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Cancer Chemotherapy and Pharmacology Dec 2006Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that...
Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120-6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414-8422, 2005). Here we explored the ability of the wild type (WT) R482-as well as the mutant G482-and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed.
Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Antineoplastic Agents; Biological Transport; Cell Line; Cell Proliferation; Cell Survival; Cisplatin; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Neoplasm; Fluorouracil; Folic Acid Antagonists; Heterocyclic Compounds, 3-Ring; Humans; Indoles; Lipids; Molecular Structure; Mutation; Neoplasm Proteins; Paclitaxel; Pyrimethamine; Pyrimidines; Quinazolines; Rhodamines; Transfection; Trimetrexate
PubMed: 16612649
DOI: 10.1007/s00280-006-0230-9 -
Molecular Pharmacology Mar 1996We investigated the enzyme kinetic and antifolate inhibitory properties of human dihydrofolate reductase enzyme with mutations at position 22. Leu-22 was changed to...
We investigated the enzyme kinetic and antifolate inhibitory properties of human dihydrofolate reductase enzyme with mutations at position 22. Leu-22 was changed to isoleucine, methionine, phenylalanine, and tyrosine to generate the various mutant enzymes. The overall catalytic efficiency (kcat/Km) for methionine and phenylalanine mutants was reduced approximately 3-fold and >6-fold for isoleucine and tyrosine mutants. An arginine mutant (L22R) was also expressed but had a dramatically reduced catalytic potential (kcat>250-fold lower than wild-type) and therefore was not studied in detail. The Ki for antifolates, methotrexate, aminopterin, and trimetrexate are more dramatically affected (increased) than the Km for dihydrofolate, particularly for phenylalanine and tyrosine mutants. One remarkable feature is that the phenylalanine mutant is as potently inhibited by piritrexim as is the wild-type human enzyme, although the Ki values for methotrexate and aminopterin were increased 88- and 118-fold, respectively. This is likely related to different positioning of the methoxyphenyl side chain of piritrexim relative to the side chains of other compounds tested. A Chinese hamster cell line harboring the L22F mutant also demonstrated an increased sensitivity of piritrexim relative to antifolates.
Topics: Animals; Base Sequence; CHO Cells; Cells, Cultured; Cricetinae; Enzyme Inhibitors; Folic Acid Antagonists; Genetic Variation; Humans; Isoleucine; Kinetics; Leucine; Methotrexate; Molecular Sequence Data; Mutagenesis; Mutation; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase
PubMed: 8643082
DOI: No ID Found -
Human Gene Therapy Nov 1997Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of... (Comparative Study)
Comparative Study
Retroviral transduction of antifolate-resistant variants of human dihydrofolate reductase (hDHFR) into cells can increase their resistance to the cytotoxic effects of these drugs. We evaluated the ability of wild-type hDHFR and 20 mutant enzymes (13 with single-amino acid substitutions, 7 with two substitutions) to prevent growth inhibition in antifolate-treated CCRF-CEM cells. The wild-type enzyme and all of the variants significantly protected transduced cells from trimetrexate (TMTX)-induced growth inhibition. However, only half of the variants conferred more protection than does the wild-type enzyme. For the variants tested, the observed protective effect was higher for TMTX than for methotrexate (< or =7.5-fold increased resistance), piritrexim (< or =16-fold), and edatrexate (negligible). Transduction of the variants L22Y-F31S and L22Y-F31R led to the greatest protection against TMTX (approximately 200-fold). Protection from loss of cell viability was similar to protection from growth inhibition. The protection associated with a particular mutant hDHFR did not result from the level of expression: Efficient protection resulted from low affinity of the variant for antifolates, reasonable catalytic activity, and good thermal stability. Clones isolated from a polyclonal population of transduced cells varied by as much as 30-fold in their resistance to TMTX, the resistance differences depending on hDHFR expression levels.
