RESUMEN
Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Organofosfonatos/farmacología , Fosfopiruvato Hidratasa/antagonistas & inhibidores , Pirrolidinonas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Organofosfonatos/química , Fosfopiruvato Hidratasa/metabolismo , Pirrolidinonas/química , Relación Estructura-ActividadRESUMEN
Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Unión al ADN/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Histonas/química , Isoxazoles/química , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Biotinilación , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Isoxazoles/síntesis química , Isoxazoles/farmacología , Cinética , Ligandos , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Docilidad , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfonamidas/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacología , metaminobenzoatos/síntesis química , metaminobenzoatos/química , metaminobenzoatos/farmacologíaRESUMEN
Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapeutics for many diseases, including cancer, in clinical trials. One PARP inhibitor, olaparib (Lynparza, AstraZeneca), was recently approved by the FDA to treat ovarian cancer with mutations in BRCA genes. BRCA1 and BRCA2 have essential roles in repairing DNA double-strand breaks, and a deficiency of BRCA proteins sensitizes cancer cells to PARP inhibition. Here we show that the receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907 (PARP1 pTyr907 or pY907). PARP1 pY907 increases PARP1 enzymatic activity and reduces binding to a PARP inhibitor, thereby rendering cancer cells resistant to PARP inhibition. The combination of c-Met and PARP1 inhibitors synergized to suppress the growth of breast cancer cells in vitro and xenograft tumor models, and we observed similar synergistic effects in a lung cancer xenograft tumor model. These results suggest that the abundance of PARP1 pY907 may predict tumor resistance to PARP inhibitors, and that treatment with a combination of c-Met and PARP inhibitors may benefit patients whose tumors show high c-Met expression and who do not respond to PARP inhibition alone.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Anilidas/farmacología , Animales , Bencimidazoles/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Crizotinib , Humanos , Técnicas In Vitro , Indoles/farmacología , Células MCF-7 , Ratones , Trasplante de Neoplasias , Fosforilación/efectos de los fármacos , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Pirazoles/farmacología , Piridinas/farmacología , Quinolinas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias del Colon/metabolismo , Proteína Adaptadora GRB2/metabolismo , Sistema de Señalización de MAP Quinasas , Neoplasias de la Próstata/metabolismo , Multimerización de Proteína , Femenino , Proteína Adaptadora GRB2/química , Células HEK293 , Humanos , Masculino , Fosforilación , Transducción de Señal , Proteínas Son Of Sevenless/metabolismo , Análisis de Matrices Tisulares , Tirosina/metabolismo , Dominios Homologos srcRESUMEN
Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines IL-4 and IL-13 and is activated in allergic airway disease. We are developing phosphopeptide mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues on IL-4 or IL-13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of genes contributing to asthma. Structure-affinity relationship studies showed that phosphopeptides based on Tyr631 from IL-4Rα bind with weak affinity to STAT6, whereas replacing the pY+3 residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set of phosphatase-stable, cell-permeable prodrug analogues inhibited cytokine-stimulated STAT6 phosphorylation in both Beas-2B human airway cells and primary mouse T-lymphocytes at concentrations as low as 100 nM. IL-13-stimulated expression of CCL26 (eotaxin-3) was inhibited in a dose-dependent manner, demonstrating that targeting the SH2 domain blocks both phosphorylation and transcriptional activity of STAT6.
Asunto(s)
Fosfopéptidos/farmacología , Factor de Transcripción STAT6/efectos de los fármacos , Dominios Homologos src/efectos de los fármacos , Animales , Asma/genética , Linfocitos T CD4-Positivos/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-13/biosíntesis , Interleucina-4/biosíntesis , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Profármacos , Ratas , Receptores de Interleucina-3/efectos de los fármacos , Receptores de Interleucina-4/efectos de los fármacos , Relación Estructura-Actividad , Activación Transcripcional/efectos de los fármacos , Tirosina/química , Tirosina/metabolismoRESUMEN
Members of the plakophilin-catenin sub-family (Pkp-1, -2, and -3) facilitate the linkage of desmosome junctional components to each other (e.g. desmosomal cadherins to desmoplakin) and the intermediate-filament cytoskeleton. Pkps also contribute to desmosomal stabilization and the trafficking of its components. The functions of Pkps outside of the desmosome are less well studied, despite evidence suggesting their roles in mRNA regulation, small-GTPase modulation (e.g. mid-body scission) during cell division, and cell survival following DNA damage. Pkp-catenins are further believed to have roles in the nucleus given their nuclear localization in some contexts and the known nuclear roles of structurally related catenins, such as beta-catenin and p120-catenin. Further, Pkp-catenin activities in the nuclear compartment have become of increased interest with the identification of interactions between Pkp2-catenin and RNA Pol III and Pkp1 with single-stranded DNA. Consistent with earlier reports suggesting possible nuclear roles in development, we previously demonstrated prominent nuclear localization of Pkp3 in Xenopus naïve ectoderm ("animal cap") cells and recently resolved a similar localization in mouse embryonic stem cells. Here, we report the association and positive functional interaction of Pkp3 with a transcription factor, Ets variant gene 1 (ETV1), which has critical roles in neural development and prominent roles in human genetic disease. Our results are the first to report the interaction of a sequence-specific transcription factor with any Pkp. Using Xenopus laevis embryos and mammalian cells, we provide evidence for the Pkp3:ETV1 complex on both biochemical and functional levels.