The influence of glycerol on the binding of methotrexate to Mycobacterium tuberculosis dihydrofolate reductase: a molecular modelling study

The enzyme, dihydrofolate reductase (DHFR), from Mycobacterium tuberculosis (mt-DHFR) is believed to be a potential drug target for the treatment of tuberculosis. The co-crystal structure of mt-DHFR bound with glycerol (GOL), NAPDH and methotrexate (MTX) reveals a GOL binding site on the enzyme. This GOL binding site could be very important for the design of novel, selective mt-DHFR inhibitors, because this binding site is absent on human DHFR (h-DHFR). We have performed molecular dynamic simulations and free energy calculations to evaluate the binding affinity of GOL and its free energy contribution to the binding of MTX to mt-DHFR. The results showed that GOL does not bind tightly to mt-DHFR. Although GOL itself contributed free energy on MTX binding to mt-DHFR, GOL also increased the flexibilities of MTX, so that MTX cannot maintain strong electronic interactions with ARG32 and ARG60, which caused the total binding free energy to decrease. These data suggest that GOL binding is weak and it could be expelled from the binding site, to allow inhibitors containing appropriate side chains to bind. This observation can be used to inform future drug design studies, especially those aimed at improving drug selectivity against h-DHFR.     

Towards understanding the mechanisms of molecular recognition by computer simulations of ligand-protein interactions

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.     

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine

We have prepared liposomes containing methotrexate-gamma-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-gamma-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects of MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N5-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N5-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.     

Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli

Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and from a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of the inhibitor methotrexate (MTX). Although the Asp27----Asn substitution causes only small changes in the association rate constants (kon) for the formation of binary and ternary (with NADPH) complexes, the dissociation rate constants for these complexes (koff) are increased for the mutant enzyme by factors of about 5- and 100-fold, respectively, at pH 7.65. In binding experiments, the initial MTX binary and ternary complexes of the mutant enzyme were found to undergo relatively rapid isomerization (kobs approximately 17 and 145 s-1, respectively). Although such rapid isomerization of complexes of WT-ECDHFR could not be detected in binding experiments, evidence of a slow isomerization (k = 4 x 10(-3) s-1) of the ternary WT-ECDHFR.MTX.NADPH complex was obtained from progress of inhibition experiments. This slow isomerization increases binding of MTX to WT-ECDHFR only 2.4-fold (much less than previously estimated). From presently available data, we could not determine the contribution of the rapid isomerization of complexes to the binding of MTX to the mutant enzyme. The Asp27----Asn substitution increases the overall dissociation constant (KD) 9-fold for the binary complex and 85-fold for the ternary complex. When it is also taken into account that a proton ultimately derived from the solvent must be added to MTX bound to the WT enzyme, but not to MTX bound to the mutant enzyme, these increases in KD for the mutant enzyme correspond to decreases in binding energy for MTX of 3.9 and 5.2 kcal/mol at pH 7.65 for the binary and ternary complexes, respectively.     

Inhibition of cell proliferation with antibody-targeted liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine

We have prepared liposomes containing methotrexate-γ-dimyristoylphosphatidylethanolamine (MTX-DMPE liposomes), to which protein A was covalently coupled, permitting specific association of these liposomes in vitro with murine cells preincubated with relevant protein A-binding monoclonal antibodies. In the absence of antibody the presence of externally-oriented methotrexate (MTX) in MTX-DMPE liposomes did not result in greater binding to cells than liposomes made without MTX-γ-DMPE. Derivation of methotrexate with phospholipid permits enhanced drug-liposome association. These liposomes are more resistant than conventional liposomes to repeated cycles of freezing and thawing. MTX-DMPE liposomes are comparable to antibody-targeted liposomes made with encapsulated water-soluble methotrexate both with respect to specific binding to target cells and drug effect. The inhibitory effects off MTX-liposomes, as well as free MTX, were reversible by either thiamin pyrophosphate (Tpp) or N⁵-formyltetrahydrofolate (F-THF), while the effects of MTX-DMPE liposomes were reversed only by N⁵-formyltetrahydrofolate. This suggests that the toxicity of non-targeted MTX-liposomes may be due to leakage of the encapsulated MTX. The absence of an effect of thiamin pyrophosphate on non-targeted MTX-DMPE liposomes indicates that they do not enter into the cell via the normal folate transport system.     

