RESUMEN
We used small molecule screening to discover compounds and mechanisms for overcoming E6 oncogene-mediated drug resistance. Using high-throughput screening in isogenic cell lines, we identified compounds that potentiate doxorubicin's lethality in E6-expressing colon cancer cells. Such compounds included quaternary ammonium salts, protein synthesis inhibitors, 11-deoxyprostaglandins, and two additional classes of compounds-analogs of 1,3-bis(4-morpholinylmethyl)-2-imidazolidinethione (a thiourea) and acylated secondary amines that we named indoxins. Indoxins upregulated topoisomerase IIalpha, the target of doxorubicin, thereby increasing doxorubicin lethality. We developed a photolabeling strategy to identify targets of indoxin and discovered a nuclear actin-related protein complex as a candidate indoxin target.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas Oncogénicas Virales/metabolismo , Alprostadil/análogos & derivados , Alprostadil/química , Alprostadil/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular , Doxorrubicina/farmacología , Etilenotiourea/análogos & derivados , Etilenotiourea/química , Etilenotiourea/farmacología , Colorantes Fluorescentes , Humanos , Proteínas Oncogénicas Virales/genética , Compuestos Orgánicos/química , Compuestos Orgánicos/farmacología , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/farmacologíaRESUMEN
Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.
Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Piperazinas/farmacología , Transducción de Señal/efectos de los fármacos , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Quinasas raf/metabolismo , Proteínas ras/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , Fosforilación/efectos de los fármacos , Piperazinas/toxicidad , Sensibilidad y EspecificidadRESUMEN
Developing small molecule inhibitors of oncoproteins, which are activated in tumor cells, is a newly popular strategy for cancer-related drug discovery. The complementary strategy, developing small molecules that restore the function of missing tumor suppressors, is much more difficult to realize. However, in this issue of Cancer Cell, Kau et al. report the discovery of small molecules that reverse some cellular consequences of the loss of the tumor suppressor PTEN.
Asunto(s)
Anticarcinógenos/farmacocinética , Proteínas de Unión al ADN/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores Citoplasmáticos y Nucleares , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Anticarcinógenos/metabolismo , Proteínas de Peces , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead , Humanos , Carioferinas/metabolismo , Metabolismo de los Lípidos , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Supresoras de Tumor/metabolismo , Proteína Exportina 1RESUMEN
Chemical genetics is an emerging approach for studying biological systems using chemical tools. This strategy aims to reveal the macromolecules responsible for regulating biological systems; thus, the approach shares much in common with genetics. In both strategies, one must (a) develop an assay that reports on a biological process of interest, (b) perturb this process systematically (with mutations or small molecules), and (c) determine the target of each perturbation to reveal macromolecules (i.e., proteins and genes) regulating the process of interest. In this review, we discuss advances and challenges in this field that have emerged over the past four years. Several technologies have converged, raising the hope that it may be possible to systematically apply chemical probes to biological processes.
Asunto(s)
Biología Molecular/tendencias , Técnicas Químicas Combinatorias , Biblioteca de Genes , Ligandos , Sustancias Macromoleculares , Biblioteca de Péptidos , FenotipoRESUMEN
Synthetic methodology was developed to construct amino acid-[2]rotaxanes that have phenylalanine and 3,5-di-tert-butylbenzene as blocking groups and dibenzo-24-crown-8, derivatized with either N-acetylargininyl or a carboxylic group, as the ring. A relative measure of the intramolecular interaction energies between the functional groups in DMSO/water mixtures is obtained by comparing their pK(a) values. Rotaxane structures were investigated through 2D NMR analysis and molecular dynamics simulations. Association constants for complexes of amino acids and rotaxanes in various protonation states were determined in a variety of solvent systems by (1)H NMR analysis. The unique intracomponent interactions that exist in the rotaxanes and their ability to act as artificial receptors are discussed.
Asunto(s)
Aminoácidos/química , Química Orgánica/métodos , Catálisis , Simulación por Computador , Éteres Cíclicos/química , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Fenilalanina/química , Rotaxanos , Relación Estructura-ActividadRESUMEN
A host-[2]rotaxane was constructed by converting a diaminophenylcalix[4]arene into a [2]rotaxane using the DCC-rotaxane method (Zehnder, D.; Smithrud, D. B. Org. Lett. 2001, 16, 2485-2486). N-Ac-Arg groups were attached to the dibenzo-24-crown-8 ring of the rotaxane to provide a convergent functional group. To demonstrate the advantage provided by the rotaxane architecture for recognition of guests that contain a variety of functional groups, association constants (K(A)) for N-Ac-Trp, indole, N-Ac-Gly, fluorescein, 1-(dimethylamino)-5-naphthalenesulfonate, and pyrene bound to the [2]rotaxane were determined by performing (1)H NMR and fluorescence spectroscopic experiments. The host-[2]rotaxane had the highest affinity for fluorescein with a K(A) = 4.6 x 10(6) M(-)(1) in a 98/2 buffer (1 mM phosphate, pH 7)/DMSO solution. A comparison of K(A) values demonstrates that both the aromatic pocket and ring of the host-[2]rotaxane contribute binding free energy for complexation. Association constants were also derived for the same guests bound to the diaminophenylcalix[4]arene and to a diphenylcalix[4]arene that contained arginine residues displayed in a nonconvergent fashion. The host-[2]rotaxane provides higher affinity and specificity for most guests than the host with divergent N-Ac-Arg groups of the one that only has an aromatic pocket. For example, the K(A) for the complex of the host-[2]rotaxane and fluorescein in the DMSO/water mixture is more than 2 orders of magnitude greater than association constants derived for the other hosts.