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1.
J Biol Chem ; 290(25): 15812-15824, 2015 Jun 19.
Article in English | MEDLINE | ID: mdl-25944913

ABSTRACT

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD(+) biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD(+) biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500-3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.


Subject(s)
Carbohydrate Metabolism , Cytokines/metabolism , NAD/metabolism , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Nicotinamide Phosphoribosyltransferase/metabolism , Sugar Phosphates/metabolism , Acrylamides/pharmacology , Cytokines/antagonists & inhibitors , Cytokines/genetics , Enzyme Inhibitors/pharmacology , Humans , Mass Spectrometry , NAD/genetics , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Neoplasms/genetics , Neoplasms/pathology , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Nicotinamide Phosphoribosyltransferase/genetics , Piperidines/pharmacology , Sugar Phosphates/genetics
2.
Mol Cancer Ther ; 12(4): 481-90, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23371859

ABSTRACT

LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non-small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response.


Subject(s)
Deoxycytidine/analogs & derivatives , Deoxyuridine/analogs & derivatives , Prodrugs/administration & dosage , Administration, Metronomic , Administration, Oral , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Deoxycytidine/chemistry , Deoxycytidine/pharmacology , Deoxyuridine/administration & dosage , Deoxyuridine/chemistry , Disease Models, Animal , Dose-Response Relationship, Drug , Gene Expression , HCT116 Cells , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Prodrugs/chemistry , Valproic Acid/pharmacology , Xenograft Model Antitumor Assays , Gemcitabine
3.
Clin Cancer Res ; 19(5): 1159-68, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23325581

ABSTRACT

PURPOSE: The oral prodrug of gemcitabine LY2334737 is cleaved systemically to gemcitabine; the mechanism responsible for hydrolysis is unknown. LY2334737 cytotoxicity was tested in the NCI-60 panel; mining of microarray expression data identified carboxylesterase (CES) as a top hydrolase candidate. Studies examined whether CES is responsible for hydrolysis and whether cellular CES expression confers prodrug sensitivity. EXPERIMENTAL DESIGN: Human recombinant CES isozymes were assayed for LY2334737 hydrolysis. Stable CES-overexpressing HCT-116 transfectants and a SK-OV-3 knockdown were prepared. Cell lines were tested for drug sensitivity and CES expression by quantitative real time-PCR (qRT-PCR), Western blotting, and immunohistochemical staining. Bystander cytotoxicity studies were conducted with GFP-tagged PC-3 cells as the reporter cell line. Therapeutic response of the HCT-116 transfectants was evaluated in xenografts. RESULTS: Of 3 human CES isozymes tested, only CES2 hydrolyzed LY2334737. Five cell lines that express CES2 responded to LY2334737 treatment. LY2334737 was less cytotoxic to a SK-OV-3 CES2 knockdown than parental cells. The drug response of CES2-transfected HCT-116 cells correlated with CES2 expression level. Bystander studies showed statistically greater PC-3-GFP growth inhibition by LY2334737 when cells were cocultured with CES2 and not mock transfectants. Oral treatment of xenograft models with 3.2 mg/kg LY2334737 once a day for 21 days showed greater tumor growth inhibition of CES2 transfectant than the mock transfectant (P ≤ 0.001). CONCLUSIONS: CES2 is responsible for the slow hydrolysis of LY2334737. Because intact prodrug circulates at high plasma levels after oral LY2334737 administration, improved response rates may be observed by tailoring LY2334737 treatment to patients with CES2 tumor expression.


Subject(s)
Antimetabolites, Antineoplastic/chemistry , Carboxylesterase/metabolism , Deoxycytidine/analogs & derivatives , Deoxyuridine/analogs & derivatives , Neoplasms/drug therapy , Prodrugs/pharmacology , Blotting, Western , Bystander Effect , Carboxylesterase/antagonists & inhibitors , Carboxylesterase/genetics , Deoxycytidine/chemistry , Deoxyuridine/pharmacology , Female , Humans , Hydrolysis , Molecular Structure , Neoplasms/enzymology , Neoplasms/pathology , RNA, Small Interfering/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Gemcitabine
4.
J Biol Chem ; 288(5): 3500-11, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23239881

ABSTRACT

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD(+), essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD(+) for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.


