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1.
Prostate ; 46(4): 298-306, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11241552

ABSTRACT

BACKGROUND: Prostate-specific antigen (PSA), a member of the kallikrein family of serine proteases, is a chymotrypsin-like glycoprotein produced by the prostate epithelium. Elevated serum PSA (> 4 ng/ml) is a tumor marker for prostatic cancer and benign prostatic hypertrophy; increasing serum PSA over time is indicative of metastatic disease. It has been suggested that PSA may contribute to tumor metastasis through degradation of extracellular matrix glycoproteins, as well as cleavage of IGF binding protein-3, a modulator of IGF-1. To elucidate the role of PSA in the development and progression of prostatic cancer, it is necessary to have a reliable, cost-effective source of enzymatically active protein. Previous efforts to express recombinant PSA (rPSA) produced inactive proPSA, or mixtures of active and inactive PSA requiring activation by removal of the propeptide. We describe the expression of active recombinant mature PSA in yeast. METHODS: Stable chromosomal integration of a construct consisting of the yeast alpha-factor signal sequence preceding the mature PSA sequence resulted in secretion of rPSA. The rPSA was purified from the yeast cell culture supernatant to homogeneity by strong cation-exchange chromatography, and characterized by SDS-PAGE, Western analysis, electrospray mass spectrometry, N-glycanase digestion, N-terminal amino acid sequencing, and inactivation by a PSA-specific inhibitor. RESULTS: We report the production of active, mature rPSA in Pichia pastoris. Two forms of rPSA varying slightly in glycosylation were identified. The specific activity of the rPSA was equal to that of human seminal plasma PSA (0.56 micromol/min mg) as determined using a chromogenic substrate. CONCLUSIONS: Large-scale production of active rPSA will be useful in the exploration of PSA effects on tumor cell proliferation, migration and metastasis. In addition, a large supply of enzyme should facilitate the discovery of novel inhibitors for in vitro and in vivo evaluation, and may provide a reproducible source of rPSA for use as a standard in diagnostic testing.


Subject(s)
DNA, Neoplasm/metabolism , DNA, Recombinant/metabolism , Genetic Vectors , Pichia , Prostate-Specific Antigen/biosynthesis , Prostatic Neoplasms/immunology , Amidohydrolases/metabolism , Blotting, Western , DNA Primers , DNA, Neoplasm/genetics , DNA, Recombinant/genetics , Gene Expression Regulation, Fungal , Gene Expression Regulation, Neoplastic , Humans , Male , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Polymerase Chain Reaction/methods , Prostate-Specific Antigen/genetics , Prostatic Neoplasms/genetics , Spectrometry, Mass, Electrospray Ionization
2.
Prog Drug Res ; 55: 213-33, 2000.
Article in English | MEDLINE | ID: mdl-11127964

ABSTRACT

The early demonstrations that prostate cancer was hormone-sensitive initiated a therapeutic strategy of hormone ablation that is still in use today. Although chemical or surgical castration reduces androgen stimulation of the androgen receptor (AR) and produces tumor regression, little survival benefit is achieved. Patients with metastatic cancer eventually relapse as their tumors progress to hormone independence. The AR is a member of the steroid receptor family; however, it manifests many unique features including: N-terminal, C-terminal interactions and antiparallel dimerization, unique N-terminal domains for co-factor recruitment, AR-specific co-activators and upstream promoter/enhancer response elements that amplify AR-mediated responses. The AR is regulated by phosphorylation and cross-talk with several signaling pathways, including MAP kinases, PKA and PKC. Non-genomic effects of AR to regulate transcription factors elk-1 and -2 have also been demonstrated. These unique features suggest mechanisms by which novel therapeutics might target and influence AR-mediated actions. Progress in this direction has been realized with the recent synthesis of non-steroidal androgen agonists that may have tissue-selective effects.


Subject(s)
Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Receptors, Androgen/drug effects , Animals , Humans , Male
3.
Semin Oncol ; 26(2 Suppl 6): 42-7, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10598554

ABSTRACT

The pyrrolopyrimidine-based antifolate, N-¿4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]benzoyl¿glutamic acid, LY231514 (MTA) has demonstrated antitumor activity in a broad array of human tumors, including breast cancer, colon cancer, non-small cell lung cancer, head and neck cancer, pancreatic cancer, and other solid tumors. The biochemical basis of this activity was explored by measuring activation of MTA by polyglutamation and the activity of MTA to inhibit several folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase (GARFT). The enzyme folylpolyglutamate synthase (FPGS) activated MTA very efficiently. Using FPGS from two different species, Km values below 2 micromol/L and high relative first order rate constants, k' (Vmax/Km) of 6.4 and 13.7 compared with another substrate, lometrexol, were obtained. The formation of polyglutamates of several antifolates were compared in vitro at high and low substrate concentrations. At low MTA concentrations, tetraglutamated and pentaglutamated MTA were the predominant forms identified after a 24-hour incubation period. In contrast, only diglutamyl methotrexate and a mixture triglutamylated, tetraglutamylated, and pentaglutamylated forms of the GARFT inhibitor lometrexol were formed under the same conditions. At higher substrate concentrations (20 micromol/L, 24 hours), greater amounts of each product were formed. The major metabolites, however, were triglutamated MTA or triglutamated lometrexol, while only diglutamyl methotrexate was recovered. Thus, MTA was an excellent substrate for FPGS and it was efficiently metabolized to highly polyglutamated species by this enzyme. The activity of MTA and its polyglutamated metabolites to inhibit several folate-dependent enzymes was measured. In vitro, MTA and its polyglutamates were potent, tight-binding inhibitors of several folate-dependent enzymes, including thymidylate synthase, dihydrofolate reductase, and GARFT. Preliminary cell-based assays (CCRF-CEM) demonstrated inhibition of the purine de novo pathway by MTA, consistent with its multitargeted mechanism of action against tumor cells. The combined effects of activation of MTA to highly polyglutamated metabolites and the potency of these polyglutamates to inhibit multiple folate-dependent enzymes provide a mechanistic basis for understanding the broad antitumor activity of this compound against many human tumor types.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Enzyme Inhibitors/pharmacology , Folic Acid Antagonists/pharmacology , Glutamates/pharmacology , Guanine/analogs & derivatives , Peptide Synthases/metabolism , Animals , Glutamates/metabolism , Guanine/metabolism , Guanine/pharmacology , Humans , Methotrexate/metabolism , Methotrexate/pharmacology , Pemetrexed , Polyglutamic Acid/metabolism , Purines/antagonists & inhibitors , Purines/biosynthesis , Tetrahydrofolates/metabolism , Tetrahydrofolates/pharmacology
4.
Semin Oncol ; 26(2 Suppl 6): 48-54, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10598555