Method for the determination of blood methotrexate by high performance liquid chromatography with online post-column electrochemical oxidation and fluorescence detection

Methotrexate (MTX) has been widely used at low dose for the treatment of different diseases including rheumatoid arthritis. MTX might be present in plasma in free form, and in blood cells in methotrexate polyglutamate (MTXPG). A rapid and sensitive HPLC method was developed for the determination of plasma MTX level, whole-blood MTX level, and whole-blood total MTX (MTX+MTXPG) level. To determine plasma MTX level or whole-blood MTX level, a 0.2-ml aliquot of plasma or whole blood (after a freeze-thaw cycle to break blood cells) was well mixed with 0.8 ml methanol and centrifuged. To determine whole-blood total MTX level, a 0.1-ml aliquot of whole blood (after a freeze-thaw cycle) was mixed with 80 microl ascorbic acid (114 mM) and incubated at 37 degrees C for 2h to enzymatically convert the MTXPG to MTX. Then 20 microl NaOH solution (0.5M) and 0.8 ml methanol were added and mixed well. After centrifugation, a 0.5-ml aliquot of the supernatant was evaporated to dryness and re-dissolved in 0.2 ml hydrochloric acid (10mM). Methylene chloride (0.2 ml) was added and mixed well. After centrifugation, the top aqueous layer was injected to HPLC for analysis. After the MTX was eluted from the HPLC column, it was electrochemically oxidized and detected by a fluorescence detector. Recoveries of spiked MTX at ppb (ng/ml) level were between 87.9 and 118% with within-day relative standard deviation less than 5.2% and day-to-day relative standard deviation less than 9.8%. The limit of detection (LOD) and limit of quantitation (LOQ) of the described method were 1.2 and 2.6 ng/ml, respectively.     

α-Chymotrypsin catalyses the synthesis of methotrexate oligomers

The enzymatic synthesis of methotrexate (MTX) catalysed by α -chymotrypsin was studied for the first time. The proteolytic enzyme displayed activity for the synthesis of MTX oligomers composed by 6 repeating units (DP_avg = 1.5). For longer oligomers, molecular dynamics simulations confirmed that as the oligomeric chain grows its accommodation in the enzymes’ active site is hindered, which is evidenced by a decrease of the binding energy associated. The full characterization of the oligomers produced was performed by nuclear magnetic resonance (NMR, ¹H and ¹³C), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF), electrospray ionization (ESI) and differential scanning calorimetry (DSC).     

Uptake of methotrexate linked to an anti-EL4-lymphoma antibody by EL4 cells

Summary To study the mechanism of tumor inhibition, the uptake of methotrexate (MTX) covalently linked to a rabbit IgG antibody against a tumor-associated antigen on the surface of mouse EL4 lymphoma cells (AELG) has been compared with the uptake of free MTX and of MTX covalently linked to normal rabbit IgG (NRG). When EL4 cells were incubated at 37°C with 10 M free MTX uptake leveled off after 30 min, at 30 pmol/mg protein. In contrast, uptake of both conjugates under these conditions continued throughout an observation period of 6 h. At 6 h the net uptake of MTX bound to AELG was 40 pmol/mg protein and that of MTX bound to NRG was 24 pmol/mg protein. These results show that both MTX-AELG and MTX-NRG conjugates are taken up by EL4 cells. The rate at which EL4 cells took up bound MTX was much slower than that of free MTX but, at 6 h, the net uptake of MTX-AELG exceeded that of the free drug. Abbreviations used in this paper: AELG, antiEL4 IgG; NRG, normal rabbit IgG; MTX, methotrexate; PBS, 0.01 M sodium phosphate, pH 7.1, containing 0.145 M sodium chloride     

