Protein Ligand Dissociation Rate Measurement

The biological efficacy of a drug candidate depends critically on the rate at which it dissociates from its therapeutically relevant target biomolecule. As described in greater detail below, the binding affinity of a small molecule for its receptor also depends upon its dissociation rate (or "off-rate"). Therefore, within a series of compounds having comparable association rates ("on-rates"), compounds with slower off-rates have, by definition, greater affinity for their protein target. Also, the more slowly a compound dissociates from its receptor, the more time it spends on the receptor effecting its desired biological outcome, and the less time it is subject to metabolism, excretion, or off-target binding and undesired side-effects. Therefore, for highly potent compounds in the advanced stages of a medicinal chemistry optimization program, compounds of similar potency can be ranked according to off-rate as a secondary measure of their potential efficacy. Also, very slow dissociation kinetics can contribute to slow clearing of a drug, which can be problematic in the event of adverse reactions such as an undesired allergic response. Therefore, methods to accurately determine the dissociation kinetics for protein-ligand interactions are of great value to the drug discovery process.

This section describes the theoretical principles underlying an ALIS-based method for determining protein-ligand dissociation rates for single ligands and for ligands which are components of mixtures. The basis of the method resembles a ''cold quench'' radioligand technique common to receptor biology, where a large excess of an inhibitor (or quench) ligand of equal or better affinity than the compound under study is added to an equilibrated protein-ligand binding reaction. As soon as any protein-ligand complex spontaneously dissociates, the freed protein is quenched by the excess of inhibitor so no protein-ligand complex can re-form. Therefore, the concentration of the protein-ligand complex will diminish with time after addition of an excess of inhibitor, and the rate of its diminution, which depends on the protein-ligand dissociation kinetics, can be measured by ALIS. The method is demonstrated by simultaneously measuring the protein-ligand dissociation rates of a number of ligands to the Zap-70 kinase.

The single-site equilibrium binding of a small molecule ligand S with its receptor E can be expressed as the chemical reaction shown here:

The dissociation rate (or "off-rate") of the protein-ligand complex ES is characterized by the first-order rate constant koff and depends on the concentration of protein-ligand complex [ES]:

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