Elettra | XRD2 Our endocannabinoid system is activated by an endogenous molecule - named “anandamide” (AEA) which is the main biological agonist of the cannabinoid receptors CB1 and CB2. These central and peripheral (G protein-coupled) receptors are expressed widely in the nervous system and immune system, respectively, and their activation causes effects similar to those induced by the active ingredients of the plant Cannabis sativa, comprising well known pain control and positive actions on appetite, pleasure and mood. Anyhow the endogenous molecule does not induce physiologically the psychotropic effects characteristic of the plant Δ9-tetrahydrocannabinol (THC). Our body produces other AEA-analogue bioactive compounds such as PEA (palmitoylethanolamide) that act as signaling molecules to elicit a broad range of key protective roles in defense against diseases. In addition, specific membrane enzymes are involved in their biosynthesis (mainly the membrane phospholipase D, NAPE-PLD) and degradation (e.g., the fatty acid amide hydrolase, FAAH). Major efforts have been made in the last thirty years worldwide to develop active compounds targeting the endocannabinoid system, especially modulators directed against the CB receptors and inhibitors of FAAH. However, preclinical and clinical findings have shown that synthesized direct agonists of CB receptors, also more potent and efficacious than the plant THC to contrast pain, are at strong risk to induce tachycardia, vomiting, heart and kidney damage and seizures. Anti-obesity CB1 antagonists (e.g., rimonabant) have been withdrawn in Europe because long-term daily use is linked to dysphoric side effects, while FAAH inhibitors (proposed as safer alternatives to raise endogenous AEA levels for pain management) similarly can lead to undesirable side effects. The membrane zinc-phospholipase NAPE-PLD is widely expressed in all body tissues, especially brain, gastrointestinal tract, heart, kidney and genital system. It hydrolyzes specifically a family of membrane glycerophospholipids (N-acylphosphatidylethanolamines, NAPE) that are synthesized in higher amounts in membranes during cellular stress, inflammation and tissue damage, conferring stability and fusiogenic properties to the membrane lipid bilayer. Previously, we succeed in solving the first structure of human NAPE-PLD in its apo-form using x-ray data collected at the Synchrotron Elettra [DOI: 10.1016/j.str.2014.12.018] (pdb id 4qn9). Surprisingly, results showed that the protein has evolved during evolution to bind mammals’ bile acid (BA) cofactors starting from a highly conserved ancestral fold. Bound BAs structurally enhance protein dimer assembly, promote the interaction with membrane and NAPE substrates, and are essential for enzyme catalysis that generates anandamide and the other bioactive ethanolamides. Essentially, our study revealed that NAPE-PLD links the marked protective effects of our endogenous lipid amides (anandamide and similar molecules) to the important physiology of BAs, involved in the digestion and absorption of nutrients as well as in the metabolism and crosstalk between our body and microbiota. To address the need for agents that can exploit the endocannabinoid system for therapy, we recently screened approved drugs and chemical probes against NAPE-PLD. We identified the ligand hydrochlorothiazide (HCTZ) with affinity in the sub-µM range in presence of NAPE-PLD bound to BAs. Importantly, HCTZ is a drug of the class of thiazide diuretics, first-line treatment worldwide for hypertension, used daily by million people in the world and highly effective to consistently lower arterial blood pressure, reduce cardiovascular events and heart failure. Despite these drugs have been used for over sixty years since their introduction in clinics, the identity of their molecular target and the mechanism of action by which these drugs chronically reduce vascular resistance had not been well explained. The complex structure between NAPEPLD and HCTZ obtained by x-ray at the beamline XRD2 of Elettra (pdb id 8pc4), and that between NAPE-PLD and a model of lipid bilayer by single particle cryo-electron microscopy (Figs. 1 a, b), together with preclinical studies in hypertensive rats, demonstrated that NAPE-PLD is indeed a renal and extrarenal target of the thiazides. NAPEPLD can account for both their acute (diuresis and accompanying decrease of plasma volume) and chronic (reduction of arterial pressure through vascular dilation) therapeutic effects, as well as associated metabolic abnormalities at high doses (e.g., hyperlipidemia and hyperglycemia) [original paper]. Importantly, results have also shown that thiazide diuretics bind to the ~9 Å-wide dimer internal channel of NAPE-PLD stabilizing the protein dimer assembly in a competitive manner with the cell cofactor pyridoxal 5’- phosphate (PLP) (pdb id 8p90) (Fig. 1 b). This channel of NAPE-PLD can create membranepores as dynamic conductive pathways through which the charged PLP can diffuse through cell membranes and subcellular compartments (e.g., mitochondria), a key physiological transport process of mammalian cells that had remained mysteries (Fig. 1 b,c). Overall, our results shed light on the mechanism of cure of hypertension and will be useful for developing more efficacious medications for the management of cardiovascular and neurovascular risk factors (Fig. 1 c). The fact that thiazide medications promote therapeutic effects that involve directly the target NAPE-PLD reveals not only a novel player in the treatment of hypertension, but also an innovative way to modulate efficaciously the endocannabinoid system in therapy. Targeting of our endocannabinoid system in therapy Elettra Highlights 2024–2025 72
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