Bioluminescence detection composition and bioluminescence detection method using multifunctional probes

Abstract

Provided herein are materials and methods for performing bioluminescence assays using bifunctional probes. Specifically, the present disclosure provides compositions and methods for detecting and / or quantifying biomolecules and / or assaying cellular processes associated with the biomolecules using bifunctional probes capable of binding to the biomolecules and generating bioluminescent and / or fluorescent signals.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 946,237, filed December 10, 2019, which is hereby incorporated by reference in its entirety.

[0002] Provided herein are materials and methods for performing bioluminescence assays using multifunctional probes (e.g., bifunctional, trifunctional, etc.). In particular, the present disclosure provides compositions and methods for detecting and / or quantifying biomolecules and / or assaying cellular processes associated with such biomolecules using multifunctional probes capable of binding to such biomolecules and generating bioluminescent and / or fluorescent signals. [Background technology]

[0003] Cell-based assays are an essential part of evaluating cellular responses to various environmental stimuli, particularly in the context of drug discovery, because they offer a simple, efficient, and cost-effective alternative to animal experiments. Because the results of cell-based assays typically depend on cellular responses to drugs, compounds, external stimuli, etc., the use of cultured cells is a key factor for generating consistent and accurate experimental results. Currently, the majority of cell-based assays use traditional two-dimensional (2D) monolayer cells cultured on a flat, rigid substrate. While 2D cell culture is a valuable method for cell-based research, it has several limitations. For example, because almost all cells in the in vivo environment are surrounded by other cells and extracellular matrix (ECM) in a three-dimensional (3D) fashion, 2D cell culture does not fully consider the cells' natural 3D environment. However, experiments performed using co-cultured cells in both 2D and 3D cell culture formats are hampered by the technical challenges of specifically labeling a cell population and / or a set of cellular proteins to generate accurate data reflecting the cell's response to environmental stimuli. It is beneficial to measure the response of specific cell populations within a heterogeneous mixture of cells, such as measuring the death of target cancer cells caused by cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells bearing chimeric antigen receptors (CARs). Nonspecific release of labeled probes from live cells often limits the usefulness of these forms of investigation to short-term measurements rather than more physiologically relevant timescales. Therefore, there is a need for cell-based assays and systems that facilitate specific labeling of cells and / or proteins within the context of a coculture environment. Summary of the Invention

[0004] In some embodiments, provided herein is a compound of formula (I): AXB (I) Compositions are provided comprising a compound of the formula: wherein A is a capture agent, X is a linker, and B is a biomolecule reactive group, or a salt thereof. In some embodiments, A is a covalent substrate for an enzyme. In some embodiments, A comprises a haloalkyl group. In some embodiments, A is a group of the formula -(CH2) n In some embodiments, A has the formula -X, where n is 4, 5, 6, 7, or 8, and X is a halogen. In some embodiments, A has the formula -(CH)-Cl. In some embodiments, B is a protein reactive group capable of forming a covalent bond with an amino group on a protein. In some embodiments, B is selected from succinimidyl ester, maleimide, isocyanate, isothiocyanate, pentafluorophenyl ester, and tetrafluorophenyl ester. In some embodiments, B is a succinimidyl ester. In some embodiments, B is maleimide. In some embodiments, the linker comprises one or more groups independently selected from an alkylene group, an arylene group, an -O- group, an -NH- group, a carbamate ester group, and a -C(O)- group. In some embodiments, the linker has the formula: [ka] In some embodiments, the linker comprises a carbon or nitrogen atom substituted with a second capture agent A'. In some embodiments, A' is attached to the carbon or nitrogen atom by a second linker (Linker'). In some embodiments, the Linker' comprises one or more groups independently selected from an alkylene group, an arylene group, an -O- group, an -NH- group, a carbamate group, and a -C(O)- group. In some embodiments, A' comprises a haloalkyl group. In some embodiments, A' has the formula -(CH)-Cl. In some embodiments, the linker comprises a carbon or nitrogen atom substituted with a fluorophore. In some embodiments, the fluorophore is attached to the carbon or nitrogen atom by a linker (Linker"). In some embodiments, the Linker" comprises one or more groups independently selected from an alkylene group, an arylene group, an -O- group, an -NH- group, a carbamate group, and a -C(O)- group. In some embodiments, the compound is cell-permeable. In some embodiments, the compound is [ka] In some embodiments, the compound is cell-impermeable.

[0005] In some embodiments, provided herein is a method for labeling a cell, comprising contacting the cell with an effective amount of a multifunctional probe herein, wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or within the cell, thereby labeling the cell.

[0006] In some embodiments, provided herein are cells labeled with an effective amount of the multifunctional probes herein, wherein the cells are derived from a carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, melanoma, brain or spinal cord tumor, germ cell tumor, neuroendocrine tumor, or carcinoid tumor.

[0007] In some embodiments, provided herein are methods comprising: (a) contacting a cell with an effective amount of a multifunctional probe comprising the multifunctional probe described herein, wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or within the cell; (b) contacting the cell with a pair of capture / detection reagents, wherein a first capture / detection reagent comprises (i) one of the complementary peptide or polypeptide components of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent, and a second capture / detection reagent comprises (i) the other of the complementary peptide or polypeptide component of the bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent or one of the binders capable of directly binding to a cell-associated biomolecule on the cell; (c) contacting the cell with a substrate for the bioluminescent complex; and (d) monitoring and / or detecting bioluminescence. In some embodiments, the method further comprises removing unbound probe from contact with the cells. In some embodiments, the unbound probe is removed from contact with the cells by washing the cells. In some embodiments, the unbound probe is removed from contact with the cells by centrifugation. In some embodiments, the unbound probe is removed from contact with the cells between steps (a) and (b). In some embodiments, the method further comprises placing the cells in a mixed cell population with unlabeled cells. In some embodiments, the method further comprises exposing the cells to a stimulus or condition. In some embodiments, the stimulus or condition causes cell death and / or permeabilization of the cells. In some embodiments, the cells are exposed to the stimulus or condition between steps (a) and (b). In some embodiments, the cells are exposed to the stimulus or condition between steps (b) and (c). In some embodiments, the cells are exposed to the stimulus or condition between steps (c) and (d).In some embodiments, the method further includes binding the capture / detection reagent to the capture agent displayed on any biomolecule on the cell or released from the cell and / or directly to a cell-associated biomolecule on the cell, whereby the capture / detection reagent, comprising the complementary peptide and polypeptide components of the bioluminescent complex, binds to an adjacent capture agent and / or biomolecule, thereby forming the bioluminescent complex. In some embodiments, the biomolecule-reactive group nonspecifically forms a covalent bond with a cell-associated biomolecule on or within the cell. In some embodiments, the probe is cell-permeable and capable of labeling a biomolecule within the cell. In some embodiments, the probe is cell-impermeable and capable of labeling a biomolecule on the surface of the cell.

[0008] In some embodiments, the first capture / detection reagent comprises (i) a complementary polypeptide component of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent, and the second capture / detection reagent comprises (i) a complementary peptide component of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent.

[0009] In some embodiments, the first capture / detection reagent comprises (i) a complementary polypeptide component of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent, and the second capture / detection reagent comprises (i) the other of the complementary peptide component or complementary polypeptide component of the bioluminescent complex and (ii) a binding agent capable of directly binding to a cell-associated biomolecule on the cell. In some embodiments, the binding agent is an antibody or antibody fragment. In some embodiments, the binding agent is a biomolecule or a small molecule. In some embodiments, the binding agent is annexin V.

[0010] In some embodiments, the first capture / detection reagent comprises (i) a complementary peptide or polypeptide component of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent, and the second capture / detection reagent comprises (i) the other of the complementary peptide or polypeptide or peptide components of the bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent, a binder capable of directly binding to a cell-associated biomolecule on the cell, and a fluorophore. In some embodiments, the capture agent is a haloalkyl group, and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate. In some embodiments, the biomolecule-reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular protein of the cell. In some embodiments, the probe is cell-permeable. In some embodiments, the modified dehalogenase enzyme comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 16. In some embodiments, the complementary peptide or polypeptide components both comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17 and are capable of forming an enhanced bioluminescence complex when contacted with each other. In some embodiments, the complementary peptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NO: 10, and the complementary polypeptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NO: 9.In some embodiments, a complementary system comprises two peptide components and one polypeptide component, where one or more of the peptide and polypeptide components are not fused to a capture agent and together comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when contacted together, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the complementary peptide and polypeptide components together comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, along with one or more additional peptide and polypeptide components not fused to a capture agent, and when contacted together, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the complementary peptide component and the additional peptide component together comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 14, and the complementary polypeptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 12 or 19. In some embodiments, the complementary peptide component and the additional peptide component comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 11 and 13. In some embodiments, the peptide component and the polypeptide component do not efficiently form a bioluminescent complex unless contacting them with each other facilitates binding of the capture agent to the capture factor. In some embodiments, efficient formation of a bioluminescent complex includes a complex that produces bioluminescence above background levels.In some embodiments, the amount of bioluminescent complex formation (and detectable bioluminescent signal) increases upon contacting the peptide and polypeptide components with each other via binding of the capture agent to the capture agent. In some embodiments, the bioluminescent complex exhibits a significant increase in bioluminescence in the presence of an appropriate substrate when compared to the bioluminescence of either the individual peptide or polypeptide components, or any pair of peptide and polypeptide components, in the presence of the appropriate substrate. In some embodiments, the substrate of the bioluminescent complex is coelenterazine or a coelenterazine analog. In some embodiments, the stimulus or condition is a therapeutic agent, an immunotherapeutic agent, or a chemotherapeutic agent. In some embodiments, bioluminescence is monitored using a luminometer.

[0011] In some embodiments, provided herein are methods comprising: (a) contacting a cell with an effective amount of a multifunctional probe described herein, wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or within the cell; and (b) removing unbound probe from contact with the cell. In some embodiments, the unbound probe is removed from contact with the cell by washing the cell. In some embodiments, the unbound probe is removed from contact with the cell by centrifugation. In some embodiments, the method further comprises placing the cell in a mixed cell population with unlabeled cells. In some embodiments, the method further includes (c) contacting the cell with a pair of capture / detection reagents, wherein a first capture / detection reagent comprises (i) one of the complementary peptide or polypeptide components of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent, and a second capture / detection reagent comprises (i) the other of the complementary peptide or polypeptide components of the bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent and one of the binding agents capable of directly binding to a cell-associated biomolecule on the cell; (d) contacting the cell with a substrate for the bioluminescent complex; and (e) monitoring and / or detecting bioluminescence. In some embodiments, the method further includes exposing the cell to a stimulus or condition. In some embodiments, the stimulus or condition causes cell death and / or permeabilization of the cell. In some embodiments, the method further comprises the step of binding the capture / detection reagent to the capture agent displayed on any biomolecule on the cell or released from the cell, and / or directly to a cell-associated biomolecule on the cell, wherein the capture / detection reagent comprising the complementary peptide and polypeptide components of the bioluminescent complex binds to adjacent capture agents and / or biomolecules, thereby forming the bioluminescent complex.In some embodiments, the biomolecule reactive group nonspecifically forms a covalent bond with a cell-associated biomolecule on or within the cell. In some embodiments, the probe is cell-permeable and capable of labeling a biomolecule within the cell. In some embodiments, the probe is cell-impermeable and capable of labeling a biomolecule on the surface of the cell. In some embodiments, the capture agent is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate. In some embodiments, the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular protein of the cell. In some embodiments, the biomolecule reactive group is a maleimide capable of forming a covalent bond with a thiol on an intracellular or extracellular protein of the cell. In some embodiments, the probe is cell-permeable. In some embodiments, the modified dehalogenase enzyme comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 16. In some embodiments, the complementary peptide component and complementary polypeptide component both comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17 and are capable of forming an enhanced bioluminescence complex when contacted with each other. In some embodiments, the complementary peptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NO: 10, and the complementary polypeptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NO: 9.In some embodiments, a complementary system comprises two peptide components and one polypeptide component, wherein one or more of the peptide and polypeptide components are not fused to a capture agent and together comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when contacted together, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the complementary peptide and polypeptide components together comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, along with one or more additional peptide and polypeptide components not fused to a capture agent, and when contacted together, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the complementary peptide component and the additional peptide component together comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 14, and the complementary polypeptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 12 or 19. In some embodiments, the complementary peptide component and the additional peptide component comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 11 and 13. In some embodiments, the peptide component and the polypeptide component do not efficiently form a bioluminescent complex unless contacting them with each other facilitates binding of the capture agent to the capture factor. In some embodiments, efficient formation of a bioluminescent complex includes a complex that produces bioluminescence above background levels.In some embodiments, the amount of bioluminescent complex formation (and detectable bioluminescent signal) increases upon contacting the peptide and polypeptide components with each other via binding of the capture agent to the capture agent. In some embodiments, the bioluminescent complex exhibits a significant increase in bioluminescence in the presence of an appropriate substrate when compared to the bioluminescence of either the individual peptide or polypeptide components, or any pair of peptide and polypeptide components, in the presence of the appropriate substrate. In some embodiments, the substrate of the bioluminescent complex is coelenterazine or a coelenterazine analog. In some embodiments, the stimulus or condition is a therapeutic agent, an immunotherapeutic agent, or a chemotherapeutic agent. In some embodiments, bioluminescence is monitored using a luminometer.

[0012] In some embodiments, provided herein are methods comprising: (a) contacting a cell with an effective amount of a multifunctional probe described herein, wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or within the cell; (b) contacting the cell with a first capture / detection reagent comprising (i) a complementary polypeptide component of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent; (c) contacting the cell with a second capture / detection reagent comprising (i) a complementary peptide component of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent, wherein the polypeptide component and the peptide component are capable of forming a bioluminescent complex when brought into proximity with one another; (d) contacting the cell with a substrate for the bioluminescent complex; and (e) monitoring and / or detecting bioluminescence. In some embodiments, the method further comprises removing unbound probe from contact with the cell. In some embodiments, the unbound probe is removed from contact with the cells by washing the cells. In some embodiments, the unbound probe is removed from contact with the cells by centrifugation. In some embodiments, the unbound probe is removed from contact with the cells between steps (a) and (b). In some embodiments, the method further comprises placing the cells in a mixed cell population with unlabeled cells. In some embodiments, the method further comprises exposing the cells to a stimulus or condition. In some embodiments, the stimulus or condition causes cell death and / or permeabilization of the cells. In some embodiments, the cells are exposed to the stimulus or condition between steps (a) and (b). In some embodiments, the cells are exposed to the stimulus or condition between steps (c) and (d). In some embodiments, the cells are exposed to the stimulus or condition between steps (d) and (e).In some embodiments, the cells die and / or become permeable, exposing and / or releasing biomolecules from the cells. In some embodiments, the capture / detection reagent, including the complementary peptide and polypeptide components of the bioluminescent complex, binds to an adjacent capture agent and / or biomolecule, thereby forming the bioluminescent complex. In some embodiments, the biomolecule-reactive group nonspecifically forms a covalent bond with a cell-associated biomolecule on or within the cell. In some embodiments, the probe is cell-permeable and capable of labeling a biomolecule within the cell. In some embodiments, the probe is cell-impermeable and capable of labeling a biomolecule on the surface of the cell. In some embodiments, the capture agent is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate. In some embodiments, the biomolecule-reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular biomolecule (e.g., a protein) within the cell. In some embodiments, the biomolecule reactive group is a maleimide capable of forming a covalent bond with a thiol on an intracellular or extracellular biomolecule (e.g., a protein) of the cell. In some embodiments, the modified dehalogenase enzyme comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 16. In some embodiments, the complementary peptide component and complementary polypeptide component both comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and are capable of forming an enhanced bioluminescence complex when contacted with each other.In some embodiments, the complementary peptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 10, and the complementary polypeptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 9. In some embodiments, the complementary system comprises two peptide components and one polypeptide component, one or more of which are not fused to a capture agent and both comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when contacted with each other, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the complementary peptide component and complementary polypeptide component, together with one or more additional peptide and polypeptide components not fused to the capture agent, comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when contacted together, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the complementary peptide component and additional peptide component together comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 14, and the complementary polypeptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 12 or 19. In some embodiments, the complementary peptide component and the additional peptide component comprise sequences having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NOs: 11 and 13.

[0013] In some embodiments, provided herein are methods comprising: (a) contacting a cell with an effective amount of a multifunctional probe described herein, wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or within the cell; (b) contacting the cell with a first capture / detection reagent comprising (i) a complementary polypeptide or peptide component of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent; (c) contacting the cell with a second non-covalent binding / detection reagent comprising (i) a complementary peptide or peptide component of a bioluminescent complex and (ii) a binding agent capable of directly binding to a biomolecule on the cell, wherein the polypeptide and peptide components are capable of forming a bioluminescent complex when brought into proximity with one another; (d) contacting the cell with a substrate for the bioluminescent complex; and (e) monitoring and / or detecting bioluminescence. In some embodiments, the method further comprises removing unbound probe from contact with the cells. In some embodiments, the unbound probe is removed from contact with the cells by washing the cells. In some embodiments, the unbound probe is removed from contact with the cells by centrifugation. In some embodiments, the unbound probe is removed from contact with the cells between steps (a) and (b). In some embodiments, the method further comprises placing the cells in a mixed cell population with unlabeled cells. In some embodiments, the method further comprises exposing the cells to a stimulus or condition. In some embodiments, the stimulus or condition causes cell death and / or permeabilization of the cells. In some embodiments, the cells are exposed to the stimulus or condition between steps (a) and (b). In some embodiments, the cells are exposed to the stimulus or condition between steps (c) and (d). In some embodiments, the cells are exposed to the stimulus or condition between steps (d) and (e).In some embodiments, the cells die and / or become permeable, exposing and / or releasing biomolecules from the cells. In some embodiments, the capture / detection reagent and non-covalent binding / detection moiety, comprising complementary peptide and polypeptide components of the bioluminescent complex, bind to adjacent capture agents and / or biomolecules, thereby forming the bioluminescent complex. In some embodiments, the biomolecule-reactive group nonspecifically forms a covalent bond with a cell-associated biomolecule on or within the cell. In some embodiments, the probe is cell-permeable and capable of labeling a biomolecule within the cell. In some embodiments, the probe is cell-impermeable and capable of labeling a biomolecule on the surface of the cell. In some embodiments, the binding agent is an antibody or antibody fragment. In some embodiments, the binding agent is a biomolecule or small molecule. In some embodiments, the binding agent is annexin V. In some embodiments, the capture agent is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate. In some embodiments, the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular protein of the cell. In some embodiments, the biomolecule reactive group is a maleimide capable of forming a covalent bond with a thiol on an intracellular or extracellular protein of the cell. In some embodiments, the modified dehalogenase enzyme comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 16. In some embodiments, the complementary peptide component and complementary polypeptide component both comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and are capable of forming an enhanced bioluminescence complex when contacted with each other.In some embodiments, the complementary peptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 10, and the complementary polypeptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 9. In some embodiments, the complementary system comprises two peptide components and one polypeptide component, one or more of which are not fused to a capture agent and both comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when contacted with each other, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the complementary peptide component and the complementary polypeptide component(s) that are not fused to a capture agent both comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when they are contacted with each other, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the complementary peptide component and the additional peptide component both comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 14, and the complementary polypeptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 12 or 19. In some embodiments, the complementary peptide component and the additional peptide component comprise sequences having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NOs: 11 and 13.

[0014] In some embodiments, provided herein are methods comprising: (a) contacting a cell population with an effective amount of a multifunctional probe described herein, wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or within the cell; (b) removing unbound probe and dead cells from a population of labeled live cells; (c) immobilizing and permeabilizing the population of labeled live cells; (d) contacting the immobilized and permeabilized cell population with a first capture / detection reagent comprising (i) a complementary polypeptide component of a bioluminescent complex and (ii) a capture agent capable of stably binding to the capture agent; (e) contacting the immobilized and permeabilized cell population with a second capture / detection reagent comprising (i) a complementary peptide component of a bioluminescent complex and (ii) a fluorophore, wherein the polypeptide component and the peptide component are capable of forming a bioluminescent complex; and (f) detecting luminescence and / or fluorescence. In some embodiments, luminescence and / or fluorescence are detected by fluorescence imaging, flow cytometry, and / or luminometry. In some embodiments, the capture agent is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate. In some embodiments, the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular protein of the cell. In some embodiments, the modified dehalogenase enzyme comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 16. In some embodiments, the complementary peptide component and complementary polypeptide component both comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and are capable of forming an enhanced bioluminescence complex when contacted with each other.In some embodiments, the complementary peptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NO: 11, and the complementary polypeptide component comprises a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NO: 9.

[0015] In some embodiments, provided herein are kits comprising a multifunctional probe comprising the multifunctional probe described herein and a pair of capture / detection reagents, each capture / detection reagent comprising a fusion of (i) a capture agent capable of stably binding to the capture agent and (ii) a complementary peptide or polypeptide component of a bioluminescent complex. In some embodiments, the complementary peptide and polypeptide components together comprise 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to the sequence of SEQ ID NO: 17. In some embodiments, the first capture / detection reagent comprises a complementary peptide comprising 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 11, and the second capture / detection reagent comprises a complementary peptide comprising 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 13. In some embodiments, the kit further comprises a complementary polypeptide comprising 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 12 or 19. In some embodiments, the capture agent comprises 70% or greater (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween) sequence identity to SEQ ID NO: 16. In some embodiments, the kit further comprises a coelenterazine substrate or a coelenterazine analog substrate.

[0016] In some embodiments, provided herein are kits comprising: (a) a first capture / detection reagent comprising (i) a capture agent capable of stably binding to the capture agent and (ii) a peptide component capable of forming a bioluminescent complex with a complementary polypeptide component; and (b) a second capture / detection reagent comprising (i) a capture agent capable of stably binding to the capture agent and (ii) a polypeptide component capable of forming a bioluminescent complex with the peptide component. In some embodiments, the capture agent comprises 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 16. In some embodiments, the peptide component and complementary polypeptide component both comprise 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to the sequence of SEQ ID NO: 17. In some embodiments, the kit further comprises a coelenterazine substrate or a coelenterazine analog substrate. In some embodiments, the peptide comprises 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 10, and the complementary polypeptide comprises 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 9.

