Structure-guided inhibitor design for human FAAH by interspecies active site conversion

Abstract

The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-A crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.

Fig. 1.

Mechanism of covalent inhibition of FAAH by PF-750. The catalytic triad Ser–Ser–Lys is shown.

Fig. 2.

Structure of the h/rFAAH protein bound to PF-750 inhibitor (purple sticks) and modeled to integrate into the membrane in a monotopic manner. The monomeric subunits of the FAAH dimer have been distinguished with green and blue coloring. Humanized residues are shown as violet spheres (carbon in violet, oxygen in red, and sulfur in yellow).

Fig. 3.

Active site of h/rFAAH in complex with PF-750. (A) The density found at the active site, shown as white 2σ contoured meshes, was modeled as the covalent inhibitor PF-750, shown as purple sticks. The residues surrounding the inhibitor (5-Å radius) are shown in cyan (conserved residues) and violet (humanized residues) sticks. A white diagram representation of the h/rFAAH backbone surrounds the active site. (B) Structural analysis of PF-750 bound to FAAH. The weak H bonds between F192 and F381 and the π-ring of the quinoline moiety are shown as yellow dashed lines. The following moieties are shown in stick representation: rFAAH residues (yellow), PF-750 (purple), h/rFAAH chimera mutated (violet), and conserved (cyan) residues. The rFAAH and h/rFAAH backbones surrounding the active site are shown in yellow and cyan, respectively.

Fig. 4.

Expansion of the membrane access channel of FAAH when bound to PF-750 compared with MAP. (A) Conformational switch of F432 in MAP-rFAAH (Left) vs. PF-750-h/rFAAH (Right) structures. The channels/solvent-filled spaces are shown in dark shadows, and the protein-filled spaces are shown as light areas. MA channel, membrane access channel; AB channel, acyl-binding channel. (B) Opening of MA channel in the PF-750–h/rFAAH structure (Right) compared with the MAP-rFAAH structure (Left). The monomers of each structure have been superposed by using the program Coot.