Analysis of the Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene sequence

Phosphoinositide-specific phospholipase Cgamma (PLCgamma) is a key regulatory enzyme that binds to the phosphoryl-tyrosine residues in the cytoplasmic domain of certain activated receptors and catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] forming IP3 and diacylglycerol (DAG) in response to several mitogenic factors. Previously, we determined that microinjected PLCgamma induces DNA synthesis in G0-arrested NIH 3T3 cells, suggesting the possibility that PLCgamma may have an oncogenic potential. In this report, we demonstrate that overexpression of PLCgamma in NIH 3T3 cells results in altered growth properties and cellular transformation. The PLCgamma/3T3 transfectants do not require serum growth factors to proliferate, display anchorage-independent growth in soft agar and induce tumors when transplanted into nude mice. These findings suggest that overexpression of PLCgamma facilitates the transformation of NIH 3T3 cells. Furthermore, PLCgamma expression and activity have been shown to be elevated in many human tumors. Thus, PLCgamma signaling may contribute to the promotion and/or progression of human cancers.     

Kenyan Plasmodium falciparum field isolates: correlation between pyrimethamine and chlorcycloguanil activity in vitro and point mutations in the dihydrofolate reductase domain

In the present work ecto-phosphatase activity in Herpetomonas muscarum muscarum has been characterized using live parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 4.27 nmol Pi/mg of protein.min. A pH curve was generated, in which these intact flagellates showed the highest phosphatase activity at pH 6.5. Classical inhibitors for acid phosphatase, such as sodium orthovanadate, sodium tartrate, and ammonium molybdate, were used in the experiments and showed different patterns of inhibition. Lithium fluoride, aluminum chloride, and fluoroaluminate complexes were also tested. Although lithium fluoride and fluoroaluminate complexes were capable of inhibiting the phosphatase activity, aluminum chloride stimulated this enzyme. Cytochemical analysis showed the localization of this enzyme on the parasite surface. This ecto-phosphatase activity was also significantly diminished when the parasites were treated with 10(-6) M platelet-activating factor (PAF), a potent phospholipid mediator that promoted cellular differentiation in this parasite.     

Cycloguanil and its parent compound proguanil demonstrate distinct activities against Plasmodium falciparum malaria parasites transformed with human dihydrofolate reductase

The lack of suitable antimalarial agents to replace chloroquine and pyrimethamine/sulfadoxine threatens efforts to control the spread of drug-resistant strains of the malaria parasite Plasmodium falciparum. Here we describe a transformation system, involving WR99210 selection of parasites transformed with either wild-type or methotrexate-resistant human dihydrofolate reductase (DHFR), that has application for the screening of P. falciparum-specific DHFR inhibitors that are active against drug-resistant parasites. Using this system, we have found that the prophylactic drug cycloguanil has a mode of pharmacological action distinct from the activity of its parent compound proguanil. Complementation assays demonstrate that cycloguanil acts specifically on P. falciparum DHFR and has no other significant target. The target of proguanil itself is separate from DHFR. We propose a strategy of combination chemotherapy incorporating the use of multiple parasite-specific inhibitors that act at the same molecular target and thereby maintain, in combination, their effectiveness against alternative forms of resistance that arise from different sets of point mutations in the target. This approach could be combined with traditional forms of combination chemotherapy in which two or more compounds are used against separate targets.     

Inducible nitric-oxide synthase generates superoxide from the reductase domain

In the absence of L-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O-2). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O-2 formation in macrophages. However, there has been a lack of direct evidence of superoxide generation from the purified iNOS, and it was previously hypothesized that significant O-2 production does not occur. Moreover, the mechanism and enzyme site responsible for O-2 generation is unknown. To determine whether iNOS produces O-2 and to identify the mechanism of this process, we performed electron paramagnetic resonance measurements on purified iNOS using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. In the presence of NADPH, prominent O-2 adduct signals were detected from iNOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of L-arginine decreased this O-2 formation, whereas its enantiomer D-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O-2 signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O-2 generation. Furthermore, strong O-2 generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O-2, and this mainly occurs at the flavin-binding sites of the reductase domain.     

