|

 

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

Chemistry & BiologyArticle Discovery and Characterization of aHighly Selective FAAH Inhibitorthat Reduces Inflammatory Pain Kay Douglas S. Johnson,Mauro Mileni,David Jonathan Z. Michele K. McKinney,Eranthie Weerapana,Nalini Marya Liimatta,Sarah E. Scott Cory Satwik Kamtekar,Keshab Yanhua Stephen Keri Van Raymond C. Stevens,and Benjamin F. Cravatt1Pfizer Global Research and Development, Groton, CT 06340, USA2Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA3Pfizer Global Research and Development, Chesterfield, MO 63017, USA4The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute,10550 North Torrey Pines Road, La Jolla, CA 92037, USA5Pfizer Global Research and Development, Ann Arbor, MI 48105, USA6Pfizer Global Research and Development, Cambridge, MA 02139, USA*Correspondence: (K.A.), (R.C.S.), (B.F.C.)DOI 10.1016/j.chembiol.2009.02.013 also produce myriad side effects, including impairments incognition and motor control, that limit their broad clinical utility.
Endocannabinoids are lipid signaling molecules that In contrast, inhibitors of the principal AEA-degrading enzyme regulate a wide range of mammalian behaviors, fatty acid amide hydrolase (FAAH) ( including pain, inflammation, and cognitive/emotional ) and FAAH(/) mice have been found state. The endocannabinoid anandamide is princi- to display analgesia ( pally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is ), anti-inflammation anxiolysis ( currently much interest in developing FAAH inhibitors ), and antidepression to augment endocannabinoid signaling in vivo. Here, without disruptions in motility, cogni- we report the discovery and detailed characterization tion, or body temperature of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm findings suggest that FAAH/AEA signaling pathways regulate that PF-3845 is a covalent inhibitor that carbamylates a discrete subset of the behavioral processes affected by direct FAAH's serine nucleophile. PF-3845 selectively CB1 agonists. The selective pharmacological blockade of FAAH inhibits FAAH in vivo, as determined by activity-based has, therefore, emerged as an exciting strategy by which to protein profiling; raises brain anandamide levels for discern the endogenous functions of AEA-mediated endocan- up to 24 hr; and produces significant cannabinoid nabinoid pathways and gauge their therapeutic potential.
Several classes of FAAH inhibitors have been reported, including reversibly (e.g., a-ketoheterocycles pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.
]) and irreversibly (e.g., carbamates [fluorophosphonates ]) acting agents. These inhibitors each possess distinct sets of attributes and defi-ciencies that reflect some of the most challenging aspects of The endogenous cannabinoid (endocannabinoid) system is FAAH inhibitor development. Reversible inhibitors, such as the composed of two G protein-coupled receptors, CB1 and CB2; a-ketoheterocycle OL-135, have been found to display excellent their cognate lipid ligands N-arachidonoyl ethanolamine (anan- selectivity for FAAH relative to other serine hydrolases in damide [AEA]) and 2-arachidonoylglycerol (2-AG); and the meta- mammalian proteomes, but they produce only transient eleva- bolic enzymes responsible for AEA and 2-AG biosynthesis and tions in AEA in vivo ). The submaximal efficacy of reversible FAAH inhibitors may be due to their rapid ). CB1 and CB2 are also activated by metabolism, as well as to the fact that a near-complete (>85%) D9-tetrahydrocannabinol (THC), the psychoactive substance blockade of FAAH activity is required to maintain elevated AEA in marijuana (THC and other direct CB1 levels in vivo ). Another challenge with devel- agonists have long been recognized to possess medicinally oping reversible inhibitors is that inhibition of FAAH leads to beneficial properties, such as pain relief; however, these agents elevated levels of several N-acyl ethanolamine (NAE) substrates, Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved 411 Chemistry & Biology A Highly Efficacious and Selective FAAH Inhibitor Table 1. Potency, Defined by kinact/Ki Values, Comparison of FAAH Inhibitors PF-3845, PF-750, and URB597 kinact (s1) The kinact and Ki values were obtained as described in . Values are averages ± SD of three independent exper-iments.
a which can diminish the efficiency and potency of the inhibitor analogs in which the quinoline group of PF-750 was replaced by mass-action competition with the substrate ( by a biaryl ether group This medicinal chemistry effort Irreversible inhibitors, such as the carbamate URB597 culminated in the discovery of PF-3845 (compound 10, (), produce a more complete blockade of a biaryl ether piperidine that displayed a 10- to 20-fold higher FAAH in vivo, but they also inhibit other serine hydrolases in kinact/Ki value for FAAH compared to PF-750 or the well-studied peripheral tissues, including several carboxylesterases ( carbamate FAAH inhibitor URB597 ). Key features of PF-3845 that contribute to enhanced Considering that FAAH is a serine hydrolase potency include the p-trifluoromethyl substituent on the biaryl and that there are > 200 members of this enzyme ether moiety and substitution of the aniline leaving group of class in the human proteome, inhibitor selectivity represents a PF-750 with 3-aminopyridine.
major challenge.
