Comparison of solution structures of dihydrofolate reductases and enzyme-ligand complexes using cross-reacting antibodies

Polyclonal antibodies against dihydrofolate reductase (DHFR) from the human lymphoblastoid cell line WIL-2/M4 were used as probes to compare the antigenic structures in solution of native DHFRs obtained from a broad range of species and their complexes with substrate, cofactor, and folate antagonist inhibitors. All these antibodies could bind to the denatured human DHFR, indicating that they were specific for the primary structure of this enzyme. Denatured chicken liver and L1210 murine leukemic DHFRs competed for all of the antibodies that bound to the human enzyme, although less effectively than the denatured human enzyme, showing the presence of similar epitopes among the vertebrate enzymes. However, both direct binding and competition experiments showed low antibody cross-reactivities with native chicken liver (8%) and murine (10%) DHFRs, suggesting differences in the disposition of similar epitopes in these enzymes. The lactobacillus casei DHFR showed a low amount (less than 2%) of cross-reactivity with the antibodies while the same antibodies did not cross-react with the Escherichia coli enzyme. DHFR from soybean seedlings competed for a large proportion (70%) of the anti-human DHFR antibodies, indicating a close similarity in the antigenic structures of plant and animal DHFRs. Binary complexes of the L. casei, avian, murine, and human DHFRs with dihydrofolate, methotrexate (MTX), trimethoprim (TMP), NADPH, and NADP+ all showed significantly lower antibody binding capacity as compared with the corresponding free enzymes. Further, these ligands inhibited antibody binding to the enzyme to varying degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid

Isoniazid is a key drug used in the treatment of tuberculosis. Isoniazid is a pro-drug, which, after activation by the katG-encoded catalase peroxidase, reacts nonenzymatically with NAD(+) and NADP(+) to generate several isonicotinoyl adducts of these pyridine nucleotides. One of these, the acyclic 4S isomer of isoniazid-NAD, targets the inhA-encoded enoyl-ACP reductase, an enzyme essential for mycolic acid biosynthesis in Mycobacterium tuberculosis. Here we show that the acyclic 4R isomer of isoniazid-NADP inhibits the M. tuberculosis dihydrofolate reductase (DHFR), an enzyme essential for nucleic acid synthesis. This biologically relevant form of the isoniazid adduct is a subnanomolar bisubstrate inhibitor of M. tuberculosis DHFR. Expression of M. tuberculosis DHFR in Mycobacterium smegmatis mc(2)155 protects cells against growth inhibition by isoniazid by sequestering the drug. Thus, M. tuberculosis DHFR is the first new target for isoniazid identified in the last decade.     

Circular-dichroism studies of ligand binding to dihydrofolate reductase from Lactobacillus casei MTX/R.

Circular-dichroism spectra (200--450 nm) were recorded for Lactobacillus casei MTX/R dihydrofolate reductase and its complexes with substrates, inhibitors and coenzymes. These spectra are compared with those reported by others for dihydrofolate reductase from other sources. The binding of NADP+ or NADPH is associated with the perturbation of one or more aromatic amino acid residues, and there is marked enhancement of the negative c.d. band at 340 nm arising from the dihydronicotinamide chromophore of NADPH. The substrates folate and dihydrofolate give rise to substantial extrinsic c.d. bands on binding, which show a number of specific differences between enzymes from different sources. The binary complexes between the enzyme and the inhibitors methotrexate or trimethoprim also show strong c.d. bands, and these are qualitatively very similar for all dihydrofolate reductases studied so far. The ternary complexes between enzyme, NADPH and trimethoprim or methotrexate are very different from the sum of the spectra of the binary complexes. Trimethoprim leads to the disappearance of the 340 nm c.d. band of bound NADPH, whereas in the methotrexate--NADPH--enzyme ternary complex a "couplet" c.d. spectrum is observed at long wavelengths. Analysis of this latter feature suggests that it arises from a direct interaction between the dihydronicotinamide and pteridine rings in the ternary complex.     

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 A resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 A 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.     

Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase–dihydrofolate reductase

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS–DHFR specific inhibitors.     

Nuclear magnetic resonance study of the state of protonation of inhibitors bound to mutant dihydrofolate reductase lacking the active-site carboxyl

