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Toshihisa Ishikawa

Publications and source records attributed to Toshihisa Ishikawa.

At least 19 recordsLinked to original sources
PubMed2007

Prediction of drug-induced intrahepatic cholestasis: in vitro screening and QSAR analysis of drugs inhibiting the human bile salt export pump.

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Identification of two biologically crucial hydroxyl groups of (-)-epigallocatechin gallate in osteoclast culture.

(-)-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.

Animals↗
PubMed2006

Functional validation of the genetic polymorphisms of human ATP-binding cassette (ABC) transporter ABCG2: identification of alleles that are defective in porphyrin transport.

The ATP-binding cassette (ABC) transporter ABCG2 has been implicated to play a significant role in the response of patients to medication and/or the risk of diseases. To clarify the possible physiological or pathological relevance of ABCG2 polymorphisms, we have functionally validated single nucleotide polymorphisms (SNP) of ABCG2. In the present study, based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Because porphyrins are considered to be endogenous substrates for ABCG2, we have investigated the porphyrin transport activity of those variant forms in vitro. We herein provide evidence that the variants Q126stop, F208S, S248P, E334stop, and S441N are defective in porphyrin transport, whereas F489L exhibited impaired transport, approximately 10% of the activity observed for the wild type. Furthermore, Flp-In-293 cells expressing those variants were photosensitive. Thus, among those genetic polymorphisms of ABCG2, at least the hitherto validated alleles of Q126stop, S441N, and F489L are suggested to be of clinical importance related to the potential risk of porphyria.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

A new strategy of high-speed screening and quantitative structure-activity relationship analysis to evaluate human ATP-binding cassette transporter ABCG2-drug interactions.

The human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR1/ABCP) plays a critical role in cellular protection against xenobiotics as well as pharmacokinetics of drugs in our body. In the present study, we aimed to analyze the quantitative structure-activity relationship (QSAR) latently residing in ABCG2-drug interactions. We first established standard methods for expression of human ABCG2 in insect cells, quality control of plasma membrane samples by using electron microscopy techniques, and high-speed screening of ABCG2 inhibition with test compounds. Plasma membrane vesicles prepared from ABCG2-expressing Sf9 cells were used as a model system to measure the ATP-dependent transport of [3H]methotrexate (MTX). Forty-nine different therapeutic drugs and natural compounds were tested for their ability to inhibit ABCG2-mediated MTX transport. Based on their inhibition profiles, we performed QSAR analysis using chemical fragmentation codes deduced from the structures of test compounds. Multiple linear regression analysis delineated a relationship between the structural components and the extent of ABCG2 inhibition, allowing us to identify one set of structure-specific chemical fragmentation codes that are closely correlated with the inhibition of ABCG2 transport activity. Based on the QSAR analysis data, we predicted the potency of gefitinib to inhibit ABCG2. The validity of our QSAR-based prediction for gefitinib was examined by actual experiments. Our kinetic analysis experiments suggest that the ABCG2-ATP complex binds gefitinib. The present study provides a new strategy for analyzing ABCG2-drug interactions. This strategy is considered to be practical and useful for the molecular designing of new ABCG2 modulators.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

A SNP in the ABCC11 gene is the determinant of human earwax type.

Human earwax consists of wet and dry types. Dry earwax is frequent in East Asians, whereas wet earwax is common in other populations. Here we show that a SNP, 538G --> A (rs17822931), in the ABCC11 gene is responsible for determination of earwax type. The AA genotype corresponds to dry earwax, and GA and GG to wet type. A 27-bp deletion in ABCC11 exon 29 was also found in a few individuals of Asian ancestry. A functional assay demonstrated that cells with allele A show a lower excretory activity for cGMP than those with allele G. The allele A frequency shows a north-south and east-west downward geographical gradient; worldwide, it is highest in Chinese and Koreans, and a common dry-type haplotype is retained among various ethnic populations. These suggest that the allele A arose in northeast Asia and thereafter spread through the world. The 538G --> A SNP is the first example of DNA polymorphism determining a visible genetic trait.

