Screening Of New Ligands Targeting Melanocortin Receptors And The Study On Their Weight Reducing Effects

Obesity,a disease of metabolic abnormality, not only raises the burden of different systems in the body, but also leads to the genesis of many kinds of diseases, therefore becomes the great threaten to public health. Many kinds of drugs have been developed for the therapy of obesity, but their toxicity and adverse effects limited their clinical use. Scientists are now endeavouring to develop new drugs for obesity.Melanocortin receptors (MCR), including MC1-5R, are a subfamily of G-protein coupled receptor superfamily. Among them, MC3R and MC4R are mainly distributed in central nervous system, and exert important psychological effects in modulating appetite as well as energy equilibrium, and could be taken as a new target for weight-reduction. As the endogenous ligand for MCR, melanocortin (melanophore stimulating hormone, MSH) can activate MCR and produce various biological effects, including pigmentation, anti-inflammation, steroidogenesis, energy homeostasis, feeding behavior and sexual behavior.In this study, membrane proteins were prepared from hypothalamus of SD rats, where there is expressed with high-density of MC3R/MC4R. [125I]-NDP-α-MSH was used as radioligand and competitive ligand binding assay was used to evaluate the binding affinity of 36 novelα-MSH analogues to MC3R/MC4R. 7 chemicals including LK-SPL-009, LK-SPL-024 were found to have high binding affinity for MC3R/MC4R. HEK293, which does not express MCR itself, was used to construct cell lines that stably expressed with MC4R. The cell line was proved to express MC4R stably by Western blot. LANCE? cAMP384 Kit was used to determine the effect of chemicals to stimulate the release of cAMP.α-MSH significantly stimulated the release of cAMP, EC50 of which was 2.65nM. Similar toα-MSH, LK-SPL-009 and LK-SPL-024 both stimulated the release of cAMP, EC50 of which were 4.03nM and 4.84nM, respectively. This result indicated that both LK-SPL-009 and LK-SPL-024 were the agonist of MC4R, and the activity for MC4R was similar toα-MSH.After the Kunming mice were raised for 6 weeks with high lipid food, the body weight increased significantly compared with the mice raised with normal food. After the mice were intracerebroventricularly injected withα-MSH for 14 continuous days, the body weight of the mice raised with high lipid food decreased for 24.4%, at the same time, the foodtake, the total plasma cholesterol, glucose, and triglyceride in the blood of the mice were all decreased compared with the mice treated with Normal Saline. Similar toα-MSH, LK-SPL-009 and LK-SPL-024 induced decrease not only in the body weight of the mice for 27.0% and 16.3%, but also the foodtake, the total plasma cholesterol, glucose and triglyceride in the blood as well. Furthermore,α-MSH and LK-SPL-024 induced increase in the leptin and insluin in the blood. The effects of both LK-SPL-009 and LK-SPL-024 were dose-dependent.In all, we have successfully set up a system for the screening of MCR agonists analogues. We have obtained 7 chemicals with relatively high binding affinity for MCR. LK-SPL-009 and LK-SPL-024 were proved to be the agonists for MC4R and could decrease the body weight of overweighted mice. The therapeutic effects of LK-SPL-009 and LK-SPL-024 on obesity deserve further study.