Subject(s)
Enzyme Inhibitors/pharmacology , NAD/biosynthesis , Neoplasms/metabolism , Neoplasms/pathology , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Acrylamides/pharmacology , Adenosine Triphosphate/deficiency , Adenosine Triphosphate/metabolism , Animals , Carbon Isotopes , Cell Death/drug effects , Cell Line, Tumor , Citric Acid Cycle/drug effects , Female , Glycolysis/drug effects , Humans , Isotope Labeling , Mice , Mice, SCID , Nicotinamide Phosphoribosyltransferase/metabolism , Pentose Phosphate Pathway/drug effects , Piperidines/pharmacology , Serine/biosynthesis , Xenograft Model Antitumor Assays
5.
Cancer Res ; 65(15): 6593-600, 2005 Aug 01.
Article in English | MEDLINE | ID: mdl-16061639

ABSTRACT

The splicing factor SPF45 (RBM17) is frequently overexpressed in many solid tumors, and stable expression in HeLa cells confers resistance to doxorubicin and vincristine. In this study, we characterized stable transfectants of A2780 ovarian carcinoma cells. In a 3-day cytotoxicity assay, human SPF45 overexpression conferred 3- to 21-fold resistance to carboplatin, vinorelbine, doxorubicin, etoposide, mitoxantrone, and vincristine. In addition, resistance to gemcitabine and pemetrexed was observed at the highest drug concentrations tested. Knockdown of SPF45 in parental A2780 cells using a hammerhead ribozyme sensitized A2780 cells to etoposide by approximately 5-fold relative to a catalytically inactive ribozyme control and untransfected cells, suggesting a role for SPF45 in intrinsic resistance to some drugs. A2780-SPF45 cells accumulated similar levels of doxorubicin as vector-transfected and parental A2780 cells, indicating that drug resistance is not due to differences in drug accumulation. Efforts to identify small molecules that could block SPF45-mediated drug resistance revealed that the selective estrogen receptor (ER) modulators tamoxifen and LY117018 (a raloxifene analogue) partially reversed SPF45-mediated drug resistance to mitoxantrone in A2780-SPF45 cells from 21-fold to 8- and 5-fold, respectively, but did not significantly affect the mitoxantrone sensitivity of vector control cells. Quantitative PCR showed that ERbeta but not ERalpha was expressed in A2780 transfectants. Coimmunoprecipitation experiments suggest that SPF45 and ERbeta physically interact in vivo. Thus, SPF45-mediated drug resistance in A2780 cells may result in part from effects of SPF45 on the transcription or alternate splicing of ERbeta-regulated genes.


Subject(s)
Drug Resistance, Multiple/physiology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , RNA-Binding Proteins/biosynthesis , Selective Estrogen Receptor Modulators/pharmacology , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Doxorubicin/pharmacokinetics , Doxorubicin/pharmacology , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm , Estrogen Receptor beta/metabolism , Etoposide/pharmacokinetics , Etoposide/pharmacology , Female , Humans , Mitoxantrone/pharmacokinetics , Mitoxantrone/pharmacology , Ovarian Neoplasms/genetics , Pyrrolidines/pharmacology , RNA Splicing , RNA Splicing Factors , RNA, Catalytic/genetics , RNA, Catalytic/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Tamoxifen/pharmacology , Thiophenes/pharmacology , Transfection
6.
Mol Cancer Ther ; 4(5): 855-63, 2005 May.
Article in English | MEDLINE | ID: mdl-15897250

ABSTRACT

5'-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5'-fluoro-2'-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5'-fluoro-2'-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were approximately 9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 micromol/L [(3)H]5-FdUMP, [(3)H]5-FUMP, [(3)H]dUMP, and not [(3)H]5-FUdR, or [(3)H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a K(m) of 1.1 mmol/L and V(max) of 439 pmol/min/mg protein. Uptake of 250 micromol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3',5'-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 micromol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Floxuridine/metabolism , Fluorouracil/pharmacology , Multidrug Resistance-Associated Proteins/metabolism , Adenosine Triphosphate/pharmacology , Antimetabolites, Antineoplastic/pharmacokinetics , Apoptosis/drug effects , Biological Transport/drug effects , Cells, Cultured , Fluorouracil/pharmacokinetics , Humans , Kidney/cytology , Kidney/drug effects , Kidney/metabolism , Phosphorylation/drug effects , Thioguanine/pharmacology , Thymidylate Synthase/metabolism
7.
Biochem Pharmacol ; 67(6): 1111-21, 2004 Mar 15.
Article in English | MEDLINE | ID: mdl-15006547

