Protein complexes for proximity-based labeling of intracellular microenvironments

Abstract

Described herein are protein complexes and related methods for proximity-based labeling of various intracellular or intercellular microenvironments. In some embodiments, a protein complex comprises a protein substrate having a ubiquitin E3 ligase associated therewith wherein a transition metal complex is conjugated to the protein substrate. In some embodiments, one or more intermediate molecule species (e.g., small molecules and / or proteins) are located between the ubiquitin E3 ligase and the protein substrate.

Description

[0001] Related application data

[0002] This application claims priority under Article 8 of the Patent Cooperation Treaty to U.S. Provisional Patent Application Serial No. 63 / 428,899, filed on November 30, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to compositions and methods for proximity-based labeling within cells, and in particular to protein complexes for proximity-based labeling of various intracellular microenvironments. Background Art

[0004] Protein proximity labeling has become a powerful method for analyzing protein interaction networks. The ability to label related or bystander proteins by proximity labeling has important implications for further understanding the cellular environment and biological role of the protein of interest. Current proximity labeling methods all involve the use of enzyme-generated reactive intermediates that label neighboring proteins at a few selected amino acid residues through diffusion or physical contact. Although the technology has had a transformative impact, these reactive intermediates, such as phenoxyl radicals (t 1 / 2 >100 μs) or biotin-AMP via biotin ligase (t 1 / 2 The inherent stability of these enzymes (>60s) promotes diffusion away from their point of origin. Consequently, the reactive intermediates generated by these enzymes present challenges for analysis within a compact microenvironment. Furthermore, the large enzyme size, the dependency of labeling on certain amino acids, and the inability to temporally control these labeling systems pose additional challenges for analysis within confined spatial regions. Given these limitations, new proximity-based labeling approaches are needed. Summary of the Invention

[0005] In view of the aforementioned shortcomings, protein complexes and related methods are described herein for proximity-based labeling of various intracellular or intercellular microenvironments. In some embodiments, the protein complex comprises a protein substrate having an ubiquitin E3 ligase associated therewith, wherein a transition metal complex is coupled to the protein substrate. In some embodiments, one or more intermediate molecular species (e.g., small molecules and / or proteins) are located between the ubiquitin E3 ligase and the protein substrate.

[0006] In addition, the transition metal complex can be coupled to the protein substrate through a variety of mechanisms or structures. In some embodiments, the transition metal complex is bonded to a haloalkane dehalogenase, which is coupled to the protein substrate. Alternatively, the transition metal complex is coupled to the protein substrate through protein trans-splicing, and the transition metal complex is initially coupled to a cleavage-type intein. The cleavage-type intein carrying the transition metal complex can be an N-intein or a C-intein. In a further embodiment, the transition metal complex is bonded to a derivatization or non-natural amino acid of the protein substrate. For example, the suitable click chemistry portion of the transition metal complex and / or derivatization / non-natural amino acid can be selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide. In some embodiments, the transition metal complex has the following formula (I):

[0007]

[0008] Where M is a transition metal;

[0009] wherein A, D, E, G, Y and Z are independently selected from C and N;

[0010] where R 3 to R 7 each represents one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - 、-C(O)OR 8 and -R 9 OH group, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is an alkyl group;

[0011] where R 1 is selected from the group consisting of a direct bond, an alkylene group, an alkenylene group, a cycloalkylene group, a cycloalkenylene group, an arylene group, a heteroalkylene group, a heteroalkenylene group, a heterocyclolene group, and a heteroarylene group;

[0012] wherein L is an optional linking moiety selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; and

[0013] R 2 Selected from alkynyl, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, hydroxyl, carboxyl, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 、-OS(O2)R 9 , thiol, biotin, hydroxylamine and halogenated alkyl, wherein R 8 and R9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl and N-succinimidyl ester; wherein X - is a counter ion, and n is an integer from 0 to 20.

[0014] On the other hand, methods for characterizing protein degradation or proteolysis are described herein. In some embodiments, the method for characterizing protein degradation comprises: providing a protein substrate having a transition metal complex associated therewith; and activating a protein marker into a reactive intermediate using the transition metal complex. The reactive intermediate is coupled to a ubiquitin E3 ligase to form a complex with the protein substrate. The transition metal complex can be associated with the protein substrate through any structure described herein. The transition metal complex can have an electronic structure for transferring energy to the protein marker to produce a reactive intermediate. In some embodiments, the energy transfer is a Dexter energy transfer or an electron transfer. In some embodiments, the energy transfer to the protein marker can originate from an excited state of the electronic structure of the transition metal complex. For example, the excited state of the complex can be a singlet excited state or a triplet excited state. Alternatively, the energy transfer (including electron transfer) to the protein marker can originate from the ground state of the electronic structure of the transition metal complex. The transition metal complex can have formula (I) provided above.

