Melanin concentrating hormone (MCH): Synthesis and bioactivity studies of MCH fragment analogues

Peptides, Vol. 10, pp. 349-354. ©Pergamon Press plc, 1989. Printed in the U.S.A.

0196-9781/89 $3.00 + .00

Melanin Concentrating Hormone (MCH): Synthesis and Bioactivity Studies of MCH Fragment Analogues T E R R Y O. M A T S U N A G A , * A N A M A R I A D E L A U R O C A S T R U C C I , t M A C E. H A D L E Y $ A N D V I C T O R J. H R U B Y *t

Departments of *Chemistry and .~Anatomy, University of Arizona, Tucson, AZ 85721 and the "l'Departamento de Fisiologia, Instituto de Bioci~ncias, Universidade de Sro Paulo CPl1176, Sro Paulo, 05499, Brazil R e c e i v e d 8 A u g u s t 1988

MATSUNAGA, T. O., A. M. L. CASTRUCCI, M. E. HADLEY AND V. J. HRUBY. Melanin concentrating hormone (MCH): Synthesis and bioactivity studies of MCH fragment analogues. PEPTIDES 10(2) 349-354, 1989.--Nineteen analogues of melanin concentrating hormone (MCH) were synthesized and tested for their skin-lightening activities in the in vitro eel skin (Synbranchus marmoratus) bioassay. All the analogues synthesized were fragments of the native sequence: Asp-Thr-Met-Arg-C~s-Met-Val-GlyArg-Val-Tyr-Arg-Pro-C~,s-Trp-Glu-Val with sequential elimination of substituents from both the carboxy- and amino-termini. All the analogues that contained tryptophan in position 15 were found to be full agonists and equipotent to MCH. In the absence of Trp tS, full agonist activity was maintained but potency was reduced ten-fold or more. The minimal fragment analogue possessing equipotency to the parent peptide, MCH, was the MCH(5-15) sequence. These observations coupled with results from work reported previously by our laboratories suggest the importance of the Trp ~5 residue for interaction with the MCH receptor in this assay system. Melanin concentrating hormone (MCH) Fish skin bioassay

Analogue synthesis

MELANIN concentrating hormone (MCH) is a heptadecapeptide that stimulates the perinuclear (centripetal) aggregation of melanin granules (melanosomes) within teleost melanocytes, resulting in a blanching of the skins. The existence of this hormone was first proposed by Enami (7) and first isolated in crude form from fish pituitary gland extracts by Imai (14). The peptide was then characterized in pure form from chum salmon extracts by Kawauchi et al. (16). It is a cyclic basic peptide with the following sequence: Asp-Thr-Met-Arg-C~,s-Met-Val-Gly-Arg-Val-Tyr-ArgPro-C'-~s-Trp-Glu-Val. Baker and Rance found that the highest concentrations of the hormone appeared to be localized in the neurointermediate lobe of the pituitary gland of teleost fish, although it was found in the hypothalamus as well (1). Wilkes and co-workers accomplished the total synthesis of the peptide and demonstrated that in addition to its MCH activity, it could also elicit MSH-like activity in frog and lizard skins, exhibiting about 1//6oo the potency of native ct-MSH (21,22). This MSH-like activity of MCH has subsequently been confirmed by other investigators (6,13). Utilizing the fish (eel) skin bioassay (4), the synthetic hormone has been shown to be maximally active at 10- ~o M with a minimal effective activity of 10 -~2 M (4). The fragment sequence, MCH(5-17), was shown to be equipotent with MCH while the