ABSTRACT

The mechanism of action of an antifolate may be investigated using a variety of experimental methods. These include experiments in a cell culture setting to observe possible protection against drug effects afforded by the end products of metabolic pathways, assessing the activity of purified target enzymes in the presence of the antifolate, and, finally, the measurement of drug effects on intracellular folate and nucleoside triphosphate pools. The current discussion is focused on studies using CCRF-CEM leukemia cells that were designed to compare and contrast mechanisms of action of the antifolates methotrexate, which is primarily a dihydrofolate reductase inhibitor, raltitrexed, a thymidylate synthase inhibitor, LY309887, a glycinamide ribonucleotide formyltransferase inhibitor, and MTA (multitargeted antifolate), which is a novel antifolate antimetabolite. The results of these studies support the hypothesis that MTA affects multiple enzymatic targets and has a distinct mechanism of action from methotrexate, raltitrexed, and LY309887.


Subject(s)
Acid Anhydride Hydrolases/drug effects , Antimetabolites, Antineoplastic/pharmacology , Deoxyribonucleotides/metabolism , Enzyme Inhibitors/pharmacology , Folic Acid Antagonists/pharmacology , Glutamates/pharmacology , Guanine/analogs & derivatives , Thymidylate Synthase/antagonists & inhibitors , Thymine Nucleotides/metabolism , Acid Anhydride Hydrolases/metabolism , Folic Acid/drug effects , Folic Acid/metabolism , Guanine/pharmacology , Humans , Methotrexate/pharmacology , Nucleoside-Diphosphate Kinase/metabolism , Nucleoside-Triphosphatase , Pemetrexed , Quinazolines/pharmacology , Ribonucleotides/metabolism , Tetrahydrofolates/pharmacology , Thiophenes/pharmacology , Tumor Cells, Cultured
5.
Adv Enzyme Regul ; 39: 313-27, 1999.
Article in English | MEDLINE | ID: mdl-10470381

ABSTRACT

The macrocyclic bisindolylmaleimide, LY333531, selectively inhibits protein kinase C beta 1 and beta 2 isoforms with an approximate IC50 of 5 nanomolar. The efficacy of LY333531 administered alone and in combination with cytotoxic cancer therapies in models of non-small cell lung carcinoma and brain tumors was determined in vivo. In the Lewis lung carcinoma, administration of LY333531 enhanced the activity of paclitaxel and fractionated radiation and, to a lesser degree, carboplatin and gemcitabine. In the human T98G glioblastoma multiforme xenograft, the addition of LY333531 to treatment with carmustine (BCNU) resulted in enhanced tumor response in a nodule grown subcutaneously and increased life-span in animals bearing an intracranial tumor from 37 days in the control animals to 64 days in the BCNU treated animals, and to 104 days in the LY333531 plus BCNU treated animals with 4 out of 5 animals being long-term survivors.


Subject(s)
Enzyme Inhibitors/therapeutic use , Isoenzymes/antagonists & inhibitors , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/enzymology , Protein Kinase C/antagonists & inhibitors , Animals , Antineoplastic Agents/administration & dosage , Brain Neoplasms/drug therapy , Carcinoma, Non-Small-Cell Lung/drug therapy , Combined Modality Therapy , Drug Therapy, Combination , Enzyme Inhibitors/administration & dosage , Female , Glioblastoma/drug therapy , Humans , Indoles/administration & dosage , Indoles/therapeutic use , Lung Neoplasms/drug therapy , Lung Neoplasms/secondary , Male , Maleimides/administration & dosage , Maleimides/therapeutic use , Mice , Mice, Inbred C57BL , Mice, Nude , Neoplasm Transplantation , Neoplasms, Experimental/radiotherapy , Protein Kinase C beta , Transplantation, Heterologous
6.
Cancer Res ; 59(15): 3671-6, 1999 Aug 01.
Article in English | MEDLINE | ID: mdl-10446980

