WO2024151471A1

WO2024151471A1 - High-throughput assay based on ligand-biased structural dynamics response

Info

Publication number: WO2024151471A1
Application number: PCT/US2024/010333
Authority: WO
Inventors: James Inglese; Patricia Karen DRANCHAK
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2023-01-09
Filing date: 2024-01-04
Publication date: 2024-07-18

Abstract

Disclosed herein are assays for detecting interaction of a target of interest (TOI) with a compound. Specifically, the disclosed assays are based on the idea that compound-induced change in the structural conformation ensemble distribution environment of a label attached to the TOI may be used to detect interaction of the compound with a TOI. The label may be part of a complementary reporter system that uses a substrate to produce a fluorescent or chemiluminescent signal. Alternatively, the label may be detected directly by fluorescence.

Description

40574-117 -1- HIGH-THROUGHPUT ASSAY BASED ON LIGAND- BIASED STRUCTURAL DYNAMICS RESPONSE BACKGROUND OF THE INVENTION [0001] Ligand binding assays are fundamental to the methods of biology, drug discovery, clinical diagnostics, and environmental toxicology. Depending on the nature and knowledge surrounding the macromolecular target also referred to here as the target of interest (TOI), whether a receptor, enzyme, regulatory subunit or scaffold, or a sequence-defined nucleic acid folded into a complex tertiary structure, assay strategies can vary widely. [0002] The ability to measure, for example, a ligand’s interaction with an enzyme TOI generally requires a functional measurement (e.g., kinase activity) and for some enzymes can require hard to obtain or unstable substrates (e.g., air-sensitive reduced folate cofactors), complex enzymatic coupling strategies (e.g., iPGM or MMCoA mutase), high-throughput screening (HTS)-incompatible post-assay derivatization steps (e.g., MtbCM), transmembrane enzymes and their reactions (e.g., hDHHCs), or receptors requiring elaborate cellular systems (e.g., GPCRs). Such issues often complicate or preclude a viable path for studying protein interactions and/or identifying new potential drugs. [0003] Competitive ligand binding approaches utilizing spectroscopically sensitive fluorophores, among other electrochemical and biophysical techniques do not rely on catalytic turnover of substrate by the TOI. However, these methods generally require a moderate to high affinity ligand that can be conjugated to the molecular beacon. There are relatively few techniques, such as surface plasmon resonance (SPR), which can measure a direct ligand interaction without the use of a functional output or labeled ligand, although for SPR the TOI is generally “labeled” to the sensor chip surface which often involves an added sequence tag and/or chemical modification to the TOI to allow adherence to the chip surface. While powerful, the differential mass dependence of SPR and comparatively low throughput can limit widespread general applications to ligand discovery and are generally not widely used for this purpose. One of the few direct binding assays for ligand discovery is fragment-based screening. However this primarily X-ray or NMR-based technique, is 40574-117 -2- limited to specialized methods, particularly TOI proteins that are soluble at high concentrations and for NMR TOI proteins of relatively low molecular mass. [0004] Assay sensitivity is limited by signal background and TOI concentration. Assays incorporating background signal decay (containing a time-resolution component), such as time-resolved fluorescence (TRF) approaches, ushered in unprecedented sensitivity on par with radioligand binding assays of the time. However, both radioligand and early TRF assays required separation of the receptor-ligand complex from unbound ligand and were therefore limited by the dissociation constant of the complex, suitable only for interactions that remained sufficiently intact during the separation phase of the assay. As well, assays requiring a media removal or sampling step are difficult to configure for HTS, particularly in low volume 384- and 1536-well format plates. Microplate washers can be used for aspiration of media from a cell monolayer. However selective testing of extracellular media for an antigen or enzyme activity can require specialized methods or reagent development. [0005] Homogenous time-resolved fluorescence (HTRF) or bioluminescent resonance energy transfer (or BRET) assay designs overcame the need for a separation step but required a second labeled component to enable a FRET element coupled to time-resolution. Even in the few cases where a single labeled ligand or probe can enable a competitive or direct binding assay, as in fluorescent polarization (FP) and thermal shift (e.g., Thermofluor), the fluorescently labeled ligand or probe must be bound at between 50-80 % to achieve a usable anisotropy or fluorescent enhancement signal, respectively, which can impact assay sensitivity and require substantial TOI protein depending on the ligand KD. [0006] Thus, while numerous configurations of detection technologies exist, none are universally applicable to a broad range of biological TOIs or even within a specific TOI class. There is still a need for a simple and highly sensitive detection assay that can easily be configured for use in HTS. The present disclosure addresses this need by providing a novel approach to directly measuring ligand binding to a TOI protein, even those with unknown function, in a high throughput manner. [0007] SUMMARY [0008] One aspect of the disclosure is a method of detecting interaction of a compound with a TOI, comprising: a) contacting the compound with: i) the TOI, wherein the TOI comprises a label, the label being a first element of a sensor-reporter system; ii) a 40574-117 -3- second element of the sensor-reporter system that interacts with the label; and optionally, iii) a third element of the sensor-reporter system; and, b) detecting the status of a first signal, if any, produced by interaction of the first and second elements, and optionally the substrate, of the sensor-reporter system, thereby detecting interaction, if any, of the compound with the TOI molecule. In certain aspects, detecting the status of the first signal may comprise detecting the presence or absence of the first signal. In certain aspects, failing to detect the first signal in the absence of a compound, and detecting the presence of the first signal in the presence of the compound indicates interaction of the compound with the TOI. In certain aspects, detecting the first signal in the absence of a compound, and detecting the absence of the first signal in the presence of the compound indicates interaction of the compound with the TOI. In certain aspects, the method may comprise comparing the status of the first signal with the status of a second signal, if any, from a second reaction mixture identical to the first reaction mixture but lacking the compound, wherein a significant difference in the status of the first and second signals indicates interaction of the compound with the TOI. [0009] In certain aspects, the method may comprise detecting the status of the second signal in the second reaction mixture. In certain aspects, the status of the first signal may comprise the presence or absence of the first signal, and the status of the second signal may comprise the presence or absence of the second signal. In certain aspects, the presence of the first signal and the absence of the second signal indicates interaction of the compound with the TOI. In certain aspects, absence of the first signal and the presence of the second signal indicates interaction of the compound with the TOI. In certain aspects, the status of the first signal may comprise the level of the first signal, and the status of the second signal may comprise the level of the second signal, and a significant difference in the levels of the first and second signals indicates interaction of the compound with the TOI. In certain aspects, a level of the first signal that is significantly greater than the level of the second signal indicates interaction of the compound with the TOI. In certain aspects, a level of second signal that is significantly greater than the level of the first signal indicates interaction of the compound with the TOI. [0010] One aspect of the disclosure is a method of detecting interaction of a compound with a TOI, comprising detecting a significant difference, if any, between: a) the status of a first signal produced by interaction of the TOI, wherein the TOI comprises a label, the label being the first element of a sensor-reporter system, with a second element 40574-117 -4- and, optionally, a third element of a sensor-reporter system, in the absence of the compound; and, b) the status of a second signal produced by interaction of the labeled TOI with the second element and, optionally the third element, of the sensor-reporter system in, the presence the compound, wherein production of the first and second signals by the sensor-reporter system does not comprise resonance energy transfer, and wherein detection of a significant difference in the status of the first and second signals indicates interaction of the compound with the TOI. [0011] One aspect of the disclosure is a method of detecting interaction of a compound with a TOI, comprising detecting a significant difference between the status of a first signal produced by a first reaction mixture comprising the TOI in the absence of the compound, wherein the TOI is joined to a label, the label being a first element in a sensor- reporter system, and the status of a second signal produced by a second reaction mixture identical to the first but comprising the compound, wherein the first and second reaction mixtures comprise a second element of the sensor-reporter system and, optionally, a third element of the sensor-reporter system, wherein production of the first and second signals does not comprise resonance energy transfer, and wherein detection of a significant difference between the levels of the first and second signals indicates interaction of the compound with a TOI molecule. In certain aspects, the first and second reaction mixtures may be physically distinct reaction mixtures. In certain aspects, the second reaction mixture may be produced from the first reaction mixture by adding the compound to the first reaction mixture. [0012] One aspect of the is a method of detecting interaction of a compound with a TOI, comprising: a) contacting a labeled TOI, the label being a first element of a sensor-reporter system, with a second element of the sensor-reporter system, and, optionally, a third element of the sensor-reporter system, thereby forming a reaction mixture; b) detecting a first signal, if any, produced by interaction of the first and second elements, and optionally the third element, of the sensor-reporter system; c) contacting the compound with the reaction mixture; d) detecting a second signal, if any, produced by interaction of the first and second elements, and optionally the third element, of the sensor- reporter system; and, e) comparing the first and second signals, wherein detection of a significant difference between the first and second signals indicates interaction of the compound with the TOI. In these aspects, detecting a significant difference may comprise detecting the presence or absence the first and second signals. In these aspects, detecting a 40574-117 -5- significant difference in the presence or absence of the first and second signals indicates interaction of the compound with the TOI. [0013] In these aspects, the status of the first signal may comprise the presence or absence of the first signal, and the status of the second signal may comprise the presence or absence of the second signal. In these aspects, detecting a significant difference between the presence or absence of the first and second signals indicates interaction of the compound with the TOI. In these aspects, detecting the absence of the first signal and the presence of the second signal indicates interaction of the compound with the TOI. In these aspects, detecting the presence of the first signal and the absence of the second signal indicates interaction of the compound with the TOI. In these aspects, the status of the first signal may comprise the level of the first signal, and the status of the second signal may comprise the level of the second signal. In these aspects, detecting a significant difference between the level of the first and second signals indicates interaction of the compound with the TOI. In these aspects, detecting that the level of the first signal is significantly greater than the level of the second signal indicates interaction of the compound with the TOI. In these aspects, detecting that the level of the first signal is significantly less than the level of the second signal indicates interaction of the compound with the TOI. [0014] In certain aspects, if interaction of the compound and the TOI is detected, the compound may be identified as a compound that interacts with the TOI. In certain aspects, the method may comprise determining KD of the compound, wherein if the KD of the compound for the TOI is at least 10^-5, at least 10^-6, at least 10^-7, at least 10^-8, at least 10^-9, at least 10^-10, or at least 10^-11, identifying the compound as a compound that binds the TOI. In certain aspects, the TOI may consist of a single molecule or a complex comprising two or more molecules. The TOI may comprise a biological molecule, which may be a protein or a nucleic acid molecule. In certain aspects, the label may be joined to the biological molecule. [0015] In certain aspects, the sensor-reporter system may comprise a complementation system comprising the label, the second element and/or the third element. The label may comprise a peptide, and the second element may be a complementing polypeptide that binds to the peptide, and the third element may be a substrate, wherein binding of the complementing polypeptide to the label produces a peptide/complementing polypeptide complex having enzymatic activity, and wherein the label/complementing polypeptide complex acts on the substrate to produce a detectable signal. The peptide and 40574-117 -6- the complementary protein may be from a bioluminescent protein, which may be luciferase or a photoprotein, and the detectable signal may be emitted light. In certain aspects the bioluminescent protein may be selected from the group consisting of Renilla luciferase, Gaussia luciferase, Nanoluc® luciferase, Cypridina luciferase, Firefly luciferase, Click- beetle luciferase, Dinoflagellate luciferase, Euphausiid luciferase, bacterial luciferase, and fungal luciferase. [0016] In certain aspects, the peptide and the complementary polypeptide may be from an enzyme that acts on the substrate to produce a colorimetric or chemiluminescent reaction. The enzyme may be selected from the group consisting of β- galactosidase, β-glucuronidase, β-lactamase, alkaline phosphatase, acetylesterase, esterase 2 from Alicyclobacillus acidocaldarius, acetylesterase, DHFR, and β-N- acetylglucosaminidase. The label may comprise a peptide, and the second element may be a complementing polypeptide that binds to the peptide, and the third element may be a photon beam, wherein binding of the complementing polypeptide to the label produces a peptide/complementing polypeptide complex, and wherein impingement of the photon beam on the peptide/complementing protein complex results in production of a detectable signal. In certain aspects, the peptide and the complementing protein may be from a fluorescent protein, which may be selected from the group consisting of a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP), a cyan fluorescent protein (CFP), an orange fluorescent protein (OFP), and a red fluorescent protein (RFP), or mutant forms of any fluorescent protein enhancing or altering its natural excitation, emission, or quantum yield. [0017] In certain aspects, the TOI molecule may comprise a protein and the peptide label may be joined to the carboxy-terminal end of the TOI protein or the amino-terminal end of the TOI protein. In certain aspects, the peptide label may be inserted into the amino acid sequence of the TOI protein. In certain aspects, the peptide label may be joined to the sidechain of an amino acid within the sequence of the TOI protein. The label may be joined to an amino acid introduced into the sequence of the TOI protein. The introduced amino acid may comprise a reactive side group. The introduced amino acid may be selected from the group consisting of cysteine, lysine, aspartic acid, glutamic acid, arginine, tyrosine, methionine, histidine, tryptophan, or reactive non-natural amino acid sidechain. 