New small-molecule inhibitors of dihydrofolate reductase inhibit Streptococcus mutans.

Streptococcus mutans is a major aetiological agent of dental caries. Formation of biofilms is a key virulence factor of S. mutans. Drugs that inhibit S. mutans biofilms may have therapeutic potential. Dihydrofolate reductase (DHFR) plays a critical role in regulating the metabolism of folate. DHFR inhibitors are thus potent drugs and have been explored as anticancer and antimicrobial agents. In this study, a library of analogues based on a DHFR inhibitor, trimetrexate (TMQ), an FDA-approved drug, was screened and three new analogues that selectively inhibited S. mutans were identified. The most potent inhibitor had a 50% inhibitory concentration (IC50) of 454.0±10.2nM for the biofilm and 8.7±1.9nM for DHFR of S. mutans. In contrast, the IC50 of this compound for human DHFR was ca. 1000nM, a >100-fold decrease in its potency, demonstrating the high selectivity of the analogue. An analogue that exhibited the least potency for the S. mutans biofilm also had the lowest activity towards inhibiting S. mutans DHFR, further indicating that inhibition of biofilms is related to reduced DHFR activity. These data, along with docking of the most potent analogue to the modelled DHFR structure, suggested that the TMQ analogues indeed selectively inhibited S. mutans through targeting DHFR. These potent and selective small molecules are thus promising lead compounds to develop new effective therapeutics to prevent and treat dental caries.

Comparison of methods for evaluating drug-drug interaction.

The goal of the present report is to compare several published methods of analyzing drug-drug interaction data. The compared methods are the curve-shift analysis, isobologram, combination index, and universal surface response analysis, and the comparison was based on analysis of published cytotoxicity data of combinations of two anti-folate agents. Major findings are as follows. The curve shift analysis enabled the inspection of the experimental data and visual evaluation of the approximate parallelism between the dose response curves. Isobologram analysis provided the range of concentration ratios where maximal synergy was obtained. The combination index analysis readily provided quantitative estimation of the extent of synergy or antagonism. The universal surface response method summarized drug-drug interaction in a single parameter, facilitating comparison of larger arrays of combinations. Only the curve shift analysis and the universal surface response method yielded a statistical estimate of differentiation between synergy, additivity, and antagonism. In summary, curve shift analysis, isobolograms, combination index analysis, and the universal response surface method are useful methods for analyzing drug-drug interaction, and provide complementary information.

Large diversity in transport-mediated methotrexate resistance in human leukemia cell line CCRF-CEM established in a high concentration of leucovorin.

To elucidate the mechanism(s) of methotrexate (MTX) resistance as a possible reason underlying treatment failure in high-dose MTX regimens combined with leucovorin (LV) rescue, we established MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of excess LV, and characterized their properties. Continuous exposure of the cells to escalating concentrations of MTX up to 20 microM in the presence of 1000 nM LV resulted in establishment of three MTX-resistant sublines with a wide disparity of resistance degree over a 4 logarithmic range (approximately 40-, 900- and 44,000-fold, respectively). Transmembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these sublines was not different from that of the parent cells. A cell growth study in various concentrations of LV showed that cells with higher resistance to MTX required more LV for optimal growth. In parallel with the resistance levels, there was an increase in mRNA expression of dihydrofolate reductase gene and a decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of the RFC1 gene in all 3 sublines revealed a point mutation in codon 47 (TCC-->TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF-CEM cells in the presence of 1000 nM LV resulted in the establishment of heterogeneous cell populations with a wide range of transport-mediated MTX resistance, which was associated with differential alterations of RFC gene. These cell lines may serve as models for investigation of the molecular mechanism(s) underlying refractory tumors in high-dose MTX regimens with LV rescue.

Development of a yeast assay for rapid screening of inhibitors of human-derived Pneumocystis carinii dihydrofolate reductase.

