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Analyze binding kinetics with HTRF: determine kon and koff by association experiments on the PHERAstar

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. On the one hand, slow dissociation constants can be beneficial to increase the duration of drug action (as for example Tiotropium for COPD treatment). 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 PHERAstar microplate reader provided highest temporal resolution and thus allowed kinetic measurement of Spiperone-d2 binding to the dopamine receptor D2. The acquired data served to determine kon, KD and koff.

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