Chemoproteomics and Phosphoproteomics Profiling Reveals Salvianolic Acid A as a Covalent Inhibitor of mTORC1.

Salvianolic acid A (SAA), a major active ingredient of Salvia miltiorrhiza Bunge (Danshen), displays strong antiproliferative activity against cancer cells. However, their protein targets remain unknown. Here, we deconvoluted the protein targets of SAA using chemoproteomics and phosphoproteomics. By using alkynylated SAA as a probe, we discovered that SAA is a covalent ligand that can modify cellular proteins via its electrophilic α,ß-unsaturated ester moiety. The subsequent chemoproteomics profiling revealed that 46 proteins were covalently modified by SAA, including Raptor, a subunit of mTORC1 for recruiting substrates for mTORC1. Although gene ontology enrichment analysis of these proteins suggested that SAA displays a promiscuous protein interaction, phosphoproteomics profiling revealed that the SAA modulated phosphoproteins were mainly enriched in the signaling pathways of PI3K-Akt-mTOR, which is closely related to cell growth and proliferation. This was confirmed by the biochemical assay with purified mTORC1, a Western blot assay with phospho-specific antibodies, and a cellular thermal shift assay. Our work discovered that SAA is a covalent ligand for protein modification and mTORC1 is one of its targets. Moreover, our work demonstrated that the integrative profiling of chemoproteomics and phosphoproteomics can be a powerful tool for target deconvolution for bioactive natural products.

Target proteins profiling of irreversible kinase inhibitor pelitinib and discovery of degradation of PRDX4 by label free chemoproteomics.

Cell-based methods for profiling the kinase inhibitor selectivity are badly needed, especially for the irreversible kinase inhibitors. Here we reported a chemoproteomics approach for profiling the target proteins of irreversible kinase inhibitor with label free quantitative proteomics by using iodoacetamide alkyne as a chemical probe. In total 41 proteins were identified in high confidence (fold change 3.5, p value < 0.05) including PRDX4, STAT3, E2 conjugating enzymes UBE2L3, UBE2K, UBE2N, UBE2V1 and UBE2Z as well as E3 ligase TRIM 25. We validated the interaction between pelitinib and PRDX4 with a cell-based assay, and discovered that pelitinib can induce the degradation of PRDX4. The discovery was confirmed by biochemical assay, cellular thermal shift assay and miRNA knockdown experiment. Our data suggested that pelitinib can be a covalent molecular glue inducing the degradation of PRDX4. In addition, our work demonstrated that identification of the interactions between ligand and ubiquitylation associated proteins by chemoproteomics profiling can be used as a new strategy for identifying molecular glue degraders.

Discovery of AHCY as an Off-Target of Doxorubicin by Integrative Analysis of Photoaffinity Labeling Chemoproteomics and Untargeted Metabolomics.

Target identification is critically important for understanding the mechanism of action of drugs. Here, we reported a new strategy for deconvolution of drug targets (or off-targets) with photoaffinity labeling chemoproteomics in combination with untargeted metabolomics by using doxorubicin (DOX) as a model. The DOX-derived photoaffinity probes were prepared and applied to capture DOX-interacting proteins in living cells. The captured DOX-interacting proteins were then identified by label-free quantitative proteomics. Totally, 151 significant proteins were identified with high confidence (fold change >4, p-value < 0.005). The gene ontology enrichment analysis suggested that the proteins were mainly involved in carbon metabolism, citrate cycle, fatty acid metabolism, and metabolic pathways. Therefore, untargeted metabolomics was applied to quantify the significantly altered metabolites in cells upon drug treatment. The pathway enrichment analysis suggested that DOX mainly interrupted with the processes of pyrimidine and purine metabolism, carbon metabolism, methionine metabolism, and phosphatidylcholine biosynthesis. Integrative analysis of chemoproteomics and metabolomics indicated that adenosylhomocysteinase (AHCY) is a new target (off-target) of DOX leading to the accumulation of S-adenosyl homocysteine. This deduced DOX target was confirmed by the cellular thermal shift assay, affinity competitive pull-down assay, biochemical assay, and siRNA knock down experiments. Our result suggested that AHCY is the uncovered off-target of DOX.