40574-117 -7- [0018] In certain aspects, the TOI molecule may comprise a nucleic acid and the peptide label is attached onto a nucleic acid base or sugar within the sequence of the nucleic acid. [0019] In certain aspects, the sensor-reporter system may comprise the label and the second element. The label may comprise a photoluminescent molecule and the second element may be a photon stream. The photoluminescent molecule may comprise a lanthanide, such as lanthanide cryptate, or a quantum dot. The lanthanide cryptate may comprise bis-bipyridine macrocycle. In certain aspects, detection of the signal may comprise time-resolved lanthanide luminescence. [0020] One aspect of the disclosure is use of a method of the disclosure to identify a compound that binds to the TOI molecule. [0021] One aspect of the disclosure is use of the method of the disclosure to identify a potential therapeutic compound. [0022] One aspect of the disclosure is use of the method of the disclosure to determine the K_D of a compound for a TOI. [0023] One aspect of the disclosure is a kit comprising the first, second, and optionally, third element of a sensor-reporter system of the disclosure and instructions for detecting an interaction of a compound with a TOI molecule according to methods of the disclosure. The kit may comprise at least one reagent for producing the labeled TOI molecule. [0024] BRIEF DESCRIPTION OF THE DRAWINGS [0025] FIG.1A depicts the conceptualization of Structural Dynamics Response (SDR) as macromolecule motion attenuated by ligand-associated frequency dampening. FIG.1B depicts the practical implementation and principle behind the SDR assay. Here, the protein target of interest (TOI) (left) undergoes vibrational and secondary structure motion in its unbound apo state. Upon ligand binding (center) sufficient structural dynamic freezing allows efficient capture of the α-peptide sequence, here at the protein TOI C- terminus, by the complementary ω-fragment to reconstitute a functional sensor- reporter protein (right). Concomitant addition of the sensor-reporter substrate (hexagon) results in greater signal output (far right arrows) from the ligand-bound protein TOI relative to any sensor-reporter protein that would reconstitute from the apo TOI form. 40574-117 -8- [0026] FIGS.2A & 2B illustrate phosphoglycerate mutase (PGM) coupled-enzyme assays. FIG.2A shows the isomerization of phosphoglycerate catalyzed by cofactor-independent PGMs (iPGMs). In the catalytic mechanism of iPGM, a protein phosphoserine intermediate facilitates the transfer of the phosphate between the 3- and 2- positions of glycerate. FIG.2B illustrates coupling enzymes used in the assay of PGMs. Coupling enzymes and their corresponding substrates are: enolase/2PG; pyruvate kinase/PEP; lactate dehydrogenase/pyruvate and NADH, and luciferase/ATP and luciferin. The products of the lactate dehydrogenase reaction are NAD⁺, the formation of which is measured by decreased absorbance at 340 nm (absorbance mode), whereas ATP is measured by firefly luciferase oxidation of luciferin (LH2) generating light luminescence (hu), oxyluciferin (L) and CO2 (luminescence mode). [0027] FIGS.3A-3F illustrate the structural dynamics response (SDR) assay concept applied to C. elegans iPGM (C. e. iPGM) and a macrocyclic peptide ligand of C. e. iPGM, Ipglycermide Ce-2. FIG.3A illustrates an SDR assay using purified C- terminally HiBiT (α-peptide or label) tagged C. e. iPGM. The equilibrium between apo (i) and Ipglycermide Ce-2-bound C. e. iPGM (ii) can be distinguished by the addition of LgBiT (ω-fragment or second component) to reconstitute NLuc (sensor-reporter complexes, iii and iv), the activity of which is determined from its furimazine (FMZ)- dependent bioluminescence. FIG.3B shows the comparative luminescence from various C. e. iPGM-HiBiT concentrations and resulting S:B (*). Assay buffer containing LgBiT and FMZ without (grey) or with (white) C. e. iPGM-HiBiT in a fixed ratio (see Table 1). FIG. 3C shows saturation binding of Ce-2 to 0.1, 0.5 or 1 nM iPGM. FIG.3D shows concentration response curves (CRCs) depicting binding of various ipglycermide macrocyclic peptide inhibitors of iPGM measured by conventional coupled-assay approach (open symbols) and SDR NLuc output (closed symbols). FIG.3E shows relative detection sensitivity of SDR (NLuc output) compared to a functional couple-enzyme for co-factor independent phosphoglycerate mutase (iPGM) for ipglycermide Ce-2 binding. Data obtained with C. elegans cofactor-independent phosphoglycerate mutase containing a C- terminal HiBiT (iPGM-HiBiT). FIG.3F shows concentration response curves determined from 0.5 ($), 1 (!), and 5 nM (#) C. e. iPGM-HiBiT for Ce-2d (FIG.3F) ranged from EC₅₀ =0.5 – 5 nM. Error bars represent the standard deviation of two technical replicate wells. 40574-117 -9- [0028] FIGS.4A-4C illustrate the SDR assay applied to dihydrofolate reductase (DHFR). FIGS.4A & 4B show SDR assay-derived saturation binding curves for methotrexate (MTX) binding to a range of human DHFR-HiBiT concentrations (0.5, 2.5, 10 and 20 nM) in the absence (FIG.4A) or presence of 5 μM NADPH (FIG.4B), to yield an MTX KD of 24 nM!120 nM, or 90 pM! ~10 nM, respectively. FIG.4C shows concentration response curves determined from 10 nM DHFR-HiBiT (", pEC50 -6.86 ±0.41) or 20 nM DHFR-HiBiT in the presence of 5 μM NADPH (!, pEC₅₀ -7.96 ±0.20). [0029] FIG.5 SDR assay correlation with other assay formats. Correlation between pEC50 from the SDR assay and the pIC50 from either the coupled enzyme assay for C. e. iPGM or a fluorescent protein (FP)-based competition binding assay for DHFR. The heavy dotted 45-degree line indicates a 1:1 correspondence between the assay formats for the given inhibitor, while the lighter lines indicate 10- and 100-fold differences. [0030] FIGS.6A-6C illustrate cell lysate-based SDR assays. FIG.6A illustrates that TOIs or sentinel-U-peptide proteins can be introduced into a cellular context by the standard approaches, i. linearized plasmid transfection, ii. Flip-in recombination, and iii. genome editing using CRISPR/Cas9 mediated homologous recombination. FIG.6B illustrates a lysate SDR assay configuration using plated cells expressing a TOI-U-peptide protein where ligand can be added to cells just prior to or after cell lysis followed by k- fragment, substrate-mediated sensor-reporter output. FIG.6C shows an example of lysate SDR C. e. iPGM-HiBiT assay gain-of-signal output for a titration of an iPGM cyclic peptide ligand, Ipglycermide Ce-2d (!, pEC507.76 ±0.04, bottom graph) vs. the inhibitory effect of the same ligand in a standard iPGM coupled enzyme assay using 5 nM C. e. iPGM (", pIC₅₀8.76 ±0.03, top graph). [0031] FIG.7 lists some examples of additional TOIs that may be studied using the SDR assay. It also lists exemplary ligands and sensor-reporter systems for each TOI. [0032] FIGS 8A & 8B illustrate a G protein-coupled receptor (GPCR)- based SDR assay. FIG.8A illustrates that ligand binding results in intracellular loop and C- terminus reorganization to alter the position and dynamics of the C-terminal U-peptide (“label”) to affect the efficiency of sensor formation from U-peptide complementation of the added k-fragment. In FIG.8B, because the GPCR is assayed as a plasma membrane preparation which may allow ligand-dependent coupling of heterotrimeric G proteins or 40574-117 -10- GRK-dependent phosphorylation (if ATP is added) and subsequent V-arrestin binding. These protein-protein interactions could enhance the SDR output or add additional levels of pharmacology to probe. For example, the GPCR-U-peptide may act as a sentinel for the indirect detection of inhibitors of GRK activity. [0033] FIG.9A-B illustrates the cAMP-dependent regulation of PKA-U- peptide by R1U for two possible SDR assay outputs. FIG.9A depicts an SDR assay system based on a PKA-U-peptide /R1U interaction, where terminally labeled U-peptide PKA is proposed to be more effectively able to complement the k-fragment when complexed with R1U. Thus, binding of cAMP, releasing R1U would make the label less accessible, resulting in decreased, or loss-of-signal (lower plot) by the sensor-reporter. FIG.9B, alternatively, the assay may be structured such that binding of cAMP would make the U-peptide label more accessible, resulting an increase in, or gain-of, -signal (lower plot) from the sensor-reporter. [0034] FIGS.10A-10E illustrate a method to surface label a protein TOI to enable an SDR assay. Using a library design approach, surface residues (e.g., lysine) distributed over the TOI surface are identified and individually encoded with cysteine residues, selected, and expressed for example in E. coli, with subsequent purification facilitated by a C- or N-terminal His tag (FIG.10A). Individual Cys-modified TOIs can be characterized for activity and ligand binding (FIG.10B) or taken directly to U-peptide labeling using a thiol-reactive label which can be used to attached, for example, an U- peptide, an environmentally sensitive fluorophore, or lanthanide cryptate (FIG.10C). Cys- modified TOIs are separated from, for example, unreacted U-peptide by Ni-NTA chromatography, excess imidazole is removed by dialysis, protein concentration is determined and TOIs are used in SDR assays. SDR activity can result in a range (a-f) of possible response outputs from the sensor-reporter complex (FIG.10D). C. e. iPGM is shown in FIG.10E as a specific example of how a maleimide-conjugated HiBiT peptide would be used to modify specific surface-engineered cysteine residues to create an SDR assay independent of either a C- or N-terminal HiBiT sequence. [0035] FIGS.11A-11B provide an overview of the generalized SDR assay concept based on the development of an SDR assay signal from a labeled TOI – ligand interaction, and specific SDR labels and sensor-reporter outputs. A TOI containing an SDR label is contacted with a solution containing test ligand (compound), or without the 40574-117 -11- test ligand (compound). The 2^nd component (and optionally 3^rd component) is added to create the SDR sensor-reporter output (FIG.11A). Specific examples of an SDR assay are shown using labels that are based on U-complementation from an enzyme or fluorescent protein sensor-reporter, or a lanthanide cryptate time-resolved fluorescence sensor-reporter. Example of the ligand (compound)-dependent outputs are shown in the compound concentration – SDR activity plot, where gain-of-signal outputs are illustrated by curves a., b., c. and d., while loss-of-signal outputs are represented by curves e. and f (FIG.11B). [0036] FIG.12 shows the results of an SDR proof-of-concept assay using an N-terminal fusion of HiBiT to firefly luciferase (N-HiBiT-FLuc). The left axis indicates FLuc enzymatic luminescence RLU with increasing concentrations of the FLuc inhibitor PTC124. The right axis indicates SDR-dependent RLU with increasing PTC124 concentration in the presence (solid squares) or absence (open squares) of 10 aM ATP. [0037] FIGS.13A-13H show representative concentration response curves (CRCs) for various clades of compounds from a library of 1,221 compounds enriched for FLuc inhibitors screened in the FLuc enzymatic luminescence assay (solid circles, !) and in the SDR assay in the presence (solid squares, #) or absence (open squares, !) of 10 aM ATP. [0038] FIG.14 shows CRCs for SDR output of imatinib binding to N- HiBiT-ABL1 kinase domain using the functional enzyme assay (!) with Kinase Glo Plus from Promega (KGP) or the SDR assay in the presence (#) or absence ($) of 80 aM ATP cofactor, respectively. [0039] FIGS.15A-15H show representative CRCs from a kinase inhibitor library SDR qHTS. Left axis, functional enzyme assay measuring ATP turnover in the presence of a peptide substrate, right axis, SDR assay in the presence (#) or absence ($) of 80 aM ATP. [0040] FIG.16 shows a heatmap displaying enzymatic IC₅₀ or SDR₅₀ activity from the full kinase inhibitor library, for a KGP assay or a SDR assay ±ATP cofactor, respectively. Annotated kinase inhibitor targets (group column) include databases including ABL1 as a target (!) or not including ABL1 (!). [0041] FIG.17 shows a correlation plot comparing the enzymatic pIC₅₀ to pSDR50 (without ATP) with symbols colored according to database annotation. AL, ALW- II-41-27; FR, FRAX-486; P1, PD-180970; P2, PD-173955; NS, NSC762948. 