Subject(s)
Aspartate-tRNA Ligase/antagonists & inhibitors , Brugia malayi/drug effects , Brugia malayi/enzymology , Filaricides/pharmacology , RNA, Transfer, Amino Acyl/antagonists & inhibitors , Resorcinols/pharmacology , Streptomyces/metabolism , Animals , Filaricides/chemistry , Filaricides/isolation & purification , Magnetic Resonance Spectroscopy , Molecular Structure , Resorcinols/chemistry , Resorcinols/isolation & purification , Survival AnalysisABSTRACT
Lymphatic filariasis (LF) is a vector borne infectious disease caused by the nematode Wuchereria bancrofti, Brugia malayi, and Brugia timori. Over 120 million people are affected by LF in the world, of which two-thirds are in Asia. The infection restricts the normal flow of lymph from the infected area resulting in swelling of the extremities and causing permanent disability. As the available drugs for the treatment of LF are becoming ineffective due to the development of resistance, there is an urgent need to find new leads for drug development. In this study, asparaginyl-tRNA synthetase (AsnRS; PDB ID: 2XGT) essential for the protein bio-synthesis in the filarial nematode was used to carry out virtual screening (VS) of plant constituents from traditional Chinese medicine (TCM) database. Docking as well as E-pharmacophore based VS were carried out to identify the hits. The top scoring hits, Agri 1 (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one-3-O-beta-D-glucopyranoside) and Agri 2 (5,7-dihydroxy-2-propylchromone 7-O-beta-D-glucopyranoside), constituents of Agrimonia pilosa, were selected for molecular dynamics (MD) simulation study for 10 ns. MD simulation showed that both the glycosides Agri 1 and Agri 2 were forming stable interactions with the target protein. Moreover, docking and MD simulation of the lead A (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one; Mol. Wt.: 304.25; CLogP: 3.07) and lead B (5,7-dihydroxy-2-propylchromone; Mol. Wt.: 220.22; CLogP: 3.02), the aglycones of Agri 1 and Agri 2, respectively, were carried out with the target AsnRS. The in silico investigations of the aglycones suggest that the lead B could be a suitable fragment-like lead molecule for anti-filarial drug discovery.
Subject(s)
Aspartate-tRNA Ligase/antagonists & inhibitors , Brugia malayi/drug effects , Databases, Pharmaceutical , Drugs, Chinese Herbal/pharmacology , Elephantiasis, Filarial/drug therapy , Enzyme Inhibitors/pharmacology , Filaricides/pharmacology , Molecular Docking Simulation , Molecular Dynamics Simulation , RNA, Transfer, Amino Acyl/antagonists & inhibitors , Wuchereria bancrofti/drug effects , Animals , Aspartate-tRNA Ligase/genetics , Aspartate-tRNA Ligase/metabolism , Binding Sites , Brugia malayi/enzymology , Computer-Aided Design , Drug Design , Drugs, Chinese Herbal/chemistry , Elephantiasis, Filarial/diagnosis , Elephantiasis, Filarial/parasitology , Enzyme Inhibitors/chemistry , Filaricides/chemistry , Humans , Ligands , Molecular Structure , Molecular Targeted Therapy , Protein Binding , Protein Conformation , RNA, Transfer, Amino Acyl/genetics , RNA, Transfer, Amino Acyl/metabolism , Structure-Activity Relationship , Wuchereria bancrofti/enzymologyABSTRACT
Lymphatic filariasis is caused by the Brugia malayi parasite. Three new congeners of the depsipeptide WS9326A (1), WS9326C (2), WS9326D (3), and WS9326E (4), were isolated from Streptomyces sp. 9078 by using a B. malayi asparaginyl-tRNA synthetase (BmAsnRS) inhibition assay. WS9326D specifically inhibits the BmAsnRS, kills the adult B. malayi parasite, and does not exhibit significant general cytotoxicity to human hepatic cells, representing a new lead scaffold for antifilarial drug discovery.
