Affinity measurement of strong ligands with NMR spectroscopy: Limitations and ways to overcome them.

NMR spectroscopy is currently extensively used in binding assays for hit identification, but its use in dissociation constant determination is more limited when compared to other biophysical techniques, in particular for tight binders. Although NMR is quite suitable for measuring the binding strength of weak to medium affinity ligands with dissociation constant K > 1 μM, it has some limitations in the determination of the binding strength of tight binders (K < 1 μM). A theoretical analysis of the binding affinity determination of strong ligands using different types of NMR experiments is provided and practical guidelines are given for overcoming the limitations and for the proper set-up of the experiments.

Efficient Screening of Target-Specific Selected Compounds in Mixtures by F NMR Binding Assay with Predicted F NMR Chemical Shifts.

Ligand-based F NMR screening is a highly effective and well-established hit-finding approach. The high sensitivity to protein binding makes it particularly suitable for fragment screening. Different criteria can be considered for generating fluorinated fragment libraries.

(R)-α-Trifluoromethylalanine as a F NMR Probe for the Monitoring of Protease Digestion of Peptides.

Fluorinated non-natural amino acids are useful tools for improving the bioavailability of peptides but can also serve as fluorinated probes in F NMR-based enzymatic assays. We report herein that the use of the non-natural α-quaternarized (R)-α-trifluoromethylalanine ((R)-α-TfmAla) provides convenient and accurate monitoring of trypsin proteolytic activity and increases resistance towards pepsin degradation.

Hydrogen Bond Acceptor Propensity of Different Fluorine Atom Types: An Analysis of Experimentally and Computationally Derived Parameters.

The propensity of organic fluorine acting as a weak hydrogen bond acceptor (HBA) in intermolecular and intramolecular interactions has been the subject of many experimental and theoretical studies often reaching different conclusions. Over the last few years, new and stronger evidences have emerged for the direct involvement of fluorine in weak hydrogen bond (HB) formation. However, not all the fluorine atom types can act as weak HBA.

Fluorine NMR functional screening: from purified enzymes to human intact living cells.

The substrate- or cofactor-based fluorine NMR screening, also known as n-FABS (n fluorine atoms for biochemical screening), represents a powerful method for performing a direct functional assay in the search of inhibitors or enhancers of an enzymatic reaction. Although it suffers from the intrinsic low sensitivity compared to other biophysical techniques usually applied in functional assays, it has some distinctive features that makes it appealing for tackling complex chemical and biological systems. Its strengths are represented by the easy set-up, robustness, flexibility, lack of signal interference and rich information content resulting in the identification of bona fide inhibitors and reliable determination of their inhibitory strength.