DETERMINING SMALL MOLECULE-PROTEIN AND PROTEIN-PROTEIN INTERACTIONS

Abstract

Provided herein are methods, systems, kits, and compositions useful for determining small molecule-protein interactions and protein-protein interactions. The photo-click tags provided herein can be conjugated to a small molecule or amino acid analog to provide compounds that can be integrated into a protein through photo-conjugation, allowing for identification of a small molecule-protein interaction or protein-protein interaction to elucidate the small molecules mechanism of action or the protein targeted by the small molecule. In some embodiments, the photo-click tags comprise a photo-conjugation moiety and a click chemistry handle, allowing for the attachment of various functional groups (e.g., affinity tags) to the small molecule or amino acid analog.

Description

BACKGROUND OF THE INVENTION

Small molecule regulation of the proteome constitutes one of the oldest methods of therapy known to humankind. However, despite entering an era of target-based therapy and personalized medicine, parallel advances in understanding the precise molecular outcomes of small molecule therapeutics remain under-developed. Currently, the state of the art involves attachment of small molecules to affinity handles and immobilization on a solid support, followed by in vitro affinity purification. Alternatively, target-based drug discovery evaluates the affinity of small molecules for a specific purified protein. These approaches fail to reveal the structural basis of the interaction (e.g., binding site), changes to protein-protein interactions (PPIs), and broader system-wide (e.g., proteome, interactome, or genome) effects. As a result, the vast majority of protein interactors are silently overlooked in current mechanism of action studies, leading to missed opportunities and incomplete toxicology profiles.

The primary bottleneck is the lack of an approach to globally reveal proteomic interactions (e.g., protein-protein interactions (PPIs)) mediated by a small molecule. Protein-protein interactions are implicated in thousands of diseases, and thus it is imperative to understand both normal and abnormal PPIs. For example, this gap is particularly notable in the case of the immunomodulatory drugs (IMIDs), thalidomide, lenalidomide, and pomalidomide, a class of pluripotent therapeutics that lack a defined mechanism of action. These, as well as other, IMIDs have been the focus of extensive time-consuming mechanism of action studies, which have inspired popular conclusions about the importance of enantioselectivity in drug discovery due to their single stereocenter. However, the IMIDs are rapidly racemized and metabolized in plasma, making any attempt at characterizing the phenotype behind a specific isoform inconclusive. A complete understanding of the role of IMID isoforms (e.g., R-lenalidomide versus S-lenalidomide) demands a molecular approach. A method to directly map the small molecule interactome has the potential to accelerate drug discovery by providing structural insight and instant validation of the binding interaction between a small molecule and its one or more target proteins, yet such global characterization is rarely performed due to the under-developed state of the current technology.

SUMMARY OF THE INVENTION

The present disclosure provides, in some aspects, compositions, techniques, methods, systems, and kits capable of mapping the direct and indirect effects of small molecules within the proteome. Some aspects of the present disclosure provide a small photo-click tag that enables efficient capture, enrichment, and characterization of small molecule binding sites on proteins. These small photo-click chemical tags can be incorporated into a variety of small molecules, including small molecule drugs, such as immunomodulatory drugs (e.g., lenalidomide, thalidomide, and pomalidomide) and non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., naproxen, celecoxib, and indomethacin). The small size of these photo-click chemical tags ensures that small molecule binding does not perturb the native small molecule- or protein-protein interactions. After treatment of the whole cell proteome with the photo-click modified small molecule, the resulting interactions (e.g., small molecule-protein or protein-protein interactions) are captured (i.e., “frozen” in place) (see, e.g., FIG. 7 and FIG. 8). These interactions can be affinity enriched for characterization, for example, by mass spectrometry (MS) (e.g., mass-independent or isotope-targeted mass spectrometry). In addition to revealing the binding site of the small molecule, thus helping to elucidate its mechanism of action, the photo-click modified small molecules can also be used to reveal downstream effects of the small molecule on cellular pathways by mapping protein-protein interactions (see, e.g., FIG. 1). In addition to confirming mechanisms of action of small molecules through their interaction with known target proteins, this approach can be used to identify transient complexes formed with previously unidentified target proteins. This technique is typically referred to throughout the present disclosure as Small Molecule Interactome Mapping by Photo-Affinity Labeling (SIM-PAL), or Minimally-Interfering Photo-Affinity Labeling (MI-PAL) when the smallest exemplified photo-click chemical tags provided herein are employed. The binding site identification method provided by SIM-PAL and MI-PAL is a strategy capable of precisely defining binding preferences between enantiomers and metabolites of a small molecule of interest. Additionally, amino acid analogs comprising a photo-click tag enable the measurement of protein-protein interaction (PPI) perturbations in the presence of the small molecule of interest, allowing for the determination of a global “interactome” in the presence of the small molecule (e.g., drug).

Thus, in one aspect, the present disclosure provides photo-click tags that are readily incorporated into a small molecule scaffold. In general, the multi-functional photo-click tags comprise two functional moieties: a photo-conjugation moiety that can covalently capture the protein target (e.g., an enzyme that binds to a small molecule of interest, a receptor, an antibody, etc.), and a biocompatible handle (e.g., a click chemistry handle) for functionalization with a reporter molecule or affinity tag (e.g., a biotinylated affinity tag). In some embodiments, the photo-click tag comprises (a) a photo-conjugation moiety, and (b) a click chemistry handle. In some embodiments, the photo-conjugation moiety is a diazirine moiety. In some embodiments, the photo-click tag comprises (a) a diazirine moiety and (b) a click chemistry handle. In some embodiments, the click chemistry handle is an alkyne. In some embodiments, the photo-click tag comprises (a) a diazirine moiety and (b) an alkyne. In some embodiments, the photo-conjugation moiety forms a reactive species upon irradiation with light (i.e., forms an activated photo-click tag). In some embodiments, the diazirine forms a reactive carbene species upon irradiation with light. In some embodiments, the light has a wavelength of between about 10 nm and 400 nm. In some embodiments, the reactive species reacts with a C—C, C—H, N—H, or O—H bond of a protein. In some embodiments, a new covalent bond is formed between the activated photo-click tag and the protein.

In some embodiments, the photo-click tag comprises the structure:

wherein Y, M, X, n, and m are as defined herein.

In some embodiments, the photo-click tag is of the formula

In another aspect, the present disclosure provides compounds that are covalently linked to any of the photo-click tags provided herein (i.e., photo-click conjugated compounds). In some embodiments, the compound is of Formula (I):

wherein S is a small molecule, P is a photo-click tag, and L is a linker, as provided herein.

In some embodiments, L comprises an ester, an ether, an amine, or an amide.

In some embodiments, S is an antibiotic, an anti-proliferative agent, an anti-cancer agent, a chemotherapeutic agent, an anti-angiogenesis agent, an anti-inflammatory agent, an immunosuppressant, an immunomodulatory agent, an anti-bacterial agent, an anti-viral agent, a cardiovascular agent, a cholesterol-lowering agent, an anti-diabetic agent, an anti-allergic agent, a contraceptive agent, or a pain-relieving agent. In some embodiments, S is an anti-inflammatory agent, or derivative thereof. In some embodiments, S is an immunomodulatory agent, or derivative thereof. In some embodiments, S is a chemotherapeutic agent, or derivative thereof.

In some embodiments, S is a steroid, or derivative thereof.

In some embodiments, the compound comprises one or more isotopically labeled atoms. In some embodiments, the isotopically labeled atom is ¹⁵N, ¹³C, ¹⁹F, or ²H.

In yet another aspect, provided herein are methods for using the photo-click tags and compounds conjugated to photo-click tags provided herein to determine, analyze, and evaluate small molecule-protein or protein-protein interactions. In some embodiments, the proteins are present in a cell, allowing for the determination of these interactions in the native cellular environment.

In one aspect, provided herein is a method for identifying a target protein of a small molecule. In some embodiments, the method comprises: (i) providing a compound comprising the small molecule connected to a photo-click tag via a linker, wherein the photo-click tag comprises (a) a diazirine moiety, and (b) a click chemistry handle; (ii) activating the diazirine moiety by irradiating the compound of (i) with a specific wavelength of light; (iii) contacting the target protein with the activated compound of (ii); (iv) forming a complex through a photo-induced covalent bond between the activated compound of (ii) and the protein; and (v) identifying the complex produced in (iv) that is bound to the compound, thereby identifying the target protein of the small molecule.

In another aspect, provided herein is a method for identifying the binding site of a small molecule on a protein. In some embodiments, the method comprises (i) providing a compound comprising the small molecule connected to a photo-click tag via a linker, wherein the photo-click tag comprises (a) a diazirine moiety, and (b) a click chemistry handle; (ii) activating the diazirine moiety by irradiating the compound of (i) with a specific wavelength of light; (iii) contacting the protein with the activated compound of (ii); (iv) forming a complex through a photo-induced covalent bond between the activated compound of (ii) and the protein; (v) digesting the protein of the complex into constitutive peptides in the presence of a protease; and (vi) identifying the one or more peptides produced in (iv) that is bound to the compound, thereby identifying the protein binding site of the small molecule.

In another aspect, provided herein is a method for identifying an interaction between a first protein and a second protein in a cell. In some embodiments, the method comprises: (i) providing the cell with an amino acid analog, wherein the amino acid analog comprises a photo-click tag comprising (a) a diazirine moiety, and (b) a click chemistry handle, and wherein the amino acid analog is incorporated into the first protein and/or the second protein during protein synthesis; (ii) activating the diazirine moiety of the amino acid analog by irradiating the cell with a specific wavelength of light, (iii) contacting the first protein or the second protein with the activated compound of (ii); (iv) forming a protein-protein complex through a photo-induced covalent bond between the activated amino acid analog of the first protein and an amino acid in the second protein; and (v) identifying the complex produced in (ii) that comprises the amino acid analog covalently linking the first protein and the second protein, thereby identifying the first protein and the second protein involved in the interaction.

Also provided herein are kits comprising any of the photo-click tags or photo-click tagged compounds provided herein. In addition, also provided herein are kits for use in performing any of the methods provided herein.

The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which constitute a part of this specification, illustrate several exemplary embodiments of the invention and together with the description, serve to explain certain principles of the invention. The embodiments disclosed in the drawings are exemplary and do not limit the scope of this disclosure.

FIG. 1 is a generalized, non-limiting schematic showing how direct and indirect effects of a small molecule (lenalidomide is exemplified) on the whole cell proteome can be studied using the technology disclosed herein, such as SIM-PAL and MI-PAL.

FIG. 2 is a generalized, non-limiting schematic outlining the MI-PAL global binding site mapping strategy.

FIG. 3 shows immunomodulatory drugs (IMIDs) as a non-limiting example to illustrate the importance of chirality in drug discovery. Despite this, the current mechanism of action of these IMIDs, for example, lenalidomide, remains incomplete and demands elucidation by a molecular approach, such as SIM-PAL or MI-PAL.

FIGS. 4A-4C shows a general schematic summarizing the gap in current technologies for analyzing small molecule-protein or protein-protein interactions. FIG. 4A illustrates the types of changes induced by small molecule binding to a target protein. FIG. 4B summarizes the current state of the druggable proteome, with a particular focus on the large percentage of the druggable proteome that is characterized as “understudied.” Adapted from Griffith et al. (2013) DGIdb: mining the druggable genome. Nature Methods 10, 1209-1210. FIG. 4C shows the predicted impact of technologies, such as SIM-PAL or MI-PAL as described herein, in uncovering druggable interactions that are currently considered “understudied” due to the lack of capable technology. SIM-PAL and MI-PAL represent strategies that are broadly translatable to any small molecule of interest.

FIG. 5 shows a generalized, non-limiting schematic outlining the SIM-PAL global binding site mapping strategy.

FIGS. 6A-6B shows a generalized strategy for developing photo-click conjugated small molecules (e.g., lenalidomide) as mechanistic probes. FIG. 6A shows examples of minimally interfering photo-click probes that have been developed herein, along with the current standard for photo-click probes (See, Li et al. (2013) Design and Synthesis of Minimalist Terminal Alkyne-Containing Diazirine Photo-Crosslinkers and Their Incorporation into Kinase Inhibitors for Cell- and Tissue-Based Proteome Profiling. Agnew Chem Int Ed. 52, pp. 8551-8556). These smaller photo-click tags may lead to less perturbation of the small molecule and/or protein binding target, have superior photo-crosslinking properties, and/or have superior click chemistry rates compared to the current, larger photo-click probes. FIG. 6B shows improved copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) properties of the minimally interfering, electronically tuned “MI-PAL” photo-click tag. Asterisks (*) indicate isotopically labeled atoms.

FIG. 7 shows a generalized strategy for applying the minimally-interfering “MI-PAL” photo-click tag to map small molecule binding sites in the whole multiple myeloma cell proteome. These results show that the binding and interactions of R-versus S-enantiomers can be elucidated using the SIM-PAL or MI-PAL technique described herein.

FIG. 8 shows a generalized, non-limiting schematic to study the effect on downstream protein-protein interactions in the presence of a small molecule (e.g., lenalidomide) or combination therapy. This method uses a photo-click labeled amino acid derivative to capture (e.g., “freeze”) and identify protein-protein interactions, allowing for the generation of a proteome linkage map.

FIG. 9 shows a generalized, non-limiting schematic outlining the SIM-PAL global binding site mapping strategy using exemplary photo-click probes. See, e.g., Flaxman H A and Woo C M (2017) Mapping the Small Molecule Interactome by Mass Spectrometry. Biochemistry, 57(2), pp. 186-193.

FIG. 10 shows a generalized, non-limiting schematic outlining system-level binding and pathway analysis in the presence of a small molecule of interest, such as a drug or drug candidate. SIM-PAL analysis can be used to predict new drug targets (i.e., proteins) or aid in the optimization of existing candidate molecules based on information, such as binding affinity, binding site size, and binding site conformation, given by the SIM-PAL analysis.

FIG. 11 shows potential, non-limiting applications of the SIM-PAL or MI-PAL technology. This technology can be used to discover and engineer molecular interactions (e.g., small molecule-protein interactions).

FIGS. 12A-12C show a general strategy to profile the NSAID interactome using SIM-PAL. FIG. 12A outlines the process in which photo-NSAIDs are applied in vivo (i.e., in cellulo) and conjugated to protein binding partners by photo-irradiation. Conjugated proteins are tagged by probe 10 using copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and enriched to separately obtain the protein interactome and conjugated peptides representing binding site hotspots. Conjugated peptides are analyzed by isotope-targeted mass spectrometry (MS). FIG. 12B shows structures of NSAIDs naproxen (1), celecoxib (2), and indomethacin (3), their photo-NSAID analogs 4-6, and the negative controls tag 7, the orthogonal compound photo-glutarimide 8, and the celecoxib analog 9. COX-2 IC₅₀ by ELISA is shown below each structure. FIG. 12C shows the structure of the cleavable biotin azide probe 10. Probe 10 is prepared as a 1:3 mixture of stable ¹²C:¹³C isotopes (highlighted in red).

FIGS. 13A-13E show a comparative analysis of NSAIDs and photo-NSAIDs with recombinant COX-2. FIG. 13A shows a fluorescence image of photo-NSAID binding to COX-2 and competitive displacement by the respective NSAID. COX-2 (125 ng) was incubated with photo-NSAIDS with or without the parent molecule, photo-crosslinked, and clicked with TAMRA-azide. FIG. 13B shows the docking structure of photonaproxen (4, red) and naproxen (1, blue) with COX-2. FIG. 13C shows the docking structure of photo-celecoxib (5, green) and celecoxib (2, blue) with COX-2. FIG. 13D shows the docking structure of photo-indomethacin (6, yellow) and indomethacin (3, blue). FIG. 13E shows the docking structure of diazirine tag 7 (purple) with COX-2. White box indicates part of COX-2 that is enlarged in sections 13B-13D. COX-2 (1 μg) was separately conjugated to each of the photo-NSAIDs (10 μM) or the diazirine tag 7 (10 μM), tryptically digested, and analyzed on an Orbitrap Elite. Conjugated peptides observed by MS are highlighted for each photo-NSAID. Docking structures were either the lowest desolvation energy or highest interface area size binding models created by Patchdock (October, 2017). Structure of COX-2 from PDB: 5KIR.

FIGS. 14A-14E show the photo-NSAID (250 μM) protein interactome. FIG. 14A shows protein enrichment of the photo-NSAIDs against the tag 7 based on PSMs. Proteins that were not statistically significant across the two biological replicates are displayed at a fold change of zero. FIG. 14B shows the overlap across the Jurkat photo-NSAID enriched proteome. FIG. 14C shows proteomic overlap between enriched proteins from Jurkat and K562 cells. FIG. 14D shows sub-cellular localization of the 1034 enriched proteins. FIG. 14E shows selected protein interaction networks captured by photo-NSAIDs. Color scheme: red=significantly enriched; pink=greater than 2-fold enriched; grey=identified in data; white=member of the protein complex not identified in data. Dashed border indicates direct observation of at least one conjugated peptide. Protein interaction networks were built using The Comprehensive Resource of Mammalian Protein Complexes (CORUM) as a reference. Half maximal effective concentration (EC₅₀) values for inhibition of the NF-κB pathway as determined by a NF-κB luciferase reporter described in Example 1.

FIGS. 15A-15E show the evaluation of photo-NSAID binding site hotspots identified by SIM-PAL. FIG. 15A shows precursor pattern distribution (MS1) and database assignment (MS2) for a histone H2A peptide conjugated to each of the photo-NSAIDs. FIG. 15B shows a cluster diagram of histone protein complexes with at least one observed binding site to a photo-NSAID. High confidence interactions between two clusters made using STRING. FIG. 15C shows the structure of the nucleosome (PDB: 2CV5). Peptides from histone H2A (red) and histone H2B (blue) that were conjugated by all three photo-NSAIDs are highlighted. FIG. 15D shows competitive displacement of photo-NSAIDs by the parent compound. Jurkat cell lysates conjugated to the indicated compound with or without the parent compound were clicked with the biotin probe 10, captured on streptavidin-agarose, and probed for histone H2A, H2B, Ku70, NPM1, or NF-κB p65. FIG. 15E shows a cellular thermal shift assay performed on Jurkat cells in the presence of the indicated compound, probed for histone H2A.

FIGS. 16A-16D show inhibition of COX-2 by (FIG. 16A) photo-naproxen (4) and naproxen (1), (FIG. 16B) photo-celecoxib (5) and celecoxib (2), and (FIG. 16(C) photo-indomethacin (6) and indomethacin (3). FIG. 16D shows COX-2 IC₅₀ values for photo-NSAIDs and NSAIDs. IC₅₀ values were determined as described by the ELISA-based COX-2 inhibitor screening assay and performed in triplicate. Data is representative of two biological replicates.

FIGS. 17A-17D show MTT cell viability assay for photo-NSAIDs and NSAIDs in Jurkat cells. Dose-response curves for (FIG. 17A) photo-naproxen (4) and naproxen (1), (FIG. 17B) celecoxib (2), photo-celecoxib (5) and celecoxib analog 9, and (FIG. 17C) photo-indomethacin (6) and indomethacin (3). FIG. 17D shows IC₅₀ values for NSAIDs and photo-NSAIDs in Jurkat cells. All dose-response curves were obtained in triplicate.

FIGS. 18A-18C show photo-NSAID dose-dependence and photo-irradiation-dependent labeling of Jurkat proteins. FIG. 18A shows dose-dependent labeling of Jurkat cells with the photo-NSAIDs, the tag 7, or photo-glutarimide 8. Jurkat cells were incubated with the small molecule at the indicated concentration for 1 hour at 37° C. and photoirradiated (30 minutes, 4° C.). Irradiated cells were lysed, clicked with tetramethylrhodamine-azide (TAMRA-azide), and visualized on gel. Coomassie blue staining shows equal protein loading. FIG. 18B shows anti-biotin Western blot from 250 μM of photo-NSAID or DMSO control treated Jurkat cells with (+) or without (−) UV irradiation for photo-naproxen (4), photo-celecoxib (5), and photo-indomethacin (6). Ponceau S staining shows equal amount of protein loading. FIG. 18C shows competition assay titration with photo-celecoxib (5) and the competitor celecoxib (2) at 1:1, 1:10, and 1:100 molar ratios. Jurkat cells were incubated with the tag 7, photo-celecoxib (5), or co-treated with photo-celecoxib (5) and the parent compound celecoxib (2) at 1:1, 1:10, and 1:100 molar ratios for 1 hour at 37° C., followed by photoirradiation (30 minutes, 4° C.) and cell lysis. Cell lysates were adjusted to 1.5 mg/mL, and were then clicked with 200 μM biotin azide probe 10, 300 μM copper (II) sulfate, 600 μM BTTP, and 2.5 mM freshly-prepared sodium ascorbate for 2 hours at 24° C. with rotation, followed by analysis by Western blotting using HRP-streptavidin conjugate. Ponceau S staining shows equal amount of protein loading.

FIG. 19 shows an anti-biotin Western blot for enriched small molecule conjugated proteins from Jurkat cells. Jurkat cells were treated with a photo-NSAID or the tag 7, photo-irradiated and clicked with the cleavable biotin azide probe 10 (click). Biotinylated proteins were enriched on streptavidin-agarose beads, and the beads were washed (1% RapiGest, 6M urea, PBS). The biotin-depleted whole proteome (supernatant) and the beads (capture) were analyzed by gel. Ponceau S staining shows protein loading.

FIGS. 20A-20C show photo-NSAID interactions with COX-2 in Jurkat cells. FIG. 20A shows a Western blot probed for COX-2 of photo-NSAID captured proteins and competitive displacement by the parent compound in Jurkat whole cell lysates. Jurkat cells were incubated with photo-NSAIDs [250 μM of photo-naproxen (4) and photoindomethacin (6); 50 μM of photo-celecoxib (5)] in the presence or absence of the parent compound (1:10 molar ratio) for 2 hours and photo-irradiated. Photo-irradiated proteins were lysed and clicked with the biotin azide probe 10 (load) and enriched on streptavidin-agarose (capture). FIG. 20B shows a cellular thermal shift assay probed for COX-2 in Jurkat cells in the presence of 50 μM of the tag 7, the celecoxib analog 9, celecoxib (2), or photo-celecoxib (5). Data are representative of two independent biological replicates. FIG. 20C shows quantification of COX-2 signal in cellular thermal shift assay using ImageJ and GraphPad Prism software. The solid lines represent the best fits of the data to the Boltzmann sigmoid equation, resulting in an apparent T_agg of 27.6±0.6° C. for the tag 7, and 40.9±1.0° C. for the celecoxib analog 9, whereas both celecoxib (2) and photo-celecoxib (5) stabilized COX-2 at 50.5±0.6° C. and 52.2±1.0° C., respectively.

FIGS. 21A-21C show proteomics data reproducibility and protein overlap of analyzed compounds. FIG. 21A shows relative protein abundance using peptide spectral matching (PSM) based label free quantification across the two Jurkat biological replicates collected at 250 μM photo-NSAIDs. FIG. 21B shows the overlap across one biological replicate of the Jurkat photo-NSAID enriched proteome at 50 μM photo-NSAIDs. FIG. 21C shows the proteomic overlap between enriched proteins from Jurkat cells exposed to 50 μM photo-celecoxib (5) or the celecoxib analog 9.

FIG. 22 shows an anti-biotin Western blot for enriched small molecule conjugated proteins from K562 cells. K562 cells were treated with a photo-NSAID or the tag 7, photo-irradiated and clicked with the cleavable biotin azide probe 10 (click). Biotinylated proteins were enriched on streptavidin-agarose beads, and the beads were washed (1% RapiGest, 6 M urea, PBS). The biotin-depleted whole proteome (supernatant) and the beads (capture) were analyzed by gel. Ponceau S staining shows protein loading.