Subject(s)
Aspartate-tRNA Ligase/antagonists & inhibitors , Brugia malayi/immunology , Filariasis/immunology , Hepatocytes/drug effects , Lactones , Peptides, Cyclic , RNA, Transfer, Amino Acyl/antagonists & inhibitors , Animals , Brugia malayi/enzymology , Humans , Lactones/chemistry , Lactones/isolation & purification , Lactones/pharmacology , Molecular Structure , Peptides, Cyclic/chemistry , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacology , Stereoisomerism , Streptomyces/chemistryABSTRACT
Lymphatic filariasis is caused by the parasitic nematodes Brugia malayi and Wuchereria bancrofti, and asparaginyl-tRNA synthetase (AsnRS) is considered an excellent antifilarial target. The discovery of three new tirandamycins (TAMs), TAM E (1), F (2), and G (3), along with TAM A (4) and B (5), from Streptomyces sp. 17944 was reported. Remarkably, 5 selectively inhibits the B. malayi AsnRS and efficiently kills the adult B. malayi parasite, representing a new lead scaffold to discover and develop antifilarial drugs.
Subject(s)
Aminoglycosides/chemistry , Anti-Bacterial Agents/chemistry , Aspartate-tRNA Ligase/antagonists & inhibitors , Brugia malayi/drug effects , Enzyme Inhibitors/chemistry , RNA, Transfer, Amino Acyl/antagonists & inhibitors , Streptomyces/chemistry , Animals , Brugia malayi/enzymology , Enzyme Inhibitors/pharmacology , Models, Molecular , Molecular StructureABSTRACT
The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.
Subject(s)
Mitochondria/drug effects , Oxazolidinones/pharmacology , Peptidyl Transferases/drug effects , Protein Synthesis Inhibitors/pharmacology , RNA, Ribosomal/drug effects , Software , Acetamides , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Binding Sites/drug effects , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/pharmacology , Cytoplasm/drug effects , Cytoplasm/enzymology , Drug Resistance/genetics , Escherichia coli/drug effects , Escherichia coli/enzymology , Humans , Linezolid , Mitochondria/enzymology , Models, Molecular , Molecular Structure , Mutation/genetics , Oxazolidinones/chemistry , Peptidyl Transferases/metabolism , Protein Synthesis Inhibitors/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Ribosomal/metabolism , RNA, Ribosomal, 23S , RNA, Transfer, Amino Acyl/antagonists & inhibitors , RNA, Transfer, Amino Acyl/metabolism , Staining and Labeling , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymologyABSTRACT
SLIDE software, which models the flexibility of protein and ligand side chains while docking, was used to screen several large databases to identify inhibitors of Brugia malayi asparaginyl-tRNA synthetase (AsnRS), a target for anti-parasitic drug design. Seven classes of compounds identified by SLIDE were confirmed as micromolar inhibitors of the enzyme. Analogs of one of these classes of inhibitors, the long side-chain variolins, cannot bind to the adenosyl pocket of the closed conformation of AsnRS due to steric clashes, though the short side-chain variolins identified by SLIDE apparently bind isosterically with adenosine. We hypothesized that an open conformation of the motif 2 loop also permits the long side-chain variolins to bind in the adenosine pocket and that their selectivity for Brugia relative to human AsnRS can be explained by differences in the sequence and conformation of this loop. Loop flexibility sampling using Rigidity Optimized Conformational Kinetics (ROCK) confirms this possibility, while scoring of the relative affinities of the different ligands by SLIDE correlates well with the compounds' ranks in inhibition assays. Combining ROCK and SLIDE provides a promising approach for exploiting conformational flexibility in structure-based screening and design of species selective inhibitors.
