Synthesis and biological evaluation of 6-aryl-6H-pyrrolo[3,4-d]pyridazine derivatives: high-affinity ligands to the alpha 2 delta subunit of voltage gated calcium channels

A novel class of 6-aryl-6H-pyrrolo[3,4-d]pyridazine ligands for the alpha2delta subunit of voltage-gated calcium channels has been described. Substitutions in the aryl ring of the molecule were generally not tolerated, and resulted in diminished binding to the alpha2delta subunit. Modifications to the pyridazine ring revealed numerous permissive substitutions, and detailed SAR studies were carried out in this portion of the molecule. Replacement of the pyridazine ring methyl group with an aminomethyl functionality provided greatly improved potency over the initial lead. The initial lead compound displayed good rat pharmacokinetic properties, and was shown to be efficacious in the Chung model for neuropathic pain in rats.     

Systematic structure-function analysis of the small GTPase Arf1 in yeast

Members of the ADP-ribosylation factor (Arf) family of small GTPases are implicated in vesicle traffic in the secretory pathway, although their precise function remains unclear. We generated a series of 23 clustered charge-to-alanine mutations in the Arf1 protein of Saccharomyces cerevisiae to determine the portions of this protein important for its function in cells. These mutants display a number of phenotypes, including conditional lethality at high or low temperature, defects in glycosylation of invertase, dominant lethality, fluoride sensitivity, and synthetic lethality with the arf2 null mutation. All mutations were mapped onto the available crystal structures for Arf1p: Arf1p bound to GDP, to GTP, and complexed with the regulatory proteins ArfGEF and ArfGAP. From this systematic structure-function analysis we demonstrate that all essential mutations studied map to one hemisphere of the protein and provide strong evidence in support of the proposed ArfGEF contact site on Arf1p but minimal evidence in support of the proposed ArfGAP-binding site. In addition, we describe the isolation of a spatially distant intragenic suppressor of a dominant lethal mutation in the guanine nucleotide-binding region of Arf1p.     

Cytochrome P450 3A4-mediated oxidative conversion of a cyano to an amide group in the metabolism of pinacidil

The conversion of nitriles to amides is generally considered to be a hydrolytic process that does not involve redox chemistry. We demonstrate here that cytochrome P450 (CYP) is responsible for the conversion of the cyano group of pinacidil to the corresponding amide. The reaction in human liver microsomes was NADPH-dependent and was nearly completely inhibited by an anti-CYP3A4 antibody. Incubations of pinacidil with recombinant CYP enzymes confirm that CYP3A4 is the principal catalyst of this reaction. The kinetics of pinacidil amide formation by CYP3A4 yielded an apparent K(m) of 452 +/- 33 microM and k(cat) of 0.108 min(-1) (k(cat)/K(m) = 0.238 mM(-1).min(-1)). Incubation of pinacidil with CYP3A4 in the presence of (18)O(2) or H(2)(18)O showed that the amide carbonyl oxygen derived exclusively from molecular oxygen. The CYP3A4-mediated reaction also was supported by hydrogen peroxide when incubations were carried out in the absence of cytochrome P450 reductase and NADPH. The reaction can be explained by a nucleophilic attack of a deprotonated ferric peroxide intermediate (Fe(3+)-O-O(-)) on the carbon atom of the -C triple bond N triple bond to form an Enz-Fe(III)-O-O-C(=NH)R intermediate, followed by cleavage of the O-O bond to give pinacidil amide. This nucleophilic addition of an Fe(3+)-O-O(-) intermediate to a -C=N pi-bond in a P450 system resembles the analogous reaction catalyzed by the nitric oxide synthases.     

Transgenic plants with altered ethylene biosynthesis or perception

The plant hormone ethylene is an essential signaling molecule involved in many plant processes including: germination, flower development, fruit ripening and responses to many environmental stimuli. Moreover, large increases in ethylene levels occur during plant stress responses, fruit ripening and flower wilting. Manipulation of ethylene biosynthesis or perception allows us to modulate these processes and thereby create plants with more robust and/or desirable traits, giving us a glimpse into the role of ethylene in the plant. Here, recent and landmark advances in genetic alteration of members of the ethylene pathway in plants and the physiological consequences of these alterations are examined.     

Epsilon-tubulin is required for centriole duplication and microtubule organization

Centrosomes nucleate microtubules and serve as poles of the mitotic spindle. Centrioles are a core component of centrosomes and duplicate once per cell cycle. We previously identified epsilon-tubulin as a new member of the tubulin superfamily that localizes asymmetrically to the two centrosomes after duplication. We show that recruitment of epsilon-tubulin to the new centrosome can only occur after exit from S phase and that epsilon-tubulin is associated with the sub-distal appendages of mature centrioles. Xenopus laevis epsilon-tubulin was cloned and shown to be similar to human epsilon-tubulin in both sequence and localization. Depletion of epsilon-tubulin from Xenopus egg extracts blocks centriole duplication in S phase and formation of organized centrosome-independent microtubule asters in M phase. We conclude that epsilon-tubulin is a component of the sub-distal appendages of the centriole, explaining its asymmetric localization to old and new centrosomes, and that epsilon-tubulin is required for centriole duplication and organization of the pericentriolar material.     

Synthesis and SAR of benzyl and phenoxymethylene oxadiazole benzenesulfonamides as selective beta3 adrenergic receptor agonist antiobesity agents

Benzyl and phenoxymethylene substituted oxadiazoles are potent and orally bioavailable beta3 adrenergic receptor (AR) agonists. The 4-trifluormethoxy substituted 5-benzyl oxadiazole 5f has an EC50 of 8 nM in the beta3 AR agonist assay with 100-fold selectivity over beta1 and beta2 AR binding inhibition activity. Its oral bioavailability in dogs is 30 +/- 4%, with a half-life of 3.8 +/- 0.4 h. In the anesthetized rhesus, 5f evoked a dose-dependent glycerolemia (ED50Gly = 0.15 mg/kg). Under these conditions a heart rate increase of 15% was observed at a dose level of 10 mg/kg.     

Soil inorganic carbon storage pattern in China

Soils with pedogenic carbonate cover about 30% (3.44 × 10⁶ km²) of China, mainly across its arid and semiarid regions in the Northwest. Based on the second national soil survey (1979–1992), total soil inorganic carbon (SIC) storage in China was estimated to be 53.3±6.3 PgC (1 Pg=10¹⁵ g) to the depth investigated to 2 m. Soil inorganic carbon storages were 4.6, 10.6, 11.1, and 20.8 Pg for the depth ranges of 0–0.1, 0.1–0.3, 0.3–0.5, and 0.5–1 m, respectively. Stocks for 0.1, 0.3, 0.5, and 1 m of depth accounted for 8.7%, 28.7%, 49.6%, and 88.9% of total SIC, respectively. In contrast with soil organic carbon (SOC) storage, which is highest under 500–800 mm yr⁻¹ of mean precipitation, SIC storage peaks where mean precipitation is <400 mm yr⁻¹. The amount and vertical distribution of SIC was related to climate and land cover type. Content of SIC in each incremental horizon was positively related with mean annual temperature and negatively related with mean annual precipitation, with the magnitude of SIC content across land cover types showing the following order: desert, grassland >shrubland, cropland >marsh, forest, meadow. Densities of SIC increased generally with depth in all ecosystem types with the exception of deserts and marshes where it peaked in intermediate layers (0.1–0.3 m for first and 0.3–0.5 m for latter). Being an abundant component of soil carbon stocks in China, SIC dynamics and the process involved in its accumulation or loss from soils require a better understanding.     

小黄皮叶挥发油的化学成分

利用水蒸气蒸馏法提取小黄皮叶挥发油,运用毛细管气相色谱-质谱联用法对挥发油进行了分析,分离出40个峰,鉴定了其中的37种成分,所鉴定成分占挥发油总量的99.87%,其主要化学成分为单萜及倍半萜类化合物。     

Discovery of a piperazine urea based compound as a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist

We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models.     

Role of ribosomal protein RPS2 in controlling let-7a expression in human prostate cancer

We discovered that an inverse relationship exists in the expression of ras/c-myc and ribosomal protein RPS2 with pre-let-7a-1/let-7a/let-7f miRNA and prostate tumor cell malignancy. Nonmalignant IBC-10a cells expressed low levels of ras/RPS2 and elevated pre-let-7a-1/let-7a/let-7f miRNA, whereas the reverse occurred in malignant PCa-20a and PC-3ML cells. Stable transfection of IBC-10a cells with pBABE.ras and pBABE.RPS2 induced ras, c-myc, and RPS2 expression, whereas the levels of let-7a/let-7f miRNA dropped to near zero. Conversely, in pBABE.pre-let-7a-1 transfected PCa-20a and PC-3ML clones, let-7a/let-7f increased whereas ras, RPS2, and c-myc dropped greater than 5-fold. Electrophoretic mobility shift assays, antibody "supershift" assays and immunoprecipitation assays revealed that RPS2 specifically binds pre-let-7a-1 to block RNA processing. Immunoflourescent studies and Northern blots confirmed that RPS2 complexes with pre-let-7a-1 (i.e., in episomal structures) to block processing to let-7a/let-7f, indicating RPS2 may prevent let-7a miRNA expression to indirectly promote oncogene expression. Functional studies further showed that the colony-forming ability (CFA) and invasive activities of IBC-10a cells were significantly enhanced in pBABE-ras.IBC-10a and pBABE-RPS2-IBC-10a clones. Conversely, with the "knockdown" of ras and RPS2 in malignant PC-3ML cells (i.e., in pLKO.TRC.shRNA.ras.PC3-ML, pLKO.TRC.shRNA.RPS2.PC-3ML transfected cells), there was both a loss of these functions and a loss of tumorigenesis in SCID mice. Likewise, with the overexpression of let-7a/let-7f in pBABE.pre-let-7a-1.PC-3ML clones (and PCa-20a clones), CFAs, invasive activities in vitro, and tumorigenesis in vivo were significantly reduced. These results show for the first time that RPS2 blocks pre-let-7a-1 processing to enable ras and c-myc expression and the transformation of primary tumor cells.