Potent inhibition of human folylpolyglutamate synthetase by a phosphinic acid mimic of the tetrahedral reaction intermediate

A phosphorous-containing pseudopeptide folate analog (Valiaeva et al., J Org Chem 2001;66:5146-54) was designed to mimic the tetrahedral intermediate formed in the ATP-dependent reaction catalyzed by folylpolyglutamate synthetase (FPGS). This analog, methotrexate-phosphinate (MTX-phosphinate; 4-amino-4-deoxy-10-methylpteroyl-L-Glu-gamma-[psi(P(O)(OH)-CH(2))]glutarate), is a highly potent (K(is), 3.1+/-0.5 nM), competitive inhibitor of recombinant human cytosolic FPGS. Within experimental limits, FPGS inhibition was not time-dependent, and preincubation of FPGS, inhibitor, and ATP did not potentiate the inhibition. These results suggest that slow phosphorylation to produce a more potent inhibitor form is not involved. MTX-phosphinate was not growth inhibitory to human CCRF-CEM leukemia cells at 1 microM (70-fold above the concentration of MTX giving 50% growth inhibition), probably because of poor transport. Because of its exceedingly high potency as an FPGS inhibitor, MTX-phosphinate represents a lead structure from which cell-permeable analogs may be developed to test the hypothesis that FPGS inhibition is therapeutically efficacious.     

Further studies on the interaction of nonpolyglutamatable aminopterin analogs with dihydrofolate reductase and the reduced folate carrier as determinants of in vitro antitumor activity

Thirteen structural analogs of the potent nonpolyglutamatable dihydrofolate reductase inhibitor N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) with modifications in the side chain, the para-aminobenzoyl moiety, or the 9,10-bridge were evaluated for the ability to inhibit human recombinant dihydrofolate reductase (DHFR), to utilize the reduced folate carrier (RFC) for influx, and to inhibit the growth of CCRF-CEM human leukemia cells in culture. In spectrophotometric assays of the kinetics of the reduction of dihydrofolate by DHFR in the presence of NADPH, these compounds had K(i) values ranging from 0.2 to 1.3pM, and thus were not greatly different in potency from the parent drug PT523. By comparison, the K(i) values of aminopterin (AMT), methotrexate (MTX), and 10-ethyl-10-deazaaminopterin (EDX) were 3.7, 4.8, and 11pM. In assays of competitive inhibition of [3H]MTX influx into CCRF-CEM cells, the K(i) values ranged from 0.21 to 7.3 micro M, as compared with 0.71, 5.4, and 1.1 micro M for PT523, AMT, and EDX. The K(t) for MTX was also re-analyzed and found to be 4.7 micro M, in better agreement with the literature than our previously reported value of 7.1 micro M. The IC(50) values of these compounds as inhibitors of the growth of CCRF-CEM cells after 72hr of drug exposure ranged from 0.53 to 55nM, and were qualitatively consistent with the other results.     

Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.     

Lack of an effect of breast cancer resistance protein (BCRP/ABCG2) overexpression on methotrexate polyglutamate export and folate accumulation in a human breast cancer cell line

Accumulation of methotrexate (MTX) and its polyglutamates (PGs) has been recognized as an important factor in MTX efficacy. We have previously described a multidrug-resistant human breast cancer cell line, MCF7/MX, that exhibits reduced accumulation of total MTX as well as MTX-PGs, and that is resistant to continuous MTX exposure [Volk EL, Rohde K, Rhee M, McGuire JJ, Doyle LA, Ross DD, et al. Methotrexate cross-resistance in a mitoxantrone selected multidrug-resistant MCF7 breast cancer cell line is due to enhanced energy-dependent drug efflux. Cancer Res 2000;60:3514-21]. These cells express high levels of the breast cancer resistance protein (BCRP/ABCG2) that has been shown to actively transport MTX and short-chain MTX-PGs in vitro. However, the effect of BCRP on MTX-PG accumulation in intact cells was unclear. Here, we show that MTX transport by BCRP is required for the observed lower levels of MTX-PGs in the resistant cells. When BCRP was inhibited with fumitremorgin C, or in cells expressing a mutated form of BCRP that is unable to transport MTX, MTX-PG accumulation was similar or even higher than that in the parental cells that do not express BCRP. Concomitantly, there was increased inhibition of thymidylate synthase. It had previously been suggested that BCRP-mediated efflux of MTX-PGs contributed to the reduced MTX-PG accumulation. However, we found no evidence of BCRP-mediated efflux of MTX-PGs from intact cells, suggesting that direct efflux of MTX-PGs does not play a major role in MTX resistance. Together, these data show that BCRP overexpression can cause a reduction in total MTX accumulation as well as a reduction in the proportion of long-chain MTX-PGs. In contrast, BCRP overexpression did not affect natural folate accumulation or the relative distribution of folylpolyglutamates in the resistant, as compared to the parental, cells. Thus, it appears that BCRP overexpression affects the metabolism of the antifolate MTX, but not that of natural folates, although indirect effects cannot be excluded.     

