Binding of a non-peptide ligand to M₁ muscarinic acetylcholine receptors by fluorescence polarisation.

Figure 1: Schematic of small ligand, receptor competition binding assay.

Homogeneous, radioactive techniques such as Scintillation Proximity Assay (SPA) have been very successful in increasing the throughput of receptor binding assays (1). Nevertheless, there is an increasing demand for non-radioactive, fluorescence-based technologies that can be applied to receptor ligand interactions. This work describes a polarisation assay using fluor-labeled telenzepine amine congener (TAC; kindly provided by the NIMH Chemical Synthesis Programme), which is an antagonist of the M1 muscarinic acetylcholine receptor (2).

The affinities of the fluor-labeled telenzepine derivatives were determined by radioactive competition binding assays, using membranes prepared from CHO cells expressing the M₁ muscarinic receptor (CHO M₁ membranes), prior to their use in homogeneous polarisation assays (data not shown).

In polarisation assays, the binding of the fluor-labeled ligand to the receptor results in an increase in the polarisation signal when compared to that of the ligand alone. On the addition of unlabeled competitor ligands, the high polarisation signal is reduced as there is competition between the unlabeled and fluor-labeled ligands for the receptor binding site (Figure 1).

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Methods

Direct binding assays were performed in black opaque 96-well (maximum working volume 300 ml) microtitre plates (Dynex Technologies Inc., Chantilly, Virginia, USA). Varying amounts of the CHO M₁ membrane protein were added with 10 ml fluor-labeled telenzepine (final assay concentrations: Cy™5 telenzepine, 3 nM; Cy3B telenzepine, 0.8 nM; fluorescein telenzepine, 1.25 nM) in a total volume of 200 ml assay buffer (50 mM Tris pH 7.5 containing 0.1% BSA, 5 mM MgCl₂ for Cy5 and Cy3B ligands; 50 mM Tris pH 7.5 containing 0.05% g-globulin, 5 mM MgCl₂ for fluorescein ligand). Incubations were performed at 22^oC, for approximately 3-4 hours, with shaking. Determinations were then made in a Jolley Research and Consulting Fluorolite FPM-2™ reader at the following wavelengths:- excitation 610 nm, emission 670 nm for Cy5; excitation 540 nm, emission 590 nm for Cy3B; excitation 485 nm, emission 535 nm for fluorescein.

In competition experiments, CHO M₁ membrane preparations (50 mg) were added to the wells of a 96-well plate (as above) with varying amounts of unlabeled competitor (atropine or TAC). 10 ml aliquots of Cy3B telenzepine (final assay concentration 0.8 nM) or Cy5 telenzepine (final assay concentration 3 nM) were then added to give a final assay volume of 200 ml in assay buffer (50 mM Tris pH 7.5 containing 0.1% BSA, 5 mM MgCl₂). Incubations were performed at 22^oC for approximately 3-4 hours, with shaking, prior to determination as described above.

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Results

Figure 2: labeled-Telenzepine binding to the CHO M1 cell membranes

Binding of the peptide ligands, labeled with either Cy5 (— ), Cy3B (—) or fluorescein (—), to increasing amounts of the Grb-2 protein are shown in Figure 2. Values are expressed as means + SEM (n=3) and are expressed as specific polarization obtained over that seen in the presence of 100 mM unlabeled phosphopeptide ligand. By analysis of the data using the method of Ohnishi et al (1998), J. Biol. Chem., 273, pp.10210-10215, the KD for the peptides were 333 nM (fluorescein), 250 nM (Cy3B) and 166 nM (Cy5). Each ligand was also assessed as a competitor in an equivalent SPA assay where these observations were also confirmed and found to be of similar affinity to the [3H]13-mer phosphopeptide SPA ligand (approx 400 nM). For reference "start" values for polarization were 62 mP (fluorescein ligand), 105 mP (Cy3B ligand) and 236 mP (Cy5 ligand).

Figure 3: M1 muscarinic receptor competition assays

Data from the competition binding assay is shown in Figure 3. Cy3B telenzepine was incubated with the CHO M1 receptor preparation in the presence of either unlabeled TAC competitor (—) or atropine antagonist (—); Cy5 telenzepine was incubated in the same way in the presence of either unlabeled TAC (—) or atropine (—). Values are means ± SEM (n=4) and expressed as specific polarisation obtained over and above that observed in the presence of 100 mM unlabeled atropine. For reference, the maximum and minimum polarisation values for the assays were: 369 and 291 mP (Cy5 ligand), 290 and 110 mP (Cy3B ligand) respectively.In repeated experiments, the IC50 values were determined and found to be 4.0 nM (Cy3B telenzepine) and 2.6 nM (Cy5 telenzepine) for atropine; 28 nM (Cy3B telenzepine) and 7.7 nM (Cy5 telenzepine) for TAC.

In similar experiments, using 50 mg CHO M₁ membrane and the fluorescein ligand, competition curves could not be obtained due to excessive NSB. On changing the buffer conditions (50 mM Tris pH7.5 containing 0.05% g-globulin, 5 mM MgCl₂), some data was obtained but the signal was poor; maximum and minimum polarisation values for this assay were 79 and 53 mP respectively. In further experiments using the Cy3B telenzepine, reduced levels of the CHO M₁ membrane preparation could be used; equivalent to those used in a typical filter binding assay. In all cases a membrane preparation from control CHO cells, not expressing the M₁ receptor, gave no specific signal over NSB values (data not shown).

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References

• Cook, N.D., et al., Drug Discovery Today, 1, 287-294 (1996).
• Karton, Y., et al., Bioconjugate Chem., 3, 234-240 (1992).