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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. Highlights: We identified the targets of artemisinin at different stages of the intraerythrocytic cycle of Plasmodium falciparum using a photoaffinity probe of artemisinin.Artemisinin can interact with parasite proteins in vivo through both covalent and noncovalent mechanisms.Artemisinin may exert its antimalarial effects by interfering with the parasite's protein synthesis, glycolysis, and oxidative homeostasis pathways.This study provides fresh insights into the mechanisms underlying artemisinin's antimalarial effects and its protein targets. [ABSTRACT FROM AUTHOR]

Facing the sudden outbreak of coronavirus disease 2019 (COVID‐19), it is extremely urgent to develop effective antiviral drugs against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Drug repurposing is a promising strategy for the treatment of COVID‐19. To identify the precise target protein of marketed medicines, we initiate a chemical biological program to identify precise target of potential antivirus drugs. In this study, two types of recombinant human coronavirus SARS‐CoV‐2 RdRp protein capturing probes with various photoaffinity labeling units were designed and synthesized based on the structure of FDA‐approved drugs stavudine, remdesivir, acyclovir, and aladenosine. Fortunately, it was found that one novel photoaffinity probe, RD‐1, could diaplayed good affinity with SARS‐CoV‐2 RdRp around the residue ARG_553. In addition, RD‐1 probe also exhibited potent inhibitory activity against 3CLpro protease. Taken together, our findings will elucidate the structural basis for the efficacy of marketed drugs, and explore a rapid and efficient strategy of drug repurposing based on the identification of new targets. Moreover, these results could also provide a scientific basis for the clinical application of marketed drugs. [ABSTRACT FROM AUTHOR]

Using chemoproteomic techniques, we first identified EIF2AK2, eEF1A1, PRDX3 and VPS4B as direct targets of berberine (BBR) for its synergistically anti-inflammatory effects. Of them, BBR has the strongest affinity with EIF2AK2 via two ionic bonds, and regulates several key inflammatory pathways through EIF2AK2, indicating the dominant role of EIF2AK2. Also, BBR could subtly inhibit the dimerization of EIF2AK2, rather than its enzyme activity, to selectively modulate its downstream pathways including JNK, NF-κB, AKT and NLRP3, with an advantage of good safety profile. In EIF2AK2 gene knockdown mice, the inhibitory IL-1β, IL-6, IL-18 and TNF-α secretion of BBR was obviously attenuated, confirming an EIF2AK2-dependent anti-inflammatory efficacy. The results highlight the BBR's network mechanism on anti-inflammatory effects in which EIF2AK2 is a key target, and inhibition of EIF2AK2 dimerization has a potential to be a therapeutic strategy against inflammation-related disorders.

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. ToC. [Display omitted] • An NAD + mimic containing a diazirine and an alkyne on adenine. • A dually modified NAD + for PARP-catalyzed ADP-ribosylation. • A photoaffinity probe to crosslink and label ADP-ribosylation-binding proteins. [ABSTRACT FROM AUTHOR]

Target identification of bioactive compounds is important for understanding their mechanisms of action and provides critical insights into their therapeutic utility. While it remains a challenge, unbiased chemoproteomics strategy using clickable photoaffinity probes is a useful and validated approach for target identification. One major limitation of this approach is the efficient synthesis of appropriately substituted clickable photoaffinity probes. Herein, we describe an efficient and consistent method to prepare such probes. We further employed this method to prepare a highly stereo-congested probe based on naturally occurring triterpenoid betulinic acid. With this photoaffinity probe, we identified tropomyosin as a novel target for betulinic acid that can account for the unique biological phenotype on cellular cytoskeleton induced by betulinic acid.

