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John K. Buolamwini - One of the best experts on this subject based on the ideXlab platform.
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CoMFA and CoMSIA 3D-QSAR studies on S6-(4-nitrobenzyl)Mercaptopurine Riboside (NBMPR) analogs as inhibitors of human equilibrative nucleoside transporter 1 (hENT1)
Bioorganic & medicinal chemistry letters, 2008Co-Authors: Amol Gupte, John K. BuolamwiniAbstract:Abstract 3D-QSAR (CoMFA and CoMSIA) studies were performed on human equlibrative nucleoside transporter (hENT1) inhibitors displaying Ki values ranging from 10,000 to 0.7 nM. Both CoMFA and CoMSIA analysis gave reliable models with q2 values >0.50 and r2 values >0.92. The models have been validated for their stability and robustness using group validation and bootstrapping techniques and for their predictive abilities using an external test set of nine compounds. The high predictive r2 values of the test set (0.72 for CoMFA model and 0.74 for CoMSIA model) reveals that the models can prove to be a useful tool for activity prediction of newly designed nucleoside transporter inhibitors. The CoMFA and CoMSIA contour maps identify features important for exhibiting good binding affinities at the transporter, and can thus serve as a useful guide for the design of potential equilibrative nucleoside transporter inhibitors.
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Novel C2-Purine Position Analogs of NitrobenzylMercaptopurine Riboside as Human Equilibrative Nucleoside Transporter 1 Inhibitors
Bioorganic & medicinal chemistry, 2007Co-Authors: Amol Gupte, John K. BuolamwiniAbstract:Abstract Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. S 6 -(4-nitrobenzyl)Mercaptopurine Riboside (NBMPR) is a prototype inhibitor of the human equilibrative nucleoside transporter (hENT1), and is a high affinity ligand with a K d of 0.1–1.0 nM. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel C 2 -purine position substituted analogs of NBMPR at the hENT1. The aim of this research was to understand the substituent requirements at the C 2 -purine position of NBMPR. Structure–activity relationships (SAR) indicate that increasing the steric bulk at the C 2 -purine position of NBMPR led to a decrease in binding affinity of these ligands at the hENT1. New high affinity inhibitors were identified, with the best compound, 2-fluoro-4-nitrobenzyl Mercaptopurine Riboside (7), exhibiting a K i of 2.1 nM. This information, when coupled with the information obtained from other structure–activity relationship studies should prove useful in efforts aimed at modeling the NMBPR and analogs pharmacophore of hENT1 inhibitors.
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nucleoside transport inhibitors structure activity relationships and potential therapeutic applications
Current Medicinal Chemistry, 1997Co-Authors: John K. BuolamwiniAbstract:A survey of structure-activity relationships and potential therapeutic applications of nucleoside transport inhibitors is presented. Among the two equilibrative (facilated diffusion), and five concentrative (sodium-dependent) nucleoside transporters identified in mammalian cells, only the equilibrative transporters (es and ei) and one concentrative transporter (cs) can be effectively blocked by one or more of the nucleoside transport inhibitors discovered to date. A structurally diverse array of compounds have been shown to exert nucleoside transport inhibitory activity to varying degrees. The most important of these are i) nucleoside analogs of which S 6 -(4-nitrobenzyl)Mercaptopurine Riboside (NBMPR, nitrobenzylthioinosine) is the prototype, ii) pyrimidopyrimidine and pteridine derivatives of which dipyridamole (persantine) is the prototype, and iii) alkyl- and cycloalkyldiamine and piperazine calcium channel antagonists of which dilazep and lidoflazine are the representatives, respectively. All of these are effective inhibitors of the es transporter, but dipyridamole is also a potent inhibitor of the ei transporter with variable activity depending on the cell type. Surprisingly, NBMPR and dipyridamole are also potent inhibitors of the newly identified cs concentrative transporter in fresh leukemia cells from patients. Not only does the es inhibitory potency of these compounds depend on tissue type, but it also varies widely among different mammalian species. Nucleoside transport inhibitors have potential for therapeutic uses in 1) adenosine potentiation in cardioprotection and cerebroprotection in ischemic heart disease and stroke, respectively, 2) the modulation of the effects of antimetabolite anticancer and antiviral agents, and 3) host tissue protection in chemotherapy with cytotoxic nucleosides. Additional aeras of potential therapeutic application of NT inhibitors include kidney transplantation, analgesia and hypertension. Most of the compounds in the present repertoire of potent NT inhibitors do not meet the requisite pharmacological profiles for successful clinical application, which calls for the discovery of better inhibitors. Advances are being made in the molecular cloning and functional expression of nucleoside tranporters that augur well for future drug design efforts