40574-117 -12- [0042] FIGS.18A & 18B show Antifolate selectivity and affinity for purified and lysate dihydrofolate reductase (DHFR) using SDR. FIG.18A shows NADPH co-factor dependent SDR saturation MTX binding for 5 and 0.5 nM DHFR. FIG.18B shows NADPH titration across 0.5 nM DHFR. [0043] FIG.19 shows that DHFR-HiBiT cellular lysate titrated from 100- to 2000-fold is potently inhibited by methotrexate. [0044] DETAILED DESCRIPTION OF THE INVENTION [0045] The present disclosure relates to assays for detecting interaction of a compound with a target of interest (TOI), such as a protein, a protein complex, or a nucleic acid molecule. More specifically, the disclosure provides methods of detecting binding of a compound to a TOI by detecting production, or alteration, of a detectable signal. Assays and method of the disclosure are based on the idea that interaction (e.g., binding) of a compound, also referred to as a ligand, with a TOI may induce alterations in the ensemble of structural conformations representing the TOI (FIG.1A). Due to such ligand-induced alterations of structural ensembles, a label (e.g., U-peptide, FIG.1B) attached to the TOI may experience a different environment in the ligand bound TOI relative to the environment experienced in the unbound TOI (e.g., apo protein TOI, FIG. 1B, FIG.8A and FIG.9A-B). For example, a conformational change in the TOI resulting from interaction (e.g., binding) of a compound or ligand may result in the label being more exposed, and thus more accessible to the surrounding environment relative to the exposure observed in the unbound TOI (e.g., U-peptide, FIG.8A and FIG.9B). Alternatively, compound-induced conformational changes may cause the label to be less exposed, and thus less accessible (e.g., U-peptide, FIG.9A). The inventors have discovered that such compound-induced change in the structural conformation ensemble distribution environment of the label may be used to detect interaction of a compound with a TOI. More specifically, the inventors have discovered that compound-induced changes to the structural conformation or vibrational motion ensemble distribution environment of the TOI may produce alterations in the presence, level, or direction of a signal resulting from the presence of the label on the TOI. Current detection systems are not designed for detecting compound or ligand-induced changes (e.g., structural changes, vibrational motion dampening or arrest, etc.) in TOIs (such as proteins), but rather are based on functional aspects of the TOI, or competitive binding assays dependent on a labeled TOI ligand, or 40574-117 -13- thermally induced ligand-dependent changes in the TOI. For example, fluorescence resonant energy transfer methods require multiple labels, thereby increasing complexity and the consequent chances of affecting the natural structure and/or function of the TOI. Similarly, two -hybrid type systems require that individual elements of a complementation reporter system be attached to separate compounds, such as proteins, that interact to bring the individual reporter system elements together, resulting in production of a signal. The methods disclosed herein provide a significant improvement over such systems. First, they use a single, small label attached to a TOI, thereby simplifying the system and lessening the effect of the label on the TOI. Next, the compound being tested for interaction with a TOI is not covalently linked to any element of the sensor reporter system. In fact, in complementation reporter systems used in the disclosed methods, at least one element need not be, and preferably is not, covalently linked to a non-requisite molecule, such as a non- requisite protein or peptide. As used herein, a non-requisite molecule is a molecule that is not required for elements of the sensor-reporter system to form a complex capable of producing detectable signal. For example, in aspects in which the sensor-reporter system comprises an enzyme-based complementation system, in which the reporter enzyme is split into first and second elements, each element consisting of a portion of the amino acid sequence of the enzyme, the second element may comprise only those sequences necessary to bind the first element, thereby re-creating the functional enzyme. Thus, in SDR assays of the disclosure using complementation reporter systems, only one element (e.g., the small label) need be attached to a molecule, namely the TOI. The second element of the reporter system is not covalently joined to a non-requisite molecule and need only comprise those sequences necessary for the second element to bind the label, thereby forming a complex capable of producing a detectable signal. This concept is illustrated in FIGS.1B, 3A and 11B. In addition, in certain aspects methods of the disclosure may not comprise surface plasmon emission. Finally, the inventors have discovered that the disclosed methods may be significantly more sensitive than current systems for detecting compound binding to a TOI when the KD is below the level of sensitivity of the detection technology of the standard methods. Thus, a method of the disclosure may generally be practiced by contacting a labeled TOI with a compound under conditions suitable for production of label-related, detectable signal, and detecting the signal, or change therein, thereby detecting interaction of the compound with the TOI. Depending on the design of the assay, detection of the signal may comprise detecting the presence, absence, gain or loss of the 40574-117 -14- signal, or an increase or decrease in the level of the signal. Such changes, relative to a basal signal resulting from the label in the presence or absence of the compound, indicate the compound interacts with the TOI. Specific variations of this general assay are disclosed herein. [0046] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims. [0047] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. As such, the terms "a", "an", "one or more" and "at least one" can be used interchangeably. [0048] Similarly, the terms "comprising", "including" and "having" can be used interchangeably. As used herein, the term “comprising” may be replaced with “consisting” or with “consisting essentially of” in particular aspect, as desired. [0049] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation. [0050] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. [0051] Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-expressed basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. 40574-117 -15- [0052] Unless otherwise expressly stated, certain features of the disclosure, which are, for clarity, described in the context of separate aspects, may also be provided in combination in a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, may also be provided separately or in any suitable sub-combination. All combinations of aspects are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. [0053] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. [0054] As used herein, the term “about” means that the recited numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical value is used, unless indicated otherwise by the context, the term “about” means the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments. [0055] As used herein, the term “isolated”, with regard to a nucleic acid molecule or a polypeptide, means that the nucleic acid molecule or polypeptide is in a condition other than its native environment, such as apart from blood and/or animal tissue. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide can be in a highly purified form, i.e., greater than 95% pure or greater than 99% pure. When used in this context, the term “isolated” does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternately phosphorylated or derivatized forms. [0056] As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “polynucleotide”, or “oligonucleotide” can comprise a polymeric form of nucleotides of any length, can comprise DNA and/or RNA, and can be single- 40574-117 -16- stranded, double-stranded, or multiple stranded. One strand of a nucleic acid also refers to its complement. [0057] One aspect of the disclosure is a method of detecting interaction of a compound with a target of interest (TOI), comprising: [0058] a) contacting the compound with: [0059] i) the TOI, to which a label has been attached, the label (e.g., U- peptide) being a first element of a sensor-reporter system; [0060] ii) a second element (e.g., k-fragment) of the sensor-reporter system, wherein the second element interacts with the label to form a complex (e.g., a protein complex) capable of producing a signal; and, optionally, [0061] iii) a third element of the sensor-reporter system; and, [0062] b) detecting the status of a first signal, if any, produced by interaction of the first and second elements, and optionally the third element, of the sensor- reporter system, thereby detecting interaction, if any, of the compound with the TOI; [0063] wherein the compound is not linked to an element of the sensor- reporter system. In certain aspects, the compound is not covalently linked to any element of the sensor -reporter system. In certain aspects, the second element of the sensor-reporter system is not linked (e.g., covalently) to a non-requisite molecule, such as a non-requisite protein or peptide. In certain aspects, production of the signal by the sensor-reporter system does not comprise resonance energy transfer. [0064] In certain aspects, detecting the status of the first signal may comprise detecting a change in the status of the first signal. In certain aspects, detecting the status of the first signal may comprise detecting the presence, absence, gain, or loss of the first signal. In certain aspects, the sensor-reporter system does not produce the first signal in the absence of a compound that interacts with TOI. Thus, in certain aspects, detecting the presence, or gain, of the first signal in the presence of the compound indicates interaction of the compound with the TOI. In certain aspects, the sensor-reporter system produces the first signal in the absence of a compound that interacts with TOI. Thus, in certain aspects detecting the absence, or loss, of the first signal in the presence of the compound indicates interaction of the compound with the TOI. [0065] In certain aspects, the method may further comprise comparing the status of the first signal with the status of a second signal, if any, from a second reaction mixture identical to the first reaction mixture but lacking the compound (e.g., a control 40574-117 -17- reaction), wherein a significant difference in the status of the first and second signals indicates interaction of the compound with the TOI. [0066] In certain aspects, the status of the second signal may be obtained by detecting the status of the second signal from the second reaction mixture. In certain aspects, detecting the status of the second signal may comprise detecting the presence, or gain, or absence, or loss, of the second signal. In certain aspects, detecting a difference between the presence or absence of the first and second signals indicates interaction of the compound with the TOI. In certain aspects, the presence of the first signal and the absence of the second signal indicates interaction of the compound with the TOI. In certain aspects, the presence of the second signal and the absence of the first signal indicates interaction of the compound with the TOI. In certain aspects, failing to detect a difference between the presence or absence of the first and second signals indicates a lack of interaction of the compound with the TOI. [0067] In certain aspects, detecting the status of the second signal may comprise detecting the level of the second signal. In certain aspects, detecting a significant difference between the level of the first and second signals indicates interaction of the compound with the TOI. In certain aspects, detecting that the level of the first signal is significantly greater than the level of the second signal indicates interaction of the compound with the TOI. In certain aspects, detecting that the level of the second signal is significantly greater than the level of the first signal indicates interaction of the compound with the TOI. In certain aspects, failing to detect a difference between the levels of the first and second signals indicates a lack of interaction of the compound with the TOI. [0068] As used herein, “contacting” a compound with a TOI does not necessarily indicate that the compound and the TOI are forced into physical contact. As used herein, “contacting” means to introduce the compound and the TOI into an environment such that they are able physically come into contact. For example, contacting may mean adding the compound to a solution containing the TOI. It will be understood by those of skill in the art that “contacting” may be replaced with terms such as “introduce”, mix” and the like. [0069] As used herein, the term “interaction” refers to atoms of the compound forming bonds with atoms of the TOI. Such bonds may be covalent or non- covalent and may comprise, for example, ionic bonds, covalent bonds, hydrogen bonds and van der Waals interactions. In certain aspects, the compound may bind to the TOI. In 40574-117 -18- certain aspects, the binding affinity (K_D) between the compound and the TOI may be at least 10^-5, at least 10^-6, at least 10^-7, at least 10^-8, at least 10^-9, or at least 10^-10. [0070] As used herein, the term “compound”, refers to any molecule that can be in contact with a TOI and its ability to interact with the TOI detected. In certain aspects, the compound may be a “test compound” The term “test compound” refers to a compound, the ability of which to interact with, or bind, the TOI is unknown. The compound may be a naturally occurring (i.e., not made by the hand of man) compound, a synthetic (e.g., made by the hand of man) compound, or a combination thereof. Examples of suitable compounds include, but are not limited to, proteins, nucleic acid molecules, lipids, carbohydrates, polysaccharides, lipoproteins, organic molecules, small molecules, and combinations thereof. In certain aspects, the TOI may comprise a protein and the compound may be a ligand for the protein. [0071] As used herein, the term “target of interest (TOI)” refers to any molecule, or complex of molecules, for which it is desired to test its ability to interact with a compound. In certain aspects, the TOI may comprise, consist, or consist essentially, of, a single molecule, which may be a naturally occurring molecule or a synthetic molecule. In certain aspects, the TOI may comprise, or consist of, a biological molecule such as a protein (which may be referred to as the TOI protein), a nucleic acid molecule, a carbohydrate, a lipid, or combinations thereof. The term protein is meant to encompass full-length proteins, as well as fragments and peptides thereof. The nucleic acid molecule may comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and/or combinations thereof. In certain aspects, the TOI may comprise a complex of two or more molecules. In such a complex, the two or more molecules may comprise a biological molecule, such as proteins, nucleic acid molecules, lipids, carbohydrates, and combinations thereof. In aspects in which the TOI comprises one or more biological molecules, the TOI may be present in cells, or it may be isolated. In certain aspects, the isolated TOI may comprise a cellular membrane. For example, in certain aspects a protein, or protein complex, to be used as a TOI in a method of the disclosure may be, or comprise, a transmembrane protein. In such aspect, it may be desirable to use the TOI protein, or protein complex, in its native confirmation. To achieve this, the TOI protein, or protein complex, may be purified from the cell by lysis of the cell, followed by the use of isolation steps that leave the TOI protein, or protein complex, in its native conformation. An example of such a TOI is illustrated in FIG.8. 