1843u89 and nolatrexed

Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate to 2'-deoxythymidine-5'-monophosphate. This pathway provides the sole intracellular de novo source of 2'-deoxythymidine-5'-triphosphate; therefore, TS represents a critical target in cancer chemotherapy. 5-Fluorouracil (5-FU) was synthesized in 1957 and represents the first class of antineoplastic agents to be developed as inhibitors of TS. While 5-FU has been widely used to treat various human malignancies, its overall clinical efficacy is limited. Therefore, significant efforts have focused on the design of novel, more potent inhibitor compounds of TS. These agents fall into two main categories: folate analogs and nucleotide analogs. Five antifolate analogs are currently being evaluated in the clinic: raltitrexed, pemetrexed, nolatrexed, ZD9331, and GS7904L. Our laboratory has identified a novel mechanism of resistance that develops to TS inhibitor compounds, namely drug-mediated acute induction of new TS synthesis; this mechanism is directly controlled at the translational level. The ability of cancer cells to acutely induce the expression of TS may represent a novel mechanism for the development of cellular drug resistance. The future success of TS inhibitor compounds in the clinic may depend on novel strategies to selectively inhibit TS and on novel combination therapies to overcome cellular drug resistance.

Topics: Antineoplastic Agents; Enzyme Inhibitors; Humans; Indoles; Isoindoles; Neoplasms; Quinazolines; Randomized Controlled Trials as Topic; Thiophenes; Thymidylate Synthase

Thymidylate synthase (TS) is a critical enzyme for DNA replication and cell growth because it is the only de novo source of thymine nucleotide precursors for DNA synthesis. TS is the primary target of 5-fluorouracil (5-FU), which has been used for cancer treatment for more than 40 years. However, dissatisfaction with the overall activity of 5-FU against the major cancers, and the recognition that TS still remains an attractive target for anticancer drugs because of its central position in the pathway of DNA synthesis, led to a search for new inhibitors of TS structurally analogous to 5,10-methylenetetrahydrofolate, the second substrate of TS. TS inhibitory antifolates developed to date that are in various stages of clinical evaluation are ZD 1694 and ZD9331 (Astra-Zeneca, London, UK), (Eli Lilly, Indianapolis, IN), LY231514 (BW1843U89 (Glaxo-Wellcome, Research Triangle Park, NC), and AG337 and AG331 (Agouron, La Jolla, CA). Although each of these compounds has TS as its major intracellular site of action, they differ in propensity for polyglutamylation and for transport by the reduced folate carrier. LY231514 also has secondary target enzymes. As a result, each compound is likely to have a different spectrum of antitumor activity and toxicity. This review will summarize the development and properties of this new class of TS inhibitors.

Topics: Animals; Antimetabolites, Antineoplastic; Colorectal Neoplasms; Enzyme Inhibitors; Folic Acid; Folic Acid Antagonists; Glutamates; Guanine; Humans; Indoles; Isoindoles; Pemetrexed; Quinazolines; Thiophenes; Thymidylate Synthase

