Inositol Phosphorylceramide Synthase Inhibitors Research Articles

Evaluation of the Leishmania Inositol Phosphorylceramide Synthase as a Drug Target Using a Chemical and Genetic Approach.

The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.

Characterization of yeast mutants lacking alkaline ceramidases YPC1 and YDC1.

Humans and yeast possess alkaline ceramidases located in the early secretory pathway. Single deletions of the highly homologous yeast alkaline ceramidases YPC1 and YDC1 have very little genetic interactions or phenotypes. Here, we performed chemical-genetic screens to find deletions/conditions that would alter the growth of ypc1∆ydc1∆ double mutants. These screens were essentially negative, demonstrating that ceramidase activity is not required for cell growth even under genetic stresses. A previously reported protein targeting defect of ypc1∆ could not be reproduced and reported abnormalities in sphingolipid biosynthesis detected by metabolic labeling do not alter the mass spectrometric lipid profile of ypc1∆ydc1∆ cells. Ceramides of ypc1∆ydc1∆ remained normal even in presence of aureobasidin A, an inhibitor of inositolphosphorylceramide synthase. Moreover, in caloric restriction conditions Ypc1p reduces chronological life span. A novel finding is that, when working backwards as a ceramide synthase in vivo, Ypc1p prefers C24 and C26 fatty acids as substrates, whereas it prefers C16:0, when solubilized in detergent and working in vitro. Therefore, its physiological activity may not only concern the minor ceramides containing C14 and C16. Intriguingly, so far the sole discernable benefit of conserving YPC1 for yeast resides with its ability to convey relative resistance toward H2O2.

Role of mature sphingolipids in yeast: new tools

Sphingolipids of yeast have been described as being important for numerous cell biological phenomena such as heat resistance, endocytosis, stress resistance and many others. The genetic or pharmacological elimination of specific features or entire classes of sphingolipids has pinpointed specific sphingolipids as pivotal regulators in many processes. The report by Epstein et al. adds two new tools for such studies: a strain being completely resistant to aureobasidin A, a specific inhibitor of inositol phosphorylceramide synthase and a second strain where this synthase is deleted. The resulting phenotypes advocate new roles of complex sphingolipids in cytokinesis, lipid droplet biogenesis and cell survival.

Requirement of a specific group of sphingolipid‐metabolizing enzyme for growth of yeast Saccharomyces cerevisiae under impaired metabolism of glycerophospholipids

Sphingolipids play critical roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened for yeast mutants showing high sensitivity to Aureobasidin A, an inhibitor of inositol phosphorylceramide synthase, and found that a lack of SAC1 encoding phosphoinositides phosphatase causes high sensitivity to the inhibitor. Double mutation analysis involving the SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that csg1Δ, csg2Δ, ipt1Δ or scs7Δ causes synthetic lethality with deletion of SAC1. As previously reported, SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase complemented the growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in synthetic growth defect with the deletion of CSG1, IPT1 or SCS7. The growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1- or PSS1-repressive conditions were also complemented by overexpression of Arf-GAP AGE1, which encodes a protein related to membrane trafficking. Under SAC1-repressive conditions, scs7Δ, csg1Δ and ipt1Δ cells showed defects in vacuolar morphology, which were complemented by overexpression of each of PSS1 and AGE1. These results suggested that a specific group of sphingolipid-metabolizing enzyme is required for yeast cell growth under impaired metabolism of glycerophospholipids.

Haplofungins, novel inositol phosphorylceramide synthase inhibitors, from Lauriomyces bellulus SANK 26899 III. Absolute structure of haplofungin A

The absolute structure of haplofungin A, a potent fungal inositol phosphoceramide (IPC) synthase inhibitor from Lauriomyces bellulus SANK 26899, was determined by chiral GC/MS analysis, NMR and X-ray crystallographic analysis of the derivatives of degradation products.

Haplofungins, new inositol phosphorylceramide synthase inhibitors, from Lauriomyces bellulus SANK 26899 II. Structure elucidation

Eight new inositol phosphorylceramide synthase inhibitors: haplofungin A, B, C, D, E, F, G and H, were discovered in a culture broth of the fungus Lauriomyces bellulus SANK 26899. The planar structures for these haplofungins were elucidated by various spectroscopic analyses and a GC/MS analysis of their degradation products. All eight compounds were found to comprise an arabinonic acid moiety linked through an ester bond to a modified long alkyl chain.

Haplofungins, novel inositol phosphorylceramide synthase inhibitors, from Lauriomyces bellulus SANK 26899 I. Taxonomy, fermentation, isolation and biological activities

In the course of screening for antifungal agents, we have discovered eight novel compounds, haplofungin A, B, C, D, E, F, G and H, from a culture broth of the fungus strain Lauriomyces bellulus SANK 26899. Haplofungins are composed of an arabinonic acid moiety linked through an ester to a modified long alkyl chain and show potent inhibitory activities against fungal inositol phosphorylceramide (IPC) synthase. Haplofungin A inhibited the activity of IPC synthase from Saccharomyces cerevisiae with an IC(50) value of 0.0015 microg ml(-1). This inhibitor also suppressed the growth of Candida glabrata at the MIC value of 0.5 microg ml(-1).

Pleofungins, Novel Inositol Phosphorylceramide Synthase Inhibitors, from Phoma sp. SANK 13899. Part 1. Taxonomy, Fermentation, Isolation, and Biological Activities.

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Pleofungins, Novel Inositol Phosphorylceramide Synthase Inhibitors, from Phoma sp. SANK 13899

In the course of a screening for inositol phosphorylceramide (IPC) synthase inhibitors, the novel inhibitors pleofungins A, B, C, and D were found in a mycelial extract of a fungus, Phoma sp. SANK13899. Purification was performed by 50% methanol and ethyl acetate extraction, reversed phase open-column chromatography, and HPLC separations. Pleofungin A inhibited the IPC synthase of Saccharomyces cerevisiae and Aspergillus fumigatus at IC(50) values of 16 and 1.0 ng/ml, respectively. The inhibitor also suppressed the growth of Candida albicans, Cryptococcus neoformans, and A. fumigatus at MIC values of 2.0, 0.3, and 0.5 mug/ml, respectively. These biological properties indicate that pleofungins belong to a novel class of IPC synthase inhibitors efficacious against A. fumigatus.

IPC Synthase as a Useful Target for Antifungal Drugs

Inositol phosphorylceramide (IPC) synthase is a common and essential enzyme in fungi and plants, which catalyzes the transfer of phosphoinositol to the C-1 hydroxy of ceramide to produce IPC. This reaction is a key step in fungal sphingolipid biosynthesis, therefore the enzyme is a potential target for the development of nontoxic therapeutic antifungal agents. Natural products with a desired biological activity, aureobasidin A (AbA), khafrefungin, and galbonolide A, have been reported. AbA, a cyclic depsipeptide containing 8 amino acids and a hydroxyl acid, is a broad spectrum antifungal with strong activity against many pathogenic fungi such as Candida spp., Cryptococcus neoformans, and some Aspergillus spp. Khafrefungin, an aldonic acid ester with a C22 long alkyl chain, has antifungal activity against C. albicans, Cr. Neoformans, and Saccharomyces cerevisiae. Galbonolide A is a 14-membered macrolide with fungicidal activity against clinically important strains, and is especially potent against Cr. neoformans. These classes of natural products are potent and specific antifungal agents. We review current progress in the development of IPC synthase inhibitors with antifungal activities, and present structure-activity relationships (SAR), physicochemical and structural properties, and synthetic methodology for chemical modification.