Wayne L. Cody

α-Melanocyte stimulating hormone message and inhibitory sequences: Comparative structure-activity studies on melanocytes

We investigated the structure-activity relationships of alpha-MSH (alpha-melanocyte stimulating hormone) fragment derivatives of the generic formulae Ac-alpha-MSH(x-13)-NH2 and Ac-alpha-MSH(6-x)-NH2. The minimal C-terminal sequences required for melanotropic activity were 8-13 and 7-13, respectively, in the frog and lizard skin bioassays. The Arg8-Trp9 sequence appears to be a fundamental component of the minimal message sequences found to date such as alpha-MSH(6-9), alpha-MSH(8-13) and alpha-MSH(7-13). We discovered that Ac-alpha-MSH(7-10)-NH2 was a weak and selective alpha-MSH antagonist on the lizard skin bioassay. Analysis of alpha-MSH(7-10) analogues of the generic formula Ac-Xaa-Arg-Trp-Yaa-NH2 led to Ac-[D-Trp7,D-Phe10]alpha-MSH(7-10)-NH2, a moderately potent, specific and competitive inhibitor of alpha-MSH in both the frog and the lizard skin bioassays.

Chapter 30. Recent Progress in the Rational Design of Peptide Hormones and Neurotransmitters

Publisher Summary Peptide hormones generally produce their biological effects by interaction with membrane bound receptors, but we know little about the specific chemical and physical properties of these binding sites. Ultimately hormone-receptor interactions are three dimensional in nature, in which two complementary topological surfaces interact to produce a biological response. These three dimensional features often can be examined by conformational restriction of the peptide hormones. Virtually all peptide hormones have multiple receptors of physiological and pharmacological importance and there is overwhelming evidence that generally each of these receptors utilizes different structural and conformational properties of the hormone. In addition to being the first peptide hormone, whose structure was elucidated and then prepared by total synthesis, oxytocin (OT) was also the first peptide hormone, for which a conformation-biological activity model was proposed. Examination of the possible conformational features of arginine vasopressin (AVP) led researcher to propose a “biologically active conformation” for vasopressin at the kidney anti-diuretic receptor. Structure–activity relationships of leutienizing hormone releasing hormone (LHRH) have been examined by determining the effects of systematic modification of each residue on biological activity. Development of rational approaches to the design of peptide hormone analogs with useful biological and physical properties is still in its infancy. Yet, conformational and topological considerations already have provided new insights into the chemical–physical basis for the biological activity of these compounds.

Structural and conformational modifications of α-MSH/ACTH4-10 provide melanotropin analogues with highly potent behavioral activities

Previous studies have identified the (4-10) heptapeptide sequence as the central core of alpha-MSH/ACTH peptides required for mediation of important biological activities. In the present study, the structure-activity relationships of Nle4-substituted and Cys4,Cys10-bridged cyclic alpha-MSH analogues, which were previously shown to exhibit a wide range of melanotropic potencies from weak agonism to super potency, were examined for grooming behavioral activity in the rat following intracerebroventricular injections. The results showed that stepwise C-terminal elongation of the linear Nle4-substituted Ac-alpha-MSH4-10-NH2 increased grooming potencies of the peptides in a manner similar to their actions on melanocytes. The most interesting finding was the observation that cyclization of the inactive linear central (4-10) core of alpha-MSH (Ac-alpha-MSH4-10) to form Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 resulted in a super potent agonist in the grooming assay. However, while cyclization of the (4-10) heptapeptide produced potent agonists on grooming behavior, the structure-activity relationships were different than the frog skin bioassay. These findings support the hypothesis that appropriate structural and confirmational modifications of alpha-MSH-related peptides can produce profound effects on the bioactivities of the peptides, and suggest that different structural-conformational requirements exist for alpha-MSH interactions with its various receptors.

Potent and prolonged melanotropic activities of the α-MSH fragment analog, Ac-[Nle4, D-Phe7]-α-MSH4–9-NH2

Abstract Ac-[Nle 4 , D -Phe 7 ]-α-MSH 4–9 -NH 2 and Ac-[Nle 4 ]-α-MSH 4–9 -NH 2 , fragment analogs of the tridecapeptide, α-melanocyte stimulating hormone (α-MSH, α-melanotropin), were synthesized. The potency and prolonged activity of the analogs were compared to α-MSH in several melanotropin bioassays. The D -Phe-containing hexapeptide was 10 times more active than α-MSH in stimulating melanoma tyrosinase activity. This analog was also 10-fold more potent than α-MSH in the lizard skin bioassay and about 10-fold less active in the frog skin bioassay. The melanotropic activity of Ac-[Nle 4 , D -Phe 7 ]-α-MSH 4–9 -NH 2 was considerably prolonged compared to α-MSH in each of the bioassays. These results demonstrate that the structural requirements for superpotency and prolonged activity of [Nle 4 , D -Phe 7 ]-substituted analogs reside within this hexapeptide sequence.