FIG. 23 shows a venn diagram of statistically-enriched photo-NSAID (250 μM) protein interactome from K562 cells across two biological replicates.

FIG. 24 shows a TNF-α titration curve for NF-κB-RE-luc2P HEK293 cells treated with 5 μM of the tag 7, celecoxib (2), photo-celecoxib (5), and DMSO. The luminescence was quantified using the ONE-Glo Luciferase Assay System Reagent on a microplate reader FilterMax F3. All data were obtained in triplicate, data representative of two independent experiments.

FIG. 25 shows example spectra of photo-naproxen (4) and photo-celecoxib (5) conjugated to a peptide from histone H2B. Spectra were validated for isotopic pattern (MS1) and spectral assignment (MS2) by SEQUEST HT.

FIG. 26 shows a synthetic scheme for the synthesis of photo-NSAID analogs. Reagents and reaction conditions: (a) (i) HBr, 12 hours, reflux (ii) acetyl chloride, CH₃OH, 4 hours, reflux (95%, two steps); (b) K₂CO₃, S7, DMF, 12 hours, 24° C. (85%); (c) 1M NaOH, methanol, 4 hours, 24° C. (98%); (d) S8, ethanol, 12 hours, reflux (99%); (e) K₂CO₃, S7, DMF, 12 h, 24° C. (75%); (f) N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), triethyl amine, S6, DMAP, DMF, 12 hours, 24° C. (88%); (g) CDI, THF, 12 hours, 24° C. (95%); (h) 3-aminopiperidine-2,6-dione hydrochloride, DBU, DMF, 12 hours, 24° C. (86%); (i) phenylhydrazine, ethanol, 12 hours, reflux (92%); (j) K₂CO₃, S7, DMF, 12 hours, 24° C. (68%).

FIG. 27 shows a synthetic scheme for the synthesis of the cleavable biotin azide probe 10.

FIG. 28 shows structures of arylalkyne 1, “minimalist” tag 2, and MI-PAL (3), chemical tags that possess a diazirine functional group and alkyne reporter handle. Retrosynthesis of 3 proceeds through the ester 4 and alkyne 5.

FIG. 29 shows the synthetic scheme used for the synthesis of MI-PAL (3) from the ester 4 and the alkyne 5.

FIGS. 30A-C show photo-conjugation and CuAAC with MI-PAL (3) to the proteome. FIG. 30A shows photoconjugation of MI-PAL (3) to alpha-crystallin. FIG. 30B shows an example collision-induced dissociation (CID) mass spectra of a peptide from alpha-crystallin conjugated to MI-PAL (3). M represents the precursor mass. FIG. 30C shows visualizion by Western blot of whole cell lysates from MM.1S (multiple myeloma) or K562 (chronic mylogenous leukemia) incubated with MI-PAL (3) and photoirradiated. Photoconjugated lysates were treated with biotin-azide and visualized by Western blot.

FIG. 31 shows the synthetic scheme used for the synthesis of the MI-PAL-tagged glycine methylester 12, the MI-PAL-tagged daunorubicin 13, and the MI-PAL-tagged mitomycin 14.

FIG. 32 shows the synthetic scheme used for the synthesis of the MI-PAL-tagged naproxen analog 16 and the MI-PAL-tagged β-estradiol 17 from the iodide 15.

DEFINITIONS

Descriptions and certain information relating to various terms used in the present disclosure are collected herein for convenience.

As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents.

The term “agent” is used herein to refer to any substance, compound (e.g., small molecule, peptide, protein, nucleic acid), supramolecular complex, material, or combination or mixture thereof. A compound may be any agent that can be represented by a chemical formula, chemical structure, or sequence. Examples of agents include, e.g., small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids, polysaccharides, etc. In general, agents may be obtained using any suitable method known in the art. In some embodiments, the agent is a small molecule. In some embodiments, the agent is an antibiotic, anti-proliferative agent, an anti-cancer agent, a chemotherapeutic agent, anti-angiogenesis agent, anti-inflammatory agent, immunosuppressant, immunomodulatory agent, anti-bacterial agent, anti-viral agent, cardiovascular agent, cholesterol-lowering agent, anti-diabetic agent, anti-allergic agent, contraceptive agent, or pain-relieving agent. In some embodiments, the agent is an anti-inflammatory agent. In some embodiments, the agent is an immunomodulatory drug. In some embodiments, the agent is a chemotherapeutic agent. These compounds may be conjugated to a photo-click tag provided herein. The ordinary skilled artisan will select an appropriate method for preparing a “photo-click conjugated agent” based, e.g., on the nature of the agent (e.g., the functional groups present in the agent) and the desired photo-click tag to be used. Examples of synthetic procedures for generating photo-click conjugated agents is provided in Examples 1 and 2 (also see FIGS. 26, 31, and 32). An agent may be at least partly purified. In some embodiments, an agent may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the agent, in various embodiments. In some embodiments, an agent may be provided as a salt, ester, hydrate, or solvate. In some embodiments an agent is cell-permeable, e.g., within the range of typical agents that are taken up by cells and acts intracellularly, e.g., within mammalian cells, to produce a biological effect. Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (−)- and (+)-isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated. Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates (e.g., with water (i.e., hydrates) or common solvents) and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable.

The term “amide,” as used herein, refers to the group —C(═O)N(R^X1)—, wherein each R^X1 is independently hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, or mono- or di-heteroarylamino. In some embodiments, R^X1 is hydrogen. In some embodiments, a photo-click tagged compound herein comprises a small molecule covalently linked to a photo-click tag by an amide.

The term “amine,” as used herein, refers to the group —N(R^X1)₂—, wherein each R^X1 is independently hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, or mono- or di-heteroarylamino. In some embodiments, R^X1 is hydrogen. In some embodiments, a photo-click tagged compound herein comprises a small molecule covalently linked to a photo-click tag by an amine.

The term “anti-inflammatory agent” refers to any compound (e.g., small molecule) that exhibits anti-inflammatory properties when administered to a subject in need thereof. The anti-inflammatory agent may be a steroidal anti-inflammatory agent or a nonsteroidal anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug. The term “nonsteroidal anti-inflammatory drug” or “NSAID” as used herein refers to any compound (e.g., small-molecule) that can be classified as an NSAID. In general, an NSAID is a small molecule that reduces pain, reduces fever, prevents blood clots, and/or decreases inflammation, and the like. In general, NSAIDs work by inhibiting the activity of cyclooxygenase enzymes (COX-1 and/or COX-2). In cells, these enzymes are involved in the synthesis of key biological mediators, namely prostaglandins, which are involved in inflammation, and thromboxanes, which are involved in blood clotting. There are two types of NSAIDs available: non-selective and COX-2 selective. Non-selective NSAIDs inhibit the activity of both COX-1 and COX-2. In some embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent. The term “steroidal anti-inflammatory agent” refers to any natural or synthetic agent that comprises the core structure of a steroid and exhibits an anti-inflammatory property when administered to a subject in need thereof.

In some embodiments, the reaction used to generate a photo-click conjugated agent is a click chemistry reaction. In some embodiments, the reaction used to conjugate the photo-click conjugated agent to a target protein is a click chemistry reaction. In some embodiments, the reaction used to conjugate (i.e., “click”) a label onto the photo-click tag is a click chemistry reaction. It is to be understood that any “click chemistry” reaction known in the art can be used to this end. Click chemistry is a chemical philosophy introduced by Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining small units together. See, e.g., Kolb, Finn, and Sharpless, Angew Chem Int Ed (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395. The term “click chemistry” does not refer to a specific reaction or set of reaction conditions, but instead refers to a class of reactions (e.g., coupling reactions). Exemplary coupling reactions (some of which may be classified as “click chemistry”) include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-yne addition; imine formation; and Michael additions (e.g., maleimide addition). Examples of click chemistry reactions can be found in, e.g., Kolb, H. C.; Finn, M. G. and Sharpless, K. B. Angew. Chem. Int. Ed 2001, 40, 2004-2021. Kolb, H. C. and Sharpless, K. B. Drug Disc. Today, 2003, 8, 112-1137; Rostovtsev, V. V.; Green L. G.; Fokin, V. V. and Sharpless, K. B. Angew. Chem. Int. Ed. 2002, 41, 2596-2599; Tomoe, C. W.; Christensen, C. and Meldal, M. J. Org. Chem. 2002, 67, 3057-3064. Wang, Q. et al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Lee, L. V. et al. J. Am. Chem. Soc. 2003 125, 9588-9589; Lewis, W. G. et al. Angew. Chem. Int. Ed. 2002, 41, 1053-41057; Manetsch, R. et al., J. Am. Chem. Soc. 2004, 126, 12809-12818; Mocharla, V. P. et al. Angew. Chem., Int. Ed. 2005, 44, 116-120; each of which is incorporated by reference herein. In some embodiments, the click chemistry reaction involves a reaction with an alkyne moiety comprising a carbon-carbon triple bond (i.e., an alkyne handle). In some embodiments, the click chemistry reaction is a copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. A CuAAC reaction generates a 1,4-disubstituted-1,2,3-triazole product (i.e., a 5-membered heterocyclic ring). See, e.g., Hein J E and Fokin V V (2010) Copper-catalyzed azide-alkyne cycloaddition (CuAAC) and beyond: new reactivity of copper (I) acetylides. Chem Soc Rev, 39(4), pp. 1302-1315; the contents of which is incorporated by reference herein. For example, as shown in Examples 1 and 2, a label (e.g., a biotinylated affinity label) comprising an azide moiety can be conjugated to a photo-click tag comprising an alkyne using a CuAAC reaction. In some embodiments, the click chemistry reaction is a photo-click reaction. As used herein, a “photo-click” reaction is a reaction in which a compound is activated upon irradiation with a specific wavelength of UV light (i.e., between about 10 nm and about 400 nm). The activated compound can then rapidly form a covalent bond with the nearest target molecule (e.g., chemical bonds present in a protein). In some embodiments, the compound comprises a diazirine, which forms a reactive carbene upon irradiation with UV light. The activated carbene can then react with a nearby C—C, C—H, N—H, O—H, or X—H (X=heteroatom) bond present in a protein, thus conjugating the diazirine to the protein. See, e.g., Dubnisky L et al (2011) Diazirine based photoaffinity labeling. Bioorg Med Chem 20, 554-570; which is incorporated herein by reference. The use of diazirines as photo-reactive crosslinking reagents, or photo-click reagents, has been described in, e.g., Sinz A. (2007) Investigation of Protein-Ligand Interactions by Mass Spectrometry. Chem Med Chem, 2, pp. 425-431; and Brunner J. (1993) New photolabeling and crosslinking methods. Annu Rev Biochem, 62, pp. 483-514; the entire contents of each of which are incorporated herein by reference.

The term “compound” as used herein encompasses any small molecule, peptide, nucleic acid, protein, or derivative thereof that can bind to and/or modulate (e.g., increase the activity of, decrease the activity of) a target of interest (e.g., a protein, e.g., an enzyme, receptor, reporter protein, etc.)). In some embodiments, the compound is a small molecule.

The term “ester,” as used herein, refers to the group —C(═O)O—. In some embodiments, a photo-click tagged compound herein comprises a small molecule covalently linked to a photo-click tag by an ester.

The term “ether,” as used herein, refers to the group —O—. In some embodiments, a photo-click tagged compound herein comprises a small molecule covalently linked to a photo-click tag by an ether.

The term “immunomodulatory drug” or “IMID” refers to any compound (e.g. small molecule) that can be classified as an immunomodulatory drug. In general, an immunomodulatory drug is an anti-inflammatory or antineoplastic compound that can modulate an immune response. Most immunomodulatory drugs are structurally and functionally similar to thalidomide. Thalidomide can exist as two enantiomers, R-thalidomide and S-thalidomide. Each enantiomer may exhibit a different function or interact with a specific drug target (e.g., protein). Thus, immunomodulatory drugs are often small molecule derivatives of R-thalidomide or S-thalidomide. Exemplary, non-limiting immunomodulatory drugs are (R/S)-lenalidomide, (R/S)-pomalidomide, and (R/S)-apremilast. In some embodiments, the immunomodulatory drug is thalidomide. In some embodiments, the immunomodulatory drug is lenalidomide. In some embodiments, the immunomodulatory drug is S-lenalidomide. In some embodiments, the immunomodulatory drug is R-lenalidomide. In some embodiments, the immunomodulatory drug is pomalidomide. In some embodiments, the immunomodulatory drug is S-pomalidomide. In some embodiments, the immunomodulatory drug is R-pomalidomide. In some embodiments, the immunomodulatory drug is apremilast. In some embodiments, the immunomodulatory drug is S-apremilast. In some embodiments, the immunomodulatory drug is R-apremilast.

The term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a small molecule and a photo-click tag. A linker may be, for example, a bond, an amino acid sequence, a peptide, or a polymer of any length and composition. In some embodiments, the linker is an organic molecule, functional group, polymer, or chemical moiety.

The term “photo-conjugation moiety,” as used herein, refers to any moiety that forms a reactive intermediate upon irradiation with light (i.e., “photo-activation”). These moieties may be referred to as “photoreactive” moieties. Examples of photoreactive moieties are aryl azides, azido-methyl-coumarins, benzophenones, anthraquinones, diazo compounds, diazirines, and psoralen derivatives. For example, an aryl azide (e.g., phenyl azide, ortho-hydroxyphenyl azide, meta-hydroxyphenyl azide, tetrafluorophenyl azide, ortho-nitrophenyl azide, and meta-nitrophenyl azide) forms a reactive nitrene groups that can initiate addition reactions with double bonds, insertion into C—H and N—H bonds, or ring expansion in the presence of a nucleophile (e.g., a primary amine). In general, aryl azides are photoactived upon irradiation with UV light with a wavelength of about 250 nm to about 350 nm. Photo-conjugation moieties, their uses, and chemical reactions are discussed in Bioconjugate Techniques, 3^rd Ed. (2013) by Hermanson; which is incorporated by reference herein.

The term “protease” refers to any enzyme capable of hydrolyzing a peptide bond. In general, a proteases catalyzes the hydrolysis of peptide bonds (i.e., digests the protein) through a unique mechanism based on the catalytic residue present in the active site of the protease. Exemplary, non-limiting proteases and their catalytic residues are serine proteases, which use a serine alcohol, cysteine proteases, which use a cysteine thiol, threonine proteases, which use a threonine secondary alcohol, aspartic proteases, which use an aspartate carboxylic acid, glutamic proteases, which use a glutamate carboxylic acid, metalloproteases, which use a metal (e.g., zinc), and asparagine peptide lyases, which use an asparagine to perform an elimination reaction and do not require water. In some embodiments, the protease is a serine protease. In some embodiments, the serine protease is trypsin. In some embodiments, the serine protease is chymotrypsin. In some embodiments, the protease is an aspartic protease. In some embodiments, the aspartic protease is pepsin. In some embodiments, one protease is used to digest a protein. In some embodiments, more than one protease is used to digest a protein.

The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. In some embodiments, a protein comprises a homodimer or a heterodimer. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein comprises a ligand binding domain. In some embodiments, a protein comprises an active site (e.g., site of biological or enzymatic activity). In some embodiments, a protein comprises an allosteric site (e.g., site of a protein that can bind to a ligand that can be remote from an active site). In some embodiments, the protein is an enzyme. In some embodiments, the protein is a receptor. In some embodiments, the protein is a reporter protein (e.g., GFP). Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. In some embodiments, the protein is present in vitro. In some embodiments, the protein is present in vivo. In some embodiments, the protein is present in a cell. In some embodiments, the protein is present in a cell lysate. In some embodiments, the protein is present in a whole proteome (i.e., the entire complement of proteins that is or can be expressed by a cell, tissue, or organism).

As used herein, the term “salt” refers to any and all salts, and encompasses pharmaceutically acceptable salts.

The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N⁺(C_1-4 alkyl)₄⁻ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

The term “sample” may be used to generally refer to an amount or portion of something (e.g., a protein). A sample may be a smaller quantity taken from a larger amount or entity; however, a complete specimen may also be referred to as a sample where appropriate. A sample is often intended to be similar to and representative of a larger amount of the entity of which it is a sample. In some embodiments a sample is a quantity of a substance that is or has been or is to be provided for assessment (e.g., testing, analysis, measurement) or use. The “sample” may be any biological sample including tissue samples (such as tissue sections and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In some embodiments a sample comprises cells, tissue, or cellular material (e.g., material derived from cells, such as a cell lysate, or fraction thereof). A sample of a cell line comprises a limited number of cells of that cell line. In some embodiments, a sample may be obtained from an individual who has been diagnosed with or is suspected of having a disease.

The term “small molecule,” as used herein, is an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and/or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. In some embodiments, the small molecule is an NSAID, or a derivative thereof. In some embodiments, the small molecule is an immunomodulatory drug, or a derivative thereof. In some embodiments, the small molecule is a chemotherapeutic agent, or a derivative thereof.

The term “steroid,” as used herein, refers to any compound with a core structure comprising four fused rings, wherein three of the four rings are cyclohexane rings and one of the four rings is a cyclopentane ring. Steroids can be classified functionally, such as corticosteroids or sex steroids. In some embodiments, the corticosteroid is a glucocorticoid. In some embodiments, the corticosteroid is a mineralocorticoid. In some embodiments, the steroid is an androgen, an estrogen, or a progestogen. In some embodiments, the androgen is testosterone. In some embodiments, the estrogen is estradiol or beta-estradiol. In some embodiments, the progestogen is progesterone. Steroids can also be classified based on their chemical composition (i.e., the number of carbon atoms present in the steroid). Exemplary classifications are cholestanes (27 carbon atoms, e.g., cholesterol), cholanes (24 carbon atoms, e.g., cholic acid), pregnanes (21 carbon atoms, e.g., progesterone), androstanes (19 carbon atoms, e.g., testosterone), and estranges (18 carbon atoms, e.g., estradiol).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Small molecules are the most prominent class of agents that are used as therapeutics for the treatment of a variety of diseases and conditions, accounting for nearly 90% of the therapeutics available in the pharmaceutical market. Many agents (e.g., small molecules) elicit cell-type specific pharmacology that may increase therapeutic efficacy or yield off target toxicity. However, direct observation of the structures that underpin the global molecular associations between the proteome and even common therapeutics, such as the non-steroidal anti-inflammatory drugs (NSAIDs), remain little understood. Presented herein are photo-click tags, compounds, and methods useful for mapping the small molecule interactome of a small molecule of interest. These methods generally involve labeling of a target protein with a photo-click tag that is conjugated to the small molecule of interest, and may be refered to throughout the present disclosure as Small Molecule Interactome Mapping by Photo-Affinity Labeling (SIM-PAL), or Minimally-Interfering Photo-Affinity Labeling (MI-PAL) when the smallest exemplified photo-click chemical tags provided herein are employed. These photo-click tags, photo-conjugated compounds, and methods can be applied in vivo to identify binding sites or protein-protein interactions within the complex cellular environment. For example, SIM-PAL uses (1) photochemical conjugation of small molecules throughout the whole proteome and (2) enrichment of the conjugated peptides for (3) targeted mass spectrometry-based assignment. In addition, these photo-click tags, photo-conjugated compounds, and methods amenable to map global binding sites for virtually any agent of interest.

Photo-Click Tags

The development of small, multi-functional photo-click tags that are readily incorporated into a small molecule scaffold has accelerated target identification for non-covalent agents that interact with one or more proteins in the proteome. In general, the multi-functional photo-click tags comprise two functional moieties: a photo-conjugation moiety that can covalently capture the protein target (e.g., an enzyme that binds to a small molecule of interest, a receptor, an antibody, etc.), and a biocompatible handle (e.g., a click chemistry handle) for functionalization with a reporter molecule or affinity tag (e.g., a biotinylated affinity tag). A biocompatible handle (e.g., a click chemistry handle) may be designed to study biological processes in their native environment (e.g., in living cells). The photo-conjugation moiety may comprise, for example, an aryl azide, benzophenone, or diazirine moiety, which can generate a short-lived, highly reactive intermediate (e.g., a nitrene, carbene, or diradical intermediate) that covalently attaches itself to a nearby biomolecule (e.g., a protein, nucleic acid, lipid). The biocompatible handle may be a click chemistry handle, for example, an alkyne, an alkene (e.g., a strained alkene or an activated alkene), a cyano moiety (—C≡N), or additional suitable click chemistry handles known in the art. Taken together, the photo-conjugation moiety and the click chemistry handle provide a multi-functional “photo-click tag”. The photo-click tag may comprise the structure:

wherein

M is a photo-conjugation moiety;

Y is a click chemistry handle;

X is —OH, —NH₂, or a halogen; and

m and n are each independently integers between 1-10, inclusive.

In some embodiments, m is 0, 1, or 2. In some embodiments, n is 0, 1, or 2. In some embodiments, m and n taken together provide a photo-click tag fewer than 10 carbons in length (i.e., a C_1-10 alkyl), not including the click chemistry handle. In some embodiments, m and n taken together provide a photo-click tag fewer than 10 carbons in length (i.e., a C_1-7 alkyl), not including the click chemistry handle. In some embodiments, the photo-click tag comprises a C_1-10 alkyl. The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (“C_1-10 alkyl”). Examples of C_1-6 alkyl groups include methyl (C₁), ethyl (C₂), propyl (C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C₆) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈), and the like. In some embodiments, the photo-click tag comprises a C_3-7 alkyl, not including the click chemistry handle. In some embodiments, the photo-click tag comprises a C₃ alkyl, not including the click chemistry handle. In some embodiments, the photo-click tag comprises a C₄ alkyl, not including the click chemistry handle. In some embodiments, the photo-click tag comprises a C₅ alkyl, not including the click chemistry handle. In some embodiments, the photo-click tag comprises a C₆ alkyl, not including the click chemistry handle. In some embodiments, the photo-click tag comprises a C₇ alkyl, not including the click chemistry handle. For example, in some embodiments, m is 1 and n is 1 to provide a photo-click tag that is 3 carbon atoms in length (C₃ alkyl). Without wishing to be bound by any particular theory, photo-click tags with small scaffolds could provide the added benefit of reduced perturbation of small molecule binding to a protein target when the small molecule is bound to the photo-click tag. For example, integration of a small photo-click tag can preserve the native interaction between the small molecule conjugated to the photo-click tag and the target protein(s). See Examples 1 and 2.

In some embodiments, X is —OH. In some embodiments, X is —NH₂. In some embodiments, X is halogen. In some embodiments, X is chlorine (Cl). In some embodiments, X is iodine (I). In some embodiments, X is fluorine (F). In some embodiments, X is bromine (Br).

