Reactive Affinity Probe Interaction Discovery Platform

Abstract

Disclosed are methods, assays, and kits for identifying ligands for biomolecules such as proteins, lipids, carbohydrates, or nucleic acids. The disclosed methods can be quantitative binding assays for the target, which can circumvent the challenges of target purification and provide a systematic approach to discovering and targeting allosteric binding sites.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 915,310, filed October 15, 2019. [Background technology]

[0002] Identifying "druggable" targets and corresponding therapeutic agents are two fundamental challenges in drug discovery research. The pharmacological actions of many biomolecules, such as proteins, remain inaccessible because their endogenous or exogenous regulators have not yet been discovered. Therefore, tools to explore the physiological functions and pharmacological potential of these biomolecules, whether endogenous or surrogate ligands, are of paramount importance.

[0003] The discovery of pharmacological modulators for biomacromolecules of interest is often achieved by screening libraries of molecules capable of binding to or modulating the function of purified biomacromolecules. However, many classes of biomacromolecules, including multi-transmembrane proteins, are difficult to purify from cells and must be screened in their native cellular environment. Such cell-based screens require the selection of surrogate readouts, such as cell viability, signal transduction, and gene expression. Compound screening using cell-based surrogate readouts presents two challenges: (i) many hits exert their pharmacological effects indirectly through off-target effects; and (ii) many true ligands that alter other aspects of the macromolecule's function are overlooked. A technology that allows the direct identification of small molecule binders for biomacromolecules of interest in intact cells would address these outstanding challenges and represent a breakthrough in this field. Summary of the Invention [Means for solving the problem]

[0004] The present invention provides a method and system for screening candidate ligands for biomolecules. The Reactive Affinity Probe Interaction Discovery (RAPID) technology is a quantitative in situ binding assay for a target that can circumvent the challenges of target purification and provide a systematic approach for the discovery and targeting of allosteric binding sites.

[0005] Disclosed herein, in certain embodiments, is a method for identifying a ligand, the method comprising: (a) contacting a biomolecule with a probe molecule, wherein the probe molecule comprises a binding element, a reporter group, and a reactive moiety; the probe molecule binds to the biomolecule via the binding element; the reactive moiety forms a covalent bond with the biomolecule, thereby forming a conjugate; (b) contacting the conjugate with a detectable molecule comprising a functional moiety that reacts with the reporter group, thereby forming a detectable conjugate; (c) contacting the detectable conjugate with a solid support, wherein the solid support comprises a recognition moiety; and the recognition moiety binds to the detectable conjugate, thereby forming a bound detectable conjugate; and (d) detecting the bound detectable conjugate, thereby identifying the probe molecule as a ligand of the biomolecule.

[0006] Numerous embodiments are further provided that can be applied to any aspect of the invention described herein. For example, in some embodiments, the binding element is a small molecule, a peptide, or a nucleic acid (such as RNA or DNA). In some embodiments, the binding element is a component of a library that includes multiple binding elements. In some embodiments, the library includes a library of small molecule fragments that can be defined as satisfying the following three rules: molecular weight ≦ 300 Da, cLogP ≦ 3, hydrogen bond donors ≦ 3, and hydrogen bond acceptors ≦ 3. Exemplary libraries include: ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, AnalytiCon's FRGx, AnCoreX's TCI-Frag, ASINEX's Bio Building Blocks, Charles River's BioFocus 3D, Emerald Bio's Fragments of Life (FOL), Enamine Fragment Library, IOTA Diverse 1500, BIONET fragment library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Vitas-M Laboratory's Allium, or Zenobia fragment library. In some embodiments, the biomolecule is a protein. In some embodiments, the reactive moiety forms a covalent bond with an amino acid of the protein. In some embodiments, the biomolecule is a lipid, carbohydrate, or nucleic acid (such as RNA or DNA). In some embodiments, the biomolecule comprises an epitope tag, for example, FLAG, 6xHis, HA, c-myc, glutathione-S-transferase, Strep-tag, maltose binding protein, chitin binding protein, S-tag, V5 tag, or AviTag.In some embodiments, the reporter group comprises an azadibenzocyclooctyne, a thiol, an alkene, an alkyne, an azide, a tetrazine, a trans-cyclooctene, a (diphenylphosphino)aryl, a (diphenylphosphino)alkyl, or an activated ester (e.g., a hydroxybenzotriazole (HOBt) ester). In some embodiments, the reactive moiety is a photocrosslinker group, a sulfonyl fluoride (or sulfonyl fluoride), a fluorosulfate, a Michael acceptor moiety, a leaving group moiety, or a moiety that forms a covalent bond with a nucleophilic moiety in the side chain of a naturally occurring alpha amino acid (e.g., the thiol group of cysteine, the amino group of lysine, the hydroxyl group of serine or threonine, or the phenolic group of tyrosine). In some embodiments, the detectable molecule comprises digoxigenin, nickel NTA (nitrilotriacetic acid), a chromophore, or a luminophore. In some embodiments, the chromophore comprises a non-fluorochrome chromophore, a quencher, an absorbing chromophore, a fluorophore, an organic dye, an inorganic dye, a metal chelate, or a fluorescent enzyme substrate. In some embodiments, the detectable molecule is biotin. Biotin can be attached to a streptavidin conjugate, such as an HRP, SulfoTag, a fluorophore, or a metal chelate. In some embodiments, the functional moiety comprises an azadibenzocyclooctyne, a thiol, an alkene, an alkyne, an azide, a tetrazine, a trans-cyclooctene, a (diphenylphosphino)aryl, a (diphenylphosphino)alkyl, or an activated ester (e.g., a hydroxybenzotriazole (HOBt) ester). In some embodiments, the solid support is a membrane, glass, plastic, a synthetically prepared polymer, an Eppendorf tube, a well of a multiwell plate, or a surface plasmon resonance chip. In some embodiments, the recognition moiety is an antibody, a DNA-binding protein, an RNA-binding protein, a carbohydrate-binding protein, or a lipid-binding protein.In some embodiments, the antibody is against a biomolecule and / or an epitope tag.