ABSTRACT

The proliferation rate of HT29 colon carcinoma cells was decreased by the multitargeted antifolate (MTA), LY231514. This effect correlated with a buildup of cells near the G1-S interface after 24 h of incubation, and a synchronized progression of the population through S phase during the next 24 h. MTA treatment (0.03-3 microM) was minimally cytotoxic (20-30%) to HT29 cells after a 24-h exposure, and no dose response was observed. In contrast, the nucleoside analogue gemcitabine (GEM) was cytotoxic (IC50, 0.071 +/- 0.011 microM; IC90, 0.648 +/- 0.229 microM) after a 24-h exposure. We hypothesized that pretreatment of these cells with MTA would increase the potency of GEM by synchronizing the population for DNA synthesis. The cytotoxicity of GEM increased 2-7-fold when MTA was administered 24 h before GEM (IC50, 0.032 +/- 0.009 microM; IC90, 0.094 +/- 0.019 microM). In addition, an increase in cell kill for the combination compared with GEM alone (IC99, 12 microM for GEM alone; IC99, 0.331 microM for combination) was observed. No increase in potency or cell kill was observed when the two compounds were added simultaneously. MTA pretreatment also potentiated the cytotoxicity of a 1-h exposure to GEM. These cell-based observations were extended to evaluate the schedule-dependent interaction of these two agents in vivo using a nude mouse HT29 xenograft tumor model. At the doses tested, MTA alone (100 mg/kg) had a marginal effect on tumor growth delay, whereas GEM (80 mg/kg) produced a statistically significant tumor growth delay. In combination, the increase in tumor growth delay was greatest when MTA was administered before GEM, compared with simultaneous drug administration or the reverse sequence, e.g., GEM followed by MTA. The effect of sequential administration of MTA followed by GEM was greater than additive, indicating synergistic interaction of these agents. Thus, in vitro, MTA induced cell cycle effects on HT29 cells that resulted in potentiation of the cytotoxicity of GEM. In vivo, combination of these two drugs also demonstrated a schedule-dependent synergy that was optimal when MTA treatment preceded GEM.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Cycle/drug effects , Colonic Neoplasms/drug therapy , Animals , Antimetabolites, Antineoplastic/administration & dosage , Antimetabolites, Antineoplastic/pharmacology , Antimetabolites, Antineoplastic/toxicity , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/toxicity , Cell Death/drug effects , Cell Division/drug effects , Colonic Neoplasms/pathology , DNA Replication/drug effects , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine/toxicity , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Synergism , Folic Acid Antagonists/administration & dosage , Folic Acid Antagonists/pharmacology , Folic Acid Antagonists/toxicity , Glutamates/administration & dosage , Glutamates/pharmacology , Glutamates/toxicity , Guanine/administration & dosage , Guanine/analogs & derivatives , Guanine/pharmacology , Guanine/toxicity , HT29 Cells , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Pemetrexed , Gemcitabine
7.
Bioorg Med Chem Lett ; 9(1): 75-8, 1999 Jan 04.
Article in English | MEDLINE | ID: mdl-9990460
8.
J Pharmacol Exp Ther ; 287(1): 315-21, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9765352

ABSTRACT

5,6,7,8-Tetrahydro-N5,N10-carbonylfolic acid (LY354899) has been demonstrated to inhibit the dehydrogenase activity of C1-tetrahydrofolate synthase. This compound was only moderately antiproliferative toward CCRF-CEM lymphocytic leukemia cells in culture, but induced apoptosis after long incubation times. Slightly greater potency was observed in CEM cells adapted to grow in low folate media. Cell cycle alterations induced by LY354899 were unique relative to antifolates that inhibit either the purine or thymidine de novo biosynthetic pathways. Based on the observed changes in DNA content, we hypothesized that inhibition of the dehydrogenase resulted in two temporally distinct events: the first was a purineless-like effect and the second was a thymineless-like effect that resulted in apoptosis. To test this hypothesis, we combined LY354899 with the purine salvage metabolite, hypoxanthine. This combination resulted in an earlier and more dramatic apoptotic response, indicating that the thymineless effect had been potentiated. Biochemical analysis of ribo- and deoxyribonucleoside triphosphates confirmed that inhibition of the dehydrogenase activity initially resulted in decreased pools of deoxypurines and deoxypyrimidines, followed 16 hr later by an increase in deoxyadenosine triphosphate (dATP) and a further decrease in deoxythymidine triphosphate (dTTP). These studies demonstrate that the inhibition of the dehydrogenase activity of C1-tetrahydrofolate synthase may represent a viable target for the development of novel antifolates. The results are discussed in terms of deoxypurine and deoxypyrimidine biosynthesis.


Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Folic Acid/analogs & derivatives , Hypoxanthine/pharmacology , Methylenetetrahydrofolate Dehydrogenase (NADP)/antagonists & inhibitors , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Division/drug effects , Deoxyadenine Nucleotides/metabolism , Drug Synergism , Folic Acid/pharmacology , Humans , Thymine Nucleotides/metabolism , Tumor Cells, Cultured
9.
Adv Enzyme Regul ; 38: 135-52, 1998.
Article in English | MEDLINE | ID: mdl-9762351