Subject(s)
Aspartate-tRNA Ligase/antagonists & inhibitors , Aspartate-tRNA Ligase/chemistry , Brugia malayi/enzymology , Enzyme Inhibitors/chemistry , Filaricides/chemistry , RNA, Transfer, Amino Acyl/antagonists & inhibitors , RNA, Transfer, Amino Acyl/chemistry , Animals , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/classification , Humans , Ligands , Models, Molecular , Protein ConformationABSTRACT
Selenocysteine transfer RNA (tRNA([Ser]Sec)) is a central molecule in the production of selenium-containing proteins, and may play a role in the regulation of their biosynthesis. Selenium concentration influences both the levels of tRNA([Ser]Sec) and the relative abundance of two isoforms. To study the mechanism by which selenium affects tRNA([Ser]Sec) levels, Chinese hamster ovary (CHO) cells were treated with the transcription inhibitor, actinomycin D, and tRNA([Ser]Sec) levels were determined by Northern blotting, primer extension and reverse-phase column chromatography. Turnover of tRNA([Ser]Sec) in CHO cells was faster than the total tRNA population. Supplementation of the culture media with selenium reduced turnover of tRNA([Ser]Sec), but did not influence turnover of a randomly selected serine tRNA. Inhibition of transcription with actinomycin D resulted in a relative increase in the abundance of the isoform containing methylcarboxymethyl-5'-uridine-2'-O-methylribose in the wobble position of the anticodon. Primer extension studies, which permitted the independent evaluation of the tRNA([Ser]Sec) arising from the introduced mouse gene and that derived from the host CHO gene, indicated an accelerated decline in tRNA([Ser]Sec) derived from both the transfected and the native gene. These results provide additional insight into the levels of regulation that control the translation of selenium containing proteins in mammalian cells.
Subject(s)
RNA, Transfer, Amino Acyl/antagonists & inhibitors , Selenium/pharmacology , Selenocysteine/genetics , Animals , CHO Cells , Cricetinae , Dactinomycin/pharmacology , Gene Dosage , Gene Expression Regulation/drug effects , Glutathione Peroxidase/genetics , Mice , Nucleic Acid Synthesis Inhibitors/pharmacology , Protein Isoforms/metabolism , Protein Synthesis Inhibitors/pharmacology , RNA Stability , RNA, Transfer/chemistry , RNA, Transfer, Amino Acyl/metabolism , Tissue DistributionABSTRACT
A 2'-O-methyl oligonucleotide complementary to 18 nucleotides in the dihydrouridine stemloop of Escherichia coli tRNA(Cys) has been shown to stably bind to the tRNA. The binding inhibits aminoacylation of the tRNA by cysteine tRNA synthetase. The same oligonucleotide sequence but with the DNA deoxy backbone does not bind to the tRNA. This provides the basis for the design and test of a series of 2'-O-methyl oligonucleotides for their ability to bind to E. coli tRNA(Cys) and inhibit aminoacylation. We show here that different regions of the tRNA have different sensitivities to oligonucleotides. A 10-mer that targets G15 forms a stable complex with the tRNA. The Kd of the complex is several orders of magnitude lower than that of the tRNA-synthetase complex. Measurements of dissociation rate constants indicate that the stronger affinity of the 10-mer to tRNA(Cys) is due to a significantly slower rate of dissociation (by a factor of 10(6)) than that of the synthetase from the tRNA. Only a stoichiometric amount of the 10-mer is necessary to completely inhibit aminoacylation. Because tRNA aminoacylation is fundamental to cell growth, these results provide the rationale for the 10-mer and its derivatives as pharmaceutical agents that target specific cell growth.
Subject(s)
Oligonucleotides/chemical synthesis , RNA, Transfer, Amino Acyl/antagonists & inhibitors , Base Sequence , Escherichia coli , Kinetics , Methylation , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides/metabolism , RNA, Transfer, Amino Acyl/chemistry , RNA, Transfer, Amino Acyl/metabolism , Structure-Activity RelationshipABSTRACT
The effect of biologically inactive and active tRNA conformers on heat inactivation of leucyl-tRNA synthetase was investigated. The data presented show that inactive tRNA conformers seem to form complexes with leucyl-tRNA synthetase, but the thermal stability of the enzyme involved in the complex with inactive and active tRNA conformers is rather different.
Subject(s)
Hot Temperature , Liver/enzymology , Nucleic Acid Conformation , RNA, Transfer, Amino Acyl/antagonists & inhibitors , RNA, Transfer/pharmacology , Animals , In Vitro Techniques , Kinetics , Nucleic Acid Renaturation , RNA, Transfer, Amino Acyl/metabolism , RabbitsABSTRACT
Sulfochlorantine containing active chlorine was shown to produce a general toxic action inactivating a number of Escherichia coli enzyme systems involved in protein biosynthesis. DNA synthesis catalysed by calf thymus DNA polymerase alpha was repressed by 50% and the synthesis of aminoacyl-tRNA catalysed by aminoacyl-tRNA synthetases was repressed by 70% in the cell-free systems at the lethal concentration of the bactericide (0.01% and higher). Sulfochlorantine produced the strongest inhibiting action on the ribosomal step of protein biosynthesis, inhibiting the poly(U)-directed polyphenylalanine formation by 95% at the sublethal concentration of the bactericide (0.005%).