Inhibition of invasion and intraerythrocytic development of Plasmodium falciparum by kinase inhibitors

We have examined the effects of seven protein kinase inhibitors (staurosporine, genistein, methyl 2,5-dihydroxycinnamate, tyrphostins B44 and B46, lavendustin A and R03) on the erythrocytic cycle of the malaria parasite, Plasmodium falciparum. One (staurosporine) strongly inhibits serine/threonine kinases, but the remainder all exhibit a strong preference for tyrosine kinases. We have been able to discriminate between effects on invasion and on intraerythrocytic development. All reagents impeded development of intraerythrocytic parasites, though at widely differing concentrations, from the sub-micromolar to the millimolar. Several inhibitors, including staurosporine, also reduced invasion. The phosphatase inhibitor, okadaic acid, had a strong inhibitory effect both on invasion and development. The regulation of malaria development by phosphorylation or dephosphorylation reactions at several points in the blood-stage cycle is implied.     

Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques

Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much more circumscribed mode of initiation in this locus. This dichotomy has raised the issue whether the patterns of replicating DNA on 2-D gels have been misinterpreted and, in some cases, may represent such noncanonical replication intermediates as broken bubbles or microbubbles. In an accompanying study (R. F. Kalejta and J. L. Hamlin, Mol. Cell. Biol. 16:4915-4922, 1996), we have shown that broken bubbles migrate to unique positions in three different gel systems and therefore are not likely to be confused with classic replication intermediates. Here, we have applied a broken bubble assay developed from that study to an analysis of the amplified DHFR locus in CHO cells. This assay gives information about the number and positions of initiation sites within a fragment. In addition, we have analyzed the DHFR locus by a novel stop-and-go-alkaline gel technique that measures the size of nascent strands at all positions along each arc in a neutral/neutral 2-D gel. Results of these analyses support the view that the 2-D gel patterns previously assigned to classic, intact replication bubbles and single-forked structures indeed correspond to these entities. Furthermore, potential nascent-strand start sites appear to be distributed at very frequent intervals along the template in the intergenic region in the DHFR domain.     

Super in vitro synergy between inhibitors of dihydrofolate reductase and inhibitors of other folate-requiring enzymes: the critical role of polyglutamylation

The combined action among polyglutamylatable and nonpolyglutamylatable antifolates, directed against dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GARFT), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), and thymidylate synthase (TS), in human ileocecal HCT-8 cells was examined in a 96-well plate growth inhibition assay (96-h continuous drug exposure). An interaction parameter, alpha, was estimated for each of 95 experiments by fitting a seven-parameter model to data with weighted nonlinear regression. In a representative experiment, raising the folic acid concentration in the medium dramatically increased the Loewe synergy for the combination of trimetrexate (TMTX) and the GARFT inhibitor AG2034 (from a mean alpha +/- SE of 1.50 +/- 0.25 at 2.3 microM folic acid to 146 +/- 20 at 78 microM folic acid). Enhancements were also found for combinations of TMTX with the GARFT inhibitors AG2032, Lometrexol, and LY309887, the AICARFT inhibitor AG2009, and the TS inhibitors LY231514 and Tomudex but not with the GARFT inhibitor LL95509 or with the TS inhibitors AG337, ZD9331, and BW1843U89. Replacing TMTX with methotrexate in two-drug mixtures decreased the intensity of Loewe synergy. Examination of isobolograms at different effect levels revealed informative reproducible changes in isobol patterns. No two-drug combinations among inhibitors of GARFT, AICARFT, and TS exhibited Loewe synergy at either 2.3 or 78 microM folic acid. Thus, the ideal requirement for the folic acid-enhanced synergy is that a nonpolyglutamylatable DHFR inhibitor be combined with a polyglutamylatable inhibitor of another folate-requiring enzyme. A hypothesis to explain this general phenomenon involves the critical role of folylpoly-gamma-glutamate synthetase and the effect of the DHFR inhibitor in decreasing the protection by folic acid of cells to the other antifolates.     