Human-derived Pneumocystis carinii dihydrofolate reductase (DHFR) was expressed in a Saccharomyces cerevisiae strain whose growth depends on complementation by this enzyme. We utilized a quantitative assay to measure the sensitivity of this yeast strain to DHFR inhibitors. This assay should be useful for identifying new inhibitors of human-derived P. carinii DHFR.

Implications for improved high-dose methotrexate therapeutic effects in cultured human breast cancer and bone marrow cells.

The cytotoxicity of high-dose methotrexate (MTX), 10 and 100 microM, and 5-fluorouracil (5-FU) combinations is independent of sequence in human MDA-MB-436 breast carcinoma cells. The growth inhibitory effects of 10 and 100 microM MTX are 22.54+/-1.56% and 16.20+/-0.74%, respectively, of the control rate. When the MTX and 5-FU concentrations are 10 microM, antiproliferative effects of MTX 2 hr before 5-FU (MTX/5-FU) and 5-FU 2 h before MTX (5-FU/MTX) are 25.17+/-1.23% and 25.60+/-1.28% of the control rate, respectively. The percentage of control rates for 5-FU alone is 94.89+/-1.35%. The growth rates of MDA-MB-436 cells in 100 microM MTX and 10 microM 5-FU are 15.19+/-0.62% (MTX/5-FU) and 16.53+/-0.85% (5-FU/MTX) of the control rate. The growth of cancer cells in the presence of 5-FU alone is 93.82+/-1.69% of the control rate. A comparison of the cell-killing effects of MTX and the nonpolyglutamable antifolate trimetrexate (TMQ) alone and in combination with 5-FU was performed to indirectly explore the role of polyglutamylation in breast cancer and bone marrow cells. The comparisons were made in equitoxic concentrations (10 microM) of MTX and TMQ and the time of exposure was the same. The inhibitory effects of TMQ, TMQ/5-FU, and 5-FU/TMQ in breast cancer cells were identical, but significantly less than MTX, MTX/5-FU, and 5-FU/MTX. The interaction between TMQ and MTX, TMQ/5-FU and MTX/5-FU, and 5-FU/TMQ and 5-FU/MTX was quantitatively similar in bone marrow. (Significant protection occurred in bone marrow cells exposed to 5-FU/TMQ and 5-FU/MTX.) Because the effects of 5-FU/MTX and 5-FU/TMQ on bone marrow were the same, it is unlikely that polyglutamylation plays a significant role in the protective effects of 5-FU. However, the greater inhibitory effect of MTX or MTX and 5-FU combinations, when compared with TMQ or TMQ and 5-FU, suggests that polyglutamylation of MTX may contribute to the cytotoxicity of this antifolate to breast cancer cells. Hence, these studies suggest that a priming and nontoxic dose of 5-FU before high-dose MTX sustains MTX cytotoxicity in breast cancer and protects against MTX toxicity to bone marrow progenitor cells.

Clustering of mutations in the first transmembrane domain of the human reduced folate carrier in GW1843U89-resistant leukemia cells with impaired antifolate transport and augmented folate uptake.

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.

Where do we stand with 5-fluorouracil?

For nearly four decades, 5-fluorouracil (5-FU) has been the mainstay of treatment for colorectal cancer. Due to the lack of other agents with significant activity, tremendous efforts have been undertaken to increase the efficacy of 5-FU by investigating alternative schedules of delivery and biomodulation. However, bolus 5-FU in combination with folinic acid (FA), either as the Mayo Clinic or Roswell Park protocol, still represents the standard treatment for adjuvant and first-line palliative chemotherapy of colorectal cancer. In a recent meta-analysis, infusional protocols of 5-FU demonstrated increased response rates (14% to 22%) and a marginal, but significant survival benefit of 3 weeks (11.3 to 12.1 months). In view of the much higher costs and complicated management of infusional 5-FU regimens, this marginal survival benefit does not yet allow protracted 5-FU application to be defined as standard therapy. However, protracted 5-FU infusion in combination with radiation can be considered standard therapy as adjuvant treatment of rectal cancer, since it has demonstrated a significant increase in survival. In the future, oral 5-FU prodrugs may be substituted for infusional 5-FU. Furthermore, current data indicate that 5-FU will also be an essential component of combination chemotherapy protocols with the new active agents oxaliplatin, irinotecan, and raltitrexed. Preclinical studies show synergistic antitumor activity of 5-FU with these agents, which corresponds well with clinical response rates of 50% in untreated and 15% to 25% in 5-FU-refractory patients. Moreover, 5-FU-based pro-drug-active drug systems serve as excellent models for tumor-targeted gene therapy.

