Mechanism-based inhibition of human folylpolyglutamate synthetase: design, synthesis, and biochemical characterization of a phosphapeptide mimic of the tetrahedral intermediate.

Folylpolyglutamate synthetase (FPGS) catalyzes anATP-dependent ligation reaction that results in the synthesis of poly(gamma-glutamate) metabolites of folates and some antifolates. We have synthesized and characterized the prototype of a new class of mechanism-based FPGS inhibitor in which a phosphonate moiety mimics the tetrahedral intermediate formed during the ligation reaction. This phosphonate, 4-amino-4-deoxy-10-methyl-pteroyl-L-glutamyl-gamma-[Psi¿P(O)(OH)-O¿] glutarate (4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate), is not a substrate for human FPGS, but is a linear, competitive inhibitor (Kis = 46 nM) with respect to methotrexate as the variable substrate. Inhibition is not time-dependent and preincubation of FPGS with this phosphonate does not increase the degree of inhibition, suggesting that it is not a slow, tight-binding inhibitor involving a time-dependent isomerization, EI --> EI*. Substructures containing the phosphonate moiety but lacking the pterin are much less inhibitory to FPGS, indicating that a significant portion of the inhibitor binding energy is derived from the pterin moiety, a feature also observed in substrate binding. 4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate is also an analog of a proposed tetrahedral intermediate in the reaction catalyzed by gamma-glutamyl hydrolase (gamma-GH), another enzyme of importance in controlling folate homeostasis in cells. This intermediate would arise from direct attack of H2O on the dipeptide, 4-NH2-10-CH3-Pte-L-Glu-gamma-L-Glu. The fact that 4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate is not an inhibitor of gamma-GH strongly suggests that hydrolysis of poly-gamma-glutamates catalyzed by gamma-GH does not involve the direct attack of water at the scissile amide bond. Methotrexate, its gamma-glutamyl dipeptide metabolite, and 4-NH2-10-CH3-Pte-L-Glu-gamma-[Psi¿P(O)(OH)-O¿]glutarate are equipotent as inhibitors of human dihydrofolate reductase (the primary target of methotrexate), but the phosphonate does not significantly inhibit another important folate-dependent enzyme, thymidylate synthase. Thus, the phosphonate moiety in this analog represents an important new lead in the development of FPGS inhibitors.

Potent inhibition of human folylpolyglutamate synthetase by suramin.

Suramin, a bis-hexasulfonated napthylurea, was studied as an inhibitor of human folylpolyglutamate synthetase (FPGS), a crucial enzyme in folate metabolism. Suramin is a more potent (IC50, 0.9 microM) inhibitor of FPGS partially purified from CCRF-CEM human leukemia cells than is bromosulfophthalein (IC50, 17 microM), the first reported nonsubstrate-analog inhibitor of FPGS (J. J. McGuire et al., Adv. Exptl. Med. Biol. 163, 199, 1983). FPGS inhibition by suramin is reversed by bovine serum albumin (which binds suramin). Suramin is a noncompetitive inhibitor with aminopterin (K(ii) = 0.9 microM; K(is) = 1.1 microM) and glutamic acid (K(ii) = 1.0 microM; K(is) = 5.2 microM) as the variable substrates; suramin inhibition tends toward being competitive with respect to the third FPGS substrate, ATP (K(ii) = 3.4 microM; K(is) = 0.35 microM), since the major effect is on its K(m). Suramin is a much less potent inhibitor of two other folate-dependent enzymes, dihydrofolate reductase (IC50, 38 microM; methotrexate (MTX), 0.6 nM) and thymidylate synthase (IC50, 87 microM; MTX, 48 microM). The effects of suramin on growth of CCRF-CEM cells and a MTX-resistant subline (R30dm) expressing low levels of FPGS activity were determined. R30dm is slightly collaterally sensitive to suramin consistent with FPGS inhibition contributing to the cytotoxic mechanism. These data, and those of Rideout et al. (Int. J. Cancer 61, 840, 1995), demonstrating that the reduced folate carrier system of CCRF-CEM is inhibited, suggest that inhibition of folate metabolism could be involved in the mechanism of action of suramin.

Biological properties of fluoroglutamate-containing analogs of folates and methotrexate with altered capacities to form poly (gamma-glutamate) metabolites.

Fluoroglutamate-containing analogs of folates and methotrexate (MTX) with altered capacities for poly (gamma-glutamate) metabolism were synthesized to probe the biological roles of polyglutamates. Compared to folic acid, DL-e,t-gamma-fluorofolic acid, a compound that is a poor substrate for polyglutamylation, was approximately 25-fold less potent in promoting growth of folate-depleted H35 rat hepatoma cells. DL-beta,beta-Difluorofolic acid, a compound that forms diglutamates more readily than does folic acid, was at least equivalent to folic acid in potency. Leucovorin (LV), a reduced folate, was 30-fold more potent than folic acid in promoting growth, whereas the analogous form of DL-e,t-gamma-fluorofolate, DL-e,t-gamma-fluoroleucovorin (DL-e,t-gamma-FLV) was only 4-fold more potent than folic acid. Both LV and DL-e,t-gamma-FLV protected or "rescued" cells from the growth inhibitory effects of MTX; however a 37- to 46-fold higher concentration of the fluoro analog was required. Folic acid, DL-e,t-gamma-fluorofolic acid, LV, and DL-e,t-gamma-FLV each potentiated the growth inhibitory effect of 5-fluoro-2'-deoxyuridine on CCRF-CEM human leukemia cells; higher concentrations of fluorinated analogs again were required. Stereochemically pure L-t-gamma-fluoromethotrexate (L-t-gamma-FMTX), a poor substrate for polyglutamylation, was evaluated as a cell growth inhibitor. In continuous exposure, L-t-gamma-FMTX), was 7-fold less potent than MTX as an inhibitor of CCRF-CEM growth. Results with these fluorinated folate and MTX analogs offer insight into the importance of polyglutamate metabolism to these biological and pharmacological effects.

Synthesis and biological evaluation of N alpha-(4-amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithine.

N alpha-(4-Amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithi ne (AMPte-DL-4,4-F2Orn, 4) was synthesized and evaluated as an inhibitor of human folypoly-gamma-glutamate synthetase (FPGS), dihydrofolate reductase (DHFR), and cell growth. Synthesis of 4 involved the use of a protected form of DL-4,4-difluoroornithine 9 which was derived from DL-4,4-difluoroglutamic acid. Biological activities of 4 were compared directly to those of the corresponding nonfluorinated compound N alpha-(4-amino-4-deoxy-10-methylpteroyl)-L-ornithine (AMPte-L-Orn, 3). Although the fluorinated analogue is a potent inhibitor of DHFR, it is a poor inhibitor of FPGS. However, the compound is transported across the cell membrane and inhibits cell growth, presumably due to the inhibition of DHFR. The data obtained with the fluorinated analogue are in contrast to those of the corresponding nonfluorinated compound 3, which is a potent inhibitor of both FPGS and DHFR but shows very low cytotoxicity due to poor transport.

Synthesis and biological activity of folic acid and methotrexate analogues containing L-threo-(2S,4S)-4-fluoroglutamic acid and DL-3,3-difluoroglutamic acid.

The stereospecific syntheses of L-threo-gamma-fluoromethotrexate (1t) and L-threo-gamma-fluorofolic acid (3t) are reported. Compounds 1t and 3t have no substrate activity with folylpoly-gamma-glutamate synthetase isolated from CCRF-CEM human leukemia cells, and compound 1t inhibits human dihydrofolate reductase at similar levels as methotrexate. The synthesis of DL-3,3-difluoroglutamic acid (6) and its incorporation into DL-beta,beta-difluorofolic acid (4) are also reported. Compound 4 acts as a better substrate for human CCRF-CEM folylpoly-gamma-glutamate synthetase than folic acid (V/K = ca. 7-fold greater). Thus, replacement of the glutamate moiety of methotrexate and folic acid with 4-fluoroglutamic acid and 3,3-difluoroglutamic acid results in folates and antifolates with altered polyglutamylation activity.

