Patent Application
20220306683
The present invention relates to proximity-based labeling techniques and, in particular, to compositions and methods permitting high resolution labeling of proteins in cellular environments.
Protein proximity labeling has emerged as a powerful approach for profiling protein inter-action networks. The ability to label associated or bystander proteins through proximity labeling can have important implications on further understanding the cellular environment and biological role of a protein of interest. Current proximity labeling methods all involve the use of enzyme-based generation of reactive intermediates that label neighboring proteins on a few select amino acid residues through diffusion or physical contact. Despite the transformative impact of this technology, the inherent stability of these reactive intermediates such as phenoxy radicals (t1/2>100 μs) through peroxidase activation or biotin-AMP (t1/2>60 s) through biotin ligases can promote diffusion far from their point of origin. As a result, these enzyme-generated reactive intermediates pose a challenge to profiling within tight micro-environments. Furthermore, the large enzyme size, the dependency on certain amino acids for labeling, and the inability to temporally control these labeling systems present additional challenges for profiling within confined spatial regions. Given these limitations, new approaches for proximity-based labeling are needed.
In one aspect, compositions and methods are described herein for providing a microenvironment mapping platform operable to selectively identify various features, including protein-protein interactions on cellular membranes. In some embodiments, a composition comprises a catalyst, and a protein labeling agent, wherein the catalyst activates the protein labeling agent to a reactive intermediate. The catalyst, in some embodiments, can have electronic structure for permitting energy transfer to the protein labeling agent to form the reactive intermediate. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate. If a protein or other biomolecule is not within the diffusion radius, the reactive intermediate is quenched by the surrounding aqueous or aqueous-based environment. As described further herein, the diffusion radius of the reactive intermediate can be tailored to specific microenvironment mapping considerations, and can be limited to the nanometer scale. In some embodiments, for example, the diffusion radius can be less than 10 nm or less than 5 nm. Moreover, in some embodiments, the reactive intermediate can have a half-life of less than 5 ns. In some embodiments, a protein labeling agent can be functionalized with a marker, such as biotin or luminescent markers for aiding in analysis. Any catalyst operable to participate in energy transfer with the protein labeling agent to provide the reactive intermediate can be employed. In some embodiments, transition metal catalyst is used. Alternatively, non-transition metal organocatalyst may be used. Energy transfer from the catalyst to the protein labeling agent can occur via a variety of mechanisms described further herein, including Dexter energy transfer.
In another aspect, conjugates for proximity-based labeling are described herein. A conjugate comprises a catalyst coupled to a biomolecular binding agent. The catalyst can have electronic structure for energy transfer to a protein labeling agent for generation of a reactive intermediate as described above. The biomolecular binding agent, in some embodiments, can be used to selectively locate or target the catalyst to a specific environment for mapping. The biomolecular binding agent, for example, locate the catalyst in the desired cellular environment for proximity labeling and associated analysis. The biomolecular binding agent can comprise a protein, polysaccharide, nucleic acid, or lipid, in some embodiments. In some instances, the biomolecular binding agent can comprise a multivalent display system comprising a protein, polysaccharide, nucleic acid, or lipid. Moreover, the biomolecular binding agent can also be a small molecule ligand with a specific binding affinity for a target protein.
In another aspect, systems for proximity-based labeling are described herein. A system, in some embodiments, comprises a conjugate including a catalyst coupled to a biomolecular binding agent, and a protein labeling agent activated by the catalyst for binding to a protein. The conjugate and protein labeling agent can have any composition and/or properties described above and in the following detailed description.
In a further aspect, methods of proximity-based labeling are described herein. A method of proximity-based labeling comprises providing a catalyst, and activating a protein labeling agent to a reactive intermediate with the catalyst. The reactive intermediate couples or bonds to a protein. In some embodiments, the catalyst is coupled to a biomolecular binding agent to selectively locate or target the catalyst to a specific environment for protein mapping in conjunction with the protein labeling agent. The catalyst, conjugate, and protein labeling agent can have composition and/or properties described above and in the following detailed description.
These and other embodiments are further described in the following detailed description.
