibodies”, Methods in Molecular Biology, Eds. Saerens and Muyldermans, 2012, Vol. 911. Immunoglobulin monovariable domains typically contain a single amino acid chain, which can be considered to contain four “framework sequences” or FRs and three “complementation-determining regions” or CDRs (as defined above). It should be clear that the frame regions of immunoglobulin monovariable domains may also contribute to antigen binding (Desmyter et al. 2002; Korotkov et al. 2009).

[0064] As further described herein, the total number of amino acid residues in the VHH, nanobody, or confobody may be in the range of 110-120, preferably 112-115, and most preferably 113. However, it should be noted that there are no particular limitations on the length and / or size of any portion, fragment, analog, or derivative of the VHH or nanobody (as further described herein), provided that such portion, fragment, analog, or derivative meets the further requirements outlined herein and is also preferably suitable for the purposes described herein.

[0065] In this application, amino acid residues / positions in the variable domains of the immunoglobulin heavy chain will be indicated by Kabat numbering (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91), as in the article Riechmann and Muyldermans, J. Immunol. Methods 2000 June 23; 240 (1-2): 185-195 (see, for example, the article in that publication). Figure 2 This applies to the VHH domain of camels. For example, see also international application WO2108 / 134235. Figure 1 It provides a table listing some amino acid positions in VHH and their numbers according to several alternative numbering systems (e.g., Aho and IMGT. Note: Unless otherwise expressly stated, for the purposes of this specification and claims, the Kabat number is definitive for the amino acid residues / positions in VHH, nanobodies, or ConfoBody; other numbering systems are for reference only).

[0066] Regarding CDRs, as is well known in the art, various rules exist for defining and describing CDRs of VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the location of structural loop regions). See, for example, http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and claims, while CDRs according to Kabat may also be mentioned, CDRs are most preferably defined based on the Abm definition (which is based on the AbM antibody modeling software from OxfordMolecular), as this is considered the optimal compromise between the Kabat and Chothia definitions. See again http: / / www.bioinf.org.uk / abs / .

[0067] Therefore, in this specification and claims, unless otherwise expressly stated herein, all CDRs or VHHs, nanobodies or confobodies are defined in accordance with the Abm rules.

[0068] It should be noted that the immunoglobulin monovariable domain (ISVD) acting as a binding agent is not limited in its broadest sense to a specific biological source or a specific preparation method. The term "immunoglobulin monovariable domain" or "ISVD" encompasses variable domains from various sources, including mouse, rat, rabbit, donkey, human, shark, and camelid variable domains. According to a specific embodiment, the immunoglobulin ISVD is derived from shark antibodies (so-called immunoglobulin neoantigen receptors or IgNARs), more specifically from naturally occurring heavy-chain shark antibodies (without light chains), and is referred to as a VNAR domain sequence. Preferably, the immunoglobulin ISVD is derived from camelid antibodies. More preferably, the immunoglobulin ISVD is derived from naturally occurring heavy-chain camelid antibodies (without light chains), and is referred to as a VHH domain sequence or nanobody.

[0069] According to a particularly preferred embodiment, the binding agent of the present invention is an immunoglobulin monovariable domain, which is a nanobody (as further defined herein, and including but not limited to VHH). As used herein, the term "nanobody" (Nb) is a single-domain antigen-binding fragment. It specifically refers to a monovariable domain derived from naturally occurring heavy-chain antibodies and is known to those skilled in the art. Nanobodies are generally derived from heavy-chain-only antibodies (without light chains) found in camelids (Hamers-Casterman et al., 1993; Desmyter et al., 1996), and are therefore commonly referred to as VHH antibodies or VHH sequences. Camelids include Old World camelids (Bactrian camel (Camelus bactrianus) and Dromedary camel (Camelus dromedarius)) and New World camelids (e.g., alpaca (Lamapaccos), llama (Lama glama), lama guanicoe (Lama guanicoe), and llama (Lama vicugna)). Nanobody® and Nanobodies® are registered trademarks of Ablynx NV (Belgium).For a further description of VHHs or nanobodies, please refer to the book “Single-Domain Antibodies,” Methods in Molecular Biology, edited by Saerens and Muyldermans, 2012, Vol. 911, with particular the chapter by Vincke and Muyldermans (2012), and a non-limiting list of patent applications mentioned as general background art, including: Vrije Universiteit Brussel’s WO 94 / 04678, WO 95 / 04079, WO 96 / 34103; Unilever’s WO 94 / 25591, WO 99 / 37681, WO00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1 134 231 and WO 02 / 48193; Vlaams Institute for Biotechnologie (VIB) WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; Ablynx NV 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 / 122825, and further published patent applications by Ablynx N.V. As is known to those skilled in the art, nanobodies are characterized in particular by the presence of one or more camelid "signature residues" in one or more framework sequences (according to Kabat numbering), such as those described in Table A-3 on page 75 of WO 08 / 020079 (incorporated herein by reference). It should be noted that the nanobodies of the present invention are not limited in their broadest sense to specific biological sources or specific methods of preparation.For example, nanobodies can typically be obtained by: (i) isolating the VHH domain of a naturally occurring heavy chain antibody; (ii) expressing a nucleotide sequence encoding a naturally occurring VHH domain; (iii) “humanizing” a naturally occurring VHH domain or expressing a nucleic acid encoding such a humanized VHH domain; (iv) “camelizing” a naturally occurring VH domain from any animal species, particularly from mammalian species such as humans, or expressing a nucleic acid encoding such a camelized VH domain; (v) “camelizing” a “domain antibody” or “Dab” as described in the art, or expressing a nucleic acid encoding such a camelized VH domain; (vi) using synthetic or semi-synthetic techniques for preparing proteins, peptides, or other amino acid sequences known per se; (vii) preparing a nucleic acid encoding a nanobodies by using nucleic acid synthesis techniques known per se, and then expressing the resulting nucleic acid; and / or (8) any combination of one or more of the above. Further descriptions of nanobodies, including humanization and / or camelification, can be found in, for example, WO08 / 101985 and WO08 / 142164, and further described herein. A specific class of nanobodies that bind conformational epitopes of natural targets is called Xaperones, and is specifically conceived here. Xaperone™ is a trademark of VIB and VUB (Belgium). Xaperone™ is a camel-family single-domain antibody that can restrict drug targets to unique, disease-associated, druggable conformations.

[0070] Within the scope of this invention, the term "immunoglobulin single variable domain" also encompasses "humanized" or "camelized" variable domains, particularly "humanized" or "camelized" nanobodies. For example, "humanization" and "camelization" can be achieved by providing nucleotide sequences encoding either a naturally occurring VHH domain or a VH domain, and then altering one or more codons in the nucleotide sequences in a manner known per se, wherein the altered codons are performed in such a way that the new nucleotide sequences encode either the "humanized" or "camelized" immunoglobulin single variable domain of this invention. This nucleic acid can then be expressed in a manner known per se to provide the desired immunoglobulin single variable domain of this invention. Alternatively, the amino acid sequences of the desired humanized or camelized immunoglobulin single variable domain of this invention can be designed based on the amino acid sequences of either the naturally occurring VHH domain or the VH domain, and then synthesized de novo using techniques known per se for peptide synthesis. Furthermore, based on naturally occurring VHH domains or the amino acid or nucleotide sequences of VH domains, nucleotide sequences encoding the desired humanized or camelified immunoglobulin single variable domains of the present invention can be designed, then synthesized de novo using techniques known per se for nucleic acid synthesis, and then the resulting nucleic acid can be expressed in a manner known per se to provide the desired immunoglobulin single variable domain of the present invention. Other suitable methods and techniques for obtaining the immunoglobulin single variable domains of the present invention and / or the nucleic acids encoding them from naturally occurring VH sequences or preferably VHH sequences are apparent to those skilled in the art, and may, for example, include combining one or more portions of one or more naturally occurring VH sequences (e.g., one or more FR sequences and / or CDR sequences), one or more portions of one or more naturally occurring VHH sequences (e.g., one or more FR sequences or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in a suitable manner to provide the nanobodies of the present invention or the nucleotide sequences or nucleic acids encoding them.

[0071] According to specific embodiments of the invention, the receptor-stabilizing binder can bind at either an ortho- or allosteric site. In other specific embodiments, the receptor-stabilizing binder can be an active conformation-selective binder or an inactive conformation-selective binder, by binding at either an ortho- or allosteric site. Generally, a conformation-selective binder that stabilizes the receptor's active conformation will increase or enhance the receptor's affinity for active conformation-selective ligands (e.g., agonists, more specifically, full agonists, partial agonists, or biased agonists) compared to a receptor without a binder (or in the presence of a mimic binder). Furthermore, a binder that stabilizes the receptor's active conformation will decrease the receptor's affinity for inactive conformation-selective ligands (e.g., inverse agonists) compared to a receptor without a binder (or in the presence of a mimic binder). Conversely, compared to receptors without a binder (or with a mimic binder), binders stabilizing the receptor's inactive conformation will enhance the receptor's affinity for inverse agonists and decrease the receptor's affinity for agonists (partial, complete, partial, or biased agonists). The increase or decrease in affinity for the ligand can be directly measured and / or calculated from the decrease or increase in EC50, IC50, Kd, ​​K, or any other measure of affinity or potency known to those skilled in the art. Particularly preferred are binders stabilizing the receptor's specific conformation capable of increasing or decreasing the affinity for conformationally selective ligands 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 more, upon binding to the receptor. It should be understood that the affinity measurement of conformationally selective ligands that trigger / inhibit specific signal transduction pathways can be performed using the following substances: any type of ligand, including natural ligands, small molecules and bioproducts; normal ligands and allosteric modulators; single compounds and compound libraries; lead compounds or fragments; etc.

[0072] As used herein, the term "affinity" refers to the degree to which a ligand (as further defined herein) binds to a target protein, shifting the equilibrium between the target protein and the ligand toward the presence of a complex formed through their binding. Thus, for example, when a GPCR and ligand are combined at relatively equal concentrations, a high-affinity ligand will bind to the available antigen on the GPCR, shifting the equilibrium toward a high concentration of the resulting complex. The dissociation constant is typically used to describe the affinity between the ligand and the target protein. Typically, the dissociation constant is below 10. -5 M. Preferably, the dissociation constant is less than 10. -6 M, more preferably less than 10 -7 M. Most preferably, the dissociation constant is less than 10. -8M. Other ways to describe the affinity between a ligand (including small molecule ligands) and its target protein are by using a binding constant (Ka), an inhibition constant (Ki) (also known as an inhibitory constant), or by indirectly assessing the efficacy of the ligand by measuring the half-maximal inhibitory concentration (IC50) or the half-maximal effective concentration (EC50). Within the scope of this invention, the ligand can be a binding agent that binds to a conformational epitope on a GPCR, preferably an immunoglobulin, such as an antibody, or an immunoglobulin fragment, such as VHH or a nanobody. It should be understood that within the scope of this invention, the term "affinity" is used in the context of binding agents that bind to conformational epitopes of a target GPCR, particularly immunoglobulins or immunoglobulin fragments such as VHH or nanobodies, and in the context of test compounds that bind to a target GPCR (more particularly to ortho- or allosteric sites of the target GPCR) (as further defined herein).

[0073] As used in this article, the term "specificity" refers to the ability of a protein or other binding agent, particularly an immunoglobulin or immunoglobulin fragment such as VHH or nanobodies, to preferentially bind to one antigen relative to different antigens, and does not necessarily imply high affinity.

[0074] As used herein, the term "specifically bind" generally refers to the ability of a binder, particularly an immunoglobulin such as an antibody, or an immunoglobulin fragment such as a VHH or nanobody, to preferentially bind to a specific antigen present in a homogeneous mixture of different antigens. In some embodiments, specific binding interactions will distinguish between desired and undesired antigens in a sample, exceeding by about 10 to 100 times or more (e.g., exceeding about 1000 times or 10,000 times) in some embodiments. In the context of a conformational profile of GPCRs, the term specifically refers to the ability of a binder (as defined herein) to preferentially recognize and / or bind to a specific conformational state of a GPCR compared to another conformational state.

[0075] Furthermore, it should be understood that in this specification and the appended claims, a protein, ligand, compound, binding domain, binding unit, or other chemical entity is referred to as "binding" to another protein, ligand, compound, binding domain, binding unit, or other chemical entity, epitope, or binding site, and such binding is preferably "specific" binding as defined herein. Moreover, preferably, such binding is "selective binding" as defined herein.

[0076] As used herein, the term "conformation-selective binder" in the context of this invention refers to a binder that binds to a target protein in a conformation-selective manner. A binder that selectively binds to a specific conformation or conformational state of a protein refers to a binder that binds to proteins in a subset of conformations or conformational states with a higher affinity than other conformations or conformational states that the protein may assume. Those skilled in the art will recognize that a binder that selectively binds to a specific conformation or conformational state of a protein will stabilize or retain the protein in that specific conformation or conformational state. For example, an active conformation-selective binder will preferentially bind to GPCRs in their active conformational state and will not bind, or to a lesser extent, to GPCRs in their inactive conformational state, and will therefore have a higher affinity for said active conformational state; or vice versa. The terms "specific binding," "selective binding," "preferential binding," and their grammatical equivalents are used interchangeably herein. The terms "conformation specificity" or "conformation selectivity" are also used interchangeably herein.

[0077] As used herein, the term “stable” or its grammatically equivalent as defined above refers to the increased stability of a protein (as described herein) or receptor (also as described herein) in terms of structure (e.g., conformational state) and / or specific biological activities (e.g., intracellular signaling activity, ligand-binding affinity, etc.). This increased stability in terms of structure and / or biological activity can be readily determined by functional assays of activity (e.g., Ca2+ release, cAMP generation or transcriptional activity, β-repressor protein recruitment, etc.) or ligand binding, or by physical methods such as X-ray crystallography, NMR, or spin labeling. The term “stable” also includes an increase in the thermal stability of the receptor under non-physiological conditions induced by denaturants or denaturing conditions. As used herein, the terms “thermostabilize” or “thermostabilizing” refer to the functional, rather than thermodynamic, properties of the receptor and the protein’s resistance to irreversible denaturation induced by thermal and / or chemical methods (including, but not limited to, heating, cooling, freezing, chemical denaturants, pH, detergents, salts, additives, proteases, or temperature). Irreversible denaturation leads to the irreversible unfolding of the functional conformation of a protein, loss of biological activity, and aggregation of denatured proteins. Regarding increased thermal stability, this can be easily determined by measuring ligand binding or by using spectroscopic methods (such as fluorescence, CD, or light scattering), which are sensitive to unfolding at elevated temperatures. Preferably, the binder is capable of increasing stability, as measured by an increase in the thermal stability of the protein or receptor in its functional conformational state at at least 2°C, at least 5°C, at least 8°C, more preferably at least 10°C, or 15°C, or 20°C. Regarding increased stability to detergents or liquid release agents, the protein or receptor is typically incubated for a specified time in the presence of the test detergent or liquid release agent, and stability is optionally determined at elevated temperatures as described above using, for example, ligand binding or spectroscopic methods. Alternatively, the binder is capable of increasing the stability of the functional conformational state of the protein or receptor to extreme pH values. Regarding extreme pH values, typical test pH values ​​will be selected within, for example, the range of 6 to 8, 5.5 to 8.5, 5 to 9, 4.5 to 9.5, more specifically the range of 4.5 to 5.5 (low pH) or 8.5 to 9.5 (high pH). As used herein, the terms “thermo-stabilize” or “thermo-stabilizing” apply to proteins or receptors embedded in lipid particles or lipid layers (e.g., lipid monolayers, lipid bilayers, etc.) and proteins or receptors dissolved in detergents.

[0078] In addition to the above, regarding the functional conformational state of GPCRs, the term "stabilizing" or "stabilized" refers to the retention or maintenance of the GPCR protein within a subset of possible conformations that may be present due to the interaction between the GPCR and the binding agent according to the invention. In this context, a binding agent that selectively binds to a specific conformation or conformational state of a protein refers to a binding agent that binds to a protein in a subset of conformations or conformations with a higher affinity than other conformations or conformations that the protein may assume. Those skilled in the art will recognize that a binding agent that specifically or selectively binds to a specific conformation or conformational state of a protein will stabilize that specific conformation or conformational state and its associated activity. Further details are provided herein.

[0079] As used herein, the terms “compound,” “test compound,” “candidate compound,” or “drug candidate compound” describe any naturally occurring or synthetic molecule tested in an assay (e.g., a screening assay or a drug discovery assay). Therefore, these compounds include both organic and inorganic compounds. These compounds include polynucleotides, lipids, or hormone analogs characterized by low molecular weight. Other biopolymeric organic test compounds include small peptides or peptide-like molecules (peptide mimics) containing about 2 to about 40 amino acids, and larger polypeptides containing about 40 to about 500 amino acids, such as antibodies, antibody fragments, or antibody conjugates. Test compounds can also be protein scaffolds. For high-throughput purposes, test compound libraries, such as combined or randomized libraries providing a sufficiently diverse range, can be used. Examples include, but are not limited to, natural compound libraries, allosteric compound libraries, peptide libraries, antibody fragment libraries, synthetic compound libraries, fragment-based libraries, phage display libraries, etc. Further detailed descriptions can be found in the specification.

[0080] As used herein, the term "ligand" refers to a molecule that specifically binds to a protein (e.g., a GPCR) as referred to herein. Ligands can be, but are not limited to, polypeptides, lipids, small molecules, antibodies, antibody fragments, nucleic acids, and carbohydrates. Ligands can be synthetic or naturally occurring. Ligands also include "natural ligands," which are ligands that are endogenous natural ligands of natural GPCRs. In the context of this invention, when the protein is a transmembrane protein such as a GPCR, the ligand can bind to the protein at a ligand-binding site exposed to the intracellular environment when the protein is in its native cellular environment (i.e., the ligand can be an "intracellular ligand"), or when the protein is in its native cellular environment, the ligand can bind to the protein at a ligand-binding site exposed to the extracellular environment (i.e., the ligand can be an "extracellular ligand"). Ligands can be agonists, partial agonists, inverse agonists, antagonists, or allosteric modulators, and can bind at either an ortho- or allosteric site. In certain implementations, the ligand may be a “conformation-selective ligand” or a “conformation-specific ligand,” meaning that such ligands bind to proteins or GPCRs in a conformation-selective manner. As further described herein, conformation-selective ligands bind to a specific conformation of a protein with higher affinity than other conformations the protein may adopt. For illustrative purposes, agonists are examples of active conformation-selective ligands, while inverse agonists are examples of inactive conformation-selective ligands. For clarity, neutral antagonists are not considered conformation-selective ligands because they do not distinguish between different conformations of GPCRs.

[0081] As used herein, “orthogonal ligand” refers to ligands (natural and synthetic) that bind to the active site of a GPCR, and is further classified according to their potency, or in other words, their role in signal transduction via 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 maximally stimulates the protein; even at saturation concentrations, partial agonists do not induce full activity. Partial agonists can also act as “blockers” by preventing the binding of more robust agonists. An “antagonist,” also known as a “neutral antagonist,” is a ligand that binds to the receptor without stimulating any activity. Antagonists are also called “blockers” because they prevent the binding of other ligands, thereby blocking agonist-induced activity. Furthermore, an “inverse agonist” is an antagonist that, in addition to blocking agonist action, reduces the receptor’s basal or constitutive activity to below that of unliganded proteins.

[0082] The ligands used in this paper can also be “biased ligands” that have the ability to selectively stimulate a subset of receptor signaling activity, such as selectively activating G-proteins or β-repressor proteins in the case of GPCRs. Such ligands are called “biased ligands,” “biased agonists,” or “functionally selective agonists.” More specifically, ligand bias can be imperfect bias, characterized by ligand stimulation of multiple receptor activities with different relative potencies (non-absolute selectivity) for different signals; or it can be perfect bias, characterized by ligand stimulation of one receptor protein activity without any stimulation of another known receptor protein activity.

[0083] Another type of ligand is called an allosteric regulator. As used herein, “allosteric regulator” or other “allosteric modulator,” “allosteric ligand,” or “effect molecule” refers to a ligand that binds to the allosteric site of a GPCR (i.e., a regulatory site that is physically different from the active site of the protein). Unlike ortho-allosteric ligands, allosteric regulators are non-competitive because they bind to receptor proteins at different sites and alter their function, even when endogenous ligands are also binding. Allosteric regulators that enhance protein activity are referred to herein as “allosteric activators” or “positive allosteric regulators” (PAMs), while allosteric regulators that reduce protein activity are referred to herein as “allosteric inhibitors” or other “negative allosteric regulators” (NAMs).

[0084] As used herein, the terms “determine,” “measure,” “evaluate,” and “determine” are used interchangeably and include both quantitative and qualitative determinations.

[0085] The term "antibody" is intended to refer to an immunoglobulin or any fragment thereof capable of binding to an antigen. The term "antibody" also refers to single-chain antibodies and antibodies having only one binding domain.