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Solution NMR Conformational Analysis of the Potent Equilibrative Sensitive (ES) Nucleoside Transporter Inhibitor, S 6-(4-Nitrobenzyl)Mercaptopurine Riboside (NBMPR)
Nucleosides and Nucleotides, 1997Co-Authors: John K. Buolamwini, Joseph J. BarchiAbstract:Abstract High resolution NMR analysis involving one-dimensional (1-D) 1H and nuclear Overhauser (NOE) difference spectroscopy was applied to solutions of NBMPR in DMSO-d 6. Coupling constants were obtained at different temperatures between 285 and 353 K, and used to analyze the rotamer preferences about the C-4′-C-5′ bond. The results revealed a rotamer distribution about the χ tortion angle that favors the high-anti range, a preponderance of the γ+ rotamer (at ∼64 %) with respect to the γ torsion angle, and a higher population of the south (S) conformer, which was favored by as little as the 4 % to as much as 31 % over the north (N) conformer as calculated by the program PSEUROT 6.2. The high-anti glycosidic torsion orientation appears to be the major conformational difference between the solution structure of NBMPR determined in this study and the structure previously observed in the solid state.
C Brouwer - One of the best experts on this subject based on the ideXlab platform.
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thiopurine metabolism and identification of the thiopurine metabolites transported by mrp4 and mrp5 overexpressed in human embryonic kidney cells
Molecular Pharmacology, 2002Co-Authors: Peter R Wielinga, John D Schuetz, Glen Reid, Liesbeth Van Deemter, E E Challa, I M Van Der Heijden, M De Haas, Annemieke Kuil, E Groeneveld, C BrouwerAbstract:Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-Mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against Mercaptopurines, which has been attributed to extrusion of Mercaptopurine metabolites by these transporters. We have analyzed the Mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl Mercaptopurine Riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of Mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.
Amol Gupte - One of the best experts on this subject based on the ideXlab platform.
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CoMFA and CoMSIA 3D-QSAR studies on S6-(4-nitrobenzyl)Mercaptopurine Riboside (NBMPR) analogs as inhibitors of human equilibrative nucleoside transporter 1 (hENT1)
Bioorganic & medicinal chemistry letters, 2008Co-Authors: Amol Gupte, John K. BuolamwiniAbstract:Abstract 3D-QSAR (CoMFA and CoMSIA) studies were performed on human equlibrative nucleoside transporter (hENT1) inhibitors displaying Ki values ranging from 10,000 to 0.7 nM. Both CoMFA and CoMSIA analysis gave reliable models with q2 values >0.50 and r2 values >0.92. The models have been validated for their stability and robustness using group validation and bootstrapping techniques and for their predictive abilities using an external test set of nine compounds. The high predictive r2 values of the test set (0.72 for CoMFA model and 0.74 for CoMSIA model) reveals that the models can prove to be a useful tool for activity prediction of newly designed nucleoside transporter inhibitors. The CoMFA and CoMSIA contour maps identify features important for exhibiting good binding affinities at the transporter, and can thus serve as a useful guide for the design of potential equilibrative nucleoside transporter inhibitors.
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Novel C2-Purine Position Analogs of NitrobenzylMercaptopurine Riboside as Human Equilibrative Nucleoside Transporter 1 Inhibitors
Bioorganic & medicinal chemistry, 2007Co-Authors: Amol Gupte, John K. BuolamwiniAbstract:Abstract Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. S 6 -(4-nitrobenzyl)Mercaptopurine Riboside (NBMPR) is a prototype inhibitor of the human equilibrative nucleoside transporter (hENT1), and is a high affinity ligand with a K d of 0.1–1.0 nM. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel C 2 -purine position substituted analogs of NBMPR at the hENT1. The aim of this research was to understand the substituent requirements at the C 2 -purine position of NBMPR. Structure–activity relationships (SAR) indicate that increasing the steric bulk at the C 2 -purine position of NBMPR led to a decrease in binding affinity of these ligands at the hENT1. New high affinity inhibitors were identified, with the best compound, 2-fluoro-4-nitrobenzyl Mercaptopurine Riboside (7), exhibiting a K i of 2.1 nM. This information, when coupled with the information obtained from other structure–activity relationship studies should prove useful in efforts aimed at modeling the NMBPR and analogs pharmacophore of hENT1 inhibitors.
