L1210 Dihydrofolate Reductase Research Articles

Synthesis and antifolate evaluation of the aminopterin analogue with a bicyclo[2.2.2]octane ring in place of the benzene ring

N-[4-[[2,4-diamino-6-pteridinyl)methyl]amino]bicyclo[2.2.2]octane-1-carbonyl]- l-glutamic acid ( 1) was synthesized and tested for antifolate activity. N-(4-Aminobicyclo[2.2.2]octane-1-carbonyl- l-glutamic acid dimethyl ester ( 6), the side chain precursor to subject compound 1, was synthesized readily via reported bicyclo[2.2.2]octane-1,4-dicarboxylic acid monoethyl ester ( 2). The side chain precursor 6 was alkylated by 6-(bromomethyl)-2,4-pteridinediamine ( 7). Subsequent ester hydrolysis then afforded 1. Antifolate and antitumor evaluation of 1 verses L1210 dihydrofolate reductase (DHFR) and three tumor cell lines (L1210, S180, and HL60) showed it to be ineffective. Although compound 1 was very similar to aminopterin structurally, the bicyclo[2.2.2]octane ring system in place of the phenyl ring in the p-aminobenzoate moiety effectively negates the stoichiometric binding displayed by many classical DHFR inhibitors bearing appropriate aromatic ring systems in the side chain.

Design and synthesis of histidine analogues of folic acid and methotrexate as potential folylpolyglutamate synthetase inhibitors.

Folypolyglutamate synthetase (FPGS) is responsible for the conversion of naturally occurring folates and antifolates to their poly-gama-glutamyl derivatives, which are the forms required for intracellular retention of folates and are also the preferred substrates (cofactors) for most folate-dependent enzymes. Folate and methotrexate analogues 6 and 4, with L-histidine in place of L-glutamate, were designed and synthesized as potential FPGS inhibitors. Target compound 5, the N tau-(carboxymethyl)-L-histidine derivative of 4, was also prepared. Compounds 4 and 5 inhibited the growth of L1210 cells (IC50 values: 0.091 and 0.15 microM, respectively) and were potent inhibitors of L1210 dihydrofolate reductase. No significant inhibition of FPGS by 4, 5, or 6 was observed at the high pH of the standard enzyme assay. This could be the consequence of a lack of protonation of the basic side chains, which is likely to be required for FPGS inhibitory activity. The observed cytotoxicity indicates that partial protonation of the imidazole ring permits cellular uptake of the analogues.

Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region.

N alpha-(4-Amino-4-deoxy-10-methylpteroyl)-N epsilon-(4-azido-5- [125I]iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the 125I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme.

The structure of mouse L1210 dihydrofolate reductase-drug complexes and the construction of a model of human enzyme

The structure of mouse L1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0 Å resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 Å resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X-ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty-one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.

L1210 dihydrofolate reductase. Kinetics and mechanism of activation by various agents.

Dihydrofolate reductase from a methotrexate-resistant subline (R6) of L1210 mouse leukemia cells is activated (i.e. has its catalytic activity increased severalfold) by treatment with (a) sulfhydryl-modifying agents (p-chloromercuribenzoate (pCMB) or 5,5'-dithiobis(2-nitrobenzoic acid], (b) salts (KCl or NaCl), or (c) chaotropes (urea or guanidinium hydrochloride). With b or c activation is rapid (less than 10 s), but with a the process is much slower; at 25 degrees C, pseudo first-order rate constants for activation by excess pCMB or 5,5'-dithiobis(2-nitrobenzoic acid) are 0.45 and 0.08 min-1, respectively. Activation can also be monitored by conformational changes in the protein as indicated by enhanced fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate or by increased intrinsic fluorescence of tryptophan residues in the enzyme. Pseudo first-order rate constants for the pCMB-induced conformational change, measured by these fluorimetric procedures (0.45 min-1 and about 0.4 min-1, respectively), are in good agreement with the value obtained from the increase in catalytic activity. The rate of modification of the single cysteine residue in the enzyme by excess 14C-labeled pCMB, however, is faster than the rate of activation, indicating that the conformational change follows derivatization and is the rate-limiting step in the overall process. Activated forms of the enzyme are more labile to thermal denaturation or proteolysis than the untreated enzyme; the former process, however, is retarded by the presence of bovine serum albumin. Activation by the various agents is considered to involve a common mechanism in which interaction of the enzyme with the agents is followed by conformational changes in the enzyme, producing a series of forms that differ in microstructure, catalytic activity, and lability.

