Receptor Binding and Functional Assays

Cell culture. CHO cells stably expressing the hY2R were cultured as described else-where.¹⁰

Buffers. The buffer for the Y2R binding studies on CHO cells was prepared by the addition of BSA (1 %) and bacitracin (100 μg · L^-1) to a buffer (pH 7.4) consisting of HEPES (25 mM), CaCl2 × 2 H2O (2.5 mM), MgCl2 × 6 H2O (1 mM). BSA and bacitracin were omitted in saturation binding, kinetic and competition binding experiments with non-peptide fluorescent antagonists. For the displacement of 5.4 and 5.16 with pNPY only BSA was omitted. The loading buffer (pH 7.4) for the determination of the mobilization of intracellular Ca²⁺ in CHO cells was prepared by dissolving NaCl (120 mM), KCl (5 mM), MgCl₂ × 6 H₂O (2 mM), CaCl₂ × 2 H₂O (1.5 mM), HEPES (25 mM), and glucose (10 mM).

5.5.6.2 Fura-2 Calcium Assay and Radioligand Competition Binding Experiments The fura-2 assay was performed as previously described using a LS50 B spectro-fluorimeter from Perkin Elmer (Überlingen, Germany).⁴² Radioligand competition binding experiments with [³H]-UR-PLN196 were performed for the determination of Ki values of the fluorescent ligands 5.4 and 5.16. as described in Chapter 6.

5.5.6.3 Flow Cytometry

All flow cytometric binding assays were performed with a FACSCalibur^TM flow cytometer (Becton Dickinson, Heidelberg, Germany), equipped with an argon laser (488 nm) and a red diode laser (635 nm).^{10, 38, 43} All binding assays were performed in a final volume of 500 µL containing labeled ligand and unlabeled peptide or antagonist as needed.

Selectivity assay. The selectivity of the new Y2R antagonists for human NPY Y2 over Y1, Y4 and Y5 receptors was investigated by flow cytometric binding assays on Cy5-pNPY (Y1R, Y5R), and Cy5-[K⁴]-hPP (Y4R) according to previously described methods.^10,

38, 44

The compounds were tested at two concentrations (1 µM and 10 µM) in duplicate.

Competition binding assay with Cy5-pNPY. The binding assay was performed as described elsewhere¹⁰ with the following adaptations. Instrument settings were: FSC:

E-1, SSC: 350 V, Fl-4: 800 V, Flow: HI; measurement stopped when 10000 gated events were counted. The experiments were performed using 490 µL of cell suspension, 5 µL of Cy5-pNPY (final concentration 5 nM) and 5 µL of test compound (100-fold concentrated). If indicated, 5 µL of Dy-635-pNPY (final concentration 10 nM) was used as fluorescent ligand. Incubation time was 90 min at room temperature. Ki values were obtained from 2-3 independent experiments.

Displacement of fluorescent ligands 5.4, and 5.15-5.17. The cells were seeded three days prior to the experiment (90-95 % confluency), trypsinized and resuspended in Ham´s F12 medium, containing 10 % fetal calf serum for the inactivation of trypsin.

After centrifugation at 1,000 rpm for 5 min, the cells were suspended in binding buffer without BSA to a density of 2-3 · 10⁶ cells/mL. Bacitracin (0.1 g/L) was added in case of competition binding with pNPY in order to prevent protease-mediated degradation of the peptide. The experiments were performed with the respective fluorescent ligand (c (5.4) = 40 nM, c (5.15) = 100 nM, c (5.16) = 60 nM, c (5.17) = 40 nM ) in the presence and absence of various competitors at different concentrations (10^–10 to 10^–4 M). Non-specific binding was determined in the presence of 10 µM BIIE 0246. The samples were incubated in siliconized reaction vessels for 30 min (90 min for pNPY) at room temperature before flow cytometer analysis. Instrument settings for fluorescent ligand 5.4: FSC: E-1, SSC: 360 V, Fl-2: 550 V, Fl-3: 800 V, Flow: HI;

measurement stopped when 10000 gated events were counted; Instrument settings for fluorescent ligands 5.15-5.17: FSC: E-1, SSC: 350 V, Fl-4: 600 V, Flow: HI;

measurement stopped when 10000 gated events were counted.