[0015] The diffusion radius of the reactive intermediate can be adjusted to adapt to specific microenvironment location analysis (based on adjacent labeling) considerations and can be limited to nanometer scale. Therefore, the reactive intermediate will react or cross-link with the protein or other biomolecules within the diffusion radius, or if there is no protein or biomolecule, it will be quenched by the surrounding environment. The high resolution of the local environment of the protein substrate can be located by the synergistic effect between the transition metal complex and the protein labeling agent. In this way, the ubiquitin E3 ligase that forms a complex with the protein substrate can be marked or measured. Other molecular species that form the complex can also be elucidated using labeling agents, including small molecules and / or proteins that recruit or are connected to the protein substrate by the ubiquitin E3 ligase.

[0016] In some embodiments, the method further comprises coupling a small molecule to a protein substrate, which small molecule provides for attachment of the ubiquitin E3 ligase to the protein substrate, thereby generating a protein complex. In some embodiments, a reactive intermediate can also be coupled to a small molecule. Structural information derived from ubiquitin E3 ligases that form complexes with protein substrates and / or small molecules can be used to develop one or more additional small molecules that can be used to recruit ubiquitin E3 ligases to different protein substrates. Molecular libraries can be constructed for various E3 ligases and / or other molecules.

[0017] On the other hand, protein complexes and systems for localizing and analyzing the intracellular protein microenvironment are described herein. In some embodiments, the protein complex comprises a transition metal complex bonded to a haloalkane dehalogenase, which is coupled to a protein. Alternatively, the transition metal complex is coupled to the protein via protein trans-splicing, with the transition metal complex initially coupled to a cleavage-type intein. The cleavage-type intein carrying the transition metal complex can be an N-intein or a C-intein. In further embodiments, the transition metal complex is bonded to a derivatized or non-natural amino acid of the protein. Suitable click chemistry moieties of the transition metal complex and / or the derivatized amino acid can be selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkynes, and azides. The transition metal complex can have formula (I) herein.

[0018] As described above, protein labeling agents or biomolecule labeling agents can work with transition metal complexes to generate reactive intermediates that can label proteins and / or other chemical and / or biomolecule species in the local microenvironment of the protein complex.

[0019] In another aspect, methods for analyzing the local microenvironment of intracellular proteins 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 into a reactive intermediate using the transition metal complex. The reactive intermediate is coupled to a protein or other biomolecule within a predetermined radius of the protein complex. The protein complex, transition metal complex, and protein labeling agent can have any of the compositions and / or properties described herein.

[0020] These and other embodiments are further described in the detailed description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are transition metal complexes described herein, according to some embodiments.

[0022] Figure 2 Shown is the binding of a transition metal complex described herein to a protein by a haloalkane dehalogenase, according to some embodiments.

[0023] Figure 3 Methods for conjugating a transition metal complex to a protein substrate via an unnatural amino acid and subsequently labeling with a protein labeling agent are shown according to some embodiments.

[0024] Figure 4 Shows the use of Figure 3 The results of protein substrate labeling with substrate-binding transition metal complexes.

[0025] Figure 5 and Figure 6 Shown is the validation of the split-intein splicing strategy using histone H3.1 and an iridium transition metal complex as described herein.

[0026] Figure 7 Conceptually illustrates the mapping analysis of the ERa interactome following estradiol and fulvestrant binding, according to some embodiments.

[0027] Figures 8 to 11 Conceptually illustrates the identification of species involved in the ERa degradation pathway as elucidated by the proximity labeling method described herein, according to some embodiments.

[0028] Figure 12 It is shown that, according to some embodiments, information obtained from the protein interactome mapping analysis methods described herein is used to develop E3 ligase activators and molecular glues.

[0029] Figure 13 is a volcano plot detailing species identified in ERa interactome mapping analysis following the combination of estradiol and fulvestrant, according to some embodiments. DETAILED DESCRIPTION

[0030] By referring to the following specific embodiments and examples and their description before and after, the embodiments described herein can be more easily understood. However, the elements, devices and methods described herein are not limited to the specific embodiments presented in the specific embodiments and examples. It should be appreciated that these embodiments are merely illustrations of the principles of the present invention. Without departing from the spirit and scope of the present invention, many modifications and adaptations will be apparent to those skilled in the art.

[0031] definition

[0032] The term "alkyl" as used herein, alone or in combination, refers to a straight or branched chain saturated hydrocarbon group optionally substituted with one or more substituents. For example, an alkyl group may be C1-C 30 or C1-C 18 .

[0033]

[0046] The term "alkenyl," as used herein, alone or in combination, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond and optionally substituted with one or more substituents.

[0034]

[0066] The term "alkynyl," as used herein, alone or in combination, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond and optionally substituted with one or more substituents.

