Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain

Abstract

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

Figure 1

Crystal structure of a PF-3845-h/rFAAH complex. (A) Active site image of PF-3845-h/rFAAH complex, showing the S241-carbamylated adduct and six residues that have been mutated in h/rFAAH. (B) Overlap of the crystal structures of the PF-3845 (gray) and PF-750 (green) complexes with h/rFAAH, showing 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). (C) 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 2

In vivo selectivity analysis for PF-3845 and URB597 as determined by competitive ABPP. (A) Gel profiles of FP-rhodamine labeled brain serine hydrolase activities from mice treated with PF-3845 or URB597 (10 mg/kg, i.p.) for the indicated times. Note that selective blockade of FP-rhodamine labeling of FAAH is observed by both inhibitors. Representative additional brain serine hydrolases are designated based on previous ABPP studies (Blankman et al., 2007; Nomura et al., 2008). (B) Gel profiles of FP-rhodamine-labeled liver serine hydrolase activities from mice treated with PF-3845 or URB597 (10 mg/kg, i.p., 2 hr). Note that URB597, but not PF-3845, blocks FP-rhodamine labeling of several liver serine hydrolase activities (bracket), which have previously been identified as carboxylesterases (Alexander and Cravatt, 2005; Zhang et al., 2007). For both (A) and (B), fluorescent gel images are shown in grayscale. For (B), a lower scanning intensity is shown due to the highly abundant activity signals for carboxylesterases present in the liver proteome.

Figure 3

Direct analysis of in vivo protein targets of alkyne analogues of PF-3845 and URB597 by CC-ABPP. (A) Structure of PF3845yne, an alkyne analogue of PF-3845. (B) Structure of JP104, an alkyne analogue of URB597 (Alexander and Cravatt, 2005). (C & D) Gel profiles of CC-ABPP experiments where brain (C) or liver (D) proteomes from PF3845yne- and JP104-treated mice (10 mg/kg, i.p., 2 hr) were treated with a rhodamine-azide tag under CC conditions and analyzed by in-gel fluorescence scanning (shown in grayscale). PF3845yne selectively labels FAAH in both brain and liver tissue [60 kDa band absent in FAAH(-/-) mice], while JP104 labels several additional proteins in liver [protein bands present in both FAAH(+/+) and (-/-) mice]. Note that the 55 kDa protein band observed in liver proteome from PF-3845-treated FAAH(-/-) mice was also detected in liver proteomes from vehicle-treated FAAH(+/+) and (-/-) mice and therefore likely represents a background protein cross-reactive with the azide-rhodamine tag.

Figure 4

Brain levels of FAAH substrates and inhibitors. (A-C) Brain levels of AEA (A), PEA (B), and OEA (C) measured at the indicated time points following treatment with PF-3845 or URB597 (10 mg/kg, i.p.). *p < 0.05; **p < 0.01 for inhibitor- versus vehicle-treated groups. n = 4 mice/group. (D) Brain levels of PF-3845 and URB597 measured at the indicated time points following inhibitor treatment.

Figure 5

Anti-hyperalgesic effects of PF-3845 in the CFA model of inflammatory pain. (A) PF-3845 produces a dose-dependent reduction of mechanical allodynia (hyperalgesia) in rats (black bars). The effect of the non-steroidal anti-inflammatory drug naproxen (10 mg/kg, p.o., hatched bar) is shown for comparison. Paw withdrawal thresholds were measured 4 hours post-administration of drugs (B and C) PF-3845-treated rats show significant blockade of FAAH activity (B) and elevated AEA levels (C) in brain tissue and blood leukocytes/plasma. All FAAH activity and NAE measurements were determined at 4 hr following drug treatment and were significantly different between PF-3845- and vehicle-treated groups (p < 0.001, n = 3 rats/group). (D) Time-course for anti-hyperalgesic effects of PF-3845. (E) Blockade of anti-hyperalgesic effects of PF-3845 (10 mg/kg, p.o.) by CB1 and CB2 antagonists (SR141716 and SR144528, respectively; 10 and 30 mg/kg, i.p., respectively administered 1 hr prior to treatment with PF-3845). Note that neither the CB1 nor CB2 antagonist displayed significant effects on mechanical allodynia in rats not treated with PF-3845 (hatched bars). ^#p<0.001, for PF-3845- versus vehicle-treated groups. *p < 0.05; **p < 0.01; ; ***p<0.001, for vehicle-PF-3845 versus CB1/CB2 antagonist-PF-3845 treated groups. n = 8 rats/group.