[0017] In some embodiments, provided herein are (a) a first capture / detection reagent comprising a fusion of (i) a capture agent capable of stably binding to the capture agent and (ii) a first peptide component capable of forming a bioluminescent complex with a complementary peptide component and a complementary polypeptide component; and (b) a second capture / detection reagent comprising a fusion of (i) a capture agent capable of stably binding to the capture agent and (ii) a second peptide component capable of forming a bioluminescent complex with a complementary peptide component and a complementary polypeptide component. and a capture / detection reagent comprising: a first peptide component and a second peptide component both comprising at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 14; and a polypeptide component comprising at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 12 or 19. In some embodiments, the capture agent comprises 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 16.

[0018] In some embodiments, methods are described herein that include: (a) contacting a cell with an effective amount of a probe comprising the compound of claim 21, wherein the biomolecule reactive group forms a covalent bond with a biomolecule on the surface of the cell; (b) contacting the cell with a first capture / detection reagent comprising (i) a capture agent capable of stably binding to the capture agent and (ii) a first complementary peptide or polypeptide component of a bioluminescent complex; and (c) contacting the cell with (i) a second biomolecule reactive group capable of stably binding to a second biomolecule and (ii) a second complementary peptide or polypeptide component of the bioluminescent complex. (d) contacting the cell with a second capture / detection reagent comprising a capture agent and a second biomolecule component; (d) contacting the cell with a substrate for the bioluminescent complex; (e) exposing the cell to a stimulus or condition; (f) binding the capture / detection reagent to the capture agent and a second biomolecule, respectively, wherein binding of adjacent capture / detection reagents results in the formation of the bioluminescent complex; and (g) monitoring and / or detecting bioluminescence in the presence of the bioluminescent substrate, wherein the amount of bioluminescence is proportional to the amount of biomolecule on the outer surface of the cell.In some embodiments, described herein are methods of (a) contacting a cell with an effective amount of a probe comprising the compound of claim 21, wherein the biomolecule-reactive group forms a covalent bond with a biomolecule on the surface of the cell; (b) washing the cell to remove unbound probe from the cell; (c) contacting the cell with a first capture / detection reagent comprising (i) a capture agent capable of stably binding to the capture agent and (ii) a first complementary peptide or polypeptide component of a bioluminescent complex; and (d) contacting the cell with (i) a second biomolecule capable of stably binding to a second biomolecule. (i) contacting the cell with a second capture / detection reagent comprising a reactive group / linking group and (ii) a second complementary peptide or polypeptide component of the bioluminescent complex; (e) exposing the cell to a stimulus or condition; (f) contacting the cell with a substrate for the bioluminescent complex; (g) binding the capture / detection reagent to the capture agent and a second biomolecule, respectively, where binding of adjacent capture / detection reagents results in the formation of the bioluminescent complex; and (h) monitoring and / or detecting bioluminescence in the presence of a bioluminescent substrate. In some embodiments, the capture agent is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate. In some embodiments, the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an extracellular biomolecule of the cell. In some embodiments, the biomolecule reactive group is a maleimide capable of forming a covalent bond with a thiol on an extracellular biomolecule of the cell. In some embodiments, the modified dehalogenase enzyme comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or ranges therebetween) sequence identity to SEQ ID NO:16.In some embodiments, the complementary peptide and polypeptide components both comprise sequences having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when contacted together, are capable of forming an enhanced bioluminescence complex. In some embodiments, the complementary system comprises two peptide and one polypeptide component, one or more of which are not fused to a capture agent, and both comprise sequences having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when contacted together, are capable of forming an enhanced bioluminescence complex by binding of the capture agent to the capture agent. In some embodiments, the complementary peptide and polypeptide components, together with one or more additional peptide and polypeptide components that are not fused to a capture agent, comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when contacted together, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the complementary peptide and polypeptide components, together with one or more additional peptide or polypeptide components fused to an additional capture agent, comprise a sequence having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 17, and when contacted together, the capture agent is capable of binding to the capture agent to form an enhanced bioluminescence complex. In some embodiments, the peptide and polypeptide components do not efficiently form a bioluminescent complex unless contacted with each other to facilitate binding of the capture agent to the capture factor.In some embodiments, efficient formation of a bioluminescent complex includes a complex that produces bioluminescence above background levels. In some embodiments, the amount of bioluminescent complex formation (and detectable bioluminescent signal) is increased by contacting the peptide and polypeptide components with each other through binding of the capture agent to the capture agent. In some embodiments, the bioluminescent complex exhibits a significant increase in bioluminescence in the presence of a suitable substrate compared to the bioluminescence of any combination of the peptide and polypeptide components in the presence of the suitable substrate. In some embodiments, the substrate of the bioluminescent complex is coelenterazine or a coelenterazine analog. In some embodiments, the stimulus or condition results in release of the second biomolecule from the cell. In some embodiments, the stimulus or condition results in cell death, membrane permeabilization, active transport, an immune response, enhanced diffusion, secretion from the cell, or release in vesicles. In some embodiments, bioluminescence is monitored using a luminometer.

[0019] In some embodiments, provided herein are methods comprising: (a) contacting a cell with an effective amount of a multifunctional probe described herein, wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or within the cell; (b) contacting the cell with a capture / detection reagent comprising a capture agent capable of stably binding to the capture agent and a complementary peptide or polypeptide component of a bioluminescent complex; (c) contacting the cell with a detection reagent comprising a fluorophore linked to the other of the complementary peptide and polypeptide components of the bioluminescent complex; (d) contacting the cell with a substrate for the bioluminescent complex; (e) binding the capture / detection reagent to the capture agent and allowing the detection reagent to form a bioluminescent complex with the capture / detection reagent; and (f) monitoring and / or detecting fluorescence from the fluorophore and / or bioluminescence from the bioluminescent complex in the presence of a bioluminescent substrate. In some embodiments, the complementary peptide comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 11, and the complementary polypeptide comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 9. In some embodiments, the method further comprises exposing the cell to a stimulus or condition capable of causing cell death.

[0020] In some embodiments, provided herein is a kit comprising: (a) a multifunctional probe comprising a biomolecule-reactive group linked to a capture agent; (b) a first capture agent fused to a polypeptide component of a bioluminescent complex; (c) a second capture agent or biomolecule-binding agent fused to a peptide component of a bioluminescent complex; and (d) a substrate for the bioluminescent complex, wherein when the peptide and polypeptide components are brought / positioned in appropriate proximity to one another, the capture agent binds to the adjacent capture agent, thereby forming a bioluminescent complex, and the bioluminescent complex emits light in the presence of the substrate.

[0021] In some embodiments, provided herein are methods that include contacting a cell or a sample containing a cell with a component of a kit described herein.

[0022] In some embodiments, provided herein is a kit comprising: (a) a multifunctional probe comprising a biomolecule-reactive group linked to a capture agent; (b) a first capture agent fused to a polypeptide component of a bioluminescent complex; (c) a fluorophore linked to the peptide component of the bioluminescent complex; and (d) a substrate for the bioluminescent complex, wherein the peptide and polypeptide components form the bioluminescent complex when co-localized with each other without external facilitation, and the bioluminescent complex emits light in the presence of the substrate. In some embodiments, the peptide component comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NO:11, and the polypeptide component comprises at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a range therebetween) sequence identity to SEQ ID NO:9.

[0023] In some embodiments, provided herein is a kit comprising: (a) a multifunctional probe comprising a biomolecule-reactive group linked to a capture agent; (b) a first capture agent fused to a polypeptide or peptide component of a bioluminescent complex; (c) a target-specific binding agent fused to the other of the polypeptide or peptide components of the bioluminescent complex; and (d) a substrate for the bioluminescent complex, wherein when the peptide and polypeptide components are brought / positioned in appropriate proximity to one another, the capture agent and target-specific binding agent bind to the adjacent capture agent and target, thereby forming the bioluminescent complex, and the bioluminescent complex produces luminescence in the presence of the substrate.

[0024] In some embodiments, provided herein is a kit comprising: (a) a multifunctional probe comprising a biomolecule-reactive group linked to a capture agent; (b) a first capture agent fused to a first peptide component of a bioluminescent complex; (c) a second capture agent fused to a second peptide component of the bioluminescent complex; (d) a polypeptide component of the bioluminescent complex; and (e) a substrate for the bioluminescent complex, wherein the peptide and polypeptide components are such that, when the peptide components are brought / positioned appropriately in proximity to one another, the capture agent binds to the adjacent capture agent, thereby forming the bioluminescent complex, and the bioluminescent complex produces luminescence in the presence of the substrate. [Brief explanation of the drawings]

[0025] [Figure 1A]Figure 1 shows a representative schematic diagram of a cell-based assay that can be performed using the multifunctional probes described herein. Cells are covalently labeled with a cell-permeable or cell-impermeable bifunctional probe containing, for example, a succinimidyl ester group for covalent labeling of multiple cellular targets (biomolecules) and a chloroalkane ligand (for covalent binding to the HaloTag protein). Note: Plasma membrane proteins represent less than 5% of total cellular protein; therefore, most cell-permeable probes bind to intracellular targets. Cells are harvested by centrifugation, followed by discarding the supernatant, to remove unbound free probe. Premixing labeled cells with unlabeled cells allows for detailed monitoring of their viability by measuring the release of labeled biomolecules upon cell death or by detecting the amount of viable labeled cells remaining in the sample. For example, a mixture of labeled tumor cells and unlabeled natural killer cells can be used to monitor NK-mediated tumor cell death. [Figure 1B]Representative schematic diagrams of cell-based assays that can be performed using the multifunctional probes described herein. Detection of labeled targets during cell death. (1) HaloTag-LgBiT and HaloTag-SmBiT bind to exposed chloroalkane ligands on the labeled target. When the two complementary BiT partners are in close proximity, a luminescent signal is generated. (2) HaloTag-LgBiT binds to exposed chloroalkane ligands on a labeled biomolecule. Direct binding of an SmBiT-labeled binding moiety (e.g., an antibody or other binding partner of a biomolecule) to a specific cellular biomolecule (e.g., a protein) to the biomolecule in close proximity to HaloTag-LgBiT generates a luminescent signal in the presence of a luminescent substrate (e.g., furimazine). (3) Specific example of (2) in 1B, where HaloTag-LgBiT binds to exposed chloroalkane ligands on a labeled biomolecule. In this case, the SmBiT-labeled binding moiety is annexin V-SmBiT, which noncovalently binds to phosphatidylserine (PS), a biomolecule exposed at the plasma membrane. When a HaloTag-LgBiT-labeled target is brought into close proximity to the annexin V-SmBiT-bound PS, a luminescent signal is generated by the luminescent substrate. [Figure 1C] FIG. 1 is a representative schematic diagram of a cell-based assay that can be performed using the multifunctional probes described herein. [Figure 2A] FIG. 1 is a representative schematic diagram of a method for labeling target cells with multifunctional probes of the present disclosure. [Figure 2B] FIG. 1 shows the results of experiments performed to detect bioluminescence in various cell types labeled with the multifunctional probes described above. [Figure 3A] FIG. 1 shows representative results of experiments performed to test antibody-dependent cellular cytotoxicity (ADCC) in Daudi cells labeled with multifunctional probes and detected using bioluminescent detection components. [Figure 3B]FIG. 1 shows representative results of experiments performed to test antibody-dependent cellular cytotoxicity (ADCC) in Daudi cells labeled with multifunctional probes and detected using bioluminescent detection components. [Figure 3C] FIG. 1 shows representative results of experiments performed to test antibody-dependent cellular cytotoxicity (ADCC) in SKBR3 cells labeled with a multifunctional probe and detected using bioluminescent detection components. [Figure 4] FIG. 1 shows representative results of experiments performed to test target cell lysis using bispecific T cell engagers (BiTEs) in cells labeled with multifunctional probes and detected using bioluminescent detection components. [Figure 5] FIG. 1 shows representative results of experiments performed to test antibody-dependent cellular cytotoxicity (ADCC) kinetics in Daudi cells (co-cultured with PBMCs) labeled with multifunctional probes and detected using bioluminescent detection components. [Figure 6A] FIG. 1 is a representative schematic diagram showing the use of a multifunctional probe of the present disclosure to assess cell death based on endogenous phosphatidylserine (PS) translocation. [Figure 6B] FIG. 1 shows representative results for cells using complementary and non-complementary bioluminescent components. [Figure 7A] FIG. 1 shows representative results of experiments performed to detect both fluorescence and luminescence using HaloTag-HiBiT and fluorescently tagged LgBiT (LgBiT-Dy605) in cells labeled with a multifunctional chloroalkane probe. [Figure 7B] FIG. 1 shows representative results of experiments performed to detect both fluorescence and luminescence using HaloTag-HiBiT and fluorescently tagged LgBiT (LgBiT-Dy605) in cells labeled with a multifunctional chloroalkane probe. [Figure 8]Figure 1 contains representative results of cell thiol group labeling using a maleimide ligand containing a chloroalkane linker for HaloTag conjugation. Labeled cells were mixed with unlabeled cells at the indicated ratios and detected using HaloTag-HiBiT and TAMRA-conjugated LgBiT. Figure 1A shows a representative dot blot (top; y-axis = nuclei +, x-axis = TAMRA +) and histogram (bottom; y-axis = cell number, x-axis = TAMRA +) measured by flow cytometry. Figure 1B shows an XY plot comparing the predicted percentage of labeled cells (x-axis) with the measured percentage of labeled cells (y-axis). [Figure 9] FIG. 1 shows the relative amount of light generated by hepatocyte spheroids labeled with 5, 10, and 20 μM maleimide / chloroalkane bifunctional probes in the presence of HaloTag-SmBiT and HaloTag-LgBiT. [Figure 10] Figure 1 shows the relative amount of light emitted by different densities of A549 control (unlabeled) cells (light bars) or cells prelabeled with bifunctional probes (dark bars) in the presence of HaloTag-SmBiT and HaloTag-LgBiT. [Figure 11] Figure 1 shows the relative amount of light emitted by different densities of A549 control (unlabeled) cells (light bars) or cells prelabeled with the bifunctional probe (dark bars) in the presence of anti-LDH antibodies prelabeled with HaloTag-LgBiT and HaloTag-SmBiT. DETAILED DESCRIPTION OF THE INVENTION

[0026] Embodiments of the present disclosure provide materials and methods for performing bioluminescence assays using multifunctional (e.g., bifunctional, trifunctional, etc.) probes. In particular, the present disclosure provides compositions and methods for detecting and / or quantifying biomolecules and / or assaying cellular processes associated with the biomolecules using multifunctional (e.g., bifunctional, trifunctional, etc.) probes capable of binding to the biomolecules and generating bioluminescent and / or fluorescent signals.

[0027] The presently disclosed inventive subject matter addresses an unmet need to provide bioassays, systems, and methods for detecting responses of specific (e.g., pre-labeled) cell populations, particularly in co-culture systems containing other, unlabeled cells. The presently disclosed assays, systems, and methods facilitate the measurement and / or quantification of cellular processes / responses (and / or biomolecules associated with cellular processes), particularly within mixed populations of cells. Currently, there are no available bioassays that use cell-permeable multifunctional probes to covalently label intracellular proteins in live cells and then detect their release (e.g., by using NanoLuc® Binary Technology (NanoBiT), NanoTrip™ Technology, HaloTag® Technology, etc.). Features of the assays, systems, and methods of the present disclosure include, but are not limited to, their universality and adaptability to multiple cell types; the ability to quantify changes in real time using standard plate readers; and amenability to miniaturization and automation through the ability to add detection reagents in a single "add-and-read" assay format (e.g., homogeneous assays) without the need to wash away components that reduce assay sensitivity (e.g., to reduce background bioluminescence). Furthermore, embodiments of the present disclosure enable covalent labeling of intracellular proteins without toxicity issues and with the sensitivity required to detect their release from cells in a quantitative manner using standard plate readers. Embodiments of the present disclosure also minimize nonspecific leakage of probes from live cells, providing the sensitivity and robustness of bioluminescence detection required to track labeled cellular processes / responses over long periods of time.

[0028] The section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0029] 1. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0030] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended, transitional phrases, terms, or words that do not exclude additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Many embodiments herein are described using open-ended "comprising" language. Such embodiments encompass multiple closed-ended "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such language. The present disclosure contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0031] With respect to the recitation of numerical ranges herein, each numerical value of the same degree of precision therebetween is expressly contemplated. For example, for the range of 6 to 9, the numerical values ​​7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numerical values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0032] As used herein, the term "alkyl" refers to an alkyl group having 1 to 16 carbon atoms (C1-C 16 alkyl), e.g., 1 to 14 carbon atoms (C1 to C 14 alkyl), 1 to 12 carbon atoms (C1 to C 12 alkyl), 1 to 10 carbon atoms (C1 to C 10 "alkyl" refers to a straight or branched saturated hydrocarbon chain containing 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), or 1 to 4 carbon atoms (C1-C4 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0033] As used herein, the term "alkylene" refers to an alkylene group having 1 to 10 carbon atoms (C1-C 10

[0033] It refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 6 carbon atoms (C1-C6 alkylene), for example. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, and CH(CH3)CH2CH2CH2CH2-.

[0034] The term "aryl," as used herein, refers to a phenyl group or a bicyclic or tricyclic aromatic fused ring system. Bicyclic fused ring systems are exemplified by a phenyl group attached to the parent molecular moiety and fused with a phenyl group. Tricyclic fused ring systems are exemplified by a phenyl group attached to the parent molecular moiety and fused with two other phenyl groups. Representative examples of bicyclic aryls include, but are not limited to, naphthyl. Representative examples of tricyclic aryls include, but are not limited to, anthracenyl and phenanthrenyl.

[0035] As used herein, the term "arylene" refers to a divalent aryl group, such as a phenylene group.

[0036] The term "halogen" or "halo" as used herein means F, Cl, Br, or I.

[0037] The term "haloalkyl" as used herein refers to an alkyl group, as defined herein, in which one or more hydrogen atoms are replaced with halogen. For example, 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms may be replaced with halogen, or all hydrogen atoms may be replaced with halogen. Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, perfluoroethyl, 2-fluoro-2-methylpropyl, 3,3,3-trifluoropropyl, 4-chlorobutyl, 5-chloropentyl, 6-chlorohexyl, 7-chloroheptyl, and 8-chlorooctyl.

[0038] "Bioluminescence" refers to the generation and emission of light by a chemical reaction catalyzed by or enabled by an enzyme, protein, protein complex, or other biological molecule (e.g., a bioluminescent complex). In typical embodiments, a substrate of a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted by the bioluminescent entity to an unstable form, which subsequently causes the substrate to emit light.

[0039] As used herein, a "capture agent" refers to a molecular entity that forms a covalent or stable non-covalent interaction (eg, bond) with a corresponding "capture agent."

[0040] As used herein, a "biomolecule reactive group" refers to a molecular entity that nonspecifically forms a covalent or stable noncovalent interaction (e.g., bond) with a biomolecule (e.g., protein, peptide, lipid, polynucleotide, etc.). As used herein, a "protein reactive group" refers to a molecular entity that nonspecifically forms a covalent or stable noncovalent interaction (e.g., bond) with a protein (e.g., binds to a protein side chain without significantly favoring the identity of the protein).

[0041] "Complementary" refers to the characteristic of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that can hybridize, dimerize, or otherwise form a complex with each other. For example, "complementary peptides and polypeptides" can combine to form a complex. Complementary elements may require assistance (e.g., from interactors) to form a complex, for example, to position the elements in the proper conformation for complementation, to colocalize the complementary elements, to lower the interaction energy for complementation, etc.

[0042] A "complex" refers to an assembly or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with one another. In one embodiment, "contact," or more specifically, "direct contact," means that two or more molecules are in sufficient proximity such that attractive non-covalent interactions, such as van der Waals forces, hydrogen bonding, ionic interactions, and hydrophobic interactions, dominate the interaction of the molecules. In such an embodiment, a complex of molecules (e.g., peptides and polypeptides) forms under assay conditions such that the complex is thermodynamically favored (e.g., compared to the unassembled or uncomplexed state of its component molecules). As used herein, the term "complex," unless otherwise specified, refers to an assembly of two or more molecules (e.g., peptides, polypeptides, or combinations thereof).

[0043] As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications to its amino acid sequence and exhibits an altered domain structure compared to a pure or parent antibody. Such a derivative may still incorporate an amino acid sequence capable of specifically binding to a target (antigen) as well as the general domain configuration present in a natural antibody. Common examples of antibody derivatives are antibodies bound to other polypeptides, rearranged antibody domains, or antibody fragments. Such derivatives may also contain at least one additional compound, such as a covalently or non-covalently linked protein domain. The linkage may be based on gene fusion by methods known in the art. The additional domain present in the antibody-containing fusion protein may preferably be linked by a flexible linker (preferably a peptide linker) containing multiple hydrophilic peptide-linked amino acids of sufficient length to bridge the distance between the C-terminus of the additional protein domain and the N-terminus of the antibody, or vice versa. The antibody may be linked to an effector molecule having biological activity or a structure suitable for selective binding to, for example, a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical, a peptide, a protein, or a drug.

[0044] A "fragment" refers to a peptide or polypeptide derived by cleavage or "fragmentation" of a larger whole entity (e.g., a protein, polypeptide, enzyme, etc.), or a peptide or polypeptide prepared to have a similar sequence to such. A fragment is thus a subsequence of the whole entity (e.g., protein, polypeptide, enzyme, etc.) from which the fragment is generated and / or designed. A peptide or polypeptide that is not a subsequence of an existing whole protein is not a fragment (e.g., not a fragment of an existing protein). A peptide or polypeptide that is "not a fragment of an existing bioluminescent protein" is an amino acid chain that (1) physically existed prior to the design and / or synthesis of the peptide or polypeptide, and (2) is not a subsequence of a protein (e.g., natural or synthetic) that exhibits substantial bioluminescent activity.

