Interaction of polyglutamyl derivatives of methotrexate, 10-deazaaminopterin, and dihydrofolate with dihydrofolate reductase.

Polyglutamyl derivatives of methotrexate (MTX) and 10-deazaaminopterin (10-DAM) containing a total of one through six glutamate residues (Glu residues) were tested as inhibitors of dihydrofolate reductase (DHFR) derived from sheep, chicken, and beef liver. The ability of dihydropteroylpentaglutamate to antagonize the inhibitory activity of these analogues was also studied. The most striking effects were seen with sheep liver DHFR, where polyglutamylation of MTX causes stepwise decreases in the concentration required for 50% inhibition (IC50) with each additional Glu residue until MTX with a total of six Glu residues has an IC50 value 1/3 that of MTX. With 10-DAM the pattern is more complex. The IC50 values increase with addition of Glu residues until a maximum is reached with 10-DAM having a total of three Glu residues which has a value twice that of 10-DAM. 10-DAM with a total of four Glu residues and 10-DAM with a total of five Glu residues have progressively lower IC50 values, the latter being equipotent with 10-DAM. With dihydropteroylpentaglutamate as substrate instead of dihydrofolate, the IC50 values are increased 2- to 5-fold for both MTX and 10-DAM derivatives. The results obtained with chicken liver and beef liver DHFR are generally similar to those described for the sheep liver enzyme, but the effects of polyglutamylation are less pronounced. The addition of 0.2 M KCl to the assay system reduces the differences in inhibitory potency of the polyglutamyl derivatives with all three enzymes tested. We conclude that polyglutamylation can alter the interaction of folate analogues and dihydrofolate with DHFR.

A comparison of the inhibitory action of 5-(substituted-benzyl)-2,4-diaminopyrimidines on dihydrofolate reductase from chicken liver with that from bovine liver.

Forty-four 5-(substituted-benzyl)-2,4-diaminopyrimidines have been tested as inhibitors of chicken and bovine liver dihydrofolate reductase. The chicken enzyme is, on the average, about 10 times less easily inhibited than bovine enzyme. Substituents which show the greatest selectivity are 4-NHCOCH3, 3-OC4H9, 3-I, 3-CF3-4-OCH3, and 3,4,5-(OCH3)3. The inhibition constants have been used to formulate quantitative structure-activity relationships for comparative purposes.

Inhibition of chicken liver dihydrofolate reductase by 5-(substituted benzyl)-2,4-diaminopyrimidines. A quantitative structure-activity relationship and graphics analysis.

The inhibition of chicken liver dihydrofolate reductase by a series of substituted benzylpyrimidines has been investigated. From the inhibition constants a quantitative structure-activity relationship has been formulated. This mathematical model is compared with molecular graphics models constructed from the X-ray crystallographic coordinates of trimethoprim and 5-(3,4-dimethoxy-4-isopropenylbenzyl)-2,4- diaminopyrimidine bound to the enzyme. There is good correspondence between the two types of models.

On the structure selectivity problem in drug design. A comparative study of benzylpyrimidine inhibition of vertebrate and bacterial dihydrofolate reductase via molecular graphics and quantitative structure-activity relationships.

Quantitative structure-activity relationships (QSAR) have been derived for the action of 68 5-(substituted benzyl)-2,4-diaminopyrimidines on dihydrofolate reductase (DHFR) from Lactobacillus casei and chicken liver. The QSAR are analyzed with respect to the stereographics models of the active sites of the enzymes and found to be in good agreement. Using these QSAR equations, we have attempted to design new trimethoprim-type antifolates having higher selectivity for the bacterial enzyme. The general problem of developing selective inhibitors is discussed.

Crystallography, quantitative structure-activity relationships, and molecular graphics in a comparative analysis of the inhibition of dihydrofolate reductase from chicken liver and Lactobacillus casei by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazine s.

The inhibition of dihydrofolate reductase from chicken liver and from Lactobacillus casei has been studied with 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazines. It was found that for the chicken enzyme, inhibitor potency for 101 triazines was correlated by the following equation: log 1/Kiapp = 0.85 sigma tau' - 1.04 log (beta X 10 sigma tau' + 1) + 0.57 sigma + 6.36. The parameter tau' indicates that for certain substituents, tau = 0. In the case of the L. casei DHFR results, meta and para derivatives could not be included in the same equation. For 38 meta-substituted compounds, it was found that log 1/Kiapp = 0.38 tau'3-0.91 log (beta X 10 tau'3 + 1) + 0.71I + 4.60 and for 32 para-substituted phenyltriazines log 1/Kiapp = 0.44 tau'4-0.65 log (beta tau'4 + 1') - 0.90 upsilon + 0.69I + 4.67. In the L. casei equation, I is an indicator variable for substituents of the type CH2ZC6H4-Y and ZCH2C6H4-Y, where Z = O, NH, S, or Se. The parameter upsilon is Charton's steric parameter, which is similar to Taft's Es. The mathematical models obtained from correlation analysis are compared with stereo color graphics models.

