Capture and Identification of a Novel Protein in the Necrotic Cell Death Pathway and Toward Discovery of Inhibitors of Human Group III Secreted Phospholipase A2 and Lessons Learned from In Vivo Mouse Studies of Secreted Phospholipase A2 Clearance from Extra-Cellular Medium by the M-type Receptor

Abstract

Chapter I. Capture and Identification of a Novel Protein in the Necrotic Cell Death Pathway The serine hydrolase inhibitors Pyrrophenone and KT195 were found to inhibit cell death induced by A23187 and H2O2 in mouse lung fibroblast cells. The effect of Pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A2 and α/β-hydrolase domaincontaining (ABHD) 6, respectively, but represents off-target effects. To identify the target/s of Pyrrophenone and KT195, several activity-based protein profiling strategies were employed which were dependent upon the synthesis of derivatives of both compounds. Ultimately, a strategy employing the use of an alkynyl derivative of KT195 to label the protein target followed by click chemistry with biotin azide, enrichment on streptavidin beads, and tryptic peptide analysis by mass spectrometry was able to identify ABHD2, a poorly characterized enzyme, as the protein target. The results identified a novel mechanism for regulating cell death induced by A23187 and H2O2 that involves the serine hydrolase ABHD2. Chapter II. Toward Discovery of Inhibitors of Human Group III Secreted Phospholipase A2 The development of inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) is an important undertaking since this enzyme has been shown to play a role in mast cell function and maturation. Currently, no potent inhibitors of hGIII-sPLA2 have been reported. Herein, we describe the adaptation of a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. The substrate is a BODIPY fluorophore containing phospholipid analogue dispersed as a minor component in non-fluorescent phospholipid vesicles to more closely resemble the natural substrate of hGIII-sPLA2. Cleavage of the BODIPY substrate leads to PLA2 dependent increase in fluorescence. The assay was optimized in both 96-well and 1536-well plate formats and uses optical detection at a wavelength outside of the UV range so as to minimize false-positive hits that result from quenching of the fluorescence. A high-throughput screen was successfully carried out on a library of 370,276 small molecules. Additionally, a panel of known sPLA2 inhibitors was screened using the 96-well plate format. Several hits were discovered, and the resulting data has been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate. Chapter III. Lessons Learned from In Vivo Mouse Studies of Secreted Phospholipase A2 Clearance from Extra-Cellular Medium by the M-type Receptor The in vivo biological functions of many mammalian secreted phospholipase A2s (sPLA2s) remain to be elucidated, especially with respect to their non-catalytic activity. Among sPLA2 binding proteins, the best known target is the M-type receptor which is hypothesized to play a role in the removal sPLA2s from extracellular fluids such as blood. The binding properties of the full set of mouse sPLA2s to the mouse M-type receptor have been analyzed in vitro. Of interest, mGIIA-sPLA2, and mGX-sPLA2 were found to be high-affinity ligands of the M-type mouse receptor. To study the in vivo molecular mechanism by which secreted phospholipases A2 are removed from blood, mice containing or lacking the M-type receptor were injected i.v. with secreted phospholipase A2 protein that bore a radioiodine tag as a tracer. The clearance and biodistribution of the radiolabeled sPLA2 was analyzed by radioactive monitoring following the collection of successive blood samples, total excreta collection, and isolation of key organs upon euthanasia. The discovery of in vivo deiodination prompted the reimagining of methods to incorporate radioiodine into the sPLA2 proteins. Though we were ultimately unable to complete the study, our efforts detail approaches to avoid in future attempts.