Pharmacological therapies for monogenic obesity caused by MC4R dysfunction

Abstract

PhDMutations in the melanocortin-4 receptor (MC4R) are the most common cause of monogenic obesity. The majority of MC4R mutations are predicted to cause the receptor to aberrantly fold. Misfolded MC4R fails to traffic to the plasma membrane (PM) and is retained in the endoplasmic reticulum (ER). Recent studies with other G-protein coupled receptors have shown that stabilisation of misfolded receptor, by pharmacological chaperones, promotes trafficking to the cell surface where the receptor may be functional. The objective of this thesis was to develop a rapid throughput cell culture based assay to monitor MC4R trafficking to the PM and to screen chemical chaperones and inducers and inhibitors of endogenous molecular chaperones, for the ability to promote folding and cell surface expression of mutant MC4R. The work presented here confirmed that clinically occurring MC4R mutants S58C, N62S, P78L, D90N, L106P, C271Y and P299H are intracellularly retained in HEK 293 cells. The cell culture assay was used to screen a number of compounds, which have been previously reported to act as chemical chaperones by stabilising protein folding. Treatment with 4-phenyl butyric acid (4-PBA) and trehalose increased total cellular levels of wild-type and mutant MC4R. The benzoquinone ansamycin, geldanamycin, has been identified as a potent inhibitor of Hsp90 activity and an inducer of the heat shock response. Geldanamycin treatment altered the cell surface expression of wild-type and mutant MC4R. Furthermore, over expression of Hsp90 co-chaperone Aha1, also effected MC4R processing. Over-expression of Hsp70 has been shown to promote the trafficking of other aberrantly folded proteins. Over-expression of Hsc70 increased trafficking levels of wild-type and mutant MC4R and promoted mutant MC4R functional expression. In conclusion this data suggests using compounds that stabilise protein folding and/or targeting endogenous molecular chaperone machineries may have efficacy for altering cell surface expression of mutant MC4R