[0007] In some embodiments, step (d) comprises detecting the bound detectable conjugate via ELISA, Western blot, immunofluorescence assay, fluorometric assay, fluorometric microvolume assay technology (FMAT), or cell subcellular dying. In some embodiments, the method is performed on a crude cell extract containing the biomolecule, on a liposomal preparation of protein containing the biomolecule, on an isolated organelle containing the biomolecule, on a purified protein preparation containing the biomolecule, or in situ. In some embodiments, the method is a cell-based assay. In some embodiments, the biomolecule is expressed in cells. In some embodiments, the cells are engineered to express the biomolecule. In some embodiments, the cells are lysed prior to step (b). In some embodiments, step (d) further comprises quantifying the amount of bound detectable conjugate. In some embodiments, step (a) further comprises a substrate for the biomolecule. In some embodiments, the amount of bound detectable conjugate formed in the presence of a substrate for the biomolecule is less than the amount of bound detectable conjugate formed in the absence of the substrate (i.e., the probe molecule is a substrate-competitive probe). In other embodiments, the amount of bound detectable conjugate formed in the presence of a substrate for the biomolecule is greater than the amount of bound detectable conjugate formed in the absence of the substrate (i.e., the probe molecule is a substrate-cooperative probe). [Brief explanation of the drawings]

[0008] [Figure 1] We present a general protocol for RAPID, a high-throughput screening in cell binding assays for ligand discovery and target engagement. [Figure 2A] Figures 2A-2C show that RAPID identifies orthosteric and allosteric ligands as starting points or probes for target occupancy. Figure 2A shows that the degree of covalent modification of the creatine transporter by a subset of the RAPID library is highly reproducible. [Figure 2B] Screening of 2000 RAPs against the creatine transporter SLC6A8 ± the substrate analog β-guanidinopropionic acid (β-GPA) is shown. Dots that fall off the diagonal are either substrate-competitive or substrate-cooperative. [Figure 2C] We demonstrate that two RAPs identified from the screen inhibit or enhance the covalent modification of targets in a dose-dependent manner as a function of GPA concentration, with IC50 / EC50 values ​​consistent with the known inhibition constant of β-GPA (~30 μM). [Figure 3] Figure 1 shows covalent inactivation of the creatine transporter SLC6A8 by the reactive affinity probe JN-1724 and protection by coadministration with the competitor β-GPA. Cells were treated with 100 μM JN-1724 with or without 1 mM β-GPA for 30 minutes and then exposed to 365 nm light for 6 minutes. Cells were washed, and the remaining transport activity of SLC6A8 was measured in a creatine uptake assay. [Figure 4]Mass spectrometry data showing covalent modification of SLC6A8 by the reactive affinity probe JN-1724, competed by co-administration with β-GPA. Cells were treated with 20 μM JN-1724 with or without 1 mM β-GPA for 30 minutes, followed by 6 minutes of irradiation with 365 nm light. Cells were lysed, biotin was clicked on, and biotinylated proteins were affinity purified with streptavidin, digested with trypsin, and identified by tandem mass spectrometry with TMT quantification. SLC6A8 (pointed by arrow) was one of the identified proteins, and co-administration with 1 mM β-GPA reduced enrichment levels by 80%. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention provides a method and system for screening candidate ligands for biomolecules. Reactive Affinity Probe Interaction Discovery (RAPID) is a quantitative in situ binding assay for targets that circumvents the challenges of target purification and provides a systematic approach for discovering and targeting allosteric binding sites. RAPID technology enables the direct identification of small molecule binders for biopolymers of interest in intact cells.

[0010] Small molecules are powerful tools for investigating protein function and can serve as leads for novel therapeutics. However, most human proteins lack small molecule ligands, and entire protein classes are considered undruggable. The methods disclosed herein can identify small molecule probes for biomolecules, such as proteins, that have proven difficult to target using high-throughput screening of complex compound libraries. While reversible binding ligands are commonly pursued, covalent fragments offer an alternative route to small molecule probes, including those that can access regions of proteins that are difficult to target by binding affinity alone.

[0011] Disclosed herein, in certain embodiments, is a method for identifying a ligand, comprising: (a) contacting a biomolecule with a probe molecule; the probe molecule comprising a binding element, a reporter group, and a reactive moiety; the probe molecule binds to the biomolecule via the binding element; the reactive moiety forms a covalent bond with the biomolecule, thereby forming a conjugate; (b) contacting the conjugate with a detectable molecule comprising a functional moiety that reacts with the reporter group, thereby forming a detectable conjugate; (c) contacting the detectable conjugate with a solid support; the solid support comprising a recognition moiety; and the recognition moiety binds to the detectable conjugate, thereby forming a bound detectable conjugate; and (d) detecting the bound detectable conjugate, thereby identifying the probe molecule as a ligand of the biomolecule.

[0012] In some embodiments, probe molecules rely on intrinsic chemical reactivity with protein residues. Probe molecules may possess reactive moieties, such as photoreactive elements, that convert reversible small molecule-protein interactions into stable covalent adducts upon ultraviolet (UV) irradiation. Probe molecules may also possess reporter groups, such as alkynes, which function as sterically minimal surrogate reporters that allow for later conjugation to azide tags via copper-catalyzed azide-alkyne cycloaddition (CuAAK or "click") chemistry. Probe molecules may also possess binding elements that target the probe to proteins that recognize specific structural features.

[0013] Published U.S. Patent Applications Nos. 2017 / 0115303 and 2016 / 0252509 describe examples of probe molecules; each of these publications is incorporated herein by reference in its entirety, and in particular with respect to the inhibitors of the complement pathway described therein.

[0014] definition Scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art unless otherwise defined. Generally, the nomenclature and techniques used in connection with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0015] The term "culturing" refers to the in vitro propagation of cells or organisms on or in various types of media. It is understood that the progeny of cells propagated in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. "Expansion" refers to any growth or division of cells.

[0016] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant reduction. In some embodiments, "reduce," "reduce," or "inhibition" typically refers to a reduction of at least 10% compared to a reference level (e.g., the absence of a given ligand), and may include, for example, a reduction of at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduce" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level.

