Protein complexes for proximity-based labeling of the intracellular microenvironment

Abstract

Described herein are protein complexes and related methods for proximity-based labeling of various intracellular or intercellular microenvironments. In some embodiments, the protein complex comprises a protein substrate with an associated ubiquitin E3 ligase, wherein a transition metal complex is coupled to the protein substrate. In some embodiments, one or more intermediate molecular species, such as small molecules and / or proteins, are present between the ubiquitin E3 ligase and the protein substrate.

Description

[Technical Field]

[0001] Related application data This application claims priority pursuant to Article 8 of the Patent Cooperation Treaty to U.S. Provisional Patent Application No. 63 / 428,899, filed November 30, 2022, which is incorporated herein by reference in its entirety.

[0002] Field The present invention relates to compositions and methods for intracellular proximity-based labeling, and in particular to protein conjugates for proximity-based labeling of diverse intracellular microenvironments. [Background technology]

[0003] Protein proximity labeling has emerged as a powerful approach for profiling protein interaction networks. The ability to label associated or nearby proteins through proximity labeling can have important implications for further understanding the cellular environment and biological role of a protein of interest. Current proximity labeling methods all involve the use of enzyme-generated reactive intermediates that label nearby proteins on several selected amino acid residues through diffusion or physical contact. Despite the novel impact of this technology, the conversion of these reactive intermediates, such as phenoxy radicals (t), through peroxidase activation, has not been well documented. 1 / 2 >100 μs), or biotin-AMP via biotin ligase (t 1 / 2 The inherent stability of enzymes (>60 s) can facilitate diffusion far away from their point of origin. As a result, these enzyme-generated reactive intermediates pose challenges to profiling within close microenvironments. Furthermore, the large size of enzymes, their dependence on specific amino acids for labeling, and the inability to temporally control these labeling systems pose further challenges to profiling within confined spatial regions. Given these limitations, new approaches for proximity-based labeling are needed. Summary of the Invention

[0004] In consideration of the above-mentioned disadvantages, the present invention provides a protein complex and related methods for proximity-based labeling of various intracellular or intercellular microenvironments. In some embodiments, the protein complex comprises a protein substrate with an associated ubiquitin E3 ligase, wherein a transition metal complex is coupled to the protein substrate. In some embodiments, one or more intermediate molecular species, such as small molecules and / or proteins, are present between the ubiquitin E3 ligase and the protein substrate.

[0005] Furthermore, the transition metal complex may be coupled to the protein substrate through a variety of mechanisms or architectures. In some embodiments, the transition metal complex is bound to a haloalkane dehalogenase, which in turn is coupled to the protein substrate. Alternatively, the transition metal complex is coupled to the protein substrate through protein trans-splicing, where the transition metal complex is first coupled to a split intein. The split intein carrying the transition metal complex may be an N-intein or a C-intein. In further embodiments, the transition metal complex is coupled to a derivatized or unnatural amino acid of the protein substrate. Suitable click chemistry moieties of the transition metal complex and / or the derivatized / unnatural amino acid may be selected from the group consisting of, for example, DBCO, BCN, TCO, tetrazine, alkyne, and azide. In some embodiments, the transition metal complex is represented by formula (I) herein: [ka] wherein M is a transition metal; A, D, E, G, Y and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, and the 1 to 4 optional ring substituents are each independently selected from alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O - , -C(O)OR 8, and -R 9 OH; R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloaklylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2)R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is a counter ion, and n is an integer from 0 to 20. It is of the type.

[0006] In another aspect, methods for characterizing protein degradation are described herein. In some embodiments, the methods for characterizing protein degradation include providing a protein substrate having an associated transition metal complex and activating a protein labeling agent with the transition metal complex to a reactive intermediate. The reactive intermediate couples with a ubiquitin E3 ligase to form a complex with the protein substrate. The transition metal complex may be associated with the protein substrate by any of the architectures described herein. The transition metal complex may have an electronic structure for transferring energy to the protein labeling agent to generate the reactive intermediate. In some embodiments, the energy transfer is Dexter energy transfer or electron transfer. In some embodiments, the energy transfer to the protein labeling agent may occur from an excited state of the transition metal complex electronic structure. The excited state of the complex may be, for example, a singlet excited state or a triplet excited state. Alternatively, the energy transfer to the protein labeling agent, including electron transfer, may occur from the ground state of the transition metal complex electronic structure. The transition metal complex may be of formula (I) provided above.

[0007] The diffusion range of reactive intermediates can be tailored to specific microenvironment mapping (proximity-based labeling) considerations and can be limited to the nanometer scale. Thus, reactive intermediates will react with or crosslink proteins or other biomolecules within their diffusion range, or, if no proteins or biomolecules are present, be quenched by the surrounding environment. The cooperative forces between the transition metal complex and the protein labeling agent can map the environment near the protein substrate with high resolution. In this manner, ubiquitin E3 ligases that form complexes with protein substrates can be labeled or determined. Other molecular species that form complexes, including small molecules and / or proteins that recruit or ligate ubiquitin E3 ligases to protein substrates, can also be elucidated with the labeling agent.

