Fatty acid amide hydrolase as a potential therapeutic target for the treatment of pain and CNS disorders

Abstract

BACKGROUND: Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes without showing the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. OBJECTIVES: This review highlights advances in the development of FAAH inhibitors of different mechanistic classes and their in vivo efficacy. Also highlighted are advances in technology for the in vitro and in vivo selectivity assessment of FAAH inhibitors employing activity-based protein profiling (ABPP) and click chemistry-ABPP, respectively. Recent reports on structure-based drug design for human FAAH generated by protein engineering using interspecies active site conversion are also discussed. METHODS: The literature searches of Medline and SciFinder databases were used. CONCLUSIONS: There has been tremendous progress in our understanding in FAAH and development of FAAH inhibitors with in vivo efficacy, selectivity, and drug like pharmacokinetic properties.

Figure 1

Structures of two principal endocannabinoids, N-arachidonoyl ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG). AEA is hydrolyzed to arachidonic acid and ethanolamine by FAAH. MAGL is a principal enzyme that hydrolyzes 2-AG to arachidonic acid and glycerol.

Figure 2

(A) Structural domains of FAAH. Primary sequence analysis reveals a predicted NH₂-terminal transmembrane domain (purple), an amidase signature sequence rich in glycine and serine residues (green), a polyproline sequence predicted to interact with Homer and SH3 domain-containing proteins (blue), and a monotopic membrane binding domain that enables FAAH to bind the membrane (red). (B) Hydrolytic mechanism of amide and ester substrates involving catalytic triad of FAAH (shown for amides). (1) Lys142, initially in a deprotonated state, (2). abstracts a proton from Ser217, which in turn abstracts a proton from the Ser241 nucleophile. (3). Attack of the nucleophile on the substrate carbonyl is proposed to occur in a coupled manner with proton donation from Ser217 to the nitrogen atom of the amide substrate. This latter step requires the concurrent donation of a proton from Lys142 to Ser217, resulting in (4) the formation of an acyl−enzyme intermediate where both Lys142 and Ser217 have returned to their initial protonation states. (5) Deacylation results in release of the free fatty acid product.

Figure 3

Crystal structure of PF-750-h/rFAAH and PF-3845-h/rFAAH complexes. (A) Overlap of the crystal structures of the PF-3845 (gray) and PF-750 (green) complexes with h/rFAAH, showing the S241-carbamylated adduct and the different modes of binding that lead to distinct conformations for the F432 residue that toggles between the membrane access (MA) channel (F432 in gray) and AB pocket (F432 in green). (B) Overlap of the crystal structures of PF-750-h/rFAAH and MAP-rFAAH complexes. The MAP adduct (yellow) is shown to indicate its different arrangement compared to the PF-750 adduct (purple and blue). Potential steric hindrance between I491 (yellow sticks) from the rFAAH structure (PDB code 1MT5) and the inhibitor PF-750 (purple and blue) from the h/rFAAH structure. The residue V491 from the h/rFAAH structure is shown in violet sticks. The spheres indicate van der Waal's radii of carbon in position 4 (purple) and distal carbon of the I491 side chain (yellow). (C) Structual analysis of PF-750 bound to FAAH. The weak H-bonds beween 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 residues (violet), and conserved (cyan) residues. (D) Overlap of crystal structures of PF-3845-h/rFAAH and MAP-rFAAH complexes, showing similar binding modes for PF-3845 (gray) and MAP (blue).

Figure 3

Crystal structure of PF-750-h/rFAAH and PF-3845-h/rFAAH complexes. (A) Overlap of the crystal structures of the PF-3845 (gray) and PF-750 (green) complexes with h/rFAAH, showing the S241-carbamylated adduct and the different modes of binding that lead to distinct conformations for the F432 residue that toggles between the membrane access (MA) channel (F432 in gray) and AB pocket (F432 in green). (B) Overlap of the crystal structures of PF-750-h/rFAAH and MAP-rFAAH complexes. The MAP adduct (yellow) is shown to indicate its different arrangement compared to the PF-750 adduct (purple and blue). Potential steric hindrance between I491 (yellow sticks) from the rFAAH structure (PDB code 1MT5) and the inhibitor PF-750 (purple and blue) from the h/rFAAH structure. The residue V491 from the h/rFAAH structure is shown in violet sticks. The spheres indicate van der Waal's radii of carbon in position 4 (purple) and distal carbon of the I491 side chain (yellow). (C) Structual analysis of PF-750 bound to FAAH. The weak H-bonds beween 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 residues (violet), and conserved (cyan) residues. (D) Overlap of crystal structures of PF-3845-h/rFAAH and MAP-rFAAH complexes, showing similar binding modes for PF-3845 (gray) and MAP (blue).

Figure 4

Substrate-derived FAAH inhibitors.

Figure 5

α-Ketoheterocycle FAAH inhibitors.

Figure 6

Carbamate FAAH inhibitors.

Figure 7

Urea FAAH inhibitors

Figure 8

Additional FAAH inhibitors.

Figure 9

Competitive activity-based protein profiling (ABPP). To determine the selectivity of an inhibitor against serine hydrolases, a proteome is reacted with inhibitor and subsequently labeled with a rhodamine-tagged fluorophosphonate. Reacted proteomes are then analyzed by 1-D SDS-PAGE. A decrease in fluorescent intensity of the probe in the presence of inhibitor indicates a target.

Figure 10

Direct analysis of in vivo protein targets by CC-ABPP. Covalent inhibitors that were converted into activity-based probes via addition of a bio-othorgonal ‘handle’ (alkyne group) are dosed to animals and given time to react with protein targets. Probe-labeled proteins are then captured and identified from proteomes (isolated tissues) using bio-orthogonal chemistry and LC-MS–based proteomic methods, respectively. Using this approach, off-target reactivity at 1 and 10 mg/kg is shown.