Recognition of cellular target binding by bioactive agents using intracellular bioluminescence resonance energy transfer

Abstract

To provide a system for detecting and analyzing intracellular bond of a bioactive agent to a cell target.SOLUTION: A system includes: (a) a bioactive agent bound to a fluorescent body; (b) polynucleotide for expressing protein fusion in a cell, in which the polynucleotide contains (i) target protein and (ii) luciferase, the bioactive agent is capable of making non-covalent bond with the target protein when interacting with the target protein, where an emission spectrum of the luciferase overlaps an excitation spectrum of the fluorescent body; and (c) a substrate of luciferase.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61 / 736,429, filed December 12, 2012, U.S. Provisional Patent Application No. 61 / 794,461, filed March 15, 2013, and U.S. Provisional Patent Application No. 61 / 880,048, filed September 19, 2013, the entireties of each of which are incorporated herein by reference. Technical Field

[0002] The present invention provides compositions and methods for the detection and analysis of intracellular binding of bioactive agents to cellular targets. In particular, provided herein are cellular targets that are fused to fluorophores, bioluminescent reporters, or bioactive agents that are bound to portions, components, or subunits of bioluminescent reporters, and methods for detecting and analyzing the interaction of a bioactive agent with a cellular target. [Background technology]

[0003] The interaction of molecular species with cellular targets is of critical importance to the understanding of cellular physiology and the development of therapeutic interventions such as new synthetic and biological drugs. Methods are needed to accurately and efficiently determine target engagement, particularly in living cells where such interactions mediate phenotypic responses. The ability to efficiently interrogate target engagement has broad implications for the discovery process, from screening treatments to optimizing hit screening for drug leads, and the discovery and characterization of therapeutically relevant cellular targets. Summary of the Invention [Problem to be solved by the invention]

[0004] Phenotypic-based screening of small molecule libraries plays an important role in the field of drug discovery. Using this screening approach, compound libraries are screened for their ability to elicit a phenotypic response, e.g., alleviation of disease symptoms, without prior knowledge of the underlying cellular target. Although this approach can be used to identify bioactive agents, e.g., small molecules, capable of modulating cellular physiology, determining the biologically relevant targets of these small molecule hits is a major technical challenge. In addition, small molecules that promote a desired phenotypic response may pose a burden in vivo due to off-target interactions. It is also important to identify off-target interactions (e.g., reduced affinity) in order to predict drug selectivity and minimize potential side effects. Today, most of the methods used to identify targets of bioactive agents rely on enrichment of these targets from complex cell lysates using "bifunctionalized" compounds that have a selective moiety (e.g., bioactive agent or related compound) and a sorting moiety (e.g., affinity tag or solid support). This enrichment is based on the binding properties of the compounds, where the intrinsic affinity of these compounds for the target is insufficient, so compound analogs are designed to bind the target covalently (e.g., photocrosslinking). With either approach, the effectiveness and specificity of the target separation is important, and the failure rate of these methods is high. The failure of these approaches can be due to insufficient capture of the target or high background due to nonspecific capture. Contributing factors to this failure include compounds that bind multiple targets with low to moderate affinity, with these relatively weak interactions being difficult to detect, the lack of robust, direct, and unbiased techniques to characterize the detected interactions, the inability to perform target separation within the native cellular environment where the interactions may be affected, only limited information is provided about the binding potential of intracellular targets, and the high background of false positive interactions due to nonspecific binding to solid supports or functionalized small molecules. [Means for solving the problem]

[0005] In one embodiment, the invention provides a BRET assay system comprising: (a) a bioactive agent conjugated to a chromophore (e.g., a fluorophore); (b) a cellular target fused to a bioluminescent reporter; and (c) a substrate for the bioluminescent reporter. In one embodiment, the bioactive agent is a small molecule. In one embodiment, the bioactive agent is an inhibitor of protein function, e.g., an enzyme inhibitor or a receptor inhibitor. In one embodiment, the chromophore is a fluorophore. In one embodiment, the fluorophore is a carboxyrhodamine analog. In one embodiment, the bioluminescent reporter comprises a polypeptide having at least 70% sequence identity to SEQ ID NO.1 (e.g., 75% identity...80% identity...85% identity...90% identity, 95% identity...98% identity...99% identity). In one embodiment, (b) is a cellular target fused to a portion, or subunit, or component of the bioluminescent reporter. In some embodiments, (b) is a cellular target that is fused to a polypeptide that requires interaction with another polypeptide to produce luminescence. In some embodiments, (a) and (b) are present in a cell. In some embodiments, (b) is expressed intracellularly, e.g., as a fusion protein with a protein of interest, e.g., the cellular target. In some embodiments, the cellular target is composed of two or more components, subunits, or polypeptides, e.g., the cellular target is a protein complex. In some embodiments, the bioluminescent reporter is composed of two or more components, subunits, or polypeptides, e.g., the bioluminescent reporter is a protein complex. In some embodiments, (a) is added extracellularly and enters the cell. In some embodiments, (a) is present both intracellularly and in the medium surrounding the cell. In some embodiments, (a) is present bound to the cell and in the medium surrounding the cell. In some embodiments, the amount of (a) present in the surrounding medium is much greater than the amount in the cell or bound to the cell, e.g., at least 2-fold greater, at least 5-fold, 10-fold, 30-fold, or 100-fold greater. In certain embodiments, the cellular target is a binding partner of a bioactive agent. In certain embodiments, the emission spectrum of the bioluminescent reporter overlaps with the absorption spectrum of the fluorophore.In some embodiments, upon binding of the bioactive agent to the cellular target, conversion of the substrate to a reaction product by the bioluminescent reporter results in excitation of the fluorophore by BRET and emission of light from the fluorophore. In some embodiments, (a) is one of a library of drugs or compounds conjugated to a chromophore. In some embodiments, (a) is one of a library of drugs or compounds conjugated to a fluorophore. In some embodiments, (b) is one of a plurality of potential cellular targets fused to a bioluminescent reporter. In some embodiments, (b) is a library of cells expressing one of a plurality of potential cellular targets fused to a bioluminescent reporter.

[0006] In some embodiments, the invention provides methods for detecting, analyzing, characterizing, etc., binding of a bioactive agent to a cellular target. In some embodiments, the cellular target may be a primary drug target. In some embodiments, the cellular target is a burden of off-targets that may cause unwanted side effects in vivo. In some embodiments, binding of a bioactive agent to a cellular target may have no discernible biological effect. In some embodiments, the invention provides a cell comprising one or more (e.g., each of) (a) a bioactive agent conjugated to a chromophore (e.g., a fluorophore), (b) a cellular target fused to a bioluminescent reporter, and (c) a substrate for the bioluminescent reporter. In some embodiments, binding of the bioactive agent to the cellular target is non-covalent. In some embodiments, the chromophore is a fluorophore. In one embodiment, the invention provides a method for detecting an interaction between a bioactive agent and a cellular target, comprising: (a) expressing intracellularly a fusion of the cellular target with a bioluminescent reporter that emits energy at a first wavelength (e.g., range of wavelengths, spectral power distribution, etc.); (b) contacting the cell with the bioactive agent bound to a fluorophore, where the fluorophore receives energy at the first wavelength and emits energy at a second wavelength (e.g., range of wavelengths, spectral power distribution, etc.); (c) contacting the cell with a substrate for the bioluminescent reporter; and (d) detecting energy at the second wavelength, where the presence of the energy at the second wavelength indicates an interaction of the bioactive agent with the cellular target.

[0007] In one embodiment, the invention provides a method for detecting an interaction between a bioactive agent and a cellular target, comprising: (a) providing a fusion of the cellular target and a bioluminescent reporter; (b) contacting the fusion with the bioactive agent conjugated to a chromophore (e.g., a fluorophore); (c) contacting the fusion with a substrate for the reporter; and (d) detecting a change in the spectral distribution of emitted light compared to the fusion contacted with the substrate without the chromophore. In some embodiments, the present invention provides a method for detecting an interaction of a second bioactive agent with a cellular target, comprising: (a) providing a fusion of the cellular target with a bioluminescent reporter; (b) contacting the fusion with both a first bioactive agent conjugated to a chromophore (e.g., a fluorophore) and the second bioactive agent; (c) contacting the fusion with a substrate for the reporter; and (d) detecting a change in the spectral power of emitted light compared to the fusion contacted with the first bioactive agent and the substrate without the second bioactive agent. In some embodiments, the change in the spectral power of emitted light is due to displacement of the first bioactive agent by the second bioactive agent. In some embodiments, the displacement is competitive displacement. In some embodiments, the change in the spectral power of emitted light is used to assess the binding ability of the cellular target with a bioactive agent. In some embodiments, the second bioactive agent is one of a plurality of bioactive agents. In some embodiments, the change in the spectral power of emitted light is used to assess the relative binding ability of a plurality of bioactive agents to a cellular target. In some embodiments, the first and second bioactive agents are synthetic molecules. In some embodiments, the cellular target is a living cell, a permeabilized cell, or a cell lysate. In some embodiments, the change in the spectral power of the emitted light is measured by measuring the ratio of light intensities at two different wavelengths or two different wavelength ranges. In some embodiments, the change in the spectral power is detected over time. In some embodiments, the chromophore is a fluorophore.

[0008] In one embodiment, the invention provides a BRET assay system comprising: (a) a bioactive agent conjugated to a fluorophore; (b) a first interaction partner fused to a structurally complementary peptide of the bioluminescent reporter; (c) a second interaction partner fused to a structurally complementary polypeptide of the bioluminescent reporter; and (d) a substrate for the bioluminescent reporter, wherein the first and second interaction partners interact to form an interaction complex, and wherein the first interaction partner, the second interaction partner, and / or the interaction complex are binding partners for the bioactive agent. In one embodiment, the first interaction partner and the second interaction partner are proteins or polypeptides that interact to form a protein complex. In one embodiment, the first interaction partner and the second interaction partner are joined by interaction of structurally complementary polypeptides. In one embodiment, the structurally complementary polypeptides are joined by interaction of the first interaction partner and the second interaction partner. In one embodiment, the interaction of the first interaction partner and the second interaction partner is measured by an increase in the amount of light emitted. In some embodiments, the first and second interaction partners form an interaction complex in the presence or absence of a bioactive agent. In some embodiments, the interaction complex is a binding partner for the bioactive agent, but neither the first nor the second interaction partner alone is a binding partner for the bioactive agent. In some embodiments, one of the interaction partners is a binding partner for the bioactive agent, but the other is not. In some embodiments, the formation of the interaction complex requires binding of the bioactive agent to the interaction partner. In some embodiments, the interaction complex formation is independent of binding of the bioactive agent. In some embodiments, the invention provides a BRET assay system that includes (a) a bioactive agent conjugated to a fluorophore, (b) a cellular target fused to a structurally complementary peptide of a bioluminescent reporter, (c) a structurally complementary polypeptide of the bioluminescent reporter, and (d) a substrate for the bioluminescent reporter. In some embodiments, the complementary peptide and polypeptide of the bioluminescent reporter cooperate to generate an active bioluminescent reporter enzyme.In certain embodiments, binding of a bioactive agent to a cellular target and pairing of complementary peptides and polypeptides results in conversion of a substrate to a reaction product by a bioluminescent reporter enzyme, which results in excitation of the fluorophore by BRET and fluorescence emission from the fluorophore.

[0009] In one embodiment, the invention provides an assay system that includes (a) a bioactive agent conjugated to a fluorophore, (b) a first binding partner fused to a complementary peptide of the bioluminescent reporter, (c) a second binding partner fused to a complementary polypeptide of the bioluminescent reporter, and (c) a substrate for the bioluminescent reporter. In one embodiment, the complementary peptide and polypeptide of the bioluminescent reporter cooperate to generate an active bioluminescent reporter enzyme. In one embodiment, if the first and second binding partners interact, the bioactive agent binds to the interaction partner, resulting in pairing of the complementary peptide and polypeptide, and conversion of the substrate to a reaction product by the bioluminescent reporter enzyme, resulting in excitation of the fluorophore by BRET and fluorescence emission from the fluorophore.

[0010] In some embodiments, the bioactive agent is conjugated to a fluorescent or luminescent quencher (e.g., Dabcyl). In this embodiment, titration of the unbound bioactive agent results in a gain in signal.

