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— Photosensitized L-biopterin induces the transfer of a hydrogen atom from the dihydronico-tinamide moiety of NADPH to the biopterin ring. Sensitization occurs through the triplet state of both the lactim and lactam tautomers of L-biopterin. Quenching kinetic analysis to measure the bimolecular rate constant demonstrated a greater reactivity for the lactim tautomer. Recombinant human dihydro-folate reductase enzyme, for which these molecules are substrate and cofactor, enhanced the rate constant of the photosensitized H• transfer to the apparent lactim tautomer by eight times to 1.6 × 1010M-1s-1. When NADP+ replaced NADPH at the active site, no enhanced photoreduction of biopterin was observed, implying that the hydrogen atom comes from the reduced nicotinamide group and not as a result of protein conformational changes. This reduction at the active site represents a photoinitiated H• transfer in protein between substrate and cofactor.

Previous NMR studies on the ternary complex of human dihydrofolate reductase (hDHFR) with methotrexate (MTX) and NADPH detected six long-lived bound water molecules. Two of the water molecules, WatA and WatB, stabilize the structure of the protein while the other four, WatC, WatD, WatE and WatF, are involved in substrate binding and specificity. WatE may also act as a proton shuttle during catalysis. Here, the contributions of individual residues to the binding of these water molecules are investigated by performing NMR experiments on ternary complexes of mutant enzymes, W24F, E30A and E30Q. W24 and E30 are conserved residues that form hydrogen bonds with WatE in crystal structures of DHFR. Nuclear Overhauser effects (NOEs) are detected between WatE and the protein in all the mutant complexes, hence WatE still has a long lifetime bound to the complex when one of its hydrogen-bonding partners is deleted or altered by mutagenesis. The NOEs for WatE are much weaker, however, in the mutants than in wild-type. The NOEs for the other water molecules in and near the active site, WatA, WatC, WatD and WatF, also tend to be weaker in the mutant complexes. Little or no change is apparent in the NOEs for WatB, which is located outside the active site, farthest from the mutated residues. The decreased NOE intensities for the bound water molecules could be caused by changes in the positions and/or lifetimes of the water molecules. Chemical shift and NOE data indicate that the mutants have structures very similar to that of wild-type hDHFR, with possible conformational changes occurring only near the mutated residues. Based on the lack of structural change in the protein and evidence for increased structural fluctuations in the active sites of the mutant enzymes, it is likely that the NOE changes are caused, at least in part, by decreases in the lifetimes of the bound water molecules.

Nuclear magnetic resonance (NMR) spectra for [2-amino,3-15N2]folate and [2-13C]folate complexed with human dihydrofolate reductase, and for complexes of similarly labeled dihydrofolate, show that the N-3 proton of bound folate or dihydrofolate exchanges slowly with solvent and that the bound substrates are in the imino-keto tautomeric form. Previously proposed schemes for substrate protonation that require bound substrate to be in the enolic tautomer are therefore unlikely. The NMR spectra for bound folate are unchanged by raising the pH from 7 to 9.5, whereas those for free folate show marked changes due to ionization for the N-3 proton. The fraction of bound folate with the N-3 proton ionized at pH 9.5 is therefore very small, and the rate constant for the dissociation of the ionized species must be at least 320 times faster than for the protonated species. Comparison of NMR spectra over the pH range 5 to 7 gives no indication of a change in ionization state of the Glu30 carboxyl group over this pH range. This raises doubts about whether the apparent pKa of approximately 6 that describes pH dependence of hydride transfer is due to ionization of this carboxyl group.

gamma-Glutamyl hydrolase is a ubiquitous enzyme that has the capacity to cleave gamma-glutamyl bonds of cellular folyl- and antifolylpoly-gamma-glutamates. This study has revealed that the enzyme is secreted by primary cultures of rat hepatocytes and by H35 hepatoma cells. It was found that more than 99% of the total enzyme from H35 cells accumulated in the medium after 48 hr incubation with the serum-free medium. The cells were shown to remain intact during the secretion period since lactate dehydrogenase, dihydrofolate reductase and lysosomal hydrolases other than gamma-glutamyl hydrolase were retained within the cell. When PteGlu5 (folylGlu4) is used as a substrate the initial product is PteGlu (folate), and there is no appearance of intermediate chain length pteroyl polyglutamates. Therefore, the secreted and cellular gamma-glutamyl hydrolase from hepatoma cells appears to be an endopeptidase. Polyclonal antibodies to the poly-gamma-glutamate substrates of the enzyme were prepared and characterized. The antibodies recognize the structural differences between alpha- and gamma-glutamyl linkages but appear equally active with PteGlu5 and its analogs such as 4-NH2-10-CH3PteGlu5 and pABAGlu5. The affinity of the antibodies is related to the gamma-glutamyl structure since L-glutamic acid, folate or p-aminobenzoic acid are inactive with the antibodies. Furthermore, poly-gamma-glutamate has lower affinity for the antibodies than the poly-gamma-glutamate derivatives of PteGlu, 4-NH2-10-CH3PteGlu or pABA.

The new folate antagonist, 5-fluoro-5,8-dideazaisoaminopterin was synthesized in four steps beginning with 2,4-diamino-5-fluoroquinazoline. It was found to be a potent inhibitor of human dihydrofolate reductase. Against L1210 leukemia in mice, 5-fluoro-5,8-dideazaisoaminopterin was equiactive with methotrexate at approximately one half of the total dose employed.

A series of thirty eight 2,4-diaminoquinazolines having diverse substitution patterns on the aromatic ring was evaluated for inhibitory activity against dihydrofolate reductase (DHFR) obtained from a human lymphoblast cell line. Many of these compounds were also evaluated as inhibitors of rat liver DHFR under the same experimental conditions. In most instances the results obtained with each enzyme were comparable indicating that the rodent enzyme is a suitable model for the human DHFR as far as the determination of I50 values is concerned. The results demonstrate that relatively simple 5-substituted- or 5,6-disubstituted-2,4-diaminoquinazolines can be potent DHFR inhibitors. The presence of a nonpolar substituent at position 7 or 8 was highly detrimental to inhibitory potency.

