High-Throughput Screening Assay for Sphingosine Kinase Inhibitors in Whole Blood Using RapidFire(R) Mass Spectrometry (original) (raw)
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SLAS Discovery, 2011
To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire® mass spectrometry system.
Methods in molecular biology (Clifton, N.J.), 2012
Sphingosine-1-phosphate (S1P) and the enzyme primarily responsible for its production, sphingosine kinase-1 (SphK-1), are thought to be dysregulated in multiple human diseases including cancer, multiple sclerosis (MS), diabetes, neurological diseases, fibrosis, and certain pathologies associated with impaired angiogenesis such as, age-related macular degeneration (AMD). Antibody-based techniques to identify and localize S1P and SphK-1 within cells and tissue specimens represent powerful tools not only to understand the biological role of these molecules but also to validate these unique in-class targets in multiple state diseases. Consequently, the potential applications of these molecules for therapy and diagnostic purposes are currently under investigation. Here, we describe two staining procedures for identification of S1P and SphK-1 in human frozen tissue samples and the challenges encountered in the process of localization in tissue samples of lipid molecules, such as S1P.
Sphingosine Kinase Inhibitors and Cancer: Seeking the Golden Sword of Hercules
Cancer Research, 2011
There is considerable evidence that sphingosine kinases play a key role in cancer progression, which might involve positive selection of cancer cells that have been provided with a survival and growth advantage as a consequence of overexpression of the enzyme. Therefore, inhibitors of sphingosine kinase represent a novel class of compounds that have potential as anticancer agents. Poor inhibitor potency is a major issue that has precluded successful translation of these compounds into the clinic. However, recent discoveries have shown that sphingosine kinase 1 is an allosteric enzyme and that some inhibitors offer improved effectiveness by inducing proteasomal degradation of the enzyme or having nanomolar potency. Herein, we provide a perspective about these recent developments and highlight the importance of translating basic pharmacologic and biochemical findings on sphingosine kinase into new drug discovery programs for treatment of cancer. Cancer Res; 71(21); 6576-82. Ó2011 AACR.
Synthesis of selective inhibitors of sphingosine kinase 1
Chemical Communications, 2013
Sphingosine kinase isoform 1 (SK1) inhibitors may serve as therapeutic agents for proliferative diseases, including hypertension. We synthesized a series of sphingosine-based SK1-selective inhibitors, the most potent of which is RB-005 (IC 50 = 3.6 lM), which also induced proteasomal degradation of SK1 in human pulmonary arterial smooth muscle cells.
Synthesis and evaluation of sphingoid analogs as inhibitors of sphingosine kinases
Bioorganic & Medicinal Chemistry, 2005
Sphingosine 1-phosphate (S1P), a product of sphingosine kinases (SphK), mediates diverse biological processes such as cell differentiation, proliferation, motility and apoptosis. In an effort to search and identify specific inhibitors of human SphK, the inhibitory effects of synthetic sphingoid analogs on kinase activity were examined.
Research Square (Research Square), 2022
Introduction: Overexpression of sphingosine kinase 1 (SphK1) is casually associated with many types of cancer and inhibitors of SphK1 sensitize tumors to chemotherapy. SphK1 is expressed as two major isoforms, SphK1a and SphK1b. To date, no information has been reported on the SphK1 isoform expression pro le and its clinical relevance. The purpose of this study was to examine the expression pro le of the SphK1a and SPhK1b isoforms in human cancer and noncancer tissues and cell lines and to explore its clinical relevance. Methods: We used PCR to qualitatively examine the expression pro le of these two isoforms in breast, liver, and prostate cancer tissues plus paired adjacent tissues, and in 11 cancer and normal cell lines (breast, cervical, bone, prostate, colon, brain, and mesothelioma tumors and benign and human kidney cells). Results: We found that SphK1a was ubiquitously expressed in all cancer cells and tissues tested; in contrast, SphK1b was only expressed in selective cell types in breast, prostate, and lung cancer. Conclusion: Our data suggest that SphK1a is important for generic SphK1/S1P functions and SphK1b mediates specialized and / or unique pathways in a speci c type of tissues and could be a biomarker of cancer. This discovery is important for future SphK1-related cancer research and may have clinical implications in drug development associated with SphK1-directed cancer treatment.
In vitroanti-leukaemia activity of sphingosine kinase inhibitor
British Journal of Haematology, 2009
In vitro anti-leukaemia activity of sphingosine kinase inhibitor Several studies have highlighted the role of sphingolipid metabolism in various diseases, ranging from neurodegenerative to cardiovascular disorders and different forms of cancer. In particular, augmentation of intracellular levels of sphingosine kinase 1 (SPHK1) and of its product Sphingosine-1-Phosphate (S1P) contributes to cell growth, survival and transformation in models of solid tumours as well as of leukaemias (
Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2006
Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the "sphingolipid rheostat", producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases.