A High Throughput LC-MS Platform for the Discovery of Autotaxin Inhibitors
thesisposted on 01.11.2017, 00:00 by Yongchao Li
The bioactive group of lipids, the eicosanoids, which includes lysophospholipids and their derivatives, are lipid mediators formed from arachidonic acid. Class 1 lipid mediators include sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA) and endocannabinoids. LPA binds to specific receptors which take part in various biological processes, including brain development, embryo implantation, hair growth, and inflammation. Produced by the enzymatic activity of extracellular autotoxin (ATX), LPA is involved in asthma pathogenesis. Additionally, LPA enhances the production of cytokines and chemotaxis in lymphocytes, which can lead to contractility and proliferation of airway smooth muscle cells as well as changing inflammatory signaling in the epithelial cells of the bronchus. Together, all these results indicate that LPA participates in the molecular pathogenesis of asthma. Our hypothesis is that the ATX-LPA axis plays a critical role in the pathogenesis of asthma and makes it an ideal target for an effective anti-asthma treatment. To test our hypothesis, a human model (an IRB-approved protocol for sub-segmental broncho-provocation with allergen, SBP-AG, to induce localized allergic inflammation in human volunteers), and a mouse model (mice subjected to a triple allergen house dust mite, ragweed and aspergillus allergen; DRA) has been developed to explore the mechanism of ATX-LPA axis on the pathogenesis of asthma. To further explore the function of LPA in asthma, we used a transgenic mouse with ATX over-expression (ATX-Tg) to study the effect of triple allergen challenge compared to that of wild type mice. LPA receptor 2 knock-out mice (LPA2-/-) were also used to carry out additional experiments to provide further evidence to support our hypothesis. The development of sensitive, efficient and high-throughput methods to study the ligand-enzyme binding affinity and their potential to inhibit ATX activity is essential to identify novel ATX inhibitors. ATX can hydrolyze phosphatidylcholine (LPC) into two products, choline and LPA, and both products can be used to measure ATX activity. Previously, several in vitro biochemical assays based on radiometry or fluorescence had been used to screen compounds for potential ATX inhibition by measuring its enzymatic products, and these tested one compound at a time for binding or inhibition. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) also can be used to measure LPA, but most of the previous LC-MS based methods were used to detect naturally occurring LPA in biological fluids (i.e. plasma, bronchoalveolar lavage). Although not previously applied to ATX inhibitor screening, mass spectrometry-based bioassays are useful in the discovery of protein ligands and enzyme inhibitors. For example, pulsed ultrafiltration (PUF) LC-MS based methods, invented in our laboratory, have been used for screening of combinatorial library mixtures and natural product extracts in order to identify ligands for a wide variety of macromolecular targets including quinone reductase-2, cyclooxygenase-2, estrogen receptors, and retinoid X receptor. The main advantages of PUF-LCMS based screening over optical or radioactive detection methods are high throughput, low cost, fewer false positives, and no need to modify either the ligand or the targets by attaching a radiolabel, chromophore or fluorophore.