Experimental setup

2.2.2.1.1 Testing for inhibitory and enhancing activity Day 1

Transfected 1321N1 astrocytoma cells were seeded in Corning 3340 black 96 well plates with clear bottom at a concentration of 40 000 cells/well. In order to let the cells attach to the bottom, the plate was incubated overnight at 37°C and 5 % CO2.

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Calcium-4 and Calcium-5 dye is prepared according to the instructions provided by Molecular Devices. The explorer kit requires the dilution of component A (dye) with 10 ml of component B (assay buffer). The bulk kit needs the dilution of the dye in 10ml HBSS buffer to prepare a stock solution of Calcium-5 dye. 1 ml of the stock solution is then diluted ad 10 ml with HBSS buffer for the final assay dye. The solutions were mixed thoroughly for at least two minutes. The medium of the cells in the assay plate is removed and cells are incubated with 100 µl dye/well at 37°C in the dark for one hour.

Fluo-4 AM 3 µM solution is prepared as described in chapter 2.1.5. Cells are incubated with 40 µl dye/well for one hour in the dark at room temperature while shaking at 200 rpm. After discarding the dye and washing with 50 µl HBSS, cells were incubated with 180 µl HBSS buffer/well supplemented with various concentrations of test compounds at room temperature for 30 minutes. The compound dilutions were freshly prepared for each experiment. The pipetting scheme for preparation of compound dilutions is presented in chapter 2.1.4. 178 µl of HBSS and 2 µl of each compound dilution were added to the respective well, rendering a final DMSO concentration of 1 %/well.

The EC50 and EC80 values of ATP were determined prior to any testing of potential antagonists every time a freshly defrozen cell line was used. This is necessary to compensate for any changes in receptor expression due to the stress cells have to endure because of the freezing process.

The EC50 and EC80 values of ATP remained unchanged for P2X1, P2X2, P2X3 and P2X4, but a decrease was observed after defreezing for cells transfected with the P2X7 receptor. The ATP concentration used for stimulation was increased from 1 mM to 2 mM. The former concentration was used for the POM experiments, the latter for the drug library experiments and the selectivity experiments of anthraquinone derivatives.

The 10 mM stock solutions of POMs, except the five polyoxotungstates from Prof. Wei Wang from University of Tianjin, China, were prepared in HBSS buffer due to insufficient solubility in organic solvents. The stock solutions of the polyoxotungstate derivatives from China were prepared in DMSO. PV5, P8W48, KB1 and KB2, could not be dissolved completely neither in buffer nor in DMSO at such high concentrations, therefore less concentrated stock solutions in HBSS were prepared for those compounds (1 mM for PV5, KB1 and KB2, and 0.1 mM for P8W48). The respective pipetting schemes are presented in chapters 2.1.6.6, 2.1.6.7 and 2.1.6.8.

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Each well was incubated with 20 µl of respective solution on top of 160 µl of buffer supplemented with 2 µl of DMSO to reach the final DMSO concentration of 1 %/well to allow the testing of higher compound concentrations of these stock solutions.

Each well incubated with test compound was stimulated with the same agonist concentration for determination of inhibitory potency. The physiological agonist ATP was the used agonist for all P2X receptor subtypes. The concentration prepared for stimulation matched the EC80 value determined previously for each receptor cell line by injecting various concentrations of ATP after incubating with 1 % DMSO instead of test compounds. The ATP dilutions used for determination of EC50 and EC80 depended on the transfected receptor tested. Dilution concentrations and preparation for each receptor are summarized in chapter 2.1.6.1. At least seven different concentrations were tested for dose-response experiments.

Pure HBSS buffer was injected as negative control. All solutions for injection were provided in a V-profile microtest plate loaded with 40 µl/well to prevent loss of volume. Further control solutions tested in duplicates in each experiments are ATP (positive control, standard stimulation concentration, preincubation with 1 % DMSO instead of test compound), carbachol 100 µM (preincubation with 1 % DMSO) and a standard antagonist provided in high enough concentrations to guarantee complete inhibition of the receptor (added for preincubation instead of test compound). The antagonist used depended on the receptor cell line tested in the respective experiment. The used antagonists and their preparation are summarized in chapter 2.1.6.5. Carbachol is an agonist at muscarinic acetylcholine receptors. These receptors are physiologically expressed in 1321N1 astrocytoma cells and impart the release of Ca²⁺ ions from intracellular storage organs. This provides an increase of Ca²⁺-dependent fluorescence independent from the transfected P2X receptor and renders a positive control to prove the functionality of the assay principle and the vitality of the cells.

Fluorescence intensity was measured after 30 minutes incubation with test compound or DMSO before and after injection of agonist using the fluorescent imaging plate reader NOVOstar®.

20 µl of agonist were injected into each well, rendering a total assay volume of 200 µl/well. The fluorescence in each well is measured for a total time of 35.6 seconds. In between injection of two wells, the injection needle is flushed with Purelab® Plus water to prevent contamination.

The program settings used in each experiment are summarized in Table 2.1.

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Table 2.1:Advanced settings of NOVOstar® for measurement of calcium influx using fluorescent dyes Fluo-4, Calcium-4 and Calcium-5.

Measurement parameter Fluo-4, Calcium-4 and Calcium-5

Excitation wavelength 485 nm, bandwidth 25 nm

Emission wavelength 520 nm, bandwidth, 20 nm

Number of flashes (Validation)

10 (10)

Gain variable

Timeframe 1 (2) 0-4 s (11.6-35.6 s)

Number of intervals 60 (4)

Time interval 0.4 (1.0)

Injection time 11.6 s

Injection volume 20 µl

Pump speed 65 µl/s

Position delay 0.2 s

Temperature RT

Basic fluorescence 22 000-28 000 fluorescent counts

Washing steps after injection 3

Washing solution Purelab® Plus

Some compounds showed ATP-induced receptor enhancing properties during initial experiments. The concentration-dependency of the effect was determined analog to the determination of inhibitory potency by preincubating cells with various concentrations of test compounds for 30 minutes prior to the stimulation by one single concentration of agonist ATP.

The concentration selected for cell stimulation represents the respective EC50 concentration of ATP. Additional experiments with fully stimulated receptors were conducted for P2X3 receptors for further characterization. The ATP concentration used in these experiments was 10 µM. All concentrations and preparation of solutions are summarized in chapter 2.1.6.2.

2.2.2.1.2 Testing for agonistic activity

Most of the test compounds were examined for their inhibitory potency, but some indicated agonistic properties during initial screening experiments. The experimental setup was modified for further investigation. Cells were incubated without test compound for 30 minutes and then injected with various concentration of test compound as agonist, instead of preincubating with the test compound and then stimulating with agonist. ATP was used as a positive control.

-51- 2.2.2.1.3 Determination of inhibition mechanism

The determination of inhibition mechanism became of great interest for several potent hits.

Cells were incubated for 30 minutes with a single concentration of selected test compound based on the previously determined inhibitory dose-response curves. Five different concentrations of test compound were tested in duplicates on one 96 well plate of cells, plus duplicates DMSO 1 % as positive control. Cells were stimulated with various ATP concentrations to create shift curves. The nature of the shift provides an indication how the test compounds affects receptor activation upon agonist presence. When full activation of receptor is reached even under influence of high inhibitor concentration, and the EC50 value of agonist is shifted to higher concentrations than the control, competitive receptor inhibition is indicated. The same EC50 value, but a decrease of agonist potency upon full stimulation points to an allosteric inhibition mechanism of the antagonist.