3.4 Potency of amino- and desaminoanthraquinone derivatives
3.4.2 Enhancement of ATP potency
3.4.2.2 Further investigations of enhancement effect of anthraquinone derivatives
-79-
3.4.2.2 Further investigations of enhancement effect of anthraquinone derivatives
-80-
physiologically possible level. The same effect is indicated for YB160, but a successful concentration-response relationship could not be obtained.
Figure 3.10: Structures and mean dose-response curves (mean ± SEM, n = 3-4) of (A) YB087 and (B) YB149 stimulated with either (1) EC50 ( , 0.05 µM) or (2) maximal response concentration of ATP ( , 10 µM).
Contrary to expectations and to the first experiments with ATP 0.05 µM, YB087 and YB149 showed inhibition at concentrations higher than 5 µM at the P2X3 receptor when stimulated with 10 µM ATP. A similar effect can be seen for YB160 to a lesser extent. This indicates that the compound seemed to inhibit the P2X3 receptor when used in higher concentrations. It is possible that all three ATP binding pockets are occupied by test compound when high concentrations of YB087, YB149 and YB160 are used. This would prevent ATP from binding and subsequently activating the receptor. As the concentration and occupancy of binding pockets decreases, the remaining anthraquinone molecules bound to the receptor enhance the affinity
10-10 10-9 10-8 10-7 10-6 10-5 10-4 0
50 100 150 200 250 300
[YB 087], M
Fluorescence increase (%)
10-10 10-9 10-8 10-7 10-6 10-5 10-4 0
50 100 150 200 250 300
[YB149], M
Fluorescence increase (%)
(A1) YB087, stimulated with ATP 0.05 µM (A2) YB087, stimulated with ATP 10 µM
(B2) YB149, stimulated with ATP 10 µM (B1) YB149, stimulated with ATP 0.05 µM
10-10 10-9 10-8 10-7 10-6 10-5 10-4 0
25 50 75 100 125 150
[YB 087], M
Fluorescence increase (%)
10-10 10-9 10-8 10-7 10-6 10-5 10-4 0
25 50 75 100 125 150
[YB 149], M
Fluorescence increase (%)
-81-
of ATP and therefore may cause the observed enhancement of maximal ATP effect. If the concentration is decreased further, it becomes too low to enhance the ATP-induced fluorescence, and the maximal ATP effect returns to the normal level. It is possible that the ATP concentration used for receptor stimulation somehow altered the receptor function, since this described phenomenon was not observed under receptor stimulation by the EC50 concentration of ATP.
Figure 3.11: Structures and mean dose-response curves (mean ± SEM, n = 3-4) of (A) YB038 and (B) YB160 stimulated with either (1) EC50 ( , 0.05 µM) or (2) maximal response concentration of ATP ( , 10 µM)
Compounds YB087, YB149 and YB160 were used in a patch clamp experiment in the group of PD Dr. med. Ralf Hausmann from the Institute of Toxicology and Pharmacology in Aachen in order to verify these observations. The results of these experiments are presented in Figure 3.12 and Table 3.14.
10-10 10-9 10-8 10-7 10-6 10-5 10-4 0
20 40 60 80 100 120 140 160 180
[YB038], M
Fluorescence increase (%)
10-10 10-9 10-8 10-7 10-6 10-5 10-4 0
25 50 75 100 125 150
[YB038], M
Fluorescence increase (%)
10-10 10-9 10-8 10-7 10-6 10-5 10-4 0
20 40 60 80 100 120 140 160 180 200 220
[YB160], M
Fluorescence increase (%)
10-10 10-9 10-8 10-7 10-6 10-5 10-4 0
20 40 60 80 100 120 140 160 180
[YB160], M
Fluorescence increase (%)
(A1) YB038, stimulated with ATP 0.05 µM (A2) YB038, stimulated with ATP 10 µM
(B1) YB160, stimulated with ATP 0.05 µM (B2) YB160, stimulated with ATP 10 µM
-82-
10- 7 10- 6 10- 5 10- 4 10- 3 0
50 100 150 200
250 YB087
YB149 YB160
[compound], M
control current (%)
Figure 3.12: Mean dose response curves of YB087, YB149 and YB160 at the P2X3 receptor obtained by patch clamp experiments (MW ± SEM). The data was provided by PD Dr. Ralf Hausmann. Each concentration was measured at 4-6 times.
The bell-shaped regression was selected for data analysis. The first EC50 value (EC50Enhancement) describes the first inflection point on the left side, the IC50 value represents the second inflection point on the right side of the bell curve. The effect of each concentration was measured between four to six times. The same observation can be seen as already described for Figure 3.10 and Figure 3.11 A1-B1, respectively. It also supports the theory that not only the compound concentration but also the ATP amount used for cell stimulation has influence on the conducted fluorescence signal.
Table 3.14: Results of bell-shaped non-linear regression analysis and respective confidence intervals of concentration-response experiments using patch clamp experiments at the P2X3 receptor. Each data point used in the analysis was measured 4-6 times.
