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Synthesis of 153N-6 analogues and structure-function analysis at murine melanocortin-1 (MC1) receptors
U G Sahm1, G W Olivier, C W Pouton
1Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, UK.
Abstract:
153N-6 (H-[Met5,Pro6,D-Phe7,D-Trp9,Phe10]-MSH(5-13)) has emerged as the most potent antagonist of alpha-MSH activity on Xenopus laevis melanophores, from a library of 32 360 peptides based on alpha-MSH(5-13) [22]. A recent report has confirmed our observation that 153N-6 also binds to mammalian melanocortin receptors. Here we report the receptor-binding affinities and biologic activities of 153N-6 and 17 selected alpha-MSH analogues at the native MCI receptor expressed by murine B16 melanoma cells. Our intention is to determine the structural requirements for agonism and competitive antagonism of melanocortin activity at the MC1-R and to discover more potent antagonists. 153N-6 was able to inhibit the action of native alpha-MSH and the potent synthetic agonist, [Nle4,D-Phe7]alpha-MSH, at the murine MC1-R. However, the Ki of 153N-6 was 439 times higher than that of alpha-MSH and 4475 times higher than that of [Nle4,D-Phe7]alpha-MSH; too high to allow 153N-6 to be considered as a practical antagonist for use in vivo (Ki of 153N-6 = 9.0 X 10(-6) M). Because Met4 is an important component of alpha-MSH binding at the MC1-R, we investigated alpha-MSH(1-13) and alpha-MSH(4-13) analogues to produce compounds with higher MC1-R-binding affinity than 153N-6. The binding affinity of 153N-6 was not significantly different from alpha-MSH(5-13), but it was 232 times lower than alpha-MSH(4-13). Coupling of H-Nle (as an isosteric replacement for Met) or acetyl-Nle to the N-terminus of 153N-6 raised the binding affinity by a factor of 46, but this and all full-length alpha-MSH analogues with Met or Nle in position 4 were full agonists of the MC1-R. A full-length alpha-MSH(1-13) derivative of 153N-6 with Ala4 did not exhibit significantly greater binding affinity than 153N-6 and appeared to be a partial agonist at the MC1-R in the cAMP assay. These data suggest that Met4 is an important determinant of the intrinsic efficacy of melanocortins as well as their binding affinity at the MCI-R. Pro6 and Phe10 (with respect to alpha-MSH) were found to be the most influential substitutions that determined the antagonist activity of 153N-6.
Insights
The peptide 153N-6 shows weak antagonist activity at the melanocortin 1 receptor (MC1-R). Structural modifications, particularly at position 4, are crucial for enhancing binding affinity and determining agonist or antagonist properties.
Area of Science:
- Biochemistry
- Pharmacology
- Molecular Biology
Background:
- Alpha-melanocyte-stimulating hormone (α-MSH) regulates melanophores and has roles in mammalian systems.
- 153N-6 is a potent α-MSH antagonist in Xenopus laevis but its activity at mammalian melanocortin receptors requires further investigation.
- The melanocortin 1 receptor (MC1-R) is a key target for understanding melanocortin activity.
Purpose of the Study:
- To evaluate the receptor-binding affinities and biological activities of 153N-6 and related α-MSH analogues at the murine MC1-R.
- To elucidate the structural requirements for agonism and antagonism at the MC1-R.
- To identify more potent MC1-R antagonists.
Main Methods:
- Radioligand binding assays to determine receptor affinities (Ki values).
- Cell-based assays (e.g., cAMP assays) to assess biological activity (agonism/antagonism).
- Systematic modification of α-MSH analogues, focusing on positions 4, 6, and 10.
Main Results:
- 153N-6 inhibited α-MSH and [Nle4,D-Phe7]α-MSH at the MC1-R, but with high Ki values (9.0 X 10(-6) M), indicating low affinity.
- 153N-6 binding affinity was significantly lower than α-MSH(4-13) but comparable to α-MSH(5-13).
- Modifications at the N-terminus (H-Nle or acetyl-Nle) increased binding affinity but resulted in full agonists. Methionine at position 4 (Met4) is critical for both binding affinity and intrinsic efficacy.
Conclusions:
- 153N-6 is not a practical in vivo antagonist for MC1-R due to its low binding affinity.
- Methionine at position 4 is a key determinant of melanocortin binding affinity and efficacy at the MC1-R.
- Specific substitutions, such as Pro6 and Phe10, significantly influence antagonist activity, providing insights for designing novel MC1-R modulators.