Antibody for detection and quantitation of membrane-associated folate-binding protein from Lactobacillus casei

Lactobacillus casei cells contain a 25 kDa, membrane-associated, folate-binding protein (fbp), which is a component of the folate transport system. Polyclonal antibody to fbp (anti-fbp) has been prepared, and conditions have been established for detection and quantitation of the protein. Anti-fbp did not block [ 3H]folate transport or binding in L. casei cells. As judged by Western blots, the antibody reacted only with fbp on sodium dodecyl sulfate electrophoretograms of Triton X-100 extracts of L. casei membranes. Anti-fbp showed no cross-reactivity with L. casei dihydrofolate reductase, L. casei 5,10-methenyltetrahydrofolate synthetase, L1210 dihydrofolate reductase, rat liver dihydrofolate reductase, or L1210 folate-binding protein. Enzyme-linked immunosorbent assay measurements indicated the presence of an fbp in membranes of Lactobacillus salivarius and two transport-defective sublines of L. casei. Anti-fbp was used to demonstrate (a) selective extraction, with n-butanol, of fbp from a mixture of Tritonsolubilized L. casei membrane proteins; (b) repression of fbp in membranes of L. casei cells grown on high levels of folate; and (c) localization of fbp by electron microscopy, using anti-fbp in conjunction with goat anti-rabbit IgG gold conjugate, in L. casei membranes.

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°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 (C 18 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-methyltetrahydrofolate, 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 ( K i = 4 μ m ) of L1210 dihydrofolate reductase and of the folate transport system (50% inhibition at ca. 200 μ m) of L1210 cells.

ChemInform Abstract: Quinazoline Antifolates Inhibiting Thymidylate Synthase: Benzoyl Ring Modifications.

AbstractDie N‐Propargyl‐benzoylglutaminsäureester (I) werden mit dem Brommethylchinazolin (II) zu den N‐Propargyl‐5,8‐dideazafolsäuren (III) umgesetzt, die auf Inhibierung der L1210‐Thymidylat‐Synthase und der Dihydrofolat‐Synthase sowie auf Inhibierung des Wachstums von L12lO‐Zellen geprüft werden.

Quinazoline antifolates inhibiting thymidylate synthase: benzoyl ring modifications.

Four new analogues of the antifolate N10-propargyl-5,8-dideazafolic acid were prepared that were substituted in the benzoyl ring. The 2'-chloro and 2'-methyl analogues were prepared from the appropriately substituted p-nitrobenzoic acids. The route to the 3'-chloro and 3',5'-dichloro analogues was by chlorination of diethyl N10-propargyl-5,8-dideazafolate and diethyl N-[4-(prop-2-ynylamino)benzoyl]-L-glutamate, respectively, using sulfuryl chloride. The compounds were tested for their inhibition of purified L1210 thymidylate synthase (TS), for their inhibition of purified L1210 dihydrofolate reductase (DHFR), and for their inhibition of the growth of L1210 cells in culture. The 2'-chloro substituent reduced the TS inhibition by twofold and the 2'-methyl substituent reduced it by 20-fold; the 3'-chloro and 3',5'-dichloro derivatives were very poor inhibitors. The substituents only slightly affected the DHFR inhibition. None of the compounds improved upon N10-propargyl-5,8-dideazafolic acid in inhibiting the growth of L1210 cells in culture.

Inhibition of dihydrofolate reductase, methotrexate transport, and growth of methotrexate-sensitive and -resistant L1210 leukemia cells in vitro by 5-substituted 2,4- diaminoquinazolines

A series of eighteen 2,4-diaminoquinazoline analogues of folic, isofolic, pteroic and isopteroic acids having various substituents at position 5 was studied. Each compound was evaluated as an inhibitor of L1210 dihydrofolate reductase, methotrexate influx into L1210 leukemia cells, and growth of methotrexate-sensitive and -resistant L1210 cells in vitro. Bridge reversal at positions 9 and 10 reduced the effectiveness of the classical analogues only with regard to the inhibition of the drug-sensitive cells as compared to methotrexate (MTX). Absence of the glutamate moiety adversely affected the potency of the compounds, particularly when coupled with reversal of the 9, 10-bridge. However, the presence of-Cl at position 5 restored significantly the potency of these compounds. The pteroate and isopteroate analogue ethyl esters were generally more effective inhibitors of cell growth than their non-esterified counterparts. Regarding the effects of substituents at position 5, the data suggest that -Cl > -CH 3 > -H for inhibition of methotrexate transport and growth of methotrexate-sensitive L1210 cells. The 5-Cl pteroate analogue and its corresponding ethyl ester were highly effective as growth inhibitors of methotrexate-resistant, transport-defective, L1210 cells in vitro.