[0011] In one embodiment, the invention provides for imaging of any of the systems described herein (e.g., BRET imaging, by charge-coupled device camera, etc.) to identify and / or identify the location of fluorescence (e.g., intracellular, extracellular, etc.) and / or the BRET signal resulting from the presence and / or interaction of components of the system (e.g., bioactive agents, cellular targets, etc.). In some embodiments, the systems and methods described herein (see Example 23 for an exemplary embodiment) are useful for measuring the intracellular selectivity and affinity of bioactive agents to a panel of putative cellular targets. In some embodiments, the effect on BRET is monitored as the bioactive agent / cellular target complex is competitively inhibited. In some embodiments, affinity is inferred by IC50 values ​​generated via competitive disruption. In some embodiments, the affinity of inhibitors to individual regions of the cellular target is determined via fusing bioluminescent reporter genes to discretized regions of the cellular target. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 shows a schematic representation of an embodiment of the invention: (A) A fluorophore-conjugated bioactive agent and a cellular target bound to a bioluminescent reporter. (B) Binding of the bioactive agent to the cellular target. (C) Addition of a reporter substrate results in BRET. (D) Displacement by excess unbound bioactive agent results in BRET and loss of fluorescence. [Diagram 2] FIG. 2 shows a graph comparing the BRET profiles of two different fluorophore-SAHA derivatives as BRET acceptors for the NANOLUC-HDAC6 fusion in living cells. [Figure 2-2] FIG. 2 shows a graph comparing the BRET profiles of two different fluorophore-SAHA derivatives as BRET acceptors for the NANOLUC-HDAC6 fusion in living cells. [Diagram 3] Figure 3 shows graphs depicting the effect of dilution of expression on assay performance. The first graph demonstrates the effect of reducing expression on the BRET assay window using competitive displacement of fluorophore-conjugated SAHA from NANOLUC-fused HDAC6. The second graph shows the observed compound potency (EC50) for drug tracer binding to the NanoLuc-histamine HI receptor along with the effect of reducing expression on the BRET assay window. [Figure 3-2]Figure 3 shows graphs depicting the effect of dilution of expression on assay performance. The first graph demonstrates the effect of reducing expression on the BRET assay window using competitive displacement of fluorophore-conjugated SAHA from NANOLUC-fused HDAC6. The second graph shows the observed compound potency (EC50) for drug tracer binding to the NanoLuc-histamine HI receptor along with the effect of reducing expression on the BRET assay window. [Figure 4] FIG. 4 shows a graph demonstrating the cellular environment requirements for prodrug processing. [Diagram 5] FIG. 5 shows a graph demonstrating the ability of the use of permeabilization agents to enhance the entry of impermeable drug tracers. [Figure 5-2] FIG. 5 shows a graph demonstrating the ability of the use of permeabilization agents to enhance the entry of impermeable drug tracers. [Figure 6] FIG. 6 shows a graph depicting the competitive displacement of fluorophore-conjugated BIRB-derivatives from NANOLUC-fused p38. [Figure 7] FIG. 7 shows a graph depicting the measurement of the reaction kinetics of fluorophore-conjugated BIRB derivatives from NANOLUC-fused p38. [Figure 8] FIG. 8 shows exemplary fluorophore-conjugated bioactive agents and other compounds that may be used in the embodiments described herein. [Figure 8-2] FIG. 8 shows exemplary fluorophore-conjugated bioactive agents and other compounds that may be used in the embodiments described herein. [Figure 9] FIG. 9 shows a comparison of p38 fusions with Renilla luciferase and NanoLuc luciferase in embodiments described herein. [Figure 9-2] FIG. 9 shows a comparison of p38 fusions with Renilla luciferase and NanoLuc luciferase in embodiments described herein. [Figure 10] FIG. 10 shows that when expression of the NANOLUC fusion is close to endogenous levels, high affinity interaction with the drug tracer is achieved at optimal S / B ratios. [Figure 10-2] FIG. 10 shows that when expression of the NANOLUC fusion is close to endogenous levels, high affinity interaction with the drug tracer is achieved at optimal S / B ratios. [Figure 11] FIG. 11 shows the dose response curves of NanoLuc-p38alpha binding to the BIRB-TOM conjugate compared to the BIRB-TMR conjugate. [Figure 12] FIG. 12 shows the dose response curve of PKCalpha-NanoLuc binding to a BIM-TOM conjugate compared to a BIM-TMR conjugate. [Figure 13] FIG. 13 shows a graph depicting the detection of a specific BRET response in living cells. [Figure 14] FIG. 14 shows the affinity of PBI-4838 for Jnk2, p38 beta and p38 alpha. [Figure 14-2] FIG. 14 shows the affinity of PBI-4838 for Jnk2, p38 beta and p38 alpha. [Figure 14-3] FIG. 14 shows the affinity of PBI-4838 for Jnk2, p38 beta and p38 alpha. [Figure 15] FIG. 15 shows the affinity of PBI-4838 for Jnk2, p38 beta and p38 alpha. [Figure 15-2] FIG. 15 shows the affinity of PBI-4838 for Jnk2, p38 beta and p38 alpha. [Figure 15-3] FIG. 15 shows the affinity of PBI-4838 for Jnk2, p38 beta and p38 alpha. [Figure 16] FIG. 16 shows the relative affinity of PBI-4966 for wild-type versus mutant BRD4. [Figure 16-2] FIG. 16 shows the relative affinity of PBI-4966 for wild-type versus mutant BRD4. [Figure 17] FIG. 17 shows that structurally distinct compounds can be identified by competitive displacement of drug tracers using the methods of the present invention. [Figure 17-2]FIG. 17 shows that structurally distinct compounds can be identified by competitive displacement of drug tracers using the methods of the present invention. [Figure 17-3] FIG. 17 shows that structurally distinct compounds can be identified by competitive displacement of drug tracers using the methods of the present invention. [Figure 18] FIG. 18 demonstrates the ability to monitor drug / target interactions intracellularly using fluorophore-drug conjugates. [Figure 19] FIG. 19 shows a graph depicting a specific BRET response in TNT. [Figure 20] Figure 20 shows a graph depicting the measurement of binding of fluorescently labeled cytokines to NanoLuc-EGFR. The affinity of the therapeutic antibodies, Vectibix, Erbitux and Herceptin, is also demonstrated by competitive displacement of the cytokines. [Figure 20-2] Figure 20 shows a graph depicting the measurement of binding of fluorescently labeled cytokines to NanoLuc-EGFR. The affinity of the therapeutic antibodies, Vectibix, Erbitux and Herceptin, is also demonstrated by competitive displacement of the cytokines. [Figure 21] FIG. 21 contains wavelength scans for a complementary polypeptide (SEQ ID NO:5) complemented with a complementary peptide (SEQ ID NO:4) alone or with a complementary peptide conjugated to TMR (SEQ ID NO:4), demonstrating efficient energy transfer. [Figure 21-2] FIG. 21 contains wavelength scans for a complementary polypeptide (SEQ ID NO:5) complemented with a complementary peptide (SEQ ID NO:4) alone or with a complementary peptide conjugated to TMR (SEQ ID NO:4), demonstrating efficient energy transfer. [Figure 22] FIG. 22 contains a wavelength scan for NanoLuc fused to HaloTag (NL-HT) and a complementary polypeptide (SEQ ID NO:6) complemented with a complementary peptide conjugated to fluorescent non-chloro-TOM (NCT) dye (PBI-5074). [Figure 23] FIG. 23 shows a schematic diagram of a ternary interaction, in which energy transfer by a structurally complementary peptide and a bioluminescent protein polypeptide complex can be used to measure the interaction of interaction partners. In this schematic diagram, a GPCR (first interaction partner) fused to a complementary polypeptide of a bioluminescent protein and a GPCR interacting protein (second interaction partner) fused to a complementary peptide of a bioluminescent protein generate a bioluminescent complex when they interact (to generate an interaction complex). This allows the measurement of a binary interaction. If a small molecule GPCR ligand bearing a fluorescent moiety for energy transfer interacts with this system, energy transfer occurs. Thus, binary protein-protein interactions and ternary drug-protein-protein interactions can be measured in the same experiment. [Figure 24] FIG. 24 shows graphs demonstrating that BRET data between cell-expressed fusions of 11S peptide and BRD4 and exogenously added NCT conjugated to BRD4 ligand is dependent on the presence of PEP-80 (structural complement to 11S): (A) donor intensity for grafting, (B) acceptor intensity. [Diagram 25] FIG. 25 shows graphs demonstrating that BRET data between cell-expressed fusions of 11S peptide and BRD4 and exogenously added NCT conjugated to iBET is dependent on the presence of PEP-80 (structural complement to 11S): (A) IμM PEP-80, (B) no PEP-80 control. [Figure 26] FIG. 26 shows that the BRET signal (acceptor and donor intensity) is dependent on the facilitating 11S-BRD4 / 114-Histone H3.3 complex. [Figure 27] FIG. 27 shows that a fluorescent BRD4 ligand (iBET-NCT / PBI-4966) can displace the 11S-BRD4 / 114-Histone H3.3 complex. [Figure 27-2]FIG. 27 shows that a fluorescent BRD4 ligand (iBET-NCT / PBI-4966) can displace the 11S-BRD4 / 114-Histone H3.3 complex. [Figure 28] FIG. 28 shows non-fluorescent iBET-151 IC50 competitive displacement of the 11S-BRD4 / 114-Histone H3.3 complex. [Figure 29] FIG. 29 shows that class I HDACs (HDACs 1, 2, 3, and 8) generate specific BRET signals with SAHA-TOM, as predicted based on the reported affinity and selectivity of SAHA for these respective targets. [Figure 29-2] FIG. 29 shows that class I HDACs (HDACs 1, 2, 3, and 8) generate specific BRET signals with SAHA-TOM, as predicted based on the reported affinity and selectivity of SAHA for these respective targets. [Diagram 30] Figure 30 shows that class IIb HDACs (HDAC6 and 10), as well as distinct regions of HDAC6, undergo specific BRET with SAHA-TOM, as predicted based on the reported affinity and selectivity of SAHA for these respective targets. [Figure 30-2] Figure 30 shows that class IIb HDACs (HDAC6 and 10), as well as distinct regions of HDAC6, undergo specific BRET with SAHA-TOM, as predicted based on the reported affinity and selectivity of SAHA for these respective targets. [Diagram 31] FIG. 31 shows that class IIa HDACs do not generate significant BRET with SAHA-TOM, as predicted based on the reported selectivity of SAHA over class I and IIb HDACs. [Figure 31-2] FIG. 31 shows that class IIa HDACs do not generate significant BRET with SAHA-TOM, as predicted based on the reported selectivity of SAHA over class I and IIb HDACs. [Figure 31-3]FIG. 31 shows that class IIa HDACs do not generate significant BRET with SAHA-TOM, as predicted based on the reported selectivity of SAHA over class I and IIb HDACs. [Figure 31-4] FIG. 31 shows that class IIa HDACs do not generate significant BRET with SAHA-TOM, as predicted based on the reported selectivity of SAHA over class I and IIb HDACs. [Figure 31-5] FIG. 31 shows that class IIa HDACs do not generate significant BRET with SAHA-TOM, as predicted based on the reported selectivity of SAHA over class I and IIb HDACs. [Diagram 32] Figure 32 shows the results of competitive displacement of SAHA for individual HDAC-NanoLuc fusions complexed with SAHA-TOM as determined via BRET. From these concentration-response curves, IC-50 values ​​can be calculated, as shown in the table next to the graph. [Diagram 33] Figure 33 (left) shows the conversion between IC-50 and Ki values ​​for individual HDAC-NanoLuc fusions as determined via BRET. The Cheng-Prusoff equation was used for this conversion. Figure 33 (right) shows a diagram depicting the relative affinity of SAHA for individual HDAC / NanoLuc fusion proteins as determined via BRET. [Diagram 34] FIG. 34 shows BRET imaging of intracellular HDAC10-NLuc / PBI-4968+ / -SAHA complexes in the presence or absence of SAHA as a specificity control. [Diagram 35] FIG. 35 shows BRET imaging of intracellular NLuc-HDAC6 / PBI-4968+ / -SAHA complexes in the presence or absence of SAHA as a specificity control. [Diagram 36] FIG. 36 shows a dot plot analysis of BRET imaging studies of SAHA-bound intracellular SAHA-TOM (PBI-4968) with HDAC10 and HDAC6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention provides compositions and methods for the detection and analysis of intracellular binding of bioactive agents to cellular targets. In particular, provided herein are bioactive agents conjugated to chromophores, such as fluorophores, potential cellular targets fused to bioluminescent reporter proteins, and methods for detecting and analyzing the interaction of the bioactive agents with the cellular targets therewith (see FIG. 1).

[0014] The interaction of a first entity (e.g., a bioactive agent) with a second entity (e.g., a cellular target) may be detected, characterized, quantified, analyzed, etc., through detection / measurement of a signal resulting from signal transduction (e.g., energy transfer (e.g., by fluorescence, light energy, resonance, BRET, etc.)) between a third entity (e.g., a fluorophore) that is connected, fused, bound, linked, etc. to the first entity, and a fourth entity (e.g., a bioluminescent reporter protein) that is connected, fused, bound, linked, etc. to the second entity. The interaction and / or binding of the first and second entities may bring the third and fourth entities into sufficient proximity to allow signal transduction (e.g., energy transfer) from one to the other. In one embodiment, the fourth entity (e.g., a bioluminescent reporter protein) emits energy (e.g., upon interaction with its substrate) that is absorbed by a third entity (e.g., a fluorophore) causing the third entity to emit a distinct energy (e.g., light of a different wavelength) from the fourth entity. In this embodiment, upon addition of a substrate of the fourth entity (e.g., a bioluminescent reporter) to the system, detection of the energy emitted by the third entity (e.g., the light emission maximum of the third entity) indicates an interaction of the first and second entities. In one embodiment, the duration, kinetics, affinity, strength and / or specificity of binding of the first and second entities is detected, measured, quantified, determined, interrogated, etc. based on measuring the signal output of the fourth entity (e.g., a bioluminescent reporter protein) under various conditions.

[0015] In a particular embodiment, a cellular target fused to a bioluminescent reporter protein and a bioactive agent bound to a chromophore, such as a fluorophore, are provided (e.g., intracellularly, extracellularly, in a lysate, ex vivo, etc.). A substrate for the bioluminescent reporter protein is added to the system. If an interaction (e.g., binding) occurs between the bioactive agent and the cellular target, the bioluminescent reporter protein and the chromophore (e.g., fluorophore) are brought into sufficient proximity for BRET to occur and a detectable signal is emitted from the chromophore (e.g., fluorophore).

