A high-throughput sphingomyelinase assay using natural substrate

Abstract

Sphingomyelinases are a group of hydrolases that cleave sphingomyelin, a common component of plasma membranes, to form ceramide and phosphocholine. Ceramide is a second messenger that is present in virtually all cell types and regulates a variety of cellular functions such as proliferation, differentiation, apoptosis, and inflammation response. Inhibition of sphingomyelinase activity to reduce ceramide concentrations has recently emerged as a potential therapeutic approach for several diseases including atherosclerosis, pathogen infections, inflammation, diabetes, and obesity. To effectively screen compound collections for the identification of new sphingomyelinase inhibitors, we have developed a high-throughput assay utilizing the natural substrate sphingomyelin in 1,536-well plate format. The assay has a signal-to-basal ratio of 6.1-fold in pH 5.0 buffer and 4.3-fold in pH 6.5 buffer, indicating a robust assay for compound library screening. A screen of ~300,000 compounds using this assay led to the identification of eight compounds as sphingomyelinase inhibitors (IC50s=1.7 to 38.2 μM) that exhibited different activities between the natural substrate assay and profluorescence substrate assay. The results demonstrate the robustness and effectiveness of the natural substrate sphingomyelinase assay for screening sphingomyelinase inhibitors.

High-Throughput Viability Assay Using an Autonomously Bioluminescent Cell Line with a Bacterial Lux Reporter

“…These assays have been used to assess compound toxicity against diverse cell types, including mammalian, bacterial, and protozoan cells, during lead discovery. 5,6 In addition, they can be used as counterscreens in parallel with other assays to eliminate false positives that arise in HTS campaigns. 4 A common assay to determine cell viability in HTS is the ATP content assay, which relies on cellular ATP in live cells as the limiting reagent in a luciferase enzyme reaction.…”

“…The compound library was prepared and handled as previously described. 5 Briefly, the LOPAC is composed of 1280 unique chemical entities that are pharmacologically active. This library has been extensively used in HTS assay development to evaluate assay performance.…”

“…In addition to its use as a reporter gene for cell-based screening assays, firefly luciferase has been used in the adenosine triphosphate (ATP) content assay for measurement of cell viability in mammalian cells and pathogens, including bacteria, fungi, and protozoa. 5,6 Recently, a bacterial luciferase gene cassette has been transfected into mammalian cells that exhibits constitutive and continuous bioluminescence in live cells. [7][8][9] Unlike the ATP content assay using the firefly luciferase, this bacterial luciferase reporter system does not require exogenous substrate addition because it synthesizes the substrate in live cells.…”

A high-throughput screening assay for assessing the viability of Cryptococcus neoformans under nutrient starvation conditions

“…The compound library was prepared and handled as previously described [9]. Briefly, the powder samples of Library of Pharmacologically Active Compounds (LOPAC) containing 1,280 known compounds were purchased from Sigma-Aldrich.…”

A Robust Liposomal Platform for Direct Colorimetric Detection of Sphingomyelinase Enzyme and Inhibitors

“…A quick and convenient method to determine SMase activity and inhibition is highly desirable in the development of point-of-care sensors for disease detection and treatment. However, current SMase activity and inhibition assays rely on complicated multistep enzymatic reactions and expensive equipment to perform read-outs − and the use of radioactive markers or non-natural substrates, and often measure SMase concentration rather than activity . Similarly, a simple assay to screen for SMase inhibitors rapidly would allow the as-yet untapped potential for SMase inhibitors as therapeutics to be realized.…”