5-Deazafolate and 6-deazatetrahydrofolate (DATHF) analogues with the glutamic acid side chain replaced by homocysteic aicd (HCysA), 2-amino-4-phosphonobutanoic acid (APBA), and ornithine (Om) were synthesized as part of a larger program directed toward inhibitors of folylpolyglutamate synthetase (FPGS) as probes of the FPGS active site and as potential therapeutic agents. The tetrahydro compounds were also of intereat as non-polyglutamatable inhibitors of the purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFT). Reductive coupling of W-acetamido-6-formylpyrido[2,3-d]pyrimidin-4(3H)-one with 4-aminobenzoic acid, followed by NIOformyhtion, mixed anhydride condensation of the reaultant N2-acetyl-No-formyl-5deazapteroic acid with bhomocysteic acid, and removal of the W-acetyl and No-formyl groups with NaOH, afforded N-(5-deazapteroyl)-~-homocysteic acid (5-dPteHCysA). Mixed anhydride condensation of W-acetyl-No-formyl-5-deazapteroic acid with methyl ~~2-amino-4(dietho~hosp~yl)butanoic acid, followed by consecutive treatment with MeaiBr and NaOH, yielded D,L-2- [ (5-deazapteroyl)amino]-4-phoephonobutanoic acid (BdPteAPBA). Treatment with NaOH alone led to retention of one ethyl eater group on the phosphonate moiety. Catalytic hydrogenation of h"-acetyl-No-formyl-5-deazapteroic acid followed by mixed anhydride condensation with methyl L-homocystsate and deproktion with NaOH afforded N-(5,6,7,8tetrahy~~5-deazapteroyl)-~-hom~teic acid (5-dH4PteHCysA). Similar chemistry starting from methyl ~,~-2-amino-4-(diethoxyphosphinyl)butanoic acid and methyl A@-(benzyloxycarbonyl)-L-ornithinate yielded D,L2-[(5-deaza-5,6,7,8-tetrahydropteroyl)amino]-4-phosphonobu~oic acid (5-dH4Pte-APBA) and N"-(bdeaza5,6,7,&tetrahydropteroyl)-~-ornithine (B-dH,PteOrn), respectively. The 5-deazafolate analogues were inhibitors of mouse liver FPGS, and the DATHF analogues inhibited both mouse FPGS and mouse leukemic cell GARFT. Analogues with HCysA and monoethyl APBA side chains were less active as FPGS inhibitors than those containing an unesteritied -pPO(OH), group, and their interaction with the enzyme was noncompetitive against variable folyl substrate. In contrast, Orn and APBA analogues obeyed competitive inhibition kinetics and were more potent, with Ki values as low as 30 nM. Comparison of the DATHF analogues as GARFT inhibitors indicated that the Om side chain diminished activity relative to DAW, but that the compounds with ysulfonate or y-phosphonate substitution retained activity, with Ki values in the submicromolar range. The best GARFT inhibitor was the 5-dH4PteAPBA diaetereomer mixture, with a Ki of 47 nM versua 65 nM for DATHF. None of the compounds showed activity against cultured WI-L2 or CEM human leukemic lymphoblasts at concentrations of up to 100 pM. We conclude that linking the Meazapteroyl moiety to these amino acid side chains previously found inhibitory to FPGS enhances binding to FPGS, and that negatively charged groups at the yposition of the DATHF analogues allow maintenance of GARFT inhibition. However, inefficient cellular uptake is an obstacle to the use of these potential dual inhibitors of FPGS and GARFT as therapeutic agents.

Substrate and inhibitor binding to dihydrofolate reductase (DHFR) primarily involves residues in the amino-terminal half of the enzyme; however, antibody binding studies performed in this laboratory suggested that the loop region located in the carboxyl terminus of human DHFR (hDHFR; residues 140-186) is involved in conformational changes that occur upon ligand binding and affect enzyme function (Ratnam, M., Tan, X., Prendergast, N.J., Smith, P.L. & Freisheim, J.H. (1988) Biochemistry 27, 4800-4804). To investigate this observation further, site-directed mutagenesis was used to construct deletion mutants of hDHFR missing 1 (del-1), 2 (del-2), 4 (del-4), and 6 (del-6) residues from loops in the carboxyl terminus of the enzyme. The del-1 mutant enzyme has a two-amino acid substitution in addition to the one-amino acid deletion. Deletion of only one amino acid resulted in a 35 decrease in the specific activity of the enzyme. The del-6 mutant enzyme was inactive. Surprisingly, the del-4 mutant enzyme retained a specific activity almost 33 that of the wild type. The specific activity of the del-2 mutant enzyme was slightly higher (38 wild-type activity) than that of the del-4 mutant. All three active deletion mutants were much less stable than the wild-type enzyme, and all three showed at least a 10-fold increase in Km values for both substrates. The del-1 and del-2 mutants exhibited a similar increase in KD values for both substrate and cofactor. The three active deletion mutants lost activity at concentrations of activating agents such as KCl, urea, and p-hydroxymercuribenzoate that continued to stimulate the wild-type enzyme. Antibody binding studies revealed conformational differences between the wild-type and mutant enzymes both in the absence and presence of bound folate. Thus, although the loops near the carboxyl terminus are far removed from the active site, small deletions of this region significantly affect DHFR function, indicating that the loop structure in mammalian DHFR plays an important functional role in its conformation and catalysis.

Dihydrofolate reductase is an intracellular target enzyme for folate antagonists, including the anticancer drug methotrexate. In order to design novel drugs with altered binding properties, a detailed description of protein-drug interactions in solution is desirable to understand the specificity of drug binding. As a first step in this process, heteronuclear three-dimensional NMR spectroscopy has been used to make sequential resonance assignments for more than 90% of the residues in human dihydrofolate reductase complexed with methotrexate. Uniform enrichment of the 21.5-kDa protein with 15N was required to obtain the resonance assignments via heteronuclear 3D NMR spectroscopy since homonuclear 2D spectra did not provide sufficient 1H resonance dispersion. Medium- and long-range NOE's have been used to characterize the secondary structure of the binary ligand-enzyme complex in solution.

A series of thirty eight 2,4-diaminoquinazolines having diverse substitution patterns on the aromatic ring was evaluated for inhibitory activity against dihydrofolate reductase (DHFR) obtained from a human lymphoblast cell line. Many of these compounds were also evaluated as inhibitors of rat liver DHFR under the same experimental conditions. In most instances the results obtained with each enzyme were comparable indicating that the rodent enzyme is a suitable model for the human DHFR as far as the determination of I50 values is concerned. The results demonstrate that relatively simple 5-substituted- or 5,6-disubstituted-2,4-diaminoquinazolines can be potent DHFR inhibitors. The presence of a nonpolar substituent at position 7 or 8 was highly detrimental to inhibitory potency.