[Preparation of luminescent silica nanoparticles with immobilized metal ion affinity for labeling phosphorylated proteins in Western Blot].

Protein phosphorylation is an important type of post-translational protein modification. In Western Blot experiment, the assay of phosphoproteins need special phospho antibodies, which are expensive, difficult to preserve, poorly reproducible. To this end, the immobilized metal ion affinity luminescent silica nanoparticles for instead of phospho antibodies were prepared. A layer of polymer was created on the surface of the silica nanoparticles via co-polymerization to protect the nanoparticles and to functionalize them with the immobilized metal ion affinity property to specifically label the phosphorylated proteins in Western Blot assays. The affinity luminescent silica nanoparticles were prepared with the following procedure. First, the sol-gel precursor fluorescein isothiocyanate-3-aminopropyltriethoxysilane (FITC-APTES) with the fluorescent moiety was prepared by modifying APTES with FITC. The luminescent silica nanoparticles (FITC@SiO2) were synthesized using the Stöber synthesis method in a reversed microemulsion. Briefly, 123.2 mL of cyclohexane, 25.6 mL of n-hexanol, and 5.44 mL of deionized water were ultrasonically mixed, and then 28.3 g of Triton X-100 were added and the mixture was magnetically stirred for 15 min to form a clear and transparent microemulsion system. Within 10 min, 0.8 mL of FITC-APTES precursor, 1.6 mL of tetraethoxysilane (TEOS), and 0.96 mL of concentrated ammonia (25%-27%, mass fraction) were added to the microemulsion, and the mixture was stirred at 24 ℃ for 24 h. After the reaction, the microemulsion system was destroyed by adding 200 mL of ethanol. The resulting FITC@SiO2 luminescent silica nanoparticles were centrifuged, and washed three times with ethanol. After dryness, the FITC@SiO2 nanoparticles were modified with methacryloxy-propyltrimethoxysilane (MPS) to introduce the double bonds for further modification. The functional monomer nitrilotriacetic acid (NTA) and glycidyl methacrylate (GMA) were copolymerized on the surface of the nanoparticles to convert FITC@SiO2-MPS to FITC@SiO2-MPS-GMA-NTA. The polymer coating of the silica nanoparticles was not only able to protect the silica from hydrolysis, but also to introduce the functional groups of nitrilotriacetic acid, which can chelate with metal ions. Elemental analysis demonstrated that the NTA groups had been bonded to the surface of the nanoparticles via copolymerization. The polymerization did not affect the morphology and fluorescence properties of the nanoparticles. The FITC@SiO2-MPS-GMA-NTA nanoparticles were activated with three different metal ions Zr4+, Fe3+, and Ti4+, for the enrichment of phosphorylated peptides derived form α-casein tryptic digestion. HPLC-MS analysis indicated that the FITC@SiO2-MPS-GMA-NTA-Ti 4+ nanoparticles are the best for the enrichment of phosphorylated peptides. The FITC@SiO2-MPS-GMA-NTA-Ti4+ nanoparticles were used for labelling the phosphorylated proteins in Western Blot experiment. The electrophoretic band of α-casein could be clearly labeled with the FITC@SiO2-MPS-GMA-NTA-Ti 4+ nanoparticles, while the bovine albumin band could not be labelled. This indicates that the luminescent FITC@SiO2-MPS-GMA-NTA-Ti4+nanoparticles can be used to label the phosphorylated proteins in Western Blot experiments.

Screening inhibitors for blocking UHRF1-methylated DNA interaction with capillary electrophoresis.

