RESUMEN
In order to obtain compounds with modified 2-APB activities, we synthesized number of 2-APB analogues and analyzed their inhibitory activities for SOCE. The IC50 of 2-APB for SOCE inhibition is 3 µM while IC50 of some of our 2-APB analogues range 0.1-10 µM. The adducts of amino acids with diphenyl borinic acid have strong inhibitory activities. By using these compounds, we will be able to regulate intracellular Ca(2+) concentration and consequent cellular processes more efficiently than with 2-APB.
Asunto(s)
Compuestos de Boro/química , Compuestos de Boro/farmacología , Señalización del Calcio/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Células CHO , Cricetulus , Cardiopatías/tratamiento farmacológico , Cardiopatías/metabolismoRESUMEN
Ras proteins are of importance in cell proliferation, and hence their mutated forms play causative roles in many kinds of cancer in different tissues. Inhibition of the Ras-depalmitoylating enzyme acyl protein thioesterases APT1 and -2 is a new approach to modulating the Ras cycle. Here we present boronic and borinic acid derivatives as a new class of potent and nontoxic APT inhibitors. These compounds were detected by extensive library screening using chemical arrays and turned out to inhibit human APT1 and -2 in a competitive mode. Furthermore, one of the molecules was demonstrated to inhibit Erk1/2 phosphorylation significantly.
Asunto(s)
Boro/química , Boro/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Tioléster Hidrolasas/antagonistas & inhibidores , Animales , Boro/toxicidad , Perros , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/toxicidad , Humanos , Lipoilación/efectos de los fármacos , Células de Riñón Canino Madin Darby , Proteínas ras/antagonistas & inhibidores , Proteínas ras/metabolismoRESUMEN
Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expansion of the polyglutamine (polyQ) stretch in huntingtin (htt). Previously, it has been shown that inhibition of the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) activity reduced aggregation of pathogenic polyQ proteins. Experimentally, this effect was achieved by modification of the intracellular IP3 levels or by application of IP3R1 inhibitors, such as 2-aminoethyl diphenylborinate (2-APB). Unfortunately, there are certain concerns about the 2-APB specificity and cytotoxicity. Moreover, a direct link between IP3R1 and polyQ aggregation has not been shown yet. In this study we show, that down-regulation of the IP3R1 levels by shRNA reduced the aggregation of mutant htt. We tested 2-APB analogs in an attempt to identify less toxic and more IP3R1-specific compounds and found that the effect of these analogs on the reduction of the mutant htt aggregation did weakly correlate with their inhibitory action toward the IP3-induced Ca(2+) release (IICR). Their effect on aggregation was not correlated with the store-operated Ca(2+) entry (SOCE), which is another target of the 2-APB related compounds. Our findings suggest that besides functional contribution of the IP3R inhibition on the mutant htt aggregation there are additional mechanisms for the anti-aggregation effect of the 2-APB related compounds.
Asunto(s)
Compuestos de Boro/química , Compuestos de Boro/farmacología , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Péptidos/metabolismo , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Técnicas de Silenciamiento del Gen , Proteína Huntingtina , Masculino , Ratones , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , Bibliotecas de Moléculas PequeñasRESUMEN
Potent transglutaminase inhibitors were obtained from disulfide compounds, cystamine, dimethyl cystine, and dimethyl homocystine. The disulfide bond and thiophene ring play an important role in inhibitory activity of synthesized aryl ß-amino ketones.
Asunto(s)
Inhibidores Enzimáticos/química , Cetonas/química , Transglutaminasas/antagonistas & inhibidores , Disulfuros/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Cetonas/síntesis química , Cetonas/farmacología , Relación Estructura-Actividad , Tiofenos/química , Transglutaminasas/metabolismoRESUMEN
Store-operated calcium entry (SOCE) is an important mechanism for replenishing intracellular calcium stores and for sustaining calcium signaling. We developed a method for synthesis of bisboron compounds that have two borinic acids or their esters in one molecule. These compounds are analogues of 2-APB, which is widely used as a membrane-permeable SOCE inhibitor. Further, we examined the effect of the newly synthesized bisboron compounds on SOCE in Jurkat T cells. All the bisboron compounds showed strong inhibitory activity on SOCE, with IC50 values of less than 1 microM, which were 20-45 times lower than observed with 2-APB.