[0035] The term "aryl," as used herein, alone or in combination, refers to an aromatic monocyclic or multicyclic ring system optionally substituted with one or more ring substituents.

[0036] 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, such as nitrogen, boron, oxygen, and / or sulfur.

[0037] The term "heterocycle," as used herein, alone or in combination, refers to a monocyclic or polycyclic ring system in which one or more atoms in the ring system are elements other than carbon (e.g., boron, nitrogen, oxygen, and / or sulfur or phosphorus), and wherein 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.

[0038] The term "cycloalkyl," as used herein, alone or in combination, refers to a non-aromatic monocyclic or multicyclic ring system optionally substituted with one or more ring substituents.

[0039] The term "heterocycloalkyl," as used herein, alone or in combination, refers to a non-aromatic monocyclic or multicyclic ring system in which one or more atoms in the ring system is an element other than carbon, such as boron, nitrogen, oxygen, sulfur, or phosphorus (alone or in combination), and wherein the ring system is optionally substituted with one or more ring substituents.

[0040] The term "alkoxy," as used herein, alone or in combination, refers to the group RO-, where R is alkyl, alkenyl, or aryl as defined above.

[0041] The term "halogen" as used herein, alone or in combination, refers to an element of Group VIIA of the Periodic Table of the Elements (halogen). Depending on the chemical environment, a halogen can be in a neutral or anionic state.

[0042] Terms not specifically defined herein are given their ordinary meanings in the art.

[0043] I. protein complex

[0044] This article describes protein complexes and related methods for labeling various intracellular or intercellular microenvironments based on proximity. In certain embodiments, the protein complex comprises a protein substrate having an ubiquitin E3 ligase associated therewith, wherein a transition metal complex is coupled to the protein substrate. In certain embodiments, one or more intermediate molecule species (e.g., small molecules and / or proteins) are located between the ubiquitin E3 ligase and the protein substrate. Small molecules and / or proteins can act as a linker between the ubiquitin E3 ligase and the protein substrate in the formation of a ternary complex. For example, small molecules and / or proteins can exhibit a recognition motif that binds or recruits the ubiquitin E3 ligase to the protein substrate. In certain embodiments, small molecules and / or proteins are attached to the ubiquitin E3 ligase before forming a ternary complex with the protein substrate. Alternatively, small molecules and / or proteins are attached to the protein substrate before forming a ternary complex with the ubiquitin E3 ligase. In other embodiments, the protein substrate and the ubiquitin E3 ligase can initially form an interaction, and small molecules and / or proteins are subsequently combined to form a ternary complex.

[0045] As described herein, transition metal complexes can activate protein labeling agents into reactive intermediates, wherein the reactive intermediates are coupled 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 diazomethane or azide. As described herein, transition metal complexes can form a variety of reactive intermediates that allow labeling of a wide range of biomolecule species within the radius of the ternary complex. Therefore, in some embodiments, the reactive intermediates can also be coupled to small molecules and / or proteins that bind to the ubiquitin E3 ligase and the protein substrate. Thus, the ubiquitin E3 ligase and / or small molecules / proteins of the ternary complex can be labeled with protein labeling agents. Coupling transition metal complexes to protein substrates can allow analysis of the local microenvironment of the protein substrate, including identification of biomolecule species involved in the degradation or proteolysis of the protein substrate. In this way, proteolytic pathways can be further elucidated and structural motifs can be identified for the development of a library of small molecule species capable of binding the ubiquitin E3 ligase to the protein substrate and other proteins of interest.

[0046] In an alternative embodiment, the protein complex comprises a protein substrate having an associated ubiquitin E3 ligase, wherein the transition metal complex is coupled to the ubiquitin E3 ligase. In such an embodiment, the protein substrate can be labeled with a protein labeling agent via a reactive intermediate. In addition, any small molecule and / or protein that forms a ternary complex with the substrate and the ubiquitin E3 ligase can also be labeled with a protein labeling agent. Coupling the transition metal complex with the ubiquitin E3 ligase can allow analysis of the local microenvironment of the E3 ligase. In this way, proteins that interact with the E3 ligase for ubiquitination can be identified. Such identification can discover previously unknown interactions, thereby expanding the use of E3 ligases to a wider range of protein degradation applications.

[0047] In some embodiments, the protein complexes described herein are located in the intracellular environment. For example, in some embodiments, the protein complex is located in the nucleus of a cell. Alternatively, the protein complex can be located in the cytoplasm or cell membrane of a cell. The transition metal complex can interact with the protein substrate or E3 ligase through covalent bonding. In other embodiments, the transition metal complex can interact with the protein substrate or E3 ligase through electrostatic interactions and / or van der Waals interactions.

