The first measure of a candidate compound's efficacy in a drug discovery program is its specific binding affinity to a desired biomolecular target. Therefore, the development of improved methods to accurately determine the absolute binding affinities of drug-like small molecules to their receptors is an active and fruitful area of research. Most methods for absolute affinity quantitation (as compared to relative affinity measurements based on displacement of a known inhibitor) rely on titration of a receptor by a ligand and readout of a signal corresponding to formation of the receptor-ligand complex. In the case of spectroscopic methods, the readout is based on emission or absorption of electromagnetic radiation; for thermophysical methods such as isothermal calorimetry, the readout is based on emission of heat. In ALIS, the protein-ligand complex concentration is determined from the MS signal measured for the ligand after its dissociation from the complex. This section describes a straightforward ALIS-based titration method to quantify the binding affinities between unlabelled small molecules and their native protein targets.
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