40574-117 -19- [0072] As used herein, a “sensor-reporter system” refers to a system comprising two or more elements that interact to produce a detectable signal. Sensor- reporter systems of the disclosure comprise a label, which is the first element of the sensor- reporter system, and at least a second element that interacts with the label to produce a complex capable of producing the detectable signal. Depending on the signal being produced, the sensor-reporter system may comprise a third element, such as a substrate that interacts with other elements of the sensor-reporter system (e.g., the label and the second element) to produce the detectable signal. One example of a sensor reporter system is a protein complementation system, which may be a bioluminescent complementation system (e.g., HiBiT Protein Tagging System). Such a system uses a protein having enzymatic activity and relies on the ability of fragments of the protein (enzyme) to bind together and reconstitute the enzymatic function of the original, intact protein. Such a system comprises a tag (aka, label), which is a small portion (e.g., 11 amino acids) from the enzymatic protein. Because the label is small, it can be attached to a target of interest (TOI). When the label binds to the shortened portion of the enzymatic protein (i.e., the full-length protein minus the label portion), the resulting protein (aka protein complex) exhibits the same enzymatic function as the original intact protein. Such a system allows the label to be used as a tag for the presence of the protein to which the label is attached. In a bioluminescent complementation system, the enzymatic activity has bioluminescent activity, allowing detection of the reconstituted protein complex by measurement of bioluminescent light. In certain aspects, methods of the disclosure may comprise combining one or more elements of the sensor-reporter system in reaction mixtures comprising for example, the TOI, the compound, or a combination thereof. [0073] As used herein, the terms “signal” and “detectable signal” may be used interchangeably and refer to a product produced as a result of interaction of the elements of the sensor-reporter system, at least one element of which is present in a reaction mixture of the disclosure. In certain aspects, the product is detectable by the eye of an observer, and/or a detection device external or internal to reaction mixtures of the disclosure. In certain aspects, the signal may comprise emission of radiation, which may include, but is not limited to, light produced, for example, by luminescence (including bioluminescence), fluorescence, and/or phosphorescence. In certain aspects, the signal may comprise production of a colored substance or change in the color of a colored substance, due to, for example, enzymatic and/or chemical modifications of a substrate. 40574-117 -20- [0074] As used herein, “label” (aka tag) refers to a molecule that is chemically linked to the TOI, thereby forming a labeled TOI, and that participates in the production of the signal by interacting with other elements of a sensor-reporter system. The label may comprise any molecule that interacts with elements of a sensor-reporter system to produce a signal. Examples of labels suitable for use in methods of the disclosure include, but are not limited to, proteins, peptides, and fluorescent molecules. The type of label joined to the TOI will depend on the type of sensor-reporter system used to produce the signal. In certain aspects, the label may be a fragment a protein that may possess catalytic activity. [0075] As used herein, the phrase “significant difference” is used to refer to the amount of difference detected between the status of first and second (and optionally more) signals and reflects an effect, or lack thereof, of a compound on a TOI. For example, the absence of a signal (i.e., the inability to detect a signal) from a first reaction mixture containing a sensor-reporter system, and detection of a signal from the same reaction mixture upon addition of a compound, or from a second reaction mixture containing the sensor-reporter system, would be considered a significant difference. When used with regard to comparing levels of signals obtained from different reaction mixtures, a significant difference refers to a difference in signal level of at least 20%. In certain aspects, the difference may be at least 30%, at least 40%, a least 50%, at least 75%, or at least 100%. In certain aspects of the disclosure, a significant difference refers to a difference in signal level of at least 1.5X, at least 2X, at least 3X, at least 4X, at least 5X, or at least 10X. In certain aspects, a significant difference is a change in signal (e.g., at least 10% or at least 20%, above that of a control reaction.) [0076] One aspect of the disclosure is a method of detecting interaction of a compound with a TOI, the method comprising detecting a significant difference, if any, between [0077] a) the status of a first signal produced by interaction of a TOI comprising a label, the label being a first element of a sensor-reporter system, with a second element, and optionally a third element, of the sensor-reporter system, in the absence of the compound, and b) the status of a second signal produced by interaction of the labeled TOI with the second element, and optionally the third element, of the sensor- reporter system, in the presence of the compound, wherein detection of a significant 40574-117 -21- difference in the status of the first and second signals indicates interaction of the compound with the TOI; and, [0078] wherein the compound is not linked to an element of the sensor- reporter system. In certain aspects, the compound is not covalently linked to any element of the sensor -reporter system. In certain aspects, the second element of the sensor-reporter system is not linked (e.g., covalently) to a non-requisite molecule, such as a non-requisite protein or peptide. In certain aspects, production of the signal by the sensor-reporter system does not comprise resonance energy transfer. [0079] One aspect of the disclosure is a method of detecting interaction of a compound with a TOI, the method comprising detecting a significant difference between i) the status of a first signal produced by a first reaction mixture comprising the TOI in the absence of the compound, wherein the TOI comprises a label, the label being the first element of a sensor-reporter system, and ii) the status of a second signal produced by a second reaction mixture identical to the first but comprising the compound, wherein the first and second reaction mixtures comprise a second element of the sensor-reporter system and, optionally, a third element of the sensor-reporter system, wherein detection of a significant difference between the levels of the first and second signals indicates interaction of the compound with a TOI molecule; and, wherein the compound is not linked to an element of the sensor-reporter system. In certain aspects, the compound is not covalently linked to any element of the sensor -reporter system. In certain aspects, the second element of the sensor-reporter system is not linked (e.g., covalently) to a non-requisite molecule, such as a non-requisite protein or peptide. In certain aspects, production of the signal by the sensor-reporter system does not comprise resonance energy transfer. [0080] In these aspects, the status of the first signal may comprise the presence or absence of the first signal. In certain aspects, the status of the second signal may comprise the presence or absence of the second signal. In these aspects, detecting a significant difference may comprise detecting the presence or absence of the first and/or second signal. In certain aspects, a significant difference between the presence or absence of first and second signals indicates interaction of the compound with the TOI. In certain aspects, absence of the first signal and the presence of the second signal indicates interaction of the compound with the TOI. In certain aspects, the presence of the first signal and absence of the second signal indicates interaction of the compound with the TOI. In 40574-117 -22- certain aspects, a lack of a significant difference between the presence or absence of the first and second signals indicates a lack of interaction of the compound with the TOI. [0081] In these aspects, detecting the status of the first signal may comprise determining the level of the first signal. In certain aspects, detecting the status of the second signal may comprise determining the level of the second signal. In these aspects, detecting a significant difference may comprise determining the levels of the first and second signals and comparing the levels of the first and second signals. In certain aspects, detecting a significant difference between the levels of the first and second signals indicates interaction of the compound with the TOI. In certain aspects, a level of second signal that is significantly greater than a level of first signal indicates interaction of the compound with the TOI. In certain aspects, a level of second signal that is significantly less than a level of first signal indicates interaction of the compound with the TOI. In certain aspects, a lack of significant difference between the level of the first and second signals indicates a lack of interaction of the compound with the TOI. [0082] In these aspects, the method may comprise detecting the first and second signals from two reaction mixtures, one of which is derived from the other, or it may comprise detecting the first and second signals from two physically separate reaction mixtures. For example, the status of the first signal may be detected from a first reaction mixture comprising a labeled TOI and associated sensor-reporter system elements, in the absence of the compound, and the status of the second signal may be detected from a second reaction mixture identical to the first but comprising the compound. The status of the signals from each reaction mixture may then be compared. Differences between the status of the signals (e.g., presence, absence, level) would indicate interaction of the compound with the TOI. Alternatively, the first signal may be detected from a reaction mixture comprising a labeled TOI and associated sensor-reporter system elements, in the absence of the compound, after which the compound may be added to the reaction mixture. Any signal detected post-addition would be considered the second signal. The signals detected from each reaction mixture may then be compared, and a significant difference between the first and second signals would indicate interaction of the compound with the TOI. Lack of a significant difference between the first and second signals would indicate lack of interaction of the compound with the TOI. In certain aspects, comparing the first and second signals would detect any change in the presence or level of the signal following addition of the compound. 40574-117 -23- [0083] One aspect of the disclosure is a method of detecting interaction of a compound with a TOI, comprising: [0084] a) contacting a labeled TOI, the label being a first element of a sensor-reporter system, with a second element of the sensor-reporter system, and, optionally, a third element of the sensor-reporter system, wherein the sensor-reporter system does not comprise resonance energy transfer, thereby forming a reaction mixture; [0085] b) detecting the status of a first signal, if any, produced by interaction of the first and second elements, and optionally the third element, of the sensor- reporter system; [0086] c) contacting the reaction mixture with the compound; [0087] d) detecting the status of a second signal, if any, produced by interaction of the first and second elements, and optionally the third element, of the sensor- reporter system, in the presence of the compound; and, [0088] e) comparing the first and second signals, wherein detection of a significant difference between the first and second signals indicates interaction of the compound with the TOI; [0089] wherein the compound is not linked to an element of the sensor- reporter system. In certain aspects, the compound is not covalently linked to any element of the sensor -reporter system. In certain aspects, the second element of the sensor-reporter system is not linked (e.g., covalently) to a non-requisite molecule, such as a non-requisite protein or peptide. In certain aspects, production of the signal by the sensor-reporter system does not comprise resonance energy transfer. [0090] In certain aspects, the status of the first signal may comprise the presence or absence of the first signal. In certain aspects, the status of the second signal may comprise the presence or absence of the second signal. In certain aspects, detection of a significant difference may comprise detecting the presence, absence, gain or loss of the first and/or second signal. In certain aspects, a significant difference in the presence, absence, gain or loss of the first or second signal indicates interaction of the compound with the TOI. In certain aspects, absence of the first signal and presence of the second signal indicates interaction of the compound with the TOI. In certain aspects, the presence of the first signal and absence of the second signal indicates interaction of the compound with the TOI. In certain aspects, a lack of a significant difference between the first and second signals indicates a lack of interaction of the compound with the TOI. 40574-117 -24- [0091] In certain aspects, detecting the status of the first signal may comprise detecting the level of the first signal. In certain aspects, detecting the status of the second signal may comprise detecting the level of the second signal. In certain aspects, detecting the significant difference may comprise determining the levels of the first and second signals and comparing the levels of the first and second signals. In certain aspects, a level of second signal significantly greater than the level of first signal indicates interaction of the compound with the TOI. In certain aspects, a level of second signal significantly less than the level of the first signal indicates interaction of the compound with the TOI. In certain aspects, lack of a significant difference between the level of the first and second signals indicates a lack of interaction of the compound with the TOI. [0092] One aspect of the disclosure is a method of identifying a compound that interacts with a TOI, comprising performing a method of the disclosure using a test compound, wherein if interaction of the test compound with the TOI is detected, identifying the test compound as a compound that interacts with the TOI. In certain aspects, methods of the disclosure may be used to determine the K_D of the compound. In