[0045] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that contains at least a portion of the antigen-binding region or variable region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, variable light chain, variable heavy chain, diabody, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134, incorporated herein by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage (e.g., papain and pepsin digestion of antibodies) of intact antibodies produced by recombinant DNA techniques or chemical polypeptide synthesis. For example, an "Fab" fragment contains one light chain and one C heavy chain. H1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fab'" fragment contains one light chain and a C H1 Domain and C H2An Fab' fragment comprises one heavy chain containing an additional constant region extending between the heavy and light chain domains. Interchain disulfide bonds can form between the two heavy chains of the Fab' fragment to form a "F(ab')2" molecule. An "Fv" fragment contains variable regions from both the heavy and light chains but lacks the constant region. A single-chain Fv (scFv) fragment contains the variable regions of the heavy and light chains connected by a flexible linker to form a single polypeptide chain containing an antigen-binding region. Exemplary single-chain antibodies are discussed in detail in WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, which are incorporated herein by reference in their entireties. In certain instances, a single variable region (e.g., heavy or light chain variable region) may retain the ability to recognize and bind to an antigen. Those skilled in the art will recognize other antibody fragments.

[0046] As used herein, an "isolated polynucleotide" can mean a polynucleotide that, by its origin, is not associated with all or part of a polynucleotide that is naturally present with the isolated polynucleotide; that is operably linked to a polynucleotide to which the isolated polynucleotide is not naturally linked; or that is not present in nature as part of a larger sequence (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof).

[0047] "Non-luminescent" refers to an entity (e.g., a peptide, polypeptide, complex, protein, etc.) that exhibits the characteristic of not emitting a detectable amount of light in the visible light spectrum (e.g., in the presence of a substrate). For example, an entity may be referred to as non-luminescent if it does not emit detectable light in a given assay. As used herein, the term "non-luminescent" is synonymous with the term "substantially non-luminescent." For example, a non-luminescent polypeptide is substantially non-luminescent, e.g., less than 10-fold (e.g., 100-fold, 200-fold, 500-fold, 1 x 10) compared to a complex of the polypeptide and its non-luminescent complementary peptide. 3 1 / 10, 1 x 10 41 / 10, 1 x 10 5 1 / 10, 1 x 10 6 1 / 10, 1 x 10 7 In some embodiments, an entity is "non-luminescent" if any emission is sufficiently small so as not to cause background interference with a particular assay.

[0048] "Non-luminescent peptide" and "non-luminescent polypeptide" refer to a peptide or polypeptide that exhibits substantially no luminescence (e.g., in the presence of a substrate), or an amount that is below noise, or 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×10) less than a significant signal (e.g., a luminescent complex) under standard conditions (e.g., physiological conditions, assay conditions, etc.) and using common equipment (e.g., a luminometer). 3 times 1×10 4 times 1×10 5 times 1×10 6 times 1×10 7 The term "bioluminescent complex" refers to peptides and polypeptides that exhibit a specific binding activity (e.g., a specific binding site ...

[0049] As used herein, "cell-impermeable" refers to a compound or moiety that is unable to cross a cell membrane to the extent that an effective amount of the compound or moiety can be delivered intracellularly.

[0050] As used herein, "cell-permeable" refers to a compound or moiety that is able to cross a cell membrane to the extent that an effective amount of the compound is delivered into the cell.

[0051] As used herein, "coelenterazine" refers to naturally occurring ("natural") coelenterazine. As used herein, the term "coelenterazine analog" or "coelenterazine derivative" includes those described in WO2003 / 040100; U.S. Application No. 12 / 056,073 (paragraph

[0086] ); U.S. Patent No. 8,669,103; WO2012 / 061529; U.S. Patent Publication No. 2017 / 0233789; and U.S. Patent Publication No. 2018 / 0030059 (the disclosures of which are incorporated herein in their entireties), in addition to furimazine. "Coelenterazine" refers to synthetic (e.g., derivatives or variants) and naturally occurring analogs thereof, including coelenterazine-N, coelenterazine-F, coelenterazine-H, coelenterazine-HCP, coelenterazine-CP, coelenterazine-C, coelenterazine-E, coelenterazine-FCP, bis-deoxycoelenterazine ("coelenterazine-HH"), coelenterazine-I, coelenterazine-ICP, coelenterazine-V, and 2-methylcoelenterazine. In some embodiments, coelenterazine analogs include pro-substrates such as those described in U.S. Patent Application No. 12 / 056,073, U.S. Publication No. 2012 / 0707849, and U.S. Publication No. 2014 / 0099654, which are incorporated herein by reference in their entireties.

[0052] As used herein, unless otherwise specified, "peptide" and "polypeptide" refer to a polymeric compound of two or more amino acids joined through a backbone by peptide amide bonds (-C(O)NH-). The term "peptide" typically refers to short amino acid polymers (e.g., chains having fewer than 25 amino acids), while the term "polypeptide" typically refers to longer amino acid polymers (e.g., chains having more than 25 amino acids).

[0053] A "pre-existing protein" refers to an amino acid sequence that physically existed before a particular event or date. A "peptide that is not a fragment of a pre-existing protein" is a short chain of amino acids that is not a fragment or subsequence of a protein (e.g., synthetic or naturally occurring) that physically existed before the design and / or synthesis of the peptide.

[0054] As used herein, the terms "sample," "test sample," "specimen," "subject-derived sample," and "patient sample" can be used interchangeably and may be a sample of blood, such as whole blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample may be used directly as obtained from the patient, or may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the properties of the sample in any of the ways discussed herein or otherwise known in the art.

[0055] "Sequence identity" refers to the degree to which two polymeric sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequence composition of monomeric subunits. The term "sequence similarity" refers to the degree to which two polymeric sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymeric sequences. For example, similar amino acids share similar biophysical characteristics and can be classified into families, e.g., acidic (e.g., aspartic acid, glutamic acid), basic (e.g., lysine, arginine, histidine), nonpolar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). "Percent sequence identity" (or "percent sequence similarity") is calculated by: (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); (2) determining the number of positions containing identical (or similar) monomers (e.g., identical amino acids present in both sequences, similar amino acids present in both sequences) to calculate the number of matching positions; (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); and (4) multiplying the result by 100 to calculate the percent sequence identity or sequence similarity. For example, if peptide A and peptide B are both 20 amino acids long and have identical amino acids at all but one position, then the sequence identity between peptide A and peptide B is 95%. If the amino acids at non-identical positions share similar biophysical properties (e.g., both are acidic), then the sequence similarity between peptide A and peptide B is 100%. As another example, if peptide C is 20 amino acids long, peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to some amino acids in peptide C, then peptide C and peptide D have 70% sequence identity, although peptide D has 93.3% sequence identity over the optimal comparison window of peptide C.As used herein, for purposes of calculating "percent sequence identity" (or "percent sequence similarity"), any gap in the aligned sequences is treated as a mismatch at that position.

[0056] As used interchangeably herein, the terms "subject" and "patient" refer to any vertebrate, including, but not limited to, mammals and humans. In some embodiments, the subject may be human or non-human. The subject or patient may be undergoing some form of treatment. As used herein, "mammal" refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates such as chimpanzees and other ape and monkey species; livestock such as cows, sheep, pigs, goats, llamas, camels, and horses; domestic mammals such as dogs and cats; and laboratory animals, including rodents such as mice, rats, rabbits, and guinea pigs. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, both male and female, are intended to be encompassed within the scope of this term.

[0057] A "subsequence" refers to a peptide or polypeptide that has 100% sequence identity with another, larger peptide or polypeptide. The subsequence is a perfect sequence match for a portion of the larger amino acid chain.

[0058] As used herein, "substantially" means that the described property, parameter, and / or value need not be obtained exactly, but rather that deviations or variations, including, for example, tolerances, measurement errors, limitations in measurement precision, and other factors known to those of skill in the art, may occur in an amount that does not eliminate the effect intended to be provided by the property. A substantially absent property or characteristic (e.g., substantially non-luminescent) may be a property or characteristic that is within the noise range, below background, below the detection capability of the assay being used, or a small percentage (e.g., less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, less than 0.00001%, less than 0.000001%, less than 0.0000001%) of a significant property (e.g., the luminescence intensity of a bioluminescent protein or bioluminescent complex).

[0059] As used herein, the term "variant" is used to describe a peptide or polypeptide that differs in amino acid sequence due to an amino acid insertion, deletion, or conservative substitution but retains at least one biological activity. "SNP" refers to a variant that is a single nucleotide polymorphism. Representative examples of "biological activity" include the ability to bind to a specific antibody or the ability to stimulate an immune response. As used herein, the term "variant" is also used to describe a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative amino acid substitutions (e.g., replacing an amino acid with a different amino acid with similar properties, such as hydrophilicity, degree and distribution of charged regions, etc.) are generally recognized in the art as involving minor changes. These minor changes can be identified in part by considering the hydropathic index of the amino acid, as understood in the art. The hydropathic index of the amino acid is based on consideration of the hydrophobicity and charge of the amino acid. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in the retention of biological function of a protein. In the context of a peptide, consideration of amino acid hydrophilicity allows for the calculation of the peptide's greatest local average hydrophilicity, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitutions with amino acids having similar hydrophilicity values ​​can allow peptides to retain biological activity, such as immunogenicity, as understood in the art. Substitutions can be made with amino acids whose hydrophilicity values ​​are within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are believed to depend on the relative similarity of the amino acids, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties, particularly those of their side chains.

[0060] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. For example, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. The meaning and scope of such terms must be clear, except that in the event of any potential ambiguity, the definitions set forth herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall encompass the plural, and plural terms shall encompass the singular.

[0061] In biology, it is useful to measure the viability of specific cell populations within a heterogeneous mixture of cells, particularly the death of target cancer cells caused by cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells. One widely used method for such applications is the chromium release assay, which involves loading cells with radioactive chromium and monitoring its release upon cell death. Similar approaches have been developed to avoid the use of radioactive materials, such as those involving preloading cells with a fluorescent dye and measuring the release of the dye into the medium upon cell death. In summary, a major challenge with such approaches is the nonspecific release of labeled probes from live cell populations, which limits their use to short-term measurements (e.g., 2-4 hours). The probes released from dead cells often need to be separated from live cells before detection, which makes these types of assays technically challenging to use in higher-throughput "add-and-read" formats (e.g., homogeneous assays), which can increase assay variability. To address these challenges, alternative assays have been developed, such as introducing reporter genes into cells of interest. In some instances, the introduction of bioluminescent reporters addresses the leakage problem and provides a highly sensitive, quantitative approach, but this also requires time-consuming cell engineering, which can be difficult when working with primary cells. Therefore, there remains a need for a minimally leaky, radioactive-free approach to uniformly, sensitively, and quantitatively detect specific cell death in mixed cell cultures without the need for cell engineering.

[0062] 2. Multifunctional Probes Embodiments of the present disclosure include multifunctional probes that can be used to detect biomolecules of interest (e.g., proteins, peptides, lipids, polynucleotides, etc.). In some embodiments, the multifunctional probes are cell-permeable (e.g., when administered extracellularly, they can bind to intracellular biomolecules). In some embodiments, the multifunctional probes are cell-impermeable (e.g., when administered extracellularly, they cannot enter the cell and can only bind to extracellular biomolecules). In some embodiments, the multifunctional probes can be linked to biomolecules, allowing for easy detection of the biomolecules via conjugation of the probe to one or more bioluminescent peptides or polypeptides. In some embodiments, bioluminescent detection of the biomolecule indicates a cellular response including biomolecule release (e.g., cell death or apoptosis). In some embodiments, the multifunctional probes can be linked to biomolecules, allowing for easy fluorescent detection and / or imaging via conjugation of the probe to one or more bioluminescent fluorescently labeled peptides or polypeptides (e.g., HiBiT fluorescently labeled peptides). In some embodiments, the use of multifunctional probes of the present disclosure capable of generating both bioluminescent and biofluorescent signals allows for the discrimination of one cell type based, inter alia, on the presence or absence of a bioluminescent and / or biofluorescent signal, which may indicate a particular cellular response.

[0063] In some embodiments, the multifunctional probe comprises at least one capture agent (e.g., a chloroalkane group, biotin, etc.) and at least one biomolecule-reactive group (e.g., a functional group capable of forming a stable (e.g., covalent) bond with a biomolecule, such as a protein, peptide, lipid, nucleotide, etc.). For example, in some embodiments, the multifunctional probe comprises at least one protein-reactive group, such as a succinimidyl ester moiety or a maleimide group, capable of forming a stable (e.g., covalent) bond with a protein (e.g., non-specifically binding (e.g., labeling, tagging, etc.) to a protein (e.g., in a cellular environment or other complex environment)). In some embodiments, the multifunctional probe is a bifunctional probe and comprises one capture agent (e.g., a chloroalkane group) and one biomolecule-reactive group (e.g., a protein-reactive group such as a succinimidyl ester moiety). In some embodiments, a multifunctional probe, in addition to one capture agent (e.g., a chloroalkane group) and one biomolecule-reactive group, further comprises an additional functional group, such as a second capture agent, a fluorophore, etc., in which case such a probe can be considered trifunctional. In some embodiments, the various functional groups and moieties of the probes herein are joined by appropriate linkers, as described in more detail herein.

[0064] In some embodiments, the multifunctional probe is a bifunctional probe that includes two functional agents (e.g., a capture agent and a biomolecule-reactive group) attached directly (e.g., covalently attached to each other) or via a linker. In some embodiments, such linkers further include one or more functional substituents, resulting in a trifunctional or other multifunctional probe.

[0065] In some embodiments, the probes herein are bifunctional probes. For example, bifunctional probes of the present disclosure may contain succinimidyl ester (SE) and chloroalkane (Cl) functional groups. When introduced into living cells, the succinimidyl ester moiety can covalently bind to free amino groups on biomolecules (e.g., intracellular proteins, extracellular proteins, membrane proteins, lipids, etc.). When these biomolecules labeled with the bifunctional probe are released from the living cells (e.g., upon cell death), they can be detected using, for example, a modified haloalkane dehydrogenase protein that covalently binds to a chloroalkane functional group on a protein previously labeled with the bifunctional probe (e.g., HaloTag, as provided in U.S. Pat. No. 7,238,842, incorporated herein by reference in its entirety). In some embodiments, the modified haloalkane dehydrogenase protein is conjugated to one or more peptides or polypeptides capable of bioluminescence detection.

[0066] In other embodiments, the bifunctional probes of the present disclosure may use a biotin / streptavidin system. For example, the bifunctional probes may contain biotin bound to a succinimidyl ester (SE) group (or other functional group) that can covalently bind to free amino groups on biomolecules (e.g., intracellular proteins, extracellular proteins, membrane proteins, lipids, etc.). These biomolecules can then be detected by binding to complementary streptavidin bound to one or more peptides or polypeptides capable of bioluminescence. As those skilled in the art will recognize based on the present disclosure, other systems consisting of complementary binding partners can also be used.

[0067] In some embodiments, the multifunctional probes of the present disclosure can be linked to biomolecules and facilitate fluorescent detection and / or fluorescent imaging by binding to one or more fluorescently labeled peptides or polypeptides capable of bioluminescence. For example, one or more components of the NanoBiT® system (e.g., SmBiT, HiBiT, LgBiT) are labeled with a fluorescent tag or fluorophore. According to these embodiments, the fluorescent signal can be detected independently of the bioluminescent signal (e.g., generated upon formation of a bioluminescent complex / complementation). The ability to generate bioluminescent and fluorescent signals can facilitate improved detection and quantification of biomolecules and / or cells associated with biomolecules.

[0068] In some embodiments, the present disclosure provides a compound of formula (I): AXB (I) (In the formula, A is a capture factor, X is a linker, B is a biomolecule reactive group or a salt thereof.

[0069] In compounds of Formula (I), A is a capture agent, which is a molecular entity that forms a covalent bond or a stable non-covalent interaction with a specific capture agent. In some embodiments, A is a covalent substrate of an enzyme (e.g., a substrate with which a modified enzyme forms a covalent or other stable bond rather than catalyzing the conversion of the substrate to a product). In some embodiments, the substrate is recognized by a mutant enzyme and forms a covalent bond with the enzyme. In such embodiments, interaction of the substrate with a wild-type version of the enzyme results in the formation of a product and regeneration of the wild-type enzyme, while interaction of the substrate with the mutant version of the enzyme results in the formation of a covalent bond between the enzyme and the substrate. The substrate may be any substrate suitable for any mutant enzyme that has been modified to form a stable or covalent bond with a substrate that is normally only transiently bound by the enzyme.

[0070] In some embodiments, A is a haloalkyl group (e.g., C2-C 12 In such embodiments, A comprises a haloalkyl group. In such embodiments, A is a substrate for a dehalogenase, e.g., a haloalkane dehalogenase. Systems comprising mutant hydrolases (e.g., mutant dehalogenases) covalently bound to their substrates (e.g., haloalkyl substrates) are described, for example, in U.S. Pat. Nos. 7,238,842, 7,425,436, 7,429,472, and 7,867,726, each of which is incorporated herein by reference in its entirety. HALOTAG is a commercially available modified dehalogenase enzyme that forms a stable (e.g., covalent) bond (e.g., an ester bond) with its haloalkyl substrate, and is utilized in embodiments herein.

[0071] In some embodiments, A has the formula -(CH2) n In some embodiments, n is 6 and X is Cl, such that A has the formula -(CH)-C. In some embodiments, the haloalkyl group may be further substituted with substituents that do not interfere with interaction with the mutant dehalogenase.

[0072] In some embodiments, the capture agent is an "affinity molecule" and the corresponding capture agent is a "receptor" (e.g., a small molecule, protein, antibody, etc.) that selectively interacts with the affinity molecule. Examples of such pairs would include an antigen as the capture agent and an antibody as the capture agent; a small molecule as the capture agent and a protein with high affinity for the small molecule (e.g., streptavidin and biotin) as the capture agent; etc.

[0073] In the compound of Formula (I), B is a biomolecule-reactive group. In some embodiments, the biomolecule-reactive group is a group capable of reacting with a biomolecule of interest, such as a protein, peptide, lipid, or polynucleotide. In some embodiments, the biomolecule-reactive group reacts nonspecifically with one or more types of biomolecules (e.g., proteins, peptides, lipids, polynucleotides, etc.). The biomolecule can be any biomolecule associated with a cell of interest, such as an intracellular or extracellular biomolecule. In some embodiments, B is a protein-reactive group. In some embodiments, B is a functional group capable of forming a covalent bond with a protein (e.g., an intracellular protein, an extracellular protein, a membrane protein, etc.). For example, in some embodiments, B is a functional group capable of reacting with an amino acid side chain to form a covalent bond. In some embodiments, B is selected from succinimidyl ester, maleimide, isocyanate, isothiocyanate, pentafluorophenyl ester, and tetrafluorophenyl ester. In some embodiments, B is a succinimidyl ester, which can react with a free amino group of a protein, such as an amino group of a lysine side chain, to form an amide bond. In some embodiments, B is a functional group capable of forming a covalent bond with a lipid. In some embodiments, B is a functional group that acts as a suicide substrate for an enzyme, resulting in the formation of a covalent bond between the compound of Formula (I) and the enzyme.

[0074] In compounds of Formula (I), X is a linker. A wide variety of linkers can be used in compounds of Formula (I). In some embodiments, the linker includes alkylene, arylene, -O-, -NH-, carbamate, and -C(O)- groups. For example, the linker can be an ester (-C(O)O-), an amide (-C(O)NH-), a carbamate (-NHC(O)O-), a urea (-NHC(O)NH-), a phenylene (e.g., 1,4-phenylene), a linear or branched alkylene, and / or an oligo- and polyethylene glycol (-(CHCHO)x The linker can include various combinations of such groups, resulting in a linker having a -) bond, etc. In some embodiments, the linker can include two or more atoms (e.g., 2 to 200 atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 atoms, or any range therebetween (e.g., 2 to 20, 5 to 10, 15 to 35, 25 to 100, etc.)). For example, in some embodiments, the linker can be of the formula: [ka] The group includes:

[0075] In some embodiments, the linker comprises a carbon or nitrogen atom substituted with an optional substituent. In some embodiments, the optional substituent comprises a third functional group (e.g., rendering an otherwise bifunctional probe trifunctional). For example, in some embodiments, the linker may comprise a carbon or nitrogen atom substituted with a second capture agent, A'. In some embodiments, A' is a haloalkyl group (e.g., a group of formula -(CH2) n C2-C groups such as -X (where n is 4, 5, 6, 7, or 8 and X is a halogen such as Cl) 12In some embodiments, A' is any group described herein for the group A, such as a haloalkyl group. In some embodiments, A' has the formula -(CH2)6-Cl. In some embodiments, the linker comprises a carbon or nitrogen atom substituted with a fluorophore.Suitable fluorophores include fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein (e.g., FAM)); rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, or TMR); coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, and aminomethylcoumarin, or AMCA); Oregon Green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514); Texas Red, Texas Red-X, SPECTRUM RED™, SPECTRUM GREEN™, cyanine dyes (e.g., CY-3™, CY-5™, CY-3.5™, CY-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, and Alexa Fluor 680); BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665); IR dyes (e.g., IRD 40, IRD 700, IRD 800), and the like.Examples of other suitable fluorescent dyes that can be used and methods for linking or incorporating fluorescent dyes into oligonucleotides such as probes are described in R.P. Haugland, "The Handbook of Fluorescent Probes and Research Chemicals," Molecular Probes, Inc., Eugene, Oreg. (June 1992). In some embodiments, the fluorophore is carboxytetramethylrhodamine (TAMRA). In other embodiments, the linker comprises a carbon or nitrogen atom substituted with a second biomolecule-reactive group (B'). In some embodiments, B' is any group described herein for group B, such as an SE group or maleimide. In other embodiments, the linker comprises a carbon or nitrogen atom substituted with a peptide capable of forming a bioluminescent complex with one or more additional peptide or polypeptide components (e.g., NanoBiT (see, e.g., U.S. Pat. No. 9,797,889, incorporated herein in its entirety) or NanoTrip™ peptides (see, e.g., International Application No. PCT / US19 / 36844, incorporated herein in its entirety). In yet other embodiments, the linker comprises a carbon or nitrogen atom substituted with a functional moiety, such as a handle, a detectable moiety, a tag, a peptide, an epitope, or any suitable chemical group.