High-dose trimetrexate and minimal-dose leucovorin: a case for selective protection?

Our objective was to find the minimum dose of leucovorin (LV; 5-formyltetrahydrofolate) needed to potentially provide selective protection of normal tissue in patients with tumors resistant to methotrexate (MTX) by virtue of transport during prolonged therapy with high-dose trimetrexate (TMTX). Based upon the known daily requirement for folate, that tumors are often resistant to methotrexate via a transport-based mechanism, and that large doses of trimetrexate can be given with large doses of leucovorin for the treatment of patients with Pneumocystis carinii, a protocol was designed to find the minimum LV dose required to allow the administration of large doses of TMTX. Patients were treated in 28-day cycles consisting of 14 consecutive days of oral TMTX (45 mg/m2 every 12 h), followed by 14 days of rest. The dose of concurrent LV was started at 5 mg/m2 twice daily. Cohorts of patients received successive half doses of LV so long as three consecutive patients had less than or equal to grade 3 toxicity. Ten patients received 29 courses of therapy. The most common toxicities encountered were thrombocytopenia (38%), mucositis (14%), and neutropenia (10%). At a LV dose of 2.5 mg/m2, toxicities were consistently limited to less than or equal to grade 3 and only one episode of grade 4 hematological toxicity. Although there was marked interpatient variability, the minimally effective LV dose for selective protection seems to be 2.5 mg/m2. If tumors are resistant to methotrexate because of decreased transport of drug (and also folate), then the same pharmacological principle used to develop TMTX/LV for the treatment of P. carinii may be applied to treatment of some patients with cancer.

Biomodulation of Fluorouracil in colorectal cancer.

5-Fluorouracil (5-FU) remains the agent of choice for the treatment of colorectal cancer. Research has focused on the biomodulation of 5-FU in order to attempt to improve the cytotoxity and therapeutic effectiveness of this drug in the treatment of advanced colorectal cancer. Modulation of 5-FU by methotrexate (MTX), trimetrexate (TMTX), interferon-alpha (IFN-alpha), leucovorin (LV), or N-(phosphonacetyl)-L-asparte acid (PALA) has produced higher response rates than those observed with 5-FU alone. Methotrexate may improve the durability of response to or survival with 5-FU, but with inferior results compared with those in trials of 5-FU and leucovorin. Trimetrexate produces a number of responses, and further phase III trials are in progress to confirm the results of promising phase II trials with this drug. IFN-alpha has shown therapeutic efficiency when combined with 5-FU alone or with 5-FU and leucovorin, but latest studies with these combinations have shown increased toxicity. Initial single-institution phase I trials with 5-FU and PALA reported promising responses, but the latter responses with PALA were not substantiated in randomized multicenter trials. Leucovorin enhances the cytotoxic activity of 5-FU in vitro and in vivo, and several clinical trials have shown improved response rates and possible trends in improved survival when such therapy is compared with the use of 5-FU as a single-agent. More recent randomized trials have focused their attention on determining the optimal dose and schedule with this combination for producing a better clinical response with minimal toxicity. Schedules using infusional 5-FU appear to be the most active regimens when 5-FU is used as a single agent, as demonstrated by recent randomized trials. The Southwest Oncology Group (SWOG) and the Eastern Cooperative Oncology Group (ECOG) have performed separate randomized trials and have shown that the optimal regimens employ infusional 5-FU as a single agent, and that these are the least toxic regimens, perhaps more effective, and associated with a better quality of life. Future studies will focus on infusional regimens involving either short-term, high-dose protracted or long-term, low-dose protracted infusion of 5-FU, since these regimens have shown the most favorable toxicity spectrum and produced the longest survival times. Future research will also focus on the evaluation of various methods of delivery of 5-FU, including oral administration of the drug in combination with compounds that can modify its catabolism.

