K.S. Vitols

B12 — Dependent methionine synthetase as a potential target for cancer chemotherapy

Methionine synthetase, a B1 2-dependent enzyme that catalyzes the reaction between 5-methyl tetrahydrofolate and homocysteine to yield tetrahydrofolate and methionine, has been purified to homogeneity from Escherichia coli K-12. The enzyme has a molecular weight of 186,000 and contains 1 mole of B1 2 r per mole of protein. Activation of the enzyme, which involves conversion of the prosthetic group to methyl-B1 2, requires adenosyl methionine plus TPNH and two flavoproteins (molecular weights of 27,000 and 19,400, respectively). These flavoproteins have also been obtained in pure form. Knowledge of the structural characteristics and catalytic mechanism of the bacterial methionine synthetase has provided guidance for parallel studies on the enzyme from L1210 mouse leukemia cells. Interference with the action of the L1210 methionine synthetase, as a possible means of preventing cell replication, has been considered in terms of the following direct and indirect approaches: (a) use of substrate analogs, (b) inhibition of transport of 5-methyl tetrahydrofolate into the cells; (c) use of adenosyl methionine analogs; (d) use of methyl-B1 2 analogs; (e) inhibition of the conversion of B1 2 to methyl-B1 2; and (f) inhibition of the transport of B1 2 into the cells.

Inhibition of dihydrofolate reductase by methotrexate: A new look at an old problem

Dihydrofolate reductase from L1210 mouse leukemia cells has been investigated with respect to factors that affect its inhibition by Methotrexate (MTX). Kinetic analyses indicate that the enzyme can exist in multiple forms (i.e., different conformational states) varying in their Km values for NADPH and Ki values for MTX. Interaction of the enzyme with ligands (particularly NADPH and MTX) appears to induce the formation of certain of these forms. The two histidyl residues and the single cysteinyl residue of the enzyme play particularly important roles in the catalytic activity of the enzyme and its ability to bind MTX. A cysteinyl-histidyl hydrogen bond is believed to play an important role in determining conformational states of the enzyme.

Transport of methotrexate and other folate compounds: Components, mechanism and regulation by cyclic nucleotides

Abstract Transport of Methotrexate and other folate compounds into cells of L1210 mouse leukemia or Lactobacillus casei occurs via “active,” carrier-mediated processes. In the bacterial system, transport is linked to glycolysis and is unaffected by cyclic nucleotides. A folate-binding protein, believed to be the carrier in the transport process, has been solubilized by treatment of L. casei membranes with Triton X-100 in the presence of [ 3 H]folate and purified to homogeneity. The protein (MW 25,000) binds an equimolar amount of folate. Owing to a preponderance of hydrophobic amino acids, the binding protein is insoluble in aqueous media unless encased in a Triton micelle. Similar experiments suggest that L1210 cells may contain a Triton-extractable membrane protein that binds 5-methyl tetrahydrofolate and Methotrexate. Transport of Methotrexate into L1210 cells is depressed by exogenous cyclic nucleotides or compounds that elevate the level of endogenous cyclic nucleotides, such as phosphodiesterase inhibitors, adenyl cyclase stimulators, glucose, ATP, ADP and GTP. Implications of these observations for cancer chemotherapy are discussed.

Chemotherapeutic potential of methotrexate peptides.

MTX peptides in which the amino acid was linked to the alpha-carboxyl group have been prepared and examined for cytotoxicity before and after treatment with proteolytic enzymes. The alanine, aspartic acid and arginine derivatives (MTX-ala, MTX-asp and MTX-arg) were synthesized by a regio-specific route, following the general procedures of Rosowsky and Montgomery. Each compound was obtained in good yield, and purity was established by TLC, HPLC, absorbance spectra and elemental analyses. The MTX peptides were not hydrolyzed by a variety of proteolytic enzymes (e.g., trypsin, plasmin, urokinase, aminopeptidase). Pancreatic carboxypeptidase A, however, hydrolyzed MTX-ala readily, MTX-asp slowly and MTX-arg not at all. The MTX-ala and, to a lesser extent, MTX-arg were substrates for pancreatic carboxypeptidase B. MTX-arg was also hydrolyzed by the endogenous carboxypeptidase N in human serum. The cytotoxicity of these MTX peptides toward L1210 cells was measured in a microculture assay system using a tetrazolium dye. MTX-ala was weakly cytotoxic (ID50 = 2.0 x 10(-6)M) compared to MTX (ID50 = 2.4 x 10(-8)M). When MTX-ala was tested in the presence of carboxypeptidase A, the ID50 value improved to 8.5 x 10(-8)M. MTX-arg gave an ID50 of 5.0 x 10(-8)M, which was not unexpected in view of its susceptibility to hydrolysis by the carboxypeptidase activity present in the fetal calf serum of the culture medium. Inclusion of carboxypeptidase B lowered the ID50 value to 2.5 x 10(-8)M. Possible clinical uses of MTX peptides are discussed.