ABSTRACT

Extensive biochemical and pharmacological evidence indicates that LY231514 is a novel antifolate antimetabolite. LY231514 is transported into cells mainly through the reduced folate carrier system and extensively metabolized to polyglutamated forms. The polyglutamates of LY231514 inhibit at least three key folate enzymes: TS, DHFR, and GARFT, and to a lesser extent AICARFT and C1-tetrahydrofolate synthase. The combined effects of the inhibition exerted by LY231514 at each target give rise to an unusual end-product reversal pattern at the cellular level that is distinct from those of other inhibitors such as methotrexate and the quinazoline antifolates. The metabolic effects exerted by LY231514 on the folate and nucleotide pools are also quite distinct from those of MTX and LY309887. The efficient polyglutamation, longer cellular retention and the multiple folate enzyme inhibition mechanism may all have contributed directly to the exciting antitumor responses now observed in Phase I and II studies. The multitargeted inhibition mechanism of LY231514 is particularly intriguing. This new level of mechanistic insight, which has evolved from the study of LY231514, challenges the traditional concept and paradigm of antifolate drug discovery and development which focused on developing very potent and selective inhibitors of single folate enzyme targets, such as DHFR, TS or one of the enzymes along the de novo purine biosynthetic pathway. Given the complex nature of folate metabolism and the critical role of folates in maintaining the physiological functions of living systems, it is completely reasonable to suspect that agents which can interfere at multiple sites in the folate pathway may trigger and cause more biochemical imbalance in the cellular DNA and RNA synthesis of malignant cells than agents that act on a single point (Fig. 5). In conclusion, LY231514 (MTA) is a new generation antifolate antimetabolite demonstrating inhibitory activity against multiple folate enzymes including TS, DHFR and GARFT. In current phase II studies, MTA is broadly active as a single agent and is showing very encouraging antitumor activity in multiple solid tumors including colorectal, breast and non-small cell lung cancers (38-43). The every three week dosing schedule has proven to be convenient and easy to administer and the clinical toxicities of LY231514 seem to be well tolerated. More advanced and extensive clinical trials of LY231514 are currently in progress.


Subject(s)
Folic Acid Antagonists/pharmacology , Glutamates/pharmacology , Guanine/analogs & derivatives , Peptide Synthases/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Guanine/pharmacology , Humans , Hydroxymethyl and Formyl Transferases/antagonists & inhibitors , Kinetics , Liver/enzymology , Methotrexate/pharmacology , Pemetrexed , Phosphoribosylaminoimidazolecarboxamide Formyltransferase , Phosphoribosylglycinamide Formyltransferase , Pteroylpolyglutamic Acids/metabolism , Swine , Tetrahydrofolate Dehydrogenase/metabolism , Tetrahydrofolates/pharmacology , Thymidylate Synthase/antagonists & inhibitors , Tumor Cells, Cultured
10.
Br J Cancer ; 78 Suppl 3: 27-34, 1998.
Article in English | MEDLINE | ID: mdl-9717988

ABSTRACT

LY231514 (N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethy l]-benzoyl]-L-glutamic acid) is a new folate-based antimetabolite currently in broad phase II clinical evaluation. Previous in vitro studies (C. Shih et al, CancerRes 57: 1116-1123, 1997) have suggested that LY231514 could be a multitargeted antifolate (MTA) capable of inhibiting thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study compared LY231514 with methotrexate, raltitrexed and a glycinamide ribonucleotide formyltransferase inhibitor, LY309887, at 300, 100, 30 and 100 nM, respectively, for their effects on intracellular folate and at 100, 66, 20 and 30 nM respectively, for their effects on nucleoside triphosphate pools in CCRF-CEM cells. Methotrexate induced an accumulation of dihydrofolate species, together with a rapid depletion of ATP, GTP and all of the deoxynucleoside triphosphates. LY309887 caused an accumulation of 10-formyltetrahydrofolate, a rapid loss of ATP, GTP and dATP, but a slower loss in dCTP, dTTP and dGTP. Both LY231514 and raltitrexed had minimal effects on folate pools. In contrast, they caused rapid depletion of dTTP, dCTP and dGTP, but induced an accumulation of dATP at different rates, with raltitrexed doing so about 2.5 times faster. Most of the observed metabolic changes could be understood on the basis of current knowledge of folate and nucleotide metabolism. We concluded that LY231514 was distinct from methotrexate, LY309887 and raltitrexed based on their metabolic effects in CCRF-CEM cells, and that in this cell line the inhibitory effects of LY231514 were exerted primarily against the thymidylate cycle and secondarily against de novo purine biosynthesis.


Subject(s)
Antineoplastic Agents/pharmacology , Folic Acid/metabolism , Glutamates/pharmacology , Guanine/analogs & derivatives , Nucleotides/metabolism , Antimetabolites, Antineoplastic/pharmacology , Chromatography, High Pressure Liquid , Drug Screening Assays, Antitumor , Enzyme Inhibitors/pharmacology , Guanine/pharmacology , Humans , Hydroxymethyl and Formyl Transferases/antagonists & inhibitors , Methotrexate/pharmacology , Pemetrexed , Phosphoribosylglycinamide Formyltransferase , Quinazolines/pharmacology , Reproducibility of Results , Tetrahydrofolate Dehydrogenase/drug effects , Tetrahydrofolate Dehydrogenase/metabolism , Tetrahydrofolates/pharmacology , Thiophenes/pharmacology , Thymidylate Synthase/antagonists & inhibitors , Tumor Cells, Cultured
11.
Cancer Chemother Pharmacol ; 41(3): 201-9, 1998.
Article in English | MEDLINE | ID: mdl-9443636