[0076] Suitable compounds include any combination of A, X, and B (and / or A', B', or further moieties such as fluorophores) above. In some embodiments, the compounds include: [ka] is selected from.

[0077] In some embodiments, compounds of Formula (I) are cell-permeable, a property that enables them to be used in methods described herein, such as, for example, methods of using compounds of Formula (I) to covalently label intracellular proteins in live cells.

[0078] The compounds of formula (I) may be in the form of salts, which can be prepared during the final isolation and purification of the compounds or separately, for example, by reacting a basic group (e.g., an amino group) of the compounds with a suitable acid, or an acidic group (e.g., a carboxylic acid group) of the compounds with a suitable base.

[0079] Acid salts can be prepared by reacting a suitable group of the compound, such as an amino group, with a suitable acid during the final isolation and purification of the compound or separately. For example, the compound can be dissolved in a suitable solvent, such as, but not limited to, methanol and water, and treated with at least one equivalent of an acid, such as hydrochloric acid. The resulting salt can be precipitated, isolated by filtration, and dried under reduced pressure. Alternatively, the solvent and excess acid can be removed under reduced pressure to obtain the salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, paratoluenesulfonate, undecanoate, hydrochloride, hydrobromide, sulfate, phosphate, and the like. The amino groups of the above compounds may be quaternized with alkyl chlorides, bromides, and iodides, such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl, and the like.

[0080] Base addition salts can be prepared during the final isolation and purification of the compounds of this disclosure by reaction of the carboxyl group with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a metal cation, such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amines may also be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.

[0081] Compounds of formula (I) can be synthesized by a variety of methods, including those shown in Scheme 1. Scheme 1 [ka]

[0082] Routine experimentation, including appropriate manipulation of reaction conditions, ordering of reagents and synthetic routes, protection of any chemical functional groups incompatible with the reaction conditions, and deprotection at appropriate points in the reaction sequence of the above methods, is within the scope of this disclosure. Suitable protecting groups and methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art, and examples thereof can be found in P.G.M. Wuts and T.W. Greene, in Greene's book titled "Protective Groups in Organic Synthesis" (4th ed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Synthesis of compounds of the present disclosure can be achieved by methods analogous to those described in the synthetic schemes and specific examples set forth herein.

[0083] 3. Bioluminescence The present disclosure provides assays, systems, and methods for detecting and / or measuring responses of specific cell populations (e.g., previously labeled) in a co-culture system (e.g., containing unlabeled cells). According to these embodiments, the present disclosure provides materials and methods for detecting and / or quantifying biomolecules and / or biomolecule-associated cellular responses using multifunctional (e.g., bifunctional) probes linking bioluminescent / fluorescent polypeptides and / or bioluminescent / fluorescent conjugates (of peptide or polypeptide components) to biomolecules. In some embodiments, the bioluminescent / fluorescent polypeptides and / or bioluminescent / fluorescent conjugates are linked to the multifunctional probe using a protein agent (e.g., a modified haloalkane dehydrogenase protein (e.g., HaloTag)) that covalently binds to a functional group (e.g., a haloalkane) on the multifunctional probe that binds the biomolecule. In other embodiments, the protein agent can be a specific binding partner or ligand of the biomolecule (e.g., a ligand that binds to a cell surface receptor), thereby providing greater specificity for labeling cell populations that express only the biomolecule of interest. In some embodiments, the protein agent may be modified to form a covalent bond with its biomolecular binding partner upon interaction at the cell surface.

[0084] In some embodiments, provided herein are materials and methods related to bioassays for detecting biomolecules released from cells. Biomolecule release from cells can occur as a result of (e.g., in response to) a stimulus or a cellular response to a stimulus. In some embodiments, biomolecule release can occur as a result of cell death, cytotoxicity, membrane permeabilization (e.g., caused by small molecules, proteins / peptides, or electrical stimulation), active transport mechanisms, an immune response, enhanced diffusion, etc. In some embodiments, biomolecule release occurs through secretion of the biomolecule from the cell itself. In other embodiments, biomolecules are released in vesicles, including, but not limited to, exosomes, lysosomes, microvesicles, extracellular vesicles, etc. In some embodiments, biomolecules are released from vesicles secreted from cells, or the biomolecule is present on the surface of the vesicle membrane (cell-associated biomolecule). According to these embodiments, the biomolecule can be a protein, lipid, polynucleotide (e.g., DNA or RNA), or combinations thereof, present in a vesicle (e.g., exosome), present on the vesicle membrane, and / or excreted from a vesicle.

[0085] In some embodiments of the present disclosure, a biomolecule (e.g., an intracellular protein, an extracellular protein, a membrane protein, a lipid, etc.) is (non-specifically) labeled (e.g., at an amino group) with a multifunctional probe that includes (i) a biomolecule-reactive group (e.g., for binding the probe to the intracellular biomolecule) and (ii) a capture agent. When the labeled biomolecule is released from the cell (e.g., due to cell permeabilization or cell death), the capture agent becomes available for binding by the extracellular capture agent. In some embodiments, binding of the capture agent to the biomolecule-bound capture agent allows detection of the biomolecule and correlation with release of the biomolecule from the cell (e.g., cell permeabilization, cell death, etc.).

[0086] In some embodiments, a capture agent is bound (e.g., fused) to the peptide and / or polypeptide components of a bioluminescent complex, such that when the capture agent binds to a capture agent on the labeled biomolecule, the peptide and polypeptide components form a bioluminescent complex and bioluminescence can be detected. In some embodiments, the capture agent is not cell-permeable, and thus bioluminescence occurs only when the labeled protein leaks from the cell. In some embodiments, the level of bioluminescence is proportional to a stimulus or cellular response. In some embodiments, the level of bioluminescence is proportional to cell death and / or permeabilization.

[0087] In some embodiments, described herein are multifunctional (e.g., bifunctional) probes and bioluminescent polypeptides and / or luciferases derived (structurally, functionally, etc.) from Oplophorus gracilirostris, NanoLuc® luciferase (Promega), and / or other luciferases derived from Oplophorus gracilirostris. Corporation; U.S. Patent Nos. 8,557,970, 8,669,103 (incorporated herein in their entireties), NanoBiT (U.S. Patent No. 9,797,889 (incorporated herein in their entireties), NanoTrip (U.S. Provisional Application No. 62 / 684,014 (incorporated herein in their entireties), and / or other multi-part bioluminescence technologies (International Application No. PCT / US19 / 36844 (incorporated herein in their entireties)). As described herein, the bioassays can incorporate commercially available NanoLuc®-based technologies (e.g., NanoLuc® luciferase, NanoBRET, NanoBiT, NanoTrip, NanoGlo, etc.), although in other embodiments, various combinations, variations, or derivatives of commercially available NanoLuc®-based technologies are used.

[0088] NanoLuc PCT Application Nos. PCT / US2010 / 033449, U.S. Pat. No. 8,557,970, PCT / 2011 / 059018, and U.S. Pat. No. 8,669,103 (each of which is incorporated herein by reference in its entirety for all purposes) describe compositions and methods comprising bioluminescent polypeptides. Such polypeptides are used in embodiments herein and can be used in combination with the compositions, assays, and methods described herein. In some embodiments, the compositions, assays, and methods provided herein comprise a bioluminescent polypeptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity to SEQ ID NO:5. In some embodiments, any of the above-described bioluminescent polypeptides are linked (e.g., fused, chemically linked, etc.) to a modified dehalogenase (e.g., a HaloTag) or utilize another system comprised of a complementary binding partner.

[0089] b. Bioluminescent complex Native Oplophorus gracilirostris luciferase (OgLuc) and the commercially available NANOLUC luciferase (Promega Corporation) each contain ten β (beta) chain polypeptides (β1, β2, β3, β4, β5, β6, β7, β8, β9, β10). U.S. Pat. No. 9,797,889 (incorporated herein in its entirety) describes the development and use of a complementation system comprising β1-9-like polypeptides and β10-like peptides (although the actual polypeptide and peptide sequences in U.S. Pat. No. 9,797,889 differ from the corresponding sequences in NANOLUC and wild-type native OgLuc).

[0090] Multipartite complementation systems (e.g., bipartite, tripartite, etc.) have been developed by combining peptides and polypeptides that collectively correspond to the complete set of ten β (beta) chains of these luciferases. Combining these sets of complementary peptides and polypeptides results in the formation of a bioluminescent complex under appropriate conditions (e.g., facilitated by binding of a capture reagent fused to the complementary component to a capture agent). In some embodiments, the peptide and polypeptide components of the bioluminescent complex find use as detection reagents (e.g., fused to a capture agent) for detecting proteins labeled by the compositions and methods herein (which in turn detect cell death). These multipartite complementation systems are described, for example, in PCT Application No. PCT / US14 / 26354, U.S. Patent No. 9,797,889, U.S. Provisional Application No. 62 / 684,014, and International Application No. PCT / US19 / 36844 (each of which is incorporated herein by reference in its entirety for all purposes), and examples of these techniques are described below.

[0091] 1. NanoBiT and related bisection technologies PCT Application No. PCT / US14 / 26354 and U.S. Pat. No. 9,797,889 (each of which is incorporated herein by reference in its entirety for all purposes) describe compositions and methods for the assembly of bioluminescent complexes, and such complexes, and their peptide and polypeptide components, can be used in embodiments herein and in combination with the compositions, assays, and methods described herein. In some embodiments, NanoBiT and other related technologies, when assembled into complexes, can produce a significant increase in luminescence (e.g., 2-fold, 5-fold, 10 ... 2 double, 10 3 double, 10 4 The present invention utilizes peptide and polypeptide components that exhibit a high affinity for ATP (e.g., ATP-dependent ATP synthesis) and a high affinity for ATP-dependent ATP synthesis (e.g., ATP-dependent ATP synthesis) ...

[0092] In some embodiments, provided herein are polypeptide components having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO: 9. In some embodiments, the polypeptide has less than 100% (e.g., less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%) sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, and / or SEQ ID NO: 6. In some embodiments, provided herein are peptide components having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO: 10. In some embodiments, the peptide has less than 100% (e.g., less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%) sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 8. In some embodiments, provided herein are peptide components having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO: 11. In some embodiments, the peptide has less than 100% (e.g., less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%) sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 8. In some embodiments, any of the above-mentioned peptide or polypeptide components of the bioluminescent complex are linked (e.g., fused, chemically linked, etc.) to a modified dehalogenase (e.g., a HaloTag) or utilize another system comprised of a complementary binding partner.In some embodiments, provided herein are peptide components having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO: 10. In some embodiments, the peptide has less than 100% (e.g., less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%) sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 8. In some embodiments, any of the above-mentioned peptide or polypeptide components of the bioluminescent complex are linked (e.g., fused, chemically linked, etc.) to a modified dehalogenase (e.g., a HaloTag) or utilize another system of complementary binding partners.

[0093] 2. Multi-fraction NanoLuc® and Related Multi-fraction Technologies U.S. Provisional Application No. 62 / 684,014 and International Application No. PCT / US19 / 36844 (incorporated herein in their entireties for all purposes) describe compositions, systems, and methods for assembling bioluminescent complexes from three or more peptide and polypeptide components. Such complexes, and their peptide and polypeptide components, can be used in combination with the compositions, assays, and methods described herein.

[0094] In some embodiments, provided herein are polypeptide components having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO: 12 or 19. In some embodiments, the polypeptide has less than 100% (e.g., less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%) sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, and / or SEQ ID NO: 9.

[0095] In some embodiments, provided herein are peptide components having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 11. In some embodiments, the peptide has less than 100% (e.g., less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%) sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 8.

[0096] In some embodiments, provided herein are peptide components having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or ranges therebetween) sequence identity to SEQ ID NO: 13. In some embodiments, the peptide has less than 100% (e.g., less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%) sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and / or SEQ ID NO: 7.

[0097] In some embodiments, provided herein are peptide components having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO: 14. In some embodiments, the peptide has less than 100% (e.g., less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%) sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, and / or SEQ ID NO: 8. In some embodiments, any of the above-described peptide or polypeptide components are linked (e.g., fused, chemically linked, etc.) to a modified dehalogenase (e.g., a HaloTag) or utilizes another system of complementary binding partners.

[0098] c. NanoBRET PCT Application No. PCT / US13 / 74765 and U.S. Patent Application No. 15 / 263,416 (incorporated herein in their entireties for all purposes) describe bioluminescence resonance energy transfer (BRET) compositions, assays, and methods (e.g., incorporating NanoLuc®-based technology). Such compositions, assays, and methods, as well as bioluminescent polypeptides and their fluorophore-linked components, can be used in combination with the compositions, assays, and methods described herein. In some embodiments, any of NanoLuc®-based, NanoBiT®-based, and / or multi-partite NanoLuc®-based or related peptides, polypeptides, complexes, fusions, and conjugates can be used in BRET-based applications using the compositions, assays, and methods described herein.

[0099] 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 readily detectable signal in response to energy absorption (e.g., resonance energy transfer). In certain embodiments, the energy acceptor is a fluorophore or other detectable chromophore.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 dyes (Biotium), BODIPY (Invitrogen), ALEXA These include, but are not limited to, FLuoR (Invitrogen), DYLIGHT FLUOR (ThermoScientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU and SQUARE DYES (SETA BioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, Phycobilisomes) (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 (eg, a carboxyrhodamine analog), such as those described in US Patent Application No. 13 / 682,589, which is incorporated herein by reference in its entirety.In some embodiments, the fluorophore of the multifunctional probe is the acceptor in a BRET application of the technology herein.

[0100] e. Luminescent Substrates The assays and methods of the present disclosure involve the use of a luminescent substrate. Bioluminescence, as used herein, generally refers to the generation and emission of light by a chemical reaction catalyzed by or enabled by an enzyme, protein, protein complex, or other biomolecule (e.g., a bioluminescent complex). In a typical embodiment, the luminescent substrate of a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted by the bioluminescent entity to an unstable form, which then emits light. In the presence of a detection reagent (e.g., a polypeptide component(s) of a bioluminescent complex) and a substrate (e.g., coelenterazine or a coelenterazine analog), a bioluminescent signal is generated. Provided herein are compositions comprising a luminescent substrate, such as coelenterazine or an analog or derivative thereof. Exemplary coelenterazine analogs include coelenterazine-h, coelenterazine-hh, and furimazine.

[0101] In some embodiments, the substrate has the following structure: [ka] Coelenterazine has the formula:

[0102] In some embodiments, the substrate is a coelenterazine analog or derivative. Exemplary coelenterazine analogs include coelenterazine-h (2-deoxycoelenterazine, i.e., 2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one), coelenterazine-hh (dideoxycoelenterazine, i.e., 2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), and furimazine (8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), which have the following structures: [ka] It has.

[0103] Further exemplary coelenterazine analogs include coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-i, coelenterazine-icp, coelenterazine-v, 2-methylcoelenterazine, etc. In some embodiments, the compound may be a coelenterazine analog described in WO2003 / 040100, U.S. Application No. 12 / 056,073 (paragraph

[0086] ), U.S. Patent No. 8,669,103; WO2012 / 061529, U.S. Patent Publication No. 2017 / 0233789, and U.S. Patent Publication No. 2018 / 0030059 (the disclosures of which are incorporated herein by reference in their entireties). In some embodiments, the coelenterazine analog or derivative includes a pro-substrate, such as those described in U.S. Application No. 12 / 056,073, U.S. Publication No. 2012 / 0707849, and U.S. Publication No. 2014 / 0099654, which are incorporated herein by reference in their entireties. In some embodiments, the compound is furimazine.

[0104] Coelenterazine and its analogs and derivatives can suffer from challenges associated with their reconstitution in buffer systems used in many assays, such as the bioluminescent methods described herein. For example, coelenterazine or its analogs or derivatives, such as furimazine, can dissolve slowly and / or inconsistently in buffers (e.g., due to the heterogeneous microcrystalline nature of the solid material). While dissolving in an organic solvent before dilution with a buffer can provide faster and more consistent results, coelenterazine compounds can suffer from instability in organic solutions during storage, including both thermal and photoinstability. In some embodiments, the composition further comprises a polymer. As further described herein, the presence of the polymer can stabilize the compound against degradation and can improve the solubility of the compound in water or aqueous solutions.

[0105] The polymer may be a naturally occurring biopolymer or a synthetic polymer. In some embodiments, the polymer is a naturally occurring biopolymer. Suitable naturally occurring biopolymers are carbohydrates, including disaccharides (e.g., trehalose and maltose), and polysaccharides (e.g., pullulan, dextran, and cellulose). Mixtures of naturally occurring biopolymers can also be used. In some embodiments, the polymer is pullulan, which is a polysaccharide containing maltotriose repeating units. Maltotriose is a trisaccharide containing three glucose units linked via α-1,4 glycosidic bonds. The maltotriose units within the pullulan polymer are linked to each other via α-1,6 glycosidic bonds.

[0106] In some embodiments, the polymer is a synthetic polymer. The synthetic polymer may be a homopolymer, copolymer, or block copolymer (e.g., diblock copolymer, triblock copolymer, etc.). Non-limiting examples of suitable polymers include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), and poly(D,L-lactide-co-PPO-co-D,L-lactide).L-lactide), alkyl polycyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), poly(ethylene glycol), poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes (e.g., polyethylene and polypropylene), polyalkylene glycols (e.g., poly(ethylene glycol) (PEG)), polyalkylene terephthalates (e.g., poly(ethylene terephthalate)), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters (e.g., poly(vinyl acetate)), polyvinyl halides (e.g., poly(vinyl chloride) (PVC)), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses (e.g., alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, etc.), polymers of acrylic acid (“polyacrylic acid”) (e.g., poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polydioxanone and its copolymers (e.g., polyhydroxyalkanoates, polypropylene fumarate), polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, and mixtures and copolymers thereof.

[0107] In addition to the compound and the polymer, the composition may contain additional components such as a buffer, a surfactant, a salt, a protein, or any combination thereof. For example, the composition may contain a buffer such as a phosphate buffer, a borate buffer, an acetate buffer, or a citrate buffer, or other common buffers such as bicine, tricine, tris(hydroxymethyl)aminomethane (Tris), N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-(N-morpholino)ethanesulfonic acid (MES), etc.

[0108] In some embodiments, the composition may include a surfactant. Exemplary surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and zwitterionic surfactants. For example, the surfactant may be a nonionic surfactant such as sorbitan 20. In some embodiments, the composition may include sodium chloride, potassium chloride, magnesium chloride, or the like. In some embodiments, the composition may include a protein. For example, the composition may include a carrier protein to prevent surface adsorption of a luminescent enzyme that may be added in a downstream assay. In some embodiments, the protein may be bovine serum albumin (BSA).

[0109] 4. Detection Assay Embodiments of the present disclosure encompass compositions, assays, and methods for labeling cells using multifunctional (e.g., bifunctional, trifunctional, etc.) probes and detecting labeled cells using bioluminescence. In certain embodiments, the assays herein provide for detection of leakage of labeled intracellular proteins from cells. Such leakage may occur as a result of an external stimulus and / or as a result of a cellular response to an external stimulus. In accordance with these embodiments, exemplary assays and methods for use with the various embodiments described herein are described below. The following assays and methods should not be construed as limiting the full scope of the present embodiments to those described in this disclosure.

[0110] As shown in Figures 1A-1B, bioluminescence assays can be performed using cell-permeable multifunctional probes with the complementary components of NanoLuc® Binary Technology (NanoBiT). As shown in Figure 1B, the multifunctional probe can include, for example, a succinimidyl ester group for covalent labeling of intracellular proteins and a chloroalkane ligand for covalently binding to HaloTag® proteins. However, as one of skill in the art would recognize based on the present disclosure, other multifunctional probes can also be used. For example, as shown in Figure 1A, a population of cells can be labeled using a multifunctional probe capable of binding to a biomolecule located within the cells or on the extracellular surface of the cells (cell-associated biomolecule). The biomolecule can be a protein, lipid, polynucleotide (e.g., DNA or RNA), or combination thereof, present within the cells, located on the extracellular surface of the cells, and / or present in / on vesicles associated with the cells (e.g., exosomes). In some embodiments, the labeled cells can be mixed with or co-cultured with unlabeled cells. In some embodiments, a stimulus is applied to the labeled cells directly (e.g., drug treatment) or indirectly (e.g., unlabeled cells affecting labeled cells), resulting in a change in one or more aspects of the cells and / or labeled biomolecules in / on the cells, which may indicate a cellular response.

[0111] According to these embodiments, the biomolecule is released from the cell, which may occur as a result of cell death, cytotoxicity, membrane permeabilization (e.g., caused by small molecules, proteins / peptides, or electrical stimulation), active transport mechanisms, immune response, enhanced diffusion, etc. In some embodiments, biomolecule release occurs via secretion of the biomolecule from the cell itself. As shown in FIG. 1A, upon release, the biomolecule can be detected and / or quantified using a bioluminescent conjugate, a fluorescent tag, or a combination of both. For example, HaloTag® protein can be fused to LgBiT and SmBiT (e.g., HaloTag-SmBiT and HaloTag-LgBiT), and upon binding to the biomolecule via a multifunctional probe, their complementarity generates a bioluminescent signal that can be detected and / or quantified. Additionally, one or more components of the NanoBiT system (e.g., HiBiT) can be labeled with a fluorescent tag or fluorophore to facilitate detection of both bioluminescent and fluorescent signals, thereby improving signal detection and quantification (Figures 7A-7B). Furthermore, if a complementary component of the NanoBiT system (e.g., LgBiT) binds to the same target protein as an antibody, one or more components of the NanoBiT system (e.g., SmBiT) can be labeled with a specific antibody to facilitate detection of the bioluminescent signal, thereby providing the ability to detect both specific proteins and specific cellular responses (e.g., detection of cytokines secreted by immune cells in response to a stimulus). As one of skill in the art will recognize based on the present disclosure, various aspects of the above detection assays can be combined with each other and with other aspects of cell-based detection assays.