13C nuclear magnetic resonance spectra have been obtained for complexes of [2-13C]methotrexate and [2-13C]trimethoprim with wild-type dihydrofolate reductase (DHFR) from Escherichia coli and with two mutant enzymes in which aspartic acid-27 is replaced by asparagine and by serine, respectively. In both the wild-type and mutated enzymes, exchange between the free inhibitor and the enzyme-complexed inhibitor is slow on the NMR time scale; hence, despite the considerably increased dissociation constants for binary complexes with the enzymes, the dissociation rate remains small relative to the frequency separation of the resonances. In all cases but one, the pKa of an inhibitor that is complexed to enzyme differs greatly from that of the free inhibitor. However, while the pKa of both inhibitors in complexes with the wild-type enzyme is elevated to above 10, the pKa of the inhibitors complexed with the Asn-27 and Ser-27 enzymes is lowered to a value below 4. Exact determinations of bound pKa values are limited by the solubility of the enzyme and the dissociation constants of the complexes. The single exception to these general conclusions is the ternary complex of the Ser-27 DHFR with trimethoprim and NADPH. In this complex, both free and enzyme-complexed trimethoprim exhibit similar pKa values (approximately equal to 7.6). However, both the exchange between free and enzyme-complexed inhibitor and the protonation of the enzyme-complexed inhibitor are slow in the NMR time scale, so that the spectra reveal three resonances corresponding to free inhibitor, to protonated enzyme-complexed inhibitor, and to unprotonated enzyme-complexed inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of dihydrofolate reductase from soybean seedlings

Dihydrofolate reductase (DHFR; EC 1.5.1.3) was purified to homogeneity from soybean seedlings by affinity chromatography on methotrexate-aminohexyl Sepharose, gel filtration on Ultrogel AcA-54, and Blue Sepharose chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme gave a single protein band corresponding to a molecular weight of 22,000. The enzyme is not a 140,000 Da heteropolymer as reported by others. Amino acid sequence-specific antibodies to intact human DHFR and also antibodies to CNBr-generated fragments of human DHFR bound to the plant enzyme on Western blots and cross-reacted significantly in immunoassays, indicating the presence of sequence homology between the two enzymes. The plant and human enzymes migrated similarly on nondenaturing polyacrylamide electrophoretic gels as monitored by activity staining with a tetrazolium dye. The specific activity of the plant enzyme was 15 units/mg protein, with a pH optimum of 7.4. Km values of the enzyme for dihydrofolate and NADPH were 17 and 30 microM, respectively. Unlike other eukaryotic enzymes, the plant enzyme showed no activation with organic mercurials and was inhibited by urea and KCl. The affinity of the enzyme for folate was relatively low (I50 = 130 microM) while methotrexate bound very tightly (KD less than 10(-10) M). Binding of pyrimethamine to the plant enzyme was weaker, while trimethoprim binding was stronger than to vertebrate DHFR. Trimetrexate, a very potent inhibitor of the human and bacterial enzymes showed weak binding to the plant enzyme. However, certain 2,4-diaminoquinazoline derivatives were very potent inhibitors of the plant DHFR. Thus, the plant DHFR, while showing similarity to the vertebrate and bacterial enzymes in terms of molecular weight and immunological cross-reactivity, can be distinguished from them by its kinetic properties and interaction with organic mercurials, urea, KCl and several antifolates.     

Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode

Plasmid-encoded bacterial R67 dihydrofolate reductase (DHFR) is a NADPH-dependent enzyme unrelated to chromosomal DHFR in amino acid sequence and structure. R67 DHFR is insensitive to the bacterial drug trimethoprim in contrast to chromosomal DHFR. The crystal structure of Q67H mutant of R67 DHFR bound to NADP(+) has been determined at 1.15 angstroms resolution. The cofactor assumes an extended conformation with the nicotinamide ring bound near the center of the active site pore, the ribose and pyrophosphate group (PP(i)) extending toward the outer pore. The ribonicotinamide exhibits anti conformation as in chromosomal DHFR complexes. The relative orientation between the PP(i) and the nicotinamide ribose differs from that observed in chromosomal DHFR-NADP(+) complexes. The coenzyme displays symmetrical binding mode with several water-mediated hydrogen bonds with the protein besides ionic, stacking, and van der Waals interactions. The structure provides a molecular basis for the observed stoichiometry and cooperativity in ligand binding. The ternary model based on the present structure and the previous R67 DHFR-folate complex provides insight into the catalytic mechanism and indicates that the relative orientation of the reactants in plasmid DHFR is different from that seen in chromosomal DHFRs.     

Structure-based design, synthesis and preliminary evaluation of selective inhibitors of dihydrofolate reductase from Mycobacterium tuberculosis

Tuberculosis is an increasing threat, owing to the spread of AIDS and to the development of resistance of the causative organism, Mycobacterium tuberculosis, to the currently available drugs. Dihydrofolate reductase (DHFR) is an important enzyme of the folate cycle; inhibition of DHFR inhibits growth and causes cell death. The crystal structure of M. tuberculosis DHFR revealed a glycerol tightly bound close to the binding site for the substrate dihydrofolate; this glycerol-binding motif is absent from the human enzyme. A series of pyrimidine-2,4-diamines was designed with a two-carbon tether between a glycerol-mimicking triol and the 6-position of the heterocycle; these compounds also carried aryl substituents at the 5-position. These, their diastereoisomers, analogues lacking two hydroxy groups and analogues lacking the two-carbon spacing linker were synthesised by acylation of the anions derived from phenylacetonitriles with ethyl (4S,5R)-4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane-4-propanoate, ethyl (4S,5S)-4-benzyloxymethyl-2,2-dimethyl-1,3-dioxolane-4-propanoate, tetrahydrooxepin-2-one and 2,3-O-isopropylidene-d-erythronolactone, respectively, to give the corresponding alpha-acylphenylacetonitriles. Formation of the methyl enol ethers, condensation with guanidine and deprotection gave the pyrimidine-2,4-diamines. Preliminary assay of the abilities of these compounds to inhibit the growth of TB5 Saccharomyces cerevisiae carrying the DHFR genes from M. tuberculosis, human and yeast indicated that 5-phenyl-6-((3R,4S)-3,4,5-trihydroxypentyl)pyrimidine-2,4-diamine selectively inhibited M. tuberculosis DHFR and had little effect on the human or yeast enzymes.     

Structural studies on bioactive compounds. 30. Crystal structure and molecular modeling studies on the Pneumocystis carinii dihydrofolate reductase cofactor complex with TAB, a highly selective antifolate

The crystal structure of the ternary complex of NADPH, the potent antifolate [2, 4-diamino-5-?3-[3-(2-acetyloxyethyl)-3-benzyltriazen-1-yl]-4 -chloroph enyl?-6-ethylpyrimidine] (TAB, 1) and Pneumocystis carinii dihydrofolate reductase (pcDHFR), refined to 2.1 A resolution, reveals that TAB binds similar to the antifolates trimethoprim and methotrexate. These data also reveal multiple conformations for the binding geometry of TAB with two preferred orientations of the acetyloxy and benzyl groups that results from a 180 degrees rotation about the N2-N3 triazenyl bond. The methyl of the acetyloxy and benzyl ring of TAB probes large hydrophobic regions of the p-aminobenzoyl folate binding pocket of the active site, in particular the region near Phe69, which is unique to the pcDHFR sequence. These results confirm prior molecular modeling investigations of the binding of TAB to pcDHFR that identified four low-energy binding geometries, two involving rotations about the terminal N(2)-N(3) triazenyl linkage and two involving atropisomerism about the pivotal pyrimethamine-phenyl bond. The primary differences in the molecular dynamics (MD) models and those observed in this crystal complex result from small conformational changes in active-site residues on energy minimization. However, two MD models place the acetyloxy and benzyl ring groups in a region of the active site between the cofactor-binding region and the p-aminobenzoyl folate pocket; an orientation never observed in any DHFR crystal structure to date. These conformers interact with solvent near the enzyme surface and are probably not observed due to the loss of specific hydrogen bonds with the enzyme. The high species pcDHFR selectivity of TAB could be the result of ligand flexibility that enables multiple binding orientations at the enzyme active site. Further modification of the acetyloxy region of TAB could increase its potency and selectivity for pcDHFR.     

Proton nuclear magnetic resonance studies of the effects of ligand binding on tryptophan residues of selectively deuterated dihydrofolate reductase from Lactobacillus casei

We have prepared a selectively deuterated dihydrofolate reductase in which all the aromatic protons except the C(2) protons of tryptophan have been replaced by deuterium and have examined the 1H NMR spectra of its complexes with folate, trimethoprim, methotrexate, NADP+, and NADPH. One of the four Trp C(2)-proton resonance signals (signal P at 3.66 ppm from dioxane) has been assigned to Trp-21 by examining the NMR spectrum of a selectively deuterated N-bromosuccinimide-modified dihydrofolate reductase. This signal is not perturbed by NADPH, indicating that the coenzyme is not binding close to the 2 position of Trp-21. This contrasts markedly with the 19F shift (2.7 ppm) observed for the 19F signal of Trp-21 in the NADPH complex with the 6-fluorotryptophan-labeled enzyme. In fact the crystal structure of the enzyme . methotrexate . NADPH shows that the carboxamide group of the reduced nicotinamide ring is near to the 6 position of Trp-21 but remote from its 2 position. The nonadditivity of the 1H chemical-shift contributions for signals tentatively assigned to Trp-5 and -133 indicates that these residues are influenced by ligand-induced conformational changes.     