certain aspects, if the K_D of the compound for the TOI is at least 10^-5, at least 10^-6, at least 10^-7, at least 10^-8, at least 10^-9, or at least 10^-10, the compound is identified as binding the TOI. [0093] One aspect is a method of identifying a drug candidate for a druggable TOI, comprising performing a method of the disclosure using a test compound, the TOI used in the method being the druggable TOI, wherein if interaction of the test compound with the druggable TOI is detected, identifying the test compound as a compound that interacts with the TOI. In certain aspects, if the K_D of the compound for the druggable TOI is at least 10^-5, at least 10^-6, at least 10^-7, at least 10^-8, at least 10^-9, or at least 10^-10, the compound is identified as a drug candidate for the druggable TOI. Examples of druggable TOIs include, but are not limited to, dihydrofolate reductase (DHFR), cyclooxygenase (COX-1/2), phosphodiesterase (PDE), SRC tyrosine-protein kinase (SRC TK), tubulin (TUBB4A), hepatitis C virus serine protease (NS3/NS4A), type II topoisomerases (TOP2A), D-alanyl-D-alanine carboxypeptidase (DacC), beta-adrenergic receptor (ADBR), opioid receptor (OR), dopamine receptor (D2R), voltage-gated calcium channel (CACNA1S), 26S proteosome (pbt t1a), histone deacetylase (HDAC), HIV-1 protease (HIV-1 PR), TOI. [0094] In certain aspects, the sensor-reporter system in a method, use, system or kit of the disclosure may be a complementation reporter system (i.e., a protein 40574-117 -25- fragment complementation system, i.e., a complementation system). A complementation system of the disclosure comprises the label (e.g., U-peptide), the second element (e.g., k- fragment) and an optional third element (e.g., reporter substrate), see FIG.1B. In a complementation system, the label comprises a peptide (i.e., peptide label or U-peptide) that binds with high affinity to a, larger, complementing polypeptide (k-fragment). Separately, neither the peptide label or the complementing polypeptide has significant, or any, activity. However, when the peptide label and the complementing polypeptide bind together, they form a peptide/complementing polypeptide complex (i.e., sensor-reporter) having restored activity, or increased activity relative to the non-complexed peptide label and complementing polypeptide. This complex can interact with an optional third element of the reporting system to produce a detectable signal. [0095] Preferably a peptide label used in a sensor-reporter system of the disclosure is small so that it does not alter the overall natural structure and/or activity of the TOI. For example, in certain aspects the peptide may comprise between 5 and 75 amino acid, 5 and 50 amino acids, between 5 and 20 amino acids, preferably between 5 and 15 amino acids, preferably between 5 and 14 amino acids, and preferably between 5 and 12 amino acids. In certain aspects the peptide may comprise about 11 amino acid residues. In certain aspects, the TOI may comprise at least one biological molecule and the peptide may be joined to the biological molecule. In certain aspects, the biological molecule in the labeled TOI may comprise a protein and the peptide may be joined to the carboxy-terminal or amino-terminal end of the protein. Such a labeled TOI protein is beneficial, since, due to the small size of the peptide label, attachment at the carboxy-terminal or amino-terminal ends will be minimally disruptive to the proper folding and/or activity of the TOI protein. In certain aspects, the peptide label may be inserted into the sequence of the TOI protein. In certain aspects, the peptide label may be inserted into the TOI protein such that the peptide label is present on the surface of the folded TOI protein. In certain aspects, the peptide label may be inserted into a loop region of a TOI protein. In certain aspects, the peptide label may be inserted into a position in the TOI protein such that the peptide label is masked by binding of a second molecule (e.g., a regulatory subunit, see for example FIG. 8A-B and FIG.9B) to the TOI protein. In such aspects, dissociation of the second molecule from the TOI protein would render the label more accessible to other elements of a sensor- reporter system, which may result in production of a detectable signal. An example of such an arrangement is illustrated in FIG.9B. Methods of joining the peptide label to, or 40574-117 -26- inserting the peptide label into, the protein are known to those skilled in the art. For example, the peptide label may be joined to the protein by chemical means (e.g., FIG.10A- E). Alternatively, attachment or insertion of the peptide label may be achieved by producing a recombinant nucleic acid molecule in which a nucleotide sequence encoding the peptide label is joined to, or inserted within, a nucleic acid sequence encoding the protein TOI. Expression of such a recombinant nucleic acid molecule results in production of protein in which the peptide label is joined to one end of the protein, or in which the peptide label is inserted into the sequence of the protein. An example of such approaches is generally illustrated in FIG.6A. [0096] In certain aspects, a nucleotide sequence encoding the peptide label may be inserted into a genome. In such aspect, the nucleotide sequence may be fused to an exon in a gene locus so that the finally expressed protein comprise the peptide label. Methods of inserting a nucleotide sequence into a gene locus are known to those skilled in the art. For example, CRSPR/Cas9 gene editing allows the rapid modification of endogenous TOI genes in a variety of cell lines. The small size of the peptide label greatly facilitates its incorporation into a targeted gene locus via homologous recombination. Thus, the terminus of any 5’ or 3’ coding exon may be targeted to create a SDR assay compatible protein that can subsequently be assayed according to methods disclosed herein. Such protein TOIs expressed at stoichiometrically appropriate cellular levels may be more likely to be regulated as complexes in a physiologically relevant manner. These complexes may be modulated by externally added ligands (e.g., small molecules) to produce a signal. Such systems may be used for identifying molecules that influence the equilibrium of protein- protein complexes in vivo and may therefore have therapeutic value. An example of how this may be practically accomplished is generally illustrated in FIG.6A-B. [0097] In certain aspects of the disclosure utilizing a complementation sensor-reporter system, the peptide (e.g., U-peptide) and the complementing polypeptide (k-fragment) are from a protein capable of producing bioluminescence. In such a complementation system, the third element may be a substrate. Binding of the complementing polypeptide to the peptide results in a protein complex or reporter that acts on the substrate to cause bioluminescence, resulting in emission of light from the sensor- reporter system. Such a sensor-reporter system may be referred to as a bioluminescent complementation system (Dixon et al., ACS Chem. Biol.11:400-408). Any protein capable of producing bioluminescence may be used in such a system. Examples of such proteins 40574-117 -27- include, but are not limited to, luciferase and phosphoproteins. In certain aspects, the luciferase may be Renilla luciferase, Gaussia luciferase, Nanoluc^® luciferase, Cypridina luciferase, Firefly luciferase, Click-beetle luciferase, Dinoflagellate luciferase, Euphausiid luciferase, bacterial luciferase, fungal luciferase, aequorin, and berovin. Examples of useful substrates include, but are not limited to, furimazine, luciferin, coelenterazine, and derivatives thereof that produce light when acted upon by luciferase. [0098] One example of a bioluminescent complementation system is the HiBiT Protein Tagging System (i.e., the HiBiT system) disclosed is U.S Patent No. US9797890, which is incorporated herein in its entirety. The HiBiT system comprises a tag (the label) called HiBiT, which is a small peptide (VSGWRLFKKIS; SEQ ID NO:1) from Nanoluc^® luciferase that binds with high affinity to a larger subunit, called LgBiT (SEQ ID NO:2) from the same luciferase protein. The HiBiT/LgBiT complex possesses luciferase activity and will produce a luminescent signal in the presence of an appropriate substrate, such as furimazine. Thus, in certain aspects of the disclosure, the label may be a peptide from a luciferase protein, the complementing protein may comprise the large subunit (LgBiT) from the luciferase protein, and the substrate may be furimazine. In such aspect, binding of the peptide to the large subunit produces a complex having increased or restored luciferase activity, which acts on the furimazine to produce bioluminescent light that may be detected as a signal. In certain aspects, the label (i.e., the first element of a sensor reporting system) may comprise, consist of, or consist essentially of, SEQ ID NO:1 or variants thereof that are capable of forming a light producing protein. In certain aspects, the complementing protein (i.e., the second element of a sensor reporting system) may comprise, consist of, or consist essentially of, VFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKID IHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINS (SEQ ID NO:2) or variants thereof that are capable of forming a light producing protein. [0099] In certain aspects of the disclosure utilizing a complementation sensor-reporter system, the peptide and the complementing polypeptide may be from an enzyme capable of producing a colored reaction product. In such a complementation system, the third element may be a chromogenic substrate. Binding of the complementing polypeptide to the peptide results in a complex that acts on the substrate to produce a colored reaction product. Examples of such enzymes include, but are not limited to, β- 40574-117 -28- galactosidase (Broome et al., 2010, Mol. Pharm.7:60-74; RM Eglen, 2002, Assay Drug Dev Technol.1:97-104)), β-glucuronidase, alkaline phosphatase, esterase, esterase 2 from Alicyclobaccilus acidocaldarius, acetylesterase, dihydrofolate reductase (DHFR), and β-N- acetylglucosaminidase. In such aspects, the sensor-reporter system comprises the appropriate chromogenic substrate. For example, a sensor-reporter system using β- galactosidase would comprise a chromogenic substrate acted on by β-galactosidase, such as 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). Likewise, a sensor-reporter system using alkaline phosphatase would comprise a chromogenic substrate acted on by alkaline phosphatase, such as p-nitrophenylphosphate (pNPP). [0100] In certain aspects of the disclosure utilizing a complementation sensor-reporter system, the peptide and the complementing polypeptide may be from a fluorescent protein (Romei & Boxer (2019) Annu Rev Biophys.48:19-44). In such a complementation system, the third element of the sensor-reporter system may be a photon beam. Binding of the complementing polypeptide (e.g., k-fragment) to the peptide (e.g., U- peptide) results in a complex having fluorescent properties. Impingement of the photon beam on the complex results in emission of light from the sensor-reporter system, which may be detected as a signal. Any fluorescent protein may be used in such a system. Examples of such proteins include, but are not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), orange fluorescent protein (OFP), and red fluorescent protein (RFP). [0101] In certain aspects, the sensor-reporter system may be a photoluminescent sensor-reporter system comprising, or consisting of, the label and a second element. In these aspects, the label may comprise, or consist of, a photoluminescent molecule. (i.e., photoluminescent label), and the second element of the sensor-reporter system is a photon beam. In such a system, the label is attached to the TOI, and impingement of the photon beam on the label causes luminescence, resulting in the emission of light from the sensor-reporter system. Examples of photoluminescent molecules useful for practicing methods of the disclosure include, but are not limited to, lanthanides, and derivatives thereof, fluorescein, and derivatives thereof, rhodamine, and derivatives thereof, cyanine, phycoerythrin, and fluorophore dyes. In such aspects, the photoluminescent molecule may be chemically attached to any part of the TOI. For example, in aspects in which the TOI comprises a protein, the photoluminescent label may be chemically joined to the amino-terminal end of the protein, the carboxyl terminal end of 40574-117 -29- the protein, or to the side group of any amino acid residue in the protein. In such aspects, impingement of the photon beam upon the labeled TOI induces production of a fluorescent signal, which may be detected visually or using a detection apparatus. Contact of the test compound with the labeled TOI may or may not alter the presence, absence, or level of the photoluminescent signal, thereby indicating interaction, or lack thereof, of the compound with the TOI. In certain aspects, the label comprises, or consists of, a lanthanide. In certain aspects, the label comprises, or consists of, lanthanide cryptate, which may be a bis- bipyridine macrocycle. An example of such a system is illustrated in FIGS.10C-D. [0102] An advantage of the methods disclosed herein is that the label is small, thereby reducing interference with proper folding or activity of the TOI. The inventors have discovered that introducing the label at targeted locations away from positions crucial for the proper folding or activity of the TOI may further reduce such interference. For example, in certain aspects the TOI may comprise a protein, the sequence of which may be altered to contain amino acid residues to which the label may be attached. Alteration of the amino acid sequence may comprise introducing substitution mutations such that the newly introduced amino acid residues allows attachment of the label. Alternatively, alteration of the amino acid sequence may comprise introducing insertion mutation such that one or more new amino acid residues to which the label may be attached are introduced into the sequence of the protein. Preferred residues to introduce into the amino acid sequence of the protein are those capable of being joined to the label, for example through their side groups. Thus, preferred amino acid residues to introduce are those having reactive side groups. Examples of suitable amino acid residues to introduce include, but are not limited to, cysteine or lysine. An example of how this may be practically accomplished is generally illustrated in FIG.10E. [0103] Heretofore have been described various systems for detecting interaction of a compound with a TOI, using complementation reporter systems. To help elucidate and further illustrate such systems, one example of such a system will now be described in greater detail. It should be understood that such description is not meant to limit the disclosure to a specific example, and