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Ipsen 5i is a Novel Potent Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants.

Tao YX, Huang H - Front Endocrinol (Lausanne) (2014)

Bottom Line: Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling.Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S).It also did not affect trafficking of a misrouted MC3R mutant (I335S).

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University , Auburn, AL , USA.

ABSTRACT
Inactivating mutations of the melanocortin-4 receptor (MC4R) cause early-onset severe obesity in humans. Comprehensive functional studies show that most of the inactivating mutants of the MC4R are retained intracellularly. In the present study, we investigated whether a small molecule inverse agonist of the MC4R, Ipsen 5i, could act as a pharmacoperone and correct the cell surface expression and function of intracellularly retained mutant MC4Rs using multiple cell lines, including HEK293 and two neuronal cell lines. We showed that Ipsen 5i rescued the cell surface expression of all 11 intracellularly retained mutant MC4Rs studied herein in at least one cell line. Ipsen 5i functionally rescued seven mutants in all cell lines used. One mutant (Y157S) was functionally rescued in HEK293 cells but not in the two neuronal cell lines. Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling. Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S). It also did not affect trafficking of a misrouted MC3R mutant (I335S). Cell impermeable peptide ligands of the MC4R or cell permeable small molecule ligand of δ opioid receptor could not rescue misrouted mutant MC4R. In summary, we demonstrated that Ipsen 5i was a novel potent pharmacoperone of the MC4R, correcting trafficking and signaling of a significant portion (73%) of intracellularly retained mutants. Additional studies are needed to demonstrate its in vivo efficacy.

No MeSH data available.


Related in: MedlinePlus

Ipsen 5i specifically rescued mutant MC4Rs and the rescue action occurred intracellularly. (A) Structures of the ligands studied. The peptide ligands NDP-MSH and α-MSH are agonists of the MC4R whereas SHU9119 is an MC4R antagonist. Naltrexone is a small molecule antagonist of the opioid receptor. Ipsen 5i is a small molecule inverse agonist of the MC4R. (B) Neuro2a cells transiently expressing WT or C84R MC4R were treated with different ligands for 24 h (10−5 M NDP-MSH, α-MSH, SHU9119 or naltrexone or 10−6 M Ipsen 5i). Cells were washed twice and then stimulated with 10−6 M NDP-MSH for 1 h and intracellular cAMP concentrations were measured. (C) Neuro2a cells transiently expressing WT or I335S MC3Rs were treated with 10−6 M Ipsen 5i for 24 h. Then, the cell surface expression and intracellular cAMP production of MC3Rs were measured. Results are shown as mean ± SEM of at least three independent experiments. *Significantly different from the DMSO-treated control group, p < 0.05.
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Figure 8: Ipsen 5i specifically rescued mutant MC4Rs and the rescue action occurred intracellularly. (A) Structures of the ligands studied. The peptide ligands NDP-MSH and α-MSH are agonists of the MC4R whereas SHU9119 is an MC4R antagonist. Naltrexone is a small molecule antagonist of the opioid receptor. Ipsen 5i is a small molecule inverse agonist of the MC4R. (B) Neuro2a cells transiently expressing WT or C84R MC4R were treated with different ligands for 24 h (10−5 M NDP-MSH, α-MSH, SHU9119 or naltrexone or 10−6 M Ipsen 5i). Cells were washed twice and then stimulated with 10−6 M NDP-MSH for 1 h and intracellular cAMP concentrations were measured. (C) Neuro2a cells transiently expressing WT or I335S MC3Rs were treated with 10−6 M Ipsen 5i for 24 h. Then, the cell surface expression and intracellular cAMP production of MC3Rs were measured. Results are shown as mean ± SEM of at least three independent experiments. *Significantly different from the DMSO-treated control group, p < 0.05.

Mentions: To investigate whether cell impermeable peptide ligands of the MC4R or cell permeable ligands of other receptors could rescue mutant MC4Rs, we studied the effect of two MC4R peptide agonists (NDP-MSH and α-MSH), one MC4R peptide antagonist (SHU9119), and one pharmacoperone of δ opioid receptor (naltrexone) (35) (Figure 8A) on C84R MC4R. As shown in Figure 8B, NDP-MSH and α-MSH decreased the signaling of WT MC4R by approximately 50%. However, none of the four ligands rescued the signaling of C84R MC4R.