Target identification of bioactive compounds is important for understanding their mechanisms of action and provides critical insights into their therapeutic utility. While it remains a challenge, unbiased chemoproteomics strategy using clickable photoaffinity probes is a useful and validated approach for target identification. One major limitation of this approach is the efficient synthesis of appropriately substituted clickable photoaffinity probes. Herein, we describe an efficient and consistent method to prepare such probes. We further employed this method to prepare a highly stereo-congested probe based on naturally occurring triterpenoid betulinic acid. With this photoaffinity probe, we identified tropomyosin as a novel target for betulinic acid that can account for the unique biological phenotype on cellular cytoskeleton induced by betulinic acid. A new method was developed to reliably prepare stereo-hindered clickable diazirines for target identification. Tropomyosin was identified as a new target for betulinic acid. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] 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. [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor (EGFR) and HER2 are two important tyrosine kinases that play crucial roles in signal transduction pathways that regulate numerous cellular functions including proliferation, differentiation, migration, and angiogenesis. In the past 20 years, many proteomic methods have emerged as powerful methods to evaluate proteins in biological processes and human disease states. Among them, activity-based protein profiling (ABPP) is one useful approach for the functional analysis of proteins. In this study, a novel photoaffinity probe 11 was designed and synthesised to assess the target profiling of the reactive group in the photoaffinity probe 11. Biological evaluation was performed, and the results showed that the novel photoaffinity probe binds to EGFR and HER2 proteins and it hits targets by the reactive group.

To explore the molecular mechanisms of BAY R3401, four types of novel photoaffinity probes bearing different secondary tags were synthesized. Their potency for glycogenolysis was evaluated in primary human liver HL-7702 cells and HepG2 cells. Probe 2d showed the best activity in primary human liver HL-7702 cells and HepG2 cells, with IC50 values of 4.45 μM and 28.49 μM, respectively. Likewise, probe 5d showed IC50 values of 6.46 μM in primary human liver HL-7702 cells and 15.29 μM in HepG2 cells, respectively. Photoaffinity labeling experiments were also performed and protein bands larger than 170 kDa were specifically tagged by probe 2d. The results suggest that the synthesized probe 2d might be a very promising tool for the isolation of the target proteins of BAY R3401.

The antitumor and actin-depolymerizing marine macrolide aplyronine A (ApA) synergistically binds to tubulin in association with actin, and prevents spindle formation and mitosis. While the crystal structure of the actin ApA complex was solved in 2006, its interaction with the tubulin heterodimer has not been clarified. To investigate the binding modes of ApA as a unique protein–protein interaction (PPI)-inducer between these two cytoskeletal proteins, we prepared its photoaffinity acetylene and fluorescent derivatives with the aid of molecular modeling studies for probe design. Among these three derivatives, the ApA–PPA–TAMRA probe specifically photoreacted with both actin and tubulin in vitro . However, the photolabeling yield of tubulin was quite low (up to ∼1%), and one of the major side-reactions was the addition of a water molecule to the carbene species generated from an aryldiazirine moiety on the hydrophilic surface of actin. [ABSTRACT FROM AUTHOR]

A procedure to measure the serum concentration of glycogen phosphorylase during acute myocardial infarction is presented. This method was based on the synthesis of photoaffinity probes, and used the semiquantitative protein electrophoretic mobility shift technique. Three novel photoaffinity probes bearing different secondary tags were synthesized. Their potency was evaluated in an enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa). The inhibitory activity of probe 1 was only 100-fold less potent than the mother compound CP-320626. The photoaffinity labeling experiments were also performed, and a protein with molecular weight (MW) of about 90⁻100 kDa, which was consistent with the MW of GP, was clearly labeled by probe 1. A semiquantitative evaluation of the GP level in serum with probe 1 was also performed. The results showed that the protein band with a MW of about 90⁻100 kDa was tagged, and the concentration of the protein in serum was found to be between 25 and 50 ng/mL. Mass spectrometric analysis revealed that alpha-1,4 glucan phosphorylase (GPMM) was well-preserved in the bands.

HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69–75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site.

