-
The Journal of Biological Chemistry Nov 1989We describe the development of resistance to trimetrexate and piritrexim (BW 301U) by a stepwise selection protocol in Chinese hamster ovary cells. Selection in... (Comparative Study)
Comparative Study
Sequential amplification of dihydrofolate reductase and multidrug resistance genes in Chinese hamster ovary cells selected for stepwise resistance to the lipid-soluble antifolate trimetrexate.
We describe the development of resistance to trimetrexate and piritrexim (BW 301U) by a stepwise selection protocol in Chinese hamster ovary cells. Selection in trimetrexate resulted in initial resistance as a result of dihydrofolate reductase gene amplification. Several trimetrexate-resistant variants that display 250-340-fold and 25-50-fold resistance to lipophilic and hydrophilic antifolates, respectively, were established. Increased antifolate resistance was associated with a prominent overexpression of dihydrofolate reductase as determined from the elevated folate reductase activity, cellular labeling with fluorescein-methotrexate, and steady-state mRNA levels as a result of a consistent dihydrofolate reductase gene amplification. However, upon subsequent incremental increases in trimetrexate, further resistance was also associated with amplification of the multidrug resistance gene. This resulted in overexpression of P-glycoprotein and a subsequent 20-50-fold collateral resistance to pleiotropic drugs such as adriamycin, actinomycin D, vinca alkaloids, etoposide, and colchicine. In contrast, initial resistance following selection with low piritrexim concentrations resulted from an unknown mechanism(s) not involving overproduction of either dihydrofolate reductase or P-glycoprotein. This piritrexim resistance was shared with trimetrexate but not with methotrexate. Upon further selection with piritrexim, resistant variants emerge with amplified dihydrofolate reductase but not with multidrug resistance genes. These variants were subsequently resistant to both hydrophilic and lipophilic folate antagonists but retained sensitivity to pleiotropic drugs. The pattern of resistance with methotrexate, trimetrexate, and piritrexim shared a common mechanism, dihydrofolate reductase gene amplification, but differed regarding the additional amplification of the multidrug resistance gene in trimetrexate-resistant cells as well as the emergence of an additional unknown mechanism(s) of resistance to lipid-soluble antifolates upon initial selection in piritrexim.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Cell Line; Cricetinae; Drug Resistance; Fluorescein; Fluoresceins; Folic Acid Antagonists; Gene Amplification; Gene Expression; Membrane Glycoproteins; Methotrexate; Pyrimidines; Quinazolines; RNA, Messenger; Tetrahydrofolate Dehydrogenase; Trimetrexate
PubMed: 2572592
DOI: No ID Found -
Bioorganic & Medicinal Chemistry Letters Aug 2019Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii (pj) can lead to serious health consequences in patients with an immunocompromised system. Trimethoprim...
Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii (pj) can lead to serious health consequences in patients with an immunocompromised system. Trimethoprim (TMP), used as first-line therapy in combination with sulfamethoxazole, is a selective but only moderately potent pj dihydrofolate reductase (pjDHFR) inhibitor, whereas non-clinical pjDHFR inhibitors, such as, piritrexim and trimetrexate are potent but non-selective pjDHFR inhibitors. To meet the clinical needs for a potent and selective pjDHFR inhibitor for PCP treatment, fourteen 6-substituted pyrido[3,2-d]pyrimidines were developed. Comparison of the amino acid residues in the active site of pjDHFR and human DHFR (hDHFR) revealed prominent amino acid differences which could be exploited to structurally design potent and selective pjDHFR inhibitors. Molecular modeling followed by enzyme assays of the compounds revealed 15 as the best compound of the series with an IC of 80 nM and 28-fold selectivity for inhibiting pjDHFR over hDHFR. Compound 15 serves as the lead analog for further structural variations to afford more potent and selective pjDHFR inhibitors.
Topics: Folic Acid Antagonists; Humans; Models, Molecular; Pneumocystis; Pneumocystis carinii; Pyrimidines; Structure-Activity Relationship; Trimethoprim
PubMed: 31176699
DOI: 10.1016/j.bmcl.2019.06.004 -
Archiv Der Pharmazie Jun 1995Two strategies towards the synthesis of Iso-Piritrexim (12) are described. A) The Mannich-reaction of ketone 2 yields the bases 4-HCl and 5-HCl. By means of LC base 4 is...
Two strategies towards the synthesis of Iso-Piritrexim (12) are described. A) The Mannich-reaction of ketone 2 yields the bases 4-HCl and 5-HCl. By means of LC base 4 is separated and treated with in situ generated 3,3-diaminoacrylonitrile (9) to yield the 2-aminonicotinonitrile 11. The cyclocondensation of 11 with guanidine provides Iso-PTX (12). B) Reduction and oxidation of the beta-ketoester 15 leads to the beta-ketoaldehyde 17, which is cyclocondensed with 2,4,6-triaminopyrimidine (18) to yield Iso-PTX (12). In the NCl-tumor-test Iso-PTX (12) shows a moderate activity against some leukemia and lung cancer cell lines.
