Structural Insights into Mycobacterium tuberculosis Rv2671 Protein as a Dihydrofolate Reductase Functional Analogue Contributing to para-Aminosalicylic Acid Resistance.

Mycobacterium tuberculosis (Mtb) Rv2671 is annotated as a 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (AROPP) reductase (RibD) in the riboflavin biosynthetic pathway. Recently, a strain of Mtb with a mutation in the 5' untranslated region of Rv2671, which resulted in its overexpression, was found to be resistant to dihydrofolate reductase (DHFR) inhibitors including the anti-Mtb drug para-aminosalicylic acid (PAS). In this study, a biochemical analysis of Rv2671 showed that it was able to catalyze the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which explained why the overexpression of Rv2671 was sufficient to confer PAS resistance. We solved the structure of Rv2671 in complex with the NADP(+) and tetrahydrofolate (THF), which revealed the structural basis for the DHFR activity. The structures of Rv2671 complexed with two DHFR inhibitors, trimethoprim and trimetrexate, provided additional details of the substrate binding pocket and elucidated the differences between their inhibitory activities. Finally, Rv2671 was unable to catalyze the reduction of AROPP, which indicated that Rv2671 and its closely related orthologues are not involved in riboflavin biosynthesis.

Emax model and interaction index for assessing drug interaction in combination studies.

Applying the Emax model in a Lowe additivity model context, we analyze data from a combination study of trimetrexate (TMQ) and AG2034 (AG) in media of low and high concentrations of folic acid (FA). The Emax model provides a sufficient fit to the data. TMQ is more potent than AG in both low and high FA media. At low TMQ:AG ratios, when a smaller amount of the more potent drug (TMQ) is added to a larger amount of the less potent drug (AG), synergy results. When the TMQ:AG ratio reaches 0.4 or larger in low FA medium, or when the TMQ:AG ratio reaches 1 or larger in high FA medium, synergy is weakened and drug interaction becomes additive. In general, synergistic effect in a dilution series is stronger at higher doses that produce stronger effects (closer to 1-Emax) than at lower dose levels that produce weaker effects (closer to 1). The two drugs are more potent in the low compared to the high FA medium. Drug synergy, however, is stronger in the high FA medium.

Structures of dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexate.

The flagellate protozoan parasite Trypanosoma cruzi is the pathogenic agent of Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. The parasite is usually transmitted by a blood-feeding insect vector, but can also be transmitted via blood transfusion. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. There is an urgent need for the development of chemotherapeutic agents for the treatment of T. cruzi infection and therefore for the identification of potential drug targets. The dihydrofolate reductase activity of T. cruzi, which is expressed as part of a bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), is a potential target for drug development. In order to gain a detailed understanding of the structure-function relationship of T. cruzi DHFR, the three-dimensional structure of this protein in complex with various ligands is being studied. Here, the crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain are reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). These structures reveal that the enzyme is a homodimer with substantial interactions between the two TS domains of neighboring subunits. In contrast to the enzymes from Cryptosporidium hominis and Plasmodium falciparum, the DHFR and TS active sites of T. cruzi lie on the same side of the monomer. As in other parasitic DHFR-TS proteins, the N-terminal extension of the T. cruzi enzyme is involved in extensive interactions between the two domains. The DHFR active site of the T. cruzi enzyme shows subtle differences compared with its human counterpart. These differences may be exploited for the development of antifolate-based therapeutic agents for the treatment of T. cruzi infection.

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.

Super in vitro synergy between inhibitors of dihydrofolate reductase and inhibitors of other folate-requiring enzymes: the critical role of polyglutamylation.

The combined action among polyglutamylatable and nonpolyglutamylatable antifolates, directed against dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), and thymidylate synthase (TS), in human ileocecal HCT-8 cells was examined in a 96-well plate growth inhibition assay (96-h continuous drug exposure). An interaction parameter, alpha, was estimated for each of 95 experiments by fitting a seven-parameter model to data with weighted nonlinear regression. In a representative experiment, raising the folic acid concentration in the medium dramatically increased the Loewe synergy for the combination of trimetrexate (TMTX) and the GARFT inhibitor AG2034 (from a mean alpha +/- SE of 1.50 +/- 0.25 at 2.3 microM folic acid to 146 +/- 20 at 78 microM folic acid). Enhancements were also found for combinations of TMTX with the GARFT inhibitors AG2032, Lometrexol, and LY309887, the AICARFT inhibitor AG2009, and the TS inhibitors LY231514 and Tomudex but not with the GARFT inhibitor LL95509 or with the TS inhibitors AG337, ZD9331, and BW1843U89. Replacing TMTX with methotrexate in two-drug mixtures decreased the intensity of Loewe synergy. Examination of isobolograms at different effect levels revealed informative reproducible changes in isobol patterns. No two-drug combinations among inhibitors of GARFT, AICARFT, and TS exhibited Loewe synergy at either 2.3 or 78 microM folic acid. Thus, the ideal requirement for the folic acid-enhanced synergy is that a nonpolyglutamylatable DHFR inhibitor be combined with a polyglutamylatable inhibitor of another folate-requiring enzyme. A hypothesis to explain this general phenomenon involves the critical role of folylpoly-gamma-glutamate synthetase and the effect of the DHFR inhibitor in decreasing the protection by folic acid of cells to the other antifolates.