Relative stability of α-melanotropin and related analogues to rat brain homogenates

Abstract α-Melanotropin (α-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle 4 , D -Phe 7 ]- α -MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle 4 , D-Phe 7 ]- α -MSH 4–10 -NH 2 , retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle 4 , D-Phe 7 ]- α -MSH 4–11 -NH 2 is totally resistant to inactivation by rat brain homogenate. Both [Nle 4 , D -Phe 7 ]-fragments are resistant to degradation by rat serum, but [Nle 4 ]-α-MSH, Ac-[Nle 4 ]-α-MSH 4–10 -NH 2 and Ac-[Nle 4 ]-α-MSH 4–11 -NH 2 are rapidly inactivated under both conditions. The cyclic melanotropin, [ Cys 4 ,Cys 10 ]-α-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[ Cys 4 ,Cys 10 ]-α-MSH 4–10 -NH 2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[ Cys 4 ,D- Phe 7 ,Cys 10 ]-α-MSH 4–10 -NH 2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.

Cyclic Melanotropins. VI. Reverse Phase HPLC Studies

Abstract The chromatographic behavior of 17 cyclic melanocyte stimulating hormone (α-MSH, α-melanotropin) analogs were studied on two reversed phase columns (Altech and Vydac) using several mobile phases. It was observed that analogs which contain a D-amino acid were always eluted earlier than the corresponding L-amino acid-containing analogues. Substitution of penicillamine for cysteine in the 4 position led to a more lipophilic compound as expected, but when penicillamine was substituted in the 10 position, apparently a less lipophilic compound resulted. These observations can be interpreted as a result of a conformational change in the molecule caused by the particular substitution. Furthermore, decreasing the size of the intramolecular disulfide ring led to a decrease in lipophilicity (i.e. retention time). The carba modification of the disulfide bridge had a similar effect as previously seen in oxytocin and vasopressin, that is, a decrease in retention time.

Reversed-phase high-performance liquid chromatography studies of α-MSH fragments

Abstract α-Melanotropin (α-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Syr-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH 2 ), that is primarily known for its ability to stimulate melanosome dispersion within integumental melanocytes (F. J. H. Tilders, D. F. Swaab and T. B. van Wimersma Greidanus (Editors), Frontiers of Hormone Research , Vol. 4, Karger, Basel, 1977; J. Ramachandran, S. W. Farmer, S. Liles and C. H. Li, Biochem. Biophys. Acta , 428 (1976) 347). In our efforts to understand the relationship of structure and conformation to the biological activities of α-MSH, we have prepared a series of diastereoisomeric analogues based on the highly potent analogue Ac-[Nle 4 , d -Phe 7 ]-αMSH 4–11 -NH 2 (T. K. Sawyer, V. J. Hruby, B. C. Wilkes, M. T. Draelos, M. E. Hadley and J. Bergsneider, J. Med. Chem. , 25 (1982) 1022). These analogues differed only in the amino acid substituted in the seven position, which was thought to be a critical residue for the biological activity of α-MSH. The chromatographic behavior of these analogues was examined on a C 18 Vydac (16-μm) reversed-phase column with five different mobile phases. The selectivity (α) for the analogues was compared in 0.10% trifluoroacetic acid (TFA), 0.10% heptafluorobutyric acid (HFBA) and 0.25 M triethylammonium phosphate (TEAP) using either acetonitrile or methanol as the organic modifier. With only one exception all analogues substituted with a d -amino acid in the seven position were eluted prior to their l -amino acid counterparts. As expected due to the greater ionic strength, the TEAP buffer allowed the greatest selectivity for the separation of these-α-MSH analogues, but it was surprising to find that the TFA buffer had a greater influence on selectivity that the HFBA buffer with either organic modifier. The probable mechanism of retention for the α-MSH analogues in perfluoroalkanoic buffers was also investigated. In addition, the relatioship between the retention time and the hydrophobicity of the seven position substitution was examined. Although the data were somewhat limited, a lack of correlation between the hydrophobicity of the seven position residue and retention time was observed.