RESUMEN
The synthesis of a new library of 5-arylidenethiazolidinone compounds using an efficient three component reaction with thiazolidine-2,4-dione, piperidine and appropriate aldehydes is reported. This reaction is excellently high yielding, tolerant towards a variety of aldehydes and provides access to these compounds in a single step (in comparison to low yielding multistep syntheses reported in the literature). Once the reaction is complete, the desired product precipitates out of the reaction mixture and is isolated by filtration and purified by washing and recrystallization. These compounds revealed anti-proliferative activities against human breast cancer cells (MCF7 and MDA). Phenotypic profiling established the most active compound 17i (EC50 = 4.52 µM) as an apoptotic agent. A novel chemical proteomics approach identified ß-actin-like protein 2, γ-enolase and macrophage migration inhibitory factor (MMIF) as putative cellular binding partners of 17i.
Asunto(s)
Apoptosis/efectos de los fármacos , Piperidinas/química , Tiazolidinas/síntesis química , Tiazolidinas/farmacología , Técnicas de Química Sintética , Humanos , Indicadores y Reactivos/química , Células MCF-7 , Modelos Moleculares , Conformación Molecular , Tiazolidinas/químicaRESUMEN
Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD(+) synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD(+) pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, ß-lapachone (ß-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. ß-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD(+) consumption. Synergy with FK866+ß-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (â¼85%) of PDA cases. This treatment strategy simultaneously decreases NAD(+) synthesis while increasing NAD(+) consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)(+) depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, µ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD(+)-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD(+) synthesis and ß-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD(+) pools in cancer cells to sensitize them to ROS-mediated cell death by ß-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors.
Asunto(s)
Naftoquinonas/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Poli Adenosina Difosfato Ribosa/metabolismo , Acrilamidas/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Neoplasias Pancreáticas/enzimología , Piperidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias PancreáticasRESUMEN
The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract. The enteric toxins of C. perfringens share two common features: (1) they are all single polypeptides of modest (approximately 25-35 kDa) size, although lacking in sequence homology, and (2) they generally act by forming pores or channels in plasma membranes of host cells. These enteric toxins include C. perfringens enterotoxin (CPE), which is responsible for the symptoms of a common human food poisoning and acts by forming pores after interacting with intestinal tight junction proteins. Two other C. perfringens enteric toxins, epsilon-toxin (a bioterrorism select agent) and beta-toxin, cause veterinary enterotoxemias when absorbed from the intestines; beta- and epsilon-toxins then apparently act by forming oligomeric pores in intestinal or extra-intestinal target tissues. The action of a newly discovered C. perfringens enteric toxin, beta2 toxin, has not yet been defined but precedent suggests it might also be a pore-former. Experience with other clostridial toxins certainly warrants continued research on these C. perfringens enteric toxins to develop their potential as therapeutic agents and tools for cellular biology.
Asunto(s)
Toxinas Bacterianas/toxicidad , Clostridium perfringens/metabolismo , Enterotoxinas/toxicidad , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/química , Enterotoxinas/biosíntesis , Enterotoxinas/química , Humanos , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
We have determined the binding affinity for binding of the four purine nucleoside triphosphates GTP, ITP, XTP, and ATP to E-site nucleotide- and nucleoside diphosphate kinase-depleted tubulin. The relative binding affinities are 3000 for GTP, 10 for ITP, 2 for XTP, and 1 for ATP. Thus, the 2-exocyclic amino group in GTP is important in determining the nucleotide specificity of tubulin and may interact with a hydrogen bond acceptor group in the protein. The 6-oxo group also makes a contribution to the high affinity for GTP. NMR ROESY experiments indicate that the four nucleotides have different average conformations in solution. ATP and XTP are characterized by a high anti conformation, ITP by a medium anti conformation, and GTP by a low anti conformation. Possibly, the preferred solution conformation contributes to the differences in affinities. When the tubulin E-site is saturated with nucleotide, there appears to be little difference in the ability of the four nucleotides to stimulate assembly. The critical protein concentration is essentially identical in reactions using the four nucleotides. All four of the nucleotides were hydrolyzed during the assembly reaction, and the NDPs were incorporated into the microtubule. We also examined the binding of two gamma-phosphoryl-modified GTP photoaffinity analogues, p(3)-1, 4-azidoanilido-GTP and p(3)-1,3-acetylanilido-GTP. These analogues are inhibitors of the assembly reaction and bind to tubulin with affinities that are 15- and 50-fold lower, respectively, than the affinty for GTP. The affinity of GTP is less sensitive to substitutions at the gamma-phosphoryl position that to changes in the purine ring.