[0016] In some embodiments, complementary peptides and polypeptides are used that can interact (e.g., form a complex) to form a bioluminescent reporter protein (or protein complex). In some embodiments, the complementary peptide is fused to a first interaction partner and the complementary polypeptide is fused to a second interaction partner. In some embodiments, the first and second interaction partners form a complex (e.g., by binding to each other). In some embodiments, the first and second interaction partners form an interaction complex when one or both of them interact with a bioactive agent. In some embodiments, the first and second interaction partners form an interaction complex in the presence or absence of a bioactive agent. In some embodiments, the formation of the interaction complex joins the complementary peptide and polypeptide to form a bioluminescent reporter. In some embodiments, the formation of the bioluminescent reporter allows for detection of the formation of the interaction complex. In some embodiments, a fluorophore is attached to the bioactive agent. In some embodiments, when the interaction complex is formed, energy is transferred from the fluorophore to the bioluminescent reporter and the bioactive agent is attached to one of the interaction partners or the interaction complex. In certain embodiments, the fluorophore allows for detection or measurement of the interaction of the first and second interaction partners, hi certain embodiments, the fluorophore allows for detection or measurement of the binding of a bioactive agent to its binding partner (e.g., the first interaction partner, the second interaction partner, and / or the interaction complex).

[0017] In some embodiments, the complementary peptide of the bioluminescent reporter is fused to a target of interest. A bioactive agent is provided that is conjugated to a complementary polypeptide and a fluorophore to detect or measure the interaction of the bioactive agent to a target of interest. In some embodiments, the complementary peptide of the bioluminescent reporter is fused to a target of interest and the complementary polypeptide of the bioluminescent reporter protein and the bioactive agent is conjugated to a fluorophore are applied to detect the proximity of a fluorescently labeled ligand, e.g., a bioactive agent, to a protein complex (e.g., to detect selective ligand binding to a heterodimeric or homodimeric receptor) by linking members of the complex to components of a multicomponent luminescent donor. The present application can thus be used to monitor the engagement of targets within protein complexes that may have a role in disease pathology.

[0018] In some embodiments, the cellular target and bioluminescent reporter fusion are expressed within the cells in which the assay is to be performed. In some embodiments, the fusion is expressed at or near its native abundance relative to the cellular target. In some embodiments, the fluorophore-conjugated bioactive agent is added extracellularly (e.g., added to the culture medium) and enters the cell by diffusion, active transport, passive transport, endocytosis, or any suitable mechanism. In some embodiments, various amounts of the fluorophore-conjugated bioactive agent are added to the cells to assay binding kinetics, assay binding affinity, provide sufficient signal, etc.

[0019] In certain embodiments, the present invention provides compositions, methods, and systems for the detection of intracellular interactions between a bioactive agent and a cellular target (e.g., known or unknown). In some embodiments, a fusion of a bioluminescent reporter and a cellular target is expressed intracellularly. The bioactive agent is conjugated to a fluorophore and introduced into the cell (e.g., the fluorophore-bioactive agent conjugate is cell-permeable, the cells are permeabilized, etc.). A substrate for the bioluminescent reporter protein is added to the cell prior to, simultaneously with, or after addition of the bioactive agent. Detection of fluorescent emission from the fluorophore (as a result of BRET) indicates an intracellular interaction (e.g., binding) between the bioactive agent and the cellular target. In some embodiments, the cellular target fused to the bioluminescent reporter is expressed at its natural cellular abundance (e.g., relative to the native cellular target or at a level appropriate for the inherent biological function of the fused target). In some embodiments, interaction of the bioactive agent with the cellular target is detected intracellularly.

[0020] In some embodiments, the interaction between a bioactive agent and a cellular target is characterized by altering cellular or system conditions, for example, in some embodiments, a competing binder of the cellular target (e.g., unbound bioactive agent) is added to the cells to compete with the fluorophore-bound bioactive agent.

[0021] In some embodiments, a library of bioluminescent reporter protein tagged cellular targets is provided (e.g., in solution, in a lysate, immobilized on a surface, expressed intracellularly, etc.). In some embodiments, a bioactive agent is provided in situations where there is no known cellular target or where knowledge of the cellular target is uncertain or incomplete. The cellular target of the bioactive agent is determined by adding the bioactive agent to the library and determining which cellular target fusions produce BRET induced fluorescence of the fluorophore to which the bioactive agent is attached. In some embodiments, the library of bioluminescent reporter tagged cellular targets is provided as a collection of nucleic acids or vectors (e.g., plasmids, BacMam virus, lentivirus, etc.) that contain nucleic acids encoding protein fusions. In some embodiments, the bioluminescent reporter protein tagged cellular targets are expressed intracellularly. In some embodiments, the library of bioluminescent reporter tagged cellular targets is provided by translating the nucleic acid in a cell-free translation reaction. In some embodiments, the library of cellular target fusions or cells expressing cellular target fusions is provided in a microplate format. In this embodiment, the interaction of bioactive agents (e.g., as identified through a phenotypic assay or screen) with an entire library of cellular targets can be examined (e.g., intracellularly, in a lysate, in solution, etc.) in a high-throughput manner. In some embodiments, bioluminescent reporter tagged cellular targets are immobilized on a solid surface to form protein aggregates. For example, in some embodiments, in addition to a bioluminescent reporter, the cellular targets are also expressed as fusions bearing a tag or bound to a protein (e.g., HALOTAG, Promega), allowing the protein to be covalently immobilized to a solid surface (e.g., a surface that displays an appropriate ligand (e.g., HALOTAG ligand)). In certain embodiments, a library of potential bioactive agents (e.g., hit compounds or small drug-like molecules) is added to the system (e.g., array) and any pairs capable of producing BRET are identified. In some embodiments, the cellular targets of all or part of the library of bioactive agents are unknown.

[0022] In certain embodiments, the compositions, methods, and systems herein provide conjugates of a bioactive agent and an energy acceptor (e.g., fluorophore, chromophore). In some embodiments, the bioactive agent is any small molecule (e.g., >2000 Daltons, >1000 Daltons, >500 Daltons, etc.), macromolecule, synthetic molecule, or molecular complex capable of interacting with the biology of a cell. In some embodiments, the energy acceptor is an entity capable of generating, exhibiting, and / or emitting a signal (e.g., light, heat, chemical reaction, fluorescence, resonance energy, etc.) when triggered by energy absorption (e.g., resonance energy transfer). In some embodiments, the bioactive agent and energy acceptor (e.g., fluorophore, chromophore) are fused, connected, coupled, or otherwise linked by any suitable structure or mechanism (e.g., expressed as a fusion construct (e.g., with or without a peptide linker), chemically linked (e.g., directly or indirectly), enzymatically linked, linked by a linker (e.g., peptide, nucleic acid, polymer, ester bond, PEG linker, carbon chain, etc.), etc.). In some embodiments, the conjugate of the bioactive agent and energy acceptor (e.g., a bioactive agent linked to a fluorophore) is generated by non-native chemical synthesis (e.g., the deliberate performance of a chemical reaction that does not exist in natural cells). The type of linkage should not be limiting.

[0023] As used herein, the term "bioactive agent" generally refers to any physiologically or pharmacologically active substance or substance suitable for detection. In some embodiments, a bioactive agent is a potential therapeutic compound (e.g., a small molecule, peptide, nucleic acid, etc.) or drug-like molecule. In some embodiments, a bioactive agent is produced by non-natural chemical synthesis (e.g., the deliberate performance of a chemical reaction that does not exist in natural cells). Bioactive agents as used in the embodiments described herein are not limited by size or structure. In certain embodiments, libraries of bioactive agents (e.g., >10 drugs, >50 drugs, >100 drugs, >500 drugs, >1000 drugs, >5000 drugs, >10,000 drugs, >50,000 drugs, etc.) are provided. In certain embodiments, systems, methods and compositions for screening libraries of bioactive agents are provided. In certain embodiments, the invention provides a means for identifying the bioactive agent in the library that is responsible for causing, causing, inducing, etc. a phenotypic effect and / or activity. In certain embodiments, the invention provides a means for identifying the cellular target of a bioactive agent (e.g., the bioactive agent that is responsible for a phenotypic effect or activity).

[0024] As used herein, the term "energy acceptor" refers to any small molecule (e.g., a chromophore), macromolecule (e.g., an autofluorescent protein, a phycobiliprotein, a nanoparticle, a surface, etc.), or molecular complex that generates a rapidly detectable signal upon energy absorption (e.g., resonance energy transfer). In certain embodiments, the energy acceptor is a fluorophore or other detectable chromophore.Non-limiting examples of suitable fluorophores include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin, etc.), CF dye (Biotium), BODIPY (Invitrogen), ALEXA Examples of dyes include FLuoR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma-Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dye (CYANDYE, LLC), SETAU and SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, phycobilisome) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, and the like. In some embodiments, the fluorophore is a rhodamine analog (e.g., a carboxyrhodamine analog), such as those described in U.S. Patent Application Serial No. 13 / 682,589, the entirety of which is incorporated herein by reference. Some such fluorophores are described herein in Example 8.In certain embodiments, BRET efficiency is greatly enhanced compared to other fluorophores due to the technical features of rhodamine analogs (e.g., carboxyrhodamine analogs) as energy acceptors, e.g., these dyes have left-shifted EC50 and low nonspecific background, which are advantageous for use in some embodiments.

[0025] In the examples used herein, the terms "TOM" and "NonChloroTOM" (or "NCT") refer to the same type of phosphor and are used interchangeably throughout the application.

[0026] In certain embodiments, compositions, methods, and systems herein provide a fusion of a cellular target with a bioluminescent reporter protein (e.g., luciferase). In certain embodiments, the cellular target and the bioluminescent reporter protein are fused, coupled, connected, etc., by any suitable structure or mechanism (e.g., expressed as a fusion construct (e.g., with or without a peptide linker), chemically linked (e.g., covalently or non-covalently), linked to an enzyme, linked by a linker (e.g., a peptide, nucleic acid, other polymer (e.g., ester bond, PEG linker, carbon chain, etc.)), etc.). In certain embodiments, an amino acid chain (e.g., 3-100 amino acids) is used to connect the cellular target and the bioluminescent reporter protein. In certain embodiments, neither the cellular target nor the bioluminescent reporter is affected (e.g., significantly affected) in terms of structure and / or function by the presence of the fusion or linker. In certain embodiments, the linker allows for fusion without loss of activity or one or both of these components. In another embodiment, the amino acid linker properly spaces and / or orients the bioluminescent moieties that are channeled for energy transfer with the fluorophore.

[0027] In certain embodiments, the cellular target includes any suitable binding / interaction partner (e.g., receptor, enzyme, protein complex) for a bioactive agent (e.g., small molecule, protein, nucleic acid, lipid, etc.). However, in certain embodiments, knowledge of the interaction between the cellular target and the bioactive agent is not required to practice the present invention. In certain embodiments, the cellular target is a protein or protein complex that binds to or otherwise interacts (e.g., has binding affinity for) the bioactive agent. In further specific embodiments, the cellular target is a receptor protein or enzyme that binds to or otherwise interacts (e.g., has binding affinity for) the small molecule bioactive agent. The present invention is not limited by the identity, type, or class of the cellular target. In certain embodiments, libraries of hundreds, thousands, tens of thousands, or even more different cellular targets find use in the present invention. Examples of cellular targets may include complexes containing any of these with nucleic acids (e.g., DNA or RNA), polysaccharides, or polypeptides. In certain embodiments, the cellular target is a G protein-coupled receptor or a protein kinase.

[0028] In certain embodiments, the bioluminescent reporter is a luciferase. In some embodiments, the luciferase is selected from those found in Gaussia, Coleoptera (e.g., firefly), Renilla, Cypridina, Lucidum, Aequorin, mutants, portions, variations thereof, and any other luciferase enzyme suitable for the systems and methods described herein. In some embodiments, the bioluminescent reporter protein is a modified, improved luciferase enzyme from Lucidum (e.g., NANOLUC enzyme from Promega Corporation, SEQ ID NO:1, or a sequence having at least 70% identity thereto (e.g., >70%, >80%, >90%, >95%). In one embodiment, the bioluminescent reporter protein is a thermostable Photuris pennsylvanica luciferase or a sequence having at least 70% (e.g., >70%, >80%, >90%, >95%) identity thereto. Exemplary bioluminescent reporters are described, for example, in U.S. Patent Application Nos. 2010 / 0281552 and 2012 / 0174242, the entireties of which are incorporated herein by reference.

[0029] In some embodiments, the bioluminescent reporter protein comprises NANOLUC (see U.S. Patent Application Nos. 2010 / 0281552 and 2012 / 0174242, the entireties of which are incorporated by reference herein). In some embodiments, the bioluminescent reporter protein comprises a polypeptide having at least 70% (e.g., >70%, >80%, >90%, >95%) identity to SEQ ID NO:1 that retains bioluminescent characteristics. In certain embodiments, the use of NANOLUC enzyme or variants thereof provides characteristics (e.g., signal strength, brightness, high light output, narrow spectrum, etc.) that enable the use of the BRET assays described herein. In one embodiment, the high light output of NANOLUC allows for low concentrations (e.g., >1 μM, >100 nM, >10 nm, >1 nm, etc.) of assay components useful for performing assays in physiologically relevant contexts, such as DNA-encoded NANOLUC. In one embodiment, NANOLUC enables the use of BRET in the characterization of cellular targets identified in phenotypic screens.