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

Trifluorothymidine (TFT) is a fluoropyrimidine that is part of the novel combination metabolite TAS-102, in which TFT is combined with a potent thymidine phosphorylase inhibitor (TPI). TAS-102 is currently tested as an orally chemotherapeutic agent in different schedules in a phase I study. In its monophosphate form, TFT can inhibit thymidylate synthase (TS) activity after binding to the TS-nucleotide binding site leading to dTTP depletion, and in its triphosphate form TFT is incorporated into DNA, eventually leading to DNA damage. In this in vitro study, we investigated whether TFT could potentiate cytotoxicity of the antifolate-based TS inhibitors AG337 (Nolatrexed), ZD1694 (Raltitrexed) and GW1843; and whether increased TS inhibition or DNA damage would be related to this result.. The drug combinations were studied in colon cancer cell lines either grown at low or high folate conditions. Multiple drug effect analysis was performed after measuring growth inhibition when the drugs were combined (MTT Assay) and expressed as Combination Index (CI), where CI<0.9 indicates synergism, CI=0.9-1.1 indicates additivity and CI>1.1 indicates antagonism. Drug target analysis was performed using the TS in situ inhibition assay and the FADU DNA-damage assay. Cells were exposed to either the drugs alone or in combination to determine the effect on TS activity and DNA damage induction, respectively.. Three experimental procedures were used to test the interaction of the drugs: either one of the drugs was kept at a constant concentration (IC25) or two drugs were added in a 1:1 IC50-based molar ratio. The combinations of TFT with one of the antifolates in which one of the drugs was kept at a constant concentration were synergistic for all antifolates in WiDr/F cells, which grow in low folate medium (CI=0.6-0.8), but only additive to antagonistic for the cell lines growing in high folate medium: TFT-AG337: CI=0.9-2.3; TFT-ZD1694: CI=0.9-1.3; TFT-GW1843: CI=0.8-1.7. The procedure in which the two drugs were added in a 1:1 IC50-based molar ratio showed antagonism for all three combinations in all cell lines (CI>2.7). TS inhibition (14.3%) and DNA damage (8%) were more pronounced than expected (P<0.05) when TFT was combined with GW1843 in WiDr/F cells, in contrast to AG337 and ZD1694, which showed inhibiting effects as expected (additive).. The combination of TFT with the antifolates AG337, ZD1694 and GW1843 is mainly additive when the drugs are given simultaneously and this is mediated by an additive TS inhibition and DNA damage. The drug interaction may partly be dependent on the folate homeostasis since WiDr/F cells growing at low folate conditions show pronounced synergism in growth inhibition, two-sided TS inhibition and DNA damage, especially when TFT is combined with the tight-binding TS inhibitor GW1843.

Topics: Antimetabolites; Colonic Neoplasms; DNA Damage; Drug Interactions; Folic Acid; Folic Acid Antagonists; Homeostasis; Humans; Indoles; Isoindoles; Quinazolines; Thiophenes; Trifluridine; Tumor Cells, Cultured

Thymidylate synthase (TS) is an important target for cancer chemotherapy. However, several mechanisms of resistance to TS inhibitors have been described. One mechanism that may be relevant to short-term exposure to TS inhibitors occurs as a result of disruption of the autoregulatory loop, which allows TS to control its own translation. This disruption leads to up-regulation of TS protein and is generally thought to decrease efficacy. This study has investigated TS protein up-regulation using a range of TS inhibitors in both tumor and nonmalignant cell lines in vitro and in vivo. Up-regulation of TS protein showed a time-, dose-, and cell-type-specific response to treatment with ZD9331. This response was observed in W1L2 cells treated for 24 h at equitoxic doses of raltitrexed (6-fold), ZD9331 (10-fold), fluorouracil (5-fold), LY231514 (7-fold), AG337 (7-fold), and BW1843U89 (3-fold). Up-regulation was observed over a range of doses. Elevation of TS protein only persisted up to 12 h after removal of drug. The extent of induction does not depend on basal TS levels. Nontransformed human fibroblasts showed significantly greater up-regulation of TS protein than tumor cells exposed to an equitoxic dose of ZD9331. In vivo experiments using the L5178Y thymidine kinase -/- mouse lymphoma implanted into DBA2 mice also showed greater up-regulation of TS protein in normal intestinal epithelial cells compared with tumor cells. These results confirm that TS up-regulation is a common feature of TS inhibition in tumor cells and that it may occur to a greater extent in normal tissues, although the clinical implications of these findings remain to be determined.

Topics: Analysis of Variance; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Blotting, Western; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Flow Cytometry; Fluorouracil; Glutamates; Guanine; Humans; Indoles; Inhibitory Concentration 50; Isoindoles; Mice; Mice, Inbred DBA; Microscopy, Confocal; Neoplasm Transplantation; Pemetrexed; Quinazolines; Thiophenes; Thymidylate Synthase; Time Factors; Tumor Cells, Cultured; Up-Regulation

