Fatty acid amide hydrolase inhibitors display broad selectivity and inhibit multiple carboxylesterases as off-targets

Abstract

Fatty acid amide hydrolase (FAAH) is the primary regulator of several bioactive lipid amides including anandamide. Inhibitors of FAAH are potentially useful for the treatment of pain, anxiety, depression, and other nervous system disorders. However, FAAH inhibitors must display selectivity for this enzyme relative to the numerous other serine hydrolases present in the human proteome in order to be therapeutically acceptable. Here we employed activity-based protein profiling (ABPP) to assess the selectivity of FAAH inhibitors in multiple rat and human tissues. We discovered that some inhibitors, including carbamate compounds SA-47 and SA-72, and AM404 are exceptionally selective while others, like URB597, BMS-1, OL-135, and LY2077855 are less selective, displaying multiple off-targets. Since proteins around 60 kDa constitute the major off-targets for URB597 and several other FAAH inhibitors with different chemical structures, we employed the multi-dimensional protein identification technology (MudPIT) approach to analyze their identities. We identified multiple carboxylesterase isozymes as bona fide off-targets of FAAH inhibitors. Consistently, enzymatic assay confirmed inhibition of carboxylesterase activities in rat liver by FAAH inhibitors. Since carboxylesterases hydrolyze a variety of ester-containing drugs and prodrugs, we speculate that certain FAAH inhibitors, by inhibiting carboxylesterases, might have drug–drug interactions with other medicines if developed as therapeutic agents.

Introduction

The endocannabinoid anandamide is a prototypical member of endogenous fatty acid amides that serves as signaling lipid messengers. It exerts neurobehavioral, cardiovascular, and immune-regulatory effects by activating cannabinoid (CB1 and CB2) and vanilloid (VR1) receptors (Di Marzo et al., 2002). Anandamide is synthesized in a stimulus-dependent manner and its signaling function is tightly controlled by the integral membrane enzyme fatty acid amide hydrolase (FAAH), which rapidly hydrolyzes anandamide and several other fatty acid amides to their corresponding acids (McKinney and Cravatt, 2005). Targeting the endocannabinergic system by inhibiting FAAH is a promising novel approach for the treatment of several nervous system disorders including pain, anxiety, and depression, as well as inflammation and hypertension (Cravatt and Lichtman, 2003). Mice lacking FAAH have significantly increased levels of certain fatty acid amides (Cravatt et al., 2001), including anandamide, and displayed analgesic (Cravatt et al., 2001), anti-inflammatory (Massa et al., 2004) and increased responsiveness to anandamide-induced hypotension and cardiodepression (Pacher et al., 2005) phenotypes.

In addition to employing FAAH^−/− mice to evaluate the consequences of constitutive elevations in fatty acid amides, specific FAAH inhibitors would represent a valuable complementary approach and may have therapeutic utility for the treatment of a range of clinical disorders in both the central nervous system and periphery. This interest has led to the disclosure of a number of FAAH inhibitors, including the carbamates URB597 (Kathuria et al., 2003) and BMS-1 (Sit and Xie, 2002), α-ketoheterocycles OL-135 (Boger et al., 2005) and α-KH 7 (Leung et al., 2003), and α-keto oxadiazoles (Leung et al., 2005). Some anandamide re-uptake inhibitors, like AM404 (Beltramo et al., 1997, Glaser et al., 2003) and LY2077855 (Moore et al., 2005, Porter et al., 2004), also inhibit FAAH activity. Many of these FAAH inhibitors have recently been shown to produce beneficial behavioral effects in rodents. For example, URB597 produced CB1 receptor-dependent analgesia, anxiolytic, anti-depressive and anti-hypertensive effects (Batkai et al., 2004, Gobbi et al., 2005, Kathuria et al., 2003), CB2-dependent anti-inflammatory effects (Holt et al., 2005) and depressed alcohol-induced addiction (Perra et al., 2005). OL-135 and BMS-1 also had analgesic effects (Chang et al., 2006, Lichtman et al., 2004, Sit and Xie, 2002). AM404 produced antinociceptive (Beltramo et al., 1997) and hypotensive (Calignano et al., 1997) effects.

FAAH, a serine hydrolase, catalyzes the hydrolysis of its substrates using a highly conserved serine residue in its active site as the catalytic nucleophile. Many of the identified FAAH inhibitors, by possessing mechanism-dependent binding groups, exert their inhibitory effects by binding and modifying the catalytic serine residue, raising concerns that these agents may also inhibit other serine hydrolases. Therefore, evaluating the activity of FAAH inhibitors on other serine hydrolases is critical to define therapeutic potential of these agents.

However, determining the selectivity of FAAH inhibitors is a significant challenge, considering the immense size of the serine hydrolase superfamily, with more than 200 members in the human proteome. To address this problem, a proteomic strategy known as activity-based protein profiling (ABPP) was disclosed that permits the simultaneous assessment of all relevant competitive enzymes in a complex proteome (Leung et al., 2003). This approach involves preincubation of cell or tissue proteomes with FAAH inhibitors followed by addition of fluorophosphonate-based probes that were proved to react only with the catalytic serine in the enzymatic active site in an activity-dependent manner (Liu et al., 1999) and bear a reporter group (rhodamine, and biotin). The profile of proteins labeled by the reporter group is then visualized by gel electrophoresis. FAAH and off-target enzymes that are modified by inhibitors are incapable of reacting, or show reduced ability to react, with these probes, if they interfere with or compete for the same site, and this event can be detected by a reduction in signal intensity from the reporter group. The ABPP approach has been successfully employed to address the selectivity of several FAAH inhibitors (Leung et al., 2003, Lichtman et al., 2004). More recently, multi-dimensional protein identification technologies (MudPITs) were employed to analyze the identities of potential target enzymes identified by the ABPP approach (Jessani et al., 2005). This combined ABPP–MudPIT technology allows a streamlined platform for high-content functional proteomic analysis.

In this report, we studied the selectivity of representative FAAH inhibitors to proteomes prepared from rat and human tissues. We identified multiple carboxylesterase isozymes as off-targets for URB597 and several other FAAH inhibitors. Considering that many carboxylesterases hydrolyze a variety of ester-containing drugs and prodrugs (Satoh and Hosokawa, 1998), we speculate that certain FAAH inhibitors might have drug–drug interactions if developed as therapeutic agents.