RESUMO
VPS34 is a class III phosphoinositide 3-kinase involved in endosomal trafficking and autophagosome formation. Inhibitors of VPS34 were believed to have value as anticancer agents, but genetic and pharmacological data suggest that sustained inhibition of VPS34 kinase activity may not be well tolerated. Here we disclose the identification of a novel series of dihydropyrazolopyrazinone compounds represented by compound 5 as potent, selective, and orally bioavailable VPS34 inhibitors through a structure-based design strategy. A water-interacting hydrogen bond acceptor within an appropriate distance to a hinge-binding element was found to afford significant VPS34 potency across chemical scaffolds. The selectivity of compound 5 over PIK family kinases arises from interactions between the hinge-binding element and the pseudo-gatekeeper residue Met682. As recent in vivo pharmacology data suggests that sustained inhibition of VPS34 kinase activity may not be tolerated, structure-activity relationships leading to VPS34 inhibition may be helpful for avoiding this target in other ATP-competitive kinase programs.
Assuntos
Antineoplásicos , Classe III de Fosfatidilinositol 3-Quinases , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Autofagia , Endossomos , Fosfatidilinositol 3-Quinase/metabolismo , FosforilaçãoRESUMO
Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.
Assuntos
Proteínas Nucleares/metabolismo , Transdução de Sinais/fisiologia , Transducina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Sistema Livre de Células , Humanos , Camundongos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Transducina/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
Epithelial-mesenchymal transition (EMT) is an important mechanism for phenotypic conversion in normal development and disease states such as tissue fibrosis and metastasis. While this conversion of epithelia is under tight transcriptional control, few of the key transcriptional proteins are known. Fibroblasts produced by EMT express a gene encoding fibroblast-specific protein 1 (FSP1), which is regulated by a proximal cis-acting promoter element called fibroblast transcription site-1 (FTS-1). In mass spectrometry, chromatin immunoprecipitation, and siRNA studies, we used FTS-1 as a unique probe for mediators of EMT and identified a complex of 2 proteins, CArG box-binding factor-A (CBF-A) and KRAB-associated protein 1 (KAP-1), that bind this site. Epithelial cells engineered to conditionally express recombinant CBF-A (rCBF-A) activate the transcription of FSP1 and undergo EMT. The FTS-1 response element also exists in the promoters modulating a broader EMT transcriptome, including Twist, and Snail, as well as E-cadherin, beta-catenin, ZO 1, vimentin, alpha1(I) collagen, and alpha-smooth muscle actin, and the induction of rCBF-A appropriately alters their expression as well. We believe formation of the CBF-A/KAP-1/FTS-1 complex is sufficient for the induction of FSP1 and a novel proximal activator of EMT.