Topics: Aminopterin; Animals; Cell Survival; Drug Resistance, Neoplasm; Folic Acid Antagonists; Gene Expression; Genetic Variation; Growth Inhibitors; Humans; Kinetics; Methotrexate; Pyrimidines; Rabbits; Tetrahydrofolate Dehydrogenase; Thymidine; Transfection; Trimetrexate
PubMed: 9414255
DOI: 10.1089/hum.1997.8.17-2069 -
Anti-cancer Drugs Jun 1993Chinese hamster ovary (CHO) T19 cells express a stable P-glycoprotein (P-170)-dependent multidrug resistance (MDR) phenotype and display a 24- to 29-fold...
Chinese hamster ovary (CHO) T19 cells express a stable P-glycoprotein (P-170)-dependent multidrug resistance (MDR) phenotype and display a 24- to 29-fold cross-resistance to the lipophilic antifolates piritrexim (PTX) and trimetrexate (TMTX). We have examined the ability of various modulators of the MDR phenotype to sensitize T19 cells to TMTX and PTX in a clonogenic assay. An almost complete reversal of TMTX resistance in T19 cells was achieved with several modulators of the MDR phenotype whereas only a partial sensitization of T19 cells to PTX was obtained with the most potent modulator. In an attempt to explore the apparent P-170-independent locus of protection against PTX, resistant T19 sublines were isolated after stepwise selection with PTX and TMTX. Thus, T19 cells made resistant to PTX displayed a dramatic decrease in P-170 mRNA levels despite the maintenance of the parental T19 MDR gene amplification, whereas T19 cells selected for TMTX resistance exhibited a further increase in P-170 mRNA levels. Hence, the modulation experiments together with the established lipophilic antifolate-resistant T19 variants suggest that although T19 cells possess a P-170-dependent MDR phenotype and display a similar cross-resistance to TMTX and PTX, the protective pathway need not be necessarily via P-170. Rather, a pathway appears to exist that protects T19 MDR cells from the cytotoxicity of PTX without requiring a P-170 function.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; CHO Cells; Carrier Proteins; Cricetinae; Drug Interactions; Drug Resistance; Membrane Glycoproteins; Methotrexate; Phenotype; Pyrimethamine; Pyrimidines; Quinidine; RNA, Messenger; Tetrahydrofolate Dehydrogenase; Trimetrexate
PubMed: 8102912
DOI: 10.1097/00001813-199306000-00018 -
British Journal of Cancer Apr 1992Exogenous purines (greater than or equal to 10(-5)M) can modulate the cytotoxicity of methotrexate (MTX) in cultured cells, protecting cells at low MTX concentrations...
Exogenous purines (greater than or equal to 10(-5)M) can modulate the cytotoxicity of methotrexate (MTX) in cultured cells, protecting cells at low MTX concentrations (less than or equal to 8 x 10(-8) M) and markedly potentiating its effect at higher concentrations. The ability of hypoxanthine (HX) to modulate the effects of two antifolates-ICI 198583 (an inhibitor of thymidylate synthetase) and piritrexim (PTX, a lipophilic inhibitor of DHFR)-was investigated using cultured mouse leukaemic cells, L1210. HX (10(-4) M) was found to potentiate only the cytotoxicity of DHFR inhibitors (MTS and PTX), increasing cell kill by 20-70 fold to the level achieved by an equivalent concentration (10(-5) M) of ICI 198583 alone. Agarose gel electrophoresis of DNA extracted from cells exposed to antifolates for 24 h demonstrated that the chromatin was cleaved into multimers of 200 base pairs. This pattern of DNA cleavage indicates cell death via apoptosis. The degree of DNA fragmentation was found to be closely linked to cytotoxicity. DNA fragmentation increased from 50% in cells treated with 10(-5) M MTX or PTX to 70% when HX was added with the drugs, a level achieved by 10(-5)M ICI 198583 alone. HX potentiation of cytotoxicity was correlated with a substantial increase in dATP in conjunction with low dTTP pools. The specific potentiation of DHFR inhibitors by HX may be due to their inhibition of purine synthesis with a concurrent rise in PRPP levels. Addition of HX with MTX substantially raised intracellular purine levels via the salvage pathway as indicated by ribonucleotide pool measurements. ICI 198583, on the other hand, stimulated de novo purine synthesis with or without added HX. Treatment with MTX plus HX or ICI 198583 (with or without HX) caused a reduction of dTTP pools to 8% of untreated control and excess dATP accumulation. The subsequent elevation (to 300% of control) of the dATP pool may provide a signal for endonucleolytic fragmentation of DNA and subsequent cell death.
Topics: Animals; DNA Damage; Deoxyadenine Nucleotides; Drug Synergism; Folic Acid; Hypoxanthine; Hypoxanthines; In Vitro Techniques; Leukemia L1210; Methotrexate; Mice; Tumor Cells, Cultured
PubMed: 1562458
DOI: 10.1038/bjc.1992.104 -
Journal of Medicinal Chemistry Dec 1985Sets of 5-(substituted benzyl)-2,4-diaminopyrimidines and 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3-substituted phenyl)-s-triazines as well as several other antifolates... (Comparative Study)
Comparative Study
Sets of 5-(substituted benzyl)-2,4-diaminopyrimidines and 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3-substituted phenyl)-s-triazines as well as several other antifolates were tested as inhibitors of Escherichia coli dihydrofolate reductase and E. coli cell cultures both sensitive and resistant to methotrexate. From the results quantitative structure-activity relationships (QSAR) were formulated. The triazines were found to inhibit sensitive and resistant cell cultures to the same degree, but the benzylpyrimidines showed marked differences against the two types of cells. Increased hydrophobicity produced benzylpyrimidines more active against the resistant E. coli cell. Metroprine did not discriminate between the two types of cells cultures, but pyrimethamine and 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidin e (BW 301U) did. The results are compared with triazines and benzylpyrimidines acting on Lactobacillus casei and murine tumor cells sensitive and resistant to methotrexate. QSAR is shown to be an effective means for detecting receptor differences.
Topics: Biological Transport, Active; Chemical Phenomena; Chemistry; Drug Resistance, Microbial; Escherichia coli; Folic Acid Antagonists; Lacticaseibacillus casei; Mathematics; Methotrexate; Pyrimethamine; Pyrimidines; Structure-Activity Relationship; Triazines
PubMed: 3934385
DOI: 10.1021/jm00150a026 -
Journal of Medicinal Chemistry Nov 2002Seven novel 2,4-diamino-5-deaza-6,7,8,9-tetrahydropyrido[3,4-g]pteridine derivatives 3-9 with different benzyl and a benzoyl substitution at the N7 position were...
Seven novel 2,4-diamino-5-deaza-6,7,8,9-tetrahydropyrido[3,4-g]pteridine derivatives 3-9 with different benzyl and a benzoyl substitution at the N7 position were designed and synthesized, as classical and nonclassical, partially restricted, linear tricyclic 5-deaza antifolates. The purpose was to investigate the effect of conformational restriction of the C6-C9 (tau(1)) and C9-N10 (tau(2)) bonds via an ethyl bridge from the N10 to the C7 position of 5-deaza methotrexate (MTX) on the inhibitory potency against dihydrofolate reductase (DHFR) from different sources and on antitumor activity. The synthetic methodology for most of the target compounds was a concise five-step total synthesis to construct the tricyclic nucleus, 2,4-diamino-5-deaza-7H-6,7,8,9-tetrahydropyrido[3,4-g]pteridine (23), followed by regioselective alkylation of the N7 nitrogen. Biological results indicated that this partial conformational modification for the classical analogue N-[4-[(2,4-diamino-5-deaza-6,7,8,9-tetrahydropyrido[3,4-g]pteridin-7-yl)methyl]benzoyl]-L-glutamic acid 3 was detrimental to DHFR inhibitory activity as well as to antitumor activity compared to MTX or 5-deaza MTX. However, the classical analogue 3 was a better substrate for folypolyglutamate synthetase (FPGS) than MTX. These results show that a classical 5-deaza folate partially restricted via a bridge between the N10 and C7 positions retains FPGS substrate activity and that the antitumor activity of classical tricyclic analogues such as 3 would be influenced by FPGS levels in tumor systems. Interestingly, the nonclassical analogues 4-9 showed moderate to good selectivity against DHFR from pathogenic microbes compared to recombinant human DHFR. These results support the idea that removal of the 5-methyl group of piritrexim along with restriction of tau(1) and tau(2) can translate into selectivity for DHFR from pathogens.
