siRNA screen identifies QPCT as a druggable target for Huntington's disease.

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

Innovative Reversed-Phase Chromatography Platform Approach for the Fast and Accurate Characterization of Membrane Vesicles' Protein Patterns.

Outer membrane vesicles (OMVs) have been widely explored to develop vaccine candidates for bacterial pathogens due to their ability to combine adjuvant properties with immunogenic activity. OMV expresses a variety of proteins and carbohydrate antigens on their surfaces. For this reason, there is an analytical need to thoroughly characterize the species expressed at their surface: we here present a simple and accurate reversed-phase ultrahigh-performance liquid chromatography (RP-UPLC) method developed according to quality by design principles. This work provides an analytical alternative to the classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization. The higher selectivity and sensitivity of the RP-UHPLC assay allow for the identification of additional protein species with respect to SDS-PAGE and facilitate its precise relative abundance quantification. According to validation results, the assay showed high accuracy, linearity, precision, repeatability, and a limit of quantification of 1% for less abundant proteins. This performance paves the way for improved production campaign consistency while also being analytically simple (no sample pretreatment required), making it suitable for routine quality control testing. In addition, the applicability of the assay to a wider range of vesicle classes (GMMA) was demonstrated.

Endothelial cell adhesion to the extracellular matrix induces c-Src-dependent VEGFR-3 phosphorylation without the activation of the receptor intrinsic kinase activity.

RATIONALE: Integrins cooperate with growth factor receptors to promote downstream signaling for cell proliferation and migration. However, the mechanism of receptor activation is still unknown. OBJECTIVE: To analyze the mechanism of phosphorylation of the vascular endothelial growth factor receptor (VEGFR)-3 by cell adhesion. METHODS AND RESULTS: We show that VEGFR-3 phosphorylation, induced by cell attachment to the extracellular matrix, is independent from the intrinsic kinase activity of the receptor, as evidenced from phosphorylation cell adhesion experiments with a mutant kinase dead receptor or in the presence of the specific kinase inhibitor MAZ 51. Cell adhesion experiments in the presence of the c-Src inhibitor PP2 or in fibroblast triple knockout for c-Src, Yes, and Fyn (SYF) demonstrate that VEGFR-3 phosphorylation, induced by extracellular matrix, is mediated by c-Src. Kinase assays in vitro with recombinant c-Src show that VEGFR-3 is a direct c-Src target and mass spectrometry analysis identified the sites phosphorylated by c-Src as tyrosine 830, 833, 853, 1063, 1333, and 1337, demonstrating that integrin-mediated receptor phosphorylation induces a phosphorylation pattern that is distinct from that induced by growth factors. Furthermore, pull-down assays show that integrin-mediated VEGFR-3 phosphorylation activates the recruitment to the receptor of the adaptor proteins CRKI/II and SHC inducing activation of JNK. CONCLUSIONS: These data suggest that cell adhesion to extracellular matrix induces a downstream signaling using the tyrosine kinase receptor VEGFR-3 as scaffold.

Novel Hits in the Correction of ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Protein: Synthesis, Pharmacological, and ADME Evaluation of Tetrahydropyrido[4,3-d]pyrimidines for the Potential Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is a lethal genetic disease caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) with a prevalence of the ΔF508 mutation. Whereas the detailed mechanisms underlying disease have yet to be fully elucidated, recent breakthroughs in clinical trials have demonstrated that CFTR dysfunction can be corrected by drug-like molecules. On the basis of this success, a screening campaign was carried out, seeking new drug-like compounds able to rescue ΔF508-CFTR that led to the discovery of a novel series of correctors based on a tetrahydropyrido[4,3-d]pyrimidine core. These molecules proved to be soluble, cell-permeable, and active in a disease relevant functional-assay. The series was then further optimized with emphasis on biological data from multiple cell systems while keeping physicochemical properties under strict control. The pharmacological and ADME profile of this corrector series hold promise for the development of more efficacious compounds to be explored for therapeutic use in CF.