Probing the structural interactions between methotrexate and dexamethasone with muscle cystatin: a biophysical study

Abstract

Drug protein interactions have gained considerable attention over the past many years. In the current communication the association of muscle cystatin (MC) with anti-rheumatic drugs methotrexate and dexamethasone was studied by thiol proteinase inhibitor assay, ultra violet (UV) absorption, fluorescence spectroscopy, and fluorescence transform infra-red spectroscopy (FTIR). A static pattern of quenching was noticed between muscle cystatin and methotrexate (MTX). Binding constant (K_a) of methotrexate to muscle cystatin was found to be 1?×?10^?7 M^?1 and the stoichiometry of binding was calculated to be one. Fluorescence measurement of the emission quenching reveals that the quenching process of cystatin by dexamethasone (DXN) was also static. The stoichiometry of binding and binding constant was also obtained. Additional evidence regarding MTX–MC and DXN–MC was obtained from UV spectroscopy and FTIR spectroscopic results. Such spectroscopic studies would help in modelling new candidate drugs for rheumatoid arthritis based on their cystatin binding profile.

Communicated by Ramaswamy H. Sarma     

Binding of methotrexate to dihydrofolate reductase and its relation to protonation of the ligand

Stopped-flow spectrophotometry and stopped-flow fluorometry have been used to study the binding of methotrexate (MTX) and 3-deazamethotrexate (3-deazaMTX) to dihydrofolate reductase (DHFR) isoenzymes from Streptococcus faecium and from Lactobacillus casei. The absorbance change and fluorescence quenching that occur when MTX binds to DHFR isoenzyme II from S. faecium (SFDHFR II) are both biphasic and give similar apparent rate constants for both phases. The faster phase has an apparent rate constant that is dependent on MTX concentration and therefore corresponds to the initial binding reaction. From the concentration dependence it has been calculated that the association rate constant is 3.0 X 10(5) M-1 s-1 at 20 degrees C and pH 7.3, and the association constant (equilibrium constant) under these conditions is 5.8 X 10(5) M-1. By examination of the amplitude of the fast-phase absorbance change at various wavelengths, it has been determined that the absorbance change occurring in the fast phase is due to MTX protonation. Within the limits of the method it was thus not possible to detect a difference in the rates of binding and of protonation of MTX. The MTX association rate constant is pH dependent, decreasing 330-fold as the pH is decreased from 5.0 to 9.0. The data fit well to a curve generated by assuming a single ionization with a pKa of 6.0 and a pH-independent association rate constant 1000-fold greater for binding of protonated MTX to SFDHFR II than for binding of unprotonated MTX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microcalorimetric evaluation of the effects of methotrexate and 6-thioguanine on sensitive T-lymphoma cells and on a methotrexate-resistant subline.

Isothermal microcalorimetry was used in order to continuously monitor and quantitatively assess the action of two antineoplastic drugs, methotrexate (MTX) and 6-thioguanine (6-TG), on a human T-lymphoma cell line, CCRF-CEM. The results with MTX were compared with data from experiments with a MTX-resistant subline, CEM/MTX. The slope of the power-time curve after drug injection relative to that obtained during unperturbed growth, was used to construct dose-response curves. The normal cell line was characterized by a D50 value (i.e., the dose producing half the maximal response) of 0.05 microM for MTX and 0.38 microM for 6-TG. For the MTX-resistant subline the D50 value was 8 microM of MTX. Comparisons of the continuous power-time curves showed the inhibitory effect of 6-TG to be faster than that of MTX.     