[0030] In some embodiments, a substrate for a bioluminescent reporter protein is provided. In some embodiments, the bioluminescent reporter protein converts the substrate into a reaction product and emits light energy as a by-product. In some embodiments, the substrate is a substrate for a luciferase enzyme. In some embodiments, the substrate is a structural variant or derivative of coelenterazine (e.g., furimazine). In some embodiments, the substrate is a substrate for an engineered, improved luciferase enzyme from luminescent shrimp, such as, for example, NANOLUC enzyme (e.g., SEQ ID NO:1) from Promega. In some embodiments, a pro-substrate for a bioluminescent reporter protein is provided, which generates the substrate by a chemical or physical process (e.g., hydrolysis, enzymatic reaction, photocleavage, etc.). In some embodiments, the pro-substrate comprises coelenterazine, a coelenterazine derivative, a structural or functional equivalent of coelenterazine, a molecule substantially equivalent (e.g., structurally and / or functionally) to coelenterazine, or a molecule functionally or structurally similar to coelenterazine. In one embodiment, the bioluminescent reporter protein converts coelenterazine, a coelenterazine derivative, a structural or functional equivalent of coelenterazine, or a substantial equivalent of coelenterazine, to coelenteramide, a coelenteramide derivative, a structural or functional equivalent of coelenteramide, or a substantial equivalent of coelenteramide, emitting light energy as a by-product.

[0031] In some embodiments, the fluorophore and bioluminescent reporter are selected to exhibit sufficient overlap between the emission (e.g., of the bioluminescent reporter) and excitation (e.g., of the fluorophore) spectra to provide efficient energy transfer between the two (e.g., by non-radiative dipole-dipole coupling). In some embodiments, the peak emission of the bioluminescent reporter is substantially separated by the peak emission of the fluorophore, e.g., at least 80 nm, 100 nm, 120 nm, 140 nm, etc., in wavelength. In certain embodiments, the Forster distance of the fluorophore and bioluminescent reporter pair is small (e.g., >20 nm, >10 nm, >5 nm, >3 nm, etc.). In this embodiment, the short Forster distance creates a requirement that the fluorophore and bioluminescent reporter must be in close proximity for energy transfer to occur. Thus, a short Forster distance reduces aberrant and / or background signals (e.g., formed by diffusing fluorophores and / or reporters).

[0032] In certain embodiments, a sufficiently bright fluorophore and bioluminescent reporter pair is selected to allow detection of the transduction signal at the native abundance (or near native abundance) of the protein of interest and / or the cellular target fused to the bioluminescent reporter. In some embodiments, if the selected fluorophore or bioluminescent reporter produces insufficient energy (light) emission, the fusion of the cellular target and bioluminescent reporter must be overexpressed (e.g., above native abundance, above biologically relevant levels, etc.) and / or the amount of fluorophore-linked bioactive agent must be increased (e.g., above potentially toxic levels, above physiologically relevant levels, above the amount at which kinetic experiments can be performed, etc.). In some embodiments, the sufficient brightness of the bioluminescent reporter and fluorophore allows detection of the bioactive agent and cellular target interaction at a range of concentrations and ratios.

[0033] In one embodiment, compositions, methods and systems are provided for the identification of cellular targets of hit compounds emerging from a phenotypic assay or phenotypic screen. In one embodiment, following the identification of a bioactive agent capable of eliciting a phenotype, a "hit compound" conjugated to a fluorophore is used to identify a cellular target through linkage to a bioluminescent reporter protein (e.g., binding of the bioactive agent to the cellular target results in BRET between the bioluminescent reporter protein and the fluorophore). In this embodiment, a phenotype is associated with a particular bioactive agent, but the interaction partner (e.g., cellular target) for that bioactive agent is unknown or uncertain. In one embodiment, a bioactive agent conjugated to a fluorophore is capable of regenerating a phenotype, thereby ensuring that conjugation of the bioactive agent to the fluorophore (or cellular target to the bioluminescent reporter) does not affect the cell-bound swatch and / or inhibit its biological activity. In some embodiments, the use of a library of cellular targets, each fused to a bioluminescent reporter protein (e.g., NANOLUC), presents fewer obstacles compared to other assays (e.g., mass spectrometry analysis) due to the poor solubility or natural abundance of the cellular targets. In some embodiments, such assays provide a greater degree of sensitivity or specificity. In some embodiments, target discrimination through linkage to a bioluminescent reporter protein allows detection through energy transfer even when target binding is inefficient or incomplete. In some embodiments, binding of a bioactive agent (e.g., a small molecule) to a cellular target is characterized using a competitive binding assay (see Figures 1C and 1D). In some embodiments, BRET allows analysis of the binding affinity of hit compounds in living cells using competitive displacement of a fluorescent tracer (e.g., a fluorophore) that binds to the same site on the cellular target.In certain embodiments, the systems and methods described herein provide the ability to use two different methods for target identification, thus providing greater stringency for identifying cellular targets and complementary methods that address limitations in other approaches (e.g., BRET offers the advantage of maintaining the bioactive agent in equilibrium with the target throughout the study).

[0034] Some of the embodiments described herein have been found to be used in drug discovery, drug validation, drug target discovery, or drug target validation. In certain embodiments, binding interactions between a bioactive agent (e.g., a small drug-like molecule) and a cellular target are detected, validated, and / or characterized. In some embodiments, the relative binding affinity of a bioactive agent to a cellular target (e.g., in solution, in a lysate, on a surface, within a cell, etc.) can be determined by its ability to displace a bioactive agent that has been bound to a fluorophore. Specifically, a higher binding affinity of a first bioactive agent to a second bioactive agent is indicated by a lower concentration of the first bioactive agent required to displace the bound bioactive agent compared to the second bioactive agent. Displacement of the bound bioactive agent is determined by a loss or reduction in energy transfer from a bioluminescent reporter protein fused to the cellular target. In some embodiments, the concentration of bioactive agent required to displace the bound bioactive agent is used to assess the binding EC50 or inhibition constant (Ki) for the bioactive agent. In one embodiment, expression of new or modified bioactive agents is driven by the ability to displace one or more bioactive agents, each of which is linked to a fluorophore, from one or more cellular targets, each of which is fused to a bioluminescent reporter protein.

[0035] In some embodiments, a collection of compounds that may have unknown binding affinity to a cellular target may be screened for the ability to bind a target fused to a bioluminescent protein by determining the ability to displace a bioactive agent bound to a fluorophore. In some embodiments, compounds are screened for the ability to preferentially bind a first cellular target compared to a second cellular target by their ability to displace a first bound bioactive agent from a first cellular target compared to displacing the second bound bioactive agent from the second bioactive target. In some embodiments, the first and second bound bioactive agents are the same.

[0036] In some embodiments, the systems and methods described herein provide the ability to determine the affinity of a bioactive agent (e.g., a hit compound, research lead, lead compound, etc.) to wild-type and mutant versions of a cellular target, e.g., a target protein. In some embodiments, characterization of the affinity and selectivity of a fluorescently labeled bioactive agent (e.g., a drug) to a disease-relevant mutant protein may be performed intracellularly. The systems and methods may be useful for identifying bioactive agents (e.g., drugs) that selectively and specifically bind wild-type or mutant proteins.

[0037] In addition to the primary target, target of interest, and / or known targets, a bioactive agent (e.g., a hit compound) may bind to unexpected and / or unintended cellular targets (off-targets). In some cases, off-target binding of a bioactive agent is responsible for some of the therapeutic effects and / or side effects associated with administration of the bioactive agent. In certain embodiments, the systems and methods described herein provide the ability to identify off-targets of a bioactive agent. Understanding the identity and extent of off-target bioactive agent binding provides valuable information regarding the pharmacology of the drug.

[0038] In some embodiments, the systems and methods described herein provide the ability to assess the binding characteristics (e.g., EC50, Kd, ​​binding rate, environmental effects, etc.) of fluorophore-conjugated bioactive agents to cellular targets fused to bioluminescent reporters. In some embodiments, the binding characteristics relate to biochemical, physical, or phenotypic properties associated with the cellular target. In some embodiments, the kinetic characteristics of the formation or dissolution of the BRET complex can be used to infer the association or dissociation rates of the unbound bioactive agent. In some embodiments, these association / dissociation rates can be used to assess the agent residence time of individual targets within intact cells. In some embodiments, the systems and methods are useful for thermodynamic molecular mechanism of action (MMOA) studies of entropic versus enthalpic interactions. In some embodiments, the bound bioactive agents may be removed by separate agents through separate sites on the cellular target. In certain embodiments, the binding properties of the conjugated bioactive agent can be used to determine the effect of post-translational modifications (e.g., cleavage, phosphorylation, methylation, acetylation, lipidation, etc.), intracellular translocation (e.g., movement to the nucleus, mitochondria, membranes, etc.), or protein interactions (e.g., interactions with other proteins, nucleic acids, lipids, etc.) on the cellular target. In one example, the binding properties of an antibody to a cellular target are determined by its effect on the binding properties of the conjugated bioactive agent. In certain embodiments, the binding properties of the conjugated bioactive agent may be used to determine the effect of chemical modification or changes (e.g., changes in intracellular metabolism, ionic states, etc.) of the bioactive agent or the conjugated fluorophore.

[0039] In some embodiments, a cellular target may comprise one or more molecular components. For example, a target may comprise two or more polypeptides and may further comprise other natural or synthetic molecules (e.g., prosthetic groups, cofactors, metabolites, nucleic acids, lipids, carbohydrates, etc.). In some embodiments, a bioactive agent conjugated to a fluorophore first binds the molecular component, and a bioluminescent reporter is fused to a second molecular component such that a signal from the conjugated bioactive agent is generated when bound to the first molecular component and the first molecular component is adjacent to the second molecular component.

[0040] A priori knowledge of the existence of an interaction between a bioactive agent and a cellular target is not necessary to practice the present invention. In some embodiments, detection and / or characterization of unknown or previously unidentified interactions by energy transfer is provided. Advantages of the systems, compositions, and methods described herein over other methods of target discovery include a broader range of bioactive agent concentrations that can be used since (in some embodiments) they do not need to be expressed intracellularly (e.g., sufficient acceptor fluorophore can be added to generate a detectable signal), the natural protein concentration of the cellular target (e.g., overexpression is not required to obtain sufficient signal for detection), a signal that is detectable on a plate reader (e.g., high throughput detection, no imaging required), detection of interactions in cells, etc.

[0041] In some embodiments, the bioactive agent (e.g., hit compound) is conjugated to a fluorescent energy acceptor dye, such that binding of the modified agent to its luciferase-fused (e.g., NANOLUC-fused) cellular target transfers energy from NANOLUC to the acceptor dye. Such a system provides a homogenous assay that can be performed in living cells. In some embodiments, the labeled bioactive agent maintains equilibrium with the cellular target throughout the test, allowing detection of targets that interact with the hit compound with low affinity. In some embodiments, BRET allows measurement of binding affinity in living cells by competitive displacement of fluorescent tracers designed for the same binding site.

[0042] In certain embodiments, the technical features of NANOLUC as an energy donor compared to other luciferases provide a significant improvement in BRET efficiency. For example, NANOLUC is much brighter than other luciferases commonly used in BRET, allowing energy transfer to be quantified at low expression levels that are more suitable for maintaining relevant biological activity within cells. In certain embodiments, the narrow emission spectrum of NANOLUC increases the dynamic range by reducing the spectral overlap in the acceptor channels. In certain embodiments, the dynamic range can be further increased using long wavelength acceptors that emit in the near red region of the spectrum (600-650 nm). In certain embodiments, evaluation of multiple diligands during development of embodiments of the present invention revealed that rhodamine analogs (e.g., carboxyrhodamine analogs), such as those described in U.S. Patent Application Serial No. 13 / 682,589, the entirety of which is incorporated herein by reference, provide an optimal dynamic range for use with NANOLUC and / or for BRET applications described herein. EXAMPLES

[0043] Example 1 During the development of an embodiment of the present invention, experiments were performed to demonstrate the improved performance of PBI tracer conjugates and compared them to conjugates with standard dyes for drug tracer applications. In this example, a conjugate of suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylase 6 (HDAC6), and either a PBI dye (SAHA-TOM (PBI-4968), see FIG. 8) or a standard dye (SAHA-TAMRA (PBI-4967), see FIG. 8) was utilized in an intracellular BRET assay using NanoLuc-HDAC fusion protein (SEQ ID NO:3).

[0044] HEK293 cells were transfected with FuGene HD (Promega) carrying plasmid DNA encoding NanoLuc-HDAC6 fusion protein. NanoLuc-HDAC6 DNA was diluted 1:1000 in promoterless carrier DNA (pGEM3Z) to produce a final concentration of 50ng / well total DNA in a 96-well plate format (seeding density of 20,000 cells / well). After 24 hours of transfection, cells were then incubated with serially diluted tracers in the presence or absence of a molar excess of unlabeled SAHA (as a specificity control). After equilibration with the drug tracer, furimazine (coelenterazine derivative substrate for NanoLuc, Promega) was added at a concentration of 20uM and BRET was quantified on a Varioskan luminometer. Specific BRET signals were calculated by subtracting the nonspecific signal (in the presence of unlabeled SAHA) from the signal generated by the SAHA tracer alone.

[0045] The results in Figure 2 show that the SAHA-TOM (PBI-4968) conjugate / tracer produced a superior and specific BRET signal compared to SAHA-TMR (PBI-4967) and also showed a left-shifted EC 50The advantage of the PBI-4968TOM dye over other commonly used fluorophores as drug conjugates for BRET applications is that it can be applied in competitive binding assays or target identification (chemoproteomics) screening using the NANOLUC fusion protein library.

