Rapid methods for screening low molecular mass compounds non-covalently bound to proteins using size exclusion and mass spectrometry applied to inhibitors of human cytomegalovirus protease.

Abstract

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 candidates which inhibit the protease by non-covalently binding to it. The assay 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 chromatography) and/or a microconcentrator (ultrafiltration). The spin column selectively passed the high molecular mass (M(r)) protease and trapped low M(r) molecules. Alternatively, the microconcentrator passed low M(r) molecules and retained the protease. If the inhibitor bound non-covalently to the protease, both the inhibitor and protease passed through the spin column (or were retained by the microconcentrator). Electrospray ionization mass spectrometry was used to assay the spin column eluate (or the microconcentrator retentate) and to characterize the amounts of protease and inhibitor based on known standards. An advantage of these techniques is that a mixture containing inhibitors can be analyzed in the presence of the protease, and inhibitors with the greatest binding affinity can be identified. Non-covalent binding specificity was demonstrated using spin columns by comparing the binding affinity of inhibitors using several mutants of cytomegalovirus protease. The techniques described are applicable to the rapid screening of compound libraries for selecting substances which bind non-covalently to a known protein.