Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied inhibition of growth of the malaria parasite Plasmodium falciparum in in vitro culture using antisense (AS) oligodeoxynucleotides (ODNs) against different target genes. W2 and W2mef strains of drug-resistant parasites were exposed to AS ODNs over 48 hr, and growth was determined by microscopic examination and [3H]hypoxanthine incorporation. At ODN concentrations of 1 microM, phosphorothioate (PS) ODNs inhibited growth in a target-independent manner. However, between 0.5 and 0.005 microM, ODNs against dihydrofolate reductase, dihydropteroate synthetase, ribonucleotide reductase, the schizont multigene family, and erythrocyte binding antigen EBA175 significantly inhibited growth compared with a PS AS ODN against human immunodeficiency virus, two AS ODNs containing eight mismatches, or the sense strand controls (P < 0.0001). The IC50 was approximately 0.05 microM, whereas that for non-sequence-specific controls was 15-fold higher. PS AS ODNs against DNA polymerase alpha showed less activity than that for other targets, whereas a single AS ODN against triose-phosphate isomerase did not differ significantly from controls. We conclude that at concentrations below 0.5 microM, PS AS ODNs targeted against several malarial genes significantly inhibit growth of drug-resistant parasites in a nucleotide sequence-dependent manner. This technology represents an alternative method for identifying malarial genes as potential drug targets.
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PMID:Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides. 855 72

Plasmodium falciparum causes the most severe form of malaria that is fatal in many cases. Emergence of drug resistant strains of P. falciparum requires that new drug targets be identified. This review considers in detail enzymes of the glycolytic pathway, purine salvage pathway, pyrimidine biosynthesis and proteases involved in catabolism of haemoglobin. Structural features of P. falciparum triosephosphate isomerase which could be exploited for parasite specific drug development have been highlighted. Utility of P. falciparum hypoxanthine-guanine-phosphoribosyltransferase, adenylosuccinate synthase, dihydroorotate dehydrogenase, thymidylate synthase-dihydrofolate reductase, cysteine and aspartic proteases have been elaborated in detail. The review also briefly touches upon other potential targets in P. falciparum.
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PMID:Metabolic enzymes as potential drug targets in Plasmodium falciparum. 929 79

The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene; hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10 degrees C heat shock (37 to 47 degrees C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for Escherichia coli that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).
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PMID:Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. 1171 67

High-throughput and virtual screening are widely used to discover novel leads for drug design. On examination, many screening hits appear non-drug-like: they act noncompetitively, show little relationship between structure and activity, and have poor selectivity. Attempts to develop these peculiar molecules into viable leads are often futile, and much time can be wasted on the characterization of these "phony" hits. Despite their common occurrence, the mechanism of action of these promiscuous molecules remains unknown. To investigate this problem, 45 diverse screening hits were studied. Fifteen of these were previously reported as inhibitors of various receptors, including beta-lactamase, malarial protease, dihydrofolate reductase, HIV Tar RNA, thymidylate synthase, kinesin, insulin receptor, tyrosine kinases, farnesyltransferase, gyrase, prions, triosephosphate isomerase, nitric oxide synthase, phosphoinositide 3-kinase, and integrase; 30 were from an in-house screening library of a major pharmaceutical company. In addition to their original targets, 35 of these 45 compounds were shown to inhibit several unrelated model enzymes. These 35 screening hits included compounds, such as fullerenes, dyes, and quercetin, that have repeatedly shown activity against diverse targets. When tested against the model enzymes, the compounds showed time-dependent but reversible inhibition that was dramatically attenuated by albumin, guanidinium, or urea. Surprisingly, increasing the concentration of the model enzymes 10-fold largely eliminated inhibition, despite a 1000-fold excess of inhibitor; a well-behaved competitive inhibitor did not show this behavior. One model to explain these observations was that the active form of the promiscuous inhibitors was an aggregate of many individual molecules. To test this hypothesis, light scattering and electron microscopy experiments were performed. The nonspecific inhibitors were observed to form particles of 30-400 nm diameter by both techniques. In control experiments, a well-behaved competitive inhibitor and an inactive dye-like molecule were not observed to form aggregates. Consistent with the hypothesis that the aggregates are the inhibitory species, the particle size and IC(50) values of the promiscuous inhibitors varied monotonically with ionic strength; a competitive inhibitor was unaffected by changes in ionic strength. Unexpectedly, aggregate formation appears to explain the activity of many nonspecific inhibitors and may account for the activity of many promiscuous screening hits. Molecules acting via this mechanism may be widespread in drug discovery screening databases. Recognition of these compounds may improve screening results in many areas of pharmaceutical interest.
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PMID:A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. 1193 26

Effects of ligand binding on protein dynamics are studied via molecular dynamics (MD) simulations on two different enzymes, dihydrofolate reductase (DHFR) and triosephosphate isomerase (TIM), in their unliganded (free) and liganded states. Domain motions in MD trajectories are analyzed by collectivities and rotation angles along the principal components (PCs). DHFR in the free state has well-defined domain rotations, whereas rotations are slightly damped in the binary complex with nicotinamide adenine dinucleotide phosphate (NADPH), and remarkably distorted in the presence of NADP(+) , showing that NADP(+) is solely responsible for the loss of correlation of the domains in DHFR. Although mean square fluctuations of MD simulations in the same PC subspaces are similar for different ligation states, linear stochastic time series models show that backbone flexibility along the first five PCs is decreased upon NADPH and NADP(+) binding in subpicosecond scale. This shows that mobility of the protein along the PCs is closely related with intraminimum dynamics, and alterations in ligation states may change the intraminimum dynamics significantly. Low vibrational frequencies of the alpha-carbon atoms of DHFR are determined from the time series models of a larger number of low indexed PCs, and it is found that number of modes in the lowest frequencies is reduced upon ligand binding. A similar result is obtained for TIM in the unliganded and dihydroxyacetone phosphate bound states. We suggest that stochastic time series modeling is a promising method to be used in determining subtle perturbations in protein dynamics.
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PMID:Effect of ligand binding on the intraminimum dynamics of proteins. 2073 Jul 77

Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.
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PMID:Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules. 2427 25