Evaluation of potent inhibitors of dihydrofolate reductase in a culture model for growth of Pneumocystis carinii.

Many antifolates are known to inhibit dihydrofolate reductase from murine Pneumocystis carinii, with 50% inhibitory concentrations (IC50s) ranging from 10(-4) to 10(-11) M. The relationship of the potency against isolated enzyme to the potency against intact murine P. carinii cells was explored with 17 compounds that had proven selectivity for or potency against P. carinii dihydrofolate reductase. Pyrimethamine and one analog were inhibitory to P. carinii in culture at concentrations two to seven times the IC50s for the enzyme, suggesting that the compounds may enter P. carinii cells in culture. Methotrexate was a potent inhibitor of P. carinii dihydrofolate reductase, but the concentrations effective in culture were more than 1,000-fold higher than IC50s for the enzyme, since P. carinii lacks an uptake system for methotrexate. Analogs of methotrexate in which chlorine, bromine, or iodine was added to the phenyl ring had improved potency against the isolated enzyme but were markedly less effective in culture; polyglutamation also lowered the activity in culture but improved activity against the enzyme. Substitution of a naphthyl group for the phenyl group of methotrexate produced a compound with improved activity against the enzyme (IC50, 0.00019 microM) and excellent activity in culture (IC50, 0.1 microM). One trimetrexate analog in which an aspartate or a chlorine replaced two of the methoxy groups of trimetrexate was much more potent and was much more selective toward P. carinii dihydrofolate reductase than trimetrexate; this analog was also as active as trimetrexate in culture. These studies suggest that modifications of antifolate structures can be made that facilitate activity against intact organisms while maintaining the high degrees of potency and the selectivities of the agents can be made.

Characterization of cross-resistance to methotrexate in a human breast cancer cell line selected for resistance to melphalan.

We have demonstrated previously decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan (MelR MCF-7). Cross-resistance studies of MelR MCF-7 cells revealed that this cell line was 6.7-fold cross-resistant to methotrexate, but only 2-fold resistant to trimetrexate. Methotrexate transport studies in MelR MCF-7 cells showed a 2-fold decrease in initial methotrexate uptake and a 2-fold decrease in the Vmax for methotrexate uptake in the resistant cells. Methotrexate resistance in MelR MCF-7 cells was also associated with a decrease in non-effluxable methotrexate following incubation with radiolabeled drug for 24 hr. Characterization of intracellular methotrexate after accumulation for 24 hr demonstrated decreased levels of free methotrexate in MelR MCF-7 cells. Analysis of methotrexate polyglutamate formation in MelR MCF-7 cells indicated that the decrease in non-effluxable, non-protein-bound methotrexate was associated with a 3-fold decrease in higher order methotrexate polyglutamate formation. No difference was noted in folylpolyglutamate synthetase activity between the resistant and parental cell lines. Therefore, the observed decrease in methotrexate polyglutamate formation in MelR MCF-7 cells appeared to result from decreased availability of substrate. There was no evidence of any alteration in the amount of the catalytic activity of dihydrofolate reductase in MelR MCF-7 cells compared with parental MCF-7 (WT MCF-7) cells; moreover, the binding affinity of dihydrofolate reductase for methotrexate and the percentage of protein-bound methotrexate were similar in both cell lines. In addition, the total amounts of thymidylate synthase protein and thymidylate synthase catalytic activity in MelR MCF-7 cells were unchanged. Thus, acquired methotrexate resistance in MCF-7 cells selected for resistance to melphalan appears to result from down-regulation of methotrexate uptake.

Expression of variant dihydrofolate reductase with decreased binding affinity to antifolates in MOLT-3 human leukemia cell lines resistant to trimetrexate.