Polyethylene glycol conjugates of methotrexate varying in their molecular weight from MW 750 to MW 40000: synthesis,characterization, and structure-activity relationships in vitro and in vivo

Poly(ethylene glycol)s (PEGs) are potential drug carriers for improving the therapeutic index of anticancer agents. In this work, the anticancer drug methotrexate (MTX) was activated with N,N'-dicyclohexylcarbodiimide (DCC) and coupled to amino group bearing PEGs of MW 750, 2000, 5000, 10 000, 20,000, and 40,000. First, the activation process of MTX with DCC in the presence and absence of N-hydroxysuccinimide was analyzed through HPLC. Preincubation of methotrexate with DCC alone at 0 degrees C proved to be favorable with respect to the amount of activated species and the formation of byproducts. MTX-PEG conjugates were synthesized according to this procedure, isolated through size-exclusion chromatography, and characterized through analytical HPLC, MALDI-TOF spectrometry, and gel permeation chromatography. In a cell-free assay, all of the drug polymer conjugates inhibited the target enzyme of MTX, dihydrofolate reductase (DHFR), to a similar extent, but were not as active as free MTX. Additionally, incubation of the MTX-PEG40000 conjugate for 6 days at 37 degrees C in phosphate buffered saline (pH 7.4), in cell-conditioned medium, or in human serum revealed no significant release of methotrexate. These results, taken together, indicate that release of MTX from polymer conjugates is not necessary for an effective interaction with the active site of dihydrofolate reductase. Evaluation of the in vitro cytotoxicity of the MTX-PEG conjugates in two adherent and three suspension human tumor cell lines revealed that the IC(50) values of the tested compounds increased with the size of the drug-polymer conjugates. The most effective compound tested in these assays was the free drug MTX itself (IC(50) value ranging from approximately 0.01 to 0.05 microM), while the IC(50) values of the polymer conjugates were higher (IC(50) value for MTX-PEG750, 2000 and 5000: approximately 0.6-3 microM; for MTX-PEG10000 and 20000: approximately 2-7 microM; and for MTX-PEG40000: > 6 microM). Subsequently, MTX-PEG5000, MTX-PEG20000, and MTX-PEG40000 were evaluated in a human mesothelioma MSTO-211H xenograft model, and their antitumor effects were compared with free methotrexate and the albumin conjugate MTX-HSA, a conjugate that is currently in phase II clinical trials. In contrast to the in vitro results, the high molecular weight MTX-PEG conjugates exhibited the highest in vivo antitumor activity: At a dose of 40 and 80 mg/kg MTX-PEG5000 was less active than MTX at its optimal dose of 100 mg/kg; MTX-PEG20000 at a dose of 40 mg/kg showed antitumor efficacy comparable to MTX, but MTX-PEG40000 at a dose of 20 mg/kg was superior to MTX and demonstrated antitumor activity of the same order as MTX-HSA (20 mg/kg).     

Simultaneous changes in various mechanisms that mediate the cell incorporation of folate compounds account for low levels of resistance to methotrexate.

Changes in the mechanisms of folate incorporation were studied in cells treated with low concentrations of methotrexate in order to evaluate their contribution to the development of resistance to antifolate drugs. The uptake of methotrexate via reduced-folate system, the membrane-associated high-affinity folate binding capacity and the activity, levels and affinity for methotrexate of dihydrofolate reductase were measured in L5178 murine leukemic lymphoblasts and in a subline, MTX/R16, 16 times more resistant to methotrexate which was isolated after a short exposure to the antifolate. Various simultaneous changes were characterized in MTX/R16 cells which co-participated in the development of resistance: a decreased affinity of the carrier for methotrexate uptake via the reduced-folate system of entry, the increase of dihydrofolate reductase activity and levels and a two-fold increased expression of a membrane-associated high-affinity folate-binding protein (mFBP). The increase of the mFBP expression, besides ensuring the growth of resistant cells by its contribution to the reduced folate intake, also participates in the methotrexate resistance by the internalization of folate cofactor which would compete with methotrexate hindering the effective inhibition of dihydrofolate reductase by the antifolate.     