16 illustrates the experimental set up and results, including the sulfonamide-iridium conjugate and biotin-tagged diazirine, for selective labeling of carbonic anhydrase with small-molecule based conjugates according to some embodiments.
Embodiments described herein can be understood more readily by reference to the following detailed description and examples and their previous and following descriptions. Elements, apparatus and methods described herein, however, 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 of skill in the art without departing from the spirit and scope of the invention.
The term “alkyl” as used herein, alone or in combination, refers to a straight or branched saturated hydrocarbon group optionally substituted with one or more substituents. For example, an alkyl can be C1-C30 or C1-C18.
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.
The term “heteroaryl” as used herein, alone or in combination, refers to an aromatic monocyclic or multicyclic 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.
The term “heterocycle” as used herein, alone or in combination, refers to an mono- or multicyclic 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 wherein the ring system is optionally substituted with one or more ring substituents. The heterocyclic ring system may include aromatic and/or non-aromatic rings.
The term “alkoxy” as used herein, alone or in combination, refers to the moiety RO—, where R is alkyl, alkenyl, or aryl defined above.
The term “halo” as used herein, alone or in combination, refers to elements of Group VIIA of the Periodic Table (halogens). Depending on chemical environment, halo can be in a neutral or anionic state.
Terms not specifically defined herein are given their normal meaning in the art.
Compositions are described herein for providing microenvironment mapping platforms operable to selectively identify various features, including protein-protein interactions on cellular membranes. In some embodiments, a composition comprises a catalyst, and a protein labeling agent, wherein the catalyst activates the protein labeling agent to a reactive intermediate. The catalyst, in some embodiments, can have electronic structure for permitting energy transfer to the protein labeling agent to form the reactive intermediate. In some embodiments, for example, the catalyst engages in Dexter energy transfer with the protein labeling agent. The energy transfer can proceed via single electron transfer, in some embodiments.
The energy transfer to the protein labeling agent can originate from an excited state of the catalyst electronic structure, in some embodiments. The excited state of the catalyst, for example, can be a singlet excited state or triplet excited state. The excited state of the catalyst can be generated by one or more mechanisms, including energy absorption by the catalyst. In some embodiments, the catalyst is a photocatalyst, wherein the excited state is induced by absorption of one or more photons. In other embodiments, the catalyst may be placed in an excited state by interaction with one or more chemical species in the surrounding environment. Alternatively, the energy transfer to the protein labeling agent, including electron transfer, may originate from a ground state of the catalyst electronic structure.
Energy transfer, including electron transfer, to the protein labeling agent forms a reactive intermediate of the protein labeling agent. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate. If a protein or other biomolecule is not within the diffusion radius, the reactive intermediate is quenched by the surrounding aqueous or aqueous-based environment. The diffusion radius of the reactive intermediate can be tailored to specific microenvironment mapping (proximity-based labeling) considerations, and can be limited to the nanometer scale. In some embodiments, for example, the diffusion radius of the reactive intermediate can be less than 10 nm, less than 5 nm, less than 4 nm, less than 3 nm, or less than 2 nm prior to quenching in an aqueous environment. Accordingly, the reactive intermediate will react or crosslink with a protein or other biomolecule within the diffusion radius or be quenched by the aqueous environment if no protein or biomolecule is present. In this way, high resolution of the local environment can be mapped via concerted effort between the catalyst and protein labeling agent. Additionally, the reactive intermediate can exhibit a t1/2 less than 5 ns, less than 4 ns, or less than 2 ns prior to quenching, in some embodiments. In additional embodiments, the diffusion radius can be extended to between 5-500 nm though extension of the reactive intermediate half-life.
Any catalyst-protein labeling agent combination exhibiting the foregoing electronic structure properties for energy transfer and reactive intermediate generation and associated protein or biomolecule binding can be employed for microenvironment mapping. In some embodiments, the catalyst is a transition metal complex. The transition metal complex, in some embodiments, can exhibit a long-lived triplet excited state (T1) facilitating energy transfer to the protein labeling agent. The T1 state can have t1/2 of 0.2-2 μs, for example. Transition metal complexes described herein can be photocatalytic and, in some embodiments, absorb light in the visible region of the electromagnetic spectrum. Absorption of electromagnetic radiation can excite the transition metal complex to the Si state followed by quantitative intersystem crossing to the T1 state. The transition metal catalyst can subsequently undergo short-range Dexter energy transfer to a protein labeling agent, and returned to the ground state, S0. The energy transfer to the labeling agent activates the labeling agent for reaction with a protein or other biomolecule. The T1 state of the transition metal complex can be greater than 60 kcal/mol, in some embodiments. The metal center, for example, can be selected from transition metals of the platinum group. The metal center can be iridium, in some embodiments.