Thus, in one aspect, provided herein are photo-click tags comprising (a) a photo-conjugation moiety, and (b) a click chemistry handle. In some embodiments, the photo-conjugation moiety is a diazirine moiety. In some embodiments, the photo-click tag comprises (a) a diazirine moiety, and (b) a click chemistry handle. In some embodiments, the click chemistry handle is an alkyne. In some embodiments, the photo-click tag comprises (a) a diazirine moiety, and (b) an alkyne. An “alkyne” is a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C_2-10 alkynyl”). In some embodiments, an alkyne group has 2 to 9 carbon atoms (“C_2-9 alkyne”). In some embodiments, an alkyne has 2 to 8 carbon atoms (“C_2-8 alkyne”). In some embodiments, an alkyne has 2 to 7 carbon atoms (“C_2-7 alkynyl”). In some embodiments, an alkyne has 2 to 6 carbon atoms (“C_2-6 alkyne”). In some embodiments, an alkyne has 2 to 5 carbon atoms (“C_2-5 alkyne”). In some embodiments, an alkyne has 2 to 4 carbon atoms (“C_2-4 alkyne”). In some embodiments, an alkyne has 2 to 3 carbon atoms (“C_2-3 alkyne”). In some embodiments, an alkyne has 2 carbon atoms (“C₂ alkyne”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butyne) or terminal (such as in 1-butyne). Examples of C_2-4 alkynes include, without limitation, ethyne (C₂), 1-propyne (C₃), 2-propyne (C₃), 1-butyne (C₄), 2-butyne (C₄), and the like. Examples of C_2-6 alkynes include the aforementioned C_2-4 alkyne groups as well as pentyne (C₅), hexyne (C₆), and the like. Additional examples of alkynes include heptyne (C₇), octyne (C₈), and the like. Unless otherwise specified, each instance of an alkyne is independently unsubstituted (an “unsubstituted alkyne”) or substituted (a “substituted alkyne”) with one or more substituents. In certain embodiments, the alkyne is an unsubstituted C_2-10 alkyne. In certain embodiments, the alkyne is an unsubstituted C₂ alkyne. In certain embodiments, the alkyne is a substituted C_2-10 alkyne.

In some embodiments, any of the photo-click tags provided herein may be optionally substituted with one or more electronegative atoms. In some embodiments, the photo-click tag is optionally substituted with one electronegative atom. In some embodiments, the photo-click tag is optionally substituted with two electronegative atoms. The electronegative atoms may be attached to the same carbon, or the electronegative atoms may be attached to two different carbons, as valency permits. In some embodiments, one or more of the electronegative atoms is fluorine. In some embodiments, the photo-click tag is optionally substituted with one fluorine atom. In some embodiments, the photo-click tag is optionally substituted with two fluorine atoms. The fluorine atoms may be attached to the same carbon, or the fluorine atoms may be attached to two different carbons, as valency permits. Placing the one or more fluorine atoms in proximity to the diazirine moiety on the photo-click tag may improve photo-conjugation and/or click chemistry reaction kinetics, as photochemical carbene intermediates and click chemistry reaction kinetics are dependent on electronic substituent effects. See, e.g., Brunner J. et al. (1980) J Biol Chem, 255, pp. 3313-3318. Without wishing to be bound by any particular theory, electronically tuning the photo-click tag by placing one or more fluorine substituents on the carbon atom adjacent to the diazirine moiety could provide a more reactive photo-click tag, thereby increasing its utility as a photo-click tag, particularly in in vivo applications or applications in whole proteomes where there are a large number of proteins and other biomolecules present (e.g., in a cell, tissue, or organism). In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-conjugation moiety (M) is an aryl azide, azido-methyl-coumarin, benzophenone, anthraquinone, diazo compound, diazirine, or psoralen derivative. In some embodiments, the photo-conjugation moeity (M) is a diazirine moeity.

Diazirines are a class of organic small molecules comprising a carbon bound to two nitrogen atoms, wherein the two nitrogen atoms are double bonded to one another, thus forming a three-membered heterocyclic ring. In some embodiments, the diazirine moiety comprises the structure:

Upon irradiation with a specific wavelength of light (e.g., ultraviolet light), a diazirine forms a reactive carbene species (see Scheme I below). In some embodiments, the reactive carbene species comprises the structure

upon irradiation with a specific wavelength of light. In some embodiments, the reactive carbene species comprises at least two free electrons. In some embodiments, the specific wavelength of light is between about 10 nm and about 400 nm. In some embodiments, the specific wavelength of light is between about 355 nm and about 365 nm. In some embodiments, the specific wavelength of light is about 355 nm. In some embodiments, the specific wavelength of light is about 365 nm.

In some embodiments, the reactive carbene species reacts with a C—C, C—H, N—H, or O—H bond of a protein. Upon insertion of the reactive carbine species into a C—C, C—H, N—H, or O—H bond of a protein, a new covalent bond between the protein and photo-click tag is formed, thereby linking the photo-click tag to the protein (see, e.g., FIG. 30). This process may be referred to as photoaffinity labeling (PAL) (Dubinsky et al. (2011) Diazirine based photoaffinity labeling. Bioorg Med Chem, 20, pp. 554-570). A non-limiting example of a photo-conjugation of a photo-click tag, or a compound conjugated to a photo-click tag, to a target protein is shown below in Scheme II. The reaction in Scheme II can be performed in vitro or in vivo.

[S represents a small molecule conjugated to a group comprising a diazirine moiety (e.g., a photo-click tag), R represents an oxygen, nitrogen, or carbon atom in amino acid in the target protein]

In some embodiments, the photo-click tag is of the formula:

wherein,

X is —OH, —NH₂, or a halogen.

In some embodiments, X is —OH. In some embodiments, X is —NH₂. In some embodiments, X is halogen. In some embodiments, X is chlorine (Cl). In some embodiments, X is iodine (I). In some embodiments, X is fluorine (F). In some embodiments, X is bromine (Br). A person of skill in the art will recognize the appropriate X group for use in a photo-click tag in accordance with the present disclosure based on the functional groups present in the agent to be conjugated to the photo-click tag. For example, FIG. 26 shows exemplary synthetic schemes that can be used to conjugate a photo-click tag wherein X is —NH₂ to a small molecule comprising an —OH group, thereby generating a photo-conjugated agent comprising a —O— linker between the small molecule and the photo-click tag.

In some embodiments, wherein X is I, the photo-click tag is of the formula:

In some embodiments, wherein X is —NH₂, the photo-click tag is of the formula:

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, the photo-click tag is of the formula:

In some embodiments, for example, when the photo-click tag is associated with a small molecule of interest, the photo-click tag is coupled to the small molecule through a linker, as described below. Thus, in some embodiments, the photo-click tag is of the formula:

In some embodiments, the photo-click tag is less than about 10 Å in length. In some embodiments, the photo-click tag is less than about 9.5 Å in length. In some embodiments, the photo-click tag is less than about 9 Å in length. In some embodiments, the photo-click tag is less than about 8 Å in length. In some embodiments, the photo-click tag is less than about 7 Å in length. In some embodiments, the photo-click tag is less than about 6 Å in length. In some embodiments, the photo-click tag is between about 6 Å and about 9.5 Å in length. In some embodiments, the photo-click tag is between about 6 Å and about 8 Å in length. In some embodiments, the photo-click tag is between about 6 Å and about 7 Å in length. In some embodiments, the photo-click tag is about 7 Å in length. In some embodiments, the photo-click tag is about 6.9 Å in length.

Compounds Comprising Photo-Click Tags

As discussed above, the photo-click tags presented herein comprise a photo-conjugation moiety that can covalently capture (i.e., bind) the protein target (e.g., an enzyme that binds to a small molecule of interest, a receptor, an antibody, etc.), and a biocompatible handle (e.g., a click chemistry handle) for functionalization with a reporter molecule or affinity tag (e.g., a biotinylated affinity tag). Without wishing to be bound by any particular theory, the affinity can be used to enrich the target protein of interest, and aid in identifying the target protein bound to the photo-click tagged agent of interest. When attached to a compound (e.g., small molecule) of interest, these “photo-click tagged compounds” can be used to map the interactions of the compound of interest with the target molecule (e.g., a protein). These photo-click tagged compounds are useful, for example, for mapping the interactions of the compound in a whole proteome, or for mapping the proteomic interactions mediated by the compound in vivo. Some compounds (e.g., small molecule drugs) can exist as stereoisomers (e.g., epimers, diastereomers, or enantiomers), wherein each stereoisomer may exhibit a different activity (e.g., bind to different target protein). In some embodiments, a compound exists as an enantiomer, wherein the S- and R-enantiomer interact differently with the same target protein. In some embodiments, a compound exists as an enantiomer, wherein the S- and R-enantiomer interact with unique target proteins. Thus, the compounds and methods of the present invention can be useful in determining the action of a specific enantiomer or other stereoisomer of a small molecule (e.g., an immunomodulatory drug). For example, the orientation of each enantiomer may place the photo-conjugation (e.g., diazirine) moiety in close proximity to one or more specific amino acids of the target protein (e.g., in the active site, in an allosteric site). A difference in the amino acids bound by the photo-conjugation moiety could indicate enantiomer-specific binding and interactions with the target protein.

Most small molecules can be covalently coupled to a photo-click tag provided herein by employing the appropriate organic chemistry reaction to couple the small molecule to the photo-click tag, for example, as shown in FIGS. 26, 70, and 71. Conjugation of the photo-click tag to the small molecule can be achieved by formation of an ester, ether, amine or amide to link the small molecule to the photo-click tag. For example, as shown in FIG. 26, an ether bond is formed when a photo-click probe comprising a free amine (NH₂) group reacts with a free hydroxyl (OH) group on the small molecule. As another example, an amide bond is formed when a photo-click probe comprising a free amine (NH2) group reacts with a free carboxylic acid (—COOH) group on the small molecule (FIG. 26). A person of ordinary skill in the art will be able to determine the appropriate reaction conditions to use for coupling a small molecule to a photo-click tag (e.g., a triisopropyl ether (TIPS) protected photo-click tag) based on the functional groups present in the small molecule of interest, or portion of the small molecule that binds to a target protein or proteins. The photo-click tag should be placed on the molecule so as to not interfere with the small molecule-protein binding interaction. Thus, the resulting photo-click tagged compound may comprise the entire small molecule, or a portion of the small molecule. Where the photo-tagged compound comprises only a portion of the small molecule, this portion of the small molecule should be sufficient for maintaining the interaction between the small molecule and the target protein(s).

Thus, in some aspects, provided herein are compounds comprising the structure of Formula (I):

wherein

S is a small molecule, or derivative thereof;

P is a photo-click tag comprising (a) a photo-conjugation moiety (M), and (b) a click chemistry handle; and

L is a linker.

In some embodiments, the click chemistry handle is an alkyne. Thus, in some embodiments, P is

In some embodiments, the photo-conjugation moiety (M) is an aryl azide, azido-methyl-coumarin, benzophenone, anthraquinone, diazo compound, diazirine, or psoralen derivative. In some embodiments, the photo-conjugation moiety (M) is a diazirine moiety. In some embodiments, P comprises (a) a diazirine moiety, and (b) a click chemistry handle. In some embodiments, the click chemistry handle is an alkyne. In some embodiments, P comprises (a) a diazirine moiety, and (b) an alkyne.

In some embodiments, P is

In some embodiments, P is

In some embodiments, P optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, P is

In some embodiments, P optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, P is

In some embodiments, P optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, P is

In some embodiments, L is a bond. In some embodiments, L comprises an ester, an ether, an amine, or an amide. In some embodiments, L comprises an ester. In some embodiments, L comprises an ether. In some embodiments, L comprises an amine. In some embodiments, L comprises an amide. In some embodiments, L is

In some embodiments, L is

In some embodiments, L is

some embodiments, L is

In some embodiments, L is

In some embodiments, L is

In some embodiments, S is an antibiotic, an anti-proliferative agent, an anti-cancer agent, a chemotherapeutic agent, an anti-angiogenesis agent, an anti-inflammatory agent, an immunosuppressant, an immunomodulatory agent, an anti-bacterial agent, an anti-viral agent, a cardiovascular agent, a cholesterol-lowering agent, an anti-diabetic agent, an anti-allergic agent, a contraceptive agent, or a pain-relieving agent.

In some embodiments, S is an anti-inflammatory agent, or derivative thereof. In some embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory drug (NSAID), or derivative thereof. Exemplary, non-limiting examples of NSAIDs for use herein are aspirin (acetylsalicylic acid), diflunisal (dolobid), salicylic acid and other salicylates, salsalate (disalcid), ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone (bute), mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib, nimesulide, clonixin, licofelone, and derivatives thereof. In some embodiments, the NSAID is selected from the group consisting of acetaminophen, aspirin, bromefenac sodium, celecoxib, diclofenac, diclofenac potassium, diclofenac sodium, diflunisal, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, ketoprofen, magnesium salicylate, meclofenamate sodium, mefenamic acid, nabumetone, naproxen, naproxen sodium, oxaproxin, oxyohebutazone, phenylbutazone, piroxicam, rofecoxib, salsalate, sodium salicylate, sulindac, tometin sodium, valdexocib, and derivatives thereof.

In some embodiments, the NSAID is naproxen, or derivative thereof. In some embodiments, the NSAID is celecoxib, or derivative thereof. In some embodiments, the NSAID is indomethacin, or derivative thereof.

In some embodiments, the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent, or derivative thereof. In some embodiments, the steroidal anti-inflammatory agent is a corticosteroid, or derivative thereof. Exemplary, non-limiting corticosteroids suitable for use herein are 21-acetoxypregnenolone, alclometasone, alclometasone dipropionate, algestone, amcinonide, beclomethasone, betamethasone, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone valerate, budesonide, chloroprednisone, ciclesonide, clobetasol, clobetasol-17-propionate, clobetasone-17-butyrate, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortisone acetate, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, dexamethasone sodium phosphate, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, fluocinonide, fluocinolone, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluocortolone caproate, fluocortolone pivalate, fluorometholone, flunisolide, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, hydrocortisone acetate, hydrocortisone-17-aceponate, hydrocortisone-17-buteprate, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, loteprednol, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone, mometasone furoate, paramethasone, paramethasone acetate, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, tixocortol pivalate, triamcinolone, triamcinolone acetonide, triamcinolone alcohol, triamcinolone benetonide, triamcinolone hexacetonide, and derivatives thereof.

In some embodiments, S is an immunomodulatory drug, or derivative thereof. In some embodiments, the immunomodulatory drug is thalidomide, lenalidomide, pomalidomide, or derivative thereof. In some embodiments, the lenalidomide is R-lenalidomide. In some embodiments, the lenalidomide is S-lenalidomide. In some embodiments, the lenalidomide is R-pomalidomide. In some embodiments, the lenalidomide is S-pomalidomide.

In some embodiments, the compound is of the formula:

or a pharmaceutically acceptable salt thereof, wherein represents a bond of un-specified stereochemistry.

In some embodiments, the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, S is a chemotherapeutic agent, or derivative thereof. Exemplary chemotherapeutic agents include, but are not limited to, anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g. goscrclin and leuprolide), anti-androgens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g. busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide), platinum containing compounds (e.g. cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mitomycin), anti-metabolites, DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonuclotide reductase inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g. verapamil), Ca²⁺ ATPase inhibitors (e.g. thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin, aminopterin, hexamethyl melamine, and derivatives thereof.

In some embodiments, the chemotherapeutic agent is daunorubicin, or derivative thereof. In some embodiments, the chemotherapeutic agent is mitomycin, or derivative thereof.

In some embodiments, the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

In some embodiments, S is a steroid. In some embodiments, the steroid is a sex steroid, or derivative thereof. In some embodiments, the sex steroid is an estrogen, or derivative thereof. In some embodiments, the estrogen is estradiol or beta-estradiol, or derivative thereof.

In some embodiments, the compound is of the formula:

or a pharmaceutically acceptable salt thereof.

Any of the compounds presented herein can comprise one or more isotopically labeled atoms. In some embodiments, the isotopically labeled atom is ¹⁵N, ¹³C, ¹⁹F, or ²H. For example, as shown in FIG. 6A, nitrogen atoms labeled with astericks (*) indicate ¹⁵N nitrogen atoms. Isotopic labeling of one or more atoms in the compound can facilitate identification of the compound through biophysical methods that are sensitive to isotopes, such as NMR. For example, NMR can be used to confirm the identity and structure of the appropriate photo-click tag or photo-click tagged compound product generated by the synthesis reaction (see, e.g., Examples 1 and 2). In addition, isotope-targeted mass spectrometry (MS) can be used to identify and assign the constitutive peptides of the target protein that are conjugated to the photo-click tagged compound (Example 1).

In another aspect, provided herein are amino acid analogs comprising the structure of Formula (II-a):

wherein

P is a photo-click tag comprising (a) a photo-conjugation moiety (M), and (b) a click chemistry handle.

In another aspect, provided herein are amino acid analogs comprising the structure of Formula (II-b):

wherein

P is a photo-click tag comprising (a) a photo-conjugation moiety (M), and (b) a click chemistry handle.

In general, L-amino acids are incorporated into proteins in mammalian (e.g., human) cells. While L-amino acids represent all of the amino acids found in proteins during translation in the ribosome, D-amino acids are found in some proteins produced by enzyme posttranslational modifications after translation and translocation to the endoplasmic reticulum. Thus, in some embodiments, the amino acid analog comprises the structure of Formula (II-a). Alternatively, in some embodiments, the amino acid analog comprises the structure of Formula (II-b).

In some embodiments the click chemistry handle is an alkyne. Thus in some embodiments, P is

In some embodiments, the photo-conjugation moiety (M) is an aryl azide, azido-methyl-coumarin, benzophenone, anthraquinone, diazo compound, diazirine, or psoralen derivative. In some embodiments, the photo-conjugation moiety (M) is a diazirine moiety. In some embodiments, P comprises (a) a diazirine moiety, and (b) a click chemistry handle. In some embodiments, the click chemistry handle is an alkyne. In some embodiments, P comprises (a) a diazirine moiety, and (b) an alkyne.

In some embodiments, P is

In some embodiments, P is

In some embodiments, P optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, P is

In some embodiments, P optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, P is

In some embodiments, P optionally comprises one or more electronegative atoms (e.g., fluorine).

In some embodiments, P is

In some embodiments, the amino acid analog is of the formula:

Also encompassed by the disclosure are kits. The kits provided may comprise a photo-click tag, a compound of Formula (I), or an amino acid analog of Formula (II-a) or (II-b), described herein; and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, the kit comprises a photo-click tag provided herein. In some embodiments, the kit comprises a compound of Formula (I) provided herein. In some embodiments, the kit comprises an amino acid analog of Formula (II-a) provided herein. In some embodiments, the kit comprises an amino acid analog of Formula (II-b) provided herein. In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a photo-click tag or photo-tagged compound described herein. In certain embodiments, a kit described herein further includes instructions for using the kit. In certain embodiments, the kits and instructions provide for carrying out a method described herein.

Methods for Determining Small Molecule-Protein and Protein-Protein Interactions

As described above the photo-click tags and photo-click tagged compounds described herein are useful for mapping the direct and indirect effects of small molecules within the proteome. Some aspects of the present disclosure provide a small photo-click tag, wherein the photo-click tag is linked to a small molecule, that enables efficient capture, enrichment, and characterization of small molecule binding sites on a target protein or proteins. These methods can also be applied to determine the target protein of a small molecule, wherein the mechanism of action and/or the protein targeted by the small molecule is not known or well characterized. In addition, the methods and compositions provided herein are useful in determining a protein-protein interaction map, for example, in the presence of a small molecule to determine how the small molecule impacts cellular signaling and/or protein-protein interactions within a cell.

In one aspect, provided herein is a method for identifying the target protein of a small molecule. In some embodiments, the method comprises: (i) providing a compound comprising the small molecule connected to a photo-click tag via a linker, wherein the photo-click tag comprises (a) a photo-conjugation moiety and (b) a click chemistry handle; (ii) activating the photo-conjugation moiety by irradiating the compound of (i) with a specific wavelength of light; (iii) contacting the target protein with the activated compound of (ii); (iv) forming a complex through a photo-induced covalent bond between the activated compound of (ii) and the protein; and (v) identifying the complex produced in (iv) that is bound to the compound, thereby identifying the target protein of the small molecule. In some embodiments, the photo-conjugation moiety is a diazirine moiety. In some embodiments, the photo-click tag comprises (a) a diazirine moiety and (b) a click chemistry handle. In some embodiments, the click chemistry handle is an alkyne. In some embodiments, the photo-click tag comprises (a) a diazirine moiety and (b) an alkyne.

In another aspect, provided herein is a method for identifying the binding site of a small molecule to a protein. In some embodiments, the method comprises: (i) providing a compound comprising the small molecule connected to a photo-click tag via a linker, wherein the photo-click tag comprises (a) a photo-conjugation moiety, and (b) a click chemistry handle; (ii) activating the photo-conjugation moiety by irradiating the compound of (i) with a specific wavelength of light; (iii) contacting the protein with the activated compound of (ii); (iv) forming a complex through a photo-induced covalent bond between the activated compound of (ii) and the protein; (v) digesting the protein of the complex into constitutive peptides in the presence of a protease; and (vi) identifying the one or more peptides produced in (v) that are bound to the compound of (i), thereby identifying the protein binding site of the small molecule. In some embodiments, the photo-conjugation moiety is a diazirine moiety. In some embodiments, the photo-click tag comprises (a) a diazirine moiety, and (b) a click chemistry handle. In some embodiments, the click chemistry handle is an alkyne. In some embodiments, the photo-click tag comprises (a) a diazirine moiety, and (b) an alkyne.

In yet another aspect, provided herein is a method for identifying an interaction between a first protein and a second protein in a cell. In some embodiments, the method comprises: (i) providing a cell with an amino acid analog, wherein the amino acid analog comprises a photo-click probe comprising (a) a photo-conjugation moiety and (b) a click chemistry handle, and wherein the amino acid analog is incorporated into the first protein and/or the second protein during protein synthesis; (ii) activating the photo-conjugation moiety of the amino acid analog by irradiating the cell with a specific wavelength of light; (iii) contacting the first protein or the second protein with the activated compound of (ii); (iv) forming a protein-protein complex through a photo-induced covalent bond between the activated amino acid analog of the first protein and an amino acid in the second protein; and (v) identifying the complex produced in (ii) that comprises the amino acid analog covalently linking the first protein and the second protein, thereby identifying the first protein and the second protein involved in the interaction. In some embodiments, the photo-conjugation moiety is a diazirine moiety. In some embodiments, the photo-click tag comprises (a) a diazirine moiety and (b) a click chemistry handle. In some embodiments, the click chemistry handle is an alkyne. In some embodiments, the photo-click tag comprises (a) a diazirine moiety and (b) an alkyne. In some embodiments, the method further comprises digesting the protein-protein complex into constitutive peptides in the presence of a protease; and identifying a conjugated peptide that comprises a peptide comprising the amino acid analog covalently linking a peptide of the first protein to a peptide of the second protein. In some embodiments, the method further comprises contacting the cell with a small molecule, wherein the cell is contacted with the small molecule before the activating of step (ii). In some embodiments, the first protein and the second protein identified in the presence of the small molecule are compared to the first protein and the second protein identified in the absence of the small molecule. In some embodiments, a difference in the first protein and the second protein in the presence of the small molecule compared to the first protein and the second protein in the absence of the small molecule indicates modulation of a protein-protein interaction in the cell in the presence of a small molecule. In some embodiments, the first protein or the second protein is enriched in the presence of a small molecule. In some embodiments, the enrichment is at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to the amount of the first protein or the second protein in the absence of the small molecule. In some embodiments, the small molecule is an antibiotic, an anti-proliferative agent, an anti-cancer agent, a chemotherapeutic agent, an anti-angiogenesis agent, an anti-inflammatory agent, an immunosuppressant, an immunomodulatory agent, an anti-bacterial agent, an anti-viral agent, a cardiovascular agent, a cholesterol-lowering agent, an anti-diabetic agent, an anti-allergic agent, a contraceptive agent, or a pain-relieving agent. In some embodiments, the small molecule is an anti-inflammatory agent, an immunomodulatory drug, a chemotherapeutic agent, or a derivative thereof. In some embodiments, the small molecule is an anti-inflammatory agent, or derivative thereof. In some embodiments, the small molecule is an immunomodulatory drug, or derivative thereof. In some embodiments, the small molecule is a chemotherapeutic agent, or derivative thereof. In some embodiments, the small molecule is a steroid, or derivative thereof.

In some embodiments, the diazirine moiety forms a reactive carbene species upon irradiation with a specific wavelength of light. In some embodiments, the reactive carbene species reacts with a C—C, C—H, N—H, or O—H bond of a protein. Upon insertion of the reactive carbene species into a C—C, C—H, N—H, or O—H bond of a protein, a new photo-induced covalent bond between the protein and photo-click tag is formed, thereby linking the compound comprising the photo-click tag to the protein to generate a complex. In some embodiments, the specific wavelength of light is between about 10 nm and about 400 nm. In some embodiments, the specific wavelength of light is between about 355 nm and about 365 nm. In some embodiments, the specific wavelength of light is about 355 nm. In some embodiments, the specific wavelength of light is about 365 nm.

In some embodiments, the photo-click tag of (i) is of the formula:

before irradiation with light. In some embodiments, the diazirine moiety

forms the reactive carbene species

upon irradiation with light. In some embodiments, the photo-click tag of (ii) comprising the activated diazirine moiety is of the formula:

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the photo-click tag of (i) is of the formula

before irradiation with light, and is of the formula

after irradiation with light. In some embodiments, the light has a wavelength between about 10 nm and about 400 nm. In some embodiments, the light has a wavelength between about 355 nm and about 365 nm. In some embodiments, the light has a wavelength of about 355 nm. In some embodiments, the light has a wavelength of about 365 nm.

Some embodiments of the method comprise contacting the protein conjugated to the compound comprising the small molecule and photo-click tag or the protein-protein complex formed in the presence of an amino acid analog with a protease to digest the proteins into constitutive peptides. Without wishing to be bound to any particular theory, protease digestion is a step often employed before analysis and identification of the protein using mass spectrometry (MS). Digestion of the protein into constitutive peptides allows for MS analysis of individual peptides, which can allow for the identification of peptides bound to photo-click tag and thus the small molecule or protein partner (i.e., protein-protein interaction partner) of interest. This method is also used, for example, to identify protein post-translational modifications. See, e.g., Gundry R L, et al. (2009) Preparation of Proteins and Peptides for Mass Spectrometry Analysis in a Bottom-Up Proteomics Workflow. Curr Protoc Mol Biol, doi:10.1002/0471142727.mb1025s88; which is incorporated by reference herein. In some embodiments, the protease is a serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, metalloprotease, or an asparagine peptide lyase. In some embodiments, the protease is a serine protease. In some embodiments, the serine protease is trypsin. In some embodiments, the serine protease is chymotrypsin. In some embodiments, the protease is an aspartic protease. In some embodiments, the aspartic protease is pepsin. In some embodiments, one protease maybe used to digest a protein. In some embodiments, more than one protease maybe used to digest a protein. In some embodiments, the digesting of step (v) is performed under conditions suitable for the protease to catalyze the cleavage of peptide bonds in the protein. Upon cleavage with a protease, the protein, which is covalently bound to the photo-tagged compound, is digested into constitutive peptides based on the specificity of the chosen protease or combination of proteases, thus providing a plurality of peptides comprising peptides that are covalently bound to the photo-tagged compound (i.e., conjugated peptides) and native peptides (i.e., peptides that are not covalently bound to the photo-tagged compound).

In some embodiments, the step of identifying comprises performing mass spectrometry to identify the protein that is bound to the compound. Mass spectrometry (MS) has been used for the identification of protein-ligand interactions, see, for example, Sinz A. (2007) Investigation of Protein-Ligand Interactions by Mass Spectrometry. Chem Med Chem, 2, 425-431. In particular, peptide mixtures are generally separated before introduction into the mass spectrometer, such as through liquid chromatography (LC) or capillary electrophoresis (CE). In some embodiments, the mass spectrometry used in the step of identifying is intact mass spectrometry. Intact mass spectrometry may also be referred to as “native mass spectrometry”, as this MS method generally involves preservation of the native protein or complex shape and structure. See, e.g., Doerr A. (2012) Mass spectrometry of intact protein complexes. Nature Methods, 10, doi: 10.1038/nmeth.2298.

In some embodiments, when the method involves a digesting step, the identifying step comprises performing mass spectrometry to identify the peptides that are bound to the compound. In some embodiments, the mass spectrometry used in the identifying step is mass-independent mass spectrometry (MS). Without wishing to be bound by any particular theory, mass-independent MS generally involves the mass-independent assignment of peptides, which uses a pattern-searching algorithm to direct tandem MS analysis to isotopically labeled species (e.g., a compound comprising one or more isotopically labeled atoms). See, e.g., Woo C M, et al. (2015) Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis. Nat Methods, 12(6), 561-567. Mass-independent MS can overcome traditional MS barriers in detection of low abundance species by enhancing the detection of the species of interest (e.g., the isotopically labeled species) even in a background of unmodified species (e.g., unmodified peptides that do not comprise an isotopically labeled atoms). Mass-independent mass spectrometry may also be referred to interchangeable as isotope-targeted mass spectrometry.

In some embodiments, the identification of the peptide or protein species of interest (e.g., the peptide or protein conjugated to the photo-click tagged compound) can be enhanced by performing an enrichment step before the identifying step to increase the abundance (i.e., enrich) the conjugate (e.g., the protein conjugated to the photo-click tagged compound, the peptide conjugated to the photo-click tagged compound, the protein-protein complex conjugated by the amino acid analog, or a peptide of the first protein conjugated to a peptide of the second protein by the amino acid analog) present in the sample for analysis. Thus, in some embodiments, the method further comprises a step of enriching the conjugate formed when the photo-activated compound covalently binds to a protein. In some embodiments, the step of enriching is performed before the digesting step. In some embodiments, the step of enriching is performed after the digesting step. In some embodiments, the step of enriching is performed without digesting the protein before or after the enriching step. In some embodiments, the step of enriching comprises covalently attaching a label to the click chemistry handle of the photo-click tag. In some embodiments, the label comprises a click chemistry handle that can be conjugated (i.e., “clicked”) to the click chemistry handle of the photo-click tag. In some embodiments, the label comprises an azide (N₃) moiety. In some embodiments, the label is covalently attached to the click chemistry handle of the photo-click tag using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction conditions. The CuAAC is a click chemistry reaction that involves the copper-catalyzed reaction of an azide with an alkyne to form a 5-membered heteroatom ring. An exemplary scheme outlining a CuAAC reaction is shown below in Scheme III.

While a CuAAC reaction requires an alkyne and an azide, a person of ordinary skill in the art is capable of determining appropriate reaction conditions for attaching a label to a click chemistry handle, wherein the label and the click chemistry handle do not comprise an azide or an alkene. See, e.g., Hein, C D et al (2008) Click Chemistry, a Powerful Tool for Pharmaceutical Sciences. Pharm Res, 25, 2216-2230, which is incorporated by reference herein.

In some embodiments, the label comprises an affinity tag. An affinity tag can be used to efficiently separate the protein or peptides covalently linked to the photo-click tagged compound (i.e., conjugated protein or conjugated peptides) from native proteins or peptides (i.e., proteins or peptides that are not covalently linked to the photo-click tagged compound). The term “affinity tag” refers to any moiety that can be used to separate a species of interest (e.g., a protein or peptide) from a complex mixture. Exemplary affinity tags include, but are not limited to, peptide tags (e.g., AviTag (GLNDIFEAQKIEWHE), Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL), polyglutamate tag (EEEEEE), E-tag (GAPVPYPDPLEPR) FLAG-tag (DYKDDDDK), HA-tag (YPYDVPDYA), His-tag (5-10 histidines, e.g., HHHHHH), Myc-tag (EQKLISEEDL). NE-tag (TKENPRSNQEESYDDNES), S-tag (KETAAAKFERQHMDS), SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), Softag 1 (SLAELLNAGLGGS), Softag 3 (TQDPSRVG), Strep-tag (Strep-tag II: WSHPQFEK), TC tag (CCPGCC), Ty tag (EVHTNQDPLD), V5 tag (GKPIPNPLLGLDST), VSV-tag (YTDIEMNRLGK), Xpress tag (DLYDDDDK), and derivatives thereof, covalent peptide tags (e.g., Isopeptag (TDKDMTITFTNKKDAE), SpyTag (AHIVMVDAYKPTK), SnoopTag (KLGDIEFIKVNK), SnoopTagJr (KLGSIEFIKVNK), DogTag (DIPATYEFTDGKHYITNEPIPPK), and derivatives thereof), protein tags (e.g., BCCP (Biotin Carboxyl Carrier Protein), Glutathione-S-transferase-tag, Green fluorescent protein-tag, HaloTag, Maltose binding protein-tag, Nus-tag, Thioredoxin-tag, Fc-tag, Designed Intrinsically Disordered tags, chitin binding protein (CBP), thioredoxin (TRX), and derivatives thereof), biotin, avidin (e.g., streptavidin), carbohydrates (e.g., lectins), and glutathione. Affinity tags are often added to a species of interest (e.g., a protein or peptide) to facilitate separation, for example, using affinity chromatography, by taking advantage of the specific interaction between two moieties (e.g., antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid). In some embodiments, the affinity tag is biotin. In some embodiments, the label is of the formula:

In some embodiments, the method further comprises separating the protein or peptides covalently linked to the photo-click tagged compound, wherein the photo-click tagged compound is covalently attached to the label. In some embodiments, when the label comprises biotin, the conjugated protein or conjugated peptide can be separated from native proteins or peptides by streptavidin-coupled beads. Once the conjugated protein or conjugated peptide is captured by the interaction between the biotin of the label and the streptavidin of the beads and separated from the native proteins or peptides, the conjugated protein or conjugated peptide can be cleaved from the beads, for example, using acidic cleavage conditions, thereby releasing the conjugated protein or conjugated peptide and generating an enriched sample for identification of the protein or peptide covalently bound to the photo-click tag. Additional suitable label/derivative-bead combinations will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure.

In some embodiments, the protein is present in a whole proteome. In some embodiments, the protein is present in vitro. For example, the methods can be performed on a sample consisting essentially of the protein of interest (e.g., the protein that binds to a small molecule of interest), such as in a purified protein sample. The methods can also be performed on a sample comprising a mixture of proteins in a sample (e.g., a cell lysate). In some embodiments, the protein is present in a cell lysate. In some embodiments, the protein is present in vivo. In some embodiments, the protein is present in a cell. In some embodiments, the cell is a mammalian (e.g., human) cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a human cell derived from a subject with a disease (e.g., cancer).

EXAMPLES

In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods, compositions, and systems provided herein and are not to be construed in any way as limiting their scope.

Example 1—Small Molecule Interactome Mapping by Photo-Affinity Labeling (SIM-PAL) Reveals Binding Site Hotspots for the NSAIDs

Many therapeutics elicit cell-type specific polypharmacology that may increase therapeutic efficacy or yield off target toxicity. However, direct observation of the structures that underpin the global molecular associations between the proteome and even common therapeutics, such as the non-steroidal anti-inflammatory drugs (NSAIDs), remain little understood. Presented here is a platform termed small molecule interactome mapping by photo-affinity labeling (SIM-PAL) and apply it to the in vivo characterization of the NSAID interactome to yield identification of specific binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, the NSAID interactome consisting of over 1000 enriched proteins was identified and nearly 200 conjugated peptides representing direct binding interactions in Jurkat and K562 cells were directly characterized. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-1, proteasome). These data further revealed a binding site hotspot on histones H2A and H2B where the three photo-NSAIDs, but not fragment-based small molecules, interacted. The binding interaction of the NSAIDs stabilized COX-2 and histone H2A by cellular thermal shift assay. Since protein-protein stabilization is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.

INTRODUCTION

Polypharmacology, wherein one drug interacts with multiple protein targets, is a common phenomenon in drug discovery. Polypharmacology manifests in increased efficacy when properly exploited or tragic unanticipated off-target effects when not fully understood. Many pharmaceuticals in diverse therapeutic areas possess either known or uncharacterized polypharmacology,¹ such as the nonsteroidal anti-inflammatory drugs (NSAIDs),² the immunomodulatory drugs,³ or the opioids.⁴ Proteomic differences across cell types are the basis for molecular interactions that culminate in an observed phenotype,⁵ suggesting that with a map of the protein-ligand interaction network throughout the whole proteome, these polypharmacology outcomes may eventually be predicted.^6,7

A method to directly map the small molecule interactome has the potential to accelerate drug discovery by providing structural insight and instant validation of the binding interaction, yet such global characterization is rarely performed. Common analytical methods to structurally reveal small molecule binding sites, such as X-ray crystallography or NMR spectroscopy, are constrained to the measurement of stable interactions between a single compatible protein and small molecule pair in vitro. Global proteomic profiles are now commonly obtained using mass spectrometry (MS), and with a small molecule affinity purification strategy can be used to identify binding proteins.⁸ However, the vast majority of proteomics studies stop short of obtaining direct structural evidence for the molecular interaction, due to inherent challenges in mapping the binding sites of a small molecule on the whole proteome. Small molecule interactions occur over a range of concentrations that require a general mechanism for capture and enrichment prior to MS analysis. The chemistry selected to capture the binding event must be rapid and general for unbiased covalent bond formation at the small molecule binding site. Yet, the demand for a general chemical strategy to covalently conjugate a small molecule locally to the protein interaction site poses great challenges to spectral assignment by database searching. Database searching methods are not adapted to the computational complexity yielded by amino acid residue-agnostic modifications to the whole proteome. Without the complexity of the whole proteome, binding sites of small molecules to defined protein isolates can be determined by application of photo-affinity labeling (PAL) to conjugate the small molecule to the protein prior to MS analysis.⁹

Translation of MS-based binding site identification from a single protein to the whole proteome thus requires (1) a selective chemical workflow to isolate the conjugated peptide and (2) a targeted MS technique for confident characterization. PAL covalently conjugates small molecules to the proteome for stringent enrichment of interacting proteins.¹⁰ Application of cleavable enrichment handles enables recovery of the small molecule-conjugated peptide.9 Critically, a targeted MS strategy, wherein unique isotopic markers are installed specifically to the small molecule conjugated peptide, provides an orthogonal handle for detection and validation that proves transformative during database assignment of peptides carrying heterogeneous modifications by MS.¹¹ Recent strides have enabled the identification of fragment-based small molecule ligands to the whole proteome.¹²

The knowledge gap caused by the lack of a small molecule interactome map extends to common pharmaceuticals like the nonsteroidal anti-inflammatory drugs (NSAIDs). The NSAIDs are potently suppress inflammation, pain, and fever and have been further explored as potential treatments for cancer^2,13 and Alzheimer's disease.¹⁴ NSAID mechanisms have been primarily characterized through inhibition of the enzymes cyclooxygenase-1 and -2 (COX-1, COX-2, respectively).¹⁵ Inhibition of COX-2, the primary cyclooxygenase involved in inflammation, prevents the production of prostaglandins, thereby reducing inflammation. However, a wealth of biomedical evidence points to broader COX-2-independent mechanisms of NSAIDs involved in anti-cancer activity for which a molecular basis remains poorly defined.^16-18 Prior studies suggest that specific NSAIDs inhibit the nuclear factor-κB (NF-κB) pathway¹⁹ and caspases.²⁰ A detailed understanding of broader NSAID mechanisms is constrained by the absence of a global understanding of NSAID-protein interactions and their underlying structures.

Herein, the development of a platform termed small molecule interactome mapping by photo-affinity labeling (SIM-PAL) and its application to the NSAIDs is reported. SIM-PAL is designed to directly characterize the protein interactions and binding site hotspots of a small molecule in a whole cell proteome using a PAL-based enrichment strategy coupled to isotope-targeted MS (FIG. 12A). Our platform involves: (1) photo-conjugation of NSAID derivatives in cells, (2) enrichment and isotopic recoding of NSAID-labeled peptides, and (3) isotope-targeted assignment of the conjugated peptides. Photo-NSAID derivatives are effective reporters of NSAID binding sites with recombinant COX-2 and the global whole cell proteome, as shown herein. Photo-NSAIDs displayed overlapping protein interactions in Jurkat and K562 cell lines. By virtue of direct characterization of the conjugated peptide, we localized the photo-NSAIDs to a specific binding hotspot on the interface of histones H2A and H2B. Histone H2A was stabilized by interacting with the NSAIDs by cellular thermal shift assay. SIM-PAL revealed the precise binding interactions for the photo-NSAIDs via an approach that is readily translated to broad classes of small molecules.

Results

Development of Photo-NSAIDs as Reporters of NSAID Binding Sites

Three NSAIDs, naproxen (1), celecoxib (2), and indomethacin (3), were conjugated to diazirine-based photo-affinity labels (“photo-NSAIDs”) to serve as reporters for NSAID binding sites (FIG. 12B). These NSAIDs were selected for their different structure-activity relationship (SAR) between COX-1 and COX-2.^21,22 Naproxen (1) is a nonselective COX-1 and COX-2 inhibitor, but is commonly employed for chronic use due to low rates of gastrointestinal side effects.²³ Celecoxib (2) is a selective COX-2 inhibitor developed by Pfizer, yet possesses off-target cardiovascular and gastrointestinal complications.²⁴ Indomethacin (3) is a member of the indole class of NSAIDs and possesses known COX-2-independent anti-inflammatory mechanisms.¹⁶ The design of photo-NSAIDs were based upon previous SAR studies^25,26 and the crystal structure between mouse COX-2 and indomethacin (3).²⁷ During our studies, a crystal structure of a NSAID with human COX-2 was reported.²⁸ In addition to photo-NSAIDs 4-6, the tag 7,²⁹ a structurally orthogonal photoglutarimide 8, and a celecoxib analog 9, was developed to assess selectivity of the binding site identification assay (FIG. 26).

All three photo-NSAIDs maintained COX-2 inhibition by ELISA (FIG. 12B. FIGS. 16A-16D), although some variation in activity was observed. Photo-celecoxib (5) was the most potent (IC₅₀=36.6 nM) and photo-naproxen (4) was the least potent (IC₅₀=36.0 μM). All photo-NSAIDs possessed antiproliferative properties within 1.3-1.4-fold of their parent compound in Jurkat cells (FIGS. 17A-17D). Elimination of the sulfonamide from celecoxib (2) to give the analog 9 is known to reduce COX-2 inhibition²⁶ and we found additionally attenuated anti-proliferative activity of the analog 9 in activated Jurkat cells (FIGS. 17A-17D). Furthermore, all photo-NSAIDs were competitively displaced from recombinant COX-2 by the native NSAID (FIG. 13A). COX-2 was separately incubated with each of the photo-NSAIDs with or without a 100-fold excess of the parent compound as a competitor.¹⁰ The samples were photo-irradiated, tagged with the fluorophore TAMRA-azide by copper-mediated azide alkyne cycloaddition (CuAAC), or click chemistry, and fluorescently visualized to reveal selective and reversible binding of COX-2 to the photo-NSAIDs. The tag 7 did not produce observable conjugation to COX-2 by fluorescence.

Photo-NSAIDs Possess Known and Transient Binding Sites with COX-2

Recombinant COX-2 was used to validate fragmentation patterns of conjugated NSAIDs to a protein and determine binding site selectivity for each of the photo-NSAIDs. Small molecule modification on a peptide may perturb MS fragmentation pathways in unexpected ways, rendering the spectra unable to be assigned by database searching. To evaluate this possibility, photo-NSAIDs (10 μM) were incubated with recombinant COX-2 for 30 minutes and photo-irradiated. The irradiated samples were appended to the cleavable biotin azide probe 10 to simulate the conjugated species ultimately observed after enrichment (FIG. 12C). The probe 10 is a multifunctional probe developed to possess a biotin affinity enrichment handle, an acid-labile diphenylsilane, and a stable isotope-coded azidoacetate for click chemistry and isotope-targeted MS. We previously established the compatibility of a similar cleavable probe scaffold in targeted MS experiments.¹¹ Following click chemistry, the samples were trypsin-digested and the probe was cleaved in situ (2% formic acid). The resulting peptides were analyzed by LC-MS/MS. MS data was searched by SEQUEST and Byonic against recombinant COX-2 with the photo-NSAID as a modification on any amino acid (Table 2). Due to the nature of photochemical conjugation, a binding site will be represented by multiple conjugated peptide structures and potentially multiple surrounding peptides in the MS data. All peptide spectral matches (PSMs) assigned to a conjugated peptide were manually validated.

Photo-NSAIDs were readily assigned by database searching. Manual inspection of these PSMs indicated that in the case the photo-NSAIDs 4-6, no irregular fragmentation pathways were observed. As expected, at a consistent dose of 10 μM across all photo-NSAIDs, photo-celecoxib (5) possessed the highest number of observed conjugated peptides (seven conjugated peptides from 14 PSMs), including within the active site of COX-2. Analysis of COX-2 treated by photonaproxen (4) and photo-indomethacin (6) revealed six conjugated peptides across 19 PSMs and eight PSMs, respectively (Table 2). By contrast, the tag 7 was found conjugated to one peptide, which was not marked by the photo-NSAIDs. Within each peptide, the specific conjugation site localized to a range of 2-4 amino acid residues that reflect the specificity of the photochemical conjugation event.

The photo-NSAIDs, the parent compounds, and the tag 7 were structurally minimized [Gaussian 16, basis set: HF 3-21g(d)] and individually docked in the crystal structure of human COX-2 (PDB: 5KIR).²⁸ Docking was performed using Patchdock, a molecular docking algorithm based on shape complementarity using rigid structures.³⁰ Structures with the lowest desolvation energy or highest interface area size docked the photo-NSAID or parent compound to the same interaction site, although the orientations within each pair of compounds differed (FIGS. 13B-13D). The docked structure of photo-naproxen (4, red), photo-celecoxib (5, green), and photo-indomethacin (6, yellow) overlaid with their respective parent compound (blue). Conjugated amino acid residues within 5 Å of the docked photo-NSAID are highlighted in FIGS. 13B-13D. The orientation of each of the photo-NSAIDs positioned the diazirine in close proximity to one or two specific amino acids on a conjugated peptide observed by MS. Other marked residues were located on solvent exposed areas of the protein. As photochemistry captures dynamic processes and the photochemical tag is structurally flexible, the additional conjugation events on recombinant COX-2 may represent transient interactions with COX-2 in vitro, which may not be observed in cellulo. The tag 7 was additionally docked and, in combination with the observed MS data, was shown to transiently bind to an orthogonal region of COX-2 (FIG. 13E). No conjugated peptides from amino acids near to the canonical binding site were observed.

Characterization of the NSAID Interactome in Jurkat and K562 Cells

Confident that photo-NSAIDs were producing defined linkages with COX-2 that recapitulated NSAID activity, we next sought to characterize global NSAID interactions within the whole cell proteome. Photo-NSAIDs, the tag 7, and the photo-glutarimide 8 were added to activated Jurkat T cells as a model system for inflammation. Small molecule dose-dependent and photo-irradiation-dependent labeling of the proteome peaked at 250 μM. Competitive displacement of the tightest COX-2 binder photo-celecoxib (5) by the parent compound occurred at a 1:10 molar ratio (FIGS. 18A-18C). Jurkat cells were stimulated with phorbol myristyl acetate and ionomycin for 18 hours prior to photo-NSAID exposure.³¹ The stimulated Jurkat cells were exposed to each compound (250 μM, 1 hour) and photo-irradiated in situ to conjugate the small molecule to the proteome. The resulting NSAID-conjugated proteins were enriched using the probe 10 in a biotin-dependent manner (FIG. 19). COX-2 from Jurkat cells was enriched by photo-NSAIDs and this enrichment was abrogated by competition with the parent compound, as indicated by Western blot (FIG. 20A). Cellular thermal shift assay, an orthogonal mechanism to validate protein-ligand interactions,³² showed that COX-2 was stabilized by celecoxib (2) and photo-celecoxib (5) by over 10° C. relative to the tag 7 (FIG. 20B-C).

