Characterization of the novel broad-spectrum kinase inhibitor CTx-0294885 as an affinity reagent for mass spectrometry-based kinome profiling.

Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.

Intensity of Nematode Infection in Children Aged 3 to 5 Years Living in Mukuru Kwa Njenga Slum Settlement, Nairobi, Kenya.

BACKGROUND: The burden of nematode infections is high mostly in children below 5 years old, with clinical manifestations ranging from mild to painful symptoms due to severe infections that end up suppressing the immune system of the infected children. The occurrence of these infections is highest in areas of extreme poverty. This study evaluated the intensity of nematode infections and assessed the status of deworming in children aged 3 to 5 years living in Mukuru slum settlement, Nairobi County, Kenya. Methodology. A total of 172 children aged between 3 and 5 years were sampled across the 5 major villages of Mukuru Slum settlement: Kwa Njenga, Vietnum, Wapewape, Kwa Reuben, and Motomoto. Community health workers administered questionnaires on the deworming history of children. Stool samples were collected, macroscopically examined, and microscopically analysed using Kato-Katz technique to assess the intensity of infection. The intensities of nematode infections were expressed as eggs per gram (epg) of faeces. RESULTS: The point prevalence of nematode infection among the 98 children in the 1st sampling was 25.5% with a mean infection intensity of 5424 epg, whereas among the 74 children sampled in 2nd sampling, 47.3% had nematode infection with a mean infection intensity of 12384 epg. The average nematode infection for the 172 participants was 34.9% with a mean intensity of 17808 epg. The highest number of children infected with nematodes was in the village of Wapewape where 34 participants were examined and 36.3% were infected with a mean intensity of 3216 epg. Kwa Reuben and Vietnum villages had the same prevalence values of 32.4% where 34 participants in each village had a mean intensity of 3624 epg and 4512 epg, respectively. In both samplings, more than 80% of children had been dewormed more than 6 months prior to the study. Ascaris lumbricoides was the only species of intestinal nematodes identified to be present in the stool samples of children in this study, whereas Trichuris trichiura and hookworm infections were found to be absent. The intensity of infection was not dependent on age or gender.

Development of a novel high-throughput assay for the investigation of GlyT-1b neurotransmitter transporter function.

The glycine transporter (GlyT-1b) is a Na(+)/Cl(-)-dependent electrogenic transporter which mediates the rapid re-uptake of glycine from the synaptic cleft. Based on its tissue distribution, GlyT-1 has been suggested to co-localise with the NMDA receptor where it may modulate the concentration of glycine at its co-agonist binding site. This data has led to GlyT-1 inhibitors being proposed as targets for disorders such as schizophrenia and cognitive dysfunction. Radiolabelled uptake assays (e.g. [(3)H]glycine) have been traditionally used in compound screening to identify glycine transporter inhibitors. While such an assay format is useful for testing limited numbers of compounds, the identification of novel glycine uptake inhibitors requires a functional assay compatible with high-throughput screening (HTS) of large compound libraries. Here, the authors present the development of a novel homogenous cell-based assay using the FLIPR membrane potential blue dye (Molecular Devices) and FLEXstation. Pharmacological data for the GlyT-1 inhibitors Org 24598 and ALX 5407 obtained using this novel electrogenic assay correlated well with the conventional [(3)H]-glycine uptake assay format. Furthermore, the assay has been successfully miniaturised using FLIPR(3) and therefore has the potential to be used for high-throughput screening.