[0008] In some embodiments, the method further includes coupling a small molecule to the protein substrate, where the small molecule provides for attachment of the ubiquitin E3 ligase to the protein substrate, thereby generating a protein complex. A reactive intermediate may also be coupled to the small molecule in some embodiments. Structural information obtained from the ubiquitin E3 ligase complexed with the protein substrate and / or small molecule may be used to develop one or more additional small molecules that can be engineered to recruit the ubiquitin E3 ligase to different protein substrates. A library of molecular glues may be constructed for a variety of E3 ligases and / or other molecules.

[0009] In another aspect, a protein complex and system for mapping intracellular protein microenvironments are described herein. In some embodiments, the protein complex comprises a transition metal complex bound to a haloalkane dehalogenase, and the haloalkane dehalogenase is coupled to the protein. Alternatively, the transition metal complex is coupled to the protein through protein trans-splicing, where the transition metal complex is first coupled to a split intein. The split intein carrying the transition metal complex may be an N-intein or a C-intein. In a further embodiment, the transition metal complex is coupled to a derivatized or unnatural amino acid of the protein. Suitable click chemistry moieties of the transition metal complex and / or the derivatized amino acid may be selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide. The transition metal complex may be of formula (I) herein.

[0010] The protein or biomolecule labeling agent can act in conjunction with the transition metal complex to generate a reactive intermediate as described above, which can label proteins and / or other chemical and / or biomolecular species in the local microenvironment of the protein complex.

[0011] In another aspect, methods for profiling the microenvironment surrounding an intracellular protein are described herein. In some embodiments, the methods include forming a protein complex comprising a transition metal complex coupled to an intracellular protein, and activating a protein labeling agent with the transition metal complex to a reactive intermediate. The reactive intermediate couples to a protein or other biomolecule within a predetermined range of the protein complex. The protein complex, transition metal complex, and protein labeling agent may have any of the compositions and / or properties described herein.

[0012] These and other embodiments are further described in the detailed description that follows. [Brief explanation of the drawings]

[0013] [Figure 1] Illustrative of the transition metal complexes described herein, according to some embodiments. [Figure 2] 1 illustrates the binding of a transition metal complex described herein to a protein through a haloalkane dehalogenase, according to some embodiments. [Figure 3] 1 illustrates the attachment of a transition metal complex to a protein substrate through an unnatural amino acid and subsequent labeling with a protein labeling agent, according to some embodiments. [Figure 4] 4 illustrates the results of protein substrate labeling using the substrate-bound transition metal complexes of FIG. 3. [Figure 5] 1 illustrates validation of a split intein splicing strategy using histone H3.1 and the iridium transition metal complexes described herein. [Figure 6] 1 illustrates validation of a split intein splicing strategy using histone H3.1 and the iridium transition metal complexes described herein. [Figure 7] 1 conceptually illustrates mapping of the ERα interactome after estradiol and fulvestrant binding, according to some embodiments. [Figure 8] 1 conceptually illustrates the identification of species involved in the ERα degradation pathway as revealed by the proximity labeling method described herein, according to some embodiments. [Figure 9] 1 conceptually illustrates the identification of species involved in the ERα degradation pathway as revealed by the proximity labeling method described herein, according to some embodiments. [Figure 10] 1 conceptually illustrates the identification of species involved in the ERα degradation pathway as revealed by the proximity labeling method described herein, according to some embodiments. [Figure 11] 1 conceptually illustrates the identification of species involved in the ERα degradation pathway as revealed by the proximity labeling table described herein, according to some embodiments. [Figure 12] 1 illustrates the use of information obtained from the protein interactome mapping methods described herein for the development of E3 ligase activators and molecular glues, according to some embodiments. [Figure 13] 1 is a volcano plot detailing species identified in ERα interactome mapping after binding of estradiol and fulvestrant, according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0014] The embodiments described herein may be more readily understood by reference to the following detailed description and examples, as well as the preceding and following descriptions. However, the elements, devices, and methods described herein are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

[0015] definition The term "alkyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain saturated hydrocarbon group, optionally substituted with one or more substituents. For example, alkyl refers to C1 to C6 30 or C1~C 18 may be.

[0016] The term "alkenyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon double bond and optionally substituted with one or more substituents.

[0017] The term "alkynyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon triple bond and optionally substituted with one or more substituents.

[0018] The term "aryl," as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system optionally substituted with one or more ring substituents.

[0019] The term "heteroaryl," as used herein, alone or in combination, refers to an aromatic monocyclic or polycyclic ring system in which one or more of the ring atoms is an element other than carbon, for example, nitrogen, boron, oxygen, and / or sulfur.