[0086] As used herein, the term "complementarity-determining region" or "CDR" in the context of antibodies refers to the variable region (also abbreviated as VH and VL, respectively) of the H (heavy) or L (light) chain and contains an amino acid sequence capable of specifically binding to an antigenic target. These CDR regions explain the fundamental specificity of an antibody for a particular antigenic determinant structure. Such regions are also referred to as "hypervariates." CDRs represent non-adjacent amino acid segments within a variable region, but regardless of species, the positions of these key amino acid sequences within the variable heavy and light chain regions have been found to be similar in position to those in the amino acid sequence of the variable chain. All classic antibodies have three CDR regions in both the variable heavy and light chains, each region being non-adjacent to the other for the corresponding light (L) and heavy (H) chains (designated L1, L2, L3, HI, H2, H3). Immunoglobulin monovariable domains, particularly nanobodies, typically contain a single amino acid chain that can be considered to contain four "frame sequences or regions" or FRs and three "complementarity-determining regions" or CDRs. The nanobody has three CDR regions, each of which is not adjacent to the other regions (designated CDR1, CDR2, and CDR3). As described herein, the Kabat numbering system will be followed to denote the amino acid position / residue CDRs in VHH, nanobody, or ConfoBody, and the frame and CDRs are defined based on Abm definitions (unless otherwise explicitly stated).

[0087] Generally, for the purposes of this disclosure and its appended claims, the compounds of the present invention will be considered “modifiers” of the target, or “modifiers” of the target (and / or signal transduction, pathway, mechanism of action and / or the biological, physiological and / or pharmacological functions relating to the target), when the presence of the compound in a suitable assay or model (i.e., at a suitable amount or concentration, such as a biologically active amount or concentration) alters the suitable or expected reading of the assay or model (i.e., at least a suitable value or parameter that can be determined using the assay or model) by at least 0.1%, such as at least 1%, such as at least 10% and up to 50% or more. Again, the modulation can result in an increase or decrease of the value or parameter (i.e., an increase or decrease of the percentage given in the preceding sentence). Furthermore, the compounds of the present invention are preferably capable of modulating the target, signal transduction, pathway, mechanism of action and / or the biological, physiological and / or pharmacological functions in a dose-dependent manner, i.e., within or beyond at least one concentration range of the compound used in the assay or model.

[0088] The method of the present invention is generally carried out using an apparatus comprising at least the following elements (all as further defined herein):

[0089] A boundary layer separating the first and second environments;

[0090] Translayer proteins;

[0091] The first ligand of a translayer protein present in the first environment (as defined herein);

[0092] Second ligands of translayer proteins present in (as defined herein) the second environment; and

[0093] A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0094] The elements are arranged relative to each other in a manner further described herein (and, where applicable, operatively connected and / or associated with each other).

[0095] In particular, in the apparatus 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” (also as further described herein, and different from the first fusion protein), and such first fusion protein, such second fusion protein (in its various forms as described herein), nucleotide sequences and / or nucleic acids encoding them, and cells, cell lines or other host cells or host organisms that express (and particularly suitably express, as described herein) or are (suitably) able to express the first and / or second fusion proteins (and preferably both) form a further aspect of the present invention.

[0096] In particular, the apparatus for carrying out the method of the present invention may include at least the following elements:

[0097] - A boundary layer separating the first and second environments;

[0098] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0099] - A translayer protein that is appropriately fused or connected (directly or through a suitable linker or spacer) to one of the binding members of the binding pair (i.e., forming a first fusion protein);

[0100] - The first ligand of the translayer protein present in the first environment; and

[0101] - The second ligand of the translayer protein present in the second environment;

[0102] The elements are arranged relative to each other in a manner further described herein (and, where applicable, operatively connected and / or associated with each other). In particular, the second member of the binding pair may be part of a second fusion protein (which is distinct from the first fusion protein comprising the translayer protein and the first binding member of the binding pair), which is further described herein.

[0103] More specifically, the apparatus for carrying out the method of the present invention may include at least the following elements:

[0104] - A boundary layer separating the first and second environments;

[0105] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0106] - A first fusion protein comprising a translayer protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in the second environment);

[0107] - A second fusion protein comprising a protein capable of directly or indirectly binding to a translayer protein and another binding member of the binding pair, the second fusion protein being present in a second environment; and

[0108] -The first ligand of the translayer protein present in the first environment;

[0109] The elements are arranged relative to each other in a manner further described herein (and, where applicable, operatively connected and / or associated with each other).

[0110] It should be noted that in this specification and claims, when it is said that a ligand, binding domain, binding unit, or other compound or protein is “capable of binding” to another protein or compound, such binding is most preferably “specific binding” as further defined herein. Furthermore, 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 other 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 appropriately connected to each other, either directly or through suitable spacers or linkers.

[0111] For the purposes of this specification and claims, a protein (e.g., binding domain, binding unit, or ligand) is referred to as "directly or indirectly" bound to a translayer protein if: (i) the protein itself binds to (and / or is able to bind to) the translayer protein (e.g., binds to an epitope or binding site on the translayer protein, as further described herein); or if (ii) the protein binds to (and / or is able to bind to) a ligand or protein, wherein the ligand or protein binds to (and / or is able to bind to) the translayer protein; or if (iii) the protein binds to (and / or is able to bind to) a protein complex, wherein the protein complex comprises a ligand or protein that binds to (and / or is able to bind to) the translayer protein. In the case of (i), the protein is referred to herein as "directly" bound to the translayer protein, while in the cases of (ii) and (iii), the protein is referred to herein as "indirectly" bound to the translayer protein. Furthermore, when a protein binds to a protein complex comprising a ligand or protein that binds to a translayer protein, the protein may bind to the ligand or protein or to any other portion, epitope, or binding site of the complex.

[0112] Therefore, in one aspect of the invention, the protein binding the translayer protein is selected from (i) binding domains, binding units, or other proteins that bind (and / or are capable of binding) epitopes or binding sites on the translayer protein; (ii) binding domains, binding units, or other proteins that bind (and / or are capable of binding) ligands or proteins that bind (and / or are capable of binding) the translayer protein; and (iii) binding domains, binding units, or other proteins that bind (and / or are capable of binding) protein complexes comprising ligands or proteins that bind (and / or are capable of binding) the translayer protein. In each such case, the binding domain, binding unit, or other protein is preferably as further described herein.

[0113] Specifically, the protein that directly or indirectly binds to the translayer protein may be selected from (i) ISVDs that bind (and / or are able to bind) epitopes or binding sites on the translayer protein; (ii) ISVDs that bind (and / or are able to bind) ligands or proteins that bind (and / or are able to bind) the translayer protein; and (iii) ISVDs that bind (and / or are able to bind) protein complexes containing ligands or proteins that bind (and / or are able to bind) the translayer protein. Again, in each such case, such ISVDs are preferably as further described herein.

[0114] In a further aspect of the invention, the apparatus for performing the method of the invention may include at least the following elements:

[0115] - A boundary layer separating the first and second environments;

[0116] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0117] - A first fusion protein comprising a translayer protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in the second environment);

[0118] - A second fusion protein comprising a protein capable of directly binding (as defined herein) to a translayer protein and another binding member of the binding pair, the second fusion protein being present in a second environment; and

[0119] -The first ligand of the translayer protein present in the first environment;

[0120] The elements are arranged relative to each other in a manner further described herein (and operatively connected and / or associated with each other where applicable). In this aspect of the invention, the protein that can directly bind (as defined herein) to a translayer protein and is present in the second fusion protein is preferably a binding domain or binding unit, and more preferably an immunoglobulin monovariable domain. It should also be understood that, in this aspect of the invention, the protein that can directly bind (as defined herein) to a translayer protein and is present in the second fusion protein serves as a second ligand.

[0121] In another aspect of the present invention, an apparatus for performing the method of the present invention may include at least the following elements:

[0122] - A boundary layer separating the first and second environments;

[0123] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0124] - A first fusion protein comprising a translayer protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in the second environment);

[0125] -The first ligand of the translayer protein present in the first environment;

[0126] - A second ligand for a translayer protein, which may optionally be part of a protein complex;

[0127] - A second fusion protein comprising a protein that can indirectly bind (as defined herein) a translayer protein and another binding member of the binding pair, the second fusion protein being present in a second environment;

[0128] The elements are arranged relative to each other in a manner further described herein (and operatively connected and / or associated with each other where applicable). In this aspect of the invention, the second ligand can be any suitable ligand (as further described herein), and the protein capable of indirectly binding (as defined herein) to a translayer protein and present in the second fusion protein is preferably a binding domain or binding unit, and more preferably an immunoglobulin monovariable 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.

[0129] It should be noted that, as further described herein, in the practice of this invention, the first ligand will typically be added to other elements of the already formed / built device of the invention as described herein, and thus the device of the invention in the absence of the first ligand (i.e. before the addition of the first ligand) forms other aspects of the invention (such as the method of adding the first ligand to the device of the invention in which the first ligand is absent or not yet present).

[0130] In this specification and claims, the term "second ligand" is used to mean a ligand, binding domain, binding unit or other chemical entity that, in the methods and apparatus described herein, directly binds to or is capable of directly binding to a translayer protein (or forms part of a protein complex that directly binds to or is capable of directly binding to a translayer protein).

[0131] As will become clear from the further description herein, the second ligand may be part of the second fusion protein, or it may be separate from the second fusion protein. In either case (i.e., whether or not the second ligand is part of the second fusion protein), the second ligand is preferably capable of binding to a conformational epitope on the translayer protein (or it may form part of a protein complex that directly binds to the translayer protein or is capable of directly binding to the translayer protein). More preferably, the second ligand (and / or the protein complex containing the second ligand) is preferably one or more functional, active, and / or druggable conformations of the translayer protein that it specifically binds to, induces the formation and / or stabilizes one or more functional, active, and / or druggable conformations of the translayer protein (and / or causes a conformational equilibrium shift of the translayer protein toward one or more such conformations); and / or it induces the formation and / or stabilizes a complex of the translayer protein, the first ligand, and the second ligand.

[0132] When the second ligand is part of the second fusion protein, it can be any ligand, binding domain, binding unit, peptide, protein, or other chemical entity capable of directly binding to translayer proteins and suitably contained within the second fusion protein. Preferably, as further described herein, when it is part of the second fusion protein, the second ligand will be a suitable binding domain or binding unit, and in particular an immunoglobulin monovariable domain.

[0133] When the second ligand separates from the second fusion protein, it can be any ligand or protein that can directly bind to a translayer protein and / or form part of a protein complex that can bind to a translayer protein. For example, as further described herein, such a second ligand can be a naturally occurring ligand of a translayer protein (e.g., a naturally occurring G-protein, such as a G-protein naturally occurring in the cell or cell line used), a semi-synthetic or synthetic analog or derivative of such a naturally occurring ligand, or a direct homolog of such a naturally occurring ligand (as described herein, a “chimeric” G-protein). Furthermore, when the second ligand is not part of the second fusion protein, the second fusion protein will contain a binding domain or binding unit that can indirectly bind (as defined herein) to a translayer protein, i.e., a binding domain or binding unit that binds to the second ligand and / or binds to a protein complex containing the second ligand. Similarly, as further described herein, such binding domains or binding units can be, in particular, immunoglobulin monovariable domains, such as camelid-derived ISVDs (or they can contain one or more such immunoglobulin monovariable domains, such as two or three such immunoglobulin monovariable domains, which may be the same or different, as further described herein).

[0134] As further described herein, in one aspect of the invention, the apparatus of the invention may be present in suitable cells or cell lines and / or the methods of the invention may be performed using suitable cells or cell lines that suitably contain (operable) the apparatus of the invention.

[0135] Therefore, as further described herein, the invention also relates to cells or cell lines suitably comprising the means of the invention and / or suitably expressing (as defined herein) or being able to suitably express elements of the means of the invention in order to provide the means of the invention (especially the means of the invention operable in said cells or cell lines). The invention also relates to cells or cell lines comprising and / or suitably expressing (as defined herein) or being able to suitably express a first fusion protein as described herein. The invention also relates to cells or cell lines comprising and / or suitably expressing or being able to suitably express a second fusion protein as described herein. In another aspect, the invention relates to cells or cell lines comprising and / or suitably expressing or being able to suitably express the first fusion protein and the second fusion protein as described herein. In aspects and embodiments where 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.

[0136] As also described herein, in one aspect of the invention, the device of the invention may be present in suitable liposomes or vesicles and / or the method of the invention may be performed using suitable liposomes or vesicles containing (operable) the device of the invention.

[0137] Therefore, as further described herein, the invention also relates to liposomes or vesicles suitably comprising elements of the device of the invention, particularly for providing an invention operable in said liposomes or vesicles. The invention also relates to liposomes or vesicles comprising a first fusion protein as described herein. The invention also relates to liposomes or vesicles in cells or cell lines comprising a second fusion protein as described herein. In yet another aspect, the invention relates to liposomes or vesicles comprising a first fusion protein as described herein and a second fusion protein as described herein. In aspects and embodiments where the second ligand does not form part of the second fusion protein, such liposomes or vesicles may also contain a suitable second ligand.

[0138] Therefore, as further described herein and illustrated by the accompanying non-limiting drawings, and depending on whether the second ligand is part of the second fusion protein, the present invention contemplates at least three preferred embodiments of the method and apparatus of the invention.

[0139] In the first such preferred embodiment ( Figure 1 In the schematic illustration, the second binding member of the binding pair will be suitably fused or connected (directly or via a suitable connector or spacer) to the second ligand. According to this preferred embodiment, the apparatus for carrying out the method of the invention may therefore include at least the following elements:

[0140] - A boundary layer separating the first and second environments;

[0141] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0142] - A translayer protein that is appropriately fused or connected (directly or via a suitable linker or spacer) to one of the binding members of the binding pair;

[0143] - The first ligand of the translayer protein present in the first environment; and

[0144] - A second ligand of a translayer protein that exists in the second environment and is appropriately fused or linked (directly or via a suitable linker or spacer) to another binding member of the binding pair;

[0145] The elements are arranged relative to each other in a manner further described herein (and, where applicable, operatively connected and / or associated with each other).

[0146] In particular, as further described herein, such a device may include the following elements:

[0147] - Separate the boundary layer between the first and second environments;

[0148] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0149] - A first fusion protein comprising a translayer protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in the second environment);

[0150] - The first ligand of the translayer protein present in the first environment; and

[0151] -A second fusion protein comprising a second ligand of a translayer protein and another binding member of the binding pair, the second fusion protein being present in a second environment;

[0152] The elements are arranged relative to each other in a manner further described herein (and, where applicable, operatively connected and / or associated with each other).

[0153] Those skilled in the art will understand that, in this first embodiment, the "second ligand" will be a binding domain, binding unit, or other protein that directly binds to (and / or is capable of binding to) an epitope or binding site on a translayer protein. Similarly, the binding domain or binding unit is preferably an immunoglobulin monovariable domain as further described herein.

[0154] In the second such preferred embodiment (schematically shown) Figure 2 In the process of binding the second binding member of the pair, the second binding member will be appropriately fused or connected (directly or via a suitable linker or spacer) to a binding domain or binding unit that does not directly bind the translayer protein but binds a second ligand (which in turn can bind the translayer protein). According to this preferred embodiment, the apparatus for carrying out the method of the invention may therefore include at least the following elements:

[0155] - A boundary layer separating the first and second environments;

[0156] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0157] A first fusion protein comprising a translayer protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in a second environment);

[0158] -The first ligand of the translayer protein present in the first environment;

[0159] - The second ligand of the translayer protein present in the second environment; and

[0160] - A second fusion protein, which is present in a second environment and contains a binding domain or binding unit capable of binding a second ligand, the binding domain or binding unit being suitably fused or connected (directly or via a suitable linker or spacer) to another binding member of the binding pair;

[0161] The elements are arranged relative to each other in a manner further described herein (and, where applicable, operatively connected and / or associated with each other).

[0162] Those skilled in the art will appreciate that, in this second embodiment, the binding domain or binding unit present in the second fusion protein will bind "indirectly" to the translayer protein, i.e., by binding to a second ligand bound to the translayer protein. Similarly, the binding domain or binding unit is preferably (and / or preferably substantially composed of) an immunoglobulin monovariable domain as further described herein. The binding domain or binding unit may also comprise or substantially consist of two or more immunoglobulin monovariable domains (e.g., two or three immunoglobulin monovariable domains), each capable (specifically) binding to a second ligand (i.e., the same epitope or binding site on the second ligand or a different epitope / binding site on the second ligand), and they may be the same or different (as further described herein), and they are suitably linked or fused to each other and to another binding member of the binding pair (optionally via a suitable linker or spacer) to form a second fusion protein suitable for the present invention. For example, but not limited to, such a binding domain or binding unit may comprise two or three copies of ConfoBody CA4437 (SEQ ID NO:4 in WO2012 / 75643 and SEQ ID No:2 herein), which are suitably linked or fused to each other and to another binding member of the binding pair (optionally via a suitable linker or spacer) to form a second fusion protein suitable for the present invention. Furthermore, in this embodiment, the second ligand may be any suitable ligand for a translayer protein as further described herein. Again, such a “multivalent” binding domain comprising two or more ISVDs should most preferably ensure that its binding to the second ligand does not substantially interfere with the ability of the second ligand to bind to the translayer protein and / or form (or facilitate the formation) of a complex between the second ligand, the translayer protein, and the first ligand.

[0163] In the third preferred embodiment (schematically shown) Figure 3In the process of binding, the second binding member of the binding pair is suitably fused or connected (directly or via a suitable connector or spacer) to the binding domain or binding unit, wherein the binding domain or binding unit does not directly bind to the translayer protein, but rather binds to a protein complex containing at least a second ligand of the translayer protein (the protein complex may bind to or be bound by the translayer protein and / or contain the translayer protein). According to this preferred embodiment, the apparatus for performing the method of the invention may therefore include at least the following elements:

[0164] - A boundary layer separating the first and second environments;

[0165] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0166] - A first fusion protein comprising a translayer protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in the second environment);

[0167] -The first ligand of the translayer protein present in the first environment;

[0168] - A protein complex containing at least a second ligand of a translayer protein, the protein complex being present in the second environment; and

[0169] - A second fusion protein, which is present in a second environment, and contains a binding domain or binding unit capable of binding a protein complex, the binding domain or binding unit being appropriately fused or connected (directly or via a suitable linker or spacer) to another binding member of the binding pair;

[0170] The elements are arranged relative to each other in a manner further described herein (and, where applicable, operatively connected and / or associated with each other).

[0171] Those skilled in the art will understand that, in this third embodiment, the binding domain or binding unit present in the second fusion protein will bind "indirectly" to the translayer protein, i.e., by binding to a protein complex containing the second ligand. Similarly, the binding domain or binding unit is preferably an immunoglobulin monovariable domain as further described herein, and the second ligand can be any suitable ligand for the translayer protein, which may be part of a protein complex further described herein.

[0172] Furthermore, the binding domain or binding unit in the second fusion protein may comprise two or more immunoglobulin monovariable domains, each capable of binding to different epitopes, portions, domains, or subunits on / within the protein complex, such as two different epitopes on a G protein complex. For example, but not limited to, when the protein complex is a heterotrimeric G protein, the binding domain or binding unit may comprise two or three different ISVDs, wherein each ISVD is 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-α subunit present in the heterotrimeric G-protein). Specific, but non-limiting, examples of such binding domains or binding units may include, for example, ConfoBody CA4435 (SEQ ID NO:1 in WO2012 / 75643 and SEQ ID NO:1 herein) and CA4437 (SEQ ID NO:4 in WO2012 / 75643 and SEQ ID NO:2 herein), which are suitably linked or fused to each other and to another binding member of the binding pair (optionally via a suitable linker or spacer) to form a second fusion protein suitable for the present invention.

[0173] Using such “multivalent” binding domains or binding units (i.e., containing two or more ISVDs) in the second fusion protein, compared to using the corresponding ISVD in a monovalent format (i.e., containing only one of the ISVDs), can also lead to improved sensitivity in the assays described herein.

[0174] More generally, apparatus for carrying out the methods of the invention in its various aspects and embodiments typically and preferably include at least the following elements:

[0175] - A boundary layer separating the first and second environments;

[0176] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0177] - A first fusion protein comprising a translayer protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in the second environment);

[0178] -The first ligand of the translayer protein present in the first environment;

[0179] - The second ligand of the translayer protein present in the second environment; and

[0180] - A second fusion protein containing another binding member of the binding pair (i.e., the other member of the binding pair is also present in the second environment);

[0181] The elements are arranged relative to each other in a manner further described herein (and, where applicable, operatively connected and / or associated with each other). In particular:

[0182] - In the first preferred embodiment described herein, the second fusion protein will include another binding member of the binding pair and a second ligand;

[0183] - In the second preferred embodiment described herein, the second fusion protein will include another binding member of the binding pair and a binding domain or binding unit capable of binding the second ligand; and

[0184] - In the third preferred embodiment described herein, the second fusion protein will include another binding member of the binding pair and a binding domain or binding unit capable of binding a protein complex containing at least the second ligand. Attached Figure Description

[0185] The invention will now be illustrated by further description herein, the following experimental section, and the accompanying non-limiting drawings. In the figures:

[0186] a) Figure 1 The first device of the present invention is schematically shown, wherein the second ligand (in) Figure 1 The middle part is represented as (4)) forming the second fusion protein (in Figure 1 In the illustrated implementation, it is part of a second member (7) of a second ligand (4), a linker (11), and a binding pair (6 / 7), and directly binds (as defined herein) to a translayer protein (2). Figure 1 In the settings shown:

[0187] - The boundary layer is represented as (1);

[0188] - The first environment is represented as [A];

[0189] - The second environment is represented by [B];

[0190] - Translayer proteins are represented as (2);

[0191] -The first ligand is represented as (3);

[0192] -The first binding site on the translayer protein (2) that is exposed to the first environment [A] and to which the first ligand (3) can bind is represented as (8);

[0193] -The second ligand is represented as (4);

[0194] - The second binding site on the translayer protein (2) that is exposed to the second environment [B] and can be bound by the second ligand (4) is represented as (9);

[0195] - A binding pair capable of generating a detectable signal is represented as (6 / 7) and consists of a first binding member (6) connected to a translayer protein (2) (directly or via a linker or spacer (10)) and a second binding member (7) connected to a second ligand (4) (directly or via a linker or spacer (11));

[0196] - The first fusion protein comprises a translayer protein (2) that is fused directly or via a linker (10) to the first binding member (6);

[0197] - The second fusion protein contains a second ligand (4) fused directly or via a linker (11) to a second binding member (7); and

[0198] - The arrangement of the first and second fusion proteins relative to each other and relative to the boundary layer (1) is such that when the second ligand (4) binds to the translayer protein (2) (i.e., directly through the binding site (9)), the first binding member (6) and the second binding member (7) can come into contact with or be close to each other (or otherwise appropriately correlated to generate a detectable signal). Figure 1 (The flashing symbol in the image represents this).