ABSTRACT

PURPOSE: The whole-body autoradiographic distribution of two radiolabeled antifolate inhibitors of GAR formyltransferase, lometrexol and LY309887, were compared in tumor-bearing mice maintained on standard diet (SD) and a low-folate diet (LFD) in order to determine the total amounts of drug that accumulated in blood, tumor, liver and kidney. The time-dependent changes in tissue distribution were evaluated over a 7-day period in order to compare the pharmacokinetic properties of both inhibitors and to assess the influence of dietary folate on this distribution. In addition, the effect of dietary folate on polyglutamation of compound accumulating in the liver was measured. The results have bearing on the potential of these two clinical agents to produce delayed toxicity in cancer patients and the use of dietary folate to modulate or prevent the development of this toxicity. METHODS: Single equimolar i.v. doses of [14C]LY309887 and [14C]lometrexol were administered to C3H mammary tumor bearing mice on SD or LFD, and the disposition of these compounds was quantitated using whole-body autoradiography. Livers were also harvested and extracted for determination of polyglutamate distribution. Animals were sacrificed both early and late (7 days) after dosing to determine the long-term retention of these compounds. RESULTS: Whole-body autoradiography revealed that the highest concentrations of both compounds were in liver and kidney. Concentrations of both compounds were two-fold higher in livers from LFD mice than in livers from SD mice. Lometrexol concentrations in liver averaged 2.8- and 2.2-fold higher than LY309887 in SD and LFD livers, respectively. In SD livers, the polyglutamate profiles of both compounds were similar, with hexaglutamates being the longest chain species detected. In LFD livers, hexaglutamates of LY309887 were observed, while hepta- and octaglutamates of lometrexol were detected after 168 h. CONCLUSIONS: The reduced hepatic retention and biochemical profile of LY309887 compared to lometrexol suggest that it may be less likely to produce delayed cumulative toxicity while still retaining antitumor activity. However, the increased hepatic accumulation observed in LFD mice emphasizes the importance of assessing and supplementing folate in cancer patients treated with this class of compounds.


Subject(s)
Enzyme Inhibitors/pharmacokinetics , Folic Acid/administration & dosage , Hydroxymethyl and Formyl Transferases/antagonists & inhibitors , Liver/metabolism , Mammary Neoplasms, Experimental/metabolism , Tetrahydrofolates/pharmacokinetics , Animals , Autoradiography , Chromatography, High Pressure Liquid , Diet , Female , Folic Acid Antagonists/pharmacokinetics , Mice , Mice, Inbred C3H , Phosphoribosylglycinamide Formyltransferase , Tissue Distribution
12.
Cancer Res ; 57(6): 1116-23, 1997 Mar 15.
Article in English | MEDLINE | ID: mdl-9067281

ABSTRACT

N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]-benzoyl]-L-glutamic acid (LY231514) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate currently undergoing extensive Phase II clinical trials. Previous studies have established that LY231514 and its synthetic gamma-polyglutamates (glu3 and glu5) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit other key folate-requiring enzymes, including dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). For example, the Ki values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In contrast, although a similar high level of inhibitory potency was observed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (Ki) for the parent monoglutamate are significantly weaker for TS (109 nM) and GARFT (9,300 nM). The effects of LY231514 and its polyglutamates on aminoimidazole carboxamide ribonucleotide formyltransferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formyltetrahydrofolate synthetase were also evaluated. The end product reversal studies conducted in human cell lines further support the concept that multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a major site of action for LY231514 at concentrations near the IC50, higher concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demonstrated that LY231514 requires polyglutamation and transport via the reduced folate carrier for cytotoxic potency. Therefore, our data suggest that LY231514 is a novel classical antifolate, the antitumor activity of which may result from simultaneous and multiple inhibition of several key folate-requiring enzymes via its polyglutamated metabolites.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Folic Acid Antagonists/pharmacology , Glutamates/pharmacology , Guanine/analogs & derivatives , Hydroxymethyl and Formyl Transferases , Tetrahydrofolate Dehydrogenase/drug effects , 5,10-Methylenetetrahydrofolate Reductase (FADH2) , Acyltransferases/antagonists & inhibitors , Aminohydrolases/antagonists & inhibitors , Formate-Tetrahydrofolate Ligase/antagonists & inhibitors , Glutamates/chemistry , Guanine/chemistry , Guanine/pharmacology , Humans , Methotrexate/pharmacology , Methylenetetrahydrofolate Dehydrogenase (NADP)/antagonists & inhibitors , Methylenetetrahydrofolate Reductase (NADPH2) , Molecular Structure , Multienzyme Complexes/antagonists & inhibitors , Oxidoreductases/antagonists & inhibitors , Pemetrexed , Phosphoribosylaminoimidazolecarboxamide Formyltransferase , Phosphoribosylglycinamide Formyltransferase , Polyglutamic Acid/pharmacology , Quinazolines/pharmacology , Tetrahydrofolates/pharmacology , Thiophenes/pharmacology , Thymidylate Synthase/antagonists & inhibitors , Tumor Cells, Cultured/drug effects
13.
Cancer Chemother Pharmacol ; 39(6): 521-31, 1997.
Article in English | MEDLINE | ID: mdl-9118464