The enriched proteomes were digested with trypsin and the released peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Tribrid with collision induced dissociation (CID) and higher energy CID (HCD) fragmentation modes. MS data were assigned by SEQUEST HT. Two biological replicates were collected for each of the photo-NSAIDs that displayed high reproducibility across the enriched proteome (>60%) and protein abundance (FIG. 21A). Using PSM-based label free quantification,³³ proteins that were greater than 2-fold enriched and statistically significant (t-test, p-value <0.05) were considered selectively enriched by the photo-NSAID relative to the control tag 7 (FIG. 14A). This analysis yielded approximately 700 proteins significantly enriched by at least one of the photo-NSAIDs (Table 3). High proteomic overlap amongst photo-NSAIDs, but not negative controls 7 and 8, was observed. Across each of the three compounds, at least 40% of the identified photo-NSAID-binding proteins were enriched by all three photo-NSAIDs and 53% of the proteins were enriched by at least two photo-NSAIDs (FIG. 14B). By comparison, 140 proteins that were statistically enriched by any photo-NSAID were also enriched by the photoglutarimide 8 (24%).

Jurkat cells were initially dosed with a concentration of 250 μM of each photo-NSAID to maximize downstream observation of conjugated peptides by MS. Although naproxen (1) enters blood plasma at concentrations that surpass 250 μM,³⁴ our IC₅₀ data against Jurkat cells showed a range of 23-216 μM across the NSAIDs and their derivatives (FIG. 17A-17D). We thus examined the NSAID interactome at 50 μM and identified 260 proteins enriched by the three photo-NSAIDs and not by the tag 7 (Table 4). At 50 μM the proteomic overlap between the photo-NSAIDs was lower, indicative of higher selectivity between the molecular structures (Figure S6B). A majority of proteins identified at 50 μM were likewise identified at 250 μM for each photo-NSAID (86-92%). The celecoxib analog 9 (50 μM) was additionally tested in activated Jurkat cells and displayed moderate proteomic overlap with photo-celecoxib (5) (FIG. 21C).

To determine the generality of these observations, photo-NSAIDs were additionally tested against K562 cells, a human chronic myeloid leukemia cell line. Several lines of evidence point to NSAID-dependent inhibition and apoptosis of K562 cells.³⁵ A total of 513 proteins were significantly enriched across two biological replicates from K562 cells, of which 42% of the proteins were enriched by at least two of the three photo-NSAIDs (FIGS. 22 and 23). Significantly enriched proteins from K562 cells possessed a moderate overlap with proteins from Jurkat cells (206 proteins), indicating a high degree of specificity across cell lines (FIG. 14C). In sum, a total of 1034 proteins were enriched from Jurkat and K562 cells by photo-NSAIDs (Table 3).

Photo-NSAIDs interacted with proteins distributed throughout the cell (FIG. 14D). Approximately two thirds of the NSAID interactome localized to the nucleus and cytoplasm. Photo-NSAIDs additionally captured proteins annotated as localized to the mitochondria (10%), endoplasmic reticulum and Golgi (11%), and membrane or secreted proteins (10%). These data are a close reflection of the natural distribution of proteins throughout the cell.³⁶ Only 30% of these proteins were previously annotated as interacting with a small molecule, let alone one of the NSAIDs (BindingDB, ChEMBL, DrugBank). This gap may be in part due to the broader range of interactions captured by photo-conjugation as compared to existing target identification strategies. Comparison of photo-NSAID interactions with proteomics profiles derived from fragment-based small molecules¹² revealed a 60% overlap. Thus, photo-NSAIDs possess a protein interaction profile that is specific to molecular structure and cell type. These data point to a broader range of molecular interactions that remain to be revealed by the reported profiling approach.

In line with evidence for capture of a broader range of associated proteins, a number of protein complexes were selectively enriched. Comparison of enriched proteins to CORUM37 revealed that NF-κB subunits (NFKB1, NFKB2) in complex with the inhibitor of NF-κB (IKKA) are directly interacting with the photo-NSAIDs, confirming their known inhibition of the NF-κB pathway² and was revalidated for celecoxib (2) and photo-celecoxib (5) via a NF-κB luciferase reporter assay (FIGS. 14E and 24). Additional protein complexes that were nearly completely enriched include the proteasome and the adaptor protein complex 2 (FIG. 14E). These proteins may have existed as complexes leading to photo-NSAID conjugation by virtue of proximity, indicative of a binding site hotspot for the photo-NSAIDs within specific families of proteins, or were enriched due to associative protein interactions. While protein complexes can be enriched through associative protein-protein interactions, our use of strong dissociative detergents to prepare cell lysates (1% Rapigest, sonication) does not typically lead to observation of protein complexes following enrichment.¹¹

Direct Photo-NSAID Interaction Mapping Reveals Binding Site Hotspots

Following tryptic digestion of the photo-NSAID proteome, the probe 10 was acid cleaved to release the conjugated peptides from the enrichment media for separate isolation. The conjugated peptides were analyzed by application of mass-independent MS. A unique isotopic signature was embedded to the probe 10 using a carbon-13-derived stable isotope ratio of 1:3 over [M:M+2] spacing to perform mass-independent MS. During click chemistry, the unique isotopic signature was exclusively transferred to the photo-NSAIDs conjugated to the proteome. The isotopic signature is therefore only found on small molecule-conjugated peptides and is used during mass-independent MS to overcome traditional MS barriers in detection of low abundance species and validation of modified peptides against a background of unmodified peptides. During data collection, the isotopically recoded species is immediately recognizable by full scan MS and may be selected for fragmentation by use of an inclusion list.^38,39 This selection process increases the fraction of isotopically recoded, small molecule-conjugated peptides selected for fragmentation.¹¹

More critically, the isotope signature played a crucial role in manual validation of database search assignments due to the ambiguity of the amino acid modification site. Database searching was performed against the SwissProt human protein database with each of the photo-NSAIDs as a modification on any amino acid residue. Small molecule conjugation to any amino acid drastically increases the size of the protein database. For example, a single modification increases the size of the fully tryptic human protein database by 60-fold, while two modifications increase the database size by 1000-fold. This exponential increase in the tryptic peptide database leads to a breakdown in false discovery rate (FDR).⁴⁰ Thus, a two-tier validation process was used for confident assignment of conjugated peptides. First, modified peptides at 5% FDR were filtered based on visual inspection of the MS2 spectral assignment. Second, each of the precursor spectra was individually validated for the isotopic signature in the MS1. Based on this analysis, 575 PSMs, corresponding to 194 individual conjugated peptides, were characterized across the photo-NSAIDs, tag 7, and photo-glutarimide 8 (Table 5). The top ten most frequently observed conjugated peptides are displayed in Table 1.

TABLE 1
Selected conjugated peptides observed with high PSM frequency
across photo-NSAIDs, the tag 7 and the photo-glutarimide 8. Presented data
is in aggregate across Jurkat and K562 cells. For the full dataset, see Table 5.
			Count of PSMs
	Conjugated Peptide	Protein (Gene)	4	5	6	7	8
1	AMGIMNSFVNDIFER	Histone H2B type 1-K	41	12	1
	(SEQ ID NO: A1)	(HIST1H2BK)
2	VGAGAPVYLAAVLEYLTAEILELAGNAAR	Histone H2A type 2-B	8	28	9
	(SEQ ID NO: A2)	(HIST2H2AB)
3	VAPEEHPVLLTEAPLNPK	Actin, cytoplasmic 1	3	14	3
	(SEQ ID NO: A3)	(ACTB)
4	VGAGAPVYMAAVLEYLTAEILELAGNAAR	Histone H2A type 2-A	2	12	5
	(SEQ ID NO: A4)	(HIST2H2AA3)
5	MSVQPTVSLGGFEITPPVVLR	Nucleophosmin	7	7	1
	(SEQ ID NO: A5)	(NPM1)
6	NLEALALDLMEPEQAVDLTLPK	X-ray repair cross-	7	3		1	2
	(SEQ ID NO: A6)	complementing 6
		(XRCC6)
7	IHFPLATYAPVISAEK	Tubulin alpha-1A		13
	(SEQ ID NO: A7)	chain (TUBA1A)
8	VGAGAPVYLAAVLEYLTAEILELAGNAARDNKK	Histone H2A type 2-B	5	8
	(SEQ ID NO: A8)	(HIST1H2AB)
9	ISGLIYEETR	Histone H4	2	10	1
	(SEQ ID NO: A9)	(HIST1H4A)
10	VETGVLKPGMVVTFAPVNVTTEVK	Elongation factor 1-	5	7
	(SEQ ID NO: A10)	alpha 1 (EEF1A1)
11	AIGAVPLIQGEYMIPCEK	Cathepsin D (CTSD)	4	2	3
	(SEQ ID NO: A11)

The individual peptides derived from a total of 150 proteins, of which over 90% of the proteins were greater than two-fold enriched, but did not necessarily pass statistical significance, by at least one of the photo-NSAIDs than by the tag 7. A number of isotopically-coded species that were either not selected or not confidently assigned by SEQUEST were also observed. Detection of enriched conjugated species using a pattern searching algorithm³⁹ revealed nearly 1000 isotopically coded precursor ions in the MS1 spectra across our photo-NSAID conjugated peptide data.

The observed interaction frequency and overlap was specific to the small molecule. Photo-celecoxib (5) and photonaproxen (4) represented the bulk of the identified interactions and were found conjugated to 93 and 85 peptides, respectively. Photo-indomethacin (6) was conjugated to 34 peptides in total. A degree of overlap between the photo-NSAID conjugated peptides was observed, where 30 peptides were identified by at least two of the three photo-NSAIDs. Of these, a single conjugated peptide on Ku70, a member of the DNA repair pathway, was observed by two photo-NSAIDs, the tag 7 and photo-glutarimide 8 (entry 6, Table 1). The remaining 14 conjugated peptides detected by the tag 7 and nine conjugated peptides from the photo-glutarimide 8 were detected exclusively by that compound.

We found a significant number of PSMs assigned to NSAID-conjugated histone peptides (FIG. 15A, FIG. 25). All directly observed histone interactions were highly interconnected and imply significantly upregulated conjugation of histone complexes by the photo-NSAIDs (FIG. 15B). These proteins were enriched in photo-NSAID proteomic data as compared to the tag 7, but were not considered statistically significant. In particular, two peptides from histone H2A and histone H2B were primarily detected. The histone H2B peptide was detected in a total of 54 PSMs across our datasets, predominantly by photo-naproxen (4, entry 1, Table 1). Histone H2A type 2-A and type 2-B were detected in a total of 62 and 19 PSMs, respectively (entries 2, 4, and 8, Table 1), predominantly in Jurkat cells. These PSMs related to peptides conjugated to photo-celecoxib (5), followed by photonaproxen (4) and to a lesser degree photo-indomethacin (6). By virtue of directly observing the conjugated peptide, photo-NSAIDs were mapped to a specific binding site hotspot around these two peptides, which are in close proximity in structures of the nucleosome (FIG. 15C).⁴¹ Conjugated peptides from nucleophosmin and elongation factor 1-alpha 1 (entries 6 and 10, Table 1) were also observed.

Validation of SIM-PAL by competition assay revealed that binding of all three photo-NSAIDs with histone H2A and H2B was competitively exchanged with the parent compound by Western blot (FIG. 15D). The interaction of photo-NSAIDs with NF-κB p65 was additionally completely displaced by the parent compound. Selective photo-conjugation and partial competition was observed when probing for Ku70 or nucleophosmin. The tag 7 and photo-NSAIDs labeled elongation factor 1-alpha 1 and rho GDP-dissociation inhibitor 2 similarly, although partial competition was still observed in some cases (data not shown). Cellular thermal shift assay in Jurkat cells showed stabilization of histone H2A by all three NSAIDs and the celecoxib analog 9 relative to the tag 7 by 10-15° C. (FIG. 15E). Photo-naproxen (4) and photo-celecoxib (5) also stabilized histone H2A, while photo-indomethacin (6) did not. These data are in line with the PSM frequency in Table 1. Characterization of these binding sites for biological function will be pursued.

DISCUSSION

The development and application of SIM-PAL to enable characterization of the small molecule interactome and direct binding interactions was described (FIG. 12A). SIM-PAL provides several key advantages, including: (1) instant validation of the interaction by virtue of direct observation, (2) measurement of a range of interactions between transient and highly specific binding events, and (3) structural information about the binding site and the small molecule structure. These advantages are highlighted by our analysis of three NSAIDs for their binding sites with a single protein (COX-2) and against the whole proteome from Jurkat and K562 cells.

We applied SIM-PAL to three NSAIDs, naproxen (1), celecoxib (2), and indomethacin (3), due to their different structures yet similar anti-inflammatory effects. Variation in their off-target toxicity and the affected COX-2-independent anti-inflammatory pathways has been reported with a limited molecular basis for these effects. For example, celecoxib was the result of a medicinal chemistry optimization for COX-2 selectivity relative to COX-1,²² yet additional COX-2 independent mechanisms have been subsequently reported.¹⁸ To illuminate the molecular driving force behind NSAID biology, we developed a set of photo-NSAIDs (4-6, FIG. 12B) that recapitulated the activity of the parent compounds and demonstrated their direct binding interaction with COX-2 (FIGS. 13A-13E). Due to the dynamic nature of PAL, we identified additional transient interactions with COX-2 in vitro and the broader proteome in cellulo. These data report on access and molecular recognition of the compounds to the proteome and afford a structural basis for further functional analysis or protein degradation strategies.⁴² A systematic study of the kinetics of PAL will provide concrete measurement of the range of transient interactions observable by SIM-PAL.

Application of the photo-activated compounds 4-8 to stimulated Jurkat and K562 cells revealed specific protein interactions that were more homologous across the three photo-NSAIDs than with the tag 7 or the photo-glutarimide 8. The photo-NSAID interactome revealed entire protein complexes of both known and novel interactions. For example, specific proteins in the NF-κB pathway are now identified as direct interactors (FIGS. 14E and 24). Enrichment of several protein complexes also indicates that the photo-conjugation event is dynamic and highly specific to the local environment of the small molecule at the time of activation. Novel interactions with the proteasome and the adaptor protein complex 2 were also notable. These results provide insight to the molecular interactions within the proteome generated by the NSAIDs, which may or may not possess direct biological function. Selection of proteins for follow up functional studies may be based on existing biological relevance, relative abundance, or dose dependence. Further studies may reveal functional relevance and the basis for some of the poorly-understood biological effects of NSAIDs.

We elected to evaluate a specific conjugation event that was observed on an interface between histone H2A and H2B in the nucleosome due to the high frequency of PSMs in our MS data. The two peptides are within 6 Å of each other in a crystal structure of the nucleosome, indicative of a binding site hotspot recognized by the photo-NSAIDs.41 While all three photo-NSAIDs were able to conjugate these peptides, photonaproxen (4) conjugation to histone H2B was observed the most frequently, while conjugation of photo-celecoxib (5) was most frequently observed conjugated to histone H2A. Comparison of these data to fragment-based small molecule interaction sites revealed that a diazirine-tagged coumarin, most structurally similar to photo-naproxen (4), likewise conjugated a similar peptide from histone H2AZ.¹² The interaction of photo-NSAIDs with histones was competitively displaced by the parent compound by Western blot (FIG. 15D). These data point to the existence of a binding site hotspot in the nucleosome. Although functional relevance cannot be inferred simply due to observation of an interaction, celecoxib (2) and photo-celecoxib (5) stabilized histone H2A by cellular thermal shift assay (FIG. 151). Stabilization of protein complexes leading to downstream signaling changes is reminiscent of other immunomodulators, including cyclosporin, FK506, and rapamycin,⁴³ and the immunomodulatory drug lenalidomide.^44,45 The photo-NSAIDs reported here are validated probes that may be applied to additional biological studies.

While the photo-NSAIDs interacted with and stabilized COX-2 in cellulo, COX-2 was only observed in PSMs at a FDR >1% that were thus removed from the final dataset (Table 3). Furthermore, conjugated peptides from only a fraction of the enriched proteins were identified. These detection differences reflect the increased challenge in seeking to perform site-specific identification of a single conjugated peptide as opposed to protein identification that may derive from multiple peptides from the same protein. Deeper analysis of the NSAID interactome may be obtained by increasing protein inputs, increasing chromatographic separation, application of additional fragmentation methods, or use of a second protease (e.g., chymotrypsin).

SIM-PAL represents the culmination of advances in chemical enrichment strategies coupled to MS technology and a computational pattern searching algorithm to lay the groundwork for rapid progress in direct structural characterization of small molecule interactions within the whole proteome. Recent work in profiling small molecule modification sites has begun to expand the number of interactions that are known to occur throughout the proteome and enable a deeper understanding of the molecular underpinnings of polypharmacology. SIM-PAL revealed the global interaction map for the three NSAIDs profiled and is readily translated to other clinically relevant agents. For example, the immunomodulatory drugs have widely established pluripotent activity and the mechanism of action is only partly understood.³ Alternatively, metformin is widely used to treat diabetes with little understanding of the underlying mechanism.⁴⁶ SIM-PAL is poised for broad application to bioactive small molecules for identification of proteomic interactions using an unbiased whole cell assay.

TABLE 2
Summary of conjugated peptides representative of binding sites of photo-NSAIDs on recombinant COX-2.
					Theo.
photo-NSAID	Position	Annotated Sequence	m/z [Da]	MH+ [Da]	MH+ [Da]
photo-	175	FlpDPQ	892.92743	1784.84758	1784.84982
naproxen	176	FlPdPQ	738.85565	1476.70403	1476.70858
	177	KFIPDpQGSNMMFAFFAQHFTH	763.76746	3814.80817	3814.79016
	178	FIPDPqGS	738.85217	1476.69707	1476.69734
	179	KFIPDPQgSNMMFAFFAQHFTH	763.76904	3814.81611	3814.79016
	179	RKFIPDPQgSNMMFA	888.42743	2663.26773	2663.27851
	179	LRRKFIPDPQgSNMMF	754.37665	2261.11539	2261.12457
	180	KFIPDPQGs	875.94287	1750.87847	1750.87268
	181	SnMMFAFFAQHFTHQFFK	1014.7923	3042.36234	3042.37822
	185	FIPDPQGSNMMFaFFA	872.06433	2614.17844	2614.17091
	188	FIPDPQGSNMMFAFFaQHFTHQ	1028.7958	3084.37278	3084.38075
	188	FIPDPQGSNMMFAFFaQHFTHQF	767.1568	3831.75489	3831.73672
	250	YQIIDGEMYPpTVK	659.99353	1977.96604	1977.95543
	250	YQIIDGEMYPpTVKDT	1089.526	2178.04473	2178.03513
	341	LSGyHFKLK	700.88739	1400.7675	1400.76128
	343	LSGYHfKLK	700.88556	1400.76384	1400.76128
	449	SQASIDQSRQMKYQSfNEYR	930.77051	2790.29697	2790.2828
	461	yESFEELTGEK	820.37549	1639.7437	1639.74139
	507	tMVEVGAPFSLK	793.90698	1586.80669	1586.81748
photo-	72	LFLKPTPnTVHYILTHFKGFWNVV	763.96851	3815.81342	3815.82412
celecoxib	79	LFLKPTPNTVHYILtHFKGFWNVV	763.56281	3813.78492	3813.80847
	101	NAIMSyVLTSR	835.3681	1669.72893	1669.73245
	101	NAIMSyVLTSR	835.3681	1669.72893	1669.73245
	165	QLPDSNEIVeK	908.90686	1816.80644	1816.81459
	165	QLPDSNEIVeK	835.87292	1670.73857	1670.73422
	166	QLPDSNEIVEk	907.90515	1814.80303	1814.79894
	248	YQIIDGEMyPPTVK	1099.9956	2198.98393	2198.98609
	248	YQIIDGEMyPPTVK	1026.9542	2052.90117	2052.90572
	320	LILIGeTIKIVIEDYV	973.80646	2919.40482	2919.40067
	446	yQSFNEYR	826.32996	1651.65264	1651.65697
	446	yQSFNEYR	753.28943	1505.57158	1505.5766
	455	rFMLKPYESFEELTGEKEMSA	979.75653	2937.25504	2937.27554
	566	GCPFTSfSVPDPELIKTVTINASSSRSGLDD	866.58209	4328.88135	4328.89089
photo-	74	LFLKPTPNTvHYILTHFK	921.81818	2763.43998	2763.45766
indomethacin	93	ILTHFKGFWNVVNNIPfLR	921.81226	2763.42221	2763.43652
	113	pPTYNADYGYK	941.91199	1882.8167	1882.82019
	339	LILIGETIKIVIEDYVQHLsGYHFKLKF	901.47186	4503.33021	4503.32663
	343	VIEDYVQHLSGYHfK	761.69812	2283.07981	2283.06763
	423	SFTRQIAGRVAGgR	962.49585	1923.98442	1923.97434
	586	TVTINASSSRs	785.87128	1570.73528	1570.73031
	597	SGLDDINPTVLl	852.92023	1704.83318	1704.82863
Amino acids that were identified as the conjugation site by SEQUEST HT searching are denoted in bold lowercase letter.
COX-2 tryptic digests were analyzed on an Orbitrap Elite and assigned to tryptic or semi-tryptic peptides using SEQUEST HT.

Lengthy table referenced here
US20200140388A1-20200507-T00001
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Lengthy table referenced here
US20200140388A1-20200507-T00002
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Lengthy table referenced here
US20200140388A1-20200507-T00003
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Supplemental Information

Methods

GENERAL EXPERIMENTAL PROCEDURES

All reactions were performed in single-neck, oven-dried, roundbottomed flasks fitted with rubber septa under a positive pressure of nitrogen, unless otherwise noted. Air- and moisture-sensitive liquids were transferred via syringe or stainless steel cannula. Organic solutions were concentrated by rotary evaporation at 30-33° C. Normal and reverse phase flash-column chromatography was performed as described by Still and co-workers.¹ Normal phase purifications employ silica gel (60 Å, 40-63 μm particle size) purchased from Silicycle (Quebec, Canada). Analytical thin-layer chromatography (TLC) was performed using glass plates pre-coated with silica gel (0.25 mm, 60 Å pore size) impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by exposure to ultraviolet light (UV), iodine (I₂), and/or submersion in ninhydrin followed by brief heating with a heat gun (10-15 seconds).

Chemical Materials.

Commercial solvents and reagents were used as received with the following exceptions. Dichloromethane and N,N-dimethylformamide were purified according to the method of Pangborn and co-workers.² Triethylamine was distilled from calcium hydride under an atmosphere of nitrogen immediately before use. 3-aminopiperidine-2,6-dione hydrochloride was obtained from Ark Pharm. RapiGest was prepared according to the method of Lee and co-workers.³ 3-[4-({Bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]propanol (BTTP) was prepared according to the method of Wu and co-workers.⁴ 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma Aldrich. Biotin-CA(PEG)₄-alcohol S14 was synthesized according to the method of Tirrell and co-workers.⁵

Biological Materials.

COX-2 polyclonal antibody (PA5-16817), histone H2A polyclonal antibody (PA5-35893) and high sensitivity streptavidin-HRP (21130) were purchased from Thermo Fisher Scientific and used in 1:100, 1:500 and 1:10,000 dilutions, respectively. Anti-Histone H2B monoclonal antibody (12364) was purchased from Cell Signaling Technology and diluted to 1:1000 in Western Blot detection. Nucleophosmin (NPM1/B23) monoclonal antibody (sc-271737), Ku-70 (XRCC6) monoclonal antibody (sc-56129), NFκB p65 monoclonal antibody (sc-8008), Ly-GDI monoclonal antibody (sc-376473), EF-1 α1 monoclonal antibody (sc-21758), Cathepsin D monoclonal antibody (sc-377299) and secondary antibody m-IgGκ BP-HRP (sc-516102) were provided by Santa Cruz, and used in 1:500, 1:200, 1:500, 1:500, 1:100, 1:200 and 1:1000 dilutions, respectively. ONE-Glo Luciferase Assay System (E6110) was purchased from Promega Corporation.

