Inhibition Of Sphingolipid Synthesis Research Articles

Ergosterol promotes aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis.

Tick-borne encephalitis virus modulates sphingolipid and phospholipid metabolism in infected human neuronal cells

The life cycle of enveloped viruses is closely linked to host-cell lipids. However, changes in lipid metabolism during infections with the tick-borne encephalitis virus (TBEV) have not been described. TBEV is a medically important orthoflavivirus, which is endemic to many parts of Europe and Asia. In the present study, we performed targeted lipidomics with HPLC-MS/MS to evaluate changes in phospholipid and sphingolipid concentrations in TBEV-infected human neuronal SK-N-SH cells. TBEV infections significantly increased phosphatidylcholine, phosphatidylinositol, and phosphatidylserine levels within 48 h post-infection (hpi). Sphingolipids were slightly increased in dihydroceramides within 24 hpi. Later, at 48 hpi, the contents of sphinganine, dihydroceramides, ceramides, glucosylceramides, and ganglioside GD3 were elevated. On the other hand, sphingosine-1-phosphate content was slightly reduced in TBEV-infected cells. Changes in sphingolipid concentrations were accompanied by suppressed expression of a majority of the genes linked to sphingolipid and glycosphingolipid metabolism. Furthermore, we found that a pharmacological inhibitor of sphingolipid synthesis, fenretinide (4-HPR), inhibited TBEV infections in SK-N-SH cells. Taken together, our results suggested that both structural and signaling functions of lipids could be affected during TBEV infections. These changes might be connected to virus propagation and/or host-cell defense.

Loss of Flot2 expression in deep cerebellar nuclei neurons of mice with Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is caused by a deficiency of the NPC1 or NPC2 gene, leading to storages of unesterified cholesterol and sphingolipids. Cerebellar ataxia is a main symptom of NPC and the deep cerebellar nuclei (DCN) is the sole signal output of the cerebellum. In this study, we explored the pathological changes in DCN neurons of Npc1 knockout mice (Npc1-). We first demonstrated that DCN neurons of Npc1- mice had prominent ganglioside GM2 accumulation in the late endosomes but not in the lysosomes. More importantly, Flot2 expression, a marker for the lipid rafts, was lost. Single-nucleus RNA sequencing analysis revealed a generalized reduction in gene expression in DCN neurons, though Camk1d, encoding one of the Ca2+/calmodulin-dependent protein kinases (CaMKs), increased in expression. We treated Npc1- mice with CaMK inhibitor KN-93, but CaMK1D expression increased further. We also fed Npc1- mice with two medications for NPC. We found that miglustat, a sphingolipid synthesis inhibitor, increased the expression of Flot2. Moreover, N-acetyl l-leucine (NALL), an experimental medicine for NPC, recovered Flot2 expression. Therefore, our data suggest that in Npc1- mice, GM2 sequestration and the loss of lipid rafts lead to cell dysfunction and symptoms of NPC.

Genetic Tools for Studying the Roles of Sphingolipids in Viral Infections.

Sphingolipids are a critical family of membrane lipids with diverse functions in eukaryotic cells, and a growing body of literature supports that these lipids play essential roles during the lifecycles of viruses. While small molecule inhibitors of sphingolipid synthesis and metabolism are widely used, the advent of CRISPR-based genomic editing techniques allows for nuanced exploration into the manners in which sphingolipids influence various stages of viral infections. Here we describe some of these critical considerations needed in designing studies utilizing genomic editing techniques for manipulating the sphingolipid metabolic pathway, as well as the current body of literature regarding how viruses depend on the products of this pathway. Here, we highlight the ways in which sphingolipids affect viruses as these pathogens interact with and influence their host cell and describe some of the manyopen questions remaining in the field.

Vti1a/b support distinct aspects of TGN and cis-/medial Golgi organization

Retrograde trafficking towards the trans-Golgi network (TGN) is important for dense core vesicle (DCV) biogenesis. Here, we used Vti1a/b deficient neurons to study the impact of disturbed retrograde trafficking on Golgi organization and cargo sorting. In Vti1a/b deficient neurons, staining intensity of cis-/medial Golgi proteins (e.g., GM130 and giantin) was increased, while the intensity of two recycling TGN proteins, TGN38 and TMEM87A, was decreased and the TGN-resident protein Golgin97 was normal. Levels and localization of DCV cargo markers, LAMP1 and KDEL were also altered. This phenotype was not caused by reduced Golgi size or absence of a TGN compartment. The phenotype was partially phenocopied by disturbing sphingolipid homeostasis, but was not rescued by overexpression of sphingomyelin synthases or the sphingolipid synthesis inhibitor myriocin. We conclude that Vti1a/b are important for distinct aspects of TGN and cis-/medial Golgi organization. Our data underline the importance of retrograde trafficking for Golgi organization, DCV cargo sorting and the distribution of proteins of the regulated secretory pathway.