[0199] b) Figure 2 The second device of the present invention is schematically shown, wherein the second ligand (in) Figure 2 The middle is represented as (4)) and the second fusion protein (in Figure 2 The illustrated implementation consists of a second member (7) of a binding domain (5), a linker (11), and a binding pair (6 / 7), wherein the binding domain (5) present in the second fusion protein indirectly binds (as defined herein, and in Figure 2 In this case, it is transmitted to the translayer protein (2) via the second ligand (4). Figure 2 In the settings shown:

[0200] - The boundary layer is represented as (1);

[0201] - The first environment is represented as [A];

[0202] - The second environment is represented by [B];

[0203] - Translayer proteins are represented as (2);

[0204] -The first ligand is represented as (3);

[0205] -The first binding site on the translayer protein (2) that is exposed to the first environment [A] and to which the first ligand (3) can bind is represented as (8);

[0206] -The second ligand is represented as (4);

[0207] - The second binding site on the translayer protein (2) that is exposed to the second environment [B] and can be bound by the second ligand (4) is represented as (9);

[0208] -The binding domain or binding unit that can bind to the second ligand (4) is represented as (5);

[0209] - A binding pair capable of generating a detectable signal is represented as (6 / 7) and consists of a first binding member (6) connected to a translayer protein (2) (directly or via a linker or spacer (10)) and a second binding member (7) connected to a binding domain or binding unit (5) (directly or via a linker or spacer (11));

[0210] - The first fusion protein comprises a translayer protein (2) that is fused directly or via a linker (10) to the first binding member (6);

[0211] - The second fusion protein includes a binding domain (5) that is fused directly or via a linker (11) to the second binding member (7); and

[0212] - The arrangement of the first and second fusion proteins relative to each other and relative to the boundary layer (1) is such that when the binding domain (5) binds to the translayer protein (2) (i.e., indirectly by binding to the second ligand (4) (which in turn binds to the translayer protein (2) via the binding site (9)), the first binding member (6) and the second binding member (7) can come into contact with or be close to each other (or otherwise appropriately correlated) to generate a detectable signal (using Figure 2 (The flashing symbol in the image represents this).

[0213] c) Figure 3 The third device of the present invention is schematically shown, wherein the second ligand (in) Figure 3 (4) is separated from the second fusion protein and forms part of a protein complex (12), which consists of the second ligand (4) and one or more other proteins (in Figure 3 In the case of [specific example], for illustrative purposes, the complex is illustrated as comprising a second ligand (4) and two other proteins (4a) and (4b), see also [reference]. Figure 3 (Illustrations). In Figure 3 In the embodiment shown, the second ligand (4) again binds to the second fusion protein (which is in Figure 3 The illustrated implementation consists of a second member (7) of a binding domain (5), a linker (11), and a binding pair (6 / 7), which is separated and indirectly bound by the binding domain (5) present in the second fusion protein (as defined herein, and in Figure 3 In the case of protein complex (12) to translayer protein (2), in the case of protein complex (12) to translayer protein (2). Figure 3In the settings shown:

[0214] - The boundary layer is represented as (1);

[0215] - The first environment is represented as [A];

[0216] - The second environment is represented by [B];

[0217] - Translayer proteins are represented as (2);

[0218] -The first ligand is represented as (3);

[0219] -The first binding site on the translayer protein (2) that is exposed to the first environment [A] and to which the first ligand (3) can bind is represented as (8);

[0220] - The second ligand is represented as (4), and forms a complex (12) with one or more other proteins (for illustrative purposes). Figure 3 In the diagram, complex (12) is represented as a complex containing three protein / subunits, namely the second ligand (4) and two other subunits (4a) and (4b), see also Figure 3 (Illustrations in the text);

[0221] - The second binding site on the translayer protein (2) that is exposed to the second environment [B] and can be bound by complex (12)(4) is represented as (9);

[0222] -The binding structural domain or binding unit that can bind to the complex (12) is represented as (5);

[0223] - A binding pair capable of generating a detectable signal is represented as (6 / 7) and consists of a first binding member (6) connected to a translayer protein (2) (directly or via a linker or spacer (10)) and a second binding member (7) connected to a binding domain or binding unit (5) (directly or via a linker or spacer (11));

[0224] - The first fusion protein comprises a translayer protein (2) that is fused directly or via a linker (10) to the first binding member (6);

[0225] - The second fusion protein includes a binding domain (5) that is fused directly or via a linker (11) to the second binding member (7); and

[0226] - The arrangement of the first and second fusion proteins relative to each other and relative to the boundary layer (1) is such that when the binding domain (5) binds to the translayer protein (2) (i.e., indirectly by binding to the complex (12) (which in turn binds to the translayer protein (2) via the binding site (9)), the first binding member (6) and the second binding member (7) can come into contact with or be close to each other (or otherwise appropriately correlated) to generate a detectable signal (using Figure 3 (The flashing symbol in the image represents this).

[0227] d) Figure 4 This is a graph showing the dose-response curve of NDP-α-MSH obtained by MC4R screening assay using CA4437 described in Example 1;

[0228] e) Figures 5A to 5C It is a graph showing the dose-response curves of the specified compounds obtained by GLP-1R screening assay using CA4437 described in Example 2;

[0229] f) Figure 6 This is a graph showing the dose-response curves of GLP-1(7-36) amides obtained by GLP-1R screening assay using CA4435 as described in Example 2;

[0230] g) Figure 7 A and 7B are graphs showing the measurement results obtained by β-2-AR screening assay using CA4437 described in Example 3;

[0231] h) Figure 8 The diagrams A through 8E show the use of CA4435 as described in Example 3 ( Figure 8 A, 8B, 8D), CA4437 Figure 8 C) and CA4435-35GS-CA4437 fusion ( Figure 8 The results obtained from the β-2-AR screening assay of E);

[0232] i) Figure 9 and 10 It is a graph showing the dose-response curves of the specified compounds obtained using the MOR screening assay described in Example 4;

[0233] j) Figure 11 This is a graph showing the measurement results obtained using the M2R screening assay described in Example 5;

[0234] k) Figure 12 This is a graph showing the measurement results obtained using the β-2AR screening assay described in Example 6;

[0235] l) Figure 13It is a graph showing the dose-response curves of the specified compounds obtained using the AT1R screening assay described in Example 7;

[0236] m) Figure 14 This is a graph showing the measurement results obtained using the AT1R screening assay described in Example 7;

[0237] n) Figure 15 The results of the compound library screening performed in Example 8 are shown;

[0238] o) Figure 16 It is a graph showing the dose-response curves of the specified compounds obtained by the screening assay using the recombinant MC4R described in Example 9;

[0239] p) Figure 17 This is a graph showing the measurement results obtained using the recombinant MC4R screening assay described in Example 9;

[0240] q) Figures 18 to 22 It is a graph showing the dose-response curves of the specified compounds obtained by screening using the recombinant OX2R described in Example 10;

[0241] r) Figure 23A Figures B and C are graphs showing the results obtained using the two recombinant APJ receptor screening assays described in Example 11. Figure 23A The results show the effects obtained using recombinant apelin receptors in ICLs with μ-opioid receptors (MOR), and Figure 23B The results obtained using recombinant apelin receptors from ICLs with β-2AR receptors are shown.

[0242] s) Figures 24 to 27 The results of the compound library screening performed in Example 12 are shown.

[0243] t) Figure 28 This is a graph showing the screening results obtained in Example 14 for a set of 78 compound fragments when tested using two assays of the present invention (both using the β-2AR-LgBiT fusion, but one assay using the CA2780-SmBiT fusion and the other using the CA4435-35GS-CA4437-LgBiT fusion). Figure 28 In the figure, the x-axis represents the results obtained using the CA4435-35GS-CA4437-LgBiT fusion, the y-axis represents the results obtained using the CA2780-SmBiT fusion, and each point represents the result obtained for one of the 78 compounds tested in both assays.

[0244] u) Figure 29A and29B This is a graph showing the screening results for the set of compound fragments obtained in Example 15 when tested using radioligand assays and the corresponding assays of the present invention. Figure 29A In B, the x-axis represents the result obtained using the assay of the present invention, and the y-axis represents the result obtained using the radioligand assay in the assay. Each point represents the result obtained for a compound when tested in both the radioligand assay and the assay of the present invention.

[0245] v) Figure 30A and 30B The graph shows the test results (100 μM and 200 μM, respectively) of the compounds mentioned in Example 16 and Table 3 in the GloSensor cAMP assay of β-2AR;

[0246] w) Figure 31A and 31B This is a graph showing a comparison of the results obtained in Example 17 when the cell-based assays of the present invention are compared with comparable membrane-based assays of the present invention;

[0247] x) Figure 32A Figure C shows the dose-response curves of apelin obtained using different VHHs (Example 18).

[0248] y) Figure 33 The dose-response curves for iperoxo against the M2 receptor produced by the assay of the present invention in Example 19 are shown in the presence and absence of LY2119620 (an allosteric modulator of the M2 receptor).

[0249] z) Figure 34 The graph obtained in Example 20 compares the results from the OX2 assay of the present invention (using recombinant OX2 fusion) and the OX2 IP-One assay, where the x-axis represents the data obtained in the assay of the present invention, the y-axis represents the data obtained in the IP-One assay, and each point represents the result of a single compound.

[0250] aa) Figure 35A and 35B This is a graph obtained in Example 21 when the assay of the present invention was used to screen a large compound library for recombinant OX2 receptor. Figure 35A The results obtained when the compound was tested at 30 µM are shown, and Figure 35B The results obtained when the compounds were tested at 200 µM are shown, where the x-axis represents the ratio of the signal obtained by the tested compound (“sample”) to the signal given by the support solvent (“blank”), and each point represents the result obtained for a single compound.

[0251] Based on the accompanying drawings and further description herein, those skilled in the art will understand that certain elements of the apparatus of the present invention (e.g., boundary layers, translayer proteins, binding pairs, any linkers, and first ligands) will be present in various aspects and embodiments of the invention, as contemplated herein. Therefore, when a detailed description of any such element (including any preference for any such element) is given herein, it should be understood that such description applies to all aspects and embodiments of the invention in which such elements are present or used, unless expressly stated otherwise herein.

[0252] In the method and apparatus of the present invention, the boundary layer (1) may be any layer suitable for separating the first environment [A] from the second environment [B] (in a suitable in vitro system or a suitable in vivo system).

[0253] For example, in a preferred aspect of the invention, the method of the invention is carried out in a suitable cell or cell line (as further described herein), and the boundary layer (1) is the cell membrane or cell wall of the cell or cell line used in the method of the invention. In this respect, the environment [A] is preferably an extracellular environment, and the environment [B] is preferably an intracellular environment. Moreover, in this respect, the first ligand (3) is preferably present in the extracellular environment, and the second ligand (4) is preferably present in the intracellular environment. Furthermore, the first and second binding members (6) and (7) and the second fusion protein are also preferably present in the intracellular environment.

[0254] In another preferred aspect of the invention, the method of the invention is carried out in suitable vesicles or liposomes (as further described herein), the boundary layer (1) being the membrane or wall of the vesicle or liposome. In this respect, the environment [A] is preferably the environment outside the vesicle or liposome, and the environment [B] is preferably the environment inside the vesicle or liposome. Furthermore, in this respect, the first ligand (3) is preferably present in the environment outside the vesicle or liposome, and the second ligand (4) is preferably present in the environment inside the vesicle or liposome. In addition, the first and second binding members (6) and (7) and the second fusion protein are also preferably present in the environment inside the vesicle or liposome.

[0255] However, it should be understood that while the invention is carried out in some preferred aspects using cells, liposomes, or other suitable vesicles, the invention is not limited in its broadest sense to the use of cells or vesicles, but can be carried out in any other suitable apparatus in which a boundary layer (1) is used to suitably separate the first environment [A] from the second environment [B]. For example, the boundary layer may also be a portion 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 pressure and / or mechanical techniques known per se).

[0256] Therefore, the boundary layer (1) can be any suitable layer, wall, or membrane, particularly a biological wall or membrane (e.g., a cell wall or cell membrane, or a portion or fragment thereof) or the wall or membrane of a liposome or other suitable vesicle. In particular, the boundary layer (1) can be a suitable lipid bilayer, such as a phospholipid bilayer. When the boundary layer (1) is the wall or membrane of a vesicle or liposome, it can be monolayer or multilayer. Furthermore, as further described herein, when the boundary layer (1) is a cell membrane or cell wall, it is preferably the wall or membrane of a cell or cell line that suitably expresses (as defined herein) a translayer protein (2) and, in particular, suitably expresses the (first) fusion protein described herein comprising the translayer protein (2).

[0257] Non-restrictive Figure 1 , 2 As illustrated in Figure 3, the boundary layer (1) contains translayer proteins (2) that cross the boundary layer (1) such that:

[0258] - The first binding site (8) of the first ligand (3) extends (as defined herein) into the first environment [A] (i.e., such that the first binding site (8) is accessible for binding by the first ligand (3) when the first ligand is present in the first environment [A]);

[0259] And it also made

[0260] - The second binding site (9) of the second ligand (4) extends (as defined herein) into the second environment [B] (i.e., such that the binding of the second ligand (4) is accessible to the second binding site (9) when the second ligand is present in the second environment [B]).

[0261] In this specification and claims, the term "translayer protein" is used to refer to a protein used (e.g., for screening) in the methods and apparatus of the present invention. In the methods and apparatus of the present invention, the translayer protein (2) is provided and / or arranged relative to a boundary layer such that it spans a boundary layer (1) such that at least a portion of the amino acid sequence of the translayer protein (2) extends from the boundary layer (1) (as defined herein) into a first environment [A], and at least one other portion of the amino acid sequence of the translayer protein (2) extends from the boundary layer (1) (as defined herein) into a second environment [B]. In this case, when a portion of the amino acid sequence of the translayer protein (2) is claimed to "extend" from the boundary layer (1) into the environment (i.e., into the first environment [A] or the second environment [B]), this should generally be understood to mean that said portion of the sequence is exposed to said environment and / or is accessible for binding to ligands, compounds or other chemical entities present in said environment. Therefore, in the methods and apparatus of the present invention, at least a portion of the amino acid sequence of the translayer protein (e.g., an epitope or binding site) should be accessible for binding to ligands, compounds, or other chemical entities present in the first environment (especially the binding of the first ligand (3)), and at least one other portion of the amino acid sequence of the translayer protein (e.g., another epitope or binding site) should be accessible for binding to ligands, compounds, or other chemical entities present in the second environment (and particularly, the binding of the second ligand (4)). In this regard, it should also be noted that the phrase “accessible for binding” should generally be understood to mean that ligands, compounds, or other chemical entities present in the relevant environment can bind to binding pockets or binding sites on or within the translayer protein, even if the actual binding site or binding pocket is located deep (deeper) in the translayer protein structure (even such that the actual binding site or binding pocket is located within a portion of the translayer protein that itself does not physically extend beyond the boundary layer). For example, refer to Chevillard’s publication (cited herein), which shows that binding sites on GPCRs of fragments used in FBDD screening techniques may be located deep within the GPCR structure (see, for example, page 1120). Figure 2 The ligands are located on the surface of the GPCR, but are still accessible for fragment binding. This paper also references teachings on GPCR structure, GPCR signaling mechanisms, and GPCR ligand binding sites from other scientific references cited in this paper.

[0262] Furthermore, 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 referred to as "present" in an environment (i.e., in 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 to 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" (i.e., not bound or anchored to any other protein or structure) or may be anchored to a boundary layer or fused to another protein (which may be anchored to the boundary layer). Similarly, a binding domain or binding unit present in the environment may be part of a larger protein or structure (e.g., a fusion protein) that may be free-floating or anchored to a boundary layer or another structure in the environment, provided that the binding domain or binding unit is accessible for binding to another domain, ligand, protein, or compound present in the environment. Furthermore, epitopes or binding sites present in the environment can be part of a larger protein or structure that can again float freely in the environment or anchor to a boundary layer or another structure, provided that the epitope or binding site is accessible for binding to another domain, ligand, protein, or compound present in the environment.

[0263] One or more portions of the translayer protein (2) extending into the first environment [A] may be any loop, epitope (linear or conformational), binding site or other portion of the amino acid sequence of the translayer protein, and similarly, one or more portions of the translayer protein extending into the second environment [B] may also be any loop, epitope (linear or conformational), binding site or other portion of the amino acid sequence of the translayer protein (but different from the portion extending into the first environment).

[0264] In a preferred aspect of the invention, the translayer protein (2) comprises at least two distinct ligand binding sites, wherein at least one first binding site extends (as defined herein) into a first environment [A] (in particular, making it accessible for binding of a first ligand (3),) and wherein at least one second binding site extends (as defined herein) into a second environment [B] (in particular, making it accessible for binding of a second ligand (4).

[0265] Typically, the transmembrane protein (2) is usually attached to and / or anchored in the boundary layer (1), for example in a manner known to (trans)membrane proteins (which are anchored in the cell wall or cell membrane in their native environment). As further described herein, this can be achieved, for example, by appropriately expressing (as defined herein) the nucleotide sequence or nucleic acid expressing the first fusion protein in a suitable host cell, such that the transmembrane protein (2) becomes appropriately anchored in the cell wall or membrane. 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, such that the transmembrane protein (2) becomes appropriately anchored in the wall or membrane of the liposomes or vesicles.

[0266] Transmembrane proteins (2) may contain one or more domains (especially one or more transmembrane domains) and are typically and preferably transmembrane proteins, such as (transmembrane) receptors.

[0267] When the transmembrane protein (2) is a transmembrane protein, it can be a bitopic membrane protein (i.e., a transmembrane protein that crosses the membrane once) or a polytopic membrane protein (i.e., a transmembrane protein that crosses the membrane twice or more). Therefore, the transmembrane protein (2) can be any known or newly discovered transmembrane protein (or its synthetic or recombinant analogues) with known or unknown biological functions and known or unknown ligands (e.g., the transmembrane protein (2)) can be a so-called “orphan” GPCR).

[0268] The transmembrane protein (2) can be an α-helical protein or a β-barrel protein, and depending on the position of the N-terminus and C-terminus of the protein relative to the boundary layer, it can be a type I, type II, type III, or type IV transmembrane protein. Preferably, a transmembrane protein is a protein having an extracellular amino terminus and an intracellular carboxyl terminus in its native cellular environment, although the invention is not limited thereto in the broadest sense.

[0269] Furthermore, when the method of the present invention is carried out in a cell, the arrangement of the N-terminus and C-terminus of the protein relative to the wall or membrane of the cell used is preferably the same as the arrangement of the terminus in the protein's native cellular environment.

[0270] When the methods of the present invention are carried out in liposomes or vesicles, the liposomes or vesicles can be: a mixture of liposomes / vesicles in which the proteins are arranged in a manner substantially the same as the proteins in their native environment relative to the cell wall or cell membrane (i.e., the N-terminus and extracellular loop extend to the exterior of the vesicle, and the C-terminus and intracellular loop extend to the interior of the vesicle); and vesicles / liposomes in which the proteins are arranged in the opposite manner. Generally, this does not affect the performance of the systems or setups described herein.

[0271] As further described herein, generally and preferably, the translaminar protein (2) will be a protein that presents (i.e., can exhibit) 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 is capable of undergoing conformational changes (particularly functional conformational changes). Specifically, the translaminar protein (2) can be a protein that can exhibit at least one functional conformation and at least one non-functional conformation (e.g., a basal conformation) and / or undergo conformational changes from a non-functional to a functional conformation; and more particularly, it can be a protein that can exhibit an active (or highly active) conformation and an inactive (or less active) conformation and / or undergo conformational changes from an inactive (or less active) conformation to an active (or highly active) conformation. The translaminar protein (2) can also be a protein that can exhibit at least one ligand-bound (particularly agonist-bound) conformation and at least one ligand-free conformation. More specifically, the translayer protein (2) can be a protein that can present at least one ligand-binding (especially agonist-binding) conformation (which is either an active conformation or a functional conformation).

[0272] As described herein, specific classes of functional conformations of (transmembrane) proteins (e.g., certain GPCRs) are referred to as / defined as “drugable conformations.” Thus, in one specific aspect, a transmembrane protein (2) can be a protein capable of presenting at least one such drugable conformation (which is typically an active conformation, although the invention is not limited to use with drugable conformations that are active conformations) and at least one non-drugable conformation (typically an inactive conformation) and / or a transmembrane protein capable of undergoing a conformational change from a non-drugable conformation to a drugable conformation.

