Mass spectrometry and immobilized enzymes for the screening of inhibitor libraries

A technique has been developed to rapidly screen enzyme inhibitor candidate s from complex mixtures, such as those created by combinatorial synthesis, Inhibitor libraries are screened by using immobilized enzyme technologies a nd electrospray ionization ion cyclotron resonance mass spectrometry. The l ibrary mixture is first sprayed into the mass spectrometer, and compounds a re identified. The library is subsequently incubated with the immobilized e nzyme of interest under the correct conditions (buffer, pH, temperature) by using an excess of enzyme to ensure a surplus of sites for ligand binding. The immobilized enzyme/inhibitor mixture is centrifuged, and an aliquot of supernatant is again analyzed by electrospray ionization mass spectrometry . Potential inhibitors are quickly identified by comparison of the spectra before and after incubation with the immobilized enzyme. Non-inhibitors sho w no change in ion intensity after incubation, whereas weak inhibitors exhi bit a visible decrease in ion abundance. Once inhibitor candidates have bee n identified, the library is reinjected into the mass spectrometer, and tan dem mass spectrometry is used to determine the structure of the inhibitor c andidates as needed. This method has been successfully demonstrated by iden tifying inhibitors of the enzymes pepsin and glutathione S-transferase from a 19- and 17-component library, respectively. It is further shown that the immobilized enzyme can be recycled and reused for continuous screening,of additional new libraries without adding additional enzyme.