Arginine-70 of human dihydrofolate reductase (hDHFR) is a highly conserved residue which X-ray crystallographic data have shown to interact with the alpha-carboxylate of the terminal L-glutamate moiety of either folic acid or methotrexate (MTX). The rationale for this study was to introduce a conservative amino acid residue change at position 70 (Arg----Lys) which might function as a titratable group and, thus, reveal possible quantitative changes in ligand binding and kinetic parameters as a function of pH. Such a mutant enzyme (R70K) has been constructed and expressed by using site-directed mutagenesis techniques. This substitution has a dramatic effect on the binding of MTX, which displays a 22,600-fold increase in the dissociation constant (KD) at pH 7.5 compared to that of the reported wild-type enzyme value. At this pH, the KD value for dihydrofolate (FAH2) for the R70K enzyme shows only a 7-fold increase over that for the wild-type hDHFR. The pH profiles of the Michaelis and dissociation constants for FAH2 and KD values for MTX for the mutant enzyme all show a 7-8-fold increase from pH 7.5 to 8.5 as compared to its wild-type counterpart. The binding of NADPH or the nonclassical inhibitor trimetrexate (TMQ) to either the wild-type or the mutant enzyme does not show such pH-dependent characteristics. Thus, since FAH2 and MTX interact with the guanidinium side chain of arginine-70 in the wild-type hDHFR, the replacement of this residue with a lysine in the R70K mutant appears to have resulted in the introduction of a titratable group with a perturbed pKa value of ca. 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)

A fluorescein derivative of the lysine analogue of folic acid, N alpha-pteroyl-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (PLF), was synthesized as a probe for dihydrofolate reductase (DHFR) and a membrane folate binding protein (m-FBP). Excitation of PLF at 282 nm and at 497 nm gave a fluorescence emission maximum at 518 nm. Binding of PLF to human DHFR or human placental m-FBP results in approximately a 20-fold enhancement in the magnitude of the fluorescence emission, suggesting that the ligand interacts with a hydrophobic region on these proteins. Additional evidence suggests that an energy transfer may occur between the pteridine and the fluorescein moieties. PLF binds to the active site of human DHFR since methotrexate (MTX) competes stoichiometrically and the denatured enzyme in the presence of PLF did not exhibit fluorescent enhancement. The dissociation constant for the fluorescein derivative with respect to human DHFR is 115 nM as compared to 111 nM for folic acid. The Ki value for the competitive inhibition of human DHFR by the fluorescent analogue of folic acid is 2.0 microM compared to 0.48 microM for folic acid. PLF was reduced to N alpha-(7,8-dihydropteroyl)-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (H2PLF) and assayed by the enzymatic conversion to the tetrahydro derivative. The Km value for human DHFR for the dihydrofolate analogue is 2.0 microM. The KD value for H2PLF to human DHFR is 47 nM as compared to 44 nM for dihydrofolate. The KD values for both H2PLF and PLF indicate that the fluorescein moiety does not significantly affect folate binding in enzyme binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

The new folate analogue, 2-desamino-2-methyl-5,8-dideazaisofolic acid, 2c , was synthesized and evaluated using a variety of biochemical and antitumor assays. For purposes of comparison, its 2-desamino, 2b , and 2-amino, 2a , counterparts, as well as N 10 -propargyl-5,8-dideazafolic acid, 1a , and the corresponding 2-desamino, 1b , and 2-desamino-2-methyl, 1c , modifications were included in these studies. Compound 2c was found to be a potent inhibitor of the growth of L1210 and MCF-7 cells in culture, being only 2-fold and 5-fold less effective than 1c , respectively. However, although analogue 2c was 189-fold less inhibitory toward L1210 thymidylate synthase (TS) than 1c , its cytotoxicity was reversed completely by thymidine alone which suggests that the compound behaves as a TS inhibitor in cells. Enzymatically synthesized polyglutamates of 2c were substantially more inhibitory toward human TS than the parent compound. Compound 2c was the most efficient substrate for mammalian folylpolyglutamate synthetase of the compounds studied having a V max / K m nearly 12-fold larger than 1c . Both 1c and 2c were effective inhibitors of the uptake of [ 3 methotrexate into MOLT-4 cells, implying that each is efficiently transported into tumor cells. These results suggest that a weak inhibitor of TS in vitro can be a potent cytotoxic agent if it can readily gain entry into target cells and be converted to polyglutamated metabolites.

Four spin-labeled inhibitors of dihydrofolate reductase (DHFR) have been synthesized, each of which has the 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) reporting group at a different distance from the 2,4-diaminopyrimidine moiety by which the inhibitors are anchored and oriented in the active site. Inhibitors in which the TEMPO group is attached by a short side chain are weakly bound to DHFR from bacteria (Streptococcus faecium and Lactobacillus casei), to the bovine enzyme and to recombinant human DHFR. However, binding is sufficiently tight, especially in the ternary complexes with NADPH, for recording of the EPR spectra of the bound ligands. The spectra indicate that when these inhibitors are bound to the enzyme the TEMPO group is highly immobilized with correlation time, tau c, 4-20ns. Inhibitors that have the reporter group attached to the glutamate moiety of methotrexate bind to all four DHFRs more tightly than the inhibitors with shorter side chains by factors of up to 10(6). However, in most complexes formed by the inhibitors with longer side chains immobilization of the TEMPO group is slight (tau c 0.2-4 ns). These results are in general agreement with predictions from X-ray crystallographic results including thermal factors but there are some unanticipated differences between some results for bacterial and eukaryotic enzymes. Three of the splin-labeled inhibitors would provide good probes for distance measurements in and around the active site of mammalian DHFR.

Prompted by recent disclosures concerning the potent antitumor activities of 5-deaza-5,6,7,8-tetrahydrofolic acid and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), we have prepared 5-deazaisofolic acid (3a) and 5-deaza-5,6,7,8-tetrahydroisofolic acid (4a). Reductive condensation of 2,6-diamino-3,4-dihydro-4-oxopyridol [2,3-d] pyrimidine with di-tert-butyl N-(4-formylbenzoyl)-L-glutamate and subsequent deprotection with trifluoroacetic acid yielded 5-deazaisofolic acid in good yield. Catalytic hydrogenation of this analogue then gave 4a. The 9-CH 3 and 9-CHO modifications of 3a and the 9-CH 3 derivative of 4a were also synthesized. Each of the new analogues was evaluated with a variety of folate-requiring enzymes as well as MCF-7 cells in culture. Compound 4a had an IC 50 of ca. 1 μM against MCF-7 cells and was nearly 100-fold less potent than DDATHF in this regard. The three oxidized isofolate analogues were all poor inhibitors of tumor cell growth