[0273] Specifically, the translayer protein (2) can be a protein that undergoes a conformational change upon binding of a ligand (especially an agonist). This conformational change upon ligand binding can be, for example, a change from an active conformation to an inactive conformation or from a functional conformation to a non-functional conformation, but is preferably a change from a non-functional conformation to a functional conformation and / or from an inactive conformation to an active conformation. In a particular aspect, it is a change from a non-drugable conformation to a druggable conformation.

[0274] For example, when the translayer protein (2) is a receptor such as a cell surface receptor (or its synthetic analogue), it can be a protein that undergoes a conformational change when a natural or synthetic (extracellular) ligand of the receptor binds to the receptor.

[0275] When the translayer protein (2) is a GPCR, the conformational change can, in a preferred but not limiting sense, be a change from a conformation that is essentially unable to bind G-proteins to a conformation that binds G-proteins (or can be bound by G-proteins).

[0276] As described herein, ligands that can induce conformational changes in translayer protein (2) from a nonfunctional state to a functional state (e.g., from an inactive state such as the basal state to the active state) are also referred to herein as “agonists” of translayer proteins. When translayer protein (2) is a GPCR, “agonists” are particularly capable of inducing conformational changes from a conformation that is essentially unable to bind G proteins to a conformation that binds G proteins.

[0277] In a preferred aspect of the invention, the translayer protein (2) is a protein that undergoes (or is capable of undergoing) a conformational change (as described herein) when bound to a first ligand (3), and conversely, the first ligand (3) is such that when it binds to the translayer protein (2), it can cause a conformational change in the translayer protein (2) (and / or the invention is used to identify such a first ligand). Similarly, in a more preferred aspect, the conformational change is from an inactive or less active state to a functional or (higher) active state, and the first ligand (3) used is such that when it binds to the translayer protein (2), it can cause a conformational change in the translayer protein from an inactive or less active state to a functional or (higher) active state. Furthermore, when the translayer protein (2) is a GPCR, in a preferred but non-limiting aspect, the conformational change after ligand binding can be a change from a conformation that is substantially unable to bind G proteins to a conformation that binds G proteins.

[0278] As further described herein, the translayer protein can be a protein capable of forming a complex with the first and second ligands. Specifically, the translayer protein can be a protein in its native environment capable of forming complexes with both intracellular and extracellular ligands. 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 native intracellular ligands of GPCRs), and such complexes are stabilized by the binding of the G protein to the intracellular conformational epitopes of the GPCR. Similarly, in this invention, the second ligand is preferably one that stabilizes the formation of the complex of the translayer protein, the first ligand, and the second ligand. For example, for this purpose, and as further described herein, when the translayer protein (2) is a GPCR, the second ligand can be a G-protein (i.e., one that transduces signals via the GPCR) associated with the GPCR in its native environment, another natively present G protein capable of binding to the GPCR and stabilizing the formation of the aforementioned complex, or a synthetic or semi-synthetic analog or derivative of the GPCR capable of binding to the GPCR and stabilizing the formation of the aforementioned complex. As also mentioned in this article, the second ligand can be a confoboody, i.e., an immunoglobulin monovariable domain (e.g., VHH or nanobody) that has been designed / generated to stabilize the formation of a complex of the confoboody, translayer protein and the first ligand.

[0279] In a preferred but non-limiting aspect of the invention, the transmembrane protein (2) will be a "seven-transmembrane protein," particularly a 7TM that acts as a receptor (e.g., a cell surface receptor). In a particularly preferred aspect, the transmembrane protein (2) may be a 7TM that signals via a G protein. Such 7TMs are also referred to in the art as GPCRs [as noted above, the terms "GPCR" and "7TM" are used interchangeably herein to include all transmembrane proteins having a 7-transmembrane domain, regardless of their intracellular signaling cascade or signal transduction mechanism, although it should be understood that throughout the specification and claims, 7TMs that signal via G proteins are a preferred aspect of the invention].

[0280] The transmembrane 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 by protein chemistry or recombinant DNA techniques generally described herein). Such a synthetic analog can be, for example, an analog of a naturally occurring transmembrane protein, wherein one or more amino acid residues or segments 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 segments of amino acid residues (e.g., segments or loops substantially corresponding to other (preferably structurally related) membrane proteins) compared to the natural sequence (in other words, containing one or more “amino acid differences” as defined herein compared to the natural sequence). Typically, the natural sequence of the naturally occurring protein used will be obtained from a species intended to be treated with the compounds of the present invention or from an animal (preferably a mammal) intended for use as an animal model to test the compounds of the present invention.

[0281] Those skilled in the art will appreciate that such synthetic analogs can be obtained using protein chemistry standard techniques and / or recombinant DNA techniques known per se. For example, when the present invention is carried out in cells as described herein, synthetic analogs can be obtained by suitably expressing the DNA sequence (or other suitable nucleotide sequence) encoding the synthetic analog in said cells.

[0282] Furthermore, as is known in the art, 7TM and other transmembrane proteins typically comprise one or more intracellular loops and one or more extracellular loops. Similarly, transmembrane proteins used in the devices of the present invention may comprise one or more loops extending (as defined herein) into a first environment and one or more loops extending (as defined herein) into a second environment. For example, when the methods of the present invention are carried out in cells, transmembrane proteins used in the devices of the present invention may comprise one or more loops extending into the intracellular environment and one or more loops extending into the extracellular environment. Similarly, when the methods of the present invention are carried out in vesicles or liposomes, transmembrane proteins used in the devices of the present invention may comprise one or more loops extending into the internal environment of the liposome or vesicle and one or more loops extending into the external environment of the liposome or vesicle. In each case, the loops extending into the first environment are most preferably arranged such that they can form functional ligand binding sites (and in particular, functional binding sites for the first ligand), and / or when the translayer protein adopts a suitable conformation, they can form functional ligand binding sites (and in particular, functional binding sites for the first ligand); and the loops extending into the second environment are most preferably arranged such that they can form functional ligand binding sites (and in particular, functional binding sites for the second ligand), and / or when the translayer protein adopts a suitable conformation (e.g., after the first ligand binds to the translayer protein), they can form functional ligand binding sites (and in particular, functional binding sites for the second ligand).

[0283] In one specific but non-limiting aspect, the loop of a translayer protein extending into one environment will substantially correspond to the extracellular loop of the translayer protein, and the loop of a translayer protein extending into other environments will substantially correspond to the intracellular loop (again, in each case, it is preferred that the extracellular loop forms a functional ligand binding site and that the intracellular loop forms another functional ligand binding site). Preferably, the loop of a translayer protein extending into a first environment [A] will substantially correspond to the extracellular loop of the translayer protein, and the loop of a translayer protein extending into a second environment [B] will substantially correspond to the intracellular loop of the translayer protein, particularly when the second environment [B] is an environment inside a cell or liposome (again, preferably, that the extracellular loop forms a functional ligand binding site extending into the first environment, and that the intracellular loop forms a different functional ligand binding site extending into the second environment).

[0284] For example, when the transmembrane protein is a transmembrane protein (e.g., 7TM), the transmembrane protein may comprise one or more extracellular loops of the transmembrane protein (particularly one or more extracellular loops of 7TM) and one or more intracellular loops of the transmembrane protein (particularly one or more intracellular loops of 7TM), more particularly such that the extracellular loops form or can form functional ligand-binding sites and that the intracellular loops form or can form different functional ligand-binding sites. Similarly, the ligand-binding sites formed by the extracellular loops will preferably extend (as defined herein) into one environment, and the ligand-binding sites formed by the intracellular loops will preferably extend (as defined herein) into other environments. In particular, when the method of the present invention is carried out in cells or liposomes, the extracellular loops will extend into an environment outside the cell or liposome, and the intracellular loops will extend into an environment inside the cell or liposome. Furthermore, preferably, the intracellular loops are arranged such that they form or can form functional ligand-binding sites for the second ligand (or in other words: in this invention, the ligand-binding site for the second ligand preferably consists of and / or contains one or more intracellular loops of a transmembrane protein. Alternatively, the ligand-binding site for the first ligand may consist of and / or contain one or more extracellular loops, but as further described herein, the actual binding / docking site for the first ligand may also be located deeper within the transmembrane protein structure).

[0285] For example, when the translayer protein is 7TM, the translayer protein may comprise three intracellular loops (i.e., three intracellular loops from 7TM) and three extracellular loops (i.e., three extracellular loops from 7TM), wherein the three intracellular loops form or may form functional ligand binding sites, and wherein the three extracellular loops form or may form different functional ligand binding sites. Again, preferably, the functional ligand binding sites formed by the three intracellular loops extend into one environment (and preferably a second environment [B]), and the functional ligand binding sites formed by the three extracellular loops extend into other environments (and preferably a second environment [A]). Furthermore, the three intracellular loops preferably form functional binding sites for a second ligand (and the three extracellular loops may form functional binding sites for a first ligand or the binding sites may be located deeper within the 7TM structure). Most preferably, the three intracellular loops will form binding sites for the second ligand extending into the second environment [B] (i.e., the environment inside the cell or liposome when the method of the present invention is carried out in a cell or liposome, respectively), and the three extracellular loops will extend into the first environment [A] (and may form functional binding sites for the first ligand, or the binding sites may be located deeper in the 7TM structure).

[0286] In one aspect of the invention, the intracellular and extracellular loops of the transmembrane protein originate from or substantially originate from the same transmembrane protein (i.e., the same or substantially the same as those present in natural transmembrane proteins). In this aspect of the invention, the transmembrane protein may have the same or substantially the same amino acid sequence as the natural transmembrane protein (which is intended to be used as a target in the screening or assay method of the invention).

[0287] In another aspect of the invention, the intracellular and extracellular loops of a transmembrane protein can originate from different transmembrane proteins. Specifically, in this aspect of the invention, the intracellular and extracellular loops can originate from different but related transmembrane proteins, for example, from two different but related 7TMs, such as two GPCRs. Specifically, in this aspect of the invention, the intracellular loop can originate from a first 7TM or GPCR, and the extracellular loop can originate from a second 7TM or GPCR different from the first 7TM or GPCR. The transmembrane domains of such chimeric proteins can originate from either the first or second 7TM or GPCR, and preferably substantially from the same GPCR, more preferably from the same GPCR as the extracellular loop (but may contain some amino acid residues from the GPCR from which the intracellular loop originates, depending on the location chosen for recombination to remove the native intracellular loop and insert a substitute intracellular loop).

[0288] In this aspect of the invention, the resulting chimeric translayer protein should still be most preferably suitable for use with the methods and apparatus of the invention. Again, in the case of 7TM, the translayer protein will comprise three intracellular loops and three extracellular loops, the three intracellular loops forming functional ligand-binding sites for a second ligand (and then selecting a second ligand such that it can bind to the ligand-binding sites (9)) formed by the intracellular loops. Similarly, the binding sites formed by the three intracellular loops will preferably extend into a second environment [B] (i.e., the environment inside the cell or liposome, respectively, when the method of the invention is performed in a cell or liposome), and the three extracellular loops will preferably extend into a first environment [A] (and may form functional binding sites for a first ligand, or the binding sites may be located deeper within the 7TM structure).

[0289] Therefore, in another aspect, the present invention relates to a device as further described herein, wherein the transmembrane protein is 7TM, comprising seven transmembrane domains, three intracellular loops, and three extracellular loops (which are connected to each other and linked in the order known as 7TM itself), namely [N-terminal sequence]-[TM1]-[IC1]-[TM2]-[EC1]-[TM3]-[IC2]-[TM4]-[EC2]-[TM5]-[IC3]-[TM6]-[EC3]-[TM7]-C-terminal sequence), wherein the intracellular loops originate from a first 7TM, and the extracellular loops originate from a second 7TM different from the first 7TM, wherein the intracellular loops form functional ligand binding sites. Preferably, the TM domains from said transmembrane protein are substantially derived from the same 7TM as the extracellular loops.

[0290] Furthermore, the intracellular loop and 7TM as a whole enable them to form functional ligand binding sites, particularly functional ligand binding sites to which (suitable) second ligands (as defined herein) can bind. These ligand binding sites preferably extend into the second environment [B].

[0291] In one specific aspect, this chimeric translayer protein contains an intracellular loop derived from a β-2-adrenergic receptor. In another specific aspect, this chimeric translayer protein contains an intracellular loop derived from a μ-opioid receptor. For some non-limiting examples of such chimeric receptors, see also the assignee's title "Chimeric proteins and methods to screen for compounds and ligands binding to GPCRs".

[0292] This application is a co-pending PCT application (binding to GPCRs) with the same international filing date and citing the same priority application as this application.

[0293] The present invention particularly relates to a device comprising such a chimeric 7TM and a second ligand capable of binding to a ligand-binding site formed by the intracellular loop.

[0294] For the remainder, the prerequisite is that a second ligand is appropriately selected to enable it to bind to the ligand-binding site (9) on the chimeric translayer protein, thereby providing an operable device of the invention (and the prerequisite is that the chimeric translayer protein itself is operable in the device of the invention), wherein such a device of the invention using the chimeric translayer protein can be substantially as further described herein.

[0295] Furthermore, such chimeric translayer proteins, the nucleotide sequences and nucleic acids encoding them, and cells, cell lines or host organisms containing such nucleotide sequences or nucleic acids and / or capable of expressing such chimeric translayer proteins constitute further aspects of the invention, as do further uses of such chimeric translayer proteins, nucleotide sequences, nucleic acids, cells, cell lines and host organisms.

[0296] Another aspect of the invention is a composition or assembly comprising at least the chimeric translayer protein and a ligand capable of binding to an intracellular loop present in the GPCR. The ligand is preferably a protein, more preferably a protein comprising or substantially composed of an immunoglobulin monovariable domain (e.g., a VHH domain), and particularly may be a confocal body (as described herein).

[0297] As described above, the chimeric translayer protein is preferably 7TM / GPCR. Furthermore, in one specific aspect, the chimeric translayer protein comprises an intracellular loop derived from a β-2-adrenergic receptor. In another specific aspect, the chimeric translayer protein comprises an intracellular loop derived from a μ-opioid receptor.

[0298] As further described in this article, and as... Figures 1 to 3 As illustrated schematically, in the apparatus of the present invention, the translayer protein (2) is typically and preferably fused or linked directly or via a suitable spacer or connector (10) to the first member (6) of the binding pair (6 / 7) to form a first fusion protein. Furthermore, 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 is also further described herein. The first fusion protein, the second fusion protein (in its various forms as described herein), the nucleotide sequence and / or nucleic acid encoding the first or second fusion protein, and the cells, cell lines, or other host cells or host organisms that express (and particularly suitably express, as described herein) or are capable of (suitably) expressing the first and / or second fusion proteins (and preferably both), and the various uses of the aforementioned substances as further described herein, form a further aspect of the invention.

[0299] The binding pair (6 / 7) used in the device of the present invention typically comprises at least two separate binding members (6) and (7), which are also referred to herein as the “first binding member” and the “second binding member”, respectively. The binding pair (6 / 7) and each of its members (6) and (7) should be such that when members (6) and (7) are in contact with or close to each other, the binding pair (6 / 7) is capable of generating a detectable signal. Such a detectable signal may be, for example, a luminescent signal, a fluorescent signal, or a chemiluminescent signal, based on a reporter gene, or based on DNA linkage. Some specific, but not limiting, examples of techniques (including binding pairs and their associated detectable signals) are based on protein complementarity, such as the NanoBit™ system, NanoLuc™ system, hGLuc system (Remy and Michnick, Nature Methods, 2006, 977), BiFC (bimolecular fluorescence complementarity), and DHFR-PCA (dihydrofolate reductase protein fragment complementarity assay); techniques based on direct interactions, such as BRET (bioluminescent resonance energy transfer), FRET (fluorescence / Foster resonance energy transfer), and BioID (proximity-dependent biotin recognition); and systems based on reporter genes (e.g., KISS / kinase substrate sensor) or proximity connectivity assays (Weilbrecht et al., Expert Review of Proteomics, 7:3, 401-409). Techniques based on protein complementarity and luminescent, fluorescent, or chemiluminescent signals (e.g., NanoLuc™ or NanoBit™) are generally preferred.

[0300] In a particularly preferred aspect, when the method of the invention is carried out in suitable cells, the first member (6) and the second member (7) of the pair (6 / 7) are preferably both polypeptides, proteins, amino acid sequences or other chemical entities that can be obtained by suitable expression (preferably in cells used in the method of the invention) of nucleic acid or nucleotide sequences encoding them.

[0301] The first and second binding members can also be part of a suitable reporter gene assay, and can be a combination of enzyme and substrate, or any other pair of domains or units that can generate a detectable signal when they come into contact with or are in close proximity to each other, such as binding pairs commonly used in experimental studies of protein-protein interactions. As mentioned above, to reduce the level of baseline / background signal, it is preferable that the two members of the binding pair do not themselves have substantial binding affinity for each other.

[0302] Some preferred, but non-limiting, examples of suitable binding pairs are pGFP and the NanoBiT® system from Promega. The latter is particularly preferred because the large and small BiTs that make up the NanoBiT® system have low affinity for each other.

[0303] The first binding member (6) may fuse with the translayer protein (2) in any suitable manner, provided that the resulting first fusion protein is such that when the second fusion protein formed by the second ligand (4) and the second member (7) binds to the translayer protein (2) through the second binding site (9), it allows the first member (6) to contact (or otherwise suitably approach) the second member (7) of the binding pair (6 / 7). Furthermore, preferably, the first binding member (6) fuses or connects with the translayer protein (2) in a manner that substantially does not affect the conformational and / or conformational changes that the translayer protein (2) may undergo under the conditions for carrying out the method of the invention.

[0304] Therefore, while the present invention does not preclude direct fusion or attachment of the first binding member (6) to the translayer protein (2), it is generally preferred that the first binding member (6) be fused or attached to the translayer protein (2) via a suitable linker (10). Flexible linkers are generally preferred, for example, having a total of 5 to 50 amino acids, preferably 10 to 30 amino acids, such as about 15 to 20 amino acids. Suitable linkers will be apparent to those skilled in the art and include GlySer linkers (e.g., 15GS linkers).

[0305] In this invention, the first and second binding members of the binding pair (6 / 7) will exist (as defined herein) in the same environment relative to the boundary layer (1), such that they can contact or be close to each other (in a manner further described herein) and, in doing so, can generate a detectable signal. Specifically, as Figure 1 , 2 As illustrated in Figure 3, the first and second binding members of the binding pair (6 / 7) will be present (as defined herein) in the same environment as the second binding site (9) on the translayer protein (2) (again, relative to the boundary layer (1)), so as to allow the second fusion protein, i.e., directly (as... Figure 1 (as shown) or indirectly (such as) Figure 2 and 3 When the binding site is reached, the first and second binding members of the binding pair (6 / 7) come into contact. For this purpose, the first binding member (6) typically attaches directly or via a linker (10) to amino acid residues / positions in / on the translayer protein (2) exposed to the same environment as the second binding site (9). As further described herein, the environment (in... Figures 1 to 3 The environment (represented as B) can be, for example, the intracellular environment (when the method of the present invention is carried out in a cell) or the environment within vesicles or lipid bodies.

[0306] In a preferred aspect of the invention, the first binding member (6) is fused directly or via a linker (10) to one end of the primary amino acid sequence of the translayer protein (2). This can be the N-terminus or C-terminus of the translayer protein (2), as long as the first binding member (6) is located on the same side of the boundary layer (1) as the second binding site (9) in the final apparatus of the invention. Thus, in the aspects of the invention carried out in cells as further described herein, and when the second binding site (9) is exposed to the intracellular environment, the first member (6) can be fused to the end of the primary amino acid sequence that terminates in the intracellular environment (typically the C-terminus in the case of 7TM).

[0307] The first fusion protein can be provided and produced using suitable protein chemistry techniques and / or recombinant DNA techniques known per se. These techniques will be clear to those skilled in the art based on the further disclosure herein and the standard manuals and other scientific references mentioned herein. When the method of the invention is carried out in cells (as further described herein), the first fusion protein is preferably provided by suitably expressing the nucleotide sequence and / or nucleic acid encoding the first fusion protein in said cells. This can again be carried out using suitable techniques known per se for recombinant DNA, and cells that suitably express or are capable of suitably expressing the first fusion protein constitute another aspect of the invention.

[0308] As further described herein, in the apparatus of the present invention, the second member (7) of the binding pair (6 / 7) typically and preferably also forms part of a fusion protein, which typically includes the second binding unit, which is fused or connected directly or via a suitable spacer or connector (11) to another ligand, protein, binding domain, or binding unit that can bind directly (as defined herein) or indirectly (as defined herein) to a translayer protein (2). For this purpose, as further described herein, the ligand, protein, binding domain, or binding unit can be, for example, a second ligand (leading to...). Figure 1 The device of the present invention, of the type schematically shown in the diagram, wherein (4) is a second ligand, is a binding domain or binding unit that can bind to the second ligand (leading to...) Figure 2 The device of the present invention, schematically shown in the diagram, wherein (4) is a second ligand and (5) is a binding domain or binding unit that binds to the second ligand, or a binding domain or binding unit that can bind to a protein complex that can bind to a translayer protein (e.g., Figure 3 As illustrated schematically, (4) is the second ligand, (12) is the protein complex containing the second ligand, and (5) is the binding domain or binding unit of the protein complex.

[0309] In the second fusion protein, the second binding member (7) is most preferably connected in a suitable manner to the other ligands, proteins, binding domains, or binding units, such suitable manner allowing the second binding member (7) to contact (or otherwise suitably approach) 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) on the translayer protein (2). For this purpose, the second binding member (7) may be directly fused or connected to the other ligands, proteins, binding domains, or binding units, but preferably they are connected via a suitable linker (11), which is preferably a flexible linker, for example having a total of 5 to 50 amino acids, preferably 10 to 30 amino acids, and typically preferably, for example, about 15 to 20 amino acids. Suitable linkers are apparent to those skilled in the art and include GlySer linkers (e.g., 15GS linkers).

