Multivalent ligands for the serotonin 5-HT4 receptor

Abstract

5-HT₄ receptors are known to form constitutive dimers in membranes. To explore whether multivalency can enhance ligand interactions and/or efficacy in 5-HT₄ receptors, the structure of the partial agonist ML10302 was modified with oligo(ethylene glycol) chains, thus generating, by a gradual approach, short and long tethered bivalent or tetravalent ligands and the corresponding spanner-linked monovalent controls. Both bivalent and tetravalent ligands displayed a 10-20-fold increase in binding affinity compared to appropriate controls, but no multivalent ligand showed greater binding energy than ML10302 itself. Furthermore, the direct assessment of receptor-Gs interaction and studies of cAMP signalling indicated that multivalency does not enhance the efficacy of ML10302.

Fig. 1. Structures of some significant serotonin 5-HT₄R ligands.

Fig. 2. Design of multivalent 5-HT₄R ligands 8a and b and 9 from 2 through monovalent 7a–e.

Fig. 3. Effect of ligands on 5-HT₄R coupling to Gs measured by BRET in SH-SY5Y cell membranes. a) All test ligands and reference compounds (1 cisapride and 2 ML10302) were tested at 10 μM in comparison with 5-HT (100 μM). The BRET signal, subtracted from the basal value, is shown as % of the 5-HT effect. Data are means with SEM bars from 6 determinations. * indicates P < 0.05 in paired t-tests evaluating the difference from 2. b) Concentration–inhibition curves of the receptor–Gs interaction induced by 100 nM 5-HT obtained with the indicated ligands. Data were fitted with a 4-parameter logistic model (solid lines). The IC₅₀ values were 34.3 nM for 7d and 2.1 nM for 8a. Data are means (n = 3) of an experiment repeated twice with similar results.