The previously undescribed 2-desamino and 2-desamino-2-methyl analogues of aminopterin (AMT) and methotrexate (MTX) were synthesized from 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile. The AMT analogues were obtained via a three-step sequence consisting of condensation with di-tert-butyl N-(4-aminobenzoyl)-L-glutamate, heating with formamidine or acetamidine acetate, and mild acidolysis with trifluoroacetic acid. The MTX analogues were prepared similarly, except that 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile was condensed with 4-(N-methylamino)benzoic acid and the resulting product was annulated with formamidine or acetamidine acetate to obtain the 2-desamino and 2-desamino-2-methyl analogues, respectively, of 4-amino-4-deoxy-N10-methylpteroic acid. Condensation with di-tert-butyl L-glutamate in the presence of diethyl phosphorocyanidate followed by ester cleavage with trifluoroacetic acid was then carried out. Retention of the L configuration in the glutamate moiety during this synthesis was demonstrated by rapid and essentially complete hydrolysis with carboxypeptidase G1 under conditions that likewise cleaved the L enantiomer of MTX but left the D enantiomer unaffected. The 2-desamino and 2-desamino-2-methyl analogues of AMT and MTX inhibited the growth of tumor cells, but were very poor inhibitors of dihydrofolate reductase (DHFR). These unexpected results suggested that activity in intact cells was due to metabolism of the 2-desamino compounds to polyglutamates.

Analogues of methotrexate (MTX) and aminopterin (AMT) modified at the gamma-position of the glutamate side chain were synthesized and evaluated as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. Condesations of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) with dimethyl DL-4-methyleneglutamate in the presence of diethyl phosphorocyanidate (DEPC) followed by alkaline hydrolysis yielded N-(4-amino-4-deoxy-N10-methylpteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneMTX). Condensation of 4-amino-4-deoxy-N10-formylpteroic acid (fAPA) with dimethyl-DL-4-methyleneglutamate by the mixed carboxylic-carbonic anhydride method yielded N-4-amino-4-deoxypteroyl)-DL-4-methyleneglutamic acid (gamma-methyleneAMT). Also prepared via DEPC coupling was a mixture of the four possible diastereomers of N-(4-amino-4-deoxy-N10-methylpteroyl)-4-cyanoglutamic acid (gamma-cyanoMTX). The requisite intermediate gamma-tert-butyl alpha-methyl 4-cyanoglutamate, as a DL-threo/DL-erythro mixture, was prepared from methyl N alpha-Boc-O-tosyl-L-serinate by reaction with sodium tert-butyl cyanoacetate followed by mild trifluoroacetic treatment to selectively remove the Boc group. The gamma-methylene derivatives of MTX and AMT are attractive because of their potential to act as Michael acceptors within the DHFR active site. gamma-CyanoMTX may be viewed as a congener of the nonpolyglutamated MTX analogue gamma-fluoroMTX. In vitro bioassay data for the gamma-methylene and gamma-cyano compounds support the idea that the active site of DHFR, already known for its ability to tolerate modification of the gamma-carboxyl group of MTX and AMT, can likewise accommodate substitution on the gamma-carbon itself.

The new folate antagonist, 5-fluoro-5,8-dideazaisoaminopterin was synthesized in four steps beginning with 2,4-diamino-5-fluoroquinazoline. It was found to be a potent inhibitor of human dihydrofolate reductase. Against L1210 leukemia in mice, 5-fluoro-5,8-dideazaisoaminopterin was equiactive with methotrexate at approximately one half of the total dose employed.

Summaryβ,γ-Methano derivatives of methotrexate (MTX) and aminopterin (AMT) were synthesized with the aim of assessing the effect of this side-chain modification on dihydrofolate reductase (OHFR) inhibition, folylpolyglutamate synthetase (FPGS) inhibition, and tumor cell growth inhibition. Mixed carboxylic-carbonic anhydride (MCA) coupling of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and dimethyl trans-α-(2-carboxycyclopropyl) glycinate, followed by alkaline hydrolysis, afforded N-( 4-amino-4-deoxy-N10-methylpteroyl)-α-( trans-2-carboxycyclopropyl)glycine (β,γ-methanoMTX) as a mixture of the four possible diastereomers with trans substitution on the cyclopropane ring. N-(4-Amino-4-deoxypteroyl)-trans-α.-(2-carboxycyclopropyl) glycine (β,γ-methanoAMT) , also as a diastereomer mixture, was obtained from 4-amino-4-deoxy-N10 - formylpteroic acid (fAPA) and dimethyl trans-α-(2-carboxycyclopropyl)-glycinate by MCA coupling and alkaline hydrolysis of the ester and N10-formyl groups. β,γ-MethanoMTX and β,γ-methanoAMT may be viewed as MTX and AMT analogues with a conformationally restricted side chain. In vitro biological activity data for these novel compounds support the view that the active site of DHFR, already known for its ability to tolerate modification of the γ-carboxyl group of MTX and AMT, can likewise accommodate substitution on the β- and γ-carbons.

All of the 181 possible overlapping hexapeptides as well as 179 octapeptides covering the amino acid sequence of human dihydrofolate reductase (hDHFR) were synthesized on polyethylene supports. The synthetic procedure of Geysen et al. (Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) J. Immunol. Methods 102, 259-274) was modified to obtain up to 100 nmol of peptide on each pin. Peptides constituting antigenic epitopes on hDHFR were identified by examining the binding of antibodies raised against both native and denatured hDHFR to these peptides by enzyme-linked immunosorbent assay. The peptides bound in a similar pattern to polyclonal antibodies against both native and denatured dihydrofolate reductase (DHFR). Six major epitopes were located corresponding to residues 27-33, 45-51, 67-74, 133-139, 153-158, and 176-181 using both hexapeptides and octapeptides. An additional epitope, constituting residues 14-21, was found by the use of octapeptides. Most of the epitopes are hydrophilic and reside largely in "loop" regions at the boundaries of secondary structural elements of hDHFR. This observation is consistent with our previous results which suggested that ligand binding at the active site of the enzyme can cause a dramatic reduction in antibody binding to DHFR due to conformational constraints in flexible loop regions in various parts of the molecule. The similarity of the immunogenic profiles of native versus denatured hDHFR indicates that the two forms of the antigen share the same amino acid sequence-specific epitopes. Competitive enzyme-linked immunosorbent assay showed that the binding of anti-hDHFR antiserum to both native and denatured hDHFR was inhibited by approximately 30% by the seven antigenic peptides, indicating that a significant proportion of the antibodies elicited by this enzyme is specific for short peptides. Besides revealing the antigenic structure of DHFR our results provide a rational basis for the design of mutant DHFRs to study the importance of loop residues in the conformational dynamics of the enzyme.

