Lipoprotein-associated phospholipase A2: utilizing potent and specific inhibitors to probe the structure function relationship

Bahnson, Brian J.Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a membrane-associated enzyme that circulates in plasma primarily bound to low-density lipoprotein (LDL). The enzyme was discovered due to its ability to catalyze the hydrolysis of the acetyl group at the sn-2 position of the diverse signaling phospholipid platelet-activating factor (PAF) and is therefore also referred to as PAF acetylhydrolase. In addition to the substrate PAF, Lp-PLA2 recognizes oxidatively fragmented phospholipids derived from polyunsaturated fatty acids at the sn-2 position as well as oxidatively fragmented phospholipids present on oxidized LDL. It is important to note that Lp-PLA2 only recognizes oxidatively fragmented phospholipids and can therefore be constitutively active without compromising cellular integrity. Due to its associations with LDL, its cleavage products and the report that an increase in Lp-PLA2 concentration or activity leads to an increase in the risk of atherosclerosis Lp-PLA2 has been reported to play an important role in the progression of heart disease. Lp-PLA2 has therefore become recognized as a viable drug target. The pharmaceutical company GlaxoSmithKline developed a specific inhibitor of Lp-PLA2, darapladib, which was recently investigated for use as an add-on treatment for patients with heart disease. The structure of Lp-PLA2 was solved at a resolution of 1.5 Å and was determined to have the classic α/β hydrolase fold. This high resolution structure was then used to develop a model that mimics the association of Lp-PLA2 in biological membranes. Using the knowledge of the structure of Lp-PLA2, combined with its membrane associations as well as disease implications, the work of this thesis was done to gain a better understanding of the structure-function relationship of the enzyme. An E. coli over-expression construct of a truncated form of Lp-PLA2 was created to resemble the sequence of protein samples of Lp-PLA2 obtained from the company ICOS Corporation, as it was this truncated version whose crystal structure was solved. Expression and purification of the recombinant Lp-PLA2, as well as point mutations created to minimize its membrane associations, were carried out using the E. coli expression construct. Activity assays using a general substrate were developed and demonstrated that there was no significant difference in specific activity, Kcat or Vmax for any of the mutants; however, there was a decrease in the KM for the Lp-PLA2 lipoprotein binding mutants. The highly pure and concentrated samples of recombinant enzyme were used for protein crystallization trials. Due to the fact that Lp-PLA2 is a drug target, high throughput inhibitor screening was carried out at the Scripps Research Institute Molecular Screening Center for Lp-PLA2 as well as with a homologous, intracellular enzyme, PAFAH-II. The highly potent preliminary hits were characterized in the lab of Dr. Benjamin Cravatt at the Scripps Research Institute and inhibitors were modified to specifically target Lp-PLA2 in vitro and in situ. The inhibited recombinant Lp-PLA2 was further studied in our lab for kinetic and biophysical properties. In order to determine the structure of darapladib and the carbamate inhibitor complexed with Lp-PLA2 crystallization trials were attempted. Though initial screening was promising, co-crystallization crystal structures were not determined. In turn, computational models of the enzyme-inhibitor complex were created to show the binding interaction. In addition, a computational model of Lp-PLA2’s interaction with its natural ligand, PAF, was modeled. Previous reports indicated that Lp-PLA2 was also carried in plasma associated with platelet derived microparticles. These microparticles are derived during platelet activation and are implicated in furthering the progression of atherosclerosis. This study confirms the presence of Lp-PLA2 in both human and mouse platelets upon activation as well as PAFAH-II using Western blot analysis. It was also confirmed that the Lp-PLA2 secreted from platelets is non-glycosylated, indicating that this enzyme was not taken up by the platelets from the plasma but was native to the platelet or its progenitor cells. The activity of the enzymes were assayed using a modified DTNB assay and activities contributed specifically to Lp-PLA2 and PAFAH-II were determined by individually blocking each form’s activity using the specific inhibitors developed through our collaboration with Scripps. It was shown that in human and mouse platelet releasate demonstrate Lp-PLA2 and PAFAH-II activity, indicating a potential role in PAFAH-II advancing the progression of atherosclerosis. In this study, we have broadened the knowledge base of Lp-PLA2 and its structure-function relationship through the development and use of potent and specific inhibitors. We have also determined new potential roles for both Lp-PLA2 and PAFAH-II in the progression of atherosclerosis due to their characterized release upon platelet activation. These roles can be explored in further studies and animal models to better understand the impact of these enzymes in disease.University of Delaware, Department of Chemistry and BiochemistryPh.D