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Chemistry (Weinheim An Der Bergstrasse,... Jan 2022Metabolic activation of the human carcinogen 1,3-butadiene (BD) by cytochrome 450 monooxygenases gives rise to a genotoxic diepoxide, 1,2,3,4-diepoxybutane (DEB). This...
Metabolic activation of the human carcinogen 1,3-butadiene (BD) by cytochrome 450 monooxygenases gives rise to a genotoxic diepoxide, 1,2,3,4-diepoxybutane (DEB). This reactive electrophile alkylates guanine bases in DNA to produce N7-(2-hydroxy-3,4-epoxy-1-yl)-dG (N7-DE-dG) adducts. Because of the positive charge at the N7 position of the purine heterocycle, N7-DEB-dG adducts are inherently unstable and can undergo spontaneous depurination or base-catalyzed imidazole ring opening to give N -[2-deoxy-D-erythro-pentofuranosyl]-2,6-diamino-3,4-dihydro-4-oxo-5-N-1-(oxiran-2-yl)propan-1-ol-formamidopyrimidine (DEB-FAPy-dG) adducts. Here we report the first synthesis and structural characterization of DEB-FAPy-dG adducts. Authentic standards of DEB-FAPy-dG and its N -labeled analogue were used for the development of a quantitative nanoLC-ESI -HRMS/MS method, allowing for adduct detection in DEB-treated calf thymus DNA. DEB-FAPy-dG formation in DNA was dependent on DEB concentration and pH, with higher numbers observed under alkaline conditions.
Topics: Butadienes; Chromatography, High Pressure Liquid; DNA; DNA Adducts; Epoxy Compounds; Formamides; Furans; Humans; Pyrimidines
PubMed: 34767297
DOI: 10.1002/chem.202103245 -
Journal of Medicinal Chemistry Oct 2021Ricin toxin A subunit (RTA) is the catalytic subunit of ricin, which depurinates an adenine from the sarcin/ricin loop in eukaryotic ribosomes. There are no approved...
Ricin toxin A subunit (RTA) is the catalytic subunit of ricin, which depurinates an adenine from the sarcin/ricin loop in eukaryotic ribosomes. There are no approved inhibitors against ricin. We used a new strategy to disrupt RTA-ribosome interactions by fragment screening using surface plasmon resonance. Here, using a structure-guided approach, we improved the affinity and inhibitory activity of small-molecular-weight lead compounds and obtained improved compounds with over an order of magnitude higher efficiency. Four advanced compounds were characterized by X-ray crystallography. They bind at the RTA-ribosome binding site as the original compound but in a distinctive manner. These inhibitors bind remotely from the catalytic site and cause local conformational changes with no alteration of the catalytic site geometry. Yet they inhibit depurination by ricin holotoxin and inhibit the cytotoxicity of ricin in mammalian cells. They are the first agents that protect against ricin holotoxin by acting directly on RTA.
Topics: Animals; Binding Sites; Chlorocebus aethiops; Crystallography, X-Ray; Dose-Response Relationship, Drug; Drug Design; Enzyme Inhibitors; Models, Molecular; Molecular Structure; Ribosomes; Ricin; Small Molecule Libraries; Structure-Activity Relationship; Surface Plasmon Resonance; Vero Cells
PubMed: 34648707
DOI: 10.1021/acs.jmedchem.1c01370 -
Nanomaterials (Basel, Switzerland) Aug 2021Ubiquitous on Earth, DNA and other nucleic acids are being increasingly considered as promising biomass resources. Due to their unique chemical structure, which is...