Analogue fragments

Synbranchus marmoratus

MCH(1-14) fragment was about IAo as potent as the native hormone (10). The MCH(5-14) fragment in which amino acid residues were removed from both amino- and carboxy-termini had only V3oothe potency of MCH although the peptide remained a full agonist. In addition, Lebl and co-workers (18) have synthesized nine MCH analogues with contracted, disulfide-linked rings. In all cases, the analogues were either totally devoid of MCH-like activity or were more than 3,000 times less active than MCH in the fish skin assay. Clearly, fragments of MCH still yield analogues with potency equal to the native hormone, although the cyclic peptide fragment ring, MCH(5-14), must be present in order to elicit agonist activity (17). This case is not unusual as other fragment analogues of native peptide hormones (i.e., ct-MSH, CCK, and somatostatin) have also been shown to maintain full equipotent activity (8, 11, 20). In our current work, we set out to sequentially synthesize and test MCH(N-17) ( N = 1, 2, 3, or 4), MCH(N-16) ( N = 1-5), MCH(N-15) (N = 1-5), and MCH(N-14) (N = 1-5) (see Table 1 for structures of the fragment analogues synthesized), to determine the minimal message fragment of the native sequence necessary for a full agonist activity. The determination of a minimal sequence with maximal activity is necessary for an understanding

~Requests for reprints should be addressed to Victor J. Hruby.

349

350

MATSUNAGA E T AL.

TABLE

1

P R I M A R Y STRUCTURES O F S O M E O F THE M C H A N A L O G U E S S Y N T H E S I Z E D

1 MCH

3

5

7

9

11

13

15

17

A s p -Thr - M e t - Arg- C~s - M e t - V a l -Gly-Arg-Val -Tyr -Arg -Pro-C~,s -Trp- Glu -Val

M C H (2-17)

Thr- M e t - Arg- C~,s - M e t - V a l -Gly-Arg -Val -Tyr -Arg -Pro-C~,s -Trp- Glu -Val

M C H (3-17)

M e t - Arg- C~s - M e t - V a l -Gly-Arg-Val -Tyr-Arg -Pro-C~s -Trp- Glu -Val

M C H (4-17)

Arg- C~,s - M e t - V a l -Gly-Arg-Val -Tyr-Arg -Pro-Cys -Trp- Glu -Val

M C H (5-17)

C~,s - M e t - V a l -Gly-Arg-Val -Tyr-Arg -Pro-C~s -Trp- Glu -Val

M C H (1-16)

A

M C H (1-15)

-Pro-Cys -Trp A s p -Thr- M e t - Arg- C~,s - M e t - V a l -Gly-Arg -Val -Tyr-Arg -Pro-C~,s C~,s - M e t - V a l -Gly-Arg-Val -Tyr-Arg -Pro-C~,s

M C H (1-14) M C H (5-14)

I ' sp -Thr - Met - Arg- Cys - M e t - V a l -Gly-Arg -Val -Tyr -Arg -Pro-Cys -Trp- Glu

A s p -Thr - Met - A r g - C~,s -Met-Val -Gly-Arg-Val -Tyr-Arg

of structure-biological activity relationships. METHOD

Nineteen analogues of MCH were synthesized (Table 1). All syntheses were performed on either a Vega Model 250 or Model 1000 peptide synthesizer, or on a hybrid semiautomated synthesizer designed in our lab. Synthesis of the MCH analogues was accomplished by a modification of the procedure of Wilkes and co-workers (21). Chloromethylated resin (Merrifield) was used as the support to introduce the first amino acid (valine, glutamic acid, tryptophan, or cysteine). Resin substitution varied from 0.3 mmole/g-0.7 mmole/g. The first amino acid was prepared by generating the cesium salt of the amino acid and attaching it to the resin with triethylamine and dimethylformamide according to the method of Gisin (9). Suitably protected Boc-amino acids were obtained either commercially (Bachem) or synthesized in our laboratories by published procedures. The coupling procedure was as follows: swell the resin with dichloromethane (DCM) (3 x 2 min); wash with ethanol (3 x 2 min); wash with DCM (3 × 2 min); deprotect with 45% TFA/2% anisole in DCM (1 × 2 min, 1 x 20 min); wash with DCM (3 × 2 rain); neutralize with 10% diisopropylethylamine in DCM (3 × 2 min); wash with DCM (3 x 2 min); couple with 3 equivalents of the appropriately protected Bocamino acid, 3 equivalents of hydroxybenzotriazole and 3 equivalents of dicyclohexylcarbodiimide (DCC); wash with DCM (3 x 2 min). Coupling reactions were monitored for completion by analyzing the resin with ninhydrin according to the method of Kaiser et al. (15). The completed resin was then dried overnight in vacuo and then treated with HF (10 ml/g), anisole (1.0 ml/g) and ethanedithiol (0.3 ml/g). The resin was stirred for one hr at 0°C. HF was removed in vacuo and the resin washed with 3 × 60 ml of ethyl acetate. The resin was suction filtered and the peptide extracted with 2 × 30 ml of 30% acetic acid followed by 2 × 30 ml of glacial acetic acid. The product was isolated by lyophilization and dissolved in 1.5 liters of degassed, argon-purged, deionized water. The pH was adjusted to 8.4 with aqueous ammonia. Cyclization was achieved through the dropwise addition of K3FeCN6 (0.01 M) until a yellow color persisted for at least 30 min or more. The solution was allowed to stir an additional 2 hr. The pH was adjusted to 4.6 with glacial acetic acid and IRA-45 anion exchange resin (HC1 form, Sigma) was added to remove unreacted ferricy-