Polyglutamylation as a factor in the trapping of 5-methyltetrahydrofolate by cobalamin-deficient L1210 cells.

Cobalamin-deficient L1210 mouse leukemia cells, developed by propagation in a medium from which cyanocobalamin was omitted and fetal bovine serum was replaced by bovine serum albumin, grew normally on excess folate or 5-formyltetrahydrofolate. These cells responded poorly, however, to 5-methyltetrahydrofolate unless exogenous cobalamin was added. A cobalamin dependency was also observed when low levels of folate or 5-formyltetrahydrofolate were used. With 5-methyltetrahydrofolate, optimal stimulation of growth was observed with free and transcobalamin II-bound cobalamin at 4,000 and 2 pM, respectively. Under cobalamin-replete conditions, the average cobalamin content was ca. 3,000 molecules/cell, and in the deficient state this value declined to < 10 molecules/cell. Optimal replication on 5-methyltetrahydrofolate required ca. 180 molecules/cell.

Transport of folate compounds, pterins and adenine in L1210 mouse leukemia cells.

L1210 mouse leukemia cells provide a convenient model for examining the mechanisms and components involved in the active transport of various metabolites and drugs. One of these transport systems exhibits a broad specificity for folate compounds, including 4-amino antagonists such as methotrexate. The primary substrate for this system is 5-methyltetra-hydrofolate (Kt = 1 microM), the principal circulating form of the vitamin in mammals. 5-Formyltetrahydrofolate (Kt = 5 microM) and Methotrexate (Kt = 5 microM) are also taken up efficiently, but folate (Kt = 100 microM) is a relatively poor substrate. Vmax for this system is ca. 15 pmoles/min/mg protein. Energy for substrate internalization is provided by an anion-exchange mechanism, and regulation appears to be mediated by cyclic AMP. The system can be inhibited irreversibly by treatment of the cells with photo-activated azido AMP or carbodiimide-activated folate compounds. The latter method allows the membrane-associated binding protein to be labeled in situ, thereby providing a means for identifying it during subsequent solubilization and purification. Guidance for this latter project is provided by previous experience in the purification to homogeneity of a similar folate-binding protein from Lactobacillus casei. L1210 cells also contain an efficient system for the transport of adenine (Kt = 20 microM; Vmax = 200 pmoles/min/mg protein). Uptake of adenine is linked with its conversion to AMP via PRPP-dependent adenine phosphori-bosyltransferase. Pterins, which have a close structural similarity to adenine (as well as to a portion of the folate molecule), are also transported into L1210 cells. Transport of [3H] 6-hydroxymethylpterin (Kt = 20 microM) was inhibited by 6-formylpterin, 6-methylpterin and 6-carboxypterin with Ki values of 42, 100 and 350 microM, respectively. Adenine (Ki = 20 microM) and various other purines were also good inhibitors of pterin transport. Present evidence indicates that adenine and pterins use separate transport systems, but isolation of the components of these systems may further delineate their interrelationships.

Biochemistry of Methotrexate: Teaching an Old Drug New Tricks

Chemotherapy, despite its shortcomings, continues to be one of the major modalities in the treatment of cancer. Most of the drugs in current use were discovered empirically, i.e., through screening programs. In recent years, however, a more rational approach for drug development has emerged, based upon a four-stage strategy: a) the first stage involves the identification of opportune targets. These are usually biochemical parameters that are closely related to cell replication, e.g., DNA (or RNA) or enzymes responsible for the synthesis of the nucleic acids and their nucleotide precursors. Illustrative of this approach is the work of Weber (1), who has developed a comprehensive program for identifying enzymes that are rate-limiting in the synthesis of purine or pyrimidine nucleotides and are present in increased amounts in tumor cells; b) Sites on the target for drug interaction are defined. For DNA, these are nucleotide sequences with enhanced susceptibility to intercalating or covalent-binding agents; specificity of these sites, however, is usually not stringent. Substrate-binding sites on enzymes offer better possibilities for specificity. Some insight into the dimensions of these sites and the amino acids that interact with ligands can be obtained by comparison of binding constants for substrates and inhibitors, by chemical modification with group-specific reagents, and by NMR measurements. Accurate three-dimensional pictures of the sites, however, require X-ray diffraction analyses of the crystalline enzymes. This latter technique has been employed in the elegant studies of Kraut and Matthews [reviewed in (2)] for visualizing the Methotrexate (MTX) binding site on dihydrofolate reductase; c) Information about the drug-binding sites, along with computer graphic modeling, then provides guidance for the chemical synthesis of compounds tailored to fit with a high degree of specificity and affinity. Montgomery, Robins, Hitchings and Elion are among the leaders in this field; d) Finally, promising compounds that interact satisfactorily with their targets are examined for other criteria necessary for clinical acceptance, viz., facile uptake by cells (via active transport or diffusion),cytotoxicity (with selectivity toward tumor cells), distribution to tumorbearing sites in the body, and favorable pharmacologic characteristics such as resistance to metabolism and/or excretion.