[0020] The term "heterocycle," as used herein, alone or in combination, refers to a monocyclic or polycyclic ring system in which one or more atoms of the ring system is an element other than carbon, such as boron, nitrogen, oxygen, and / or sulfur or phosphorus, and the ring system is optionally substituted with one or more ring substituents. Heterocyclic ring systems may include aromatic and / or non-aromatic rings, including rings with one or more points of unsaturation.

[0021] The term "cycloalkyl," as used herein, alone or in combination, refers to a non-aromatic mono- or multi-cyclic ring system, optionally substituted with one or more ring substituents.

[0022] The term "heterocycloalkyl," as used herein, alone or in combination, refers to a non-aromatic monocyclic or polycyclic ring system in which one or more of the atoms in the ring system is an element other than carbon, for example, boron, nitrogen, oxygen, sulfur, or phosphorus, alone or in combination, and the ring system is optionally substituted with one or more ring substituents.

[0023] The term "alkoxy," as used herein, alone or in combination, refers to the moiety RO--, where R is alkyl, alkenyl, or aryl as defined above.

[0024] The term "halo," as used herein, alone or in combination, refers to an element of Group VIIA of the periodic table (a halogen). Depending on the chemical environment, a halo can be in a neutral or anionic state.

[0025] Terms not specifically defined herein have their ordinary meaning in the art.

[0026] I. protein complexes Described herein are protein complexes and related methods for proximity-based labeling of various intracellular or intercellular microenvironments. In some embodiments, the protein complex comprises a protein substrate with an associated ubiquitin E3 ligase, and a transition metal complex is coupled to the protein substrate. In some embodiments, one or more intermediate molecular species, such as small molecules and / or proteins, are located between the ubiquitin E3 ligase and the protein substrate. The small molecules and / or proteins may act as linkers between the ubiquitin E3 ligase and the protein substrate in the formation of a ternary complex. The small molecules and / or proteins may, for example, exhibit a recognition motif that binds or recruits the ubiquitin E3 ligase to the protein substrate. In some embodiments, the small molecules and / or proteins are attached to the ubiquitin E3 ligase before forming a ternary complex with the protein substrate. Alternatively, the small molecules and / or proteins are attached to the protein substrate before forming a ternary complex with the ubiquitin E3 ligase. In a further embodiment, the protein substrate and ubiquitin E3 ligase may first interact, and the small molecule and / or protein subsequently bind to form a ternary complex.

[0027] As described herein, a transition metal complex can activate a protein labeling agent to a reactive intermediate, where the reactive intermediate couples to the ubiquitin E3 ligase of the ternary complex. The protein labeling agent can be any species consistent with the technical principles described herein. In some embodiments, the protein labeling agent is a diazirine or azide. As described herein, the transition metal complex can form multiple reactive intermediates, allowing for labeling of several biomolecular species within the ternary complex. Thus, in some embodiments, the reactive intermediate can also be coupled to a small molecule and / or protein that binds to the ubiquitin E3 ligase and the protein substrate. Thus, the ubiquitin E3 ligase and / or the small molecule / protein of the ternary complex can be labeled with the protein labeling agent. Having a transition metal complex coupled to a protein substrate can enable profiling of the microenvironment near the protein substrate, including identifying biomolecular species involved in the degradation of the protein substrate or protein degradation. In this manner, it is also possible to further elucidate protein degradation pathways and recognize structural motifs to develop libraries of small molecule species that enable the attachment of ubiquitin E3 ligase to protein substrates and other proteins of interest.

[0028] In another embodiment, the protein complex comprises a protein substrate with an associated ubiquitin E3 ligase, wherein a transition metal complex is coupled to the ubiquitin E3 ligase. In such an embodiment, the protein substrate may be labeled with a protein labeling agent through a reactive intermediate. Furthermore, any small molecules and / or proteins that form a ternary complex with the substrate and the ubiquitin E3 ligase may also be labeled with a protein labeling agent. Having a transition metal complex coupled to the ubiquitin E3 ligase may enable profiling of the microenvironment near the E3 ligase. In this manner, proteins that interact with the E3 ligase for ubiquitination may be identified. Such identification may also uncover previously unknown interactions, thereby expanding the use of E3 ligases for broader proteolytic applications.

[0029] In some embodiments, the protein complexes described herein are located in an intracellular environment. In some embodiments, for example, the protein complex is located in the nucleus of a cell. Alternatively, the protein complex may be localized in the cytoplasm or membrane of a cell. The transition metal complex may interact with the protein substrate or E3 ligase through a covalent bond. In other embodiments, the transition metal complex may interact with the protein substrate or E3 ligase through electrostatic and / or van der Waals interactions.

[0030] In some embodiments, the transition metal complex couples to a protein substrate or E3 ligase of a protein complex through interaction with a haloalkane dehalogenase (HaloTag). The haloalkane dehalogenase may be co-expressed with the protein substrate, for example, using standard cloning methods. The transition metal complex may be functionalized with a haloalkane moiety for covalent attachment to the haloalkane dehalogenase, resulting in coupling of the protein of interest. Figure 2 illustrates the attachment of a transition metal complex described herein to a protein via a haloalkane dehalogenase, according to some embodiments.