anide and ferrocyanide ions. The resin was then removed from solution by suction filtration. The solution was concentrated in vacuo and lyophilized to yield a cream precipitate. The product was then dissolved in a minimal volume of 30% acetic acid and loaded onto a Sephadex G-10 exclusion column (2.5 × 100 cm) for desalting and initial purification. The product was monitored at 280 nm. The appropriate fractions were collected and lyophilized to yield a cream powder. This was dissolved in distilled deionized H20 and loaded onto a 2.5 x 40 cm carboxymethylcellulose cation exchange column (Bio-Rad). The column was eluted with a linear gradient of 250 ml of 0.01 M (NHn)OAc in the mixing chamber and 250 ml of 0.7 M (NH4)OAc in the reservoir chamber. The fractions were collected in 20 ml aliquots and monitored at 280 nm. The appropriate fractions were then collected and lyophilized. The product was then dissolved in 0.1% TFA and purified on a Vydac TP-1010 semiprep reverse phase column (10 mm × 25 cm) and a Spectra-Physics 8800 HPLC system equipped with an ABI Analytical Spectroflow 757 absorbance detector and an SP4270 integrator. All precautions were taken to insure that all products isolated were not exposed in any way (glassware, columns, resins, HPLC columns) to ot-MSH or related analogues previously synthesized in this laboratory by using acid cleaned glassware and columns, and virgin resins and HPLC columns. The column was equilibrated with a 93% TFA buffer (0.1%): 7% acetonitrile mixture followed by a linear gradient to 43% acetonitrile enrichment over 30 min. The desired product was found to be the major eluting peak and always comprised about 60% to 70% of the entire fraction loaded onto the column. Lyophilization of the appropriate fractions yielded a fluffy white powder. Analytical Data

Amino acid analysis was performed at the Biotechnology Center, Department of Biochemistry of the University of Arizona. Samples were hydrolyzed with 300 txl of 3 N mercaptoethanesulfonic acid or 4 N methanesulfonic acid and reacted at 110°C for 22 hr. Samples were then diluted to a final concentration of 50 pmoles/50 txl with citrate buffer and analyzed quantitatively for amino acid composition on a Beckman 7300 amino acid analyzer. Table 2 lists the observed amino acid compositions of the synthesized peptides. Thin-layer chromatography was run on silica gel matrix (Analtech) 10 x 2.5 cm plates with glass backings. Peptides were

MCH FRAGMENT ANALOGUES

351

TABLE 2 AMINO ACID ANALYSIS OF MCH AND FRAGMENT ANALOGUES* Peptide

Gly

Glu

Thr

Val

Met

Tyr

Arg

Asp

Prot

Cyst

Trp

MCH MCH(2-17) MCH(3-17) MCH(4-17) MCH(1-16) MCH(2-16) MCH(3-16) MCH(4-16) MCH(5-16) MCH(I-15) MCH(2-15) MCH(3-15) MCH(4-15) MCH(5-15) MCH(1-14) MCH(2-14) MCH(3-14) MCH(4-14) MCH(5-14)

1.00 1.00 1.00 1.00 0.92 1.00 1.00 1.00 1.11 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