We next examined the mechanism of FAAH inhibition by Toward the goal of developing FAAH inhibitors that display PF-3845 in more detail by using an enzyme-coupled assay optimal efficacy and selectivity, we recently reported a class of with oleamide as a substrate, where stoichiometric quantities of NAD+ are formed upon the generation of ammonia from specificity (Here, we report a detailed mecha- oleamide by FAAH hydrolysis (). Linear produc- nistic, structural, and pharmacological characterization of the tion of NAD+ was observed over a 40 min time period in the FAAH piperidine urea inhibitor PF-3845. Our data indicate that absence of PF-3845 (see available online). In the PF-3845 displays an unprecedented combination of in vivo presence of PF-3845, the progress curves for oleamide hydro- activity and selectivity, designating this agent as a valuable phar- lysis by FAAH exhibited curvature (A), consistent with macological tool for studying FAAH-regulated endocannabinoid an irreversible mechanism of inhibition. The data were fit to a pseudo-first-order decay equation to determine kobs values ateach inhibitor concentration. Plotting these kobs values as a func- RESULTS AND DISCUSSION tion of PF-3845 concentration revealed saturation (B),thus supporting a two-step mechanism for FAAH inactivation Optimization of Potency of Piperidine/Piperazine Urea involving reversible binding of PF-3845 to FAAH, followed by the second chemical step that results in covalent bond forma- Recently, we ) and others have tion. Based on this model, kinact and Ki values for FAAH inhibition described a class of FAAH inhibitors that contain a piperidine/ by PF-3845 were calculated to be 0.0033 ± 0.0002 s1 and piperazine urea moiety. These agents were found to inhibit 0.23 ± 0.03 mM, respectively ). Similar progress and satu- FAAH by a covalent, irreversible mechanism involving the carba- ration curves were plotted for inhibition of FAAH by the carba- mylation of FAAH's catalytic S241 nucleophile. Lead urea inhib- mate inhibitor URB597 (C and S1D), generating kinact itors, such as PF-750 however, showed only moderate and Ki values of 0.0033 ± 0.0003 s1 and 2.0 ± 0.3 mM, respec- potency for FAAH, as determined by measurements of kinact/Ki tively (These data thus indicate that most, if not all, of values (800 M1s1). In an effort to improve the potency of the increased potency observed for PF-3845 over URB597 is piperidine/piperazine urea inhibitors, we explored a series of due to an improvement in binding affinity (Ki value).
412 Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved Chemistry & Biology A Highly Efficacious and Selective FAAH Inhibitor Table 2. Structure-Activity Relationship of Biaryl Ether Urea FAAH Inhibitors Values are averages ± SD of two independent experiments.
Crystal Structure of a PF-3845-FAAH Complex interactions between this inhibitor's 4-trifluoromethyl-2-pyridyl To gain more detailed insights into the basis for the improved group and the hydrophobic acyl chain-binding pocket of FAAH potency of PF-3845, we next determined the crystal structure (In this respect, the biaryl ether piperidine moiety of of a PF-3845-FAAH complex. Here, we used a recently PF-3845 binds in a fashion that more closely resembles the described ‘‘humanized'' rat-FAAH (h/rFAAH) protein, in which arachidonyl chain of the original methyl arachidonyl phospho- the active site of rat FAAH (rFAAH) has been mutagenically con- nate (MAP)-rFAAH crystal structure ( verted to match the active site of human FAAH (hFAAH) ( ). Previous studies have confirmed that h/rFAAH One marked difference that we previously observed between maintains the high recombinant expression levels of rFAAH the crystal structures of FAAH bound to MAP ( and the inhibitor sensitivity profile of hFAAH ) and PF-750 (was that the shorter thus providing a versatile model for structure-activity rela- PF-750 group allowed F432 to undergo a conformational shift tionship (SAR) studies relevant to the human enzyme.
that moved this residue out of the membrane access channel A crystal structure of the PF-3845-h/rFAAH complex was and into the acyl chain-binding pocket In the determined at 2.8 A˚ resolution (see for data collection PF-3845-h/rFAAH structure, F432 resides in the membrane and refinement statistics), revealing a dimeric enzyme with an access channel at a site similar to its position in the MAP-rFAAH overall fold that matched those reported in previous FAAH struc- structure Adjacent to F432, residue M436 also tures (PF-3845 was found undergoes a rotation of about 135 along the Ca-Cb axis in to be covalently attached to the catalytic S241 nucleophile both the PF-3845 and MAP-FAAH structures The ) of h/rFAAH through a carbamate linkage structural rearrangements of F432 and M436 thus appear to ), similar to the previously reported structure of h/rFAAH occur in a coordinated manner in these three structures and complexed with PF-750 ). The 3-aminopyri- establish that inhibitors can promote conformational shifts that dine leaving group was not observed in the h/rFAAH structure, alter the relative size of FAAH's acyl chain-binding pocket and but the remaining piperidine portion of the parent molecule occu- membrane access channel. The dynamic nature of this region pied the acyl chain-binding pocket A). As was observed of FAAH could facilitate entrance of lipid substrates from the in the PF-750-h/rFAAH structure a strong cell membrane into the enzyme's active site.
aromatic C-H.p interaction ) can be seen The PF-3845-FAAH structure also aided our understanding of between F192 and the phenyl aromatic ring of PF-3845 the SAR within the biaryl ether urea series by means of Monte (The 20-fold improvement in potency for PF-3845 Carlo simulations and energy calculations. For example, these appears to derive from a more extended set of van der Waals calculations suggest that the higher potency of piperidine ureas Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved 413

Chemistry & Biology A Highly Efficacious and Selective FAAH Inhibitor (compounds 6, 7, 10; over the corresponding piperazineureas (compounds 1, 3, 4; ) is likely due to unfavorableelectrostatic interactions of the protonated piperazine withinthe hydrophobic acyl chain-binding pocket. Furthermore,replacement of the central phenyl group of PF-3845 with a pyridylgroup drastically reduces inhibitor potency (compound 15;which may reflect disruption of the aromatic-CH.pinteraction with F192 caused by the higher electron withdrawingcharacter of the pyridyl nitrogen, which lessens the electrondensity of the p ring. Finally, the crystal structure providesan attractive model to explain the improved potency gainedby insertion of a trifluoromethyl group at the 4 position of the2-pyridyl ring that resides in the acyl chain-binding pocket.
Reducing the size of this substituent to a methyl or fluoro groupdecreased inhibitor potency (compounds 6 and 8, respectively;), which can be rationalized by simulations that predictsuboptimal van der Waals contacts for these smaller groups.
Conversely, moving the trifluoromethyl group to the 3 or 6 posi-tion, which also impairs potency (compounds 11 and 12, respec-tively; is predicted to introduce steric clashes with theFAAH protein.
PF-3845 Selectively Inhibits FAAH In Vivo for up to 24 hrWe next administered PF-3845 to mice (10 mg/kg, i.p.) andmonitored FAAH inactivation and substrate levels in the brainover a time course from 1 to 24 hr. For comparison, we alsoanalyzed mice treated with URB597 (10 mg/kg, i.p.). BothPF-3845- and URB597-treated mice showed rapid andcomplete inactivation of FAAH in the brain, as judged by compet-itive activity-based protein profiling (ABPP) () with the serine hydrolase-directed probefluorophosphonate (FP)-rhodamine ((A). PF-3845 showed a longer duration of action thanURB597, as judged by the rate of recovery of FP labeling ofFAAH. Whereas URB597-treated animals showed nearly wild-type levels of brain FAAH activity by 24 hr, FAAH was still mostlyinhibited (>75%) at this time point in PF-3845-treated animals(A). These ABPP studies confirmed that PF-3845 andURB597 were both highly selective for FAAH in the brain, sincenone of the other FP-reactive serine hydrolases in this tissuewere inhibited by these agents. In contrast, URB597, but notPF-3845, blocked FP labeling of several additional liver serinehydrolases (B), which have previously been identifiedas carboxylesterases ). We furthermore tested PF-3845 for the effects onFAAH-2, a recently identified FAAH homolog that is selectivelyexpressed in higher mammals ), but not rodents.
In contrast to URB597, which has been found to inhibit humanFAAH-2 with high potency ), PF-3845 showed Figure 1. Crystal Structure of a PF-3845-h/rFAAH Complex negligible activity against this FAAH variant (IC50 > 10 mM; (A) Active site image of the 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) To confirm the in vivo selectivity of PF-3845, we synthesized complexes with h/rFAAH, showing the different modes of binding that lead to distinct conformations for the F432 residue that toggles between the (which maintained high potency for FAAH (kinact/Ki membrane access (MA) channel (F432 in gray) and the acyl chain-binding value of 5230 M1s1). We then directly analyzed the protein (AB) pocket (F432 in green).
targets of PF3845yne in vivo by click chemistry (CC)-ABPP (C) Overlap of the crystal structures of PF-3845-h/rFAAH and MAP-rFAAH complexes, showing similar binding modes for PF-3845 (gray) and MAP (blue).
). For these studies, we compared the labelingprofile of PF3845yne to those of JP104 B), a previously 414 Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology A Highly Efficacious and Selective FAAH Inhibitor the former agent once again showing selective reactivity withFAAH and the latter inhibitor labeling a number of proteins thatwere found in both FAAH(+/+) and (/) mice D). Asingle faint 60 kDa labeling event was observed in liver pro-teomes from PF3845yne-treated FAAH(/) mice, but a similarsignal was also detected in vehicle-treated animals, suggestingthat this protein may represent a nonspecific target of theazide-rhodamine tag. Collectively, these data indicate thatPF-3845 inhibits FAAH in vivo with exceptional efficacy andselectivity.
FAAH Inhibition by PF-3845 Causes a Dramatic Figure 2. In Vivo Selectivity Analysis for PF-3845 and URB597 as and Sustained Elevation in AEA Determined by Competitive ABPP Previous studies with FAAH(/) mice (A) Gel profiles of FP-rhodamine-labeled brain serine hydrolase activities from and rodents treated with FAAH inhibi- 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 confirmed a key role for FAAH in regulating tissue levels of both inhibitors. Representative additional brain serine hydrolases are desig-nated based on previous ABPP studies ( AEA and other NAEs. We found that PF-3845-treated mice (10 mg/kg, i.p.) also showed dramatic (>10-fold) elevations in (B) Gel profiles of FP-rhodamine-labeled liver serine hydrolase activities from brain levels of AEA and other NAEs (N-pamitoyl etha- mice treated with PF-3845 or URB597 (10 mg/kg, i.p., 2 hr). Note that nolamine [PEA] [B] and N-oleoyl ethanolamine [OEA] URB597, but not PF-3845, blocks FP-rhodamine labeling of several liver serine [These animals also showed significant elevations hydrolase activities (bracket), which have previously been identified as carbox- in AEA, PEA, and OEA levels in liver tissue In ylesterases For both (A) and(B), fluorescent gel images are shown in grayscale. For (B), a lower scanning contrast, PF-3845-treated animals did not show changes in the intensity is shown due to the highly abundant activity signals for carboxyles- levels of 2-AG in brain or liver B), consistent with terases present in the liver proteome.
previous findings indicating that distinct enzymes regulate thisendocannabinoid in vivo ( described alkyne analog of URB597 ( We also confirmed that PF-3845 did PF3845yne and JP104 were administered to FAAH(+/+) not alter endocannabinoid (AEA or 2-AG) levels in FAAH(/) and (/) mice (10 mg/kg, i.p.), and, after 2 hr, animals were sacrificed and their brain and liver proteomes analyzed by PF-3845-induced elevations in NAEs peaked at 3 hr and CC-ABPP by using an azide-rhodamine tag. Consistent with were maintained at maximal levels for up to 7–12 hr, after which the results of our competitive ABPP experiments (), they slowly declined. This decline was faster for AEA than for PF3845yne and JP104 selectively reacted with a single protein PEA or OEA, possibly reflecting that AEA is a much preferred in mouse brain that was confirmed as FAAH based on its substrate for FAAH () and absence in FAAH(/) mice C). In liver, however, therefore more sensitive to low levels of enzyme recovery. The PF3845yne and JP104 showed strikingly different profiles, with slightly faster rate of recovery for OEA versus PEA is also Figure 3. Direct Analysis of In Vivo ProteinTargets of Alkyne Analogs of PF-3845 andURB597 by CC-ABPP(A) Structure of PF3845yne, an alkyne analog ofPF-3845.