Ubiquitous on Earth, DNA and other nucleic acids are being increasingly considered as promising biomass resources. Due to their unique chemical structure, which is different from that of more common carbohydrate biomass polymers, materials based on nucleic acids may exhibit new, attractive characteristics. In this study, fluorescent nanoparticles (biodots) were prepared by a hydrothermal (HT) method from various nucleic acids (DNA, RNA, nucleotides, and nucleosides) to establish the relationship between the structure of precursors and fluorescent properties of biodots and to optimize conditions for preparation of the most fluorescent product. HT treatment of nucleic acids results in decomposition of sugar moieties and depurination/depyrimidation of nucleobases, while their consequent condensation and polymerization gives fluorescent nanoparticles. Fluorescent properties of DNA and RNA biodots are drastically different from biodots synthesized from individual nucleotides. In particular, biodots synthesized from purine-containing nucleotides or nucleosides show up to 50-fold higher fluorescence compared to analogous pyrimidine-derived biodots. The polymeric nature of a precursor disfavors formation of a bright fluorescent product. The reported effect of the structure of the nucleic acid precursor on the fluorescence properties of biodots should help designing and synthesizing brighter fluorescent nanomaterials with broader specification for bioimaging, sensing, and other applications.
PubMed: 34578581
DOI: 10.3390/nano11092265 -
International Journal of Molecular... Jul 2021This article reviews evidence suggesting that a common mechanism of initiation leads to the development of many prevalent types of cancer. Endogenous estrogens, in the... (Review)
Review
This article reviews evidence suggesting that a common mechanism of initiation leads to the development of many prevalent types of cancer. Endogenous estrogens, in the form of catechol estrogen-3,4-quinones, play a central role in this pathway of cancer initiation. The catechol estrogen-3,4-quinones react with specific purine bases in DNA to form depurinating estrogen-DNA adducts that generate apurinic sites. The apurinic sites can then lead to cancer-causing mutations. The process of cancer initiation has been demonstrated using results from test tube reactions, cultured mammalian cells, and human subjects. Increased amounts of estrogen-DNA adducts are found not only in people with several different types of cancer but also in women at high risk for breast cancer, indicating that the formation of adducts is on the pathway to cancer initiation. Two compounds, resveratrol, and -acetylcysteine, are particularly good at preventing the formation of estrogen-DNA adducts in humans and are, thus, potential cancer-prevention compounds.
Topics: Acetylcysteine; Animals; Antioxidants; Carcinogenesis; DNA Adducts; Estradiol; Estrogens; Estrone; Humans; Quinones; Resveratrol
PubMed: 34361004
DOI: 10.3390/ijms22158238 -
Se Pu = Chinese Journal of... Mar 2021Type Ⅱ ribosome-inactivating proteins (RIPs) are an important class of protein toxins that consist of A and B chains linked by an interchain disulfide bond. The... (Review)
Review
Type Ⅱ ribosome-inactivating proteins (RIPs) are an important class of protein toxins that consist of A and B chains linked by an interchain disulfide bond. The B-chain with lectin-like activity is responsible for binding to the galactose-containing receptors on eukaryotic cell surfaces, which is essential for A-chain internalization by endocytosis. The A-chain has -glycosidase activity that irreversibly depurinates a specific adenine from 28S ribosomal RNA (28S rRNA) and terminates protein synthesis. The synergistic effect of the A-B chain inactivates the ribosome, inhibits protein synthesis, and exhibits high cytotoxicity. Ricin and abrin that are expressed by the plants and , respectively, are typical type Ⅱ RIPs. The toxicity of ricin and abrin are 385 times and 2885 times, respectively, more that of the nerve agent VX. Owing to their ease of preparation, wide availability, and potential use as a bioterrorism agent, type Ⅱ RIPs have garnered increasing attention in recent years. Ricin is listed as a prohibited substance under schedule 1A of the Chemical Weapons Convention (CWC). The occurrence of ricin-related bioterrorism incidents in recent years has promoted the development of accurate, sensitive, and rapid detection and identification technology for type Ⅱ RIPs. Significant progress has been made in the study of toxicity mechanisms and detection methods of type Ⅱ RIPs, which primarily involve qualitative and quantitative analysis methods including immunological assays, mass spectrometry analysis methods, and toxin activity detection methods based on depurination and cytotoxicity. Immunoassays generally involve the specific recognition of antigens and antibodies, which is based on oligonucleotide molecular recognition elements called aptamers. These methods are fast and highly sensitive, but for highly homologous proteins in complex samples, they provide false positive results. With the rapid development of biological mass spectrometry detection technology, techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are