We have studied the molecular basis for the resistance of human CEM leukemia cells to GW1843, a thymidylate synthase inhibitor. GW1843-resistant cells displayed a approximately 100-fold resistance to GW1843 and methotrexate but were collaterally sensitive to the lipophilic antifolates trimetrexate and AG337, which enter cells by diffusion. These cells exhibited a 12-fold decreased methotrexate influx but surprisingly had a 2-fold decreased folic acid growth requirement. This was associated with a 4-fold increased influx of folic acid, a 3.5-fold increased steady-state level of folic acid, and a 2.3-fold expansion of the cellular folate pool. Characterization of the transport kinetic properties revealed that GW1843-resistant cells had the following alterations: (a) 11-fold decreased transport K(m) for folic acid; (b) 6-fold increased transport K(m) for GW1843; and (c) a slightly increased transport V(max) for folic acid. Sequence analysis showed that GW1843-resistant cells contained the mutations Val-29 --> Leu, Glu-45 --> Lys, and Ser-46 --> Ile in the first transmembrane domain of the reduced folate carrier. Transfection of the mutant-reduced folate carrier cDNA into methotrexate transport null cells conferred resistance to GW1843. This is the first demonstration of multiple mutations in a confined region of the human reduced folate carrier in an antifolate-resistant mutant. We conclude that certain amino acid residues in the first transmembrane domain play a key role in (anti)folate binding and in the conferring of drug resistance.

Topics: Antimetabolites, Antineoplastic; Biological Transport; Blotting, Northern; Blotting, Southern; Blotting, Western; Carrier Proteins; Cell Division; Cell Membrane; Chlorides; DNA Mutational Analysis; DNA, Complementary; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Inhibitors; Exons; Folic Acid; Folic Acid Antagonists; Humans; Indoles; Inhibitory Concentration 50; Isoindoles; Kinetics; Leucovorin; Leukemia; Membrane Proteins; Membrane Transport Proteins; Methotrexate; Mutagenesis, Site-Directed; Mutation; Polymorphism, Single-Stranded Conformational; Protein Structure, Secondary; Protein Structure, Tertiary; Quinazolines; Recombinant Proteins; Reduced Folate Carrier Protein; Thymidylate Synthase; Time Factors; Transfection; Trimetrexate; Tumor Cells, Cultured

The combined cytotoxic effects of the thymidylate synthase (TS) inhibitors 5-fluorouracil (5FU) and different antifolates were studied in seven colon cancer cell lines. Growth inhibition of the antifolates, Nolatrexed, Raltitrexed, GW1843U89, or MTA in combination with 5FU, was determined and multiple drug effect analysis showed that the drugs acted mostly additively. The only synergistic interaction was found for 5FU and Nolatrexed in the LS174T cell line. Also Raltitrexed and 5FU were slightly synergistic in WiDr/F cells grown at low folate levels, but for the other cell lines grown at high folate levels this combination was more antagonistic. GW1843U89 and 5FU were mainly additive, while 5FU and MTA showed antagonism in WiDr and additivity in LS174T. The effect of the drugs at their target was evaluated by in situ TS inhibition. We observed lower TS activity in all cells when two drugs were used instead of one. Statistical analysis revealed that none of the values of the combinations was higher or lower than could be expected from the product of the effect of single drugs. We concluded that the effects on TS inhibition were additive for all 5FU/antifolate combinations in all cell lines. DNA strand break formation, as a result of TS inhibition, was measured by means of a fluorometric analysis of DNA unwinding. Raltitrexed-induced DNA damage was significantly increased by 5FU in WiDr cells [single agent: 67% double stranded (ds) DNA, combination: 39% ds DNA, P<0.0001]. In LS174T a trend for antagonistic effects was observed for combinations of MTA, GW1843U89, or Raltitrexed and 5FU. The combinations showed additive effects in WiDr/F cells. The overall conclusion of the three assays in each of the cell lines indicated that 5FU and antifolate combinations were predominantly additive in colon cancer cells.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Colonic Neoplasms; DNA Damage; Fluorouracil; Folic Acid Antagonists; Glutamates; Guanine; Humans; Indoles; Isoindoles; Pemetrexed; Quinazolines; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