The epidermal growth factor receptor (EGFR) and HER2 are two important tyrosine kinases that play crucial roles in signal transduction pathways that regulate numerous cellular functions including proliferation, differentiation, migration, and angiogenesis. In the past 20 years, many proteomic methods have emerged as powerful methods to evaluate proteins in biological processes and human disease states. Among them, activity-based protein profiling (ABPP) is one useful approach for the functional analysis of proteins. In this study, a novel photoaffinity probe11was designed and synthesised to assess the target profiling of the reactive group in the photoaffinity probe11. Biological evaluation was performed, and the results showed that the novel photoaffinity probe binds to EGFR and HER2 proteins and it hits targets by the reactive group. [ABSTRACT FROM PUBLISHER]

(−)-Agelastatin A (AglA, 1 ), a member of the pyrrole-aminoimidazole marine alkaloid (PAI) family, possesses a unique tetracyclic structure and is one of the most potent anticancer PAIs isolated to date. In efforts to expand the SAR of these agents and delineate sites that tolerate modification while retaining activity, we synthesized several derivatives and tested their anticancer activity. The cytotoxic effects of these derivatives were measured against several cancer cell lines including cervical cancer (HeLa), epidermoid carcinoma (A431), ovarian (Igrov and Ovcar3), osteosarcoma (SJSA1), acute T cell leukemia (A3), epidermoid carcinoma (A431) in addition to primary human chronic lymphocytic leukemia (CLL) cells. New positions for modification of AglA and new substitutions were explored leading to novel derivatives, 14-chloro AglA ( 3 ) and 14-methyl AglA ( 12 ), that retained activity toward various cancer cell lines with decreased toxicity toward B- and T-cells. The SAR data informed the synthesis of a trifunctional probe bearing an alkyne and a diazirine potentially useful for cellular target identification. [ABSTRACT FROM AUTHOR]

Rice exposed to organic pollutants such as polybrominated diphenyl ethers (PBDEs) usually experiences reduced biomass and increased soluble sugar content. This study showed that 2, 2′, 4, 4′-tetrabromodiphenyl ether (BDE-47) led to increased glucose, fructose, and sucrose in rice leaves, accompanied by decreased photosynthetic rate and biomass. In order to identify the key enzyme that BDE-47 interacted with, a diazirine-alkynyl photoaffinity probe was designed, and photoaffinity labeling based chemoproteomics was conducted. Among all differentially expressed proteins, fructose-1, 6-bisphosphate aldolase (FBA) involved in carbohydrate metabolism was most likely the target protein of BDE-47. Spectral techniques and molecular docking analysis further revealed that the pollutant-protein interaction was driven by hydrophobic force. BDE-47 inhibited FBA catalytic efficiency by competing with its substrate, fructose-1, 6-diphosphate (F-1, 6-P), leading to soluble sugar accumulation, photosynthetic rate decline and biomass reduction. This study unraveled the influencing mechanism of PBDEs on rice by combining the novel photoaffinity labeling-based chemoproteomics with conventional proteomics. The improved knowledge on direct interaction between organic pollutants and proteins will help alleviate the harmful effects of soil pollution on plants. [Display omitted] • Biomass and total sugar content of rice changed upon BDE-47 exposure. • Soluble sugar accumulation led to photosynthesis inhibition and total sugar change. • Carbohydrate metabolism was the main metabolic process affected by BDE-47. • Photoaffinity labeling based chemoproteomics identified BDE-47 target proteins. • Binding of BDE-47 with FBA in glycolysis caused soluble sugar accumulation. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis and evaluation of a photoaffinity probe molecule for furospinosulin-1, a hypoxia-selective growth inhibitor that we identified from marine sponge, was studied. An analogue carrying an alkyne tail showed potent hypoxia-selective inhibitory activity exceeding that of the parent molecule, and exhibited in vivo anti-tumor activity following oral administration. The alkyne moiety in the analogue was also found to be a good anchoring group for the preparation of probe molecules; a photoaffinity probe molecule having an optimized spacer length was selected through the systematic synthesis of several probes and the evaluation of their hypoxia-selective growth inhibitory activity and electrophoretic mobility shift properties. [Copyright &y& Elsevier]

