Search

The enzyme 7,8-dihydropteroate synthase (DHPS) catalyzes the condensation of para-aminobenzoic acid (pABA) with 6-hydroxymethyl-7, 8-dihydropterin-pyrophosphate to form 7,8-dihydropteroate and pyrophosphate. DHPS is essential for the de novo synthesis of folate in prokaryotes, lower eukaryotes, and in plants, but is absent in mammals. Inhibition of this enzyme's activity by sulfonamide and sulfone drugs depletes the folate pool, resulting in growth inhibition and cell death. Here, we report the 1.7 A resolution crystal structure of the binary complex of 6-hydroxymethylpterin monophosphate (PtP) with DHPS from Mycobacterium tuberculosis (Mtb), a pathogen responsible for the death of millions of human beings each year. Comparison to other DHPS structures reveals that the M. tuberculosis DHPS structure is in a unique conformation in which loop 1 closes over the active site. The Mtb DHPS structure hints at a mechanism in which both loops 1 and 2 play important roles in catalysis by shielding the active site from bulk solvent and allowing pyrophosphoryl transfer to occur. A binding mode for pABA, sulfonamides and sulfones is suggested based on: (i) the new conformation of the closed loop 1; (ii) the distribution of dapsone and sulfonamide resistance mutations; (iii) the observed direction of the bond between the 6-methyl carbon atom and the bridging oxygen atom to the alpha-phosphate group in the Mtb DHPS:PtP binary complex; and (iv) the conformation of loop 2 in the Escherichia coli DHPS structure. Finally, the Mtb DHPS structure reveals a highly conserved pterin binding pocket that may be exploited for the design of novel antimycobacterial agents., (Copyright 2000 Academic Press.)

We examined the effect of a human immunodeficiency virus (HIV)-specific immune-based therapy in Thailand, where access to antiviral drug therapy is limited. A 40-week trial was conducted with 297 asymptomatic, HIV-infected Thai subjects with CD4-cell counts greater than 300 microl/mm(3). Subjects were randomized to receive either HIV type 1 (HIV-1) immunogen (Remune; inactivated HIV-1 from which gp120 is depleted in incomplete Freund's adjuvant or adjuvant control at 0, 12, 24, and 36 weeks at five different clinical sites in Thailand. Neither group received antiviral drug therapy. The a priori primary endpoint for the trial was changes in CD4-cell counts with secondary parameters of percent changes in CD8-cell counts (percent CD4, CD8, and CD4/CD8) and body weight. Subsets of subjects were also examined for changes in plasma HIV-1 RNA levels, Western blot immunoreactivity, and HIV-1 delayed-type hypersensitivity (DTH) skin test reactivity. There was a significant difference in changes in CD4-cell counts that favored the HIV-1 immunogen-treated group compared to those for the adjuvant-treated control group (P<0.05). On average, for HIV-1 immunogen-treated subjects CD4-cell counts increased by 84 cells by week 40, whereas the increase for the control group was 38 cells by week 40. This increase in CD4-cell count was associated with increased HIV-specific immunogenicity, as shown by Western blotting and enhanced HIV-1 DTH skin reactivity. No significant differences in adverse events were observed between the groups. The results of this trial suggest that HIV-1 immunogen is safe and significantly increases CD4-cell counts and HIV-specific immunity compared to those achieved with the adjuvant control in asymptomatic HIV-1-infected subjects not taking antiviral drugs.

The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (Ki). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The Ki values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance.