ABSTRACT

PURPOSE: Cell cycle-related events in CCRF-CEM lymphocytic leukemia cells were examined subsequent to inhibition of thymidylate synthase (TS) or GAR formyltransferase (GARFT) and prior to cell death or stasis. METHODS: Cell populations were treated with the GARFT inhibitors 6R-5, 10-dideazatetrahydrofolate (Lometrexol) or LY309887, the TS inhibitor ZD1694, or the multitargeted antifolate LY231514. DNA content, nucleoside precursor incorporation and proliferating cell nuclear antigen (PCNA) expression as functions of drug treatment were assessed by multiparameter flow cytometry. Cellular respiration was measured by MTT analysis and apoptosis was detected by extraction of DNA fragments. RESULTS: Cell populations treated for up to 96h with lometrexol or LY309887 did not replicate and maintained a cell cycle distribution with distinct G1, S and G2/M regions. The number of S phase cells in treated populations was slightly elevated relative to control as measured by DNA content and PCNA. However, these cells were unable to incorporate 5-bromodeoxyuridine (BrdU). Throughout treatment, cells incubated with GARFT inhibitors maintained intact membranes and respired at a level comparable to untreated cells. In contrast, ZD1694 as well as LY231514, induced synchronization of the treatment population at the G1/S interface within 12h of drug addition. This was followed by synchronous entry of the population into S phase. After 24 h of treatment, more than 90% of the cells were capable of incorporating BrdU and stained positive for PCNA. DNA fragmentation occurred in cells treated with ZD1694 or LY231514 but not in those treated with GARFT inhibitors. In addition, the viable cells remaining after 24-48 h of treatment with ZD1694 or LY231514 were respiring at twice the level of untreated cells. CONCLUSION: These results demonstrate that the distinct endpoints of GARFT and TS inhibition are preceded by distinct cell cycle and metabolic alterations.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Cell Cycle/drug effects , Folic Acid Antagonists/pharmacology , Hydroxymethyl and Formyl Transferases , Acyltransferases/antagonists & inhibitors , Cell Line , Cell Survival/drug effects , DNA Fragmentation/drug effects , Glutamates/pharmacology , Guanine/analogs & derivatives , Guanine/pharmacology , Humans , Oxygen/metabolism , Pemetrexed , Phosphoribosylglycinamide Formyltransferase , Tetrahydrofolates/pharmacology , Thymidylate Synthase/antagonists & inhibitors
14.
Biochem Pharmacol ; 52(9): 1477-9, 1996 Nov 08.
Article in English | MEDLINE | ID: mdl-8937460

ABSTRACT

The importance of polyglutamation for the activation of natural folates and classical antifolates and recent evidence for the role of dietary folate as a biochemical modulator of antifolate efficacy led us to investigate the influence of changes in dietary folate on folylpolyglutamate synthetase (FPGS) activity. Activities were measured using lometrexol (6R-5,10-dideazatetrahydrofolic acid) as a substrate for FPGS with extracts of murine tissues, murine tumors, and human tumor xenografts from mice on standard diet or low folate diet. Tissues and tumors from mice on standard diet exhibited a 6-fold range of FPGS activity. Kidney had the lowest activity (36 pmol/hr.mg protein), followed by the human xenograft PANC-1 pancreatic carcinoma (46 pmol/hr.mg protein), liver (109 pmol/hr.mg protein), murine C3H mammary tumor (112 pmol/hr.mg protein), and the human xenograft MX-1 mammary carcinoma (224 pmol/hr.mg protein). In response to restricted dietary folate, four out of five tissues had significantly increased (25-50%) FPGS activity. Only the tumor with highest FPGS activity under standard diet conditions (MX-1 mammary) did not respond to low folate diet. The results indicate that changes in dietary folate intake can modulate FPGS activity significantly in vivo and suggest that the tissue distribution and toxicities of classical antifolates requiring polyglutamation for activation and cellular retention will be influenced significantly by folate status of the host.


Subject(s)
Folic Acid/administration & dosage , Peptide Synthases/metabolism , Animals , Diet , Female , Folic Acid/metabolism , Folic Acid Antagonists/metabolism , Humans , Mice , Mice, Inbred C3H , Neoplasm Transplantation , Neoplasms, Experimental/enzymology , Substrate Specificity , Tetrahydrofolates/metabolism , Transplantation, Heterologous
15.
Clin Cancer Res ; 2(7): 1135-41, 1996 Jul.
Article in English | MEDLINE | ID: mdl-9816279

ABSTRACT

Membrane-associated folate receptors (FRs) have been detected in many mammalian species, and multiple isoforms have been identified. The pharmacological properties of FRs from murine kidney, liver, and six murine tumors were characterized. Murine kidney expressed primarily folate-binding protein 1, analogous to human FR-alpha, whereas murine liver expressed predominantly folate-binding protein 2, analogous to human FR-beta. Five of six murine tumors expressed high-affinity FRs with pharmacological properties consistent with folate-binding protein 1 isoform expression. Restriction of dietary folate resulted in significant changes in the FR expression in most murine tissues. Kidney and tumor FRs showed a decreased affinity for folic acid, suggesting a change in isoform expression in response to a low folate diet. Density of the FR in the kidney decreased, and, in contrast, density of the FR in all tumors increased. The response of the liver to a low folate diet was unique in that there were no detectable changes in affinity or density of liver FR. Changes in dietary folate that modulate FR isoform expression may have relevance for cancer patients treated with antifolates.


Subject(s)
Carrier Proteins/analysis , Folic Acid/administration & dosage , Neoplasms, Experimental/chemistry , Receptors, Cell Surface , Animals , Female , Folate Receptors, GPI-Anchored , Humans , Kidney/chemistry , Liver/chemistry , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL
16.
Adv Enzyme Regul ; 36: 365-81, 1996.
Article in English | MEDLINE | ID: mdl-8869756