[0310] As described herein, the second ligand can be any ligand, protein, binding domain, or binding unit capable of binding to a translayer protein, i.e., via a binding site (9) (when the second ligand is part of the second fusion protein, it should most preferably also be appropriately included in the second fusion protein).

[0311] Typically, in this invention (and regardless of whether the binding site is directly or indirectly bound by the second fusion protein used in the device of the invention), the binding site (9) can be a conformational epitope on the translayer protein (2). More specifically, the binding site (9) can be a conformational epitope on the translayer protein (2) that changes its "shape" (i.e., the spatial arrangement of domains, loops, and / or amino acid residues forming the epitope) when the translayer protein (2) undergoes a conformational change, such as a conformational change from an inactive or less active state to an active, more active, and / or functional state, and / or when the first ligand binds to the translayer protein.

[0312] Preferably, the binding site (9) and the second ligand cause the affinity of the interaction between the binding site (9) and the second ligand (4) to change when the binding site (9) changes its shape, since the translayer protein (2) undergoes a conformational change. In particular, the binding site (9) and the second ligand can cause the affinity of the interaction between the binding site (9) and the second ligand (4) to increase when the translayer protein (2) undergoes a conformational change from an inactive or less active state to an active, more active, functional and / or drugable state and / or undergoes a conformational change when the first ligand (3) (especially the first ligand (3) as an agonist of the translayer protein (2)) binds to the translayer protein (2).

[0313] Specifically, the interaction between the second ligand (4) and its binding site (9) can cause the second ligand (4) to bind to the binding site (9) with a higher affinity when the translayer protein (2) is in an active, highly active and / or functional state and / or cause the second ligand (4) to bind to the binding site (9) with a higher affinity when the first ligand (3) (in particular, the first ligand (3) acts as an agonist for the translayer protein (2)) binds to the translayer protein (2). For example, the interaction between the second ligand (4) and its binding site (9) can increase the affinity of the second ligand (4) for the translayer protein (2) by 10-fold, for example, 100-fold or more, when the translayer protein (2) undergoes such a conformational change. This ranges from micromolar affinity (i.e., more than 1000 nM) when the translayer protein is in an inactive, less active, or ligand-free conformation to nanomolar affinity (i.e., less than 1000 nM, for example, less than 100 nM) when the translayer protein (2) is in a functional, active, or more active and / or ligand-binding conformation. For example, in the case of GPCRs, it is known that the affinity of the interaction between G proteins and G protein binding sites increases when a ligand (especially an agonist) binds to the extracellular binding site of a GPCR. Furthermore, WO2012 / 007593, WO2012 / 007594, WO2012 / 75643, WO 2014 / 118297, WO2014 / 122183, and WO2014 / 118297 describe a VHH domain (ConfoBody) that exhibits higher affinity for GPCRs in functional, active, or highly active and / or ligand-binding conformations compared to inactive, less active, or ligand-free conformations of translayer proteins (e.g., in nanomolar ranges for functional, active, or ligand-binding conformations, and in micromolar ranges for inactive or ligand-free conformations).

[0314] When the second ligand binds to the translayer protein (2), the second ligand itself may also undergo a conformational change. In embodiments where the second fusion protein indirectly binds to the translayer protein (2), this may also mean that the binding domain or binding unit (5) in the second fusion protein binding the second ligand (4) can be such that it has a higher affinity for the conformation adopted by the second ligand (4) when the second ligand (4) binds to the translayer protein (2) compared to the conformation adopted by the second ligand (4) when it does not bind to the translayer protein (2). For example, it is known that G proteins undergo conformational changes when binding to GPCRs, and it is possible that the VHH domain present in the second fusion protein has a higher affinity for the GPCR-binding conformation of the G protein compared to the unbound conformation of the GPCR.

[0315] In a preferred aspect, the binding site (9) is a binding site on the translayer protein (2) that serves as a binding site for a native ligand of the translayer protein when the translayer protein is in its native environment. More specifically, the binding site (9) may be a binding site on the translayer protein (2) that serves as an intracellular binding site for a native intracellular ligand of the translayer protein when the translayer protein is in its native environment. For example, when the translayer protein (2) is a receptor, the binding site (9) may be a binding site on the translayer protein (2) that serves as an intracellular binding site for one or more intracellular ligands of the translayer protein (2) involved in signal transduction when the translayer protein is in its native environment.

[0316] In a specific context, when the translayer protein (2) is a GPCR, the binding site (9) may be a binding site for a G-protein (and / or a G-protein complex). As further described herein, in this case, the second ligand may be a natural, synthetic, or recombinant protein or may be another ligand that can bind to the G-protein binding site on the GPCR.

[0317] The second ligand (4) is typically a protein or a ligand of a protein. In aspects of the invention carried out in suitable cells or cell lines, the second ligand (4) may be a protein native to the cells or cell lines used, or may be a suitable (recombinant) protein expressed in the cells or cell lines used. For example, when the second ligand (4) is not part of the second fusion protein, it may be a ligand of a translayer protein (2) naturally present in the cells or cell lines (e.g., when the translayer protein (2) is a GPCR, the second ligand (4) may be a G protein naturally expressed by the cells or cell lines used). Alternatively, the second ligand may be a protein recombinantly expressed in the cells or cell lines used, for example when the cells or cell lines do not naturally express a suitable ligand for the translayer protein (2) or when it is necessary to use a ligand different from that naturally expressed by the cells or cell lines (e.g., when it is necessary to use an analogue, derivative, or ortholog of the naturally expressed ligand, in which case the natural expression of the naturally expressed ligand in the cells or cell lines used may also be temporarily or constitutively suppressed or knocked out). When the second ligand (4) forms part of the second fusion protein, the second ligand is usually recombinantly expressed as part of the second fusion protein.

[0318] As further described herein, the second ligand (4) may be part of the second fusion protein or it may be separate from the second fusion protein. In either case (i.e., whether or not the second ligand is part of the second fusion protein), the second ligand is preferably capable of binding to a conformational epitope on the translayer protein (or is such that it is part of a protein complex that directly binds to the translayer protein or is capable of directly binding to the translayer protein). More preferably, the second ligand (and / or the protein complex containing the second ligand) preferably specifically binds to one or more functional, active, and / or druggable conformations of the translayer protein, such that it induces the formation and / or stabilizes one or more functional, active, and / or druggable conformations of the translayer protein (and / or causes a conformational equilibrium shift of the translayer protein toward one or more such conformations); and / or causes it to induce the formation and / or stabilize a complex of the translayer protein, the first ligand, and the second ligand.

[0319] When the second ligand is part of the second fusion protein, it can be any ligand, binding domain, binding unit, peptide, protein, or other chemical entity capable of directly binding to translayer proteins and suitably contained within the second fusion protein. Preferably, as further described herein, when it is part of the second fusion protein, the second ligand will be a suitable binding domain or binding unit, and in particular an immunoglobulin monovariable domain.

[0320] When the second ligand is separate from the second fusion protein, it can be any ligand or protein that can directly bind to a translayer protein and / or form part of a protein complex that can bind to a translayer protein. For example, as further described herein, such a second ligand can be a naturally occurring ligand of a translayer protein, a semi-synthetic or synthetic analog or derivative of such a naturally occurring ligand, or a direct homolog of such a naturally occurring ligand. Furthermore, when the second ligand is not part of the second fusion protein, the second fusion protein will contain a binding domain or binding unit that can indirectly bind (as defined herein) to a translayer protein, i.e., a binding domain or binding unit that binds to the second ligand and / or binds to a protein complex containing the second ligand. Similarly, as further described herein, such a binding domain or binding unit can be, in particular, an immunoglobulin monovariable domain, such as the ISVD derived from camels. As also mentioned herein, such a binding domain or binding unit may comprise two or more (e.g., two or three) ISVDs (appropriately fused or connected, optionally by appropriate linkers or spacers), which may be identical or different, and (when identical) they typically bind to the same binding site or epitope on the second ligand, or (when different) they may bind to the same or different epitopes or binding sites on the second ligand (and, when the second ligand is a protein complex such as a G protein complex, they may bind to the same or different subunits of the protein complex).

[0321] Those skilled in the art will also understand that, when 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 substantially not interfere with the binding of the second ligand to the translayer protein. For example, it preferably binds to a binding site or epitope on the second ligand that is different from the binding site on the second protein that binds the translayer protein (and preferably is also sufficiently removed from the binding site on the second protein that binds the translayer protein to avoid any major steric hindrance).

[0322] When the second ligand (4) is a naturally occurring ligand of the translayer protein (2), it can be, for example, a ligand involved in a signal transduction pathway or signal transduction involving the translayer protein (2). For example, when the second ligand (4) is a receptor, the second ligand (4) can be a naturally occurring ligand of the receptor, particularly a naturally occurring intracellular ligand of the receptor, such as an intracellular ligand that binds to an intracellular binding site on the receptor when an extracellular ligand binds to an extracellular binding site on the receptor, or an intracellular ligand that binds to an intracellular binding site on the receptor as part of a pathway providing said constituent activity when the receptor has some degree of constituent activity. Suitable examples of such natural ligands will be apparent to those skilled in the art based on the disclosure herein, and will generally depend on the translayer protein (2) used. For example, when the translayer protein (2) is 7TM or GPCR, the second ligand (4) may be a G protein (preferably), including but not limited to naturally occurring G proteins (e.g., G proteins naturally occurring in the cells or cell lines used) or synthetic or semi-synthetic analogs or derivatives of naturally occurring G-proteins (including chimeric G-proteins), all as further described herein.

[0323] As further described herein, particularly in aspects and embodiments of the invention performed 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 proteins. For example, when the translayer protein is a GPCR and the second ligand is a G protein or an analogue or derivative of a G protein, the second ligand may be part of a complex formed by said G protein and optionally one or more other proteins. A preferred, but non-limiting, example of such a complex is a G-protein trimer comprising G-α, G-β, and G-γ subunits. The complex may also comprise the translayer protein itself (e.g., GPCR and G-protein or GPCR and 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 that can bind to the second ligand or said complex. For example, in the case where the second ligand forms part of a G protein complex, the binding domain or binding unit in the second fusion protein may be a VHH domain that binds to the complex (e.g., to a subunit within the complex or to an interface between two or more of the subunits). As described herein, an example of such a VHH domain is the VHH referred to as “CA4435” (SEQ ID NO: 1 in WO2012 / 75643 and SEQ ID NO: 1 herein).

[0324] The second ligand (4) may also be a synthetic or semi-synthetic analog or derivative of such a naturally occurring ligand, for example, by the deletion, insertion, and / or substitution of a limited number of amino acid residues or fragments having a primary amino acid sequence different from that of the corresponding natural ligand. Such analogs or derivatives may again be provided using suitable techniques of recombinant DNA technology known per se, which again may in one aspect involve the expression of a nucleotide sequence or nucleic acid encoding the analog or derivative in a suitable host or host cell (preferably, as part of the whole second fusion protein, also including a second binding member (7) and any linker (11), if present). For example, when the translayer protein (2) is 7TM or GPCR, the second ligand (4) may be an analog or derivative of a G protein (preferably) which may also have one or more amino acid differences from the natural sequence (as defined herein), provided that the analog or derivative still has sufficient affinity for the translayer protein (2) to allow for the suitable use of the analog or derivative in the method of the present invention.

[0325] For example, in one specific embodiment, such an analog or derivative of a naturally occurring G-protein can be a naturally occurring G-protein in which one or more amino acid residues (and / or one or more amino acid residue segments) are replaced by one or more amino acid residues (and / or one or more amino acid residue segments) at the same or corresponding position in another naturally occurring G protein.

[0326] When the G protein is a heterotrimeric protein, such substitution of one or more amino acid residues (and / or one or more amino acid residue segments) can be present or occur in any one, two or all three subunits of the G-α, G-β and / or G-γ subunits, and especially in the G-α subunit.

[0327] For example, it is well known that there are multiple genes in humans, each encoding a different G-α subunit. G-α has multiple isoforms that can be divided into different functional subfamilies (e.g., see Flock et al., Nature, 2015, 524(7564), 173-179; and Nehme et al., PLoS One, 2017, 12(4)). Analogs or derivatives of the naturally occurring G-α subunit used in this invention can be obtained by replacing one or more amino acid residues (and / or one or more amino acid residue segments) in the naturally occurring G-α subunit (of its amino acid sequence) with one or more amino acids (and / or one or more amino acid residue segments) present at (substantially) the same or corresponding positions in another naturally occurring α subunit (which may belong to the same or different subfamily as the original subunit). Some specific, but non-limiting, examples are naturally occurring Gαs subunits in which one or more amino acid residues and / or one or more amino acid residue segments have been replaced at (substantially) the same or corresponding positions in the Gαi subunit, or naturally occurring GαS subunits in which one or more amino acid residues and / or one or more amino acid residue segments have been replaced at (substantially) the same or corresponding positions in the Gαq subunit. Typically, but not exclusively, such substituted / alternated amino acids or segments will be present at or near the C-terminus of the α-subunit.

[0328] Specific, but non-limiting, examples of such “chimeric” G-proteins and their design can also be found in the scientific literature. See, for example, the publications of Flock et al. and Nehme et al. mentioned above.

[0329] The second ligand (4) may also be another type of ligand that has been generated to bind to the binding site (9) on the translayer protein (2), and preferably binds in the manner further described herein.

[0330] In a particularly preferred aspect, when the method of the invention is carried out in suitable cells, the second ligand (4) is preferably a polypeptide, protein, amino acid sequence or other chemical entity that can be obtained by suitable expression (preferably in cells used in the method of the invention) of a nucleic acid or nucleotide sequence encoding it.

[0331] As described herein, and regardless of whether it is a naturally occurring ligand of the translayer protein (2) (e.g., a naturally occurring G protein), a synthetic or semi-synthetic analog or derivative of such a naturally occurring ligand (e.g., a chimeric G protein as described above), or another ligand (e.g., a ConfoBody as further described herein), the second ligand (4) generally enables it to bind, in particular specifically, to an epitope on the translayer protein (2), specifically binding site (9). In particular, the second ligand (4) can enable it to bind, in particular, specifically to an epitope (which would be an intracellular epitope if the translayer protein (2) is in its native cellular environment).

[0332] As described herein, the epitope (i.e., the binding site (9)) can be a linear epitope or a conformational epitope, and is preferably a conformational epitope (as described herein). For example, when the translayer protein (2) is a GPCR, the epitope can be contained in one or more amino acid residues and / or amino acid residue segments on at least one intracellular loop of the GPCR, and in particular, can be a conformational epitope formed by or containing one or more amino acid residues and / or amino acid residue segments on at least two different intracellular loops of the GPCR.

[0333] The epitope of the second ligand (4) can be, in particular, a (partial) epitope on a translayer protein (2) that participates in signal transduction mediated by the translayer protein (2). For example, the second ligand can bind to an epitope on the translayer protein (2) located within the binding site of a downstream signal transduction protein. For example, when the translayer protein (2) is a GPCR, the second ligand (4) can be a binding domain or binding unit capable of specifically binding to a conformational epitope contained in, located at, or overlapping with the G protein binding site of the GPCR.

[0334] When the translayer protein (2) used is a protein that can present / exist in two or more conformations (e.g., basal state / conformation, active state / conformation and / or inactive state / conformation) and / or can undergo conformational changes (especially functional conformational changes), the second ligand (4) is preferably such that it can bind, in particular specifically bind, the functional conformational state of the translayer protein (2).

[0335] In a particularly preferred aspect, the second ligand (4) enables, upon binding to the translayer protein (2), to stabilize and / or induce the functional and / or active conformational state of the translayer protein (2) (and / or to shift the conformational equilibrium of the translayer protein (2) from an inactive or less active state to a more active state), to enable the translayer protein to enter a more druggable conformation (and / or to shift the conformational equilibrium of the translayer protein (2) from a less druggable conformation to a more druggable conformation), to alter the conformation of the protein (on the associated binding pocket) to make it more amenable or accessible for binding the first ligand (3) or generally increasing the affinity between the first ligands (3) (and / or to shift the conformational equilibrium of the protein to such a conformation), and / or to induce and / or stabilize the formation of a complex comprising the second ligand, the translayer protein, and the first ligand (and / or to shift the conformational equilibrium of the translayer protein (2) to the formation of such a complex), or any combination thereof.

[0336] Therefore, when the translayer protein (2) is a GPCR, the second ligand (4) can be such that it binds, in particular, to stabilize and / or induce the functional conformational state of the GPCR, more preferably the active conformational state of the GPCR. The second ligand (4) preferably also causes, when it binds to an agonist (e.g., when it is bound by a first ligand (3) acting as a protein or GPCR agonist), to preferentially / specifically bind the protein or GPCR compared to a conformational state in which the protein or GPCR is not bound by any first ligand (3) or is not bound by a ligand (3) acting as a reverse agonist; and / or causes it to increase the affinity of the protein or GPCR for at least one compound or ligand acting as a protein or GPCR agonist (i.e., at least two times, particularly at least five times, more preferably at least ten times).

[0337] As described above, a class of preferred compounds used as second ligands in this invention (particularly when the second ligand is contained in the second fusion protein) are generally described in WO2012 / 007593, WO2012 / 007594, WO2012 / 75643, WO2014 / 118297, WO2014 / 122183 and WO2014 / 118297, and contain a VHH domain (Confobody) capable of stabilizing the GPCR in the desired conformation.

[0338] Furthermore, WO2012 / 75643 discloses numerous VHH domains that can indirectly bind GPCRs, i.e., by binding to G proteins or G protein complexes. Some preferred, but non-limiting, examples of these are VHHs called “CA4435” (SEQ ID NO:1 in WO2012 / 75643 and SEQ ID NO:1 herein), which can bind to G protein complexes, and VHHs called “CA4437” (SEQ ID NO:4 in WO2012 / 75643 and SEQ ID NO:2 herein), which can bind to G proteins. Such VHH domains may suitably be incorporated into a second fusion to provide a second fusion protein that can indirectly bind GPCRs by binding to G proteins or G protein complexes.

[0339] Therefore, in a preferred aspect of the invention, the second fusion protein comprises at least one such VHH or ConfoBody and a second binding member (7).

[0340] Typically, in this invention, when the second fusion protein binds directly or indirectly (as defined herein) to the translayer protein (2), the first binding member (6) and the second binding member (7) will be in close proximity to each other. Specifically, when the second ligand (4) present in the second fusion protein directly binds to the translayer protein (2), or when the binding domain or binding unit (5) present in the second fusion protein indirectly binds to the translayer protein, i.e., when the binding domain or binding unit (5) binds to the second ligand (4), or... Figure 3 In the illustrated embodiment, upon binding to the protein complex (12), the first and second binding members will be close to each other, and the second ligand (4) or the protein complex (12) will subsequently bind to or be bound by the translayer protein (2). It will be apparent to those skilled in the art that, preferably, the first and second binding members themselves should not have high affinity for each other, so that their association (and the generation of a detectable signal) is primarily driven by the proximity of the first and second binding members, since the second ligand binds (directly or indirectly) to the translayer protein and is substantially not, or only to a lesser extent, driven by 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 will generally provide a baseline for the detectable signal, which should not substantially interfere with the determination of the present invention, since the readings of the determination focus primarily on any changes in the detectable signal, such as when the first ligand is added to the apparatus of the present invention that does not yet contain the first ligand (more generally, it should also be noted that for some uses of the methods and apparatus of the present invention, it may be preferable to have a certain level of baseline signal, since the readings may also include a reduction in signal compared to the baseline).

[0341] Therefore, in general, in this invention, the detectable signal (or any change in the signal) generated by the first and second binding members will be proportional to the amount of the second fusion protein directly or indirectly bound to the translayer protein (2). This will in turn depend on the binding interaction between the second ligand (4) and the translayer protein (in particular, between the second ligand and one or more specific conformations that the translayer protein may present, such as functional, active, and / or druggable conformations) and / or any change in the binding interaction (in particular, any change in the binding interaction that is a result of a conformational change in the translayer protein and / or a shift in the conformational equilibrium of the translayer protein, for example, due to the binding of the first ligand to the translayer protein and / or the formation of a complex between the first ligand, the translayer protein, and the second ligand).

[0342] This document includes methods for identifying and creating various components of the aforementioned devices and compositions, as well as methods for assembling such devices and compositions. Such methods can be combined with and form part of any determinations and methods used to measure or determine one or more properties of the first ligand.

[0343] By way of non-limiting examples, a method for determining one or more properties of a first ligand as described herein may include one or more steps aimed at determining a second ligand: binding a translayer protein, specifically binding a translayer protein, specifically binding a domain of a translayer protein located in a second environment, being a conformationally selective binder of the translayer protein, stabilizing the conformation of the translayer protein, stabilizing the inactive conformation of the translayer protein, stabilizing the functional, active, and / or drugatable conformation of the translayer protein, and / or stabilizing a complex of the translayer protein and the first ligand.

