Pharmacological chaperone action in humanized mouse models of MC4R-linked obesity
Patricia René, Damien Lanfray, Denis Richard, Michel Bouvier
Patricia René, Damien Lanfray, Denis Richard, Michel Bouvier
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Resource and Technical Advance Metabolism Therapeutics

Pharmacological chaperone action in humanized mouse models of MC4R-linked obesity

Abstract

MC4R mutations represent the largest monogenic cause of obesity, resulting mainly from receptor misfolding and intracellular retention by the cellular quality control system. The present study aimed at determining whether pharmacological chaperones (PCs) that restore folding and plasma membrane trafficking by stabilizing near native protein conformation may represent valid therapeutic avenues for the treatment of melanocortin type 4 receptor–linked (MC4R-linked) obesity. To test the therapeutic PC potential, we engineered humanized MC4R (hMC4R) mouse models expressing either the WT human MC4R or a prevalent obesity-causing mutant (R165W). Administration of a PC able to rescue cell surface expression and functional activity of R165W-hMC4R in cells restored the anorexigenic response of the R165W-hMC4R obese mice to melanocortin agonist, providing a proof of principle for the therapeutic potential of MC4R-targeting PCs in vivo. Interestingly, the expression of the WT-hMC4R in mice revealed lower sensitivity of the human receptor to α–melanocyte-stimulating hormone (α-MSH) but not β-MSH or melanotan II, resulting in a lower penetrance obese phenotype in the WT-hMC4R versus R165W-hMC4R mice. In conclusion, we created 2 new obesity models, a hypomorphic highlighting species differences and an amorphic providing a preclinical model to test the therapeutic potential of PCs to treat MC4R-linked obesity.

Authors

Patricia René, Damien Lanfray, Denis Richard, Michel Bouvier

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Figure 6

Characterization of PC UM0130866 compound.

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Characterization of PC UM0130866 compound. (A) Evaluation of antagonist ...
(A) Evaluation of antagonist potency of compound UM0130866 on WT-hMC4R. HEK293T cells stably expressing the WT-hMC4R were preincubated for 1 hour with increasing concentrations of compound UM0130866, followed by the addition of 25 nM NDP-α-MSH, concentration corresponding to EC50, for 30 minutes. cAMP accumulation was then measured using a competitive immunoassay based on homogeneous time-resolved fluorescence (HTRF) technology (cAMP dynamic-2 kit from cis-bio). Data are expressed as percentage of inhibition compared with maximal cAMP response of WT-hMC4R–expressing cells upon 25 nM NDP-α-MSH exposure. Data are the means ± SEM of 5 independent experiments. (B) Evaluation of PC potency on R165W-hMC4R. HEK293T cells stably expressing the R165W-hMC4R were treated with increasing concentrations of UM0130866 for 15 hours. Cells were then washed and incubated 1 hour at 37°C with 3 μM of NDP-α-MSH and cAMP accumulation was measured. Data are expressed as percentage of activation compared with maximal cAMP response of WT-hMC4R–expressing cells upon a saturated concentration for NDP-α-MSH (3 μM) in basal condition. Data are the means ± SEM (n = 7). (C) Effect on trafficking to the cell surface of WT- and R165W-hMC4R. HEK293T cells transiently expressing WT- (triangle) or mutant R165W- (square) hMC4R, tagged at their C-terminal with RlucII were incubated in the absence (open symbols) or presence (filled symbols) of 10 μM UM0130866 over time. Cell surface expression level was then measured by BRET between the receptor-RlucII (donor of energy) and rGFP-CAAX (acceptor of energy anchored at the cell surface). Inset: schematic representation of the cell surface expression BRET assay. Data are the mean ± SD of 2 independent experiments.