[0048] In some embodiments, the transition metal complex is coupled to a protein substrate or E3 ligase of a protein complex through interaction with a haloalkane dehalogenase (HaloTag). For example, a haloalkane dehalogenase can be co-expressed with a protein substrate using standard cloning procedures. The transition metal complex can be functionalized with a haloalkane moiety for covalent binding to the haloalkane dehalogenase, resulting in coupling of the protein of interest. Figure 2 Shown is the binding of a transition metal complex described herein to a protein by a haloalkane dehalogenase, according to some embodiments.

[0049] Alternatively, the transition metal complex can be bonded to a derivatized amino acid or non-natural amino acid of a protein substrate or E3 ligase. Suitable click chemistry moieties for the transition metal complex and / or non-natural amino acid can be selected from the group consisting of DBCO, BCN, TCO, tetrazine, alkyne, and azide. Figure 3Validation of this binding strategy using cereblon (CRBN) is shown. Transfected cells were used to express CRBN using BCNK. An iridium complex containing a tetrazine moiety was introduced to bind the iridium complex to CRBN. The CRBN modulator CC-885 was added to bind CRBN, and diazo-biotin was added as a protein labeling agent. The iridium complex was irradiated to form a reactive intermediate by energy transfer to the diazo-biotin, as further described herein. The reactive intermediate binds to biomolecular species within a predetermined radius of CRBN-Ir. Figure 4 Results of labeling studies are shown. In the presence of CRBN and CC-885, CRBN and GSPT1 were enriched. In the absence of CC-885, only CRBN was enriched.

[0050] Furthermore, in some embodiments, the transition metal complex is coupled to a protein substrate or an E3 ligase via protein trans-splicing, and the transition metal complex is initially coupled to a cleaved intein. The cleaved intein carrying the transition metal complex can be an N-intein or a C-intein. Figure 5 and Figure 6 Shown is the validation of the split-intein splicing strategy using histone H3.1 and an iridium transition metal complex as described herein.

[0051] In some embodiments, the transition metal complex has the following Formula I:

[0052]

[0053] Wherein M is a transition metal;

[0054] wherein A, D, E, G, Y and Z are independently selected from C and N;

[0055] where R 3 to R 7 each represents one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - 、-C(O)OR 8 and -R 9 OH group, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is an alkyl group;

[0056] where R 1 is selected from the group consisting of a direct bond, an alkylene group, an alkenylene group, a cycloalkylene group, a cycloalkenylene group, an arylene group, a heteroalkylene group, a heteroalkenylene group, a heterocyclolene group, and a heteroarylene group;

[0057] wherein L is an optional linking moiety selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; and

[0058] R 2 Selected from alkynyl, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, hydroxyl, carboxyl, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 、-OS(O2)R 9 , thiol, biotin, hydroxylamine and halogenated alkyl, wherein R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl and N-succinimidyl ester; wherein X - is a counterion, and n is an integer from 0 to 20. As provided in Formula I, the linking moiety L is optional and thus may not be present in some embodiments of the transition metal complex.

[0059] It is understood that in the absence of the optional substituent R 3 to R 7 In the case of , hydrogen occupies the position on the aryl ring of Formula I. In addition, in some embodiments, the counterion (X - ) can be selected from tetraalkyl borate, tetrafluoroborate, tetraphenyl borate, PF6 - and chlorine.

[0060] The polarity of the transition metal complex can be determined by the 3 to R 7 In some embodiments, for example, R 3 to R 7 One or more of are selected to exhibit hydrophobic, lipophilic or non-polar characteristics. In some embodiments, for example, R 3 to R 7 One or more of the groups may be alkyl, fluorine, or fluoroalkyl. The transition metal complexes described herein that exhibit hydrophobic, lipophilic, or non-polar properties may be suitable for placement in or entry into the intracellular environment. According to the principles described herein, transition metal complexes can penetrate cell membranes for localization and analysis of proteins within the intracellular environment. Thus, such transition metal complexes are cell permeable. Figure 1 Various transition metal complexes described herein are shown. In some embodiments, L is a 2 The polyethylene glycol (PEG) portion of the amide is attached. Figure 1 In some embodiments, for example, R 2Contains halogenated alkanes or click chemistry moieties, including but not limited to BCN, DBCO, TCO, tetrazines, alkynes, and azides. Figure 1 As shown, R 2 These click chemistries can be coupled directly to the linker (L) or through a heteroatom, an aryl group, or a carbonyl group.