(B) Structure of JP104, an alkyne analog ofURB597 (C and D) Gel profiles of CC-ABPP experiments inwhich (C) brain or (D) liver proteomes fromPF3845yne- and JP104-treated mice (10 mg/kg,i.p., 2 hr) were treated with a rhodamine-azidetag under CC conditions and analyzed by in-gelfluorescence scanning (shown in grayscale).
PF3845yne selectively labels FAAH in both brainand liver tissue (60 kDa band absent in FAAH(/)mice), whereas JP104 labels several additionalproteins in liver (protein bands present in bothFAAH(+/+) and (/) mice). Note that the 55 kDaprotein band observed in liver proteome fromPF-3845-treated FAAH(/) mice was also de-tected in liver proteomes from vehicle-treatedFAAH(+/+) and (/) mice and therefore likelyrepresents a background protein that is cross-reactive with the azide-rhodamine tag.
Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved 415 Chemistry & Biology A Highly Efficacious and Selective FAAH Inhibitor Figure 4. Brain Levels of FAAH Substrates and Inhibitors(A–C) Brain levels of (A) AEA, (B) PEA, and (C) OEA measured at the indicated time points after 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 after inhibitor treatment.
consistent with the relative activity that FAAH displays for these antiallodynic effects in the CFA model. Alternatively, the kinetics substrates in vitro (). Despite FAAH's preference of FAAH blockade may have been slower at lower doses of for hydrolyzing AEA, significant elevations in this NAE (as well as PF-3845, which could reduce efficacy in pain models that require PEA and OEA) were still observed at 24 hr posttreatment with sustained elevations in AEA. It is also noteworthy that the PF-3845, consistent with the substantial level of FAAH inhibition maximal elevations observed for AEA in PF-3845-treated maintained at this time point (A). URB597-treated mice animals were similar in brain and plasma (10-fold), whereas also showed elevations in NAEs, but these increases were OEA and PEA were increased to a lower extent in plasma more transient, with AEA levels returning to near-baseline levels (3-fold) compared to brain (10-fold). This might suggest the by 7 hr posttreatment A–4C). The different rates of existence of alternative degradative () (or recovery of both FAAH activity and NAE levels biosynthetic []) pathways for AEA versus (in mice treated with PF-3845 versus URB597 OEA/PEA in peripheral tissues.
likely reflect the distinct half-lives of these inhibitors in vivo, since Considering that mice treated with PF-3845 showed high higher relative brain levels of PF-3845 were maintained over the (>10-fold) elevations in AEA that lasted for at least 7 hr, we time course analysis D).
next evaluated the duration of action of this inhibitor in the CFAmodel from 2–8 hr postadministration. PF-3845 (10 mg/kg, PF-3845 Produces Cannabinoid Receptor-Dependent p.o.) exhibited significant inhibition of mechanical allodynia at Reductions in Inflammatory Pain all time points tested D), although this effect appeared We next assessed whether PF-3845 was efficacious in a rat to diminish in magnitude by 8 hr. This loss in activity could reflect model of inflammatory pain. Subcutaneous injection of complete gradual recovery of FAAH activity in PF-3845-treated rats, as Freund's adjuvant (CFA) into the plantar surface of the hind paw may be reflected in the decline in AEA levels that was observed produced a significant decrease in mechanical paw weight at a similar time point in mice treated with this inhibitor (see threshold (PWT) at 5 days postinjection A). PF-3845 A). However, further time course studies in rats will be (1–30 mg/kg, oral administration [p.o.]) caused a dose- required to accurately estimate the duration of action of dependent inhibition of mechanical allodynia with a minimum PF-3845 in these animals. We next examined the involvement effective dose (MED) of 3 mg/kg (rats were analyzed at 4 hr post- of cannabinoid receptors in the PF-3845-induced antiallodynia.
dosing with PF-3845). At higher doses (10 and 30 mg/kg), Specific antagonists for central CB1 (SR141716) and peripheral PF-3845 inhibited pain responses to an equivalent, if not greater, CB2 (SR144528) receptors each partially, but significantly, degree than the nonsteroidal anti-inflammatory drug naproxen reduced the antiallodynia activity of PF-3845 (E).
(10 mg/kg, p.o.) (We next determined the relationship Notably, dual treatment with SR141716 and SR144528 between the in vivo efficacy and modulation of FAAH activity and produced a near-complete ablation of the efficacy of PF-3845 substrates (i.e., NAEs) by PF-3845 in central (brain) and periph- in the CFA model (These data indicate that PF-3845 eral systems (peripheral blood leukocytes/plasma, liver). Robust, blocks inflammatory pain responses in the CFA model by near-complete inhibition of FAAH activity ( a cannabinoid receptor-dependent mechanism that likely with concomitant elevations in AEA (and other NAEs involves both CB1 and CB2 receptors, as has been shown for (Figures B, S4C, and ) were observed in brain, peripheral other FAAH inhibitors blood leukocytes/plasma, and liver from PF-3845-treatedanimals. Interestingly, we observed a slightly left-shifted dose- response profile for measurements of FAAH activity and NAEs Endocannabinoids, like other lipid signaling molecules, are compared to efficacy in the CFA model. For instance, at 1 mg/kg, thought to be produced on demand by neurons, rather than PF-3845 was found to produce a near-complete blockade of being stored in synaptic vesicles like classical neurotransmitters FAAH activity (B) and close to maximal elevations in AEA but efficacy was not observed at this dose of uptake pathways may exist for endocannabinoids ( inhibitor (These data may indicate the need to fully block FAAH (and fully elevate AEA levels) in order to observe the hydrophobic nature of these lipids 416 Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved

Chemistry & Biology A Highly Efficacious and Selective FAAH Inhibitor Figure 5. Antihyperalgesic 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 nonsteroidal anti-inflammatorydrug naproxen (10 mg/kg, p.o., hatched bar) is shown for comparison. Paw-withdrawal thresholds were measured 4 hr postadministration of drugs.
