Atherosclerosis-linked diseases, such as coronary heart disease and aneurysms, are major causes of death world-wide. Defective efferocytosis, the lack of noninflammatory clearance of apoptotic cells by macrophages, is implicated in the development of unstable atherosclerotic plaques. Identifying efferocytosis-impairing factors and appropriate countermeasures may lead to novel atherosclerosis treatments. N-acylethanolamines (NAEs) have been shown to improve efferocytosis and inhibit atherosclerosis by binding several receptors to exert protective effects. N-Acyl-Phosphatidylethanolamine Hydrolyzing Phospholipase D (NAPE-PLD) forms NAEs and phosphatidic acid by hydrolyzing N-acyl-phosphatidylethanolamines (NAPEs). Decreased Napepld expression is linked to necrotic plaque formation, while increasing NAPE-PLD activity using small molecule NAPE-PLD activators enhances efferocytosis by BMDM in vitro, suggesting that NAPE-PLD regulates efferocytosis and atherosclerotic plaque development. Further optimization is needed for use of these activators in in vivo studies on atherosclerosis. The precise binding sites for and the mechanisms by which small molecule NAPE-PLD activators enhance enzymatic activity remain unclear. Elucidation would enable rationale design of more potent activators. Furthermore, the lack of a robust high-throughput cellular assay for assessing NAPE-PLD activity has hindered screening for potentially more potent activators. To address these issues, our group employed a combination of in silico drug docking, photo-affinity labeling with LC/MS, protein complex crosslinking, and cellular fluorescence assays. Our molecular models and LC/MS data indicate that activators bind near the NAPE-PLD catalytic site. Moreover, crosslinking studies suggest that at least in vitro, recombinant NAPE-PLD is primarily monomeric with relatively little enzyme in the homodimeric form previously shown to be required for activity. Finally, we developed a high-throughput assay using HEK293 cells optimized for screening NAPE-PLD activity in the presence or absence of activators, facilitating future screening of NAPE-PLD activators.

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