Human thymidylate synthase (TS) contains three highly conserved residues Ile-108, Leu-221, and Phe-225 that have been suggested to be important for cofactor and antifolate binding. To elucidate the role of these residues and generate drug-resistant human TS mutants, 14 variants with multiple substitutions of these three hydrophobic residues were created by site-directed mutagenesis and transfected into mouse TS-negative cells for complementation assays and cytotoxicity studies, and the mutant proteins expressed and characterized. The I108A mutant confers resistance to raltitrexed and Thymitaq with respective IC50 values 54- and 80-fold greater than wild-type but less resistance to BW1843U89 (6-fold). The F225W mutant displays resistance to BW1843U89 (17-fold increase in IC50 values), but no resistance to raltitrexed and Thymitaq. It also confers 8-fold resistance to fluorodeoxyuridine. Both the kinetic characterization of the altered enzymes and formation of antifolate-resistant colonies in mouse bone marrow cells that express mutant TS are in accord with the IC50 values for cytotoxicity noted above. The human TS mutants (I108A and F225W), by virtue of their desirable properties, including good catalytic function and resistance to antifolate TS inhibitors, confirm the importance of amino acid residues Ile-108 and Phe-225 in the binding of folate and its analogues. These novel mutants may be useful for gene transfer experiments to protect hematopoietic progenitor cells from the toxic effects of these drugs.

Topics: Animals; Antimetabolites, Antineoplastic; Binding Sites; Bone Marrow Cells; Drug Resistance; Folic Acid; Folic Acid Antagonists; Granulocytes; Humans; Indoles; Isoindoles; Isoleucine; Kinetics; Macrophages; Mice; Models, Molecular; Mutagenesis, Site-Directed; Quinazolines; Recombinant Proteins; Stem Cells; Thiophenes; Thymidylate Synthase

Resistance to some (lipophilic) antifolates has been associated with P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). A possible relationship with non-P-gp MDR has not been established. We studied resistance to antifolates in SW-1573 human lung carcinoma cells, a P-gp overexpressing variant SW-1573/2R160 and a multidrug resistance protein (MRP) overexpressing variant SW-1573/2R120. In this study, thymidylate synthase (TS) inhibitors with different properties concerning the efficiency of membrane transport and the efficiency of polyglutamylation were tested for cross-resistance in SW-1573/2R120 and SW-1573/2R160 cells. Growth inhibition patterns in this cell line panel were measured by the Sulforhodamine B (SRB) assay. Resistance factors for TS inhibitors were: 2.4 and 0.4 for 5-fluorouracil (5FU), 18.8 and 8.8 for ZD1694, 17 and 0.7 for AG337, and 40 and 8.3 for BW1843U89 in SW-1573/2R160 and SW-1573/2R120, respectively. This study showed changes in the TS enzyme kinetics during the induction of doxorubicin resistance in both SW-1573 variants, resulting in 2-fold lower Km values for 2'-deoxyuridine-5'-monophosphate (dUMP) in both resistant variants compared to the parental cell line. TS activity, TS protein induction and TS mRNA expression all had 2-fold increased in the SW-1573/2R120 compared to the SW-1573/2R160. 3H-MTX influx was 2-fold lower in SW-1573/2R160 cells compared to SW-1573/2R120 and SW-1573 cells. In the SW-1573/2R160 cell line, an aberrant intracellular trafficking towards the target TS was observed, compared to SW-1573/2R120 and SW-1573 cells as measured by the TS in situ assay. The rate of TS inhibition by the TS inhibitors used in this study was similar in all cell lines. In conclusion, collateral sensitivity to 5FU and the lipophilic AG337 and cross-resistance to other antifolates were observed in non-P-gp MDR SW-1573/2R120 cells, as well as resistance to all antifolates in P-gp SW-1573/2R160 cells. The mechanism of resistance in SW-1573/2R160 cells possibly involves reduced influx and changes in intracellular trafficking routes. For the SW-1573/2R120 cell line, several changes related to the TS enzyme possibly play a role in the observed cross-resistance and collateral sensitivity pattern.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Western; Cell Division; Drug Resistance, Multiple; Fluorodeoxyuridylate; Fluorouracil; Folic Acid Antagonists; Gene Expression Regulation; Humans; Indoles; Isoindoles; Methotrexate; Peptide Synthases; Polymerase Chain Reaction; Quinazolines; RNA, Messenger; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured