The endocannabinoid hydrolase FAAH is an allosteric enzyme.
Allosteric Regulation
/ drug effects
Allosteric Site
/ drug effects
Amidohydrolases
/ antagonists & inhibitors
Animals
Arachidonic Acids
Benzamides
/ pharmacology
Biocatalysis
/ drug effects
Carbamates
/ pharmacology
Catalytic Domain
/ drug effects
Drug Design
Endocannabinoids
/ metabolism
Enzyme Assays
Humans
Hydrolysis
/ drug effects
Kinetics
Molecular Dynamics Simulation
Mutation
Polyunsaturated Alkamides
Protein Subunits
/ antagonists & inhibitors
Rats
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
10 02 2020
10 02 2020
Historique:
received:
26
09
2019
accepted:
22
01
2020
entrez:
12
2
2020
pubmed:
12
2
2020
medline:
21
11
2020
Statut:
epublish
Résumé
Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that in vivo controls content and biological activity of N-arachidonoylethanolamine (AEA) and other relevant bioactive lipids termed endocannabinoids. Parallel orientation of FAAH monomers likely allows both subunits to simultaneously recruit and cleave substrates. Here, we show full inhibition of human and rat FAAH by means of enzyme inhibitors used at a homodimer:inhibitor stoichiometric ratio of 1:1, implying that occupation of only one of the two active sites of FAAH is enough to fully block catalysis. Single W445Y substitution in rat FAAH displayed the same activity as the wild-type, but failed to show full inhibition at the homodimer:inhibitor 1:1 ratio. Instead, F432A mutant exhibited reduced specific activity but was fully inhibited at the homodimer:inhibitor 1:1 ratio. Kinetic analysis of AEA hydrolysis by rat FAAH and its F432A mutant demonstrated a Hill coefficient of ~1.6, that instead was ~1.0 in the W445Y mutant. Of note, also human FAAH catalysed an allosteric hydrolysis of AEA, showing a Hill coefficient of ~1.9. Taken together, this study demonstrates an unprecedented allosterism of FAAH, and represents a case of communication between two enzyme subunits seemingly controlled by a single amino acid (W445) at the dimer interface. In the light of extensive attempts and subsequent failures over the last decade to develop effective drugs for human therapy, these findings pave the way to the rationale design of new molecules that, by acting as positive or negative heterotropic effectors of FAAH, may control more efficiently its activity.
Identifiants
pubmed: 32041998
doi: 10.1038/s41598-020-59120-1
pii: 10.1038/s41598-020-59120-1
pmc: PMC7010751
doi:
Substances chimiques
Arachidonic Acids
0
Benzamides
0
Carbamates
0
Endocannabinoids
0
Polyunsaturated Alkamides
0
Protein Subunits
0
cyclohexyl carbamic acid 3'-carbamoylbiphenyl-3-yl ester
0
Amidohydrolases
EC 3.5.-
fatty-acid amide hydrolase
EC 3.5.1.-
anandamide
UR5G69TJKH
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2292Commentaires et corrections
Type : ErratumIn
Références
Piomelli, D. The molecular logic of endocannabinoid signalling. Nat. Rev. Neurosci. 4, 873–84 (2003).
doi: 10.1038/nrn1247
Maccarrone, M., Guzmán, M., Mackie, K., Doherty, P. & Harkany, T. Programming of neural cells by (endo)cannabinoids: from physiological rules to emerging therapies. Nat. Rev. Neurosci. 15, 786–801 (2014).
doi: 10.1038/nrn3846
Lu, H.-C. & Mackie, K. An Introduction to the Endogenous Cannabinoid System. Biol. Psychiatry 79, 516–525 (2016).
doi: 10.1016/j.biopsych.2015.07.028
Maccarrone, M. et al. The Endocannabinoid System and Its Relevance for Nutrition. Annu. Rev. Nutr, https://doi.org/10.1146/annurev.nutr.012809.104701 (2010).
Ahn, K., McKinney, M. K. & Cravatt, B. F. Enzymatic pathways that regulate endocannabinoid signaling in the nervous system. Chem. Rev. 108, 1687–707 (2008).
doi: 10.1021/cr0782067
Maccarrone, M. et al. Endocannabinoid signaling at the periphery: 50 years after THC. Trends Pharmacol. Sci. 36, 277–96 (2015).
doi: 10.1016/j.tips.2015.02.008
Maccarrone, M., Dainese, E. & Oddi, S. Intracellular trafficking of anandamide: new concepts for signaling. Trends Biochem. Sci, https://doi.org/10.1016/j.tibs.2010.05.008 (2010).
Hermanson, D. J., Gamble-George, J. C., Marnett, L. J. & Patel, S. Substrate-selective COX-2 inhibition as a novel strategy for therapeutic endocannabinoid augmentation. Trends in Pharmacological Sciences 35, 358–367 (2014).
doi: 10.1016/j.tips.2014.04.006
Rodríguez De Fonseca, F. et al. An anorexic lipid mediator regulated by feeding. Nature, https://doi.org/10.1007/s10773-014-2266-7 (2001).