Effect of Rosa canina Methanol Extract on Membrane Trafficking in Different Niemann‐Pick C1 Phenotypes

Niemann Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder that is caused by mutations in Niemann‐Pick C1 (NPC1) or Niemann‐Pick C2 (NPC2) genes ultimately leading to progressive neurological deterioration. 95% of NPC cases arise from mutations in the NPC1 gene. NPC1 protein mediates the intracellular cholesterol trafficking and plays a crucial role in maintaining lipid homeostasis. Mutations in NPC1 are associated with defective cholesterol mobilization and an accumulation of unesterified cholesterol and subsequently sphingomyelin and glycosphingolipids in the late endo‐lysosomes. N‐butyldeoxynojirimycin, known as Miglustat, is one of the approved therapeutic options. It is an inhibitor of sphingolipid synthesis and is used in substrate reduction therapy. Nevertheless, the efficiency of Miglustat varies among NPC patients depending on the type of the mutation and moreover its accompanied by several side effects, such as diarrhea. The focus of this study is to find an alternative therapy that presents less side effects. Rosa canina L. methanol extract (RCME) has been shown to improve the protein trafficking and maintain cholesterol homeostasis. Here, we used skin‐derived fibroblasts from patients harbouring homozygote or compound heterozygote mutations to assess the effects of RCME on the trafficking of NPC1 and cellular cholesterol contents as compared to wild type cells. For this purpose, the cells were treated for 24 h with RCME (100 µg/ml) with and without Miglustat (100 µM) and the trafficking of NPC1 between the endoplasmic reticulum (ER) and the Golgi was examined by endoglycosidase H treatment. This treatment, which discriminates between mannose‐rich N‐linked glycosylated proteins and mature complex glycosylated forms, demonstrated a significant enhancement of the trafficking of a NPC1 mutants between the ER and the Golgi upon RCME treatment as compared to Miglustat. This trafficking behaviour was observed with those NPC1 mutants that are not entirely blocked in the ER and exit the ER at a slow rate. Markedly, the high cholesterol levels measured in the patients’ fibroblasts are substantially reduced upon RCME treatment, while almost similar cholesterol levels were maintained in the wild type cells. Impaired cellular cholesterol trafficking is associated with partial distortion of the lipid membrane composition, particularly of the cholesterol‐ and sphingolipids‐enriched membrane microdomains or lipid rafts (LRs). Our data show that RCME and also Miglustat maintained a normal LRs distribution as assessed by the LR marker flotillin 2.We conclude that RCME possess the capacity to reverse adverse effects caused by mutations in the NPC1gene including impaired NPC1 trafficking, elevated cholesterol levels and lipid raft alterations. All this together render the constituents of RCME, including quercetin, a promising agents in NPC therapy.

Lipid Raft Disruption Alters Human Follicle Stimulating Hormone Receptor Signaling