Polymorphisms in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes of Plasmodium falciparum and in vivo resistance to sulphadoxine/pyrimethamine in isolates from Tanzania

The efficacy of sulphadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of mutations associated with the active site sequence in the Plasmodium falciparum genes coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is associated with in vitro resistance to pyrimethamine and sulphadoxine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results show that alleles of DHPS (436-alanine, 437-alanine and 540-lysine) were significantly reduced in prevalence on day 7 after S/P treatment. In this area, a DHPS with 436-serine, 437-glycine and 540-glutamate appears to play a major role in resistance to S/P in vivo. Evidence for the influence of mutations in the DHFR gene in this investigation is not clear, probably because of the high prevalence of 'resistance-related' mutations at day 0 in the local parasite population. For apparently the same reason, it was not possible to show a statistical association between S/P resistance and the presence of particular polymorphisms in the DHFR and DHPS genes before treatment.     

Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production

Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2.-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2.- during catalytic turnover in the presence of L-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2.- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2.- production by nNOS and the CYS-331 mutant is CaM-dependent and that O2.- production can be modulated by substrates and inhibitors of nNOS. O2.- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2.-, but that the reductase domain is both necessary and sufficient for O2.- production.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors lovastatin and simvastatin inhibit in vitro development of Plasmodium falciparum and Babesia divergens in human erythrocytes

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors lovastatin and simvastatin inhibit the in vitro intraerythrocytic development of Plasmodium falciparum and Babesia divergens, with concentrations inhibiting parasite growth by 50% in the ranges of 10 to 20 and 5 to 10 micrograms.ml-1, respectively. For P. falciparum, the 50% inhibitory concentrations were in the same range whatever the chloroquine susceptibility of the strains tested (strain F32/Tanzania [chloroquine susceptible] or FcB.1/Columbia [resistant]). The stage-dependent susceptibility of P. falciparum to simvastatin was studied by subjecting synchronized cultures to 6-h pulses of drug throughout the 48-h erythrocytic life cycle. The most important inhibitory effects were observed between the 12th and 30th hours of the cycle, corresponding to the trophozoite stage. This period precedes the S phase and the nuclear divisions. Parasites in the newly formed ring stage (time zero to the 6th hour of the cycle) and the schizont stage (30th to 48th hour of the cycle) were weakly or not susceptible to simvastatin pulses.     

Topological mutation of Escherichia coli dihydrofolate reductase

The human large intestine contains a large and diverse population of bacteria. Certain genera, namely Bifidobacterium and Lactobacillus, are thought to exert health-promoting effects. Prebiotics such as fructooligosaccharides (FOS) have been shown to stimulate the growth of endogenous bifidobacteria. In this study, changes of lactic acid producing bacteria in continuous culture fermentors (semi-defined, anaerobic medium containing 5 g 1(-1) FOS, dilution rate of 0.1 h-1, pH 5.5) were followed over a 21 d period after inoculation with blended human faeces from four healthy adults. Samples were also taken every 3 d for influent/effluent FOS, short chain fatty acid (SCFA), lactate and microbiological analyses. Results showed that SCFA concentrations decreased abruptly 1 d after inoculation while lactate concentrations increased. Classical methods of enumeration using selective media showed that the proportion of total culturable count represented by bifidobacteria and lactobacilli increased from 11.9% on day 1 to 98.1% on day 21. However, molecular methods using genus-specific 16S rRNA oligonucleotide probes indicated that the bifidobacterial population maintained a level between 10 and 20% of total 16S rRNA during the first 6 d and disappeared rapidly when the maximum concentration of lactate was reached. Lactobacilli, which were initially present in low numbers, increased until day 9 and remained at high levels (20-42% of total 16S rRNA) to day 21, with the exception of day 18. Although FOS has usually been regarded as a selective substrate for bifidobacteria, these observations suggest that: (1) lactobacilli are also able to use FOS, (2) lactobacilli can out-compete bifidobacteria in continuous culture at pH 5.2-5.4 when FOS is the primary carbon and energy source, and (3) bifidobacteria can grow faster on FOS than lactobacilli under controlled conditions.     

