RESUMEN
In this account, we report the development of a series of substituted cinnamic anilides that represents a novel class of mitochondrial permeability transition pore (mPTP) inhibitors. Initial class expansion led to the establishment of the basic structural requirements for activity and to the identification of derivatives with inhibitory potency higher than that of the standard inhibitor cyclosporine-A (CsA). These compounds can inhibit mPTP opening in response to several stimuli including calcium overload, oxidative stress, and thiol cross-linkers. The activity of the cinnamic anilide mPTP inhibitors turned out to be additive with that of CsA, suggesting for these inhibitors a molecular target different from cyclophylin-D. In vitro and in vivo data are presented for (E)-3-(4-fluoro-3-hydroxy-phenyl)-N-naphthalen-1-yl-acrylamide 22, one of the most interesting compounds in this series, able to attenuate opening of the mPTP and limit reperfusion injury in a rabbit model of acute myocardial infarction.
Asunto(s)
1-Naftilamina/análogos & derivados , Acrilamidas/química , Anilidas/química , Cinamatos/química , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Daño por Reperfusión Miocárdica/tratamiento farmacológico , 1-Naftilamina/síntesis química , 1-Naftilamina/química , 1-Naftilamina/farmacología , Acrilamidas/síntesis química , Acrilamidas/farmacología , Anilidas/síntesis química , Anilidas/farmacología , Animales , Calcio/metabolismo , Cinamatos/síntesis química , Cinamatos/farmacología , Femenino , Masculino , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Dilatación Mitocondrial/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Conejos , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Snake presynaptic neurotoxins with phospholipase A(2) activity are potent inducers of paralysis through inhibition of the neuromuscular junction. These neurotoxins were recently shown to induce exocytosis of synaptic vesicles following the production of lysophospholipids and fatty acids and a sustained influx of Ca(2+) from the medium. Here, we show that these toxins are able to penetrate spinal cord motor neurons and cerebellar granule neurons and selectively bind to mitochondria. As a result of this interaction, mitochondria depolarize and undergo a profound shape change from elongated and spaghetti-like to round and swollen. We show that snake presynaptic phospholipase A(2) neurotoxins facilitate opening of the mitochondrial permeability transition pore, an inner membrane high-conductance channel. The relative potency of the snake neurotoxins was similar for the permeability transition pore opening and for the phospholipid hydrolysis activities, suggesting a causal relationship, which is also supported by the effect of phospholipid hydrolysis products, lysophospholipids and fatty acids, on mitochondrial pore opening. These findings contribute to define the cellular events that lead to intoxication of nerve terminals by these snake neurotoxins and suggest that mitochondrial impairment is an important determinant of their toxicity.
Asunto(s)
Mitocondrias/metabolismo , Neuronas/metabolismo , Fosfolipasas A2/metabolismo , Animales , Calcio/química , Venenos Elapídicos/química , Ácidos Grasos/química , Hidrólisis , Lisofosfolípidos/química , Mitocondrias/efectos de los fármacos , Modelos Biológicos , Neurotoxinas/química , Permeabilidad , Ratas , Ratas Wistar , SerpientesRESUMEN
We have investigated the mitochondrial effects of BH3I-2', Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2' (half-maximal uncoupling at about 80 nm) > Chelerythrine (half-maximal uncoupling at about 2 microm) > HA14-1 (half-maximal uncoupling at about 20 microm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.
Asunto(s)
Barbitúricos/farmacología , Benzopiranos/farmacología , Ligandos , Proteínas Proto-Oncogénicas c-bcl-2/química , Alcaloides , Animales , Antineoplásicos/farmacología , Apoptosis , Barbitúricos/química , Benzofenantridinas , Benzopiranos/química , Caspasa 9 , Caspasas/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Clorambucilo/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Células HeLa , Humanos , Potenciales de la Membrana , Microscopía Fluorescente , Mitocondrias/metabolismo , Fenantridinas/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirimidinas/química , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Estaurosporina/farmacología , Factores de Tiempo , Vidarabina/análogos & derivados , Vidarabina/farmacología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Methylglyoxal and synthetic glyoxal derivatives react covalently with arginine residue(s) on the mitochondrial permeability transition pore (PTP). In this study, we have investigated how the binding of a panel of synthetic phenylglyoxal derivatives influences the opening and closing of the PTP. Using both isolated mitochondria and mammalian cells, we demonstrate that the resulting arginine-phenylglyoxal adduct can lead to either suppression or induction of permeability transition, depending on the net charge and hydrogen bonding capacity of the adduct. We report that phenylglyoxal derivatives that possess a net negative charge and/or are capable of forming hydrogen bonds induced permeability transition. Derivatives that were overall electroneutral and cannot form hydrogen bonds suppressed permeability transition. When mammalian cells were incubated with low concentrations of negatively charged phenylglyoxal derivatives, the addition of oligomycin caused a depolarization of the mitochondrial membrane potential. This depolarization was completely blocked by cyclosporin A, a PTP opening inhibitor, indicating that the depolarization was due to PTP opening. Collectively, these findings highlight that the target arginine(s) is functionally linked with the opening/closing mechanism of the PTP and that the electric charge and hydrogen bonding of the resulting arginine adduct influences the conformation of the PTP. These results are consistent with a model where the target arginine plays a role as a voltage sensor.
Asunto(s)
Arginina/química , Canales Iónicos/química , Animales , Calcio/química , Línea Celular Tumoral , Ciclosporina/química , Células HeLa , Humanos , Enlace de Hidrógeno , Membranas Intracelulares/metabolismo , Potenciales de la Membrana , Mitocondrias/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Dilatación Mitocondrial , Modelos Químicos , Permeabilidad , Fenilglioxal/química , Unión Proteica , Conformación Proteica , Ratas , Ratas Wistar , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de TiempoRESUMEN
Deoxynucleoside triphosphates (dNTPs) used for mitochondrial DNA replication are mainly formed by phosphorylation of deoxynucleosides imported into mitochondria from the cytosol. We earlier obtained evidence for a mitochondrial 5'-nucleotidase (dNT2) with a pronounced specificity for dUMP and dTMP and suggested that the enzyme protects mitochondrial DNA replication from excess dTTP. In humans, accumulation of dTTP causes a mitochondrial genetic disease. We now establish that dNT2 in vivo indeed is located in mitochondria. The native enzyme shows the same substrate specificity and affinity for inhibitors as the recombinant dNT2. We constructed ponasterone-inducible cell lines overproducing dNT2 with and without the green fluorescent protein (GFP) linked to its C terminus. The fusion protein occurred in mitochondria mostly in an inactive truncated form, with only a short C-terminal fragment of dNT2 linked to GFP. No truncation occurred when dNT2 and GFP were not linked. The cell mitochondria then contained a large excess of active dNT2 with or without the mitochondrial presequence. After removal of ponasterone overproduced dNT2 disappeared only slowly from the cells, whereas dNT2-mRNA was lost rapidly. Overproduction of dNT2 did not lead to an increased excretion of pyrimidine deoxyribonucleosides, in contrast to overproduction of the corresponding cytosolic deoxynucleotidase, suggesting that the mitochondrial enzyme does not affect overall cellular deoxynucleotide turnover.