title: Rational design of fatty acid amide hydrolase inhibitors that act by covalently bonding to two active site residues.
abstract: The design and characterization of α-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two α-ketooxazoles (3 and 4) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of 2 (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent noncompetitive inhibition, the cocrystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrates that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged over the 1-6 h time course monitored.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Katerina
authors.initials: K
authors.lastname: Otrubova
authors.forename: Monica
authors.initials: M
authors.lastname: Brown
authors.forename: Michael S
authors.initials: MS
authors.lastname: McCormick
authors.forename: Gye W
authors.initials: GW
authors.lastname: Han
authors.forename: Scott T
authors.initials: ST
authors.lastname: O'Neal
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
authors.forename: Aron H
authors.initials: AH
authors.lastname: Lichtman
authors.forename: Dale L
authors.initials: DL
authors.lastname: Boger
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors*; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001923:Brain Chemistry / Q000187:drug effects; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006930:Hyperalgesia / Q000188:drug therapy; D007202:Indicators and Reagents; D007700:Kinetics; D050356:Lipid Metabolism / Q000187:drug effects; D051379:Mice; D008958:Models, Molecular; D008968:Molecular Conformation; D009437:Neuralgia / Q000188:drug therapy; D050050:Protein Carbonylation; D051381:Rats; D011994:Recombinant Proteins; D012235:Rhodamines; D013379:Substrate Specificity; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D012235:Rhodamines; D000099252:Fatty Acid Amide Hydrolases