discovery of an α-melanotropin antagonist effective in vivo

Pergamon

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Peptides, Vol. 15, No. 4, pp. 627-632, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved

0196-9781/94 $6.00 + .00

Discovery of an c -Melanotropin Antagonist Effective In Vivo

A N A M A R I A DE L. C A S T R U C C I , *l W A D E C. S H E R B R O O K E , t T O M I K. SAWYER,z~ 2 D O U G L A S J. STAPLES,:~ M A R I A C A R O L I N A B. T U M A * A N D M A C E. H A D L E Y §

*Departamento de Fisiologia, Universidade de Sdo Paulo, S~o Paulo, Brasil, fSouthwestern Research Station, American Museum of Natural History, Portal, AZ, ¢The Upjohn Company, Kalamazoo, MI,

and §Department of Anatomy, University of Arizona, Tucson, AZ

Received 5 May 1993

CASTRUCCI, A. M. L., W. C. SHERBROOKE, T. K. SAWYER, D. J. STAPLES, M. C. B. TUMA AND M. E. HADLEY. Discovery of an e~-rnelanotropin antagonist effective in vivo. PEPTIDES 15(4) 627-632, 1994.--A hybrid analogue, H-His-D-Arg- Ala-Trp-D-Phe-Lys-NH2, was designed based upon the primary structures of a growth hormone-releasing peptide analogue, [His ~,Lysf]GHRP, and the MSH fragment, Ac-c~-MSH(6-1 I)-NHz. In vitro studies demonstrated the a-MSH antagonistic efficacy of the analogue in the lizards Sceloporus jarrovii and Urosaurus ornatus. In live white background-adapted S. jarrovii previously injected with the antagonist (10 nmol/5 g b.wt.), maximal skin darkening induced by a-MSH was reduced to 50%. In white background-adapted U. ornatus, previous injection of the analogue (1 nmol/5 g b.wt.) totally abolished the response to a-MSH and depressed to 50% the maximal response elicited by the superpotent MSH analogue, [NIe4,D-Phe7]a-MSH.

a-Melanocyte-stimulating hormone Lizard melanocytes

Growth hormone-releasing factor MSH receptor Antagonistic activity

c~-MELANOCYTE-STIMULATING hormone (c~-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly- Lys-Pro-Val-NH2, synthesized and released by the pars inter- media of the vertebrate pituitary. The hormone is produced after several cleavages of pro-opiomelanocortin (POMC), which also produces other peptides such as ACTH, endorphins, /3-MSH, and 3'-MSH (34). Melanocyte-stimulating hormone stimulates skin pigmentation (darkening) in most vertebrates. Recently, it has been reported that c~-MSH, as well as other POMC products, is also locally produced by Langerhans cells, keratinocytes, and melanocytes in mammalian skin (26-28,32). In the skin, it may subserve autocrine and paracrine functions such as regulation of cell proliferation and melanogenesis, c~-Melanocyte-stimulating hormone also plays a role in extrapigmentary functions: memory, attention, and learning enhancement (4,34); antipyretic activity (30,33); nerve sprouting and regeneration (17,35,36); and immune system regulation (5,14).

The design and synthesis of MSH analogues have provided extensive knowledge of melanocyte and central nervous system melanotropic receptors, of peptide/receptor interaction and requirements, and of signaling transduction mechanisms (9,10,25,37,38). The minimal agonistic message sequence on integumental melanocytes of frog (Rana pipiens) and lizard (Anolis carolinensis) has been shown to be His6,Phe7,ArgS,Trp 9

(9,25). Nonbiodegradable, superpotent melanotropin analogues with residual agonistic activity, such as [Nlea,D-PheV]a-MSH and related fragment analogues, have proven useful for comparative structure-activity studies of melanotropin receptors both in vitro and in vivo (1,6-8,11,20,23,24,31,39). These analogues have become potent tools for medical and therapeutic purposes (13,15,29). On the other hand, behavioral studies have demonstrated the efficacy of a diverse, stereochemical modification (D-Lys 8) of the message sequence in 4-10 analogues. The mod- ified fragments exhibited enhanced agonism in central nervous system receptors (22,37).

Recently, design strategies have been advanced to identify peptides exhibiting antagonistic activity at the MSH receptor (3,12,40,41). For example, relative to the primary structures of a growth hormone-releasing peptide (GHRP) analogue I (Fig. 1) and of MSH(6-1 1) analogue II (Fig. 1), a series of hybrid derivatives (III-V, Fig. 1) have been reported to exhibit potent in vitro competitive antagonistic activity in the frog, Rana pipiens, and the lizard, Anolis carolinensis, melanocytes (12,40,41).