[0046] While this example demonstrates the binding of drug tracers to known high affinity targets, in another embodiment, such tracers are combined with a library of NANOLUC fusion proteins to profile relative drug affinity and specificity among a family of targets. In this embodiment, the profiling approach helps identify drugs with heterogeneous binding profiles that may be associated with undesirable drug side effects in vivo.

[0047] Example 2 During development of embodiments of the present invention, experiments were conducted that demonstrated the unexpected advantage of low expression levels of NanoLuc for BRET applications.

[0048] A) HEK293 cells were transfected with varying amounts of NanoLuc-HDAC6 DNA using Fugene HD and seeded in a 96-well plate format. For transfection, NanoLuc-HDAC6 DNA was diluted in promoterless carrier DNA (pGEM3Z). The final DNA concentration / well was maintained at 50 ng / well, but NanoLuc-HDAC6 DNA was diluted 1:10, 1:100 and 1:1000 (see Fig. 8 for a seeding density of 20,000 cells / well in a 96-well format). After 24 hours of transfection, cells were treated with serially diluted SAHA (PBI-4968, see Fig. 8) conjugates in the presence of a fixed concentration (1 μM) of SAHA-TOM. After 2 hours of incubation, furimazine was added to 20 uM and BRET was detected on a Varioskan luminometer. The results in FIG. 3 demonstrate that an unexpectedly high dilution of DNA encoding the NanoLuc fusion was required to achieve an adequate S / B ratio for BRET applications.

[0049] B) DNA encoding a NanoLuc-HistamineHI (GPCR) fusion protein was transfected into HEK293 cells with varying amounts of DNA ranging from undiluted to 1:10,000 (diluted into promoterless carrier DNA (pGEM3Z as above) to maintain a constant amount of DNA / transfection as above). 24 hours after transfection, cells were treated with serial dilutions of mepyramine-bodipy-633 (CellAura) and equilibrated for 2 hours. Cells were then treated with furimazine at a concentration of 20uM and BRET was detected with a Varioskan luminometer.

[0050] The results in Figure 3B show that extensive dilution of the DNA encoding the NanoLuc fusion is necessary to generate a high affinity interaction with optimal signal to background. The ability to dilute the NanoLuc fusion protein to very low levels is beneficial in a variety of BRET applications, including competitive binding assays or target identification (chemoproteomics) screening of NanoLuc fusion proteins.

[0051] Example 3 During the development of embodiments of the present invention, experiments were performed to demonstrate the predictive value of intracellular BRET measurements to quantitate prodrug binding compared to traditional biochemical (activity-based) formats. An example of this involves the natural prodrug, FK228, which requires the reducing environment of the cytoplasm in order to become active and able to bind to HDAC6.

[0052] HEK293 cells were transfected with DNA encoding NanoLuc-HDAC6 at a 1:1000 dilution with carrier DNA as described above. After 24 hours of transfection, cells were incubated with serially diluted natural prodrug (FK-228) in the presence of a fixed concentration (1 μM) of SAHA-TOM (PBI-4968) conjugate. For comparison, a biochemical, activity-based HDAC6 assay was performed in parallel. Briefly, three-fold dilutions of FK-228 (Selleckchem Cat. No. S3020) were performed at 100X in 100% DMSO in wells of a 96-well plate. Five μL aliquots of this 100X / 100% DMSO titration series were added to 245 μL of HDAC-Glo™ I / II Assay Buffer (Promega) alone or 245 μL of HDAC-Glo™ I / II Assay Buffer supplemented with 2X (0.5 mM) DTT to create a 2X / 2% DMSO master intermediate titration series of FK-228 in wells of a 96-well plate. From this master intermediate titration series, five replicates were transferred from the wells of a white 384-well assay plate (Corning 3673). A 5 μL addition of 2X (2 nM) HDAC 6 (BPS Bioscience catalog number 50006) was added to all wells for a final concentration of InM HDAC6 / well. Ten μL of the enzyme / inhibitor mix was allowed to preincubate for 45 minutes at room temperature. Final Detection Reagent (Promega) was added to all wells (20 μL final assay volume) in a volume equal to HDAC-Glo™ I / II (10 μL) and luminescence was measured after 10 min incubation at room temperature. The final concentration of HDAC-Glo™ I / II substrate for all test conditions was 50 μM. Data were plotted (sigmoidal dose response - varying slope) using Prism™ software from GraphPad (no DTT: filled circles; 0.25 mM DTT: open squares).

[0053] The results in Figures 4A and 4B show the requirements of the cellular environment to measure binding of a prodrug that must be processed by the cell to become active.

[0054] Example 4 During development of embodiments of the present invention, experiments were performed to demonstrate the use of permeabilization agents to introduce impermeable drug tracers into cells.

[0055] HEK293 cells were transfected with DNA encoding PKC alpha (PKCa)-NanoLuc or NanoLuc-PKC alpha at a 1:1000 dilution with promoterless carrier DNA as described above. 24 hours after transfection, cells were treated with or without digitonin to a final concentration of 50ug / mL. Cells were then treated with serial dilutions of staurosporine-PBI-dye conjugate (PBI-5129, see Figure 8). Cells were co-cultured in the presence or absence of unconjugated staurosporine at 5μM as a specificity control for binding. After 2 hours of equilibration with the tracer, furimazine was added to a final concentration of 20uM and the BRET ratio was measured with a Varioskan luminometer (Figures 5A and 5B).

[0056] In a second experiment, identically transfected PKCa-NanoLuc cells were treated with serial dilutions of digitonin prior to treatment with a fixed concentration of staurosporine-TOM (PBI-5129, see Figure 8) tracer (5uM final concentration) in the presence or absence of 5uM unlabeled staurosporine (specificity control) (Figure 5C).

[0057] 5A-C demonstrate the ability to use permeabilization agents to enhance entry of impermeable drug tracers and their application to BRET-based chemical proteomic screening of a NanoLuc™ fusion protein library.

[0058] Example 5 During development of embodiments of the present invention, experiments were performed to demonstrate the use of NanoLuc / BRET to measure relative drug affinity in living cells. In an embodiment, similar experiments are designed to optimize leads from high-throughput chemical screens.

[0059] HEK293 cells were transfected with DNA encoding the NanoLuc-p38 fusion protein (final concentration of 50 ng DNA / well in 96-well format). 24 hours after transfection, cells were treated with serial dilutions of BIRB-796 or PBI-4835 in the presence of 0.5 uM PBI-4838 (BIRB conjugate derivatives, see, e.g., US 13 / 682,589, which is incorporated herein by reference in its entirety). JPEG0007676487000001.jpg6277

[0060] After 2 hours of equilibration, furimazine was added to a concentration of 20 uM and BRET was measured with a Varioskan luminometer.

[0061] Figure 6 demonstrates that the dose-response BRET curves highly confirmed the known affinity of BIRB-796 relative to PBI-4835 ("BIRB derivative"). The ability to measure the relative affinity of unlabeled drugs can be applied to HTS screening, lead optimization, or chemical proteomics applications. In one embodiment, similar experiments are configured to characterize optimized hit compounds from high-throughput chemical screening (e.g., compounds with lower IC50 values ​​for tracer displacement exhibit higher binding efficiency to the target of interest).

[0062] Example 6 During development of embodiments of the present invention, experiments were performed to demonstrate the use of NanoLuc / BRET to monitor the kinetics of drug binding in living cells. In one embodiment, similar experiments are configured to optimize leads from high throughput chemical screens.

[0063] HEK293 cells were transfected with DNA encoding a NanoLuc-p38 fusion (at a final concentration of 50 ng DNA / well in a 96-well format). After 24 hours of transfection, cells were pretreated for 2 hours with protected furimazine to a final concentration of 20 uM (PBI-4378, see FIG. 8). BRET was measured over time in a Varioskan luminometer set at 37° C. After a short pre-readout, cells were stimulated with varying concentrations of PBI-4838 (in the presence or absence of 1 μM BIRB796 as a specificity control). The dose- and time-dependent increase in BRET was then kinetically monitored for 4 hours.

[0064] In a separate experiment, transfected cells were pretreated with 20 μM PBI-4377 and 1 μM PBI-4838 to generate a stable BRET signal. TIFF0007676487000002.tif72150After a short pre-readout, cells were stimulated with 1 μM unlabeled BIRB-796 and competitive displacement was monitored in real time by BRET.

[0065] The results in Figures 7A-B support the use of BRET to measure kinetics of target engagement in living cells. This format can be utilized for HTS or chemical proteomic screening of NanoLuc fusion proteins in living cells. In one embodiment, similar experiments are configured to characterize lead compounds optimized from high-throughput chemical screening (e.g., compounds that alter the kinetics of association or dissociation with a target of interest). In addition to providing a measure of relative compound affinity, this kinetic BRET assay could provide a quantitative assessment of target residence time in biochemical or intact cells.

[0066] Example 7 The following examples relate to the use of specific NANOLUC substrates (see FIG. 8) or enzyme components to identify the subcellular localization of drug binding events using BRET.

[0067] Physical separation of the complementary NANOLUC enzyme polypeptide or peptide allows for the separation of the NANOLUC (donor) signal into the extracellular space. For example, a small signal peptide is genetically linked to a cell surface receptor. Upon exogenous addition of a large signal polypeptide to the cell culture medium, the donor signal is separated into the extracellular space. This signal separation enhances the signal / background generated in various BRET applications, including drug tracer binding / displacement.

[0068] In one embodiment, where an impermeable NANOLUC substrate is applied, a full-length NANOLUC protein fusion is utilized.

[0069] Example 8 The following provides synthetic schemes for exemplary compounds that find use in embodiments of the present invention.

[0070] Boc-protected SAHA amine 7-Trityloxycarbamoylheptanoic acid (200 mg, 463 μmol) was mixed with 4-[(N-Boc)aminomethyl]aniline (113 mg, 510 μmol), HBTU (352 mg, 927 μmol) and triethylamine (194 uL, 1.4 mmol) in 3 ml DMF. The reaction was stirred overnight and then adsorbed onto Celite and the product was obtained by column chromatography eluting with a gradient of 0->100% ethyl acetate in heptane. M+H calculated: 635.3; found: 635.9

[0071] SAHA amine Suberoyl(4-[(N-Boc)aminomethyl]anilide)hydroxamic acid (286 mg, 450 mmol) was dissolved in 2 ml DCM with 0.25 ml TIS. Trifluoroacetic acid (0.9 ml) was then added and the reaction was stirred for 30 min. The solvent was removed under reduced pressure and the crude reaction product was purified by preparative HPLC or used without further purification.

[0072] PBI-4967 SAHA-TAMRA The crude reaction product of suberoyl[4-(aminomethyl)anilide]hydroxamic acid (27 mg) was mixed with 4 mg (7.6 μmol) of tetramethylrhodamine 6-succinimidyl ester in 1 mL of DMF with 5 drops of TEA. After 30 min, the reaction was diluted with HO and MeCN and the products were separated by preparative HPLC (5->60% MeCN in 0.1% aqueous TFA).

[0073] The appropriate fractions were lyophilized to yield the desired product as a magenta solid. M+H calculated: 706.3; found: 706.6.

[0074] PBI-4968 SAHA-TOM TIFF0007676487000004.tif7782Suberoyl[4-(aminomethyl)anilide]hydroxamic acid crude reaction product (8 mg) was mixed with 5 mg (7.6 μmol) of TOM 6-succinimidyl ester in 0.8 mL of DMF with 3 drops of TEA. After 30 min, the reaction was diluted with HO and MeCN and the product was isolated by preparatory HPLC (5->60% MeCN in 0.1% aqueous TFA). The appropriate fractions were lyophilized to dryness to yield the desired product. M+H calculated: 838.4; found: 838.7

[0075] Staurosporine-amine Staurosporine p-nitrophenylcarbamate (3 mg, 4.8 μmol) was dissolved in 0.5 mL DMF and treated with excess cadaverine. The reaction was warmed in a 70° C. oil bath for 2 h, then diluted with HO, acidified with formic acid, and subjected to preparative high-performance liquid chromatography in 10 mM aqueous NHOAc, eluting with 25% to >75% MeCN. The appropriate fractions were lyophilized to give a slightly yellow solid. M+H calculated: 595.3; Found: 595.5

[0076] PBI-5129 Staurosporine-TOM TIFF0007676487000005.tif4689 Staurosporine 5-aminopentylcarboxamide (5 mg, 8.4 μmol) was dissolved in 1 ml DMF and treated with triethylamine and TOM 6-SE (4 mg, 6 μmol). The reaction was monitored by analytical HPLC. After completion of the reaction, MeCN and H2O were added and the TEA was neutralized by addition of a small amount of AcOH. Preparative HPLC (25% to >100% MeOH in 10 mM NHOAc) and subsequent concentration afforded 1.8 mg of a deep blue solid. M+H calculated: 1139.5; found: 1139.8.

[0077] Dasatinib-TOM TOM Pentylamine TIFF0007676487000006.tif6058TOM 6-carboxylic acid (26 mg, 46 μmol) was stirred in 1 ml DMF containing 2 equivalents of triethylamine and treated with TSTU (17 mg, 56.5 μmol, 1.2 equivalents) and the reaction was monitored by HPLC. After 40 min, the reaction was added to a solution of cadaverine (94 mg, 0.92 mmol, 20 equivalents) in 0.5 ml DMF and stirred for 20 min. The reaction was then neutralized by the addition of TFA and diluted with MeCN and water. Preparative HPLC (25->100% MeCN in 0.1% aqueous TFA) followed by lyophilization afforded the desired product as a purple solid. LCMS: ((M+2H) / 2) calculated: 324.4; found: 324.3.

