The ALIS system enables several useful techniques for studying protein-ligand interactions, and is generally applicable to a broad range of protein classes, including serum proteins, kinases, and GPCRs. The methods described here require neither tagging of the ligands nor the existence of a biochemical assay, as they rely purely on the MS readout of an affinity selection experiment for their implementation.
ALIS-based titration experiments yield an absolute measure of protein-ligand binding affinities without assumptions regarding ligand depletion or other simplifications. No competitor ligand is necessary for the method's implementation, and it can readily measure the affinity of active-site or allosteric ligands to a receptor. Also, the titration method can be used to validate that a protein synthesized and purified by biochemical techniques retains specific ligand binding ability, and that the binding affinity correlates with that expected from orthogonal methods to confirm proper protein folding.
The ALIS ACE50 method enables the simultaneous classification of ligands of dissimilar structure according to their binding site. This capability can assist the development of structure-activity relationships and understanding protein-ligand interactions in multi-domain or multi-subunit targets, even in the absence of atomic resolution structure data. As shown in the examples above, the ACE50 method enables the triage of multiple hits arising from high-throughput screening according to binding site and target-specific binding affinity, and facilitates combinatorial library-based structural optimization of these hits to high-affinity lead compounds. This method is especially useful as a tool for the study of allosteric ligands, facilitating the advancement of compounds with improved target specificity engendered by binding at sites distinct from those conserved within protein families .
Dissociation rate measurement using ALIS mimics the radioligand quench method; however, because the ALIS readout is MS-based, it is readily adapted to mixture-based analysis. This feature facilitates medicinal chemistry optimization of protein-ligand off-rates using combinatorial synthesis techniques. The ability to measure the effect of allosteric ligands on the dissociation rate of an active site ligand is also demonstrated, and this ability highlights the advantages of using ALIS for the in-depth study of protein-ligand interactions.
While the methods described in this chapter have been optimized for affinity selection-MS using continuous SEC, they are readily adaptable to spin-column, gel permeation, or other well validated and highly accessible two-stage AS-MS designs. The use of AS-MS for studying protein-ligand interactions, especially for the discovery of ligands from pools of compounds, has been reported by a number of experts in the pharmaceutical industry and academia over the past decade. Due to the advantages offered by AS-MS, it can be anticipated that these techniques will be increasingly applied by medicinal and synthetic organic chemists, biochemists, analytical chemistry experts and other researchers throughout the pharmaceutical discovery community.
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