RESUMO
This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos , Proliferação de Células , Ensaios de Triagem em Larga Escala , Transdução de Sinais , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Sinalização YAP/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Proliferação de Células/efeitos dos fármacos , Camundongos , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Linhagem Celular Tumoral , Fosfoproteínas/metabolismo , Fosfoproteínas/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Descoberta de Drogas , Camundongos Nus , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Fenótipo , Relação Estrutura-Atividade , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
BACKGROUND: Several pathogenic bacteria utilize receptors of the CEACAM family to attach to human cells. Binding to different members of this receptor family can result in uptake of the bacteria. Uptake of Neisseria gonorrhoeae, a gram-negative human pathogen, via CEACAMs found on epithelial cells, such as CEACAM1, CEA or CEACAM6, differs mechanistically from phagocytosis mediated by CEACAM3, a CEACAM family member expressed selectively by human granulocytes. PRINCIPAL FINDINGS: We find that CEACAM1- as well as CEACAM3-mediated bacterial internalization are accompanied by a rapid increase in phosphatidylinositol-3,4,5 phosphate (PI(3,4,5)P) at the site of bacterial entry. However, pharmacological inhibition of phosphatidylinositol-3' kinase (PI3K) selectively affects CEACAM1-mediated uptake of Neisseria gonorrhoeae. Accordingly, overexpression of the PI(3,4,5)P phosphatase SHIP diminishes and expression of a constitutive active PI3K increases CEACAM1-mediated internalization of gonococci, without influencing uptake by CEACAM3. Furthermore, bacterial uptake by GPI-linked members of the CEACAM family (CEA and CEACAM6) and CEACAM1-mediated internalization of N. meningitidis by endothelial cells require PI3K activity. Sensitivity of CEACAM1-mediated uptake toward PI3K inhibition is independent of receptor localization in cholesterol-rich membrane microdomains and does not require the cytoplasmic or the transmembrane domain of CEACAM1. However, PI3K inhibitor sensitivity requires the Ig(C2)-like domains of CEACAM1, which are also present in CEA and CEACAM6, but which are absent from CEACAM3. Accordingly, overexpression of CEACAM1 Ig(C2) domains blocks CEACAM1-mediated internalization. CONCLUSIONS: Our results provide novel mechanistic insight into CEACAM1-mediated endocytosis and suggest that epithelial CEACAMs associate in cis with other membrane receptor(s) via their extracellular domains to trigger bacterial uptake in a PI3K-dependent manner.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Espaço Extracelular/metabolismo , Regiões Constantes de Imunoglobulina/química , Neisseria gonorrhoeae/metabolismo , Neisseria meningitidis/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Aderência Bacteriana , Linhagem Celular , Endocitose , Células Endoteliais/microbiologia , Células Endoteliais/patologia , Interações Hospedeiro-Patógeno , Humanos , Inositol Polifosfato 5-Fosfatases , Microdomínios da Membrana/metabolismo , Proteínas Mutantes/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Monoéster Fosfórico Hidrolases/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Defence mechanisms against intracellular bacterial pathogens are incompletely understood. Our study characterizes a type I IFN-dependent cell-autonomous defence pathway directed against Legionella pneumophila, an intracellular model organism and frequent cause of pneumonia. We show that macrophages infected with L. pneumophila produced IFNß in a STING- and IRF3- dependent manner. Paracrine type I IFNs stimulated upregulation of IFN-stimulated genes and a cell-autonomous defence pathway acting on replicating and non-replicating Legionella within their specialized vacuole. Our infection experiments in mice lacking receptors for type I and/or II IFNs show that type I IFNs contribute to expression of IFN-stimulated genes and to bacterial clearance as well as resistance in L. pneumophila pneumonia in addition to type II IFN. Overall, our study shows that paracrine type I IFNs mediate defence against L. pneumophila, and demonstrates a protective role of type I IFNs in in vivo infections with intracellular bacteria.