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

Abstract

P.W. (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). pp. 1553-1561. Further information on publisher's website:https://doi.org/10. 1016/j.biocel.2010.06.008 Publisher's copyright statement: NOTICE: this is the author's version of a work that was accepted for publication in International journal of biochemistry and cell biology. Additional information:Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl) 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.3

Exploring Leishmania major Inositol Phosphorylceramide Synthase (LmjIPCS): Insights into the ceramide binding domain

“…These observation are consistent with the predicted reaction mechanism, 23 in that analogues that lack the primary (C1) hydroxyl group (e.g. 18ax) showed no evidence of phosphorylation and the presence of an inositol head group.…”

“…Collectively, these results suggest that the free-amino group of sphingosine contributes strongly to the observed inhibition by these compounds potentially through an electrostatic interaction with a positively charged ammonium salt that would be formed at physiological relevant pH. 23 Similar observations have been previously reported, e.g. the inhibitory effect of sphingosine analogues on the S. cerevisiae phosphatidate phosphatase, 52 a member of the LPP enzyme superfamily believed to share a mechanism of action with the sphingolipid synthases.…”

“…Previously we have demonstrated that whilst N-acetyl-D-erythro-sphingosine was an acceptor substrate, D-erythro-sphingosine was not. 23 Similarly here, MS analyses revealed that whilst N-octanoyl-3-deoxyceramide (26ax) was turned over by the enzyme, its derivative containing a free amine (26an) was not, and functioned as an inhibitor with an IC 50 of 14.7 µM ( Figure 4B). Collectively, these results suggest that the free-amino group of sphingosine contributes strongly to the observed inhibition by these compounds potentially through an electrostatic interaction with a positively charged ammonium salt that would be formed at physiological relevant pH.…”

“…23 In this system each compound was incubated with the labelled acceptor substrate NBD-C 6 -ceramide, and the donor substrate PI in the presence of LmjIPCS. The amount of labelled product, inositol phosphoryl-NBD-C 6 -ceramide (NBD-C 6 -IPC), formed was then quantified.…”

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases

“…However, photoaffinity label ing experiments with diazirine-containing phospholipid analogs (40) combined with a thorough analysis of enzyme kinetics will be necessary to distinguish the above two mechanisms. The latter will be a challenging task [see e.g., Mina et al (41)], because for a bifunctional enzyme like SMS2, it involves systematic variation of the concentra tions of three different substrates (i.e., ceramide, PC, and PE) when only the forward reactions are considered. …”