Screening of Ligand Binding on Melatonin Receptor Using Non-Peptide Combinatorial Libraries

Abstract

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.

From peptide libraries to optimized nonpeptide ligands in the search for S‐farnesyltransferase inhibitors

“…The decreasing number of congeners and the increasing concentration of the actives in the following steps, refines the selection and finally leads to the most potent analog(s). This is in contrast to what was observed in a recent model study of nonpeptide ligands, in which small mixtures of one single active component and a variable number of inactives were screened in a binding assay (22). In the latter case, the effective dilution of the single active species had to be taken into account, which limited the acceptable number of compounds in the mixtures as a function of the potency of the active species and the detection power of the binding assay.…”

New Radioligands for Describing the Molecular Pharmacology of MT1 and MT2 Melatonin Receptors

“…For DIV880, the process was a little different. The compound is an iodinated analog of a bromo-compound that resulted from a large screening process, similar to the compounds described elsewhere [11,12]. This compound was specific to MT 2 , with a pK i 2 logs “better” for MT 2 than for MT 1 .…”

“…The aim was to broaden the panel of available tools for studying these receptors [3,10]. By conducting several series of high throughput screening HTS campaigns [11,12], we found a MT 2 -specific partial agonist, DIV880. The K i of this compound is 2 logs less potent with MT 1 than MT 2 .…”

Recombinant Human Melatonin Receptor MT1 Isolated in Mixed Detergents Shows Pharmacology Similar to That in Mammalian Cell Membranes

“…These findings validated the quality of the purified material, indicating that it may be of particular interest for primary screening of MT1-binding molecules. Purified MT1 samples could be very helpful for limiting the false positive rate usually experienced with classical screenings, since no cellular or membranous artifacts would be present to interfere with the assay , . Obviously, this assay format would be unable to indicate the G-protein coupling and signaling properties of the compounds, which are known to be important, particularly in the melatoninergic system [19].…”