Structure-activity relationships of triazolopyridine oxazole p38 inhibitors: identification of candidates for clinical development

The synthesis, structure-activity relationship, in vivo activity, and metabolic profile for a series of triazolopyridine-oxazole based p38 inhibitors are described. The deficiencies of the lead structure in the series, CP-808844, were overcome by changes to the C4 aryl group and the triazole side-chain culminating in the identification of several potential clinical candidates.     

Early detection of voice diseases via a web-based system

Voice is the result of the coordination of the whole pneumophonoarticulatory apparatus. The analysis of the voice allows the identification of the diseases of the vocal apparatus and currently is carried out from an expert doctor through methods based on the auditory analysis. The paper presents a web-based system for the acquisition and automatic analysis of vocal signals. Vocal signals are submitted by the users through a simple web-interface and are analyzed in real-time by using state-of-the art signal processing techniques, providing first-level information on possible voice alterations. The system offers different analysis functions to the doctors that may analyze suspected cases in detail. The system is currently being tested in the otorhinolaryngologist setting to carry out mass prevention via screening at a regional scale.     

Salivary gland-specific gene expression in the malaria vector Anopheles gambiae

Molecular studies on the tissue-specific gene expression in the salivary glands of Anopheles gambiae may provide useful tools for the development of new strategies for the control of the most efficient malaria vector in the sub-Saharan Africa. We summarize here the results of a recent investigation focused on the isolation of secreted factors and putative receptors from the salivary glands of An. gambiae. Using the Signal Sequence Trap technique we have identified the first cDNAs specifically expressed in the An. gambiae salivary glands. Among these, four are exclusively expressed in female glands and encode factors presumably involved in blood-feeding, whereas two other cDNAs seem to be expressed both in male and in female glands and are likely implicated in sugar-feeding. Homologues of genes previously identified in the yellow fever mosquito Aedes aegypti, like the apyrase and D7, as well as novel salivary gland-specific cDNAs, were identified. The isolation and characterization of promoter sequences from the corresponding genes may prove useful for the expression of anti parasitic agents in the salivary glands of transgenic mosquitoes.     

Identification of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of Met kinase

An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in in vitro potency. The amide moiety could be replaced with acylurea and malonamide substituents to give compounds with improved potency in the Met-driven GTL-16 human gastric carcinoma cell line. Acylurea pyrrolotriazines with substitution at C-5 demonstrated single digit nanomolar kinase activity. X-ray crystallography revealed that the C-5 substituted pyrrolotriazines bind to the Met kinase domain in an ATP-competitive manner.     

Identification of symbiotically defective mutants of Lotus japonicus affected in infection thread growth

During the symbiotic interaction between legumes and rhizobia, the host cell plasma membrane and associated plant cell wall invaginate to form a tunnel-like infection thread, a structure in which bacteria divide to reach the plant root cortex. We isolated four Lotus japonicus mutants that make infection pockets in root hairs but form very few infection threads after inoculation with Mesorhizobium loti. The few infection threads that did initiate in the mutants usually did not progress further than the root hair cell. These infection-thread deficient (itd) mutants were unaffected for early symbiotic responses such as calcium spiking, root hair deformation, and curling, as well as for the induction of cortical cell division and the arbuscular mycorrhizal symbiosis. Complementation tests and genetic mapping indicate that itd2 is allelic to Ljsym7, whereas the itdl, itd3, and itd4 mutations identified novel loci. Bacterial release into host cells did occur occasionally in the itdl, itd2, and itd3 mutants suggesting that some infections may succeed after a long period and that infection of nodule cells could occur normally if the few abnormal infection threads that were formed reached the appropriate nodule cells.     

Green fluorescent protein-based system for analysis of E-selectin-mediated adhesion

Numerous cell-based or cell-free systems for study of selectin adhesion use radiolabeled tracers. However, in addition to handling problems associated with the use of radioisotopes, these assays have difficulty relating a number of counts to a number of adherent cells. Here, we describe an assay that uses the natural fluorescence of the green fluorescent protein (GFP) to measure binding of cells to E-selectin. We elaborated an adhesion system composed of a cell monolayer expressing E-selectin ligand to which monodispersed fluorescent Chinese hamster ovary (CHO) cells expressing E-selectin are added. Due to GFP autofluorescence, adhered cells can be easily distinguished from cell monolayers by fluorescence microscopy, and adhesion can be measured by cytofluorometry. We applied this GFP-based adhesion assay to measure the adherence of a pancreatic tumor cell line and found that the binding parameters of these cells satisfy a number of E-selectin-specific criteria.     

The cytosolic and transmembrane domains of the beta 1,6 N-acetylglucosaminyltransferase (C2GnT) function as a cis to medial/Golgi-targeting determinant

The beta 1,6 N-acetylglucosaminyltransferase (C2GnT) has been recently mapped to the cis/medial-Golgi compartment. To analyze the Golgi-targeting determinants of C2GnT, we constructed various deletion mutants of the enzyme fused to the enhanced green fluorescent protein (EGFP) and localized these proteins by fluorescence microscopy in living cells. We found that the N-terminal peptide encompassing amino acids 1 to 32 represents the minimal Golgi-targeting signal sufficient to localize EGFP to the same compartment as the full-length C2GnT. This peptide makes up the cytoplasmic and the transmembrane domains of the enzyme and was referred to as CTd (cytoplasmic and transmembrane domains). We compared the Golgi-targeting efficiency of the C2GnT-derived CTd with its homologous domains from other glycosyltransferases, including the H-type alpha(1,2)-fucosyltransferase (FucTI), the polypeptide N-acetylgalactosaminyltransferase-I (GalNAcT-I), the alpha(1,3)-fucosyltransferase VII (FucTVII), and the alpha(2,6)-sialyltransferase (ST6Gal-I) and found that the Golgi-targeting determinants of these glycosyltransferases were also composed of their cytosolic and transmembrane domains. To investigate whether the CTd of C2GnT could serve as a cis to medial Golgi-specific signal, we tested its ability to mislocalize two late-Golgi acting glycosyltransferases FucTI and FucTVII. We show that fusing the C2GnT-derived CTd with the catalytic domain of FucTVII resulted in a complete mislocalization of the enzyme to the C2GnT compartment, with a parallel alteration of sialyl-Lewis x synthesis and P-selectin binding. The intracellular distribution and activity of FucTI, however, were not affected. Thus, CTds of either early or late-Golgi acting glycosyltransferases represent the Golgi-targeting domains of these enzymes. In addition, we show that C2GnT-derived CTd can function as a cis/medial Golgi-targeting determinant.