Download a homogenous flow cytometric method for the combined

A HOMOGENOUS FLOW CYTOMETRIC METHOD FOR THE COMBINED
DETERMINATION OF ACTIVITY AND AFFINITY OF NPY Y1 RECEPTOR
LIGANDS
Erich Schneider, Elvira Schreiber, Günther Bernhardt and Armin Buschauer
Department of Pharmaceutical and Medicinal Chemistry II, University of Regensburg,
Germany
The pharmacological characterization of ligands acting on G-protein coupled
receptors (GPCRs) requires the determination of both affinity and activity. The
combination of binding assay and functional test using the same measurement setup
would considerably facilitate the process of drug evaluation. As part of a project to
develop fluorescence-based methods for the investigation of ligand - GPCR
interactions we studied the applicability of flow cytometry. NPY receptors were
selected as a model. Here we report on a new flow cytometric method for the
combined determination of equilibrium binding constants and activity of NPY Y1
receptor ligands in a homogenous assay using human erythroleukemia (HEL) cells.
Cyanine dyes, which are excitable at 635 nm and emit in the Fl-4 channel of the flow
cytometer (6618 nm), were used for fluorescence labelling as the autofluorescence
of living cells is negligible at this wavelength. The fluorescent Ca2+ indicator Fluo-4
allows the detection of Ca2+ responses at 53015 nm (Fl-1) after excitation at 488 nm.
For the flow cytometric measurements pNPY was labelled with the cyanine dye Dy630, and HEL cells (constitutively expressing the NPY Y1 receptor) were loaded with
Fluo-4. A combined determination of affinity and activity was performed with the Y1
receptor antagonist BIBP 3226 and related argininamides synthesized by our working
group. The data correlated well with the results of radioligand binding and the
spectrofluorimetric Fura-2 assay. For example with BIBP 3226 we obtained Ki values
of 2.74 nM and 1.54 nM, determined by flow cytometry and radioligand binding
assay, respectively. The flow cytometric measurement of the inhibitory effect of BIBP
3226 on Ca2+ mobilization revealed a Kb value of 2.21 nM. With the conventional
spectrofluorimetric assay we determined a Kb of 1.55 nM.
Provided that fluorescence labelled tracer ligands with sufficiently high activity and
affinity are available, the assay presented here can be easily applied to other GPCRs.
With the advent of microtiter plate based sample delivery devices for flow cytometers
this fast and reliable assay could be adopted to the pharmacological investigation of
libraries of substances.
(Supported by the DFG, Graduiertenkolleg GRK 760)