Asunto(s)
Nucleótidos de Purina/metabolismo , Tubulina (Proteína)/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Inosina Trifosfato/química , Inosina Trifosfato/metabolismo , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , Nucleótidos de Purina/química , Ribonucleótidos/química , Ribonucleótidos/metabolismoRESUMEN
BALB/c mice resolve Leishmania donovani infection in the liver over an 8-12-week period. However, after an initial phase of 2-4 weeks where increases in parasite load are not readily detectable, parasite numbers in the spleen begin to increase reaching maximum levels at 16 weeks post-infection. Thereafter, parasite replication in the spleen is controlled and BALB/c mice maintain this residual parasite load in the spleen for many months, without further increase. We evaluated functions of CD11C+ splenic dendritic cells throughout the course of L. donovani infection in the spleen of BALB/c mice. Unlike the dendritic cell (DC)-specific antigen DEC-205, CD11C was not up-regulated on macrophages during visceral leishmaniasis. No appreciable impairment of splenic DC functions was observed when this antigen-presenting cell subset was purified from 30-day post-infected mice. Significant impairment in inducing allogeneic mixed lymphocyte reaction (MLR) and presenting L. donovani antigens or keyhole limpet haemocyanin (KLH) to specific T cells was observed with CD11C+ splenic DC purified from 60-day post-infected mice. Functional impairment of splenic DC at 60 days post-infection correlated with their reduced surface expression of major histocompatibility complex (MHC) class II molecules, impairment of interleukin-12 (IL-12) production and to their ability to suppress interferon-gamma (IFN-gamma) production by Leishmania antigen-primed T cells. Of interest, the impairment of splenic DC in presenting Leishmania antigens or KLH to specific T cells was corrected at 120 days post-infection, and correlated with their up-regulation of MHC class II expression, IL-12 production, induction of IFN-gamma by Leishmania antigen-primed T cells and the onset of control over splenic parasite replication in vivo. These results indicate that functional integrity of DC may be important in controlling L. donovani infection.
Asunto(s)
Células Dendríticas/inmunología , Integrina alfaXbeta2/análisis , Leishmania donovani/crecimiento & desarrollo , Leishmaniasis Visceral/inmunología , Bazo/inmunología , Animales , Presentación de Antígeno , Antígenos Bacterianos/inmunología , Técnicas de Cultivo de Célula , Antígenos de Histocompatibilidad Clase II/metabolismo , Interleucina-12/metabolismo , Leishmaniasis Visceral/parasitología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Bazo/parasitología , Staphylococcus aureus/inmunologíaRESUMEN
We recently reported that Leishmania donovani infect the human T-cell line in vitro. To examine whether primary human T cells could be infected by this parasite, a direct interaction of the peripheral blood T cells with L. donovani was examined. The percentage of gamma/delta+ T cells was markedly increased when in vitro generated normal human T-cell blasts were cultured with L. donovani amastigotes. About 30% of the gamma/delta+ T cells in the parasite exposed T-cell blasts expressed parasite antigens intracellularly without detectable intracellular parasites. Parasite exposed T-cell blasts had a reduced surface expression of HLA-DR and were lysed by the sorted CD56+ cells. In contrast, neither L. donovani amastigotes nor T-cell blasts exposed to heat killed amastigotes and/or were sensitive to the NK cell-mediated lysis. Of interest is that about 10% CD3+ peripheral blood T cells in two out of three Indian Kala-azar patients tested expressed intracellular L. donovani antigens.
Asunto(s)
Células Asesinas Naturales/inmunología , Leishmania donovani/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos de Protozoos/análisis , Antígenos de Protozoos/inmunología , Complejo CD3/análisis , Antígeno CD56/análisis , Citotoxicidad Inmunológica , Humanos , Líquido Intracelular/inmunología , Leishmania donovani/crecimiento & desarrollo , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/inmunología , Activación de LinfocitosRESUMEN
In isolated preparations of the urinary bladder detrusor of the house musk shrew Suncus murinus (order: insectivora; family: Soricidae), electrical field stimulation (0.5-32 pulses/s) evoked neurogenic contractile responses that were markedly attenuated by atropine (1 microM). The non-cholinergic component was reduced but not abolished by the P2-purinoceptor antagonist, suramin (300 microM). Thus, neuromuscular transmission in the suncus urinary bladder is effected by cholinergic and purinergic nerves together with an as-yet unidentified component. Using immunohistochemical methods, the suncus urinary bladder was seen to be supplied by nerves containing neuropeptide Y, tyrosine hydroxylase, vasoactive intestinal polypeptide, galanin, substance P, calcitonin gene-related peptide and type I nitric oxide synthase. The pattern of responses to electrical field stimulation was more similar to that of humans and Old World primates, than to that of rodents or lagomorphs. The pattern of innervation of the bladder wall, in terms of the distribution of populations containing a given neuropeptide, was very similar to that in humans. Hence, Suncus murinus may provide a novel species for modelling the neuropharmacology of the human bladder, and also for studying the evolution of autonomic innervation.