Pharmacological Tools

In general, the term “pharmacological tool” defines all types of compounds, which are useful for a detailed pharmacological exploration of a certain target. In case of GPCRs, pharmacological tools are compounds, preferentially antagonists, which bind to a monomeric or oligomeric receptor subtype selectively and with high affinity and provide information about the receptor, for instance, expression, localization, distribution, function, as well as ligand-receptor interactions including binding mode, binding kinetics, etc.

Hence, bivalent antagonists, fluorescence- and radioligands are attractive tools for the characterization of GPCRs and for the investigation of GPCR ligands. In the following, radio- and fluorescence-based techniques commonly applied for the investigation of ligand-receptor interactions with focus on labeled pharmacological tools for NPY receptors will be presented.

1.5.1 Radioligands and Autoradiography

[³H]- and [¹²⁵I]-labeled ligands are often utilized as standard radioligands in binding experiments and autoradiography, respectively. Tritium is a low energy β^– emitter (max. β energy 18.7 keV). Hence, this radionuclide is often applied in bioassays due to low risk of radiation exposure and simple safety precautions. Radioligand binding experiments are usually performed in order to characterize the binding affinities of novel compounds, as well as for the evaluation of binding properties of the receptor.

Binding studies of NPY receptors were performed with variously radiolabeled peptides like [³H]-propionyl-NPY.^140-141 However, selective ligands were needed for the discrimination of receptor subtypes. [³H]BIBP 3226 was reported as a highly potent, reversibly binding Y₁R selective antagonist, useful, for instance, for competition binding assays or the determination of the number of binding sites (Bmax).¹⁴² Recently, two acylguanidine bioisosteric analogues of BIBP 3226 were reported as tritiated radioligands with decreased basicity and retained Y1R affinity and selectivity. Besides saturation experiments and kinetics, these ligands were successfully applied to autoradiography.^{137, 143}

Receptor autoradiography represents a classical technique for the in vitro/ex vivo detection of receptor expression and distribution. ¹²⁵I (γ-emitter, 186 keV) has been applied especially as peptide-label in autoradiographic investigations, for instance, [¹²⁵I]-NPY and [¹²⁵I]-PYY as tools for all NPY receptor subtypes except for the Y4R,^111,

144-147 or [¹²⁵I]-PYY (3-36) for the detection of the Y2R^{111, 145}. However, there are limitations in the use of ¹²⁵I, for instance, due to the release of ¹²⁵I and metabolization/distribution of free iodine in certain tissues (e.g. thyroid gland, stomach, kidneys).¹⁴⁸

1.5.2 Positron Emission Tomography (PET)

Positron emission tomography (PET) is a powerful molecular imaging technique in medical diagnostics. PET is based on the use of short-lived positron emitting isotopes such as ¹⁸F (t1/2 = 109.7 min), ¹¹C (t1/2 = 20.4 min) or ⁶⁴Cu (t1/2 = 12.7 h). Most commonly ¹⁸F is employed as a substitute of a hydrogen atom. The van-der-Waals radii of fluorine and hydrogen are almost the same (1.35 Å vs. 1.20 Å), whereas the differences in electronic properties are very pronounced.

More than 20 nuclear reactions are known for ¹⁸F production. Proton bombardment of ¹⁸O enriched water resulting in the ¹⁸O(p,n)¹⁸F nuclear reaction is the most effective method and delivers the desired radionuclide with high molar radioactivity.¹⁴⁹ Nowadays, ¹⁸F-PET is routinely applied as a diagnostic imaging method in the field of oncology, neurology and cardiology. Most efforts were spent in the development of PET tracers for tumor imaging. For instance, 2-[¹⁸ F]fluoro-2-deoxy-D-glucose ([¹⁸F]FDG) is extensively used in diagnosis and therapy control.

[¹⁸F]FDG, a substrate of glucose transporters, is accumulated in tumor tissues due to metabolic trapping in cancer cells.¹⁵⁰

As already discussed, NPY receptors are overexpressed in various tumors and therefore, selective PET-ligands are promising pharmacological tools for cancer diagnosis and receptor imaging, respectively. A synopsis of described PET-ligands for the Y1R and the Y5R, respectively, is presented in Figure 1.14. For instance, the [¹¹ C]-PET ligand 1.7 was prepared as a Y5R antagonist with high affinity (IC50 = 1.5 nM), appropriate lipophilicity (log D7.4 = 2.79) and moderate brain penetration (brain/plasma ratio = 0.50). Furthermore, the cold analog proved Y5R selectivity over the other NPY receptors.¹⁵¹ The compounds 1.8 and 1.9 were synthesized in our group as prototypical [¹⁸F]-PET ligands for the Y1R, derived from BIBP 3226.¹⁵² 2,4-Diaminopyridine derivatives were identified as promising PET tracer candidates for the Y1R in terms of binding affinity and lipophilicity by Kameda et al.¹⁵³ and, recently, one of these compounds was selected for ¹⁸F-labeling. The resulting antagonist [¹⁸F]Y1-973 does not bind to the Y2, Y4 or Y5 receptor but exhibits Y1R binding in the subnanomolar range.¹⁵⁴ In vitro autoradiography together with in vivo PET imaging in rhesus monkey proved the applicability of the novel [¹⁸F]-ligand in animal models with potential translation to human PET studies. However, no promising candidates for PET imaging of the Y2R have been identified yet. Thus, there is urgent need for such compounds to explore the suitability of Y2R imaging.

Figure 1.14. PET-ligands for the Y1R and Y5R.

1.5.3 Fluorescent Ligand-Based Assays and Fluorescence Imaging

Fluorescence-based binding assays are preferred over radioactive assays in terms of safety precautions and waste disposal. Moreover, numerous fluorescent probes have been developed over the past two decades resulting in the design of potent fluorescence ligands for different GPCRs.^155-162 Thus, there is a wide range of tools, which broaden the scope of application of fluorescence based assays and molecular imaging in GPCR research.

Fluorescence polarization assays are based on the excitation of the sample with polarized light. Free fluorescent ligands emit non-polarized fluorescence after excitation, whereas the emission of receptor bound fluorescent ligands is polarized due to rigidization of the fluorophore in the receptor-ligand-complex. The resulting fluorescence anisotropy allows determination of ligand binding under equilibrium conditions and study of binding kinetics, respectively. As there are no washing steps required in contrast to radioligand binding assays this technique is simply adaptable to HTS.¹⁶³

Various binding assays based on fluorescence resonance energy transfer (FRET) have been developed. Briefly, FRET occurs between a “donor” fluorophore and an

“acceptor” fluorophore. As prerequisite for FRET the emission spectrum of the donor must overlap with the excitation spectrum of the acceptor and secondly, the two fluorophores have to be in close proximity to each other (usually < 10 nm).

Consequently, this phenomenon was exploited for the detection of GPCR oligomers,

e.g., in case of oxytocine receptors¹⁶⁴ and dopamine D2/somatostatin sst5 receptor heterooligomers.¹⁶⁵

1.5.3.1 Flow Cytometry

Flow cytometry provides a sensitive and quantitative method for the measurement of cellular fluorescence. The optical setup of the FACSCalibur^TM flow cytometer used in this work is presented in Figure 1.15.

The cytometer is equipped with two lasers, namely an argon laser (488 nm) and a red diode laser (635 nm). Fluorescence resulting from excitation at 488 nm is detected by the photomultipliers Fl-1, Fl-2 and Fl-3, whereas the photomultiplier Fl-4 only detects the red fluorescence emitted after excitation with the red diode laser.

Flow cytometry is widely used for the investigation of numerous cellular and cell-associated parameters, e.g. cell cycle¹⁶⁶, apoptosis¹⁶⁷, oxidant production¹⁶⁸, membrane potential¹⁶⁹, calcium elevation¹⁷⁰ and pH changes¹⁷¹. Binding assays for flow cytometric devices have been described e.g. for the chemokine receptor CXCR4¹⁷², the EGF receptor¹⁷³ or the α-factor receptor¹⁷⁴. Recently, flow cytometric equilibrium binding assays for the NPY receptors were established in our group.^95,

175-177 Herein, flow cytometry was successfully applied for the simultaneous determina-tion of binding affinities at the Y1R, Y2R and Y5R, emphasizing the applicability of this technique for HTS.¹⁷⁶

Figure 1.15. Optical setup of the FACSCalibur^TM flow cytometer. The fluorescence emission is separated from the SSC light by filters and dichroic mirrors and detected by different photomultiplier tubes (adapted from Mayer¹⁷⁸).

The main advantage of a flow cytometric binding assay compared to a classic radioligand binding assay is the fact that, similar to fluorescence polarization techniques, the separation of bound and free ligand is not required. Moreover, the sample volume of only several picoliters defined by the intersection of the laser beam

with the sample stream is very small.¹⁷⁹ Therefore, the background signal caused by free fluorophores is very low compared to the signal from the cell. Thus, binding of fluorescent ligands to GPCRs can be determined at equilibrium.

1.5.3.2 Confocal Laser Scanning Microscopy (CLSM)

The number of binding assays using CLSM is rising. For instance, fluorescent adenosine A1 receptor ligands were characterized by confocal microscopy binding studies at the single cell level.¹⁸⁰ Moreover, quantitative imaging of the native α₁ -adrenoceptor with Bodipy-labeled prazosin revealed intracellular high affinity binding sites.¹⁸¹ Recently, the fluorescence intensity distribution analysis (FIDA) was reported for GPCR-focussed high throughput screening.¹⁸² This technique is applicable to membranes of low GPCR expression levels due to a low detection limit.

Furthermore, confocal microscopy is regarded as an indispensable tool for GPCR imaging. The application of confocal microscopy for the visualization of specifically bound fluorescent peptides, e.g. Bodipy-conjugated NPY ligands or carboxy-fluorescein-NPY for the investigation of NPY receptors, has proven most powerful in studying receptor internalization and trafficking of receptor-ligand complexes into the cells.^{57, 183} Lastly, the implication of the Y1R in the regulation of intracellular Ca²⁺

within the cardiovascular system was also identified by means of confocal microscopy.¹⁸⁴