1.03 1.02 1.04 1.01 1.10 1.02 1.02 1.02 1.00 -----------

0.94 1.01 --1.00 1.00 ---0.95 0.94 ---0.99 0.85 ----

3.00 2.76 2.83 2.87 1.86 1.72 1.81 1.92 1.64 1.98 2.07 2.04 2.02 1.99 1.98 2.13 2.15 2.13 2.02

1.91 1.89 1.95 0.95 1.80 1.88 1.87 0.96 0.96 1.82 1.75 1.86 1.01 1.04 1.75 1.70 1.80 0.87 0.85

0.86 0.76 0.77 0.82 1.08 0.86 0.89 0.95 0.84 0.88 0.90 0.94 0.98 1.13 0.93 0.90 0.92 0.90 0.83

2.77 2.74 2.84 2.73 3.07 2.74 2.76 2.73 2.20 2.71 2.70 2.76 3.10 2.33 2.92 2.82 2.95 2.88 1.79

0.99 ---0,97 ----1.05 ----1.06 -----

1.07 1.11 1.09 1.05 1.08 1.10 0.95 1.01 0.90 1.13 1.01 1.12 0.88 1.03 1.09 0.99 1.13 1.03 1.07

1.70 1.64 1.70 1.64 1.81 1.83 1.85 2.02 1.74 1.79 1.87 1.77 1.89 1.91 2.00 1.78 1.84 1.83 1.84

0.93 0.97 0.86 0.82 1.01 0.90 0.94 1.02 0.96 1.00 0.99 0.92 0.97 1.01 ------

*Amino acid analysis in mercaptoethanesulfonic acid, uncorrected for decomposition, 22 hr at II0°C. tProline and half cystine compositions were determined by analysis in methanesulfonic acid and normalized to glycine. All amino acid analyses are uncorrected for decomposition.

developed in four different solvent systems: A) n-butanol:acetic acid:water:pyridine (15:3:8:10 v/v); B) n-butanol:acetic acid:water (4:1:1 v/v); C) pyridine:acetic acid:water (50:30:15 v/v); D) ethyl acetate:pyridine:acetic acid:water (5:5:1:3 v/v). Detection was by Ninhydrin spray. Observed R e values are listed in Table 3. Mass spectral analyses were performed at the Midwest Center for Mass Spectrometry in Lincoln, NE. The mode was fast atom bombardment. Samples were suspended in a matrix o f dithiothreitol, dithioerythreitol, and glycerol. The calculated and observed results are listed in Table 4.

Bioassays M C H activity o f M C H and its analogues was assessed using the in vitro fish skin bioassay detailed elsewhere (4). The teleost, Synbranchus marmoratus, an eel, was obtained from the Pantanal o f Brazil. Animals were sacrificed and the skins removed and bathed in a physiological bicarbonate solution (teleost Ringer) c o m p o s e d o f the following: NaC1, 128 raM; KCI, 2.7 raM; CaC12, 1.8 mM; N a H C O 3, 2.5 mM; glucose, 5.6 mM; pH = 7.2 (adjusted by bubbling with CO2). Skins were stretched between two P V C rings and allowed to equilibrate in R i n g e r ' s solution for one hour. A baseline light reflectance value from the skins was recorded on a Photovolt reflectometer (Model 670). A graph o f percent skin-lightening vs. -log analogue concentration was then used to quantitate the biological response. In teleost Ringer solution, melanosomes are dispersed out into the dendritic processes o f the melanophores. After 1 hr exposure o f the skins to M C H or its analogues, perinuclear aggregation o f the melanosomes occurs leading to a lightening o f the skins. The responses o f the skins to a k n o w n concentraton o f a peptide were measured by the increase in reflectance from the skins. This method is based upon previously e m p l o y e d techniques for the frog and lizard skin bioassays (3, 12,

19). The potency data o f Table 5 were analyzed by Student's t-test

TABLE 3 Rf VALUES OF MCH FRAGMENT ANALOGUES FOR DIFFERENT CHROMATOGRAPHY SOLVENTS*

MCH(1-17) MCH(2-17) MCH(3-17) MCH(4-17) MCH(1-16) MCH(2-16) MCH(3-16) MCH(4-16) MCH(5-16) MCH(1-15) MCH(2-15) MCH(3-15) MCH(4-15) MCH(5-15) MCH(1-14) MCH(2-14) MCH(3-14) MCH(4-14) MCH(5-14)

A

B

C

D

0.59 0.62 0.65 0.65 0.58 0.63 0.64 0.70 0.64 0.65 0.71 0.68 0.58 0.64 0.53 0.62 0.59 0.58 0.64

0.09 0.20 0.23 0.15 0.09 0.19 0.23 0.39 0.26 0.37 0.44 0.42 0.30 0.40 0.11 0.10 0.12 0.16 0.27

0.85 0.92 0.92 0.85 0.91 0.83 0.94 0.98 0.94 0.78 0.93 0.71 0.77 0.86 0.62 0.69 0.62 0.62 0.74

0.85 0.86 0.85 0.85 0.80 0.86 0.86 0.80 0.80 0.87 0.84 0.82 0.73 0.84 0.74 0.88 0.84 0.81 0.82

*The solvent conditions were comprised of the following: A) nbutanol:acetic acid:water:pyddine (15:3:8:10, v/v), B) n-butanol:acetic acid:water (4:1:1, v/v), C) pyridine:acetic acid:water, (50:30:15, v/v), D) ethyl acetate:pyridine:acetic acid:water (5:5:1:3, v/v).

M A T S U N A G A ET AL.

352

TABLE 4

A

MASS SPECTROMETRIC DATA--FAST ATOM BOMBARDMENT

Sample MCH(1-17) MCH(2-17) MCH(3-17) MCH(4-17) MCH(I-16) MCH(2-16) MCH(3-16) MCH(4-16) MCH(5-16) MCH( 1-151 MCH(2-15) MCH(3-15) MCH(4-15) MCH(5-15) MCH(I-14) MCH(2-14) MCH(3-14) MCH(4-14) MCH(5-14)

Molecular Weight Calculated 2097.9 1982.9 1881.8 1750.8 1998.8 1883.8 1782.8 1651.7 1495.6 1869.8 1754.7 1653.7 1522.6 1366.5 1683.7 1568.7 1467.6 1336.6 1180.5

80

Molecular Weight Observed 2098.0 1983.0 1883.0 1752.0 2000.0 1885.0 1784.0 1653.0 1497.0 1870.8 1755.8 1654.8 1524.0 1368.0 1685.0 1569.0 1468.0 1337.0 1181.0

(M/z) (M/z) MH ÷ MH ÷ MH + MH ÷ MH ÷ MH + MH + MH + MH ÷ MH ÷ MH + MH + MH + (M/z) (M/z) (M/z) (M/z)

•

MCH

////

/

o Me.,_,. 60 I

* •

/ .'~"

MCH1-16 MCH i_r4

] ~ / T/

~

0 u~

0.0

~

2o

12

111

r

i 9

I0

I

B

80

6O

~~_0 4o u

~

2o

(/)<0.05) and by analysis of variance (ANOVA) at the 95% confidence limit. i

12

RESULTS

As determined previously, MCH is effective in stimulating a

RELATIVE POTENCIES OF MCH FRAGMENT ANALOGUES AS DETERMINED BY THE IN VITRO FISH (SYNBRANCHUSMARMORATUS) SKIN BIOASSAY

MCH MCH(2-17) MCH(3-17) MCH(4~ 17) MCH(5-17) MCH( 1-16) MCH(2-16) MCH(3-16) MCH(4-16) MCH(5-16) MCH(1-15) MCH(2-15) MCH(3-15) MCH(4-15) MCH(5-15) MCH(I-14) MCH(2-14) MCH(3-14) MCH(4-14) MCH(5-14)

I

I0

I

9

I

8

-Log[Concentrotion] (M 1

FIG. 1. Dose-response curves to MCH and some analogues as determined by the in vitro fish skin bioassay (n = 161.