The role of multidrug resistance proteins MRP1, MRP2 and MRP3 in cellular folate homeostasis

Previously, we reported that the multidrug resistance proteins MRP1, MRP2 and MRP3 confer resistance to therapeutic antifolates by mediating their cellular extrusion. We now determined whether MRPs also play a role in controlling cellular homeostasis of natural folates. In MRP1, MRP2 and MRP3-transfected 2008 human ovarian carcinoma cells total cellular folate content was 32-38% lower than in 2008 cells (105+/-14pmolfolate/mgprotein) when grown in medium containing 2.3 microM folic acid (FA). Under these conditions cellular growth rates were not compromised. However, when cells were challenged under folate-depleted conditions with a short exposure (4 hr) to FA or leucovorin, MRP1 and MRP3 overexpressing cells were impaired in their growth. In contrast to wild-type cells, MRP1 transfected cells retained only 60% of the maximum growth when exposed to 500 nM leucovorin or 500 microM FA. For 2008/MRP1 and 2008/MRP3 cells FA growth stimulation capacity was dramatically decreased when, during a 4 hr exposure, metabolism into rapidly polyglutamatable and retainable dihydrofolate was blocked by the dihydrofolate reductase inhibitor trimetrexate. To retain growth under such conditions MRP1 overexpressing cells required much higher concentrations of FA (EC(50) > 500 microM) compared to 2008 cells (EC(50): 12 microM). These results suggest that down- and up-regulation of MRP1 (and MRP3) expression can influence cellular folate homeostasis, in particular when cellular retention by polyglutamylation of folates is attenuated.     

Effect of polyglutamylation of methotrexate on its accumulation and the development of resistance in the protozoan parasite Leishmania

Folates are polyglutamylated in most organisms, and in cancer cells the polyglutamylation of folates and of the antifolate methotrexate (MTX) is an important determinant of MTX susceptibility. The folylpolyglutamate synthetase (FPGS) responsible for polyglutamylation of folates was recently characterized in the parasite Leishmania. We show here that MTX is polyglutamylated in Leishmania tarentolae and that triglutamates are the predominant form. The glutamate chain length of MTX increases significantly in Leishmania cells transfected with the FPGS gene and decreases in cells with one FPGS allele disrupted. Modulation in the expression of the FPGS gene also has a profound effect on MTX susceptibility and this effect was found to be dependent on the folate concentration of the medium. In the folate-rich medium SDM-79, overexpression of FPGS will confer MTX resistance while in M-199 medium, which has much less folates, FPGS transfectants are more sensitive to MTX. Cells with one allele of FPGS disrupted are more resistant to MTX in low folate medium. The modulation of FPGS expression affects both the short-term and long-term accumulation of folate and MTX, showing a marked decrease in accumulation in the FPGS haploid mutant. This differential accumulation was mediated by decreased influx of the drug into the cell. Finally, the analysis of MTX-resistant Leishmania mutants indicated that the presence of shorter glutamate chains on MTX is correlated with MTX resistance.     

