Solid-Phase Synthesis of Heterobivalent Ligands Targeted to Melanocortin and Cholecystokinin Receptors

Abstract

Heteromultivalency provides a route to increase binding avidity and to high specificity when compared to monovalent ligands. The enhanced specificity can potentially serve as a unique platform to develop diagnostics and therapeutics. To develop new imaging agents based upon multivalency, we employed heterobivalent constructs of optimized ligands. In this report, we describe synthetic methods we have developed for the preparation of heterobivalent constructs consisting of ligands targeted simultaneously to the melanocortin receptor, hMC4R, and the cholecystokinin receptors, CCK-2R. Modeling data suggest that a linker distance span of 20-50 Å is needed to crosslink these two G-protein coupled receptors (GPCRs). The two ligands were tethered with linkers of varying rigidity and length, and flexible polyethylene glycol based PEGO chain or semi-rigid [poly(Pro-Gly)] linkers were employed for this purpose. The described synthetic strategy provides a modular way to assemble ligands and linkers on solid-phase supports. Examples of heterobivalent ligands are provided to illustrate the increased binding avidity to cells that express the complementary receptors.

Fig. 1

List of heterobivalent constructs composed of melanocortin and cholecystokinin ligands (MSH-7 and CCK-6, respectively). The heterobivalent ligands were either constructed from the semi-rigid poly(Pro-Gly) linker (compound 1–5) or from the flexible PEGO linker (compound 6–7). The legend shows the peptide sequences of MSH-7 and CCK-6 ligands and the structure of the PEGO linker (20 atoms)

Fig. 2

Synthetic route for heterobivalent ligands. The inset on left, top shows addition of PEGO sequence. The inset on bottom, right shows the structure of protected Ac-MSH-7 ligand on resin. The reagents for each step are as follows—(i) Fmoc-amino acid-OH (3 eq), Cl-HOBt (3 eq), diisopropylcarbodiimide (6 eq); (ii) 20% Piperidine/DMF; (iii) PEGO linker assembly; refer text for details. (iv) 50% Ac₂O in Pyridine; (v) TFA (91%), H₂O (3%), Thioanisole (3%), Triisopropylsilane (3%) m is 1 or 2; n is 3, 6, 9, 12, 15

Fig. 3

(a) HPLC profile of crude compound 6 at 280 nm; (b) Peptide concentration determination of compound 6 using 0.5 mM D-Tryptophan as standard (with t_R of 2.4 min in this figure) co-injected in analytical HPLC and monitored at 280 nm

Fig. 4

Representative curves from the competitive binding assay of heterobivalent ligands evaluated for their monovalent and bivalent binding by competing them against Eu-labeled NDP-α-MSH and CCK-8 ligands. Single plot IC₅₀ values were determined where data from all n measurements were pooled first and non-linear regression analysis performed. (a) Binding of Ligand 2 competed with 0.1 nM Eu-CCK8 in Hek293/CCK cells, with an IC₅₀ of 46 nM (R² = 0.90). (b) Binding of Ligand 2 competed with 0.1 nM Eu-CCK8 in Hek293/MC4R/CCK cells, with an IC₅₀ of 2.3 nM (R² = 0.89). (c) Binding of Ligand 6 competed with 0.1 nM Eu-CCK8 in Hek293/CCK cells, with an IC₅₀ of 11 nM (R² = 0.93). (d) Binding of Ligand 6 competed with 0.1 nM Eu-CCK8 in Hek293/MC4R/CCK cells, with an IC₅₀ of 0.5 nM (R² = 0.83)