[0017] The terms "increased," "increase," or "enhance," or "activate" are all used herein to generally mean an increase by a statistically significant amount; for the avoidance of doubt, the terms "increased," "increase," or "enhance," or "activate" mean an increase of at least 10% compared to a base level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% increase, including a 100% increase, or any increase between 10-100%, compared to a base level, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, or at least about 1000-fold, or more, compared to a base level.

[0018] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0019] As used herein, the term "interaction," when referring to an interaction between two molecules, refers to physical contact (e.g., binding) between the molecules. Generally, such an interaction results in an activity (which produces a biological effect) of one or both of the molecules. This activity can be a direct activity (e.g., signal transduction) of one or both of the molecules.

[0020] As used herein, "isolated protein" refers to a protein that, when isolated from a cell or produced by recombinant DNA technology, is substantially free of other proteins, cellular material, separation media, and culture medium, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide, or fusion protein is derived, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. The phrase "substantially free of cellular material" includes preparations of a target polypeptide (e.g., an immunoglobulin) or fragment thereof in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the phrase "substantially free of cellular material" includes preparations of a target protein or fragment thereof having less than about 30% (by dry weight) non-target proteins (also referred to herein as "contaminating proteins"), more preferably less than about 20% non-target proteins, even more preferably less than about 10% non-target proteins, and most preferably less than about 5% non-target proteins. When an antibody, polypeptide, peptide, fusion protein, or fragment thereof, e.g., a biologically active fragment, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0021] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0022] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcriptional regulatory sequences, operably linked means that the linked DNA sequences are contiguous, and, where necessary to link two protein-coding regions, contiguous and in reading frame. With respect to switch sequences, operably linked indicates that the sequences are capable of undergoing switch recombination.

[0023] Screening assays The present invention provides a method and system for screening candidate ligands for biomolecules. Reactive Affinity Probe Interaction Discovery (RAPID) is a quantitative in situ binding assay for targets that can circumvent the challenges of target purification and provide a systematic approach for the discovery and targeting of allosteric binding sites.

[0024] Disclosed herein, in certain embodiments, is a method for identifying a ligand, comprising: (a) contacting a biomolecule with a probe molecule; the probe molecule comprising a binding element, a reporter group, and a reactive moiety; the probe molecule binds to the biomolecule via the binding element; the reactive moiety forms a covalent bond with the biomolecule, thereby forming a conjugate; (b) contacting the conjugate with a detectable molecule comprising a functional moiety that reacts with the reporter group, thereby forming a detectable conjugate; (c) contacting the detectable conjugate with a solid support; the solid support comprising a recognition moiety; and the recognition moiety binds to the detectable conjugate, thereby forming a bound detectable conjugate; and (d) detecting the bound detectable conjugate, thereby identifying the probe molecule as a ligand of the biomolecule.

[0025] Numerous embodiments are further provided that can be applied to any aspect of the invention described herein. For example, in some embodiments, the binding element is a small molecule, peptide, or nucleic acid (such as RNA or DNA). In some embodiments, the binding element is a component of a library comprising multiple binding elements. In some embodiments, the library comprises a ChemBridge fragment library, Pyramid Platform Fragment-Based Drug Discovery, Maybridge fragment library, AnalytiCon's FRGx, AnCoreX's TCI-Frag, ASINEX's Bio Building Blocks, Charles River's BioFocus 3D, Emerald Bio's Folgments of Life (FOL), Enamine Fragment Library, IOTA Diverse 1500, BIONET fragment library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Vitas-M Laboratory's Allium, or Zenobia fragment library. In some embodiments, the biomolecule is a protein. In some embodiments, the reactive moiety forms a covalent bond with an amino acid of the protein. In some embodiments, the biomolecule is a lipid, a carbohydrate, or a nucleic acid (such as RNA or DNA). In some embodiments, the biomolecule comprises an epitope tag, such as FLAG, 6xHis, HA, c-myc, glutathione-S-transferase, Strep-tag, maltose-binding protein, chitin-binding protein, S-tag, V5 tag, or AviTag.In some embodiments, the reporter group comprises an azadibenzocyclooctyne, a thiol, an alkene, an alkyne, an azide, a tetrazine, a trans-cyclooctene, a (diphenylphosphino)aryl, a (diphenylphosphino)alkyl, or an activated ester (e.g., a hydroxybenzotriazole (HOBt) ester). In some embodiments, the reactive moiety is a photocrosslinkable group, a sulfonyl fluoride, a fluorosulfate, a Michael acceptor moiety, a leaving group moiety, or a moiety that forms a covalent bond with a nucleophilic moiety in the side chain of a naturally occurring alpha amino acid (e.g., the thiol group of cysteine, the amino group of lysine, the hydroxyl group of serine or threonine, or the phenolic group of tyrosine). In some embodiments, the detectable molecule comprises digoxigenin, nickel NTA (nitrilotriacetic acid), a chromophore, or a luminophore. In some embodiments, the chromophore comprises a non-fluorescent dye chromophore, a quencher, an absorbing chromophore, a fluorophore, an organic dye, an inorganic dye, a metal chelate, or a fluorescent enzyme substrate. In some embodiments, the detectable molecule is biotin. Biotin can be bound to a streptavidin conjugate, such as HRP, SulfoTag, a fluorophore, or a metal chelate. In some embodiments, the functional moiety comprises an azadibenzocyclooctyne, a thiol, an alkene, an alkyne, an azide, a tetrazine, a trans-cyclooctene, a (diphenylphosphino)aryl, a (diphenylphosphino)alkyl, or an activated ester (e.g., a hydroxybenzotriazole (HOBt) ester). In some embodiments, the solid support is a membrane, glass, plastic, a synthetically prepared polymer, an Eppendorf tube, a well of a multiwell plate, or a surface plasmon resonance chip. In some embodiments, the recognition moiety is an antibody, a DNA-binding protein, an RNA-binding protein, a carbohydrate-binding protein, or a lipid-binding protein. In some embodiments, the antibody is an antibody against a biomolecule and / or an epitope tag.