Topics: Animals; Antineoplastic Agents; Drug Screening Assays, Antitumor; Folic Acid Antagonists; Humans; Mannich Bases; Pyrimidines; Tumor Cells, Cultured
PubMed: 7677569
DOI: 10.1002/ardp.19953280612 -
Journal of the American Chemical Society Aug 1988
PubMed: 22148802
DOI: 10.1021/ja00226a042 -
Bioorganic & Medicinal Chemistry May 2018To combine the potency of trimetrexate (TMQ) or piritrexim (PTX) with the species selectivity of trimethoprim (TMP), target based design was carried out with the X-ray...
Targeting species specific amino acid residues: Design, synthesis and biological evaluation of 6-substituted pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors and potential anti-opportunistic infection agents.
To combine the potency of trimetrexate (TMQ) or piritrexim (PTX) with the species selectivity of trimethoprim (TMP), target based design was carried out with the X-ray crystal structure of human dihydrofolate reductase (hDHFR) and the homology model of Pneumocystis jirovecii DHFR (pjDHFR). Using variation of amino acids such as Met33/Phe31 (in pjDHFR/hDHFR) that affect the binding of inhibitors due to their distinct positive or negative steric effect at the active binding site of the inhibitor, we designed a series of substituted-pyrrolo[2,3-d]pyrimidines. The best analogs displayed better potency (IC) than PTX and high selectivity for pjDHFR versus hDHFR, with 4 exhibiting a selectivity for pjDHFR of 24-fold.
Topics: Amino Acids; Anti-Bacterial Agents; Catalytic Domain; Crystallography, X-Ray; Drug Design; Enzyme Assays; Folic Acid Antagonists; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Molecular Docking Simulation; Molecular Structure; Pneumocystis carinii; Protein Binding; Pyrimidines; Pyrroles; Sequence Homology, Amino Acid; Species Specificity; Tetrahydrofolate Dehydrogenase
PubMed: 29691153
DOI: 10.1016/j.bmc.2018.04.032 -
Seminars in Oncology Oct 1996For the past 10 years, chemotherapy for advanced urothelial carcinoma has centered on cisplatin-based combination regimens such as methotrexate, vinblastine, doxorubicin... (Review)
Review
For the past 10 years, chemotherapy for advanced urothelial carcinoma has centered on cisplatin-based combination regimens such as methotrexate, vinblastine, doxorubicin and cisplatin (MVAC). Although such regimens have provided modest improvements in response rates, time to progression and survival, this has been achieved with moderate to severe toxicity. The median survival of patients with advanced disease remains at 12 to 13 months, and attempts to increase the dose intensity of existing regimens have been unsuccessful. Increasingly, attention has turned to the identification of new active agents, and a number have been recently identified, including paclitaxel, gemcitabine, ifosfamide, trimetrexate, piritrexim, and gallium nitrate. These agents will form the basis of new combination regimens that will attempt to improve on the advances in response and survival achieved with combination chemotherapy during the past decade.
Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Humans; Urinary Bladder Neoplasms
PubMed: 8893874
DOI: No ID Found -
Seminars in Oncology Oct 1997Many novel antifolate compounds with unique pharmacologic properties are currently in clinical development. These newer antifolates differ from methotrexate, the most... (Review)
Review
Many novel antifolate compounds with unique pharmacologic properties are currently in clinical development. These newer antifolates differ from methotrexate, the most widely used and studied drug in this class, in terms of their lipid solubility and cellular transport affinity, their level of polyglutamation, and their specificity for inhibiting folate-dependent enzymes, such as dihydrofolate reductase, thymidylate synthase, or glycinamide ribonucleotide formyltransferase. The current status (ie, mechanism of action, clinical response rates, and toxicity) of some of the newer antifolate compounds presently in clinical testing, including edatrexate, piritrexim, raltritrexed, LY 231514, AG337, AG331, 1843U89, ZD 9331, and lometrexol, is reviewed.
Topics: Aminopterin; Animals; Antimetabolites, Antineoplastic; Clinical Trials as Topic; Drug Design; Enzyme Inhibitors; Folic Acid Antagonists; Glutamates; Guanine; Humans; Indoles; Isoindoles; Pemetrexed; Pyrimidines; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidylate Synthase
PubMed: 9420020
DOI: No ID Found -
The Journal of Biological Chemistry Jun 1992The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and...
