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Abstract Previous work from our group showed that tamoxifen, an oral drug that has been in use for the treatment of breast cancer for over 40 years, is active both in vitro and in vivo against several species of Leishmania, the etiological agent of leishmaniasis. Using a combination of metabolic labeling with [3H]-sphingosine and myo -[3H]-inositol, alkaline hydrolysis, HPTLC fractionations and mass spectrometry analyses, we observed a perturbation in the metabolism of inositolphosphorylceramides (IPCs) and phosphatidylinositols (PIs) after treatment of L. amazonensis promastigotes with tamoxifen, with a significant reduction in the biosynthesis of the major IPCs (composed of d16:1/18:0-IPC, t16:0/C18:0-IPC, d18:1/18:0-IPC and t16:0/20:0-IPC) and PIs (sn -1- O -(C 18:0)alkyl -2- O -(C 18:1)acylglycerol-3-HPO 4 -inositol and sn -1- O -(C 18:0)acyl-2- O -(C 18:1)acylglycerol-3-HPO 4 -inositol) species. Substrate saturation kinetics of myo -inositol uptake analyses indicated that inhibition of inositol transport or availability were not the main reasons for the reduced biosynthesis of IPC and PI observed in tamoxifen treated parasites. An in vitro enzymatic assay was used to show that tamoxifen was able to inhibit the Leishmania IPC synthase with an IC 50 value of 8.48 μM (95% CI 7.68–9.37), suggesting that this enzyme is most likely one of the targets for this compound in the parasites. Graphical abstract Image 1 Highlights • Tamoxifen alters the sphingolipid metabolism of L. amazonensis. • Tamoxifen treated parasites show a significant reduction of IPC and PI species. • Tamoxifen-treated parasites present a reduction of inositol transport. • Tamoxifen is an inhibitor of L. major 's IPC synthase in a micromolar range. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause fetal damage and abortion in both animals and humans. Sphingolipids are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Here we report the identification, isolation, and analyses of the Toxoplasma serine palmitoyltransferase, an enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA. In all eukaryotes analyzed to date, serine palmitoyltransferase is a highly conserved heterodimeric enzyme complex. However, biochemical and structural analyses demonstrated the apicomplexan orthologue to be a functional, homodimeric serine palmitoyltransferase localized to the endoplasmic reticulum. Furthermore, phylogenetic studies indicated that it was evolutionarily related to the prokaryotic serine palmitoyltransferase, identified in the Sphingomonadaceae as a soluble homodimeric enzyme. Therefore this enzyme, conserved throughout the Apicomplexa, is likely to have been obtained via lateral gene transfer from a prokaryote. [ABSTRACT FROM AUTHOR]

The tobacco whitefly, Bemisia tabaci , is a polyphagous crop pest which causes high levels of economic damage across the globe. Insecticides are often required for the effective control of this species, among which the neonicotinoid class have been particularly widely used. Deciphering the mechanisms responsible for resistance to these chemicals is therefore critical to maintain control of B. tabaci and limit the damage it causes. An important mechanism of resistance to neonicotinoids in B. tabaci is the overexpression of the cytochrome P450 gene CYP6CM1 which leads to the enhanced detoxification of several neonicotinoids. In this study we show that qualitative changes in this P450 dramatically alter its metabolic capacity to detoxify neonicotinoids. CYP6CM1 was significantly over-expressed in two strains of B. tabaci which displayed differing levels of resistance to the neonicotinoids imidacloprid and thiamethoxam. Sequencing of the CYP6CM1 coding sequence from these strains revealed four different alleles encoding isoforms carrying several amino acid changes. Expression of these alleles in vitro and in vivo provided compelling evidence that a mutation (A387G), present in two of the CYP6CM1 alleles, results in enhanced resistance to several neonicotinoids. These data demonstrate the importance of both qualitative and quantitative changes in genes encoding detoxification enzymes in the evolution of insecticide resistance and have applied implications for resistance monitoring programs. [Display omitted] • Four different alleles of the P450 CYP6CM1 were linked to resistance to neonicotinoids. • Drosophila melanogaster and insect cell line expression revealed allelic associated differences in metabolic capacity. • Further analysis revealed that the mutation A387G was responsible for the enhanced resistance to several neonicotinoids. [ABSTRACT FROM AUTHOR]

Abstract: Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent V max =2.31pmolmin−1 U−1. Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery. [ABSTRACT FROM AUTHOR]

Abstract: A short sequence involving Rh(II) mediated carbene insertion followed by Lewis acid promoted reductive acetal cleavage with Et3SiH provides a stereoselective method for the construction of 2,3,5-trisubstituted tetrahydrofuran rings. [Copyright &y& Elsevier]