Evidence for the facilitated transport of methotrexate polyglutamates into lysosomes derived from S180 cells. Basic properties and specificity for polyglutamate chain length.

Evidence is presented outlining basic properties of a previously undescribed facilitative transport system mediating transfer of methotrexate (MTX) polyglutamates from the cytoplasmic to the lysosomal compartment of the cell. These experiments were conducted using purified lysosomes prepared from murine S180 cells, and a model substrate ([3H]MTX + G1; methotrexate with 1 additional glutamyl residue) to examine biological aspects as well as pharmacological significance of this process in a tumor cell model. The data, expressed as a function of latent beta-hexosaminidase activity, a measure of lysosomal integrity, show that [3H]MTX + G1 uptake in lysosomes is temperature-dependent, is stimulated specifically by magnesium chloride and potassium chloride with maximal enhancement observed in the presence of both agents together, exhibits Michaelis-Menten saturation kinetics with Km and Vmax values of 346 +/- 39 microM and 2.8 +/- 0.3 pmol/min/unit of beta-hexosaminidase activity, respectively, and is competitively inhibited by longer chain polyglutamates with increasing effectiveness as shown by Ki values of 334 +/- 19, 201 +/- 16, 106 +/- 13, and 42 +/- 8 microM, for MTX + G1, MTX + G2, MTX + G3, and MTX + G4, respectively. In addition, uptake is inversely related to medium osmolarity indicating that the phenomenon we observe represents internalization of the [3H]MTX + G1 and not adsorption to a possible surface binding site. As a whole, the data are consistent with a single mediated transport system shared by all MTX polyglutamates for entry into lysosomes. It is our view that this transport system represents the initial step in the degradation of polyglutamates in the cell. In addition, based on a comparative analysis of the kinetics for hydrolysis and transport, we suggest that it is also the limiting step in this process and, as such, regulates the extent of degradation of the free cellular pools of these compounds.     

Molecular basis of inhibitory peptide maurotoxin recognizing Kv1.2 channel explored by ZDOCK and molecular dynamic simulations

Inhibitory peptide-channel interactions have been utilized to characterize both channels and peptides; however, the fundamental basis for these interactions remains elusive. Here, combined computation methods were employed to study the specific binding of maurotoxin (MTX) peptide to Kv1.2 channel. In the first stage, numerous predicted complexes were generated by docking an ensemble of all 35 NMR conformations of MTX to Kv1.2 channel with ZDOCK program. Then the resulted complexes were clustered and classified into four main binding modes, based on experimental information and interaction energy analysis after the energy minimization and molecular dynamics (MD) simulations. By examining the stability of the plausible candidates through unrestrained MD simulations and calculation of the binding free energies, a final reasonable MTX-Kv1.2 complex was identified, with an overall high degree of correlation between the calculation and experiment on mutational effects. In the obtained complex structure model, MTX mainly used its beta-sheet domains to associate the channel mouth instead of the well-recognized functionally important S5P linkers of Kv1.2 channel. Structure analysis characterized that the most essential Tyr(32) residue of MTX was surrounded by a "pocket" formed by many nonpolar and polar residues of Kv1.2 channel, and revealed a pore-blocking Lys(23) and an important Lys(7) stabilized by strong electrostatic interactions with Asp(379) of Kv1.2. Furthermore, a stepwise structural arrangement for both ligand and receptor was found to accompany the tighter interaction of MTX into the target channel. The starting conformation of MTX, the side-chain conformation of the most important residue Tyr(32), and proper introduction of flexibility for candidate complexes were demonstrated to be considerably important factors for obtaining the final reasonable complex structure model. All these findings should not only be helpful for identifying more plausible K(+) channel-inhibitory peptide complex structures, but also provide intrinsically valuable structural biology information to interpret binding affinities, specificities, and diversity of K(+) channel-nature toxin interactions.     