ABSTRACT

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Azides/pharmacology , Isoxazoles/pharmacology , Multidrug Resistance-Associated Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/genetics , Amino Acid Sequence , Animals , Cell Division/drug effects , Drug Resistance, Multiple , HeLa Cells , Humans , Mice , Molecular Sequence Data , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Photoaffinity Labels , Sequence Homology, Amino Acid , Species Specificity , Tumor Cells, Cultured
8.
Am J Pathol ; 163(5): 1781-90, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14578179

ABSTRACT

Our effort to identify novel drug-resistant genes in cyclophosphamide-resistant EMT6 mouse mammary tumors led us to the identification of SPF45. Simultaneously, other groups identified SPF45 as a component of the spliceosome that is involved in alternative splicing. We isolated the human homologue and examined the normal human tissue expression, tumor expression, and the phenotype caused by overexpression of human SPF45. Our analyses revealed that SPF45 is expressed in many, but not all, normal tissues tested with predominant expression in normal ductal epithelial cells of the breast, liver, pancreas, and prostate. Our analyses using tissue microarrays and sausages of tumors indicated that SPF45 is highly expressed in numerous carcinomas including bladder, breast, colon, lung, ovarian, pancreatic, and prostate. Interestingly, this study revealed that overexpression of SPF45 in HeLa, a cervical carcinoma cell line, resulted in drug resistance to doxorubicin and vincristine, two chemotherapeutic drugs commonly used in cancer. To our knowledge, this is the first study showing tumor overexpression of an alternate splicing factor resulting in drug resistance.


Subject(s)
Drug Resistance, Neoplasm/genetics , Neoplasms/metabolism , RNA-Binding Proteins/biosynthesis , Blotting, Western , Epithelium/physiology , HeLa Cells , Humans , Immunohistochemistry , Microscopy, Confocal , Neoplasms/pathology , Organ Specificity , Phenotype , Polymerase Chain Reaction , Protein Array Analysis , RNA Splicing Factors , RNA-Binding Proteins/genetics , Transfection , Up-Regulation
9.
Int J Cancer ; 103(1): 121-5, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12455064

ABSTRACT

Our study examines the ability of LY335979 (Zosuquidar trihydrochloride) to modulate 3 distinct ABC transporters that are mechanisms of drug resistance: P-glycoprotein (Pgp, ABCB1), multidrug resistance associated protein (MRP1, ABCC2) and breast cancer resistance protein (BCRP, ABCG2). Pgp-mediated resistance can be modulated by coadministration with the highly potent, selective inhibitor, LY335979. Modulation of resistance by mitoxantrone and vinorelbine, 2 drugs used to treat certain solid tumors, was examined in a 3-day cytotoxicity assay using a panel of HL60 leukemia cell lines or MCF-7 breast cancer transfectants. LY335979, at 0.5 microM, substantially reversed mitoxantrone resistance and fully reversed vinorelbine resistance of Pgp-expressing HL60/Vinc cells. However, LY335979 did not modulate drug resistance in the MRP1-expressing HL60/ADR or drug-sensitive parental HL60 cells. To ascertain if LY335979 modulates BCRP-mediated drug resistance, the sensitivity of 26-fold mitoxantrone resistant, BCRP-transfected MCF-7 cells was evaluated. Addition of 5 microM LY335979, a concentration approximately 100-fold higher than the affinity of Pgp, had little to no effect on the BCRP transfectant. [(125)I]Iodomycin photolabeled Pgp in CEM/VLB(100) membranes and was inhibited by 5 microM LY335979 and GF120918. No photolabeling of MRP or BCRP occurred in H69AR or MCF-7/BCRP membranes, respectively. These results further demonstrate that LY335979 is highly specific for Pgp and does not modulate MRP1- or BCRP-mediated resistance and can be used in combination with mitoxantrone and vinorelbine in tumor cells.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Breast Neoplasms/metabolism , Dibenzocycloheptenes/pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Quinolines/pharmacology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Doxorubicin/pharmacology , Female , Fetal Viability/drug effects , Humans , Immunoblotting , Multidrug Resistance-Associated Protein 2 , Neoplasm Proteins/metabolism , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism
10.
J Biol Chem ; 277(38): 35225-31, 2002 Sep 20.
Article in English | MEDLINE | ID: mdl-12138119

