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

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.

Mass Media and the Contagion of Fear: The Case of Ebola in America.

BACKGROUND: In the weeks following the first imported case of Ebola in the U. S. on September 29, 2014, coverage of the very limited outbreak dominated the news media, in a manner quite disproportionate to the actual threat to national public health; by the end of October, 2014, there were only four laboratory confirmed cases of Ebola in the entire nation. Public interest in these events was high, as reflected in the millions of Ebola-related Internet searches and tweets performed in the month following the first confirmed case. Use of trending Internet searches and tweets has been proposed in the past for real-time prediction of outbreaks (a field referred to as "digital epidemiology"), but accounting for the biases of public panic has been problematic. In the case of the limited U. S. Ebola outbreak, we know that the Ebola-related searches and tweets originating the U. S. during the outbreak were due only to public interest or panic, providing an unprecedented means to determine how these dynamics affect such data, and how news media may be driving these trends. METHODOLOGY: We examine daily Ebola-related Internet search and Twitter data in the U. S. during the six week period ending Oct 31, 2014. TV news coverage data were obtained from the daily number of Ebola-related news videos appearing on two major news networks. We fit the parameters of a mathematical contagion model to the data to determine if the news coverage was a significant factor in the temporal patterns in Ebola-related Internet and Twitter data. CONCLUSIONS: We find significant evidence of contagion, with each Ebola-related news video inspiring tens of thousands of Ebola-related tweets and Internet searches. Between 65% to 76% of the variance in all samples is described by the news media contagion model.

A high-throughput scintillation proximity assay for sphingosine-1-phosphate lyase.

The emergence of sphingosine-1-phosphate lyase (SPL) as a promising therapeutic target for inflammatory diseases has heightened interest in the identification of small molecules that modulate its activity. The enzymatic activity of SPL is typically measured using radiometric or fluorescence-based assays that require a lipid extraction step, or by direct quantitation of reaction products using mass spectrometry (MS). To facilitate testing large numbers of compounds to identify SPL modulators, we developed a robust scintillation proximity assay (SPA) that is compatible with high-throughput screening (HTS). This assay employs recombinant human full-length SPL in insect cell membrane preparations to catalyze the conversion of biotinylated aminosphingosine-1-[(33)P]phosphate (S1(33)P-biotin) to trans-2-hexadecenal-biotin and ethanolamine [(33)P]phosphate. To validate the SPA and confirm the fidelity of its measurement of SPL enzyme activity, we developed a Rapid-Fire MS method that quantitates nonradiolabeled S1P-biotin. In addition, we developed a simple, scalable method to produce S1(33)P-biotin in quantities sufficient for HTS. The optimized SPA screen in 384-well microplates produced a mean plate-wise Z'-statistic of 0.58 across approximately 3,000 plates and identified several distinct structural classes of SPL inhibitor. Among the inhibitors that the screen identified was one compound with an IC50 of 1.6 µM in the SPA that induced dose-dependent lymphopenia in mice.