[0344] Based on this and the further disclosure herein, those skilled in the art will understand that the methods and apparatus of the present invention can be used to measure or determine one or more properties of a first ligand (particularly properties of the first ligand relating to, influencing, and / or determining the interaction between the first ligand and the translayer protein), one or more properties of a second ligand (particularly properties of the second ligand relating to, influencing, and / or determining the interaction between the second ligand and the translayer protein), and / or one or more properties of any binding domain or binding unit present in the second fusion protein (particularly properties of the binding domain or binding unit relating to, influencing, and / or determining the interaction between the binding domain or binding unit and the translayer protein when the binding domain or binding unit binds directly to the translayer protein; or, properties relating to, influencing, and / or determining the interaction between the binding domain or binding unit and the second ligand or the complex when the binding domain or binding unit binds to the second ligand and / or a protein complex containing thereof).

[0345] More specifically, regarding the first ligand, the methods and apparatus of the present invention can be used to measure or determine the ability of a first ligand to bind to a translayer protein to induce a conformational change in the translayer protein and / or to induce a conformational equilibrium shift in the translayer protein. For example, as further described herein, the methods and apparatus of the present invention can be used to measure or determine the ability of a given first ligand to act as an agonist, antagonist, inverse agonist, inhibitor, or regulator (e.g., allosteric) modulator of a translayer protein and / or to screen or identify small molecules, proteins, or other compounds or chemical entities that are, or can act as, agonists, inverse agonists, inhibitors, or regulators (e.g., allosteric) modulators of translayer proteins. In this regard, those skilled in the art based on the disclosure herein will understand that when the methods and apparatus of the present invention are used for such purposes (i.e. for purposes relating to the first ligand), other elements typically (and preferably) used in the apparatus of the present invention (e.g., the second ligand and / or any binding domains or binding units present in the second fusion protein) will be selected such that they have known properties (i.e., their properties in relation to their use in the methods and apparatus of the present invention are known and / or have been characterized) and / or such that they have been validated for use in the methods and apparatus of the present invention.

[0346] The assays of the present invention can also be performed in the presence of a compound known to act on translayer proteins (e.g., in the presence of a known agonist, antagonist, inverse agonist, inhibitor, or regulator (e.g., an allosteric regulator) of the translayer protein) at a concentration at which the "known" compound is known to act on the translayer protein. The known compound will then typically be present in the same environment as the first ligand (i.e., the ligand whose properties are determined using the assays of the present invention). For example, in the method of the present invention, where the first ligand is added to the apparatus of the present invention where the first ligand is not yet present, the known compound may be added at substantially the same time as the first ligand, may be added separately before the first ligand, or may be added after the first ligand, and the read-out from the assay may vary depending on the order in which the first ligand and the known compound are added, and the time between the addition of the first ligand and the known compound when they are not added at substantially the same time (or vice versa). It is also possible to determine different properties of the first ligand and / or identify different first ligands with these properties by changing the order and / or timing of adding the first ligand and the known compound.

[0347] For example, but not limited to, the assays of the present invention can be performed in the presence of a known agonist of the translayer protein (i.e., in the same environment as the first ligand). In this setting, the assays of the present invention can, for example, be used to determine whether and how the first ligand is able to counteract the agonist effect of a known compound, for example, because it acts as an antagonist (therefore, in this setting, the assays of the present invention can be used to identify and / or characterize potential antagonist agonists of the translayer protein). The setting in the presence of a known agonist can also be used, for example, to identify and / or characterize the first ligand, which may act as an allosteric modulator that increases or decreases the effect of the agonist and / or as an inverse agonist of the translayer protein. Competitive assays can also be performed between the first ligand and the known compound.

[0348] The methods and apparatus of the present invention can be used to measure or determine the ability of a second ligand to bind to a translayer protein, particularly to bind to and / or stabilize a specific conformation of the translayer protein (e.g., a functional, active, or druggable conformation) and / or stabilize and / or induce the formation of a complex between the first ligand, the second ligand, and the translayer protein. For example, as further described herein, the methods and apparatus of the present invention can be used to measure or determine the ability of a given VHH as a confoy of a translayer protein, or to identify, optimize, or validate VHHs that can serve as confoys. For this purpose, typically, the VHH or candidate VHH will be present in a second fusion protein (i.e., as a second ligand) and will directly bind to the translayer protein or be tested for its ability to directly bind to the translayer protein or one or more specific conformations of the translayer protein. Also as further described herein, the methods and apparatus of the present invention can also be used to measure or determine the ability of analogues, derivatives, or orthologs of natural ligands of translayer proteins as ligands of translayer proteins (e.g., to test analogues, derivatives, or orthologs of naturally occurring G proteins as ligands of relevant GPCRs). In this case, typically, the second ligand will not be present in the second fusion protein (although a second fusion protein containing the analogue, derivative, or orthogonal homolog may also be used as the second ligand), and instead, the second fusion protein will contain a binding domain or binding unit (e.g., VHH) capable of binding the second ligand (or a complex containing it). In this regard, those skilled in the art based on the disclosure herein will appreciate that when the methods and apparatus of the present invention are used for such purposes (i.e., for purposes relating to the second ligand), other elements typically (and preferably) used in the apparatus of the present invention (e.g., the first ligand and / or any binding domain or binding unit present in the second fusion protein) will be selected such that they have known properties (i.e., their properties are known and / or have been characterized in relation to their use in the methods and apparatus of the present invention) and / or such that they have been validated for use in the methods and apparatus of the present invention.

[0349] The methods and apparatus 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 apparatus 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 VHHs that can indirectly bind to translayer proteins (which can then be used, for example, as a binding domain or binding unit in the apparatus of the present invention as described herein or for any other suitable purpose). Such methods and apparatus 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 VHHs (again, such VHHs can be used as a binding domain or binding unit in the apparatus of the present invention as described herein or for any other suitable purpose). In this regard, those skilled in the art will understand from the disclosure herein that when the methods and apparatus of the present invention are intended for such purposes (i.e., for the purpose of indirectly binding binding domains or binding units of translayer proteins), then other elements typically (and preferably) used in the apparatus of the present invention (e.g., first and second ligands) will be selected such that they have known properties (i.e., their properties in relation to their use in the methods and apparatus of the present invention are known and / or have been characterized) and / or such that they have been validated for use in the methods and apparatus of the present invention.

[0350] In this invention, generally speaking, the detectable signal will preferably be generated in response to a conformational change and / or a shift in the conformational equilibrium of the translayer protein, and more preferably proportional to it. Also as further described herein, but not limited to any particular mechanism or explanation, said conformational change and / or shift in conformational equilibrium of the translayer protein can be caused, in turn, by a first ligand binding to the translayer protein (or otherwise causing a conformational change in the translayer protein) and / or by forming a complex of the first ligand, the translayer protein, and a second ligand (the second ligand may, for example, stabilize the complex or otherwise induce or promote the formation of the complex). Therefore, more generally, in this invention, a detectable signal (or any change thereof, as further described herein) is generated in response to the presence of the first ligand in a first environment and / or in response to the binding of the first ligand to the translayer protein (or otherwise causing a conformational change and / or a shift in the conformational equilibrium of the translayer protein).

[0351] Furthermore, typically and particularly when the methods and apparatus of the present invention are used to test, optimize, and / or validate a first ligand and / or identify small molecules, proteins, ligands, or other chemical entities that can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric) of translayer proteins, the detectable signal (or any variation thereof, as further described herein) will be proportional to the amount and / or concentration of the first ligand present in a first environment (and / or to which the translayer protein is exposed) and / or the affinity of the first ligand for the translayer protein (e.g., compared to other ligands tested).

[0352] Therefore, based on the description herein, those skilled in the art will understand that, in one aspect of the invention, the methods and apparatus 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 the first ligand in the first environment. The methods and apparatus described herein can also be used to measure the amount of signal generated when different concentrations of the first ligand are present in the first environment, for example, to establish a relationship between the amount / concentration of the first ligand in the first environment and the level and / or variation of a detectable signal. The methods and apparatus described herein can also be used to determine the affinity of the first ligand for a translayer protein, for example, by comparing the signal generated in the first environment by one or more known concentrations of the first ligand with signals generated in the same apparatus by other ligands having known affinity for translayer proteins at known concentrations.

[0353] As further described herein, the methods and apparatus of the present invention can also be used to determine whether a given (first) ligand is an agonist, antagonist, inverse agonist, inhibitor, or regulator (e.g., allosteric) modulator of a translayer protein.

[0354] Those skilled in the art will also appreciate that, when using the methods and apparatus of the present invention to determine one or more characteristics of a first ligand, the apparatus of the present invention will typically be first set up or otherwise established without the presence of a first ligand, and then the apparatus will come into contact with the ligand (e.g., by adding the ligand to a first environment), after which a detectable signal (or any change therein) resulting from the presence of the first ligand will be measured (and optionally compared with a signal in the absence of the first ligand and / or having one or more reference values). Therefore, the apparatus described herein in the absence of a first ligand (e.g., before the addition of the first ligand) forms a further aspect of the invention.

[0355] Another aspect of the invention is a method for providing the apparatus of the invention as described herein, the method comprising the step of adding a first ligand to the apparatus of the invention (as described herein), the apparatus (as described herein) not (yet not yet) containing the first ligand. The apparatus thus obtained can then be used to measure or otherwise determine at least one property of the first ligand, and in particular, the properties of the first ligand can be measured or otherwise determined using the apparatus of the invention.

[0356] Those skilled in the art will understand from the disclosure herein that the apparatus of the present invention that does not contain a first ligand (i.e., the apparatus of the present invention that does not yet include a first ligand) will include at least the following elements:

[0357] - A boundary layer separating the first and second environments;

[0358] - Translayer proteins;

[0359] - Ligands present in the second environment for translayer proteins; and

[0360] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0361] The elements are arranged relative to each other in the manner further described herein (i.e., substantially in the same manner as the apparatus of the invention comprising the first ligand) and are operatively connected and / or associated with each other where applicable.

[0362] In particular, the apparatus of the present invention that does not contain a first ligand (i.e., the apparatus of the present invention that does not yet include a first ligand) will include at least the following elements:

[0363] - A boundary layer separating the first and second environments;

[0364] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0365] - A translayer protein that is appropriately fused or connected (directly or via a suitable linker or spacer) to one of the binding members of the binding pair (i.e., forming a first fusion protein); and

[0366] - The second ligand of the translayer protein present in the second environment;

[0367] The elements are arranged relative to each other in the manner further described herein (i.e., substantially in the same manner as described for the apparatus of the invention comprising the first ligand) (and, where applicable, operatively connected and / or associated with each other). In particular, the second member of the binding pair may be part of a second fusion protein (which is distinct from the first fusion protein comprising the translayer protein and the first binding member of the binding pair), which is further described herein.

[0368] More specifically, the apparatus of the present invention that does not contain a first ligand (i.e., the apparatus of the present invention that does not yet include a first ligand) will include at least the following elements:

[0369] - A boundary layer separating the first and second environments;

[0370] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0371] - A first fusion protein comprising a translayer protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in the second environment);

[0372] - A second fusion protein comprising a protein that can directly or indirectly bind to a translayer protein and another binding member of the binding pair, the second fusion protein being present in a second environment;

[0373] The elements are arranged relative to each other in the manner further described herein (i.e., substantially in the same manner as the apparatus of the invention comprising the first ligand) and are operatively connected and / or associated with each other where applicable.

[0374] Other aspects, embodiments, and preferences of the invention’s device without a first ligand are as described herein with respect to the invention’s device having a first ligand but subsequently lacking a first ligand.

[0375] Generally, once the first ligand is added as part of the method described herein, any such device of the present invention that does not contain the first ligand will become a corresponding device of the present invention that has the first ligand. Therefore, another aspect of the present invention is to provide a method for providing a device of the present invention as described herein, the method comprising the step of adding a first ligand to a device of the present invention that does not (yet has not yet) contain the first ligand (as described herein). The device thus obtained can then be used to measure or otherwise determine at least one property of the first ligand, and in particular, the properties of the first ligand that can be measured or otherwise determined using the device of the present invention.

[0376] The present invention also relates to a method for measuring or otherwise determining at least one property of a compound or ligand, the method comprising at least the following steps:

[0377] - Add the compound or ligand as a first ligand to the apparatus of the present invention that does not yet contain a first ligand; and

[0378] - Measure or otherwise determine at least one property of the compound or ligand, wherein the property is a property that can be measured or otherwise determined using the device.

[0379] In this aspect of the invention, the property is preferably a property representing the ability of a compound or ligand to bind to and / or regulate translayer proteins (e.g., affinity).

[0380] The present invention also relates to a method for measuring or otherwise determining the ability of a compound or ligand to alter a detectable signal, said detectable signal being generated by a binding pair present in the apparatus of the present invention further described herein, the method comprising at least the steps of:

[0381] - Add the compound or ligand as a first ligand to the apparatus of the present invention that does not yet contain a first ligand; and

[0382] - Determine whether the addition of the compound or ligand results in a change in a detectable signal generated by the binding pair used in the device, and optionally measure the change in the detectable signal.

[0383] Therefore, in another aspect, the present invention relates to a method comprising at least the following steps:

[0384] a) A device that includes at least the following elements:

[0385] - A boundary layer separating the first and second environments;

[0386] - Translayer proteins;

[0387] - Ligands present in the second environment for translayer proteins; and

[0388] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0389] The elements are arranged relative to each other in a manner further described herein (and, where applicable, operatively connected and / or associated with each other).

[0390] as well as;

[0391] b) Add the first ligand to the first environment.

[0392] The method preferably further includes the following steps:

[0393] c) Measure the signal generated by the binding pair and / or measure the change in the signal generated by the binding pair.

[0394] Therefore, in a more specific aspect, the present invention relates to a method comprising at least the following steps:

[0395] a) A device that includes at least the following elements:

[0396] - A boundary layer separating the first and second environments;

[0397] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0398] - A translayer protein that is appropriately fused or connected (directly or via a suitable linker or spacer) to one of the binding members of the binding pair (i.e., forming a first fusion protein); and

[0399] - The second ligand of the translayer protein present in the second environment;

[0400] The elements are arranged relative to each other in a manner further described herein (and are operatively connected and / or associated with each other where applicable);

[0401] as well as;

[0402] b) Add the first ligand to the first environment.

[0403] The method preferably further includes the following steps:

[0404] c) Measure the signal generated by the binding pair and / or measure the change in the signal generated by the binding pair.

[0405] In another specific aspect, the present invention relates to a method comprising at least the following steps:

[0406] a) A device that includes at least the following elements:

[0407] - A boundary layer separating the first and second environments;

[0408] - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal;

[0409] - A first fusion protein comprising a translayer protein and one of the binding members of the binding pair (i.e., such that the member of the binding pair is present in the second environment);

[0410] - A second fusion protein comprising a protein that can directly or indirectly bind to a translayer protein and another binding member of the binding pair, the second fusion protein being present in a second environment;

[0411] The elements are arranged relative to each other in a manner further described herein (and are operatively connected and / or associated with each other where applicable);

[0412] as well as;

[0413] b) Add the first ligand to the first environment.

[0414] The method preferably further includes the following steps:

[0415] c) Measure the signal generated by the binding pair and / or measure the change in the signal generated by the binding pair.

[0416] 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 small molecules, small peptides, biomolecules, or other chemical entities. Those skilled in the art will also appreciate that the methods according to 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 the device as a first ligand, and in particular to measure or otherwise determine the ability of said compound or ligand to induce a detectable signal change resulting from the binding pair, the ability of said compound or ligand to bind to a translayer protein, the ability of said compound or ligand to induce a conformational change in a translayer protein, and / or the ability of said compound or ligand to regulate (as defined herein) translayer proteins and / or signaling pathways and / or biological mechanisms involving translayer proteins. In particular, said methods can be used to determine whether such compounds or ligands are or can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric modulators) of translayer proteins, and / or signaling pathways and / or biological mechanisms involving translayer proteins. Furthermore, the methods and apparatus of the present invention can be used to identify and / or screen compounds or ligands that have the ability to induce changes in detectable signals generated by binding pairs, the ability to bind to translayer proteins, the ability to induce conformational changes in translayer proteins, the ability to modulate translayer proteins and the signal transduction pathways and / or biological mechanisms involved in translayer proteins, and / or the ability to act as agonists, antagonists, inverse agonists, inhibitors and / or regulators (e.g., allosteric regulators) of translayer proteins, and this use of the methods and apparatus described herein forms a further aspect of the invention.

[0417] It should also be noted that, in another aspect, the methods and apparatus of the present invention can also be used to measure or otherwise determine at least one property of the second ligand, such as the ability of the second ligand to bind to a translayer protein, the ability of the second ligand to bind to and / or stabilize a specific conformation of the translayer protein (e.g., an active and / or druggable conformation), and / or the ability of the second ligand to stabilize a complex of the translayer protein, the first ligand, and the second ligand. Typically, in this aspect of the invention, one or more first ligands having a known ability to bind to and / or modulate translayer proteins will be used to determine whether the apparatus of the present invention containing the (candidate) second ligand produces a detectable signal when the first ligand is added to the apparatus (e.g., at one or more known concentrations).

[0418] 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 (as defined herein) to translayer proteins, and particularly to binding domains or binding units that are specific and / or selective for the translayer protein conformation that occurs when the first ligand used binds to the translayer protein. Binding domains or binding units thus identified, optimized, and / or validated can be used, for example, in the apparatus of the invention (i.e., as part of a second fusion protein) and / or for inducing or stabilizing specific conformations of translayer proteins (e.g., for screening or crystallization purposes, as described in the prior art regarding conformation-specific ligands for GPCRs cited herein). Therefore, for example, this aspect of the invention can be used to identify, optimize, and / or validate ISVDs used as ConfoBody, which can then be used for the purposes described herein and / or for uses known per se for conformation-specific ISVDs. Reference again is made to other prior art cited herein.

[0419] Typically, in this aspect of the invention, the binding domain, binding unit, ligand, or other protein to be tested or verified to bind (directly) to a translayer protein is part of the second fusion protein. Therefore, the invention further relates to a method for measuring or otherwise determining at least one characteristic of the binding domain, binding unit, ligand, or other protein, the method comprising at least the following steps:

[0420] - Provides an apparatus of the present invention that does not yet include a first ligand, wherein the second fusion protein includes the binding domain, binding unit, ligand, or other protein;

[0421] - Add a first ligand to the device; and

[0422] - Determine whether the addition of the first ligand results in a change in a detectable signal generated by the binding pair used in the device, and optionally measure the change in the detectable signal.

[0423] Those skilled in the art will recognize, based on the disclosure herein, that at least one property of the binding domain, binding unit, ligand, or other protein will, in particular, be the ability of the binding domain, binding unit, ligand, or other protein to bind a translayer protein (especially the conformation of the translayer protein when the first ligand used binds to it), the ability of the binding domain, binding unit, ligand, or other protein to stabilize the conformation of the translayer protein when the first ligand used binds to it, and / or the ability of the binding domain, binding unit, ligand, or other protein to promote or induce the formation of a complex of the first ligand used, the translayer 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.

[0424] In another aspect of the invention, the apparatus described herein is again used to measure or otherwise determine at least one property of the second ligand and / or identify, optimize, and / or validate candidate second ligands, but in this respect, the second fusion protein will not contain the second ligand to be tested or the candidate second ligand, but will instead contain a binding domain or binding unit known to bind the second ligand to be tested or the candidate second ligand. In other words, in this aspect, the second fusion protein will contain a binding domain or binding unit that can indirectly (as defined herein) bind to a translayer protein, i.e., through the second ligand to be tested or the candidate second ligand or through a protein complex containing it, provided that the second ligand or complex is capable of binding to the translayer protein (and, in particular, to the conformation of the translayer protein produced when the first ligand binds to the translayer protein). As with the foregoing aspects, this aspect can also be used to identify, optimize, and / or validate (candidate) ligands for translayer proteins, such as synthetic or semi-synthetic analogs or derivatives of ligands that are naturally occurring ligands of translayer proteins. For example, when the translayer protein is a GPCR, this aspect of the invention can be used to identify, optimize, and / or verify analogs or derivatives of G proteins that are native ligands of the GPCR, or to determine whether orthologs of the original (naive) G protein of the relevant GPCR can bind to the GPCR and / or stabilize the complex of the GPCR and the first ligand used.

[0425] Therefore, the present invention further relates to a method for measuring or otherwise determining at least one property of a ligand or other protein, the method comprising at least the following steps:

[0426] - Provides an apparatus of the present invention that does not yet include a first ligand, wherein the ligand or other protein is present and / or used as a second ligand, and wherein the second fusion protein includes a binding domain or binding unit that can bind the ligand or other protein and / or bind a protein complex containing the ligand or other protein;

[0427] - Add a first ligand to the device; and

[0428] - Determine whether the addition of the first ligand results in a change in a detectable signal generated by the binding pair used in the device, and optionally measure the change in the detectable signal.

[0429] As described herein, in one particular aspect of the invention, the method of the invention is carried out using suitable cells or cell lines, wherein all elements of the device of the invention are suitably present and arranged to provide an operable device of the invention. Such cells or cell lines will suitably contain a translayer protein (2) in their cell walls or cell membranes, i.e., such that the translayer protein (2) is present and spans the cell wall or cell membrane, such that at least a portion of the amino acid sequence of the translayer protein extends (as defined herein) into the extracellular environment, and at least one other portion of the amino acid sequence of the translayer protein extends (as defined herein) into the intracellular environment. Furthermore, preferably and as further described herein, the translayer protein will form part of a first fusion protein as described herein, and the device will also contain a second fusion protein as described herein. More preferably, the extracellular environment will be a “first environment” (i.e., an environment in which the first ligand (3) is present or to which the first ligand (3) is added) and the intracellular environment will be a “second environment” (i.e., an environment in which the binding pair (6 / 7) and the second fusion protein are present).

