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.