Insulin-like growth factor (IGF)-binding proteins inhibit the biological activities of IGF-1 and IGF-2 but not des-(1-3)-IGF-1.

Late gestation is associated with insulin resistance in rats and humans. It has been reported that rats at term gestation show active hepatic gluconeogenesis and glycogenolysis, and diminished lipogenesis, despite normal or mildly elevated plasma insulin concentrations, indicating a state of resistance to the hormone action. Since autophosphorylation of the insulin receptor has been reported to play a key role in the hormone signal transduction, we have partially purified plasma-membrane liver insulin receptors from virgin and 22-day-pregnant rats and studied their binding and kinase activities. (1) Insulin binding to partially purified receptors does not appear to be influenced by gestation, as indicated by the observed KD and Bmax. values. (2) The rate of autophosphorylation and the maximal 32P incorporation into the receptor beta-subunit from pregnant rats at saturating concentrations of insulin are markedly decreased with respect to the corresponding values for virgin rats. (3) The diminished autophosphorylation rate was due to a decreased responsiveness of the kinase activity to the action of insulin. (4) Phosphorylation of the exogenous substrates casein and poly(Glu80Tyr20) by insulin-receptor kinase was also less when receptors from pregnant rats were used. These results show the existence of an impairment at the receptor kinase level of the insulin signalling mechanism that might be related to the insulin-resistant state characteristic of term gestation in rats.     

Monoclonal antibodies reacting with serogroup and serovar specific epitopes on different lipopolysaccharide subunits of Leptospira interrogans serovar pomona

Abstract Monoclonal antibodies with two kinds of specificities, produced against Leptospira interrogans serovar pomona , were studied by agglutination and immunoblotting. Antibodies reacted either exclusively with serovar pomona or with all members of the Pomona serogroup, but none of the antibodies reacted with representative serovars of other serogroups. Both antibodies recognized epitopes on purified lipopolysaccharide (LPS) from serovar pomona . In immunoblotting experiments the serogroup specific antibody recognized both the major LPS bands of 21 kDa and 26 kDa whereas the serovar specific antibodies reacted only with the 26 kDa band, thus localizing serovar specificity in the 26 kDa band and serogroup specific epitopes on at least two different LPS subunits.     

Degradation of horseradish peroxidase after microinjection into mammalian cells

Horseradish peroxidase (HRP) has been microinjected into mammalian cells in tissue culture by the erythrocyte ghost-mediated technique. This protein was selected because it can be localized and quantified after injection by cytochemical and spectrophotometric methods. HRP labeled by reductive methylation retained full catalytic activity, was efficiently loaded into erythrocyte ghosts, and did not associate to a significant degree with ghost membranes. A combination of cytochemical staining and autoradiography established that HRP injected into rat L6 myoblasts, HE(39)L human diploid fibroblasts, or HeLa cells was intracellular and uniformly distributed throughout the cell, while cell lysis techniques showed that the catalytically active HRP was not membrane bound. Inactivation of labeled HRP after injection paralleled the disappearance of the 40-kDa polypeptide, and was always more rapid than its overall degradation. This difference was associated with a pool of water-insoluble radioactivity in the injected cells. This material was of smaller molecular size than the native protein: many labeled peptides were detected in the range of 10 to 38 kDa. By the use of inhibitors of autophagic proteolysis or lysosomal function it was established that HRP degradation was not subjected quantitatively to the same regulatory processes as the average endogenous protein labeled in the same cultures.     

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc₁) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, Q_N and Q_P. The L197F mutation is located in the Q_N site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another Q_N site inhibitor, but not to strobilurin or myxothiazol, which target the Q_P site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC₅₀) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.     

The discovery of UK-369003, a novel PDE5 inhibitor with the potential for oral bioavailability and dose-proportional pharmacokinetics

This paper describes our recent efforts to design and synthesise potent and selective PDE5 inhibitors and the use of in vitro predictors of clearance, absorption and permeability to maximise the potential for dose-proportional pharmacokinetics and good oral bioavailability in man. Optimisation of the preclinical profile resulted in the identification of UK-369003 (19a) and its nomination as a clinical candidate. The clinical pharmacokinetic and safety profile has enabled us to progress the compound to test its efficacy in patients with lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) and a paper describing its efficacy has recently been published.     

Mimicking the biological activity of diazobenzo[b]fluorene natural products with electronically tuned diazofluorene analogs

Under appropriate electronic modulation, simple diazofluorene analogs recapitulate the DNA cleavage activity of kinamycin D under thiol-based reducing conditions. Achieving DNA cleavage under these reducing conditions is key to anticancer activity, as the most active compound, 1-methoxydiazofluorene, inhibits the proliferation of HeLa cells.     

Inhibitors of epidermal growth factor receptor tyrosine kinase: optimisation of potency and in vivo pharmacokinetics

The structure-activity and structure-property relationships of anilinoquinazoline inhibitors of EGFR were investigated. Strategies to lower volume of distribution and shorten half-life through structure and pKa modulation are discussed.     

Evolutionary relationships, interisland biogeography, and molecular evolution in the Hawaiian violets (Viola: Violaceae)

The endemic Hawaiian flora offers remarkable opportunities to study the patterns of plant morphological and molecular evolution. The Hawaiian violets are a monophyletic lineage of nine taxa distributed across six main islands of the Hawaiian archipelago. To describe the evolutionary relationships, biogeography, and molecular evolution rates of the Hawaiian violets, we conducted a phylogenetic study using nuclear rDNA internal transcribed spacer sequences from specimens of each species. Parsimony, maximum likelihood (ML), and Bayesian inference reconstructions of island colonization and radiation strongly suggest that the Hawaiian violets first colonized the Maui Nui Complex, quickly radiated to Kaua'i and O'ahu, and recently dispersed to Hawai'i. The lineage consists of "wet" and "dry" clades restricted to distinct precipitation regimes. The ML and Bayesian inference reconstructions of shifts in habitat, habit, and leaf shape indicate that ecologically analogous taxa have undergone parallel evolution in leaf morphology and habit. This parallel evolution correlates with shifts to specialized habitats. Relative rate tests showed that woody and herbaceous sister species possess equal molecular evolution rates. The incongruity of molecular evolution rates in taxa on younger islands suggests that these rates may not be determined by growth form (or lifespan) alone, but may be influenced by complex dispersal events.