A General NMR Method for Rapid, Efficient, and Reliable Biochemical Screening

Abstract

High-throughput screening is usually the method of drug-lead discovery. It is now well accepted that, for a functional assay, quality is more important than quantity. The ligand-based or protein-based NMR screening methodologies for detecting compounds binding to the macromolecular target of interest are now well established. A novel and sensitive NMR method for rapid, efficient, and reliable biochemical screening is presented. The method named 3-FABS (three fluorine atoms for biochemical screening) requires the labeling of the substrate with a CF(3) moiety and utilizes (19)F NMR spectroscopy for the detection of the starting and enzymatically modified substrates. The method allows for high-quality screening of large compound or natural product extract collections and for measuring their IC(50) values. Applications of this technique to the screening of inhibitors of the Ser/Thr kinase AKT1 and the protease trypsin are presented. In addition, an interesting application of 3-FABS to functional genomics is also presented.

Efficient synthetic inhibitors of anthrax lethal factor

“…Although the fluorescence-based assay is a robust technique to search for very potent inhibitors, it becomes more ambiguous in detecting weaker ligands (Ͼ100 M), possibly due to interference introduced by test compounds (normally used at high concentration) in the spectrophotometric assay. For this reason, we relied on a NMR-based enzymatic assay, which is unlikely to lead to false positives (16)(17)(18)(19)(20)(21)(22)(23). Recently, the use of 19 F-1D NMR to detect enzyme activity and inhibition both in proteases and kinases has been reported (22).…”

Fluorine NMR‐Based Screening on Cell Membrane Extracts

“…n-Fluorine atoms for biochemical screening (n-FABS) [2][3][4] is an NMR functional assay based on fluorine spectroscopy that requires the labeling of the substrate (or cofactor) of the enzyme with a fluorine-containing group and allows the direct measurement of the conversion of the substrate (S) into the product (P). n-FABS is a homogeneous assay and does not need radioactive labeling, secondary coupled chemical or enzymatic reactions, or labeled antibodies.…”

A General Approach for the Identification of Site‐Specific RNA Binders by 19F NMR Spectroscopy: Proof of Concept

“…[11][12][13] Very recently, the potential of 19 F detection for NMR-based binding and functional screenings has been recognized. Experiments such as FAXS (fluorine chemical shift anisotropy and exchange for screening) [14] and 3-FABS (three fluorine atoms for biochemical screening) [15] are particularly suitable because these methods are rarely subject to experimental artifacts. When this method is combined with cryogenic probe technology optimized for 19 F detection, the low sensitivity of the instrumentation can be diminished.…”