Search Results (13)

Following a High-Throughput Screening campaign to discover inhibitors of acid ceramidase, we report the novel and extremely potent covalent inhibitor, 1. Following resynthesis and stability monitoring, we discovered that 1 is chemically unstable and reacts with DMSO at room temperature. This mode of decomposition is likely general for this class of compound, and we urge caution for their use in drug discovery research. Full article

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell-death-promoting signaling lipid that plays a central role in therapy-induced cell death. We previously determined that acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug LCL-805 across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 μM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 μM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide. Full article

Electronegative low-density lipoprotein (LDL(−)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(−). However, these enzymes’ activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously increased in LDL(−) incubated alone at 37 °C, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(−). In addition, LDL(−) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These observations defend the existence of the CDase-like activity’s association with LDL(−). However, neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity detected in LDL(−). Full article

Dysregulation of sphingolipid metabolism plays a complex role in hematological malignancies, beginning with the first historical link between sphingolipids and apoptosis discovered in HL-60 leukemic cells. Numerous manuscripts have reviewed the field including the early discoveries that jumpstarted the studies. Many studies discussed here support a role for sphingolipids, such as ceramide, in combinatorial therapeutic regimens to enhance anti-leukemic effects and reduce resistance to standard therapies. Additionally, inhibitors of specific nodes of the sphingolipid pathway, such as sphingosine kinase inhibitors, significantly reduce leukemic cell survival in various types of leukemias. Acid ceramidase inhibitors have also shown promising results in acute myeloid leukemia. As the field moves rapidly, here we aim to expand the body of literature discussed in previously published reviews by focusing on advances reported in the latter part of the last decade. Full article

SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2–RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor. Full article

Glioblastoma (GBM) remains one of the most aggressive cancers, partially due to its ability to migrate into the surrounding brain. The sphingolipid balance, or the balance between ceramides and sphingosine-1-phosphate, contributes to the ability of GBM cells to migrate or invade. Of the ceramidases which hydrolyze ceramides, acid ceramidase (ASAH1) is highly expressed in GBM samples compared to non-tumor brain. ASAH1 expression also correlates with genes associated with migration and focal adhesion. To understand the role of ASAH1 in GBM migration, we utilized shRNA knockdown and observed decreased migration that did not depend upon changes in growth. Next, we inhibited ASAH1 using carmofur, a clinically utilized small molecule inhibitor. Inhibition of ASAH1 by carmofur blocks in vitro migration of U251 (GBM cell line) and GBM cells derived from patient-derived xenografts (PDXs). RNA-sequencing suggested roles for carmofur in MAPK and AKT signaling. We found that carmofur treatment decreases phosphorylation of AKT, but not of MAPK. The decrease in AKT phosphorylation was confirmed by shRNA knockdown of ASAH1. Our findings substantiate ASAH1 inhibition using carmofur as a potential clinically relevant treatment to advance GBM therapeutics, particularly due to its impact on migration. Full article

Vemurafenib (PLX4032), small-molecule inhibitor of mutated BRAFV600E protein, has emerged as a potent anti-cancer agent against metastatic melanoma harboring BRAFV600E mutation. Unfortunately, the effect of PLX4032 in the treatment of metastatic BRAF mutated colorectal cancer (CRC) is less potent due to high incidence of fast-developing chemoresistance. It has been demonstrated that sphingolipids are important mediators of chemoresistance to various therapies in colon cancer. In this study, we will explore the role of major regulators of sphingolipid metabolism and signaling in the development of resistance to vemurafenib in BRAF mutant colon cancer cells. The obtained data revealed significantly increased expression levels of activated sphingosine kinases (SphK1 and SphK2) in resistant cells concomitant with increased abundance of sphingosine-1-phosphate (S1P) and its precursor sphingosine, which was accompanied by increased expression levels of the enzymes regulating the ceramide salvage pathway, namely ceramide synthases 2 and 6 and acid ceramidase, especially after the exposure to vemurafenib. Pharmacological inhibition of SphK1/SphK2 activities or modulation of ceramide metabolism by exogenous C6-ceramide enhanced the anti-proliferative effect of PLX4032 in resistant RKO cells in a synergistic manner. It is important to note that the inhibition of SphK2 by ABC294640 proved effective at restoring the sensitivity of resistant cells to vemurafenib at the largest number of combinations of sub-toxic drug concentrations with minimal cytotoxicity. Furthermore, the obtained findings revealed that enhanced anti-proliferative, anti-migratory, anti-clonogenic and pro-apoptotic effects of a combination treatment with ABC294640 and PLX4032 relative to either drug alone were accompanied by the inhibition of S1P-regulated AKT activity and concomitant abrogation of AKT-mediated cellular levels of nucleophosmin and translationally-controlled tumour protein. Collectively, our study suggests the possibility of using the combination of ABC294640 and PLX4032 as a novel therapeutic approach to combat vemurafenib resistance in BRAF mutant colon cancer, which warrants additional preclinical validation studies. Full article