DL-beta,beta-difluoroglutamic acid mediates position-dependent enhancement or termination of pteroylpoly(gamma-glutamate) synthesis catalyzed by folylpolyglutamate synthetase.

Poly(gamma-glutamylation) of glutamate (L-Glu)-containing antifolates and natural folates is important in pharmacological mechanisms and in physiological processes. Based on previous work from our laboratories, we hypothesized that replacement of the L-Glu moiety in parent molecules with DL-beta,beta-difluoroglutamic acid (DL-beta,beta-F2Glu) might be a generic means of increasing polyglutamylation by both increasing the synthesis rate and decreasing the degradation rate (J. J. McGuire et al., J. Biol. Chem. 265, 14073-14079 (1990)); thus biological potency might be increased without other biochemical properties being altered. DL-beta,beta-F2Glu, synthesized by an improved route (B. P. Hart and J. K. Coward, Tetrahedron Lett. 34, 4917-4920 (1993)), has been incorporated into a methotrexate (MTX) homolog, beta,beta-difluoromethotrexate (beta,beta-F2MTX), and a folic acid (PteGlu) homolog, beta,beta-difluorofolic acid (beta,beta-PteF2Glu). Biochemical properties of beta,beta-F2MTX (e.g., inhibition of isolated dihydrofolate reductase, transport in whole cells) are similar to those of MTX except that, in accord with our hypothesis, apparent substrate efficiency for rat and human folylpolyglutamate synthetase (FPGS) is 4- to 7.5-fold higher, respectively, for beta,beta-F2MTX than for MTX. Analysis of the products synthesized by purified FPGS, however, suggests that while addition of the first gamma-Glu to beta,beta-F2MTX is highly efficient, subsequent additions occur at a negligible rate; this premise was confirmed by directly comparing the in vitro FPGS substrate activity of MTX-gamma-Glu and beta,beta-F2MTX-gamma-Glu. Furthermore, the dramatically diminished in vitro growth inhibitory potency of beta,beta-F2MTX as compared to MTX when exposure time to drug is decreased (despite otherwise similar biochemical properties) suggests that polyglutamylation is also impaired in intact cells. Similar results with FPGS have been obtained with oxidized and reduced forms of beta,beta-PteF2Glu. These data suggest that the effect of beta,beta-F2Glu on polyglutamylation by FPGS is dependent on its position relative to the point of L-Glu ligation. When beta,beta-F2Glu is the acceptor amino acid (i.e., point of attachment), ligation of Glu is enhanced; however, if beta,beta-F2Glu is one residue distal to the acceptor amino acid, further elongation is blocked.

Evolution of drug resistance in CCRF-CEM human leukemia cells selected by intermittent methotrexate exposure.

A clinically relevant mechanism of acquired methotrexate (MTX) resistance is decreased MTX polyglutamate (MTXGn) synthesis (McCloskey et al. J. Biol. Chem. 266:6181, 1991) secondary to deficient folylpolyglutamate synthetase (FPGS) activity. Earlier studies showed that this mechanism resulted after intermittent MTX treatment, but did not define its evolution in populations or its clonal frequency of occurrence and heterogeneity. We thus studied evolution of resistance in CCRF-CEM human leukemia cell populations and clones after repeated treatment with 30 microM MTX for 24 h. In populations, MTX resistance was detectable after 1 treatment and increased in degree as total cycles increased to 7. Defective MTXGn synthesis in populations was the only resistance mechanism detected, and FPGS activity in extracts decreased with treatment cycle. After 1 treatment, defective MTXGn synthesis was the major (27/48 clones) form of resistance; 18 clones were sensitive, while 1 clone with a DHFR-related change, no clones with decreased MTX uptake, and 2 complex phenotypes were observed. Sporadic clones with DHFR-mediated resistance appeared up to cycle 4, but defective MTXGn synthesis remained the major resistance mechanism. The degree of clonal resistance tended to increase with treatment cycle, but 1 clone in cycle 2 was similar to the clones from cycle 7. No change in FPGS gene copy number or restriction pattern, or FPGS mRNA level or size (2.3 Kb) was detected in populations. Decreased FPGS activity must result from decreased translation, increased protein turnover, or a point mutation affecting catalysis.

