Rescue of Melanocortin 4 Receptor (MC4R) Nonsense Mutations by Aminoglycoside‐Mediated Read‐Through

Abstract

Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycosidemediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.

Therapeutics Based on Stop Codon Readthrough

“…Another study also questioned the ability of PTC124 to suppress PTCs because it was unable to mediate readthrough using multiple readthrough reporter constructs in cultured cells (85). Furthermore, PTC124 was unable to restore functional protein expressed from endogenous mutant transcripts in cell models associated with obesity (melanocortin 4 receptor) (14), peroxisome biogenesis disorders (30), and long-QT syndrome (53). …”

Nonsense-mediated decay in genetic disease: Friend or foe?

“…These compounds have been shown in both in vitro and in vivo models to alleviate disease pathogenesis by enhancing PTC read-through [119]. Examples include cystic fibrosis (CF) [120-131], Becker and Duchenne muscular dystrophy (BMD/DMD) [128, 129, 132-140], ataxia telangiectasia [139, 141, 142], Rett syndrome (RTT) [143-146], Usher syndrome type I (USH1) [147-149], Hurler syndrome (MPS1) [150-154], Maroteaux-Lamy syndrome (MPSVI) [155], carnitine palmitoyltransferase 1A (CPT1A) [156], hemophilia [157, 158], methylmalonic acidura (MMA) [159], neuronal ceroid lipofuscinosis (NCL) [114, 160, 161], spinal muscular atrophy (SMA) [162], peroxisome biogenesis disorder (PBD) [163, 164], obesity [165], poor drug metabolism [166], and cancer [151]. …”

Obesidad monogénica humana: papel del sistema leptina-melanocortina en la regulación de la ingesta de alimentos y el peso corporal en humanos

“…Actualmente no existe tratamiento alguno para estas anomalías. Si bien, estudios recientes muestran cómo la recuperación de expresión en la superficie celular de mutantes MC4R podría tener un beneficio terapéutico dado que las mutaciones MC4R causantes de obesidad, inducen a una retención intracelular de receptores mediante un sistema de control de calidad celular 31 . Por su parte, el receptor de melanocortina-3 (MC3R), será otro importante regulador de la homeostasis energética.…”