widely used in the identification of proteins. These methods not only provide accurate information on molecular weight and structure of proteins, but also demonstrate accurate quantification. Enzyme digestion combined with mass spectrometry is the predominantly used detection method. Accurate identification of protein toxins can be achieved by fingerprint analysis of enzymatically digested peptides. For analysis of protein toxins in complex samples, abundant peptide markers are obtained using a multi-enzyme digestion strategy. Targeted mass spectrometry analysis of peptide markers is used to obtain accurate qualitative and quantitative information, which effectively improves the accuracy and sensitivity of the identification of type Ⅱ RIP toxins. Although immunoassay and mass spectrometry detection methods can provide accurate identification of type Ⅱ RIPs, they cannot determine whether the toxins will retain potency. The widely used detection methods for activity analysis of type Ⅱ RIPs include depurination assay based on -glycosidase activity and cytotoxicity assay. Both the methods provide simple, rapid, and sensitive analysis of type Ⅱ RIP toxicity, and complement other detection methods. Owing to the importance of type Ⅱ RIP toxins, the Organization for the Prohibition of Chemical Weapons (OPCW) has proposed clear technical requirements for the identification and analysis of relevant samples. We herein reviewed the structural characteristics, mechanism of action, and the development and application of type Ⅱ RIP detection methods; nearly 70 studies on type Ⅱ RIP toxins and their detection methods have been cited. In addition to the technical requirements of OPCW for the unambiguous identification of biotoxins, the trend of future development of type Ⅱ RIP-based detection technology has been explored.
Topics: Abrin; Plant Proteins; Ribosome Inactivating Proteins; Ribosomes; Ricin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 34227307
DOI: 10.3724/SP.J.1123.2020.10001 -
Computational and Structural... 2021Ribonucleotides (rNTPs) are predicted to be incorporated into the genome at a rate of up to 3 million times per cell division, making rNTPs the most common non-standard...
Ribonucleotides (rNTPs) are predicted to be incorporated into the genome at a rate of up to 3 million times per cell division, making rNTPs the most common non-standard nucleotide in the human genome. Typically, misinserted ribonucleotides are repaired by the ribonucleotide excision repair (RER) pathway, which is initiated by RNase H2 cleavage. However, rNTPs are susceptible to spontaneous depurination generating abasic ribonucleotides (rAPs), which are unable to be processed by RNase H2. Additionally, rAPs have been found in nascent RNA and coupled to R-loops. Recent work identified that base excision repair (BER) protein AP-Endonuclease 1 (APE1) is responsible for the initial processing of rAPs embedded in DNA and in R-loops. APE1 is a well characterized AP endonuclease that cleaves 5' of abasic sites, but its ability to cleave at rAPs remains poorly understood. Here, we utilize enzyme kinetics, X-ray crystallography, and molecular dynamics simulations to provide insight into rAP processing by APE1. Enzyme kinetics were used to determine pre-steady-state rates of APE1 cleavage on DNA substrates containing rAP, revealing a decrease in activity compared to cleavage at a canonical deoxy-AP substrate. Using X-ray crystallography, we identified novel contacts between the rAP and the APE1 active site. We demonstrate that the rAP sugar pucker is accommodated in the active site in a C3'-endo conformation, influencing its position and contributing to a decrease in activity compared to the deoxy-AP site. Together, this work provides molecular level insights into rAP processing by APE1 and advances our understanding of ribonucleotide processing within genomic DNA.
PubMed: 34188778
DOI: 10.1016/j.csbj.2021.05.035 -
Angewandte Chemie (International Ed. in... Sep 2021DNA-encoded compound libraries are a widely used small molecule screening technology. One important aim in library design is the coverage of chemical space through...
DNA-encoded compound libraries are a widely used small molecule screening technology. One important aim in library design is the coverage of chemical space through structurally diverse molecules. Yet, the chemical reactivity of native DNA barcodes limits the toolbox of reactions for library design. Substituting the chemically vulnerable purines by 7-deazaadenine, which exhibits tautomerization stability similar to natural adenine with respect to the formation of stable Watson-Crick pairs, yielded ligation-competent, amplifiable, and readable DNA barcodes for encoded chemistry with enhanced stability against protic acid- and metal ion-promoted depurination. The barcode stability allowed for straightforward translation of 16 exemplary reactions that included isocyanide multicomponent reactions, acid-promoted Pictet-Spengler and Biginelli reactions, and metal-promoted pyrazole syntheses on controlled pore glass-coupled barcodes for diverse DEL design. The Boc protective group of reaction products offered a convenient handle for encoded compound purification.