[0112] In some embodiments, as shown in FIG. 1B, specific cell populations may be labeled with a cell-permeable multifunctional probe containing a succinimidyl ester group for covalent labeling of intracellular proteins and a chloroalkane ligand for covalent binding to HaloTag® proteins. The succinimidyl ester reacts nonspecifically with exposed amines (e.g., of lysine residues) on cellular biomolecules (e.g., intracellular proteins). The chloroalkane ligand is then presented on the surface of such biomolecules. The pre-labeled cells can then be used to monitor cell-specific responses within a cell population (e.g., a mixed cell population) or to detect / image biomolecules. When the labeled biomolecule is released from the cell, the chloroalkane ligand on the biomolecule becomes available for extracellular binding by a modified dehalogenase (e.g., HaloTag). The dehalogenase-bound protein can be detected / imaged, and the rate or amount of biomolecule release from the labeled cell population can be monitored / quantified. In some embodiments, the rate / amount of protein release from the cells provides a measure of the rate / amount of cell permeabilization and / or cell death.

[0113] In some embodiments, the capture agent (e.g., a modified dehalogenase (e.g., a HaloTag)) is linked to or provided as a fusion with a detectable moiety. In some embodiments, the detectable moiety becomes detectable (or its signal is enhanced) when the capture agent binds to a probe-labeled biomolecule. In some embodiments, the detectable moiety becomes detectable (or its signal is enhanced) when two or more capture agents bind to capture agents located at adjacent or nearby sites on a biomolecule. For example, in certain embodiments herein, a capture agent is provided as a fusion with multiple peptide / polypeptide components necessary to form a bioluminescent complex (e.g., via NanoBiT, NanoLuc® multi-part components, or other multi-part technologies (U.S. Provisional Application No. 62 / 684,014, International Application No. PCT / US19 / 36844, PCT Application No. PCT / US14 / 26354, and / or U.S. Patent No. 9,797,889, which are incorporated herein by reference in their entireties)). When the capture agent binds to an adjacent capture factor displayed on a labeled protein, a bioluminescent complex of the components is formed, and the resulting bioluminescence is read, indicating biomolecule release, cell permeabilization, and / or cell death. In some embodiments, the peptide / polypeptide components cannot efficiently form a bioluminescent complex without facilitating (e.g., bringing them into close proximity to the dehalogenase to which they are fused).

[0114] In some embodiments, the disclosed bifunctional probes and bioluminescent conjugates are used to measure cell death / apoptosis in target cell populations within mixed cell cultures. Biomolecules (e.g., proteins) within the target cells can be labeled with cell-permeable multifunctional (e.g., bifunctional) probes. When the labeled protein is released into the medium (e.g., due to protein leakage), components of NanoBiT, NanoLuc®, and / or related multi-segmented technology fused to the HaloTag® protein (components 1 and 2) bind to chloroalkane ligands on the biomolecule, bringing them into close proximity to generate an active luciferase conjugate. Upon addition of an appropriate substrate (e.g., coelenterazine, a coelenterazine analog, NanoGlo, etc.), the light emitted is directly proportional to the amount of biomolecule released from the dead cells (Figure 1A).

[0115] As shown in Figure 1B, in some embodiments, the multifunctional (e.g., bifunctional) probe comprises a succinimidyl ester (SE) and a chloroalkane (Cl) functional group. When introduced into living cells, the succinimidyl ester moiety covalently binds to free amino groups on biomolecules (e.g., intracellular proteins). When these labeled biomolecules are released from living cells upon cell death, they can be detected using the HaloTag® protein (a modified haloalkane dehydrogenase) that covalently binds to the chloroalkane functional group of proteins previously labeled with the bifunctional probe. In one embodiment, quantitative detection of released labeled biomolecules uses HaloTag®-SmBiT and HaloTag®-LgBiT fusion proteins. Detection is facilitated when two succinimidyl ester-chloroalkane ligands bind nonselectively to amino groups on proteins that are located in close proximity. The positioning of the two succinimidyl ester-chloroalkane ligands relative to the protein brings the HaloTag®-SmBiT component and the complementary HaloTag®-LgBiT component into sufficiently close proximity to form a complementary bioluminescent peptide / polypeptide complex. In the presence of luciferase substrate, light is generated in direct proportion to the amount of labeled protein released from dead or dying cells, which is directly proportional to the number of dead or dying target cells. At sufficiently high labeling densities, bioluminescence complementation occurs to the extent necessary for sensitive detection.

[0116] Target cells can include, but are not limited to, any cells from any source that can be labeled using the materials and methods provided herein. In some embodiments, the target cells are derived from a patient. In some embodiments, the target cells are of a cell type commonly used in cell culture experiments and / or clinical settings. In some embodiments, the target cells are derived from carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, melanoma, brain or spinal cord tumor, germ cell tumor, neuroendocrine tumor, or carcinoid tumor. In some embodiments, the target cells are any cancerous or non-cancerous primary cell. In some embodiments, the target cells are stem cells or stem cell-derived cells from a variety of different sources, including, but not limited to, bone marrow, embryonal blast cyst or ovarian capsule, spleen, blood, including peripheral blood and umbilical cord blood, adipose tissue, and other tissues and organs. In some embodiments, the target cells are hematopoietic stem cells, endothelial progenitor cells, embryonic stem cells, or mesenchymal stem cells.

[0117] The cell-permeable bifunctional probes of the present disclosure offer the ability to pre-label specific cell types of interest, including, but not limited to, primary cells or cells isolated from patient samples, without the need to genetically engineer the cells. This type of specific cell labeling allows for tracking the viability of the labeled cell population using sensitive and quantitative bioluminescence techniques. Changes can be measured as an endpoint or in real time using a convenient "add and read" format, with high throughput and suitable for many different assay platforms.

[0118] Embodiments herein are not limited to the HaloTag, SmBiT, and LgBiT components shown in FIG. 1B. Rather, other capture agents (e.g., other modified dehalogenases (see, e.g., U.S. Pat. Nos. 7,238,842, 7,425,436, 7,429,472, and 7,867,726, each of which is incorporated herein by reference in its entirety) and complementary detection systems (e.g., other bipartite, tripartite, and multipartite bioluminescent complex systems (see, e.g., U.S. Provisional Application No. 62 / 684,014, International Application No. PCT / US19 / 36844, PCT Application No. PCT / US14 / 26354, and / or U.S. Pat. No. 9,797,889, each of which is incorporated herein by reference in its entirety)) are also used in embodiments herein. Capture reagents may also include fluorescent proteins or fragments of fluorescent proteins or non-fluorescent subunits that form fluorescent moieties upon complementation. For example, fluorescent proteins or fragments of fluorescent proteins or non-fluorescent subunits include those described in Feng et al., (Nature Communications, vol. 8, “Improved split fluorescent proteins for endogenous protein labeling” (2017)), Foglieni et al., (Scientific Reports, vol. 7, “Split GFP technologies to structurally characterize and quantify functional biomolecular interactions of FTD-related proteins” (2017)), and Koraichi et al., (Journal of Cell Science, vol. 131, “High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation” (2018)).

[0119] As one of skill in the art will recognize based on the present disclosure, the methods and assays herein are capable of detecting any cellular response that directly or indirectly leads to the release of intracellular biomolecules from cells and / or involves the detection of biomolecules on the extracellular surface of cells (cell-associated biomolecules). In some embodiments, the cellular response occurs as a result of the cell's own response to a physiological stimulus (e.g., apoptosis induced by CAR T cells). In other embodiments, the cellular response occurs as a result of an experimental manipulation to release the biomolecule from the cell (e.g., treatment with a cell permeabilizing agent). In some embodiments, the release or exposure of the biomolecule may occur as a result of cell death, cytotoxicity, membrane permeabilization (e.g., caused by small molecules, proteins / peptides, or electrical stimulation), active transport mechanisms, immune responses, enhanced diffusion, etc. In some embodiments, the release or exposure of the biomolecule occurs via secretion of the biomolecule itself from the cell. In other embodiments, the biomolecule is released in vesicles, including, but not limited to, exosomes, lysosomes, microvesicles, extracellular vesicles, etc.

[0120] In some embodiments, the methods, assays, materials, and reagents described herein find use in assays to determine the effectiveness of a particular therapeutic or therapeutic agent for killing abnormal cells (e.g., cancer cells, tumor cells, etc.). For example, provided herein are methods for assaying the sensitivity of biopsied tumor cells to a chemotherapeutic agent. Also provided are methods for determining the drug and / or chemotherapeutic agent sensitivity of a target cell type (e.g., within a mixed cell population) in vitro. In some embodiments, biomolecules (e.g., proteins) of a target population of cells are labeled with a multifunctional reagent described herein. The labeled cells are subjected to a therapeutic or therapeutic agent, and the assays described herein are used to monitor the effectiveness of the agent in killing the target cells. In some embodiments, the assay uses patient-specific cells (e.g., obtained by biopsy) to determine the effectiveness of a therapeutic / therapeutic agent for a particular patient. In other embodiments, the assay uses a cell line or other model cell population to test the general effectiveness of a therapeutic / therapeutic agent. In some embodiments, the target cells are mixed and / or cultured with other, unlabeled cells prior to administration of the therapeutic / therapeutic agent. In some embodiments, the assays herein allow for monitoring cell death of target cells in the presence of other non-target cells (e.g., in cell culture, in situ, in vitro, etc.).

[0121] In some embodiments, the methods, assays, materials, and reagents herein find use in assays to determine the toxicity of a particular agent or condition to a cell population. For example, the toxicity of a particular chemical can be assayed to determine its safety, and the toxicity of a therapeutic agent can be assayed on cell types adjacent to the treatment site (e.g., cells adjacent to a tumor) or on cell types that become exposed to the therapeutic agent as a result of administering the agent to treat a disease or disorder.

[0122] In some embodiments, the target cells can be any suitable type of cell whose death a researcher, clinician, or other user desires to monitor. The cells can be solid tumor cells (e.g., from cell culture, biopsy from a subject, etc.), non-solid cancer cells, non-cancerous cells, healthy human cells, cells from model animals, cell lines, etc.

[0123] In some embodiments, the methods, assays, materials, and reagents herein find use in drug discovery. In some embodiments, potential drugs are tested against one or more cell types to determine their toxicity (e.g., against non-target cells) and / or their efficacy (e.g., in killing target cells). The methods herein are suitable for high-throughput assays, for example, to test the ability of a library of drugs to kill cells. In some embodiments, one or more steps of the methods herein are performed by a robotic or otherwise automated system.

[0124] In some embodiments, the methods, assays, materials, and reagents provided herein can be used to evaluate the efficacy of potential therapeutic agents against target cells (e.g., cancer cells). For example, effector cells, such as effector B cells, effector T cells (e.g., cytotoxic T lymphocytes), natural killer (NK) cells, or peripheral blood mononuclear cells (PBMCs), can be cocultured with pre-labeled tumor cells, and the ability of these effector cells to target the pre-labeled tumor cells can be assessed by measuring / quantifying the bioluminescent signal generated upon activation of the effector cells (e.g., treatment with an antibody to induce ADCC) and subsequent tumor cell lysis (e.g., apoptosis). Representative results demonstrating these effects are shown in Figures 3-5 of the present disclosure. According to these embodiments, the methods, assays, materials, and reagents provided herein can be used to identify novel therapeutic antibodies that promote ADCC in target cells.

[0125] In some embodiments, evaluating the effectiveness of effector cells in inducing apoptosis of target cells may include obtaining target and / or effector cells directly from a subject as a potential basis for therapy (e.g., T cell therapy). For example, tumor cells from a subject may be pre-labeled with a multifunctional probe of the present disclosure and then co-cultured with T cells isolated from the patient. In some embodiments, the T cells are engineered to recognize the tumor cells (e.g., engineered to express a chimeric antigen receptor (CAR) that binds to an antigen expressed on the surface of the tumor cells). Once co-culture is performed, the ability of the engineered T cells to target and lyse tumor cells can be measured / quantified based on the amount of bioluminescent signal generated by apoptotic tumor cells. In some embodiments, the T cells are not isolated from the subject, but from a healthy donor (e.g., allogeneic T cell therapy). According to these embodiments, the methods, assays, materials, and reagents provided herein can be used to identify novel therapeutic T cell therapeutics that can be used to target cancer cells in a subject.

[0126] 5. Kit Embodiments of the present disclosure also include kits comprising the various components described herein. Embodiments of the present disclosure may include kits comprising a multifunctional probe (e.g., a bifunctional probe, a trifunctional probe, a tetrafunctional probe, etc.) and one or more components of a bioluminescent complex (e.g., each component fused or linked to a capture agent). In some embodiments, the kit may also comprise a luminescent substrate. The kit may comprise a container and / or instructions. The kit may also comprise at least one of a DNA molecule, an RNA molecule, a peptide, a polypeptide, a protein, or any combination or derivative thereof. In some embodiments, the kit comprises a donor DNA template comprising a sequence encoding a peptide or polypeptide (e.g., HiBiT, LgBiT) or a modified dehalogenase protein (e.g., HaloTag).

[0127] In some embodiments, the kit may include various detection reagents, including, but not limited to, a container containing a multifunctional probe (e.g., a bifunctional probe, a trifunctional probe, a tetrafunctional probe, etc.), a container containing a first component of a bioluminescent complex (e.g., fused to a capture agent (e.g., HaloTag)), a container containing a second component of a bioluminescent complex (e.g., fused to a capture agent (e.g., HaloTag)), and, optionally, a container containing a third component of a bioluminescent complex (e.g., fused to a capture agent (e.g., HaloTag)). In some embodiments, the components of a bioluminescent complex are provided in a single container or in separate containers. The kit may also include a luminescent substrate (e.g., Nano-Glo® Luciferase Assay Substrate) and digitonin. The kit may also include various buffers and other reagents necessary to perform a bioluminescent bioassay.

[0128] 6. Modularity of Components Experiments conducted during the development of embodiments herein, as well as previous work on some of the components of the assays, kits, and the like, demonstrate the modularity of the various components. For example, the biomolecule reactive groups (e.g., succinimidyl esters, maleimides, isocyanates, isothiocyanates, pentafluorophenyl esters, tetrafluorophenyl esters, and the like) can be attached (e.g., directly, via a variety of different linkers, and the like) to various capture agents (e.g., haloalkyl groups). Similarly, the capture agents (e.g., HaloTags or structurally or functionally related dehalogenases) can be fused or linked to various other components of the assays and kits herein (e.g., bioluminescent polypeptides, peptide or polypeptide components of multipartite complementation systems (e.g., bipartite (e.g., NanoBiT), tripartite (e.g., NanoTrip), and the like), fluorophores, and the like) without altering the function of the capture agent. The peptide or polypeptide components of the multipartite complementation system (e.g., bipartite (e.g., NanoBiT), tripartite (e.g., NanoTrip), etc.) can be fused or otherwise linked to a variety of other components, such as fluorophores, capture agents (e.g., HaloTag or structurally or functionally related dehalogenases), and antibodies or antibody fragments that retain the ability to form active bioluminescent complexes.

[0129] In light of the modular nature of the components described herein, any combination of such components (e.g., in both kits, assays, as fusions, as linked pairs, etc.) is contemplated and within the scope of the present specification.

[0130] In some embodiments, the kit comprises a multifunctional probe comprising a biomolecule-reactive group (e.g., succinimidyl ester, maleimide, isocyanate, isothiocyanate, pentafluorophenyl ester, tetrafluorophenyl ester, etc.) linked to a capture agent (e.g., a haloalkyl group); a capture agent (e.g., HaloTag or a structurally or functionally related dehalogenase) fused to a polypeptide component of a bioluminescent complex; and a capture agent (e.g., HaloTag or a structurally or functionally related dehalogenase) fused to a peptide component of a bioluminescent complex (e.g., a peptide that exhibits lower affinity for the polypeptide component (e.g., requiring enhanced complex formation)); the peptide and polypeptide components form a bioluminescent complex when appropriately brought / positioned in proximity to one another. In some embodiments, the kit further comprises a substrate for the bioluminescent complex. In some embodiments, assays utilizing the above-described components are provided.

[0131] In some embodiments, the kit comprises a multifunctional probe comprising a biomolecule-reactive group (e.g., succinimidyl ester, maleimide, isocyanate, isothiocyanate, pentafluorophenyl ester, tetrafluorophenyl ester, etc.) linked to a capture agent (e.g., a haloalkyl group); a capture agent (e.g., HaloTag or a structurally or functionally related dehalogenase) fused to a polypeptide component of a bioluminescent complex; and a fluorophore linked to a peptide component of a bioluminescent complex (e.g., a peptide exhibiting high affinity for the polypeptide component); the peptide and polypeptide components, upon colocalization, form a bioluminescent complex. In some embodiments, the kit further comprises a substrate for the bioluminescent complex. In some embodiments, assays utilizing the above-described components are provided.

[0132] In some embodiments, the kit comprises a multifunctional probe comprising a biomolecule-reactive group (e.g., succinimidyl ester, maleimide, isocyanate, isothiocyanate, pentafluorophenyl ester, tetrafluorophenyl ester, etc.) linked to a capture agent (e.g., a haloalkyl group); a capture agent (e.g., HaloTag or a structurally or functionally related dehalogenase) fused to a polypeptide component of a bioluminescent complex; and an antibody (or antibody fragment or other suitable specific binding moiety, aptamer, etc.) fused to a peptide component of a bioluminescent complex (e.g., a peptide that exhibits lower affinity for the polypeptide component (e.g., requiring enhanced complex formation)); the peptide and polypeptide components form a bioluminescent complex when appropriately brought / positioned in proximity to one another. In some embodiments, the kit further comprises a substrate for the bioluminescent complex. In some embodiments, assays utilizing the above-described components are provided.

[0133] In some embodiments, the kit comprises a multifunctional probe comprising a biomolecule-reactive group (e.g., succinimidyl ester, maleimide, isocyanate, isothiocyanate, pentafluorophenyl ester, tetrafluorophenyl ester, etc.) linked to a capture agent (e.g., a haloalkyl group); a capture agent (e.g., HaloTag or a structurally or functionally related dehalogenase) fused to a first peptide component of a bioluminescent complex; a capture agent (e.g., HaloTag or a structurally or functionally related dehalogenase) fused to a second peptide component of a bioluminescent complex; and a polypeptide component of the bioluminescent complex; wherein the capture agent binds to the capture agent such that, when the peptide components are appropriately proximate / positioned to one another, the first peptide component, the second peptide component, and the polypeptide component form a bioluminescent complex. In some embodiments, the kit further comprises a substrate for the bioluminescent complex. In some embodiments, an assay utilizing the above-described components is provided. [Example]

[0134] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and discernible, and can be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having thus described the present disclosure in detail, the present disclosure will be more clearly understood by reference to the following examples. However, the following examples are intended merely to illustrate certain aspects and embodiments of the present disclosure and should not be construed as limiting the scope of the present disclosure. The disclosures of all journal citations, U.S. patents, and publications referenced herein are hereby incorporated by reference in their entirety.

[0135] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0136] Example 1 Compound synthesis 4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoic acid [ka] To a solution of terephthalic acid (2.97 g, 17.9 mmol) in anhydrous DMF (40 mL) was added diisopropylethylamine (12.5 mL, 71.5 mmol), followed by HATU (hexafluorophosphate azabenzotriazole tetramethyluronium, 3.40 g, 8.94 mmol). The solution was stirred for 10 minutes, and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-amine (2.0 g, 8.94 mmol) was slowly added. The resulting reaction mixture was then stirred overnight. The solution was extracted with ethyl acetate and acetic acid solution (2 M) and washed with saturated brine. After drying over sodium sulfate, the organic solvent was evaporated, and the residue was purified by flash chromatography. 1H NMR (400 MHz, CDCl3) δ 8.11 (m, 2H), 7.85 (m, 2H), 6.83 (s, 1H), 3.70-3.59 (m, 8H), 3.52-3.45 (m, 4H), 1.75-1.69 (m, 2H), 1.58-1.54 (m, 2H), 1.43-1.33 (m, 4H); MS m / z 371 [M + H].

[0137] 2,5-dioxopyrrolidin-1-yl 4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate [ka] To a solution of 4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoic acid (1.02 g, 2.76 mmol) in dichloromethane was added diisopropylethylamine (0.96 ml, 5.51 mmol). Subsequently, TSTU (N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate, 0.91 g, 3.03 mmol) was added and the reaction mixture was stirred for 20 minutes. The solvent was evaporated and the residue was purified by flash chromatography. 1 H NMR (400 MHz, DMSO) δ 8.84 (t, J = 8.0Hz, 1H), 8.20 (m, 2H), 8.08 (m, 2H), 3.62-3.54 (m, 6H), 3.50-3.43 (m, 4H), 3.38-3.30 (m, 2H), 2.91 (s, 4H), 1.71-1.64 (m, 2H), 1.49-1.42 (m, 2H), 1.39-1.22 (m, 4H); MS m / z 468 [M + H].

[0138] N 2 ,N 6 -Bis(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoyl)lysine [ka] To a solution of 2,5-dioxopyrrolidin-1-yl 4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoic acid (0.11 g, 0.23 mmol) in anhydrous DMF was added diisopropylethylamine (0.09 g, 0.7 mmol). Subsequently, a solution of L-lysine (0.017 g, 0.12 mmol) in PBS buffer (100 mM, pH 7.4) was added, and the reaction mixture was stirred overnight. The resulting solution was purified by preparative HPLC to give the product. MS m / z 854 [M+H].