The transition metal complex can have any composition and structure consistent with the foregoing principles of excitation and energy transfer to a protein labeling agent. In some embodiments, a photocatalytic transition metal complex is hexacoordinate. Transition metal photocatalyst of proximity-based labeling compositions described herein, in some embodiments, are of Formula (I):
wherein M is a transition metal;
wherein A, D, E, G, Y and Z are independently selected from C and N;
wherein R1-R6 each represent one to four optional ring substituents, each of the one to four optional ring substituents independently selected from the group consisting of alkyl, heteroalkyl, haloalkyl, halo, hydroxy, alkoxy, amine, amide, ether, —C(O)O−, —C(O)OR7, and —R8OH, wherein R7 is selected from the group consisting of hydrogen and alkyl, and R8 is alkyl; and
wherein X− is a counterion. It is understood that hydrogen occupies positions on the aryl rings of Formula (I) in the absence of optional substituents R1-R6. In some embodiments, counterion (X−) can be selected from tetraalkylborate, tetrafluoroborate, tetraphenylborate, PF6−, and chloride. In some embodiments, M is selected from transition metals of the platinum group. The metal center can be iridium.
Ligands of the transition metal complex of Formula (I) can comprise one or more pyridine moieties, in some embodiments. In some embodiments, one or more ligands comprise bipyridine or derivatives thereof. Ligands of the transition metal complex can also be selected from the species provided in Table I.
In some embodiments, the photocatalytic transition metal complex of Formula (I) is [Ir(dF(CF3)ppy)2(dtbbpy)](PF6), [Ir(dF(CF3)ppy)2(bpy)](PF6), or derivatives thereof.
Additionally, ligands of photocatalytic transition metal complexes described herein, including complexes of Formula (I), can be modified with one or more polar or hydrophilic moieties for enhancing solubility of the transition metal complexes in aqueous or aqueous-based environments. Suitable moieties can include, but are not limited to, carboxyl, hydroxyl, and/or alkylene oxide moieties, such as polyethylene glycol.
As described further herein, transition metal complexes can incorporate a reactive functionality for coupling a biomolecular binding agent. In some embodiments, for example, a transition metal catalyst, including transition metal photocatalyst of Formula (I), can comprise one or more click chemistry moieties including, but not limited to, BCN (bicyclononyne, bicyclo[6.1.0]nonyne), DBCO (dibenzo-bicyclo-octyne), TCO (trans-cyclooctene), tetrazine, alkyne, and azide.
Catalyst operable for energy transfer to a protein labeling agent for producing a reactive intermediate can comprise non-transition metal catalyst, in some embodiments. Various organocatalyst for use in proximity-based labeling compounds and methods described herein can include a thioxanthone, phenothiazine, flavin, phenoxazine, benzophenothiazine, coumarin, acetophenone, or a benzophenone group, in some embodiments. In some cases, an organocatalyst comprises a triarylmethane group, rose Bengal, porphyrin, chlorin, bacteriochlorin, methylene blue, an acridine dye, a xanthene dye, or an arylmethane dye. The non-transition metal organocatalyst can have any electronic structure and/or other properties of transition metal complexes described herein, including singlet and/or triplet excited states for energy transfer to the protein labeling agent. Energy transfer from the organocatalyst to the protein labeling agent may occur via any mechanism described herein, such as Dexter energy transfer or single electron transfer. In some embodiments, an organocatalyst is a photocatalyst, wherein the excited state for energy transfer to the protein labeling agent is generated by absorption of one or more photons by the organocatalyst. Organocatalyst, for example, may absorb light in the visible region of the electromagnetic spectrum.
Protein labeling agents receive energy transfer from the catalyst to form a reactive intermediate. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate. Diffusion radii of reactive intermediates are described above. Specific identity of a protein labeling agent can be selected according to several considerations, including identity of the catalyst, the nature of the reactive intermediate formed, lifetime and diffusion radius of the reactive intermediate.
For example, in embodiments wherein the catalyst is a transition metal photocatalyst, the protein labeling agent can be a diazirine. Triplet energy transfer from the excited state photocatalyst can promote the diazirine to its triplet (T1) state. The diazirine triplet under-goes elimination of N2 to release a free triplet carbene, which undergoes picosecond-timescale spin equilibration to its reactive singlet state (t1/2<1 ns) which either crosslinks with a nearby protein or is quenched in the aqueous environment.
Any diazirine consistent with the technical principles discussed herein, including the reaction mechanism of
In some embodiments wherein the catalyst is a transition metal photocatalyst, the protein labeling agent can be an azide. Triplet energy transfer from the excited state photocatalyst can promote nitrene formation from the azide. The reactive nitrene either crosslinks with a nearby protein or is quenched in the aqueous environment. Any azide operable to undergo energy transfer with eth transition metal photocatalyst for nitrene formation can be employed. In some embodiments, an azide is an aryl azide.
In some embodiments wherein the catalyst is an organocatalyst, the protein labeling agent can comprise one or more moieties for receiving single electron transfer from the organocatalyst. Single electron transfer to the protein labeling agent can generate a reactive radical having diffusion radius and/or lifetime described herein for reacting with a protein in the local environment of the organocatalyst. For example, an organocatalyst can be a flavin photocatalyst. The excited flavin photocatalyst can undergo single electron transfer with phenol moieties to generate a reactive phenoxy radical.
In another aspect, conjugates for proximity-based labeling are described herein. A conjugate comprises a catalyst coupled to a biomolecular binding agent. The catalyst coupled to the biomolecular binding agent can comprise any catalyst described herein, including the transition metal catalysts and organocatalysts detailed in Section I above. Moreover, the biomolecular binding agent can comprise a protein, polysaccharide, nucleic acid, or lipid, in some embodiments. In some instances, the biomolecular binding agent can comprise a multivalent display system comprising a protein, polysaccharide, nucleic acid, or lipid. In some embodiments, the biomolecular binding agent can be a small molecule ligand with a specific binding affinity for a target protein. The biomolecular binding agent can be employed to locate the catalyst in the desired extracellular environment for proximity labeling and associated analysis. Accordingly, specific identity of the biomolecular binding agent can be selected according to the chemical and/or steric requirements of the desired target site for placement of the catalyst in the proximity based labeling process. Any biomolecular target site can be chosen, and target sites are not limited in the present disclosure. In some embodiments, target sites can be proteins for studying protein-protein interactions, including interaction with cellular membrane receptors. In some embodiments, for example, the biomolecular binding agent is an antibody, such as a secondary antibody for interacting with a primary antibody bound to the desired antigen. In other embodiments, the biomolecular binding agent is a ligand with specificity for a protein receptor of the cellular membrane, such as a G protein-coupled receptor.
The biomolecular binding agent can be bonded to the catalyst. In some embodiments, the catalyst comprises a reactive handle or functionality for coupling the biomolecular binding agent. In some embodiments, for example, a catalyst can comprise one or more click chemistry moieties including, but not limited to, BCN, DBCO, TCO, tetrazine, alkyne, and azide.
In another aspect, systems for proximity-based labeling are described herein. A system, for example, comprises a conjugate including a catalyst coupled to a biomolecular binding agent, and a protein labeling agent activated by the catalyst for binding to a protein. The conjugate can comprise any catalyst and biomolecular binding agent described herein, including the embodiments detailed in Section II above. The catalyst, for example, can have electronic structure to activate the protein labeling agent to a reactive intermediate via energy transfer. Moreover, the protein labeling agent can comprise any of the labeling agents described herein, including the protein labeling agents set forth in Section I above. Specific identity of the conjugate and associated protein labeling agent can be selected according to several considerations, such as the chemical nature and/or steric requirements of the biological environment to be mapped with the proximity-based labeling system.
Systems for proximity-based labeling described herein can be employed in various applications. In some embodiments, the systems enable target identification, wherein the conjugate and associated protein labeling agent permit identification of one or more molecules in a biological context by proteomics. Additionally, systems comprising the conjugate and protein labeling agent facilitate interactome mapping. Targeting a conjugate and protein labeling agent allows detection and identification of one or more molecules and neighboring interactors in a biological context by proteomics. Identification of such molecules by systems described herein can permit enrichment and/or purification of such molecules and neighboring interactors. Additionally, systems comprising a conjugate and protein labeling agent further enable detection and identification of one or more molecules in a biological context via microscopy.
In another aspect, methods of proximity-based labeling are described herein. A method of proximity-based labeling comprises providing a conjugate comprising a catalyst coupled to a biomolecular binding agent, activating a protein labeling agent to a reactive intermediate with the catalyst, and coupling the reactive intermediate to a protein. The conjugate can comprise any catalyst and biomolecular binding agent described herein, including the embodiments detailed in Section II above. Moreover, the protein labeling agent can comprise any of the labeling agents described herein, including the protein labeling agents set forth in Section I above. Specific identity of the conjugate and associated protein labeling agent can be selected according to several considerations, such as the chemical nature and/or steric requirements of the biological environment to be mapped with the proximity-based labeling system. In some embodiments of proximity-based labeling, the catalyst can be provided in the absence of a biomolecular binding agent.
Methods described herein can be employed to map various biological environments, including local areas of cellular membranes and/or the local extracellular environment. The conjugate comprising the catalyst and biomolecular binding agent may be targeted to a specific local region of a cellular membrane, such as a receptor of interest. Activation of the protein labeling agent can identify protein(s) and/or other molecules in the targeted local region. Notably, the activated protein labeling agent can also identify or label molecules associated with another cell in contact with the targeted cellular region. Therefore, intercellular interactions and intercellular environments can be elucidated and mapped with systems and methods described herein. The foregoing methods enable interactome mapping, and the identification of one or more molecules and neighboring interactors in a biological context by proteomics. Identification of such molecules by methods described herein can permit enrichment and/or purification of such molecules and neighboring interactors.
These and other embodiments are further illustrated by the following non-limiting examples.
It was demonstrated that carbenes generated through photocatalytic diazirine sensitization could label proteins. As illustrated in
Biotinylation of BSA was detected for Sample 2. When a solution of BSA and biotinylated diazirine probe was irradiated with 450 nm light (Sample 3), the degree of biotinylation was less than 0.5%, establishing that the diazirine presents minimal background signal at this wavelength. However, in the presence of water soluble photocatalyst 3, catalyst dependent biotinylation of BSA was observed. Photocatalytic labeling of BSA was further confirmed through intact protein mass spectrometry. Unlike prior enzyme-based labeling methodologies, this approach requires continuous delivery of visible-light to sustain diazirine sensitization via the photocatalyst 3 for protein labeling. This feature was exploited to demonstrate how turning the light source on or off affords fine temporal control over the labeling process, as illustrated in
A secondary antibody-photocatalyst conjugate was prepared as a general entry point for spatially targeted photocatalytic proximity labeling on cell surfaces. A goat anti-mouse (Gt/α-Ms) antibody was first decorated with azide groups through reaction with azidobutyric acid N-hydroxysuccinimide ester, and then conjugated to alkyne-bearing iridium catalyst via click chemistry, resulting in an antibody-photocatalyst ratio of 1:6. The iridium catalyst is the iridium complex 3 illustrated in
Next, to address protein targeted labeling on a surface, a model system was prepared containing human Fc-tagged vascular endothelial growth factor receptor 2 (VEGFR2) and epidermal growth factor receptor (EGFR) proteins attached to α-human immunoglobulin-G (IgG) agarose beads (
Antibody-targeted photocatalytic diazirine activation (i.e. μMap) was applied to the surface of live cells. For these experiments, addition of the antibody to the cell surface 5 was maintained at 4° C. to limit antibody-mediated protein crosslinking. CD45, a highly abundant tyrosine phosphatase on T cell surfaces involved in antigen receptor signaling was selected as an initial target. Western blot analysis of CD45-targeted μMap on Jurkat cells showed light and time dependent protein biotinylation compared to the isotype targeting control (
With proof of concept for cell surface labeling in hand, it was explored whether μMap could differentiate between spatially separated microenvironments on the same cell membrane. To this end, CD29 and CD47 were selected as ideal targets with no known co-spatial association on the cell surface. Indeed, μMapping of CD45, CD29, or CD47 on Jurkat cells resulted in the enrichment of unique sets of proteins, which included both known (CD29:CD49D, CD45:CD45AP:CD2) and previously unknown interactors (
The selectivity of compositions and methods described herein was also harnessed to investigate the proximal protein interactome of PD-L1 in B cells. As has been well established, PD-L1 plays an important role in cancer cells as an immune checkpoint ligand that can accelerate tumor progression via suppression of T cell activity. In the event, PD-L1 targeted μMap revealed CD30, a member of the tumor necrosis factor receptor family, and CD300A, an immune inhibitory receptor 5 (
To further validate the enriched subset of proteins identified by PD-L1 μMapping, 10 targeted labeling of these two highly enriched proteins were performed. Targeted μMapping of these proteins within the PD-L1 microenvironment should, therefore, afford similar enrichment lists, verifying their spatial association. Indeed, it was found that α-CD30, α-CD300A, and α-PD-L1 directed μMapping identified the same set of 12 surface receptors (
It is well recognized that the development of new therapeutic oncology strategies will require an understanding of the underlying mechanisms of intercellular communication, particularly within the context of T-cell activation and differentiation. Furthermore, given that localization of PD-L1 is found within the T cell/antigen presenting cell (APC) immunosynapse (i.e. at the interface between two immunointeractive cells), it was hypothesized that PD-L1 directed μMapping with conjugates and methods described herein should lead to not only biotinylation of a PD-L1 expressing APC surface (cis-labeling), but also to the biotinylation of the adjacent synaptic T cell (trans-labeling) (
PD-L1- and CD45-targeted μMapping was evaluated in a two-cell system composed of PD-L1-expressing JY-B-lymphocytes as the antigen presenting cell and Jurkat T-lymphocytes uniquely expressing PD-1 and the CD45RO isoform. Given that immune cell-APC interactions are driven by the binding of multiple receptor types (e.g. adhesion, co-stimulatory/co-repressor, and T cell receptor (TCR)-major histocompatibility complex (WIC) 5, staphylococcal enterotoxin D (SED) was employed to facilitate WIC class II and TCR engagement and promote B cell/T cell immune synapse formation and signaling (
Conjugates comprising flavin photocatalyst in conjunction with phenol-based protein labeling agents can be employed in proximity-based labeling compositions and methods described herein.
To first ascertain the capability of a small molecule-iridium conjugate to direct labeling towards a specific protein, a simple two-protein biochemical assay was designed. An equimolar ratio of the target protein, carbonic anhydrase (CA), with bovine serum albumin (BSA), as a competitor protein were chosen. Following this hypothesis, irradiation of the mixture in the presence of a biotin-tagged diazirine and sulfonamide-iridium conjugate would lead to selective labelling of CA over BSA. Analysis of labeling ratios by immunoblotting with streptavidin would provide an indication of both reaction efficiency and selectivity. Cognisant of the effect that the iridium catalyst may play on ligand binding, the catalyst-ligand conjugate was prepared with a PEG3 (triethylene glycol) linker to spatially separate the two components. Gratifyingly, after irradiation with 450 nm light for 10 minutes in the presence of biotin-peg3-diazirine, a 3.5:1 labeling in favor of the target protein CA was observed.
In contrast, when labeling was performed in the presence of an unconjugated photocatalyst, BSA was selectively biotinylated over CA in a ratio of 5:1. Taken together, this comprises a 3-fold increase in labeling selectivity when using the Ir-ligand conjugate, providing confidence in this approach for targetID. Importantly, this selectivity was completely ablated when the targeted experiment was performed with an excess of the unconjugated sulfonamide ligand, confirming that the observed selectivity was the result of a ligand protein binding event. Moreover, significant enrichment of CA when performing the labelling in HEK293T cell lysate was found, validating the compatibility of this methodology with the most complex of biological settings.