A gene transfer strategy for making bone marrow cells resistant to trimetrexate.

Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y-containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers.

Trial of sequential trimetrexate, fluorouracil, and high-dose leucovorin in previously treated patients with gastrointestinal carcinoma.

PURPOSE: Trimetrexate (TMTX) is a dihydrofolate reductase inhibitor, which, like methotrexate (MTX), has been shown to potentiate fluorouracil (FU) cytotoxicity by increasing phosphoribosylpyrophosphate (PRPP) levels. We investigated the safety and efficacy of a sequential TMTX/FU/leucovorin (LV) combination. PATIENTS AND METHODS: Forty-one patients with advanced gastrointestinal carcinoma (mostly colorectal) received variable doses of TMTX followed 24 hours later by FU/LV (500 mg/m2 of each drug). Almost all patients had received previous chemotherapy. The initial 19 patients were treated on a 3-week-on/1-week-off schedule without any significant toxicity; the remaining patients were treated for 6 consecutive weeks followed by a 2-week rest period. TMTX was escalated in 30-mg/m2 increments from 20 to 110 mg/m2 in separate patient cohorts. When the 110-mg/m2 dose of TMTX was reached, the FU dose was escalated from 500 mg/m2 to 600 mg/m2. RESULTS: The partial response (PR) rate in assessable patients with colorectal cancer (all previously treated) was 20% (seven of 35; 95% confidence interval, 7% to 33%), and with other gastrointestinal cancers was one of four patients. Median survival has not been reached with a median follow-up of 13.5 months. The maximum-tolerated dose (MTD) was 110 mg/m2 for TMTX, 500 mg/m2 for FU, and 500 mg/m2 for LV on a 6-weeks-on/2-weeks-off cycle. The principal toxicities were grade 3 or 4 diarrhea, which occurred in 17% of patients, and hypersensitivity reactions, which occurred in 26% of patients. CONCLUSION: TMTX can be administered at maximal doses in combination with FU and LV without increasing toxicity. The PR rate of 20% in advanced colorectal carcinoma patients previously treated with chemotherapy is encouraging and merits further study.

Clinical pharmacokinetics and pharmacology of trimetrexate.

Trimetrexate represents one of a number of new antimetabolites that have been studied in malignant, rheumatological and infectious disease. Methotrexate, the classical antifolate agent, is active in a broad spectrum of clinical settings, but its use is limited ny pre-existing or acquired cellular resistance. Trimetrexate is an agent that does not require uptake by the folate carrier transport system, a major mechanism of cellular resistance both in vitro and in vivo. Both dihydrofolate reductase inhibition and high performance liquid chromatography (HPLC) assays can be used to determine drug concentrations. Clearance of trimetrexate has been reported to follow biphasic or triphasic patterns. Elimination is primarily by biotransformation with less than 5% of the drug excreted renally in an unchanged form. Both active and inactive metabolites have been found, but the precise metabolic pathways have yet to be defined. The role of trimetrexate in the treatment of Pneumocystis carinii pneumonia is limited to compassionate use, as clinical studies have shown cotrimoxazole (trimethoprim-sulfamethoxazole) to be superior to trimetrexate. However, in a wide spectrum of malignant processes, trimetrexate appears to have a role either as a high-dose single agent, with calcium folinate (leucovorin calcium) rescue, or in combination with other antineoplastic agents. However, further trials are needed to fully establish the efficacy of trimetrexate in these settings. Increased knowledge of the pattern of resistance for individual tumours and tumour types may result in trimetrexate becoming more widely used clinically.

Leucovorin protection against repeated daily dose toxicity of trimetrexate in rats.

Repeated high doses of trimetrexate (TMX), a potent non-classical antifolate, have been administered as an experimental treatment for life-threatening Pneumocystis carinii infections in man. This therapy includes the coadministration of leucovorin, a reduced folate cofactor, to prevent antifolate toxicity in the host. The purpose of this investigation was to assess possible toxicologic sequelae of this combination regimen in an animal model. TMX at daily oral doses of 25, 35, and 45 mg/kg produced dose-related myelosuppression, thymic lymphoid depletion, seminiferous tubular atrophy, and degenerative lesions of the gastrointestinal tract. Mortality observed with TMX alone occurred earlier at higher doses and was specifically associated with severe degenerative enteropathy of the cecum. Oral leucovorin doses of 1, 5, 20, or 50 mg/kg administered twice daily, at the time of TMX administration and 6 hr later, protected against TMX lethality and target organ toxicity in a dose-related manner. Leucovorin was only partially protective against TMX-induced macrocytic anemia and the degree of protection was not dose-related. Leucovorin protection against cecal enteropathy was associated with increased DNA synthetic rates and higher mitotic activity of cecal epithelium than those in rats administered TMX alone. Importantly, the combination of daily administration of high dose TMX for 4 weeks with protective coadministration of leucovorin did not result in target organ toxicities that differed from TMX alone.

Leucovorin enhances cytotoxicity of trimetrexate/fluorouracil, but not methotrexate/fluorouracil, in CCRF-CEM cells.

BACKGROUND: Increased response rates in studies of patients with colon cancer have indicated that the cytotoxic effects of fluorouracil (5-FU) are potentiated by leucovorin (LV) and by methotrexate (MTX). However, preliminary studies using a sequential combination of MTX, LV, and 5-FU showed no additional potentiation. PURPOSE: We hypothesized that the lack of additional cell kill with this combination could be due to competition of LV with MTX for cellular uptake and reduced folate polyglutamylation. We have tested this possibility by comparing the cytotoxicity of drug combinations containing MTX with that of drug combinations containing trimetrexate (TMTX), an antifolate that does not compete with LV for uptake or polyglutamylation. METHODS: Human lymphocytic leukemia CCRF-CEM cells were exposed to MTX or TMTX for 24 hours and to 5-FU during the last 4 hours of antifolate exposure. LV was administered 30 minutes before 5-FU. RESULTS: After 20 hours of exposure to TMTX or MTX, intracellular levels of phosphoribosyl pyrophosphate were elevated to a similar degree, and these levels did not decrease after a 30-minute exposure to LV. No additional cell kill was observed when LV was added to the MTX/5-FU combination, but cytotoxicity was enhanced when LV was added to the TMTX/5-FU combination. CONCLUSIONS: This study supports the hypothesis that the lack of additional cell kill when high-dose LV is added to the MTX/5-FU combination may be due to competition of MTX with LV for cellular uptake and/or competition of MTX or its polyglutamates with polyglutamylation of reduced folates. Inasmuch as TMTX does not compete with LV and reduced folates for uptake and polyglutamylation, the synergy obtained with the combination of TMTX plus 5-FU and high-dose LV further supports this hypothesis.

Mechanisms of natural resistance to antifolates in human soft tissue sarcomas.