Abstract: Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells. [Copyright &y& Elsevier]

Abstract: Quercetin is a flavonoid natural product, that is, found in many foods and has been found to have a wide range of medicinal effects. Though a number of quercetin binding proteins have been identified, there has been no systematic approach to identifying all potential targets of quercetin. We describe an O7-biotinylated derivative of quercetin (BioQ) that can act as a photoaffinity proteomics reagent for capturing quercetin binding proteins, which can then be identified by LC–MS/MS. BioQ was shown to inhibit heat induction of HSP70 with almost the same efficiency as quercetin, and to both inhibit and photocrosslink to CK2 kinase, a known target of quercetin involved in activation of the heat shock transcription factor. BioQ was also able to pull down a number of proteins from unheated and heated Jurkat cells following UV irradiation that could be detected by both silver staining and Western blot analysis with an anti-biotin antibody. Analysis of the protein bands by trypsinization and LC–MS/MS led to the identification of heat shock proteins HSP70 and HSP90 as possible quercetin target proteins, along with ubiquitin-activating enzyme, a spliceosomal protein, RuvB-like 2 ATPases, and eukaryotic translation initiation factor 3. In addition, a mitochondrial ATPase was identified that has been previously shown to be a target of quercetin. Most of the proteins identified have also been previously suggested to be potential anticancer targets, suggesting that quercetin’s antitumor activity may be due to its ability to inhibit multiple target proteins. [Copyright &y& Elsevier]

Abstract: Allosamidins, metabolites of Streptomyces with strong inhibitory activities toward family 18 chitinases, show a variety of biological activities in various organisms. We prepared photoaffinity and biotinylated probes of allosamidin and demethylallosamidin, the N-demethyl derivative that shows much stronger anti-asthmatic activity than allosamidin. Mild acid hydrolysis of allosamidins afforded mono-amine derivatives, which were amidated to prepare probes with a photoactivatable aryl azide and/or biotin moieties. The derivatives with an N-acyl group at C-2 of the D-allosamine residue at the non-reducing end of allosamidins inhibited Trichoderma chitinase as strongly as the original compounds. Since the target of allosamidins in asthma is unclear, photoaffinity probes were used to analyze allosamidin-binding proteins in bronchoalveolar lavage (BAL) fluid in IL-13-induced asthmatic mice. Ym1, a chitinase-like protein, was identified as the main allosamidin-binding protein among proteins whose expression was upregulated by IL-13 in BAL fluid. Binding of allosamidins with Ym1 was confirmed by the experiments with photoaffinity probes and recombinant Ym1. [Copyright &y& Elsevier]

A convenient synthesis is described of a derivative of L-proline called photoproline, containing a diazirine group at position-4 of the pyrrolidine ring, starting from L-4-hydroxyproline. The use of Fmoc-L-photoproline in the synthesis of a cyclic peptidomimetic antibiotic demonstrates that this photoprobe can be incorporated into synthetic peptides using solid-phase Fmoc chemistry. Photoproline may be of wide value in the preparation of diverse peptide-based photoaffinity probes. [ABSTRACT FROM AUTHOR]

Abstract: Herein we describe the further improvement of our in-house developed firefly bioluminescence assay system for the determination of inhibition of protein phosphatase (PP). The advantage with the new system is higher sensitivity as well as being time and sample efficient. The inhibition activity of tautomycin with PP1γ was determined using the upgraded test system and K i was found to be 4.5nM, which compare favorably with the activity reported previously by others using different methods. The test system was then used in order to determine the activity of nine tautomycin (TTM) photoaffinity probes. One of the TTM photoaffinity probes (anti-10) was found to possess higher activity than the natural product itself with a K i of 3.4nM, while the remaining photoaffinity probes were found to possess K i in the range of 8.0–213nM. [Copyright &y& Elsevier]