Topics: Animals; Antineoplastic Agents; Cell Division; Cell Line; Drug Resistance, Neoplasm; Escherichia coli; Folic Acid Antagonists; Heterocyclic Compounds, 3-Ring; Humans; Methotrexate; Molecular Conformation; Peptide Synthases; Pneumocystis; Recombinant Proteins; Species Specificity; Structure-Activity Relationship; Substrate Specificity; Tetrahydrofolate Dehydrogenase; Toxoplasma; Tumor Cells, Cultured
PubMed: 12408727
DOI: 10.1021/jm0202369 -
Journal of Medicinal Chemistry Jul 1995Six previously unknown 2,4-diamino-6-(anilinomethyl)- and 2,4-diamino-6-[(N-methylanilino)-methyl]pyrido[3,2-d]pyrimidines (5-10) were synthesized from...
Six previously unknown 2,4-diamino-6-(anilinomethyl)- and 2,4-diamino-6-[(N-methylanilino)-methyl]pyrido[3,2-d]pyrimidines (5-10) were synthesized from 2,4-diamino-6-(bromomethyl)-pyrido[3,2-d]pyrimidine hydrobromide (11.HBr) by treatment with the appropriate aniline or N-methylaniline in dimethylformamide at room temperature, with or without NaHCO3 present. Compounds 5-10 were tested as inhibitors of dihydrofolate reductase from Pneumocystis carinii, Toxoplasma gondii, and rat liver as a part of a larger effort directed toward the discovery of lipophilic nonclassical antifolates combining high enzyme selectivity and high potency. Of the six analogues tested, the most potent and selective against T. gondii DHFR was 2,4-diamino-6-[(3',4',5'-trimethoxy-N-methylanilono)methyl]pyrido[ 3,2-d d pyrimidine (7), which had an IC50 of 0.0047 microM against this enzyme as compared with 0.026 microM against the rat liver enzyme. The potency of 7 against T. gondii DHFR was similar to that of trimetrexate (TMQ, 1) and piritrexim (PTX, 2) but was > 500-fold greater than that of trimethoprim (TMP, 3). However, while 7 was more selective than either TMQ (19x) or PTX (63x) against this enzyme, its selectivity in comparison with TMP was 8-fold lower. 2,4-Diamino-6-[3',4',5'-trimethoxyanilino)methyl]pyrido[3,2-d]pyri midin e (6) was 17-fold less active than 7 and was also less selective. 2,4-Diamino-6-[(3',4'-dichloro-N-methylanilino)methyl]pyrido[3, 2-d]pyrimidine (10) had an IC50 of 0.022 microM against P. carinii DHFR and was comparable in potency to TMQ and PTX. The species selectivity of 10 for P. carinii versus rat liver DHFR was greater than that of either TMQ (21-fold) or PTX (31-fold). On the other hand, even though 10 was slightly more active than TMQ against the P. carinii enzyme, its selectivity was 7-fold lower than that of TMP. Thus, the goal of combining high enzyme binding activity, which is characteristic of the fused-ring compounds TMQ and PTX, with high selectivity for T. gondii and P. carinii DHFR versus rat liver DHFR, which is characteristic of the monocyclic compound TMP, remained unmet in this limited series.
