Structural insights into human NAPE-PLD interactions

The membrane-associated enzyme NAPE-PLD (N-acyl phosphatidylethanolamine specific-phospholipase D) generates the endogenous cannabinoid arachidonylethanolamide (anandamide) and other lipid signalling amides that play key roles in several physiological pathways, including appetite, lifespan, stress, and pain response. Earlier, our structural and biophysical studies showed that the interaction between the dimeric protein and bile acid cofactors turns the enzyme on. This discovery casted an interesting light on NAPE-PLD as a point where lipid amide signalling, and bile acid physiology converge. In the last years, the therapeutic potential of NAPE-PLD has come to light in the field of emotional behaviour, neurodegenerative and metabolic diseases, paving the way for the development of specific small molecule ligands. However, due to the delicate behaviour of this membrane target and its challenging crystallization process, no structure in complex with specific small molecules have been reported so far, making the process of ligand discovery extremely difficult. Using the label-free technique of surface plasmon resonance, here we report the discovery of different classes of novel ligands that bind NAPE-PLD in the low micromolar range. We successfully managed to determine the first crystal structures of NAPE-PLD in complex with these molecules, providing useful insights into its function and modulation. Importantly, we first detected and validated by X-ray diffraction the interaction of the membrane target with the compound pyridoxal-5’-phosphate (PLP), which is the active form of vitamin B6, involved in the cellular metabolism of amino acids and lipids, as well as in the biosynthesis of neurotransmitters. In addition, we have successfully determined the structure of NAPE-PLD in complex with the thiazide diuretic hydrochlorothiazide (HCTZ), a drug that has been used for more than half a century to treat hypertension. Finally, we determined the complex structure with the compound naphthalenetrisulfonic acid. Our results characterize at atomic level the binding mode of these ligands and suggest the potential involvement of NAPE-PLD in the control of hypertension.