Platinum-folate compounds: synthesis, properties and biological activity

Cis-diamminediaquaplatinum(II)-ion, the biologically active form of the anticancer agent Cisplatin, reacted readily with tetrahydrofolate at pH 7 and 37 degrees C to produce a stable complex. The reaction was monitored spectrophotometrically by the change in absorbance maximum from 298 nm (tetrahydrofolate) to 275 nm (complex); occurrence of isobestic points at 282 and 327 nm indicated that a single product was formed. Purity of platinum-tetrahydrofolate, after isolation in ca. 70% yield, was established by TLC and HPLC. Elemental analysis, absorbance spectra at various pH values and nmr spectra provided evidence that the diammine platinum moiety was bridged across the N-5 and N-10 positions of tetrahydrofolate. Complexation also occurred with 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, Methotrexate and aminopterin, but not with folate or 7,8-dihydrofolate. Biological implications of these observations have been investigated. Intracellular folates in L1210 cells have been identified and quantitated via reverse phase HPLC (C18 column; tetrabutylammonium phosphate as the pairing ion) and changes in the levels of these compounds, after exposure of cells to Cisplatin, have been measured. Platinum derivatives of tetrahydrofolate or other reduced folates were not found, but there was a decrease in the level of 5,10-methenyltetrahydrofolate, accompanied by an increase in 5-formyl and 10-formyltetrahydrofolate (and perhaps tetrahydrofolate). The chemical interaction of the diaqua form of Cisplatin with Methotrexate resulted in decreased uptake of the latter by L1210 cells. The platinum complex of tetrahydrofolate was a reasonably good inhibitor (Ki = 4 microM) of L1210 dihydrofolate reductase and of the folate transport system (50% inhibition at ca. 200 microM) of L1210 cells.

Enzymatic activation of 5-formyltetrahydrofolate via conversion to 5, 10-methenyltetrahydrofolate.

The ATP-dependent conversion of 5-formyltetrahydrofolate (folinate) to the 5,10-methenyl derivative, catalyzed by 5,10-methenyltetrahydrofolate synthetase (EC 6.3.3.2), is of considerable importance in cancer chemotherapy, since it provides the basis for the administration of folinate to counteract the deleterious effects of high-dose Methotrexate regimens. Methenyltetrahydrofote synthetase has been purified 10,000-fold from L. casei using sequential affinity chromatography on immobilized folinate and ATP. The monomeric enzyme is homogeneous upon SDS-polyacrylamide gel electrophoresis, has a molecular weight of 23,000 (confirmed by gel filtration), and contains a single cysteine residue. The turnover number is ca. 250 min-1, and the Km values at pH 6 for 5-formyltetrahydrofolate and Mg-ATP are 0.6 and 1.0 microM, respectively; the equilibrium constant is 0.7-1.0 mM. Methotrexate, 5-methyltetrahydrofolate, and folate are not inhibitory. The mechanism for the reaction is proposed to involve phosphorylation of the formyl group to create an enol phosphate; subsequent attack on the methenyl carbon by N-10 would generate a tetrahedral intermediate, with release of the phosphate providing the driving force for ring closure.

Overview: Rational Basis for Development of Fluoropyrimidine/5-Formyltetrahydrofolate Combination Chemotherapy

Fluorodeoxyuridylate (FdUMP) and thymidylate synthase (TS) are one of the better understood systems of drug-target interaction in cancer chemotherapy. Isolation and characterization of TS (initially from Lactobacillus casei and later from a variety of other sources), cloning and sequencing of the gene, determination of the 3-D structure of the enzyme by X-ray diffraction, and elucidation of the structure of both the catalytic intermediate and the enzyme-inhibitor complex have revealed critical parameters of the target at the molecular level. Potentiation of FdUMP binding by 5,10-methylenetetrahydrofolate (CH2-FH4), discovered at the enzymatic level, has been exploited to increase the clinical effectiveness of fluoropyrimidines. CH2-FH4 can be generated from folate, 5-methyltetrahydrofolate, or 5-formyltetrahydrofolate (citrovorum factor, CF); the latter is the compound of choice for therapeutic regimens. Transformation of CF to CH2-FH4 can occur via two pathways: (a) CF----5,10-methyltetrahydrofolate----CH2-FH4; or (b) CF----tetrahydrofolate----CH2-FH4. The relative importance of these pathways in various cells is not yet clear. The role of CH2-FH4 in FdUMP toxicity, and its central position in folate coenzyme-dependent C1 metabolism, emphasize the need for development of methods to quantitate intracellular levels of this compound.