RESUMEN
The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Fabaceae/química , Guanidinas/farmacología , Leucemia de Células T/metabolismo , Monoterpenos/farmacología , Extractos Vegetales/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Fluoresceínas/metabolismo , Guanidinas/química , Guanidinas/aislamiento & purificación , Humanos , Leucemia de Células T/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Monoterpenos/química , Monoterpenos/aislamiento & purificación , Fitoterapia , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Relación Estructura-Actividad , Verapamilo/farmacología , Verapamilo/uso terapéuticoRESUMEN
The vector-mediated introduction of cDNA into mammalian cells by calcium phosphate co-precipitation or permeation with lipofectamine is widely used for the integration of cDNA into genomic DNA. Such integration, however, of cDNA occurs randomly at unpredictable sites in the host's chromosomal DNA, and the number of integrated recombinant DNAs is not controllable. To overcome this problem, we developed the Flp-In method to integrate one single copy of cDNA encoding the human ABC transporter ABCG2 into FRT-tagged genomic DNA. Examination of more than 20 metaphase spreads for both fluorescence in situ hybridization (FISH) mapping and multicolor-FISH analysis revealed that ABCG2 cDNA was incorporated into the telomeric region of the short arm on one of chromosomes 12 in Flp-In-293 cells. By using those cells, we investigated the effect of genetic polymorphisms and post-translational modifications of human ABC transporter ABCG2 on the protein expression and degradation. On the basis of our experience, it has been concluded that the Flp recombinase system provides a useful tool to quantitatively analyze the protein stability and endoplasmic reticulum (ER)-associated degradation of proteins like the ABC transporter. This system is also applicable for similar studies of the biogenesis of other proteins using the secretory pathway as well as proteins with other cellular localizations.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , ADN Nucleotidiltransferasas/metabolismo , Expresión Génica , Marcación de Gen/métodos , Mutagénesis Insercional/genética , Mutación/genética , Proteínas de Neoplasias/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Humanos , Immunoblotting , Datos de Secuencia Molecular , Reproducibilidad de los ResultadosRESUMEN
Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.
Asunto(s)
Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Medicamentos bajo Prescripción/farmacocinética , Animales , Simulación por Computador , Árboles de Decisión , Aprobación de Drogas , Interacciones Farmacológicas , Humanos , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Medicamentos bajo Prescripción/efectos adversosRESUMEN
Drug-induced hepatotoxicity is one of the major problems encountered in drug discovery and development. Selection of a candidate compound for pre-clinical studies in the drug discovery process is a critical step that can determine the speed and expenditure of clinical development. Because inhibition of human adenosine triphosphate-binding cassette transporter ABCB11 (SPGP/bile salt export pump) has severe consequences, which include intrahepatic cholestasis and hepatotoxicity, resulting from exposure to toxic xenobiotics or drug interactions, in vitro screening methods are necessary for quantifying and characterizing the inhibition of ABCB11. In line with such initiatives, we developed methods for in vitro high-speed screening and quantitative structure-activity relationship (QSAR) analysis to investigate the interaction of ABCB11 with a variety of compounds. We identified one set of chemical fragmentation codes closely linked with inhibition of ABCB11. Furthermore, the high-speed screening method enables us to analyze the kinetics of ABCB11-inhibition by test compounds and to distinguish competitive and non-competitive inhibitors. Troglitazone and novobiocin were found to be competitive inhibitors to taurocholate, whereas porphyrins were non-competitive inhibitors. Kinetics-based classification of inhibitors is considered important to improve the accuracy of our QSAR analysis. The present mini-review addresses technical pitfalls and improvements for high-speed screening and QSAR analysis in the ABCB11 inhibition study.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Cromanos/farmacología , Evaluación Preclínica de Medicamentos , Humanos , Control de Calidad , Relación Estructura-Actividad Cuantitativa , Tiazolidinedionas/farmacología , TroglitazonaRESUMEN
Impacts of genetic polymorphisms of the ATP-binding cassette (ABC) transporter BCRP/MXR1/ABCP (ABCG2) on drug response have been implicated; however, the hitherto reported data involve some inconsistencies. To re-evaluate the effect of single nucleotide polymorphisms (SNP) of ABCG2 in vitro, we created a total of seven variant cDNAs (V12M, Q141K, F208S, S248P, F431L, S441N and F489L) by site-directed mutagenesis and stably expressed each of them in Flp-In-293 cells using the Flp recombinase system. Multicolor fluorescence in situ hybridization mapping analysis revealed that one single copy of ABCG2 cDNA was incorporated into the telomeric region of chromosome 12p. It was proven that mRNAs of those integrated ABCG2 variants were expressed evenly in Flp-In-293 cells. However, the protein expression levels varied among those variants. In particular, expression of the F208S and S441N variants was markedly low, suggesting the instability of these variant proteins. Drug resistance profiles of Flp-In-293 cells expressing two major SNP variants (V12M and Q141K) toward the drug SN-38 demonstrated that the IC50 value (drug concentrations producing a 50% reduction of cell growth) for Q141K was approximately 50% of that for wild type. The contributions of the minor SNP variants (F208S, S248P, F431L, S441N and F489L) to drug resistance toward SN-38, mitoxantrone, doxorubicin, daunorubicin or etoposide were significantly lower than wild type. Based on our functional validation, the above-mentioned non-synonymous polymorphisms as well as acquired mutants (R482G and R482T) of ABCG2 were classified into four groups. Furthermore, new camptothecin analogs synthesized by our research group had potent effects in circumventing ABCG2-mediated drug resistance without any influence from major non-synonymous polymorphisms.