We report on the in vivo and in vitro a -MSH antagonistic activity of H-His,D-Arg,Ala,Trp,D-Phe,Lys-NH2 (III, Fig. 1) in two lizards, Sceloporus jarrovii and Urosaurus ornatus.

Requests for reprints should be addressed to Ana Maria de L. Castrucci, Departamento de Fisiologia, Inst. Bioci~ncias, Universidade de Sao Paulo, C.P. 11176, S~o Paulo, 05422-900, Brasil.

2 Current address: Parke-Davis Pharmaceutical Research Division/Warner-Lambert Co., Ann Arbor, MI 48106.

627

628 CASTRUCCI ET AL.

I His 6 - Phe-Arg-Trp - Gly-Lys 11 II H-His-_D-Trp-AIa-Trp-_D-Phe-Lys-NH 2 III H-His-_D-Arg-AIa-Trp-_D-Phe-Lys-NH 2 IV H-His-_D-Trp-Arg-Trp-_D-Phe-Lys-NH 2 V H-His - Phe-Arg-Trp-_D-Phe-Lys-NH 2

FIG. 1. Primary structures of MSH(6-11) (I), a GHRP analogue (II), and three hybrid MSH-GHRP analogues (Ill-V). Amino acids in bold indicate residues different from the 6-11 sequence of a-MSH.

METHOD

Peptide Synthesis

The hybrid analogue H-His-D-Arg-Ala-Trp-D-Phe-Lys-NH: was prepared by solid-phase peptide synthesis, purified by sem- ipreparative, reversed-phase high performance liquid chroma- tography, and characterized by fast atom bombardment-mass spectrometry and amino acid analysis, as previously described (40).

Bioassays

The lizards Sceloporus jarrovii and Urosaurus ornatus were collected in the Chiricahua Mountains, Arizona. After decapi- tation, the dorsal skins were excised and prepared for in vitro bioassay as described elsewhere (9). In the absence of stimuli the melanosomes (melanin granules) aggregate in the perinuclear region within melanocytes, leading to lightening of the skins (basal level). The addition of a-MSH, or other melanotropic agonists, elicits dose-dependent melanosome migration into the dendritic processes of the pigment cell, resulting in skin darkening. Changes in skin color were monitored with a Photovolt reflectometer and calculated as percent change from the initial basal readings. Dose-response curves to a-MSH were determined in the absence and in the presence of the antagonist, after a preincubation of 1 h. Equipotent doses of a-MSH eliciting half- maximal darkening responses (ECso) were graphically determined for each experimental condition. The ECsos from dose- response curves determined under the various experimental

procedures were compared by the Student's t-test and considered as significantly different when p < 0.05. The pA2 value (that is, the negative logarithm of the molar concentration of the antagonist that requires twice the concentration of a-MSH to elicit equivalent darkening responses) was determined through the analysis of dose-response curves to a-MSH in the presence of at least three different concentrations of the antagonist (42).

In vivo bioassays were performed as already reported for the lizard Anolis carolinensis (6). Briefly, after weighing, lizards were allowed to adapt to white background (skin lightening) for 24 h. The animals were then injected IP (0.1 ml/5 g b.wt.) with either saline or the antagonist (1 or 10 nmol/5 g b.wt.) immediately prior to equivalent volume injections of a-MSH (0.4 or 1 nmol/5 g b.wt.) or [Nle4,D-PheT]a-MSH (0.1 nmol). Reflec- tance measurements were taken with the Photovolt reflectometer immediately before the injections (basal reading), after 30 min, and every hour from then on until the darkened animals (saline plus melanotropin-injected controls) had lightened back to the initial reflectance values.

RESULTS

a-Melanocyte-stimulating hormone induced a dose-dependent darkening of Sceloporus jarrovii and Urosaurus ornatus skins in vitro (Figs. 2 and 3). The ECsos, not significantly different, were 4 × l0 -]° M a n d 2 × 10 -t° M, respectively.

The dose-response curve to a-MSH in S. jarrovii skins was significantly shifted about fivefold to the right, and the maximal response was depressed in the presence of H-His-D-Arg-Aia-Trp- D-Phe-Lys-NH2 at l0 -5 M (Fig. 2). Because maximal responses to a-MSH were not achieved in the presence of the antagonist at concentrations higher than 10 -6 M, neither ECsos nor the pA2 could be determined in S. jarrovii skin bioassay.