Subject(s)
Enzyme Inhibitors/pharmacology , Escherichia coli/drug effects , Hydantoins/pharmacology , Polyphosphates/pharmacology , Bacterial Proteins/antagonists & inhibitors , Catalysis , DNA Polymerase II/antagonists & inhibitors , DNA, Bacterial/antagonists & inhibitors , Drug Combinations/pharmacology , Escherichia coli/enzymology , RNA, Transfer, Amino Acyl/antagonists & inhibitors , Ribosomes/drug effects , Ribosomes/enzymologyABSTRACT
The effect of the alkaloid sparteine on arginyl-tRNA formation was studied. It was demonstrated that sparteine sulfate in the concentration range 10-60 mM inhibits the charging reaction when amino acid, ATP and tRNA are used as variable substrates. The mode of action is different for all pattern of inhibition for all varied substrates is generally uncompetitive. A pattern of inhibition for all varied substrates is generally uncompetitive. A non-competitive mechanism for amino acid and tRNA was observed at low sparteine concentration, but in the case of ATP it is also uncompetitive.
Subject(s)
RNA, Transfer/metabolism , Sparteine/pharmacology , Alkaloids/pharmacology , Arginine-tRNA Ligase/metabolism , RNA, Transfer, Amino Acyl/antagonists & inhibitorsSubject(s)
Acyltransferases/antagonists & inhibitors , Aminoacyltransferases , Polyamines/pharmacology , Prostate/enzymology , Animals , Cytosol/enzymology , In Vitro Techniques , Male , N-Formylmethionine/antagonists & inhibitors , Putrescine/pharmacology , RNA, Transfer, Amino Acyl/antagonists & inhibitors , Rats , Spermidine/pharmacology , Spermine/pharmacologyABSTRACT
The effect of hemin, phosphorylated sugars, adenosine 3',5'-monophosphate (cyclic AMP) and a number of purines on a specific initiator tRNA deacylase activity in rabbit reticulocytes has been investigated. In the concentration range established to be optimal for maximal stimulation of translation (5.5-30.0 microM), hemin produces a 20-82% inhibition of Met-tRNAfMet deacylation. In contrast, all phosphorylated sugars tested, with the exception of fructose 1,6-diphosphate, are without effect. High concentrations of cyclic AMP (2-4 mM) also significantly inhibit the deacylase activity. The role of hemin and Met-tRNAfMet deacylase in the control of peptide initiation are discussed.
Subject(s)
Acyltransferases/antagonists & inhibitors , Aminoacyltransferases , Cyclic AMP/pharmacology , Heme/analogs & derivatives , Hemin/pharmacology , Sugar Phosphates/pharmacology , Acyltransferases/blood , Animals , Fructosediphosphates/pharmacology , In Vitro Techniques , N-Formylmethionine/antagonists & inhibitors , N-Formylmethionine/blood , Nucleotides/pharmacology , Peptide Chain Initiation, Translational/drug effects , Protein Biosynthesis/drug effects , RNA, Transfer, Amino Acyl/antagonists & inhibitors , RNA, Transfer, Amino Acyl/blood , Rabbits , Reticulocytes/metabolismABSTRACT
Inhibitor studies of the only known eukaryotic methionyl-tRNA transformylase (10-formyltetrahydrofolate:L-methionyl-tRNA N-transformylase, EC 2.1.2.9) were carried out. All the natural pteroylglutamic acid derivatives examined, with the exception of pteroylglutamic acid, are inhibitors. The most effective is 5-methyltetrahydrofolate (5-CH3-H4PteGlu) (KI = 3 . 10(-6) M), which is the only noncompetitive inhibitor of the enzyme. All the other derivatives tested are competitive, and H4PteGlu shows a cooperative inhibition. These and other data obtained with pteroylglutamic analogues show that, in contrast to the bacterial enzyme, Euglena transformylase is also inhibited by compounds without a fully reduced pyrazine ring and is very sensitive to compounds with a methyl group in position 5 or 10 of the pteridine ring.