PubMed: 34153170
DOI: 10.1002/anie.202104348 -
Biochemistry Jun 2021is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. uses the unusual pathway with aminofutalosine (AFL) as an intermediate for...
is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. uses the unusual pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (MTAN) is the direct path for producing downstream metabolites in the pathway. However, genomic analysis indicates is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (AFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and -adenosylhomocysteine. The / value for AFL is 6.8 × 10 M s, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for AFLDA and demonstrated inhibitory effects on growth. Supplementation with futalosine partially restored growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-AFLDA and with MTCF at the catalytic sites show a catalytic site Zn or Fe as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp hydroxyl group of the transition state analogue. Metabolomics analysis revealed that AFLDA has intracellular activity and is inhibited by MTCF. The pathway in bifurcates at aminofutalosine with MTAN producing adenine and depurinated futalosine and AFLDA producing futalosine. Inhibition of cellular MTAN or AFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.
Topics: Catalytic Domain; Crystallography, X-Ray; Deoxyadenosines; Helicobacter pylori; Models, Molecular; N-Glycosyl Hydrolases; Nucleosides; Purine-Nucleoside Phosphorylase; Substrate Specificity; Thionucleosides; Vitamin K 2
PubMed: 34077175
DOI: 10.1021/acs.biochem.1c00215 -
International Journal of Biochemistry... 2021Alkaline phosphatase is an enzyme that converts para-nitrophenyl phosphate to para-nitrophenol (yellow coloured) in 2-amino, 2-methyl, 1-propanol buffer at pH 10.5....
Alkaline phosphatase is an enzyme that converts para-nitrophenyl phosphate to para-nitrophenol (yellow coloured) in 2-amino, 2-methyl, 1-propanol buffer at pH 10.5. However, when this protocol is applied to the in vitro cellular model systems to estimate alkaline phosphatase activity, it tends to generate clumps of genomic DNA, leading to inaccurate pipetting for protein estimation. The aim of the study was to introduce minor modifications in the existing protocol to make it simple, cost-effective, with minimal labor-intensive procedures while estimating alkaline phosphatase activity in cellular model systems. The genomic DNA clumps were dissolved by depurination (adding 0.2 N HCl) and fragmentation (adding 0.2 N NaOH) during enzyme estimation. Moreover, these minor modifications have been standardized and optimized extensively by using serum samples (rich source of alkaline phosphatase), hFOB/ER9 (human Fetal osteoblastic cell) and HepG2 cells. Our results suggest that the modification incorporated in previously published method was robust enough to estimate ALP activity and protein concentration accurately. There was no significant variation in ALP activity estimated after modification (P > 0.05). This innovative approach could be beneficial for a researcher by providing an easy, cost effective and less labor-intensive solution for estimation of enzymatic activity in cellular model systems.
PubMed: 33824775
DOI: No ID Found -
Toxins Mar 2021International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and...
International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin in various sample matrices are considered necessary actions for risk assessment during a suspected exposure. This study reports a portable electrochemical assay for detecting active ricin based on the adenine electro-oxidation released from herring sperm DNA substrate by its catalytic action. Also, kinetic parameters were calculated, and the values were of 3.14 µM and 2107 min. A linear response was found in optimized experimental conditions for ricin concentrations ranging from 8 to 120 ng/mL, and with a detection limit of 5.14 ng/mL. This proposed detection strategy emphasizes the possibility of field detection of active ricin in food matrices and can be applied to other endonucleolytic activities.
Topics: Adenine; Animals; DNA; Electrochemical Techniques; Fishes; Kinetics; Male; Reproducibility of Results; Ricin; Spermatozoa; Substrate Specificity
PubMed: 33810228
DOI: 10.3390/toxins13040238