[0031] Alternatively, transition metal complexes may be conjugated to derivatized or unnatural amino acids of protein substrates or E3 ligases. Suitable click chemistry moieties for transition metal complexes and / or unnatural amino acids may be selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide. Figure 3 illustrates the validation of such a conjugation strategy with cereblon (CRBN). Transfected cells were used to express CRBN with BCNK. An iridium complex containing a tetrazine moiety was introduced to conjugate the iridium complex to CRBN. The CRBN modulator CC-885 was added to bind to CRBN, and diazo-biotin was added as a protein labeling agent. As further described herein, the iridium complex was irradiated to form a reactive intermediate through energy transfer to diazo-biotin. The reactive intermediate binds to a biomolecular species within a predetermined range of CRBN-Ir. Figure 4 illustrates the results of a labeling study. In the presence of CRBN and CC-885, CRBN and GSPT1 are enriched. In the absence of CC-885, only CRBN is enriched.

[0032] Furthermore, in some embodiments, the transition metal complex is coupled to a protein substrate or E3 ligase through protein trans-splicing, and the transition metal complex is first coupled to a split intein. The split intein carrying the transition metal complex can be an N-intein or a C-intein. Figures 5 and 6 illustrate validation of the split intein splicing strategy with histone H3.1 and the iridium transition metal complexes described herein.

[0033] In some embodiments, the transition metal complex has Formula I: [ka] wherein M is a transition metal; A, D, E, G, Y and Z are independently selected from C and N; R 3 ~R 7each represent 1 to 4 optional ring substituents, and the 1 to 4 optional ring substituents are each independently selected from alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O - , -C(O)OR 8 , and -R 9 OH; R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O2)R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is a counter ion, and n is an integer from 0 to 20. As provided in Formula I, the linking moiety L is optional and therefore may be absent in some embodiments of the transition metal complex.

[0034] Optional Substituent R 3 ~R 7 It is understood that in the absence of a counterion (X), hydrogen occupies the positions on the aryl ring of formula I. Additionally, in some embodiments,- ) is tetraalkylborate, tetrafluoroborate, tetraphenylborate, PF6 - and chlorine.

[0035] The polarity of the transition metal complex is R 3 ~R 7 It is also possible to tailor the R to a particular cellular environment through the selection of R. 3 ~R 7 One or more of R are selected to exhibit hydrophobic, lipophilic, or non-polar properties. 3 ~R 7 One or more of may be alkyl, fluoro, or fluoroalkyl. Transition metal complexes described herein that exhibit hydrophobic, lipophilic, or non-polar properties may also be suitable for placement or passage into an intracellular environment. Transition metal complexes may be capable of passing through cell membranes to map proteins in the intracellular environment in accordance with the principles described herein. Thus, such transition metal complexes are cell-permeable. Figure 1 illustrates various transition metal complexes described herein. In some embodiments, L is selected from R 2 amide combined with a polyethylene glycol (PEG) moiety for linkage to the PEG moiety. As provided in Figure 1, the PEG moiety may be substituted with an alkylene moiety. In some embodiments, for example, R 2 R comprises a haloalkane or click chemistry moiety including, but not limited to, BCN, DBCO, TCO, tetrazine, alkyne, and azide. As exemplified in Figure 1, R 2 These click chemistries may be coupled directly to the linker (L) or through a heteroatom, aryl, or carbonyl.

[0036] The transition metal complex may have an electronic structure that allows energy transfer to the protein labeling agent to generate a reactive intermediate. In some embodiments, the energy transfer is Dexter energy transfer or electron transfer. In some embodiments, energy transfer to the protein labeling agent may occur from an excited state of the transition metal complex electronic structure. The excited state of the complex may be, for example, a singlet excited state or a triplet excited state. In some embodiments, the transition metal complex of Formula I may exhibit a long-lived triplet excited state (T1) that facilitates energy transfer to the protein labeling agent. The T1 state may have a t of, for example, 0.2 to 2 μs. 1 / 2 The transition metal complexes described herein may be photocatalytic and, in some embodiments, absorb light in the visible or infrared region of the electromagnetic spectrum. Absorption of electromagnetic radiation excites the transition metal complex to the S1 state, which can then undergo quantitative intersystem crossing to the T1 state. The transition metal complex can subsequently return to the ground state, S0, via short-range Dexter energy transfer to the protein labeling agent. Energy transfer to the labeling agent activates the labeling agent for reaction with proteins or other biomolecules in the local environment of the protein substrate. The T1 state of the transition metal complex may, in some embodiments, be greater than 60 kcal / mol. The metal center may be selected, for example, from transition metals of the platinum group. In some embodiments, the metal center may be iridium.