Jashvant D. Unadkat - One of the best experts on this subject based on the ideXlab platform.
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Residues Met89 and Ser160 in the human equilibrative nucleoside transporter 1 affect its affinity for adenosine, guanosine, S6-(4-nitrobenzyl)-Mercaptopurine Riboside, and dipyridamole.
Molecular pharmacology, 2004Co-Authors: Christopher J. Endres, Jashvant D. UnadkatAbstract:The human equilibrative nucleoside transporter 1 (hENT1) is an important modulator of the physiological action of adenosine. We identified amino acid residues involved in adenosine transport using a yeast-based assay to rapidly screen and identify randomly generated hENT1 mutants that exhibited decreased sensitivity to inhibition of adenosine transport by various hENT1 competitive inhibitors. We identified Met89 and Ser160 as important in the affinity of hENT1 for various substrates and inhibitors. Mutation to Met89Cys or Ser160Cys significantly (p < 0.05) increased the S6-(4-nitrobenzyl)-Mercaptopurine Riboside (NBMPR) IC50 values by approximately 4- and 6-fold, respectively (42 +/- 13 and 65 +/- 1.6 nM) compared with the wild-type transporter (11 +/- 0.7 nM). The double mutant Met89Cys/Ser160Cys synergistically increased the NBMPR IC50 value to approximately 19-fold of that of the wild-type transporter. In contrast, compared with wild-type hENT1, the sensitivity to dipyridamole inhibition was significantly (p < 0.05) increased by only the Ser160Cys (approximately 2.6-fold) or the double mutant Met89Cys/Ser160Cys (approximately 4.7-fold) but not by the Met89Cys mutant. Mutation to Met89Cys or Ser160Cys increased the Km of adenosine (approximately 8- and 3-fold) and the Ki of guanosine (approximately 6- and 2-fold). The double mutant increased both the Km value of adenosine and the Ki value of guanosine by approximately 8-fold and seemed to confer no additional reduction in adenosine or guanosine affinity than that by mutation of Met89 alone. Together, these data indicate that transmembrane domains (TMDs) 2 (Met89) and 4 (Ser160) of hENT1 interact and are important in conferring sensitivity to NBMPR. In contrast, Ser160 and Met89 of hENT1, respectively, play a dominant role in conferring sensitivity to dipyridamole and adenosine/guanosine affinity.
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Mutation of leucine-92 selectively reduces the apparent affinity of inosine, guanosine, NBMPR [S6-(4-nitrobenzyl)-Mercaptopurine Riboside] and dilazep for the human equilibrative nucleoside transporter, hENT1.
The Biochemical journal, 2004Co-Authors: Christopher J. Endres, Dhruba J Sengupta, Jashvant D. UnadkatAbstract:We developed a yeast-based assay for selection of hENT1 (human equilibrative nucleoside transporter 1) mutants that have altered affinity for hENT1 inhibitors and substrates. In this assay, expression of hENT1 in a yeast strain deficient in adenine biosynthesis (ade2) permits yeast growth on a plate lacking adenine but containing adenosine, a hENT1 substrate. This growth was prevented when inhibitors of hENT1 [e.g. NBMPR [S6-(4-nitrobenzyl)-Mercaptopurine Riboside], dilazep or dipyridamole] were included in the media. To identify hENT1 mutants resistant to inhibition by these compounds, hENT1 was randomly mutagenized and introduced into this strain. Mutation(s) that allowed growth of yeast cells in the presence of these inhibitors were then identified and characterized. Mutants harbouring amino acid changes at Leu92 exhibited resistance to NBMPR and dilazep but not dipyridamole. The IC50 values of NBMPR and dilazep for [3H]adenosine transport by one of these mutants L92Q (Leu92-->Gln) were approx. 200- and 4-fold greater when compared with the value for the wild-type hENT1, whereas that for dipyridamole remained unchanged. Additionally, when compared with the wild-type transporter, [3H]adenosine transport by L92Q transporter was significantly resistant to inhibition by inosine and guanosine but not by adenosine or pyrimidines. The Km value for inosine transport was approx. 4-fold greater for the L92Q mutant (260+/-16 mM) when compared with the wild-type transporter (65+/-7.8 mM). We have identified for the first time an amino acid residue (Leu92) of hENT1 that, when mutated, selectively alters the affinity of hENT1 to transport the nucleosides inosine and guanosine and its sensitivity to the inhibitors NBMPR and dilazep.