The generality of small-molecule based catalyst conjugates as a platform with regard to ligand-directed targeting identification of proteins was further investigated. Small molecule-iridium conjugates could be readily prepared in an operationally straight-forward manner using a coppercatalyzed azide-alkyne click reaction (CuAAC) between iridiumalkyne of
It was also sought to establish if other targeting modalities, in addition to small-molecules, would be compatible with catalytic labelling technology described herein. The α-helical stapled cyclic peptide ATSP7041, which targets the E3 ligase MDM2, could be readily conjugated to the photocatalyst through a modified azido-lysine. Significant enrichment (3-fold) of the target protein compared to the free photocatalyst control was observed, as provided in
PPIs are essential to cell function and comprise a challenging class of targets for small molecule drug discovery. This challenge arises, in part, from the transient nature of these interactions which can make them difficult to detect biochemically. However, a number of prominent small molecule ligands are known to bind to protein complexes or to proteins that function through dynamic complexes. A method that could effectively distinguish the components of protein complexes would, therefore, prove an important tool for the investigation of PPIs. It was initially set out to study the small molecule AT7519, which binds to cyclin dependent kinase 2 (CDK2). It is well-established that CDK2 forms a PPI with the protein cyclin A, which contributes to the regulation of the cell cycle. As illustrated in
It was next set about exploring the rapamycin/mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, leading to suppression of the immune response. Notably, upon irradiation, both proteins were enriched compared to controls (FKBP12: 2-fold, mTOR: 12-fold), despite any disruption to binding efficacy caused by the pendant Ir-catalyst, as illustrated in
It was questioned whether the tight labeling of the μMap platform with conjugates and labeling agents described herein could be exploited to identify ligand binding sites through a combination of Western blotting and MS2 analysis. To best explore this, the three-protein complex Calcineurin A/FKBP12/Calcineurin B was chosen, which binds to the macrolide tacrolimus. Upon synthesis of a range of tacrolimus-Ir conjugates with different linker lengths, it was found that short linkers led to labeling directly around the binding site of the complex, allowing footprinting of the molecular recognition site. However, with increasing tether length the labeling radius increased to include other members of the protein complex, changing the primary site of labeling. MS2 analysis of the PEG3 linker supported this data, showing labeled residues directly adjacent to the binding site (
A four-protein complex was then examined where the neighboring proteins do not directly interact with the small molecule ligand, to demonstrate that labeling of protein interactors through space can be achieved. For this, the E3 ligase complex CRBN/DDB1/Cul4a/RBX1 was chosen. In line with previous examples described herein, when a short tether was employed, only CRBN the primary target was labeled, however with increasing tether length, the neighboring proteins in the E3 ligase complex could be captured with this technology. Proteomic analysis of the labeling reaction employing a PEG3 linker showed a molecular footprint of the small molecule binding site on CRBN in addition to labeled sites on the neighboring Cul4a, that presumably arise from secondary protein-protein interactions (
The adenosine receptor A2a (ADORA2A) as an exemplary membrane target. This GPCR has become an important target for immunotherapy, but critically, has never been identified through live cell chemoproteomics. Using a reported ligand for ADORA2A, A2a, an Ir-conjugate (A2α-Ir) was synthesized and a tethered diazirine-conjugate, as described by Yao (A2α-Dz) (
Human GPR40 receptor (hGPR40) was also subjected to labeling with compositions and methods described herein. A hGPR40-Ir conjugate was employed along with a protein labeling agent of biotinylated diazirine (Diaz-PEG3-Bt) (
Various embodiments of the invention have been described in fulfillment of the various objects of the 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 invention.
The present application claims priority pursuant to Article 8 of the Patent Cooperation Treaty to U.S. Provisional Patent Application Ser. No. 62/858,539 filed Jun. 7, 2019 and U.S. Provisional Patent Application Ser. No. 62/982,576 filed Feb. 27, 2020, each of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/036285 | 6/5/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62858539 | Jun 2019 | US | |
| 62982576 | Feb 2020 | US |