[0037] In other embodiments, the complex may be placed in an excited state by interaction with one or more chemical species in the surrounding environment. Alternatively, energy transfer to the protein labeling agent, including electron transfer, may occur from the ground state of the complex electronic structure. Energy transfer from the protein labeling agent to produce a reactive intermediate allows for profiling of the microenvironment near the protein substrate of the ternary complex, as described herein.

[0038] II. Methods and systems for profiling protein degradation or proteolysis In another aspect, methods for characterizing protein degradation or proteolysis are described herein. In some embodiments, the methods for characterizing protein degradation include providing a protein substrate having an associated transition metal complex and activating a protein labeling agent with the transition metal complex to a reactive intermediate. The reactive intermediate couples with a ubiquitin E3 ligase to form a complex with the protein substrate. The transition metal complex may associate with the protein substrate by any architecture described herein, including the mechanisms described in Section I above. Furthermore, the transition metal complex may be of Formula I herein and may have any of the properties described in Section I above.

[0039] As described herein, the transition metal complex may have an electronic structure that allows energy transfer to the protein labeling agent to generate a reactive intermediate. In some embodiments, the energy transfer is Dexter energy transfer or electron transfer. In some embodiments, energy transfer to the protein labeling agent may occur from an excited state of the transition metal complex electronic structure. The excited state of the complex may be, for example, a singlet excited state or a triplet excited state. The excited state of the transition metal complex may occur through one or more mechanisms, including energy absorption by the complex. In some embodiments, the transition metal complex is a photocatalyst, where the excited state is induced by the absorption of one or more photons. In other embodiments, the transition metal complex may be placed in the excited state by interaction with one or more chemical species in the surrounding environment. Alternatively, energy transfer to the protein labeling agent, including electron transfer, may occur from the ground state of the transition metal complex electronic structure. The transition metal complex may be of Formula I, provided above.

[0040] The diffusion range of the reactive intermediate may be tailored to specific microenvironment mapping (proximity-based labeling) considerations and may be limited to the nanometer scale. In some embodiments, for example, the diffusion range of the reactive intermediate may be less than 100 nm, less than 50 nm, less than 10 nm, less than 5 nm, less than 4 nm, less than 3 nm, or less than 2 nm before quenching in the surrounding environment. In some embodiments, the diffusion range may be between 0.5 nm and 10 nm. Thus, the reactive intermediate will react with or crosslink with proteins or other biomolecules within its diffusion range, or, if no proteins or biomolecules are present, will be quenched by the surrounding environment. In this manner, the cooperative forces between the transition metal complex and the protein labeling agent allow the environment near the protein substrate to be mapped with high resolution. Furthermore, in some embodiments, the reactive intermediate will react with or crosslink with proteins or other biomolecules within its diffusion range before quenching. 1 / 2 The reactive intermediate may exhibit, for example, a t of less than 1 to 5 ns. 1 / 2 In further embodiments, the diffusion range can be extended to 5-500 nm through an increase in the half-life of the reactive intermediate. For example, in some embodiments, the reactive intermediate can have a half-life of 1-100 μs or longer. In some embodiments, more than one species of protein labeling agent type can be used in the systems described herein. For example, two or more species with different diffusion ranges can be used in the system. In such embodiments, the different diffusion ranges can allow for labeling at different distances from the transition metal complex, thereby further mapping the interactome of the protein substrate of the ternary complex.

[0041] In some embodiments, the protein labeling agent may be diazirine. Triplet energy transfer from the excited-state photocatalyst may promote the diazirine to its triplet (T1) state. The diazirine triplet undergoes elimination of N2 to release a free triplet carbene, which undergoes spin equilibration on the picosecond time scale to its reactive singlet state (t 1 / 2<1 ns), which crosslinks with nearby proteins or is quenched in an aqueous environment. In some embodiments, the extinction coefficient of the transition metal complex is 3-5 orders of magnitude greater than that of the diazirine.

[0042] Any diazirine consistent with the technical principles discussed herein can be used. Diazirine sensitization can be extended to, for example, a variety of p- and m-substituted aryltrifluoromethyldiazirines, which carry valuable payloads for microscopy and proteomics applications, including free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. Diazirines may be functionalized with markers such as biotin. In some embodiments, the marker is desthiobiotin. Markers can aid in the identification of proteins labeled with protein labeling agents. Markers can be useful, for example, in assay results via Western blots and / or other analytical techniques. In addition to biotin and desthiobiotin, markers can include alkyne, azide, FLAG tags, fluorophores, and chloroalkane functionalities.

[0043] In further embodiments in which the transition metal complex is a photocatalyst, the protein labeling agent may be an azide. Triplet energy transfer from the excited-state photocatalyst may promote nitrene formation from the azide. The reactive nitrene either crosslinks with nearby proteins or is quenched in an aqueous environment. Any azide operable to undergo energy transfer with a transition metal photocatalyst to form a nitrene may be used. In some embodiments, the azide is an aryl azide.