ABSTRACT

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent transporter of structurally diverse organic anion conjugates. The protein also actively transports a number of non-conjugated chemotherapeutic drugs and certain anionic conjugates by a presently poorly understood GSH-dependent mechanism. LY475776is a newly developed (125)I-labeled azido tricyclic isoxazole that binds toMRP1 with high affinity and specificity in a GSH-dependent manner. The compound has also been shown to photolabel a site in the COOH-proximal region of MRP1's third membrane spanning domain (MSD). It is presently not known where GSH interacts with the protein. Here, we demonstrate that the photactivateable GSH derivative azidophenacyl-GSH can substitute functionally for GSH in supporting the photolabeling of MRP1 by LY475776 and the transport of another GSH-dependent substrate, estrone 3-sulfate. In contrast to LY475776, azidophenacyl-[(35)S] photolabels both halves of the protein. Photolabeling of the COOH-proximal site can be markedly stimulated by low concentrations of estrone 3-sulfate, suggestive of cooperativity between the binding of these two compounds. We show that photolabeling of the COOH-proximal site by LY475776 and the labeling of both NH(2)- and COOH- proximal sites by azidophenacyl-GSH requires the cytoplasmic linker (CL3) region connecting the first and second MSDs of the protein, but not the first MSD itself. Although required for binding, CL3 is not photolabeled by azidophenacyl-GSH. Finally, we identify non-conserved amino acids in the third MSD that contribute to the high affinity with which LY475776 binds to MRP1.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Azides/metabolism , Glutathione/metabolism , Isoxazoles/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Animals , Cell Line , Humans , Iodine Radioisotopes , Mice , Photoaffinity Labels , Radioligand Assay , Sulfur Radioisotopes
11.
J Biol Chem ; 277(32): 28690-9, 2002 Aug 09.
Article in English | MEDLINE | ID: mdl-12034727

ABSTRACT

Substrates transported by the 190-kDa multidrug resistance protein 1 (MRP1) (ABCC1) include endogenous organic anions such as the cysteinyl leukotriene C(4). In addition, MRP1 confers resistance against various anticancer drugs by reducing intracellular accumulation by co-export of drug with reduced GSH. We have examined the properties of LY475776, an intrinsically photoactivable MRP1-specific tricyclic isoxazole modulator that inhibits leukotriene C(4) transport by this protein in a GSH-dependent manner. We show that [125I]LY475776 photolabeling of MRP1 requires GSH but is also supported by several non-reducing GSH derivatives and peptide analogs. Limited proteolysis revealed that [(125)I]LY475776 labeling was confined to the 75-kDa COOH-proximal half of MRP1. More extensive proteolysis generated two major 125I-labeled fragments of approximately 56 and approximately 41 kDa, and immunoblotting with regionally directed antibodies showed that these fragments correspond to amino acids approximately 1045-1531 and approximately 1150-1531, respectively. However, an approximately 33-kDa COOH-terminal immunoreactive fragment was not labeled, inferring that the major [125I]LY475776-labeling site resides approximately between amino acids 1150-1250. This region encompasses transmembrane (TM) segments 16 and 17 at the COOH-proximal end of the third membrane spanning domain of the protein. [125I]LY475776 labeling of mutant MRP1 molecules with substitutions of Trp(1246) in TM17 were reduced >80% compared with wild-type MRP1, confirming that TM17 is important for LY475776 binding. Finally, vanadate-induced trapping of ADP inhibited [125I]LY475776 labeling, suggesting that ATP hydrolysis causes a conformational change in MRP1 that reduces the affinity of the protein for this inhibitor.