Mary V. Relling - One of the best experts on this subject based on the ideXlab platform.
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HPLC determination of thiopurine nucleosides and nucleotides in vivo in lymphoblasts following Mercaptopurine therapy.
Clinical chemistry, 2002Co-Authors: Thierry Dervieux, Yaqin Chu, Ching-hon Pui, William E. Evans, Mary V. RellingAbstract:Background: Mercaptopurine is a prodrug requiring intracellular activation to thiopurine nucleotides to exert antileukemic effect. We developed a reversed-phase liquid chromatographic assay for the quantification of Mercaptopurine, thioguanine, and methylMercaptopurine nucleoside and nucleotide concentrations in the target tissue, the leukemic lymphoblast. Methods: Leukemic blasts were isolated from peripheral blood and bone marrow by a standard Ficoll-hypaque procedure. Proteins were removed by ultrafiltration in the presence of dithiothreitol. Thiopurine ribonucleotides were converted into their respective ribonucleosides by treatment of ultrafiltrate with acid phosphatase. Thiopurine nucleosides and bases were measured by direct injection of ultrafiltrate into the chromatographic system. Thiopurine nucleotide concentrations were calculated by subtracting the thiopurine nucleoside concentrations measured after treatment with acid phosphatase from those measured after direct injection of ultrafiltrate in the chromatographic system. Analytes were separated on a C18 Supelco column with ammonium phosphate-methanol eluent coupled with ultraviolet detection. Results: CVs for intra- and interday precision were 1.1–14% (median, 4.9%), and recovery of added analyte was 89–126% (median, 105%) at low and high concentrations of analytes, except for Mercaptopurine Riboside. The median signal for each of the five metabolites in lymphoblast samples was 98% (range, 80–106%) of that in water. Detection limits for thiopurine bases and nucleosides ranged from 0.5 to 4.5 pmol/5 × 106 cells. Conclusions: This method is suitable for measurement of thiopurine metabolite concentrations in lymphoblasts in children with acute lymphoblastic leukemia following a single dose of intravenous Mercaptopurine.
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Assay of 6-Mercaptopurine and its metabolites in patient plasma by high-performance liquid chromatography with diode-array detection.
Journal of chromatography. B Biomedical sciences and applications, 1999Co-Authors: Yuen Yi Hon, Yaqin Chu, Matthijs E. C. Van De Poll, Mary V. RellingAbstract:Abstract A reversed-phase high-performance liquid chromatography (HPLC) method was developed to determine 6-Mercaptopurine (MP) and seven of its metabolites (6-thioguanine, 6-thioxanthine, 6-Mercaptopurine Riboside, 6-thioguanosine, 6-thioxanthine Riboside, 6-methylMercaptopurine and 6-methylMercaptopurine Riboside) simultaneously in human plasma. A volume of 100 μl of plasma was used. Protein was removed from the sample by a simple and easy ultrafiltration step and ultrafiltrate was directly injected onto the HPLC system. Analytes were detected and confirmed with a diode-array detector before quantitation at 295 and 330 nm. The limit of detection for the analytes ranged from 20 to 50 n M . For the majority of patients receiving a 1 g/m 2 MP intravenous infusion, MP and all metabolites except 6-thioguanine and 6-methylMercaptopurine Riboside were present. This method serves as useful tool to characterize pharmacokinetics and pharmacodynamics of MP in oncology patients, and the small volume of plasma lends itself to pediatric studies.