[0061] The transition metal complex may have an electronic structure for transferring energy to a protein labeling agent to produce a reactive intermediate. In some embodiments, the energy transfer is a Dexter energy transfer or an electron transfer. In some embodiments, the energy transfer to the protein labeling agent may originate from an excited state of the electronic structure of the transition metal complex. For example, the excited state of the complex may be 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. For example, the T1 state may have a t of 0.2-2 μs. 1 / 2 . The transition metal complexes described herein can be photocatalytic and, in some embodiments, absorb light in the visible or infrared region of the electromagnetic spectrum. The absorption of electromagnetic radiation can excite the transition metal complex to the S1 state, followed by quantitative intersystem crossing to the T1 state. The transition metal complex can then undergo short-range Dexter energy transfer to the protein labeling agent and return to the ground state S0. The energy transfer to the labeling agent activates the labeling agent to react with proteins or other biomolecules in the local environment of the protein substrate. In some embodiments, the T1 state of the transition metal complex can be greater than 60 kcal / mol. For example, the metal center can be selected from a transition metal of the platinum group. In some embodiments, the metal center can be iridium.

[0062] In other embodiments, the complex can be in an excited state by interacting with one or more chemical species in the surrounding environment. Alternatively, energy transfer (including electron transfer) to the protein labeling agent can originate from the ground state of the complex's electronic structure. As described herein, energy transfer to the protein labeling agent to generate a reactive intermediate allows analysis of the microenvironment local to the protein substrate of the ternary complex.

[0063] II. Methods and systems for analyzing protein degradation or proteolysis

[0064] In another aspect, methods for characterizing protein degradation or proteolysis are described herein. In some embodiments, the method for characterizing protein degradation comprises: providing a protein substrate having a transition metal complex associated therewith; and activating a protein labeling agent into a reactive intermediate using the transition metal complex. The reactive intermediate is coupled to a ubiquitin E3 ligase to form a complex with the protein substrate. The transition metal complex can associate with the protein substrate via any structure described herein, including the mechanism described in Section 1 above. In addition, the transition metal complex can have Formula I herein and have any of the properties described in Section 1 above.

[0065] As described herein, the transition metal complex can have an electronic structure for transferring energy to the protein labeling agent to produce a 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 can originate from an excited state of the transition metal complex electronic structure. For example, the excited state of the complex can be a singlet excited state or a triplet excited state. The excited state of the transition metal complex can be generated by one or more mechanisms, including energy absorption by the complex. In some embodiments, the transition metal complex is a photocatalyst, wherein the excited state is induced by absorbing one or more photons. In other embodiments, the transition metal complex can be in an excited state by interacting with one or more chemical species in the surrounding environment. Alternatively, the energy transfer (including electron transfer) to the protein labeling agent can originate from the ground state of the transition metal complex electronic structure. The transition metal complex can have Formula I provided above.

[0066] The diffusion radius of reactive intermediates can be adjusted to adapt to specific microenvironment location analysis (based on adjacent labels) considerations and can be limited to nanometer scale. In some embodiments, for example, before quenching in the surrounding environment, the diffusion radius of reactive intermediates can be less than 100nm, less than 50nm, less than 10nm, less than 5nm, less than 4nm, less than 3nm or less than 2nm. In some embodiments, the diffusion radius can be 0.5nm to 10nm. Therefore, reactive intermediates will react or crosslink with the protein or other biomolecules in the diffusion radius, or if there is no protein or biomolecule, they are quenched by the surrounding environment. In this way, the high resolution of the protein substrate local environment can be carried out location analysis by the synergistic effect between transition metal complexes and protein labeling agents. In addition, in some embodiments, reactive intermediates can show a t less than 5ns, less than 4ns or less than 2ns before quenching. 1 / 2 For example, reactive intermediates can exhibit t values less than 1–5 ns. 1 / 2. In other embodiments, by extending the half-life of the reactive intermediate, the diffusion radius can be extended to between 5-500 nm. 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 type of protein labeling agent can be used in the system described herein. For example, two or more types with different diffusion radii can be used in the system. In such embodiments, different diffusion radii can allow labeling at different distances from the transition metal complex, thereby further positioning the interaction group of the protein substrate of the ternary complex for analysis.

[0067] In some embodiments, the protein labeling agent can be diazomethane. Triplet energy transfer from an excited-state photocatalyst can promote diazomethane into its triplet state (T1). The diazomethane triplet state undergoes elimination of N2 to release a free triplet carbene, which undergoes spin equilibrium on a picosecond timescale to its reactive singlet state (t 1 / 2 <1 ns), the singlet state is either cross-linked to nearby proteins or quenched in an aqueous environment. In some embodiments, the extinction coefficient of the transition metal complex is 3 to 5 orders of magnitude greater than the extinction coefficient of diazomethane.

[0068] Any diazomethane is in accordance with the technical principles discussed herein. For example, diazomethane sensitization can be extended to a variety of p- and m-substituted aryl trifluoromethyl diazomethanes, which provide valuable payloads for microscopy and proteomics applications, including free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. Diazomethane can be functionalized with markers such as biotin. In certain embodiments, the marker is desthiobiotin. The marker can help identify proteins labeled by protein markers. For example, the marker can be used to determine the results by Western blotting and / or other analytical techniques. In addition to biotin and desthiobiotin, the marker can include alkynes, azides, FLAG tags, fluorophores, and chloroalkane functional groups.

