Low throughput screening

Because there are no nonpeptidic antagonists for the hY4 receptor known so far, a small library of known available drugs (∼80) was screened for inhibition of the aequorin signal elicited with 200 nM rPP. Compounds which inhibited the luminsecence signal by more than 50 % at a concentration of 10 µM were further analyzed and additionally tested in a flow cytometric binding assay with P388-S99A-hY4-K23 cells. The antipsychotic drug chlorprothixene, the antidiabetic agent glibenclamide and the H1-antihistamine terfenadine were able to inhibit the luminescence signal at concentrations in the micromolar range (see Fig. 102a). But in the flow cytometric binding assay, the compounds were not able to displace 10 up to a concentration of 100 µM (Fig. 102b) indicating that they do not bind to the same binding site as the labeled ligand. Therefore, it is more likely that these compounds act via other receptor signaling mechanisms present in the cells. Glibenclamide is known to block K⁺-channels and therefore induces the opening of voltage-gated Ca²⁺-channels in the B-cells of the pancreas. An increase in intracellular calcium in the transfected CHO cells would discharge a fraction of active aequorin and therefore reduce the luminescence signal elicited by the agonist rPP simulating an antagonism in the functional assay.

c (compound) [µM]

0,001 0,01 0,1 1 10 100 1000

% of max. luminescence signal

0

Guanidine-type histamine H2 receptor agonists were the first nonpeptidic NPY Y1

receptor antagonists described in the literature (Michel and Motulsky, 1990).

Stimulated by the early discoveries in the Y1 antagonist field, several recently synthesized novel H2 agonists were tested for activity at Y4 receptors in the aequorin assay. Preincubation with the compounds PG 55B, AK 49 and AK 59 reduced the luminescence signal elicited by 200 nM rPP (Fig. 103a). The IC50 values were 10.5 ± 1.1 µM (PG 55B), 60.7 ± 7.3 µM (AK 49) and 83.9 ± 4.2 µM (AK 59). But in contrast to the compounds chlorprothixene, glibenclamide and terfenadine, the H2 agonists were also able to displace the fluorescent ligand 10 from its binding sites at P388-S99A-hY4-K23 cells (Fig. 103b) even though with high Ki values of 28.0 ± 5.6 µM (PG

a b

Fig. 102: Selected compounds tested in the aequorin assay inhibiting the luminescence signal elicited with 200 nM rPP using CHO-hY4-K13b-qi5-K8-mtAEQ-E11 cells (panel a; mean values ± SEM, n=3) and in the flow cytometric binding assay competing with 20 nM cy5-[K⁴]-hPP using P388-S99A-hY₄ cells (panel b; mean values ± SEM, n=1).

c (compound) [µM]

Fig. 103: Selected compounds tested in the aequorin assay inhibiting the luminescence signal elicited with 200 nM rPP using CHO-hY4-K13b-qi5-K8-mtAEQ-E11 cells (panel a; mean values ± SEM, n=3) and in the flow cytometric binding assay competing with 10 nM cy5-[K⁴]-hPP using P388-S99A-hY4 cells (panel b; mean values ± SEM,n=1-3).

55B), 28.5 ± 10.2 µM (AK 49) and 64.4 ± 54.4 µM (AK 59). Concentrations above 300 µM could not be tested because there were too few gated cells indicating a toxic effect on the P388 cells. The separate building blocks of the compounds were also tested in the flow cytometric binding assay and, surprisingly, even the common parent compound of the guanidine-type H2 agonists, imidazoylpropylgunanidine, displaced the fluorescent ligand 10 from its binding sites with a calculated Ki value of 33.0 ± 4.1 µM. By contrast, the free acids representing the variable parts of the compounds, PG 15, cyclohexaneproprionic acid and AK 1, did not compete with 10 for binding at the hY4 receptor (Fig. 103b).