The analogue at 10 -5 M displaced to the right about 20-fold the dose-response curve to a-MSH in U. ornatus skins (ECso = 4 × l 0 -9 M, Fig. 3). Two other dose-response curves to a-MSH were determined in U. ornatus skins in the presence of lower concentrations of the antagonist. The ECsos were fivefold (ECso = 10 -9 M) and twofold (ECso = 4 × 10 -]° M) higher in the presence of the blocker at l0 6 M and l0 -7 M, respectively

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t he an tagon i s t 10 -6M t t ~ a r t t agc t~s t 10 -5M

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T

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FIG. 2. Dose-response curves to a-MSH in the absence and in the presence of 10 -6 and 10 -5 M H-His-D-Arg-Ala-Trp-D-Phe-Lys-NH2, as determined in the in vitro Sce- loporusjarrovii skin bioassay. Each point is the mean _+ SE darkening response of the skins (n = 5) to the hormone at the concentrations noted.

MELANOTROPIN ANTAGONISTIC ACTIVITY 629

60

U :40

i; 'el0

~:to

- - • - - ~ - | - - A - - l U ~ 4 A - | - - • - - A L I q l A - N I H - - O - - I L I H I A - i l N I

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11

9

to 9 8 7

- l o g [concen t ra t ion ] (M)

FIG. 3. Dose-response curves to a-MSH in the absence and in the presence of 10 -7, 10 -6, and l0 -5 M H-His-o-Arg-Ala-Trp-D-Phe-Lys-NH2, as determined in the in vitro Urosaurus ornatus skin bioassay. Each point is the mean _+ SE darkening response of the skins (n = 6-12) to the hormone at the concentrations noted.

(Fig. 3). The PA2 for H-His-D-Arg-AIa-Trp-D-Phe-Lys-NH2 was determined to be 7.0 in U. ornatus melanocytes.

After 24 h of white background adaptation, the injection of 0.4 nmol a-MSH/5 g b.wt. induced maximal in vivo skin darkening in S. jarrovii (Fig. 4). White background-adapted S. jarrovii previously injected with 10 nmol antagonist/5 g b.wt. exhibited half-maximal in vivo darkening, compared to the controls, in response to the same amount of injected c~-MSH (Fig. 4).

In white background-adapted U. ornatus 1 nmol a-MSH/5 g b.wt. was necessary to produce maximal in vivo response (Fig. 5), which was totally prevented in U. ornatus previously injected with 1 nmol antagonist/5 g b.wt. (Fig. 5).

Injections of 10 pmol/5 g b.wt. of the superpotent c~-MSH analogue, [NIe4,D-PheT]c~-MSH, elicited maximal and prolonged

in vivo skin darkening in U. ornatus (Fig. 6). If the animals previously received 1 nmol antagonist/5 g b.wt., a half-maximal response was achieved with the [Nle4,D-Phe 7] analogue, and its residual (prolonged) activity was abolished (Fig. 6).

DISCUSSION

Extensive studies during the last 15 years on the structure- activity of a-MSH have led to a better understanding of the receptor requirements for signal transduction on pigment cells and neurons (23,24,37). Much less has been achieved, however, on the design of competitive antagonists to a-MSH (16). A series of MSH inhibitors has long been known, such as melatonin (21), melittin (18), and calcium-chelating agents (19). But these

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a f te r t h e a n t a g o n i s t 1 0 n m o l / S g b .w.

T ~ O ~ T.

I I I I I I 1 1

SO 60 90 ltO 180 180 210 240

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T IME ( rain. )

FIG. 4. In vivo demonstration of partial blockade of maximal darkening response to 0.4 nmol of a-MSH/5 g b.wt. induced by 10 nmol of H-His-D-Arg-Ala-Trp-D-Phe-Lys-NH2/5 g b.wt., in Sceloporusjarrovii (n = 6).


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l I i I I I I i I I I I I L I

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FIG. 5. In vivo demonstration of total blockade of maximal darkening response to 1.0 nmol of a-MSH/5 g b.wt. induced by 1.0 nmol of H-His-D-Arg-Ala-Trp-D-Phe-Lys-NHj 5 g b.wt., in Urosaurus ornatus (n = 6).

agents most certainly do not act on the melanotropin receptor, and likely interfere with transductional and/or posttransduc- tional events.