[0026] In some embodiments, step (d) comprises detecting the bound detectable conjugate via ELISA, Western blot, immunofluorescence assay, fluorometric assay, fluorometric microvolume assay technique (FMAT), or subcellular staining. In some embodiments, the method is performed on a crude cell extract containing the biomolecule, on a liposomal preparation of protein containing the biomolecule, on an isolated organelle containing the biomolecule, on a purified protein preparation containing the biomolecule, or in situ. In some embodiments, the method is a cell-based assay. In some embodiments, the biomolecule is expressed in cells. In some embodiments, the cells are engineered to express the biomolecule. In some embodiments, the cells are lysed prior to step (b). In some embodiments, step (d) further comprises quantifying the amount of bound detectable conjugate. In some embodiments, step (a) further comprises a substrate for the biomolecule. In some embodiments, the amount of bound detectable conjugate formed in the presence of a substrate for the biomolecule is less than the amount of bound detectable conjugate formed in the absence of the substrate (i.e., the probe molecule is a substrate-competitive probe), while in other embodiments, the amount of bound detectable conjugate formed in the presence of a substrate for the biomolecule is greater than the amount of bound detectable conjugate formed in the absence of the substrate (i.e., the probe molecule is a substrate-cooperative probe).

[0027] As used herein, the terms "probe" or "test compound" or "candidate agent" refer to an agent or collection of agents (e.g., compounds) being screened for their ability to affect cells. Test compounds can include a wide variety of compounds, including chemical compounds, mixtures of chemical compounds, e.g., polysaccharides, organic or inorganic small molecules (e.g., molecules with molecular weights of less than 2000 Daltons, less than 1000 Daltons, less than 1500 Daltons, less than 1000 Daltons, or less than 500 Daltons), biopolymers, e.g., peptides, proteins, peptide analogs, and their analogs and derivatives, peptidomimetics, nucleic acids, nucleic acid analogs, and derivatives; extracts made from biological materials such as bacterial, plant, fungal, or animal cells or tissues; and natural or synthetic compositions.

[0028] Depending on the particular embodiment being performed, the probes can be provided free in solution or attached to a carrier, or solid support (e.g., beads). Many suitable solid supports can be used to immobilize the probes. Examples of suitable solid supports include agarose, cellulose, dextran (e.g., commercially available as Sephadex or Sepharose), carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resins, plastic film, polyamine methyl vinyl ether maleic acid copolymer, glass beads, amino acid copolymers, ethylene-maleic acid copolymer, nylon, silk, and the like. Furthermore, for the methods described herein, probes can be screened individually or in groups. Group screening is particularly useful when the hit rate of effective probes is expected to be low enough that more than one positive result is not expected for a given group.

[0029] Small molecule properties that correlate with good lead compounds are known in the art. Lipinski's Rule of Five provided the initial framework for developing orally bioavailable drug candidates. These rules were strengthened by the discovery that the number of rotatable bonds (NROT) is a critical parameter, with a maximum of 7 appearing optimal for oral bioavailability. Polar surface area (PSA) can also be another key property; a PSA of 110-140 Å 2 Passively absorbed molecules with a low oral bioavailability are likely to have low oral bioavailability. Recently, the term "lead-like" has been introduced for molecules identified from HTS campaigns that are suitable for optimization and have relatively "scaled-down" properties compared to Lipinski's values. The literature has addressed the problems faced by compounds discovered by screening drug-sized compound libraries. Recently, a novel alternative approach has emerged, termed "fragment-based" drug discovery (Carr, R. and Jhoti, H. (2002) Structure-based screening of low-affinity compounds. Drug Discovery Today 7, 522-527; Erlanson, DA et al. (2000) Site-directed ligand discovery. Proc. Natl. Acad. Sci. USA 97, 9367-72; Vetter, D. (2002) Chemical microarrays, fragment diversity, label-free imaging by plasmon resonance—a chemical genomics approach. J. Cell. Biochem. 39, 79-84). Using this method, hits identified generally follow the "Rule of Three," which can be a useful rule for constructing fragment libraries for lead generation.

[0030] This approach begins with fragment libraries (molecular weights between 100 and 250 Da) that are screened by high-throughput X-ray crystallography. These fragments probe key binding interactions in the protein but are small enough to minimize the possibility of unfavorable interactions (electronic or steric) that could prevent efficient binding (Hann, M. et al. (2001) Molecular complexity and its impact on the probability of finding leads for drug discovery. J. Chem. Inf. Comput. Sci. 41, 856-864). The binding mode of these small ligands in the protein is then defined by interpretation of electron density maps. X-ray crystallography is highly effective at identifying weak interactions (μM to mM), allowing fragment hits to be identified that do not have measurable activity in biological assays. Fragment libraries can be constructed to sample chemical diversity or target specific interactions in proteins. Screening both types of fragment libraries against kinases and proteases, followed by optimization of hits into potent lead compounds, reveals that successful hits exhibit specific physicochemical properties.

[0031] Analysis of a diverse set of fragment hits showed that such hits, on average, appeared to follow the "rule of three": molecular weight <300, number of hydrogen-bond donors ≤3, number of hydrogen-bond acceptors ≤3, and ClogP ≤3. Furthermore, it was suggested that NROT (≤3) and PSA (≤60) could also be useful criteria for fragment selection. These data suggest that the "rule of three" could be useful when constructing fragment libraries for efficient lead discovery.

[0032] Many small molecule libraries are known in the art and commercially available. These small molecule libraries can be screened using the screening method described herein. A chemical library or compound library is a collection of stored chemicals that can be used in combination with the method described herein to screen candidate drugs for specific effects. A chemical library contains information about the chemical structure, purity, quantity, and physicochemical properties of each compound. Compound libraries can be commercially available from, for example, Enzo Life Sciences, Aurora Fine Chemicals, Exclusive Chemistry Ltd., ChemDiv, ChemBridge, TimTec Inc., AsisChem, and Princeton Biomolecular Research.