[0139] N 2 ,N 6 -Bis(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoyl)lysine 2,5-dioxopyrrolidin-1-yl [ka] N 2 ,N 6 To a solution of N,N,N',N'-bis(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoyl)lysine (31 mg, 0.036 mmol) in anhydrous DMF was added diisopropylethylamine (0.014 g, 0.11 mmol). Subsequently, TSTU (N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate, 0.013 g, 0.043 mmol) was added, and the reaction mixture was stirred for 20 minutes. The resulting solution was then purified by preparative HPLC to give the above product. MS m / z 951 [M+H].

[0140] N-(4-aminobutylamino)-3-oxo-3H-spiro[isobenzofuran-1-9'-xanthene]-5-carboxamide [ka] To a solution of 5-carboxytetramethylrhodamine (72.8 mg, 169.1 μmol) in anhydrous DMF (10 mL) was added diisopropylethylamine (60 μL, 338.2 μmol). TSTU (N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate, 56.01 mg, 186.0 μmol) was then added, and the reaction mixture was stirred for 20 minutes. Diaminobutane (14.87 mg, 168.7 μmol) was slowly added, and the reaction mixture was stirred for 20 minutes. The reaction mixture was purified using preparative HPLC. MS m / z 501 [M+H].

[0141] N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 -(4-(tert-butoxycarbonyl)benzoyl)lysine [ka] To a solution of 4-(tert-butoxycarbonyl)benzoic acid (500 mg, 2.3 mmol) in anhydrous DMF (20 mL) was added TSTU (N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate, 677.3 mg, 2.25 mmol). Diisopropylethylamine (883.8 μL, 6.75 mmol) was then slowly added, and the reaction mixture was stirred for 15 min. 6 -(((9H-Fluoren-9-yl)methoxy)carbonyl)lysine (994.69 mg, 2.70 mmol) was added, and the resulting reaction mixture was stirred overnight. The solution was extracted with ethyl acetate and washed with saturated brine. After drying over sodium sulfate, the organic solvent was evaporated, and the residue was purified by flash chromatography. 1H NMR (400 MHz, DMSO) δ 12.62 (s, 1H), 8.72-8.79 (d, 1H), 7.98 (s, 4H), 7.84-7.91 (d, 2H), 7.64-7.71 (d, 2H), 7.36-7.41 (t, 2H), 7.26-7.35 (m, 3H), 4.32-4.42 (m, 1H), 4.23-4.31 (m, 2H), 4.15-4.23 (m, 1H), 3.18 (d, 2H), 1.71-1.88 (m, 2H), 1.57 (s, 9H), 1.35-1.50 (m, 3H), 1.34-1.14 (m, 1H); MS m / z 573 [M + H].

[0142] N 2 -(4-(tert-butoxycarbonyl)benzoyl-N 6 -(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoyl)lysine [ka] N 6 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 2 To a solution of -(4-(tert-butodicarbonyl)benzoyl)lysine (599.0 mg, 1.05 mmol) in anhydrous DMF (10 mL) was added 3,4,6,7,8,9,10,10a-octahydropyrimido[1,2-a]azepine (625.7 μL, 4.18 mmol). The reaction mixture was stirred for 30 minutes. A separate solution of 2,5-dioxopyrrolidin-1-yl 4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzoate (490.5 mg, 1.05 mmol) in anhydrous DMF (2 mL) was then added dropwise to the reaction mixture and stirred for 2 hours. The reaction mixture was purified using preparative HPLC. MS m / z 704 [M+H].

[0143] tert-Butyl 4-((1-((4-(3',6'-bis(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1-9'-xanthene]-5-carboxamido)butyl)amino)-6-(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzamido)-1-oxohexan-2-yl)carbamoyl)benzoate [ka] N 2 -(4-(tert-butoxycarbonyl)benzoyl-N 6 To a solution of -(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbammoyl)benzoyl)lysine (44.0 mg, 62.48 μmol) in anhydrous DMF (10 mL) was added TSTU (N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (20.7 mg, 68.7 μmol). Subsequently, diisopropylethylamine (32.7 μL, 187.4 μmol) was added slowly, and the reaction mixture was stirred for 20 minutes. N-(4-aminobutylamino)-3-oxo-3H-spiro[isobenzofuran-1-9'-xanthene]-5-carboxamide (31.28 mg, 62.5 μmol) was added, and the reaction mixture was stirred for 1 hour. The reaction mixture was purified using preparative HPLC. 1H NMR (400 MHz, MeOD4) δ 8.54 (d, 1H), 8.04 (m, 1H), 8.01 (m, 1H), 7.99 (d, 1H), 7.90-7.97 (m, 2H), 7.85 (s, 4H), 7.32 (dd, 1H), 7.23 (d, 2H), 6.96-7.03 (m, 2H), 6.89-6.93 (m, 2H), 4.54 (dd, 1H), 3.63-3.74 (m, 5H), 3.55-3.63 (m, 5H), 3.52-3.55 (m, 2H), 3.45-3.51 (m, 5H), 3.41 (t, 2H), MS m / z 1187 [M + H].

[0144] 4-((1-((4-(3',6'-bis(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1-9'-xanthene]-5-carboxamido)butyl)amino)-6-(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzamido)-1-oxohexan-2-yl)carbamoyl)benzoic acid [ka] A solution of 1:1 TFA:DCM (6 mL) was prepared and tert-butyl 4-((1-((4-(3',6'-bis(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-l-9'-xanthene]-5-carboxamido)butyl)amino)-6-(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzamido)-1-oxohexan-2-yl)carbamoyl)benzoate was added slowly. The reaction mixture was stirred for 40 minutes. The reaction mixture was concentrated and dried under high vacuum overnight. MS m / z 1130 [M+H].

[0145] 2,5-Dioxopyrrolidin-1-yl 4-((1-((4-(3',6'-bis(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxamido)butyl)amino)-6-(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzamido)-1-l-oxohexan-2-yl)carbamoyl)benzoate [ka] To a solution of 4-((1-((4-(3',6'-bis(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1-9'-xanthene]-5-carboxamido)butyl)amino)-6-(4-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamoyl)benzamido)-1-oxohexan-2-yl)carbamoyl)benzoic acid (7.7 mg, 6.8 μmol) in anhydrous DMF (2 mL) was added TSTU(N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (2.1 mg, 6.8 μmol). Diisopropylethylamine (2.4 μL, 13.6 μmol) was then added slowly, and the reaction mixture was stirred for 10 minutes. The reaction mixture was purified using preparative HPLC. 1H NMR (400 MHz, MeOD4) δ 8.55 (d, 1H), 8.13-8.19 (m, 2H), 7.99-8.07 (m, 3H), 7.86 (d, 4H), 7.34 (d, 1H), 7.21-7.26 (d, 2H), 6.97-7.02 (dd, 2H), 6.93 (t, 2H), 4.60 (s, 1H), 3.63-3.73 (m, 9H), 3.52-3.62 (m, 9H), 3.40-3.52 (m, 9H), 3.37-3.39 (m, 3H), 3.14-3.16 (m, 2H), 2.93 (s, 4H), MS m / z 1227.6 [M + H].

[0146] Example 2 Cell Labeling and Detection Methods Generally, the assay protocol involves two parts: labeling cells using bioluminescence techniques and measuring the release of labeled protein from dead cells. In some embodiments, the cells can be labeled at a final probe concentration of 0.1-100 μM, or in some cases 0.5-10 μM. Higher probe concentrations generally improve labeling efficiency but may adversely affect cells. To achieve the required assay performance (sensitivity, linearity, and assay window) without significantly affecting cell physiology, the labeling conditions and assay device must be optimized based on the cell type and experimental conditions. An example of optimization is shown in Table 1 below.

[0147] Next, the bifunctionally labeled probe was thawed at room temperature and mixed. This probe was typically contained in DMSO. The bifunctional probe was added to the cells at a final concentration of approximately 0.5–10 μM. The cells were then mixed by gentle pipetting and incubated at 37°C for 30 minutes. To inactivate unreactive free labeled probe, 10x the volume of prewarmed (37°C) cell culture medium containing 10% FBS was added, and the cells were incubated for an additional 5 minutes. The labeled cells were then spun down, and the labeling medium was removed. The cells were resuspended in fresh cell culture medium at the concentration required for the experiment.

[0148] In some embodiments, one or more components of the NanoBiT system can be used to measure cell death of pre-labeled cell populations in mixed cell cultures. An exemplary protocol for using this assay in a 96-well plate with 100 μL / well, i.e., 50 μL sample + 50 μL detection reagent + 25 μL Nano-Glo® Luciferase Assay Substrate, is described herein.

[0149] Labeled cells (1,250–10,000 cells / well) were seeded in 50 μl of appropriate medium along with experimental treatment conditions (e.g., drug, effector cells, etc.). Wells without cells were included as negative controls to measure background luminescence of the medium. Separate wells containing prelabeled cells were included to measure maximum release of labeled protein. The detection reagent was assembled: approximately 1 ml of detection reagent buffer (2x) was combined with approximately 1.25 μl of detection component 1 and 1.25 μl of detection component 2. If maximum release measurements were required, the required amount of prepared detection reagent (50 μl per sample) was removed and digitonin was added at a concentration of 0.12 mg / ml (e.g., 6 μl added to 1 ml of detection reagent).

[0150] Approximately 50 μL of prepared detection reagent was added to all assay wells and negative control (medium) wells. Detection reagent containing digitonin was added to control wells to measure maximum release. Cells were placed in a tissue culture incubator and treatment continued for up to 24 hours. Nano-Glo® Luciferase Assay Substrate was prepared by diluting the substrate (1:50) in Assay Medium. The assay plate was then removed from the tissue culture incubator and equilibrated to room temperature for approximately 5 minutes. Approximately 25 μL of prepared substrate was added to the assay wells, mixed, and luminescence was read.

[0151] The cytotoxicity rate (%) was calculated using the following formula: Percent Cytotoxicity = 100 x (experimental RLU - medium background) / (maximum cytotoxicity [digitonin control] - medium background) can be calculated using

[0152] The background and luminescence signal of the assay vary with different media. Most experiments were performed using 10% serum. Serum-free media may occasionally result in higher background; adding BSA to a final concentration of 0.1% may reduce the background and increase the assay window.

[0153] The non-destructive nature of the assays and methods described herein makes them compatible with multiple other fluorescent and luminescent assays. Furthermore, these assays and methods can be multiplexed with various viability / virulence assays, such as CellTiter-Glo and LDH-Glo, as well as other functional assays, including flow analysis.

[0154] Most standard plate readers are designed to measure luminescence and are suitable for the assays described herein. Some instruments do not require gain adjustment, while others require optimization of gain settings to achieve sensitivity and dynamic range. Opaque, white, multi-well plates are commonly used and are compatible with luminometers (e.g., Corning Costar® Item No. 391796 well or Costar® Item No. 3570384 well plates). The light signal is attenuated in black plates. Clear plates are not recommended for luminescence readings. The relative light units (RLU values) shown in the figures will vary depending on the plate and luminometer. However, this does not affect assay performance as long as the signal is above the instrument background and within the linear range of instrument detection (live vs. dead cells, calculated signal is above assay background).

[0155] For assay optimization, it may be advantageous to label cells at various probe concentrations and establish a cell titration curve. Various viability / toxicity assays can also be used to assess the effect of labeling on cell viability. Here, we demonstrate an example of assay optimization using Raji and K562 cells. As shown in Table 1, both Raji and K562 cells were labeled at 5 μM and showed an increase in light output upon cell death. However, labeling efficiency, toxicity, assay linearity, and assay window varied depending on the cell line and labeling conditions. K562 cells showed more robust labeling at 5 μM compared to 2 μM, demonstrating assay linearity up to 5,000 cells / well. Labeling K562 at 2 μM extended the assay linearity range up to 20,000 cells / well, but reduced the assay window from 18-fold to 7-8-fold. Importantly, using CellTiter-Glo, no decrease in cell viability was measured, and the increase in light output within the linear range of the assay for live cells compared to the media control was less than twofold (a general indication that targeting was not toxic). Labeling Raji cells at 5 μM reduced cell viability measured by CellTiter-Glo by more than 40% (data not shown), which also corresponded to a significant increase in the luminescence signal in live cells compared to the media control (more than twofold increase from the media-only control). Lowering the labeling concentration to 1 μM allowed Raji cells to be labeled with an acceptable assay window of more than fivefold and linearity up to 20,000 cells / well without significantly affecting cell viability.

[0156] For assay optimization, it may be advantageous to label cells at various probe concentrations and establish a cell titration curve. Various viability / toxicity assays can also be used to assess the effect of labeling on cell viability. Here, we demonstrate an example of assay optimization using Raji and K562 cells. As shown in Table 1, both Raji and K562 cells were labeled at 5 μM and showed an increase in light output upon cell death. However, labeling efficiency, toxicity, assay linearity, and assay window varied depending on the cell line and labeling conditions. K562 cells showed more robust labeling at 5 μM compared to 2 μM, demonstrating assay linearity up to 5,000 cells / well. Labeling K562 at 2 μM extended the assay linearity range up to 20,000 cells / well, but reduced the assay window from 18-fold to 7-8-fold. Importantly, using CellTiter-Glo, no decrease in cell viability was measured, and the increase in light output within the linear range of the assay for live cells compared to the media control was less than twofold (a general indication that targeting was not toxic). Labeling Raji cells at 5 μM reduced cell viability measured by CellTiter-Glo by more than 40% (data not shown), which also corresponded to a significant increase in the luminescence signal in live cells compared to the media control (more than twofold increase from the media-only control). Lowering the labeling concentration to 1 μM allowed Raji cells to be labeled with an acceptable assay window of more than fivefold and linearity up to 20,000 cells / well without significantly affecting cell viability. [Table 1]

[0157] K562 and Raji cells were labeled with different probe concentrations, as shown in Table 1. A no-labeling control was included for toxicity assessment. After labeling, cells were resuspended in the appropriate medium, counted, and seeded into wells of a 96-well plate in two-fold serial dilutions starting at 20,000 cells / well. Detection reagent, with or without digitonin, was added as described above, and the plate was placed in a tissue culture incubator. After incubation, Nano-Glo® Luciferase Assay Substrate was added to the samples, and luminescence was read. After luminescence reading, an equal volume of CellTiter-Glo was added to the samples to measure changes in viability. Data represent the raw relative light units (RLU) of live and dead (digitonin-treated cells) at various cell densities. The linear range of the assay is underlined. Dead / Live is the ratio between the luminescence signal of dead and live cells, calculated after subtracting the assay background (medium control). [Table 2]

[0158] Example 3 Bioluminescence detection of cells labeled with bifunctional probes As shown in Figures 2A-2B, various cells were labeled with the bifunctional probes of the present disclosure. Figure 2A is a representative schematic of the method used to label the various target cells identified in Figure 2B. As shown in Figure 2B, the cell-permeable bifunctional probes of the present disclosure (SE-Cl probes) were successfully taken up by all cells tested. Robust luminescence was detected from labeled proteins released after cell death, as quantified using the NanoBiT system, while little luminescence was detected in the medium and live cells.

[0159] According to these experiments, the various cell types tested were harvested by centrifugation at 300g for 5 minutes, followed by aspirating the medium and resuspending in PBS. The bifunctional probe described above was then added to the medium and incubated at 37°C for 30 minutes. Pre-warmed (37°C) complete medium containing 10% serum was then added to the cells and incubated at 37°C for 5 minutes. The labeled cells were then harvested by centrifugation at 300g for 5 minutes, excess medium was removed, and the labeled cells were then resuspended in the desired medium. NanoBiT detection reagent was then added to the labeled cells and incubated at 37°C for 30 minutes before reading luminescence (RLU).

[0160] Example 4 Bifunctional probes for testing antibody-dependent cellular cytotoxicity As shown in Figures 3A-3C, experiments were performed to test antibody-dependent cellular cytotoxicity (ADCC) in Daudi cells (Figures 3A-3B) and SKBR3 cells (Figure 3C) labeled with bifunctional probes of the present disclosure. Daudi cells and SKBR3 cells were labeled with 1 μM and 10 μM of labeled probe, respectively, and co-cultured with peripheral blood mononuclear cells (PBMCs; Cellular Technologies Limited) at a 20:1 E:T ratio (100,000 effectors to 5,000 targets) with serial dilutions of the indicated antibodies for 5 hours at 37°C, after which Nano-Glo® Luciferase Assay Substrate was added. Figure 3A shows the mean raw relative light units (RLU) of the assay for background (media + detection), labeled target cells alone (T), labeled target cells mixed with effector cells (T + E), T + E with rituximab, and maximum emission (MR). The curves in Figures 3B and 3C were generated using a four-parameter variable slope algorithm in Prism after subtracting the assay background (RLU [target cells + effector cells + detection components] - RLU [media + detection components]). The calculated EC values ​​were 6.8 ng / mL and 0.11 μg / mL for rituximab and Herceptin, respectively.

[0161] Example 5 Bispecific T cell engager (BiTE) assay As shown in Figure 4, experiments to test target cell lysis were performed on cells labeled with bifunctional probes using bispecific T cell engagers (BiTEs) and detected using bioluminescent detection components. BiTEs are a group of engineered bispecific monoclonal antibodies that are being investigated for use as anticancer agents because they are used to direct the cytotoxic activity of T cells toward target cells (e.g., cancer cells). Raji target cells (1.67 μM labeled probe) and TALL-104 effector cells were cocultured at a 2.5:1 ratio (12,500 effectors to 5,000 targets) in 96-well assay plates, as indicated. Blincyto was serially diluted to the indicated concentrations, and the assay plate was incubated at 37°C for 6 hours before the addition of Nano-Glo® luciferase assay substrate. Curves were generated after subtracting the assay background as described above. EC50 = 5.38 pM. S / B = BiTE / without BiTE. Percent antibody-dependent target cytolysis (%) = 100 × (RLU [target + effector + antibody] - RLU [target + effector]) / (RLU [target + effector + digitonin] - RLU [target + effector]).

[0162] Example 6 ADCC assay kinetics As shown in Figure 5, experiments were performed to examine the kinetics of antibody-dependent cellular cytotoxicity (ADCC) in Daudi cells labeled with bifunctional probes and detected using bioluminescent detection components (co-cultured with PBMCs). Labeled Daudi cells were co-cultured with PBMCs (Cellular Technologies Limited) at an E:T ratio of 20:1 (100,000 effectors to 5,000 targets) using serial dilutions of the indicated antibodies in the presence of NanoGlo® Vivazine™ live cell substrate. Assay plates were incubated at 37°C and read at the indicated times. Curves were generated in Prism using a four-parameter variable slope algorithm without background subtraction.

[0163] Example 7 Cell death assay As shown in Figures 6A-6B, the multifunctional probes of the present disclosure can be used to detect and / or quantify various cellular responses, including cell death based on endogenous phosphatidylserine (PS) translocation (Figure 6A). Cells are labeled with the multifunctional probe and mixed with HaloTag-BiT and complementary Annexin V-BiT. In healthy cells, HaloTag-BiT binds to a chloroalkane (CA) ligand (labeled biomolecule) exposed on the extracellular surface of the cell, while the PS does not bind to Annexin V-BiT. However, in unhealthy cells, PS translocates to the outer leaflet of the plasma membrane, where Annexin V-BiT can bind, thus indicating the early stages of apoptosis. As the plasma membrane begins to disassemble, creating gaps, Annexin V-BiT can enter the cell and bind to PS in the inner leaflet. When two complementary BiT partners are in functional proximity (e.g., Annexin V-LgBiT and HaloTag-SmBiT), a luminescent signal is generated. Figure 6B contains representative data in live and dead cells labeled with SE-CA using non-complementary (first and second bars from the left) and complementary (third and fourth bars from the left) BiT pairs. These results demonstrate the ability of the disclosed multifunctional probes to distinguish cell populations based on physiologically relevant responses.

[0164] Example 8 Dual detection of fluorescence and luminescence As shown in Figures 7A-7B, an experiment was conducted to detect both fluorescence and luminescence using the multifunctional probes of the present disclosure. In this embodiment, cells were labeled with the indicated concentrations of multifunctional chloroalkane probes and detected using HaloTag-HiBiT (high affinity) and LargeBiT (LgBiT) tagged with the fluorophore, Dy605 (red). Figure 7A contains representative fluorescence images of cells stained with LgBiT-Dy605 (top row) or HaloTag-HiBiT + LgBiT-Dy605 (bottom row). Cell nuclei were co-stained with Hoechst (blue). Figure 7B contains representative quantitative data corresponding to the relative intensity of luminescence.

[0165] Example 9 Dual detection of fluorescence and luminescence In the following example, we used a cell-permeable bifunctional maleimide-chloroalkane-labeled probe, PBI-8158. This probe irreversibly binds to thiol groups, rather than free amines as in other previously described probes. Proteins in cells were labeled with the probe, and the release of the protein-bound probe was detected using HaloTag-BiT. [ka]

[0166] In this experiment, labeled and unlabeled cells were mixed at various ratios (100, 80, 60, 40, 20, and 0%) to test whether the fraction of labeled cells could be detected within the mixed population using flow cytometry. The mixed cells were fixed with 4% paraformaldehyde and subsequently permeabilized with 0.1% Triton in PBS. This permeabilization step allows BiT to enter the cells. HaloTag-HiBiT was added to the cells and allowed to bind to the chloroalkane linker for 2 hours at 37°C. The cells were then washed with PBS containing 0.1% Tween 20 (PBST) to remove unbound HiBiT. LgBiT conjugated to TAMRA was added to the cells and incubated for 30 minutes at room temperature, followed by washing with PBST. Nuclei were stained using Hoechst (1:2500 in PBS). Single cells positive for Hoechst were gated, and TAMRA-positive cells were detected using a BDLSR Fortessa cytometer. Figure 8 contains representative results of labeling cellular thiol groups using a maleimide ligand containing a chloroalkane linker for HaloTag attachment.