The binding of the compounds was further confirmed in a flow cytometric assay using CHO-hY4-K13b-qi5-K8-mtAEQ-E11-K11 cells and 3 nM of 10 (Fig. 104). The compounds were tested up to a concentration of 300 µM but no complete binding curves could be measured because the CHO cells could not be gated after incubation with higher concentrations of compounds. No reliable IC50 values could be determined for PG 55B and AK 59 but the calculated Ki values for AK 49 (68.2 ± 27.0 µM) and the imidazoylpropylguanidine (71.7 ± 12.5 µM) were in the same range as determined with P388-S99A-hY4-K23 cells.

c (antagonist) [µM]

1 10 100 1000

bound Cy5-K4hPP [%]

0 20 40 60 80 100 120

PG 55B AK 49 AK 59

imidazoylpropylguanidine

Because of the preincubation of the cells in the presence of the test compounds prior to the aequorin assay, a potential luminescence signal elicited immediately after addition of the compounds would not be detected whereas active aequorin would be consumed. This would result in a decreased luminescence signal when the agonist is added pretending an antagonistic effect of the compound.

Therefore, the compounds AK 49, PG 55B and MF 1 (a Y1 receptor antagonist) were tested in the spectrofluorimetric fura-2 calcium assay.

Fig. 104: Flow cytometric binding assay with CHO-hY4-K13b-qi5-K8-mtAEQ-E11-K11 cells.

Competition of selected compounds with 3 nM cy5-[K⁴]-hPP (mean values ± SEM, n=3).

As shown in Fig. 105, the addition of the solvent ethanol did not induce an increase in intracellular calcium concentration. Subsequent addition of 100 nM rPP elicited the calcium signal. Also the addition of 50 µM AK 49 did not release a calcium response, but the calcium signal subsequently released by rPP was reduced compared to the control experiment, indicating an antagonistic effect of AK 49. Addition of 50 µM PG 55B or MF 1 elicited a strong calcium signal. After the increase in intracellular calcium concentration, the cells were still excitable with the Y4 receptor agonist rPP indicating that the hY4 receptors were not desensitized after the first rise in intracellular calcium concentration. This behavior suggests that the increase in intracellular calcium concentration caused by PG 55B and MF1 is not mediated by the hY4 receptor, but is a result of other signaling pathways, e.g. direct G-protein activation.

To confirm this observation, the compounds PG 55B, AK 49 and MF1 were tested for their agonistic potency in the aequorin assay. As shown in Fig. 107a, 30 µM of MF 1 elicited a luminescence signal whereas the compound PG 55B was not able to induce a luminescence signal up to the highest concentration tested (100 µM). This is in contrast to the results of the fura-2 assay (see Fig. 105) where the addition of 50 µM of PG 55B led to an increase in intracellular calcium concentration. The compound AK 49 was found to be inactive in the aequorin agonist assay up to a concentration of 100 µM. Addition of the cells to 300 µM of AK 49 induced a weak

time [s]

Fig. 105: Calcium responses of CHO-hY4 -K13b-qi5-K8-mtAEQ-E11-K11 cells after addition of 50 µM compound (first arrow) and subsequently 100 nM rPP (second arrow; in case of PG 55B, the addition of rPP was delayed).

luminescence signal, which was not inhibited by preincubation of the cells with 20 µM of the H2 antagonist ranitidine, indicating that this signal was not mediated by the H2

receptor.

Taken together, the compound AK 49 did not induce an increase in intracellular calcium concentration up to a concentration of 50 µM in the fura-2 assay and did not elicit a luminescence signal in the aequorin assay up to a concentration of 100 µM.

Instead, it reduced the calcium signal elicited with 100 nM rPP in the fura-2 assay and suppressed the luminescence signal elicited with 200 nM rPP in the aequorin assay with an IC50 value of 60.7 µM. cy5-[K⁴]-hPP was displaced at the hY4 receptor by AK 49 in flow cytometric binding assays using hY4-expressing CHO- and P388 cells with calculated Ki values of 68.2 µM resp. 28.5 µM. Therefore, AK 49 could be a starting point for the search for new nonpeptidic Y4 antagonists.