RESUMEN
The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.
Asunto(s)
Oligonucleótidos Antisentido/farmacología , Oligonucleótidos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Sinergismo Farmacológico , Ensayos Analíticos de Alto Rendimiento , Humanos , Membranas Intracelulares/efectos de los fármacos , Ratones , Ratones Transgénicos , Oligonucleótidos/análisis , ARN Interferente Pequeño/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/toxicidadRESUMEN
A series of racemic balanol analogues with modification of the benzamido moiety of balanol have been synthesized and evaluated for their inhibitory activities against human protein kinase C isozymes (PKC-alpha, -beta I, -beta II, -gamma, -delta, -epsilon, and -eta). The structural modification includes replacement of the 4-hydroxyphenyl group with variously substituted phenyl rings, substitution of the amide linkage with a sulfonamide or an ester, and replacement of the 4-hydroxyphenyl substructure with a hydroxyl substituted indole or a hydroxybenzyl group. in general, these analogues were found to be less potent than balanol, but a number of analogues were identified with improved isozyme selectivity. The structure-activity relationship studies of these analogues also indicated that (1) the optimal general PKC inhibition requires a free 4-hydroxyl group in the benzamido portion of the molecule, (2) the amide linkage of the benzamido moiety is important for PKC inhibition, and (3) the conformation associated with the benzamido moiety seems to have a profound effect on PKC inhibition. The requirement of a free 4-hydroxyl group in conjunction with an appropriate conformation of the benzamido moiety for optimal PKC inhibition suggests that the 4-hydroxyphenyl group may be involved in a specific inhibitor-enzyme interaction important for PKC inhibition.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Animales , Baculoviridae/genética , Inhibidores Enzimáticos/química , Humanos , Isoenzimas/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , Proteína Quinasa C/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Espectrofotometría Infrarroja , Spodoptera , Relación Estructura-ActividadRESUMEN
Balanol is a potent protein kinase C (PKC) inhibitor that is structurally composed of a benzophenone diacid, a 4-hydroxybenzamide, and a perhydroazepine ring. A number of balanol analogs in which the perhydroazepine moiety is replaced have been synthesized and their biological activities evaluated against both PKC and cAMP-dependent kinase (PKA). The results suggested that the activity and the isozyme/kinase selectivity of these compounds are largely related to the conformation about this nonaromatic structural element of the molecules.
Asunto(s)
Azepinas/síntesis química , Azepinas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Hidroxibenzoatos/síntesis química , Hidroxibenzoatos/farmacología , Isoenzimas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Conformación Molecular , Relación Estructura-ActividadRESUMEN
Both isotopic and nonisotopic assay methodologies are employed in high-throughput screening for drug discovery. Recent advances in cell-based and in vitro biochemical assays will be reviewed, with special emphasis on detection technologies amenable to automated 'mix and read' procedures in high-throughput screening. A major trend is the advent of homogenous assay systems which employ fluorescence resonance energy transfer, fluorescence polarization, and fluorescence correlation spectroscopy. Cell-based assay systems have also become popular in high-throughput screens in which active compounds that directly modulate the disease target are identified. Colorimetric and amperometric methods have also been described recently, but are yet to be adapted widely in high-throughput screens.
Asunto(s)
Diseño de Fármacos , Preparaciones Farmacéuticas/análisis , Colorimetría/métodos , Transferencia de Energía , Radioquímica/métodos , Espectrometría de Fluorescencia/métodosRESUMEN
The oligomeric stilbenes (+)-alpha-viniferin (1), miyabenol C (2), and kobophenol A (3) have been isolated from Caragana sinica (Buchoz) Rehd (Leguminosae). (+)-alpha-Viniferin (1) and miyabenol C (2) exhibited protein kinase C inhibitory activity at low micromolar concentrations. (+)-alpha-Viniferin inhibited keratinocyte proliferation (0.4 microM) and free radical release in whole blood (47 microM), in vitro, and may be useful in treating hyperproliferative or inflammatory skin diseases.
Asunto(s)
Medicamentos Herbarios Chinos/química , Proteína Quinasa C/antagonistas & inhibidores , Estilbenos/farmacología , Células Cultivadas , Medicamentos Herbarios Chinos/farmacología , Humanos , Espectroscopía de Resonancia Magnética , Estilbenos/aislamiento & purificaciónRESUMEN
Betulinic acid [1] and platanic acid [2], isolated from the leaves of Syzigium claviforum, were found to be inhibitors of HIV replication in H9 lymphocyte cells. Evaluation of anti-HIV activity with eight derivatives of 1 revealed that dihydrobetulinic acid [3] was also a potent inhibitor of HIV replication. The C-3 hydroxy group and C-17 carboxylic acid group, as well as the C-19 substituents, contribute to enhanced anti-HIV activity. The inhibitory activity of these compounds against protein kinase C (PKC) was also examined, since a correlation between anti-HIV and anti-PKC activities has been suggested. However, there was no apparent correlation between anti-HIV activity and the inhibition of PKC among these compounds.
Asunto(s)
Antivirales/aislamiento & purificación , VIH-1/efectos de los fármacos , Plantas Medicinales/química , Triterpenos/aislamiento & purificación , Antivirales/farmacología , Células Cultivadas , VIH-1/fisiología , Humanos , Linfocitos/microbiología , Triterpenos Pentacíclicos , Proteína Quinasa C/antagonistas & inhibidores , Triterpenos/farmacología , Replicación Viral/efectos de los fármacos , Ácido BetulínicoRESUMEN
We have previously shown that some ellagitannins are potent inhibitors of protein kinase C (PKC). On the basis of this finding, several series of hexahydroxybiphenyl derivatives of ellagic acid were synthesized as simple analogs of these ellagitannins and were evaluated for their inhibitory effect against PKC. Compounds 23 and 26 were found to be potent inhibitors of PKC, while hexakis-(benzyloxy)biphenyl derivatives exhibited weak anti-PKC activity.