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Polimorfismo de Nucleótido Simple/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/clasificación , Adenosina Trifosfatasas/metabolismo , Camptotecina/química , Camptotecina/farmacología , Línea Celular , Cromosomas Humanos/genética , ADN Complementario/genética , Resistencia a Medicamentos , Expresión Génica/efectos de los fármacos , Genoma Humano/genética , Glutamina/genética , Glutamina/metabolismo , Humanos , Hibridación Fluorescente in Situ , Estructura Molecular , Mutación/genética , Proteínas de Neoplasias/clasificación , Valina/genética , Valina/metabolismoRESUMEN
Drug-induced intrahepatic cholestasis is one of the major causes of hepatotoxicity, which often occur during the drug discovery and development process. Human ATP-binding cassette transporter ABCB11 (sister of P-glycoprotein/bile salt export pump) mediates the elimination of cytotoxic bile salts from liver cells to bile, and, therefore, plays a critical role in the generation of bile flow. The authors have recently developed in vitro high-speed screening and quantitative structure-activity relationship analysis methods to investigate the interaction of ABCB11 with a variety of compounds. Based on the extent of inhibition of the bile salt export pump, the authors analysed the quantitative structure-activity relationship to identify chemical groups closely associated with the inhibition of ABCB11. This approach provides a new tool to predict compounds with a potential risk of drug-induced intrahepatic cholestasis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Colestasis Intrahepática/inducido químicamente , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Relación Estructura-Actividad Cuantitativa , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Ácidos y Sales Biliares/antagonistas & inhibidores , Ácidos y Sales Biliares/metabolismo , Colestasis Intrahepática/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Humanos , Preparaciones Farmacéuticas/química , Valor Predictivo de las PruebasRESUMEN
(-)-Epigallocatechin gallate (EGCG) induces cell death of osteoclasts in an Fe(2+)- and H(2)O(2)-dependent manner. In the present study, we further explore the cytotoxic mechanism of EGCG using four EGCG analogues. Molecules methylated at position 4' in the B ring (EGCG-4'-O-Me) or at position 4'' in the D-ring (EGCG-4''-O-Me) showed markedly decreased cytotoxicity to osteoclasts, indicating that hydroxyl groups at these two positions of EGCG are crucial for inducing cell death of osteoclasts. EGCG-4'-O-Me also showed the lowest Fe(3+)-reducing activity among five EGCGs. The Fe(3+)-reducing activity of EGCG was enhanced under conditions whereby protonated EGCG levels were increased, indicating that the protonated status of EGCG was involved in the Fe(3+)-reducing activity. The hydroxyl group at position 4'' in the D-ring was shown by quantum chemical calculation to be preferentially deprotonated among all of the hydroxyl groups in EGCGs. It was also shown that the highest occupied molecular orbital (HOMO) was localized to the B-ring of EGCGs, except for EGCG-4'-O-Me. We report here that the HOMO on the B-ring plays crucial roles in both the Fe(3+)-reducing activity of EGCG and the cytotoxicity of EGCG to osteoclasts, while deprotonation of the hydroxyl group at position 4'' in the D-ring plays a supplementary role.
Asunto(s)
Catequina/análogos & derivados , Osteoblastos/química , Animales , Animales Recién Nacidos , Catequina/análisis , Catequina/química , Catequina/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Hierro/química , Masculino , Ratones , Osteoblastos/efectos de los fármacos , Oxidación-ReducciónRESUMEN
The vector-mediated introduction of cDNA into mammalian cells by calcium phosphate co-precipitation or permeation with lipofectamine is widely used for the integration of cDNA into genomic DNA. However, integration of cDNA into the host's chromosomal DNA occurs randomly at unpredictable sites, and the number of integrated recombinant DNAs is not controllable. To investigate the effect of genetic polymorphisms of ABCG2 on the protein expression and the drug resistance profile, we developed the Flp-In method to integrate one single copy of ABCG2 variant-cDNA into FRT-tagged genomic DNA. More than 20 metaphase spreads were examined for both fluorescence in situ hybridization (FISH) mapping and multicolor-FISH analysis, and it has been revealed that ABCG2 cDNA was incorporated into the telomeric region of the short arm on one of chromosomes 12 in Flp-In-293 cells. Based on the currently available SNP data for human ABCG2, we have created a total of seven variants by site-directed mutagenesis and stably expressed them in Flp-In-293 cells. While mRNAs of those integrated ABCG2 variants and wild type were evenly expressed in Flp-In-293 cells, the protein expression levels of F208S and S441N variants were found to be markedly low. It is suggested that the protein instability due to enhanced degradation resulted in the low levels of their protein expression. Thus, the Flp recombinase system would provide a useful tool to validate the effect of nonsynonymous SNPs on the protein stability and post-translational modification of ABCG2.