Analyze binding kinetics with HTRF: determine koff by dissociation measurements on the PHERAstar and calculate kon

Nicolas Pierre, Thomas Roux Cisbio Bioassays, Codolet, France 05/2019

Drugs are characterized by their equilibrium constant KD which describes the affinity of the compound to its target. It refers to half the ligand concentration of saturated binding. However, this dissociation constant only applies to the equilibrium state and neglects the importance of the speed of association and dissociation. The optimal kinetic binding of a drug differs with the target of interest and related disease. On the one hand, slow dissociation constants can be beneficial to increase the duration of drug action. On the other hand, it has been hypothesized for specific targets that fast dissociations could decrease on target side effects.
Here, we present the determination of the association and dissociation rate of Spiperone-d2 binding to the dopamine receptor D2 (D2R), a G-protein coupled receptor (GPCR) and target of antipsychotic drugs. The speed of the PHERAstar offers a high temporal resolution that is absolutely required to monitor the binding dynamics and subsequently calculate the on- and off-rates.

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