Fu, J., Oveisi, F., Gaetani, S., Lin, E. & Piomelli, D. Oleoylethanolamide, an endogenous PPAR-α agonist, lowers body weight and hyperlipidemia in obese rats. Neuropharmacology, https://doi.org/10.1016/j.neuropharm.2005.02.013 (2005).
Bachur, N. R., Masek, K., Melmon, K. L. & Udenfriend, S. Fatty acid amides of ethanolamine in mammalian tissues. J. Biol. Chem (1965).
Huang, S. M. et al. Identification of a New Class of Molecules, the Arachidonyl Amino Acids, and Characterization of One Member That Inhibits Pain. J. Biol. Chem, https://doi.org/10.1074/jbc.M107351200 (2001).
Di Marzo, V. & Maccarrone, M. FAAH and anandamide: is 2-AG really the odd one out? Trends Pharmacol. Sci. 29, 229–33 (2008).
doi: 10.1016/j.tips.2008.03.001
Bracey, M. H., Hanson, M. A., Masuda, K. R., Stevens, R. C. & Cravatt, B. F. Structural adaptations in a membrane enzyme that terminates endocannabinoid signaling. Science 298, 1793–6 (2002).
doi: 10.1126/science.1076535
Mileni, M. et al. Structure-guided inhibitor design for human FAAH by interspecies active site conversion. Proc. Natl. Acad. Sci. 105, 12820–12824 (2008).
doi: 10.1073/pnas.0806121105
McKinney, M. K. & Cravatt, B. F. Structure and function of fatty acid amide hydrolase. Annu. Rev. Biochem. https://doi.org/10.1146/annurev.biochem.74.082803.133450 (2005).
Palermo, G. et al. Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets. PLOS Comput. Biol. 11, e1004231 (2015).
doi: 10.1371/journal.pcbi.1004231
Boger, D. L. et al. Discovery of a potent, selective, and efficacious class of reversible alpha-ketoheterocycle inhibitors of fatty acid amide hydrolase effective as analgesics. J. Med. Chem. 48, 1849–56 (2005).
doi: 10.1021/jm049614v
McKinney, M. K. & Cravatt, B. F. Structure-based design of a FAAH variant that discriminates between the N-acyl ethanolamine and taurine families of signaling lipids. Biochemistry 45, 9016–22 (2006).
doi: 10.1021/bi0608010
Dainese, E. et al. Membrane lipids are key modulators of the endocannabinoid-hydrolase FAAH. Biochem. J. 457 (2014).
Centonze, D., Finazzi-Agrò, A., Bernardi, G. & Maccarrone, M. The endocannabinoid system in targeting inflammatory neurodegenerative diseases. Trends Pharmacol. Sci. 28, 180–187 (2007).
doi: 10.1016/j.tips.2007.02.004
Cravatt, B. F. et al. Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase. Proc. Natl. Acad. Sci. https://doi.org/10.1073/pnas.161191698 (2001).
Booker, L. et al. The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice. Br. J. Pharmacol. 165, 2485–96 (2012).
doi: 10.1111/j.1476-5381.2011.01445.x
Bisogno, T. & Maccarrone, M. Latest advances in the discovery of fatty acid amide hydrolase inhibitors. Expert Opin. Drug Discov. 8, 509–22 (2013).
doi: 10.1517/17460441.2013.780021
Mallet, C., Dubray, C. & Dualé, C. FAAH inhibitors in the limelight, but regrettably. Int. J. Clin. Pharmacol. Ther. 54, 498–501 (2016).
doi: 10.5414/CP202687
Edan, G. & Kerbrat, A. Inhibitor of Fatty Acid Amide Hydrolase — Learning from Tragic Failures. N. Engl. J. Med. 376, 392–394 (2017).
doi: 10.1056/NEJMc1615417
Palermo, G., Rothlisberger, U., Cavalli, A. & De Vivo, M. Computational insights into function and inhibition of fatty acid amide hydrolase. Eur. J. Med. Chem. 91, 15–26 (2015).
doi: 10.1016/j.ejmech.2014.09.037
Palermo, G. et al. Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility, Gating Residues and Multiple Binding Pockets. PLoS Comput. Biol, https://doi.org/10.1371/journal.pcbi.1004231 (2015).