TABLE 5

Peptide

lil

Potency Relative to MCH* 1.0 1.0 1.0 1.0 1.0 (10) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.1 [0.07 (10)] 0.07 0.05 0.023 0.014 [0.003 (I0)]

*The potency data were analyzed by Student's t-test (p<0.05) and by analysis of variance (ANOVA) at the 95% confidence limit.

significant melanosome aggregation within integumental melanocytes of the teleost fish, Synbranchus marmoratus, at a concentration as low as 10-J2 M. We had previously determined that MCH(5-17) was equipotent to MCH (101. Not surprising, therefore, was the observation that sequential deletions of the Nterminal sequence yielding the peptides MCH(2-17), M C H ( 3 171, and MCH(4-17) did not significantly lower the potencies of the analogues relative to that of MCH (Table 5). We have previously shown that deletion of the C-terminal tripeptide resulted in an analogue, MCH(1-14), which had about V~o the potency of MCH. This observation suggested that one or more amino acids of the tripeptide sequence, Trp-Glu-Val, were essential for full potency of the peptide. Removal of the terminal amino acid, Va117, or the penultimate amino acid, Glu 16, did not diminish the activity or the potency of the resulting analogues. Removal of Trp at position 15, however, resulted in at least a 10-fold decrease in activity (Fig. 11. These results demonstrate the importance of Trp j5 for full MCH-like potency. Figure 1 shows a plot of percent skin-lightening vs. -log analogue concentration. It appears that all analogues tested displayed full agonist activity. As can be seen, all of the analogues containing Trp j5 were equipotent when compared to MCH(1-17). Thus, removal of Val and Glu from the C-terminus did not affect the potency of the analogue regardless of the presence or absence of residues 1, 2, 3, or 4 on the N-terminus. In turn, the presence or absence of residues 1-4 did not affect potency as long as the MCH(5-15) sequence was intact. It will be noted that the

MCH FRAGMENT ANALOGUES

353

TABLE 6 SEQUENCE COMPARISON OF SOME SYNTHETIC MCH ANALOGUES 5 MCH(5-14)

7

11

13

15

17

C y s - M e t - V a l - G l y - A r g - V a l - Tyr-Arg-Pro-C~s

MCH(10-17) [Ala 5, CysS] M C H ( 5 - 1 7 )

9

C);s- Tyr-Arg-Pro-C~s-Trp-Glu-Val Ala-Met-Val-C~s-Arg-Val-Tyr-Arg-Pro-C~s-Trp-Glu-Val

MCH(5-14) exhibited I/7o the potency of MCH in comparison to the 1/3oopotency reported previously (5,10). The sequential addition of Arg 4, Met 3, Thr 2, and Asp I to the amino terminus of MCH(5-14) resulted in a progressive increase in potency. This increase amounted to a change from VTothe potency for MCH(514) to VJo the potency for MCH(1-14). Amino acid residue 4 (Arg) is a potentiating factor since MCH(4--14) was about 2-fold more potent than MCH(5-14). Clearly the residues on both the aminoand carboxy-termini independently increase potency. CONCLUSIONAND DISCUSSION We have synthesized all the possible MCH sequence fragments that contain the intact disulfide ring structure and have found that the MCH(5-15) fragment appears to be the minimal sequence that is equipotent to MCH when tested in the in vitro fish skin bioassay. These results, combined with those reported previously from our laboratory (10), point to the importance of the Trp 15 position for maintenance of full (equipotent) agonistic activity in MCH. Upon elimination of the Trp ~5 moiety, potency was always decreased 10-fold or more depending upon the amino acid substitution (or lack thereof) bonded to the Cys 5 N-terminus. These results are consistent with the previous suggestion by Castrucci and co-workers that the MCH potency is predominantly a function of the C-terminus of the hormone (5). These results suggest that the indole ring of tryptophan may be important in aiding the fit of MCH into its receptor pocket, thus facilitating binding. As well, the presence of the Trp moiety may induce favorable conformational changes in the ring, aiding fit. Since MCH(5-14) and MCH(1-14) are both full agonists, it appears that Trp ~5 is not an absolute requirement for full message transduction but, rather, is facilitory to binding with the membrane receptor. Clearly, we can say that the first four amino acids of the N-terminus are not necessary for the full activation of the fish melanocyte receptor nor are the C-terminal Glu 16 and Va117 residues when Trp 15 is present. Interestingly, however, in the absence of Trp 15 we have observed a progressive increase in potency upon sequential addition of residues 1-4 of the Nterminus (Table 5). This observation might suggest that these amino-termini residues may have, at one time, been contributory to binding potency but have now become evolutionarily vestigial in nature. Based on this study and the previous studies from our laboratory (10), we conclude that MCH(5-15) is the minimal sequence needed to elicit an equipotent response to the native hormone in the skin-lightening eel skin assay. From previous reports of either significant reduction of potency or complete elimination of activity upon contraction of the MCH ring, it appears that the ten amino

