Competitionbased Binding Site Determination and Affinity Ranking in Mixtures

The location on a target protein at which a potential drug lead binds is a key determinant of its biological efficacy. For example, the mechanistic basis of many therapeutic compounds, especially those that target enzymes, involves in vivo competition by the drug with another ligand or cofactor for a particular binding site on the protein target [53]. Therefore, the ability to characterize the binding site of a small molecule ligand with respect to known cofactors, substrates, or other small molecule drugs having known binding sites is of paramount importance in the drug discovery process. Techniques to classify ligands according to binding site are especially important for protein targets where no atomic-resolution structural data is available (for example, from NMR or x-ray crystallo-graphic analysis), including GPCRs and other membrane-associated proteins.

The ability of a known competitor ligand to displace a target-bound library member - as measured by ALIS - reveals the binding site classification and affinity ranking of mixture components [36]. In practice, affinity selection experiments are performed with samples containing a constant concentration of the li-gand(s) of interest and serially increasing concentrations of a competitor ligand. In these experiments, the ALIS responses of the ligands and the competitor reflect the equilibrium concentrations of each protein-ligand complex.

The competitor used in these experiments may be either a known ligand or MS-sensitive substrate or cofactor for the target of interest, a representative chosen from multiple ligand classes discovered in a high-throughput screening cam-

Fig. 3.9 ALIS-MS results for the titration of 5 mM HSA with warfarin in the presence of a 5 mm concentration of its stable isotope-labeled congener warfarin-D6. Increasing concentrations of warfarin reduce the response of warfarin-D6 due to isosteric binding competition. Reprinted from [39] with permission from the American Chemical Society.

paign, or the progenitor of a series of structural analogs synthesized for affinity ranking. If two ligands bind at different sites, the method can yield their absolute binding affinity and a quantitative assessment of the degree of allosteric coopera-tivity between them.

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