Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed—the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE systems made from botulinum neurotoxin aptamers selected by ASExp have been investigated. The results of this investigation indicate that ASExp can be used to rapidly select aptamers for the DCE sensing system.     

Reductive metabolism of 1,1,1,2-tetrachloroethane and related chloroethanes by rat liver microsomes

The susceptibility of polychlorinated ethanes to reductive metabolism was evaluated by measuring the amount of each compound consumed during anaerobic incubations with rat live microsomes; 1,1,1,2-tetrachloroethane, pentachloroethane and hexachloroethane were metabolized extensively, 1,1,1,2-tetrachloroethane and the trichloroethanes were metabolized very slowly and the dichloroethanes were not metabolized at a detectable rate. The electron affinity of the chloroethanes was determined by measuring electrochemical half-wave reduction potentials. Chloroethanes with an E1/2 of - 1.35 V or less negative were reduced readily in microsomes while those with an E1/2 equal to or more negative than -1.90 V were not good substrates for enzymatic reduction. Metabolites produced from 1,1,1,2-tetrachloroethane in vitro were 1,1-dichloroethylene (DCE) and 1,1,2-trichloroethane (TCEA) and the ratio DCE/TCEA was about 25:1. These conversions were NADPH-dependent and were inhibited by air, CO and metyrapone. In the presence of SKF 525-A, DCE formation was inhibited by 47%. Microsomes from untreated or beta-naphthoflavone-treated rats were 70-90% less active than microsomes from phenobarbital-treated rats. The Km was 0.50 mM and the Vmax was 66 nmol min-1 mg-1 protein for DCE formation. The results are consistent with the proposal that 1,1,1,2-tetrachloroethane is reduced by hepatic cytochrome(s) P-450 to a free radical intermediate which, for the most part, remains closely associated with the enzyme, is reduced further and undergoes beta-elimination of a chloride ion to form DCE. The occurrence of this reductive pathway in vivo was demonstrated by the quantitation of DCE and TCEA in blood from rats treated with 1,1,1,2-tetrachloroethane.     

In vitro microsome- and cytosol-mediated binding of 1,2-dichloroethane and 1,2-dibromoethane with DNA

Metabolic activation of 1,2-dichloroethane (DCE) and 1,2-dibromoethane (DBE) to forms able to bind covalently with DNA occurs in vitroeither by wat of microsomal or cytosolic pathways. The involvement of these two pathways is variable with respect to species or compound tested. Rat enzymes are generally more efficient than mouse enzymes in bioactivating haloalkanes and DBE is more reactive than DCE. This parallels both the previous report on in vivocomparative interaction and the higher genotoxicity of DBE.Abbreviations DCE dichloroethane - DBE dibromoethane - PB phenobarbitone - ANOVA analysis of variance Supported by grants from Ministero Pubblica Instruzione and from Ministero della Sanità (Contract No. 500.4/RSC/135/L/2353), Rome, Italy.     

1.5 Mb deletion of chromosome 4p16.3 associated with postnatal growth delay,psychomotor impairment,epilepsy, impulsive behavior and asynchronous skeletal development

Several Wolf-Hirschhorn syndrome patients have been studied, mouse models for a few candidate genes have been constructed and two WHS critical regions have been postulated, but the molecular basis of the syndrome remains poorly understood.     

The parmodulin NRD-21 is an allosteric inhibitor of PAR1 Gq signaling with improved anti-inflammatory activity and stability

Novel analogs of the allosteric, biased PAR1 ligand ML161 (parmodulin 2, PM2) were prepared in order to identify potential anti-thrombotic and anti-inflammatory compounds of the parmodulin class with improved properties. Investigations of structure-activity relationships of the western portion of the 1,3-diaminobenzene scaffold were performed using an intracellular calcium mobilization assay with endothelial cells, and several heterocycles were identified that inhibited PAR1 at sub-micromolar concentrations. The oxazole NRD-21 was profiled in additional detail, and it was confirmed to act as a selective, reversible, negative allosteric modulator of PAR1. In addition to inhibiting human platelet aggregation, it showed superior anti-inflammatory activity to ML161 in a qPCR assay measuring the expression of tissue factor in response to the cytokine TNF-alpha in endothelial cells. Additionally, NRD-21 is much more plasma stable than ML161, and is a promising lead compound for the parmodulin class for anti-thrombotic and anti-inflammatory indications.     

Discovery of benzimidazole derivatives as orally active renin inhibitors: Optimization of 3,5-disubstituted piperidine to improve pharmacokinetic profile

We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio.     

1，2-二氯乙烷对大鼠肝细胞内游离钙离子浓度的影响

目的了解1,2-二氯乙烷染毒24h对大鼠肝细胞的损伤及其机理。方法运用显微荧光术测定了大鼠肝细胞内游离钙离子浓度,同时,测定了大鼠肝细胞培养上清液乳酸脱氢酶（LDH）活力作为大鼠肝细胞受损的指标。结果所有剂量组的1,2-二氯乙烷的大鼠肝细胞内游离钙离子浓度与对照组比较差异均无显著性（P〉0．05）;LDH活力仅在浓度为5mmol／L的1,2-二氯乙烷染毒组与对照组比较差异也无显著性（P〉0．05）;而1,2-二氯乙烷其他染毒组（即浓度为10mmol／L和20mmol／L）与对照组比较差异均有显著性（P〈0．01）。结论较高浓度（10mmol／L和20mmol／L）的1,2-二氯乙烷能损伤大鼠肝细胞,损伤的途径不是通过破坏肝细胞内钙稳态机制。     

A novel complexity-to-diversity strategy for the diversity-oriented synthesis of structurally diverse and complex macrocycles from quinine

Recent years have witnessed a global decline in the productivity and advancement of the pharmaceutical industry. A major contributing factor to this is the downturn in drug discovery successes. This can be attributed to the lack of structural (particularly scaffold) diversity and structural complexity exhibited by current small molecule screening collections.Macrocycles have been shown to exhibit a diverse range of biological properties, with over 100 natural product-derived examples currently marketed as FDA-approved drugs. Despite this, synthetic macrocycles are widely considered to be a poorly explored structural class within drug discovery, which can be attributed to their synthetic intractability.Herein we describe a novel complexity-to-diversity strategy for the diversity-oriented synthesis of novel, structurally complex and diverse macrocyclic scaffolds from natural product starting materials. This approach exploits the inherent structural (including functional) and stereochemical complexity of natural products in order to rapidly generate diversity and complexity. Readily-accessible natural product-derived intermediates serve as structural templates which can be divergently functionalized with different building blocks to generate a diverse range of acyclic precursors. Subsequent macrocyclisation then furnishes compounds that are each based around a distinct molecular scaffold. Thus, high levels of library scaffold diversity can be rapidly achieved. In this proof-of-concept study, the natural product quinine was used as the foundation for library synthesis, and six novel structurally diverse, highly complex and functionalized macrocycles were generated.     

The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. Activation by the isolated perfused rat liver.

In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.