Folate concentration dependent transport activity of the Multidrug Resistance Protein 1 (ABCC1)

The Multidrug Resistance Protein MRP1 (ABCC1) can confer resistance to a variety of therapeutic drugs. In addition, MRP1/ABCC1 mediates cellular export of natural folates, such as folic acid and l-leucovorin. In this study we determined whether cellular folate status affected the functional activity of MRP1/ABCC1 mediated efflux of an established substrate, the anthracycline daunorubicin (DNR). As a model system we used the human ovarian carcinoma cell line 2008wt, and its MRP1/ABCC1 transfected subline 2008/MRP1. Both types of these moderate- and high-MRP1/ABCC1 expressing cells displayed efflux of DNR when maintained in standard culture media (2.3microM folic acid). The initial total cellular DNR efflux rate in 2008/MRP1 cells was approximately 2-fold higher compared to 2008wt cells. This efflux consisted of MRP1/ABCC1 mediated transport, possibly non-MRP1 mediated transport, as well as passive diffusion. Benzbromarone, a specific MRP1 inhibitor, decreased the initial efflux rate in 2008/MRP1 cells (4-fold) and in 2008wt cells (2-fold). When 2008/MRP1 cells were challenged for 2 days in folate-free medium, total cellular DNR efflux was decreased to 43% of the initial efflux rate under folate-rich conditions. In 2008wt cells DNR efflux was decreased to 84% of the folate-rich conditions. Benzbromarone did not inhibit DNR efflux after the folate-free period in both cell lines. Repletion of folate by a 2-24hr exposure to 2.5microM l-leucovorin or folic acid resulted in a complete restoration of DNR efflux. In contrast, expression of MRP1/ABCC1 protein was not changed significantly during the folate-free period or the repletion-period, nor were cellular ATP or ADP pools. In conclusion, this study demonstrates that the cellular folate status can influence the transport activity of MRP1/ABCC1. These results have potentially important implications in the understanding of the (patho-)physiological roles of MRP1/ABCC1, and possibly other ABC transporter proteins in cellular folate homeostasis and drug resistance.     

Induction of resistance to the multitargeted antifolate Pemetrexed (ALIMTA) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression

Pemetrexed (ALIMTA, MTA) is a novel thymidylate synthase (TS) inhibitor and has shown activity against colon cancer, mesothelioma and nonsmall-cell lung cancer. We induced resistance to Pemetrexed in the human colon cancer cell line WiDr by using a continuous exposure to stepwise increasing Pemetrexed concentrations (up to 20 microM) as well as a more clinically relevant schedule with intermittent exposure (up to 50 microM) for 4 hr every 7 days, resulting in WiDr variants WiDr-cPEM and WiDr-4PEM, respectively. However, using the same conditions, it was not possible to induce resistance in the WiDr/F cell line, a variant adapted to growth under low folate conditions. Mechanisms of resistance to Pemetrexed were determined at the level of TS, folylpolyglutamate synthetase (FPGS) and reduced folate carrier (RFC). WiDr-4PEM and WiDr-cPEM showed cross-resistance to the polyglutamatable TS inhibitor Raltitrexed (6- and 19-fold, respectively) and the nonpolyglutamatable TS-inhibitor Thymitaq (6- and 42-fold, respectively) but not to 5-fluorouracil. The ratios of TS mRNA:beta actin mRNA in WiDr-4PEM and WiDr-cPEM were 5-fold (P=0.01) and 18-fold (P=0.04) higher, respectively, compared to WiDr (ratio: 0.012). In addition, TS protein expression in the resistant WiDr variants was elevated 3-fold compared to WiDr, while the catalytic activity of TS with 1 microM dUMP increased from 30 pmol/hr/10(6) cells in WiDr cells to 2201 and 7663 pmol/hr/10(6) cells in WiDr-4PEM and WiDr-cPEM, respectively. The activity of FPGS was moderately decreased, but not significantly different in all WiDr variants. Finally, no evidence was found that decreased catalytic activity of RFC was responsible for the obtained Pemetrexed resistance. Altogether, these results indicate that resistance to Pemetrexed in the colon cancer cell line WiDr was solely due to upregulation of TS of which all related parameters (mRNA and protein expression and TS activity) were increased, rather than alterations in FPGS or RFC activity.