Nematicidal alkaloids and related compounds produced by the fungus Penicillium cf. simplicissimum.
(9/765)
A new nematicidal alkaloid, peniprequinolone (1), together with the known alkaloids penigequinolones A and B (2a, 2b), 3-methoxy-4-hydroxy-4-(4'-methoxyphenyl)quinolinone (3), and 3-methoxy-4,6-dihydroxy-4-(4'-methoxyphenyl)quinolinone (4), were isolated from Penicillium cf. simplicissimum (Oudemans) Thom. Cyclopenin (5) and a compound (6a/6b) structurally related to cyclopenin also were isolated from the fungus, and their structures were established by spectroscopic analysis. The biological activities of 1, 2, 3, 4, and 5 were examined by a bioassay with root-lesion nematodes. (+info)
In vivo monitoring of intracellular ATP levels in Leishmania donovani promastigotes as a rapid method to screen drugs targeting bioenergetic metabolism.
(10/765)
A method for the rapid screening of drugs targeting the bioenergetic metabolism of Leishmania spp. was developed. The system is based on the monitoring of changes in the intracellular ATP levels of Leishmania donovani promastigotes that occur in vivo, as assessed by the luminescence produced by parasites transfected with a cytoplasmic form of Phothinus pyralis luciferase and incubated with free-membrane permeable D-luciferin analogue D-luciferin-[1-(4,5-dimethoxy-2-nitrophenyl) ethyl ester]. A significant correlation was obtained between the rapid inhibition of luminescence with parasite proliferation and the dissipation of changes in mitochondrial membrane potential (DeltaPsi(m)) produced by buparvaquone or plumbagin, two leishmanicidal inhibitors of oxidative phosphorylation. To further validate this test, a screen of 14 standard leishmanicidal drugs, using a 50 microM cutoff, was carried out. Despite its semiquantitative properties and restriction to the promastigote stage, this test compares favorably with other bioenergetic parameters with respect to time and cell number requirements for the screening of drugs that affect mitochondrial activity. (+info)
DNA transformation of Leishmania infantum axenic amastigotes and their use in drug screening.
(11/765)
Protocols for DNA electroporation in Leishmania promastigote cells are well established. More recently, in vitro culture of axenic Leishmania amastigotes became possible. We have established conditions for DNA transformation of axenically grown Leishmania infantum amastigotes. Parameters for DNA electroporation of Leishmania axenic amastigotes were systematically studied using luciferase-mediated transient transfection. Cell lines expressing stable luciferase activity were then selected, and their ability to be used in an in vitro drug screening procedure was determined. A model was established, using axenic amastigotes expressing luciferase activity, for rapidly determining the activity of drugs directly against both axenic and intracellular amastigotes. For intracellular amastigotes, the 50% effective concentrations of pentamidine, sodium stibogluconate (Pentostam), meglumine (Glucantime), and potassium antimonyl tartrate determined with the luciferase assay were 0.2 microM (0.12 microg/ml), 55 microg/ml, 95 microg/ml, and 0.12 microg/ml, respectively; these values are in agreement with values determined by more labor-intensive staining methods. We also showed the usefulness of luciferase-expressing parasites for analyzing drug resistance. The availability of luciferase-expressing amastigotes for use in high-throughput screening should facilitate the search for new antileishmanial drugs. (+info)
Potent anti-Trypanosoma cruzi activities of oxidosqualene cyclase inhibitors.
(12/765)
Trypanosoma cruzi is the protozoan agent that causes Chagas' disease, a major health problem in Latin America. Better drugs are needed to treat infected individuals. The sterol biosynthesis pathway is a potentially excellent target for drug therapy against T. cruzi. In this study, we investigated the antitrypanosomal activities of a series of compounds designed to inhibit a key enzyme in sterol biosynthesis, oxidosqualene cyclase. This enzyme converts 2,3-oxidosqualene to the tetracyclic product, lanosterol. The lead compound, N-(4E,8E)-5,9, 13-trimethyl-4,8, 12-tetradecatrien-1-ylpyridinium, is an electron-poor aromatic mimic of a monocyclized transition state or high-energy intermediate formed from oxidosqualene. This compound and 27 related compounds were tested against mammalian-stage T. cruzi, and 12 inhibited growth by 50% at concentrations below 25 nM. The lead compound was shown to cause an accumulation of oxidosqualene and decreased production of lanosterol and ergosterol, consistent with specific inhibition of the oxidosqualene cyclase. The data demonstrate potent anti-T. cruzi activity associated with inhibition of oxidosqualene cyclase. (+info)
In vitro generation of novel pyrimethamine resistance mutations in the Toxoplasma gondii dihydrofolate reductase.