Asunto(s)
Ácidos Borínicos/síntesis química , Compuestos de Boro/síntesis química , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Ácidos Borínicos/química , Ácidos Borínicos/farmacología , Compuestos de Boro/química , Compuestos de Boro/farmacología , Canales de Calcio , Humanos , Concentración 50 Inhibidora , Células Jurkat , Estructura Molecular , Transducción de Señal/efectos de los fármacosRESUMEN
Aryl beta-aminoethyl ketones were discovered as potent inhibitors of tissue transglutaminase. Heteroaryl-like thiophene groups and N-benzyl N-t-butyl aminoethyl group are critical to the strong inhibitory activity of aryl beta-aminoethyl ketones.
Asunto(s)
Cetonas/química , Transglutaminasas/antagonistas & inhibidores , Animales , Cobayas , Cetonas/metabolismo , Cetonas/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Transglutaminasas/metabolismoRESUMEN
Store-operated calcium entry (SOCE) or calcium release-activated calcium current (I(CRAC)) is a critical pathway to replenish intracellular calcium stores, and plays indispensable roles in cellular functions such as antigen-induced T lymphocyte activation. Despite the importance of I(CRAC) in cellular functions, lack of potent and specific inhibitor has limited the approaches to the function of I(CRAC) in native cells. 2-Aminoethyl diphenylborinate (2-APB) is a widely used SOCE/I(CRAC) inhibitor, while its effect is rather unspecific. In the attempt to develop more potent and selective compounds here we identified two structurally isomeric 2-APB analogues that are 100-fold more potent than 2-APB itself. One of the 2-APB analogues activates and inhibits endogenous SOCE depending on the concentration while the other only inhibits it. The 2-APB analogue inhibits store depletion-mediated STIM1 clustering as well as heterologously expressed CRAC current. Together with the observation that, unlike 2-APB, the analogue compounds failed to activate CRACM3/Orai3 current in the absence of STIM, our results suggest that inhibition and activation of SOCE/I(CRAC) by the 2-APB analogues is mediated by STIM.
Asunto(s)
Compuestos de Boro/farmacología , Señalización del Calcio/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Compuestos de Boro/química , Células CHO , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Pollos , Cricetinae , Cricetulus , Células HeLa , Humanos , Activación del Canal Iónico/fisiología , Células Jurkat , Masculino , Proteínas de la Membrana/genética , Ratones , Proteínas de Neoplasias/genética , Técnicas de Placa-Clamp , Molécula de Interacción Estromal 1RESUMEN
Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles.
Asunto(s)
Compuestos de Boro/administración & dosificación , Canales de Calcio/efectos de los fármacos , Calcio/metabolismo , Membrana Celular/metabolismo , Activación del Canal Iónico/fisiología , Animales , Células CHO , Canales de Calcio/fisiología , Membrana Celular/efectos de los fármacos , Cricetinae , Cricetulus , Activación del Canal Iónico/efectos de los fármacos , Tasa de Depuración Metabólica/efectos de los fármacosRESUMEN
Intracellular calcium [Ca(2+)](i) is mobilized in many cell types in response to activation of phosphoinositide (PIP(n)) signaling pathways involving PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3). To further explore the relationship between increases in intracellular PIP(n) concentrations and mobilization of [Ca(2+)](i), each of the seven phosphorylated phosphoinositides (PIP(n)s) were delivered into cells and the metabolism and physiological effects of the exogenously administered PIP(n)s were determined. The efficient cellular delivery of fluorophore-tagged and native PIP(n)s was accomplished using histone protein, neomycin, and dendrimeric polyamines. PtdIns(4,5)P(2) fluorophore-tagged analogs with short- and long-acyl chains were substrates for cellular enzymes in vitro and for phospholipases in stimulated fibroblasts. PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2), each induced calcium mobilization rapidly after exogenous addition to fibroblasts. PtdIns(3,4,5)P(3) induced a significant, but smaller increase in intracellular calcium. These observations suggest that PIP(n)s other than PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) may have direct roles in signaling involving [Ca(2+)](i).
