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.
Highlights
Over the last decade, the fragment-based approach (FBA) has become an established and efficient method for hit identification and optimization as an appealing alternative to highthroughput screening (HTS).[1]
fatty acid amide hydrolase (FAAH) is a 63 kDa protein whose structure was first solved by Bracey et al in 2002,[16] expressed in E. coli as a truncated functional form of the enzyme (DTM-FAAH) which lacks 29 residues at the N terminus
The insertion of an N-terminal fusion protein increases the amount of membrane protein that is heterologously expressed, and maltose-binding protein (MBP) is effective at promoting overexpression of soluble well-folded proteins.[17]
Summary
The fragment-based approach (FBA) has become an established and efficient method for hit identification and optimization as an appealing alternative to highthroughput screening (HTS).[1] The central idea in FBA is to identify fragments that bind to target proteins from a relatively small library of compounds (100–1000) complying with the rule of three.[2] Such libraries are able to cover the chemical space more thoroughly than libraries of larger molecules and are more likely to contain cores that fit into the target binding site.[3] Due to their low molecular weight, fragments usually bind weakly to the target macromolecules (mm–mm range). In order to detect such weak binding, sensitive biophysical techniques such as NMR, fluorescence spectroscopy, surface plasmon resonance, and X-ray crystallography[4] have been applied. FBAs have been successfully applied against soluble protein targets, and several compounds have advanced to [a] C.
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