Characterization of an R-plasmid dihydrofolate reductase with a monomeric structure

A plasmid-encoded dihydrofolate reductase that originated in a clinical isolate of Salmonella typhimurium (phage type 179) moderately resistant to trimethoprim has been isolated and characterized. The dihydrofolate reductase (called type III) was purified to homogeneity using a combination of gel filtration, hydrophobic chromatography, and methotrexate affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions indicated that the enzyme is a 16,900 molecular weight monomeric protein. Kinetic analyses showed that trimethoprim is a relatively tight binding inhibitor (Ki = 19 nM) competitive with dihydrofolate. The enzyme is also extremely sensitive to methotrexate inhibition (Ki = 9 pM) and has a high affinity for dihydrofolate (Km = 0.4 microM). The sequence of the first 20 NH2-terminal residues of the protein shows 50% homology with the trimethoprim-sensitive chromosomal Escherichia coli dihydrofolate reductase and suggests that the two enzymes may be closely related. This is the first example of a plasmid encoding for a monomeric dihydrofolate reductase only moderately resistant to trimethoprim, and a resistance mechanism, dependent in part on the high dihydrofolate affinity of the type III enzyme, is proposed.     

Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.     

Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase

Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a,b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified by using a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (Ki values) for trimethoprim were 1.00 and 1.08 microM for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the Vmax increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KD for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and enzyme-NADPH complexes were measured by stopped-flow techniques.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and biological evaluation of a fluorescent analogue of folic acid

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)     

Interactions of 5-deazapteridine derivatives with Mycobacterium tuberculosis and with human dihydrofolate reductases

There are major differences between the structures of human dihydrofolate reductase (hDHFR) and Mycobacterium tuberculosis dihydrofolate reductase (mtDHFR). These differences may allow us to design more selective mtDHFR inhibitors. In this paper we study the reactions of six different compounds derived from 5-deazapteridine with human and bacterial enzymes. Results suggest that the addition of hydrophobic groups to the aminophenyl ring would increase mtDHFR-inhibitor affinity and selectivity.     

Gammaherpesviruses encode functional dihydrofolate reductase activity

We overexpressed and purified from Escherichia coli the dihydrofolate reductase (DHFR) of the gammaherpesviruses human herpesvirus 8 (HHV-8), herpesvirus saimiri (HVS), and rhesus rhadinovirus (RRV). All three enzymes proved catalytically active. The K(m) value of HHV-8 DHFR for dihydrofolate (DHF) was 2.02+/-0.44 microM, that of HVS DHFR was 4.31+/-0.56 microM, and that of RRV DHFR is 7.09+/-0.11 microM. These values are approximately 5-15-fold higher than the K(m) value reported for the human DHFR. The K(m) value of HHV-8 DHFR for NADPH was 1.31+/-0.23 microM, that of HVS DHFR was 3.78+/-0.61 microM, and that of RRV DHFR was 7.47+/-0.59 microM. These values are similar or slightly higher than the corresponding K(m) value of the human enzyme. Methotrexate, aminopterin, trimethoprim, pyrimethamine, and N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), all well-known folate antagonists, inhibited the DHFR activity of the three gammaherpesviruses competitively with respect to DHF but proved markedly less inhibitory to the viral than towards the human enzyme.     

13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid.

13C NMR studies of 13C-labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9-13C]- and [2,4a,6-13C]methotrexate (MTX) and [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP+); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180 degrees difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4-NH2 group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP+ or NADPH to the E. coli DHFR-MTX complex results in a single set of 13C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.     

High-throughput screening and sensitized bacteria identify an M. tuberculosis dihydrofolate reductase inhibitor with whole cell activity

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a bacterial pathogen that claims roughly 1.4 million lives every year. Current drug regimens are inefficient at clearing infection, requiring at least 6 months of chemotherapy, and resistance to existing agents is rising. There is an urgent need for new drugs that are more effective and faster acting. The folate pathway has been successfully targeted in other pathogens and diseases, but has not yielded a lead drug against tuberculosis. We developed a high-throughput screening assay against Mtb dihydrofolate reductase (DHFR), a critical enzyme in the folate pathway, and screened a library consisting of 32,000 synthetic and natural product-derived compounds. One potent inhibitor containing a quinazoline ring was identified. This compound was active against the wild-type laboratory strain H37Rv (MIC(99)?=?207 μM). In addition, an Mtb strain with artificially lowered DHFR levels showed increased sensitivity to this compound (MIC(99)?=?70.7 μM), supporting that the inhibition was target-specific. Our results demonstrate the potential to identify Mtb DHFR inhibitors with activity against whole cells, and indicate the power of using a recombinant strain of Mtb expressing lower levels of DHFR to facilitate the discovery of antimycobacterial agents. With these new tools, we highlight the folate pathway as a potential target for new drugs to combat the tuberculosis epidemic.     

Purification and properties of recombinant Pneumocystis carinii dihydrofolate reductase

Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography. The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence. The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7. The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl. Urea at 2.5 M also increased the enzyme activity twofold. Kinetic analysis of the purified enzyme revealed that the K_m values for dihydrofolate and NADPH were 1.8 and 1.4 μM, respectively, and that the k_cat was 70 s⁻¹. Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P. carinii DHFR and showed little selectivity over the human DHFR. Trimetrexate and piritrexim were much more potent inhibitors of the P. carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR.