RESUMEN
Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.
Asunto(s)
Fenotiazinas/química , Fenotiazinas/farmacología , Empalmosomas/efectos de los fármacos , Empalmosomas/metabolismo , Empalme Alternativo , Humanos , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Precursores del ARN/genética , Precursores del ARN/metabolismo , Factores de Empalme de ARN/química , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Empalmosomas/genética , Factor de Empalme U2AF/química , Factor de Empalme U2AF/genética , Factor de Empalme U2AF/metabolismoRESUMEN
Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Osteoblastos/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína p53 Supresora de Tumor/genética , Antineoplásicos/química , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/genética , Caspasa 7/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Descubrimiento de Drogas , Células HEK293 , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Especificidad de Órganos , Osteoblastos/metabolismo , Osteoblastos/patología , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Estaurosporina/farmacología , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/deficienciaRESUMEN
A palladium(II)-catalyzed norbornene-mediated regioselective alkylation at the C-2 indole position of N-tert-butyloxycarbonyl (Boc)-protected (S)-tryptophan ethyl ester is reported. The protocol employs mild reaction conditions and is tolerant of a range of functional groups. The reaction proceeds without racemization at the stereogenic center of the amino acid.
Asunto(s)
Norbornanos/química , Compuestos Organometálicos/química , Paladio/química , Triptófano/química , Alquilación , Catálisis , Estructura Molecular , EstereoisomerismoRESUMEN
Step-economical syntheses of annulated 1,2,3-triazoles were accomplished through copper-catalyzed intramolecular direct arylations in sustainable one-pot reactions. Thus, catalyzed cascade reactions involving [3 + 2]-azide-alkyne cycloadditions (CuAAC) and C-H bond functionalizations provided direct access to fully substituted 1,2,3-triazoles with excellent chemo- and regioselectivities. Likewise, the optimized catalytic system proved applicable to the direct preparation of 1,2-diarylated azoles through a one-pot C-H/N-H arylation reaction.
RESUMEN
The ruthenium(II) carboxylate complex [Ru(O(2)CMes)(2)(p-cymene)] enabled efficient direct arylations of unactivated C-H bonds with easily available, inexpensive phenols. Extraordinary chemoselectivity of the well-defined ruthenium catalyst set the stage for challenging C-H/C-O bond functionalizations to occur under solvent-free conditions as well as in water, and allowed first direct C-H bond arylations with user-friendly diaryl sulfates as electrophiles.
Asunto(s)
Ácidos Carboxílicos/química , Fenoles/química , Rutenio/química , Catálisis , Cimenos , Estructura Molecular , Monoterpenos/química , Agua/químicaRESUMEN
Regioselective syntheses of 1,4,5-trisubstituted 1,2,3-triazoles were accomplished by three different strategies, relying on (i) the interception of stoichiometrically formed 5-cuprated-1,2,3-triazoles, (ii) the use of stoichiometrically functionalized alkynes or (iii) catalytic C-H bond functionalizations. This perspective article summarizes progress in this research area until June 2010.