(B and C) PF-3845-treated rats show significant blockade of (B) FAAH activity and elevated (C) AEA levels in brain tissue and blood leukocytes/plasma. All FAAHactivity and NAE measurements were determined at 4 hr after drug treatment and were significantly different between PF-3845- and vehicle-treated groups(p < 0.001, n = 3 rats/group).
(D) Time course for antihyperalgesic effects of PF-3845 (10 mg/kg, p.o.).
(E) Blockade of antihyperalgesic effects of PF-3845 (10 mg/kg, i.p.) by CB1 and CB2 antagonists (SR141716 and SR144528, respectively; 10 and 30 mg/kg, i.p.,respectively, administered 1 hr prior to measurement of paw-withdrawal thresholds). Note that neither the CB1 nor the CB2 antagonist displayed significanteffects 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.
also grants them the ability to freely diffuse across cell PF-3845 also induces elevations in other noncannabinoid membranes. This feature assigns primary responsibility for NAEs (e.g., OEA, PEA), which may have additional effects on signal termination to degradative enzymes, such as FAAH. The inflammation and other physiological processes pharmacological blockade of FAAH has, thus, emerged as Structural and kinetic studies indicate that the a strategy by which to elevate endocananbinoid signaling in vivo and to study the behavioral consequences. Over the described inhibitors (URB597, PF-750) likely originates from past decade, several classes of FAAH inhibitors have been more extended van der Waals contacts with the hydrophobic described that vary in their mechanism of action, potency, selec- FAAH acyl chain-binding tunnel. This premise is also supported tivity, and in vivo efficacy. Arguably, the most well-studied FAAH by SAR data generated with a set of biaryl ether analogs of inhibitor to date is URB597 a carbamate PF-3845, in which the introduction of polar groups within that irreversibly inhibits FAAH with high selectivity in the nervous certain regions of the inhibitor scaffold severely impaired system (However, URB597 activity (e.g., compound 16, ).
displays some notable shortcomings, including the inactivation In summary, the properties of PF-3845 defined in this study of other serine hydrolases in peripheral tissues ( indicate that this agent should be considered a frontline inhibitor ) and a relatively limited duration for the pharmacological investigation of FAAH-regulated endo- of action in vivo. Development of a FAAH inhibitor that displays cannabinoid pathways. Differentiating properties of PF-3845 an optimal combination of efficacy and selectivity has, therefore, include its high target selectivity, extended duration of action, remained an intensely pursued objective.
and excellent oral bioavailability (>80%, data not shown). These We have described herein the detailed characterization of an features should make PF-3845 particularly well suited for advanced piperidine urea inhibitor of FAAH, PF-3845. Our data studying the role of the endocannabinoid system in both central argue that PF-3845 displays remarkable selectivity for FAAH and peripheral physiological processes, as well as chronic path- and robust efficacy in vivo, as reflected in sustained elevations ological syndromes (e.g., neuropathic pain, depression) that of brain AEA that correlate with cannabinoid receptor- may require sustained elevations in endocannabinoid tone for Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved 417 Chemistry & Biology A Highly Efficacious and Selective FAAH Inhibitor Monte Carlo Simulations of FAAH Inhibitor Adducts The program ICM-Pro (Molsoft, L.L.C.) was used to simulate covalent docking The endocannabinoid system regulates a number of impor- of biaryl ether urea inhibitors into the active site of the h/rFAAH as described in. As a positive control, we performed tant physiological processes in mammals and is the prin- the docking of the PF-3845 ligand, which provided a conformation that closely cipal site of action for THC, the psychoactive ingredient in matched the conformation observed in the PF-3845-FAAH crystal structure.
marijuana. THC and other direct agonists of the CB1receptor have potentially useful medicinal properties, such Competitive ABPP Studies as pain relief, but these activities are accompanied by PF-3845 or URB597 (neat) were dissolved at 1 mg/ml by sonication and vor- a variety of untoward side effects, including impairment in texing directly into a solution of 18:1:1 v/v/v saline:emulphor:ethanol. MaleC57Bl/6J mice (6–12 weeks old, 20–28 g) were intraperitoneally (i.p.) adminis- motor control and cognitive function. In contrast, blockade tered PF-3845, URB597, or an 18:1:1 v/v/v saline:emulphor:ethanol vehicle at of endocannabinoid-metabolizing enzymes such as FAAH a volume of 10 ml/g weight (10 mg/kg final dose). After the indicated amount of has been shown to reduce pain sensation, inflammation, time, mice were anesthetized with isofluorane and sacrificed by decapitation.
and anxiety/depression without substantial effects on Brains were removed and hemisected along the midsagittal plane, and each motility or cognition. Despite considerable advances in the half was then flash frozen in liquid N2. One half of the brain was homogenized development of FAAH inhibitors, agents that combine high in PBS (pH 7.4, 2 ml), diluted to 1 mg protein/ml; treated with FP-rhodamine(1 mM, 0.5 hr); and analyzed by gel-based ABPP by following previously target selectivity with exceptional in vivo efficacy are still described methods (). The other half of the brain needed. Here, we report the discovery and detailed charac- was used for lipid and drug measurements as detailed in terization of PF-3845, a piperidine urea FAAH inhibitor that satisfies these criteria. PF-3845 was shown by mechanisticand structural studies to covalently inactivate FAAH by car- bamylation of the enzyme's serine nucleophile. PF-3845 CC-ABPP studies were performed by following previously described proce- completely inhibited FAAH activity, but did not react with dures ) and as detailed in .
other serine hydrolases in vivo, as determined by activity-based proteomics; raised brain levels of the endocannabi- Measurement of FAAH Activity and Lipid Levels from Rat Tissues noid AEA for up to 24 hr; and produced significant CB1/ and Blood Leukocytes CB2-dependent antihyperalgesic effects in the CFA model Preparation of rat tissue (brain and liver) and blood leukocytes for measuring of inflammatory pain. From these data, we conclude that FAAH activity and lipid levels is described in PF-3845 is a highly selective and efficacious inhibitor of FAAH that should prove to be a valuable pharmacological Analysis of PF-3845 Effects in the CFA Model of Inflammatory Pain tool for investigating the function of the endocannabinoid Male Sprague-Dawley rats (200–250 g) were used for all experiments.