The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylases in which N10-formyltetrahydrofolate is the one-carbon donor. We have found that the antifolates methotrexate (MTX) and piritrexim (PTX) completely block the de novo purine pathway in mouse L1210 leukemia cells growing in culture but with only minor accumulations of GAR and AICAR to less than 5% of the polyphosphate derivatives of N-formylglycinamide ribotide (FGAR) which accumulate when the pathway is blocked completely by azaserine. This azaserine-induced accumulation of FGAR polyphosphates is completely abolished by MTX, indicating that inhibition of the pathway is at or before GAR transformylase (reaction 3; Lyons, S. D., and Christopherson, R. I. (1991) Biochem. Int. 24, 187-197). Three h after the addition of MTX (0.1 microM), cellular 5-phosphoribosyl-1-pyrophosphate has accumulated 3.4-fold while 6-methyl-mercaptopurine riboside (25 microM) induces a 6.3-fold accumulation. These data suggest that amido phosphoribosyltransferase catalyzing reaction 1 of the pathway is the primary site of inhibition. In support of this conclusion, we have found that dihydrofolate-Glu5, which accumulates in MTX-treated cells, is a noncompetitive inhibitor of amido phosphoribosyltransferase with a dissociation constant of 3.41 +/- 0.08 microM for interaction with the enzyme-glutamine complex in vitro. Folate-Glu5, MTX-Glu5, PTX, dihydrotriazine benzenesulfonyl fluoride, and AICAR also inhibit amido phosphoribosyltransferase.
Topics: Amidophosphoribosyltransferase; Aminoimidazole Carboxamide; Animals; Azaserine; Folic Acid Antagonists; Leukemia, Experimental; Methotrexate; Methylthioinosine; Mice; Purines; Pyrimidines; Ribonucleotides; Tumor Cells, Cultured
PubMed: 1597445
DOI: No ID Found -
Protein Expression and Purification 1991Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity...
Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography. The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence. The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7. The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl. Urea at 2.5 M also increased the enzyme activity twofold. Kinetic analysis of the purified enzyme revealed that the Km values for dihydrofolate and NADPH were 1.8 and 1.4 microM, respectively, and that the kcat was 70 s-1. Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P. carinii DHFR and showed little selectivity over the human DHFR. Trimetrexate and piritrexim were much more potent inhibitors of the P. carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR.
Topics: Cloning, Molecular; Escherichia coli; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Pneumocystis; Recombinant Proteins; Tetrahydrofolate Dehydrogenase
PubMed: 1821803
DOI: 10.1016/1046-5928(91)90088-z -
Acta Crystallographica. Section F,... Jun 2015To further define the interactions that enhance the selectivity of binding and to directly compare the binding of the most potent analogue...
To further define the interactions that enhance the selectivity of binding and to directly compare the binding of the most potent analogue {N(6)-methyl-N(6)-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine; compound 26} in the series of bicyclic pyrido[2,3-d]pyrimidine analogues of piritrexim (PTX) with native human (h), Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) dihydrofolate reductase (DHFR) enzymes, the crystal structures of hDHFR complexed with N(6)-methyl-N(6)-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 22), of hDHFR complexed with compound 26 and of pcDHFR complexed with N(6)-methyl-N(6)-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 24) are reported as ternary complexes with NADPH. This series of bicyclic pyrido[2,3-d]pyrimidines were designed in which there was a transposition of the 5-methyl group of PTX to the N9 position of the pyrido[2,3-d]pyrimidine. It was hypothesized that the N9-methyl group would preferentially interact with Ile123 of pcDHFR (and Ile123 of pjDHFR), but not with the shorter Val115 in hDHFR. Structure-activity data for this series of antifolates revealed that a trifluoro derivative (26) was the most selective against pjDHFR compared with mammalian DHFR (h/pj = 35.7). Structural data for the hDHFR-26 complex revealed that 26 binds in a different conformation from that observed in the pcDHFR-26 complex. In the hDHFR-26 complex the trifluorophenyl ring of 26 occupies a position near the cofactor-binding site, with close intermolecular contacts with Asp21, Ser59 and Ile60, whereas this ring in the pcDHFR-26 complex is positioned away from the cofactor site and near Ile65, with weaker contacts with Ile65, Phe69 and Ile123. Comparison of the intermolecular contacts between the N9-methyl group with Val115/Ile123 validates the hypothesis that the N9-methyl substituent preferentially interacts with Ile123 compared with Val115 of hDHFR, as the weaker contact with Val115 in the hDHFR structure is consistent with its weaker binding affinity compared with pcDHFR. The results for the structures of hDHFR-22 and pcDHFR-24 show that their inhibitor-binding orientation is similar to that observed in pcDHFR-26 and the pcDHFR variant (F69N) reported previously. The naphthyl moiety of 24 makes several intermolecular contacts with the active-site residues in pcDHFR that help to stabilize the binding, resulting in a more potent inhibitor.
Topics: Amino Acid Motifs; Anti-Bacterial Agents; Catalytic Domain; Crystallization; Crystallography, X-Ray; Folic Acid Antagonists; Halogenation; Humans; Models, Molecular; Molecular Sequence Data; NADP; Pneumocystis carinii; Protein Binding; Pyrimidines; Recombinant Proteins; Species Specificity; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase
PubMed: 26057816
DOI: 10.1107/S2053230X15008468