Using Electrospray Ionization FTICR Mass Spectrometry To Study Competitive Binding of Inhibitors to Carbonic Anhydrase

We report a method based on mass spectrometry for the characterization of noncovalent complexes of proteins with mixtures of ligands; this method is relevant to the study of drug leads and may be useful in screening libraries for tight-binding compounds. This study describes the competitive binding of inhibitors derived from para-substituted benzenesulfonamides to bovine carbonic anhydrase II (BCAII, EC 4.2.1.1) using this technique. Relative binding constants and structural information for a mixture of inhibitors can be obtained in a single experiment using ESI-FTICR-MS. The work demonstrates that ESI-MS has significant potential for measuring relative binding affinities and characterizing the structures of ligands associated noncovalently to proteins. We have detected noncovalent complexes in the gas phase for ligands having values of K sub(b) as low as 1.7 x 10 super(6) M super(-1) in solution. The technique also allowed identification of tightbinding ligands from small libraries. The structures of inhibitors having similar masses can be identified by the high-resolution and multistep dissociation mass spectrometry of which FTICR is uniquely capable. This range of capabilities for ESI-FTICR-MS should be widely useful in medicinal chemistry.