EC50Enhancement
[µM]
IC50 [µM] Maximal current increase (%)
Concentration of maximal current increase [µM]
YB087 8.00 18.8 116 11.2
YB149 6.12 12.7 123 8.07
YB160 11.9 21.2 104 14.7
An increase of maximal receptor stimulation possible under ATP influence could only be observed for YB038 (see Figure 3.11 A1 and A2). This indicates that YB038 somehow interacts differently with the P2X3 receptor than the other compounds YB087, YB149 and YB160. In an
-83-
attempt to get further insight in the mechanism of action, all four compounds were tested in another experiment analog to the determination of inhibition mechanism for antagonists, where cells were preincubated with certain concentrations of anthraquinone derivative selected based on their EC50Enhancement values, and subsequently stimulated with various concentrations of ATP (for experimental setup see chapter 2.2.2.1.3). The results are presented in Figure 3.13 and Figure 3.14.
It was expected that under the influence of different concentrations of the respective anthraquinone compound, either the maximal level of fluorescence increase reached by full receptor stimulation would be enhanced or the EC50 value of ATP would be shifted to the left towards lower concentrations.
As already observed in the previous experiments, the addition of high concentrations (10 µM and 5 µM) of YB087, YB149 and YB160 abolished any agonistic ATP activity (YB087) or visibly lowered it (YB149 and YB160, see Figure 3.13 and Figure 3.14). The decrease was statistical significant for YB087 and YB149. A slight enhancement of maximal ATP effect could be observed at concentrations lower than 5 µM, but it was nowhere nearly as high as detected in the initial concentration-response experiments (see Figure 3.10 A1 and B1 and Figure 3.11 B1). None of the slight increases were statistically significant, same as for the EC50 values of ATP. The results indicate that the enhancement of the ATP effect does not repose on an increase of ATP affinity to the P2X3 receptor, and seems to be dependent on the ATP concentration used for receptor stimulation, as observed previously (see again Figure 3.10 and Figure 3.11). Since high concentrations of anthraquinone enhancer lead to inhibition of the receptor, it is possible that this effect only occurs when not all binding pockets are occupied.
A decrease of EC50 value could be observed for YB038, but the difference was not statistically significant from the EC50 value of the ATP control curve, and it could only be detected for the YB038 concentrations 5 µM and 1 µM (see higher pEC50 values in Figure 3.14 A2). Under the influence of YB038 0.5 µM, the EC50 value increased contrary to expectations, but the enhancement of fluorescence increase was the highest detected for all four compounds in this experiment. Due to the inconsistency of the results, a final conclusion about the mechanism how YB038 increases the ATP-induced fluorescence signal was still not possible, although all experiments indicate that the mechanism of action is different from the other three compounds used for further examination. A possible explanation would be the altercation of the ion pore by so that more calcium ions are capable of passing and therefore increasing the maximal ATP effect. This could also explain the further increase of maximum possible ATP-induced fluorescence signal demonstrated in Figure 3.10 and Figure 3.11.
-84-
Figure 3.13: (1) Mean ATP dose-response curves, (2) ATP pEC50 values and (3) enhancement of maximal ATP effect characterizing the influence scaffold G anthraquinone derivatives (A) YB038 and (B) YB160. Cells were stimulated with ATP 0.0001-10 µM after preincubation with 0.1 µM ( ), 0.5 µM ( ), 1 µM ( ), 5 µM ( ) or 10 µM (×) of test compound, respectively. Buffer containing 1 % DMSO without compound was used as control ( ). The unpaired Student’s t-test for determination of significant differences of means was conducted for pEC50 and enhanced maximal ATP effect. Statistical significance (p < 0.05) is marked with (*), highly statistical significant differences (p < 0.001) with (***).Data is presented as mean ± SEM from 3-8 independent experiments.
10-10 10-8 10-6 10-4 -25
0 25 50 75 100 125 150
[ATP], M
Fluorescence increase (%)
ATP
+ 0.1 µM+ 0.5 µM+ 1 µM
+ 5 µM
+ 10 µM 6.5
6.7 6.9 7.1 7.3 7.5
pEC50
10-10 10-8 10-6 10-4 -75
-50 -25 0 25 50 75 100 125 150
[ATP], M
Fluorescence increase (%)
ATP
+ 0.1 µM+ 0.5 µM+ 1 µM
+ 5 µM
+ 10 µM 6.0
6.5 7.0 7.5 8.0 8.5 9.0
pEC50
ATP + 0.1
µM + 0.5
µM + 1
µM + 5
µM + 10 µM -150
-130 -110 -90 -70 -50 -30 -10 10 30 50
*
***
enhanced max. ATP effect (%)
ATP + 0.1
µM + 0.5
µM + 1
µM + 5
µM + 10 µM -120
-100 -80 -60 -40 -20 0 20 40
*
enhanced max. ATP effect (%) ***
(A) YB087 (A1)
(B) YB149 (B1)
(A2) (A3)
(B2)
(B3)
(B3)
-85-
Figure 3.14: (1) Mean ATP dose-response curves, (2) ATP pEC50 values and (3) enhancement of maximal ATP effect characterizing the influence scaffold G anthraquinone derivatives (C) YB038 and (D) YB160. Cells were stimulated with ATP 0.0001-10 µM after preincubation with 0.1 µM ( ), 0.5 µM ( ), 1 µM ( ), 5 µM ( ) or 10 µM (×), respectively. Buffer containing 1 % DMSO without compound was used as control ( ). The unpaired Student’s t-test for determination of significant differences of means was conducted for EC50 and enhanced maximal ATP effect (p<
0.05). Statistical significance (p< 0.05) is marked with (*). Data is presented as mean ± SEM from 3-8 independent experiments.