Over 6.7 million people struggle with infertility each year. Studying signaling by reproductive hormones in fertility can allow us to gain a better understanding of the signaling pathways that must function correctly for proper fertility. Some infertility is due to incorrect human follicle stimulating hormone receptor (hFSHR) function. When follicle stimulating hormone (FSH) binds to hFSHR this begins a signaling cascade where the end product is the maturation of sperm by Sertoli cells in men, and egg development and production of estrogen through stimulation of granulosa cells in women. It has been determined that hFSHR is localized to microdomains of the cell membrane called lipid rafts which are characterized by higher concentrations of sphingolipids and cholesterol. This composition and structure makes them less fluid than non‐raft membranes, and it is believed that lipid rafts regulate signaling of many proteins that reside in the rafts, including hFSHR. It was hypothesized that if the lipid rafts in cell membranes were removed then hFSHR signaling would be altered. To remove lipid rafts HEK293 cells stably expressing hFSHR were treated with sphingolipid synthesis inhibitors (Myriocin or Fumonisin B1) or a cholesterol withdrawing drug (Methyl‐β‐cyclodextrin). Removing either the sphingolipids or cholesterol results in disruption of the lipid rafts. hFSHR signaling from the cells with disrupted lipid rafts were compared to wild type hFSHR signaling by western blot. Two pathways were investigated: cAMP production and the activation of p44/42 MAP kinase (ERK1/2). cAMP production was measured indirectly by detecting phosphorylated cAMP Response Element Binding protein (p‐CREB) and p44/42 MAPK signaling was measured by directly detecting the phosphorylation of the kinase (p‐p44/42). It was found that treatment with the lipid raft disrupting agents resulted in increased basal cAMP production (as measured by pCREB activation). However, time‐dependent hFSH stimulation was decreased compared to cells with intact lipid rafts. In contrast, p‐p44 signaling in the drug treated cells was not altered compared to untreated cells. These results suggest that G protein mediated activation of adenylyl cyclase to produce cAMP is lipid raft dependent while p44/42 MAPK activation is not. Further understanding of how lipid raft residency allows for functional hFSHR signaling would allow for the development of new treatments and pharmaceuticals for men and women struggling with infertility.

Sphingolipid synthesis inhibitor fumonisin B1 causes verticillium wilt in cotton

Verticillium wilt caused by Verticillium dahliae is a major disease of cotton. Acidic protein-lipopolysaccharide complexes are thought to be the toxins responsible for its symptoms. Here, we determined that the sphingolipid biosynthesis inhibitor fumonisin B1 (FB1) acts as a toxin and phenocopies the symptoms induced by V. dahliae. Knocking out genes required for FB1 biosynthesis reduced V. dahliae pathogenicity. Moreover, we showed that overexpression of a FB1 and V. dahliae both downregulated gene, GhIQD10, enhanced verticillium wilt resistance by promoting the expression of brassinosteroid and anti-pathogen genes. Our results provide a new strategy for preventing verticillium wilt in cotton.

Plasma membrane effects of sphingolipid-synthesis inhibition by myriocin in CHO cells: a biophysical and lipidomic study

Suppression of a specific gene effect can be achieved by genetic as well as chemical methods. Each approach may hide unexpected drawbacks, usually in the form of side effects. In the present study, the specific inhibitor myriocin was used to block serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid synthetic pathway, in CHO cells. The subsequent biophysical changes in plasma membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) became lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated GPL than the non-treated ones, although they achieved it only partially, their plasma membranes remaining slightly more fluid and more penetrable than those from the control cells. The good agreement between results obtained with very different tools, such as genetically modified and chemically treated cells, reinforces the use of both methods and demonstrates that both are adequate for their intended use, i.e. the complete and specific inhibition of sphingolipid synthesis in CHO cells, without apparent unexpected effects.

Vacuolar Protein-Sorting Receptor MoVps13 Regulates Conidiation and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

Magnaporthe oryzae (synonym Pyricularia oryzae) is a filamentous fungal pathogen that causes major yield losses in cultivated rice worldwide. However, the mechanisms of infection of M. oryzae are not well characterized. The VPS13 proteins play vital roles in various biological processes in many eukaryotic organisms, including in the organization of actin cytoskeleton, vesicle trafficking, mitochondrial fusion, and phagocytosis. Nevertheless, the function of the Vps13 protein in plant pathogenic fungi has not been explored. Here, we analysed the biological functions of the Vps13 protein in the development and pathogenicity of M. oryzae. Deletion mutants of MoVps13 significantly reduced the conidiation and decreased the rate of fungal infection on hosts. Moreover, the loss of MoVps13 resulted in defective cell wall integrity (CWI) and plasma membrane (PM) homeostasis when treated with chemicals for inducing cell wall stress (200 mg/mL Congo Red or 0.005% SDS) and sphingolipid synthesis inhibitors (2 μM myriocin or 2 μM amphotericin B). This indicated that MoVps13 is also involved in cell wall synthesis and sphingolipid synthesis. Through immunoblotting, autophagic flux detection, co-localization, and chemical drug sensitivity assays, we confirmed the involvement of Movps13 in ER-phagy and the response to ER stress. Additionally, we generated the C-terminal structure of MoVps13 with high accuracy using the alphaflod2 database. Our experimental evidence indicates that MoVps13 is an important virulence factor that regulates the pathogenicity of M. oryzae by controlling CWI, lipid metabolism and the ER-phagy pathway. These results have expanded our knowledge about pathogenic fungi and will help exploration for novel therapeutic strategies against the rice blast fungus.