Subject(s)
Azides/pharmacology , DNA-Binding Proteins/metabolism , Glutathione/metabolism , Isoxazoles/pharmacology , Multidrug Resistance-Associated Proteins , Antibodies, Monoclonal/metabolism , Binding Sites , Cell Membrane/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , HeLa Cells , Humans , Immunoblotting , Leukotriene C4/metabolism , Light , Models, Chemical , MutS Homolog 3 Protein , Mutation , Protein Binding/drug effects , Protein Conformation , Transfection , Tumor Cells, Cultured , Vanadates/pharmacology
12.
Mol Pharmacol ; 61(5): 964-73, 2002 May.
Article in English | MEDLINE | ID: mdl-11961113

ABSTRACT

P-glycoprotein (P-gp) is an efflux transporter involved in limiting the oral bioavailability and tissue penetration of a variety of structurally divergent molecules. A better understanding of the structural requirements of modulators of P-gp function will aid in the design of therapeutic agents. Toward this goal, three-dimensional quantitative structure-activity relationship (3D-QSAR) models were generated using in vitro data associated with inhibition of P-gp function. Several approaches were undertaken with multiple iterations, yielding Catalyst 3D-QSAR models being able to qualitatively rank-order and predict IC(50) values for P-gp inhibitors excluded from the model in question. The success of these validations suggests that a P-gp pharmacophore for 27 inhibitors of digoxin transport in Caco-2 cells consisted of four hydrophobes and one hydrogen bond acceptor. A second pharmacophore generated with 21 inhibitors of vinblastine binding to plasma membrane vesicles derived from CEM/VLB(100) cells contained three ring aromatic features and one hydrophobic feature. A third pharmacophore generated with 17 inhibitors of vinblastine accumulation in P-gp expressing LLC-PK1 cells contained four hydrophobes and one hydrogen bond acceptor. A final pharmacophore was generated for inhibition of calcein accumulation in P-gp expressing LLC-PK1 cells and found to contain two hydrophobes, a ring aromatic feature, and a hydrogen bond donor. The similarity of features for the pharmacophores of P-gp inhibitors of digoxin transport and vinblastine binding suggest some commonality in their binding sites. Utilization of such models may prove to be of value for prediction of molecules that may modulate one or more P-gp binding sites.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Caco-2 Cells , Ergonovine/chemistry , Ergonovine/pharmacology , Fluoxetine/chemistry , Fluoxetine/pharmacology , Humans , Models, Molecular , Oxytocics/chemistry , Oxytocics/pharmacology , Protein Conformation , Selective Serotonin Reuptake Inhibitors/chemistry , Selective Serotonin Reuptake Inhibitors/pharmacology , Structure-Activity Relationship , Swine , Transfection , Vinblastine/chemistry , Vinblastine/pharmacology
13.
Mol Pharmacol ; 61(5): 974-81, 2002 May.
Article in English | MEDLINE | ID: mdl-11961114

ABSTRACT

Using in vitro data, we previously built Catalyst 3-dimensional quantitative structure activity relationship (3D-QSAR) models that qualitatively rank and predict IC(50) values for P-glycoprotein (P-gp) inhibitors. These models were derived and tested with data for inhibition of digoxin transport, calcein accumulation, vinblastine accumulation, and vinblastine binding. In the present study, 16 inhibitors of verapamil binding to P-gp were predicted using these models. These inhibition results were then used to generate a new pharmacophore that consisted of one hydrogen bond acceptor, one ring aromatic feature, and two hydrophobes. This model predicted the rank order of the four data sets described previously and correctly ranked the inhibitory potency of a further four verapamil metabolites identified in the literature. The degree of similarity in rank ordering prediction by these inhibitor pharmacophore models generated to date confirms a likely overlap in the sites to which the three P-gp substrates used in these studies (verapamil, vinblastine, and digoxin) bind. Alignment of the three substrate probes indicated that they are likely to bind the same or overlapping sites within P-gp. Important features on these substrates include multiple hydrophobic and hydrogen bond acceptor features, which are widely dispersed and in agreement among most of the five inhibitor pharmacophores we have described so far. These 3D-QSAR models will be useful for future prediction of likely substrates and inhibitors of P-gp.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Verapamil/pharmacology , Vinblastine/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Caco-2 Cells , Humans , Models, Molecular , Protein Conformation , Structure-Activity Relationship , Verapamil/chemistry , Vinblastine/chemistry
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