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Proceedings of the National Academy of... Feb 1996P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the...
P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Animals; Antibodies, Monoclonal; Biological Transport; Carrier Proteins; Cell Line; Clone Cells; Colony-Forming Units Assay; Drug Resistance, Multiple; Fibroblasts; Flow Cytometry; Folic Acid Antagonists; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Methotrexate; Mice; Neoplasm Proteins; Phleomycins; Pyrimidines; Recombinant Proteins; Retroviridae; Transfection; Vinblastine
PubMed: 8577747
DOI: 10.1073/pnas.93.3.1238 -
Antimicrobial Agents and Chemotherapy Oct 1994We have developed a new micromethod to study the effect of drugs on microsporidia, using MRC5 fibroblasts infected by 10(5) spores of Encephalitozoon cuniculi. After 3... (Comparative Study)
Comparative Study
We have developed a new micromethod to study the effect of drugs on microsporidia, using MRC5 fibroblasts infected by 10(5) spores of Encephalitozoon cuniculi. After 3 days of incubation with various concentrations of drugs, parasitic foci were counted in stained cultures. The inhibition of microsporidial growth exceeding 90% with albendazole (0.005 microgram/ml), fumagillin (0.001 microgram/ml), 5-fluorouracil (3 micrograms/ml), and sparfloxacin (30 micrograms/ml) was observed. Chloroquine, pefloxacin, azithromycin, and rifabutin were partially effective, at high concentrations. Arprinocid, metronidazole, minocycline, doxycycline, itraconazole, and difluoromethylornithine were not evaluable, since concentrations that inhibited microsporidia were also toxic for fibroblasts. Pyrimethamine, piritrexim, sulfonamides, paromomycin, roxithromycin, atovaquone, and flucytosine were ineffective. Our results confirm that albendazole and fumagillin have marked activity against E. cuniculi and show the antimicrosporidial activity of 5-fluorouracil and sparfloxacin. These data may form the basis for treatment of Encephalitozoon hellem and Septata intestinalis infections and represent an attempt to identify drugs effective against Enterocytozoon bieneusi.
Topics: Animals; Cell Line; Dogs; Dose-Response Relationship, Drug; Encephalitozoon cuniculi
PubMed: 7840584
DOI: 10.1128/AAC.38.10.2440 -
The International Journal of... Mar 2004Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate... (Comparative Study)
Comparative Study
Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-14C]orotate (OA) is quantitatively converted to [2-14C]OMP and then [2-14C]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102+/-11 microM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N(alpha)(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2-d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3-12-fold. These data indicate that LTX induces the most potent inhibition of APRT.
Topics: Amidophosphoribosyltransferase; Antineoplastic Agents; Cell Line, Tumor; Enzyme Inhibitors; Folic Acid Antagonists; Humans; Leukemia; Molecular Structure; Phosphoribosyl Pyrophosphate; Pyrimethamine
PubMed: 14687931
DOI: 10.1016/j.biocel.2003.08.014 -
The Journal of Biological Chemistry Mar 1992We have studied the discrepancy in the degree of methotrexate (MTX) resistance that exists between two clonal cell lines, mouse 3T6 R50 cells and Chinese hamster ovary...
We have studied the discrepancy in the degree of methotrexate (MTX) resistance that exists between two clonal cell lines, mouse 3T6 R50 cells and Chinese hamster ovary B11 0.5 cells that overexpress comparable levels of dihydrofolate reductase, yet exhibit a 100-fold difference in MTX resistance while maintaining similar sensitivity to the lipophilic antifolates trimetrexate and piritrexim. These data suggested that R50 cells may possess additional mechanism(s) of antifolate resistance, such as MTX transport alteration. Flow cytometric analysis using fluorescein methotrexate revealed comparable levels of fluorescein MTX displacement with lipophilic antifolates in viable R50 and B11 0.5 cells, but marked insensitivity of R50 cells to MTX competition, thus suggesting a poor uptake of MTX into R50 cells. Analysis of the kinetic parameters of dihydrofolate reductase from R50 cells neither showed alterations in enzyme affinities for various antifolates nor in the Michaelis constant for folic acid and NADPH nor a change in the pH activity optimum. R50 cell-free extracts contained wild-type levels of folylpoly-gamma-glutamyl synthetase activity. However, following metabolic labeling with [3H]MTX, no MTX polyglutamates could be detected in R50 cells. We conclude that the high level of MTX resistance in R50 cells is multifactorial, including overexpression of dihydrofolate reductase, reduced MTX transport, and possibly altered formation of MTX polyglutamates. The potential interactions between the different modalities of MTX resistance in R50 cells are being discussed.
