Photoaffinity Probe Research Articles

An NAD+ with Dually Modified Adenine for Labeling ADP-Ribosylation-Specific Proteins

Protein adenosine diphosphate (ADP)-ribosylation participates in various pivotal cellular events. Its readers and erasers play key roles in modulating ADP-ribosylation-based signaling pathways. Unambiguous assignments of readers and erasers to individual ADP-ribosylated proteins provide insightful knowledge on ADP-ribosylation biology and require the development of tools and technologies for this goal. Herein, we report the design and the synthesis of a nicotinamide adenine dinucleotide (NAD+) carrying a photoactive and a clickable group. Functioning as a substrate for poly-ADP-ribosylation (PARylation), this NAD+ mimic with dually modified adenine enables covalent crosslinking and labeling of proteins bound to PARylation, representing a new photoaffinity probe for studying this critical post-translational modification.

Crafting Molecular Tools for 15‐Lipoxygenase‐1 in a Single Step

Small molecule modulators are powerful tools for selectively probing and manipulating proteins in native biological systems. However, the development of versatile modulators that exhibit desired properties is hindered by the lack of a rapid and robust synthetic strategy. Here, we develop a facile and reliable one‐step methodology for the generation of multifunctional toolboxes encompassing a wide variety of chemical modulators with different desired features. These modulators bind irreversibly to the protein target via a selective warhead. Key elements are introduced onto the warhead in a single step using multi‐component reactions. To illustrate the power of this new technology, we synthesized a library of diverse modulators designed to explore a highly challenging and poorly understood protein, human 15‐lipoxygenase‐1. Modulators made include; activity‐based/photoaffinity probes, chemosensors, photocrosslinkers, as well as light‐controlled and high‐affinity inhibitors. The efficacy of our compounds was successfully established through the provision of on demand inhibition and labeling of our target protein in vitro, in cellulo and in vivo; thus, proving that this technology has promising potential for applications in many complex biological systems.

Profiling sorghum-microbe interactions with a specialized photoaffinity probe identifies key sorgoleone binders in Acinetobacter pittii.

Interactions between plants and soil microbial communities that benefit plant growth and enhance nutrient acquisition are driven by the selective release of metabolites from plant roots, or root exudation. To investigate these plant-microbe interactions, we developed a photoaffinity probe based on sorgoleone (sorgoleone diazirine alkyne for photoaffinity labeling, SoDA-PAL), a hydrophobic secondary metabolite and allelochemical produced in Sorghum bicolor root exudates. We applied SoDA-PAL to the identification of sorgoleone-binding proteins in Acinetobacter pittii SO1, a potential plant growth-promoting microbe isolated from sorghum rhizosphere soil. Competitive photoaffinity labeling of A. pittii whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins, including putative transporters, transcriptional regulators, and a subset of proteins with predicted enzymatic functions. We performed computational protein modeling and docking with sorgoleone to prioritize candidates for experimental validation and then confirmed binding of sorgoleone to four of these proteins in vitro: the α/β fold hydrolase SrgB (OH685_09420), a fumarylacetoacetase (OH685_02300), a lysophospholipase (OH685_14215), and an unannotated hypothetical protein (OH685_18625). Our application of this specialized sorgoleone-based probe coupled with structural bioinformatics streamlines the identification of microbial proteins involved in metabolite recognition, metabolism, and toxicity, widening our understanding of the range of cellular pathways that can be affected by a plant secondary metabolite.IMPORTANCEHere, we demonstrate that a photoaffinity-based chemical probe modeled after sorgoleone, an important secondary metabolite released by sorghum roots, can be used to identify microbial proteins that directly interact with sorgoleone. We applied this probe to the sorghum-associated bacterium Acinetobacter pittii and showed that probe labeling is dose-dependent and sensitive to competition with purified sorgoleone. Coupling the probe with proteomics and computational analysis facilitated the identification of putative sorgoleone binders, including a protein implicated in a conserved pathway essential for sorgoleone catabolism. We anticipate that discoveries seeded by this workflow will expand our understanding of the molecular mechanisms by which specific metabolites in root exudates shape the sorghum rhizosphere microbiome.