that detection systems may use any component disclosed here, or equivalents thereof known it the art. [0104] One aspect of the disclosure is method of detecting interaction of a compound with a target of interest (TOI), the method comprising: [0105] a) introducing the compound to a reaction mixture comprising: 40574-117 -30- [0106] i) the TOI, the TOI comprising a peptide comprising a first portion of a light-producing protein; [0107] ii) a second portion of the light producing protein, wherein interaction of the first portion and the second portions forms a protein complex capable of producing light; and, [0108] iii) a substrate for the light producing, wherein reaction of the substrate with the protein complex results in the production of light; [0109] b) detecting the status of light produced by the reaction mixture, if any, thereby detecting interaction, if any, of the compound with the TOI. [0110] In certain aspects, production of light does not comprise resonance energy transfer. In certain aspects, the compound is not covalently linked to the first or second portion of the light producing protein. In certain aspects, the second portion of the light producing protein is not linked to a non-requisite molecule. In certain aspects, detecting the status of light may comprise detecting the presence, absence, gain, or loss of a light signal. In certain aspects, the protein complex may not produce light in the absence of a compound that interacts with TOI. Thus, in certain aspects, detecting the presence of light in the presence of the compound indicates interaction of the compound with the TOI. In certain aspects, the protein complex may not produce light in the absence of a compound that interacts with TOI. Thus, in certain aspects detecting the absence of light in the presence of the compound indicates interaction of the compound with the TOI. [0111] In certain aspects, the method may further comprise comparing the status of light from the first reaction mixture with the status of light from a second reaction mixture identical to the first reaction mixture but lacking the compound (e.g., a control reaction), wherein a significant difference in the status of light produced by the first and second signals indicates interaction of the compound with the TOI. [0112] In certain aspects, the status of light from the second reaction mixture may be obtained by detecting the presence, absence, gain, loss, or level of light produced by the second reaction mixture. In certain aspects, detecting a difference between the presence or absence of light from the first and second reaction mixtures indicates interaction of the compound with the TOI. In certain aspects, the presence of light in the first reaction mixture and the absence of light in the second reaction mixture indicates interaction of the compound with the TOI. In certain aspects, the presence of light in the second reaction mixture and the absence of light in the first reaction mixture indicates 40574-117 -31- interaction of the compound with the TOI. In certain aspects, failing to detect a difference between the presence or absence of light in the first and second reaction mixtures indicates a lack of interaction of the compound with the TOI. [0113] In certain aspects, detecting the status of light in the second reaction mixture may comprise detecting the level of the second signal. In certain aspects, detecting a significant difference between the level of light produced by the first and second reaction mixtures indicates interaction of the compound with the TOI. In certain aspects, detecting that the level of light produced by the first reaction mixture is significantly greater than the level or light produced by the second reaction mixture indicates interaction of the compound with the TOI. In certain aspects, detecting that the level of light produced by the second reaction mixture is significantly greater than the level of light produced by the first reaction mixture indicates interaction of the compound with the TOI. In certain aspects, failing to detect a difference between the level of light produced by the first and second reaction mixtures indicates a lack of interaction of the compound with the TOI. [0114] One aspect of the disclosure is method of detecting interaction of a compound with a target of interest (TOI), the method comprising: [0115] a) determining a first status of light, if any, produced from a reaction mixture comprising: [0116] i) the TOI, to which has been attached a peptide label comprising a first portion of a light-producing protein; [0117] ii) a second portion of the light producing protein, wherein interaction of the first portion and the second portions forms a protein complex capable of producing light; and, [0118] iii) a substrate for the light producing protein, wherein reaction of the substrate with the protein complex results in the production of light; [0119] b) introducing the compound to the reaction mixture of a); [0120] c) determining a second status of light, if any, produced by the reaction mixture of b); [0121] wherein a significant difference in the first status of light and the second status of light indicates that the compound interacts with the TOI. In certain aspects, the compound is not covalently linked to the first or second portion of the light producing 40574-117 -32- protein. In certain aspects, the second portion of the light producing protein is not linked to a non-requisite molecule. In certain aspects, production of light does not comprise resonance energy transfer. In certain aspects, the second portion of the light producing protein is not attached (e.g., covalently) to a non-heterologous protein or molecule. [0122] In certain aspects, the status of light comprises the presence of absence of light. In certain aspects, if the reaction mixture produces light prior to the addition of compound, and the reaction mixture comprising the compound fails to produce light, then it is determined that the compound interacts with the TOI. In certain aspects, if the reaction mixture does not produce light prior to the addition of compound, and the reaction mixture comprising the compound produces light, then it is determined that the compound interacts with the TOI. In certain aspects, if the presence or absence of light does not significantly differ before and after addition of the compound to the reaction mixture, it is determined that the compound does not interact the TOI. In certain aspects, if the first status comprises a level of light that differs from the level of light in the second status, the test compound binds the TOI. [0123] In certain aspects, if the level of light produced by the reaction mixture prior to the addition of the compound is significantly lower than the level of light produced by the reaction mixture following addition of the compound, it is determined that the compound interacts the TOI. In certain aspects, if the level of light produced by the reaction mixture prior to the addition of the compound is significantly higher than the level of light produced by the reaction mixture following addition of the compound, it is determined that the compound interacts the TOI. In certain aspects, if the level of light produced by the reaction mixture does note significantly differ before and after addition of the compound to the reaction mixture, it is determined that the compound does not interact with the TOI. [0124] One aspect of the disclosure is a method of detecting interaction of a compound with a target of interest (TOI), the method comprising: [0125] a) determining the status of light produced from a first reaction mixture comprising: [0126] i) the TOI, to which has been attached a peptide label comprising a first portion of a light-producing protein; 40574-117 -33- [0127] ii) a second portion of the light producing protein, wherein interaction of the first portion and the second portions forms a protein complex capable of producing light; and, [0128] iii) a substrate for the light producing protein, wherein reaction of the substrate with the protein complex results in the production of light; [0129] b) determining the status of light produced from a second reaction mixture identical to the first reaction mixture except that it comprises the compound, wherein if the status of light from the first reaction mixture is significantly different from the status of light from the second reaction mixture, determining that the compound interacts with the TOI. In such a method, if the status of light from the first reaction mixture does not significantly differ from the status of light from the second reaction mixture, it is determined that the compound does not interact with the TOI. In certain aspects, the compound is not covalently linked to the first or second portion of the light producing protein. In certain aspects, the second portion of the light producing protein is not linked to a non-requisite molecule. In certain aspects, production of light does not comprise resonance energy transfer. [0130] In certain aspects, detecting the status of light may comprise detecting the presence, absence, gain, or loss of a light signal. In certain aspects, the protein complex may not produce light in the absence of a compound that interacts with TOI. Thus, in certain aspects, detecting the presence of light in the presence of the compound indicates interaction of the compound with the TOI. In certain aspects, the protein complex may not produce light in the absence of a compound that interacts with TOI. Thus, in certain aspects detecting the absence of light in the presence of the compound indicates interaction of the compound with the TOI. [0131] In certain aspects, detecting a difference between the presence or absence of light from the first and second reaction mixtures indicates interaction of the compound with the TOI. In certain aspects, the presence of light in the first reaction mixture and the absence of light in the second reaction mixture indicates interaction of the compound with the TOI. In certain aspects, the presence of light in the second reaction mixture and the absence of light in the first reaction mixture indicates interaction of the compound with the TOI. In certain aspects, failing to detect a difference between the presence or absence of light in the first and second reaction mixtures indicates a lack of interaction of the compound with the TOI. 40574-117 -34- [0132] In certain aspects, detecting a significant difference between the level of light produced by the first and second reaction mixtures indicates interaction of the compound with the TOI. In certain aspects, detecting that the level of light produced by the first reaction mixture is significantly greater than the level or light produced by the second reaction mixture indicates interaction of the compound with the TOI. In certain aspects, detecting that the level of light produced by the second reaction mixture is significantly greater than the level of light produced by the first reaction mixture indicates interaction of the compound with the TOI. In certain aspects, failing to detect a difference between the level of light produced by the first and second reaction mixtures indicates a lack of interaction of the compound with the TOI. [0133] In these aspects, the light producing protein may a luminescent protein or a fluorescent protein. In these aspects, the light-producing protein may be a luciferase protein. In these aspects, the peptide label may comprise a portion of a bioluminescent protein, which may comprise SEQ ID NO:1. In these aspects the second portion of the light producing protein may comprise SEQ ID NO:2. In these aspects, interaction of the test compound with the TOI may comprise binding the TOI. In these aspects, the TOI may comprise a protein or a protein complex. [0134] One aspect of the disclosure is use of a method of the disclosure to identify a compound that interacts with a TOI. In such aspects, the compound used in a method of the disclosure is a test compound, the ability of which to interact with a TOI is unknown. If the results of the method indicate that the test compound interacts with the TOI (as determined by alteration in effect of the compound on the presence, absence, or level of signal produced by a reaction mixture of the disclosure), then the test compound is identified as a compound that interacts with the TOI. [0135] In certain aspects of the disclosure, the TOI, the compound and/or one or more of the elements of the senor-reporter system may be in a solution (e.g., a buffered solution) suitable for allowing interaction of TOI, the compound and/or one or more of the elements of the senor-reporter system. In certain aspects, the TOI, the compound and/or one or more of the elements of the senor-reporter system may be bound, either directly or through a linker, to a surface. Examples of such surfaces include, but are not limited to, a plate, a tube, a sensor surface, such as an optical surface or an electrically conductive surface, or a bead. 40574-117 -35- [0136] One aspect of the disclosure is a system for conducting a method of the disclosure. In certain aspects, the system may detect interaction of a compound with a TOI. In certain aspects, the system may comprise, consist of, or consist essentially of, i) a TOI to which has been attached a label, the label being a first element of a sensor-reporter system; a second element of the sensor-reporter system, wherein the second element interacts with the label to form a complex capable of producing a detectable system; and, optionally, a third element of the sensor reporter system; wherein the second element is not linked to a non-requisite molecule.. In certain aspects, the second element interacts with the label to form a protein that interacts with the third element to produce the detectable signal. In certain aspects, production of the signal does not comprise resonance energy transfer. In certain aspects, the elements of the system may be in solution. In certain aspects, one or more of the elements of the system may be attached, either directly or through a linker molecule, to a surface. In certain aspects, the system may include a device for detecting a signal produced by the system. Examples of such devices include, but are not limited to, a luminescence detector, a fluorescence detector, which may include an excitation system, or an absorbance reader. In certain aspects, the sensor reporter system may be a complementation sensor reporter system. In certain aspects, the complementation report system may comprise a light-producing protein. In certain aspects, the second element may interact with the label to form a protein complex capable of producing detectable signal. In certain aspects, the protein complex may comprise a fluorescent of luminescent protein, and the detectable signal may comprise light. In certain aspects, the optional third element may comprise a substrate for the light producing protein. In certain aspects, the label