Site-directed mutagenesis has been used to delete 2 residues (Gly45-Lys46) from a flexible "loop" region between residues 40 and 46 of human dihydrolate reductase. Steady-state kinetic studies show that the Km values for the deletion mutant enzyme for both dihydrofolate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) as well as the pH rate profile are virtually identical to that of the wild type. In contrast, the Vmax value of the mutant enzyme is decreased 2.5-fold. The results suggest that the loop region may play a role in the catalytic efficiency but not necessarily in the binding of substrates. Agents such as KCl, urea, and organomercurials at concentrations which show activating effects on the wild-type human dihydrofolate reductase have little or no effect on the deletion mutant. Competitive enzyme-linked immunosorbent assay experiments using peptide-specific antibodies against cyanogen bromide fragments generated from human dihydrofolate reductase show that the binding of folate, NADPH, and methotrexate, either in binary or in ternary complexes with the wild-type enzyme, causes a striking reduction in the binding of the antibodies. Compared with wild type, the binding of these ligands with the deletion mutant enzyme causes much less inhibition (2-16-fold less) in the binding of all three antibodies. The altered properties of the mutant enzyme can be explained on the basis of a need for the flexible loop 40-46 for reversible protein unfolding during activation and also for conformational changes induced by ligand binding, thus "communicating" the effects of ligand binding.

Dihydrofolate reductase (DHFR) is an intracellular target enzyme for folate antagonist drugs, including methotrexatte. In order to compare the binding of methotrexate to human DHFR in solution with that observed in the crystalline state, NMR spectroscopy has been used to determine the conformation of the drug bound to human DHFR in solution. In agreement with what has been observed in the crystalline state, NOE's identified protein and methotrexate protons indicate that methotrexate binds in a non-productive orientation. In contrast to what has been reported for E. coli DHFR in solution, only one bound conformation of methotrexate is observed.

cDNAs encoding the bifunctional dihydrofolate reductase-thymidylate synthase from Glycine max were isolated and sequenced. The 1794 base full length cDNA contains a single open reading frame of 1593 bases. The predicted size of the encoded protein is 530 amino acids with a molecular weight of 59,707. The protein has two domains: a 226 residue DHFR domain in the N-terminus, which is over 30% identical to human DHFR or the DHFR domain of protozoal DHFR-TS, and a 304 residue thymidylate synthase (TS) domain, which is over 60% identical to eukaryotic TS enzymes. The whole protein sequence is greater than 75% identical to DHFR-TS sequences from two other plants, Daucus carota and Arabidopsis thaliana. The sequence of two tryptic peptides obtained from DHFR preparations matched the predicted amino acid sequence, one peptide lying in the DHFR domain and the other in the TS domain. These results indicate that DHFR and TS exist in a bifunctional polypeptide in Glycine max. The coding region of the cDNA was inserted downstream of the T7 promoter and translation initiation signals in the vector pET-3a. This construct (pDR-TS) was transformed into Escherichia coli BL21 (DE) [plysS] which produces T7 RNA polymerase upon induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of the bifunctional enzyme was confirmed by detection of both DHFR and TS activities. The purified enzyme has a subunit molecular mass of 60 kDa. This is the first report of expression of a plant DHFR-TS cDNA.

A series of eight previously undescribed 2,4-diaminothieno[2,3-d]pyrimidine analogues of the potent dihydrofolate reductase (DHFR) inhibitors trimetrexate (TMQ) and piritrexim (PTX) were synthesized as potential drugs against Pneumocystis carinii and Toxoplasma gondii, which are major causes of severe opportunistic infections in AIDS patients. 2,4-Diamino-5-methyl-6-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 3,4,5-trimethoxy or 2,5-dimethoxy substitution in the aryl/aralkyl moiety and 2,4-diamino-5-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 2,5-dimethoxy substitution in the aryl/aralkyl moiety were obtained by reaction of the corresponding 2-amino-3-cyanothiophenes with chloroformamidine hydrochloride. The aryl group in the 5,6-disubstituted analogues was either attached directly to the hetero ring or was separated from it by one or two carbons, whereas the aryl group in the 5-monosubstituted analogues was separated from the hetero ring by two or three carbons. 2-Amino-3-cyano-5-methyl-6-(aryl/alkyl)thiophene intermediates for the preparation of the 5,6-disubstituted analogues were prepared from omega-aryl-2-alkylidene-malononitriles and sulfur in the presence of a secondary amine, and 2-amino-3-cyano-4-(aryl/aralkyl)thiophene intermediates for the preparation of the 5-monosubstituted analogues were obtained from omega-aryl-1-chloro-2-alkylidenemalononitriles and sodium hydrosulfide. Synthetic routes to the heretofore unknown ylidenemalononitriles, and the ketone precursors thereof, were developed. The final products were tested in vitro as inhibitors of DHFR from Pneumocystis carinii, Toxoplasma gondii, rat liver, beef liver, and Lactobacillus casei. A selected number of previously known 2,4-diaminothieno[2,3-d]pyrimidines lacking the 3,4,5-trimethoxyphenyl and 2,5-dimethoxyphenyl substitution pattern of TMQ and PTX, respectively, were also tested for comparison. None of the compounds was as potent as TMQ or PTX, and while some of them showed some selectivity in their binding to Pneumocystis carinii and Toxoplasma gondii versus rat liver DHFR, this effect was not deemed large enough to warrant further preclinical evaluation.

The mechanism of protonation of the dihydropterin ring prior to hydride transfer in the catalytic mechanism of dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADP oxidoreductase, E.C.1.5.1.3) remains controversial. Several proposals have emerged. One follows protonation of 04 followed by tautomerism1 which may be accompanied by required conformational changes.2 Another3 proposes a proton transfer from glu30 in chicken liver DHFR to N5 via 04 and the intervening water molecule. A different scheme4,5 for protonation involves the formation of the enol structure of 04.

The compound 5,8-dideazaisofolic acid (IAHQ), 1a, was found to posses modest antitumor activity against a variety of human and murine tumor cells both in vitro and in vivo. 1–5 Its lack of potency was attributed to slow influx into target cells as demonstrated with [3H] IAHQ in the presence of HCT-8 human colon adenocarcinoma cells in culture.6 The analogues 2-desamino-5,8-dideazaisofolic acid, 1b, and 2-desamino-2-methyl-5,8-dideazaisofolic acid, 1c, were prepared subsequently and found to be 6-and 44-fold more cytotoxic toward L1210 cells than 1a 7,8 This activity enhancement correlated well with increased affinity for the reduced folate transporter as measured by competition for the uptake of [3H] MTX into MOLT-4 cells.8

The folate analogue 5,8-dideazaisofolic acid (IAHQ), 1a, has demonstrated antitumor activity against a variety of mammalian tumor cells both in vitro and in vivo.1–5 Its derivative, 2-desamino-2-methyl-5,8-dideazaisofolic acid, 1b, was found to be 20-and 44-fold more cytotoxic toward MCF-7 and L1210 cells in culture, respectively.6 In order to evaluate the effects of other substituents located at position five of the quinazoline nucleus upon antitumor activity, we have prepared 5-fluoro-5,8-dideazaisofolic acid, 2a, and desamino-2-methyl-5-fluoro-5,8-dideazaisofolic acid, 2c, together with their 9-methyl derivatives, 2b and 2d. This report describes the chemistry and preliminary biological evaluation of these new compounds.