More recently, based on conformational modeling and various design strategies, several MSH-related peptides were shown to selectively and competitively inhibit a-MSH melanosome dispersing actions on frog (Rana pipiens) and lizard (Anolis carolinensis) melanocytes (3,40,41). One of these peptides, H-His- D-Arg-Ala-Trp-D-Phe-Lys-NH2, was chosen for additional testing in vitro and in vivo in two other lizard species, Sceloporusjarrovii and Urosaurus ornatus. The darkening potencies of a-MSH on in vitro skins of both species were nearly identical. On the other hand, when the dose-response curve to the hormone was determined in the presence of the MSH antagonist at 10 -5 M, its inhibitory potency was greater in U. ornatus than in S. jarrovii.

Therefore, U. ornatus skins in vitro were used to determine the antagonist PA2 to be 7.0, demonstrating that the antagonist was markedly more effective in U. ornatus than in Anolis carolinensis (pA2 = 5.0) (41). These in vitro results demonstrate that despite interspecies variation in potency, the MSH antagonist exhibits competitive inhibition of a-MSH on a variety of melanocyte receptors studied to date.

In the in vivo assays, the difference between the potencies of a-MSH needed to elicit maximal darkening of S. jarrovii (0.4 nmol/5 g b.wt.) and U. ornatus (1 nmol/5 g b.wt.) was not sta- tistically significant. Again, the antagonist was more potent (more than tenfold) in U. ornatus than in S. jarrovii. A striking difference in both in vitro and in vivo antagonism potency among different lizard species contrasts with the similarity among lizard species in the agonistic potency of c~-MSH.

- 0 - (NI44,D-Piw7 ]MphI-MSH 1 0 1OmOI/Sg b . w .

6O

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a f t e r t h e a n t a g o n i s t 1 n m o l / S g b . w .

TIME ( rain. )

FIG. 6. In vivo demonstration of partial blockade of maximal darkening response to 10 pmol of [Nle 4, D-Phe 7] a-MSH/5 g b.wt. induced by 1.0 nmol of H-His-D-Arg-Ala-Trp-o- Phe-Lys-NH2/5 g b.wt., in Urosaurus ornatus (n = 7-8).

MELANOTROPIN ANTAGONISTIC ACTIVITY 631

[NIe4,D-Phe7]a-MSH is a very potent analogue o fa -MSH on various melanocyte systems, including the lizard Anolis carol# nensis, and it exhibits residual in vitro and in vivo activity (20,23,24). The analogue is typically 10-100 times more potent than a-MSH, and this is also true for U. ornatus. The in vivo prolonged effects may result, to some extent, from its resistance to serum enzymes (2,7,8,11), as related to the replacement of L- Phe 7 by D-Phe 7 in the active site, and subsequent prevention of cleavage by endopeptidases. Furthermore, the [Nle4,D-Phe 7] analogue is more lipophilic than the native hormone, and may be able to interact more effectively with lipid components of biological membranes. This may result in further availability of the peptide, even after removal of the hormone by circulation (in vivo) or by rinsings (in vitro), and additionally account for its superpotency and residual effects. Nevertheless, despite the su- peragonist-like characteristics of [NIe4,D-Phe7]a-MSH, its maximal darkening response was half blocked, and the residual effect was abolished after previous injection of the MSH antagonist. This result demonstrates that, although at a higher (100 times) concentration, the MSH antagonist III is capable of competing with [Nle4,D-Phe7]a-MSH.

To our knowledge, this is the first report of in vivo antagonism of a-MSH using a designed MSH analogue. Its potent in vivo inhibition of a-MSH-induced darkening (equimolar concentrations of hormone and antagonist), and of [Nle4-D-Phe 7] analogue effects in U. ornatus, suggests that the two D-amino acid modifications of III may have provided it with moderate binding affinity to the melanotropin receptor as well as potential resistance to serum enzyme degradation.

The in vivo antagonistic activity of H-His-D-Arg-Ala-Trp-D- Phe-Lys-NH2 affords a variety of future research opportunities. Compared to fairly nonspecific MSH antagonists (e.g., melittin), which act on signal transduction pathways common to several peptide hormones, the competitive inhibitory nature of MSH antagonist III provides for MSH receptor selectivity therapeutic applications.

ACKNOWLEDGEMENTS

This work was partially supported by FAPESP (89/3780-6 and 92/ 0707-9) and CNPq (500443/90-4), Brasil. A.M.L.C. is thankful to the American Museum of Natural History for the facilities provided during her stays at the Southwestern Research Station, in Portal, AZ.

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discovery of an α-melanotropin antagonist effective in vivo

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