ABSTRACT

We have studied the molecular effects of a LFD in a murine model in order to better define the biochemical changes associated with folate deficiency. In addition, we have demonstrated the effect of a LFD on the pharmacokinetic profile and therapeutic activity and toxicity of lometrexol. These studies showed increased density of FR in tumors implanted in LFD mice and a decrease in the affinity of these receptors for folic acid. The results suggest that tumors can compensate for low folate bioavailability by up-regulation of a second FR with slightly lower affinity for folic acid. The higher density of this FR would provide greater capacity for garnering serum folate. FPGS activity increased in several tumors and liver and kidney of LFD mice. The increase in this enzyme activity would result in enhanced polyglutamation of folates and classical antifolates and thus increased cellular retention. Consistent with these changes in liver FPGS, mice injected i.v. with a single dose of lometrexol accumulated significantly more drug in liver and tumors of LFD animals compared to SD mice. Also, higher liver concentrations of lometrexol persisted longer in LFD mice. Polyglutamate analysis showed that longer polyglutamate forms appeared earlier in liver of LFD mice. After 7 days, longer polyglutamyl forms were recovered from liver of LFD mice (octa- and hepta-glutamyl lometrexol) compared to those on SD. A comparison of the efficacy and toxicity of lometrexol in C3H mammary tumor-bearing mice showed that in mice on LFD, lometrexol treatment produced a delayed toxicity with an LD50 of 0.1-0.3 mg/kg, a 3000-fold increase in lethality compared to SD mice. Supplementation of mice with folic acid restored anti-tumor activity and increased the therapeutic dose-range over which efficacy could be assessed. These studies support the use of folic acid supplementation for cancer patients treated with antifolate therapy in order to prevent the biochemical changes in FR and FPGS associated with folate deficiency, prevent delayed toxicity to GARFT inhibitors and enhance the therapeutic potential of this class of drugs.


Subject(s)
Carrier Proteins/metabolism , Diet , Hydroxymethyl and Formyl Transferases , Peptide Synthases/metabolism , Receptors, Cell Surface , Tetrahydrofolates/metabolism , Tetrahydrofolates/toxicity , Acyltransferases/metabolism , Animals , Carrier Proteins/drug effects , Enzyme Inhibitors/pharmacology , Folate Receptors, GPI-Anchored , Folic Acid/metabolism , Folic Acid/pharmacology , Kidney/chemistry , Kidney/metabolism , Liver/chemistry , Liver/metabolism , Mice , Neoplasms/enzymology , Phosphoribosylglycinamide Formyltransferase , Polyglutamic Acid/metabolism , Protein Binding , Tumor Cells, Cultured
17.
Invest New Drugs ; 14(3): 287-94, 1996.
Article in English | MEDLINE | ID: mdl-8958184

ABSTRACT

Lometrexol, a tight-binding antifolate inhibitor of the purine de novo enzyme glycinamide ribonucleotide formyltransferase (GARFT), was the first GARFT inhibitor to be investigated clinically. Unexpected observations of delayed cumulative toxicity prompted a search for a second generation antimetabolite with a more favorable biochemical, pharmacological and toxicological profile. LY309887, 6R-2',5'-thienyl-5, 10-dideazatetrahydrofolic acid, had 9-fold greater potency to inhibit GARFT (Ki = 6.5 nM) compared to lometrexol. Like lometrexol, LY309887 was activated by folpolyglutamate synthetase, however, it had a lower first order rate constant. In vitro and in vivo data were consistent with these observations: polyglutamation of LY309887 was less extensive compared to lometrexol and livers of mice accumulated fewer polyglutamates of LY309887 than polyglutamates of lometrexol. The affinities of these two compounds for isoforms of human folate receptors (FR) were compared. Lometrexol had a 6-fold higher affinity for FR alpha than LY309887 and both compounds had higher affinity for the alpha isoform compared to the beta isoform. The selectivity of LY309887 for FR alpha (beta (Ki)/ alpha (Ki) = 10.5) was twice that of lometrexol's (beta / alpha = 5.0). Lometrexol and LY309887 were potent cytotoxic compounds against the human leukemia cell line CCRF-CEM with IC50's of 2.9 nM and 9.9 nM, respectively. In vivo, LY309887 was more potent than lometrexol at inhibiting tumor growth in the C3H mammary murine tumor model and several tumor xenografts. Excellent efficacy was achieved by both compounds in several colon xenografts. In two pancreatic human xenografts, LY309887 achieved greater efficacy than lometrexol. In summary, the biochemical and pharmacological properties of lometrexol and LY309887 support the hypothesis that these antifolates will have clinical activity against human solid tumors. LY309887 is a second generation GARFT inhibitor with biochemical and pharmacological properties which distinguish it from lometrexol and suggest that it will have broad antitumor activity, a different pharmacokinetic profile and produce less toxicity than lometrexol in cancer patients.


Subject(s)
Acyltransferases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Folic Acid Antagonists/pharmacology , Hydroxymethyl and Formyl Transferases , Tetrahydrofolates/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Survival/drug effects , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Female , Folic Acid Antagonists/chemistry , Humans , Mice , Mice, Inbred C3H , Mice, Nude , Molecular Structure , Phosphoribosylglycinamide Formyltransferase , Structure-Activity Relationship , Tetrahydrofolates/chemistry , Transplantation, Heterologous , Tumor Cells, Cultured
18.
Mol Pharmacol ; 48(2): 326-33, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7651366

ABSTRACT

The metabolism of 5,10-dideazatetrahydrofolate (DDATHF [lometrexol]) to polyglutamate derivatives by folylpoly-gamma-glutamate synthetase (FPGS) plays a central role in the activity of this compound as an antineoplastic agent. The availability of a series of DDATHF derivatives differing in structure throughout the molecule has allowed a study of the structural requirements for substrate activity with mouse liver and hog liver FPGS. Kinetics of the polyglutamation reaction in vitro have been related to the potency of these compounds as inhibitors of the growth of human CEM leukemic cells. The structure-activity relationships for enzyme from both sources were nearly identical. FPGS from both species showed a broad acceptance for structural changes in the pyridopyrimidine ring, in the phenyl group, and in the intermediate bridge region, with structural changes in these regions being reflected in changes in Km for FPGS but much more modest alterations in Vmax. The data suggested that the phenyl ring was not contributing to any pi-pi hydrophobic interactions. It appeared to function primarily in maintaining a favorable distance between the pyridopyrimidine ring and the glutamate side chain. The lowest Km values were found for DDATHF analogs in which there were small alterations at the 10 position, e.g., 5-deazatetrahydrofolate, 10-methyl-DDATHF, and 10-formyl-5-deazatetrahydrofolate; the first-order rate constants for these substrates were the highest in this series, an indication of the efficiency of polyglutamation at low substrate concentrations. After correction for the intrinsic inhibitory activity of the parent DDATHF analog as an inhibitor of the target enzyme, the first-order rate constants for FPGS were found to be predictive of the potency of tumor cell growth inhibition for most of the compounds in this structural series.


