Abstract:To determine the specificity and internalization kinetics of non-peptide small molecule ML00253764 rescuing the obesity-causing MC4R mutants，the cell model for the stable expression of melanocortin receptors and their mutants were established. The results by flow cytometry（FACS）showed that ML00253764 resulted in the increase of the membrane surface expression of MC4R deficient mutation receptor N62S and P78L by 43% and 34%，respectively，while almost no effects to other melanocortin receptor and their mutants，demonstrating this rescue mediated by ML00253764 was MC4R receptor- and mutant- specific. Further study showed that the internalization of rescued receptors was similar to the wild type（WT）receptor，and there was no significant difference in rate of internalization t1/2. Both endogenous ligands α-MSH and AgRP had no rescue effect on the membrane surface expression of MC4R deficient mutation receptor，proving that action way of ML00253764 was different from that of above two，and what mutant receptors were rescued resulted from the specificity of pharmacological chaperone with cell membrane permeability.