may comprise a first portion of a light-producing protein and the second element may comprise second portion of a light producing protein. In certain aspects, the label may comprise SEQ ID NO:1 and/or the second element may comprise SEQ ID NO:2. In certain aspects, the system may comprise a compound, wherein the compound is not covalently linked to any element of the sensor reporter system. [0137] One aspect of the disclosure is use of a system or method of the disclosure to identify a potential therapeutic compound. [0138] One aspect of the disclosure is use of a system or method of the disclosure to determine the K_D of a compound for a TOI. [0139] One aspect of the disclosure is a kit comprising the first, second, and optionally, third element of a sensor-reporter system of the disclosure and instructions 40574-117 -36- for detecting interaction of a compound with a TOI molecule according to a method of the disclosure. In certain aspects, the kit may comprise at least one reagent for producing a labeled TOI. In certain aspects, the kit may comprise a system of the disclosure. [0140] This written description uses examples to disclose the invention, including the best mode and methods to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. Examples [0141] Example 1. Binding of phosphoglycerate mutase by macrocyclic peptides [0142] Post-translational modifications regulate many aspects of protein function by means of controlling activity through, for example, phosphorylation, localization through lipid modification, and cellular lifetime through ubiquitination. Ligand binding likewise can have significant impact on regulation of protein function and proteostasis, through allosteric activation and protein half-life stabilization. While ligand binding has been widely recognized as having dramatic effects on the cellular consequences of the ligand bound vs. apo protein, this phenomenon has not been appreciated to have correspondingly impactful effects outside of a cellular context. This example demonstrates that this phenomenon can be exploited to enable a powerful new approach to the detection of ligand binding. [0143] Phosphoglycerate mutase (PGM) catalysis utilizes a transiently formed active site created by inward phosphatase and transferase domain motions to sequester its phosphoglycerate substrate (FIG.2A). To assay PGM, coupling enzymes are required to process 2-PG to a detectable signal (FIG.2B) using either an absorbance or luminescence output. Previous work on the co-factor independent (iPGM) parasitic forms of PGM resulted in the discovery of a potent ipglycermide class of iPGM inhibitors from RNA-encoded macrocyclic peptide (MCP) libraries using affinity selection methods. Co- crystallographic analysis of an C. e. iPGM-ipglycermide complex revealed the MCP bound to the interface of the two enzyme domains, essentially freezing the iPGM into an inactive 40574-117 -37- confirmation (Yu et al. (2017) Nature Comm.8:14932; Wiedmann et al. (2021) J. Biol. Chem.296:100628). This discovery led to the hypothesis that domain freezing upon MCP ligand binding would present a sufficiently different conformational ensembles of the protein, and that such conformational changes could be exploited by differential alpha complementation. In short, it was theorized that ligand binding would influence alpha complementation in a sensor-reporter system such as the HiBiT – LgBiT NLuc system. [0144] To test this idea, a version of C. e. iPGM containing a C-terminal 11 amino acid HiBiT peptide (U-peptide) fusion (FIG.3A, i-iv) was designed, expressed, and purified. Nanoluciferase (NLuc) luminescence produced by the large k-fragment (LgBiT) of NLuc and fumarizine substrate in the presence or absence of HiBiT labeled iPGM protein was then measured across varying concentrations of MCP (Ce-2 or Ce2-d). A large difference between the background luminescence detected from the non- complemented LgBiT NLuc fragment (FIG.3B, grey bars) and the luminescence detected from complementation with the iPGM-HiBiT (FIG.3B, white bars) was observed. In fact, a 50-fold signal-to-background was seen between 50 pM and 0.5 nM iPGM-HiBiT (FIG. 3B). Moreover, unanticipated, and remarkable detection of saturation binding for ipglycermide Ce-2 was observed (FIG 3C). In addition, the rank order of potency and selectivity for a family of Ce-2, Ce-2d, Sa-D2 and Sa-D3 ipglycermides (FIG 3D) were readily observed using SDR. The sensitivity of the assay was sufficient to allow the sub- nanomolar KD of Ce-2 to be accurately determined (FIG.3E). Further, as shown in FIGS. 3D & 3F, this assay design can also be used to evaluate concentration response curves for protein-ligand interactions. Thus, this binding assay provides a simpler and more sensitive method for detecting binding of iPGM to MCPs compared with the relatively complex and restricted sensitivity coupled enzyme assay used previously. [0145] Exploring the assay sensitivity further, the high affinity ipglycermide Ce-2 was determined by surface plasmon resonance kinetics to have a 38 pM KD. Here, a CRC for Ce-2 was observed using as little as 20 pM iPGM-HiBiT (FIG.3E), 60-fold below that needed in the enzyme-coupled assay. [0146] Example 2. Dihydrofolate reductase binding by methotrexate [0147] To determine if the SDR assay concept is generalizable to proteins beyond the highly dynamic iPGM enzyme, a structurally and catalytically unrelated enzyme, dihydrofolate reductase (DHFR) which catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to produce 5,6,7,8- tetrahydrofolate (THF), was 40574-117 -38- investigated. This conversion is a central reaction of one-carbon metabolism and de novo synthesis of purines, pyrimidines, and amino acids. Typically, an NADPH to NADP⁺ absorbance output has been used to monitor the conversion of H₂F to H₄F, both of which are notoriously air sensitive making the assay challenging and impractical to conduct as an HTS assay. For a demonstration of an SDR assay, human DHFR with HiBiT at its C- terminus, was expressed and purified. Saturation binding using methotrexate (MTX), a potent inhibitor of DHFR, was measured by titrating MTX either in the absence of NADPH (FIG.4A) or in the presence of 5 µM NADPH (FIG.4B). across a range of DHFR concentrations (0.5 – 20 nM). It is well documented that MTX forms a tight-binding ternary complex with NADPH and DHFR, in an ordered binding event, whereby an NADPH-DHFR binary complex facilitates MTX binding. [0148] Saturation binding of MTX to DHFR was observed with either no NADPH (KD^MTX= 24!120 nM) or with DHFR preincubated with 5 µM NADPH (KD^MTX= 0.1!10 nM) (FIG.4A & 4B). The specificity of binding was confirmed by not only the appropriate K_D values, but also the ~10-fold higher affinity of MTX for NADPH-bound DHFR, which can be seen in the concentration response curve plot (FIG.4C). [0149] Example 3. Assay Correlation [0150] To further assess the relative sensitivity and accuracy of SDR compared to standard assay formats utilized for the TOI classes examined here, a comparison of the SDR pEC50 to the pIC50 for a selection of inhibitors for either C. e. iPGM or human DHFR (FIG.5) was conducted. For iPGM, the potency of several ipglycermides (Ce-2, Ce-2d and Ce-2d Y7A) obtained from SDR was compared to results obtained using the coupled enzyme assay (FIGS.2A & 2B). For DHFR, the comparison was made using a fluorescence polarization (FP) assay developed using commercially available MTX-fluorescein (MTX-FL), which required approximately 5 nM DHFR and 10 nM MTX-FL to obtain sufficient assay signal. The general trend was that SDR provided a more sensitive measure of highly potent inhibitor binding constants, most likely because of the lower TOI protein concentration from which a signal can be observed in SDR. This is particularly clear with DHFR antifolate inhibitors, appearing as much as 100-fold more potent in the SDR than the FP assay, which is limited by the higher levels of MTX- fluorescein and DHFR need to configure the assay. [0151] Example 4. SDR using cell lysates 40574-117 -39- [0152] The SDR assay cases examined thus far utilized purified protein. Given the sensitivity of the SDR assay, its use in a cellular lysate context was explored. Using a human cell line engineered to recombinantly express C. e. iPGM-HiBiT stably from a random integration event (FIG.6A, i), two general protocols for measuring binding of MCP to iPGM using cell lysates was developed (FIG.6B). In the first protocol, cells were grown in 1536-well plates (FIG.6B, step 1a) and just prior to addition of Nano-Glo HiBiT lytic detection luciferase reagent, a titration of ipglycermide Ce-2d was added to the wells via pin-tool transfer (FIG.6B, step 2). In the second protocol, C. e. iPGM-HiBiT expressing cells were grown in bulk and lysate prepared (FIG.6B, step 1b) prior to addition to the 1536-well plate, followed by addition of an ipglycermide Ce-2d titration (FIG.6B, step 2). The binding of Ce-2d, measured as a gain-of-signal response by the SDR assay (shown here for the second protocol), is compared to its inhibition of iPGM activity (IC₅₀) in the coupled enzyme assay (FIG.6C, top). A rightward shift in the SDR EC₅₀ (FIG. 6C, bottom) suggests that the enzyme concentration in the cell extract was higher than the 5 nM [iPGM] used in the coupled enzyme assay. Nonetheless, this experiment clearly demonstrated that the SDR assay can be used in crude cell lysates opening the possibility to measure the ligand-mediated binding for TOI proteins that would be difficult to express and purify, or that are only functional /regulated in a cellular context. [0153] Example 5. General SDR assay protocol [0154] A general SDR assay protocol was developed based on the aforementioned Examples. An example of the protocol is shown in Table 1.

40574-117 -40- Table 1. Exemplary α-peptide TOI SDR assay protocol for purified, lysate or cell sourced TOI

Notes: (1a) 100 pM – 30 nM target-HiBiT in assay buffer (30 mM Tris-HCl pH 8, 5 mM MgSO4, 20 mM KCl, 0.1% BSA). (1b) cellular lysate expressing target-HiBiT, 100 ug/mL in assay buffer. (1c) cells expressing target-HiBiT, 2000 cells/well in cellular growth media (2) Control ligands, stock 10 mM, 16 pt. 1:3 titration in duplicate or vehicle (DMSO); Compounds transfer by Pintool. (4) Prepare Nano-Glo HiBiT lytic detection luciferase reagent by diluting LgBiT protein 1:100 + 1:50 Nano-Glo HiBiT lytic substrate (Furimazine) in Nano-Glo HiBiT Lytic Buffer (Prepare Fresh). (6) ViewLux settings: 1-60 s exposure; gain=medium to high.; speed=slow; binning=2X [0155] Table 1 illustrates the simplicity of this assay concept whether being carried out using a purified TOI protein (Table 1, Step 1a), a TOI-containing cellular lysate (Table 1, Step 1b), or the use of a cell line containing the TOI (Table 1, Step 1c). Each context is followed by the same compound treatment and assay protocol. Given the 1536-well compatibility of the method, the SDR assay protocol may be used for any TOI and can be conducted in HTS formats such as the quantitative HTS (qHTS) format that is optimally practiced in the 1536-well plate format. [0156] Example 6. Ligand Binding Using firefly luciferase as a target. [0157] As a further proof-of-concept target, an U-peptide (HiBit comprising SEQ ID NO:1) was appended to either the N- or C-terminus of firefly luciferase (FLuc), an ATP cofactor-dependent monooxygenase having a broad, well-characterized and accessible ligand pharmacology. For initial characterization of SDR, the 11 amino acid HiBiT tag (U-peptide) and complementary 18 kDa LgBiT subunit (k-fragment), which together reconstitute functional Nanoluciferase (NLuc) enzyme, comprised the U- complementation component. Using bacterially expressed and purified N-HiBiT-FLuc potent inhibition of enzymatic activity was demonstrated (FIG.12, solid circles) using PTC124, an aryl carboxylate that undergoes tight-binding to FLuc upon adenylation by the 40574-117 -41- enzyme (24). The corresponding SDR assay measurements were made either in the absence or presence of ATP cofactor, as U-complementation is ATP-independent, thus permitting conditions not possible using the catalytic activity of FLuc as the basis of the assay. These circumstances permitted both an ATP-independent low affinity (FIG.12, open squares) and ATP-dependent high affinity (FIG.12, solid squares) binding of PTC124 to be directly detected. From the FLuc enzyme assay we observe a pIC50 of 8.22 ^0.02 for PTC124, where the corresponding SDR pEC50 values are 8.39 ^0.06 and 6.81 ^0.09, with and without ATP, respectively. A unique characteristic of the SDR response is an opposite gain-of-signal output accompanying ligand binding compared to the loss-of-signal typical for inhibitors of an enzymatic assay. [0158] Example 7. Mechanistic structure activity relationships (SAR) from SDR. [0159] From the PubChem data repository (AID 2309) a library of 1,221 compounds was assembled based on diverse FLuc inhibitor chemotypes and associated analogs having a range of inhibitory potencies and mechanisms allowing compounds to be grouped in a structure activity relationship (SAR). The comparative 11-point quantitative HTS (qHTS) outputs from both the FLuc enzyme assay and the ATP-dependent SDR assay highlight the mirrored and varied efficacy displayed in the SDR compared to inhibition of a functional enzyme activity output allowing SAR to be derived from SDR. Chemotype hierarchal clustering based on Tanimoto (TT) similarity score above 0.8 defined 29 clades (A-C’). [0160] Example 8. ATP cofactor-dependent ligand binding. [0161] The salient features of the data set mentioned above include the chemotype dependence on ATP (clade F (FIG. 13A), clade S (FIG. 13E), clade T (FIG. 13F) and A’ (FIGS.13G & H)), and well-defined ATP-independent SAR of clades O (FIG.13B) and P (FIGS. 13C & 13D). Compounds in clades O and P likely occupy the FLuc ATP binding site, supported in part, by the close overlap of the ^ATP SDR output-driven concentration response curves (clades O (FIG. 13B and P (FIGS. 13C & 13D)), and particularly in the structural resemblance of the pyrazolo pyrimidine core (clade P) to the adenine heterocycle of ATP. This pyrazolo pyrimidine contains three points of variation encoding 40 compounds, where the SDR tracks closely with the functional enzyme assay. Among the ATP-dependent SDR responses are the aryl carboxylate-containing oxadiazoles (27 aryl carboxylates of the 35 clade T members, reminiscent of adenylate-forming PTC124). 