Novel 6,5-fused ring heterocyclic antifolates: biochemical and biological characterization.

Six novel antifolates with 2,4-diaminopyrimidine-fused five-membered rings containing either pyrrole or cyclopentene rings were characterized at the cellular and biochemical level. Five of these antifolates were more growth inhibitory to the CCRF-CEM human leukemia cell line than methotrexate [MTX; drug concentration effective at inhibiting cell growth by 50% relative to untreated control (EC50), 12 nM], the antifolate used in the clinic, and two were more potent than 10-ethyl-10-deazaaminopterin (EC50, 2.7 nM); similar patterns of response were obtained in the FaDu and A253 squamous carcinoma cell lines. In addition, the growth inhibitory potency of these antifolates was generally less dependent on exposure time than was MTX. Growth inhibitory effects could be reversed by leucovorin, indicating an antifolate mechanism. These antifolates targeted dihydrofolate reductase (DHFR) based on direct human DHFR inhibition assays [drug concentration inhibiting enzyme activity by 50% (IC50), 0.6-28 nM; MTX IC50, 0.8 nM] and the cross-resistance of MTX-resistant CCRF-CEM cells containing elevated DHFR. Inhibition of human thymidylate synthase was generally weak. These 6,5-fused ring heterocyclic antifolates utilized the reduced folate/MTX transporter for uptake, based on the cross-resistance of MTX uptake-impaired CCRF-CEM cells, and were efficient substrates for this uptake system, based on inhibition of [3H]MTX uptake (IC50, 0.3-5.8 microM; aminopterin IC50, 2.6 microM). These analogues were substrates for CCRF-CEM folylpolyglutamate synthetase, with several being among the most active substrates now known (highest Vrel/Km 0.73; MTX and 10-ethyl-10-deazaaminopterin, 0.013 and 0.24, respectively). Substrate activity for murine intestinal folylpolyglutamate synthetase was also assayed, and a different specificity pattern was observed. These new antifolates are apparently not substrates for aldehyde oxidase. Analogues containing the fused cyclopentene ring are preferred to those containing the fused pyrrole ring based on growth inhibitory potency, effectiveness against decreased uptake mutants and apparent affinity for transport, and inhibition of DHFR. In addition, fused cyclopentene-containing analogues are efficiently polyglutamylated. The data indicate that antifolates with 2,4-diaminopyrimidine-fused five-membered rings, especially those containing the fused cyclopentene ring, are an important new class of antifolates which warrant further exploration at the synthetic and preclinical levels.

Cross-resistance studies of folylpolyglutamate synthetase-deficient, methotrexate-resistant CCRF-CEM human leukemia sublines.

CCRF-CEM human leukemia sublines resistant to short-term methotrexate (MTX) exposure as a result of decreased folylpolyglutamate synthetase (FPGS) activity were examined for their response to other cytotoxic agents. The R3/7 and R30dm sublines display 25 and 1%, respectively, of the FPGS activity of CCRF-CEM cells as measured with MTX in vitro. Response to agents in outgrowth experiments was examined under both continuous exposure (120 h, where MTX resistance is not observed) and short-term (6-14.5 h) exposure. During continuous exposure to various classes of agents, cross-resistance of R3/7 and R30dm that correlated with FPGS level was not observed, although some minor (< or = 3-fold) stochastic variations in sensitivity were noted. These agents included actinomycin D, Adriamycin, etoposide, vincristine, cisplatin, cytosine arabinoside, 5-fluorouracil, and some other antifolates. Cross-resistance during continuous exposure that did correlate with FPGS level was noted, however, to glutamate-containing thymidylate synthase inhibitors (including ICI D1694) and, to a minor extent, to 6-mercaptopurine and 5-fluorodeoxyuridine. Slight collateral sensitivity during continuous exposure that apparently correlated with FPGS level was noted to the lipid-soluble antifolate trimetrexate and to 5,8-dideazapteroyl-L-ornithine, an FPGS-specific inhibitor. In short-term exposures (where MTX resistance of the sublines is observed), the resistant sublines displayed sensitivity or cross-resistance to each agent that was qualitatively similar to that observed for the same agent in continuous exposure. Because of the requirement for reduced folates in the anti-DNA mechanism of action of fluoropyrimidines and the current clinical use of leucovorin (LV) to enhance their effects, the interaction of LV and fluoropyrimidines was examined. The results suggest that even highly FPGS-deficient cells are as sensitive to the effects of LV modulation as are wild-type cells even at fluoropyrimidine exposure times as short as 4 h.