Various alterations of the dihydrofolate reductase (DHFR) gene are involved in resistance. In order to understand the mechanism that induce such gene alterations in human leukemia cells, we studied the expression products of DHFR gene in trimetrexate (TMQ)- and/or methotrexate (MTX)-resistant sublines derived from a MOLT-3 human leukemia cell line. A 200-fold TMQ-resistant subline (MOLT-3/TMQ200) expressed the mutated DHFR mRNA, with a base change (T-->C) at the second position of codon 31, as well as the wild type gene. A MTX-resistant subline derived from MOLT-3/TMQ200 (MOLT-3/TMQ200-MTX500) showed a further increase in the expression of the mutated DHFR mRNA, compared to MOLT-3/TMQ200, with a marked decrease of expression of the wild type DHFR mRNA, which is confirmation of amplification of the mutated DHFR gene. By contrast, a 10,000-fold MTX-resistant subline (MOLT-3/MTX10,000) over-expressed the wild type DHFR mRNA, which is confirmation of amplification of the wild type gene. Increased levels of the DHFR enzyme in these sublines were proportional to expression levels of the DHFR mRNA. The DHFR enzyme expressed in MOLT-3/TMQ200-MTX500 cells showed a 40-fold increase in the Ki values for both MTX and TMQ, compared with values for the wild type DHFR expressed in both MOLT-3/MTX10,000 and its parent cell line. These findings suggest that the altered DHFR gene, which was introduced in MOLT-3 cells by exposure to TMQ, gave rise to a variant enzyme with reduced affinity to antifolates, and that complex DHFR alterations confer drug-resistant phenotypes in antifolate-resistance. Structural difference between the antifolates could be important in the introduction of the differential DHFR gene alterations in the antifolate resistance.

Conformational analysis of the lipophilic antifolate trimetrexate.

The conformational properties of the lipophilic antifolate trimetrexate (TMQ) were calculated and compared to the structurally-analogous prototypical antifolate methotrexate (MTX) using both empirical force-field and AM1 quantum mechanical methods. The conformational preferences of TMQ and MTX are diametrically opposed with respect to the bridge-system set of torsion angles tau 1, tau 2: TMQ prefers gauche, trans while MTX prefers approximately trans, gauche. These predictions are consistent with the observed crystal structures of TMQ (i.e., tau 1 = 79 degrees, tau 2 = 178 degrees) and of DHFR-bound MTX (i.e., tau 1 = -157 degrees, tau 2 = 57 degrees in L. casei). The crystal structure of MTX.4H2O deviates from this pattern with tau 1 closer to cis (i.e., 39 degrees) than the predicted trans, yet this near-cis conformation is driven by intermolecular hydrogen-bonding and electrostatic forces operative in the MTX crystal. As a consequence of these strong intermolecular forces, MTX incurs 1.8 kcal/mole in conformational-strain energy in its crystalline form. In contrast, TMQ experiences virtually no conformational strain in its crystalline form. This disparity is attributed to two distinctions between TMQ and MTX: (i) MTX crystallizes as a zwitterion while TMQ crystallizes as the free base, and (ii) the hydrophilic glutamate tail in MTX is replaced by three lipophilic trimethoxy groups in TMQ. The corresponding conformational-strain energy of DHFR-bound MTX is 2.0 kcal/mole while that of DHFR-bound TMQ is only 0.65 kcal/mole based on the assumption that the latter adopts the same bridge conformation as the former. This cost in conformational-strain energy for TMQ and MTX is paid at the expense of their respective free energies of binding of DHFR. Consequently, the present study offers the possibility of designing a new class of antifolates which are conformationally strain-free when bound to DHFR and thereby more effective as chemotherapeutic agents.

Determinants of trimetrexate lethality in human colon cancer cells.

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.

Methotrexate and gamma-tert-butyl methotrexate transport in CEM and CEM/MTX human leukemic lymphoblasts.

