A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase

Mina, J.G.; Mosely, J.A.; Ali, H.Z.; Shams-Eldin, H.; Schwarz, R.T.; Steel, P.G.; Denny, P.W.

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 Vmax = 2.31 pmol min−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.

Citation

Mina, J., Mosely, J., Ali, H., Shams-Eldin, H., Schwarz, R., Steel, P., & Denny, P. (2010). A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase. International Journal of Biochemistry and Cell Biology, 42(9), 1553-1561. https://doi.org/10.1016/j.biocel.2010.06.008

• Journal Article Type: Article
• Acceptance Date: Jun 9, 2010
• Online Publication Date: Jun 16, 2010
• Publication Date: Sep 1, 2010
• Deposit Date: Jun 11, 2010
• Publicly Available Date: Jul 20, 2010
• Journal: International Journal of Biochemistry and Cell Biology
• Print ISSN: 1357-2725
• Electronic ISSN: 1878-5875
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 42
• Issue: 9
• Pages: 1553-1561
• DOI: https://doi.org/10.1016/j.biocel.2010.06.008
• Public URL: https://durham-repository.worktribe.com/output/1543761