Epigenetic inheritance in mammals relies in part on propagation of DNA methylation patterns throughout development. UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) is required for maintenance the methylation pattern. It was reported that UHRF1 is overexpressed in a number of cancer types, and its depletion has been established to inhibit growth and invasion of cancer cells. It has been considered as a new therapeutic target for cancer. In the present work, we described a method for screening inhibitors for blocking the formation of UHRF1-methylated DNA (mDNA) complex by using nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM). A recombinant UHRF1 with the SRA domain (residues 408-643), a fluorescently labeled double strand mDNA (12 mer) and a known inhibitor mitoxantrone were employed for proof of concept. The method allows to measure the dissociation constant (Kd) of the UHRF1-mDNA complex as well as the rate kinetic constant for complex formation (kon) and dissociation (koff). A small chemical library composed of 60 natural compounds were used to validate the method. Sample pooling strategy was employed to improve the screening throughput. The merit of the method was confirmed by the discovery of two natural products proanthocyanidins and baicalein as the new inhibitors for blocking the formation of UHRF1-mDNA complex. Our work demonstrated that CE represents a straightforward and robust technique for studying UHRF1-mDNA interaction and screening of the inhibitors.

Probing Protein-Protein Interactions with Label-Free Mass Spectrometry Quantification in Combination with Affinity Purification by Spin-Tip Affinity Columns.

We describe an affinity purification-mass spectrometry (AP-MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)-Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor protein FYCO1. Totally, 96 proteins including seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments.

Targeting USP9x/SOX2 axis contributes to the anti-osteosarcoma effect of neogambogic acid.

SOX2 has been viewed as a critical oncoprotein in osteosarcoma. Emerging evidence show that inducing the degradation of transcription factors such as SOX2 is a promising strategy to make them druggable. Here, we show that neogambogic acid (NGA), an active ingredient in garcinia, significantly inhibited the proliferation of osteosarcoma cells with ubiquitin proteasome-mediated degradation of SOX2 in vitro and in vivo. We further identified USP9x as a bona fide deubiquitinase for SOX2 and NGA directly interacts with USP9x in cells. Moreover, knockdown of USP9x inhibited the proliferation and colony formation of osteosarcoma cells, which could be rescued by overexpression of SOX2. Consistent with this, knockdown of USP9x inhibited the proliferation of osteosarcoma cells in a xenograft mouse model. Collectively, we identify USP9x as the first deubiquitinating enzyme for controlling the stability of SOX2 and USP9x is a direct target for NGA. We propose that targeting the USP9x/SOX2 axis represents a novel strategy for the therapeutic of osteosarcoma and other SOX2 related cancers.

Screening of inhibitors against histone demethylation jumonji domain-containing protein 3 by capillary electrophoresis.

Jumonji domain-containing proteins (JMJDs) play an important role in the epigenetic regulation of gene expression. Aberrant regulation of histone modification has been observed in the progression of a variety of diseases, such as neurological disorders and cancer. Therefore, discovery of selective modulators of JMJDs is very attractive in new drug discovery. Herein, a simple capillary electrophoresis (CE) method was developed for screening of inhibitors against JMJD3. A known JMJD3 inhibitor GSK-J1, 5-carboxyfluorescein labeled substrate peptide with an amino acid sequence of KAPRKQLATKAARK(me3)SAPATGG (truncated from histone H3), as well as a small chemical library composed of 37 purified natural compounds and 30 natural extracts were used for method development and validation. The separation of substrate from its demethylated product was achieved by addition of polycation hexadimethrine bromide (HDB) in the running buffer. The enzyme activity was thus assayed accurately through separating the demethylated product from the substrate and then measuring the peak area of the product. The enzyme inhibition can be read out by comparing the peak area of the demethylated product obtained in the present of inhibitors and that of the negative control in the absence of any inhibitor. The merit of the method is proved by discovering two new JMJD3 inhibitors: salvianic acid A and puerarin 6''-O-xyloside.

Second messenger Ap4A polymerizes target protein HINT1 to transduce signals in FcεRI-activated mast cells.

Signal transduction systems enable organisms to monitor their external environments and accordingly adjust the cellular processes. In mast cells, the second messenger Ap4A binds to the histidine triad nucleotide-binding protein 1 (HINT1), disrupts its interaction with the microphthalmia-associated transcription factor (MITF), and eventually activates the transcription of genes downstream of MITF in response to immunostimulation. How the HINT1 protein recognizes and is regulated by Ap4A remain unclear. Here, using eight crystal structures, biochemical experiments, negative stain electron microscopy, and cellular experiments, we report that Ap4A specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. The polymerization interface overlaps with the area on HINT1 for MITF interaction, suggesting a possible competitive mechanism to release MITF for transcriptional activation. The mechanism depends precisely on the length of the phosphodiester linkage of Ap4A. These results highlight a direct polymerization signaling mechanism by the second messenger.

Quinacrine Depletes BCR-ABL and Suppresses Ph-Positive Leukemia Cells.

Drug resistance to TKIs and the existance of CML leukemia stem cells is an urgent problem. In this study, we demonstrate that quinacrine (QC) induces apoptosis in BCR-ABL positive CML and acute lymphoblastic leukemia (ALL) cells. Interestingly, QC inhibits the colony formation of primary CD34+ progenitor/stem leukemia cells from CML patients. QC targets RNA polymerase I, which produces ribosomal (r)RNA, involving in protein translation process. Also, QC treatment prolongs CML-like mice survival and inhibits K562 tumor growth in vivo. In conclusion, we demonstrate that QC depletes BCR-ABL protein and suppresses Ph-positive leukemia cells in vitro and in vivo.

Determining the affinity of anti-mitotic compounds binding to colchicine binding site of tubulin by affinity probe capillary electrophoresis.

The colchicine binding site of tubulin is often used to screen the anti-mitotic compounds, which are widely used as anti-cancer therapies. In the present work, an affinity probe capillary electrophoresis (APCE) method was developed for determining the affinity of anti-mitotic compounds. To this end, a fluorescently labeled affinity probe, 5-carboxyfluorescein-colchicine (F-colchicine), was prepared for the affinity competition experiment. The probe can form a stable complex with tubulin with the binding stoichiometry of 0.75, and the dissociation constant Kd of the complex was determined as 5.7 × 10-5 mol/L. In the affinity competition experiment, F-colchicine was incubated with tubulin and the test compound in the solution. The F-colchicine-tubulin complexes and free F-colchicine were quickly separated by CE and the concentration of free F-colchicine was accurately determined with the laser induced fluorescence detection. The affinity constant of the tested compound can be measured with the affinity competition binding curve. The enantiomers of the anti-mitotic compound were evaluated by using the method. The binding affinity of the enantiomers displayed an enantioselective manner. Compared to other affinity binding assay methods, our method is more straightforward, more accurate, and more cost-effective.

Novel Triapine Derivative Induces Copper-Dependent Cell Death in Hematopoietic Cancers.

Triapine, an iron chelator that inhibits ribonucleotide reductase, has been evaluated in clinical trials for cancer treatment. Triapine in combination with other chemotherapeutic agents shows promising efficacy in certain hematologic malignancies; however, it is less effective against many advanced solid tumors, probably due to the unsatisfactory potency and pharmacokinetic properties. In this report, we developed a triapine derivative IC25 (10) with potent antitumor activity. 10 Preferentially inhibited the proliferation of hematopoietic cancers by inducing mitochondria reactive oxygen species production and mitochondrial dysfunction. Unlike triapine, 10 executed cytotoxic action in a copper-dependent manner. 10-Induced up-expression of thioredoxin-interacting protein resulted in decreased thioredoxin activity to permit c-Jun N-terminal kinase and p38 activation and ultimately led to the execution of the cell death program. Remarkedly, 10 showed good bioavailability and inhibited tumor growth in mouse xenograft models. Taken together, our study identifies compound 10 as a copper-dependent antitumor agent, which may be applied to the treatment of hematopoietic cancers.

[Preparation of immobilized trypsin micro monolithic column and its application to rapid digestion of trace proteins].

A method for the preparation of a micro monolithic column with immobilized trypsin was developed for the rapid and efficient digestion of proteins. The micro monolithic column was prepared by photo-polymerization inside the tip of 20 µL pipette. The polymerization solution was composed of the functional monomers 4-pentenoic succinimide ester (PAS) and 2-hydroxyethyl methacrylate (HEMA), crosslinker pentaerythritol triacrylate (PETA) dissolved in a ternary porogenic system comprising dimethyl sulfoxide (DMSO), formyldimethylamine (DMF) and 1-dodecanol. Immobilization of trypsin was achieved by a chemical reaction between the amino group and succinimide. The effects of the active ester content in the polymerization mixture and the volumes of the monolithic bed on the column capacities of the immobilized trypsin were systematically investigated. The digestion efficiency as well as the stability and repeatability of the immobilized trypsin were systematically investigated by using standard proteins cytochrome C and bovine serum albumin. The experimental results indicated that high digestion efficiency with great reproducibility between batches and the digestion procedure could be obtained within 10 min. The trypsin immobilized columns were applied to the digestion of proteins extracted from 1×105 human acute promyelocytic leukemia (NB4) cells and human acute T-cell leukemia (Jurkat T) cells. A total of 2489 and 2572 proteins were readily identified by Nano-LC-MS/MS analysis. The quantity ratio of the identified proteins increased 2.2% and 6.1%, respectively, compared to the case of in-solution digestion, demonstrating the robustness of the trypsin immobilized micro column and its potential application to proteome studies.

Screening of break point cluster region Abelson tyrosine kinase inhibitors by capillary electrophoresis.

In the present study, a capillary electrophoresis (CE) method was developed for screening of inhibitors against the break point cluster region Abelson tyrosine kinase (BCR-ABL). The screening method was established by using 5-carboxyfluorescein labeled peptide substrate of BCR-ABL (F-ABLS), a known BCR-ABL tyrosine kinase inhibitor dasatinib, as well as a small chemical library consisting of 37 natural products. Thus, the inhibition of BCL-ABL kinase by small inhibitors was assayed by a CE system equipped with the laser induced fluorescence detector. The yield of phosphorylated product could be precisely measured through the separation by CE. The method is competent for enzymatic inhibition assay as well as the measurement of the inhibition kinetics. For screening BCR-ABL tyrosine kinase inhibitors, the hits were readily identified once the peak area of the phosphorylated products was reduced in comparison with the negative control. By taking the advantage of the screening method, luteolin and epicatechin gallate were discovered as the new BCR-ABL inhibitors.

Screening and Identifying a Novel ssDNA Aptamer against Alpha-fetoprotein Using CE-SELEX.

Alpha-fetoprotein (AFP) is a liver cancer associated protein and has long been utilized as a serum tumor biomarker of disease progression. AFP is usually detected in HCC patients by an antibody based system. Recently, however, aptamers generated from systematic evolution of ligands by exponential enrichment (SELEX) were reported to have an alternative potential in targeted imaging, diagnosis and therapy. In this study, AFP-bound ssDNA aptamers were screened and identified using capillary electrophoresis (CE) SELEX technology. After cloning, sequencing and motif analysis, we successfully confirmed an aptamer, named AP273, specifically targeting AFP. The aptamer could be used as a probe in AFP immunofluorescence imaging in HepG2, one AFP positive cancer cell line, but not in A549, an AFP negative cancer cell line. More interesting, the aptamer efficiently inhibited the migration and invasion of HCC cells after in vivo transfection. Motif analysis revealed that AP273 had several stable secondary motifs in its structure. Our results indicate that CE-SELEX technology is an efficient method to screen specific protein-bound ssDNA, and AP273 could be used as an agent in AFP-based staining, diagnosis and therapy, although more works are still needed.

Screening of epidermal growth factor receptor inhibitors in natural products by capillary electrophoresis combined with high performance liquid chromatography-tandem mass spectrometry.

A method for screening of inhibitors to epidermal growth factor receptor (EGFR) in natural product extracts with capillary electrophoresis (CE) in conjunction with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is reported. The method was established by employing 5-carboxyfluorescein labeled substrate peptide, two commercially available EGFR inhibitors OSI-744 and ZD1839, and a small chemical library consisted of 39 natural product extracts derived from the Traditional Chinese Medicines. Biochemical assay of crude natural product extracts was carried out by using CE equipped with a laser induced fluorescence detector. The CE separation allowed an accurately quantitative measurement of the phosphorylated product, hence the measurement of the enzymatic activity as well as the inhibition kinetics. The hits are identified if the peak area of the phosphorylated product is reduced in comparison with the negative control. The active constituents in the natural product extract were then identified by an assay-guided isolation with HPLC-MS/MS system. With the method, the flavonoids component of the Lycopus lucidus extract, namely quercetin and rutin were identified to be the active ingredients. Their IC50 values were determined as 0.88 µM and 10.1 µM, respectively. This result demonstrated a significant merit of our method in the identification of the bioactive compounds in natural products.

Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography-tandem mass spectrometry.

In this study, capillary electrophoresis (CE) combined with HPLC-MS/MS were used as a powerful platform for screening of inhibitors of mammalian target of rapamycin (mTOR) in natural product extracts. The screening system has been established by using 5-carboxyfluorescein labeled substrate peptide F-4EBP1, a known mTOR inhibitor AZD8055, and a small chemical library consisted of 18 natural product extracts. Biochemical screening of natural product extracts was performed by CE with laser induced fluorescence detection. The CE separation allowed a quantitative measurement of the phosphorylated product, hence the quantitation of enzymatic inhibition as well as inhibition kinetics. The hits are readily identified as long as the peak area of the phosphorylated product is reduced in comparison with the negative control. Subsequent assay-guided isolation of the active natural product extract was performed with HPLC-MS/MS to track the particular active components. The structures of the identified active components were elucidated by the molecular ions and fragmentation information provided by MS/MS analysis. The CE-based assay method only requires minute pure compounds, which can be readily purified by HPLC. Therefore, the combination of CE and HPLC-MS/MS provides a high-throughput platform for screening bioactive compounds from the crude nature extracts. By taking the advantage of the screening system, salvianolic acid A and C in extract of Salvia miltiorrhiza were discovered as the new mTOR inhibitors.

[Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry].

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein novel magnetic nanoparticles with an average size around 135 nm in diameter were prepared for the enrichment of glycoproteins in human serum. The prepared magnetic nanoparticles possessed uniform core/shell/shell structure which was composed of 8 nm magnetite internal core and double layers consisting of silica and poly glycidyl methacrylate (GMA). The latter was constructed by seed polymerization. Modified by a polyethylene hydrophilic linker, it made the surfaces of the magnetic nanoparticles highly hydrophilic so as to reduce the nonspecific adsorption of proteins. We examined affinity purification of glycoprotein in diluted human serum using our prepared magnetic nanoparticles with immobilization of concanavalin A (MNP @ ConA). The enriched proteins were reduced, alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chromatography. Protein identification was realized with nano-high performance liquid chromatography-orbitrap mass spectrometry. A total of 80 proteins were identified, among them 76 proteins were found to be glycoproteins by use of bioinformatic tools. /3-2-Glycoprotein 1 present in serum at low mass concentration around 0.000 01 g/L was also identified. This demonstrates the capability of magnetic nanoparticle for recovering minute amounts of glycoproteins from a fluid exhibiting a dynamic concentration range more than 12 orders of magnitude. Overall, MNP @ ConA has been proven to be an efficient alternative to currently available immobilization supports.

Screening of protein kinase inhibitors in natural extracts by capillary electrophoresis combined with liquid chromatography-tandem mass spectrometry.

We report a capillary electrophoresis method in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for screening of protein kinase inhibitors (PKIs) in natural extracts. Protein kinase A (PKA), substrate 5-carboxyfluorescein-labeled kemptide (CLK) and inhibitor H-89 were employed for the method development and validation. Enzymatic inhibition assay was performed with electrophoretically mediated microanalysis technique. Once the bioactivity of a natural extract was confirmed, an assay-guided isolation and structure elucidation using LC-MS/MS were accomplished to identify the compounds which are responsible for the observed bioactivity. Totally 33 natural extracts were screened with the method, and baicalin in the extract of Radix Scutellariae was identified to be a new PKI of PKA. This result demonstrated the practical applicability of our method in screening of PKIs from natural products.

Self-resistance to an antitumor antibiotic: a DNA glycosylase triggers the base-excision repair system in yatakemycin biosynthesis.

Resistance is (not) futile: The yatakemycin biosynthetic gene cluster involves the ytkR2 gene, which encodes a protein with homology to a recently discovered bacterial DNA glycosylase. Genetic validation in vivo, biochemical assays, and in vitro mutagenesis studies revealed that YtkR2 confers resistance for the bacteria by specifically recognizing and cleaving the YTM-modified base (see scheme).