[0044] The cooperative forces between the transition metal complex and the protein labeling agent can map the environment near the protein substrate with high resolution. In this manner, the ubiquitin E3 ligase that forms a complex with the protein substrate can be determined. Other molecular species that form complexes or are in the diffusion range of reactive intermediates can also be elucidated with the labeling agent, including small molecules that recruit or link the ubiquitin E3 ligase to the protein substrate.

[0045] In some embodiments, the method further includes coupling a small molecule to the protein substrate or to the E3 ligase, where the small molecule recruits the ubiquitin E3 ligase for attachment, providing a protein complex. The small molecule may, in some embodiments, be a synthetic molecule, protein, or peptide. Reactive intermediates may also, in some embodiments, be coupled to the small molecule. Structural information obtained from the ubiquitin E3 ligase complexed with the protein substrate and / or small molecule may be used to develop one or more additional small molecules that can be engineered to recruit / attach the ubiquitin E3 ligase to different protein substrates of interest. A library of molecular glues may be constructed for various E3 ligases and / or other molecules. Protein substrates with coupled transition metal complexes along with protein labeling agents provide a system for analyzing the local interactome of protein substrates, including their interactions with E3 ligases. In some embodiments, the protein substrate is a receptor.

[0046] Furthermore, in some embodiments, a method for characterizing protein degradation or proteolysis begins with a ubiquitin E3 ligase having an associated transition metal complex, and activates a protein labeling agent with the transition metal complex to a reactive intermediate. The reactive intermediate is coupled to a protein substrate to form a complex with the E3 ligase. The transition metal complex may associate with the E3 ligase by any of the architectures described herein, including the mechanisms described in Section I above. Having a transition metal complex coupled to a ubiquitin E3 ligase may enable profiling of the microenvironment near the E3 ligase. In this manner, proteins that interact with the E3 ligase for ubiquitination may be identified. Such identification may also uncover previously unknown interactions, thereby expanding the use of E3 ligases for broader proteolytic applications.

[0047] E3 ligases with coupled transition metal complexes described herein, along with protein labeling agents, provide a system for analyzing the local interactome of E3 ligases, including their interactions with diverse proteins.

[0048] III. Methods for mapping intracellular protein interactomes In another aspect, methods for profiling the microenvironment near an intracellular protein are described herein. In some embodiments, the methods include forming a protein complex comprising a transition metal complex coupled to an intracellular protein and activating a protein labeling agent with the transition metal complex to a reactive intermediate. The reactive intermediate couples to a protein or other biomolecule within a predetermined range of the protein complex. The protein complex, transition metal complex, and protein labeling agent may have any of the compositions and / or properties described in Sections I and II above. For example, the transition metal complex may bind to the intracellular protein by any of the mechanisms or architectures described in Section I above. In some embodiments, the reactive intermediates coupled to one or more proteins are involved in one or more degradation pathways of the intracellular protein.

[0049] In some embodiments, the intracellular protein is a receptor. In these embodiments, the protein labeling agent is activated after a molecule binds to the receptor. The molecule binding to the receptor may be the binding of a receptor activator. Alternatively, the molecule binding may be the binding of a receptor inhibitor or degrader. The protein labeling agent may also be activated without any binding to the receptor.

[0050] The aforementioned principles are illustrated schematically in Figures 7-12. Figure 7 illustrates interactome mapping of estrogen receptor alpha (ERα) after separate binding of estradiol and the selective estrogen receptor degrader, fulvestrant. As generally provided in Figure 7, ERα interactome mapping upon estradiol binding may reveal the presence of well-studied oncogenes, oncoproteins, and / or oncogene regulators. In contrast, fulvestrant binding may reveal the presence of tumor suppressor genes, proteins known to suppress oncogenesis, and / or proteins involved in the ERα degradation pathway. Interactome mapping upon fulvestrant binding may identify, for example, protein species JJJ and MMM, known to interact with the ERα degradation machinery, including the appropriate E3 ligase complex for ERα ubiquitination, as illustrated in Figures 8 and 9, respectively. Estradiol binding may also reveal species that interact with or play a role in the ERα degradation machinery, as illustrated in Figures 10 and 11. In Figure 10, a protein species BBB can be identified that is known to post-translationally modify ERα, leading to ERα degradation. The species BBB can be, for example, a kinase involved in the phosphorylation-mediated degradation of ERα. Furthermore, an E3 ligase not previously known to degrade ERα can be elucidated, as shown in Figure 11. Interactome mapping can be used to develop targeted E3 ligase activators for the treatment of breast cancer, as illustrated in Figure 12, and for the development of other molecular glues that degrade proteins of interest. Example 2 herein further illustrates these principles.

[0051] These and other embodiments are further illustrated in the following non-limiting examples. [Example]

[0052] Example 1 - Coupling of transition metal complexes to proteins The protein-transition metal complex conjugate in Figure 2 was prepared as follows. Cells (HEK243T stably expressing HaloTag-G3BP1) were seeded and grown to 95% confluence before labeling. The cells were incubated for 1 hour in DMEM medium containing 5 μM of the iridium photocatalyst in Figure 2. During this period, the iridium photocatalyst crosses the cell membrane and becomes conjugated to the HaloTag-G3BP1 protein. The cells were then incubated for 1 hour in fresh DMEM medium to eliminate or minimize noise from any unbound iridium catalyst.