Topics: Animals; Blotting, Northern; Blotting, Southern; CHO Cells; Cell Line; Cell Survival; Clone Cells; Cricetinae; DNA; Dose-Response Relationship, Drug; Drug Resistance; Electrophoresis, Gel, Two-Dimensional; Fibroblasts; Flow Cytometry; Folic Acid Antagonists; Hydrogen-Ion Concentration; Methotrexate; Mice; Protein Biosynthesis; Proteins; Pyrimidines; RNA; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase; Trimetrexate
PubMed: 1372892
DOI: No ID Found -
Antimicrobial Agents and Chemotherapy Oct 1989Three sulfonamides and four dihydrofolate reductase inhibitors were tested alone and in combination to determine their in vitro effects on two strains of Toxoplasma...
Three sulfonamides and four dihydrofolate reductase inhibitors were tested alone and in combination to determine their in vitro effects on two strains of Toxoplasma gondii grown in MRC5 fibroblast tissue culture. Toxoplasma growth was quantitated by an enzyme immunoassay performed directly on the fixed cultures, and linear regression models were used to quantify the relationship between the optical density values generated by the enzyme-linked immunosorbent assay and the concentrations of the antimicrobial agents in the culture medium. The cytopathic effects of antimicrobial agents on T. gondii were examined in Giemsa-stained cultures. Sulfonamides and dihydrofolate reductase inhibitors exhibited similar patterns of inhibition, consisting of an important increase of the inhibitory effect within a narrow range of concentrations. Sulfadiazine, sulfamethoxazole, and sulfisoxazole were all found to have important inhibitory effects on T. gondii; the 50% inhibitory concentrations estimated from the regression models were 2.5 micrograms/ml for sulfadiazine, 1.1 micrograms/ml for sulfamethoxazole, and 6.4 micrograms/ml for sulfisoxazole. This inhibition of growth was associated with a reduction of the number of parasitized cells and intracellular parasites that were morphologically normal. With dihydrofolate reductase inhibitors, including pyrimethamine, trimethoprim, trimetrexate-glycuronate, and piritrexim, a strong inhibition of Toxoplasma growth was observed, which was associated with striking morphological changes of the parasites. The 50% inhibitory concentrations were 0.04 microgram/ml for pyrimethamine, 2.3 micrograms/ml for trimethoprim, 0.16 ng/ml for trimetrexate-glycuronate, and 6.9 ng/ml for piritrexim. When sulfonamides and dihydrofolate reductase inhibitors were used in combination, a synergistic effect was observed with sulfadiazine combined with pyrimethamine, trimetrexate-glycuronate, and piritrexim; sulfisoxazole combined with pyrimethamine; and trimethoprim combined with sulfamethoxazole. These results were analyzed in comparison with human pharmacokinetics data.
Topics: Animals; Enzyme-Linked Immunosorbent Assay; Folic Acid Antagonists; Microbial Sensitivity Tests; Pyrimethamine; Pyrimidines; Quinazolines; Sulfamethoxazole; Sulfisoxazole; Toxoplasma; Trimethoprim; Trimetrexate
PubMed: 2531568
DOI: 10.1128/AAC.33.10.1753 -
Naunyn-Schmiedeberg's Archives of... Mar 1997We have previously reported that the histaminergic system is involved in the control of pain perception, and that substances able to enhance histamine brain levels, such...
We have previously reported that the histaminergic system is involved in the control of pain perception, and that substances able to enhance histamine brain levels, such as the histamine-N-methyltransferase inhibitor, metoprine, induce antinociception. In the present study, in order to corroborate the idea of inducing antinociception by inhibiting histamine catabolism, the effects of a noncompetitive histamine-N-methyltransferase inhibitor. SKF 91488, were studied in rodents by means of tests inducing three different kinds of noxious stimuli: thermal (mouse hot plate), chemical (mouse abdominal constrictions) and mechanical (rat paw pressure). The ability to react to noxious stimuli was assessed by the rota-rod test. In addition, a competitive inhibitor of the histamine catabolism enzyme, BW 301 U, was studied in the hot plate test. SKF 91488 (30, 50 and 100 micrograms per animal i.c.v.) raised dose-dependently the pain threshold in all three tests. To verify whether SKF 91488-induced antinociception is due to inhibition of histamine-N-methyltransferase, (R)-alpha-methylhistamine, described to block histamine release and synthesis by stimulating the histamine H3-autoreceptor and activating the negative feed-back mechanism, was used. When administered at doses which do not alter the pain threshold per se, 0.5 microgram per rat i.c.v. or 10 mg kg-1 i.p. in mice, (R)-alpha-methylhistamine was able to antagonize significantly the antinociceptive effect induced by 30 micrograms per animal i.c.v. of SKF 91488. BW 301 U (30 and 100 mg kg-1 i.p.) showed a dose-dependent, long-lasting antinociception, which was also antagonized by pretreatment with (R)-alpha-methylhistamine. The present data show that the antinociceptive effect previously described for metoprine is not restricted to this molecule, but is also shared by other histamine-N-methyl-transferase inhibitors. This generalization provides further evidence to the importance of the histaminergic system in pain control mechanisms.