[0069] In another embodiment where the transition metal complex is a photocatalyst, the protein labeling agent can be an azide. Triplet energy transfer from the excited state photocatalyst can promote the formation of a nitrene from the azide. The reactive nitrene either cross-links with nearby proteins or is quenched in an aqueous environment. Any azide that can transfer energy with the transition metal photocatalyst and form a nitrene can be used. In some embodiments, the azide is an aryl azide.

[0070] The synergistic effect between transition metal complexes and protein labeling agents can be used to perform high-resolution localization analysis of the local environment of protein substrates. In this way, ubiquitin E3 ligases that form complexes with protein substrates can be determined. Other molecular species that form complexes or are within the diffusion radius of reactive intermediates can also be elucidated using labeling agents, including small molecules that recruit or attach ubiquitin E3 ligases to protein substrates.

[0071] In some embodiments, the method further includes coupling a small molecule to a protein substrate or coupling the small molecule to an E3 ligase, wherein the small molecule recruits the attachment of the ubiquitin E3 ligase to provide a protein complex. In some embodiments, the small molecule can be a synthetic molecule, a protein, or a peptide. In some embodiments, a reactive intermediate can also be coupled to a small molecule. Structural information derived from ubiquitin E3 ligases that form complexes with protein substrates and / or small molecules can be used to develop one or more additional small molecules that can be used to recruit / attach ubiquitin E3 ligases to different protein substrates of interest. Molecular gel libraries can be constructed for various E3 ligases and / or other molecules. Protein substrates with transition metal complexes coupled thereto are combined with protein labeling agents to provide a system for exploring the local interaction group (including interactions with E3 ligases) of protein substrates. In some embodiments, the protein substrate is a receptor.

[0072] In addition, in some embodiments, a method for characterizing protein degradation begins with a ubiquitin E3 ligase having a transition metal complex associated therewith, and using the transition metal complex to activate a protein labeling agent into a reactive intermediate. The reactive intermediate is coupled to the protein substrate to form a complex with the E3 ligase. The transition metal complex can associate with the E3 ligase via any structure described herein, including the mechanism described in Section 1 above. Coupling the transition metal complex to the ubiquitin E3 ligase can allow analysis of the local microenvironment of the E3 ligase. In this way, proteins that interact with the E3 ligase for ubiquitination can be identified. Such identification can discover previously unknown interactions, thereby expanding the use of E3 ligases to a wider range of protein degradation applications.

[0073] E3 ligases with transition metal complexes coupled thereto, combined with protein labeling agents, provide a system for probing the local interactome of E3 ligases, including interactions with various proteins.

[0074] III. Methods for mapping and analyzing the protein interactome in cells

[0075] On the other hand, this paper describes a method for analyzing the local microenvironment of intracellular proteins. In some embodiments, the method includes: forming a protein complex comprising a transition metal complex coupled to an intracellular protein, and utilizing the transition metal complex to activate a protein labeling agent into a reactive intermediate. The reactive intermediate is coupled to a protein or other biomolecule within a predetermined radius of the protein complex. The protein complex, the transition metal complex, and the protein labeling agent can have any composition and / or characteristics described in Sections I and II above. For example, the transition metal complex can bind to the intracellular protein through any mechanism or structure described in Section I above. In some embodiments, the reactive intermediate is coupled to one or more proteins involved in one or more degradation pathways of the intracellular protein.

[0076] In some embodiments, the intracellular protein is a receptor. In such embodiments, the protein marker is activated after molecular binding to the receptor. The molecular binding to the receptor can be binding to a receptor activator. Alternatively, the molecular binding can be binding to an inhibitor or degrader of the receptor. The protein marker can also be activated in the absence of any binding to the receptor.

[0077] Figures 7 to 12 The aforementioned principle is schematically illustrated in FIG. Figure 7 Figure 2 shows the interaction map analysis of estrogen receptor α (ERα) after binding of estradiol and the selective estrogen receptor degrader fulvestrant. Figure 7 As generally provided in the literature, ERα interactome mapping analysis following estradiol binding can reveal the presence of well-studied oncogenes, oncoproteins, and / or regulators of oncogenes. Conversely, fulvestrant binding can reveal the presence of tumor suppressors, proteins known to inhibit carcinogenesis, and / or proteins involved in the ERα degradation pathway. For example, fulvestrant interactome mapping analysis following fulvestrant binding can identify protein classes JJJ and MMM that are known to interact with the ERα degradation machinery, including the relevant E3 ligase complex for ERα ubiquitination, respectively. Figure 8 and Figure 9 Estradiol binding can also reveal species that interact or function in the ERα degradation mechanism, such as Figure 10 and Figure 11 As shown. Figure 10 In this study, protein species BBB can be identified that are known to post-translationally modify ERα and lead to ERα degradation. For example, species BBB can be kinases involved in phosphorylation-mediated ERα degradation. In addition, previously unknown E3 ligases that degrade ERα can be elucidated, such as Figure 11 This interactome mapping analysis can be used to develop targeted E3 ligase activators for the treatment of breast cancer and for the development of other molecular glues to degrade proteins of interest, such as Figure 12 Example 2 in this article further illustrates these principles.