Sphingolipid Inhibitors as an Alternative to Treat Candidiasis Caused by Fluconazole-Resistant Strains.

Candida species are fungal pathogens known to cause a wide spectrum of diseases, and Candida albicans and Candida glabrata are the most common associated with invasive infections. A concerning aspect of invasive candidiasis is the emergence of resistant isolates, especially those highly resistant to fluconazole, the first choice of treatment for these infections. Fungal sphingolipids have been considered a potential target for new therapeutic approaches and some inhibitors have already been tested against pathogenic fungi. The present study therefore aimed to evaluate the action of two sphingolipid synthesis inhibitors, aureobasidin A and myriocin, against different C. albicans and C. glabrata strains, including clinical isolates resistant to fluconazole. Susceptibility tests of aureobasidin A and myriocin were performed using CLSI protocols, and their interaction with fluconazole was evaluated by a checkerboard protocol. All Candida strains tested were sensitive to both inhibitors. Regarding the evaluation of drug interaction, both aureobasidin A and myriocin were synergic with fluconazole, demonstrating that sphingolipid synthesis inhibition could enhance the effect of fluconazole. Thus, these results suggest that sphingolipid inhibitors in conjunction with fluconazole could be useful for treating candidiasis cases, especially those caused by fluconazole resistant isolates.

Regulation of sphingolipid synthesis by the G1/S transcription factor Swi4

SBF (Swi4/Swi6 Binding Factor) complex is a crucial regulator of G1/S transition in Saccharomyces cerevisiae. Here, we show that SBF complex is required for myriocin resistance, an inhibitor of sphingolipid synthesis. This phenotype was not shared with MBF complex mutants nor with deletion of the Swi4p downstream targets, CLN1/CLN2. Based on data mining results, we selected putative Swi4p targets related to sphingolipid metabolism and studied their gene transcription as well as metabolite levels during progression of the cell cycle. Genes which encode key enzymes for the synthesis of long chain bases (LCBs) and ceramides were periodically transcribed during the mitotic cell cycle, having a peak at G1/S, and required SWI4 for full transcription at this stage. In addition, HPLC-MS/MS data indicated that swi4Δ cells have decreased levels of sphingolipids during progression of the cell cycle, particularly, dihydrosphingosine (DHS), C24-phytoceramides and C24-inositolphosphoryl ceramide (IPC) while it had increased levels of mannosylinositol phosphorylceramide (MIPC). Furthermore, we demonstrated that both inhibition of de novo sphingolipid synthesis by myriocin or SWI4 deletion caused partial arrest at the G2/M phase. Importantly, our lipidomic data demonstrated that the sphingolipid profile of WT cells treated with myriocin resembled that of swi4Δ cells, with lower levels of DHS, IPC and higher levels of MIPC. Taken together, these results show that SBF complex plays an essential role in the regulation of sphingolipid homeostasis, which reflects in the correct progression through the G2/M phase of the cell cycle.

Sphingosine Promotes Embryo Biomass in Upland Cotton: A Biochemical and Transcriptomic Analysis.

Sphingolipids are essential membrane components and signal molecules, but their regulatory role in cotton embryo growth is largely unclear. In this study, we evaluated the effects of treatment with the sphingolipid synthesis inhibitor fumonisin B1 (FB1), the serine palmityl transferase (SPT) inhibitor myriocin, the SPT sphingolipid product DHS (d18:0 dihydrosphingosine), and the post-hydroxylation DHS product PHS (t18:0 phytosphingosine) on embryo growth in culture, and performed comparative transcriptomic analysis on control and PHS-treated samples. We found that FB1 could inhibit cotton embryo development. At the five-day ovule/embryo developmental stage, PHS was the most abundant sphingolipid. An SPT enzyme inhibitor reduced the fresh weight of embryos, while PHS had the opposite effect. The transcriptomic analysis identified 2769 differentially expressed genes (1983 upregulated and 786 downregulated) in the PHS samples. A large number of transcription factors were highly upregulated, such as zinc finger, MYB, NAC, bHLH, WRKY, MADS, and GRF in PHS-treated samples compared to controls. The lipid metabolism and plant hormone (auxin, brassinosteroid, and zeatin) related genes were also altered. Our findings provide target metabolites and genes for cotton seed improvement.