[0430] Therefore, in another respect, the present invention relates to the method or apparatus as described herein, wherein the boundary layer (2) is the cell wall or membrane.

[0431] As also described herein, when the method of the present invention is carried out in cells or suitable cell lines, the cells or cell lines used are preferably such that they suitably express one or more, and preferably all, of the following elements of the device of the present invention:

[0432] - A first fusion protein comprising a translayer protein (2) and a first binding member (6);

[0433] - A second fusion protein containing a second binding member (7) and a protein that can bind directly or indirectly (as defined herein) to a translayer protein (2);

[0434] and / or

[0435] - When the second fusion protein binds indirectly to the translayer protein (2), the second ligand (4) and / or the protein constituting the protein complex (12) are involved.

[0436] In the context of a cell or cell line expressing one or more elements of the apparatus of the present invention, and more generally in the context of this specification and claims, the term “suitably expressed” means that a cell or cell line expresses or is able to express (i.e., under the conditions for carrying out the method of the present invention) a nucleotide sequence or nucleic acid encoding said element, such that when such element is expressed, it can be used as an operable part of the apparatus of the present invention. For example, with respect to a translayer protein (2), this means that the translayer protein is expressed as part of a first fusion protein such that the expressed translayer protein (2) is suitably anchored or otherwise incorporated into the cell wall or cell membrane of the cell such that the translayer protein crosses the cell wall or cell membrane, wherein at least a portion of the amino acid sequence of the translayer protein extends (as defined herein) into the extracellular environment and at least one other portion of the amino acid sequence of the translayer protein extends (as defined herein) into the intracellular environment. Regarding the first and second fusion proteins, “appropriate expression” means that the first and second fusion proteins are expressed such that (and most preferably in the intracellular environment) the first and second binding members of the binding pair (6 / 7) can come into contact with or be close to each other when the second fusion protein binds directly or indirectly to the translayer protein (2) in a manner further described herein.

[0437] Any suitable expression of each such element of the device of the present invention may be transient or constitutive, provided that all the necessary elements of the device of the present invention are present in sufficient quantity and operablely at the point in time when the cell is used to perform the method of the present invention.

[0438] In one aspect of the invention, in one embodiment of the invention, wherein the second fusion protein indirectly binds to the translayer protein (i.e., wherein the second ligand (4) is not part of the second fusion protein), the cells or cell lines used are preferably such that they naturally express the second ligand (4) and / or proteins constituting the protein complex (12). For example, but not limited to, in this aspect of the invention, when the translayer protein (2) is a G PCR, the second ligand (4) may be a G protein naturally expressed by the cells or cell lines used, and / or the protein complex (12) may be a protein trimer comprising the G-α subunit, G-β subunit, and G-γ subunit G naturally expressed by the cells or cell lines 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 (and particularly intracellular ligands) of the translayer protein (2) and / or naturally express one or more ligands that can be used as the second ligand of the translayer protein (2), depending on the translayer protein (2) being used or screened.

[0439] The cell or cell line can be any cell or cell line suitable for the methods and apparatus 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 HEK 293T.

[0440] Suitable techniques for transient or stable expression of desired proteins in such cells or cell lines so that translayer proteins (2) are properly anchored to the cell wall or cell membrane of said cells are clear to those skilled in the art and include, for example, techniques involving the use of suitable transfection reagents, such as X-tremeGENE™ or polyethyleneimine (PEI) from SigmaAldrich.

[0441] When the present invention is carried out using cells or cell lines that appropriately express one or more elements of the device of the present invention, the method of the present invention generally further includes the step of culturing or maintaining the cells under conditions that allow the cells or cell lines to appropriately express the elements.

[0442] Therefore, in another aspect, the present invention relates to cells or cell lines comprising fusion proteins, said fusion proteins comprising a translayer 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, said binding pair comprising at least the binding domain or binding unit as the first binding member and another binding domain or binding unit as the second binding member, wherein said first and second binding members of the binding pair enable them to generate a detectable signal when in contact with or in close proximity to each other. The invention also relates to cells or cell lines that express or are capable of expressing (i.e., under suitable conditions) such fusion proteins.

[0443] Such cells or cell lines may be as further described herein, and preferably express or are capable of expressing the fusion protein in such a manner that the translayer protein is incorporated into and crosses the cell wall or cell membrane of the cell or cell line. More preferably, at least a portion of the amino acid sequence of the translayer protein extends (as defined herein) into the extracellular environment, and at least one other portion of the amino acid sequence of the translayer protein extends (as defined herein) into the intracellular environment. More preferably, in said cells or cell lines, a first binding member of the binding pair is present (as defined herein) in the intracellular environment of the cell and / or the cell or cell line expresses or is capable of expressing the fusion protein such that, upon such expression, the first binding member is present (as defined herein) in the intracellular environment of the cell.

[0444] Furthermore, the translayer protein present in the fusion protein is preferably, as further described herein, and more preferably, has at least two ligand-binding sites, one of which extends (as defined herein) into the extracellular environment and the other of which extends (as defined herein) into the intracellular environment. Moreover, as described herein, the translayer protein is preferably such that it undergoes a conformational change from one conformation to another (and particularly, from a substantially inactive or less active conformation to an active or more active conformation) when a ligand binds to the ligand-binding site on the translayer protein, and especially when a ligand present in the extracellular environment binds to the ligand-binding site on the translayer protein present in the extracellular environment (as defined herein). As further described herein, the translayer protein is preferably further stabilized in a functional and / or active (or more active) conformation by binding to an intracellular binding site on the translayer protein via a suitable ligand, binding domain, or binding unit (e.g., the ConfoBody or a native ligand of the translayer protein described herein, such as a native intracellular ligand) and / or a binding site on the translayer protein that serves as an intracellular binding site when the translayer protein is in its native environment, and / or when the translayer protein is present in the cells or cell lines used in this invention, and preferably both). This is particularly true in drugable conformations and / or ligand-binding conformations, and more particularly in agonist-binding conformations. In particular, also as described herein, the translayer protein may be able to form a complex when a first ligand binds to an extracellular binding site and a second ligand binds to an intracellular binding site. More specifically, as described herein, translayer proteins may be able to form complexes in which the translayer protein is in a functional or active conformation induced by a first ligand binding to an extracellular binding site, wherein the active or functional conformation is stabilized by the binding of a second ligand to an intracellular binding site, the second ligand being capable of stabilizing the functional, active, or ligand-bound conformation and / or the complex. In a preferred but non-limiting aspect, the translayer protein is a transmembrane protein, particularly 7TM. Furthermore, the members of the binding pair and any linkers used may be as further described herein.

[0445] In another aspect, the present invention relates to cells or cell lines comprising fusion proteins comprising proteins capable of (directly or indirectly, as described herein) binding to translayer proteins (as described herein), the proteins being fused directly or via suitable linkers to a binding domain or binding unit that is a first binding member of a binding pair, the binding pair comprising at least a first binding member and the binding domain or binding unit as a second binding member, wherein the first and second binding members of the binding pair enable them to generate a detectable signal when in contact with or in close proximity to each other. The invention also relates to cells or cell lines that express or are capable of expressing (i.e., under suitable conditions) such fusion proteins.

[0446] The proteins present in the fusion protein and capable of binding to transmembrane proteins are preferably as further described herein with respect to proteins that may be present in the second fusion protein. Furthermore, members of the binding pair and any linkers used may be as further described herein. Also as stated herein, the protein may bind directly (as described herein) or indirectly (as described herein). Again, in this respect, the transmembrane proteins that the protein can bind to are preferably also as further described herein, and may be, in particular, transmembrane proteins, more particularly 7TM.

[0447] As described herein, when a protein present in the fusion protein directly binds to a translayer protein, it preferably specifically binds to one or more functional, active, and / or druggable conformations of the translayer protein, inducing and / or stabilizing one or more functional, active, and / or druggable conformations of the translayer protein (and / or causing conformational equilibrium shift of the translayer protein towards one or more such conformations); and / or inducing and / or stabilizing a complex of the protein, the translayer protein, and another ligand of the translayer protein (all as further described herein). Furthermore, when a protein present in the fusion protein directly binds to a translayer protein, the protein preferably binds to an intracellular binding site on the translayer protein. The intracellular binding site on the translayer protein may be a binding site on the translayer protein that serves as an intracellular binding site when the translayer protein is in its native environment, and / or a binding site on the translayer protein that serves as an intracellular binding site when the translayer protein is present in the cells or cell lines used in this invention (and preferably both).

[0448] Furthermore, when the protein present in the fusion protein binds directly to the translayer protein, it is preferably a VHH domain or a binding domain or binding unit derived from the VHH domain, particularly a ConfoBody (as described herein).

[0449] As described herein, when the protein present in the fusion protein binds indirectly to the translayer protein, it is preferably such that it can bind to a ligand that can bind to the translayer protein. When the second ligand does not form part of the second fusion protein, the ligand may be as described herein with respect to "second ligand". Again, the ligand is preferably such that it binds specifically to one or more functional, active, and / or druggable conformations of the translayer protein, such that it induces the formation and / or stabilizes one or more functional, active, and / or druggable conformations of the translayer protein (and / or causes a conformational equilibrium shift of the translayer protein toward one or more such conformations); and / or causes it to induce the formation and / or stabilize a complex of the ligand, the translayer protein, and another ligand of the translayer protein (all as further described herein). Furthermore, the ligand is preferably such that it can bind to an intracellular binding site on the translayer protein. The intracellular binding site on the translayer protein can be a binding site on the translayer protein that serves as an intracellular binding site when the translayer protein is in its native environment, and / or a binding site on the translayer protein that serves as an intracellular binding site when the translayer protein is present in the cells or cell lines used in this invention (and preferably both). Furthermore, as described herein, the ligand can also be part of a protein complex that can bind to the translayer protein (i.e., to the intracellular binding site on the translayer protein), in which case the protein present in the fusion protein can also bind to the protein complex.

[0450] Furthermore, when the protein present in the fusion protein indirectly binds to the translayer protein, it is preferably a VHH domain or a binding domain or binding unit derived from the VHH domain. In a preferred aspect, when the protein present in the fusion protein indirectly binds to the translayer protein, and the translayer protein is a GPCR, the ligand binding 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 G-α, G-β, and G-γ subunits. Furthermore, the G protein may be native to the cells or cell lines used, or may be a suitable analogue or derivative of a native G protein (as described herein and recombinantly expressed in the cells or cell lines) or a suitable orthogonal homolog of a native G protein of the cells or cell lines used (again, recombinantly expressed in the cells or cell lines used).

[0451] Regardless of whether the protein present in the fusion protein directly or indirectly binds to a translayer protein, the cell or cell line preferably expresses or is capable of expressing the fusion protein in its intracellular environment. Another aspect of the invention relates to such cells or cell lines containing such fusion proteins in their intracellular environment.

[0452] In another aspect, the present invention relates to cells or cell lines comprising a first fusion protein and a second fusion protein, wherein:

[0453] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0454] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0455] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, which is fused directly or via a suitable linker to a second binding member of the binding pair.

[0456] The present invention also relates to cells or cell lines that express or are able to express (i.e., under suitable conditions) such first and second fusion proteins.

[0457] The present invention particularly relates to cells or cell lines comprising a first fusion protein and a second fusion protein, wherein:

[0458] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0459] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0460] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0461] - When the second fusion protein binds to a translayer protein that forms part of the first fusion protein (directly or indirectly, as described herein), the first and second binding members of the binding pair may come into contact with or be close to each other.

[0462] The present invention also relates to cells or cell lines comprising a first fusion protein and a second fusion protein, wherein:

[0463] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0464] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0465] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0466] - The first and second binding members of the binding pair are present in the intracellular environment of the cell (as defined herein).

[0467] The present invention also relates to cells or cell lines comprising a first fusion protein and a second fusion protein, wherein:

[0468] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0469] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0470] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0471] - When the second fusion protein binds (directly or indirectly, as described herein) to a translayer protein that forms part of the first fusion protein, the cell or cell line is able to generate a detectable signal (and in particular a detectable signal generated by the first and second binding members of the binding pair).

[0472] The present invention also relates to cells or cell lines comprising a first fusion protein and a second fusion protein, wherein:

[0473] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0474] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0475] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0476] - When a ligand of a translayer protein present in the extracellular environment binds to the translayer protein, the cell or cell line produces a detectable signal and / or a change in a detectable signal (and in particular a detectable signal and / or such a change in signal produced by the first and second binding members of the binding pair).

[0477] In one particular aspect, the present invention relates to cells or cell lines comprising a first fusion protein and a second fusion protein, wherein:

[0478] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0479] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0480] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0481] - When an agonist of a translayer protein present in the extracellular environment binds to the translayer protein, the cell or cell line produces a detectable signal and / or a change in a detectable signal (and in particular a detectable signal and / or such a change in signal produced by the first and second binding members of the binding pair).

[0482] Furthermore, such cells or cell lines containing or expressing such first and second fusion proteins may, as further described herein, and preferably express or be able to express the first fusion protein in such a manner that the translayer protein is incorporated into and crosses the cell wall or cell membrane of the cell or cell line, more preferably such that at least a portion of the amino acid sequence of the translayer protein extends (as defined herein) into the extracellular environment, and at least one other portion of the amino acid sequence of the translayer protein extends (as defined herein) into the intracellular environment.

[0483] The cells or cell lines are preferably configured to express or be able to express the first and second fusion proteins such that, during such expression, when the second fusion protein binds (directly or indirectly, as described herein) to a translayer protein forming part of the first fusion protein, the first and second binding members of the binding pair can come into contact with or be close to each other. It will be apparent to those skilled in the art that such cells or cell lines will express the first and second fusion proteins in such a manner that, during such expression, the first and second binding members of the binding pair will be present (as defined herein) in the same environment relative to the cell wall or cell membrane. Preferably, the cells or cell lines are configured to express or be able to express the first and second fusion proteins such that, during such expression, both the first and second binding members of the binding pair will be present (as defined herein) in the intracellular environment of the cell. This also generally means that the cells or cell lines are preferably configured to express or be able to express the second fusion protein in their intracellular environment.

[0484] Furthermore, in aspects of the invention relating to the expression or ability to express such first and second fusion proteins, translayer proteins, proteins that can directly or indirectly bind to translayer proteins, members of binding pairs, and any adapters used may be as further described herein.

[0485] In a further aspect, the invention also relates to methods, and particularly to assays or screening methods using the cells or cell lines described herein. As further described herein, such assays and screening methods can be particularly used to identify compounds and other chemical entities that bind (and particularly specifically bind) to translayer proteins, modulate translayer proteins, and / or modulate the signaling, signaling pathways, and / or biological or physiological activities involving translayer proteins, their signaling, and / or their signaling pathways. Therefore, the cells and cell lines described herein can be used in methods for identifying compounds or other chemical entities that can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric) of translayer proteins.

[0486] This invention also relates to the use of the cells or cell lines described herein, particularly in assay and screening methods and techniques. The methods and uses of the apparatus of this invention can be further described herein, and will generally also include the step of culturing or maintaining the cells under conditions that allow the cells or cell lines to adequately express the desired fusion protein or protein.

[0487] Similarly, in all these respects, such cells, cell lines, and their uses are preferred as further described herein.

[0488] In another aspect of the invention, the method of the invention is carried out using suitable liposomes or vesicles, wherein all elements of the device of the invention are suitably present and arranged to provide an operable device of the invention. Such liposomes or vesicles will suitably contain a translayer protein (2) in their walls or membranes, i.e., such that the translayer protein (2) is present and spans the walls or membranes of the liposome or vesicle, such that at least a portion of the amino acid sequence of the translayer protein extends (as defined herein) into the environment outside the liposome or vesicle, and at least one other portion of the amino acid sequence of the translayer protein extends (as defined herein) into the environment inside the liposome or vesicle. Furthermore, preferably and as further described herein, in the aspect of the invention carried out in liposomes or vesicles, the environment outside the liposome or vesicle will be a “first environment” (i.e., an 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 will be a “second environment” (i.e., an environment in which the binding pair (6 / 7) and the second fusion protein are present).

[0489] Therefore, in another aspect, the present invention relates to the method or apparatus as described herein, wherein the boundary layer (2) is the wall or membrane of a liposome or other (suitable) vesicle.

[0490] As also described herein, when the method of the present invention is carried out in liposomes or vesicles, the liposomes or vesicles preferably suitably include (i.e., in a manner that provides an operable device of the present invention) the following elements of the device of the present invention:

[0491] - A first fusion protein comprising a translayer protein (2) and a first binding member (6);

[0492] - A second fusion protein containing a second binding member (7) and a protein that can bind directly or indirectly (as defined herein) to a translayer protein (2);

[0493] and / or

[0494] - When the second fusion protein binds indirectly to the translayer protein (2), the second ligand (4) and / or the protein constituting the protein complex (12) are involved.

[0495] Liposomes or vesicles containing the said elements can typically be provided by forming liposomes or vesicles in the presence of the relevant elements of the device of the present invention, such that the elements are suitably incorporated into the liposomes or vesicles. This can typically be carried out by methods and techniques known per se for forming liposomes or vesicles, preferably in a suitable aqueous buffer or another suitable aqueous medium. Such methods may also include the step of separating liposomes or vesicles in which the elements of the desired device of the present invention are suitably and operably incorporated from all desired elements that do not contain the device and / or from vesicles or liposomes in which the elements do not form the operable device of the present invention. Device elements incorporated into liposomes or vesicles can be provided in a manner known per se, for example by recombinant expression of a suitable host cell or host organism, followed by isolation and purification of the expressed elements.

[0496] Typically, in aspects of the invention carried out in liposomes or vesicles, where the second ligand does not form part of the second fusion protein, a sufficient amount of the second ligand should be provided and appropriately contained within the vesicles or liposomes.

[0497] Liposomes or vesicles can be any liposomes or vesicles suitable for the methods and devices of the present invention, including but not limited to liposomes based on 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), dioleoylphosphatidylethanolamine (DOPE), or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC). Liposomes and vesicles can also be liposomes or vesicles comprising and / or based on (e.g., reconstructed from) one or more membrane fractions obtained from cells expressing desired elements of the devices of the present invention.

[0498] Therefore, in another aspect, the present invention relates to liposomes or vesicles comprising a fusion protein comprising a translayer 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 another binding domain or binding unit as the second binding member, wherein the first and second binding members of the binding pair enable them to generate a detectable signal when they are in contact with or in close proximity to each other. The invention also relates to a method of providing such liposomes or vesicles, the method comprising at least the steps of incorporating such a fusion protein into the liposome or vesicle and / or forming the liposome or vesicle in the presence of said fusion protein.

[0499] As further described herein, the liposomes or vesicles preferably cause the translayer protein to be anchored or otherwise suitably incorporated into and across the wall or membrane of the liposome or vesicle, more preferably causing at least a portion of the amino acid sequence of the translayer protein to extend (as defined herein) into an environment outside the liposome or vesicle, and at least one other portion of the amino acid sequence of the translayer protein to extend (as defined herein) into an environment within the liposome or vesicle. More preferably, the first binding member of the binding pair is present (as defined herein) in an environment within the liposome or vesicle.

[0500] Furthermore, the translayer protein present in the fusion protein is preferably, as further described herein, and more preferably, has at least two ligand-binding sites, one of which extends (as defined herein) into the environment outside the liposome or vesicle, and the other of which extends (as defined herein) into the environment inside the liposome or vesicle. Moreover, as described herein, the translayer protein is preferably such that when a ligand binds to the ligand-binding site on the translayer protein, and particularly when a ligand present in the external environment of the liposome or vesicle binds to the ligand-binding site on the translayer protein present in the external environment of the liposome or vesicle, it undergoes a conformational change from one conformation to another (and particularly, from a substantially inactive or less active conformation to an active or more active conformation). As further described herein, the translayer protein is preferably further stabilized in a functional and / or active (or more active) conformation by binding to an intracellular binding site on the translayer protein via a suitable ligand, binding domain, or binding unit (e.g., the ConfoBody or the native ligand of the translayer protein described herein) with the binding site on the translayer protein (which may be the binding site on the translayer protein as an intracellular binding site when the translayer protein is in its native environment, and / or the binding site on the translayer protein in the internal environment of the liposome or vesicle used in this invention, and preferably both) through binding to a binding site on the translayer protein in the internal environment of the liposome or vesicle, and preferably both). Specifically, as also described herein, the translayer protein may be able to form a complex when a first ligand binds to a binding site in the external environment of the liposome or vesicle (as defined herein) and a second ligand binds to a binding site in the internal environment of the liposome or vesicle (as defined herein). More specifically, as described herein, translayer proteins may be able to form complexes in which the translayer protein is in a functional or active conformation induced by the binding of a first ligand to a binding site present in the external environment of a liposome or vesicle (as defined herein), wherein the active or functional conformation is stabilized by the binding of a second ligand to a binding site present in the internal environment of a liposome or vesicle (as defined herein), the second ligand being capable of stabilizing the functional, active, or ligand-bound conformation and / or the complex. In a preferred but non-limiting aspect, the translayer protein is a transmembrane protein, particularly 7TM. Furthermore, the members of the binding pair and any linkers used may be as further described herein.

[0501] In another aspect, the present invention relates to liposomes or vesicles comprising a fusion protein comprising a protein capable of (directly or indirectly, as described herein) binding to a translayer protein (as described herein), the protein being 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 a first binding member and the binding domain or binding unit as a second binding member, wherein the first and second binding members of the binding pair enable them to generate a detectable signal when they are in contact with or in close proximity to each other. The invention also relates to a method of providing such liposomes or vesicles, the method comprising at least the steps 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.

