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FAAH-catalyzed C-C bond cleavage of a new multi-target analgesic drug Alessia Ligresti, Cristoforo Silvestri, Rosa Maria Vitale, Jose Martos, Fabiana Piscitelli, Jenny Wang, Marco Allarà, Robert Carling, Livio Luongo, Francesca Guida, Anna Illiano, Angela Amoresano, Sabatino Maione, Pietro Amodeo, David Woodward, Vincenzo Di Marzo, and Gennaro Marino ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00315 • Publication Date (Web): 18 Sep 2018 Downloaded from http://pubs.acs.org on September 19, 2018
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FAAH-catalyzed C-C bond cleavage of a new multi-target analgesic drug Alessia Ligresti1*, Cristoforo Silvestri1, Rosa Maria Vitale1, Jose L. Martos2, Fabiana Piscitelli1, Jenny W. Wang3, Marco Allarà1, Robert W. Carling2, Livio Luongo4, Francesca Guida4, Anna Illiano6, Angela Amoresano6, Sabatino Maione4, Pietro Amodeo1, David F. Woodward3, Vincenzo Di Marzo1,5*, Gennaro Marino6,7 Author affiliations: 1
National Research Council of Italy, Institute of Biomolecular Chemistry, Endocannabinoid
Research Group, Italy 2
Discovery Department, Selcia Limited, Essex, United Kingdom
3
Department of Biological Sciences, Allergan Inc., Irvine, USA§
4
Department of Experimental Medicine, Pharmacology Division, University of Campania, Italy
5
Canada Excellence Research Chair on the Microbiome-Endocannabinoidome Axis in Metabolic
Health, Universitè Laval, Quebec City, Canada 6
Department of Chemical Sciences, University of Naples “Federico II”, Italy
7
University “Suor Orsola Benincasa”, Italy
*
Corresponding authors:
[email protected];
[email protected] Author information notes: §
Current affiliation: JeniVision Inc., Irvine, USA
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ABSTRACT The discovery of extended catalytic versatilities is of great importance in both the chemistry and biotechnology fields. Fatty acid amide hydrolase (FAAH) belongs to the amidase signature superfamily and is a major endocannabinoid inactivating enzyme using an atypical catalytic mechanism involving hydrolysis of amide and occasionally ester bonds. FAAH inhibitors are efficacious in experimental models of neuropathic pain, inflammation and anxiety, among others. We report a new multi-target drug, AGN220653, containing a carboxyamide-4-oxazole moiety and endowed with efficacious analgesic and anti-inflammatory activities, which are partly due to its capability of achieving inhibition of FAAH, and subsequently increasing the tissue concentrations of the endocannabinoid anandamide. This inhibitor behaves as a non-competitive, slowly reversible inhibitor. Autoradiography of purified FAAH incubated with AGN220653, opportunely radiolabeled, indicated covalent binding followed by fragmentation of the molecule. Molecular docking suggested a possible nucleophilic attack by FAAH-Ser241 on the carbonyl group of the carboxyamide-4-oxazole moiety, resulting in the cleavage of the C-C bond between the oxazole and the carboxyamide moieties, instead of either of the two available amide bonds. MRM-MS analyses only detected the Ser241-assisted formation of the carbamate intermediate, thus confirming the cleavage of the aforementioned C-C bond. Quantum mechanics calculations were fully consistent with this mechanism. The study exemplifies how FAAH structural features and mechanism of action may override the binding and reactivity propensities of substrates. This unpredicted mechanism could pave the way to the future development of a completely new class of amidase inhibitors, of potential use against pain, inflammation and mood disorders.
Keywords: FAAH mechanism, C-C bond cleavage, multi-target inhibitors
Fatty acid amide hydrolase (FAAH) is an atypical serine hydrolase, which embodies a catalytic mechanism for the hydrolysis of amide and ester substrates. As the enzyme responsible for the hydrolysis of the endocannabinoid N-arachidonoyl-ethanolamine (anandamide, AEA) and related anti-inflammatory molecules1, FAAH has been proposed as a therapeutic target for the treatment of neuropathic pain, inflammation and mood disorders. Indeed, AEA, via different mechanisms including peripheral and/or spinal activation of cannabinoid CB1 and CB2 receptors, or activation ACS Paragon Plus Environment
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and/or desensitization of transient receptor potential vanilloid type-1 (TRPV1) channels, exerts antinociceptive and anti-hyperalgesic actions in various animal models of inflammatory and neuropathic pain2 . Furthermore, AEA also inhibits anxiety and depression in experimental models3. Accordingly, selective FAAH inhibitors have been developed as potential analgesics, anxiolytics and anti-depressants4. However, when tested in phase II clinical trials, an irreversible FAAH inhibitor failed to alleviate pain in patients with osteoarthritis5. Recently, new strategies focusing on targeted antagonism of selected prostanoid receptors have been developed6-7 resulting in efficacious compounds acting through the simultaneous blockade of the action of pro-inflammatory prostanoids at DP1, DP2, EP1, EP4, FP and TP receptors, while sparing the more benign EP2, EP3 and IP receptors. Based on this background, and on the increasing demand for rationally designed multitarget drugs, we aimed at including the chemical requirements for FAAH inhibition into the aforementioned prostanoid pan-antagonists through their progressive chemical modification. Having achieved this aim, we discovered, using molecular modeling, biochemistry, quantum mechanics and mass spectrometry approaches, that these new multi-target molecules inhibit FAAH by favoring an unprecedented catalytic behavior. This unique mechanism may now open previously unexplored avenues in the fields of biochemistry and medicinal and physical chemistry, with potential impact on therapeutic drug development.
RESULTS AND DISCUSSION Rational design of combined pan-PG antagonists hosting the capability of inhibiting FAAH in a slowly reversible manner We previously patented8 and reported7 a novel scaffold (AGN211377) with a unique polypharmacology and therapeutically ideal prostanoid receptor pan-antagonism profile at FP, TP, DP, EP1 and EP2 receptors that spares EP2, EP3 and IP receptors6.
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Figure 1. (a) AEA levels (pmol/mg of wet tissue) in mouse brains treated with AGN220653 (3mg/kg, i.p.) for different time points (30 min, 1h, 4h and 24h). Data are expressed as mean ± SEM (n=4 per group). Student t test *p