Fumonisin B1-Induced Changes in Cotton Fiber Elongation Revealed by Sphingolipidomics and Proteomics.

Sphingolipids are essential biomolecules and membrane components, but their regulatory role in cotton fiber development is poorly understood. Here, we found that fumonisin B1 (FB1)—a sphingolipid synthesis inhibitor—could block fiber elongation severely. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we detected 95 sphingolipids that were altered by FB1 treatment; of these, 29 (mainly simple sphingolipids) were significantly increased, while 33 (mostly complex sphingolipids) were significantly decreased. A quantitative analysis of the global proteome, using an integrated quantitative approach with tandem mass tag (TMT) labeling and LC-MS/MS, indicated the upregulation of 633 and the downregulation of 672 proteins after FB1 treatment. Most differentially expressed proteins (DEPs) were involved in processes related to phenylpropanoid and flavonoid biosynthesis. In addition, up to 20 peroxidases (POD) were found to be upregulated, and POD activity was also increased by the inhibitor. To our knowledge, this is the first report on the effects of FB1 treatment on cotton fiber and ovule sphingolipidomics and proteomics. Our findings provide target metabolites and biological pathways for cotton fiber improvement.

Cystic Fibrosis Defective Response to Infection Involves Autophagy and Lipid Metabolism.

Cystic fibrosis (CF) is a hereditary disease, with 70% of patients developing a proteinopathy related to the deletion of phenylalanine 508. CF is associated with multiple organ dysfunction, chronic inflammation, and recurrent lung infections. CF is characterized by defective autophagy, lipid metabolism, and immune response. Intracellular lipid accumulation favors microbial infection, and autophagy deficiency impairs internalized pathogen clearance. Myriocin, an inhibitor of sphingolipid synthesis, significantly reduces inflammation, promotes microbial clearance in the lungs, and induces autophagy and lipid oxidation. RNA-seq was performed in Aspergillusfumigatus-infected and myriocin-treated CF patients’ derived monocytes and in a CF bronchial epithelial cell line. Fungal clearance was also evaluated in CF monocytes. Myriocin enhanced CF patients’ monocytes killing of A. fumigatus. CF patients’ monocytes and cell line responded to infection with a profound transcriptional change; myriocin regulates genes that are involved in inflammation, autophagy, lipid storage, and metabolism, including histones and heat shock proteins whose activity is related to the response to infection. We conclude that the regulation of sphingolipid synthesis induces a metabolism drift by promoting autophagy and lipid consumption. This process is driven by a transcriptional program that corrects part of the differences between CF and control samples, therefore ameliorating the infection response and pathogen clearance in the CF cell line and in CF peripheral blood monocytes.

Inhibition of sphingolipid synthesis improves outcomes and survival in GARP mutant wobbler mice, a model of motor neuron degeneration.

Numerous mutations that impair retrograde membrane trafficking between endosomes and the Golgi apparatus lead to neurodegenerative diseases. For example, mutations in the endosomal retromer complex are implicated in Alzheimer's and Parkinson's diseases, and mutations of the Golgi-associated retrograde protein (GARP) complex cause progressive cerebello-cerebral atrophy type 2 (PCCA2). However, how these mutations cause neurodegeneration is unknown. GARP mutations in yeast, including one causing PCCA2, result in sphingolipid abnormalities and impaired cell growth that are corrected by treatment with myriocin, a sphingolipid synthesis inhibitor, suggesting that alterations in sphingolipid metabolism contribute to cell dysfunction and death. Here we tested this hypothesis in wobbler mice, a murine model with a homozygous partial loss-of-function mutation in Vps54 (GARP protein) that causes motor neuron disease. Cytotoxic sphingoid long-chain bases accumulated in embryonic fibroblasts and spinal cords from wobbler mice. Remarkably, chronic treatment of wobbler mice with myriocin markedly improved their wellness scores, grip strength, neuropathology, and survival. Proteomic analyses of wobbler fibroblasts revealed extensive missorting of lysosomal proteins, including sphingolipid catabolism enzymes, to the Golgi compartment, which may contribute to the sphingolipid abnormalities. Our findings establish that altered sphingolipid metabolism due to GARP mutations contributes to neurodegeneration and suggest that inhibiting sphingolipid synthesis might provide a useful strategy for treating these disorders.

