4 DEVELOPMENT OF FUNCTIONAL ASSAYS FOR THE HUMAN NPY Y 2
4.2 Aequorin assay
4.2.3.7 Antagonism of BIIE0246
Concentration-response curves of pNPY were determined in the presence of the Y2 -selective antagonist BIIE0246. The cells were preincubated with increasing concentrations of the antagonist for 1 h before injection of increasing concentrations of the agonist.
The concentration-response curves were shifted to the right with a concomitant depression of the maximum indicating mixed competitive-noncompetitive antagonism.
This is in contrast to the competitive antagonism proposed for BIIE0246 (Dumont et al., 2000; Weiser et al., 2000). On the other hand, El Bahh and co-workers (El Bahh et al., 2002) described an apparently insurmountable antagonism in hippocampal slices. Moreover, an insurmountable antagonism was found in the rat colon bioassay (Dumont et al., 2000). El Bahh and co-workers suggested that BIIE0246 is a competitive antagonist, but because of its lipophilicity, the compound is enriched in
Fig. 66: Concentration-response curves of pNPY in the presence of BIIE0246 (2). CHO-hY2 -K9-qi5-K9-mtAEQ-A7 cells were preincubated with 2 for 1 h.
Increasing concentrations of the antagonist led to a rightward shift and to a depression of the maximum of the luminescence signal. (mean values ± SEM, n=3).
Fig. 65: mRNA expression of transfected cells. PCR was performed only in presence of primer pairs of Gqi5 and β-actin.
A: K1 cells. B: CHO-hY2-K9 cells C: CHO-hY2 -K9-qi5-K9 cells D: CHO-hY2-K9-qi5-K9-mtAEQ-A7 cells M: marker.
the membrane providing a much higher local concentration near the receptors compared to the concentration in the organ bath, simulating an insurmountable antagonism.
Surprisingly, there was no depression of the concentration-response curves in the aequorin assay observed without preincubation of the cells with the antagonist. (Fig.
67). After injection of the cell suspension to a mixture of agonist and antagonist higher concentrations of antagonist were needed to shift the concentration-response curves rightward. Schild regressions revealed a pA2 value of 6.37 which is in dramatic contrast to the Ki value determined in the flow cytometric binding assay.
However, comparing these data one has to consider that the binding assay was performed at equilibrium. Additionally, the strong adsorption of 2 to the 96-well plate (see Fig. 32) has to be regarded and usually much more data points are required for reliable calculations when performing a Schild plot. Nevertheless, these data indicate that the apparent mixed antagonism can be separated into two mechanisms with different kinetics.
c (pNPY) [nM]
10-2 10-1 100 101 102 103 104 105
fractional luminescence [%]
0 10 20 30 40 50 60
0 µM 0,3 µM 1 µM 3 µM 10 µM
Therefore, the dissociation kinetics of BIIE0246 was measured. The cells were incubated with 5 nM cy5-pNPY for 2 h until equilibrium was reached (see Fig. 19).
The bound fluorescence was recorded in channel 4 with the flow cytometer, and after 2 min 100 nM of 2 was injected to the cell suspension. The displacement of bound cy5-pNPY by 2 was followed recording the cell-bound fluorescence as shown in Fig.
68.
Fig. 67: Concentration-response curves of pNPY in the presence of the indicated concentrations of 2. CHO-hY2 -K9-qi5-K9-mtAEQ-A7 cells were injected into a mixture of agonist and antagonist (mean values ± SEM, n=3)
time [min]
0 10 20 30 40 50 60
fluorescence FL-4
200 250 300 350 400
averaged raw data fit to one rate constant fit to two rate constants
The kinetics of displacement of cy5-pNPY by 2 can be described more precisely with a two-exponential fit (R=0.994) compared to a function which assumes only one dissociation rate constant (R=0.979). As cy5-pNPY is an agonist, this ligand distinguishes between precoupled and uncoupled receptors, and might bind to as well as dissociate from the two different receptor states with different kinetics.
Another explanation is the presence of two different binding sites for the antagonist 2.
The antagonist binds faster to the binding site of the labeled ligand as a competitive antagonism occurs when agonist and antagonist are added at the same time (Fig.
66). Binding to another allosteric binding site is slower indicated by a second dissociation rate constant and the presence of a mixed competitive-noncompetitive antagonism after preincubation of 2 with the cells.
In addition, the antagonist (100 nM) displaces only 44 % of the specific binding (unspecific binding was 44 [RFU], determined in presence of 1 µM pNPY). This is in contrast to the binding data determined with CHO-hY2-K9 cells (in competition with 5 nM cy5-pNPY, see section 3.2.3.1) where the same concentration of 2 displaced more than 90 % of the specifically bound cy5-pNPY (Fig. 20b). The data suggest that cy5-pNPY binds partially in an irreversible binding mode to the hY2 receptor.
Recently, similar results were described by Dautzenberg and Neysari analyzing the binding kinetics of [125I]-NPY and [125I]-PYY to membranes of hY2-expressing SMS-KAN and HEK293 cells (Dautzenberg and Neysari, 2005). After preincubation with the radioligand (120 min), not more than 20 % of bound [125I]-NPY or [125I]-PYY could be displaced from the receptor by addition of 10 µM NPY, PYY or BIIE0246. This irreversible binding mechanism was not observed for the hY1 or mY5 receptor. In addition, in a FLIPR assay using HEK293 cells stably expressing the hY2 receptor
Fig. 68: Dissociation kinetics of 2 replacing cy5-pNPY at the hY2
receptor recorded by flow cytometry.
Data are best fitted with a model using two rate constants (red) compared to a model with one rate constant (blue).
and the chimeric Gqi9 protein, simultaneous addition of the agonist PYY and the antagonist 2 revealed a competitive antagonism whereas an insurmountable antagonism (with a minimal rightward shift of the EC50 values) was observed after 30 min preincubation in presence of the antagonist. Dautzenberg suggested that the observed insurmountable effect of 2 is due to a lack of dissociation from its receptor sites (Dautzenberg and Neysari, 2005). Hence, the dissociation kinetics of the hY2
receptor has to be considered in binding and functional assays. When available, measurements of binding kinetics should be performed with a labeled antagonist instead of labeled agonist in order to exclude different binding kinetics to different receptor states as well as dynamic processes as desensitization and internalization during the binding process.