Dihydrofolate reductases from chicken liver and Lactobacillus casei bind Cibacron blue F3GA in different modes and at different sites.

The binding of Cibacron blue F3GA to dihydrofolate reductase (EC 1.5.1.3) from chicken liver an amethopterin-resistant Lactobacillus casei has been studied by difference spectroscopy. The blue dye binds to the enzyme from each species in a specific fashion with a 1:1 stoichiometry. However, the mode of interaction of the dye with the enzyme and the site of interaction on the enzyme are very different for the avian and bacterial enzymes. The dye seems to bind in an almost totally "electrostatic mode" at the dihydrofolate binding site to the chicken liver enzyme and is displaced from the enzyme only by dihydrofolate, folate, or methotrexate and not at all by NADPH. In contrast, the binding of the dye to the bacterial enzyme is characterized by a totally "apolar interaction" at a site partially overlapping both the NADPH site and the methotrexate/dihydrofolate site. NADPH can displace the dye only partially and methotrexate is more efficient than NADPH in displacing the dye. Both NADPH and methotrexate are needed for a total displacement of the dye from the bacterial enzyme. We propose that the blue dye is capable of binding specifically to any protein possessing a cluster of aromatic and other apolar groups and/or geometrically spaced positively charged groups for proper interaction with the aromatic rings and/or sulfonate groups of the dye molecule. The so-called specificity of the blue dye for the nucleotide binding proteins is thus a special case of the above mentioned requirements and not diagnostic of the "dinucleotide fold."

Interaction of methotrexate, folates, and pyridine nucleotides with dihydrofolate reductase: calorimetric and spectroscopic binding studies.

The thermodynamic parameters, deltaG, deltaH, and deltaS characterizing the tight binding of methotrexate, folates, and pyridine nucleotides to chicken liver dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) have been determined from calorimetric and fluorescence measurements. At 25 degrees the binding of NADPH and NADP+ is characterized by small negative enthalpies and large positive entropies whereas the binding of the folates and methotrexate is accompanied by large negative enthalpies and small negative entropies. In addition, the enthalpy of methotrexate-enzyme interaction demonstrates a proton transfer associated with binding; this is not the case with folate and dihydrofolate, thus confirming the conclusions drawn from the observed difference spectra characteristic of the interaction of methotrexate and substrates with the enzyme. The implications of these results are discussed in terms of the nature of the binding process, conformational changes in the enzyme, and the nature of the active site region.

Crystal structures of chicken liver dihydrofolate reductase: binary thioNADP+ and ternary thioNADP+.biopterin complexes.

The role of the 3'-carboxamide substituent of NADPH in the reduction of pteridine substrates as catalyzed by dihydrofolate reductase (EC 1.5.1.3, DHFR) has been investigated by determining crystal structures at 2.3 A of chicken liver DHFR in a binary complex with oxidized thionicotinamide adenine dinucleotide (thioNADP+) and in a ternary complex with thioNADP+ and biopterin. These structures are isomorphous with those previously reported for chicken liver DHFR [Volz, K.W., Matthews, D.A., Alden, R.A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. ThioNADPH, which has a 3'-carbothioamide substituent in place of a 3'-carboxamide, functions very poorly as a coenzyme for DHFR [Williams, T. J., Lee, T. K., & Dunlap, R. B. (1977) Arch, Biochem. Biophys. 181, 569-579; Stone, S. R., Mark, A., & Morrison, J. F. (1984) Biochemistry 23, 4340-4346]. Comparisons show that, while NADP+ and NADPH bind to DHFR with the pyridine ring and 3'-carboxamide coplanar, the thioamide group is twisted by 23 degrees from the pyridine plane in both the binary and ternary complexes. This twist appears to be due to steric conflict between the thioamide sulfur atom and both the pyridine ring at C4 and the adjacent protein backbone at Ala-9. It results in an unfavorably close contact between the sulfur and the biopterin pteridine ring (0.9 A less than the van der Waals separation) which, on the basis of the refined structure, greatly destabilizes the binding of biopterin.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of bovine and chicken liver dihydrofolate reductases and its relationship to a specific cysteine residue in their NH2-terminal amino acid sequences.

The enzymatic activity of bovine liver dihydrofolate reductase is activated approximately 1.5- to 2.5-fold on treatment with organic mercurials. In contrast to the almost instantaneous reaction and high degree of activation (approximately 10-fold) observed with chicken liver dihydrofolate reductase, the beef liver enzyme requires relatively specific conditions of pH, temperature, preincubation times, and presence of substrate to exhibit this degree of activation. It is also demonstrated that both chicken liver and beef liver dihydrofolate reductases (and perhaps all of the animal dihydrofolate reductases) contain a single sulfhydryl group within the 11 or so amino acids of the NH2-terminal sequence and that this is the site of mercurial interaction and activation. On the other hand, this sulfhydryl group and the characteristic activation does not occur in the corresponding bacterial reductases. It is suggested that although the reactive sulfhydryl group may be characteristic of animal dihydrofolate reductases, it is not directly required for substrate binding or the catalytic mechanism per se.

Primary structure of chicken liver dihydrofolate reductase.

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.

Crystal structure of chicken liver dihydrofolate reductase complexed with NADP+ and biopterin.

The 2.2-A crystal structure of chicken liver dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a ternary complex with NADP+ and biopterin (a poor substrate). The space group and unit cell are isomorphous with the previously reported structure of chicken liver DHFR complexed with NADPH and phenyltriazine [Volz, K. W., Matthews, D. A., Alden, R. A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. The structure contains an ordered water molecule hydrogen-bonded to both hydroxyls of the biopterin dihydroxypropyl group as well as to O4 and N5 of the biopterin pteridine ring. This water molecule, not observed in previously determined DHFR structures, is positioned to complete a proposed route for proton transfer from the side-chain carboxylate of E30 to N5 of the pteridine ring. Protonation of N5 is believed to occur during the reduction of dihydropteridine substrates. The positions of the NADP+ nicotinamide and biopterin pteridine rings are quite similar to the nicotinamide and pteridine ring positions in the Escherichia coli DHFR.NADP+.folate complex [Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263-3277], suggesting that the reduction of biopterin and the reduction of folate occur via similar mechanisms, that the binding geometry of the nicotinamide and pteridine rings is conserved between DHFR species, and that the p-aminobenzoylglutamate moiety of folate is not required for correct positioning of the pteridine ring in ground-state ternary complexes. Instead, binding of the p-aminobenzoylglutamate moiety of folate may induce the side chain of residue 31 (tyrosine or phenylalanine) in vertebrate DHFRs to adopt a conformation in which the opening to the pteridine binding site is too narrow to allow the substrate to diffuse away rapidly. A reverse conformational change of residue 31 is proposed to be required for tetrahydrofolate release.

Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim.

Refined crystal structures are reported for complexes of Escherichia coli and chicken dihydrofolate reductase containing the antibiotic trimethoprim (TMP). Structural comparison of these two complexes reveals major geometrical differences in TMP binding that may be important in understanding the stereo-chemical basis of this inhibitor's selectivity for bacterial dihydrofolate reductases. For TMP bound to chicken dihydrofolate reductase we observe an altered binding geometry in which the 2,4-diaminopyrimidine occupies a position in closer proximity (by approximately 1 A) to helix alpha B compared to the pyrimidine position for TMP or methotrexate bound to E. coli dihydrofolate reductase. One important consequence of this deeper insertion of the pyrimidine into the active site of chicken dihydrofolate reductase is the loss of a potential hydrogen bond that would otherwise form between the carbonyl oxygen of Val-115 and the inhibitor's 4-amino group. In addition, for TMP bound to E. coli dihydrofolate reductase, the inhibitor's benzyl side chain is positioned low in the active-site pocket pointing down toward the nicotinamide-binding site, whereas, in chicken dihydrofolate reductase, the benzyl group is accommodated in a side channel running upward and away from the cofactor. As a result, the torsion angles about the C5-C7 and C7-C1' bonds for TMP bound to the bacterial reductase (177 degrees, 76 degrees) differ significantly from the corresponding angles for TMP bound to chicken dihydrofolate reductase (-85 degrees, 102 degrees). Finally, when TMP binds to the chicken holoenzyme, the Tyr-31 side chain undergoes a large conformational change (average movement is 5.4 A for all atoms beyond C beta), rotating down into a new position where it hydrogen bonds via an intervening water molecule to the backbone carbonyl oxygen of Trp-24.