166100-37-4Relevant articles and documents
Development of Fragment-Based n-FABS NMR Screening Applied to the Membrane Enzyme FAAH
Lambruschini, Chiara,Veronesi, Marina,Romeo, Elisa,Garau, Gianpiero,Bandiera, Tiziano,Piomelli, Daniele,Scarpelli, Rita,Dalvit, Claudio
, p. 1611 - 1619 (2013)
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.
Novel analogues of arachidonylethanolamide (anandamide): Affinities for the CB1 and CB2 cannabinoid receptors and metabolic stability
Lin, Sonyuan,Khanolkar, Atmaram D.,Fan, Pusheng,Goutopoulos, Andreas,Qin, Ce,Papahadjis, Demetris,Makriyannis, Alexandras
, p. 5353 - 5361 (2007/10/03)
Several analogues of the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide) were synthesized and evaluated in order to study (a) the structural requirements for high-affinity binding to the CB1 and CB2 cannabinoid receptors and (b) their hydrolytic stability toward anandamide amidase. The series reported here was aimed at exploring structure-activity relationships (SAR) primarily with regard to stereoelectronic requirements of ethanolamido headgroup for interaction with the cannabinoid receptor active site. Receptor affinities, reported as K(i) values, were obtained by a standard receptor binding assay using [3H]CP- 55,940 as the radioligand, while stability toward the amidase was evaluated by comparing the K(i) of each analogue in the presence and absence of phenylmethanesulfonyl fluoride (PMSF), a serine protease blocker and inhibitor of anandamide amidase. Introduction of a methyl group in the 1'- and 2'-positions or substitution of the ethanolamido headgroup with a butylamido group gave analogues with vastly improved biochemical stability. This is accomplished in some cases with increased receptor affinity. Conversely, oxazolyl and methyloxazolyl headgroups led to low-affinity analogues. Substitution of the hydroxyl group with electronegative substituents such as fluoro, chloro, allyl, and propargyl groups significantly increased receptor affinity but did not influence the biochemical stability. The 2'-chloro analogue of anandamide was found to have the highest affinity for CB1. Additionally, reversing the positions of the carbonyl and NH in the amido group produces retro-anandamides possessing considerably higher metabolic stability. Replacement of the arachidonyl tail with oleyl or linoleyl results in analogues with low affinities for both receptors. All of the analogues in this study showed high selectivity for the CB1 receptor over the peripheral CB2 receptor. The most potent analogues were tested for their ability to stimulate the binding of [35S]GTPγS to G- proteins and were shown to be potent cannabimimetic agonists. The results are discussed in terms of pharmacophoric features affecting receptor affinity and enzymatic stability.