Rats were given free access to food and water and were maintained on a12/12 hr light/dark cycle. For the inflammatory pain model, 0.15 ml CFA EXPERIMENTAL PROCEDURES (Sigma) was injected into the plantar surface of the left hind paw of the rat.
The CFA injection immediately induces local inflammation, paw swelling, and pain, which persist for at least 2 weeks postinjection. To assess mechan- ical allodynia, mechanical paw-withdrawal thresholds (PWTs) were measured synthesized as described previously ). URB597 and JP104 by using a set of Von Frey Hairs on day 5 postinjection as illustrated by the were purchased from Cayman Chemicals (Ann Arbor, MI). Inhibitors were Dixon Up and Down Method ). Animals that exhibit the pain criteria stored as dry powders at room temperature and dissolved in DMSO to prepare of 9 g or less were then placed on study. Test compound was administered via concentrated stock solutions for the in vitro potency measurements.
either oral or i.p. routes at the indicated concentrations (mg/kg) with the dosing SR141716 and SR144528, selective CB1 and CB2 receptor antagonists, vehicle 5% N,N-dimethylacetamide and 95% 2-hydroxypropyl-b-cyclodextrin were synthesized at Pfizer. Polystyrene 96- and 384-microplates were (40%) (Sigma) in water). PWTs were evaluated again at 4 hr postdosing. PWT purchased from Rainin and Evergreen Scientific (Los Angeles, CA), respec- measurements were averaged, and statistical comparisons between groups tively. All reagents used were the highest quality commercially available.
were made by using analysis of variance and unpaired t tests. The Pfizer Insti-tutional Animal Care and Use Committee reviewed and approved the animaluse in these studies. The animal care and use program is fully accredited by Synthesis of PF-3845 and Structural Analogs the Association for Assessment and Accreditation of Laboratory Animal The synthesis of PF-3845 and other biaryl ether piperidine/piperazine ureas was performed as described in .
SUPPLEMENTAL DATA FAAH Expression and Purification All three forms of FAAH (hFAAH, rFAAH, and h/rFAAH) were expressed and Supplemental Data include Supplemental Experimental Procedures, one purified by following previously described procedures ( table, and five figures and can be found with this article online at and as described in .
Determination of Inhibitor Potency Inhibitor potencies were determined by following previously described proce-dures and as described in We thank Kimberly Masuda and Jessica P. Alexander for technical assistance with ABPP studies; Max Totrov from Molsoft for helpful suggestions anddiscussions on the docking and simulation work; Steve Kesten for the scale- h/rFAAH Crystallography up of PF-3845; Mike Walters, Michael Connolly, Yuntao Song, Mark Morris, Crystallization and structure determination were performed as described in and Sue Kesten for early chemistry contributions to the biaryl ether series; and Zhigang Wang and Daniel Everdeen for their assistance with the 418 Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved Chemistry & Biology A Highly Efficacious and Selective FAAH Inhibitor measurement of inhibitor potencies. This work was supported in part by the Fegley, D., Gaetani, S., Duranti, A., Tontini, A., Mor, M., Tarzia, G., and Pio- National Institutes of Health (DA017259), the Helen L. Dorris Institute for the melli, D. (2005). Characterization of the fatty acid amide hydrolase inhibitor Study of Neurological and Psychiatric Disorders in Children and Adolescents, cyclohexyl carbamic acid 30-carbamoyl-biphenyl-3-yl ester (URB597): effects and The Skaggs Institute for Chemical Biology.
on anandamide and oleoylethanolamide deactivation. J. Pharmacol. Exp.
Ther. 313, 352–358.
Received: December 22, 2008 Fowler, C.J. (2006). The cannabinoid system and its pharmacological manip- Revised: February 11, 2009 ulation–a review, with emphasis upon the uptake and hydrolysis of ananda- Accepted: February 23, 2009 mide. Fundam. Clin. Pharmacol. 20, 549–562.
Published: April 23, 2009 Glaser, S.T., Kaczocha, M., and Deutsch, D.G. (2005). Anandamide transport:a critical review. Life Sci. 77, 1584–1604.
Gobbi, G., Bambico, F.R., Mangieri, R., Bortolato, M., Campolongo, P., Soli-nas, M., Cassano, T., Morgese, M.G., Debonnel, G., Duranti, A., et al. (2005).
Ahn, K., Johnson, D.S., Fitzgerald, L.R., Liimatta, M., Arendse, A., Stevenson, Antidepressant-like activity and modulation of brain monoaminergic transmis- T., Lund, E.T., Nugent, R.A., Nomanbhoy, T.K., Alexander, J.P., and Cravatt, sion by blockade of anandamide hydrolysis. Proc. Natl. Acad. Sci. USA 102, B.F. (2007). Novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity. Biochemistry 46, 13019–13030.
Hillard, C.J., and Jarrahian, A. (2003). Cellular accumulation of anandamide: Ahn, K., McKinney, M.K., and Cravatt, B.F. (2008). Enzymatic pathways that consensus and controversy. Br. J. Pharmacol. 140, 802–808.
regulate endocannabinoid signaling in the nervous system. Chem. Rev. 108, Holt, S., Comelli, F., Costa, B., and Fowler, C.J. (2005). Inhibitors of fatty acid amide hydrolase reduce carrageenan-induced hind paw inflammation in Alexander, J.P., and Cravatt, B.F. (2005). Mechanism of carbamate inactiva- pentobarbital-treated mice: comparison with indomethacin and possible tion of FAAH: implications for the design of covalent inhibitors and in vivo func- involvement of cannabinoid receptors. Br. J. Pharmacol. 146, 467–476.
tional probes for enzymes. Chem. Biol. 12, 1179–1187.