Thymidylate synthase (5,10-methyltetrahydrofolate: dUMP C-methyl-transferase, EC 2.1.1.45) (TS) catalyzes the conversion of deoxyuridylate to thymidylate. Because of its important role in the synthesis of DNA precursors, this enzyme has been extensively studied. The primary structure of thymidylate synthase is highly conserved as revealed by comparison of amino acid sequences of the avian1, mammalian2, and bacterial3 enzymes. Most of these enzymes are homodimers made up of about 35 kDa subunits. However, the enzyme from protozoal parasite, Leishmania major is contained in a bifunctional polypeptide with both thymidylate synthase and dihydrofolate reductase (DHFR) activity. Little is known about TS from plants, although there has been a report of the isolation of a protein complex containing both TS and DHFR activities4. Whether plant TS is a bifunctional enzyme with DHFR or the two enzymes are non-covalently associated can be addressed by cDNA cloning and sequencing. Isolation and expression of the plant TS gene will facilitate characterization of the enzyme at the molecular level, providing biochemical evidence for the study of macromolecule evolution.

The reduced-folate/methotrexate transporter represents the major avenue of methotrexate uptake into mammalian cells1–3. This transport system has a high affinity for methotrexate (MTX) (Km = 1–3 μM) and reduced folates with substitutions at position 5 (3–26μM)4, but a correspondingly low affinity for folic acid (Km = 200–400 μM)1. Considerable progress has been made towards elucidation of MTX transport. The development of folate analogs containing chemically or photpdynamically activatable groups has allowed the identification of specific proteins that could be involved in the carrier-mediated transport of MTX in both murine and human systems5–6. Our laboratory has attempted to study the membrane component of the transporter protein from human leukemic CCRF-CEM cells by cotransfection of mouse Ltk cells with the herpesvirus thymidine kinase gene and genomic DNA from the CCRF-CEM-7A cell line. The resulting mousehuman cell lines may be able to be used to study the protein’s regulation. This paper will discuss the creation and partial characterization of the hybrid cell lines.

Two membrane folate receptor (MFR) isoforms are present in human tissues i.e. MFR-1 (e.g. placenta) and MFR-2 (e.g. placenta, KB cells, CaCo-2 cells). MFR-1 was expressed in COS-1 cells and the resulting protein had the same polypeptide molecular weight as the native protein. The affinities of (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, and 5,10-dideazatetrahydrofolate as well as folic acid and methotrexate to MFR-1, MFR-2 and placental MFR (MFR-1 plus MFR-2) were determined in terms of the Ki values for their competitive inhibition of the binding of [3H]folic acid to these proteins. The results indicated a striking difference in the stereospecificity of MFR-1 and MFR-2 for reduced folate coenzymes; MFR-2 preferentially bound to the physiological (6S) diastereoisomers and MFR-1 bound preferentially to the unphysiological (6R) diastereoisomers, while dideazatetrahydrofolate did not show significant stereospecificity for MFR-1. Furthermore, MFR-2 displayed significantly (2- to 100-fold) greater affinities for all the compounds tested compared to MFR-1. Purified placental MFR, a natural source of MFR-1 which contains variable amounts of MFR-2, showed intermediate Ki values for the compounds tested compared with MFR-1 and MFR-2 and stereospecificities similar to MFR-1. These observations demonstrate striking differences in the ligand binding sites of MFR-1 and MFR-2 which could potentially be exploited in the design of MFR isoform specific antifolates.

There is marked pH dependence of the rate constant (koff) for tetrahydrofolate (H4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31----Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH approximately 5, and this accounts for the marked decrease in kcat for this variant as the pH is decreased from 7 to 5. This decreased kcat at low pH is not seen for most DHFRs. koff for dissociation of folate, dihydrofolate (H2folate), and H4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of koff in the range pH 5-7 is well described by a pKa of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), koff increases further as the pH is raised, and this relation is governed by a second pKa which is close to the pKa for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases koff. Evidence is presented that the dependence of pH on koff for hDHFR accounts for the shape of the kcat versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.

The 5-deaza and 5,8-dideaza analogues of N alpha-pteroyl-L-ornithine (Pter-Orn), the 5-deaza, 8-deaza, and 5,8-dideaza analogues of N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-Orn), and the N delta-carboxymethyl derivative of N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-ornithine (mAPA-Orn) were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of tumor cell growth in culture. Reductive amination of 2-acetamido-6-formylpyrido[2,3-d]pyrimidine-4(3H)-one with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate followed by removal of the blocking groups afforded the 5-deaza analogue of Pter-Orn, whereas N-alkylation of methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate with 2-amino-6-(bromomethyl)quinazolin-4(3H)-one and deprotection gave the corresponding 5,8-dideaza analogue. Reductive coupling of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile and 4-aminobenzoic acid followed by reaction with 95-97% formic acid yielded 4-amino-4-deoxy-5-deaza-N10-formylpteroic acid, which on condensation with methyl N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection gave the 5-deaza analogue of APA-Orn. A similar sequence starting from 2,4-diamino-quinazoline-6-carbonitrile led to the corresponding 5,8-dideaza compound, whereas treatment of 2,4-diamino-pyrido[3,2-d]pyrimidine-6-methanol with phosphorus tribromide followed by condensation with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection afforded the 8-deaza analogue. For the preparation of the N delta-carboxymethyl derivative of mAPA-Orn, N alpha-(benzyloxycarbonyl)-L-ornithine was subjected to N delta-monoalkylation with glyoxylic acid and sodium cyanoborohydride, followed by N delta-acylation with ethyl trifluoroacetate, N alpha-deprotection by hydrogenolysis, condensation with 4-amino-4-deoxy-N10-methylpteroic acid, and N delta-deprotection by gentle treatment with ammonia. The 2,4-diamino derivatives all inhibited the growth of tumor cells in culture, with IC50 values of 0.2-2 microM, and inhibited purified DHFR with IC50 values of 0.02-0.08 microM. Deletion of ring nitrogens and N delta-carboxymethylation both increased potency in the cell growth assay; however, the ornithine derivatives were less potent than aminopterin or methotrexate.