Topics: Analgesics; Animals; Dimaprit; Dose-Response Relationship, Drug; Enzyme Inhibitors; Folic Acid Antagonists; Histamine Agonists; Histamine N-Methyltransferase; Injections, Intraventricular; Male; Methylhistamines; Mice; Muscle Contraction; Pain Measurement; Postural Balance; Psychomotor Performance; Pyrimidines; Rats; Rats, Wistar
PubMed: 9089666
DOI: 10.1007/pl00004954 -
Journal of Molecular Biology Jul 2002The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been...
The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR.
Topics: Amino Acid Sequence; Animals; Catalytic Domain; Crystallography, X-Ray; Folic Acid Antagonists; Humans; Hydrogen Bonding; In Vitro Techniques; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; NADP; Protein Conformation; Pyrimidines; Recombinant Proteins; Sequence Homology, Amino Acid; Tetrahydrofolate Dehydrogenase; Toxoplasma
PubMed: 12096917
DOI: 10.1016/s0022-2836(02)00469-2 -
International Journal For Parasitology May 1993A variety of anti-folate compounds have been tested for their ability to inhibit the growth of Babesia bovis as measured by the incorporation of [3H]hypoxanthine into...
A variety of anti-folate compounds have been tested for their ability to inhibit the growth of Babesia bovis as measured by the incorporation of [3H]hypoxanthine into the parasite's nucleic acids. Inhibitors of folate synthesis (including 7-methylguanosine and several sulpha drugs) were without effect but several structural analogues of folate were toxic. The most potent folate analogues were the lipophilic compounds piritrexim and trimetrexate, each causing 50% inhibition of [3H]hypoxanthine incorporation (IC50) at a concentration of 2.9 nM; other classical anti-folates such as pyrimethamine, methotrexate and trimethoprim were at least 100-fold less effective with IC50 values of 1.2, 0.29 and 0.50 microM, respectively. From these results we conclude that B. bovis does not synthesize folate de novo under cell culture conditions. However, the toxic effects of piritrexim and trimetrexate suggest that dihydrofolate reductase (DHFR) activity is essential for the parasite, most probably because of the role of this enzyme in the synthesis of thymidine nucleotides via thymidylate synthase.
Topics: Animals; Babesia bovis; Folic Acid; Folic Acid Antagonists; Pyrimethamine; Pyrimidines; Quinazolines; Trimethoprim; Trimetrexate
PubMed: 8359989
DOI: 10.1016/0020-7519(93)90016-r -
Journal of Molecular Modeling Sep 2012Pneumocystis carinii is typically a non-pathogenic fungus found in the respiratory tract of healthy humans. However, it may cause P. carinii pneumonia (PCP) in people...
Pneumocystis carinii is typically a non-pathogenic fungus found in the respiratory tract of healthy humans. However, it may cause P. carinii pneumonia (PCP) in people with immune deficiency, affecting mainly premature babies, cancer patients and transplant recipients, and people with acquired immunodeficiency syndrome (AIDS). In the latter group, PCP occurs in approximately 80% of patients, a major cause of death. Currently, there are many available therapies to treat PCP patients, including P. carinii dihydrofolate reductase (PcDHFR) inhibitors, such as trimetrexate (TMX), piritrexim (PTX), trimethoprim (TMP), and pyrimethamine (PMT). Nevertheless, the high percentage of adverse side effects and the limited therapeutic success of the current drug therapy justify the search for new drugs rationally planned against PCP. This work focuses on the study of pyrimidine inhibitors of PcDHFR, using both CoMFA and CoMSIA 3D-QSAR methods.
Topics: Catalytic Domain; Folic Acid Antagonists; Humans; Inhibitory Concentration 50; Models, Molecular; Pneumocystis carinii; Pyrimidines; Quantitative Structure-Activity Relationship; Static Electricity; Tetrahydrofolate Dehydrogenase
PubMed: 22527273
DOI: 10.1007/s00894-012-1399-y -
Advances in Enzyme Regulation 1985We have provided a rationale for the clinical use of a new lipid-soluble folate antagonist, BW 301U, in terms of its potential for killing several classes of... (Review)
Review
We have provided a rationale for the clinical use of a new lipid-soluble folate antagonist, BW 301U, in terms of its potential for killing several classes of methotrexate-resistant cells. As part of a Phase I evaluation of this agent we studied normal bone marrow from cancer patients and their metabolic susceptibility to either BW 301U or to MTX and then repeated the observations at the end of five days of BW 301U infusions. Both inhibitors were roughly comparable at equimolar concentrations prior to therapy, but a relative resistance developed to MTX after BW 301U treatment. Such findings were replicated in an in vitro HL-60 cell culture system that was exposed to BW 301U. Some possible mechanisms for this unusual collateral resistance are discussed.
Topics: Animals; Antineoplastic Agents; Bone Marrow; Cell Division; Cell Line; DNA; Drug Resistance; Folic Acid Antagonists; Humans; Leukemia, Experimental; Methotrexate; Mice; Models, Biological; Phenotype; Pyrimethamine; Pyrimidines
PubMed: 3915188
DOI: 10.1016/0065-2571(85)90086-x