[0053] Example 2 - ERα interactome mapping considering estradiol / fulvestrant binding The local interactome of ERα following the binding of estradiol and fulvestrant was investigated according to the compositions and methods described herein.

[0054] At 80% confluence, 10 cm plates of MCF7 cells stably expressing FLAG-CfaC-HA-ESR1 were cultured in phenol red-free DMEM supplemented with 10% v / v charcoal-stripped FBS, 1% penicillin / streptomycin, and 1% L-glutamine. After 3 days of culture, cells were treated with 10 μM TAK243 for 1 hour, then co-treated with 100 nM estradiol or 100 nM fulvestrant for 4 hours before harvesting. The resulting cell pellet was washed twice with DPBS and then hypotonicly lysed for 10 minutes on ice using 0.6 mL of RSB buffer (10 mM Tris buffer, 15 mM NaCl, 1.5 mM MgCl2, Roche Complete EDTA-free protease inhibitors, pH 7.6, supplemented with 100 nM estradiol or 100 nM fulvestrant). After isolation of crude nuclei using centrifugation at 400 g for 5 min at 4°C, the nuclei were resuspended in 1 mL of RSE buffer and homogenized using 10 strokes with a loose-pestle Dounce homogenizer.

[0055] Nuclei were again pelleted at 400 g for 5 min at 4°C and resuspended in 0.6 mL of cross-linking buffer (20 mM HEPES, 1.5 mM MgCl, 150 mM KCl, Roche Complete EDTA-free protease inhibitors, pH 7.6, supplemented with 100 nM estradiol or 100 nM fulvestrant), then centrifuged at 400 g for 5 min at 4°C. At this point, pellets equivalent to two 10 cm plates of the same treatment type were combined and resuspended using 400 μL of cross-linking buffer containing 0.3 μM CfaN-Ir. Trans-splicing was allowed to occur for 1 h with rotation at 37°C. The resulting pellet was washed three times with 400 μL of crosslinking buffer, followed by the addition of 500 μM biotin-diazirine in 200 μL of crosslinking buffer. Irradiation was performed for 3 minutes at 4°C using 100% blue light intensity in a Penn PhD Photoreactor M2. The irradiated nuclei were washed twice more with 400 μL of crosslinking buffer, and then lysed on ice for 10 minutes with LB3 buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% sodium lauroyl sarcosinate, Roche Complete EDTA-free protease inhibitor, pH 7.5). The pellet was sonicated twice using a Branson probe-top sonicator at 35% amplitude for 10 seconds and then centrifuged at 18,000 g for 15 minutes at 4°C. The supernatant was used as the nuclear lysate, and the protein concentration was determined using the BCA assay.

[0056] All replicates were diluted to 0.5 mg / mL using binding buffer (25 mM Tris, 150 mM NaCl, 0.2% v / v NP-40, pH 7.5). Lysates were loaded onto 25 μL of prewashed magnetic Sepharose streptavidin beads for 18 hours at 4°C with rotation. The beads were then washed three times with 1% w / v SDS in DPBS, twice with 1 M NaCl in DPBS, once with 10% ethanol in DPBS, and three times with 50 mM ammonium bicarbonate in water. The washed beads were transferred to a fresh Lo-bind tube using 500 μL of 50 mM ammonium bicarbonate, and as much supernatant as possible was removed. The resulting bead pellet was resuspended in 30 μL of 50 mM ammonium bicarbonate containing 0.4 μg of trypsin and incubated overnight at 37°C with rotation. The resulting supernatant was collected using centrifugation and filtered with 0.22 μM cellulose acetate using a Spin-X centrifuge tube filter. The resulting eluate was ready for mass spectrometry analysis using a TimsTOF.

[0057] Figure 13 is a volcano plot detailing the species identified in the interactome mapping after binding of estradiol and fulvestrant. The volcano plot is a composite of results obtained from independent runs of binding estradiol and fulvestrant.

[0058] Various embodiments of the present invention have been described in fulfillment of various objectives of the present invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the present invention.

Claims

1. 1. A protein complex comprising: a protein substrate having an associated ubiquitin E3 ligase, wherein a transition metal complex is coupled to the protein substrate. The protein complex.

2. 2. The protein complex of claim 1, wherein the transition metal complex binds to a haloalkane dehalogenase, and the haloalkane dehalogenase couples to the protein substrate.

3. The protein complex of claim 1 , wherein the transition metal complex binds to an unnatural amino acid of the protein substrate.

4. The protein of claim 3, wherein the unnatural amino acid comprises a click chemistry moiety.

5. The protein complex of claim 4, wherein the unnatural amino acid and the transition metal complex are coupled by click chemistry.

6. 2. The protein complex of claim 1, wherein the transition metal complex couples to the protein substrate through protein trans-splicing, and the transition metal complex is first coupled to a split intein.

7. 7. The protein complex of claim 6, wherein the split intein is an N-intein.

8. 7. The protein complex of claim 6, wherein the split intein is a C-intein.

9. The protein complex of claim 1, further comprising one or more small molecules between the protein substrate and the E3 ligase.

10. The transition metal complex has the formula (I): 【Chemistry 1】 wherein M is a transition metal; A, D, E, G, Y and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, wherein the 1 to 4 optional ring substituents are each independently selected from alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O - , -C(O)OR 8 , and -R 9 OH; R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is a counterion, and n is an integer from 0 to 20. The protein complex of claim 1 ,

11. 1. A method for characterizing protein degradation, comprising: providing a protein substrate having an associated transition metal complex; activating a protein labeling agent with said transition metal complex to a reactive intermediate; coupling the reactive intermediate to a ubiquitin E3 ligase to form a complex with the protein substrate; The method comprising:

12. 12. The method of claim 11, further comprising coupling a small molecule to the protein substrate, wherein the small molecule recruits the attachment of the ubiquitin E3 ligase to provide a protein complex.

13. 13. The method of claim 12, further comprising developing one or more additional small molecules that can be engineered to recruit the ubiquitin E3 ligase to different protein substrates.

14. The transition metal complex has the formula: 【Chemistry 2】 wherein M is a transition metal; A, D, E, G, Y and Z are independently selected from C and N; R 3 ~R 7 each represent 1 to 4 optional ring substituents, wherein the 1 to 4 optional ring substituents are each independently selected from alkyl, heteroalkyl, haloalkyl, haloalkenyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O - , -C(O)OR 8 , and -R 9 OH; R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is alkyl; R 1 is selected from the group consisting of a direct bond, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroalkylene, heteroalkenylene, heterocyclene, and heteroarylene; L is an optional linking moiety selected from the group consisting of amide, ester, sulfonamide, sulfonate, carbamate, and urea; R 2 is an alkyne, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkylnyl, heterocyclyl, hydroxy, carboxyl, halo, alkoxy, maleimide, -C(O)H, -C(O)OR 8 , -OS(O 2 )R 9 , thiol, biotin, oxyamine, and haloalkyl, and R 8 and R 9 is independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl, and N-succinimidyl esters; X - is a counterion, and n is an integer from 0 to 20. The method of claim 11,

15. 13. The method of claim 12, wherein the small molecule is also labeled with the protein labeling agent through the reactive intermediate.

16. 12. The method of claim 11, wherein the transition metal complex binds to a haloalkane dehalogenase, and the haloalkane dehalogenase couples to the protein substrate.

17. 12. The method of claim 11, wherein the transition metal complex binds to an unnatural amino acid of the protein substrate.

18. 18. The method of claim 17, wherein the unnatural amino acid comprises a click chemistry moiety.

19. 12. The method of claim 11, wherein the transition metal complex is coupled to the protein substrate through protein trans-splicing, and the transition metal complex is first coupled to a split intein.

20. The method of claim 11 , wherein the protein substrate is a receptor.

21. 1. A method for profiling the microenvironment near an intracellular protein, comprising: forming a protein complex comprising a transition metal complex coupled to the intracellular protein; activating a protein labeling agent with said transition metal complex to a reactive intermediate; coupling said reactive intermediate to a protein or other biomolecule within a predetermined range of said protein complex; The method comprising:

22. 22. The method of claim 21, wherein forming the protein complex comprises coupling the transition metal complex to a haloalkane dehalogenase, the haloalkane dehalogenase being coupled to the intracellular protein.

23. 22. The method of claim 21, wherein the protein complexation comprises coupling the metal complex to an unnatural amino acid of the intracellular protein.

24. 24. The method of claim 23, wherein the transition metal complex is coupled to the unnatural amino acid via click chemistry.

25. 22. The method of claim 21, wherein the transition metal complex is coupled to the intracellular protein through protein trans-splicing, and wherein the transition metal complex is first coupled to a split intein.

26. 26. The method of claim 25, wherein the split intein is an N-intein.

27. 26. The method of claim 25, wherein the split intein is a C-intein.

28. 22. The method of claim 21, wherein the reactive intermediate couples to a protein involved in a degradation pathway of the intracellular protein.

29. 29. The method of claim 28, wherein the reactive intermediate couples to an E3 ligase of the intracellular protein.

30. 22. The method of claim 21, wherein the intracellular protein is a receptor.

31. 31. The method of claim 30, wherein the protein labeling agent is activated after molecular binding to the receptor.

32. 32. The method of claim 31 , wherein the molecular binding comprises binding to an activator of the receptor.

33. 32. The method of claim 31 , wherein the molecular binding comprises binding to an inhibitor or degrader of the receptor.

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