Previous work utilizing proteomic and immunohistochemical analyses has identified that high levels of acid ceramidase (AC) expression confers a poorer response to neoadjuvant treatment in locally advanced rectal cancer. We aimed to assess the radiosensitising effect of biological and pharmacological manipulation of AC and elucidate the underlying mechanism. AC manipulation in three colorectal cancer cell lines (HT29, HCT116 and LIM1215) was achieved using siRNA and plasmid overexpression. Carmofur and a novel small molecular inhibitor (LCL521) were used as pharmacological AC inhibitors. Using clonogenic assays, we demonstrate that an siRNA knockdown of AC enhanced X-ray radiosensitivity across all colorectal cancer cell lines compared to a non-targeting control siRNA, and conversely, AC protein overexpression increased radioresistance. Using CRISPR gene editing, we also generated AC knockout HCT116 cells that were significantly more radiosensitive compared to AC-expressing cells. Similarly, two patient-derived organoid models containing relatively low AC expression were found to be comparatively more radiosensitive than three other models containing higher levels of AC. Additionally, AC inhibition using carmofur and LCL521 in three colorectal cancer cell lines increased cellular radiosensitivity. Decreased AC protein led to significant poly-ADP ribose polymerase-1 (PARP-1) cleavage and apoptosis post-irradiation, which was shown to be executed through a p53-dependent process. Our study demonstrates that expression of AC within colorectal cancer cell lines modulates the cellular response to radiation, and particularly that AC inhibition leads to significantly enhanced radiosensitivity through an elevation in apoptosis. This work further solidifies AC as a target for improving radiotherapy treatment of locally advanced rectal cancer. Full article

Racemic chiral O,N-heterocycles containing 2-arylchroman or 2-aryl-2H-chromene subunit condensed with morpholine, thiazole, or pyrrole moieties at the C-3-C-4 bond were synthesized with various substitution patterns of the aryl group by the cyclization of cis- or trans-3-aminoflavanone analogues. The 3-aminoflavanone precursors were obtained in a Neber rearrangement of oxime tosylates of flavanones, which provided the trans diastereomer as the major product and enabled the isolation of both the cis- and trans-diastereomers. The cis- and trans-aminoflavanones were utilized to prepare three diastereomers of 5-aryl-chromeno[4,3-b][1,4]oxazines. Antiproliferative activity of the condensed heterocycles and precursors was evaluated against A2780 and WM35 cancer cell lines. For a 3-(N-chloroacetylamino)-flavan-4-ol derivative, showing structural analogy with acyclic acid ceramidase inhibitors, 0.15 μM, 3.50 μM, and 6.06 μM IC₅₀ values were measured against A2780, WM35, and HaCat cell lines, and apoptotic mechanism was confirmed. Low micromolar IC₅₀ values down to 2.14 μM were identified for the thiazole- and pyrrole-condensed 2H-chromene derivatives. Enantiomers of the condensed heterocycles were separated by HPLC using chiral stationary phase, HPLC-ECD spectra were recorded and TDDFT-ECD calculations were performed to determine the absolute configuration and solution conformation. Characteristic ECD transitions of the separated enantiomers were correlated with the absolute configuration and effect of substitution pattern on the HPLC elution order was determined. Full article

Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH₂, led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE₂, which correlated with the upregulation of numerous NF_kB-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu. Full article

Farber disease is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments for Farber disease are clinically available, and affected patients have a severely shortened lifespan. We have recently reported a novel acid ceramidase deficiency model that mirrors the human disease closely. Acid sphingomyelinase is the enzyme that generates ceramide upstream of acid ceramidase in the lysosomes. Using our acid ceramidase deficiency model, we tested if acid sphingomyelinase could be a potential novel therapeutic target for the treatment of Farber disease. A number of functional acid sphingomyelinase inhibitors are clinically available and have been used for decades to treat major depression. Using these as a therapeutic for Farber disease, thus, has the potential to improve central nervous symptoms of the disease as well, something all other treatment options for Farber disease can’t achieve so far. As a proof-of-concept study, we first cross-bred acid ceramidase deficient mice with acid sphingomyelinase deficient mice in order to prevent ceramide accumulation. Double-deficient mice had reduced ceramide accumulation, fewer disease manifestations, and prolonged survival. We next targeted acid sphingomyelinase pharmacologically, to test if these findings would translate to a setting with clinical applicability. Surprisingly, the treatment of acid ceramidase deficient mice with the acid sphingomyelinase inhibitor amitriptyline was toxic to acid ceramidase deficient mice and killed them within a few days of treatment. In conclusion, our study provides the first proof-of-concept that acid sphingomyelinase could be a potential new therapeutic target for Farber disease to reduce disease manifestations and prolong survival. However, we also identified previously unknown toxicity of the functional acid sphingomyelinase inhibitor amitriptyline in the context of Farber disease, strongly cautioning against the use of this substance class for Farber disease patients. Full article

Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system. With its overall dismal prognosis (the median survival is 14 months), GBMs demonstrate a resounding resilience against all current treatment modalities. The absence of a major progress in the treatment of GBM maybe a result of our poor understanding of both GBM tumor biology and the mechanisms underlying the acquirement of treatment resistance in recurrent GBMs. A comprehensive understanding of these markers is mandatory for the development of treatments against therapy-resistant GBMs. This review also provides an overview of a novel marker called acid ceramidase and its implication in the development of radioresistant GBMs. Multiple signaling pathways were found altered in radioresistant GBMs. Given these global alterations of multiple signaling pathways found in radioresistant GBMs, an effective treatment for radioresistant GBMs may require a cocktail containing multiple agents targeting multiple cancer-inducing pathways in order to have a chance to make a substantial impact on improving the overall GBM survival. Full article

Glioblastoma is the most common, malignant primary tumor of the central nervous system. The average prognosis for life expectancy after diagnosis, with the triad of surgery, chemotherapy, and radiation therapy, is less than 1.5 years. Chemotherapy treatment is mostly limited to temozolomide. In this paper, the authors review an emerging, novel drug called acid ceramidase, which targets glioblastoma. Its role in cancer treatment in general, and more specifically, in the treatment of glioblastoma, are discussed. In addition, the authors provide insights on acid ceramidase as a potential druggable target for glioblastoma. Full article