Evaluation of Pharmacological Rescue of Melanocortin-4 Receptor Nonsense Mutations by Aminoglycoside
Friederike Höpfner,
Sarah Paisdzior,
Nanina Reininghaus,
Iqra Sohail,
Patrick Scheerer,
Paolo Annibale,
Heike Biebermann,
Peter Kühnen
Affiliations
Friederike Höpfner
Institute of Experimental Pediatric Endocrinology, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Charité—Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany
Sarah Paisdzior
Institute of Experimental Pediatric Endocrinology, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Charité—Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany
Nanina Reininghaus
Institute of Experimental Pediatric Endocrinology, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Charité—Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany
Iqra Sohail
Max Delbrück Center, Robert-Rössle-Straße 10, 13092 Berlin, Germany
Patrick Scheerer
Group Protein X-ray Crystallography and Signal Transduction, Institute of Medical Physics and Biophysics, Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Virchowweg 6, 10117 Berlin, Germany
Paolo Annibale
Max Delbrück Center, Robert-Rössle-Straße 10, 13092 Berlin, Germany
Heike Biebermann
Institute of Experimental Pediatric Endocrinology, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Charité—Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany
Peter Kühnen
Institute of Experimental Pediatric Endocrinology, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Charité—Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany
The melanocortin-4 receptor (MC4R) is critical for central satiety regulation, therefore presenting a potent target for pharmacological obesity treatment. Melanocortin-4 receptor mutations prevalently cause monogenetic obesity. A possibility of overcoming stop mutations is aminoglycoside-mediated translational readthrough. Promising results were achieved in COS-7 cells, but data for human cell systems are still missing, so uncertainty surrounds this potential treatment. In transfected HEK-293 cells, we tested whether translational readthrough by aminoglycoside Geneticin combined with high-affinity ligand setmelanotide, which is effective in proopiomelanocortin or leptin receptor deficiency patients, is a treatment option for affected patients. Five MC4R nonsense mutants (W16X, Y35X_D37V, E61X, W258X, Q307X) were investigated. Confocal microscopy and cell surface expression assays revealed the importance of the mutations’ position within the MC4R. N-terminal mutants were marginally expressed independent of Geneticin treatment, whereas mutants with nonsense mutations in transmembrane helix 6 or helix 8 showed wild-type-like expression. For functional analysis, Gs and Gq/11 signaling were measured. N-terminal mutants (W16X, Y35X_D37V) showed no cAMP formation after challenge with alpha-MSH or setmelanotide, irrespective of Geneticin treatment. Similarly, Gs activation was almost impossible in W258X and Q307X with wild-type-like cell surface expression. Results for Gq/11 signaling were comparable. Based on our data, this approach improbably represents a therapeutic option.