[0033] Without limitation, compounds can be tested at any concentration that can affect cells relative to a control for a suitable period of time. In some embodiments, compounds are tested at concentrations ranging from about 0.01 nM to about 100 mM, from about 0.1 nM to about 500 μM, from about 0.1 μM to about 20 μM, from about 0.1 μM to about 10 μM, or from about 0.1 μM to about 5 μM.

[0034] Compound screening assays can be used in high-throughput screening, a process in which a library of compounds is tested for a given activity. High-throughput screening seeks to screen large numbers of compounds rapidly and in parallel. For example, using microtiter plates and automated assay equipment, a laboratory can perform 100,000 or more assays per day in parallel.

[0035] Following the screening assay, a subsequent assay can be performed to further identify whether the identified test compound has desirable properties for the intended use. For example, following the screening assay, a second assay selected from the group consisting of measuring bioavailability, toxicity, or pharmacokinetics can be performed, but is not limited to these methods.

[0036] The present invention also encompasses kits for identifying the ligands described herein. The kits of the present invention may also include instructional materials disclosing or describing the use of the disclosed kits or probes in the methods of the disclosed inventions provided herein. The kits may also include additional components to facilitate the particular application for which the kit is designed. For example, the kits may additionally include means for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as sheep anti-mouse-HRP, etc.) and reagents required for control (e.g., a control biological sample or standard). The kits may further include buffers and other reagents approved for use in the methods of the disclosed invention. Non-limiting examples include agents for reducing nonspecific binding, such as carrier proteins or detergents.

[0037] A "kit" is any product (e.g., a package or container) containing at least one reagent, such as a probe or small molecule, for specifically detecting and / or affecting the expression of a marker of the present invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. The kit may contain one or more reagents necessary to express a composition useful in the methods of the present invention. In certain embodiments, the kit may further include a reference standard. Those skilled in the art can envision many such controls, including, but not limited to, common molecules. The reagents in the kit may be provided in individual containers or as a mixture of two or more reagents in a single container. Additionally, instructions describing how to use the composition may be included in the kit. [Example]

[0038] Example 1: RAPID protocol for screening RAP libraries against affinity-tagged targets Preparation of capture plates Greiner HiBind plates (Sigma-Aldrich catalog number: M4561-40EA) were coated with capture antibody. Protein A (Thermo, catalog number: 101100) was reconstituted at 5 mg / mL in 50% glycerol / PBS and diluted 1:500 in coating buffer (Thermo, BupH™ Carbonate-Bicarbonate Buffer Packs). Plates were washed once with 100 μL of coating buffer and placed side-by-side in groups of six. 50 μL of Protein A was added to each well of each plate. The stamped plates were placed in a 4°C deli refrigerator overnight. The following morning, the Protein A-coated plates were washed twice with 100 μL of coating buffer. 200 μL of Superblock (Thermo Fisher, catalog number 37515) was added to each well of each plate and incubated at room temperature for 1 hour. After blocking the capture plates for 2 hours, all plates were washed three times with PBS / T. 135 mL of 1 μg / mL capture antibody in SuperBlock buffer was prepared. 50 μL of the prepared antibody was added to each well of the capture plate and incubated at room temperature for 1 hour.

[0039] Preparation of lysate plates For the HEK293T cell line, 7.5M cells were used per 96-well plate. Twenty-four 96-well plates were coated with poly-D-lysine (Sigma-Aldrich, catalog number P7280-5MG) and washed. Then, 75K cells were plated per well and incubated overnight to allow cells to attach. The RAP dosing plates were reconstituted with cell imaging medium at 4x the desired final concentration. For each cell plate, remove the medium and dispense 200 μL of cell imaging medium (CIM) (Thermo, catalog number A14291DJ). Next, for each plate, remove 200 μL of CIM and immediately add 75 μL of CIM. After washing the cells and dispensing 75 μL of CIM into each plate, 25 μL of reconstituted RAP was added and incubated in a 37°C cell incubator for 30 minutes. While the cells are incubating, prepare lysis buffer (~10 mL / plate) by adding 12.5 mL of 20% DDM (Anatrace, catalog number D31025GM), 250 mL of Hepes-buffered saline, and five complete protease tablets (Sigma Aldrich, catalog number 4693132001). Irradiate the plates with a UV crosslinker (Spectrolinker, catalog number 1195T76). After 3 minutes of irradiation, remove the medium and wash the cells with 200 μL of CIM to remove excess RAP. Then, remove the CIM from each plate and add 100 μL of lysis buffer. Incubate the plates at room temperature for 1 hour to complete lysis.

[0040] For a total of 140 mL of click mix, prepare 30 mL of each reagent to conjugate the reporter biotin to the RAP alkyne via copper-catalyzed azide-alkyne cycloaddition. This corresponds to 600 mg of THPTA (Click Chemistry Tools, Catalog No. 1010-5G), 600 mg of ascorbate (Sigma-Aldrich, Catalog No. 11140-50G), 120 mg of copper sulfate (Sigma-Aldrich, Catalog No. 451657-10G), and 750 μL of 10 mM picolyl-biotin-azide (Click Chemistry Tools, Catalog No. 1167-100). Dissolve each reagent individually in 30 mL of water and mix the reagents in the following order just before initiating the click reaction: THPTA -> THPTA -> copper (turns blue) -> ascorbate (turns clear). Add 40 μL of click mix to each well of all plates. After 1 hour of incubation, remove the capture antibody from the capture plate by washing three times with 300 μL of PBS-T (Boston BioProducts, catalog number IBB-171). Quench the click reaction by adding 10 μL of 0.5 M EDTA (Sigma Aldrich, catalog number 324506-100ML) to each well. Transfer 100 μL of lysate to the corresponding capture plate and incubate at room temperature for at least 1 hour. After the 1 hour capture incubation, wash the plate five times with 300 μL of PBS / T.