[0167] Example 10 Detection in pre-labeled cells in 3D culture Primary hepatocytes were harvested and prelabeled with a bifunctional probe containing a maleimide moiety conjugated to a chloroalkane linker for conjugation with HaloTag protein. Liver spheroids were allowed to form in ultra-low-attachment 96-well tissue culture plates for 7 days. After 7 days, the medium was removed, and the spheroids were fixed using 4% paraformaldehyde. The spheroids were then permeabilized using 0.1% Triton X-100 in PBS and subsequently washed three times with 0.1% bovine serum albumin (BSA) in PBS. For detection, HaloTag-SmBiT and HaloTag-LgBiT (50 nM each in 0.1% BSA in PBS) were added to the labeled spheroids. After 2 hours of incubation at 37°C, the Nano-Glo® Live Cell Assay System was added, and the plates were read using a GloMax luminometer with 0.5-second integration. The data shown in Figure 9 are the relative amounts of luminescence generated by liver spheroids labeled with 5, 10, and 20 μM of the bifunctional probe.

[0168] Example 11 Detection of live cells prelabeled using HaloTag-LgBiT and HaloTag-SmBiT A549 cells were harvested and prelabeled with a bifunctional probe containing a succinimidyl ester group (A) or a maleimide moiety (B) conjugated to a chloroalkane linker for conjugation with HaloTag protein. After removing the medium and washing, live cells were fixed using 4% paraformaldehyde and then permeabilized using PBS containing 0.1% Triton X-100. For detection, HaloTag-SmBiT and HaloTag-LgBiT (200 nM each in PBS containing 0.1% BSA) were added to the labeled cells. After 2 hours of incubation at 37°C, the Nano-Glo® Live Cell Assay System was added, and the plates were read using a GloMax luminometer with 0.5-second integration. The data in Figure 10 show the relative amount of luminescence generated in different densities of A549 control (unlabeled) cells (light bars) or cells prelabeled with the bifunctional probe (dark bars).

[0169] Example 12 Detection of prelabeled live cells using antibodies against specific cellular proteins prelabeled with HaloTag-LgBiT and SmBiT In this example, HaloTag-LgBiT binds to exposed chloroalkane ligands on the labeled target, and a luminescent signal is generated when an antibody against a specific cellular protein, prelabeled with SmBiT, binds to the protein in close proximity to the HaloTag-LgBiT.

[0170] A549 cells were harvested and prelabeled with a bifunctional probe containing a maleimide moiety conjugated to a chloroalkane linker for binding to the HaloTag protein. After removing the medium and washing, live cells were fixed using 4% paraformaldehyde and then permeabilized using PBS containing 0.1% Triton X-100. For detection, anti-LDH antibodies prelabeled with HaloTag-LgBiT (200 nM) and HaloTag-SmBiT (2 μg / ml) were added to the labeled cells. After 2 hours of incubation at 37°C, the Nano-Glo® Live Cell Assay System was added, and the plates were read using a GloMax luminometer with 0.5-second integration. The data in Figure 11 show the relative amount of luminescence generated in different densities of A549 control (unlabeled) cells (light bars) or cells prelabeled with the bifunctional probe (dark bars). Another aspect of the present invention may be as follows. [1] Formula (I): AXB (I) (In the formula, A is a capture factor, X is a linker, B is a biomolecule reactive group or a salt thereof. [2] The compound or salt thereof according to [1] above, wherein A is a covalent substrate for an enzyme. [3] The compound or salt thereof according to [2] above, wherein A contains a haloalkyl group. [4] A is a compound of the formula -(CH 2 ) n-X (In the formula, n is 4, 5, 6, 7, or 8, X is a halogen. The compound or salt thereof according to [3] above, [5] A is a compound of the formula -(CH 2 ) 6 The compound or salt thereof according to any one of [1] to [4] above, which has —Cl. [6] The compound or salt thereof according to any one of [1] to [5] above, wherein B is a protein-reactive group capable of forming a covalent bond with an amino group on a protein. [7] The compound or salt thereof according to any one of [1] to [5] above, wherein B is selected from succinimidyl ester, maleimide, isocyanate, isothiocyanate, pentafluorophenyl ester, and tetrafluorophenyl ester. [8] The compound or salt thereof according to [7] above, wherein B is a succinimidyl ester. [9] The compound or salt thereof according to any one of [1] to [8] above, wherein the linker comprises one or more groups independently selected from an alkylene group, an arylene group, an -O- group, an -NH- group, a carbamate ester group, and a -C(O)- group.

[10] The linker has the formula: TIFF0007745550000021.tif32164 The compound or salt thereof according to [9] above, which contains the group:

[11] The compound or salt thereof according to any one of [1] to

[10] above, wherein the linker contains a carbon or nitrogen atom substituted with a second capture factor A'.

[12] The compound according to

[11] above, wherein A' is bonded to the carbon atom or nitrogen atom by a second linker (Linker').

[13] The compound according to

[12] above, wherein the linker' comprises one or more groups independently selected from an alkylene group, an arylene group, an -O- group, an -NH- group, a carbamate ester group, and a -C(O)- group.

[14] The compound or salt thereof according to any one of the above

[11] to

[13] , wherein A' contains a haloalkyl group.

[15] A' is a compound of the formula -(CH 2 ) 6 The compound or salt thereof according to

[14] above, which has —Cl.

[16] The compound or salt thereof according to any one of [1] to

[15] above, wherein the linker contains a carbon atom or nitrogen atom substituted with a fluorophore.

[17] The compound according to

[16] , wherein the fluorophore is attached to the carbon atom or nitrogen atom by a linker (linker").

[18] The compound according to

[17] above, wherein the linker comprises one or more groups independently selected from an alkylene group, an arylene group, an -O- group, an -NH- group, a carbamate group, and a -C(O)- group.

[19] The compound or salt thereof according to any one of [1] to

[18] above, which is cell-permeable. 〔20〕 TIFF0007745550000022.tif145164 The compound or salt thereof according to [1] above, selected from:

[21] The compound or salt thereof according to any one of [1] to

[18] above, which is cell-impermeable.

[22] A method for labeling cells, comprising contacting the cells with an effective amount of the compound according to any one of [1] to

[21] , wherein the biomolecule reactive group forms a covalent bond with a cell-associated biomolecule on or within the cells, thereby labeling the cells.

[23] Cells labeled with an effective amount of the compound according to any one of [1] to

[21] above.

[24] The cell according to

[23] above, which is derived from a carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, melanoma, brain or spinal cord tumor, germ cell tumor, neuroendocrine tumor, or carcinoid tumor.

[25] (a) contacting a cell with an effective amount of a probe comprising the compound according to any one of [1] to

[21] above, wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or in the cell; (b) treating the cells with a set of capture / detection reagents, a first capture / detection reagent comprising: (i) one of the complementary peptide or complementary polypeptide components of a bioluminescent complex; (ii) a capture agent capable of stably binding to the capture factor; Including, a second capture / detection reagent, (i) the other of the complementary peptide or polypeptide components of the bioluminescent complex; (ii) a capture agent capable of stably binding to the capture agent or a binding agent capable of directly binding to a cell-associated biomolecule on the cell; and contacting the sample with said set of capture / detection reagents comprising: (c) contacting the cell with a substrate for the bioluminescent complex; (d) monitoring and / or detecting bioluminescence; A method comprising:

[26] The method according to

[25] , further comprising the step of removing unbound probe from the contact with the cell.

[27] The method of

[26] , wherein the unbound probe is removed from contact with the cells by washing the cells.

[28] The method of

[26] , wherein the unbound probe is removed from contact with the cells by centrifugation.

[29] The method of

[26] , wherein the unbound probe is removed from contact with the cell between steps (a) and (b).

[30] The method according to

[25] , further comprising placing the cells in a mixed cell population with unlabeled cells.

[31] The method of

[25] , further comprising exposing the cells to a stimulus or condition.

[32] The method according to

[31] , wherein the cells undergo cell death and / or become permeable upon stimulation or conditions.

[33] The method according to

[31] , wherein the cells are exposed to the stimulus or condition between steps (a) and (b).

[34] The method according to

[31] , wherein the cells are exposed to the stimulus or condition between steps (b) and (c).

[35] The method according to

[31] , wherein the cells are exposed to the stimulus or condition between steps (c) and (d).

[36] The method of

[25] , further comprising a step of binding the capture / detection reagent to the capture factor displayed on any biomolecule on the cell or released from the cell, and / or directly binding the capture / detection reagent to a cell-associated biomolecule on the cell, wherein the capture / detection reagent, comprising complementary peptide and polypeptide components of the bioluminescent complex, binds to adjacent capture factors and / or biomolecules, thereby forming the bioluminescent complex.

[37] The method according to

[25] , wherein the biomolecule reactive group non-specifically forms a covalent bond with a cell-associated biomolecule on or within the cell.

[38] The method according to

[25] , wherein the probe is cell-permeable and capable of labeling a biomolecule within the cell.

[39] The method according to

[25] , wherein the probe is cell-impermeable and capable of labeling a biomolecule on the surface of the cell.

[40] The first capture / detection reagent comprises: (i) complementary polypeptide components of a bioluminescent complex; and (ii) a capture agent capable of stably binding to the capture factor; Including, a second capture / detection reagent, (i) the complementary peptide component of a bioluminescent complex; and (ii) a capture agent capable of stably binding to the capture factor; The method according to

[25] , comprising:

[41] The first capture / detection reagent comprises: (i) complementary polypeptide components of a bioluminescent complex; and (ii) a capture agent capable of stably binding to the capture factor; Including, a second capture / detection reagent, (i) the other of the complementary peptide or polypeptide components of the bioluminescent complex; (ii) a binding agent capable of directly binding to a cell-associated biomolecule on said cell; The method according to

[25] , comprising:

[42] The method of

[41] , wherein the binding agent is an antibody or an antibody fragment.

[43] The method according to

[41] , wherein the binding agent is a biomolecule or a small molecule.

[44] The method according to

[43] , wherein the binding agent is annexin V.

[45] The first capture / detection reagent is (i) a complementary peptide or polypeptide component of a bioluminescent complex; and (ii) a capture agent capable of stably binding to the capture factor; Including, a second capture / detection reagent, (i) the complementary peptide or polypeptide or peptide component of the bioluminescent complex; and (ii) a capture agent capable of stably binding to the capture agent, a binding agent capable of directly binding to a cell-associated biomolecule on the cell, and a fluorophore; The method according to

[25] , comprising:

[46] The method according to

[25] , wherein the capture factor is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate.

[47] The method of

[25] , wherein the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular protein of the cell.

[48] ​​The method according to

[25] , wherein the probe is cell-permeable.

[49] The method described in

[46] , wherein the modified dehalogenase enzyme comprises at least 70% sequence identity to SEQ ID NO: 16.

[50] The method described in

[25] , wherein the complementary peptide component and the complementary polypeptide component both comprise sequences having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, are capable of forming an enhanced bioluminescence complex.

[51] The method described in

[50] , wherein the complementary peptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 10, and the complementary polypeptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 9.

[52] The method described in

[25] , wherein the complementary system comprises two peptide components and one polypeptide component, at least one of which is not fused to a capture agent and both comprise a sequence having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, the capture agent binds to the capture factor, thereby forming an enhanced bioluminescence complex.

[53] The method described in

[52] , wherein the complementary peptide component and the further peptide component both comprise a sequence having at least 70% sequence identity to SEQ ID NO: 14, and the complementary polypeptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 12.

[54] The method according to

[53] , wherein the complementary peptide component and the additional peptide component comprise sequences having at least 70% sequence identity to SEQ ID NOs: 11 and 13.

[55] The method of any one of

[50] and

[51] , wherein the peptide component and polypeptide component do not efficiently form a bioluminescent complex unless they are brought into contact with each other, thereby promoting binding of the capture agent to the capture factor.

[56] The method according to any one of

[25] to

[55] above, wherein the bioluminescent complex exhibits a significant increase in bioluminescence in the presence of an appropriate substrate, compared to the bioluminescence of any of the individual peptide and polypeptide components, and any pair of peptide and polypeptide components, in the presence of the appropriate substrate.

[57] The method of

[25] , wherein the substrate of the bioluminescent complex is coelenterazine or a coelenterazine analog.

[58] The method according to

[25] , wherein the stimulus or condition is a therapeutic agent, an immunotherapeutic agent, or a chemotherapeutic agent.

[59] The method according to

[25] , wherein bioluminescence is monitored using a luminometer.

[60] (a) contacting a cell with an effective amount of a probe comprising the compound according to any one of [1] to

[21] , wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or in the cell; (b) removing unbound probe from contact with said cells; and A method comprising:

[61] The method of

[60] , wherein the unbound probe is removed from contact with the cells by washing the cells.

[62] The method of

[60] , wherein the unbound probe is removed from contact with the cells by centrifugation.

[63] The method of

[60] , further comprising placing the cells in a mixed cell population with unlabeled cells.

[64] (c) subjecting the cells to a set of capture / detection reagents, a first capture / detection reagent comprising: (i) one of the complementary peptide or complementary polypeptide components of a bioluminescent complex; (ii) a capture agent capable of stably binding to the capture factor; Including, a second capture / detection reagent, (i) the other of the complementary peptide or polypeptide components of the bioluminescent complex; (ii) a capture agent capable of stably binding to the capture agent, and one of a binding agent capable of directly binding to a cell-associated biomolecule on the cell. and contacting the sample with said set of capture / detection reagents comprising: (d) contacting the cell with a substrate for the bioluminescent complex; (e) monitoring and / or detecting bioluminescence; The method according to

[60] , further comprising:

[65] The method of

[60] , further comprising exposing the cells to a stimulus or condition.

[66] The method according to

[60] , wherein the cells undergo cell death and / or become permeable upon stimulation or conditions.

[67] The method of

[60] further comprising a step of directly binding the capture / detection reagent to the capture factor displayed on any biomolecule on the cell or released from the cell, and / or to a cell-associated biomolecule on the cell, wherein the capture / detection reagent comprising the complementary peptide component and complementary polypeptide component of the bioluminescent complex binds to an adjacent capture factor and / or biomolecule, thereby forming the bioluminescent complex.

[68] The method according to

[60] , wherein the biomolecule reactive group non-specifically forms a covalent bond with a cell-associated biomolecule on or within the cell.

[69] The method according to

[60] , wherein the probe is cell-permeable and capable of labeling a biomolecule within the cell.

[70] The method according to

[60] , wherein the probe is cell-impermeable and capable of labeling a biomolecule on the surface of the cell.

[71] The method according to

[60] , wherein the capture factor is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate.

[72] The method of

[60] , wherein the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular protein of the cell.

[73] The method according to

[60] , wherein the probe is cell-permeable.

[74] The method described in

[71] , wherein the modified dehalogenase enzyme comprises at least 70% sequence identity to SEQ ID NO: 16.

[75] The method described in

[60] , wherein the complementary peptide component and the complementary polypeptide component both comprise sequences having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, are capable of forming an enhanced bioluminescence complex.

[76] The method described in

[75] , wherein the complementary peptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 10, and the complementary polypeptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 9.

[77] The method described in

[60] , wherein the complementary system comprises two peptide components and one polypeptide component, at least one of which is not fused to a capture agent and both comprise a sequence having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, the capture agent binds to the capture factor, thereby forming an enhanced bioluminescence complex.

[78] The method described in

[77] , wherein the complementary peptide component and the further peptide component both comprise a sequence having at least 70% sequence identity to SEQ ID NO: 14, and the complementary polypeptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 12.

[79] The method described in

[78] , wherein the complementary peptide component and the additional peptide component comprise sequences having at least 70% sequence identity to SEQ ID NOs: 11 and 13.

[80] The method according to any one of

[60] to

[79] , wherein the peptide component and the polypeptide component do not efficiently form a bioluminescent complex unless they are brought into contact with each other, thereby promoting binding of the capture agent to the capture factor.

[81] The method according to any one of

[60] to

[80] above, wherein the bioluminescent complex exhibits a significant increase in bioluminescence in the presence of an appropriate substrate, when compared to the bioluminescence of any of the individual peptide and polypeptide components, and any pair of peptide and polypeptide components, in the presence of the appropriate substrate.

[82] The method of

[60] , wherein the substrate of the bioluminescent complex is coelenterazine or a coelenterazine analog.

[83] The method described in

[60] , wherein the stimulus or condition is a therapeutic agent, an immunotherapeutic agent, or a chemotherapeutic agent.

[84] The method according to

[60] , wherein bioluminescence is monitored using a luminometer.

[85] (a) contacting a cell with an effective amount of a probe comprising the compound according to any one of [1] to

[21] , wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or in the cell; (b) removing the cells; (i) complementary polypeptide components of a bioluminescent complex; and (ii) a capture agent capable of stably binding to the capture factor; contacting the antibody with a first capture / detection reagent comprising: (c) removing the cells; (i) the complementary peptide component of a bioluminescent complex; and (ii) a capture agent capable of stably binding to the capture factor; wherein the polypeptide component and the peptide component are capable of forming a bioluminescent complex when brought into proximity with one another; and (d) contacting the cell with a substrate for the bioluminescent complex; (e) monitoring and / or detecting bioluminescence; A method comprising:

[86] The method of

[85] , further comprising removing unbound probe from the contact with the cell.

[87] The method of

[86] , wherein the unbound probe is removed from contact with the cells by washing the cells.

[88] The method of

[86] , wherein the unbound probe is removed from contact with the cells by centrifugation.

[89] The method of

[86] , wherein the unbound probe is removed from contact with the cell between steps (a) and (b).

[90] The method of

[86] , further comprising placing the cells in a mixed cell population with unlabeled cells.

[91] The method described in

[85] , further comprising exposing the cells to a stimulus or condition.

[92] The method according to

[91] , wherein the cells undergo cell death and / or become permeable upon stimulation or conditions.

[93] The method of

[91] , wherein the cells are exposed to the stimulus or condition between steps (a) and (b).

[94] The method of

[91] , wherein the cells are exposed to the stimulus or condition between steps (c) and (d).

[95] The method of

[91] , wherein the cells are exposed to the stimulus or condition between steps (d) and (e).

[96] The method according to

[91] , wherein the cells undergo cell death and / or become permeable, thereby exposing and / or releasing biological molecules from the cells.

[97] The method described in

[96] , wherein the bioluminescent complex is formed by binding of a capture / detection reagent comprising complementary peptide and polypeptide components of the bioluminescent complex to an adjacent capture factor and / or biological molecule.

[98] The method according to

[85] , wherein the biomolecule reactive group non-specifically forms a covalent bond with a cell-associated biomolecule on or within the cell.

[99] The method according to

[85] , wherein the probe is cell-permeable and capable of labeling a biomolecule within the cell.

[100] The method according to

[85] , wherein the probe is cell-impermeable and capable of labeling a biomolecule on the surface of the cell.

[101] The method according to

[85] , wherein the capture factor is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate.

[102] The method of

[85] , wherein the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular protein of the cell.

[103] The method described in

[101] , wherein the modified dehalogenase enzyme comprises at least 70% sequence identity to SEQ ID NO: 16.

[104] The method described in

[85] , wherein the complementary peptide component and the complementary polypeptide component both comprise sequences having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, are capable of forming an enhanced bioluminescence complex.

[105] The method described in

[104] , wherein the complementary peptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 10, and the complementary polypeptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 9.

[106] The method described in

[85] , wherein the complementary system comprises two peptide components and one polypeptide component, at least one of which is not fused to a capture agent and both comprise a sequence having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, the capture agent binds to the capture factor, thereby forming an enhanced bioluminescence complex.

[107] The method described in

[106] , wherein the complementary peptide component and the further peptide component both comprise a sequence having at least 70% sequence identity to SEQ ID NO: 14, and the complementary polypeptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 12.

[108] The method described in

[107] , wherein the complementary peptide component and the additional peptide component comprise sequences having at least 70% sequence identity to SEQ ID NOs: 11 and 13.

[109] (a) contacting a cell with an effective amount of a probe comprising the compound according to any one of [1] to

[21] , wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or in the cell; (b) removing the cells; (i) a complementary polypeptide or peptide component of a bioluminescent complex; and (ii) a capture agent capable of stably binding to the capture factor; contacting the antibody with a first capture / detection reagent comprising: (c) removing the cells; (i) a complementary peptide or polypeptide component of a bioluminescent complex; and (ii) a binding agent capable of directly binding to a biomolecule on the cell; wherein the polypeptide component and the peptide component are capable of forming a bioluminescent complex when brought into proximity with one another; (d) contacting the cell with a substrate for the bioluminescent complex; (e) monitoring and / or detecting bioluminescence; A method comprising:

[110] The method according to

[109] , further comprising removing unbound probe from the contact with the cell.

[111] The method of

[110] , wherein the unbound probe is removed from contact with the cells by washing the cells.

[112] The method of

[110] , wherein the unbound probe is removed from contact with the cells by centrifugation.

[113] The method of

[110] , wherein the unbound probe is removed from contact with the cell between steps (a) and (b).

[114] The method according to

[109] , further comprising placing the cells in a mixed cell population with unlabeled cells.

[115] The method described in

[109] , further comprising exposing the cells to a stimulus or condition.

[116] The method according to

[115] , wherein the cells undergo cell death and / or become permeable upon stimulation or conditions.

[117] The method according to

[115] , wherein the cells are exposed to the stimulus or condition between steps (a) and (b).

[118] The method of

[115] , wherein the cells are exposed to the stimulus or condition between steps (c) and (d).

[119] The method of

[115] , wherein the cells are exposed to the stimulus or condition between steps (d) and (e).

[120] The method according to

[116] , wherein the cells undergo cell death and / or become permeable, thereby exposing and / or releasing biological molecules from the cells.

[121] The method described in

[120] , wherein the capture / detection reagent and non-covalent binding / detection moiety comprising the complementary peptide and polypeptide components of the bioluminescent complex bind to adjacent capture factors and / or biological molecules, thereby forming the bioluminescent complex.

[122] The method according to

[109] , wherein the biomolecule reactive group non-specifically forms a covalent bond with a cell-associated biomolecule on or within the cell.

[123] The method according to

[109] , wherein the probe is cell-permeable and capable of labeling a biomolecule within the cell.

