We have developed a high throughput ultrafiltration affinity screening method coupled to MS (affinity selection/mass spectrometry; ASMS), which works with any soluble target and small molecule library (including natural products)1. ASMS is amenable to parallelization, efficient and robust enough to allow study of many targets against very large libraries on the basis of affinity, yet designed to identify target-specific binders over a broad range of affinities, and it provides both rank ordering and affinity measurements of bound ligand(s). Because we work at excess protein, relative to individual compounds, the protein concentration drives the binding reaction. Also, assay stringency is both adjustable and dependent on that protein concentration. Furthermore, we have developed a computational method to remove promiscuous compounds that bind non-selectively to proteins in general, greatly reducing our ''false positive'' hit rates. We have demonstrated the validity of ASMS with numerous targets and screening paradigms [10, 37], establishing it as a very powerful drug discovery tool.
We recently reported the discovery of a new class of inhibitors to an essential Streptococcus pneumoniae cell wall biosynthesis enzyme, MurF, by our novel affinity screening method 1. The strategy involved screening very large mixtures of diverse small organic molecules against the protein target on the basis of equilibrium binding, followed by iterative ultrafiltration steps and ligand identification by mass spectrometry. Hits from any affinity-based screening method often can be relatively non-selective ligands, sometimes referred to as ''nuisance'' or ''promiscuous'' compounds. Ligands selective in their binding affinity for the MurF
1) Sections of text and figures are included with permission from Sage Publications and Corwin Press.
target were readily identified through electronic subtraction of an empirically determined subset of promiscuous compounds in the library without subsequent selectivity panels. The complete strategy for discovery and identification of novel specific ligands can be applied to all soluble protein targets and a wide variety of ligand libraries.
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