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate and is essential for the synthesis of thymidylate, purines and several amino acids. Inhibition of the enzyme's activity leads to arrest of DNA synthesis and cell death. The enzyme has been studied extensively as a drug target for bacterial, protozoal and fungal infections, and also for neoplastic and autoimmune diseases. Here, we report the crystal structure of dihydrofolate reductase from Mycobacterium tuberculosis, a human pathogen responsible for the death of millions of human beings per year. Three crystal structures of ternary complexes of M. tuberculosis DHFR with NADP and different inhibitors have been determined, as well as the binary complex with NADP, with resolutions ranging from 1.7 to 2.0 A. The three DHFR inhibitors are the anticancer drug methotrexate, the antimicrobial trimethoprim and Br-WR99210, an analogue of the antimalarial agent WR99210. Structural comparison of these complexes with human dihydrofolate reductase indicates that the overall protein folds are similar, despite only 26 % sequence identity, but that the environments of both NADP and of the inhibitors contain interesting differences between the enzymes from host and pathogen. Specifically, residues Ala101 and Leu102 near the N6 of NADP are distinctly more hydrophobic in the M. tuberculosis than in the human enzyme. Another striking difference occurs in a region near atoms N1 and N8 of methotrexate, which is also near atom N1 of trimethoprim, and near the N1 and two methyl groups of Br-WR99210. A glycerol molecule binds here in a pocket of the M. tuberculosis DHFR:MTX complex, while this pocket is essentially filled with hydrophobic side-chains in the human enzyme. These differences between the enzymes from pathogen and host provide opportunities for designing new selective inhibitors of M. tuberculosis DHFR., (Copyright 2000 Academic Press.)

The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosis dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of Escherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. leprae dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both M. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.

Objective: To examine the effect of treatment with an inactivated, gp120-depleted, HIV-1 immunogen (Remune) in 30 Thai subjects infected with HIV-1 subtype E., Design: Sixty-week open-label study., Methods: Thirty HIV-positive volunteers with CD4 cell counts > or = 300 x 10(6)/l were given intramuscular injections of Remune into the triceps muscle on day 1 and then at weeks 4, 8, 12, 24, 36, 48 and 60., Results: Treatment with Remune was well-tolerated and augmented HIV-1-specific immune responses. Furthermore, subjects had a significant increase in CD4 cell count (P < 0.0001), CD4 cell percentage (P < 0.0001), CD8 cell percentage (P < 0.0001), and body weight (P < 0.0001) compared with pretreatment levels. Fourteen subjects with detectable viral load at day 1 showed a decrease at week 60 (P=0.04). Retrospective Western blot analysis showed 23 subjects with increased intensity of antibody bands and 15 patients showed development of new reactivities to HIV proteins, especially towards p17 and p15., Conclusion: These results indicate that HIV-specific immune-based therapeutic approaches such as Remune should be further examined in countries with different clades of HIV-1 and where access to antiviral drug therapies is limited.

The safety and immunogenicity of REMUNE, an HIV-specific immune based therapy for HIV infection, was evaluated in a cohort of 30 HIV infected subjects in Thailand. This therapy utilizes a gp120 depleted inactivated virus (HZ321), which exhibits a high degree of conservation with the core antigens of both type B' and E strains of HIV, the predominant Thailand isolates. The treatment was well tolerated, with no serious adverse events reported over the course of the 4-month trial. Treatment in which four doses were administered with REMUNE appeared to boost HIV-specific immune responses, with approximately 75% of the treated subjects demonstrating an increase in either the repertoire or the intensity of the serological response to HIV as measured by Western blot. CD4%, viral load, and weight remained stable over the course of the 4-month study relative to baseline values. Viral subtyping of this cohort revealed a predominance of type 'E'. These data suggest that REMUNE is safe and immunogenic in seropositive Thai subjects and supports further study of the therapeutic potential of REMUNE to treat HIV-1 infection.

The codon for serine residue 108 of the Plasmodium falciparum dihydrofolate reductase gene was replaced with those for the other 19 amino acids. Except for the Lys108 mutant, which was not expressed, all other substitutions yielded DHFR mutants which were expressed in Escherichia coli as inactive inclusion bodies. Nine of the mutants--Asn108, Thr108, Gly108, Ala108, Gln108, Cys108, Val108, Leu108, and Met108--yielded active DHFR upon refolding of the protein from the inclusion bodies. The remaining mutants--IIe108, Arg108, Pro108, Asp108, His108, Tyr108, Phe108, Trp108, and Glu108--did not exhibit detectable DHFR activity on refolding. The Asn108 mutant had almost unperturbed kinetic parameters but conferred resistance to pyrimethamine and cycloguanil; other active mutants showed poorer DHFR activity. We purified and characterized four mutants which produced highest DHFR activity, i.e., the Gln108, Gly108, Cys108, and Ala108 mutants. These mutant enzymes had kcat/K(m) values ranging from 7 to 22% of the wild-type enzyme. While DHFRs from Gly108, Cys108, and Ala108 mutants were as susceptible to pyrimethamine and cycloguanil as the wild type, the Gln108 mutation conferred high resistance to both inhibitors. Our data suggest that residue 108 is important for antifolate binding, and that the Ser108 to Asn108 mutation was selected in nature because of (i) the need for only a single base change, (ii) its good activity, and (iii) its resistance to antifolates.

Specificity is an important aspect of structure-based drug design. Distinguishing between related targets in different organisms is often the key to therapeutic success. Pneumocystis carinii is a fungal opportunist which causes a crippling pneumonia in immunocompromised individuals. We report the identification of novel inhibitors of P. carinii dihydrofolate reductase (DHFR) that are selective versus inhibition of human DHFR using computational molecular docking techniques. The Fine Chemicals Directory, a database of commercially available compounds, was screened with the DOCK program suite to produce a list of potential P. carinii DHFR inhibitors. We then used a postdocking refinement directed at discerning subtle structural and chemical features that might reflect species specificity. Of 40 compounds predicted to exhibit anti-Pneumocystis DHFR activity, each of novel chemical framework, 13 (33%) show IC50 values better than 150 microM in an enzyme assay. These inhibitors were further assayed against human DHFR: 10 of the 13 (77%) bind preferentially to the fungal enzyme. The most potent compound identified is a 7 microM inhibitor of P. carinii DHFR with 25-fold selectivity. The ability of molecular docking methods to locate selective inhibitors reinforces our view of structure-based drug discovery as a valuable strategy, not only for identifying lead compounds, but also for addressing receptor specificity.

Single and multiple mutations at residues 16, 51, 59, 108, and 164 of Plasmodium falciparum dihydrofolate reductase (pfDHFR) have been linked to antifolate resistance in malaria. We prepared and characterized all seven of the pfDHFR mutants found in nature, as well as six mutants not observed in nature. Mutations involving residues 51, 59, 108, or 164 conferred cross resistance to both the antifolates pyrimethamine and cycloguanil, whereas mutation of residue 16 specifically conferred resistance to cycloguanil. The antifolate resistance of enzyme mutants found in nature correlated with in vivo antifolate resistance; however, mutants not found in nature were either poorly resistant or had insufficient catalytic activity to support DNA synthesis. Thus, specific combinations of multiple mutations at target residues were selected in nature to optimize resistance. Further, the resistance of multiple mutants was more than the sum of the component single mutations, indicating that residues were selected for their synergistic as well as intrinsic effects on resistance. Pathways inferred for the evolution of pyrimethamine-resistant mutants suggested that all multiple mutants emerged from stepwise selection of the single mutant, S108N. Thus, we propose that drugs targeted to both the wild-type pfDHFR and S108N mutant would have a low propensity for developing resistance, and hence could provide effective antimalarial agents.

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 Km values for dihydrofolate and NADPH were 1.8 and 1.4 microM, respectively, and that the kcat 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.

Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc2(6) and trimethoprim-resistant mutant mc2(26) was purified to homogeneity. In crude extracts, the specific activity of the enzyme from the trimethoprim resistant strain was comparable to that from the sensitive strain. The DHFR from both sources was purified using affinity chromatography on MTX-Sepharose followed by Mono Q FPLC. The enzyme has an apparent molecular mass of 23 kDa from gel filtration on Sephadex G-100 and from SDS-PAGE. Amino terminal sequence analysis showed homology with DHFRs from a subset of other gram-positive organisms. The purified enzyme from the trimethoprim-sensitive organism exhibited Km values for H2folate and NADPH of 0.68 +/- 0.2 microM and 21 +/- 4 microM, respectively. The Km values for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 +/- 0.4 microM and 5.3 +/- 1.5 microM, respectively. A kcat of 4.5 sec-1 was determined for the DHFR from both sources. The enzyme from both sources was competitively inhibited by pyrimethamine and trimethoprim. The Ki value of trimethoprim, for the enzyme from the drug-resistant organism was about six-fold higher than for the enzyme from drug-sensitive strain. Our data suggest that mutation of DHFR contributes to trimethoprim resistance in the mc2(26) strain of M. smegmatis.