2,4-Diaminopyrido[3,2-d]pyrimidine inhibitors of dihydrofolate reductase from Pneumocystis carinii and Toxoplasma gondii

Six previously unknown 2,4-diamino-6-(anilinomethyl)- and 2,4-diamino-6-[(N-methylanilino)-methyl]pyrido[3,2-d]pyrimidines (5-10) were synthesized from 2,4-diamino-6-(bromomethyl)-pyrido[3,2-d]pyrimidine hydrobromide (11.HBr) by treatment with the appropriate aniline or N-methylaniline in dimethylformamide at room temperature, with or without NaHCO3 present. Compounds 5-10 were tested as inhibitors of dihydrofolate reductase from Pneumocystis carinii, Toxoplasma gondii, and rat liver as a part of a larger effort directed toward the discovery of lipophilic nonclassical antifolates combining high enzyme selectivity and high potency. Of the six analogues tested, the most potent and selective against T. gondii DHFR was 2,4-diamino-6-[(3',4',5'-trimethoxy-N-methylanilono)methyl]pyrido[ 3,2-d d pyrimidine (7), which had an IC50 of 0.0047 microM against this enzyme as compared with 0.026 microM against the rat liver enzyme. The potency of 7 against T. gondii DHFR was similar to that of trimetrexate (TMQ, 1) and piritrexim (PTX, 2) but was > 500-fold greater than that of trimethoprim (TMP, 3). However, while 7 was more selective than either TMQ (19x) or PTX (63x) against this enzyme, its selectivity in comparison with TMP was 8-fold lower. 2,4-Diamino-6-[3',4',5'-trimethoxyanilino)methyl]pyrido[3,2-d]pyri midin e (6) was 17-fold less active than 7 and was also less selective. 2,4-Diamino-6-[(3',4'-dichloro-N-methylanilino)methyl]pyrido[3, 2-d]pyrimidine (10) had an IC50 of 0.022 microM against P. carinii DHFR and was comparable in potency to TMQ and PTX. The species selectivity of 10 for P. carinii versus rat liver DHFR was greater than that of either TMQ (21-fold) or PTX (31-fold). On the other hand, even though 10 was slightly more active than TMQ against the P. carinii enzyme, its selectivity was 7-fold lower than that of TMP. Thus, the goal of combining high enzyme binding activity, which is characteristic of the fused-ring compounds TMQ and PTX, with high selectivity for T. gondii and P. carinii DHFR versus rat liver DHFR, which is characteristic of the monocyclic compound TMP, remained unmet in this limited series.     

Identification of potent inhibitors of Plasmodium falciparum plasmepsin II from an encoded statine combinatorial library

An encoded 13,020-member combinatorial library was synthesized containing a statine core. Evaluation of this library with plasmepsin II, an aspartyl protease required for hemoglobin metabolism in the malaria parasite, led to the identification of potent and selective inhibitors as well as novel structure-activity relationships.     

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria

We have exploited the recently developed ability to trans- fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine-resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24-fold above that seen for the transfectant expressing the wild-type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.     

A new procedure for improving the predictiveness of CoMFA models and its application to a set of dihydrofolate reductase inhibitors

A new automated procedure to improve the predictive quality of CoMFA models for both training and test sets is described. A model of greater consistency is generated by performing small reorientations of the underlying molecules for which too low activities are calculated. In order to predict activities of test compounds, the most similar molecules in the previously optimized model are identified and used as a basis for the prediction. This method has been applied to two independent sets of dihydrofolate reductase inhibitors (80 compounds each, serving as training sets), resulting in a significant increase of the cross-validated r2 value. For both models, the predictive r2 value for a test set consisting of 70 compounds was improved substantially.     

Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase

We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochemical properties of the recombinant protein. This enzyme is involved in the de novo synthesis of deoxythymidine 5'-phosphate and is critical for cell growth. High levels of T. maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which inhibit the growth of non-recombinant cells. The enzyme was purified to homogeneity in the following two steps: heat treatment followed by affinity chromatography or cation-exchange chromatography. Most of the biochemical properties of T. maritima DHFR resemble those of other bacterial or eukaryotic DHFRs, however, some are unique to T. maritima DHFR. The pH optima for activity, Km for substrates, and polypeptide chain length of T. maritima DHFR are similar to those of other DHFRs. In addition, the secondary structure of T. maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs. Interestingly, T. maritima DHFR exhibits some characteristics of eukaryotic DHFRs, such as a basic pI, an excess of positively charged residues in the polypeptide chain and activation of the enzyme by inorganic salts and urea. Unlike most other DHFRs which are monomeric or part of a bifunctional DHFR-thymidylate synthase (TS) enzyme, T. maritima DHFR seems to generally form a dimer in solution and is also much more thermostable than other DHFRs. It may be that dimer formation is a key factor in determining the stability of T. maritima DHFR.     

Drug-resistant Plasmodium falciparum malaria in the eastern Transvaal

In March 1993, a study was undertaken in the Komatipoort/Malelane area to monitor the in vitro sensitivity of Plasmodium falciparum to antimalarial drugs currently in use in South Africa. Of the 12 isolates collected, 7 were successfully tested for sensitivity to chloroquine and quinine, 6 for mefloquine susceptibility, and 5 for sensitivity to Fansidar. Four of the isolates were resistant to chloroquine at RIII level, 1 at RII level, and 2 were sensitive. All isolates were found to be sensitive to both quinine and mefloquine. Results suggested possible resistance to Fansidar. These findings have implications for tourists travelling to this area.     

2,4-Diaminothieno[2,3-d]pyrimidine lipophilic antifolates as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase

Ten previously unreported 2,4-diaminothieno[2,3-d]pyrimidine lipophilic dihydrofolate reductase inhibitors were synthesized as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. Pivaloylation of 2,4-diamino-5-methylthieno[2,3-d]pyrimidine followed by dibromination with N-bromosuccinimide in the presence of benzoyl peroxide gave 2,4-bis(pivaloylamino)-6-bromo-5-(bromomethyl)thieno[2,3-d]pyrimid ine, which after condensation with substituted anilines or N-methylanilines and deprotection with base yielded 2,4-diamino-6-bromo-5-[(substituted anilino)methyl]thieno[2,3-d]pyrimidines. Removal of the 6-bromo substituent was accomplished with sodium borohydride and palladium chloride. The reaction yields were generally good to excellent. The products were tested as inhibitors of dihydrofolate reductase (DHFR) from P. carinii, T. gondii, and rat liver. Although the IC50 could not be reached for the 6-unsubstituted compounds because of their extremely poor solubility, three of the five 6-bromo derivatives were soluble enough to allow the IC50 to be determined against all three enzymes. 2,4-Diamino-5-[3,5-dichloro-4-(1-pyrrolo)anilino]methyl]- 6-bromothieno[2,3-d]pyrimidine was the most active of the 6-bromo derivatives, with an IC50 of 7.5 microM against P. carinii DHFR, but showed no selectivity for either P. carinii or T. gondii DHFR relative to the enzyme from rat liver.     

Characterization of Plasmodium falciparum glutamate dehydrogenase-soluble antigen

There are limited data on the factors associated with menopausal hot flashes, a common and potentially morbid condition. The objective of this study was to identify predictors of menopausal hot flashes. To meet this objective, 233 naturally perimenopausal or post-menopausal women (ages 45-65) attending a large urban hospital center primary care clinic, mammography unit, or women's health practice were enrolled. The women responded to a self-administered questionnaire assessing selected demographic factors, reproductive history, and behavioral factors. Sixty-seven percent of respondents experienced hot flashes, with 63% reporting frequent hot flashes (at least one hot flash per day) and 60% with hot flashes describing the hot flashes as severe. Women with hot flashes were significantly more likely to have mothers who experienced hot flashes (OR = 4.4, CI = 2.0-10.0) or to be smokers (OR = 2.0, CI = 1.2-3.5). There were no statistically significant associations between hot flashes and other selected demographic, reproductive, or behavior characteristics. These results reveal that menopausal hot flashes are associated with a maternal history of hot flashes as well as with cigarette smoking. These results may help physicians to counsel their patients about smoking cessation.