Topics: Animals; Chromatography, Thin Layer; Folic Acid Antagonists; Liver; Magnetic Resonance Spectroscopy; Pneumocystis; Pyrimidines; Rats; Spectrophotometry, Infrared; Toxoplasma
PubMed: 7629801
DOI: 10.1021/jm00014a014 -
Oncology 1984The structural features and lipid solubility of four different classes of antifolate compounds were compared for their inhibition of dihydrofolate reductase (DHFR) and...
The structural features and lipid solubility of four different classes of antifolate compounds were compared for their inhibition of dihydrofolate reductase (DHFR) and growth in a normal and methotrexate (MTX)-resistant 3T6 mouse cell line. All of the compounds have been shown previously to have antifolate activity. The resistant cell line has a 7-fold increase in DHFR activity with normal transport, but an altered affinity for MTX. All the antifolates were equally effective in inhibiting DHFR and growth in the parent cell line. Inhibition of partially purified DHFR from the resistant cells increased with changes in lipid solubility and structure of the compounds, compared to the parent DHFR. These data demonstrate that the resistant cells may be more sensitive to the structurally dissimilar antifolates than to MTX and lend importance to further development of this type of antifolate. These results suggest that these compounds may be useful in circumventing antifolate resistance due to alterations in target enzyme concentration and drug-enzyme affinity, as well as drug transport.
Topics: Adamantane; Animals; Aspartic Acid; Cell Division; Cell Line; Drug Resistance; Folic Acid Antagonists; Methotrexate; Mice; Pyrimethamine; Pyrimidines
PubMed: 6540852
DOI: 10.1159/000225851 -
Journal of Medicinal Chemistry May 2004Six previously undescribed N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepines with water-solubilizing O-carboxyalkyloxy or O-carboxybenzyloxy side chains at the...
Synthesis of 2,4-diamino-6-[2'-O-(omega-carboxyalkyl)oxydibenz[b,f]azepin-5-yl]methylpteridines as potent and selective inhibitors of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductase.
Six previously undescribed N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepines with water-solubilizing O-carboxyalkyloxy or O-carboxybenzyloxy side chains at the 2'-position were synthesized and compared with trimethoprim (TMP) and piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three of the opportunistic organisms known to cause significant morbidity and mortality in patients with AIDS and other disorders of the immune system. The ability of the new analogues to inhibit reduction of dihydrofolate to tetrahydrofolate by Pc, Tg, Ma, and rat DHFR was determined, and the selectivity index (SI) was calculated from the ratio IC(50)(rat DHFR)/IC(50)(Pc, Tg, or Ma DHFR). The IC(50) values of the 2'-O-carboxypropyl analogue (10), with SI values in parentheses, were 1.1 nM (1300) against Pc DHFR, 9.9 nM (120) against Tg DHFR, and 2.0 nM (600) against Ma DHFR. The corresponding values for the 2'-O-(4-carboxybenzyloxy) analogue (12) were 1.0 nM (560), 22 nM (21), and 0.75 nM (630). By comparison, the IC(50) and SI values for TMP were Pc, 13 000 nM (14); Tg, 2800 nM (65); and Ma, 300 nM (610). For the prototypical potent but nonselective inhibitors PTX and TMX, respectively, these values were Pc, 13 nM (0.26) and 47 nM (0.17); Tg, 4.3 nM (0.76) and 16 nM (0.50); Ma, 0.61 nM (5.4) and 1.5 nM (5.3). Thus 10 and 12 met the criterion for DHFR inhibitors that combine the high selectivity of TMP with the high potency of PTX and TMX.
Topics: Animals; Azepines; Benzazepines; Folic Acid Antagonists; Models, Molecular; Mycobacterium avium; Pneumocystis carinii; Pteridines; Rats; Solubility; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase; Toxoplasma; Water
PubMed: 15115391
DOI: 10.1021/jm030599o