[0041] Streptavidin-HRP (Cell Signaling Technologies, Catalog No. 3999S) is prepared in PBS / T by diluting the Cell Signaling Technologies (P / N) material 1:1000. 50 μL of prepared streptavidin-HRP is added to each well and incubated at room temperature for 30 minutes. After the 30-minute streptavidin incubation, the plate is washed five times with 300 μL of PBS / T. The final wash is left in the plate to prevent drying.

[0042] Load a Tecan with 200 μL tips and TMB. Add at least 135 μL of TMB (Thermo Fisher, Cat. No. N301) to the Tecan trough and open the Stamp 50 μL method. Empty plates, load them into the Tecan in the appropriate order, and run the method. Repeat this process until all plates have received TMB. Quench each plate with 50 μL of 0.2 N sulfuric acid. Read the plates sequentially on a plate reader and quantify the absorbance at 450 nm.

[0043] Identification of a substrate-sensitive binder for the creatine transporter SLC6A8 Cells expressing the tagged creatine transporter SLC6A8 were used to screen for substrate-sensitive binders. Figure 1 shows the general protocol for RAPID, a high-throughput screening in cell-based binding assays for ligand discovery and target engagement.

[0044] RAPID identifies orthosteric and allosteric ligands as starting points or probes for target occupancy. The degree of covalent modification of the creatine transporter by a subset of the RAP library is highly reproducible (Figure 2A). Screening of 2,000 RAPs against the creatine transporter SLC6A8 substrate analog, guanidinopropionic acid (GPA), identified substrate-sensitive binders (Figure 2B). Dots that fall off the diagonal are either substrate-competitive or substrate-cooperative. Two RAPs identified from the screen show dose-dependent inhibition or enhancement of covalent target modification as a function of GPA concentration (Figure 2C). The IC50 / EC50 values ​​are consistent with the known inhibition constant of GPA (~30 μM). Cells were treated with 100 μM JN-1724 with or without 1 mM β-GPA for 30 minutes and then irradiated with 365 nm light for 6 minutes. Cells were washed, and the residual transport activity of SLC6A8 was measured by a creatine uptake assay. Covalent inactivation of the creatine transporter SLC6A8 by the reactive affinity probe JN-1724 and protection by coadministration with the competitor β-GPA are shown in Figure 3.

[0045] Treatment of cells with the substrate-competitive RAP quantitatively inhibits the creatine transporter. Treatment with 100 μM JN-1724 for 30 minutes, followed by 6 minutes of cross-linking at 365 nm, was sufficient to inhibit SLC6A8 creatine transport to 5.7% of normal transport. To confirm target engagement by unbiased mass spectrometry analysis of the adductome, cells were treated with 20 μM JN-1724 for 30 minutes, with or without 1 mM β-GPA, followed by 6 minutes of irradiation at 365 nm. Cells were lysed, biotin was conjugated via a click reaction, and biotinylated proteins were affinity-purified with streptavidin, digested with trypsin, and identified by tandem mass spectrometry with TMT quantification. SLC6A8 (pointed by an arrow) was one of the identified proteins, and co-treatment with 1 mM β-GPA reduced its enrichment level by 80% (Figure 4).

[0046] Incorporation by Reference All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including definitions herein, will control.