The Regulatory Proteins Rtg1/3 Govern Sphingolipid Homeostasis in the Human-Associated Yeast Candida albicans

Integrating nutrient sensing with the synthesis of complex molecules is a central feature of metabolism. Yet the regulatory mechanisms underlying such integration are often unknown. Here, we establish that the transcription regulators Rtg1/3 are key determinants of sphingolipid homeostasis in the human fungal pathogen Candida albicans. Quantitative analysis of the C.albicans lipidome reveals Rtg1/3-dependent alterations in all complex sphingolipids and their precursors, ceramides. Mutations in the regulators render the fungus susceptible to myriocin, a sphingolipid synthesis inhibitor. Rtg1/3 exert control on the expression of several enzymes involved in the synthesis of sphingolipids' building blocks, and the regulators are activated upon engulfment of C.albicans cells by human neutrophils. We demonstrate that Rtg1p and Rtg3p are regulated at two levels, one in response to sphingolipids and the other by the nutrient sensor TOR. Our findings, therefore, indicate that the Rtg1/3 system integrates nutrient sensing into the synthesis of complex lipids.

Sphingolipid Mediators of Myocardial Pathology.

Cardiomyopathy is the leading cause of mortality worldwide. While the causes of cardiomyopathy continue to be elucidated, current evidence suggests that aberrant bioactive lipid signaling plays a crucial role as a component of cardiac pathophysiology. Sphingolipids have been implicated in the pathophysiology of cardiovascular disease, as they regulate numerous cellular processes that occur in primary and secondary cardiomyopathies. Experimental evidence gathered over the last few decades from both in vitro and in vivo model systems indicates that inhibitors of sphingolipid synthesis attenuate a variety of cardiomyopathic symptoms. In this review, we focus on various cardiomyopathies in which sphingolipids have been implicated and the potential therapeutic benefits that could be gained by targeting sphingolipid metabolism.

A sphingolipid synthesis-related protein OrmA in Aspergillus fumigatus is responsible for azole susceptibility and virulence.

Previous studies identified that the budding yeast Saccharomyces cerevisiae have two sphingolipid synthesis-related proteins, Orm1p and Orm2p, that negatively regulate the activities of SPT, which is a key rate-limiting enzyme in sphingolipid synthesis. However, little is known about whether sphingolipids in the cell membrane, which are closely related to ergosterols, could affect the efficacy of azole drugs, which target to the ergosterol biosynthesis. In this study, through genome-wide homologue search analysis, we found that the Aspergillus fumigatus genome only contains one Orm homologue, referred to as OrmA for which the protein expression could be induced by azole antifungals in a dose-dependent manner. Deletion of ormA caused hypersensitivity to azoles, and adding the sphingolipid synthesis inhibitor myriocin rescued the azole susceptibility induced by lack of ormA. In contrast, overexpression of OrmA resulted in azole resistance, indicating that OrmA is a positive azole-response regulator. Further mechanism analysis verified that OrmA is related to drug susceptibility by affecting endoplasmic reticulum stress responses in an unfolded protein response pathway-HacA-dependent manner. Lack of ormA led to an abnormal profile of sphingolipid ceramide components accompanied by hypersensitivity to low temperatures. Furthermore, deletion of OrmA significantly reduced virulence in an immunosuppressed mouse model. The findings in this study collectively suggest that the sphingolipid metabolism pathway in A. fumigatus plays a critical role in azole susceptibility and fungal virulence.

SAR Studies on Aromatic Acylhydrazone-Based Inhibitors of Fungal Sphingolipid Synthesis as Next-Generation Antifungal Agents.

Recently, the fungal sphingolipid glucosylceramide (GlcCer) synthesis has emerged as a highly promising new target for drug discovery of next-generation antifungal agents, and we found two aromatic acylhydrazones as effective inhibitors of GlcCer synthesis based on HTP screening. In the present work, we have designed libraries of new aromatic acylhydrazones, evaluated their antifungal activities (MIC80 and time-kill profile) against C. neoformans, and performed an extensive SAR study, which led to the identification of five promising lead compounds, exhibiting excellent fungicidal activities with very large selectivity index. Moreover, two compounds demonstrated broad spectrum antifungal activity against six other clinically relevant fungal strains. These five lead compounds were examined for their synergism/cooperativity with five clinical drugs against seven fungal strains, and very encouraging results were obtained; e.g., the combination of all five lead compounds with voriconazole exhibited either synergistic or additive effect to all seven fungal strains.