Cell Culture Materials.

K562 and Jurkat cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37° C. and 5% CO₂ in a water-saturated incubator. EDTA-free protease inhibitor cocktail was obtained from Roche Diagnostics. Streptavidin-agarose beads were obtained from Thermo Scientific and washed with PBS prior to use. The GloResponse NF-κB-RE-luc2P HEK293 Cell Line, a clonal derivative of Human Embryonic Kidney 293 (HEK293), was provided by Choudhary lab, and maintained in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37° C. and 5% CO₂ in a water-saturated incubator.

Chemical Instrumentation.

Proton nuclear magnetic resonance spectra (¹H NMR) were recorded at 400 or 500 MHz at 24° C., unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, δ scale) downfield from tetramethylsilane and are referenced to residual protium in the NMR solvent [CHCl₃, δ 7.26; CHD₂OD, δ 3.31; (CHD₂)(CD₃)SO, δ 2.49]. Data are represented as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, quin=quintet, m=multiplet and/or multiple resonances, br=broad, app=apparent), integration, coupling constant in Hertz, and assignment. Proton-decoupled carbon nuclear magnetic resonance spectra (¹³C NMR) were recorded at 125 MHz at 24° C., unless otherwise noted. Chemical shifts are expressed in parts per million (ppm, δ scale) downfield from tetramethylsilane and are referenced to the carbon resonances of the solvent (CDCl₃, δ 77.0, CD₃OD, δ 49.0; (CD₃)₂SO, δ 39.0). ¹³C NMR and data are represented as follows: chemical shift, carbon type. Chemical shifts are expressed in parts per million (ppm, δ scale) downfield from tetramethylsilane. Infrared (IR) spectra were obtained using a Shimadzu 8400S FT-IR spectrometer referenced to a polystyrene standard. Data are represented as follows: frequency of absorption (cm⁻¹), intensity of absorption (s=strong, m=medium, w=weak, br=broad). High-resolution mass spectrometry (HRMS) measurements were obtained at the Chemistry and Chemical Biology Department, Harvard University Mass Spectrometry Facility using a Bruker microTOF-Q II hybrid quadrupole-time of flight, Agilent 1260 UPLC-MS. Low-resolution mass spectrometry (LRMS) measurements were obtained on Waters ACQUITY UPLC equipped with SQ Detector 2 mass spectrometer. The samples were photoirradiated with UVP Blak-Ray B-100AP high-intensity UV lamp (UVP LLC, Upland, Calif.). The absorbance was measured on a multi-mode microplate reader FilterMax F3 (Molecular Devices LLC, Sunnyvale, Calif.). TAMRA-fluorescence and chemiluminescence were detected by scanning the gel on Azure Imager C600 (Azure Biosystems, Inc., Dublin, Calif.).

Experimental Procedures with COX-2

COX-2 Inhibitor Screening Assay by ELISA.

The COX-2 inhibitor screening assay kit (701080, Cayman Chemical, Ann Arbor, Mich.) was used to determine the inhibitory concentration of COX-2 in the presence of photo-NSAIDs or NSAIDs. The effect of inhibitors on COX-2-mediated prostaglandin production was measured in triplicate with several concentration gradients of photo-NSAIDs or NSAIDs. Non-specific binding (NSB), maximum binding (B0), 100% initial activity (IA), background COX-2 (BC) and blank samples, in duplicate, were also measured. A prostaglandin screening standard curve was obtained by plotting the data as ln [B/B₀/(1−B/B₀)] versus log concentrations of prostaglandin and performing a linear regression fit. The concentration of prostaglandin for each inhibitor sample was calculated by using the prostaglandin standard curve. The curve of percent inhibition by the inhibitor concentration was fitted and analyzed to determine the IC₅₀ value for inhibitors using GraphPad Prism (GraphPad Software, San Diego, Calif.).

Competitive Analysis of NSAIDs and Photo-NSAIDs with Isolated COX-2.

Recombinant human COX-2 (125 ng, 12036-H08B, Sino Biological Inc., Beijing, China) was separately incubated with each of the photo-NSAIDs with or without a 100-fold excess of the parent compound as a competitor for 2 hours at 37° C. The samples were photo-irradiated for 30 minutes at 4° C., tagged with TAMRA-azide (760765, Sigma-Aldrich, St. Louis, Mo.) by copper-click chemistry for 2 hours at 24° C., and detected by fluorescence spectroscopy (Azure Imager C600, Azure Biosystems) with Cy3 excitation.

Molecular Docking of the Structure of COX-2 with Photo-NSAIDs and the Tag 7.

The photo-NSAIDs, the parent compounds, and the tag 7 were structurally minimized using Gaussian 16.0 with a basis set of HF 3-21g(d). The energetically minimal structures were used in a geometry-based molecular docking algorithm PatchDock (https://bioinfo3d.cs.tau.ac.il/PatchDock/) to calculate docking transformations that based on a scoring function and atomic desolvation energy. The structures with the lowest desolvation energy and highest interface area size were used in FIG. 13. The receptor molecule was obtained from the crystal structure of human COX-2 (PDB: 5KIR).

In-Vitro Photolabeling of COX-2 with Photo-NSAIDs and Analysis by LC-MS/MS.

Recombinant COX-2 (1 μg) was separately incubated with each of the photo-NSAIDs (10 μM) or the tag 7 (10 μM) for 30 minutes at 37° C., and photo-irradiated for 15 minutes at 4° C. The samples were then tagged with the cleavable biotin azide probe 10 by copper-click chemistry for 3 hours at 24° C., followed by sample digestion with trypsin for 12 hours at 37° C., and probe cleavage with 2% formic acid. The resulting peptides were analyzed by LC-MS/MS. MS data was searched in Proteome Discoverer v2.2 with SEQUEST HT or Byonic v2.1 against the recombinant COX-2.

Experimental Procedures with Whole Cells

Cell growth assay (MTT) with photo-NSAIDs and NSAIDs. Jurkat cells were seeded in a 96-well flatbottomed cell culture plate (Falcon) in RPMI with 10% FBS and 1% penicillin/streptomycin at a density of 1×10⁵ cells/mL and maintained for 24 hours at 37° C. and 5% CO₂ atmosphere. Cells were treated with photo-NSAIDs or NSAIDs in triplicate at different doses and incubated for 24 hours at 37° C. MTT solution (10 μL, 5 mg/mL) was added to each well and gently mixed. The cells were incubated for 4 hours at 37° C., followed by addition of 0.1 mL isopropanol with 0.04 N HCl to each well. The resulting mixture was mixed thoroughly by repeated pipetting. The absorbance was measured within 1 hour on a multi-mode microplate reader with a wavelength of 620 nm.

In-Situ Photo-Labeling of K562 or Jurkat Cells with Photo-NSAIDs.

K562 cells or PMA/Ionomycin activated Jurkat cells (˜97% cell viability) were suspended in FBS-free RPMI at the indicated concentration of photo-NSAIDs or negative controls tag 7 the photo-glutarimide 8, or the analog 9 for 2 hours at 37° C., followed by UV irradiation at 365 nm for 30 minutes at 4° C. In competition experiments, cell lysates were co-treated with the photo-NSAIDs and their parent compounds in 10-fold excess. The photoirradiated samples were pelleted, lysed with 1% RapiGest and EDTA-free protease inhibitor and briefly sonicated with a probe tip sonicator. Sonicated cell lysates were cleared by centrifugation and the concentration of the soluble proteins was determined using the BCA protein assay. Protein concentrations were adjusted to a final concentration of 2.5 mg/mL. Cell lysates (500 μL) were reacted with pre-mixed click reagents at a final concentration of 200 μM biotin azide probe 10, 300 μM copper (II) sulfate, 600 μM BTTP and 2.5 mM freshly-prepared sodium ascorbate for 5 hours at 24° C. with rotation. The proteins were precipitated with methanol (1 mL) for 1 hour at −80° C., pelleted by centrifugation (15,000 g) for 10 minutes at 4° C., and air dried for 10 minutes at 24° C.

Enrichment of Photo-Crosslinked Proteins for LC-MS/MS.

Biotinylated protein pellets were resuspended in 1% RapiGest in PBS (400 μL) and briefly sonicated. Streptavidin-agarose resin (200 μL, washed 3×1 mL PBS) was added to the suspended proteins and incubated for 12 hours at 24° C. The beads were centrifuged for 3 minutes at 3000×g and the supernatant was removed. The beads were washed with 1% RapiGest (1 mL), urea (6M, 3×1 mL), and PBS (2×1 mL) in succession. The washed beads were resuspended in PBS (200 μL). The proteins on beads were reduced with 5 mM dithiothreitol (DTT) for 30 minutes at 24° C. and alkylated with 10 mM iodoacetamide for 30 minutes at 24° C. in the dark. The beads were pelleted by centrifugation and resuspended in 0.5 M urea/PBS (200 μL) and trypsin (1.5 μg) digested for

12 hours at 37° C. with rotation. The supernatant digest was collected and the beads were washed with PBS (200 μL) and water (2×200 μL). The washes were combined to obtain the “trypsin fraction”. Cleavage of the probe 10 and recovery of the conjugated peptide was performed in 2% formic acid in water (200 μL) for 30 minutes at 24° C. The cleavage solution was collected and the beads were washed with 80/o acetonitrile in water (400 μL) and the fractions were combined to afford the “cleavage fraction”. The trypsin and cleavage fractions were concentrated to dryness using a SpeedVac concentrator heated at 40° C., and stored at −20° C. until analysis by LC-MS/MS.

Cell Thermal Shift Assay (CETSA) with Photo-NSAIDs and NSAIDs.

Jurkat cells in 15 mL of FBSfree RPMI at a density of 2.5×10⁶ cells/mL were incubated with 50 μM of tag 7, photo-NSAIDs or NSAIDs for 2 hours in a 5% CO2 atmosphere at 37° C. The cell suspension was then collected and centrifuged for 3 minutes at 300×g and the culture medium was aspirated. The cell pellets were re-suspended and washed with PBS (2×15 mL) by centrifugation. PBS (1 mL) supplemented with EDTA-free protease inhibitors (1×) was added to each tube, and the resulting cell suspension was equally distributed into eight different 0.2 mL PCR tubes. The PCR tubes were heated over a temperature gradient (37-62° C.) for 20 minutes in a 96-well thermal cycler, followed by repeated freeze-thaw cycles (5×). The PCR tubes containing the resulting cell lysates were centrifuged at 20,000×g for 30 minutes at 4° C. to pellet cell debris together with precipitated and aggregated proteins. Each supernatant with the soluble protein fraction was transferred carefully to a new tube and analyzed by Western blot. The Western blot bands were quantified by ImageJ, and the data were fitted to obtain apparent Tagg values using the Boltzmann Sigmoid equation within GraphPad Prism.

Luciferase Reporter Assay for NF-κB Pathway.

The GloResponse NF-κB-RE-luc2P HEK293 cell line contains a luciferase gene (luc2P) under the control of a minimal TATA promoter with multiple nuclear factor-κB response elements (NF-κB-REs). A total of 10,000 NF-κB-RE-luc2P HEK293 cells per well were dispensed into a 384-well plate, and incubated respectively with 5 μM of tag 7, celecoxib (2), photocelecoxib (5) and DMSO for 1 hour at 37° C., followed by addition of two-fold serial dilutions of TNF-α to induce reporter gene expression. After 5 hours of induction in a tissue culture incubator at 37° C., luminescence was quantified using the ONE-Glo Luciferase Assay System Reagent on a multi-mode microplate reader FilterMax F3 (read for 0.5 second/well). The TNF-α titration curve was fitted and analyzed to determine the half maximal effective concentration (EC₅₀) using GraphPad Prism.

Mass Spectrometry Procedures

Mass Spectrometry Procedures.

The desalted samples were resuspended in 0.1% formic acid in water (15 μL). The sample (4.0 μL) was loaded onto a C18 trap column (3 cm, 3 μm particle size C10 Dr. Maisch 150 μm I.D) and then separated on an analytical column (Thermo Scientific Acclaim PepMap 100, 2 μm particle size, 250 mm length, 75 μm internal diameter) at 150 nL/min with a Thermo Scientific EASY-nLC 1000 system connected in line to a Thermo Scientific Orbitrap Fusion Tribrid or Orbitrap Elite. The column temperature was maintained at 50° C. The tryptic peptides were separated via a stepwise gradient from 5% to 98% of 0.1% formic acid in acetonitrile over 120 minutes (0-1 minutes, 0-5%; 1-91 minutes, 5-27%; 91-115 minutes, 27-98%; 115-120 minutes, 98%-0%). The cleavage peptides were separated via a step-wise gradient from 5% to 98% of 0.1% formic acid in acetonitrile over 130 minutes (0-1 minutes, 0-5%; 1-81 minutes, 5-28%; 81-100 minutes, 28-98%; 100-135 minutes, 98%-0%). Survey scans of peptide precursors were performed at 120K FWHM resolution (m/z=200). Tandem MS was performed on the most abundant precursors exhibiting a charge state from 2 to 5 at a resolving power settings of 15K and fragmentation energy of 36V. HCD/CID fragmentation was applied with 35% collision energy and resulting fragments detected using the normal scan rate in the ion trap.

Data Analysis Procedures.

Data analysis was performed with Proteome Discoverer v2.2 using SEQUEST HT, allowing for variable modifications (methionine oxidation: +15.995 Da; cysteine alkylation: +57.021 Da; photo-naproxen: +452.206 Da, +454.222 Da; photo-celecoxib: +543.151 Da, +545.167 Da: photo-indomethacin: +592.220 Da, +594.236 Da), two missed cleavages and mass tolerance of 10 ppm for the precursor ion, 0.02 Da and 0.6 Da for fragment ions from HCD and CID, respectively. For binding sites of photo-NSAIDs to COX-2 (Table 2), MS data was searched by SEQUEST HT and Byonic against the recombinant COX-2 with the photo-NSAID before and after click chemistry as a modification on any amino acid and assigned to tryptic or semi-tryptic peptides. For the proteomic analysis of K562 and Jurkat cells (Table 3, 4), MS/MS raw files of the tryptic digests were searched against the Swiss-Prot human database (downloaded in 2016) and a contaminant protein database. The statistically significant enriched proteome were determined according to the procedure of Washburn and co-workers.⁶ Briefly, the normalized spectral abundance factor (NSAF) was calculated as the number of spectral counts divided by the protein's length, and then divided by the sum of spectral counts for all proteins in the experiment. The zero NSAF values were replaced by the minimum measured NSAF value and the natural log transformation of NSAF values was used for evaluation with the statistical t-test. Normalized protein assignments at 1% FDR were considered statistically significantly enriched if the fold change was greater than two and the associated p-value was <0.05 (t-test) in labeled samples than in samples treated with the tag 7. For experiments where biological replicates were not obtained, enrichment was determined by subtraction of all proteins observed in the samples treated with the tag 7. MS/MS data from cleavage fractions were searched against the entire proteome identified in tryptic digests for direct binding site characterization (Table 5). All calculations were performed in Microsoft Excel and all peptide spectral matches (PSMs) at 5% FDR were manually validated for precursor isotopic pattern in the MS1 and spectral assignment in the MS2.

Synthetic Procedures

Synthesis of methyl (S)-2-(6-hydroxynaphthalen-2-yl) propanoate (S1)

Step 1:

Naproxen (1, 5.00 g, 21.7 mmol, 1 equiv) was added to aqueous HBr (48% w/w, 35 mL, 309 mmol, 14.2 equiv) in a round bottom flask fitted to a reflux condenser. The reflux condenser was connected to a vented trap containing saturated aqueous sodium bicarbonate solution. The mixture was stirred for 12 hours at reflux. The product mixture was cooled over 30 minutes to 24° C. The cooled solution was poured in deionized (DI) water (200 mL) and cooled in an ice bath until a visible white precipitate was formed. The precipitate was filtered through a fritted funnel, washed with DI water (3×30 mL) and dried for 2 hours in air.

Step 2:

The crude white powder was dissolved in methanol (40 mL). Acetyl chloride (2.30 mL, 32.6 mmol, 1.50 equivalents) was added to the resulting solution at 0° C. and the mixture was heated for 4 hours at reflux. The product mixture was cooled over 30 minutes to 24° C. and evaporated to dryness. The resulting material was azeotroped with benzene (2×20 mL) to afford compound S1 as an off-white solid (4.70 g, 95%).

R_f=0.30 (20% ethyl acetate-hexane; UV). ₁H NMR (500 MHz, DMSO-d₆): δ 9.71 (s, 1H, H₁₀), 7.73 (d, 1H, J=7.2 Hz, H₂), 7.65-7.63 (m, 2H, H₄/H₅), 7.31 (d, 1H, J=8.0 Hz, 6₆), 7.11-7.09 (m, 2H, H₁/H₃), 3.90 (q, 1H, J=7.3 Hz, H₉), 3.58 (s, 3H, H₈), 1.46 (d, 3H, J=7.3 Hz, H₇). ¹³C NMR (125 MHz, DMSO-d₆): δ 174.9 (C), 155.7 (C), 135.2 (C), 134.1 (C), 129.6 (CH), 128.1 (C), 126.8 (CH), 126.4 (CH), 126.1 (CH), 119.3 (CH), 108.9 (CH), 52.2 (OCH₃), 44.8 (CH) 18.9 (CH₃). IR (ATR-FTIR), cm⁻¹: 3348 (br), 2982 (m), 2957 (m), 2940 (m), 1703 (s), 1637 (s), 1613 (s), 1509 (s), 1486 (s), 1437 (m), 1338 (s), 1208 (m), 1171 (m), 1147 (m), 927 (m), 857 (m), 808 (m), 558 (m), 471 (m). LRMS-ESI (m/z): [M+H]⁺ calculated for C₁₄H₁₅O₃, 231.1, found, 231.2.

The ¹H NMR and ¹³C NMR of S1 prepared by the above procedures were in agreement with those reported previously.⁷

Synthesis of methyl (S)-2-(6-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethoxy)naphthalen-2-yl)propanoate (S2)

Potassium carbonate (300 mg, 2.17 mmol, 2.10 equiv) and iodide S7 (350 mg, 1.40 mmol, 1.00 equiv) was added to a solution of S1 (250 mg, 1.04 mmol, 1 equiv) in DMF (6.0 mL). The mixture was stirred for 12 hours at 35° C. The product mixture was poured into a separatory funnel containing DI water (15 mL) and diethyl ether (30 mL). The organic portion was separated, washed with brine and dried over magnesium sulfate. The magnesium sulfate was filtered off and the ethereal solution was concentrated in vacuo to afford a waxy material. The material was purified using flash-column chromatography (eluting with 0-40% ethyl acetate-hexane, two steps) to afford the diazirine S2 (310 mg, 85%).

R_f=0.37 (20% ethyl acetate-hexane; UV). ¹H NMR (500 MHz, CDCl₃): δ 7.74 (d, 1H, J=8.5 Hz, H₂), 7.71 (d, 1H, J=8.2 Hz, H₅), 7.68 (s, 1H, H₄), 7.43 (dd, 1H, J=8.5, 1.8 Hz, H₆), 7.18 (dd, 1H, J=8.9, 2.5 Hz, H₁), 7.10 (d, 1H, J=2.4 Hz, H₃), 3.97 (t, 2H, J=7.2 Hz, H₁₀), 3.87 (q, 1H, J=7.2 Hz, H₉), 3.68 (s, 3H, H₈), 2.11 (td, 2H, J=8.1, 5.0 Hz, H₁₃), 2.02 (t, 1H, J=5.0 Hz, H₁₄), 1.98 (t, 2H, J=7.2 Hz, H₁₁), 1.78 (t, 2H, J=7.2 Hz, H₁₂), 1.60 (d, 3H, J=7.1 Hz, H₇). ¹³C NMR (125 MHz, CDCl₃): δ 175.1 (C), 156.4 (C), 135.9 (C), 133.6 (C), 129.4 (CH), 129.1 (C), 127.2 (CH), 126.3 (CH), 125.9 (CH), 119.1 (CH), 106.6 (CH), 82.8 (C), 69.2 (CH), 62.6 (OCH₂), 52.1 (OCH₃), 45.4 (CH), 32.9 (CH₂), 32.7 (CH₂), 26.7 (CN₂), 18.6 (CH₃), 13.3 (CH₂). IR (ATR-FTIR), cm⁻¹: 3295 (br), 2922, 2852, 2940, 1705 (s), 1634 (s), 1468 (m), 1510 (w), 1468 (m), 1393 (m), 1209 (m), 1180 (m), 939 (m), 811 (m), 640 (m), 475 (m), 1172

(m), 1147 (m), 930 (m), 857 (m), 808 (m), 560 (m), 490 (m). HRMS-ESI (m/z): [M+H]⁺ calculated for C₂₁H₂₃N₂O₃, 351.1709; found, 351.1710.

Synthesis of (S)-2-(6-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethoxy)naphthalen-2-yl)propanoic acid [Photo-Naproxen (4)]

Compound S2 (200 mg, 570 μmol, 1 equiv) was added to a 1M NaOH in methanol (3.0 mL). The mixture was stirred for 4 hours at 24° C. The product material was concentrated in vacuo. The residue obtained was acidified to pH 2, with KHSO₄ (1M) and the resulting mixture was added to ethyl acetate. The organic portion was separated, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford photo-naproxen (4) as a waxy solid (190 mg, 98%).

¹H NMR (500 MHz, CDCl₃): δ 7.72 (m, 3H, H₂/H₄/H₅), 7.45 (dd, 1H, J=8.2, 1.8 Hz, H₆), 7.18 (dd, 1H, J=8.5, 2.4 Hz, H₁), 7.09 (d, 1H, J=2.4 Hz, H₃), 3.97 (t, 2H, J=7.0 Hz, H₉), 3.90 (t, 1H, J=7.1 Hz, H₈), 2.13 (td, 2H, J=7.9, 2.9 Hz, H₁₂), 2.02 (t, 1H, J=2.6 Hz, H₁₃), 1.98 (t, 2H, J=7.1 Hz, H₁₀), 1.80 (t, 2H, J=7.5 Hz, H₁₁), 1.62 (d, 3H, 7.2 Hz, H₇). ¹³C NMR (125 MHz, CDCl₃): δ 180.1 (C), 156.5 (C), 135.2 (C), 133.7 (C), 129.4 (CH), 129.1 (C), 127.2 (CH), 126.3 (CH), 126.1 (CH), 119.1 (CH), 106.6 (CH), 82.8 (C), 69.2 (CH), 62.6 (OCH₂), 45.2 (CH), 32.9 (CH₂), 32.7 (CH₂), 26.7 (CN₂), 18.2 CH₃), 13.3 (CH₂). IR (ATR-FTIR), cm⁻¹: 3295 (br), 2921 (w), 2852 (m), 1703 (s), 1633 (s), 1605 (s), 1468 (m), 1393 (m), 1263 (m), 1208 (m), 1180 (w), 939 (w), 853 (w), 812 (w), 641 (w), 475 (w). HRMS-ESI (m/z): [M+Na]⁺ calculated for C₂₀H₂₀N₂NaO₃, 359.1372; found, 359.1372.