[0078] These and other embodiments are further described in the following non-limiting examples.

[0079] Example 1 - Conjugation of transition metal complexes to proteins

[0080] Prepare as follows Figure 2 Protein-transition metal complex. Cells (HEK243T stably expressing HaloTag-G3BP1) were seeded and grown to 95% confluence before labeling. The cells were incubated for one hour in DMEM medium containing 5 μM Figure 2 During this time, the iridium photocatalyst crosses the cell membrane and binds to the HaloTag-G3BP1 protein. The cells are then incubated in fresh DMEM medium for one hour to remove or minimize background signal from any unbound iridium catalyst. Example 2 - ERα interactome mapping analysis based on estradiol / fulvestrant binding

[0081] The local interactome of ERa following binding of estradiol and fulvestrant was investigated according to the compositions and methods described herein.

[0082] 80% confluent MCF7 cells stably expressing FLAG-CfaC-HA-ESR1 were replaced with phenol red-free DMEM supplemented with 10% v / v carbon-bound FBS, 1% penicillin / streptomycin, and 1% L-glutamine per 10 cm culture plate. After 3 days of culture, cells were treated with 10 μM TAK243 for 1 hour and then co-treated with 100 nM estradiol or 100 nM fulvestrant for 4 hours before harvest. The resulting cell pellet was washed twice with DPBS and then hypotonic lysed on ice for 10 minutes using 0.6 mL RSB buffer (10 mM Tris buffer, 15 mM NaCl, 1.5 mM MgCl2, Roche EDTA-free protease inhibitor cOmplete, pH 7.6, supplemented with 100 nM estradiol or 100 nM fulvestrant). After crude nuclei were isolated by centrifugation at 400 g for 5 min at 4°C, the nuclei were resuspended in 1 mL of RSE buffer and homogenized by 10 strokes using a Dounce homogenizer with a loose mallet.

[0083] The nuclei were pelleted again at 400 g for 5 min at 4°C and resuspended in 0.6 mL of crosslinking buffer (20 mM HEPES, 1.5 mM MgCl2, 150 mM KCl, Roche EDTA-free protease inhibitor cOmplete, pH 7.6, supplemented with 100 nM estradiol or 100 nM fulvestrant) and then centrifuged again at 400 g for 5 min at 4°C. At this point, the pellets from two 10 cm plates of the same treatment type were combined and resuspended in 400 μL of crosslinking buffer containing 0.3 μM CfaN-Ir. Trans-splicing was allowed to occur for 1 h at 37°C with rotation. The resulting pellet was washed three times with 400 μL of crosslinking buffer before adding 500 μM biotin-diazomethane in 200 μL of crosslinking buffer and irradiated with 100% intensity blue light for 3 min at 4°C using a Penn PhD photoreactor M2. Irradiated nuclei were washed again twice with 400 μL of cross-linking buffer before lysis on ice for 10 min in 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's EDTA-free protease inhibitor cOmplete, pH 7.5). The pellet was sonicated twice for 10 seconds using a Branson probe sonicator at 35% amplitude, followed by centrifugation at 18,000 g for 15 min at 4°C. The supernatant was obtained as the nuclear lysate, and the protein concentration was determined using the BCA assay.

[0084] 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). Lysate was loaded onto 25 uL pre-washed magnetic agarose streptavidin beads and incubated at 4 °C for 18 h by end-to-end rotation. The beads were then washed 3 times with 1% w / v SDS in DPBS, washed twice with 1 M NaCl in DPBS, washed once with 10% ethanol in DPBS, and washed 3 times with 50 mM ammonium bicarbonate in water. 500 μL of 50 mM ammonium bicarbonate were used to transfer the washed beads to a new Lo-bind tube, removing as much supernatant as possible. The gained beads were resuspended in 30 μL of 50 mM ammonium bicarbonate containing 0.4 μg trypsin, and incubated overnight at 37 °C, rotating. The resulting supernatant was collected by centrifugation and filtered through 0.22 μm cellulose acetate using a Spin-X centrifuge tube filter. The resulting eluate was ready for mass spectrometry analysis using a TimsTOF.

[0085] Figure 13Figure 1 is a volcano plot detailing the species identified in the interactome mapping analysis following the combination of estradiol and fulvestrant. This volcano plot is a composite of results obtained from independent runs combining estradiol and fulvestrant.

[0086] In order to achieve the various purposes of the present invention, various embodiments of the present invention have been described. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Many modifications and adaptations thereof will be apparent to those skilled in the art without departing from the spirit and scope of the present invention.

Claims

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

2. The protein complex according to claim 1, wherein the transition metal complex is bonded to a haloalkane dehalogenase, and the haloalkane dehalogenase is coupled to the protein substrate.

3. The protein complex of claim 1, wherein the transition metal complex is bonded to a derivatized or 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 via click chemistry.

6. The protein complex of claim 1, wherein the transition metal complex is coupled to the protein substrate via protein trans-splicing, the transition metal complex being initially coupled to a cleaved intein. The protein complex according to claim 6 , wherein the split-type intein is an N-intein. The protein complex according to claim 6 , wherein the split-type 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 protein complex according to claim 1, wherein the transition metal complex has the following formula (I): Where M is a transition metal; wherein A, D, E, G, Y and Z are independently selected from C and N; where R 3 to R 7 each represents one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - 、-C(O)OR 8 and -R 9 OH group, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is an alkyl group; where R 1 is selected from the group consisting of a direct bond, an alkylene group, an alkenylene group, a cycloalkylene group, a cycloalkenylene group, an arylene group, a heteroalkylene group, a heteroalkenylene group, a heterocyclolene group, and a heteroarylene group; wherein L is an optional linking moiety selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; and R 2 Selected from alkynyl, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, hydroxyl, carboxyl, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 、-OS(O2)R 9 , thiol, biotin, hydroxylamine and alkyl halide, wherein R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl and N-succinimidyl ester; wherein X - is a counter ion, and n is an integer from 0 to 20.

11. A method for characterizing protein degradation, comprising: providing a protein substrate having a transition metal complex associated therewith; activating a protein labeling agent into a reactive intermediate using the transition metal complex; and The reactive intermediate is coupled to ubiquitin E3 ligase to form a complex with the protein substrate.

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

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

14. The method of claim 11, wherein the transition metal complex has the formula: Where M is a transition metal; wherein A, D, E, G, Y and Z are independently selected from C and N; where R 3 to R 7 each represents one to four optional ring substituents, each of the one to four optional ring substituents being independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, haloalkenyl, halogen, hydroxy, alkoxy, amine, amide, ether, -C(O)O - 、-C(O)OR 8 and -R 9 OH group, where R 8 is selected from the group consisting of hydrogen and alkyl, and R 9 is an alkyl group; where R 1 is selected from the group consisting of a direct bond, an alkylene group, an alkenylene group, a cycloalkylene group, a cycloalkenylene group, an arylene group, a heteroalkylene group, a heteroalkenylene group, a heterocyclolene group, and a heteroarylene group; wherein L is an optional linking moiety selected from the group consisting of amides, esters, sulfonamides, sulfonates, carbamates, and ureas; and R 2 Selected from alkynyl, amine, protected amine, azide, hydrazide, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, hydroxyl, carboxyl, halogen, alkoxy, maleimide, -C(O)H, -C(O)OR 8 、-OS(O2)R 9 , thiol, biotin, hydroxylamine and alkyl halide, wherein R 8 and R 9 are independently selected from the group consisting of alkyl, haloalkyl, aryl, haloaryl, N-succinimidyl and N-succinimidyl ester; wherein X - is a counter ion, and n is an integer from 0 to 20.

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

16. The method of claim 11, wherein the transition metal complex is bonded to a haloalkane dehalogenase, and the haloalkane dehalogenase is coupled to the protein substrate.

17. The method of claim 11, wherein the transition metal complex is bonded to a derivatized or unnatural amino acid of the protein substrate.

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

19. The method of claim 11, wherein the transition metal complex is coupled to the protein substrate via protein trans-splicing, the transition metal complex being initially coupled to a cleaved intein.

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

21. A method for analyzing the local microenvironment of a protein within a cell, comprising: forming a protein complex comprising a transition metal complex coupled to a protein within the cell; activating a protein labeling agent into a reactive intermediate using the transition metal complex; and The reactive intermediate is coupled to a protein or other biomolecule within a predetermined radius of the protein complex.

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

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

25. The method of claim 21, wherein the transition metal complex is coupled to the intracellular protein via protein trans-splicing, the transition metal complex being initially coupled to a cleaved intein.

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

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

28. The method of claim 21, wherein the reactive intermediate is coupled to a protein involved in a protein degradation pathway within the cell.

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

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

31. The method of claim 30, wherein the protein marker is activated upon molecular binding to the receptor.

32. The method of claim 31, wherein the molecule binds comprises an activator that binds to the receptor.

33. The method of claim 31, wherein the molecule binds comprises an inhibitor or degrader that binds to the receptor.

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