Mechanism of carbamate inactivation of FAAH: implications for the design of covalent inhibitors and in vivo functional probes for enzymes

Abstract

Fatty acid amide hydrolase (FAAH) regulates a large class of signaling lipids, including the endocannabinoid anandamide. Carbamate inhibitors of FAAH display analgesic and anxiolytic properties in rodents. However, the mechanism by which carbamates inhibit FAAH remains obscure. Here, we provide biochemical evidence that carbamates covalently modify the active site of FAAH by adopting an orientation opposite of that originally predicted from modeling. Based on these results, a series of carbamates was designed that display enhanced potency. One agent was converted into a "click chemistry" probe to comprehensively evaluate the proteome reactivity of FAAH-directed carbamates in vivo. These inhibitors were selective for FAAH in the nervous system, but they reacted with several enzymes in peripheral tissues. The experimental strategy described herein can be used to create in vivo probes for any enzyme susceptible to covalent inhibition.

Figure 1

Two Possible Binding Modes for O-Biaryl Carbamate Inhibitors of FAAH (A and B) The carbamate inhibitor URB597 was modeled into the FAAH active site in two different orientations. In (A), the O-biaryl group of URB597 is located in the acyl chain binding (ACB) channel of FAAH and mimics the location of the arachidonyl chain of FAAH substrates/inhibitors [21]. In (B), the O-biaryl group of URB597 is located in the cytoplasmic access (CA) channel of FAAH, positioning the phenolic oxygen near the S217-K142 residues of the catalytic triad (S241-S217-K142) responsible for leaving group protonation. Blue, basic; red, acidic; green, hydrophobic (see [21] for more details on FAAH crystal structure).

Figure 2

Evidence that URB532 and URB597 Covalently Inhibit FAAH by Carbamylation of the Enzyme’s S241 Nucleophile (A) Two possible modes for covalent labeling of FAAH by URB597. In the left scheme, the O-biaryl group of URB597 serves a leaving group, resulting in carbamylation of the enzyme. In the right scheme, the N-cyclohexyl group of URB597 serves a leaving group, resulting in carbonylation of the enzyme. (B-D) The MS data shown support the left scheme. (B) MALDI-MS mapping of a tryptic digest of purified recombinant FAAH, highlighting the tryptic peptide that contains the FAAH nucleophile S241 (AA213-243). (C and D) MALDI-MS mapping of a tryptic digest of FAAH pretreated with (C) URB597 and (D) URB532, highlighting new tryptic peptides with masses that correspond to the AA213-243 peptide modified by one molecule of C(O)NH-cyclohexane and C(O)NH-butane, respectively. Asterisked peptides correspond to other FAAH tryptic peptides.

Figure 3

In Vivo Proteomic Profiling of Carbamate Targets with the CC Probe JP104(A) General method for characterizing the proteome reactivity of JP104 in vivo. Mice are administered JP104 at escalating doses (e.g., 0.25-10 mg/kg, i.p.). After 1 hr, the animals are sacrificed, and their tissues are removed, homogenized, and reacted under CC conditions with an azide-modified rhodamine reporter tag (RhN₃). Labeled proteins are visualized by in-gel fluorescence scanning. (B and C) Membrane protein reactivity profiles of JP104 in various mouse tissues. A single 65 kDa JP104 target was identified in brain (RhN₃ lanes). (C) This protein was confirmed as FAAH based on its absence in FAAH^-/- brains (upper panel). The selective labeling of FAAH by JP104 in brain tissue was further supported by competitive ABPP experiments with the serine hydrolase-directed probe FP-rhodamine (FP-Rh), which showed that none of the other brain serine hydrolases reacted with JP104 (FP-Rh lanes). (B) Additional targets of JP104 were identified in liver and kidney (right panels, RhN₃ lanes). (C) These proteins represented neither FAAH nor FAAH isoforms, as their labeling persisted in FAAH^-/- tissues (middle and lower panels). Similar profiles were observed in soluble fractions of mouse kidney and liver (see Figure S2). Fluorescence images are shown in grayscale.

Figure 4

Dose Dependence of Protein Labeling by JP104 In Vivo At 1 mg/kg JP104 (i.p.), the labeling of FAAH was approximately 80% of maximum, while none of the off-target sites were labeled at greater than 20% of maximum. Data represent the average of three trials per dose of JP104.

Figure 5

Characterization of In Vivo Off-Target Sites of FAAH-Directed Carbamates (A) Labeling of recombinant CE6 and Est31 in transfected COS7 cell proteomes by JP104 (1 μM). (B) Inhibition of FP-Rh labeling of recombinant CE6 by JP104 and URB597 (100 nM FP-Rh, 0.01-100 μM JP104 and URB597). From these data sets, IC₅₀ values of approximately 50 and 200 nM were estimated for JP104 and URB597, respectively (see Table S1 for complete data on CE6 inhibition). I, inhibitor. (C) Inhibition of FAAH and CE6 by URB597 in vivo. Administration of URB597 to mice (10 mg/kg, i.p., 1 hr) blocked JP104 labeling of brain FAAH (left panel) and the 60-65 kDa liver target identified as CE6 (right panel). JP104 labeling was conducted in vitro by using membrane proteomes of tissues from URB597-treated mice and was visualized by CC with RhN₃. Equivalent inhibition of JP104 labeling of CE6 was observed in liver soluble proteomes from URB597-treated mice (data not shown). Fluorescence images are shown in grayscale.