RAPID METHODS FOR SCREENING LOW MOLECULE MASS COMPOUNDS NONCOVALENTLYBOUND TO PROTEINS USING SIZE-EXCLUSION AND MASS-SPECTROMETRY APPLIED TO INHIBITORS OF HUMAN CYTOMEGALOVIRUS PROTEASE

General and rapid methods were developed for determining the extent of non-covalent binding between small molecules and proteins, using the model system of human cytomegalovirus protease and several drug candid ates which inhibit the protease by non-covalently binding to it The as say was performed by off-line coupling of size-exclusion methods with mass spectrometry in the following manner, The protease and inhibitor were incubated together under native conditions and then subjected to separation based on size, by use of a spin column (gel permeation chro matography) and/or a microconcentrator (ultrafiltration), The spin col umn selectively passed the high molecular mass (M-r) protease and trap ped low M-r molecules, Alternatively, the microconcentrator passed low M-r molecules and retained the protease. If the inhibitor bound non-c ovalently to the protease both the inhibitor and protease passed throu gh the spin column (or were retained by the microconcentrator). Electr ospray ionization mass spectrometry was used to assay the spin column eluate (or the microconcentrator retentate) and to characterize the am ounts of protease: and inhibitor based on known standards. An advantag e of these techniques is that a mixture containing inhibitors can be a nalyzed in the presence of the protease, and inhibitors with the great est binding affinity can be identified. Non-covalent binding specifici ty was demonstrated using spin columns by comparing the binding affini ty of inhibitors using several mutants of cytomegalovirus protease, Th e techniques described are applicable to the rapid screening of compou nd libraries for selecting substances which bind non-covalently to a k nown protein. (C) 1998 John Wiley & Sons, Ltd.