RESUMEN
The malaria parasite Plasmodium falciparum has at least five putative histone deacetylase (HDAC) enzymes, which have been proposed as new antimalarial drug targets and may play roles in regulating gene transcription, like the better-known and more intensively studied human HDACs (hHDACs). Fourteen new compounds derived from l-cysteine or 2-aminosuberic acid were designed to inhibit P. falciparum HDAC-1 (PfHDAC-1) based on homology modeling with human class I and class II HDAC enzymes. The compounds displayed highly potent antiproliferative activity against drug-resistant (Dd2) or drug sensitive (3D7) strains of P. falciparum in vitro (50% inhibitory concentration of 13 to 334 nM). Unlike known hHDAC inhibitors, some of these new compounds were significantly more toxic to P. falciparum parasites than to mammalian cells. The compounds inhibited P. falciparum growth in erythrocytes at both the early and late stages of the parasite's life cycle and caused altered histone acetylation patterns (hyperacetylation), which is a marker of HDAC inhibition in mammalian cells. These results support PfHDAC enzymes as being promising targets for new antimalarial drugs.
Asunto(s)
Aminoácidos Dicarboxílicos/farmacología , Antimaláricos/farmacología , Cisteína/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Plasmodium falciparum/efectos de los fármacos , Aminoácidos Dicarboxílicos/química , Animales , Antimaláricos/química , Cisteína/análogos & derivados , Cisteína/química , Resistencia a Medicamentos , Eritrocitos/parasitología , Humanos , Modelos Moleculares , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/química , Plasmodium falciparum/enzimología , Homología de Secuencia de AminoácidoRESUMEN
Here we describe the rational design, computer-aided virtual ligand docking and synthesis of 19 nonpeptidic compounds designed to inhibit histone deacetylases and kill melanoma cells. Compounds were derived from cysteine, fused at the S-terminus to 4-butanoyl hydroxamate, and at the N-terminus to 4-(dimethylamino)benzoic acid. The latter was extended by coupling to amines to form a small library of prospective anti-cancer compounds. Four compounds were cytotoxic at sub-micromolar concentrations against cells of a particularly aggressive human melanoma (MM96L), and nine compounds showed selectivities of >or=5:1 for killing human melanoma instead of normal human fibroblast cells. The most active compounds were shown to cause hyperacetylation of histones due to inhibition of histone deacetylases. Further refinement of these compounds may produce an anti-tumor drug suitable for treating melanoma.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Cisteína/farmacología , Inhibidores Enzimáticos/farmacología , Antineoplásicos/síntesis química , Línea Celular Tumoral , Cisteína/análogos & derivados , Cisteína/síntesis química , Diseño de Fármacos , Fibroblastos/citología , Fibroblastos/metabolismo , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Melanoma/patología , Modelos QuímicosRESUMEN
We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F(1)F(0)-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F(1) and F(0) sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F(0) sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F(0) sector, most likely mediated through altered protein-protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F(1)-alpha(3)beta(3) hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur "at a distance." We consider how subtle conformational changes in OSCP may be transmitted to F(0).
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Sustitución de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Cartilla de ADN/genética , ADN de Hongos/genética , Estabilidad de Enzimas , Variación Genética , Potencial de la Membrana Mitocondrial , Proteínas de la Membrana/química , ATPasas de Translocación de Protón Mitocondriales/química , Mutagénesis Sitio-Dirigida , Fosforilación Oxidativa , Consumo de Oxígeno , Conformación Proteica , Subunidades de Proteína , Protones , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Oligomycin has long been known as an inhibitor of mitochondrial ATP synthase, putatively binding the F(o) subunits 9 and 6 that contribute to proton channel function of the complex. As its name implies, OSCP is the oligomycin sensitivity-conferring protein necessary for the intact enzyme complex to display sensitivity to oligomycin. Recent advances concerning the structure and mechanism of mitochondrial ATP synthase have led to OSCP now being considered a component of the peripheral stator stalk rather than a central stalk component. How OSCP confers oligomycin sensitivity on the enzyme is unknown, but probably reflects important protein-protein interactions made within the assembled complex and transmitted down the stator stalk, thereby influencing proton channel function. We review here our studies directed toward establishing the stoichiometry, assembly, and function of OSCP in the context of knowledge of the organization of the stator stalk and the proton channel.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Oligomicinas/farmacología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Animales , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Modelos Moleculares , Complejos Multiproteicos , Subunidades de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologíaRESUMEN
By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3-kDa subunit g of bovine mitochondrial F1F0-ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0-ATP synthase in the atp20-null strain was not affected in the absence of subunit g and levels of oligomycin-sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0-ATP synthase from mitochondrial lysates prepared from atp20-null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well-characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20-null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20-null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0-ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.