[0078] Dasatinib-TOM TIFF0007676487000007.tif53152 Dasatinib (25 mg, 51 μmol) was mixed with p-nitrophenyl chloroformate (14 mg, 69 μmol, 1.36 equiv.) and 30 uL TEA in 0.9 ml of 2:1 DMF:THF. The reaction was stirred overnight and then 35 mg of additional p-nitrophenyl chloroformate was added. After stirring for an additional 24 h, 1 / 2 the volume of the reaction was added to a solution of 7 mg (10 μmol) TOM pentylamine in 0.5 ml DMF and 30 uL TEA. The reaction was then stirred for 1 h before dilution with MeCN and water and purification by preparative HPLC. Lyophilization provided the desired product as a purple solid. MS: ((M+2H) / 2) calculated: 580.8; found: 581.0.

[0079] Purvalanol-TOM PBI 5077 TIFF0007676487000008.tif10187Purvalanol (10 mg, 23 μmol) was stirred in 1 mL DMF containing 20 mg TEA (198 μmol) and treated with 7.7 mg (25.4 μmol, 1.1 equiv.) TSTU. After 30 min, the reaction was diluted with Et2O and ½ of the total reaction volume was added to a vial containing 6 mg (9 μmol) TOM(5-aminopentyl)-6-carboxamide. The reaction was stirred for 3 h and labile organics were removed under reduced pressure. The resulting DMF solution was diluted with water and MeCN, acidified with TFA, and the product was isolated by preparative HPLC (25->100% MeCN in 0.1% aqueous TFA) followed by lyophilization to yield 3.5 mg of a purple solid. MS: M+H calculated: 1061.5, found: 1061.6.

[0080] Example 9 Use of Renilla luciferase or NanoLuc luciferase in the compound identification method of the present invention HEK293 cells were transfected with plasmid DNA encoding NanoLuc-p38alpha or RenillaLuc-p38alpha using Fugene HD (Promega) with a lipid:DNA ratio of 3:1 and seeded in 96-well plates at a density of 20,000 cells per well (generating 50 ng of DNA / well). After 24 h of transfection, the cell medium was replaced with serum-free medium (Opti-MEM) and cultured with serially diluted PBI-4838 (a BIRB796 derivative conjugated to a TOM dye) in the presence / absence of 1 μM BIRB796. Cells were cultured for 2 h at 37°C. Furimazine was added to the NanoLuc-expressing samples at a final concentration of 20 μM. Native coelenterazine was added to the RenillaLuc-expressing samples at a final concentration of 20 μM. BRET was then measured with a Varioskan luminometer equipped with a 450 nm bandpass filter and a 630 nm longpass filter. The BRET ratio was determined by dividing the signal in the 630 channel by the signal in the 450 channel.

[0081] Figure 9 demonstrates that both Renilla luciferase and NanoLuc luciferase can be used in the methods of the invention. Upon treatment of cells expressing NanoLuc-p38alpha or RenillaLuc-p38alpha with serial dilutions of PBI-4838, a dose-dependent increase in BRET is observed. Saturating concentrations of unlabeled BIRB-796 can block this BRET signal, demonstrating the specificity of binding with either luciferase fusion to p38alpha.

[0082] Example 10 Comparison of TOM-BIRB vs. TMR-BIRB for NanoLuc-p38alpha HEK293 cells were transfected with plasmid DNA encoding NanoLuc-p38alpha using Fugene HD as described above and seeded into 96-well plates (generating 50ng of DNA / well). After 24 hours of transfection, the cell medium was replaced with serum-free medium (Opti-MEM) and incubated with serial dilutions of PBI-4838 (BIRB796 derivative conjugated to TOM dye) or PBI-4836 (BIRB derivative conjugated to TMR dye) in the presence / absence of 1 μM BIRB796. Cells were incubated for 2 hours at 37°C. Furimazine was added to NanoLuc-expressing samples at a final concentration of 20 μM. BRET was then measured with a Varioskan luminometer equipped with a bandpass filter of 450 nm and a longpass filter of 630 nm. The BRET ratio was determined by dividing the signal in the 630 channel by the signal in the 450 channel.

[0083] Figure 11 demonstrates another example of the improved ability of TOM adducts over TMR adducts for binding to target proteins in a cell-based format. The dose response curve for NanoLuc-p38alpha binding shows an EC50 of 438nM for the BIRB-TOM conjugate compared to an EC50 of 450nM for the BIRB-TMR conjugate. The affinity of BIRB-TOM in this report is in good agreement with literature reports compared to BIRB-TMR (Chem Biol Drug Des 2009; 74:547-559).

[0084] Example 11 Comparison of BIM-TOM vs. BIM-TMR against PKC alpha-NanoLuc HEK293 cells were transfected with plasmid DNA encoding PKCalpha-NanoLuc (diluted 1:1000 in pGEM3Z carrier DNA) using Fugene HD as described above and seeded into 96-well plates (yielding 50ng / well of total DNA). After 24 hours of transfection, the cell medium was replaced with serum-free medium (Opti-MEM) and incubated with serial dilutions of PBI-5075 (BIM conjugated to TOM dye) or PBI-5051 (BIM conjugated to TMR dye) in the presence / absence of 5uM staurosporine. Cells were incubated for 2 hours at 37°C. Furimazine was added to NanoLuc-expressing samples at a final concentration of 20μM. BRET was then measured in a Varioskan luminometer equipped with a 450nm bandpass filter and a 630nm longpass filter. The BRET ratio was determined by dividing the signal in the 630 channel by the signal in the 450 channel. To determine the specific BRET signal, the BRET ratio at each concentration of tracer plus unlabeled staurosporine was subtracted from the BRET ratio at each concentration of tracer without unlabeled staurosporine.

[0085] Figure 12 demonstrates another example of the enhanced potency of TOM adducts relative to TMR adducts for binding to target proteins in a permeabilized cell format. The dose response curve for PKC alpha-NanoLuc binding shows an EC50 of 483 nM for the BIM-TOM conjugate compared to the extremely right-shifted potency of the BIM-TMR conjugate. The affinity of BIM-TOM in this report is in good agreement with literature reports compared to BIM-TMR (J Biol Chem. 1991 Aug 25; 266(24):15771-81.).

[0086] Example 12 Target identification in p38 / MAPK alternative routing The following example serves to demonstrate the ability to profile the selectivity of a drug tracer against a panel of putative targets within a given phylogenetic target family. A similar experimental setup may be used to identify targets of fluorescently labeled drugs using BRET in cells. This setup can ultimately lead to the identification of primary drugs as well as off-target interactors. The involvement of multiple targets may indicate drug promiscuity and potential drug side effects. This example serves to demonstrate the ability to measure BRET-mediated interactions of both primary and secondary targets.

[0087] HEK293 cells were transfected with plasmid DNA encoding N- or C-terminal NanoLuc fusions to various members of the MAPK pathway (Jnkl, Jnk2, Jnk3, p38 alpha, p38 beta, p38 gamma, p38 delta, or PKC alpha or MAPK 8, 9, 10, 14, 11, 12, or 13, PKC alpha, respectively) using Fugene HD (Promega Corporation) at a lipid:DNA ratio of 3:1, and seeded into 96-well plates at a density of 20,000 cells per well (yielding 50 ng of DNA / well). After 24 hours of transfection, the cell medium was replaced with serum-free medium (Opti-MEM) and cultured in the presence or absence of 4 μM BIRB796 and 2 μM PBI-4838. Cells were cultured at 37° C. for 2 hours. Furimazine was added to the NanoLuc-expressing samples at a final concentration of 20 μM. BRET was then measured on a Varioskan luminometer equipped with a 450 nm bandpass filter and a 630 nm longpass filter. The BRET ratio was determined by dividing the signal in the 630 channel by the signal in the 450 channel. To determine the response ratio, the BRET value from the tracer alone was divided by the BRET value from the tracer plus unmodified BIRB796.

[0088] Figure 13 shows the selectivity of PBI-4838 for Jnk2, p38 beta and p38 alpha (consistent with literature reports). p38 alpha is recognized as the primary target of BIRB796. However, interactions with targets such as Jnk2 and p38 beta could indicate potential off-target burden. As expected, PKC alpha-NanoLuc fusion bound to PBI-4838 showed a relatively small specific BRET signal due to the low affinity of BIRB796 towards this target.

[0089] Example 13 Target identification in p38 / MAPK pathway determination of affinity The following examples serve to demonstrate the ability to profile the affinity of a drug tracer to a panel of putative targets within a given phylogenetic target family. A similar experimental setup may be used to characterize the affinity of a fluorescently labeled drug to a given target using BRET in cells.

[0090] HEK293 cells were transfected with plasmid DNA encoding NanoLuc-p38alpha, NanoLuc-p38beta, Jnk2-NanoLuc, PKCalpha-NanoLuc or NanoLuc-HDAC6 at a lipid:DNA ratio of 3:1 using Fugene HD (Promega Corporation) and seeded into 96-well plates at a density of 20,000 cells per well (yielding 50ng DNA / well). After 24 hours of transfection, the cell medium was replaced with serum-free medium (Opti-MEM) and cultured with serially diluted PBI-4838 in the presence or absence of 4uM BIRB796. Cells were cultured for 2 hours at 37°C. For a separate set of samples, the transfected cells were treated with serially diluted BIRB796 in the presence of 1uM PBI-4838. Furimazine was added to the NanoLuc-expressing samples at a final concentration of 20 μM. BRET was then measured on a Varioskan luminometer equipped with a 450 nm bandpass filter and a 630 nm longpass filter. The BRET ratio was determined by dividing the signal in the 630 channel by the signal in the 450 channel.

[0091] Figures 14 and 15 demonstrate the apparent affinity of PBI-4838 for Jnk2, p38 beta and p38 alpha. As expected, PKC alpha and HDAC6 showed relatively small specific BRET signals due to the low affinity of PBI-4838 for these targets. Furthermore, the high affinity of BIRB796 for Jnk2, p38 alpha and p38 beta is demonstrated by competitive displacement in cells. Similar experiments could be configured to rank the affinity of a drug tracer (by increasing in BRET) or an unmodified drug (by competitive displacement) for a given set of targets. As mentioned above, high affinity interactions for targets such as Jnk2 and p38 beta could indicate potential off-target burden.

[0092] Example 14 Affinity determination of drug tracers for wild type versus mutant versions of target proteins The following example serves to demonstrate the ability to measure the relative affinity of a drug tracer for wild-type versus mutant target proteins. In this example, the affinity of a fluorescently labeled iBET compound (a small molecule inhibitor of BRD4 binding prevents interaction with acetylated histones) for wild-type and mutant (N140A / N433A; lacking full binding ability to iBET) BRD4 (bromodomain-containing protein 4) was determined. A similar experimental setup may be used to characterize the affinity and selectivity of a fluorescently labeled drug for disease-related mutant proteins in cells. Such experiments may be useful to identify drugs that selectively bind proteins specifically to wild-type or mutant proteins.

[0093] HEK293 cells were transfected with plasmid DNA encoding NanoLuc-BRD4 or NanoLuc-BRD4(N140A / N433A) at a lipid:DNA ratio of 3:1 using Fugene HD (Promega Corporation) and seeded in 96-well plates at a density of 20,000 cells per well (yielding 50 ng of DNA / well). After 24 h of transfection, the cell medium was replaced with serum-free medium (Opti-MEM) and incubated with serially diluted PBI-4966 (iBET conjugated to TOM dye) in the presence / absence of 10 μM iBET. Cells were incubated for 2 h at 37°C. Furimazine was added to the NanoLuc-expressing samples at a final concentration of 20 μM. BRET was then measured with a Varioskan luminometer equipped with a bandpass filter of 450 nm and a longpass filter of 630 nm. The BRET ratio was determined by dividing the signal in the 630 channel by the signal in the 450 channel.

[0094] FIG. 16 shows the relative affinity of PBI-4966 for wild type versus mutant BRD4. As expected, a right-shifted potency was observed for mutant BRD4, indicating reduced affinity for PBI-4966. Similar experiments could be constructed to rank order the affinity of drug tracers to disease-relevant mutants of a target. This principle could be extended to measure the relative affinity of drugs to wild type or mutant proteins by competitive tracer displacement. Since an increase in BRET signal represents only binding to the NanoLuc fusion, this method allows for assays that measure the engagement of a target in a complex mixture of similar antigens present in the assay sample. These antigens could include targets with similar properties, but not identical to the target of interest (e.g., distinguishing between heterologous versus endogenous analytes or mutant versus weight analytes, etc.).

[0095] Example 15 Screening a panel of compounds to determine target engagement The following examples demonstrate that the methods of the invention can be used to determine target engagement by screening panels of compounds. This screening method could be extended to larger compound libraries (e.g., LOPAC) as well.

[0096] HEK293 cells were transfected with NanoLuc-HDAC6 DNA using Fugene HD and seeded into wells of a 96-well plate. For transfection, NanoLuc-HDAC6 DNA was diluted in promoterless carrier DNA (pGEM3Z). The final DNA concentration / well remained at 50 ng / well, but NanoLuc-HDAC6 DNA was diluted 1:1000 (at a seeding density of 20,000 cells / well in a 96-well format). After 24 hours of transfection, cells were treated with serially diluted inhibitors (see Figure 17) in the presence of a fixed concentration (1 μM) of SAHA-TOM conjugate (PBI-4968). After 2 hours of incubation, furimazine was added to 20 uM and BRET was detected on a Varioskan luminometer.

[0097] The results in Figure 17 demonstrate that structurally distinct compounds can be discriminated by competitive displacement of drug tracers using the method of the present invention. Furthermore, the relative potency of various compound classes can be ascertained. The results show that apicidin binds to HDAC6 with negligible / no efficiency compared to the high binding efficiency of panobinostat or SAHA to HDAC6. This is consistent with literature reports using orthogonal assay formats. This screening method could be expanded to include high-throughput screening of large chemical libraries to identify / characterize new chemicals with inhibitor potential against a given target.

[0098] Example 16 Drug-target interaction monitoring It is commonly accepted that in the presence of fluorophores with large spectral overlap, high levels of non-specific BRET occur in media containing luciferase (Couturier, 2012). This has been described as a burden to the use of BRET for high-throughput chemical screening. The following example serves to demonstrate the ability to monitor drug / target interactions using fluorophore-drug conjugates in the presence of luciferase, NanoLuc, in media at concentrations generally recognized as problematic for BRET.

[0099] HEK293 cells were transfected with plasmid DNA encoding CDK2-NanoLuc (diluted 1:100 into pGEM3Z carrier DNA) at a lipid:DNA ratio of 3:1 using Fugene HD (Promega Corporation) and seeded into wells of a 96-well plate at a density of 20,000 cells per well (yielding 50 ng / well of total DNA). After 24 h of transfection, the cell medium was replaced with serum-free medium (Opti-MEM) and cultured with serially diluted PBI-5077 (purvalanol B conjugated to TOM dye) in the presence / absence of 10 μM purvalanol B. Cells were incubated at 37°C for 2 h.

[0100] Furimazine was added to the NanoLuc-expressing samples at a final concentration of 20 μM. BRET was then measured on a Varioskan luminometer equipped with a 450 nm bandpass filter and a 630 nm longpass filter. The BRET ratio was determined by dividing the signal in the 630 channel by the signal in the 450 channel (FIG. 18).

[0101] This example supports the use of fluorophore tracers in culture media to monitor drug / target interactions via BRET. By co-incubating samples (+ drug tracer) with Purvalanol B (unmodified drug), the non-specific BRET signal between NanoLuc and the tracer can be measured. Over the range of trace concentrations tested, this background BRET signal was low. These results demonstrate that, despite the large spectral overlap between NanoLuc (donor) and fluorophore (acceptor), it is possible to monitor specific binding between drug tracers and nanoLuc-target fusions in cell culture media.

[0102] Example 17 Target ID screening using protein accumulation The following examples demonstrate the ability to generate NanoLuc fusion sequences using cell-free expression and measure drug binding and relative affinity by BRET.

[0103] A panel of 11 kinases (N- and C-terminal NanoLuc fusions) including putative targets of BIRB796 (e.g., members of the MAPK and Jnk families), putative targets of dasatinib (e.g., Src and LCK), and an irrelevant HDAC6 negative control were expressed in the TnT™ T7 Rapid-Coupling Reticulocyte Transcription / Translation System (Promega Corporation) as recommended by the manufacturer to generate arrays with 23 different NanoLuc fusions. Each TNT™ reaction was diluted 1:100 in PBS and replicates were placed into wells of a 96-well plate. NanoLuc fusion arrays were screened with two different TOM drug tracers (BIRB-TOM; PBI-4838 and dasatinib-TOM; PBI-5170) in the presence and absence of excess unrelated drug. For each TNT™ reaction, four replicates with 2 μM BIRBTOM / four replicates with 2 μM BIRB-TOM+4 μM BIRB796 (total of 2×96-well plates) and four replicates with 1 μM Dasatinib-TOM / four replicates with 1 μM Dasatinib-TOM+5 μM Dasatinib (total of 2×96-well plates) were performed. Reactions were incubated at room temperature for 2 hours with constant mixing. After incubation, furimazine was added to a final concentration of 20 μM. BRET was then measured on a Varioskan luminometer equipped with a 450 nm bandpass filter and a 610 nm longpass filter. The BRET ratio was determined by dividing the signal in the 610 channel by the signal in the 450 channel. To determine the response ratio, the BRET value with tracer alone was divided by the BRET value with tracer+unmodified.

[0104] The results (Figure 19) demonstrated the selectivity of the BIRB-TOM tracer for JNk2, JNk3, MAPK14, MAPK11 and MAPK13, and the selectivity of the dasatinib-TOM tracer for ABL1, Src, LCK, MEK5, MAPK11 and MAPK14, which were consistent with literature reports. These results demonstrate the ability to profile drug tracers against a panel of putative targets expressed in a cell-free expression system and to identify hits. This construction of the no-wash protein NanoLuc fusion sequence can lead to the identification of the primary drug target, as well as off-target interactors.

[0105] After the above screening, the BIRB-TOM hits were further analyzed for binding affinity to the BIRB-TOM tracer and competitive displacement of the BIRB-TOM tracer with BIRB796. Binding affinity experiments to the BIRB-TOM tracer were performed by incubating triplicates of the diluted TNT reactions with serially diluted BIRB-TOM tracer in the presence / absence of 10 μM BIRB796. Reactions were incubated at room temperature with constant mixing for 2 hours, and furimazine was added to a final concentration of 20 μM before analysis as above. Competitive displacement experiments were performed by incubating triplicates of the diluted TNT reactions with serially diluted BIRB796 in the presence of 2 μM BIRB-TOM tracer. Reactions were incubated at room temperature with constant mixing for 2 hours, and furimazine was added to a final concentration of 20 μM before analysis as above. The results of the competitive displacement experiment in Figure 19 show that BIRB796 exhibited the highest affinity for MAPK14 and Jnk2, while it had low affinity for MAPK11, MAPK13, and Jnk3. Consistent with literature reports, BIRB796 efficiently binds MAPK14, but also associates with other MAPK and Jnk family members, indicating off-target burden for BIRB-796. These results demonstrate that hits expressed in cell-free expression systems and identified in protein sequence screening can be further analyzed by BRET for drug binding affinity. Furthermore, these results support the use of cell-free expression systems to generate NanoLuc protein fusions as suitable analytes for target-related applications.

[0106] Example 18 Expression levels of NanoLuc fusions The following example demonstrates that when the expression of NanoLuc fusion is close to endogenous level, it achieves high affinity interaction with drug tracer at optimal S / B ratio. As mentioned above, it was demonstrated that dilution of NanoLucHDAC6 DNA with carrier DNA at 1:1000 is necessary to achieve high affinity interaction. In this example, the expression of serial dilutions of NanoLuc HDAC6 DNA was compared with the expression of endogenous HDAC6 using Western blot analysis.

[0107] HEK293 cells, 2.5x10 5Cells / well were seeded into wells of a 6-well plate and transfected with PEI with varying amounts of NanoLuc HDAC6 DNA. NanoLuc HDAC6 DNA was diluted in non-expression carrier DNA (pCI neo). The final concentration of DNA remained at 2 μg / well, while NanoLuc HDAC6 DNA was diluted 1:0, 1:10, 1:100, 1:1000 and 1:10000. Controls were untransduced cells. After 24 hours of transfection, media was removed, cells were washed with PBS and lysed for 10 minutes with 400 μl mammalian lysis buffer (Promega) + 1:50 RQ1 DNase (Promega) + 1xRQ1 buffer. Following lysis, 133 μl of 4X SDS-loading buffer was added to each well and cell lysates were collected, analyzed on SDS-PAGE gels and electrotransferred to PVDF membranes (Invitrogen). The membrane was blocked with 5% BSA (Promega) in TBS buffer for 1 h and probed overnight at 4° C. with a 1:500 dilution of HDAC6 antibody (Millipore) in TBS supplemented with 0.1% Tween (TBST). After three washes in TBST, the membrane was incubated with anti-rabbit HRP antibody (Jackson) in TBST for 1 h, followed by five washes with TBST and one with TBS. The membrane was then incubated with ECL substrate from Promega for 1 min and scanned on an ImageQuant LAS4000 (GE). The results (FIG. 10) show that a 1:1000 dilution of NanoLuc HDAC6 DNA resulted in expression levels comparable to those of endogenous HDAC6. This is the same dilution that provided high affinity interaction with the drug tracer at the optimal S / B ratio.

[0108] Example 19 Quantification of fluorescent cytokine binding to cell surface receptor NanoLuc fusions During the development of an embodiment of the present invention, experiments were performed to demonstrate the ability to measure the relative affinity of therapeutic antibodies to cell surface receptors by competitive displacement of fluorescently labeled cytokines. In this example, commercially available TAMRA-epidermal growth factor (TMR-EGF; Life Technologies) was applied in a cellular BRET assay using a stable cell line expressing a NanoLuc-EGFR(Herl) fusion protein. Upon treatment with a therapeutic antibody known to interfere with EGF binding, the generation of a BRET signal between TMR-EGF and NanoLuc-EGFR can be prevented.

[0109] HEK293 stable cell lines were generated by transfection with pF5 NanoLuc-EGFR plasmid DNA (Promega) and FuGene HD (Promega). After transfection, stable cell lines were generated by G418 selection followed by limited dilution cloning. Clone-derived cells expressing NanoLuc-EGFR were seeded into 96-well plates at a density of 20,000 cells per well. 20 hours after seeding, cells were serum-starved by replacing the medium with serum-free OptiMEM. After starvation, cells were treated with serially diluted TMR-EGF (Life Technologies). Following equilibration with TMR-EGF, furimazine (coelenterazine derivative substrate for NanoLuc; Promega) was added at a concentration of 20 μM and BRET was quantified with a Varioskan luminometer. In subsequent experiments, antibody-mediated displacement of TMR-EGF was measured by pretreating cells with Vectibix, Erbitux or Herceptin (negative control) prior to stimulation with TMR-EGF (10 ng / ml final concentration).

[0110] The results in FIG. 20 demonstrate that BRET can be used to quantitate the binding of fluorescent cytokines to cell surface receptors bound to NanoLuc. Furthermore, the affinity of therapeutic antibodies capable of inhibiting cytokine binding can be measured by competitive displacement of fluorescent cytokines. Vectibix and Erbitux were able to inhibit TMR-EGF binding to NanoLuc-EGFR, while Herceptin (a Her2 binder with negligible affinity to Herl) was unable to inhibit TMR-EGF binding. This example demonstrates that BRET can quantitate the binding of fluorescent cytokines to known high affinity targets in a format that can be rapidly reconstituted to monitor the binding of therapeutic antibodies (e.g., Vectibix, Erbitux, etc.) or other binders (e.g., cytokine biosystems) by competitive displacement of fluorescent cytokines. This demonstrates the ability to use BRET as a technique to measure the relative potency of biologics in cell formats.

[0111] Example 20 BRET use In one embodiment, the present invention allows for the simultaneous detection / measurement of three moieties: (1) a peptide of a bioluminescent reporter protein, (2) a polypeptide of a bioluminescent reporter protein that interacts through a structural complement to form a complex, and (3) a fluorescent third moiety (e.g., a fluorescent small molecule).

[0112] A) This example demonstrates energy transfer from a bioluminescent complex formed from a structurally complementary peptide (SEQ ID NO:6) and a polypeptide (SEQ ID NO:7) to form a bioluminescent complex. A fluorescent dye was attached to the complementary peptide sequence. Alternatively, a fluorescent protein could be fused (e.g., a fusion protein) to the peptide or polypeptide (e.g., formed from a genetic construct).

[0113] E. coli lysates expressing complementary polypeptide sequences were prepared (polylysates). 40 μL of polylysate was mixed with 10 μL complementary peptide (pep) or fluorescently labeled complementary peptide (pep-TMR) and incubated for 10 min at room temperature. 50 μL of 100 μM substrate of the complex (furimazine) in 50 mM HEPES pH 7.4 was added and incubated for 30 min at room temperature. Luminescence was measured over the range of 400-700 nm on a TECAN M1000.

[0114] FIG. 21 illustrates the highly efficient energy transfer from the Poly / Pep complex (donor) to TMR (acceptor) and the corresponding red shift in the wavelength of the emitted light.

[0115] B) This example demonstrates the use of BRET for detection, such as detecting small molecule concentration or enzyme activity. Because energy transfer is highly distance dependent, the degree of energy transfer can often be related to the geometry of the system. For example, insertion of a calcium-chelating polypeptide can be used to measure calcium concentration through modulation of energy transfer.

[0116] Enzymes that also change the distance, either through conformational changes in the sensor as described above, or through cleavage of the sensor from the fluorescent moiety, can be measured by the system described herein. One structurally complementary member of the complex is bound to a structurally complementary peptide of a bioluminescent reporter protein and a fluorescent moiety to which energy is transferred when the polypeptides interact. One example of this is a peptide sensor made in which the peptide is conjugated to a fluorescent dye via a linker (e.g., DEVD caspase-3 cleavage site). When exposed to the complementary polypeptide, energy transfer is observed. When exposed to caspase-3, the fluorescent moiety is released from the complex, eliminating energy transfer, but maintaining emission at 460 nm.

[0117] The complementary polypeptide (SEQ ID NO:8) and NL-HT (NanoLuc fused to HaloTag) were purified. 20 μL of 8 pM NL-HT were mixed with 20 μL of 100 nM PBI-378 (see U.S. Patent Application Serial No. 13 / 682,589, incorporated herein by reference in its entirety) and incubated for 10 minutes at room temperature. 40 μL of NanoGlo + 100 μM furimazine were added and luminescence was measured over the range 300-800 nm on a TECAN M1000.

[0118] 20 μL of 33 ng / uL complementary polypeptide (SEQ ID NO:6) was mixed with 20 μL of approximately 500 μM PBI-5074 (fluorescent dye-linker-complementary peptide). 40 μL of NanoGlo + 100 μM furimazine was added and emission was measured over the range 300-800 nm on a TECAN M1000.

[0119] FIG. 22 illustrates the energy transfer from a complementary peptide / polypeptide complex (donor) to the TOM dye of PBI-5074 (acceptor) and the corresponding red shift in the wavelength of emitted light.

[0120] C) Three-component interactions It is also possible to measure three interacting molecules using energy transfer with structurally complementary pairs of bioluminescent reporter proteins. One example is a GPCR fused to a complementary polypeptide of a bioluminescent reporter protein and a GPCR interacting protein fused to a complementary peptide of the bioluminescent reporter protein that forms a bioluminescent complex upon interaction. This allows the measurement of a two-component interaction. Energy transfer occurs if a small molecule GPCR ligand bearing a fluorescent moiety for energy transfer interacts with the complex. Thus, two-component protein-protein interactions and ternary drug-protein-protein interactions are measured in the same experiment. Also, the fluorescent molecule only generates a signal upon interaction with the protein pair, which eliminates any signal from the ligand interacting with the inactive protein (Figure 23).

[0121] Example 21 BRET with multicomponent donors The following example demonstrates that ligand-receptor interactions can be monitored via energy transfer from a multicomponent luminescent donor, where the simultaneous presence of each donor component is required for luminescence. In this example, the donor consists of two complementary subunits, an 11S polypeptide and a PEP-80 peptide. The 11S polypeptide was genetically linked to the N-terminus of BRD4, and the fusion protein was expressed in HEK293 cells. Luminescence was generated from the fusion protein only in the presence of both furimazine () and the PEP-80 complementary polypeptide (Figure 24A). Cell lysates incubated with furimazine alone (without pep-80 peptide) did not generate significant donor or acceptor signals (Figures 24A, 25A). When a solution of a fluorescent BRD4 ligand (iBET-NCT) is applied, a dose-dependent BRET signal is generated only in the presence of the PEP-80 peptide (Figures 24B, 25A, 25B). Furthermore, this BRET signal can be reduced during competitive binding by a non-fluorescent BRD4 ligand demonstrating signal specificity (Figure 25A).

[0122] HEK293 cells were transfected with 11S-BRD4 DNA using Fugene HD and seeded in a 96-well plate format. For transfection, 11S-BRD4 DNA was diluted in promoterless carrier DNA (pGEM3Z). The final DNA concentration / well was maintained at 50ng / well, but 11S-BRD4 DNA was diluted 1:10 (seeding density of 20,000 cells / well in 96-well format). After 20 hours of transfection, cells were lysed in OptiMEM+50ug / mL digitonin in the presence or absence of 1μM PEP-80 peptide. Cells were then treated with serially diluted iBET-NCT (PBI-4966) in the presence or absence of 10μM iBET as a specificity control. After 1 hour of incubation, furimazine was added at 10 μM and BRET was detected with a Varioskan luminometer.

[0123] The concept illustrated in this example is also applicable to detecting the proximity of fluorescently labeled ligands to protein complexes by linking members of the complex to components of a multicomponent luminescent donor (e.g., detecting ligands that selectively bind to heterodimeric or homodimeric receptors). The present application could thus be used to monitor the engagement of targets in disease-relevant protein complexes.

[0124] Example 22 During the development of an embodiment of the present invention, experiments were performed to demonstrate that intracellular ligand-receptor interactions can be monitored via energy transfer from a protein complex, where light emission requires the proximity of the luminescent donor moieties to facilitate interaction between the two proteins. In this example, the donor comprises two subunits, the 11S peptide and the 114 peptide. The 11S peptide was genetically linked to the N-terminus of BRD4, and the 114 peptide was genetically linked to the C-terminus of histone H3.3. These fusion proteins were co-expressed in HEK293 cells. Light emission from the fusion proteins occurred only in the presence of both fusion constructs (Figure 26A). Cells expressing 11S-BRD4 alone, H3.3-114 alone, or various control constructs did not generate significant donor or acceptor signals (Figure 26). When a solution of a fluorescent BRD4 ligand (iBET-NCT / PBI-4966) was applied, a dose-dependent acceptor signal occurred only in samples co-transfected with 11S-BRD4 and histone H3.3-114 (Figure 27). When expressed as a BRET ratio (610 / 450), specific BRET was observed only from the facilitating complex of 11S and 114 subunits and iBET-NCT, or NANOLUC-BRD4 and iBET-NCT facilitating complex. Furthermore, this BRET signal was reduced in a dose-dependent manner through competitive binding of a non-fluorescent BRD4 ligand (IBET 151), demonstrating signal specificity (Figure 28).

[0125] HEK293 cells were transfected with BRD4 DNA fusion constructs + / - histone H3.3 DNA fusion constructs using Fugene HD and seeded in 96-well plate format. For transfection, reporter constructs were diluted in promoterless carrier DNA (pGEM3Z). Final DNA concentration / well was maintained at 50 ng / well, however, each DNA expression construct was diluted 1:10 (seeding density of 20,000 cells / well in 96-well format). After 20 h of transfection, cells were treated with serially diluted iBET-NCT (PBI-4966) in the presence or absence of 10 μM iBET151 as a specificity control. To determine whether iBET-151 was able to compete with the iBET-NCT tracer in a dose-dependent manner, transfected cells were treated with serially diluted iBET-151 in the presence of a fixed concentration of iBET-NCT / PBI-4966 tracer (2 μM). After 1 h of incubation, furimazine was added at 10 μM and BRET was detected with a Varioskan luminometer.

[0126] The concept illustrated in this example is also applicable to detect the proximity of fluorescently labeled ligands to protein complexes by linking members of the complex to components of a multicomponent luminescent donor (e.g., to detect ligands that selectively bind to heterodimeric or homodimeric receptors). This application could be used to monitor the engagement of targets within protein complexes. Furthermore, this technique could enable the identification or characterization of compounds selective for disease-relevant intracellular protein complexes.

[0127] Example 23 During the development of an embodiment of the present invention, experiments were performed to demonstrate the ability to profile the intracellular selectivity and affinity of drugs against a panel of putative targets within a given phylogenetic target family using BRET. In this example, the engagement of vorinostat (SAHA) against the entire panel of class I / II / IV (non-sirtuin) HDACs in cells is profiled. First, a fluorescent SAHA derivative (SAHA-NCT) is profiled against various NANOLUC / HDAC fusions using BRET. Once energy transfer is confirmed between each NANOLUC / HDAC fusion and the SAHA tracer, the affinity of SAHA is determined by competitive disruption of each energy transfer complex. The affinity can then be inferred by the IC50 values ​​generated in each experiment. Additionally, the affinity of inhibitors to individual regions can be determined via genetic fusion of NANOLUC to discriminated regions of the target protein.

[0128] Determination of SAHA tracer affinity to individual HDACs in cells. HeLa cells were transfected with plasmid DNA encoding NANOLUC fusions with the entire family of non-sirtuin histone deacetylases (HDACs 1-11). Cells were transfected by diluting the respective DNA constructs at a mass ratio of 1:100 into pGEM3Z carrier DNA, and lipid-DNA complexes were generated using Fugene HD (Promega Corporation) at a lipid:DNA ratio of 3:1. Transfected cells were then seeded into 96-well plates at a density of 20,000 cells per well (total yield of DNA 50 ng / well). After 24 h of transfection, the cell medium was replaced with serum-free medium (Opti-MEM) and cultured with serially diluted PBI-4968 in the presence or absence of 20 μM SAHA (the latter sample used to quantify nonspecific BRET). Cells were cultured at 37° C. for 2 h. To NANOLUC-expressing samples, furimazine was added to a final concentration of 10 μM. BRET was then measured on a BMG Clariostar luminometer equipped with a 450 nm bandpass filter and a 610 nm longpass filter. The BRET ratio was determined by dividing the signal in the 630 channel by the signal in the 450 channel. To determine the specific background-corrected BRET value, the nonspecific BRET value of each concentration of tracer was subtracted from the BRET value of the tracer alone.

[0129] Example 24 The following example serves to demonstrate the ability to measure binding of compounds to intracellular targets in single cell lysis using BRET imaging. This example shows the binding of SAHA and its fluorescent derivative, SAHA-NCT (PBI-4968), to NanoLuc-HDAC6 and HDAC10-NanoLuc in living cells.

[0130] Measurement of SAHA and SAHA-NCT binding to HPAC6 and HDAC10 in living cells by BRET imaging. HeLa cells were transfected with plasmid DNA encoding NanoLuc fusions with HDAC6 and HDAC10, respectively. Cells were transfected by diluting the respective DNA constructs at a mass ratio of 1:100 into pGEM3Z carrier DNA, and lipid-DNA complexes were generated by adding Fugene HD (Promega Corporation) at a lipid:DNA ratio of 3:1. The transfection complexes were then transferred to the cell suspension (1x10 cells) and incubated for 3 h at 4°C for 1 h. 5 NanoLuc-expressing samples were mixed at 100 μM (cells / ml) and then overlayed on glass-bottom 35 mm dishes at a density of 200,000 cells per dish (total yield of 50 ng DNA / well). After 24 h of transfection, the cell medium was replaced with serum-free medium (Opti-MEM) and incubated with 2 mM PBI-4968 in the presence or absence of 10 mM SAHA. Cells were incubated for 2 h at 37°C. Furimazine was added to a final concentration of 10 μM to NanoLuc-expressing samples. Samples were imaged using an Olympus LV200 bioluminescence microscope. To measure the binding and displacement of PBI-4968 to either NanoLuc-HDAC6 or HDAC10-Nanoluc by BRET, sequential images were acquired using a 495 nm short pass filter and a 590 nm long pass filter, respectively (Figures 34 and 35). Image sections representing individual cells were quantitatively analyzed using the image analysis program Image. BRET ratios were determined by dividing the values ​​obtained in the 590 channel by the values ​​in the 450 channel. A minimum of 100 cells were analyzed for each sample. All data were summarized in a dot plot format (Figure 36). Specific binding of SAHA and SAHA-NCT was demonstrated by comparative analysis of BRET values ​​obtained from three different samples: Sample 1 - Negative control (untreated). Sample 2 - positive control (2mM PBI-4968). Sample 3 - Tracer substitution (2mM PBI-4968 + 10mM SAHA).

[0131] These results demonstrate cellular uptake and specific binding of PBI-4968 and SAHA to distinct members of the HDAC family (HDACs 6 and 10) at the single cell level. These results suggest that the extent of uptake and binding varies significantly within cell populations. The image-based BRET assay format allows for analysis of target-specific compound binding at the single cell level to determine differences based on cell type, cell confluency, cell cycle status, and other physiological parameters that require single cell lysis for analysis.

[0132] All publications and patents discussed in this application and / or listed below are hereby incorporated by reference. Various modifications and variations of the methods and compositions of the invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with a specific preferred embodiment, it should be understood that the invention claimed in the claims should not be unduly limited to this specific embodiment. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in the relevant arts are intended to be within the scope of the following claims.

Claims

1. 1. A system comprising: (a) a bioactive agent conjugated to a fluorophore, the bioactive agent being cell permeable; (b) a polynucleotide for expressing a protein fusion in a cell, the protein fusion comprising (i) a protein of interest fused to (ii) a luciferase, wherein the bioactive agent is capable of non-covalently binding to the protein of interest upon interaction with the protein of interest, wherein the emission spectrum of the luciferase overlaps with the excitation spectrum of the fluorophore; (c) a luciferase substrate; and The system comprising:

2. The system of claim 1 , wherein the bioactive agent is a small molecule, a peptide, or a nucleic acid.

3. The system of claim 2 , wherein the nucleic acid is RNA.

4. The system of claim 2 , wherein the nucleic acid is DNA.

5. The system of claim 1 , wherein the fluorophore is a small molecule fluorophore.

6. The system of claim 5 , wherein the small molecule fluorophore is a carboxyrhodamine analogue.

7. 2. The system of claim 1, wherein the luciferase comprises a polypeptide having at least 90% sequence identity to SEQ ID No.

1.

8. The system of claim 1, wherein the luciferase substrate is coeleterazine or a coeleterazine derivative.

9. The system according to claim 8, wherein the substrate is 2-furanylmethyl-deoxy-coenterazine.

10. The system of claim 1 , wherein the conjugate of the bioactive agent and the fluorophore is added extracellularly and enters the cell.

11. The system of claim 1 , further comprising a permeabilization agent for enhancing entry of the bioactive agent bound to the fluorophore into the cell.

12. The system of claim 1 , wherein the bioactive agent is produced by non-natural chemical synthesis.

13. (i) the luciferase has a first emission spectrum having a first peak emission; (ii) the phosphor has an excitation spectrum that overlaps with the first emission spectrum; (iii) the phosphor has a second emission spectrum having a second peak emission, the second peak emission being separate from the first peak emission; The system of claim 1 .

14. 14. The system of claim 13, wherein the second peak emission is at least 80 nm away from the first peak emission.

15. 2. The system of claim 1, wherein when the bioactive agent binds to the protein of interest, conversion of the substrate to a reaction product by the luciferase results in excitation of the fluorophore and emission of fluorescence from the fluorophore by bioluminescence resonance energy transfer (BRET).

16. The system of claim 1 , wherein the polynucleotide is expressed from a vector.

17. The system of claim 16 , wherein the vector is a plasmid or a viral vector.

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