Jayamanne, A., Greenwood, R., Mitchell, V.A., Aslan, S., Piomelli, D., and Blankman, J.L., Simon, G.M., and Cravatt, B.F. (2007). A comprehensive Vaughan, C.W. (2006). Actions of the FAAH inhibitor URB597 in neuropathic profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoyl- and inflammatory chronic pain models. Br. J. Pharmacol. 147, 281–288.
glycerol. Chem. Biol. 14, 1347–1356.
Kathuria, S., Gaetani, S., Fegley, D., Valino, F., Duranti, A., Tontini, A., Mor, M., Boger, D.L., Sato, H., Lerner, A.E., Austin, B.J., Patterson, J.E., Patricelli, M.P., Tarzia, G., La Rana, G., Calignano, A., et al. (2003). Modulation of anxiety and Cravatt, B.F. (1999). Trifluoromethyl ketone inhibitors of fatty acid amide through blockade of anandamide hydrolysis. Nat. Med. 9, 76–81.
hydrolase: a probe of structural and conformational features contributing to Keith, J.M., Apodaca, R., Xiao, W., Seierstad, M., Pattabiraman, K., Wu, J., inhibition. Bioorg. Med. Chem. Lett. 9, 265–270.
Webb, M., Karbarz, M.J., Brown, S., Wilson, S., et al. (2008). Thiadiazolopiper- Boger, D.L., Sato, H., Lerner, A.E., Hedrick, M.P., Fecik, R.A., Miyauchi, H., azinyl ureas as inhibitors of fatty acid amide hydrolase. Bioorg. Med. Chem.
Wilkie, G.D., Austin, B.J., Patricelli, M.P., and Cravatt, B.F. (2000). Exception- Lett. 18, 4838–4843.
ally potent inhibitors of fatty acid amide hydrolase: the enzyme responsible for Koutek, B., Prestwich, G.D., Howlett, A.C., Chin, S.A., Salehani, D., Akhavan, degradation of endogenous oleamide and anandamide. Proc. Natl. Acad. Sci.
N., and Deutsch, D.G. (1994). Inhibitors of arachidonyl ethanolamide hydro- USA 97, 5044–5049.
lysis. J. Biol. Chem. 269, 22937–22940.
Bracey, M.H., Hanson, M.A., Masuda, K.R., Stevens, R.C., and Cravatt, B.F.
Lambert, D.M., Vandevoorde, S., Jonsson, K.O., and Fowler, C.J. (2002). The (2002). Structural adaptations in a membrane enzyme that terminates endo- palmitoylethanolamide family: a new class of anti-inflammatory agents? Curr.
cannabinoid signaling. Science 298, 1793–1796.
Med. Chem. 9, 663–674.
Brandl, M., Weiss, M.S., Jabs, A., Suhnel, J., and Hilgenfeld, R. (2001).
Leung, D., Hardouin, C., Boger, D.L., and Cravatt, B.F. (2003). Discovering C-H.pi-interactions in proteins. J. Mol. Biol. 307, 357–377.
potent and selective inhibitors of enzymes in complex proteomes. Nat. Bio- Chang, L., Luo, L., Palmer, J.A., Sutton, S., Wilson, S.J., Barbier, A.J., Breiten- technol. 21, 687–691.
bucher, J.G., Chaplan, S.R., and Webb, M. (2006). Inhibition of fatty acid amide Leung, D., Saghatelian, A., Simon, G.M., and Cravatt, B.F. (2006). Inactivation hydrolase produces analgesia by multiple mechanisms. Br. J. Pharmacol. 148, of N-acyl ethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids. Biochemistry 45, 4720–4726.
Cravatt, B.F., Giang, D.K., Mayfield, S.P., Boger, D.L., Lerner, R.A., and Gilula, Lichtman, A.H., Leung, D., Shelton, C., Saghatelian, A., Hardouin, C., Boger, N.B. (1996). Molecular characterization of an enzyme that degrades neuromo- D., and Cravatt, B.F. (2004a). Reversible inhibitors of fatty acid amide hydro- dulatory fatty-acid amides. Nature 384, 83–87.
lase that promote analgesia: evidence for an unprecedented combination of Cravatt, B.F., Demarest, K., Patricelli, M.P., Bracey, M.H., Giang, D.K., Martin, potency and selectivity. J. Pharmacol. Exp. Ther. 311, 441–448.
B.R., and Lichtman, A.H. (2001). Supersensitivity to anandamide and Lichtman, A.H., Shelton, C.C., Advani, T., and Cravatt, B.F. (2004b). Mice lack- enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide ing fatty acid amide hydrolase exhibit a cannabinoid receptor-mediated hydrolase. Proc. Natl. Acad. Sci. USA 98, 9371–9376.
phenotypic hypoalgesia. Pain 109, 319–327.
Cravatt, B.F., Saghatelian, A., Hawkins, E.G., Clement, A.B., Bracey, M.H., Liu, Y., Patricelli, M.P., and Cravatt, B.F. (1999). Activity-based protein and Lichtman, A.H. (2004). Functional disassociation of the central and periph- profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 96, 14694–14699.
eral fatty acid amide signaling systems. Proc. Natl. Acad. Sci. USA 101, Long, J.Z., Li, W., Booker, L., Burston, J.J., Kinsey, S.G., Schlosburg, J.E., Pavon, F.J., Serrano, A.M., Selley, D.E., Parsons, L.H., et al. (2009). Selective Deutsch, D.G., Omir, R., Arreaza, G., Salehani, D., Prestwich, G.D., Huang, Z., blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behav- and Howlett, A. (1997). Methyl arachidonyl fluorophosphonate: a potent irre- ioral effects. Nat. Chem. Biol. 5, 37–44.
versible inhibitor of anandamide amidase. Biochem. Pharmacol. 53, 255–260.