Abstract: Glycosyltransferases (GTs) play a crucial role in mycobacterial cell wall biosynthesis and are necessary for the survival of mycobacteria. Hence, these enzymes are potential new drug targets for the treatment of tuberculosis (TB), especially multiple drug-resistant TB (MDR-TB). Herein, we report the efficient syntheses of Araf(α 1→5)Araf, Galf(β 1→5)Galf, and Galf(β 1→6)Galf disaccharides possessing a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) unit that were prepared as fluorescent disaccharide acceptors for arabinosyl- and galactosyl-transferases, respectively. Such analogs may offer advantages relative to radiolabeled acceptors or donors for studying the enzymes and for assay development and compound screening. Additionally, analogs possessing a 5-azidonaphthalene-1-sulfonamidoethyl unit were prepared as photoaffinity probes for their potential utility in studying active site labeling of the GTs (arabinosyl and galactosyl) in Mycobacterium tuberculosis (MTB). Beyond their preparation, initial biological testing and kinetic analysis of these disaccharides as acceptors toward glycosyltransferases are also presented. [Copyright &y& Elsevier]

Isomeric azidostyrylquinolines with a 4-azidostyryl group in position 2 or 4 of the quinoline ring have been synthesized. In the neutral form the azidostyrylquinolines absorb in the near UV but the protonated species absorb in the short wavelength region of the visible spectrum. In both forms the azides are lightsensitive and decompose under the influence of light with a quantum yield close to 1. [ABSTRACT FROM AUTHOR]

Abstract: Five photoaffinity probes of tautomycin, which possess an aromatic azide with linker attached to the 2-position of tautomycin, were prepared in order to study the binding site of tautomycin with protein phosphatase 1γ. The photoaffinity probes were synthesized by selectively introducing the photolabeling units onto the 2-position of tautomycin by using oxime chemistry. [Copyright &y& Elsevier]

Abstract: A trifunctional probe (binding element–photoreactive group–affinity tag) of natural product paeoniflorin was designed and synthesized based on the previous primary structure-activity relationship. This new probe is a potential tool for labeling, purification, and identification of the target proteins. [Copyright &y& Elsevier]

Abstract: Trimetoquinol (TMQ, 1) is a potent non-selective β-adrenoceptor (β-AR) agonist possessing a tetrahydroisoquinoline (THI) structure. The binding site for 1-trimethoxybenzyl group of 1, which distinguishes it from classical catecholamines, is unknown. Affinity and photoaffinity labeled compounds are good tools to determine the exact interaction between a ligand and a specific amino acid(s) in a receptor. In this study, we designed and synthesized a series of affinity 6, 12, 18, and photoaffinity 24, 29 labeled analogues of TMQ. All of these compounds were full agonists and demonstrated an equal or greater binding affinity and functional activity as compared to TMQ on β1-, β2-, and β3-AR. Washout experiments on Chinese hamster ovary (CHO) cells expressing hu β2-AR were helpful in identifying the isothiocyanate 18 and the azide 24 as very effective affinity and photoaffinity labels at this receptor subtype. [Copyright &y& Elsevier]

Abstract: A photosensitive azide-fluoro-dehydrocoelenterazine analog (Az-F-DCT) was synthesized, starting from 4-fluorophenylacetic acid, as a photoaffinity-labeling probe in order to analyze symplectin active site. To examine the photo-reactivity of Az-F-DCT, azide-fluoro-coelenterazine analog (Az-F-CT) was used as a potent symplectin chromophore model. Photolysis of Az-F-CT in 2,2,2-trifluoroethanol afforded nitrene intermediate to give an insertion product. The structure of this product was confirmed through spectroscopic analyses particularly by using a proton/deuterium (H/D) exchange experiments with ESI-Q-TOF-MS and -MS/MS measurement. [Copyright &y& Elsevier]

