Discovery of novel sphingosine kinase-1 inhibitors. Part 2

Xiang, Yibin; Hirth, Bradford; Kane, John L.; Liao, Junkai; Noson, Kevin; Yee, Christopher; Asmussen, Gary; Fitzgerald, Maria; Klaus, Christine; Booker, Michael L.

doi:10.1016/j.bmcl.2010.06.019

Cited by 41 publications

(33 citation statements)

References 14 publications

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“…Although the performance of the SphK1 ADP 2 FP assay was robust (data not shown), the IC 50 value of 15 mM (Fig. 2) for a specific inhibitor of SphK1 (compound 54) 30 determined in the assay was significantly higher than the reported value of 68 nM in a microfluidic mobility shift assay (Caliper/PerkinElmer). 30 We also evaluated the Adapta (Life Technologies), a TR-FRET version of this assay that uses europium-labeled anti-ADP antibody and Alexa Fluor 647-labeled ADP tracer for TR-FRET readout.…”

Section: Development Of Enzyme Assayscontrasting

confidence: 52%

“…2) for a specific inhibitor of SphK1 (compound 54) 30 determined in the assay was significantly higher than the reported value of 68 nM in a microfluidic mobility shift assay (Caliper/PerkinElmer). 30 We also evaluated the Adapta (Life Technologies), a TR-FRET version of this assay that uses europium-labeled anti-ADP antibody and Alexa Fluor 647-labeled ADP tracer for TR-FRET readout. All combinations of assay signal mode (FP and TR-FRET) and recombinant SphK1 N-terminal tag (GST or 6His) displayed the same shift in potency of the compound 54 in the ADP detection assay (data not shown).…”

Section: Development Of Enzyme Assayscontrasting

confidence: 46%

“…1B). We optimized both assays in 384-well microplates for robust performance, confirmed their sensitivity to inhibition by the SphK1-selective compound 54, 30 and screened *10 6 compounds. The biochemical IMAP screen identified 5,561 concentrationresponsive hits, of which only 17 were ‡10-fold SphK1 selective over SphK2 and all of these were ATP competitive.…”

Section: Introductionmentioning

confidence: 87%

“…2), a value in better agreement with the reported value determined by microfluidic mobility shift assay. 30 We therefore selected the IMAP TR-FRET assay as our preferred HTS-compatible format for SphK1 HTS and developed an SphK2 selectivity assay in the same format.…”

Section: Development Of Enzyme Assaysmentioning

confidence: 99%

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A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay

Kashem

Kennedy

Fogarty

et al. 2016

ASSAY and Drug Development Technologies

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Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.

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Section: Development Of Enzyme Assayscontrasting

confidence: 52%

Section: Development Of Enzyme Assayscontrasting

confidence: 46%

Section: Introductionmentioning

confidence: 87%

Section: Development Of Enzyme Assaysmentioning

confidence: 99%

See 2 more Smart Citations

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay

Kashem

Kennedy

Fogarty

et al. 2016

ASSAY and Drug Development Technologies

View full text Add to dashboard Cite

show abstract

“…Another new generation selective SK1 inhibitor is Genzyme 51, with an IC 50 around 60 nM, an acceptable pharmacokinetic profile and SK2 inhibition at concentrations close to 10 µM. (Xiang et al, 2010(Xiang et al, , 2009). …”

Section: Selective Sk1 Inhibitorsmentioning

confidence: 98%

Inhibitors of sphingosine-1-phosphate metabolism (sphingosine kinases and sphingosine-1-phosphate lyase)

Sanllehí

Abad

Casas

et al. 2016

Chemistry and Physics of Lipids

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Therapeutic Strategies and Pharmacological Tools Influencing S1P Signaling and Metabolism

Vogt

Stark

2016

Med. Res. Rev.

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During the last two decades the study of the sphingolipid anabolic, catabolic, and signaling pathways has attracted enormous interest. Especially the introduction of fingolimod into market as first p.o. therapeutic for the treatment of multiple sclerosis has boosted this effect. Although the complex regulation of sphingosine-1-phosphate (S1P) and other catabolic and anabolic sphingosine-related compounds is not fully understood, the influence on different (patho)physiological states from inflammation to cytotoxicity as well as the availability of versatile pharmacological tools that represent new approaches to study these states are described. Here, we have summarized various aspects concerning the many faces of sphingolipid function modulation by different pharmacological tools up to clinical candidates. Due to the immense heterogeneity of physiological or pharmacological actions and complex cross regulations, it is difficult to predict their role in upcoming therapeutic approaches. Currently, inflammatory, immunological, and/or antitumor aspects are discussed.

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Discovery of novel sphingosine kinase-1 inhibitors. Part 2

Cited by 41 publications

References 14 publications

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay

Inhibitors of sphingosine-1-phosphate metabolism (sphingosine kinases and sphingosine-1-phosphate lyase)

Therapeutic Strategies and Pharmacological Tools Influencing S1P Signaling and Metabolism

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