acid disulfide-linked ring is an absolute requirement for maintenance of equipotent activity (18). Previous reports that Cys 5,Cys 14 acetamidomethyl protected MCH has full agonist activity but at only 0.03% (~oo) the potency of MCH suggests that the ring is important for maintenance of full biological potency. It is not clear, however, whether the bulky acetamidomethyl group, or the presence of the acyclic sequence may be responsible for impaired binding despite the presence of the exocyclic amino and carboxy amino acids that appear to be necessary for binding (2). Regardless, cyclization via the CysS-Cys ~4 sulfurs may allow exposure of the topographical features of the 6-13 sequence necessary for signal transduction. Our current results have therefore led us to focus on the residues 6-13 with regards to signal transduction. Although modifications have been few, the synthesis of ring contracted analogues by Lebl et al. have provided some interesting results (18). Of particular interest was the fact that the [ A l a s , C ~ s ~ 4 ] M C H ( 5 - 1 7 ) and the [C3~sJ°]MCH(10-17) analogues induced complete skin lightening (i.e., were full agonists), albeit at drastically reduced potency (3000- and 20,000-fold less potent than native MCH). Table 6 lists the two ring contracted analogues as well as the MCH(5-14) sequence. As can be seen, the Tyr ~I-Argl2-Pro]3-Cysl4 part of the sequence is conserved in all three analogues. As we have clearly demonstrated in this work, residues 1-4 of the amino terminus and 15-17 of the carboxy terminus are only responsible for maintaining potency in vitro. In their absence, the remaining fragment analogue, MCH(5-14), becomes less potent (1/70 the potency of MCH) but nevertheless, remains a full agonist. This would seem to suggest that the residues in positions 11-14 may in some way be critical for message transduction. Since the Cys 14 moiety is oxidized to form part of the disulfide, we suspect that its purpose is to provide the correct conformation for the molecule. This leaves the TyrArg-Pro sequence which comprises residues 11-13 which may be critical for message transduction. To date, all analogues with MCH-like activity have this part of the sequence conserved. This is consistent with work by Kawazoe and co-workers, who, upon modifications of either Tyr j~ or Arg j2 in MCH, found drastic reductions in potency (17) relative to MCH. ACKNOWLEDGEMENTS This work was supported by U.S. Public Health Service (AM 17420, V.J.H.) and the National Science Foundation (DCB-86-15603, M.E.H.), and from FAPESP 87/0851-4 and CNPq407196/87, Brazil (A.M.C.). The technical assistance of Mr. Michael Shea and the clerical assistance of Ms. Danielle DuBois are gratefully acknowledged. The authors also wish to acknowledge the Midwest Center for Mass Spectrometry, a National Science Foundation Regional Instrumentation Facility (Grant No. CEH 8211164).

354

M A T S U N A G A E T AL.

