Rapid mass spectrometric determination of preferred irreversible proteinase inhibitors in combinatorial libraries

Abstract Optimal N -iodoacetyldipeptide inactivators of hepatitis A virus 3C proteinase were identified directly from equimolar mixtures of these compounds using electrospray ionization mass spectrometry (ESI-MS). Limiting amounts of proteinase were allowed to react with the library of inhibitors and subsequently analyzed by ESI-MS to determine the mass of the adducts formed. N -iodoacetyl-Ser-Phe-NH 2 was found to be the most potent inactivator with a second order rate constant of 840 ± 90 M −1 s −1 . Fragmentation of the complexex by using cyanogen bromide and trypsin followed by liquid chromatography/ESI-MS confirmed the identity of the adduct and allowed inhibitor mass differences of as little as 6 Da to be distinguished in a single experiment. This approach allows the rapid screening and identification of preferred covalent inhibitors or intermediates from combinatorial libraries without deconvolution or resynthesis and should be applicable to irreversible inhibitors of virtually any enzyme that uses a covalent catalysis mechanism.