[0047] equivalent While specific embodiments of the subject invention have been described, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification and such variations. Finally, preferred embodiments of the present invention are described in sections. [Embodiment 1] 1. A method for identifying a ligand, comprising: (a) contacting a biomolecule with a probe molecule, wherein the probe molecule comprises a binding element, a reporter group, and a reactive moiety, wherein the probe molecule binds to the biomolecule via the binding element and the reactive moiety forms a covalent bond with the biomolecule, thereby forming a conjugate; (b) contacting the conjugate with a detectable molecule comprising a functional moiety that reacts with the reporter group, thereby forming a detectable conjugate; (c) contacting the detectable conjugate with a solid support, wherein the solid support comprises a recognition moiety, and the recognition moiety binds to the detectable conjugate, thereby forming a bound detectable conjugate; and (d) identifying the probe molecule as a ligand of the biomolecule by detecting the bound detectable conjugate; A method comprising: [Embodiment 2] 2. The method of embodiment 1, wherein the binding element is a small molecule. [Embodiment 3] 2. The method of embodiment 1, wherein the binding member is a peptide. [Embodiment 4] 2. The method of embodiment 1, wherein the binding member is a nucleic acid. [Embodiment 5] 5. The method of embodiment 4, wherein the nucleic acid is RNA or DNA. [Embodiment 6] 6. The method of any one of embodiments 1 to 5, wherein the binding member is a member of a library comprising a plurality of binding members. [Embodiment 7] 7. The method of embodiment 6, wherein the binding elements are small molecule fragments, and each small molecule fragment has at least one of the following characteristics: molecular weight≦300 Da, cLogP≦3, hydrogen bond donors≦3, and hydrogen bond acceptors≦3. [Embodiment 8] 8. The method according to any one of embodiments 1 to 7, wherein the biomolecule is a protein. [Embodiment 9] 9. The method of embodiment 8, wherein the reactive moiety forms a covalent bond with an amino acid of the protein. [Embodiment 10] 8. The method according to any one of embodiments 1 to 7, wherein the biomolecule is a lipid. [Embodiment 11] 8. The method of any one of embodiments 1 to 7, wherein the biomolecule is a carbohydrate. [Embodiment 12] 8. The method according to any one of embodiments 1 to 7, wherein the biomolecule is a nucleic acid. [Embodiment 13] 13. The method of embodiment 12, wherein the nucleic acid is RNA. [Embodiment 14] 13. The method of embodiment 12, wherein the nucleic acid is DNA. [Embodiment 15] 15. The method of any one of embodiments 1 to 14, wherein the biomolecule comprises an epitope tag. [Embodiment 16] 16. The method of embodiment 15, wherein the epitope tag is FLAG, 6xHis, HA, c-myc, glutathione-S-transferase, Strep-tag, maltose binding protein, chitin binding protein, S tag, V5 tag, or AviTag. [Embodiment 17] 17. The method of any one of embodiments 1-16, wherein the reporter group comprises an azadibenzocyclooctyne, a thiol, an alkene, an alkyne, an azide, a tetrazine, a trans-cyclooctene, a (diphenylphosphino)aryl, a (diphenylphosphino)alkyl, or an activated ester (e.g., a hydroxybenzotriazole (HOBt) ester). [Embodiment 18] 18. The method of any of embodiments 1-17, wherein the reactive moiety is a photocrosslinkable group, a sulfonyl fluoride, a fluorosulfate, a Michael acceptor moiety, a leaving group moiety, or a moiety that forms a covalent bond with a nucleophilic moiety in the side chain of a naturally occurring alpha amino acid (e.g., the thiol group of cysteine, the amino group of lysine, the hydroxyl group of serine or threonine, or the phenolic group of tyrosine). [Embodiment 19] 19. The method of any of embodiments 1-18, wherein the detectable molecule comprises digoxigenin, nickel NTA (nitrilotriacetic acid), a chromophore, or a luminophore. [Embodiment 20] 20. The method of embodiment 19, wherein the detectable molecule comprises digoxigenin. [Embodiment 21] 20. The method of embodiment 19, wherein the detectable molecule comprises nickel NTA (nitrilotriacetic acid). [Embodiment 22] 20. The method of embodiment 19, wherein the detectable molecule comprises a chromophore. [Embodiment 23] 23. The method of embodiment 22, wherein the chromophore comprises a non-fluorescent chromophore, a quencher, an absorbing chromophore, a fluorophore, an organic dye, an inorganic dye, a metal chelate, or a fluorescent enzyme substrate. [Embodiment 24] 20. The method of embodiment 19, wherein the detectable molecule comprises a luminophore. [Embodiment 25] 19. The method of any preceding embodiment, wherein the detectable molecule is biotin. [Embodiment 26] 26. The method of embodiment 25, wherein biotin is bound to a streptavidin conjugate. [Embodiment 27] 27. The method of embodiment 26, wherein the streptavidin conjugate comprises HRP, a SulfoTag, a fluorophore, or a metal chelate. [Embodiment 28] 28. The method of any one of embodiments 1-27, wherein the functional group moiety comprises an azadibenzocyclooctyne, a thiol, an alkene, an alkyne, an azide, a tetrazine, a trans-cyclooctene, a (diphenylphosphino)aryl, a (diphenylphosphino)alkyl, or an activated ester (e.g., a hydroxybenzotriazole (HOBt) ester). [Embodiment 29] 29. The method of any of embodiments 1 to 28, wherein the solid support is a membrane, glass, plastic, a synthetically prepared polymer, an Eppendorf tube, a well of a multiwell plate, or a surface plasmon resonance chip. [Embodiment 30] 30. The method of any preceding embodiment, wherein the recognition moiety is an antibody, a DNA-binding protein, an RNA-binding protein, a carbohydrate-binding protein, or a lipid-binding protein. [Embodiment 31] 31. The method of embodiment 30, wherein the recognition moiety is an antibody. [Embodiment 32] 32. The method of embodiment 31, wherein the antibody is an antibody against a biological molecule. [Embodiment 33] 32. The method of embodiment 31, wherein the antibody is an antibody against an epitope tag. [Embodiment 34] 34. The method of any of embodiments 1-33, wherein step (d) comprises detecting bound detectable conjugate by ELISA. [Embodiment 35] 34. The method of any of embodiments 1-33, wherein step (d) comprises detecting bound detectable conjugate by Western blot. [Embodiment 36] 34. The method of any one of embodiments 1-33, wherein step (d) comprises detecting bound detectable conjugate by immunofluorescence assay. [Embodiment 37] 34. The method of any of embodiments 1-33, wherein step (d) comprises detecting the bound detectable conjugate by a fluorometric assay. [Embodiment 38] 34. The method of any of embodiments 1-33, wherein step (d) comprises detecting the bound detectable conjugate by Fluorometric Micro-Volume Assay Technology (FMAT). [Embodiment 39] 34. The method of any one of embodiments 1 to 33, wherein step (d) comprises detecting bound detectable conjugate by subcellular staining. [Embodiment 40] 40. The method of any of embodiments 1 to 39, performed on a crude cell extract comprising the biomolecule. [Embodiment 41] 40. The method according to any one of embodiments 1 to 39, which is carried out on a liposomal preparation of protein comprising a biomolecule. [Embodiment 42] 40. The method according to any one of embodiments 1 to 39, which is carried out on an isolated organelle containing the biomolecule. [Embodiment 43] 40. The method of any of embodiments 1 to 39, wherein the method is performed on a purified protein preparation comprising the biomolecule. [Embodiment 44] 40. The method of any one of embodiments 1 to 39, which is carried out in situ. [Embodiment 45] 40. The method according to any one of embodiments 1 to 39, which is a cell-based assay. [Embodiment 46] 46. ​​The method of embodiment 45, wherein the biomolecule is expressed in a cell. [Embodiment 47] 47. The method of embodiment 46, wherein the cells are engineered to express a biomolecule. [Embodiment 48] 48. The method of any one of embodiments 45 to 47, wherein the cells are lysed before step (b). [Embodiment 49] 49. The method of any one of embodiments 1 to 48, wherein step (d) further comprises quantifying the amount of bound detectable conjugate. [Embodiment 50] 50. The method of embodiment 49, wherein step (a) further comprises a substrate for the biomolecule. [Embodiment 51] 51. The method of embodiment 50, wherein the amount of bound detectable conjugate formed in the presence of a substrate for the biomolecule is less than the amount of bound detectable conjugate formed in the absence of the substrate (i.e., the probe molecule is a substrate-competitive probe). [Embodiment 52] 51. The method of embodiment 50, wherein the amount of bound detectable conjugate formed in the presence of a substrate for the biomolecule is greater than the amount of bound detectable conjugate formed in the absence of the substrate (i.e., the probe molecule is a substrate-cooperative probe).