ATP-Binding Cassette Transporters↗
PubMed2006

Human ABC transporter ABCG2 in xenobiotic protection and redox biology.

Human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR/ABCP) is regarded as a member of the phase III system of xenobiotic metabolism. This efflux pump is suggested to be responsible for protecting the body from toxic xenobiotics and for removing toxic metabolites. The aim of this review article is to address new aspects of ABCG2 related to redox biology, namely the posttranslational modification (intra- and intermolecular disulfide bond formation) of ABCG2 protein and the transport of porphyrin and chlorophyll metabolites, as well as the high-speed screening and QSAR analysis method to evaluate ABCG2-drug interactions.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Evaluation of human P-glycoprotein (MDR1/ABCB1) ATPase activity assay method by comparing with in vitro transport measurements: Michaelis-Menten kinetic analysis to estimate the affinity of P-glycoprotein to drugs.

Human ABC transporter P-glycoprotein (P-gp/ABCB1) encoded by the multidrug resistance (MDR1) gene is recognized as one of the most important factors regulating pharmacokinetics of a number of clinically important drugs because of its function of extruding a wide range of structurally unrelated amphiphilic and hydrophobic drugs from the inside to the outside of cells in an ATP-driven mechanism. In the present study, we have evaluated the high-speed ATPase activity assay method by comparing with in vitro transport assay systems using MDR1-transfected MDR1-MDCK cells. Since substrate drugs were found to interfere with the photometric detection of inorganic phosphate (Pi) that was liberated according to the hydrolysis of ATP to ADP in ATPase activity assay, at first, a method in which the amount of Pi can be calculated correctly. Results demonstrate that the kinetic parameters obtained in ATPase activity assay are not necessarily correspond with those in in vitro transport assay, suggesting that these methods might detect the different processes of drug-P-gp interaction. The combining of the ATPase activity assay and in vitro transport technologies provides us the insight into mechanisms of the membrane transport of drugs by P-gp.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Nuclear receptor-mediated transcriptional regulation in Phase I, II, and III xenobiotic metabolizing systems.

Studies of the genetic regulation involved in drug metabolizing enzymes and drug transporters are of great interest to understand the molecular mechanisms of drug response and toxic events. Recent reports have revealed that hydrophobic ligands and several nuclear receptors are involved in the induction or down-regulation of various enzymes and transporters involved in Phase I, II, and III xenobiotic metabolizing systems. Nuclear receptors (NRs) form a family of ligand-activated transcription factors (TFs). These proteins modulate the regulation of target genes by contacting their promoter or enhancer sequences at specific recognition sites. These target genes include metabolizing enzymes such as cytochrome P450s (CYPs), transporters, and NRs. Thus it was now recognized that these NRs play essential role in sensing processing xenobiotic substances including drugs, environmental chemical pollutants and nutritional ingredients. From literature, we picked up target genes of each NR in xenobiotic response systems. Possible cross-talk, by which xenobiotics may exert undesirable effects, was listed. For example, the role of NRs was comprehensively drawn up in cholesterol and bile acid homeostasis in human hepatocyte. Summarizing current states of related research, especially for in silico response element search, we tried to elucidate nuclear receptor mediated xenobiotic processing loops and direct future research.

Animals↗
PubMed2006

Transport mechanism-based drug molecular design: novel camptothecin analogues to circumvent ABCG2-associated drug resistance of human tumor cells.

