Radioligand binding studies determination

In radioligand binding studies, non-linear Scatchard plots or competition curves that have abnormally steep slopes imply complex binding phenomena, possibly involving multiple receptor types or affinity states. In such cases, the thermodynamic parameters should be separately determined for each receptor type or affinity state. 

Radioligand binding studies are a rapid means to determine the affinities of reference and novel ligands for a receptor. It is less realised, however, that this technique can equally be applied as a protocol for the discrimination between agonists and antagonists. Therefore some attention in this chapter will be paid to this methodology and its biochemical background. 

Compounds 14-17 are derivatives of the corresponding silanes 10 and 12. Functional pharmacological studies (Ml-M3 receptors) and radioligand binding experiments (M1-M4 receptors) with the pure (/ )- and (S )-enantiomers of these compounds revealed very similar pA2 values as observed for the antipodes of the related analogues 10 and 1234. The highest stereoselectivity indices (functional studies) were determined for 17 [SI = 44 (Ml), 7.6 (M2), 20 (M3)]. 

The most important determinants of antagonist activity were, however, indicated to be the 4-phenyl and C3 and C5 ester substituents. These studies of Loev et al. were largely confirmed in a variety of subsequent investigations based on in vitro pharmacological and radioligand bind approaches [4]. 

The MIPs have also been utilized as the recognition elements in pseudoimmunoassays. " In this approach, MIPs are substituted for antibodies to quantify the amount of analyte in a biological sample, such as blood plasma. Most MIP immunoassays are competitive binding studies in which a radio- or fluorescent-labeled analyte is added to a mixture of the MIP and imlabeled analyte. After equilibrium is reached, some fraction of the labeled species is bound to the polymer surface and thus can be separated from the supernatant. The supernatant is then analyzed via scintillation or fluorescence techniques to determine the concentration of the original unlabeled analyte. Mosbach et al. have demonstrated that MIP-based immunoassays can rival the selectivity of antibody-based assays. Imprinted polymers for the opioid receptor ligands enkephalin and morphine were prepared and showed submicromolar (pM) level selectivity in a radioligand competition assay in aqueous buffers. The analysis... 

The thermodynamic analyses most often used, particularly fhe van t Hoff mefhod, require that measurements be made at steady-state conditions. In the case of radioligand binding determination of equilibrium constants, fhe time required for fhe protein-ligand interaction to reach steady state depends on fhe incubation temperature, and, therefore, the equilibrium constant must be determined for each temperature studied. For the most accurate results, fhe determination needs to be made at more than two temperatures in order to detect non-linearity. The integrated form of the van t Hoff equation takes fhe simple form fhat is commonly used only if ATT and AS° for the interaction are not temperature dependent otherwise, non-linearity in the van t Hoff plot can arise. Meaningful information can StiU be obtained in such cases, but more complex analysis is required. 

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