The active sites of all bacterial and vertebrate dihydrofolate reductases that have been examined have a tryptophan residue near the binding sites for NADPH and dihydrofolate. In cases where the three-dimensional structure has been determined by X-ray crystallography, this conserved tryptophan residue makes hydrophobic and van der Waals interactions with the nicotinamide moiety of bound NADPH, and its indole nitrogen interacts with the C4 oxygen of bound folate through a bridge provided by a bound water molecule. We have addressed the question of why even the very conservative replacement of this tryptophan by phenylalanine does not seem to occur naturally. Human dihydrofolate reductase with this replacement of tryptophan by phenylalanine has increased rate constants for dissociation of substrates and products and a considerably decreased rate of hydride transfer. These cause some changes in steady-state kinetic behavior, including substantial increases in Michaelis constants for NADPH and dihydrofolate, but there is also a 3-fold increase in kcat. The branched mechanistic pathway for this enzyme has been completely defined and is sufficiently different from that of wild-type enzyme to cause changes in some transient-state kinetics. The most critical changes resulting from the amino acid substitution appear to be a 50% decrease in stability and a decrease in efficiency from 69% to 21% under intracellular conditions.

The heretofore unknown y-(m-carboxyanilide) and y-(m-boronoanilide) derivatives of methotrexate (MTX) and the y-(m-carboxyanilide) derivatives of aminopterin (AMT) were prepared and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of cell growth in culture with the aim of comparing their activity with that of Nu-(4-amino-4-deoxypteroyl)-Nd-hemiphthaloyl-~-o~ith~e, a potent antifolate whose side chain likewise contains a hydrophobic aromatic ring with an acid group on the ring. All three anilides were potent DHFR inhibitors, with activity comparable to MTX and AMT. The y-(m-boronoanilide) displayed growth inhibitory potency similar to that of the hemiphthaloylornithine analogue, with an ICw of only 0.7 nM. This compound, which is the most potent of the y-amides of MTX tested to date, is also the first reported example of an antifolate with a B(OH), group in the side chain and is especially novel because of its potential to form a stable tetrahedral boronate complex by reaction with electron rich OH or NHz groups in the active site of DHFR or other folate enzymes. In antitumor assays against L1210 leukemia in mice, Nu-(4-amino-4-deoxypteroyl)-N6-hemiphthaloyl-~-ornithine gave a T/C of >263% at 20 mg/kg (qdx9) and 300% at 16 mg/kg (bidxlO), whereas maximally tolerated doses of MTX of 8 mg/kg (qdx9) and 1 mg/kg (bidxlO) gave T/C values of 213 and 188%, respectively. MTX y-(m-boronoanilide) was also active; with a T/C of 175% at 32 mg/kg (qdxg), the highest dose tested. This laboratory has been active for a number of years in the design and synthesis of analogues of the well-known anticancer drugs methotrexate (MTX) and aminopterin (AMT) for the purpose of systematically exploring the subtle relationships that exist between structure and biological activity in classical antifolates.2 In two recent papers we reported the synthesis3s4 and potent antifolate3 activity of Nu-(4-amino-4-deoxypteroy1)-Nb-hemiphthaloyl-L-ornithine (1 ), an analogue of aminopterin (AMT) with three instead of two CH2 groups in the side chain and the yCOOH group replaced by a more liphophilic hemiphthaloyl group. Compound 1 had the unusual property of being not only more toxic than MTX to MTX sensitive cells but also more toxic to MTX resistant cells than MTX was to the parental cells from which the resistant sublines were deri~ed.~ With the aim of identifying the structural features that may contribute to this unusual level of activity, we wished to examine several possible factors: (a) whether the number of CH, groups in the side chain could be decreased from three to two, (b) whether the amide could be "reversed" from NHCO to CONH, (c) whether the COOH group on the phenyl ring could be moved from the ortho to the meta position, and (d) whether this group could be replaced by another acidic group. The present paper describes the synthesis of compounds 2-4 (Chart I) the first three analogues of 1 studied in this connection. Compounds 1-4 all inhibited DHFR from human leukemic cells with a potency similar to AMT

Recent studies on the activity of antifolates have been directed at the perturbations in folate pools caused by inhibitors of dihydrofolate reductase. For the most part these studies have been restricted to the effects of methotrexate, but the effects of trimetrexate have also been examined. A constant feature of these results is the elevation of dihydrofolate which accompanies inhibition by both these agents (1–7). A variable result is the extent to which the reduced folate coenzymes are depleted following exposure to the antifolates. In several cell lines the reduction in tetrahydrofolate is quite modest (less than 40%) with the 10-formyltetrahydrofolate being maintained (1–5). In other cases such as L1210 cells in vivo there is a marked depletion of the reduced folate coenzyme pools (6). Preliminary studies with a rodent hepatoma cell line indicated that the reduced folate coenzymes are depleted by 80% or greater and this is compensated for by an increase in H2PteGlun and 10-HCO-H2PteGlun (7). The current study extends these investigations to the effect of methotrexate on the folate coenzymes in human HEPG2 hepatoma cells in vitro.

The 2.3-A crystal structure of recombinant human dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a binary complex with folate (a poor substrate at neutral pH) and also as a binary complex with an inhibitor, 5-deazafolate. The inhibitor appears to be protonated at N8 on binding, whereas folate is not. Rotation of the peptide plane joining I7 and V8 from its position in the folate complex permits hydrogen bonding of 5-deazafolate's protonated N8 to the backbone carbonyl of I7, thus contributing to the enzyme's greater affinity for 5-deazafolate than for folate. In this respect it is likely that bound 5-deazafolate furnishes a model for 7,8-dihydrofolate binding and, in addition, resembles the transition state for folate reduction. A hypothetical transition-state model for folate reduction, generated by superposition of the DHFR binary complexes human.5-deazafolate and chicken liver.NADPH, reveals a 1-A overlap of the binding sites for folate's pteridine ring and the dihydronicotinamide ring of NADPH. It is proposed that this binding-site overlap accelerates the reduction of both folate and 7,8-dihydrofolate by simultaneously binding substrate and cofactor with a sub van der Waals separation that is optimal for hydride transfer.

Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2{prime}-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K{sub m} values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K{sub m} and k{sub cat} values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K{sub m}(NADH)/K{sub m}(NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2{prime}-phosphate, makes a major contribution in terms of binding energy and differentiation of K{sub m} values for NADPH and NADH.more » Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme.« less

The role of the active site residue phenylalanine-31 (Phe31) for recombinant human dihydrofolate reductase (rHDHFR) has been probed by comparing the kinetic behavior of wild-type enzyme (wt) with mutant in which Phe31 is replaced by leucine (F31L rHDHFR). At pH 7.65 the steady-state kcat is almost doubled, but the rate constant for hydride transfer is decreased to less than half that for wt enzyme, as is the rate of the obligatory isomerization of the substrate complex that precedes hydride transfer. Although steady-state measurements indicated that the mutation causes large increases in Km for both substrates, dissociation constants for many complexes are decreased. These apparent paradoxes are due to major mutation-induced decreases in rate constants (koff) for dissociation of folate, dihydrofolate, and tetrahydrofolate from all of their complexes. This results in a mechanism proceeding almost entirely by only one of the two pathways used by wt enzyme. Other consequences of these changes are a much altered dependence of steady-state kcat on pH, inhibition rather than activation by tetrahydrofolate, absence of hysteresis in transient-state kinetics, and a decrease in enzyme efficiency under physiological conditions. The results indicate that there is no quantitative correlation between dihydrofolate binding and the rate of hydride transfer for this enzyme.

The inhibitory activity of 101 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazines against purified dihydrofolate reductase from human lymphoblastoid cell (WIL 2) has been studied. From the obtained Kiapp values, quantitative structure-activity relationships (QSAR) have been derived. The QSAR from human dihydrofolate reductase are compared with QSAR for triazines inhibiting bovine and murine tumor DHFR, as well with QSAR for their inhibitory action on murine tumor cell culture.

Dihydrofolate reductase has been isolated from methotrexate-resistant WIL2 human lymphoblastoid cells. This subline produces ca. 150 times more enzyme than the parental drug-sensitive line. The reductase has been purified to homogeneity by methotrexate affinity chromatography, gel filtration, and preparative isoelectric focusing in a yield of 65%. The enzyme has a pI = 7.7 and a molecular weight of ca. 22000. The amino-terminal 27 amino acid residues have been determined, revealing the location of the single cysteine residue at position 6. Reaction of this cysteine with p-(hydroxy-mercuri)benzoate in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) results in a 5-fold increase in enzyme activity. Other agents including KCl, urea, and thiourea also cause enzyme activation. The Km values for NADPH and dihydrofolate are 0.25 and 0.036 microM, respectively. Mercurial activation of the enzyme results in a 27-fold increase in the Km for NADPH and a 35-fold increase in the Km for dihydrofolate.

The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei is virtually completely and irreversibly inactivated by relatively low concentrations of dansyl chloride. The complete inactivation can be correlated with the dansylation of a single lysine residue and ca. 90% quenching of protein fluorescence. This quenching phenomenon appears to be due, at least in part, to energy transfer from one or more excited state tryptophan residues to the covalently attached dansyl moiety. Under identical conditions lysine is not modified when the ternary complex of enzyme-NADPH-amethopterin is dansylated. The unreactive dansyl hydroxide protects the enzyme against dansyl chloride dependent inactivation and fluorescence studies indicate a single ligand binding site (KD = 1 x 10(-4) M). It is suggested that the dimethylaminonaphthyl moiety of dansyl chloride is directed to a hydrophobic region at or near the active center of the enzyme where a particularly susceptible lysine residue reacts to form a covalent bond with the reagent.

The complete covalent structure of dihydrofolate reductase from chicken liver is described. The S-carboxymethylated protein was subjected to cleavage by cyanogen bromide which produced five fragments. Fragment CB2 contained an internal homoserine residue which was not cleaved by cyanogen bromide. Sequences and ordering of the cyanogen bromide fragments were established by means of automated sequencer analyses of the fragments and from smaller peptides generated by proteolysis with trypsin and staphylococcal protease. The covalent structure of the single polypeptide chain comprises 189 residues of molecular weight 21,651. The chicken liver enzyme is homologous to that from L1210 cells and shows regions of homology to dihydrofolate reductases from Streptococcus faecium, Escherichia coli, and Lactobacillus casei. These homologous regions in the chicken liver enzyme are primarily related to conserved amino acid residues implicated in the binding of NADPH and methotrexate by bacterial dihydrofolate reductases.

Analogues of methotrexate (MTX) and aminopterin (AMT) with aminophosphonoalkanoic, aminoalkanesulfonic, and aminoalkanephosphonic acid side chains in place of glutamate were synthesized and tested as inhibitors of folylpolyglutamate synthetase (FPGS) from mouse liver. The aminophosphonoalkanoic acid analogues were also tested as inhibitors of dihydrofolate reductase (DHFR) from L1210 murine leukemia cells and as inhibitors of the growth of MTX-sensitive (L1210) and MTX-resistant (L1210/R81) cells in culture. The optimal number of CH2 groups in aminophosphonoalkanoic acid analogues of AMT was found to be two for both enzyme inhibition and cell growth inhibition but was especially critical for activity against FPGS. Deletion of the alpha-carboxyl also led to diminished anti-FPGS activity in comparison with previously studied homocysteic acid and 2-amino-4-phosphonobutyric acid analogues. In the aminoalkanesulfonic acid analogues of MTX without an alpha-carboxyl, anti-FPGS activity was low and showed minimal variation as the number of CH2 groups between the carboxamide and sulfonate moieties was changed from one to four. In similar aminoalkanephosphonic acid analogues of MTX, anti-FPGS activity was also low, was comparable for two and three CH2 groups between the carboxamide and phosphonate moieties, and was diminished by monoesterification of the phosphonate group. These effects demonstrate that the alpha-carboxyl group of folate analogues is involved in binding to the active site of FPGS, and that an alpha-carboxyl group should be retained as part of the structure of FPGS inhibitors.

Chicken liver dihydrofolate reductase is rapidly and stoichiometrically inactivated by a substituted 4,6-diaminodihydrotriazine containing a terminal benzenesulfonylfluoride (DTBSF). The substrate dihydrofolate largely prevents the enzyme inhibition by DTBSF, whereas NADPH had no effect, indicating that the inhibitor is bound at or near the folate site. Using radiolabeled inhibitor between 1.0 and 1.2 mol was incorporated/mol of enzyme (Mr = 21,651), following treatment with 8 M urea at 75 degrees C. Digestion of the maleylated, radiolabeled inhibitor-enzyme complex with trypsin and subsequent gel filtration on Sephadex G-50 SF yielded a single major peak of radioactivity. The covalently modified limited tryptic peptide was subsequently purified to homogeneity using high performance liquid chromatography. The radiolabeled tryptic peptide had the following sequence: Asn-Glu-Tyr (DTBS)-Lys-Tyr-Phe-Gln-Arg (residues 29-36). Automated Edman degradation of this peptide revealed that the radioactivity derived from the inhibitor was released at Step 3, identifying tyrosine-31 as the specific site of covalent attachment of the affinity label.