(13/765)
Pyrimethamine is a potent inhibitor of dihydrofolate reductase and is widely used in the treatment of opportunistic infections caused by the protozoan parasite Toxoplasma gondii. In order to assess the potential role of dhfr sequence polymorphisms in drug treatment failures, we examined the dhfr-ts genes of representative isolates for T. gondii virulence types I, II, and III. These strains exhibit differences in their sensitivities to pyrimethamine but no differences in predicted dhfr-ts protein sequences. To assess the potential for pyrimethamine-resistant dhfr mutants to emerge, three drug-sensitive variants of the T. gondii dhfr-ts gene (the wild-type T. gondii sequence and two mutants engineered to reflect polymorphisms observed in drug-sensitive Plasmodium falciparum) were subjected to random mutagenesis and transfected into either wild-type T. gondii parasites or dhfr-deficient Saccharomyces cerevisiae under pyrimethamine selection. Three resistance mutations were identified, at amino acid residues 25 (Trp-->Arg), 98 (Leu-->Ser), and 134 (Leu-->His). (+info)
Biological characterization of Trypanosoma cruzi strains.
(14/765)
Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans. (+info)
Diamidine compounds: selective uptake and targeting in Plasmodium falciparum.
(15/765)
Extensive drug resistance in Plasmodium falciparum emphasizes the urgent requirement for novel antimalarial agents. Here we report potent antimalarial activity of a number of diamidine compounds. The lead compound pentamidine is concentrated 500-fold by erythrocytes infected with P. falciparum. Pentamidine accumulation can be blocked by inhibitors of hemoglobin digestion, suggesting that the drug binds to ferriprotoporphyrin IX (FPIX). All of the compounds bound to FPIX in vitro and inhibited the formation of hemozoin. Furthermore, inhibitors of hemoglobin digestion markedly antagonized the antimalarial activity of the diamidines, indicating that binding to FPIX is crucial for the activity of diamidine drugs. Pentamidine was not accumulated into uninfected erythrocytes. Pentamidine transport into infected cells exhibits an initial rapid phase, nonsaturable in the micromolar range and sensitive to inhibition by furosemide and glibenclamide. Changing the counter-ion in the order Cl(-) < Br(-) < NO(2)(-) < I(-) +info)
Genome comparison of progressively drug resistant Plasmodium falciparum lines derived from drug sensitive clone.
(16/765)
Chloroquine has been the mainstay of malaria chemotherapy for the past five decades, but resistance is now widespread. Pyrimethamine or proguanil form an important component of some alternate drug combinations being used for treatment of uncomplicated Plasmodium falciparum infections in areas of chloroquine resistance. Both pyrimethamine and proguanil are dihydrofolate reductase (DHFR) inhibitors, the proguanil acting primarily through its major metabolite cycloguanil. Resistance to these drugs arises due to specific point mutations in the dhfr gene. Cross resistance between cycloguanil and pyrimethamine is not absolute. It is, therefore, important to investigate mutation rates in P. falciparum for pyrimethamine and proguanil so that DHFR inhibitor with less mutation rate is favored in drug combinations. Hence, we have compared mutation rates in P. falciparum genome for pyrimethamine and cycloguanil. Using erythrocytic stages of P. falciparum cultures, progressively drug resistant lines were selected in vitro and comparing their RFLP profile with a repeat sequence. Our finding suggests that pyrimethamine has higher mutation rate compared to cycloguanil. It enhances the degree of genomic polymorphism leading to diversity of natural parasite population which in turn is predisposes the parasites for faster selection of resistance to some other antimalarial drugs. (+info)