Saturation binding experiments with 5.4, and 5.15-5.17. Saturation binding experiments were performed by analogy with the above mentioned competition binding assays with following adaptations. The cells were incubated with the respective fluorescent ligand in increasing concentrations (c ≈ 0.2 × KD - 10 × KD) for 30 min. Non-specific binding was determined with 100-fold excess of BIIE 0246.

Kinetics of Y2R binding of Cy5-pNPY, 5.4 and 5.16. The cells were treated as already described. Association kinetics was determined by incubation of the cells with a constant concentration of labeled ligand (c (Cy5-pNPY) = 5 nM, c (5.4) = 40 nM, c (5.16) = 60 nM). Samples were taken at different time periods and measured (For

instrument settings see the respective competition binding protocols). Non-specific binding was measured with 100-fold excess of pNPY in case of Cy5-pNPY and BIIE 0246 for the labeled non-peptide ligands, respectively.

For dissociation experiments cells were pre-incubated with the corresponding labeled ligand at the above mentioned concentrations for 30 min (Cy5-pNPY: 120 min) in binding buffer without BSA, bacitracin, except for Cy5-pNPY (buffer + 1 % BSA and 0.1 g/L bacitracin). For non-specific binding, incubation was performed in pre-sence of 100-fold excess of pNPY in case of Cy5-pNPY and BIIE 0246 for the non-peptide labeled ligands, respectively. Afterwards, cells were washed with cold binding buffer and resuspended in binding buffer consisting of pNPY and BIIE 0246 (100-fold excess), respectively. Samples were taken at different time periods and measured. (For instrument settings see the respective competition binding protocols).

5.5.6.4 Data Processing

All data to be fitted were processed with GraphPad Prism 5.

Fura-2 Assay. The ratio of fluorescence intensities at 510 nm after excitation at 340 and 380 nm was used for the calculation of the calcium concentration according to the Grynkiewicz equation.⁴⁵ At least two points between 20 and 80 % inhibition served for the calculation of IC50 values after logit-log transformation using the following equation:

logit (P) = log [P/(100-P)]

IC50 values were obtained by plotting logit (P) versus log B with the slope n according to logit (P) = nlog B–nlog IC50 by linear regression. The KB value was calculated using the Cheng-Prusoff equation⁴⁶:

KB = IC50 · [EC50/(EC50+L)]

P: mean percent inhibition with SEM < 10 %, determined from 2-3 independent experiments performed on different days;

B: antagonist concentration;

IC50: concentration of competitor producing 50 % inhibition;

EC₅₀: agonist concentration that induces 50 % of the maximum response;

L: concentration of pNPY.

Radioligand Binding Assay. IC50 values were converted to Ki values according to the Cheng-Prusoff equation⁴⁶ using the respective KD value of the radioligand.

Flow Cytometry. The geometric means of fluorescence in the corresponding fluorescence channel (Fl-2, Fl-3, Fl-4) of the gated cells were calculated using the WinMDI 2.9 software. The inhibition constant Ki was calculated using the Cheng-Prusoff equation⁴⁶:

Ki = IC50 · [KD/(KD+L)]

Ki: inhibition constant of the competitor;

KD: dissociation constant of the labeled ligand;

IC50: concentration of unlabeled competitor producing 50 % inhibition of the specific binding of the labeled ligand;

L: concentration of the labeled ligand.

Data from saturation binding were evaluated by one-site saturation fits. Rate constants (kob, koff) were analyzed by linear regressions. The association rate constant (kon) was calculated according to the following equation:

kon = (kob–koff)/L

L: concentration of the ligand;

kob: observed/macroscopic association rate constant;

koff: dissociation rate constant.