Ulrike G. Sahm

Synthesis and biological evaluation of α-MSH analogues substituted with alanine

Abstract The influence of single amino acid replacements by alanine on the binding affinity and biological activity of α-MSH in B16 murine melanoma cells has been studied systematically. α-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were determined using a radioreceptor assay. Biological activity of the analogues was determined by measuring tyrosinase stimulation. Relative activity and affinity data were generally in agreement with earlier results using terminal deletion fragments of α-MSH, but the alanine scan revealed important new insights into the role of individual residues. The three terminal amino acids at either end were not necessary for binding or activity, with amino acids 4–9 forming a core sequence required for receptor binding and triggering of the biological response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met 4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe 7 , Arg 8 , and Trp 9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.

The melanocortin (MC3) receptor from rat hypothalamus: Photoaffinity labelling and binding of alanine-substituted α-MSH analogues

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of α‐MSH, [125I‐Tyr2,Nle4,d‐Phe7,ATB‐Lys11]α‐MSH.SDS‐PAGE followed by autoradiography showed a single band at 53–56 kDA for the native receptor of 35 kDA after deglycosylated with PNGase F, consistent with the predicted cDNA. Receptor binding studies with α‐MSH, γ‐MSH and [Nle4,d‐Phe7]α‐MSH established that α‐MSH and γ‐MSH had similar affinities while [Nle4,d‐Phe7]α‐MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved ‘core sequence’ (‐Met‐Glu/Gly‐His‐Phe‐Arg‐Trp‐) of MSH/ACTH peptides. The binding affinities of alanine‐substituted analogues of α‐MSH were determined to investigate the role of individual residues in ligand—receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.

Influence of α-MSH terminal amino acids on binding affinity and biological activity in melanoma cells

The influence of the terminal amino acids of alpha-MSH on its biological action in B16 murine melanoma cells has been systematically studied. Fragments of alpha-MSH lacking various sequences of terminal residues were synthesized by solid-phase peptide synthesis and their binding affinity to melanoma cells was measured using a radioreceptor assay. Biological activity was determined by measuring both tyrosinase activity and melanogenesis. The relative affinities and activities of the fragments generally followed the same pattern as found previously in other assay systems (frog and lizard bioassay and Cloudman S91 mouse melanoma), with the three amino acids at each terminal not being essential for binding and biological activity, although the C-terminal amino acids 11-13 are more important than those in the N-terminus. The differences in biological activity between the fragments can be explained by their relative binding affinities for the receptor.

Binding and biological activity of C-terminally modified melanocortin peptides: a comparison between their actions at rodent MC1 and MC3 receptors.

Five subtypes of melanocortin receptors have to date been identified, but to date little is known about the different structural requirements for binding and biological activity at these receptors. In this study, the role of C-terminal melanocortin peptide residues in imparting selectivity for the receptor subtypes was examined. C-terminally modified analogues of alpha-MSH and gamma-MSH were synthesized and their interaction with MC1 and MC3 melanocortin receptors was investigated. This study provides further evidence for an important role of proline 12 (numbering with respect to alpha-MSH) for binding and activity at the MC1 receptor. Although the influence of C-terminal amino acids on binding and activity at MC3-R was less marked, some of them were nevertheless observed to be beneficial for the interaction with this receptor subtype.

Receptor Binding Affinities and Biological Activities of Linear and Cyclic Melanocortins in B16 Murine Melanoma Cells Expressing the Native MC1 Receptor

Cyclic α‐melanocyte‐stimulating hormone (α‐MSH) analogues produced by disulphide bridging (e.g. [Cys4,Cys10]α‐MSH) are known to be almost equipotent to the native hormone in amphibian skin bioassays and as a consequence have been proposed as a paradigm for the active conformation of native MSH at the pigment cell MC1 receptor. However this proposal has been somewhat speculative as there is no published data comparing biological activity of cyclic MSH analogues with data on receptor binding. This study addresses this problem by comparing tyrosinase stimulatory activity with their receptor binding affinity in B16 murine melanoma cells expressing the native MC1 melanocortin receptor.

Pharmacokinetics of oestrone-3-O-sulphamate.

The sulphatase pathway is thought to be the major route of oestrogen synthesis in breast tumours in postmenopausal women. There is currently considerable interest in developing a potent steroid sulphatase inhibitor to block oestrogen synthesis by this route. One of the most potent inhibitors discovered so far is oestrone-3-O-sulphamate (EMATE) which is active in vivo. In this study we report the preparation of a formulation for the administration of EMATE by the oral route. A method, using high-performance liquid chromatography (HPLC), was also established to measure concentrations of EMATE in rat plasma after its oral or i.v. administration. Using the oral formulation and HPLC assay, EMATE was readily detected in rat plasma after oral administration. Plasma EMATE concentrations were related to the dose of drug administered orally over the 10-40 mg/kg range. To examine the pharmacokinetics of EMATE, the compound (40 mg/kg, single dose) was administered either orally (in the formulation) or i.v. (in propylene glycol) with plasma samples being collected for up to 6 h. After oral administration, EMATE was rapidly absorbed, with the peak plasma concentration being detected at 30 min, after which plasma concentrations rapidly decreased. After i.v. administration a plasma EMATE concentration was detected at 1 h similar to that after oral administration. The clearance of EMATE from plasma followed a bi-phasic curve, showing an initial half-life of 30 min, followed by a slower half-life of 4 h 30 min. Little evidence was obtained for any metabolism of EMATE to oestrone. Rat liver sulphatase activity was almost completely inhibited (>99%) within 30 min of oral or i.v. administration of EMATE.

Synthesis of 153N-6 analogues and structure-function analysis at murine melanocortin-1 (MC1) receptors.

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.

Melanocortin probes for the melanoma MC1 receptor: synthesis, receptor binding and biological activity

The fate of α-melanocyte-stimulating hormone (α-MSH) subsequent to binding to the melanoma melanocortin MC1 receptor is of interest with regard to its potential use in targeting cytotoxic drugs or imaging to melanoma. Tools such as lodinated, photoaffinity-labelled, biotinylated and fluorescent melanocortins are required to study the fate of the ligand during its interaction with the receptor. In this study, a series of probes for the receptor based on the potent analogue, [Nle4, Dphe7] α-MSH, have been developed and tested for their potential usefulness. All probes contain the core melanocortin motif His-Phe-Arg-Trp. They bind the receptor readily and appear to have similar Intrinsic efficacies to the endogenous peptide, so that biological activity is regulated by their receptor binding affinity. Functional photoaffinity-labelled, biotinylated and fluorescent probes are described. The biotinylated probe binds the receptor when coupled to streptavidin, although with reduced affinity.