Molecular mechanism of activation of the immunoregulatory amidase NAAA

Abstract

Palmitoylethanolamide is a bioactive lipid that strongly alleviates pain and inflammation in animal models and in humans. Its signaling activity is terminated through degradation by N-acylethanolamine acid amidase (NAAA), a cysteine hydrolase expressed at high levels in immune cells. Pharmacological inhibitors of NAAA activity exert profound analgesic and antiinflammatory effects in rodent models, pointing to this protein as a potential target for therapeutic drug discovery. To facilitate these efforts and to better understand the molecular mechanism of action of NAAA, we determined crystal structures of this enzyme in various activation states and in complex with several ligands, including both a covalent and a reversible inhibitor. Self-proteolysis exposes the otherwise buried active site of NAAA to allow catalysis. Formation of a stable substrate- or inhibitor-binding site appears to be conformationally coupled to the interaction of a pair of hydrophobic helices in the enzyme with lipid membranes, resulting in the creation of a linear hydrophobic cavity near the active site that accommodates the ligand’s acyl chain.

Natural Potent NAAA Inhibitor Atractylodin Counteracts LPS-Induced Microglial Activation

“…To our knowledge, atractylodin is the most potent natural chemical with an IC 50 value of 2.81 ± 0.51 µM in NAAA inhibition, which is more potent than the reported NAAA inhibitors diacerein, a prodrug of the natural products rhein (IC 50 = 7.2 ± 1.10 µM) (Petrosino et al, 2015) and fucoxanthinol from brown seaweeds (IC 50 = 12.75 ± 1.12 µM) (Jin et al, 2020). The crystal structure of NAAA has recently been reported by the Nagar group (Gorelik et al, 2018). NAAA belongs to the N-terminal nucleophile (Ntn) superfamily and is activated by self-proteolysis at Cys126 in human NAAA.…”

“…To visualize the enzyme active site modified by atractylodin, we constructed a docking model based on the protein structure reported by Gorelik et al (2018). Docking studies showed that atractylodin occupied the catalytic cavity of NAAA, where the furan head group of atractylodin has a hydrophobic-related interaction with the backbone of the TRP181 and LEU152 residues of human NAAA ( Figures 4A, B).…”

“…However, the number of reported NAAA inhibitors remains limited and is mainly due to the restricted substrate-binding site of NAAA. The catalytic cavity of NAAA is suitable for accommodating only narrow and relatively straight compound backbones ( Gorelik et al., 2018 ). Furthermore, identifying specific molecular structures to fit the catalytic center of this enzyme remains a challenge for synthetic chemists.…”

Synthesis, biological evaluation, and structure activity relationship (SAR) study of pyrrolidine amide derivatives as N-acylethanolamine acid amidase (NAAA) inhibitors

“…Recently, Alexei Gorelik et al have reported the crystal structure of NAAA covalently bound to ARN726 (PDB ID: 6DY2), which allows us to study the interaction between NAAA and 4g. 21 Docking studies showed that 4g lies in the same binding pocket defined by ARN726. Briefly, the pyrrolidine ring of 4g occupies the enzyme catalytic center of NAAA, preventing the carbonyl group from forming a covalent bond with catalytic residues, e.g., Cys126.…”

“…[16][17][18][19] These electrophilic chemical groups can covalently react with the cysteine nucleophile of NAAA. [19][20][21] However, inhibitors with such structures show poor biostability, thus limiting their systemic effect. For example, β-lactone-based inhibitors can be quickly hydrolysed to the corresponding β-hydroxy acid in physiological pH buffer or rat plasma.…”