Massa, F., Marsicano, G., Hermann, H., Cannich, A., Monory, K., Cravatt, B.F., Di Marzo, V. (2008). Endocannabinoids: synthesis and degradation. Rev.
Ferri, G.L., Sibaev, A., Storr, M., and Lutz, B. (2004). The endogenous canna- Physiol. Biochem. Pharmacol. 160, 1–24.
binoid system protects against colonic inflammation. J. Clin. Invest. 113, Di Marzo, V., Bisogno, T., and De Petrocellis, L. (2007). Endocannabinoids and related compounds: walking back and forth between plant natural products McFarland, M.J., and Barker, E.L. (2004). Anandamide transport. Pharmacol.
and animal physiology. Chem. Biol. 14, 741–756.
Ther. 104, 117–135.
Dixon, W.J. (1980). Efficient analysis of experimental observations. Annu. Rev.
McKinney, M.K., and Cravatt, B.F. (2005). Structure and function of fatty acid Pharmacol. Toxicol. 20, 441–462.
amide hydrolase. Annu. Rev. Biochem. 74, 411–432.
Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved 419 Chemistry & Biology A Highly Efficacious and Selective FAAH Inhibitor Mechoulam, R. (1986). The Pharmacohistory of Cannabis sativa (Boca Raton, acid amide hydrolase inhibitor URB597 (cyclohexylcarbamic acid 30-carba- FL: CRS Press).
moylbiphenyl-3-yl ester) reduces neuropathic pain after oral administration Mileni, M., Johnson, D.S., Wang, Z., Everdeen, D.S., Liimatta, M., Pabst, B., in mice. J. Pharmacol. Exp. Ther. 322, 236–242.
Bhattacharya, K., Nugent, R.A., Kamtekar, S., Cravatt, B.F., et al. (2008).
Saghatelian, A., Trauger, S.A., Want, E.J., Hawkins, E.G., Siuzdak, G., and Structure-guided inhibitor design for human FAAH by interspecies active site Cravatt, B.F. (2004). Assignment of endogenous substrates to enzymes by conversion. Proc. Natl. Acad. Sci. USA 105, 12820–12824.
global metabolite profiling. Biochemistry 43, 14332–14339.
Moreira, F.A., Kaiser, N., Monory, K., and Lutz, B. (2008). Reduced anxiety-like Speers, A.E., and Cravatt, B.F. (2004). Profiling enzyme activities in vivo using behaviour induced by genetic and pharmacological inhibition of the endocan- click chemistry methods. Chem. Biol. 11, 535–546.
nabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) is mediated byCB1 receptors. Neuropharmacology 54, 141–150.
Speers, A.E., Adam, G.C., and Cravatt, B.F. (2003). Activity-based protein Naidu, P.S., Varvel, S.A., Ahn, K., Cravatt, B.F., Martin, B.R., and Lichtman, profiling in vivo using a copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition.
A.H. (2007). Evaluation of fatty acid amide hydrolase inhibition in murine J. Am. Chem. Soc. 125, 4686–4687.
models of emotionality. Psychopharmacology (Berl.) 192, 61–70.
Swinney, D.C. (2004). Biochemical mechanisms of drug action: what does it Nomura, D.K., Blankman, J.L., Simon, G.M., Fujioka, K., Issa, R.S., Ward, take for success? Nat. Rev. Drug Discov. 3, 801–808.
A.M., Cravatt, B.F., and Casida, J.E. (2008). Activation of the endocannabinoidsystem by organophosphorus nerve agents. Nat. Chem. Biol. 4, 373–378.
Tsuboi, K., Sun, Y.X., Okamoto, Y., Araki, N., Tonai, T., and Ueda, N. (2005).
Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, Pacher, P., Batkai, S., and Kunos, G. (2006). The endocannabinoid system as a novel member of the choloylglycine hydrolase family with structural and an emerging target of pharmacotherapy. Pharmacol. Rev. 58, 389–462.
functional similarity to acid ceramidase. J. Biol. Chem. 280, 11082–11092.
Patricelli, M.P., Lovato, M.A., and Cravatt, B.F. (1999). Chemical and muta-genic investigations of fatty acid amide hydrolase: evidence for a family of Wei, B.Q., Mikkelsen, T.S., McKinney, M.K., Lander, E.S., and Cravatt, B.F.
serine hydrolase with distinct catalytic features. Biochemistry 38, 9804–9812.
(2006). A second fatty acid amide hydrolase with variable distribution among Patricelli, M.P., Giang, D.K., Stamp, L.M., and Burbaum, J.J. (2001). Direct placental mammals. J. Biol. Chem. 281, 36569–36578.
visualization of serine hydrolase activities in complex proteome using fluores- Zhang, D., Saraf, A., Kolasa, T., Bhatia, P., Zheng, G.Z., Patel, M., Lannoye, cent active site-directed probes. Proteomics 1, 1067–1071.
G.S., Richardson, P., Stewart, A., Rogers, J.C., et al. (2007). Fatty acid amide Russo, R., Loverme, J., La Rana, G., Compton, T.R., Parrott, J., Duranti, A., hydrolase inhibitors display broad selectivity and inhibit multiple carboxyles- Tontini, A., Mor, M., Tarzia, G., Calignano, A., and Piomelli, D. (2007). The fatty terases as off-targets. Neuropharmacology 52, 1095–1105.
420 Chemistry & Biology 16, 411–420, April 24, 2009 ª2009 Elsevier Ltd All rights reserved

Source: http://chemistry.umbc.edu/files/2016/04/Ahnetal_ChemBiol-2009.pdf