Synthesis of 4-(5-hydroxy-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide S4)

Ethyl 4, 4, 4-trifluoro-3-oxobutanoate (S3, 2.00 g, 10.9 mmol, 1 equiv) and 4-hydrazineylbenzenesulfonamide (2.40 g, 10.9 mmol, 1.00 equiv) were dissolved in ethanol (10 mL) and the resulting mixture was stirred for 12 hours at reflux. The product mixture was cooled for 30 minutes to 24° C. The solvent was evaporated in vacuo and the residue was poured into DI water (15 mL). The solution was acidified to pH 2 and a white precipitate formed. The precipitate was filtered through a frit funnel, the precipitate was filtered through a fritted funnel and dried for 2 hours in air to afford the benzenesulfonamide S4 as a slight yellow powder (3.30 g, 99%).

¹H NMR (500 MHz, DMSO-d6): δ 12.86 (s, 1H, H₂), 7.96 (m, 4H, H₃/H₄), 7.46 (m, 2H, H₅), 5.97 (s, 1H, H₁). ¹³C NMR (125 MHz, DMSO-d₆): δ 154.8 (C), 142.7 (CH), 141.9 (q, 2JCF=37.2 Hz, C), 140.6 (CH), 127.3 (CH), 122.7 (q, 1JCF=267.4 Hz, CF₃), 122.3 (C), 86.4 (C). ¹⁹F NMR (375 MHz, DMSO-d₆): δ −61.5. IR (ATR-FTIR), cm⁻¹: 3449 (br), 3383 (w), 3280 (w), 2983 (w), 2940 (m), 1703 (s), 1637 (s), 1613 (m), 1589 (m), 1579 (m), 1556 (w), 1535 (m), 1436 (m), 1408 (m), 1208 (m), 1102 (m), 991 (m), 927 (w), 895 (w), 848 (w), 795 (w), 778 (m), 706 (w), 541 (w), 470 (w).

Synthesis of 4-(5-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethoxy)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide (5)

A mixture of compound S4 (90.0 mg, 286 μmol, 1 equiv), and iodo-diazirine S7 mg, 286 μmol, 1.00 equiv) was dissolved in DMF (3.0 mL) at 24° C. To this mixture, was added potassium carbonate (40.0 mg, 286 μmol, 1.00 equiv) and stirred for 12 hours at 24° C. The reaction mixture was partitioned between ethyl acetate (30 mL) and DI water (15 mL) and the organic portion was separated and washed with brine (3×15 mL). The combined organic portion was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford an off white powder. The powder was purified by flash column chromatography (30-60% ethyl acetate-hexane, three steps) to afford photo-celecoxib (5) as a white powder (92.0 mg, 75%).

R_f=0.20 (40% ethyl acetate-hexane; UV). ¹H NMR (500 MHz, CDCl₃): δ 8.05 (d, 2H, J=8.2 Hz, H₃), 8.02 (d, 2H, J=8.1 Hz, H₄), 5.95 (s, 1H, H₁), 5.13 (br, 2H, H₅), 4.04 (t, 2H, J=7.2 Hz, H₂), 2.05-2.03 (m, 5H, H₆/H₈/H₉), 1.71-1.69 (m, 2H, H₇). ¹³C NMR (125 MHz, CDCl₃): δ 154.6 (C), 143.0 (q, 2JCF=38.8 Hz, CH), 141.1 (C), 140.4 (C), 127.6 (CH), 122.5 (CH), 121.7 (q, 1JCF=267.5 Hz CF₃), 85.3 (C), 82.4 (C), 69.7 (CH), 67.6 (OCH₂), 32.4 (CH₂), 32.1 (CH₂), 26.1 (CN₂), 13.2 (CH₃). ¹⁹F NMR (375 MHz, CDCl₃): δ −63.6. IR (ATR-FTIR), cm⁻¹: 3277 (br), 1590 (s), 1563 (m), 1512 (m), 1490 (w), 1416 (m), 1380 (w), 1335 (w), 1246 (m), 1151 (m), 1101 (m), 968 (w), 908 (w), 840 (w), 743 (w), 715 (w), 626 (m), 543 (w). HRMS-ESI (m/z): [M+H]⁺ calculated for C₁₇H₁₇F₃N₅O₃S, 428.0999; found, 428.0949.

Synthesis of N-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamide (6)

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (418 mg, 2.19 mmol, 1.50 equiv), triethyl amine (400 μL, 2.98 mmol, 2.00 equiv), N, N′-dimethylaminopyridine (36.0 mg, 290 μmol, 0.20 equiv) and the amine S6 (200 mg, 1.46 mmol, 1.00 equiv) were added in sequence to a solution of indomethacin (3, 522 mg, 1.46 mmol, 1 equiv) in DMF (10 mL) at 24° C. The reaction mixture was stirred for 12 hours at 24° C. and then quenched by addition of potassium hydrogen sulfate (1M, 10 mL). The mixture was transferred into a separatory funnel containing ethyl acetate (50 mL). The organic portion was separated, washed with brine (3×10 mL) and the dried over anhydrous sodium sulfate. The sodium sulfate was filtered and the eluent was concentrated in vacuo to afford a crude residue. The crude residue was dissolved in ethyl acetate and crystallized from hexane to afford photo-indomethacin (6) as a white solid (615 mg, 88%).

¹H NMR (500 MHz, CDCl₃): δ 7.70 (d, 2H, J=8.0 Hz, H₁₁), 7.49 (d, 2H, J=7.8 Hz, H₁₂), 6.92 (s, 1H, H₁), 6.9 (d, 1H, J=9.0 Hz, H₃), 6.72 (d, 1H, J=9.0 Hz, H₂), 5.78 (s, 1H, H₁₄), 3.83 (s, 3H, H₁₃), 3.65 (s, 2H, H₄), 3.11 (t, 2H, J=7.2 Hz, H₅), 2.42 (s, 3H, H₁₀), 1.88 (dd, 2H, J=7.5, 2.4 Hz, H₈), 1.84-1.83 (m, 1H, H₉) 1.62 (t, 2H, t, J=7.0 Hz, H₆), 1.53 (t, 2H, J=7.2 Hz, H₈). ¹³C NMR (125 MHz, CDCl₃): δ 169.9 (C), 168.2 (C), 156.2 (C), 139.4 (C—Cl), 136.4 (C), 133.5 (═C—CH3), 131.1 (CH), 130.9 (CH), 130.3 (C), 129.1 (═C—NR2), 115.1 (CH), 112.6 (CH), 112.2 (C), 100.9 (CH), 82.5 (C), 69.3 (CH), 55.7 (OCH₃), 34.6 (CH₂), 32.4 (CH₂), 32.1 (CH₂), 31.8 (CH), 26.7 (CN₂), 13.3 (CH₂), 12.9 (CH₃). IR (ATR-FTIR), cm⁻¹: 3297 (br), 3066 (w), 2930 (w), 2835 (w), 1673 (s), 1650 (m), 1590 (w), 1525 (s), 1476 (m), 1455 (m), 1356 (m), 1314(s), 1222 (s), 1148 (w), 1088 (m), 1065 (w) 909 (w), 833 (w), 728 (w), 644 (w) 481(w). HRMS-ESI (m/z): [M+H]⁺ calculated for C₂₆H₂₆ClN₄O₃, 477.1693; found, 477.1660.

Synthesis of the Photo-Glutarimide 8

Step 1. Synthesis of 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl 1H-imidazole-1-carboxylate (S5)

To a solution of 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl) ethan-1-ol (200 mg, 1.45 mmol, 1 equiv) in tetrahydrofuran (10 mL) at 24° C., was added carbonyldiimidazole (950 mg, 5.80 mmol, 4.00 equiv). The mixture was allowed to stir for 12 hours at 24° C. The product mixture was concentrated in vacuo to a solid residue. The residue was directly loaded to a hexane-equilibrated silica gel column and the desired material was eluted using a 50-1000/% ethyl acetate-hexane solvent, two steps). The imidazole S5 was isolated as colorless oil after concentration of the residual solvent (320 mg, 95%).

R_f=0.15 (40% ethyl acetate-hexane; UV). ¹H NMR (500 MHz, CDCl₃): δ 8.13 (s, 1H, H₆) 7.42-7.41 (m, 1H, H₇), 7.03-7.02 (m, 1H, H₈), 4.26-4.23 (t, 2H, J=7.1 Hz, H₅), 1.99-1.96 (m, 3H, H₂/H₁), 1.91-1.88 (t, 2H, J=6.9 Hz, H₄), 1.66-1.63 (t, 2H, J=7.1 Hz, H₃). ¹³C NMR (125 MHz, CDCl₃): δ 148.3 (C), 137.1 (CH), 130.7 (CH), 117.1 (CH), 82.3 (C), 69.6 (CH), 62.9 (OCH₂), 32.1 (CH₂), 32.0 (CH₂), 26.0 (CN₂), 13.1 (CH₂). IR (ATR-FTIR), cm⁻¹: 3291 (br), 3158 (w), 3132 (m), 2960 (w), 2921 (m), 2858 (m), 1758 (s), 1588 (s), 1525 (m), 1473 (m), 1444 (m), 1404 (m), 1380 (w), 1316 (w), 1282 (w), 1240 (w), 1173 (w), 1095 (m), 1058 (m), 1003 (w), 898 (m), 833 (w), 768 (w), 749 (w), 649 (w), 598 (w), 526 (w). HRMSESI (m/z): [M+H]⁺ calculated for C₁₁H₁₃N₄O₂, 233.1039; found, 233.1064.

Step 2. Synthesis of 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl (2,6-dioxopiperidin-3-yl)carbamate (8)

1,8-Diazabicyclo[5.4.0]undec-7-ene (650 mg, 4.31 mmol, 5.30 equiv) and 3-aminopiperidine-2,6-dione hydrochloride (600 mg, 2.58 mmol, 3.14 equiv) were added in sequence to a stirred solution of 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl 1H-imidazole-1-carboxylate (200 mg, 820 μmol, 1 equiv) in DMF (10 mL). The mixture was stirred for 12 hours at 24° C. The product mixture was partitioned between ethyl acetate (30 mL) and DI water (20 mL). The organic portion was separated and the aqueous portion was extracted with ethyl acetate (2×20 mL). The combined organic portion was washed with DI water (4×10 mL) then with brine (10 mL). The solution was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford the crude material. The crude was purified by silica gel flash chromatography (using 20-60% ethyl acetate-hexanes, two steps) to afford the photo-glutarimide 8 as an oil (220 mg, 86%).

R_f=0.20 (40° % ethyl acetate-hexane). ¹H NMR (500 MHz, CDCl₃): δ 8.60 (m, 1H, H₁₀), 5.72 (m, 1H, H₆), 4.41-4.36 (m, 1H, H₇), 4.02 (t, 2H, J=5.7 Hz, H₅), 2.84-2.49 (m, 4H, H₈/H₉), 2.04-1.91 (m, 3H, H₂/H₁), 1.78 (t, 2H, J=6.2 Hz, H₄), 1.70 (t, 2H, J=7.2 Hz, H₃). ¹³C NMR (125 MHz, CDCl₃): δ 171.6 (C), 171.4 (C), 155.9 (C), 82.6 (C), 69.4 (CH), 65.1 (CH), 60.2 (OCH₂), 32.5 (CH₂), 32.2 (CH₂), 31.2 (CH₂), 25.2 (CN₂), 13.2 (CH₂). IR (ATR-FTIR), cm⁻¹: 3283 (br), 3107 (w), 2958 (w), 2918 (m), 2858 (w), 1694 (s), 1587 (s), 1527 (m), 1356 (m), 1331 (m), 1302 (m), 1197 (m), 1082 (w), 1063 (m), 1037 (m), 991 (w), 776 (m), 650 (m), 472 (m). HRMS-ESI (m/z): [M+Na]⁺ calculated for C₁₃H₁₆N₄NaO₄, 315.1069; found, 315.1076.

Synthesis of 1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-ol (S9)

Ethyl 4, 4, 4-trifluoro-3-oxobutanoate (S3, 200 μL, 1.37 mmol, 1 equiv) and phenylhydrazine (148 μL, 1.37 mmol, 1.00 equiv) were dissolved in ethanol (1.4 mL) and the resulting mixture was stirred for 12 hours at reflux. The reaction mixture was cooled to 24° C. and the solvent was evaporated in vacuo. The residue was dissolved into ethyl acetate (3 mL) and washed with 1N HCl (3×3 mL). The organic layer was dried over sodium sulfate, filtrated and concentrated in vacuo. The resulting material was washed with dichloromethane (5 mL) to afford the compound as orange solid (289 mg, 92%).

R_f=0.25 (30% ethyl acetate-hexanes; UV). ¹H NMR (500 MHz, DMSO-d₆): δ 7.71 (d, 2H, H2, J=8.0 Hz), 7.51 (dd, 2H, J=8.0 Hz, H₃), 7.38 (t, 1H, J=8.0 Hz, H₄), 5.94 (s, 1H, H₁). ¹³C NMR (125 MHz, DMSO-d₆): δ 153.7 (C), 140.4 (q, 2JCF=37.4 Hz, C), 137.7 (C), 129.1 (CH), 127.2 (CH), 122.3 (CH), 121.3 (q, 1JCF=266.9 Hz, CF₃), 85.6 (q, 3JCF=1.6 Hz, CH). ¹⁹F NMR (375 MHz, DMSO-d₆): δ −61.8. IR (ATR-FTIR), cm⁻¹: 3373 (br), 1599 (m), 1505 (m), 1491 (m), 1456 (m), 1407 (m), 1151 (s), 1119 (s), 984 (s), 758 (s), 691 (s). HRMS-ESI (m/z): [M+H]⁺ calculated for C₁₀H₈F₃N₅O, 229.0583; found, 229.0598.

Synthesis of 5-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethoxy)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole (9)

A mixture of compound S9 (38.1 mg, 167 μmol, 1 equiv) and iodo-diazirine S7 (41.4 mg, 178 μmol, 1.00 equiv) was dissolved in DMF (1.7 mL) at 24° C. Potassium carbonate (46.1 mg, 334 μmol, 2.00 equiv) was added to the resulting mixture. The reaction mixture was stirred for 12 hours at 50° C., then cooled for 30 minutes to 24° C. and partitioned between ethyl acetate (5 mL) and DI water (5 mL). The organic portion was separated and washed with brine (3×5 mL). The organic portion was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford an off white powder. The powder was purified by flash column chromatography (20% ethyl acetate-hexanes) to afford the celecoxib analog 9 as a pale yellow oil (39.4 mg, 68%).

R_f=0.58 (20% ethyl acetate-hexanes; UV). ¹H NMR (500 MHz, CDCl₃): δ 7.74 (d, 2H, J=7.6 Hz, H₃), 7.48 (dd, 2H, J=7.6, 7.6 Hz, H₄), 7.36 (t, 1H, J=7.6, 7.6 Hz, H₅), 5.92 (s, 1H, H₁), 3.99 (t, 2H, J=6.0 Hz, H₂), 2.01-1.95 (m, 5H, H₆/H₈/H₉), 1.66 (t, 2H, J=6.8 Hz, H₇). ¹³C NMR (125 MHz, CDCl₃): δ 154.0 (C), 141.8 (q, 2JCF=38.4 Hz, CH), 137.5 (C), 129.0 (CH), 127.7 (CH), 123.0 (CH), 120.9 (q, 1JCF=267.3 Hz CF₃), 84.7 (q, 3JCF=2.1 Hz, CH), 82.4 (C), 69.4 (CH), 67.1 (OCH₂), 32.6 (CH₂), 32.3 (CH₂), 26.1 (CN₂), 13.2 (CH₃). ¹⁹F NMR (375 MHz, CDCl₃): δ −63.3 ppm. IR (ATR-FTIR), cm⁻¹: 3303 (s), 1594 (m), 1562 (m), 1508 (m), 1488 (m), 1457 (m), 1243 (s), 1126 (s), 1099 (s), 968 (s), 759 (s), 639 (s) cm-1. HRMS-ESI (m/z): [M+H]⁺ calculated for C₁₇H₁₆F₃N₄O, 349.1271; found, 349.1276.

Synthesis of 2-aminoethyl 2-azidoacetate (S11)

Step 1. Acid Chloride Formation

Azidoacetic acid (S10, 154 mg, 1.49 mmol, 1 equiv) was dissolved in methanol (10 mL) at 24° C. Acetyl chloride (211 μL, 2.49 mmol, 2.00 equiv) was added dropwise to the stirred solution at 24° C. The resulting mixture was stirred for 6 hours at 24° C. The product mixture was evaporated to dryness and used directly in the following step.

Step 2. Synthesis of 2-aminoethyl 2-azidoacetate (S11)

The azido-acetyl chloride (151 mg, 1.49 mmol, 1 equiv) obtained in the previous step was dissolved in methanol (10 mL) at 24° C. Aminoethanol (450 μL, 7.47 mmol, 5.00 equiv) was added dropwise to the stirred solution at 24° C. The resulting mixture was stirred for 16 hours at 24° C. The product mixture was concentrated to dryness. The residue obtained was purified by flash-column chromatography (eluting with 10% ethyl acetate-hexanes, grading to 20% ethyl acetate-hexanes, one step) to afford 2-aminoethyl 2-azidoacetate (S11) as clear yellow oil (209 mg, 98%).

R_f=0.60 (10% methanol-dichloromethane; ninhydrin). ¹H NMR (500 Hz, CDCl₃): δ 7.01 (br s, 1H, H₁), 3.96 (s, 2H, H₅), 3.68 (t, 2H, J=5.0 Hz, H₃), 3.57 (br s, 1H, H₄), 3.41 (td, 2H, J=5.5, 5.0 Hz, H₂). ¹³C NMR (125 Hz, CDCl₃): δ 167.7 (C), 62.0 (CH₂), 52.8 (CH₂), 42.3 (CH₂). IR (ATR-FTIR), cm⁻¹: 3306 (s), 2107 (s), 1655 (s), 1548 (m). HRMS-ESI (m/z): [M+H]⁺ calculated for C₄H₉N₄O₂, 145.0726; found, 145.0720.

Synthesis of 2-aminoethyl 2-azidoacetate (S13)

Step 1. Acid Chloride Formation

Azidoacetic acid-13C2 (S12, 146 mg, 1.42 mmol, 1 equiv) was dissolved in methanol (10 mL) at 24° C. Acetyl chloride (200 μL, 2.84 mmol, 2.00 equiv) was added dropwise to the stirred solution at 24° C. The resulting mixture was stirred for 6 hours at 24° C. The product mixture was evaporated to dryness and used directly in the following step.

Step 2. Synthesis of 2-aminoethyl 2-azidoacetate (S13)

The azidoacetyl chloride-¹³C₂ (172 mg, 1.42 mmol, 1 equiv) obtained in the previous step was dissolved in methanol (10 mL) at 24° C. Aminoethanol (427 μL, 7.10 mmol, 5.00 equiv) was added dropwise to the stirred solution at 24° C. The resulting mixture was stirred for 16 hours at 24° C. The product mixture was concentrated to dryness. The residue obtained was purified by flash-column chromatography (eluting with 10% ethyl acetate-hexanes, grading to 20% ethyl acetate-hexanes, one step) to afford 2-aminoethyl 2-azidoacetate-¹³C₂ (S13) as clear yellow oil (207 mg, 99%).

R_f=0.60 (10% methanol-dichloromethane; ninhydrin). ¹H NMR (500 MHz, CDCl₃): δ 6.73 (br s, 1H, H₁), 4.17-3.87 (dd, 2H, JCH=144.1, 5.8 Hz, H₅), 3.76 (t, 2H, J=5.5 Hz, H₃), 3.48 (td, 2H, J=5.5, 2.0 Hz, H₂), 2.30 (br s, 1H, H₄). ¹³C NMR (125 MHz, CDCl₃): δ 167.7 (d, JCC=54.9 Hz, 13C), 62.1 (CH₂), 52.9 (d, JCC=54.9 Hz, 13CH₂), 42.3 (CH₂). IR (ATR-FTIR), cm⁻¹: 3272 (s), 2946 (s), 2017 (s), 1654 (s). HRMS-ESI (m/z): [M+H]⁺ calculated for C₂¹³C₂H₉N₄O₂, 147.0793; found, 147.0789.

Synthesis of the Cleavable Biotin Azide Probe 10:

Triethylamine (102 μL, 736 μmol, 20.0 equiv) and dichlorodiphenylsilane (38.8 μL, 184 μmol, 5.00 equiv) were added in sequence to a stirred solution of the biotin-CA(PEG)₄-alcohol S14⁵ (20.7 mg, 36.8 μmol, 1 equiv) in dichloromethane (370 μL). The resulting solution was stirred for 2 hours at 24° C. A 1:3 mixture of the azide S11 and the azide-¹³C₂ S13 (53.0 mg, 368 μmol, 10.0 equiv) was added in sequence to stirred solution. The resulting solution was stirred for an additional 12 hours at 24° C. The product mixture was diluted sequentially with dichloromethane (3 mL) and saturated aqueous sodium bicarbonate solution (3 mL). The resulting biphasic mixture was transferred to a separatory funnel and the layers that formed were separated. The aqueous layer was extracted with dichloromethane (3·3 mL), and the organic layers were combined. The combined organic layers were dried over sodium sulfate. The dried solution was filtered, and the filtrate was concentrated by rotary evaporation. The residue obtained was purified by flash-column chromatography (eluting with 1% methanol-dichloromethane, grading to 10% methanol-dichloromethane, 3 steps) to afford the IsoTaG azido silane probe 10 as a clear oil (16.6 mg, 51%).

R_f=0.43 (5% methanol-dichloromethane; I2). ¹H NMR (600 MHz, CD₃OD): δ 7.66 (d, 4H, J=6.6 Hz, H₁₈), 7.43 (t, 2H, J=7.2 Hz, H₂₀), 7.37 (t, 4H, J=7.8 Hz, H₁₉), 4.47 (dd, 1H, J=7.8, 5.4 Hz, H₂), 4.29 (dd, 1H, J=7.8, 4.2 Hz, H₅), 3.94 (d, 0.5H, J=4.8 Hz, H₂₃), 3.85-3.83 (m, 1.5H, H₂₃), 3.71 (t, 2H, J=6.0 Hz, H₁₃), 3.61-3.55 (m, 18H, H₁₁/H₁₂/H₁₅), 3.52 (t, 2H, J=6.5 Hz, H₂₁), 3.44-3.41 (m, 0.5H, H₂₂), 3.35 (t, 1.5H, J=5.0 Hz, H₂₂), 3.19 (dt, 1H, J=5.0, 4.5 Hz, H₆), 2.92 (dd, 1H, J=12.6, 5.4 Hz, H₁), 2.69 (d, 1H, J=12.6 Hz, H₁), 2.45 (t, 2H, J=6.0 Hz, H₁₄), 2.21 (t, 2H, J=7.5 Hz, H₁₀), 1.75-1.55 (m, 4H, H₇/H₉), 1.44 (quint, 2H, J=7.5 Hz, H₈), 1.25 (s, 6H, H₁₆/H₁₇). ¹³C NMR (151 MHz, CD₃OD): δ 176.1 (C), 174.1 (C), 170.2 (d, JCC=52.8 Hz, 13C), 166.1 (C), 136.1 (4×CH), 135.6 (2×C), 131.4 (2×CH), 128.9 (4×CH), 77.0 (CH₂), 71.6 (CH₂), 71.5 (3×CH₂) 71.3 (2×CH₂), 70.6 (CH₂), 68.4 (CH₂), 63.4 (CH), 62.7 (CH₂) 61.6 (CH), 57.0 (CH₂), 52.97 (d, Jcc=52.8 Hz, ¹³CH₂), 51.6 (CH₂), 42.6 (CH₂), 41.1 (CH₂), 40.4 (CH₂), 37.8 (CH₂), 36.7 (CH₂), 29.8 (CH₂), 29.5 (CH₂), 28.2 (2×CH₃), 26.8 (CH₂). IR (ATR-FTIR), cm⁻¹: 3296 (br), 2927 (m), 2103 (s), 1683 (s), 1644 (s), 1116 (s). HRMS-ESI (m/z): [M+Na]⁺ calculated for C₄₁H₆₂N₈O₁₀SSiNa/C₃₉¹³C₂H₆₂N₈O₁₀SSiNa, 909.3977/911.4044, found, 909.3932/911.3986.