The coding sequence of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from a moderately pyrimethamine-resistant strain (HB3) of Plasmodium falciparum was assembled in a pUC expression vector. The coding sequence possesses unique Nco1 and Xba1 sites which flank 243 bp of the DHFR gene that include all point mutations thus far linked to pyrimethamine resistance. Wild-type (3D7) and highly pyrimethamine-resistant (7G8) TS-DHFRs were made from this vector by cassette mutagenesis using Nco1-Xba1 fragments from the corresponding cloned TS-DHFR genes. Catalytically active recombinant TS-DHFRs were expressed in Escherichia coli, albeit at low levels. Both TS and DHFR coeluted upon gel filtration and copurified upon affinity and anion exchange chromatography. Gel filtration and SDS-PAGE indicated that the enzyme was a dimer with identical 67-kDa subunits, characteristic of protozoan TS-DHFRs. Amino-terminal sequencing gave 10 amino acids which perfectly matched the sequence predicted from the nucleotide sequence. The recombinant TS-DHFR was purified to homogeneity by 10-formylfolate affinity chromatography followed by Mono Q FPLC. The inhibition properties of pyrimethamine toward the purified recombinant enzymes show that the point mutations are the molecular basis of pyrimethamine resistance in P. falciparum.

The classical anti-microbial antifolates trimethoprim, pyrimethamine, and cycloguanil are poor inhibitors of purified dihydrofolate reductase (DHFR) from Leishmania major. They show no selectivity for Leishmania DHFR relative to the human enzyme, and it is not surprising that they are ineffectual as anti-leishmanial agents. Several 5-(substituted-benzyl)-2,4-diaminopyrimidines have been screened as inhibitors for purified L. major and human DHFRs. These compounds inhibit Leishmania DHFR with I50 values ranging from 0.2 to 11 microM, and show about 5 to greater than 100-fold greater selectivity for the parasite DHFR than the human enzyme. These pyrimidine analogs are more potent inhibitors of Leishmania promastigote and amastigote growth than the classical anti-microbial antifolates, and serve as lead compounds for the development of new selective antileishmanial agents.

Biochemical aspects of action of antifolates and 4-aminoquinolines and their resistance in the malaria parasites are reviewed, with emphasis on pyrimethamine and chloroquine respectively. Resistance to pyrimethamine has been shown to be associated with either an increase in the amount of parasite dihydrofolate reductase or a reduced affinity of the enzyme for drug binding, in line with the presence of a distinctive pathway for folate metabolism. The theories for drug synergism in the folate pathway are discussed with respect to resistance to pyrimethamine and its combination with sulpha drugs. The biochemical basis for chloroquine resistance is still unclear, reflecting incomplete understanding of its mechanism of action. Data implicating the role of haemozoin and other components as a putative chloroquine receptor of the parasites are reviewed, and possible explanations for resistance are discussed.

The bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) of Leishmania major has been cloned and expressed in Escherichia coli and Saccharomyces cerevisiae. The strategy involved placing the entire 1560-bp coding sequence into a parent cloning plasmid that was designed to permit introduction of unique restriction sites at the 5'- and 3'-ends. In this manner, the entire coding sequence could be easily subcloned into a variety of expression vectors. High levels of TS-DHFR gene expression were driven by tac, pL and T7 RNA pol promoters in E. coli, and the GAPDH-ADH-2 promoter in S. cerevisiae. L. major TS-DHFR also complemented TS deficiency in E. coli. In E. coli, the protein accumulated to very high levels, but most was present as inactive inclusion bodies. Nevertheless, substantial amounts were soluble; up to 2% of the soluble protein was catalytically active TS-DHFR. In the yeast systems, essentially all of the bifunctional protein was soluble and catalytically active, and crude extracts contained about 100-fold more enzyme than do extracts from wild-type L. major. The expressed TS-DHFR from yeast and E. coli was purified to homogeneity by methotrexate-Sepharose affinity chromatography. About 8.5 mg of homogeneous, catalytically active protein is obtained from a 1-L culture of yeast, and 1.5 mg was obtained from 1 L of E. coli culture. A 200-L fermentation of the yeast expression system yielded a crude extract containing over 4 g of TS-DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)