Photoaffinity probe-enabled discovery of sennoside A reductase in Bifidobacterium pseudocatenulatum

Sennoside A (SA), a typical prodrug, exerts its laxative effect only after its transformation into rheinanthrone catalyzed by gut microbial hydrolases and reductases. Hydrolases have been identified, but reductases remain unknown. By linking a photoreactive group to the SA scaffold, we synthesized a photoaffinity probe to covalently label SA reductases and identified SA reductases using activity-based protein profiling. From lysates of an active strain, Bifidobacterium pseudocatenulatum (B. pseudocatenulatum), 397 proteins were enriched and subsequently identified using mass spectrometry. Among these proteins, chromate reductase/nicotinamide adenine dinucleotide phosphate (NADPH)-dependent flavin mononucleotide (FMN) reductase/oxygen-insensitive NADPH nitroreductase (nfrA) was identified as a potent SA reductase through further bioinformatic analysis and Uniprot database screening. We also determined that recombinant nfrA could reduce SA. Our study contributes to further illuminating mechanisms of SA transformation to rheinanthrone and simultaneously offers an effective method to identify gut bacterial reductases.

Covalent Interactions of Anthocyanins with Proteins: Activity-Based Protein Profiling of Cyanidin-3-O-glucoside.

Anthocyanins are common natural pigments with a variety of physiological activities. Traditional perspectives attribute their molecular mechanism to noncovalent interactions influencing signaling pathways. However, this ignores the nature of its benzopyrylium skeleton, which readily reacts with the electron-rich groups of proteins. Here, we modified cyanidin-3-O-glucoside (C3G) via activity-based protein profiling technology by our previous synthesis route and prepared the covalent binding probe (C3G-Probe) and the noncovalent photoaffinity probe (C3G-Diazirine). The properties of C3G's covalent binding to proteins were also discovered by comparing the labeling of the two probes to the whole HepG2 cell proteome. We further explored its target proteins and enriched pathways in HepG2 and HeLa cells. Western blot analysis further confirmed the covalent binding of C3G to four target proteins: insulin-degrading enzyme, metal cation symporter ZIP14, spermatid perinuclear RNA-binding protein, and Cystatin-B. Pathway analysis showed that covalent targets of C3G were concentrated in metabolic pathways and several ribonucleoprotein complexes that were also coenriched. The results of this study provide new insights into the interaction of the naturally active molecule C3G with proteins.

Glutathione-Based Photoaffinity Probe Identifies Caffeine as a Positive Allosteric Modulator of the Calcium-Sensing Receptor.

The calcium-sensing receptor (CaSR), abundantly expressed in the parathyroid gland and kidney, plays a central role in calcium homeostasis. In addition, CaSR exerts multimodal roles, including inflammation, muscle contraction, and bone remodeling, in other organs and tissues. The diverse functions of CaSR are mediated by many endogenous and exogenous ligands, including calcium, amino acids, glutathione, cinacalcet, and etelcalcetide, that have distinct binding sites in CaSR. However, strategies to evaluate ligand interactions with CaSR remain limited. Here, we developed a glutathione-based photoaffinity probe, DAZ-G, that analyzes ligand binding to CaSR. We showed that DAZ-G binds to the amino acid binding site in CaSR and acts as a positive allosteric modulator of CaSR. Oxidized and reduced glutathione and phenylalanine effectively compete with DAZ-G conjugation to CaSR, while calcium, cinacalcet, and etelcalcetide have cooperative effects. An unexpected finding was that caffeine effectively competes with DAZ-G's conjugation to CaSR and acts as a positive allosteric modulator of CaSR. The effective concentration of caffeine for CaSR activation (<10 μM) is easily attainable in plasma by ordinary caffeine consumption. Our report demonstrates the utility of a new chemical probe for CaSR and discovers a new protein target of caffeine, suggesting that caffeine consumption can modulate the diverse functions of CaSR.

Oligonucleotide-Based Photoaffinity Probes: Chemical Tools and Applications for Protein Labeling.

A variety of proteins interact with DNA and RNA, including polymerases, histones, ribosomes, transcription factors, and repair enzymes. However, the transient non-covalent nature of these interactions poses challenges for analysis. Introducing a covalent bond between proteins and DNA via photochemical activation of a photosensitive functional group introduced onto nucleic acids offers a means to stabilize these often weak interactions without significantly altering the binding interface. Consequently, photoactivatable oligonucleotides are powerful tools for investigating nucleic acid-protein interactions involved in numerous biological and pathological processes. In this review, we provide a comprehensive overview of the chemical tools developed so far and the different strategies used for incorporating the most commonly used photoreactive reagents into oligonucleotide probes or nucleic acids. Furthermore, we illustrate their application with several examples including protein binding site mapping, identification of protein binding partners, and in cell studies.

Target fishing and mechanistic insights of the natural anticancer drug candidate chlorogenic acid

Chlorogenic acid (CGA) is a natural product that effectively inhibits tumor growth, demonstrated in many preclinical models, and phase II clinical trials for patients with glioma. However, its direct proteomic targets and anticancer molecular mechanisms remain unknown. Herein, we developed a novel bi-functional photo-affinity probe PAL/CGA and discovered mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) was one of the main target proteins of CGA by using affinity-based protein profiling (AfBPP) chemical proteomic approach. We performed in-depth studies on ACAT1/CGA interactions via multiple assays including SPR, ITC, and cryo-EM. Importantly, we demonstrated that CGA impaired cancer cell proliferation by inhibiting the phosphorylation of tetrameric ACAT1 on Y407 residue through a novel mode of action in vitro and in vivo. Our study highlights the use of AfBPP platforms in uncovering unique druggable modalities accessed by natural products. And identifying the molecular target of CGA sheds light on the future clinical application of CGA for cancer therapy.

Probing for optimal photoaffinity linkers of benzophenone-based photoaffinity probes for adenylating enzymes

The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5′-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2–8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2–8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4′-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.

Evaluation of Antibacterial Functionalized Dihydropyrimidine Photoaffinity Probes Toward Mechanism of Action Studies.

Antibiotic-resistant bacteria are a global health concern, necessitating the development of antibiotics working through new or underutilized mechanisms. Functionalized amino dihydropyrimidines have previously demonstrated potential as antibacterial agents, but they had limited potency, and their biological mechanism was not understood. To further evaluate their potential, focused libraries were prepared and screened for bacterial growth inhibition, and these compounds provided additional insights into the structure-activity relationships, allowing for the preparation of compounds that inhibited all strains of Staphylococcus aureus with an MIC of 2 μg/mL. After eliminating the proposed mechanism of dihydrofolate reductase inhibition, trifluoromethyl diazirine photoaffinity probes were synthesized to investigate their mechanism, and these were tested to ensure the photolabile group did not impact the antibacterial activity. Finally, the compounds were screened for hemolysis and mammalian cytotoxicity. While they lacked nonspecific membrane rupturing activity, many of the compounds showed significant mammalian cytotoxicity, indicating further development will be required to render them selective for bacteria.

ENL reads histone β-hydroxybutyrylation to modulate gene transcription.

Histone modifications are typically recognized by chromatin-binding protein modules (referred to as 'readers') to mediate fundamental processes such as transcription. Lysine β-hydroxybutyrylation (Kbhb) is a new type of histone mark that couples metabolism to gene expression. However, the readers that prefer histone Kbhb remain elusive. This knowledge gap should be filled in order to reveal the molecular mechanism of this epigenetic regulation. Herein, we developed a chemical proteomic approach, relying upon multivalent photoaffinity probes to capture binders of the mark, and identified ENL as a novel target of H3K9bhb. Biochemical studies and CUT&Tag analysis further suggested that ENL favorably binds to H3K9bhb, and co-localizes with it on promoter regions to modulate gene expression. Notably, disrupting the interaction between H3K9bhb and ENL via structure-based mutation led to the suppressed expression of genes such MYC that drivecell proliferation. Together, our work offered a chemoproteomics approach and identified ENL as a novel histone β-hydroxybutyrylation effector that regulates gene transcription, providing new insight into the regulation mechanism and function of histone Kbhb.

Identification and Validation of PKR as a Direct Target for the Novel Sulfonamide-Substituted Tetrahydroquinoline Nonselective Inhibitor of the NLRP3 Inflammasome.

The NLRP3 inflammasome is a critical component of the innate immune system. The persistent abnormal activation of the NLRP3 inflammasome is implicated in numerous human diseases. Herein, sulfonamide-substituted tetrahydroquinoline derivative S-9 was identified as the most promising NLRP3 inhibitor, without obvious cytotoxicity. In vitro, S-9 inhibited the priming and activation stages of the NLRP3 inflammasome. Incidentally, we also observed that S-9 had inhibitory effects on the NLRC4 and AIM2 inflammasomes. To elucidate the multiple anti-inflammatory activities of S-9, photoaffinity probe P-2, which contained a photoaffinity label and a functional handle, was developed for target identification by chemical proteomics. We identified PKR as a novel target of S-9 in addition to NLRP3 by target fishing. Furthermore, S-9 exhibited a significant anti-neuroinflammatory effect in vivo. In summary, our findings show that S-9 is a promising lead compound targeting both PKR and NLRP3 that could emerge as a molecular tool for treating inflammasome-related diseases.

Discovery of a Photoaffinity Probe that Captures the Active Conformation of the Cannabinoid CB2 Receptor.

The cannabinoid receptor type 2 (CB2R) is a G protein-coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor localization, expression and occupancy. Previously, we have reported a photoaffinity probe LEI-121 that stabilized the inactive conformation of the CB2R. Here, we report the structure-based design of a novel bifunctional probe that captures the active conformation of the CB2R upon irradiation with light. An alkyne handle was incorporated to visualize the receptor using click-chemistry with fluorophore-azides. These probes may hold promise to study different receptor conformations in relation to their cellular localization and function.

Synthesis and Photolysis Properties of a New Chloroquine Photoaffinity Probe.

A new chloroquine-derived photoaffinity probe has been prepared by a convergent synthesis from derivative of 4,7-dichloroquinoline and N1,N1-diethyl-N4-methylpentane. The features of this probe are a unique 3-azido photolabel, the pyridine ring of the quinoline, and the presence of a secondary amine at the 4-position of the quinoline. These features, particularly the 4-amino methylation, prevent triazole formation through combination of the 3-azide and the 4-amine. This undergoes facile cleavage with exposure to a medium-pressure mercury lamp with a 254 nm excitation wavelength. Trapping of the nitrene byproduct is accomplished with its reaction with N-phenylmaleimide as its cycloazidation product. The structure of a ring-opened DBU amine has been structurally characterized.

Photoaffinity probe-based antimalarial target identification of artemisinin in the intraerythrocytic developmental cycle of Plasmodium falciparum.

Malaria continues to pose a serious global health threat, and artemisinin remains the core drug for global malaria control. However, the situation of malaria resistance has become increasingly severe due to the emergence and spread of artemisinin resistance. In recent years, significant progress has been made in understanding the mechanism of action (MoA) of artemisinin. Prior research on the MoA of artemisinin mainly focused on covalently bound targets that are alkylated by artemisinin-free radicals. However, less attention has been given to the reversible noncovalent binding targets, and there is a paucity of information regarding artemisinin targets at different life cycle stages of the parasite. In this study, we identified the protein targets of artemisinin at different stages of the parasite's intraerythrocytic developmental cycle using a photoaffinity probe. Our findings demonstrate that artemisinin interacts with parasite proteins in vivo through both covalent and noncovalent modes. Extensive mechanistic studies were then conducted by integrating target validation, phenotypic studies, and untargeted metabolomics. The results suggest that protein synthesis, glycolysis, and oxidative homeostasis are critically involved in the antimalarial activities of artemisinin. In summary, this study provides fresh insights into the mechanisms underlying artemisinin's antimalarial effects and its protein targets.

Photo-affinity and Metabolic Labeling Probes Based on the Opioid Alkaloids.

The opioids are powerful analgesics yet possess contingencies that can lead to opioid-use disorder. Chemical probes derived from the opioid alkaloids can provide deeper insight into the molecular interactions in a cellular context. Here, we designed and developed photo-click morphine (PCM-2) as a photo-affinity probe based on morphine and dialkynyl-acetyl morphine (DAAM) as a metabolic acetate reporter based on heroin. Application of these probes to SH-SY5Y, HEK293T, and U2OS cells revealed that PCM-2 and DAAM primarily localize to the lysosome amongst other locations inside the cell by confocal microscopy and chemical proteomics. Interaction site identification by mass spectrometry revealed the mitochondrial phosphate carrier protein, solute carrier family 25 member 3, SLC25A3, and histone H2B as acylation targets of DAAM. These data illustrate the utility of chemical probes to measure localization and protein interactions in a cellular context and will inform the design of probes based on the opioids in the future.

Identification of Low-Density Lipoprotein Receptor-Related Protein 1 as a CXCL14 Receptor Using Chemically Synthesized Tetrafunctional Probes.

CXCL14 is a primordial CXC-type chemokine that transports CpG oligodeoxynucleotides (ODN) into endosomes and lysosomes in dendritic cells, thereby leading to the activation of the Toll-like receptor 9 (TLR9)-mediated innate immune system. However, the underlying molecular mechanism by which the CXCL14-CpG ODN complex enters cells remains elusive. Herein, we describe the chemical synthesis of CXCL14-derived photoaffinity probes and their application to the identification of target receptors for CXCL14 using quantitative proteomics. By utilizing native chemical ligation and maleimide-thiol coupling chemistry, we synthesized site-specifically modified CXCL14-based photoaffinity probes that contain photoreactive 2-aryl-5-carboxytetrazole (ACT) and a hydrazine-labile cleavable linker. CXCL14-based probes were found to be capable of binding CpG ODN to immune cells, whose bioactivities were comparable to native CXCL14. Application of CXCL14-derived probes to quantitative proteomic experiments enabled the identification of dozens of target receptor candidates for CXCL14 in mouse macrophage-derived RAW264.7 cells, and we discovered that low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for CXCL14 by competitive proteome profiling. We further showed that disruption of LRP1 affected the incorporation of the CXCL14-CpG ODN complex in the cells. Overall, this report highlights the power of synthetic CXCL14-derived photoaffinity probes combined with chemical proteomics to discover previously unidentified receptors for CXCL14, which could promote an understanding of the molecular functions of CXCL14 and the elaborate machinery of innate immune systems.

Discovery of Artemisinins as Microsomal Prostaglandins Synthase-2 Inhibitors for the Treatment of Colorectal Cancer via Chemoproteomics.

Colorectal cancer remains the second leading cause of cancer-related mortalities worldwide. While artemisinin (ART), a key active compound from the traditional Chinese medicinal herb Artemisia annua, has been recognized for its antiproliferative activity against colon cancer cells, its underlying molecular underpinnings remain elusive. Whereas promiscuity of heme-dependent alkylating of macromolecules, mainly proteins, has been seen pivotal as a universal and primary mode of action of ART in cancer cells, accumulating evidence suggests the existence of unique targets and mechanisms of actions contingent on cell or tissue specificities. Here, we employed photoaffinity probes to identify the specific targets responsible for ART's anti-colon cancer actions. Upon validation, microsomal prostaglandins synthase-2 emerged as a specific and reversible target of ART in HCT116 colorectal cancer cells, whose inhibition resulted in reduced cellular prostaglandin E2 biosynthesis and cell growth. Our discovery opens new opportunities for pharmacological treatment of colon cancer.

Resveratrol, a novel inhibitor of fatty acid binding protein 5, inhibits cervical cancer metastasis by suppressing fatty acid transport into nucleus and downstream pathways.

Because of cervical cancer (CC) metastasis, the prognosis of diagnosed patients is poor. However, the molecular mechanisms and therapeutic approach for metastatic CC remain elusive. In this study, we first evaluated the effect of resveratrol (RSV) on CC cell migration and metastasis. Via an activity-based protein profiling (ABPP) approach, a photoaffinity probe of RSV (RSV-P) was synthesized, and the protein targets of RSV in HeLa cells were identified. Based on target information and subsequent in vivo and in vitro validation experiments, we finally elucidated the mechanism of RSV corresponding to its antimetastatic activity. The results showed that RSV concentration-dependently suppressed CC cell migration and metastasis. A list of proteins was identified as the targets of RSV, through the ABPP approach with RSV-P, among which fatty acid binding protein 5 (FABP5) attracted our attention based on The Cancer Genome Atlas (TCGA) database analysis. Subsequent knockout and overexpression experiments confirmed that RSV directly interacted with FABP5 to inhibit fatty acid transport into the nucleus, thereby suppressing downstream matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) expression, thus inhibiting CC metastasis. Our study confirmed the key role of FABP5 in CC metastasis and provided important target information for the design of therapeutic lead compounds for metastatic CC.

Baicalin reduces chronic stress-induced breast cancer metastasis via directly targeting β2-adrenergic receptor

Recent studies have shown that stress can substantially facilitate breast cancer metastasis, which can be reduced by nonselective β1/β2-adrenergic receptor (β1/β2-AR) blocker. However, several side effects were identified. Thus, it is extremely warranted to explore more effective and better-tolerated β2-AR blocker. Currently, we demonstrated that baicalin (BA), a major bioactive component of Scutellaria baicalensis Georgi, could significantly attenuate stress hormones especially epinephrine (Epi)-induced breast cancer cell migration and invasion in vitro. Mechanistically, we identified that β2-AR was a direct target of BA via the drug affinity responsive target stability (DARTS) combined with mass spectrum assay, and BA photoaffinity probe with pull-down assay, which was further confirmed by a couple of biophysical and biochemical assays. Furthermore, we demonstrated that BA could directly bind to the Phe-193 and Phe-289 of β2-AR, subsequently inhibit cyclic adenosine monophosphate-protein kinase A-focal adhesion kinase (cAMP-PKA-FAK) pathway, and thus impede epithelial-mesenchymal transition (EMT), thereby hindering the metastatic progression of the chronic stress coupled with syngeneic and xenograft in vivo orthotopic and tail vein mouse model. These findings firstly identify BA as a potential β2-AR inhibitor in the treatment of stress-induced breast cancer metastasis.