Additional References for Methods

• (1) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.
• (2) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996, 15, 1518.
• (3) Lee, P. J. J.; Compton, B. J.; Patent, U.S., Ed.; Waters Investments Limited: USA, 2007; Vol. 7229539.
• (4) Wang, W.; Hong, S.; Tran, A.; Jiang, H.; Triano, R., Liu, Y.; Chen, X.; Wu, P. Chem Asian J 2011, 6, 2796.
• (5) Szychowski, J.; Mahdavi, A.; Hodas, J. J. L.; Bagert, J. D.; Ngo, J. T.; Landgraf, P.; Dieterich, D. C.; Schuman, E. M.; Tirrell, D. A. J. Am. Chem. Soc. 2010, 132, 18351.
• (6) Zybailov, B.; Mosley, A. L.; Sardiu, M. E.; Coleman, M. K.; Florens, L.; Washburn, M. P. J Proteome Res 2006, 5, 2339.
• (7) Mésangeau, C.; Pérès, B.; Descamps-François, C.; Chavatte, P.; Audinot, V.; Coumailleau, S.; Boutin, J. A.; Delagrange, P.; Bennejean, C.; Renard, P.; Caignard, D. H.; Berthelot, P.; Yous, S. Bioorg Med Chem 2010, 18, 3426.

Example 2: Synthesis of an Electronically-Tuned Minimally Interfering Alkynyl Photo-Affinity Label to Measure Small Molecule-Protein Interactions

Reported herein is the synthesis of an electronically-tuned minimally interfering photoaffinity label (MI-PAL), a compact five-carbon tag functionalized with an alkyl diazirine and alkyne handle. MI-PAL is compatible with protein photo-conjugation, click chemistry and mass spectrometry and readily installed to complex molecules for biological target identification.

The development of small multifunctional chemical tags that are readily embedded to a small molecule has accelerated target identification for non-covalent ligands. These chemical tags typically possess two primary functions: first, photo-conjugation chemistry to covalently capture the protein target and second, a reporter group for characterization of the liganded proteome.^1,2 Photo-affinity labels (PALs) like the aryl azide, benzophenone, and diazirine generate short-lived highly reactive nitrene, carbene or diradical intermediates, respectively, that insert to nearby biomolecules.^3-5 The covalent conjugation event facilitates subsequent ligand-dependent detection of the biomolecule. To allow for the greatest flexibility in detection, a biocompatible handle (e.g., alkyne) is commonly embedded to the chemical tag for versatile functionalization with a reporter molecule via copper-catalyzed azide-alkyne cycloaddition (CuAAC).⁴

Equally important is the facile integration of the chemical tag to the ligand in a minimally perturbative manner in order to preserve the native interactions of the ligand with the proteome. Thus, of the available PALs, the diazirine has seen recent application in numerous target identification studies due to its small size.^6-8 Integration of the diazirine with an alkyne reporter handle have yielded chemical tags like the aryl diazirine 1,⁹ 10.1 Å in length, or the “minimalist” tag (2),¹⁰ 9.4 Å in length (FIG. 28). The minimalist tag (2) has been applied to drug on-target¹¹ and off-target identification,^12,11 fragment based screening,¹³ and binding site mapping.^14,13

Synthesis of an electronically-tuned five carbon tag, such as the diazirine 3, would afford a smaller alkyl tag scaffold (6.9 Å)¹⁵ with universally improved photoconjugation and CuAAC properties (FIG. 28). Photochemical carbene intermediates and CuAAC reaction kinetics are strongly dependent on electronic substituent effects. Electronic stabilization of the reactive carbene by fluorine was first demonstrated with 3-trifluoromethyl-3-phenyldiazirine, which possessed superior stability and selectivity as compared to alkyl diazirines.¹⁶ Reactive carbenes formed from diazirines can produce intramolecular and intermolecular products that may differ based on the substituents adjacent to the carbene.⁵ Electronic tuning of the alkyne with fluorine likewise accelerates strain-promoted azide-alkyne cycloaddition¹⁷ and CuAAC, with measured improvements in reaction rates of 18-fold greater for the difluoropropyne relative to the dihydropropyne.¹⁸ Based on these data, we hypothesized that strategic placement of fluorine adjacent to the alkyne and diazirine functional groups would improve the essential properties required for the chemical tag and provide synthetic access to a minimally-interfering photo-affinity label (MI-PAL, 3). The tag was retrosynthetically accessed by coupling the ester 4 to a monobromodifluoroalkyne 5, followed by installation of the diazirine.

Synthesis of MI-PAL (3) commenced from the ester 4 and the monobromodifluoroalkyne 5 (FIG. 29). The nucleophilic addition of the monobromodifluoroalkyne 5 to the alphahydroxyethyl ester 4 afforded the desired difluoroketone 6 and the hydrate 7 in 77% yield as a 1:1 mixture. The hydrate 7 was formed in situ due to the electrophilicity of the difluoropropyne. A brief investigation of the scope of the initial coupling step revealed that the desired reaction was promoted by the methoxymethyl ester forming a lithium chelate complex preventing collapse to the ketone at low temperatures. The equimolar mixture of ketone 6 and hydrate 7 was then treated with hydroxylamine, followed by a sequence of tosylchloride in pyridine and ammonia in ether to install the diaziridine 8. Elaboration of the ketone 6 to the diaziridine 8 was enabled by the difluoropropyne protected with triisopropylsilane (TIPS) to prevent undesired nucleophilic or deprotonation pathways promoted by basic ammonia. Oxidation (iodine, trimethylamine) of the diaziridine 8 afforded the diazirine 9 in 65% overall yield from the mixture of 6 and 7. Acid deprotection (TMSCl, MeOH) of the diazirine 9 revealed the alcohol 10 (99%). The alcohol 10 could be further desilylated to yield MI-PAL (3) itself (66%). However, we found that MI-PAL (3) was relatively volatile (boiling point=86° C.) and thus in practice rarely removed the TIPS protecting group until after incorporation of MI-PAL (3) to a small molecule of interest. The advanced alcohol intermediate 10 was thus prepared by a four-step sequence in high overall yields (49% overall).

With MI-PAL (3) in hand, we next evaluated the photochemical and CuAAC properties bybmass spectrometry and Western blot. MI-PAL (3, 10 μM) was incubated with an isolated protein, alpha-crystallin, and photo-irradiated (365 nm) for 15 minutes (FIG. 30A). The conjugated protein was trypsin digested and analyzed by liquid chromatography-tandem mass spectrometry on an Orbitrap Elite by collision induced dissociation (CID). Tandem mass spectra of MI-PAL (3) conjugated-peptides obtained by CID displayed characteristic alkyl fluorine ion losses as diagnostic markers.¹⁹ An example peptide conjugated to MI-PAL (3) is shown in FIG. 30B. To test both photo-conjugation and CuAAC properties, 100 μM of MI-PAL (3) was incubated with MM. 1S or K562 whole cell lysates and photo-irradiated (30 minutes). The MI-PAL photo-conjugated lysates were treated by CuAAC with biotin-azide as a reporter and visualized by Western blot. A UV-specific signal due to biotinylation from samples photo-conjugated to MIPAL (3) was observed (FIG. 30C).

MI-PAL (3) was readily incorporated to a range of small molecules (FIGS. 31 and 32). The alcohol 10 was activated with carbonyldiimidazole (CDI) in quantitative yield to afford the carbamate 11 (FIG. 31). The carbamate 11 was treated with several coupling partners of increasing complexity followed by removal of the TIPS protecting group to reveal the terminal alkyne. The MI-PAL-tagged glycine methylester 12 was formed in 69% yield over two steps. Daunorubicin was readily modified by the carbamate 11 to afford the MI-PAL-tagged daunorubicin 13 in 53% yield over two steps. The alcohol 10 was additionally activated with 4-nitrophenyl chloroformate and installed to mitomycin to prepare the tagged mitomycin analog 14. In all cases, desilylation with TBAF proceeded smoothly following conjugation of MI-PAL to a small molecule. Mitomycin and the MI-PAL-tagged mitomycin 14 displayed similar anti-proliferative activity against K562 cells (EC₅₀=34.9 μM and 40.6 μM, respectively).

We additionally tested the participation of MI-PAL in direct S_N2 displacement to modify small molecule ligands (FIG. 32). The alcohol 10 was transformed to the iodide 15 in the presence of iodine and triphenylphosphine in excellent yield (99%). The iodide 15 was then elaborated to the naproxen analog 16 in the presence of cesium carbonate in N,N-dimethylformamide. In situ deprotection of the alkyne by cesium carbonate afforded the MI-PAL-tagged naproxen analog 17 in 81% yield. Mixtures of the iodide 15 and cesium carbonate with β-estradiol (18) additionally provided the MI-PAL-tagged β-estradiol 19 in 74% yield. Thus, MI-PAL is readily functionalized to a range of complex ligands to accelerate target identification.

Characterization of the small molecule interactome is dramatically accelerated by chemical tags that enable measurement of the non-covalent interaction between the ligand and its biomolecular targets. Integration of photo-activatable functional groups with a handle for CuAAC in a short chemical tag is arguably the most likely to preserve the native interactions of the small molecule with the protein targets, although the design of a reporter for a small molecule will be structure-dependent. Functional assays to compare the modified small molecule to the parent compound are necessary to validate the preservation of biological activity, including phenotype assays (e.g., cell viability) or in vitro binding assays if the target is known (e.g., fluorescence polarization, SPR, or ITC). Reported herein is the development of an electronically-tuned five carbon tag 3 as a novel minimally-interfering photo-affinity label. MIPAL (3) possesses a diazirine appended directly to a difluoropropyne that enables facile synthetic access to the tag and its essential functions in photo-conjugation and CuAAC. We demonstrated the photo-conjugation with a single protein and whole proteome, UV-dependent CuAAC with a biotin-azide reporter, and measurement by mass spectrometry, as well as incorporation of MI-PAL (3) into several complex small molecules. MI-PAL (3) thus constitutes a small electronically-tuned alkyl diazirine alkyne tag for application in non-covalent ligand target identification studies.

References

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EQUIVALENTS AND SCOPE

All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

Section headings used herein are not to be construed as limiting in any way. It is expressly contemplated that subject matter presented under any section heading may be applicable to any aspect or embodiment described herein.

LENGTHY TABLES
The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Claims

1. A photo-click tag comprising (a) a diazirine moiety, and (b) a click chemistry handle.

2. The photo-click tag of claim 1, wherein the diazirine moiety comprises the structure:

3. The photo-click tag of claim 1 or 2, wherein the click chemistry handle is an alkyne.

4. The compound of any one of claims 1-3, wherein the photo-click tag is of the formula:

5. The photo-click tag of any one of claims 1-4, wherein the photo-click tag optionally comprises one or more electronegative atoms.

6. The photo-click tag of claim 5, wherein one or more of the electronegative atoms is fluorine.

7. The photo-click tag of any one of claims 1-6, wherein the diazirine moiety forms a reactive carbene species upon irradiation with a specific wavelength of light.

8. The photo-click tag of claim 7, wherein the specific wavelength of light is between about 10 nm and about 400 nm.

9. The photo-click tag of claim 8, wherein the specific wavelength of light is between about 355 nm to about 365 nm.

10. The photo-click tag of any one of claims 7-9, wherein the reactive carbene species reacts with a C—C, C—H, N—H, or O—H bond of a protein.

11. The photo-click tag of claim 10, wherein a new covalent bond is formed between the photo-click tag and the protein.

12. The photo-click tag of any one of claims 1-11, wherein the photo-click tag is between 6 Å and 9.5 Å in length.

13. A compound comprising the structure of Formula (I):

14. The compound of claim 13, wherein L comprises an ester, an ether, an amine, or an amide.

15. The compound of claim 13 or 14, wherein L is.

16. The compound of any one of claims 13-15, wherein P is:

17. The compound of any one of claims 13-16, wherein S is an antibiotic, an anti-proliferative agent, an anti-cancer agent, a chemotherapeutic agent, an anti-angiogenesis agent, an anti-inflammatory agent, an immunosuppressant, an immunomodulatory agent, an anti-bacterial agent, an anti-viral agent, a cardiovascular agent, a cholesterol-lowering agent, an anti-diabetic agent, an anti-allergic agent, a contraceptive agent, or a pain-relieving agent.

18. The compound of claim 17, wherein S is an anti-inflammatory agent, or derivative thereof.

19. The compound of claim 18, wherein the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug (NSAID), or derivative thereof.

20. The compound of claim 19, wherein the NSAID is naproxen, celecoxib, indomethacin, or derivative thereof.

21. The compound of claim 19 or 20, wherein the compound is of the formula:

22. The compound of claim 17, wherein S is an immunomodulatory drug, or derivative thereof.

23. The compound of claim 22, wherein the immunomodulatory drug is thalidomide, lenalidomide, pomalidomide, or derivative thereof.

24. The compound of claim 23, wherein the lenalidomide is R-lenalidomide.

25. The compound of claim 23, wherein the lenalidomide is S-lenalidomide.

26. The compound of any one of claims 22-25, wherein the compound is of the formula:

27. The compound of claim 26, wherein the compound is of the formula:

28. The compound of claim 17, wherein S is a chemotherapeutic agent, or derivative thereof.

29. The compound of claim 28, wherein the chemotherapeutic agent is mitomyosin, daunorubicin, or derivative thereof.

30. The compound of claim 28 or 29, wherein the compound is of the formula:

31. The compound of any one of claims 13-16, wherein S is a steroid, or a derivative thereof.

32. The compound of claim 31, wherein the steroid is an estrogen, or a derivative thereof.

33. The compound of claim 32, wherein the estrogen is beta-estradiol, or a derivative thereof.

34. The compound of any one of claims 31-33, wherein the compound is of the formula:

35. The compound of any one of claims 13-34, wherein the compound comprises one or more isotopically labeled atoms.

36. The compound of claim 35, wherein the isotopically labeled atom is 15N, 13C, 19F, or 2H.

37. A method for identifying a target protein of a small molecule, the method comprising: (i) providing a compound comprising the small molecule connected to a photo-click tag via a linker, wherein the photo-click tag comprises (a) a diazirine moiety, and (b) a click chemistry handle;(ii) activating the diazirine moiety by irradiating the compound of (i) with a specific wavelength of light;(iii) contacting the target protein with the activated compound of (ii);(iv) forming a complex through a photo-induced covalent bond between the activated compound of (ii) and the protein, and(v) identifying the complex produced in (iv) that is bound to the compound, thereby identifying the target protein of the small molecule.

38. A method for identifying the binding site of a small molecule on a protein, the method comprising: (i) providing a compound comprising the small molecule connected to a photo-click tag via a linker, wherein the photo-click tag comprises (a) a diazirine moiety, and (b) a click chemistry handle;(ii) activating the diazirine moiety by irradiating the compound of (i) with a specific wavelength of light;(iii) contacting the protein with the activated compound of (ii);(iv) forming a complex through a photo-induced covalent bond between the activated compound of (ii) and the protein;(v) digesting the protein of the complex into constitutive peptides in the presence of a protease; and(vi) identifying the one or more peptides produced in (iv) that is bound to the compound, thereby identifying the protein binding site of the small molecule.

39. The method of claim 37 or 38, wherein the diazirine moiety forms a reactive carbene species upon irradiation with the specific wavelength of light

40. The method of any one of claims 37-39, wherein the reactive carbene species reacts with a C—C, C—H, N—H, or O—H bond of the protein.

41. The method of claim 40, wherein a new covalent bond is formed between the photo-click tag and the protein.

42. The method of any one of claims 37-41, wherein the specific wavelength of light is between about 10 nm and about 400 nm.

43. The method of claim 42, wherein the specific wavelength of light is between about 355 nm and about 365 nm.

44. The method of claim 37 or 38, wherein the click chemistry handle is an alkyne.

45. The method of claim 37 or 38, wherein the photo-click tag of (i) is of the formula:

46. The method of any one of claims 37-45, wherein the diazirine moiety

47. The method of claim 38, wherein the protease is a serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, metalloprotease, or an asparagine peptide lyase.

48. The method of claim 47, wherein the serine protease is trypsin.

49. The method of claim 37, wherein the identifying of step (v) comprises performing mass spectrometry to identify the proteins produced in (v) that are bound to the compound.

50. The method of claim 49, wherein the mass spectrometry is intact mass spectrometry.

51. The method of claim 38, wherein the digesting of step (v) is performed under conditions suitable for the protease to catalyze the cleavage of peptide bonds in the protein.

52. The method of claim 38, wherein the identifying of step (vi) comprises separating the peptides produced in (v) that are bound to the compound from peptides that are not bound to the compound.

53. The method of claim 38 or 52, wherein the identifying of step (vi) comprises performing mass spectrometry to identify the peptides produced in (v) that are bound to the compound.

54. The method of claim 53, wherein the mass spectrometry is mass-independent mass spectrometry.

59. The method of claim 37 further comprising a step of enriching the complex formed in step (v).

60. The method of claim 38 further comprising a step of enriching the complex formed in step (iv).

61. The method of claim 60, wherein the enriching is performed before the digesting of step (v).

62. The method of claim 60, wherein the enriching is performed after the digesting of step (v).

63. The method of any one of claims 59-62, wherein the enriching comprises covalently attaching a label to the click chemistry handle of the photo-click tag.

64. The method of claim 63, wherein the label comprises an affinity tag.

65. The method of claim 64, wherein the affinity tag is biotin.

66. The method of any one of claims 63-65, wherein the label comprises an azide moiety.

67. The method of any one of claims 63-66, wherein the label is of the formula:

68. The method of any one of claims 63-67, wherein the label is attached to the click chemistry handle of the photo-click tag using copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction conditions.

69. The method of claim 63-68 further comprising separating the complex using affinity chromatography.

70. The method of claim 69, wherein the affinity chromatography uses a column comprising streptavidin-coupled beads.

71. The method of claim 37 or 38, wherein the protein is present in a whole proteome.

72. The method of claim 37 or 38, wherein the protein is present in a cell.

73. The method of claim 37 or 38, wherein the protein is present in a cell lysate.

74. The method of claim 72 or 73, wherein the cell is a mammalian cell.

75. The method of claim 74, wherein the mammalian cell is a human cell.

76. The method of claim 37 or 38, wherein the protein is in vitro.

77. The method of claim 37 or 38, wherein the protein is in vivo.

78. A method for identifying an interaction between a first protein and a second protein in a cell, the method comprising: (i) providing the cell with an amino acid analog, wherein the amino acid analog comprises a photo-click tag comprising (a) a diazirine moiety, and (b) a click chemistry handle, and wherein the amino acid analog is incorporated into the first protein and/or the second protein during protein synthesis,(ii) activating the diazirine moiety of the amino acid analog by irradiating the cell with a specific wavelength of light;(iii) contacting the first protein or the second protein with the activated compound of (ii);(iv) forming a protein-protein complex through a photo-induced covalent bond between the activated amino acid analog of the first protein and an amino acid in the second protein; and(v) identifying the complex produced in (ii) that comprises the amino acid analog covalently linking the first protein and the second protein, thereby identifying the first protein and the second protein involved in the interaction.

79. The method of claim 78 further comprising digesting the protein-protein complex of (iii) into constitutive peptides in the presence of a protease; and (iv) identifying a conjugated peptide that comprises a peptide comprising the amino acid analog covalently linking a peptide of the first protein to a peptide of the second protein.

80. The method of claim 78, wherein the diazirine moiety forms a reactive carbene species upon irradiation with the specific wavelength of light

81. The method of claim 80, wherein the reactive carbene species reacts with a C—C, C—H, N—H, or O—H bond of the first protein or the second protein.

82. The method of claim 81, wherein the amino acid analog is incorporated into the first protein during protein synthesis, and wherein a new covalent bond is formed between the photo-click tag of the amino acid analog and the second protein.

83. The method of claim 81, wherein the amino acid analog is incorporated into the second protein during protein synthesis, and wherein a new covalent bond is formed between the photo-click tag of the amino acid analog and the first protein.

84. The method of any one of claims 78-83, wherein the specific wavelength of light is between about 10 nm and about 400 nm.

85. The method of claim 84, wherein the specific wavelength of light is between about 355 nm and about 365 nm.

86. The method of claim 78, wherein the click chemistry handle is an alkyne.

87. The method of claim 78, wherein the amino acid analog of (i) comprises the structure of Formula (II-a):

88. The method of claim 87, wherein P is of the formula

89. The method of any one of claims 78-88, wherein the amino acid analog of (i) is of the formula:

90. The method of any one of claims 78-89, wherein the diazirine moiety

91. The method of claim 78, wherein the protease is a serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, metalloprotease, or an asparagine peptide lyase.

92. The method of claim 91, wherein the serine protease is trypsin.

93. The method of claim 78, wherein the digesting step is performed under conditions suitable for the protease to catalyze the cleavage of peptide bonds in the protein-protein complex.

94. The method of claim 78, wherein the identifying step comprises performing mass spectrometry to identify the conjugated peptide that comprises a peptide comprising the amino acid analog covalently linked to a peptide of the second protein.

95. The method of claim 94, wherein the mass spectrometry is mass-independent mass spectrometry.

96. The method of claim 78 further comprising a step of enriching the complex formed in step (iii).

97. The method of claim 96, wherein the enriching comprises covalently attaching a label to the click chemistry handle of the photo-click tag.

98. The method of claim 97, wherein the label comprises an affinity tag.

99. The method of claim 98, wherein the affinity tag is biotin.

100. The method of any one of claims 97-99, wherein the label comprises an azide moiety.

101. The method of any one of claims 97-100, wherein the label is of the formula:

102. The method of any one of claims 97-101, wherein the label is attached to the click chemistry handle of the photo-click tag using copper (i)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction conditions.

103. The method of claim 97-102 further comprising separating the complex using affinity chromatography.

104. The method of claim 103, wherein the affinity chromatography uses a column comprising streptavidin-coupled beads.

105. The method of any one of claims 78-104 further comprising contacting the cell with a small molecule, wherein the cell is contacted with the small molecule before the activating of step (ii).

106. The method of claim 105, wherein the first protein and the second protein identified in the presence of the small molecule are compared to the first protein and the second protein identified in the absence of the small molecule.

107. The method of claim 106, wherein a difference in the first protein and the second protein in the presence of the small molecule compared to the first protein and the second protein in the absence of the small molecule indicates modulation of a protein-protein interaction in the cell in the presence of a small molecule.

108. The method of any one of claims 105-107, wherein the small molecule is an anti-inflammatory agent, an immunomodulatory drug, a chemotherapeutic agent, or a derivative thereof.

109. The method of claim 78, wherein the first protein and the second protein are present in a whole proteome.

110. The method of claim 78, wherein the first protein and the second protein are present in a cell.

111. The method of claim 78, wherein the first protein and the second protein are present in a cell lysate.

112. The method of claim 110 or 111, wherein the cell is a mammalian cell.

113. The method of claim 112, wherein the mammalian cell is a human cell.

114. The method of claim 78, wherein the first protein and the second protein are present in vivo.

115. A kit comprising the photo-click tag of any one of claims 1-12.

116. A kit comprising the compound of any one of claims 13-36.

117. A kit for use in performing the method of any one of claims 37-77.

118. A kit for use in performing the method of any one of claims 78-114.