Claims

1. 1. A method for identifying a ligand of a biomolecule, comprising: (a) contacting the biomolecule, a probe molecule, and a substrate for the biomolecule, wherein the probe molecule comprises a binding element, a reporter group, and a reactive moiety, the probe molecule binds to the biomolecule via the binding element, and the reactive moiety forms a covalent bond with the biomolecule, thereby forming a conjugate; (b) contacting the conjugate with a detectable molecule comprising a functional moiety that reacts with the reporter group, thereby forming a detectable conjugate; (c) contacting the detectable conjugate with a solid support, the solid support comprising a recognition moiety, the recognition moiety binding to the detectable conjugate, thereby forming a bound detectable conjugate; and (d) identifying the probe molecule as a ligand of the biomolecule by detecting the bound detectable conjugate; Including, the biomolecule is expressed in a cell; The above method, wherein the amount of bound detectable conjugate formed in the presence of a substrate for the biomolecule is less than the amount of bound detectable conjugate formed in the absence of the substrate (i.e., the probe molecule is a substrate-competitive probe), or the amount of bound detectable conjugate formed in the presence of a substrate for the biomolecule is greater than the amount of bound detectable conjugate formed in the absence of the substrate (i.e., the probe molecule is a substrate-cooperative probe).

2. The method of claim 1 , wherein the binding member is a small molecule fragment, a peptide, or a nucleic acid.

3. The method of claim 2, wherein the nucleic acid is RNA or DNA.

4. The method of any one of claims 1 to 3, wherein the binding member is a member of a library comprising a plurality of binding members.

5. 5. The method of claim 4, wherein the binding elements are small molecule fragments, and each small molecule fragment has at least one of the following characteristics: molecular weight < 300 Da, cLogP < 3, hydrogen bond donors < 3, and hydrogen bond acceptors < 3.

6. The method according to any one of claims 1 to 5, wherein the biomolecule is a protein, lipid, carbohydrate, or nucleic acid.

7. 7. The method of claim 6, wherein the biomolecule is a protein and the reactive moiety forms a covalent bond with an amino acid of the protein.

8. The method of claim 6, wherein the biomolecule is a nucleic acid, and the nucleic acid is RNA or DNA.

9. The method of any one of claims 1 to 8, wherein the biomolecule comprises an epitope tag.

10. 10. The method of claim 9, wherein the epitope tag is FLAG (registered trademark), 6xHis, HA, c-myc, glutathione-S-transferase, Strep-tag, maltose binding protein, chitin binding protein, S tag, V5 tag, or AviTag (registered trademark).

11. 11. The method of any one of claims 1 to 10, wherein the reporter group comprises an azadibenzocyclooctyne, a thiol, an alkene, an alkyne, an azide, a tetrazine, a trans-cyclooctene, a (diphenylphosphino)aryl, a (diphenylphosphino)alkyl, or an activated ester.

12. 12. The method of any one of claims 1 to 11, wherein the reactive moiety is a photocrosslinkable group, a sulfonyl fluoride, a fluorosulfate, a Michael acceptor moiety, a leaving group moiety, or a moiety that forms a covalent bond with a nucleophilic moiety in the side chain of a naturally occurring alpha amino acid.

13. 13. The method of claim 12, wherein the reactive moiety is a moiety that forms a covalent bond with a nucleophilic moiety in the side chain of a naturally occurring alpha amino acid, the nucleophilic moiety in the side chain being a thiol group of cysteine, an amino group of lysine, a hydroxyl group of serine or threonine, or a phenolic group of tyrosine.

14. The method of any one of claims 1 to 13, wherein the detectable molecule comprises digoxigenin, nickel NTA (nitrilotriacetic acid), a chromophore, or a luminophore.

15. 15. The method of claim 14, wherein the detectable molecule comprises a chromophore, the chromophore comprising a non-fluorescent chromophore, a quencher, an absorbing chromophore, a fluorophore, an organic dye, an inorganic dye, a metal chelate, or a fluorescent enzyme substrate.

16. The method of any one of claims 1 to 13, wherein the detectable molecule is biotin.

17. 17. The method of claim 16, wherein the biotin is bound to a streptavidin conjugate.

18. 18. The method of claim 17, wherein the streptavidin conjugate comprises HRP, SulfoTag, a fluorophore, or a metal chelate.

19. 19. The method of any one of claims 1 to 18, wherein the functional moiety comprises an azadibenzocyclooctyne, a thiol, an alkene, an alkyne, an azide, a tetrazine, a trans-cyclooctene, a (diphenylphosphino)aryl, a (diphenylphosphino)alkyl, or an activated ester.

20. 20. The method of any one of claims 1 to 19, wherein the solid support is a membrane, glass, plastic, a synthetically prepared polymer, an Eppendorf tube, a well of a multiwell plate, or a surface plasmon resonance chip.

21. 21. The method of any one of claims 1 to 20, wherein the recognition moiety is an antibody, a DNA-binding protein, an RNA-binding protein, a carbohydrate-binding protein, or a lipid-binding protein.

22. 22. The method of claim 21, wherein the recognition moiety is an antibody.

23. 23. The method of claim 22, wherein the antibody is an antibody against the biomolecule or an antibody against an epitope tag.

24. 24. The method of any one of claims 1 to 23, wherein step (d) comprises detecting the bound detectable conjugate by ELISA, Western blot, immunofluorescence assay, fluorometric assay, Fluorometric Micro-Volume Assay Technology (FMAT), or subcellular staining.

25. 25. The method of any one of claims 1 to 24, performed on a crude cell extract comprising said biomolecule, a liposomal preparation of protein comprising said biomolecule, an isolated organelle comprising said biomolecule, or a purified protein preparation comprising said biomolecule.

26. The method of any one of claims 1 to 24, which is carried out in situ.

27. The method of any one of claims 1 to 24, which is a cell-based assay.

28. 28. The method of any one of claims 1 to 27, wherein the cells are engineered to express the biomolecule.

29. 29. The method of any one of claims 1 to 28, wherein the biomolecule is a tagged creatine transporter.

30. The method of any one of claims 1 to 29, wherein the cells are lysed prior to step (b).

31. The method of any one of claims 1 to 30, wherein step (d) further comprises quantifying the amount of bound detectable conjugate.

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