Asunto(s)
Compuestos de Bifenilo/síntesis química , Dibenzoxepinas/síntesis química , Ácido Elágico/análogos & derivados , Proteína Quinasa C/antagonistas & inhibidores , Compuestos de Bifenilo/farmacología , Dibenzoxepinas/farmacología , Ácido Elágico/síntesis química , Ácido Elágico/química , Ácido Elágico/farmacología , Humanos , Estructura Molecular , Proteínas Recombinantes/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
A novel series of diaminoanthraquinones was discovered initially as protein kinase C inhibitors with IC50s in the 50-100 microM range. They exhibited potent tumor cell growth inhibitory activity in vitro without cross resistance to adriamycin. Further evaluation of two of the most active compounds NSC 639365 (3) and NSC 639366 (4) in human tumor cloning assay showed potent cytocidal activity. The results suggest therapeutical potentials against human tumors.
Asunto(s)
Antraquinonas/síntesis química , Antineoplásicos/síntesis química , Antraquinonas/química , Antraquinonas/uso terapéutico , División Celular/efectos de los fármacos , Humanos , Proteína Quinasa C/antagonistas & inhibidores , Células Tumorales Cultivadas/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
The effect of altering intracellular free Ca2+ on juvenile hormone (JH) and acid synthesis by larval and pupally-committed corpora allata (CA) of fifth stadium Manduca sexta was investigated. Larval CA required extracellular Ca2+ greater than or equal to 0.1 mM for maximal JH synthesis, while JH acid synthesis by glands after pupal commitment was independent of extracellular Ca2+. Free Ca2+ in the hemolymph ranged from 1.4 to 2.1 mM during the fifth stadium. Both calcium ionophores and caffeine, which releases Ca2+ from intracellular stores, inhibited JH synthesis by larval CA but stimulated JH acid synthesis by post-commitment CA. These results suggest that intracellular stores may be the principal source of Ca2+ for the biosynthetic activity of the post-commitment gland. Calcium channel blockers (La3+, Cd2+) and antagonists (verapamil, isradipine and nitrendipine) decreased both JH and JH acid synthesis, indicating the existence of Ca2+ channels in the CA cell membrane. Calmodulin (CaM) antagonists inhibited the activity of both larval and post-commitment CA, suggesting an integral relationship of CaM to the effects of Ca2+ on gland activity. One of these effects is the demonstrated requirement of 0.1 mM extracellular Ca2+ for allatostatin inhibition of JH I synthesis by larval CA.
Asunto(s)
Calcio/metabolismo , Corpora Allata/metabolismo , Proteínas de Insectos , Hormonas Juveniles/biosíntesis , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Calmodulina/metabolismo , Membrana Celular/metabolismo , Corpora Allata/efectos de los fármacos , Hemolinfa/química , Larva/efectos de los fármacos , Larva/metabolismo , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/metabolismo , Péptidos/fisiología , Ácido Pirrolidona Carboxílico/análogos & derivados , RadioinmunoensayoRESUMEN
When an in vitro assay system and radioimmunoassays specific for juvenile hormones (JH) I and III were used to probe the effect of day 4 last instar larval brains on JH synthesis by day 0 last instar larval corpora allata (CA) of the tobacco hornworm, Manduca sexta, a selective inhibition of JH I synthesis by the CA was observed. The nature of this inhibition suggested the presence of an allatostatin specific for the synthesis of JH I. Its occurrence in the day 4 brain was demonstrated by the ability of a crude brain extract to inhibit the CA in a dose-dependent manner. The allatostatic factor (ASF) appears to be a protein, based on its heat lability and pronase sensitivity, and it has apparent molecular weights of 6.8 and 13 kDa. Inhibition of JH I synthesis occurs within 1 min of exposure of the CA to the factor and is reversible by 6 h after this exposure. Thus it appears that a cerebral neuropeptide specifically inhibiting JH I synthesis by the CA is present in Manduca on day 4 of the last larval instar, a time when the hemolymph titer of JH must drop to ensure the occurrence of pupal commitment.
Asunto(s)
Corpora Allata/fisiología , Hormonas Juveniles/biosíntesis , Lepidópteros/fisiología , Mariposas Nocturnas/fisiología , Neuropéptidos/farmacología , Sistemas Neurosecretores/fisiología , Animales , Corpora Allata/efectos de los fármacos , Larva , Técnicas de Cultivo de Órganos , Sesquiterpenos/biosíntesisRESUMEN
When an in vitro assay system and radioimmunoassays specific for juvenile hormones (JH) I and III were used to probe the effect of co-incubating pupal brains with last instar larval corpora allata (CA) from the tobacco hornworm, Manduca sexta, a selective activation of JH III synthesis by the CA was observed. This homolog-specific activation suggested the presence of an allatotropic factor for the synthesis of JH III (JH III ATF), and its presence was demonstrated by the ability of a postmicrosomal supernatant of a day 0 pupal brain homogenate to activate the CA in vitro in a dose-dependent manner. This moiety appears to be a protein, based on its heat lability and protease sensitivity, and has an apparent molecular size of 40 kD and an isoelectric point of 5.5 JH III ATF activity is localized in specific neural tissues of the day 0 pupa, the brain and first three abdominal ganglia, with the brain containing 4 times the activity in the ganglia. The existence of this factor suggests that JH III synthesis by the CA of Manduca is regulated by a neuropeptide.