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Líquido Intracelular/metabolismo , Fosfatidilinositoles/metabolismo , Transducción de Señal/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Colorantes Fluorescentes , Histonas/metabolismo , Histonas/farmacología , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/farmacología , Ratones , Células 3T3 NIH , Fosfatidilinositoles/química , Fosfatidilinositoles/farmacología , Fosforilación , Estructura Terciaria de Proteína/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Adrenal medullary (AM) cells are exposed to high concentrations of cortical hormones, one of which is a ouabain-like substance. Thus, the effects of ouabain on catecholamine secretion and distribution of Na+,K+-ATPase alpha and beta subunits in rat and guinea-pig AM cells were examined using amperometry and immunological techniques. While exposure to 1 microm ouabain did not have a marked effect on resting secretion, it induced an increase in secretion due to mobilization of Ca2+ ions that were stored during a 4 min interval between muscarine applications. Immunocytochemistry revealed that Na+,K+-ATPase alpha1 subunit-like and beta3 subunit-like immunoreactive (IR) materials were distributed ubiquitously at the cell periphery, whereas alpha2- and beta2-like IR materials were present in restricted parts of the cell periphery. The alpha1 and alpha2 subunits were mainly immunoprecipitated from AM preparations by anti-beta3 and anti-beta2 antisera, respectively. Peripheral BODIPY-FL-InsP3 binding sites were localized below membrane domains with alpha2- and beta2-like IR materials. The results indicate that in AM cells, alpha1beta3 isozymes of Na+,K+-ATPase were present ubiquitously in the plasma membrane, while alpha2beta2 isozymes were in the membrane domain closely associated with peripheral Ca2+ store sites. This close association of the alpha2beta2 isozyme with peripheral Ca2+ store sites may account for the facilitation of mobilization-dependent secretion in the presence of 1 microm ouabain.
Asunto(s)
Médula Suprarrenal/enzimología , Células Cromafines/enzimología , Subunidades de Proteína/química , Subunidades de Proteína/fisiología , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Médula Suprarrenal/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Células Cromafines/efectos de los fármacos , Cobayas , Isoenzimas/química , Isoenzimas/fisiología , Ouabaína/farmacología , RatasRESUMEN
The localization and function of Ca(2+) stores in isolated chromaffin cells of rat adrenal medulla were investigated using confocal laser microscopy and amperometry. Binding sites for BODIPY-inositol 1,4,5-trisphosphate (IP(3)), -ryanodine (Ry), and -thapsigargin (Thap) were both perinuclear and at the cell periphery. The endoplasmic reticulum (ER), which was identified by ER Tracker dye, took up fluorescent Ry and IP(3), and the majority of BODIPY-Ry-binding area was bound by fluorescent IP(3). Under Ca(2+)-free conditions, the amount of caffeine-induced catecholamine secretion was 33% of that of muscarine-induced secretion, but muscarine induced little or no secretion after exposure to caffeine. Muscarine-induced Ca(2+) increases, as observed with fluo-3, lasted for a few tens of seconds under Ca(2+)-free conditions, whereas a caffeine-induced Ca(2+) transient diminished rapidly with a half decay time of 3s and this spike-like Ca(2+) transient was then followed by a sustained increase with a low level. These results indicate that IP(3) receptors and Ry receptors (RyRs) are present in common ER Ca(2+) storage and the lower potency of caffeine for secretion may be due to a rapid decrease in RyR channel activity to a low level.