[124] The method according to

[109] , wherein the probe is cell-impermeable and capable of labeling a biomolecule on the surface of the cell.

[125] The method of

[109] , wherein the binding agent is an antibody or an antibody fragment.

[126] The method according to

[109] , wherein the binding agent is a biomolecule or a small molecule.

[127] The method described in

[126] , wherein the binding agent is annexin V.

[128] The method of

[109] , wherein the capture factor is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate.

[129] The method of

[109] , wherein the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular protein of the cell.

[130] The method described in

[128] , wherein the modified dehalogenase enzyme comprises at least 70% sequence identity to SEQ ID NO: 16.

[131] The method described in

[109] , wherein the complementary peptide component and the complementary polypeptide component both comprise sequences having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, are capable of forming an enhanced bioluminescence complex.

[132] The method described in

[131] , wherein the complementary peptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 10, and the complementary polypeptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 9.

[133] The method described in

[109] , wherein the complementary system comprises two peptide components and one polypeptide component, at least one of which is not fused to a capture agent and both comprise a sequence having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, the capture agent binds to the capture factor, thereby forming an enhanced bioluminescence complex.

[134] The method described in

[133] , wherein the complementary peptide component and the additional peptide component both comprise a sequence having at least 70% sequence identity to SEQ ID NO: 14, and the complementary polypeptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 12.

[135] The method according to

[134] , wherein the complementary peptide component and the additional peptide component comprise sequences having at least 70% sequence identity to SEQ ID NOs: 11 and 13.

[136] (a) contacting a cell population with an effective amount of a probe comprising the compound according to any one of [1] to

[21] , wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or in the cells; (b) removing unbound probe and dead cells from the population of labeled live cells; (c) fixing and permeabilizing the population of labeled live cells; (d) The fixed and permeabilized cell population is (i) complementary polypeptide components of a bioluminescent complex; and (ii) a capture agent capable of stably binding to the capture factor; contacting the antibody with a first capture / detection reagent comprising: (e) treating the fixed and permeabilized cell population with (i) the complementary peptide component of a bioluminescent complex; and (ii) a fluorophore; wherein the polypeptide component and the peptide component are capable of forming a bioluminescent complex; and (f) detecting luminescence and / or fluorescence; A method comprising:

[137] The method according to

[136] , wherein the luminescence and / or fluorescence is detected by fluorescence imaging, flow cytometry, and / or luminescence measurement.

[138] The method of

[136] , wherein the capture factor is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate.

[139] The method of

[136] , wherein the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an intracellular or extracellular protein of the cell.

[140] The method described in

[138] , wherein the modified dehalogenase enzyme comprises at least 70% sequence identity to SEQ ID NO: 16.

[141] The method described in

[136] , wherein the complementary peptide component and the complementary polypeptide component both comprise sequences having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, are capable of forming an enhanced bioluminescence complex.

[142] The method described in

[141] , wherein the complementary peptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 11, and the complementary polypeptide component comprises a sequence having at least 70% sequence identity to SEQ ID NO: 9.

[143] A kit comprising a multifunctional probe containing the compound according to any one of [1] to

[21] above and a pair of capture / detection reagents, Each capture / detection reagent is (i) a capture agent capable of stably binding to the capture factor; (ii) a complementary peptide or polypeptide component of a bioluminescent complex; The kit further comprises a fusion product of

[144] The kit described in

[143] , wherein the complementary peptide component and the complementary polypeptide component both have 70% or more sequence identity to the sequence of SEQ ID NO: 17.

[145] The kit described in

[144] , wherein the first capture / detection reagent comprises a complementary peptide having 70% or more sequence identity to SEQ ID NO: 11, and the second capture / detection reagent comprises a complementary peptide having 70% or more sequence identity to SEQ ID NO: 13.

[146] The kit described in

[143] above, further comprising a complementary polypeptide having 70% or more sequence identity to SEQ ID NO: 12 or 19.

[147] The kit described in

[143] , wherein the capture agent has 70% or more sequence identity to SEQ ID NO: 16.

[148] The kit according to

[143] , further comprising a coelenterazine substrate or a coelenterazine analog substrate.

[149] (a)(i) a capture agent capable of stably binding to the capture factor; (ii) a peptide component capable of forming a bioluminescent complex with a complementary polypeptide component; a first capture / detection reagent comprising a fusion of (b)(i) a capture agent capable of stably binding to the capture agent; (ii) a polypeptide component capable of forming a bioluminescent complex with the peptide component; a second capture / detection reagent comprising a fusion of A kit comprising:

[150] The kit described in

[149] , wherein the capture agent has 70% or more sequence identity to SEQ ID NO: 16.

[151] The kit described in

[149] , wherein both the peptide component and the complementary polypeptide component have 70% or more sequence identity to the sequence of SEQ ID NO: 17.

[152] The kit according to

[151] , further comprising a coelenterazine substrate or a coelenterazine analog substrate.

[153] The kit described in

[151] , wherein the peptide has 70% or more sequence identity to SEQ ID NO: 10 and the complementary polypeptide has 70% or more sequence identity to SEQ ID NO: 9.

[154] (a)(i) a capture agent capable of stably binding to the capture factor; (ii) a first peptide component capable of forming a bioluminescent complex with a complementary peptide component and a complementary polypeptide component; a first capture / detection reagent comprising a fusion of (b)(i) a capture agent capable of stably binding to the capture agent; (ii) a complementary peptide component and a second peptide component capable of forming a bioluminescent complex with the complementary polypeptide component; a second capture / detection reagent comprising a fusion of A kit comprising: The kit, wherein the first peptide component and the second peptide component both comprise at least 70% sequence identity to SEQ ID NO: 14, and the polypeptide component comprises at least 70% sequence identity to SEQ ID NO: 12 or 19.

[155] The kit described in

[154] , wherein the capture agent has 70% or more sequence identity to SEQ ID NO: 16.

[156] (a) contacting a cell with an effective amount of a probe comprising the compound according to

[21] , wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on the surface of the cell; (b) removing the cells; (i) a capture agent capable of stably binding to the capture factor; (ii) a first complementary peptide or polypeptide component of a bioluminescent complex; contacting the antibody with a first capture / detection reagent comprising: (c) removing the cells; (i) a second biomolecule-reactive group capable of stably binding to a second biomolecule; and (ii) a second complementary peptide or polypeptide component of the bioluminescent complex; and contacting the second capture / detection reagent with a second capture / detection reagent comprising: (d) contacting the cell with a substrate for the bioluminescent complex; (e) exposing the cells to a stimulus or condition; (f) binding the capture / detection reagents to the capture agent and second biomolecule, respectively, where binding of adjacent capture / detection reagents results in the formation of the bioluminescent complex; (g) monitoring and / or detecting bioluminescence in the presence of a bioluminescent substrate, wherein the amount of bioluminescence is proportional to the amount of biomolecules on the outer surface of the cells; A method comprising:

[157] (a) contacting a cell with an effective amount of a probe comprising the compound according to

[21] , wherein the biomolecule reactive group forms a covalent bond with a biomolecule on the surface of the cell; (b) washing the cells to remove unbound probe from the cells; (c) removing the cells; (i) a capture agent capable of stably binding to the capture factor; (ii) a first complementary peptide or polypeptide component of a bioluminescent complex; contacting the antibody with a first capture / detection reagent comprising: (d) removing the cells; (i) a second biomolecule reactive group / binding group capable of stably binding to a second biomolecule; (ii) a second complementary peptide or polypeptide component of the bioluminescent complex; and contacting the second capture / detection reagent with a second capture / detection reagent comprising: (e) exposing the cells to a stimulus or condition; (f) contacting the cells with a substrate for the bioluminescent complex; (g) binding the capture / detection reagents to the capture agent and second biomolecule, respectively, where binding of adjacent capture / detection reagents results in the formation of the bioluminescent complex; (h) monitoring and / or detecting bioluminescence in the presence of a bioluminescent substrate; A method comprising:

[158] The method according to

[156] or

[157] , wherein the capture factor is a haloalkyl group and the capture agent is a modified dehalogenase enzyme capable of forming a covalent bond with a haloalkane substrate.

[159] The method according to

[156] or

[157] , wherein the biomolecule reactive group is a succinimidyl ester capable of forming a covalent bond with an amine on an extracellular biomolecule of the cell.

[160] The method described in

[159] , wherein the modified dehalogenase enzyme comprises at least 70% sequence identity to SEQ ID NO: 16.

[161] The method described in

[156] or

[157] , wherein the complementary peptide component and the complementary polypeptide component both comprise sequences having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, are capable of forming an enhanced bioluminescence complex.

[162] The method of

[156] or

[157] , wherein the complementary system comprises two peptide components and one polypeptide component, at least one of which is not fused to a capture agent and both comprise a sequence having at least 70% sequence identity to SEQ ID NO: 17, and when they are contacted with each other, the capture agent binds to the capture factor, thereby forming an enhanced bioluminescence complex.

[163] The method of

[156] or

[157] , wherein the complementary peptide component and the complementary polypeptide component together comprise a sequence having at least 70% sequence identity to SEQ ID NO: 17, together with one or more additional peptide or polypeptide components fused to additional capture agents, and when they are contacted with each other, the capture agent binds to the capture agent, thereby forming an enhanced bioluminescence complex.

[164] The method of

[162] or

[163] , wherein the peptide component and polypeptide component do not efficiently form a bioluminescent complex unless they are brought into contact with each other, thereby promoting binding of the capture agent to the capture factor.

[165] The method described in any one of

[157] to

[164] , wherein the bioluminescent complex exhibits a significant increase in bioluminescence in the presence of an appropriate substrate when compared to the bioluminescence of any combination of the peptide and polypeptide components in the presence of the appropriate substrate.

[166] The method of

[156] or

[157] , wherein the substrate of the bioluminescent complex is coelenterazine or a coelenterazine analog.

[167] The method according to

[156] or

[157] , wherein the stimulus or condition causes the second biological molecule to be released from the cell.

[168] The method of

[156] or

[157] , wherein the stimulus or condition results in cell death, membrane permeabilization, active transport, an immune response, enhanced diffusion, secretion from the cell, or release in vesicles.

[169] The method according to

[156] or

[157] , wherein bioluminescence is monitored using a luminometer.

[170] (a) contacting a cell with an effective amount of a probe comprising the compound according to any one of [1] to

[21] , wherein the biomolecule-reactive group forms a covalent bond with a cell-associated biomolecule on or in the cell; (b) contacting the cells with a capture / detection reagent comprising a capture agent capable of stably binding to the capture agent and a complementary peptide or polypeptide component of a bioluminescent complex; (c) contacting the cells with a detection reagent comprising a fluorophore linked to the other of the complementary peptide and polypeptide components of the bioluminescent complex; (d) contacting the cell with a substrate for the bioluminescent complex; (e) binding the capture / detection reagent to the capture agent and allowing the detection reagent to form a bioluminescent complex with the capture / detection reagent; (f) monitoring and / or detecting fluorescence from the fluorophore and / or bioluminescence from the bioluminescent complex in the presence of a bioluminescent substrate; A method comprising:

[171] The method described in

[170] , wherein the complementary peptide comprises at least 70% sequence identity to SEQ ID NO: 11 and the complementary polypeptide comprises at least 70% sequence identity to SEQ ID NO: 9.

[172] The method of

[170] , further comprising exposing the cells to a stimulus or condition capable of causing cell death.

[173] (a) a multifunctional probe comprising a biomolecule reactive group linked to a capture agent; (b) a first capture agent fused to a polypeptide component of the bioluminescent complex; (c) a second capture or biomolecule binding agent fused to the peptide component of the bioluminescent complex; and (d) a substrate for said bioluminescent complex; A kit comprising: when the peptide and polypeptide components are brought into appropriate proximity / positioning with one another, the capture agent binds to the adjacent capture factor, thereby forming a bioluminescent complex; the bioluminescent complex produces luminescence in the presence of the substrate; The kit.

[174] A method comprising contacting a cell or a sample containing a cell with a component of the kit described in

[173] .

[175] (a) a multifunctional probe comprising a biomolecule reactive group linked to a capture agent; (b) a first capture agent fused to a polypeptide component of the bioluminescent complex; (c) a fluorophore linked to a peptide component of the bioluminescent complex; (d) a substrate for said bioluminescent complex; A kit comprising: the peptide component and the polypeptide component form the bioluminescent complex when they are allowed to co-localize with each other without external facilitation; the bioluminescent complex produces luminescence in the presence of the substrate; The kit.

[176] The kit described in

[175] , wherein the peptide component has at least 70% sequence identity to SEQ ID NO: 11 and the polypeptide component has at least 70% sequence identity to SEQ ID NO: 9.

[177] A method comprising contacting a cell or a sample containing a cell with a component of the kit described in

[175] .

[178] (a) a multifunctional probe comprising a biomolecule reactive group linked to a capture agent; (b) a first capture agent fused to a polypeptide or peptide component of the bioluminescent complex; and (c) a target-specific binding agent fused to another of the polypeptide or peptide components of the bioluminescent complex; and (d) a substrate for said bioluminescent complex; A kit comprising: when the peptide and polypeptide components are brought into appropriate proximity / positioning with one another, the capture agent and target-specific binding agent bind to the adjacent capture agent and target, thereby forming the bioluminescent complex; the bioluminescent complex produces luminescence in the presence of the substrate; The kit.

[179] A method comprising contacting a cell or a sample containing a cell with a component of the kit described in

[178] .

[180] (a) a multifunctional probe comprising a biomolecule reactive group linked to a capture agent; (b) a first capture agent fused to a first peptide component of the bioluminescent complex; (c) a second capture agent fused to a second peptide component of the bioluminescent complex; and (d) a polypeptide component of a bioluminescent complex; and (e) a substrate for said bioluminescent complex; A kit comprising: the peptide and polypeptide components form the bioluminescent complex by binding the capture agent to the adjacent capture factor when the peptide components are brought / positioned appropriately close to each other; the bioluminescent complex produces luminescence in the presence of the substrate; The kit.

[181] A method comprising contacting a cell or a sample containing a cell with a component of the kit described in

[180] .

[0171] array WT OgLuc (SEQ ID NO: 1): MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPDGSLLFRVTINGVTGWRLCENILA WT OgLuc Lg (SEQ ID NO: 2): MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPD WT OgLuc β9 (SEQ ID NO: 3): GSLLFRVTIN WT OgLuc β10 (SEQ ID NO: 4): GVTGWRLCENILA NanoLuc (SEQ ID NO: 5): MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITTVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA NanoLuc Lg (SEQ ID NO: 6): MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITTVTGTLWNGNKIIDERLINPD NanoLuc β9 (SEQ ID NO: 7): GSLLFRVTINV NanoLuc β10 (SEQ ID NO: 8): GVTGWRLCERILA LgBiT (SEQ ID NO: 9):MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN SmBiT (SEQ ID NO: 10): VTGYRLFEEIL HiBiT (SEQ ID NO: 11): VSGWRLFKKIS LgTrip (3546) (SEQ ID NO: 12): MKHHHHHHVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD SmTrip9 (SEQ ID NO: 13): GSMLFRVTINS β9 / β10 dipeptide (SEQ ID NO: 14): GSMLFRVTINSVSGWRLFKKIS SmTrip10 (SEQ ID NO: 15): VSGWRLFKKIS HaloTag (SEQ ID NO: 16): MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG The base sequence of the entire bioluminescent complex (SEQ ID NO: 17): MVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPDGSMLFRVTINSVTGYRLFEEIL WT strand 9-SmBiT (SEQ ID NO: 18): GSMLFRVTINSVTGYRLFEEIL LgTrip 3546(1-8) (SEQ ID NO: 19):MVFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD

Claims

1. 1. A method for labeling a cell, comprising contacting said cell with an effective amount of a compound of formula A-X-B or a salt thereof; A is -(CH 2 ) n -halogen, n is 4 to 8; X has at least the following structure: is a linker comprising the linker is a combination of groups selected from -O-, -NH-, -C(O)-, ester (-C(O)O-), amide (-C(O)NH-), carbamate ester (-NHC(O)O-), urea (-NHC(O)NH-), phenylene, linear or branched alkylene, a group having the structure -(CH 2 ) n -halogen (n is 4 to 8) bonded to a carbon or nitrogen atom, and a fluorophore bonded to a carbon or nitrogen atom; B is a biomolecule reactive group selected from succinimidyl esters and maleimides; The method, wherein the biomolecule reactive group forms a covalent bond with a cell-associated biomolecule on or within the cell, thereby labeling the cell.

2. The cells are subjected to a set of capture / detection reagents, a first capture / detection reagent comprising one of the complementary peptide or polypeptide components of a bioluminescent complex linked to a capture agent capable of stably binding to said capture agent; contacting a second capture / detection reagent set with the set of capture / detection reagents, the second capture / detection reagent comprising the other of the complementary peptide or polypeptide components of the bioluminescent complex linked to one of a capture agent capable of stably binding to the capture agent or a binder capable of directly binding to a cell-associated biomolecule on the cell; contacting the cells with a substrate for the bioluminescent complex; Monitoring and / or detecting bioluminescence; Further comprising: the bioluminescent conjugate is a luciferase-based conjugate; The method of claim 1 , wherein the capture agent is a modified haloalkane dehydrogenase protein.

3. removing unbound probe from contact with said cells; The method of claim 1 further comprising:

4. contacting the cells with a first capture / detection reagent comprising a complementary polypeptide component of a bioluminescent complex linked to a capture agent capable of stably binding to the capture agent; contacting the cells with a second capture / detection reagent comprising a complementary peptide component of a bioluminescent complex linked to a capture agent capable of stably binding to the capture agent, wherein the polypeptide component and the peptide component are capable of forming a bioluminescent complex when brought into proximity with one another; contacting the cells with a substrate for the bioluminescent complex; Monitoring and / or detecting bioluminescence; Further comprising: the bioluminescent conjugate is a luciferase-based conjugate; The method of claim 1 , wherein the capture agent is a modified haloalkane dehydrogenase protein.

5. contacting the cells with a first capture / detection reagent comprising a complementary polypeptide or peptide component of a bioluminescent complex linked to a capture agent capable of stably binding to the capture agent; contacting the cells with a second non-covalent binding / detection reagent comprising a complementary peptide or polypeptide component of a bioluminescent complex linked to a binding agent capable of directly binding to a cell-associated biomolecule on the cells, wherein the polypeptide and peptide components are capable of forming a bioluminescent complex when brought into proximity with one another; contacting the cells with a substrate for the bioluminescent complex; Monitoring and / or detecting bioluminescence; Further comprising: the bioluminescent conjugate is a luciferase-based conjugate; The method of claim 1 , wherein the capture agent is a modified haloalkane dehydrogenase protein.

6. removing unbound probe and dead cells from the population of labeled live cells; Fixing and permeabilizing the population of labeled live cells; contacting the fixed and permeabilized cell population with a first capture / detection reagent comprising a complementary polypeptide component of a bioluminescent complex linked to a capture agent capable of stably binding to said capture agent; contacting the fixed and permeabilized cell population with a second capture / detection reagent comprising a complementary peptide component of a bioluminescent complex linked to a fluorophore, wherein the polypeptide component and the peptide component are capable of forming a bioluminescent complex; detecting luminescence and / or fluorescence; Further comprising: the bioluminescent conjugate is a luciferase-based conjugate; The method of claim 1 , wherein the capture agent is a modified haloalkane dehydrogenase protein.

7. contacting the cells with a first capture / detection reagent comprising a capture agent linked to a first complementary peptide or polypeptide component of a bioluminescent complex, the capture agent being capable of stably binding to the capture agent; contacting the cells with a second capture / detection reagent linked to a second complementary peptide or polypeptide component of the bioluminescent complex, the second capture / detection reagent comprising a second biomolecule-reactive group capable of stably binding to a second biomolecule; contacting the cells with a substrate for the bioluminescent complex; exposing the cells to a stimulus or condition; binding the capture / detection reagents to the capture agent and second biomolecule, respectively, where binding of adjacent capture / detection reagents results in the formation of the bioluminescent complex; monitoring and / or detecting bioluminescence in the presence of a bioluminescent substrate, wherein the amount of bioluminescence is proportional to the amount of biomolecules on the outer surface of the cell; Further comprising: the bioluminescent conjugate is a luciferase-based conjugate; The method of claim 1 , wherein the capture agent is a modified haloalkane dehydrogenase protein.

8. washing the cells to remove unbound probe from the cells; contacting the cells with a first capture / detection reagent comprising a capture agent linked to a first complementary peptide or polypeptide component of a bioluminescent complex, the capture agent being capable of stably binding to the capture agent; contacting the cells with a second capture / detection reagent that comprises a second biomolecule reactive / binding group that is capable of stably binding to a second biomolecule, linked to a second complementary peptide or polypeptide component of the bioluminescent complex; exposing the cells to a stimulus or condition; contacting the cells with a substrate for the bioluminescent complex; binding the capture / detection reagents to the capture agent and second biomolecule, respectively, where binding of adjacent capture / detection reagents results in the formation of the bioluminescent complex; monitoring and / or detecting bioluminescence in the presence of a bioluminescent substrate; Further comprising: the bioluminescent conjugate is a luciferase-based conjugate; The method of claim 1 , wherein the capture agent is a modified haloalkane dehydrogenase protein.

9. contacting the cells with a capture / detection reagent comprising a capture agent capable of stably binding to the capture agent linked to a complementary peptide or polypeptide component of a bioluminescent complex; contacting the cells with a detection reagent comprising a fluorophore linked to the other of the complementary peptide and polypeptide components of the bioluminescent complex; contacting the cells with a substrate for the bioluminescent complex; binding the capture / detection reagent to the capture agent and allowing the detection reagent to form a bioluminescent complex with the capture / detection reagent; monitoring and / or detecting fluorescence from said fluorophore and / or bioluminescence from said bioluminescent complex in the presence of a bioluminescent substrate; Further comprising: the bioluminescent conjugate is a luciferase-based conjugate; The method of claim 1 , wherein the capture agent is a modified haloalkane dehydrogenase protein.

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