The synthesis of a novel radioactive peptidic photoaffinity probe for the PTP-1B enzyme as well as some SAR leading to the choice of this compound as a photoaffinity probe are presented. [Copyright &y& Elsevier]

Mannosyltransferases play a crucial role in mycobacterial cell-wall biosynthesis and are potential new drug targets for the treatment of tuberculosis. Herein, we describe the synthesis of α-(1 → 2)- and α-(1 → 6)-linked mannopyranosyl disaccharides possessing a 5-azidonaphthlene-1-sulfonamidoethyl group as photoaffinity probes for active-site labeling studies of mannosyltransferases in Mycobacetrium tuberculosis. [Copyright &y& Elsevier]

Two types of photoaffinity probe, which possesses a benzophenone or a diazirine photophore on the 2-position of tautomycin, were prepared in order to prove the details of binding site to PP1. These photoaffinity probes were designed on the basis of the structure–activity relationship; thus, the diacid moiety is indispensable. The selective introduction of photolabeling units on the 2-position of tautomycin was achieved through the 2-oxime of tautomycin diacid. [Copyright &y& Elsevier]

2′3′-cyclic GMP-AMP (2′3′-cGAMP), generated by cyclic GMP-AMP synthase (cGAS) under activation by cytosolic DNA, has a vital role in innate immune response via its receptor protein stimulator of interferon genes (STING) to fight viral infections and tumors. In order to have a complete understanding of biological functions of 2′3′-cGAMP, it is important to find out whether 2′3′-cGAMP has other unrevealed binding proteins present in mammalian cells and executes unknown functions. Here we report the 2′3′-cGAMP-based photoaffinity probes that capture and isolate 2′3′-cGAMP-binding proteins. These probes enable the identification of some potential 2′3′-cGAMP-binding proteins from HeLa cells. EF1A1, an essential protein regulating protein synthesis, is further validated to associate with 2′3′-cGAMP in vitro and in cells to impede protein synthesis. Thus, our studies provide a powerful approach to enable identification of the 2′3′-cGAMP interactome, discover unknown functions of 2′3′-cGAMP, and understand its physiological/pathological roles in tumor immunity and immune-related diseases. [Display omitted] • Photoaffinity probes of 2′3′-cGAMP enable identification of its interactome • 2′3′-cGAMP associates with EF1A1 in EF1 complex in preference to free-form EF1A1 • 2′3′-cGAMP prevents GTP binding to EF1A1 in EF1 complex • 2′3′-cGAMP impedes protein synthesis independently from STING Hou et al. develop the photoaffinity probes of 2′3′-cGAMP to identify its interactome, and discover its STING-independent function to inhibit protein synthesis by association with EF1A1 in EF1 complex. Their studies provide a useful approach to understand biological functions of 2′3′-cGAMP and its roles in immune-related diseases. [ABSTRACT FROM AUTHOR]

The preparation of a phenylalanine analogue containing an azido group and its incorporation into dipeptides is described. Peptides modified in this way are taken up into barley ( Hordeum vulgare L.) scutella via the previously characterized peptide-transport system. Photoactivation of modified peptides in the presence of isolated scutella resulted in irreversible inhibition of peptide uptake in a concentration-dependent manner. Transport of other solutes which share a common mechanism of energy coupling, but which are transported via distinct carriers, was not inhibited after photo-derivatization of scutella with the modified peptides. Derivatization of isolated scutellar tissue with a C-labelled peptide analogue, resulted in incorporation of label into two proteins of M = 54000 and 41000. Scutellar tissue from early-germinating seeds, which do not show active peptide uptake, did not incorporate label into these polypeptides. It is concluded that these proteins are components of the barley peptide-transport system. [ABSTRACT FROM AUTHOR]

