Application Note


Binding kinetics determined with live cells and in high throughput


I


t is easy to understand that potential drugs must bind to their target struc- ture. Therefore, the affinity of drug can-


didates to their target is tested using differ- ent drug concentrations and at one time- point. This, however, completely neglects that molecular interactions are not static. Rather, the binding is defined by continuous association and dissociation. The parame- ters of how fast a molecule binds (associa- tion rate) and how fast it dissociates (disso- ciation rate) are referred to as binding kinetics. As these kinetics impact on the efficiency of drugs or on the occurrence and severity of side-effects, it is desired to screen drug candidates regarding their binding kinetics before proceeding with in vivo and clinical studies. A novel microplate-based method deter-


mines kinetic parameters not only in puri- fied protein solutions, but also in cellular environments. This is of paramount impor- tance to study interactions with membrane standing G-protein coupled receptors (GPCRs). The assay employs Förster reso- nance energy transfer (FRET) between a donor fluorophore and an acceptor fluo- rophore. The donor fluorophore is coupled


to the target molecule and the acceptor is bound to the drug. Only when the drug binds its target are both fluorophores close enough to allow energy transfer from donor to acceptor and consequently the acceptor fluorophore to emit light (Figure A).


Example: association and dissociation of Spiperone-d2 and


dopamine receptor D2 A pair of ligand and cells expressing the tar- get GPCR was used to demonstrate how association kinetics are measured in a microplate. Spiperone coupled to the accep- tor fluorophore d2 served as ligand and the


GPCR dopamine receptor D2 (D2R) expressed in HEK cells and coupled to the acceptor fluorophore terbium cryptate served as target. Cells were preincubated in 384-well plates and the analysis was started by addition of Spiperone-d2 at increasing


concentrations (1.55-100nM). At the same time recording of the TR-FRET signals was started and continued with one measure- ment each minute. The PHERAstar FSX microplate reader detected the signal coming from donor and acceptor fluorophore simul- taneously to guarantee high temporal reso- lution. The result of FRET measurements is given


as a ratio of acceptor signal and donor sig- nal. The ratio accordingly increases with increased binding of the ligand to the recep- tor. This is shown by the association experi- ment: at all ligand concentrations, the FRET ratio increases with time. The longer the reaction takes, the more ligand is bound until it reaches the equilibrium. For low lig- and concentration the ratio at equilibrium is low whereas it increases along with concen- tration (Figure B). The association rate as


well as the affinity (KD – equilibrium con- stant) can be derived from these association curves as detailed on BMG LABTECH’s homepage. To determine the dissociation constant,


Spiperone-d2 is subsequently displaced by an excess of non-fluorescent agonist of D2R. Dissociation and the related decrease in TR-FRET ratio was monitored in real- time (Figure C) using the PHERAstar FSX microplate reader. Subsequent analysis revealed a dissociation rate of 0.007 min-1.


Conclusion The TR-FRET-based method to determine the kinetics of drugs binding their targets does not rely on expensive instrumenta- tion, radioactivity or countless reactions for individual timepoints. Rather, a microplate reader with high temporal reso- lution in TR-FRET measurements is suffi- cient to resolve binding events and calcu- late association and dissociation rates. The simultaneous detection of two emissions (donor and acceptor) plus the possibility to measure TR-FRET kinetically make the PHERAstar FSX the instrument of choice for measuring binding kinetics.


DDW Drug Discovery World Fall 2019 13


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