Subject(s)
Hydroxymethyl and Formyl Transferases , Peptide Synthases/metabolism , Tetrahydrofolates/metabolism , Acyltransferases/antagonists & inhibitors , Animals , Cell Division/drug effects , Kinetics , Leukemia/pathology , Liver/enzymology , Mice , Phosphoribosylglycinamide Formyltransferase , Substrate Specificity , Swine , Tetrahydrofolates/chemistry , Tetrahydrofolates/pharmacology , Tumor Cells, Cultured
19.
Oncol Res ; 7(2): 97-102, 1995.
Article in English | MEDLINE | ID: mdl-7579732

ABSTRACT

Based on differential levels of membrane-associated folate binding protein (mFBP) expression, murine L1210 leukemia, human KB epidermoid carcinoma, and human IGROV1 ovarian carcinoma cells maintained under low (physiological) folate conditions (2 nM folinic acid) were used as model systems to investigate the potential role of mFBP in antifolate transport. In addition, L1210 parental cells were compared to a subline, L1210A, expressing high levels of mFBP and defective reduced folate carrier. Antifolates for which KB-derived mFBP has high affinity (5, 10-dideazatetrahydrofolic acid [DDATHF] and homo-DDATHF [0.24 and 0.78 respectively relative to folic acid]) and low affinity (methotrexate [0.002]) were chosen for this study. Protection against DDATHF/homo-DDATHF induced cytotoxicity was achieved preferentially by folic acid compared to folinic acid in IGROV1 and L1210A cells. In IGROV1 cells, cytotoxicity IC50s were increased 18- and 5.5-fold for DDATHF and homo-DDATHF respectively by 20 nM folic acid. Moreover, greater protection was observed in L1210A cells, where IC50s were increased 354- and 80-fold for these same compounds by 20 nM folic acid. Similar protection was not observed in KB cells, suggesting that KB mFBP was not functional in DDATHF transport. Although mFBP expression may be an important determinant in the cytotoxicity of antifolates for certain tumor cells, our data demonstrate a lack of correlation between levels of mFBP and function of mFBP for DDATHF transport in the models studied.


Subject(s)
Carrier Proteins/physiology , Folic Acid Antagonists/metabolism , Folic Acid Antagonists/pharmacology , Membrane Proteins/physiology , Receptors, Cell Surface , Tumor Cells, Cultured/drug effects , Animals , Antineoplastic Agents , Drug Interactions , Drug Screening Assays, Antitumor , Female , Folate Receptors, GPI-Anchored , Folic Acid/pharmacology , Humans , KB Cells/drug effects , Leucovorin/pharmacology , Leukemia L1210/drug therapy , Mice , Ovarian Neoplasms/drug therapy
20.
Cancer Res ; 54(4): 1021-6, 1994 Feb 15.
Article in English | MEDLINE | ID: mdl-8313357

ABSTRACT

Tight-binding inhibition of recombinant human monofunctional glycinamide ribonucleotide formyltransferase by Lometrexol (6R-5,10-dideazatetrahydrofolate) requires polyglutamation. LY254155 and LY222306 differ from 5,10-dideazatetrahydrofolate in the replacement of the 1',4'- phenylene moiety by a 2',5'-thiophene and a 2',5'-furan, respectively. Compared to Lometrexol, the thiophene and furan analogues had 25- and 75-fold greater inhibitory potencies against human monofunctional glycinamides ribonucleotide formyltransferase (Ki = 2.1 and 0.77 nM, respectively). The binding affinities of the thiophene and furan analogues for membrane folate-binding protein from human KB cells were 6- and 350-fold weaker than Lometrexol, respectively. Both the thiophene analogue and 5,10-dideazatetrahydrofolate inhibited the in vivo growth of murine 6C3HED lymphosarcoma, murine C3H mammary carcinoma, and human xenograft HXGC3, HC1, and VRC5 colon carcinomas by 95-100%. The thiophene analogue was efficacious against human xenograft PANC-1, a pancreatic carcinoma which was completely resistant to 5,10- dideazatetrahydrofolate. These novel antifolates represent the first monoglutamated tight-binding inhibitors of glycinamide ribonucleotide formyltransferase. By eliminating the need for polyglutamation, this class of antifolates may have clinical activity in the treatment of solid tumors expressing low levels of folylpolyglutamate synthetase or tumors resistant to antifolate therapy due to increased gamma-glutamyl hydrolase activity.


Subject(s)
Acyltransferases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Folic Acid Antagonists/pharmacology , Hydroxymethyl and Formyl Transferases , Receptors, Cell Surface , Ribonucleotides/metabolism , Tetrahydrofolates/pharmacology , Acyltransferases/metabolism , Animals , Carrier Proteins/metabolism , Female , Folate Receptors, GPI-Anchored , Humans , Leukemia/drug therapy , Leukemia/pathology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Phosphoribosylglycinamide Formyltransferase , Stereoisomerism , Structure-Activity Relationship
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