. Author manuscript; available in PMC: 2009 Jul 27.

Published in final edited form as: Angew Chem Int Ed Engl. 2008;47(9):1685–1688. doi: 10.1002/anie.200702770

Table 1.

Cumulative data for binding of heterobivalent ligands to MC4R- δ-OR dual expressing cells in the presence or absence of a heterologous blocking agent

Compound	Linker		IC₅₀ [nm] of MSH(7) ^b			IC₅₀ [nm] of Delt-II^b
		Max. linker length^a [Å]	hMCR4^d	Dual expression^e	Fold increase^c	hδ-OR^f	Dual expression^g	Fold increase^c
1	-[PG]₃-	≤13	110 ± 9	180 ± 30	0.6	150 ± 8	110 ± 40	1.3
2	-[PG]₆-	≤25	110 ± 330	39 ± 14	2.9	330 ± 110	240 ± 120	1.3
3	-[PG]₉-	≤35	260 ± 160	9.7 ± 3.3	27	210 ± 130	100 ± 50	2.1
4	-[PG]₁₂-	≤45	150 ± 70	3.1 ± 1.1	47	430 ± 200	300 ± 90	1.5
5	-[PG]₁₅-	≤55	160 ± 50	3.3 ± 1.8	48	500 ± 90	230 ± 70	2.2
6	-PEGO-[PG]₆-PEGO-	≤56	90 ± 40	2.1 ± 0.4	44	90 ± 40	70 ± 3	1.4
7	-PEGO-[PG]₁₂-PEGO-	≤76	120 ± 60	2.5 ± 1.1	47	100 ± 30	290 ± 80	0.3
8	-PEGO-[PG]₁₈-PEGO-	≤96	150 ± 40	3.5 ± 1.3	43	200 ± 90	110 ± 40	1.8

Maximal linker length estimated from modeling studies.

IC₅₀ is a concentration of compound at 50% specific binding. Monomeric hexapeptide ligand Ac-MSH(7)-NH₂ exhibits modest specific binding affinity to the hMC4R, IC₅₀ = 39 ± 4.1 nM, monomeric Deltorphin-II showed IC₅₀ = 0.42 ± 0.02 nM to the δ-OR.

Fold increase is the ratio of IC₅₀ values between monovalent and bivalent binding modes. For each heterobivalent ligand, binding was assessed by four different competitive binding assays using the dual-receptor expressing cells.

hMC4R monovalent binding mode; at hMC4R alone by competing Eu-NDP-αMSH in the presence of a saturating concentration of 10 μM Naloxone, a δ-OR blocking agent.

hMC4R heterobivalent binding mode; at the hMC4R with both receptors available for binding by competing Eu-NDP-αMSH.

At the δ-OR alone using Eu-DPLCE and a saturating concentration of 10 μM NDP-αMSH, which blocks the hMC4R.

At the δ-OR with both receptors available using Eu-DPLCE.