REFERENCES 1. Baker, B. I.; Rance, T. A. Further observations on the distribution and properties of teleost melanin concentrating hormone. Gen. Comp. Endocrinol. 50:423-431; 1983. 2. Baker, B. I.; Eberle, A. N.; Bauman, J. B.; Siegrist, W.; Girard, J. Effect of melanin concentrating hormone on pigment and adrenal cells in vitro. Peptides 6:1125-1130; 1985. 3. Bjorklund, A.; Meurling, P.; Nilsson, G.; Nobin, A. Standardization and evaluation of a sensitive and convenient assay for melanocyte stimulating hormone using Anolis skin in vitro. J. Endocrinol. 53:161-169; 1972. 4. Castrucci, A. M. L.; Hadley, M. E.; Hruby, V. J. A teleost skin bioassay for melanotropic peptides. Gen. Comp. Endocrinol. 66: 374-380; 1987. 5. Castrucci, A. M. L.; Hadley, M. E.; Wilkes, B. C.; Zechel, C.; Hruby, V. J. Melanin concentrating hormone exhibits both MSH and MCH activities on individual melanophores. Life Sci. 40:1845-1851; 1987. 6. Eberle, A. N.; Atherton, E.; Dryland, A.; Shepherd, R. C. Peptide synthesis, part 9. Solid-phase synthesis of melanin concentrating hormone using a continuous flow polyamide method. J. Chem. Soc. Perkin Trans. 1:361-367; 1986. 7. Enami, M. Melanophore-concentrating hormone (MCH) of possible hypothalamic origin in the catfish, Parasilurus. Science 121:36-37; 1955. 8. Gaudreau, P.; Morell, J. L.; St. Pierre, S.; Quirion, R.; Pert, C. B. Cholecystokinin octapeptide fragments: Synthesis and structure-activity relationship. In: Hruby, V. J.; Rich, D. H., eds. Peptides: Structure and function. Illinois: Pierce Chemical Co.; 1983:441-444. 9. Gisin, B. F. The preparation of Merrifield-resins through total esterification with cesium salts. Helv. Chim. Acta 56:1476-1482; 1973. 10. Hadley, M. E.; Zechel, C.; Wilkes, B. C.; Castrucci, A. M. L.; Visconti, M. A.; Pozo-Alonso, M.; Hruby, V. J. Differential structural requirements for the MSH and MCH activities of melanin concentrating hormone. Life Sci. 40:1139-1145; 1987. 11. Hruby, V. J.; Wilkes, B. C.; Cody, W. L.; Sawyer, T. K.; Hadley, M. E. Melanotropins: Structural, conformational, and biological considerations in the development of superpotent and superprolonged

12. 13. 14. 15. 16. 17. 18.

19. 20.

21.

22.

analogs. In: Hearn, M. T. W., ed. Peptide protein reviews, vol. 3. New York: Marcel Dekker, Inc.; 1984:1-64. Huntington, T.; Hadley, M. E. Evidence against mass action direct feedback control of melanophore-stimulating hormone (MSH) release. Endocrinology 95:472-479; 1974. Ide, H.; Kawazoe, I.; Kawauchi, H. Fish melanin concentrating hormone disperses melanin in amphibian melanophores. Gen. Comp. Endocrinol. 58:486--490; 1985. Imai, K. Extraction of melanophore concentrating hormone (MCH) from the pituitary of fishes. Endocrinol. Jpn. 5:34--48; 1958. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34:595-598; 1970. Kawauchi, H.; Kawazoe, M.; Tsubokawa, M.; Kishida, M.; Baker, B. I. Characterization of melanin concentrating hormone in chum salmon pituitaries. Nature 305:321-323; 1983. Kawazoe, 14 Kawauchi, H.; Hirano, T.; Naito, N. Structure-activity relationships of melanin-concentrating hormone. Int. J. Pept. Prot. Res. 29:714-721; 1987. Lebl, M.; Hruby, V. J.; Castrucci, A. M. L.; Visconti, M. A.; Hadley, M. E. Melanin concentrating hormone analogues: contraction of the cyclic structure. 1. Agonist activity. J. Med. Chem. 31: 949-953; 1988. Shizume, K.; Lerner, A. B.; Fitzpatrick, T. B. In vitro bioassay for the melanocyte stimulating hormone. Endocrinology 54:553-560; 1954. Veber, D. Conformational considerations in the design of somatostatin analogs showing increased metabolic stability. In: Gross, E.; Meienhofer, J., eds. Peptides: Structure and biological function. Illinois: Pierce Chemical Co.; 1979:409-419. Wilkes, B. C.; Hruby, V. J.; Sherbrooke, W. C.; Castrucci, A. M. L.; Hadley, M. E. Synthesis and biological actions of melanin concentrating hormone. Biochem. Biophys. Res. Commun. 122:613-619; 1984. Wilkes, B. C.; Hruby, V. J.; Castrucci, A. M. L.; Sherbrooke, W. C.; Hadley, M. E. Synthesis of a cyclic melanotropic peptide exhibiting both melanin-concentrating and -dispersing activities. Science 224: 1111-1113; 1984.