Activation of methotrexate-alpha-alanine by carboxypeptidase A-monoclonal antibody conjugate.

Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted enzyme and antibody. On the basis of SDS-PAGE analysis and measurement of catalytic activity, the preparation contained approximately equal amounts of 1:1 and 2:1 (enzyme:antibody) conjugates; binding activity of the conjugate (1.8 x 10(5) molecules/cell) was similar to that of unreacted antibody. In vitro cytotoxicity studies with UCLA-P3 cells demonstrated the ability of cell-bound conjugate to convert the prodrug methotrexate-alpha-alanine (MTX-Ala) to methotrexate (MTX). In the absence of conjugate, ID50 values for MTX-Ala and MTX were 8.9 x 10(-6) and 5.2 x 10(-8) M, respectively. ID50 for the prodrug improved to 1.5 x 10(-6) M with cells containing bound conjugate. This potentiation of MTX-Ala cytotoxicity by conjugate-bound CP-A, which was at least 30-fold greater than that produced by a comparable amount of free enzyme, is attributed to enhanced effectiveness of MTX generated at the cell surface as opposed to the surrounding medium. Examination of the time course of cytotoxicity over a 96-h period showed that the conjugate-prodrug combination (at 2.5 x 10(-6) M) was nearly as effective as MTX in preventing cell replication. These results demonstrate the chemotherapeutic potential of carboxypeptidase-monoclonal antibody conjugates used in conjunction with MTX peptide prodrugs.     

Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Upon exposure of primary monolayer cultures of hepatocytes and H35 hepatoma cells, methptrexate (MTX) is taken up by carrier-mediated mechanisms and converted to γ-glutamyl derivatives with one to four residues being added. Under conditions that result in 90% or greater conversion, the primary metabolite in both cell types is MTX with three additional glutamates (4-NH₂-10-CH₃PteGlu₄). When the time-dependent synthesis of MTX polyglutamates (4-NH₂-10-CH₃PteGlu₂ and higher) at extracellular concentrations of 10 and 100 μm methotrexate is measured, both cell types exhibit linear synthesis for 4 to 6 hr, at which time an apparent steady state intracellular concentration of approximately 40 μm is reached. The concentration of MTX polyglutamate synthesized is not due a restriction in MTX since the hepatocytes and H35 cells accumulated 400 and 138 μm intracellular methotrexate, respectively, after 24 h in the presence of 100 μm extracellular MTX. Examination of MTX polyglutamate formation following a 24-h incubation showed concentration dependence with respect to intra- and extracellular MTX. Saturation was reached at a medium concentration of approximately 2 μm with both cell types which corresponded to 10 to 12 μm intracellular MTX. Placement of cells at steady state in medium lacking MTX results in the rapid equilibration of all free intracellular MTX with the medium. The MTX polyglutamates leave the cell by a slow loss of intact polyglutamates and also by intracellular cleavage to MTX followed by efflux. The longer-chain-length γ-glutamyl derivatives (Glu_4–5) are more avidly retained by the cells than the shorter ones (Glu_2–3).     

Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells.

4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.     

Inhibition of Stenotrophomonas maltophilia dihydrofolate reductase by methotrexate: a single slow-binding process

Although antifolates such as trimethoprim are used in the clinical treatment of Stenotrophomonas maltophilia infection, the dihydrofolate reductase (DHFR) of this microorganism is scarcely known because it has never been isolated. Here, we describe the purification of this enzyme and kinetically characterize its inhibition by methotrexate (MTX). Upon MTX treatment, time-dependent, slow-binding inhibition was observed due to the generation of a long-lived, slowly dissociating enzyme-NADPH-inhibitor complex. Kinetic analysis revealed a one-step inhibition mechanism (K(I) = 28.9 +/- 1.9 pM) with an association rate constant (k(i)) of 3.8 x 10(7) M(-1)s(-1). Possible mechanisms for MTX binding to S. maltophilia DHFR are discussed.