A Direct in Vivo Comparison of the Melanocortin Monovalent Agonist Ac-His-DPhe-Arg-Trp-NH2 versus the Bivalent Agonist Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-NH2: A Bivalent Advantage.

Bivalent ligands targeting putative melanocortin receptor dimers have been developed and characterized in vitro; however, studies of their functional in vivo effects have been limited. The current report compares the effects of homobivalent ligand CJL-1-87, Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-NH2, to monovalent ligand CJL-1-14, Ac-His-DPhe-Arg-Trp-NH2, on energy homeostasis in mice after central intracerebroventricular (ICV) administration into the lateral ventricle of the brain. Bivalent ligand CJL-1-87 had noteworthy advantages as an antiobesity probe over CJL-1-14 in a fasting-refeeding in vivo paradigm. Treatment with CJL-1-87 significantly decreased food intake compared to CJL-1-14 or saline (50% less intake 2-8 h after treatment). Furthermore, CJL-1-87 treatment decreased the respiratory exchange ratio (RER) without changing the energy expenditure indicating that fats were being burned as the primary fuel source. Additionally, CJL-1-87 treatment significantly lowered body fat mass percentage 6 h after administration (p < 0.05) without changing the lean mass percentage. The bivalent ligand significantly decreased insulin, C-peptide, leptin, GIP, and resistin plasma levels compared to levels after CJL-1-14 or saline treatments. Alternatively, ghrelin plasma levels were significantly increased. Serum stability of CJL-1-87 and CJL-1-14 (T1/2 = 6.0 and 16.8 h, respectively) was sufficient to permit physiological effects. The differences in binding affinity of CJL-1-14 compared to CJL-1-87 are speculated as a possible mechanism for the bivalent ligand's unique effects. We also provide in vitro evidence for the formation of a MC3R-MC4R heterodimer complex, for the first time to our knowledge, that may be an unexploited neuronal molecular target. Regardless of the exact mechanism, the advantageous ability of CJL-1-87 compared to CJL-1-14 to increase in vitro binding affinity, increase the duration of action in spite of decreased serum stability, decrease in vivo food intake, decrease mice's body fat percent, and differentially affect mouse hormone levels demonstrates the distinct characteristics achieved from the current melanocortin agonist bivalent design strategy.