Acquired and intrinsic drug resistance in cancer is the major obstacle to long-term, sustained patient response to chemotherapy. Irinotecan (CPT-11) is a widely-used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I). However, overexpression of ABCG2 (BCRP/MXR/ABCP) reportedly confers cancer cells resistance to SN-38, the active form of CPT-11. To circumvent the ABCG2-associated drug resistance, we have synthesized and characterized a total of fourteen new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. While the lactone E ring is a prerequisite for anticancer activity, modifications of the A or B rings do not significantly affect Topo I inhibition activity. In this context, we have synthesized new CPT analogues with different substitutions at positions 10 or 11 of the A ring. All of the tested CPT analogues strongly inhibited the Topo I activity in a cell-free system. Accordingly, we have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from ABCG2-overexpressing cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with a hydroxyl group at position 10 or 11 of the A ring are good substrates for ABCG2 and therefore effectively extruded from cancer cells. Thus, hydrogen bond formation is considered to be involved in substrate recognition and/or transport processes of ABCG2. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Identification of cysteine residues critically involved in homodimer formation and protein expression of human ATP-binding cassette transporter ABCG2: a new approach using the flp recombinase system.

Since ABCG2 is a half-transporter in the ATP-binding cassette (ABC) transporter family, it has been suspected that ABCG2 functions as a homodimer. In the present study, we have investigated the molecular mechanism underlying homodimer formation of ABCG2. Based on the amino acid sequence of ABCG2, three cysteine residues (Cys592, Cys603, and Cys608) are expected to exist in the extracellular loop. To identify a cysteine residue(s) required for homodimer formation, we have substituted those cysteine residues to glycine by site-directed mutagenesis and stably expressed the resulting variants in Flp-In-293 cells. Substitution of the amino acid at position 603 from cysteine to glycine (C603G) completely diminished homodimer formation, whereas substitution of both Cys592 and Cys608 to glycine residues (C592G/C608G) had no effect on homodimer formation. These results strongly suggest that Cys603 is prerequisite for homodimer formation of ABCG2 via a disulfide bond. On the other hand, immunohistochemistry experiments revealed that the C592G/C608G variant is mainly located in intracellular compartments. The C592G/C608G variant exhibited lower activity of ATP-dependent methotrexate (MTX) transport, and its expression did not confer Flp-In-293 cells resistance to SN-38 or mitoxantrone. Cys592 and Cys608 appear to be important for intracellular sorting of the de novo synthesized ABCG2 protein to the plasma membrane. Taken together, cysteine residues in the extra-cellular loop are considered to play pivotal roles in homodimer formation and plasma membrane localization of ABCG2.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Genetic polymorphisms of human ABC transporter ABCG2: development of the standard method for functional validation of SNPs by using the Flp recombinase system.

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.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Molecular modeling of new camptothecin analogues to circumvent ABCG2-mediated drug resistance in cancer.

Irinotecan (CPT-11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I). However, overexpression of ABCG2 (BCRP/MXR/ABCP) reportedly confers cancer cells resistance to SN-38, the active form of CPT-11. To circumvent the ABCG2-associated drug resistance, the structure-activity-relationship (SAR) of 14 new camptothecin (CPT) analogues has been studied with respect to the substrate specificity of ABCG2. While the lactone E ring is a prerequisite for anticancer activity, modifications of the A or B rings do not significantly affect Topo I inhibition. Based on the substrate specificity of ABCG2, it is strongly suggested that CPT analogues with a hydroxyl group at position 10 or 11 of the A ring are recognized by ABCG2 and are thereby effectively extruded from cancer cells. To develop a platform for the molecular modeling to circumvent anticancer drug resistance, we have carried out quantum chemical calculations and neural network SAR analysis. Electrostatic potential iso-surfaces generated by ab initio MO calculations using restricted Hartree-Fock method have revealed that negative potential localized at positions 10 or 11 in the A ring is important for recognition by ABCG2.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design.