[0502] The proteins present in the fusion protein and capable of binding to transmembrane proteins are preferably as further described herein with respect to proteins that may be present in the second fusion protein. Furthermore, members of the binding pair and any linkers used may be as further described herein. Also as stated herein, the protein may bind directly (as described herein) or indirectly (as described herein). Again, in this respect, the transmembrane proteins that the protein can bind to are preferably also as further described herein, and may be, in particular, transmembrane proteins, more particularly 7TM.

[0503] As described herein, when a protein present in the fusion protein directly binds to a translayer protein, it is preferably such that it specifically binds to one or more functional, active, and / or druggable conformations of the translayer protein, such that it induces the formation and / or stabilizes one or more functional, active, and / or druggable conformations of the translayer protein (and / or causes a conformational equilibrium shift of the translayer protein toward one or more such conformations); and / or causes it to induce the formation and / or stabilize a complex of the protein, the translayer protein, and another ligand of the translayer protein (all as further described herein). Furthermore, when a protein present in the fusion protein directly binds to a translayer protein, the protein is preferably such that it can bind to the following binding sites: when the translayer protein is in its native state, a binding site on the translayer protein that is an intracellular binding site; and / or when the translayer protein is present in liposomes or vesicles (when said liposomes or vesicles are used in this invention), a binding site on the translayer protein present in the internal environment of the liposomes or vesicles (as defined herein), (and preferably both).

[0504] Furthermore, when the protein present in the fusion protein binds directly to the translayer protein, it is preferably a VHH domain or a binding domain or binding unit derived from the VHH domain, particularly a ConfoBody (as described herein).

[0505] As described herein, when the protein present in the fusion protein binds indirectly to the translayer protein, it is preferably such that it can bind to a ligand that can bind to the translayer protein. When the second ligand does not form part of the second fusion protein, the ligand may be as described herein with respect to "second ligand". Again, the ligand is preferably such that it binds specifically to one or more functional, active, and / or druggable conformations of the translayer protein, such that it induces the formation and / or stabilizes one or more functional, active, and / or druggable conformations of the translayer protein (and / or causes a conformational equilibrium shift of the translayer protein toward one or more such conformations); and / or causes it to induce the formation and / or stabilize a complex of the ligand, the translayer protein, and another ligand of the translayer protein (all as further described herein). Furthermore, the ligand is preferably such that it can bind to the following binding sites: a binding site on a translayer protein that is an intracellular binding site when the translayer protein is in its native state, and / or a binding site on a translayer protein present in the internal environment of a liposome or vesicle (as defined herein) when the translayer protein is present in a liposome or vesicle (when said liposome or vesicle is used in this invention), (and preferably both). Additionally, as described herein, the ligand can also be part of a protein complex that can bind to a translayer protein, in which case the protein present in the fusion protein can also bind to said protein complex.

[0506] Furthermore, when the protein present in the fusion protein indirectly binds to the translayer protein, it is preferably a VHH domain or a binding domain or binding unit derived from the VHH domain. In a preferred aspect, when the protein present in the fusion protein indirectly binds to the translayer protein, and the translayer protein is a GPCR, the ligand binding 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 G-α, G-β, and G-γ subunits.

[0507] Regardless of whether the protein present in the fusion protein directly or indirectly binds to a translayer protein, the fusion protein is preferably located in an environment within liposomes or vesicles (as defined herein). Furthermore, even when the second ligand is not part of the fusion protein, the environment within the liposomes or vesicles will also contain an appropriate amount of the second ligand.

[0508] In another aspect, the present invention relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein, wherein:

[0509] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0510] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0511] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, which is fused directly or via a suitable linker to a second binding member of the binding pair.

[0512] The present invention also relates to a method for providing such liposomes or vesicles, the method comprising at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming liposomes or vesicles in the presence of the fusion protein.

[0513] The present invention particularly relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein, wherein:

[0514] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0515] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0516] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0517] - When the second fusion protein binds to a translayer protein that forms part of the first fusion protein (directly or indirectly, as described herein), the first and second binding members of the binding pair may come into contact with or be close to each other.

[0518] Furthermore, the present invention also relates to a method for providing such liposomes or vesicles, the method comprising at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming liposomes or vesicles in the presence of the fusion protein.

[0519] The present invention also relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein, wherein:

[0520] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0521] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0522] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0523] - The first and second binding members of the binding pair are located in the environment inside the liposomes or vesicles (as defined herein).

[0524] Furthermore, the present invention also relates to a method for providing such liposomes or vesicles, the method comprising at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming liposomes or vesicles in the presence of the fusion protein.

[0525] The present invention further relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein, wherein:

[0526] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0527] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0528] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0529] - When the second fusion protein binds (directly or indirectly, as described herein) to a translayer protein that forms part of the first fusion protein, the liposomes or vesicles are able to generate detectable signals (and in particular detectable signals generated by the first and second binding members of the binding pair).

[0530] Furthermore, the present invention also relates to a method for providing such liposomes or vesicles, the method comprising at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming liposomes or vesicles in the presence of the fusion protein.

[0531] The present invention further relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein, wherein:

[0532] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0533] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0534] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0535] - When a ligand of a translayer protein present in the environment outside the liposome or vesicle binds to the translayer protein, the liposome or vesicle produces a detectable signal and / or a change in a detectable signal (and in particular a detectable signal and / or such a change in signal produced by the first and second binding members of the binding pair).

[0536] Furthermore, the present invention also relates to a method for providing such liposomes or vesicles, the method comprising at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming liposomes or vesicles in the presence of the fusion protein.

[0537] In one particular aspect, the present invention relates to liposomes or vesicles comprising a first fusion protein and a second fusion protein, wherein:

[0538] - The first fusion protein comprises a binding domain or binding unit as a first binding member of the binding pair, and the second fusion protein comprises a binding domain or binding unit as a second binding member of the binding pair, wherein the first and second binding members of the binding pair enable the generation of a detectable signal when they are in contact with or close to each other; and

[0539] - The first fusion protein comprises a translayer protein (as described herein) that is fused directly or via a suitable linker to the first binding member of the binding pair; and

[0540] - The second fusion protein comprises a protein capable of binding (directly or indirectly, as described herein) to the translayer protein, the protein being fused directly or via a suitable linker to a second binding member of the binding pair; and

[0541] When an agonist of a translayer protein present in the environment outside a liposome or vesicle binds to the translayer protein, the liposome or vesicle produces a detectable signal and / or a change in a detectable signal (and in particular a detectable signal and / or such a change in signal produced by the first and second binding members of the binding pair).

[0542] Furthermore, the present invention also relates to a method for providing such liposomes or vesicles, the method comprising at least the steps of incorporating the fusion protein into the liposomes or vesicles and / or forming liposomes or vesicles in the presence of the fusion protein.

[0543] Such liposomes or vesicles containing such first and second fusion proteins may be as further described herein, and preferably have translayer proteins that are suitably anchored to or otherwise incorporated into the wall or membrane of the liposome or vesicle and span said wall or membrane, more preferably such that at least a portion of the amino acid sequence of the translayer protein extends out (as defined herein) into the environment outside the liposome or vesicle, and at least one other portion of the amino acid sequence of the translayer protein extends out (as defined herein) into the environment inside the liposome or vesicle.

[0544] When the second fusion protein binds (directly or indirectly, as described herein) to a translayer protein forming part of the first fusion protein, the liposome or vesicle preferably allows the first and second binding members of the binding pair to be in contact with or close to each other. 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 will be present (as defined herein) in the same environment as the wall or membrane of the liposome or vesicle. Preferably, the liposome or vesicle allows both the first and second binding members of the binding pair to be present (as defined herein) in the environment within the liposome or vesicle.

[0545] Furthermore, in aspects of the invention relating to liposomes or vesicles comprising such first and second fusion proteins, translayer proteins, proteins that can directly or indirectly bind to translayer proteins, members of binding pairs, and any adapters used may be as further described herein.

[0546] In a further aspect, the invention also relates to methods, and particularly to assays or screening methods using liposomes or vesicles as described herein. As further described herein, such assays and screening methods can be particularly used to identify compounds and other chemical entities that bind (and particularly specifically bind) to translayer proteins, modulate translayer proteins, and / or modulate the signaling, signaling pathways, and / or biological or physiological activities involving translayer proteins, their signaling, and / or their signaling pathways. Therefore, liposomes or vesicles as described herein can be used in methods for identifying compounds or other chemical entities that can act as agonists, antagonists, inverse agonists, inhibitors, or modulators (e.g., allosteric modulators) of translayer proteins.

[0547] This invention also relates to the use of liposomes or vesicles as described herein, particularly in assay and screening methods and techniques. Such methods and uses can be further described herein with reference to the methods and uses of the apparatus of this invention.

[0548] Similarly, in all these respects, such liposomes or vesicles and their uses are preferred as further described herein.

[0549] Those skilled in the art will appreciate that compounds discovered, developed, generated, and / or optimized using the methods and techniques described herein can be used for any suitable or desired purpose. Such purpose is generally related to a target against which the compound is screened / generated, the signal transduction, pathway, and / or mechanism of action associated with that target, and / or the biological, physiological, and / or pharmacological function relating to said target, pathway, signal transduction, and / or mechanism of action. Typically and preferably, the compounds of the present invention will be, and / or will be selected, such that they are capable of modulating said target, signal transduction, pathway, mechanism of action, and / or said biological, physiological, and / or pharmacological function in a desired or anticipated manner. As described herein, this modulation can take any desired or anticipated form, including but not limited to upregulation and downregulation of said target, signal transduction, pathway, mechanism of action, and / or said biological, physiological, and / or pharmacological function. Therefore, the compounds of the present invention can, for example, be used as agonists, antagonists, inverse agonists, inhibitors, or another type of modulator (e.g., allosteric modulators) of said target and / or its signal transduction, pathway, mechanism of action, and / or said biological, physiological, and / or pharmacological function. All of these can be determined using appropriate in vitro, cellular, and / or in vivo assays (e.g., appropriate efficacy or potency assays) and / or appropriate animal models, depending on the specific target, signal transduction, pathway, mechanism of action, and / or the biological, physiological, and / or pharmacological function involved. Appropriate assays and models will be clear to those skilled in the art.

[0550] Generally, when the compounds of the present invention are agonists (or antagonists) of a target, they will also be agonists (or antagonists) of the signal transduction, pathway, mechanism of action, and / or the biological, physiological, and / or pharmacological functions involved in the target. However, as will be apparent to those skilled in the art, the compounds of the present invention may also (and not excluded outside the scope of the invention) be, for example, but not limited to, any kind of hypothesis or interpretation, agonists (or antagonists) of a target or its signal transduction, but their action as agonists (or antagonists) of a target or its signal transduction results in their action as antagonists (or antagonists) of aspects of the biological, physiological, and / or pharmacological functions involved in the target or signal transduction.

[0551] In one aspect of the practice of this invention, the apparatus and methods described herein will be used to test whether a compound or ligand present in the environment [A] (e.g., in the extracellular environment if the invention is carried out in cells, or in the environment outside the liposomes or vesicles if the invention is carried out in liposomes or vesicles) is capable of generating a detectable signal (i.e., in a manner that allows said compound or ligand to bind to binding sites (8) on translayer proteins (2)). Similarly, when the methods and apparatus of the invention are used to screen groups, series, or libraries of compounds or ligands, the methods and apparatus of the invention will be used to determine which compounds or ligands from said group, series, or library generate a detectable signal (i.e., “hit”).

[0552] Typically, in this invention, the detectable signal is measured by measuring the signal generated (or may be generated) by the pair (6 / 7) (i.e., the signal generated when the first member (6) and the second member (7) are in contact with each other, close to each other, or otherwise associated with each other to generate a detectable signal). It should be noted that in this invention, changes in the signal are typically measured, including those referred to herein as "generating a detectable signal".

[0553] The change can be an increase in signal compared to a baseline level (which may also be below the detection limit of the device used to measure the signal, in which case the signal will be detected in the presence of the compound's ligand (where no signal was previously measured), and this is also included in the term "increase in signal" as used herein) or a decrease in signal compared to a baseline level.

[0554] In practice of the present invention, when the transmembrane protein (2) is a GPCR or 7TM (and, as will be clear to those skilled in the art based on the disclosure herein, generally also when the transmembrane protein (2) is another transmembrane protein involved in signal transduction), an increase in signal will indicate that the compound or ligand is acting as an agonist of the receptor. Conversely, when the transmembrane protein (2) is a GPCR or 7TM (and generally also when the transmembrane protein (2) is another transmembrane protein involved in signal transduction), a decrease in signal indicates that the compound or ligand is acting as a deflector agonist of the receptor. Thus, advantageously, the methods and apparatus of the present invention can make it possible to identify agonists and deflectors of GPCRs or 7TMs (or other receptors) and / or distinguish between agonists and deflectors (or vice versa). For example, refer to the examples given in Example 6 and Figure 12 The results are shown in the image.

[0555] It should be noted that the present invention is not limited to any particular mechanism, explanation, or hypothesis regarding how the contact between the compound or ligand and the binding site (8) on the translayer protein (2) results in a change in the detectable signal. However, hypotheses will involve one or more of the following mechanisms.

[0556] As described herein, generally speaking, the translayer protein (2) will be a protein that exists in equilibrium between two or more conformations in the absence of a compound or ligand, and some of these conformations have a lower affinity (or even virtually no affinity) for the binding interaction between the translayer protein (2) (binding site (9)) and the second ligand (4) compared to the other conformations. Typically, in this invention, the level of detectable signal measured (or measurable) at a certain point in time (or within a certain time interval) will depend on how much of the second ligand (4) (i.e., the second fusion protein) binds or becomes bound to the translayer protein, because the binding of the second fusion protein to the translayer protein (2) will bring (more) second binding members (7) closer to the first binding member (6), resulting in a detectable signal (or an increase in the detectable signal compared to the background signal level, which may be present due to the binding of “free” second ligands to the binding member (6), and this background level is typically insignificant or below the detection limit).

[0557] Therefore, in general, in this invention, the shift in conformational equilibrium of a translayer protein (2) from a state having low (lower) or essentially no affinity for the second ligand (4) to a state having binding affinity for the second ligand (4) and / or a state having better binding affinity for the second ligand (4) typically results in an increase in detectable signal.

[0558] Assuming that in this invention, contact between the translayer protein (2) and a compound or ligand acting as an agonist will shift this balance to a conformational state with binding affinity for the second ligand (4) and / or a state with better binding affinity, resulting in a detectable increase in signal. For example, this could be because the presence of the agonist compound or ligand allows the formation of new conformational states that would not be formed in the absence of the compound or ligand (e.g., the formation of a complex containing the compound or ligand, the translayer protein, and the second ligand), because the agonist compound or ligand stabilizes (or generally favors) the conformational state with high (higher) affinity for the second ligand (4), and / or because the agonist compound or ligand results in a new conformation that can bind the second ligand. Any one or more of these and other mechanisms (or any combination thereof) may be involved at any time, but the overall effect will be an increase in the amount of the second ligand (4) associated with the translayer protein (2) at a certain moment (i.e. when the translayer protein (2) is in contact with the agonist compound or ligand) and / or within a certain time interval (i.e. after the translayer protein (2) has been in contact with the agonist compound or ligand),...

Claims

1. An apparatus that includes at least the following elements: - A boundary layer separating the first and second environments; - Translayer proteins; - The first ligand of a translayer protein present in the first environment; - Second ligands of translayer proteins present in the second environment; and - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal.

2. The apparatus of claim 1, wherein the first binding member of the binding pair is part of a first fusion protein containing the first binding member, wherein the first binding member is fused or connected to the translayer protein directly or via a suitable linker or spacer.

3. The apparatus according to claim 1 or 2, wherein the second ligand is a protein, ligand, binding domain, binding unit or other chemical entity that specifically binds to one or more functional, active and / or druggable conformations of a translayer protein, induces and / or stabilizes one or more functional, active and / or druggable conformations of the translayer protein (and / or causes conformational equilibrium shift of the translayer protein to one or more such conformations); and / or induces and / or stabilizes a complex of the translayer protein, the first ligand and the second ligand.

4. The apparatus according to any one of claims 1 to 3, wherein the second ligand is part of a (second) fusion protein, the (second) fusion protein comprising a second binding member of the binding pair fused or connected directly to or via a suitable linker or spacer to the second ligand.

5. The device according to any one of claims 1 to 3, wherein the second ligand is an immunoglobulin monovariable domain.

6. The apparatus according to any one of claims 1 to 3, wherein the second ligand is a naturally occurring ligand of a translayer protein or an analogue, derivative or ortholog of such a naturally occurring ligand.

7. The apparatus according to any one of claims 1 to 3 and 6, wherein the second member of the binding pair is part of a (second) fusion protein, the (second) fusion protein comprising a second binding member of the binding pair fused directly or via a suitable linker or spacer to a binding domain or binding unit capable of binding to the second ligand.

8. The device according to claim 7, wherein the binding domain or binding unit is an immunoglobulin monovariable domain.

9. The apparatus according to any one of claims 1 to 3, wherein the second ligand is part of a protein complex comprising the second ligand and one or more other proteins, the protein complex binding to or being bound to the translayer protein.

10. The apparatus according to claims 1 to 3 and 9, wherein the second member of the binding pair is part of a (second) fusion protein, the (second) fusion protein comprising a second binding member of the binding pair that is fused or connected directly or via a suitable linker or spacer to a binding domain or binding unit capable of binding to the second ligand.

11. The apparatus of claim 10, wherein the binding domain or binding unit is an immunoglobulin monovariable domain.

12. The apparatus of claim 1 or 2, wherein the second binding member of the binding pair is part of a second fusion protein comprising a second binding member of the binding pair that is fused or connected directly or via a suitable linker or spacer to a protein capable of binding directly or indirectly to the translayer protein.

13. The apparatus of any one of claims 1, 2, and 12, wherein the second binding member of the binding pair is part of a second fusion protein comprising a second binding member of the binding pair that is fused or linked directly or via a suitable linker or spacer to a protein (which is a second ligand) capable of directly binding to the translayer protein.

14. The apparatus according to any one of claims 1, 2, 12, and 13, wherein the second binding member of the binding pair is part of a second fusion protein comprising a second binding member of the binding pair fused directly or via a suitable linker or spacer to a protein (the protein being the second ligand) capable of directly binding to the translayer protein, wherein the protein capable of directly binding to the translayer protein is a protein, ligand, binding domain, binding unit, or other chemical entity that: specifically binds to one or more functional, active, and / or druggable conformations of the translayer protein; induces the formation and / or stabilizes one or more functional, active, and / or druggable conformations of the translayer protein (and / or shifts the conformational balance of the translayer protein to one or more such conformations); and / or induces the formation and / or stabilizes a complex of the translayer protein, the first ligand, and the second ligand.

15. The device according to claim 13 or 14, wherein the protein capable of directly binding to the translayer protein is an immunoglobulin monovariable domain.

16. The apparatus of any one of claims 1, 2, and 12, wherein the second binding member of the binding pair is part of a second fusion protein comprising a second binding member of the binding pair that is fused directly or via a suitable linker or spacer to a protein capable of indirectly binding to the translayer protein.

17. The apparatus of claim 16, wherein the second binding member of the binding pair is part of a second fusion protein comprising a second binding member of the binding pair fused directly or via a suitable linker or spacer to a binding domain or binding unit protein capable of binding to the second ligand.

18. The apparatus of claim 17, wherein the second binding member of the binding pair is part of a second fusion protein comprising a second binding member fused or connected directly or via a suitable linker or spacer to a binding domain or binding unit protein capable of binding the second ligand, wherein the second ligand is a protein, ligand, binding domain, binding unit, or other chemical entity that: specifically binds one or more functional, active, and / or druggable conformations of a translayer protein; induces and / or stabilizes one or more functional, active, and / or druggable conformations of a translayer protein (and / or shifts the conformational balance of the translayer protein toward one or more such conformations); and / or induces and / or stabilizes a complex of the translayer protein, the first ligand, and the second ligand.

19. The device according to claim 17 or 18, wherein the protein binding domain or binding unit protein capable of binding to the second ligand is an immunoglobulin monovariable domain.

20. The apparatus of claim 16, wherein the second binding member of the binding pair is part of a second fusion protein comprising a second binding member of the binding pair fused directly or via a suitable linker or spacer to a binding domain or binding unit protein capable of binding to a protein complex containing a second ligand, the protein complex binding to or capable of binding to a translayer protein.

21. The apparatus of claim 20, wherein the protein-binding domain or binding unit protein capable of binding to the protein complex is an immunoglobulin monovariable domain.

22. An apparatus that includes at least the following elements: - A boundary layer separating the first and second environments; - Translayer proteins; - Ligands of translayer proteins present in the second environment; and - A binding pair consisting of at least a first binding member and a second binding member, which is capable of generating a detectable signal.

23. The device according to any one of the preceding claims, wherein the boundary layer is a cell wall or a cell membrane.

24. The device according to any one of the preceding claims, wherein the boundary layer is the wall or membrane of a liposome or vesicle.

25. The apparatus according to any one of the preceding claims, wherein the translayer protein is a GPCR.

26. A method including the following steps: a) Provide the apparatus according to claim 22; and b) Add a first ligand to the first environment of the device.

27. The method of claim 26, further comprising the following steps: c) Measure the signal generated by the binding pair and / or measure the change in the signal generated by the binding pair.

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