L1210 Dihydrofolate Reductase: activation and enhancement of methotrexate sensitivity

Dihydrofolate reductase, purified to homogeneity from a subline of L1210 murine leukemia cells resistant to 10 −6 m Methotrexate, was resolved into two principal forms (1 and 2) by isoelectric focusing. These forms had pI values of 7.4 and 8.2, respectively; both stained for protein and catalytic activity, Form 1 was a single component, comprising ca. 10% of the total protein, but multiple components of 2 were observed by narrowing the pH range in the electrophoretic procedure. Urea-denatured enzyme exhibited two bands of approximately equal intensity upon isoelectric focusing. These results were interpreted to mean that the enzyme consists of a set of conformationally different forms, arising from two primary structures. Inhibition of the native enzyme by Methotrexate was polyphasic, and appreciably activity remained when the drug was present at an equimolar concentration. Various agents (KCl, H +, urea, and SH-modifiers), known to “activate” dihydrofolate reductases, produced a monophasic, stoichiometric inhibition. Activating agents appear to induce a more open (and labile) conformation of the enzyme. This leads to increased affinity for MTX accompanied, in some instances, by increased catalytic activity.

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl- l-lysine analogue of methotrexate, N α-( 4-amino-4-deoxy-10-methylpteroyl)-N ε-(5-[N,N- dimethylamino]-1-naphthalenesulfonyl]-1-naphthalenesulfonyl)- l-lysine , is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [ 3H]methotrexate transport with a K i of 7.02 μM, a value virtually identical to the K t for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl- l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.

Multiple forms of L1210 dihydrofolate reductase differing in affinity for methotrexate

Dihydrofolate reductase, purified from a Methotrexate-resistant subline (R6) of L1210 mouse leukemia cells, consists of two forms (designated 1 and 2 ) differing in affinity for the drug. Form 1 is more sensitive to inhibition by Methotrexate. Form 2 is more heat-labile, but it can be stabilized by bovine serum albumin or NADPH. Isoelectric focusing resolves 1 and 2 ; pI values are 7.4 and 8.2. Forms 1 and 2 comprise about 10 and 90% of the total protein, but 1 has at least a 2-fold higher specific activity. Binding measurements with [ 3H]Methotrexate provide K D values of ca. 1 and 27 mM for 1 and 2 .

The amino acid sequence of dihydrofolate reductase from the mouse lymphoma L1210.

The determination of the amino acid sequence of the dihydrofolate reductase (EC 1.5.1.3) from cells of the mouse lymphoma L1210 is described. The protein was cleaved by cyanogen bromide to produce the six fragments CB1 (residues 1 to 14), CB2 (residues 15 to 52), CB3 (residues 53 to 111), CB4 (residues 115 to 125), CB5 (residues 126 to 139), and CB6 (residues 140 to 186). One of the fragments, CB2, contained an internal homoserine derived from a methionine which was not cleaved by cyanogen bromide. The amino acid sequences and order of the cyanogen bromide fragments were determined by a combination of automatic and manual sequence analyses of the fragments and small peptides from tryptic, thermolytic, and Staphylococcus aureus protease digestions. The complete sequence comprises 186 residues in a single polypeptide chain of molecular weight 21,458. Comparison of the sequence of the L1210 dihydrofolate reductase with the sequences of the enzymes from Streptococcus faecium, escherichia coli RT500, and Lactobacillus casei indicates that all enzymes show some homology, which is strongest in the regions forming the substrate binding cleft.

Interaction of 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides with dihydrofolate reductase from amethopterin-resistant L1210 cells.

The 1,N6-ethenoadenine derivatives of triphosphopyridine and reduced triphosphopyridine nucleotides (TPN and TPNH) epsilon-TPN and epsilon-TPNH) have been synthesized and used as fluorescent probes to examine the pyridine nucleotide binding site of L1210 dihydrofolate reductase. Epsilon-TPNH (Km = 16.7 muM) was able to replace TPNH (Km = 3.8 muM) in the enzyme-catalyzed reduction of dihyrdofolate, and both epsilon-TPN and epsilon-TPNH formed binary complexes with the enzyme that were stable to polyacrylamide gel electrophoresis. The fluorescence of epsilon-TPN was enhanced and the emission maximum shifted from 415 to 405 nm when the nucleotide was bound to the enzyme. The ethenoadenine moiety in epsilon-TPNH behaved similarily, but the fluorescence changes were complicated by concurrent effects of binding upon the dihydronicotinamide fluorophore. Fluorescence enhancement titrations yielded values of 1.8 and 0.59 muM, respectively, for the dissociation constants of the enzyme-epsilon-TPN and enzyme-epsilon-TPNH complexes. Titration experiments based upon quenching of enzyme fluorescence gave similar values, viz., 2.1 and 0.53 muM for the dissociation constants of these complexes. Fluorimetric titration of the enzyme-TPNH complex with epsilon-TPN (or of the enzyme-TPN complex with epsilon-TPNH) failed to reveal the presence of a second pyridine nucleotide binding site. The fluorescence enhancement of enzyme-bound epsilon-TPN or dihydrofolate was quenched when amethopterin or epsilon-TPN, respectively, was added to form a ternary complex. These results provide information concerning the nature of the pyridine nucleotide binding site and its spatial relationship to the dihydrofolate/amethopterin binding site.