DL-3,3-difluoroglutamate: an enhancer of folylpolyglutamate elongation.

The effect of 3,3-difluoroglutamate (F2Glu) on the reaction catalyzed by rat liver folypolyglutamate synthetase was investigated. F2Glu was a potent, concentration-dependent inhibitor of poly(gamma-glutamylation) using [3H]Glu and either methotrexate (4-NH2-10-CH3PteGlu) or tetrahydrofolate as substrates. It was determined that F2Glu acted as an alternate substrate, but in contrast to the previously characterized alternate substrate 4-fluoroglutamate (McGuire, J.J., and Coward, J.K. (1985) J. Biol. Chem. 260, 6747-6754), it did not terminate polyglutamate chain elongation. Instead, F2Glu promoted chain elongation. Thus, synthesis of products from [3H]methotrexate containing 1 and 2 additional amino acid residues occurred at a substantially higher rate in the presence of F2Glu when compared to identical reactions in the presence of Glu; this was more pronounced for the product containing 2 additional residues. Identities of the products were established by their respective chromatographic elution positions and by limit digestion with gamma-glutamyl hydrolases. Ligation of Glu to 4-NH2-10-CH3PteGlu-gamma-(3,3-difluoroglutamate) was also enhanced. These results are consistent with F2Glu enhancing the synthesis of poly(gamma-glutamate) metabolites at the level of either the incoming amino acid (glutamate analog) or the gamma-glutamyl acceptor species. F2Glu is thus the first glutamate analog which enhances chain elongation catalyzed by folypolyglutamate synthetase.

Interaction of D, L-erythro- and D,L-threo-gamma-fluoromethotrexate with human leukemia cell dihydrofolate reductase.

Gamma-fluoromethotrexate (FMTX) is a poorly glutamylated mimic of the anti-cancer drug methotrexate (MTX) which is useful in studies of the roles of MTX poly-gamma-glutamates. A second chiral center occurs at C-4 of the 4-fluoroglutamate used to synthesize FMTX and, as a consequence, FMTX occurs as both D,L-erythro and D,L-threo diastereomers. The interaction of both diastereomers with intracellular dihydrofolate reductase has been examined in the human leukemia cell line CCRF-CEM, using a centrifugal column technique. Measurements of the rate at which radiolabel was displaced from [3H]MTX-saturated dihydrofolate reductase following suspension of the cells in unlabeled drug indicated that MTX and the erythro isomer of FMTX gave essentially the same rate of displacement; the rate of displacement by the threo isomer of FMTX was slower, but the interpretation of these data was ambiguous since the rate of transport of threo-FMTX may have been limiting. In reciprocal experiments in which dihydrofolate reductase was saturated with [3H]erythro-FMTX, the erythro isomer and MTX again behaved equivalently in terms of displacement. When dihydrofolate reductase was saturated with [3H]threo-FMTX, the radiolabel was clearly displaced at a much faster rate than either other radiolabel regardless of whether the displacing agent was MTX or the isomer. These results indicate a distinct stereospecificity for interaction of inhibitor with dihydrofolate reductase in which the threo isomer has a faster off-rate. Of the two FMTX diastereomers, the erythro isomer thus most closely mimics the properties of MTX.