Abstract: The circadian rhythmic leaf-movements in legumes, called nyctinasty, are regulated by a pair of leaf-closing and -opening factors. Recent fluorescence studies revealed that cis-p-coumaroylagmatine (1), the leaf-opening factor from genus Albizzia, specifically bound to motor cells in a joint of leaf and stem. In order to identify the receptor protein, which is expected to be involved in the plasma membrane of the cell, we designed and synthesized a photoaffinity probe (2), containing a photoreactive azide group on the aromatic ring and FLAG segment as a peptide antigen for immunoprecipitation. [Copyright &y& Elsevier]

Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (K D of 0.40 ± 0.15 µM). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100′s binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors. [ABSTRACT FROM AUTHOR]

To explore the molecular mechanisms of BAY R3401, four types of novel photoaffinity probes bearing different secondary tags were synthesized. Their potency for glycogenolysis was evaluated in primary human liver HL-7702 cells and HepG2 cells. Probe 2d showed the best activity in primary human liver HL-7702 cells and HepG2 cells, with IC50 values of 4.45 μM and 28.49 μM, respectively. Likewise, probe 5d showed IC50 values of 6.46 μM in primary human liver HL-7702 cells and 15.29 μM in HepG2 cells, respectively. Photoaffinity labeling experiments were also performed and protein bands larger than 170 kDa were specifically tagged by probe 2d. The results suggest that the synthesized probe 2d might be a very promising tool for the isolation of the target proteins of BAY R3401. [ABSTRACT FROM AUTHOR]

Honokiol, a biphenolic neolignan isolated from Magnolia officinalis, was reported to have a promising anti-inflammatory activity for the treatment of various diseases. There are many efforts on the synthesis and structure-activity relationship of honokiol derivatives. However, regioselective O-alkylation of honokiol remains a challenge and serves as a tool to provide not only some derivatives but also chemical probes for target identification and mode of action. In this study, we examined the reaction condition for regioselective O-alkylation, in which C2 and C4'-alkylated analogs of honokiol were synthesized and evaluated for inhibitory activity on nitric oxide production and cyclooxygenase-2 expression. Furthermore, we successfully synthesized a potential photoaffinity probe consisting of biotin and benzophenone based on a C4'-alkylated derivative.

A procedure to measure the serum concentration of glycogen phosphorylase during acute myocardial infarction is presented. This method was based on the synthesis of photoaffinity probes, and used the semiquantitative protein electrophoretic mobility shift technique. Three novel photoaffinity probes bearing different secondary tags were synthesized. Their potency was evaluated in an enzyme inhibition assay against rabbit muscle glycogen phosphorylase a (RMGPa). The inhibitory activity of probe 1 was only 100-fold less potent than the mother compound CP-320626. The photoaffinity labeling experiments were also performed, and a protein with molecular weight (MW) of about 90–100 kDa, which was consistent with the MW of GP, was clearly labeled by probe 1. A semiquantitative evaluation of the GP level in serum with probe 1 was also performed. The results showed that the protein band with a MW of about 90–100 kDa was tagged, and the concentration of the protein in serum was found to be between 25 and 50 ng/mL. Mass spectrometric analysis revealed that alpha-1,4 glucan phosphorylase (GPMM) was well-preserved in the bands. [ABSTRACT FROM AUTHOR]

HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69–75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site. [ABSTRACT FROM AUTHOR]

Diaminoquinazolines represent a privileged scaffold for antimalarial discovery, including use as putative Plasmodium histone lysine methyltransferase inhibitors. Despite this, robust evidence for their molecular targets is lacking. Here we report the design and development of a small-molecule photo-cross-linkable probe to investigate the targets of our diaminoquinazoline series. We demonstrate the effectiveness of our designed probe for photoaffinity labeling of Plasmodium lysates and identify similarities between the target profiles of the probe and the representative diaminoquinazoline BIX-01294. Initial pull-down proteomics experiments identified 104 proteins from different classes, many of which are essential, highlighting the suitability of the developed probe as a valuable tool for target identification in Plasmodium falciparum.