In the post-genome-sequencing era, emerging genomic technologies are shifting the paradigm for drug discovery and development. Nevertheless, drug discovery and development still remain high-risk and high-stakes ventures with long and costly timelines. Indeed, the attrition of drug candidates in preclinical and development stages is a major problem in drug design. For at least 30% of the candidates, this attrition is due to poor pharmacokinetics and toxicity. Thus, pharmaceutical companies have begun to seriously re-evaluate their current strategies of drug discovery and development. In that light, we propose that a transport mechanism-based design might help to create new, pharmacokinetically advantageous drugs, and as such should be considered an important component of drug design strategy. Performing enzyme- and/or cell-based drug transporter, interaction tests may greatly facilitate drug development and allow the prediction of drug-drug interactions. We recently developed methods for high-speed functional screening and quantitative structure-activity relationship analysis to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide a practical tool to screen synthetic and natural compounds, and these data can be applied to the molecular design of new drugs. In this review article, we present an overview on the genetic polymorphisms of human ABC transporter ABCG2 and new camptothecin analogues that can circumvent AGCG2-associated multidrug resistance of cancer.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

High-speed screening of human ATP-binding cassette transporter function and genetic polymorphisms: new strategies in pharmacogenomics.

Drug transporters represent an important mechanism in cellular uptake and efflux of drugs and their metabolites. Hitherto a variety of drug transporter genes have been cloned and classified into either solute carriers or ATP-binding cassette (ABC) transporters. Such drug transporters are expressed in various tissues such as the intestine, brain, liver, kidney, and, importantly, cancer cells, where they play critical roles in the absorption, distribution, and excretion of drugs. We developed high-speed functional screening and quantitative structure-activity relationship analysis methods to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide powerful and practical tools for screening synthetic and natural compounds, and the deduced data can be applied to the molecular design of new drugs. Furthermore, we demonstrate a new "SNP array" method to detect genetic polymorphisms of ABC transporters in human samples.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCG2: therapeutic implications.

Pharmacogenomics, the study of the influence of genetic factors on drug action, is increasingly important for predicting pharmacokinetics profiles and/or adverse reactions to drugs. Drug transporters, as well as drug metabolism play pivotal roles in determining the pharmacokinetic profiles of drugs and their overall pharmacological effects. There is an increasing number of reports addressing genetic polymorphisms of drug transporters. However, information regarding the functional impact of genetic polymorphisms in drug transporter genes is still limited. Detailed functional analysis in vitro may provide clear insight into the biochemical and therapeutic significance of genetic polymorphisms. This review addresses functional aspects of the genetic polymorphisms of human ATP-binding cassette transporters, ABCB1 and ABCG2, which are critically involved in the pharmacokinetics of drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Novel camptothecin analogues that circumvent ABCG2-associated drug resistance in human tumor cells.

Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin; CPT-11) is a widely used potent antitumor drug that inhibits mammalian DNA topoisomerase I (Topo I); however, overexpression of ABCG2 (BCRP/MXR/ABCP) can confer cancer cell resistance to SN-38, the active form of CPT-11. We have recently demonstrated that plasma membrane vesicles prepared from ABCG2-overexpressing PC-6/SN2-5H cells transported SN-38 and its glucuronide conjugate in an ATP-dependent manner (Nakatomi et al., Biochem Biophys Res Commun 2001;288:827-32). In the present study, we have characterized a total of 14 new camptothecin (CPT) analogues with respect to both the inhibition of Topo I and the substrate specificity of ABCG2. All of the tested CPT analogues, which have different substitutions at positions 10 and 11, strongly inhibited the Topo I activity in a cell-free system, as did SN-38. Their antitumor activities in the SN-38-resistant PC-6/SN2-5H2 cell line greatly varied, however, being correlated with intracellular accumulation levels. We have examined ATP-dependent transport of those CPT analogues by using plasma membrane vesicles prepared from both PC-6/SN2-5H2 cells and ABCG2-transfected HEK-293 cells. Based on the substrate specificity of ABCG2 thus evaluated, it is strongly suggested that CPT analogues with high polarity are good substrates for ABCG2 and are therefore effectively extruded from cancer cells. In this context, to circumvent ABCG2-associated drug resistance, low-polarity CPT analogues are considered to be potent lead compounds. The present study provides a practical approach to discover new CPT-based drugs for the chemotherapy of drug-resistant human cancer.

ATP Binding Cassette Transporter, Subfamily G, Mem↗