Efforts to use fresh human sarcoma cells for evaluating antifolate resistance with an in situ thymidylate synthesis assay using 5-[3H] deoxyuridine were unsuccessful because of low thymidylate synthesis activity in enzymatically disaggregated tumors. By incubating tumor cell suspensions in supplemented RPMI-1640 medium with 10% fetal bovine serum for 3 days, activity of the in situ thymidylate synthesis assay markedly increased (1.42 versus 0.03 pmol/h/10(7) cells), thus allowing 75% of samples to be evaluated for antifolate sensitivity. By criteria developed with a methotrexate-resistant and -sensitive cell line, this assay indicated that most sarcomas are naturally resistant to methotrexate (12 of 15). Natural resistance to 10-ethyl-10-deazaaminopterin and trimetrexate was also observed in 60% of the samples (nine of 15, respectively). The results from the 3-day in situ assay were confirmed by specific tests for resistance mechanisms in most sarcoma samples. The resistance mechanisms detected were impaired polyglutamylation, an increased level of dihydrofolate reductase, and amplification of this gene. These results encourage further exploration of this assay to predict response to antifolates in individual patients and to evaluate efficacy of new antifolates as candidates for clinical trial.

Rapid development of resistance to antifolates in vitro: possible clinical implication.

Within three repeated 7-day incubation periods with either methotrexate (MTX) or trimetrexate (TMTX), human colon adenocarcinoma cells (HCT-8) developed high levels of resistance to these drugs, as evidenced by approximately 20- and 50-fold increases, respectively, in the median effective doses. Similarly, within six short-term exposures (4 hours) to the same drugs, a high degree of resistance developed in the cells. Alternating 4-hour treatment cycles with MTX and TMTX did not delay the onset of resistance to these antimetabolites in the HCT-8 cells. The same strategy produced no better results than giving either MTX or TMTX alone to (C57BL/6 x DBA/2)F1 mice bearing murine leukemia P388 cells. Furthermore, HCT-8 cells resistant to short-term (4-hour) exposure to MTX were cross-resistant to the same drug given for 7 days continuously, and cells resistant to MTX given continuously for 7 days were cross-resistant to the same drug given for 4 hours. Analogous results were obtained with TMTX, indicating that, under these circumstances, changing the schedule of administration of the same agent does not overcome resistance to it. The clinical relevance of these data to prolonged adjuvant chemotherapy, as well as loco-regional and continuous-infusion chemotherapy, is discussed.

Role of substrate depletion in the inhibition of thymidylate biosynthesis by the dihydrofolate reductase inhibitor trimetrexate in cultured hepatoma cells.

The effects of the lipid-soluble dihydrofolate reductase inhibitor, trimetrexate, on the inhibition of thymidylate biosynthesis as a result of perturbation in cellular folate pools in H35 hepatoma cells in vitro has been investigated. Exposure of the cultures to increasing concentrations of trimetrexate between 2 and 20 nM causes a marked reduction in de novo thymidylate biosynthesis and a concomitant decrease in (6R)5,10-methylenetetrahydropteroylpolyglutamate (5,10-CH2H4PteGlun) from 2.0-0.2 microM, respectively. This is accompanied by an increase in H2PteGlun from 1.2 microM in control cultures to 4.7 microM in cultures exposed to 20 nM trimetrexate. The dependency of de novo thymidylate biosynthesis on intracellular 5,10-CH2H4PteGlun in trimetrexate-treated cells is compared with (a) the relationship of thymidylate biosynthesis on intracellular levels of 5,10-CH2H4PteGlun in folate-depleted cells supplemented with increments of folic acid and (b) the substrate (5,10-CH2H4PteGlun) dependence of purified thymidylate synthase from the same source. All three results are nearly identical demonstrating that trimetrexate-dependent inhibition of de novo thymidylate biosynthesis is primarily a result of substrate depletion. These results coupled with the weak inhibitory properties of H2PteGlun for thymidylate synthase Ki = 5.0 microM) suggest that H2PteGlun accumulation is not the major determinant in inhibiting thymidylate synthase following trimetrexate inhibition but under certain conditions has the potential to enhance the inhibition caused by substrate depletion.