Screening of ligand binding on melatonin receptor using non-peptide combinatorial libraries

The screening of combinatorial libraries requires a deconvolution procedure to obtain, in fine, the most active compound of the starting library. The standard screening assays used in regular molecular pharmacology, have been poorly assessed when transposed to combinatorial chemistry-related experiments, particularly those involving large numbers of chemicals in a single assay. One key issue is the effect of the inactive analogs on the identification of the active ligand in mixtures. We chose melatonin receptors to measure the apparent affinity of a single ligand when tested alone or in mixtures of non-peptide low molecular weight compounds. Using ligands with IC50 from the micro- to the picomolar range, mixed with increasingly complex mixtures of 5 to 20 or 25 inactive compounds, we analyzed the displacements from the mt1 and MT2 melatonin receptor subtypes of the radioligand 2-iodomelatonin (KD= 25 pmol/l and 200 pmol/l, respectively) . The behavior of equimolar mixtures in displacement curves led to the conclusion that the observed binding affinity reflects the dilution effect of mixing the active component with inactive compounds but does not reveal noticeable interactions which would interfere with the binding process. From the practical point of view, the concentrations of the active species in the binding assay should be large enough to displace significantly the radioligand, a requirement which may be limited by the solubility of the ligand mixtures. In contrast, previous observations with peptide libraries report that the dilution effect is often compensated by additive or synergic action of structurally related analogs, thus making possible the deconvolution of very large (typically up to 10(7) compounds) peptide libraries.