Ipsen 5i is a Novel Potent Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants.

Tao YX, Huang H - Front Endocrinol (Lausanne) (2014)

Ipsen 5i specifically rescued mutant MC4Rs and the rescue action occurred intracellularly. (A) Structures of the ligands studied. The peptide ligands NDP-MSH and α-MSH are agonists of the MC4R whereas SHU9119 is an MC4R antagonist. Naltrexone is a small molecule antagonist of the opioid receptor. Ipsen 5i is a small molecule inverse agonist of the MC4R. (B) Neuro2a cells transiently expressing WT or C84R MC4R were treated with different ligands for 24 h (10−5 M NDP-MSH, α-MSH, SHU9119 or naltrexone or 10−6 M Ipsen 5i). Cells were washed twice and then stimulated with 10−6 M NDP-MSH for 1 h and intracellular cAMP concentrations were measured. (C) Neuro2a cells transiently expressing WT or I335S MC3Rs were treated with 10−6 M Ipsen 5i for 24 h. Then, the cell surface expression and intracellular cAMP production of MC3Rs were measured. Results are shown as mean ± SEM of at least three independent experiments. *Significantly different from the DMSO-treated control group, p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 8: Ipsen 5i specifically rescued mutant MC4Rs and the rescue action occurred intracellularly. (A) Structures of the ligands studied. The peptide ligands NDP-MSH and α-MSH are agonists of the MC4R whereas SHU9119 is an MC4R antagonist. Naltrexone is a small molecule antagonist of the opioid receptor. Ipsen 5i is a small molecule inverse agonist of the MC4R. (B) Neuro2a cells transiently expressing WT or C84R MC4R were treated with different ligands for 24 h (10−5 M NDP-MSH, α-MSH, SHU9119 or naltrexone or 10−6 M Ipsen 5i). Cells were washed twice and then stimulated with 10−6 M NDP-MSH for 1 h and intracellular cAMP concentrations were measured. (C) Neuro2a cells transiently expressing WT or I335S MC3Rs were treated with 10−6 M Ipsen 5i for 24 h. Then, the cell surface expression and intracellular cAMP production of MC3Rs were measured. Results are shown as mean ± SEM of at least three independent experiments. *Significantly different from the DMSO-treated control group, p < 0.05.
Mentions: To investigate whether cell impermeable peptide ligands of the MC4R or cell permeable ligands of other receptors could rescue mutant MC4Rs, we studied the effect of two MC4R peptide agonists (NDP-MSH and α-MSH), one MC4R peptide antagonist (SHU9119), and one pharmacoperone of δ opioid receptor (naltrexone) (35) (Figure 8A) on C84R MC4R. As shown in Figure 8B, NDP-MSH and α-MSH decreased the signaling of WT MC4R by approximately 50%. However, none of the four ligands rescued the signaling of C84R MC4R.

Bottom Line: Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling.Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S).It also did not affect trafficking of a misrouted MC3R mutant (I335S).

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University , Auburn, AL , USA.

ABSTRACT
Inactivating mutations of the melanocortin-4 receptor (MC4R) cause early-onset severe obesity in humans. Comprehensive functional studies show that most of the inactivating mutants of the MC4R are retained intracellularly. In the present study, we investigated whether a small molecule inverse agonist of the MC4R, Ipsen 5i, could act as a pharmacoperone and correct the cell surface expression and function of intracellularly retained mutant MC4Rs using multiple cell lines, including HEK293 and two neuronal cell lines. We showed that Ipsen 5i rescued the cell surface expression of all 11 intracellularly retained mutant MC4Rs studied herein in at least one cell line. Ipsen 5i functionally rescued seven mutants in all cell lines used. One mutant (Y157S) was functionally rescued in HEK293 cells but not in the two neuronal cell lines. Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling. Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S). It also did not affect trafficking of a misrouted MC3R mutant (I335S). Cell impermeable peptide ligands of the MC4R or cell permeable small molecule ligand of δ opioid receptor could not rescue misrouted mutant MC4R. In summary, we demonstrated that Ipsen 5i was a novel potent pharmacoperone of the MC4R, correcting trafficking and signaling of a significant portion (73%) of intracellularly retained mutants. Additional studies are needed to demonstrate its in vivo efficacy.

No MeSH data available.


Related in: MedlinePlus