In a continuing investigation of determinants of their 200-fold methotrexate resistance and their collateral sensitivity to gamma-tert-butyl methotrexate, the ability of CEM/MTX cells to transport the two drugs was analyzed and compared with that of CEM cells. The Km and Vmax values for the influx of methotrexate into CEM cells did not differ significantly from those of CEM/MTX cells, and this was the case for gamma-tert-butyl methotrexate as well. Surface binding and influx rates were proportional to cell surface area, but differences in efflux rates and methotrexate uptake were too large to be explained on this basis. Neither methotrexate nor trimetrexate competed with gamma-tert-butyl methotrexate influx in CEM cells. However, both drugs perturbed the gamma-tert-butyl methotrexate steady state in CEM cells, resulting in slightly less uptake than with gamma-tert-butyl methotrexate alone. However, the major difference between the two cell types was in the methotrexate uptake plateau, which was much greater in the case of the parental cell line. A related observation was the more rapid efflux of methotrexate from CEM/MTX cells than from CEM cells. The poor uptake, the associated meager capacity to polyglutamylate methotrexate and the enhanced methotrexate efflux appear to be responsible for its decreased activity against CEM/MTX cells. Half-lives for gamma-tert-butyl methotrexate efflux were the same in both cell lines, allowing the drug to accumulate to cytotoxic levels despite its inability to form polyglutamates.

Intrinsic resistance to methotrexate in human soft tissue sarcoma cell lines.

A human fibrosarcoma cell line, HT-1080, and four new cell lines (HS-16, HS-28, HS-30, and HS-42) were established from untreated patients with mesenchymal chondrosarcoma, peripheral nerve sheath sarcoma, malignant hemangiopericytoma, and mixed mesodermal tumor, respectively, and were used for analysis of mechanisms of intrinsic resistance to methotrexate. All four new cell lines were resistant to methotrexate as determined by inhibition of thymidylate synthase in whole cells and by growth inhibition, as compared with HT-1080, a methotrexate sensitive cell line. Methotrexate uptake, level of dihydrofolate reductase, and inhibition of this enzyme by methotrexate in the four cell lines were comparable to HT-1080 cells. However, levels of long chain polyglutamates (glu3-5) of methotrexate achieved after a 24-h incubation with this drug were much lower in the four new cell lines as compared to the HT-1080 cell line (5- to 20-fold lower). The low levels of methotrexate polyglutamates formed is likely the major cause of intrinsic methotrexate resistance in these new sarcoma cell lines.

Enzyme studies of methotrexate-resistant human leukemic cell (K562) subclones.

Five methotrexate (MTX)-resistant K562 cell subclones (K562/MTX-1 approximately -5) were established and were examined for mechanisms of drug resistance. Impairment of MTX-polyglutamate formation, with membrane transport alteration, in the resistant cells was demonstrated in the previous studies (Koizumi, S. (1988) Jpn. J. Cancer Res. 79, 1230). Further analysis of sensitivity of the cells to trimetrexate (TMQ), which is not polyglutamated and does not require the reduced folate transporter, but is a potent inhibitor of human DHFR, revealed a modest decrease in sensitivity to TMQ (2.4- to 15-fold). Enzyme studies showed that the dihydrofolate reductase (DHFR) activities of these resistant subclones were very similar to that of the parent cells. The number of binding sites of these subclones for MTX calculated from Scatchard analysis was increased up to 7-fold in the K562/MTX-1 and -4 subclones and up to 3-fold in the other 3 subclones as compared to the parent cells. KD values of MTX for the DHFR in the K562/MTX-1 and -4 subclones also appeared to be altered relative to the parent cell line. Further, thymidylate synthase (TS) activity of the resistant subclones was reduced to 50% in K562/MTX-1 and -4 cells, and to 11-25% in the other subclones as compared to the parent cell line. These findings suggest that antifolate resistance in the newly established K562/MTX subclones in multifactorial with polyglutamation and transport defects accounting for the majority of resistance to MTX, and that alteration in the binding affinity of DHFR for MTX and diminished levels of TS may contribute to the 'residual' drug resistance to TMQ and have importance with respect to MTX.

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.

Mechanisms of sensitivity and natural resistance to antifolates in a methylcholanthrene-induced rat sarcoma.

A methylcholanthrene-induced rat sarcoma that can be propagated in vitro or in vivo was evaluated for resistance to antifolates and was found to be relatively resistant to methotrexate and 10-ethyl-10-deazaaminopterin but sensitive to trimetrexate. Rat sarcoma cell extracts contained low levels of dihydrofolate reductase activity, the target enzyme of methotrexate, and inhibition of this enzyme by these three antifolates was similar. Transport studies showed poor uptake of both methotrexate and 10-ethyl-10-deazaaminopterin. In contrast, trimetrexate achieved high intracellular levels. The poor uptake of methotrexate was not due to lack of polyglutamylation. Thus, the basis for natural resistance to methotrexate and 10-ethyl-10-deazaaminopterin, compared with trimetrexate, in this rat sarcoma cell line was due to decreased transport of these drugs.