title: Development of fragment-based n-FABS NMR screening applied to the membrane enzyme FAAH.
abstract: Despite the recognized importance of membrane proteins as pharmaceutical targets, the reliable identification of fragment hits that are able to bind these proteins is still a major challenge. Among different ¹⁹F NMR spectroscopic methods, n-fluorine atoms for biochemical screening (n-FABS) is a highly sensitive technique that has been used efficiently for fragment screening, but its application for membrane enzymes has not been reported yet. Herein, we present the first successful application of n-FABS to the discovery of novel fragment hits, targeting the membrane-bound enzyme fatty acid amide hydrolase (FAAH), using a library of fluorinated fragments generated based on the different local environment of fluorine concept. The use of the recombinant fusion protein MBP-FAAH and the design of compound 11 as a suitable novel fluorinated substrate analogue allowed n-FABS screening to be efficiently performed using a very small amount of enzyme. Notably, we have identified 19 novel fragment hits that inhibit FAAH with a median effective concentration (IC₅₀) in the low mM-μM range. To the best of our knowledge, these results represent the first application of a ¹⁹F NMR fragment-based functional assay to a membrane protein.
authors.affiliation: Department of Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova (Italy).
authors.forename: Chiara
authors.initials: C
authors.lastname: Lambruschini
authors.forename: Marina
authors.initials: M
authors.lastname: Veronesi
authors.forename: Elisa
authors.initials: E
authors.lastname: Romeo
authors.forename: Gianpiero
authors.initials: G
authors.lastname: Garau
authors.forename: Tiziano
authors.initials: T
authors.lastname: Bandiera
authors.forename: Daniele
authors.initials: D
authors.lastname: Piomelli
authors.forename: Rita
authors.initials: R
authors.lastname: Scarpelli
authors.forename: Claudio
authors.initials: C
authors.lastname: Dalvit
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000818:Animals; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005461:Fluorine / Q000737:chemistry; D054879:Halogenation; D020128:Inhibitory Concentration 50; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D051381:Rats; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D004791:Enzyme Inhibitors; D005461:Fluorine; D000099252:Fatty Acid Amide Hydrolases
keywords: FAAH; fluorine NMR screening; fragment-based approach; membrane proteins