Mei, G. et al. Closing the gate to the active site: effect of the inhibitor methoxyarachidonyl fluorophosphonate on the conformation and membrane binding of fatty acid amide hydrolase. J. Biol. Chem. 282, 3829–36 (2007).
doi: 10.1074/jbc.M605653200
Zou, H. et al. Human cyclooxygenase-1 activity and its responses to COX inhibitors are allosterically regulated by nonsubstrate fatty acids. J. Lipid Res. 53, 1336–47 (2012).
doi: 10.1194/jlr.M026856
Mor, M. et al. Cyclohexylcarbamic acid 3’- or 4’-substituted biphenyl-3-yl esters as fatty acid amide hydrolase inhibitors: synthesis, quantitative structure-activity relationships, and molecular modeling studies. J. Med. Chem. 47, 4998–5008 (2004).
doi: 10.1021/jm031140x
Ahn, K. et al. Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain. Chem. Biol. 16, 411–20 (2009).
doi: 10.1016/j.chembiol.2009.02.013
Romero, F. A. et al. Potent and selective alpha-ketoheterocycle-based inhibitors of the anandamide and oleamide catabolizing enzyme, fatty acid amide hydrolase. J. Med. Chem. 50, 1058–68 (2007).
doi: 10.1021/jm0611509
Butini, S. et al. Discovery of Potent Inhibitors of Human and Mouse Fatty Acid Amide Hydrolases. J. Med. Chem. 55, 6898–6915 (2012).
doi: 10.1021/jm300689c
Patricelli, M. P., Lashuel, Ha, Giang, D. K., Kelly, J. W. & Cravatt, B. F. Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: identification of the transmembrane domain as a site for oligomerization. Biochemistry 37, 15177–15187 (1998).
doi: 10.1021/bi981733n
Mileni, M. et al. Crystal Structure of Fatty Acid Amide Hydrolase Bound to the Carbamate Inhibitor URB597: Discovery of a Deacylating Water Molecule and Insight into Enzyme Inactivation. J. Mol. Biol. 400, 743–754 (2010).
doi: 10.1016/j.jmb.2010.05.034
Dong, L., Sharma, N. P., Jurban, B. J. & Smith, W. L. Pre-existent asymmetry in the human cyclooxygenase-2 sequence homodimer. J. Biol. Chem. 288, 28641–55 (2013).
doi: 10.1074/jbc.M113.505503
Di Venere, A. et al. Rat and human fatty acid amide hydrolases: overt similarities and hidden differences. Biochim. Biophys. Acta 1821, 1425–33 (2012).
doi: 10.1016/j.bbalip.2012.07.021
Long, J. Z., LaCava, M., Jin, X. & Cravatt, B. F. An anatomical and temporal portrait of physiological substrates for fatty acid amide hydrolase. J. Lipid Res. 52, 337–344 (2011).
doi: 10.1194/jlr.M012153
Hermanson, D. J. et al. Substrate-selective COX-2 inhibition decreases anxiety via endocannabinoid activation. Nat. Neurosci. 16, 1291–1298 (2013).
doi: 10.1038/nn.3480
Wang, Y. & Zhang, X. FAAH inhibition produces antidepressant-like efforts of mice to acute stress via synaptic long-term depression. Behav. Brain Res. 324, 138–145 (2017).
doi: 10.1016/j.bbr.2017.01.054
Faure, L. et al. Synthesis of Phenoxyacyl-Ethanolamides and Their Effects on Fatty Acid Amide Hydrolase Activity. J. Biol. Chem. 289, 9340–9351 (2014).
doi: 10.1074/jbc.M113.533315
Gattinoni, S. et al. Enol carbamates as inhibitors of fatty acid amide hydrolase (FAAH) endowed with high selectivity for FAAH over the other targets of the endocannabinoid system. ChemMedChem 5, 357–60 (2010).
doi: 10.1002/cmdc.200900472
Ramarao, M. K. et al. A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening. Anal. Biochem. 343, 143–51 (2005).
doi: 10.1016/j.ab.2005.04.032
Roughley, S. D. et al. Fatty acid amide hydrolase inhibitors. 3: tetra-substituted azetidine ureas with in vivo activity. Bioorg. Med. Chem. Lett. 22, 901–6 (2012).
doi: 10.1016/j.bmcl.2011.12.032
Dainese, E. et al. A novel role for iron in modulating the activity and membrane-binding ability of a trimmed soybean lipoxygenase-1. FASEB J. 24, 1725–1736 (2010).
doi: 10.1096/fj.09-141390
Monod, J., Wyman, J. & Changeux, J. P. On the nature of allosteric transitions: A plausible model. J. Mol. Biol, https://doi.org/10.1016/S0022-2836(65)80285-6 (1965).
Dainese, E., Di Muro, P., Beltramini, M., Salvato, B. & Decker, H. Subunits composition and allosteric control in Carcinus aestuarii hemocyanin. Eur. J. Biochem. https://doi.org/10.1046/j.1432-1327.1998.2560350.x (1998).
Harvey, M. J., Giupponi, G. & Fabritiis, G. De. ACEMD: Accelerating Biomolecular Dynamics in the Microsecond Time Scale. J. Chem. Theory Comput. 5, 1632–9 (2009).
doi: 10.1021/ct9000685