40574-117 -42- Additional chemotypes demonstrating an ATP-dependence on binding are shown in clades F (FIG.13A), T (FIG.13F) and A’ (FIGS.13G & 13H) members of which can be found to contain aryl (e.g., clade T) or alky carboxylates (e.g., clade F). An interesting category of SDR CRC profile is observed, for example, with compound 5 where SDR is strictly ATP- dependent vs an ATP affinity shifting effect (FIG.13E, clade S). [0162] Example 9. Impact of N- vs. C-terminus U-peptide. [0163] A third category of CRC profiles lack a strong SDR for certain FLuc inhibitors identified by the functional assay, for example those compounds falling between clades K and L. Upon closer examination of chemotypes populating this class are 2- phenylbenzo[d]thiazoles, structural analogs of the FLuc substrate luciferin, shown to occupy to the luciferin binding pocket on the enzyme. From previous co-crystal structure (PDB: 4E5D) it was observed that the FLuc N-terminus in close proximity to the phenyl- benzothiazole binding site potentially creating a clash between the N-terminal U-peptide. [0164] Example 10. Generality of SDR. [0165] To expand beyond the FLuc SDR assay proof-of-concept, several protein families were investigated, including kinases, isomerases, and reductases. The results confirm that an SDR can be obtained from a partially purified or crude cellular lysate allowing the study of proteins not easily accessible from bacterial overexpression systems. The first examples illustrate SDR applied to protein kinases in an ATP- and competitive ligand probe-independent format. [0166] Abelson tyrosine kinase (ABL1), first identified as the BCR–ABL1 fusion protein in patients with Philadelphia chromosome-positive human leukemia, has been the target of successful CML therapeutics. Using an N-terminal HiBiT ABL1 kinase domain the protein was expressed and purified from E. coli (26), establishing functional activity using a Kinase Glo Plus assay (KGP) by measuring tyrosine phosphorylation of the 12 residue abletide peptide substrate through ATP depletion, and demonstrated a gain-of-signal SDR from an imatinib titration (FIG.14). The IC50 values obtained with the KGP assay were then compared to the SDR EC50 (also referred to as SDR50) values using a compound library composed of 128 kinase inhibitors enriched for those annotated as having ABL1 as a target kinase. For the SDR assay the response in either the absence or presence of ATP cofactor was examined. Representative CRCs for each assay format (FIGS. 15A-15H), as with imatinib and the FLuc PoC case, give consistent gain-of-signal responses upon ligand binding for the SDR. While subtle the general effect of ATP is to right-shift the CRC, not 40574-117 -43- unexpected given the common binding site. For the N-HiBiT ABL1 kinase the SDR efficacy is not as pronounced as observed for C-terminal FLuc-HiBiT. [0167] Results from 62 kinase inhibitors from the library displaying activity in ABL1 assays are depicted in a heatmap, FIG.16, showing the inhibitor potencies obtained from the functional KGP enzyme vs SDR assays (^ATP), and kinase selectivity (i.e., group column) as determined by available databases. A correlation plot prepared from this data clearly illustrates, aside from a few exceptions, the equivalence or higher sensitivity of the SDR assay compared to the KGP assay (FIG.17). Here, the accuracy and predictive power of SDR to identify kinase inhibitors is shown, for example, by the 4-methyl-N- phenylbenzamide, ALW-II-41-27 (AL), while not annotated as an ABL kinase inhibitor, was clearly found to be a potent inhibitor of the kinase, a result supported by structural similarity to imatinib and the imatinib analog, NSC762948. Only in a single case, for PD 173955 (P2) did we observe a marked greater (100-fold) sensitivity in the KGP assay over the SDR assay. This was not likely a chemotype specific effect given two other pyrido-pyrimidinones, PD- 180970 (P1) and FRAX-486 (FR) were on the 1:1 correspondence diagonal (FIG.17). [0168] Protein Kinase A (PKA), the first protein kinase structurally elucidated, is a cAMP-regulated molecular switch responsible for mediating numerous cellular functions and signal transduction pathways through serine/threonine phosphorylation. Similarly, to the ABL1 N-terminal U-peptide fusion protein, PKA-N-HiBiT displayed a gain-of-signal SDR upon binding the isoquinoline sulfonamide H-89, an ATP-binding site PKA inhibitor, displaying a modest SDR shift in the presence of ATP and higher sensitivity versus the functional assay. Example 11. Recombinant mammalian cell lysates. The sensitivity of SDR indicates that this method may be applied to recombinant TOIs obtained from, for example, mammalian cellular extracts. To test this, DHFR-C-HiBiT obtained from a human diffuse large B-cell lymphoma cell line was explored. The recombinant DHFR was approximately 20% of the total cellular DHFR as assessed by western blot. Lysates prepared from these cells were then used directly or following dialysis as a source of DHFR-C-HiBiT. Dilution of the extract between 100- to 2,000-fold gave detectable SDR outputs allowing the SDR EC₅₀ of MTX to be measured regardless of dialysis (FIG.19). 40574-117 -44- Discussion The work disclosed herein demonstrates that using an SDR output, it is possible to measure the concentration-dependence of ligand-target interactions, previously requiring disparate functional assays, in a single straightforward format using bioluminescence generated from Nanoluciferase reconstituted by U-complementation from HiBiT/LgBiT. While other split-reporter systems are available, the superior sensitivity, low KD, and rapid onset of HiBiT/LgBiT complementation allows for the study of target proteins at nM down to pM concentrations without the requirement of chromophore maturation. Importantly, the ligand potency (EC50) or affinity (KD) for these interactions agree with previously published work, further validating the use of SDR as a quantitative approach. Thus, SDR represents an important evolution to the enzymologist toolkit for proteins formerly prohibited from HTS study due to their troublesome functional assays. Prior to the inventor’s discovery, outside of a cellular context, where ligand-mediated gene transcription and translation or protein turnover can modulate the levels of, for example a TOI-U peptide fusion, it has not been appreciated that a TOI ligand binding event could substantially modulate the degree of alpha complementation between a TOI U-peptide fusion and partner k-fragment. Although, while both thermal-shift or CETSA-based approaches respectively measure, using purified protein or in a cellular setting, the ligand-induced thermal stabilization of a TOI, neither directly nor isothermally assess this property. The inventors surprising observation, which forms the basis of SDR, that a TOI ligand binding event can substantially influence the degree of complementation between an U-peptide TOI fusion protein and the complementary k-fragment, allows for the construction of assays to enable ligand and drug discovery for proteins and enzymes previously considered intractable or for which available assays were impractical for high throughput screening. SDR does not require thermal denaturation, or other treatments (e.g., proteolysis) to discriminate the ligand-bound from control sample. The SDR assay is a mix-and-read type format, neither necessitating separation steps or specialized detectors, which readily scales to standard 384- and 1536-well format microtiter plates. [0169] The simplicity of the disclosed method has advantages over other ligand binding assay approaches (Table 2). 40574-117 -45- Table 2. Comparison of the SDR assay to common HTS assay methods

on a single label that can be genetically or chemically incorporated onto the TOI (FIG. 11A). In contrast, FRET or BRET-based methods like HTRF or LANCE, and related technologies such as Alpha Screen (Luminescent singlet oxygen channeling assay) and electrochemiluminescence (e.g., Meso Scale Discovery technology) are based on multicomponent systems where, minimally, a donor and acceptor element are required, often involving an antibody and/or labeled ligand. Such systems therefore are often not amenable to novel proteins for which antibodies or known ligands of sufficient affinity are not available. Even when ligands are accessible their labeling is not necessarily inconsequential, sometimes requiring a substantial synthetic chemistry effort. Additionally, the expense of certain donor fluorophores, such as europium (Eu⁺³) or terbium (Tb⁺³) cryptates are exorbitantly expensive, preventing routine use. The disclosed methods circumvent these issues by using a single label that is inexpensive and that is easy to link to TOI molecules (FIG.11B). However, should a label other than one compatible with alpha complementation be employed, the method could be used with, for example a time- resolved output from a lanthanide cryptate if ligand binding creates a significant environmental perturbation near the lanthanide cryptate to affect the time-resolved fluorescence (FIG.11B). Table 3 illustrates some example sensor-reporters possible with SDR. 40574-117 -46- Table 3. SDR assay TOI labels to enable sensor-reporter component

TRF, time-resolved fluorescence; 4-DAPA, 4-N,N-dimehtylaminophtalimidoalanine [0171] The examples presented herein utilized multiple TOI proteins (e.g., iPGM, DHFR, FLuc, and ABL1) to explore the generality of the SDR assay. However, the methods disclosed herein may be generalized to a wide range of protein TOIs, including the plethora of disease-implicated TOIs that have been problematic to measure such as proteins having mutation-modifying functions resulting in disease pathology, including cystic fibrosis transmembrane conductance regulator (CFTR), KCNQ1 gain-of-function mutations linked to familial atrial fibrillation, mutations of STAT3 resulting in lymphoproliferation, various hormone associated diseases of heterozygous constitutively activating FGFR gene mutations, and the potentially large number physiologic processes that mutations in G protein-coupled receptors (GPCRs) can cause, ranging from retinitis pigmentosa (RP) to diabetes insipidus. Use of the V2AR would be interesting because it is representative of GPCRs and an SDR assay configuration would allow the measurement of agonist or antagonist binding to this transmembrane receptor class independently of the nature of the signal transduction components (FIG.8A). Moreover, depending on the lysate preparation, the V2AR may be coupled to any one of its multiple transducers, the heterotrimeric G proteins, GUs, GPCR kinases, or V-arrestins, which may further influence the SDR signal depending on the nature of the receptor ligand (FIG.8B). This could also allow for an indirect measure of a compound on, for example, the kinase activity of a G- protein coupled receptor kinase (GRK). 40574-117 -47- [0172] An interesting case for a ligand-mediated protein-protein interaction is found with Protein kinase A (PKA), which is regulated by independent subunits of R1U. Binding of R1U to PKA, produces an R1U-PKA complex and causes inhibition of the catalytic activity of PKA. Binding of cAMP to R1U causes dissolution of the R1U-PKA complex, resulting in release of an active PKA protein (FIG.9). In an SDR assay using a labeled PKA protein (e.g., PKA-U-peptide), in which the label is attached to the PKA protein such that it is made inaccessible by release of R1U, binding of ligand would result in loss of a signal upon exogenous addition of cAMP (FIG.9A). In an alternate design, release of R1U by cAMP results in a more accessible U-peptide and consequent increase in signal (FIG.9B). Moreover, the label may be fused with the PKA gene in vivo (e.g., CRISPR/Cas9-mediated homologous recombination of an U-peptide fused to the 3’ PKA exon, FIG.6A, iii ) to obtain an SDR assay more likely to recapitulate cellular PKA-R1U stoichiometry potentially necessary to observe cAMP regulation of PKA. [0173] Given the counterintuitive nature of this finding, that a small label attached to a TOI can be sufficiently influenced by remote ligand binding to measurably alter its capacity to complement a sensor-reporter system, the novel and unanticipated methods disclosed herein are groundbreaking for drug and chemical probe discovery.

Claims

40574-117 -48- WHAT IS CLAIMED IS: 1. A method of detecting interaction of a compound with a target of interest (TOI), comprising: a) contacting the compound with a reaction mixture comprising: i. the TOI, wherein the TOI comprises a label comprising a first portion of an enzymatic protein, the label being the first element of a sensor-reporting system; ii. a second element of the sensor-reporter system, wherein the second element comprises a second portion of the enzymatic protein, wherein interaction of the first and second portions of the enzymatic protein forms a protein complex capable of producing a detectable signal; and, iii. a substrate for the enzymatic protein, wherein reaction of the protein complex with the substrate produces a detectable signal; and, b) detecting a change in status of the detectable signal, if any, produced by interaction of the protein complex and the substrate, thereby detecting interaction, if any, of the compound with the TOI molecule; wherein the compound is not covalently linked to the first or second element of the sensor-reporting system. 2. The method of claim 1, wherein the second element is not linked to a non- requisite molecule. 3. The method of claim 1 or 2, wherein production of the detectable signal does not comprise resonance energy transfer. 4. The method of any one of claims 1-3, wherein the enzymatic protein is a light producing protein and the detectable signal is light. 5. The method of any one of claims 1-4, wherein detecting a change in the status of the detectable signal comprises detecting a change in the presence or absence of the signal. 6. The method of any one of claims 1-5, wherein detecting a change in the status of the detectable signal comprises detecting a change in the level of the signal. 7. The method of any one of claims 1-6, wherein the enzymatic protein is a luminescent protein. 40574-117 -49- 8. The method of any one of claims 1-7, wherein the enzymatic protein is a luciferase. 9. The method of any one of claims 1-8, wherein the first portion of an enzymatic system comprises SEQ ID NO:1 and the second portion of the enzymatic protein comprises SEQ ID NO:2. 10. The method of any one of claims 1-9, wherein prior to contacting the compound with the reaction mixture, step a) comprises, determining a first status of the detectable signal, if any, produced by interaction of the protein complex and the substrate in the reaction mixture; wherein the determining step in step b) comprises determining a second status of the detectable signal, if any, produced by interaction of the protein complex and the substrate in the reaction mixture in the presence of the compound; and, wherein a significant difference in the status of the first and second signals indicates that the compound interacts with the TOI. 11. The method of any one of claims 1-10, wherein if interaction of the compound with the TOI is detected, identifying the compound as binding the TOI. 12. A kit comprising the first, second, and optionally, third element of a sensor- reporter system of the disclosure and instructions for detecting an interaction of a compound with a TOI molecule according to the method of any one of claims