Next to the advantages, like outstanding sensitivity of quantitation, the use of ra-dioligands also has some inherent disadvantages. Since handling radioisotopes in organic syntheses requires relatively high safety standards and is subject to broad legal restrictions, the production of radioligands is expensive and also restricted to a few specialized institutions. For the user this means a sizeable expense and a limited number of commercially available radioligands. Performing radioligand binding assays according to legal requirements adds further costs. Among them are those for specially equipped rooms or the disposal of radioactive waste. The nature of the radionuclide causes its own problems. Radionuclides with a short half life have to be used within a correspondingly short time, while radionuclides with a long half life generate additional problems with regard to their storage and disposal. Very often the radioactive decay leads also to destruction, raising the question of the chemical purity of the radioligand after longer storage.
Next to radioisotopes, any other label allowing the detection of a ligand with sufficient sensitivity can be employed in binding assays. The preferred markers, apart from radioligands, are ligands conjugated to a fluorophore [37-40]. Since background fluorescence causes problems when measuring the fluorescence in the presence of the receptor material , either the nonbound fluorescent marker has to be quantified in conventional binding assays after separation or, alternatively, the bound fluorescent marker after being liberated from the target. Binding assays based on time-resolved fluorescence (TRF) or fluorescence polarization (FP) [10-12] have gained particular attraction. TRF assays usually employ ligands labeled with lanthanide chelates (e.g. Eu3+ chelate) and are used for a great variety of targets [12, 13, 42-44]. Measuring the amount of bound or the nonbound marker generally requires a separation step and the addition of an enhancer solution since the matrix of the binding sample hinders optimal quantita-tion. FP allows the distinction between nonbound and bound fluorescent markers in homogenous phase based on the restricted rotation of fluorescent markers bound to a target. Commonly, fluorescent markers originating from peptides are employed for FP applications; however, recently some examples using fluorescent markers originating from small molecules have been described as well [14, 15, 45]. Other fluorescence-based methods to investigate target-ligand interactions use fluorescence correlation spectroscopy (FCS) or fluorescence resonance energy transfer (FRET) [8, 12, 46].
Despite successful examples of fluorescence-based binding assays and the relevance of radioligand binding assays in the drug discovery process, two major inherent disadvantages regarding any labeling strategy remain. First of all, labeling always means an additional synthetic effort. In addition to the labeling process, very often a new synthetic route has to be established to obtain a suitable precursor for the labeling reaction. Secondly, labeling of a ligand can decrease its affinity. Replacing a stable isotope with a radioisotope (e.g. 3H with 1H) does usually not change the ligand in this respect, whereas the substitution by a radioisotope with different electronic properties (e.g. replacing 1H with 1251) may cause a significant decrease in affinity. In the case of labeling a native ligand with a large fluorophore - compared to a radioisotope - a severe decrease in affinity can frequently be observed. This is especially true for small molecules and seriously limits the use of fluorophore-labeled ligands in binding assays [12, 41, 47].
However, the determination of affinity does not necessarily have to rely on labeled ligands. It is also possible with native ligands when using suitable detection methods, as for example nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), acoustic biosensors or calorimetry [48, 49]. A particularly versatile and sensitive detection principle for the investigation of interactions between targets and native ligands is mass spectrometry .
254 | 7 MS Binding Assays - An Alternative to Radioligand Binding 7.3
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