Int. J. Peptide Protein Res. 17,1981,239-242

@-ENDORPHIN: SYNTHESIS A N D RADIOLIGAND BINDING ACTIVITY OF ANALOGS CONTAINING CYSTINE BRIDGES

JAMES BLAKE, PASCUAL FERRARA and CHOH H A 0 LI

Hormone Research Laboratory, University of California, Son Francisco, CA, U.S.A.

Received 19 May, accepted for publication 5 June 1980

TWO anaIogs of human &endorphin containing cystine bridges between positions 21 and 26 or 14 and 26 have been synthesized and their radioligand binding activity using rat brain membranes has been determined. Both peptides showed three to four times the binding activity of human &endorphin,

Key words: human @endorphin; radioligand binding activity; solid-phase peptide synthesis.

A continuing investigation in this laboratory has been directed towards structure-activity relationships in 0-endorphin (Fig. 1; for a review see Li, 1977). Recently Blake et al. (1979) reported the synthesis of three analogs of 0-EP which incorporated cystine bridges between positions 7-26, 11-26, or 17-26. Al- though the peptide containing the C y ~ ~ - C y s ? ~ bridge showed diminished opiate activity by the guinea pig ileum assay, the other two peptides retained full activity. Immunoreactivity against rabbit antiserum to &-EP was virtually abolished in all the analogs. Subsequent to that report the peptides were assayed by the newly developed radioligand binding assay (Ferrara et al., 1979) using rat brain membranes with tritiated 0-EP (Houghten & Li, 1978) as the primary ligand. The results showed that the peptide which con- tained the C y ~ ' ~ - C y s ~ ~ bridge was signifi- cantly more active than 0-EP and the other two analogs. This led us to consider whether a slight Abbreviations: Symbols for amino acids and peptides in accordance with the IUPAC-IUB Commission on Biochemical Nomenclature. AU amino acids except glycine are of L configuration. Other abbreviations are: PEP, @endorphin; ph-EP, human @endorphin; Boc, tert.-butyloxycarbonyl

change in the position of the cysteine residues in the C y ~ ' ~ - C y s ~ ~ analog might further aug- ment the binding affinity of the analog to rat brain membranes. Thus, we report herein the synthesis and radioligand binding activity of [Cy~21-Cys2~, Phe2'. Gly3'] -&EP and [ C y ~ ~ ~ - C y s ~ ~ , Phe27, Gly3'] -&EP together with the radioligand binding activity of the three previously described analogs (Blake et al., 1979).

RESULTS AND DISCUSSION

Synthesis For synthetic convenience we decided to main- tain one cysteine residue at position 26 and to move the location of the other cysteine to either side of position 1.7. Substitution analogs have shown the importance of the Phe-18 and Lys-19 residues for high analgesic activity (Blake et al., 1980); also an omission analog suggests a contribution of the Asn-20 residue to biological activity (Li et al., 1980). Accord- ingly, we chose to substitute Ala-21 with cysteine. On the other side of position 17, it appears from omission analogs that the Leu-14 residue makes no vital contribution to biological

0367-8377/81/010239-04 $02.00/0 0 1981 Munksgaard, Copenhagen 239

J . BLAKE ET AL.

The peptide analogs [CysZ1 -CysZ6, Phe2',

Gly31] -&EP (11) were synthesized by the solid- phase method (Merrifield, 1963) as described

15 20 "y3' 1 -PqEP (I) and [cYs'4-cYs26 3 Gln-Thr-Pro-Le"-"al-Thr-Leu-Phe-Lys-Asn-

for the synthesis of 0-EP analogs with cystine bridges (Blake et al., 1979). Briefly, Boc-glycyl resin was alternatively subjected to deblocking in 55% trifluoroacetic acid/methylene chloride, neutralization with 5% diisopropylethylamine/ methylene chloride, and coupling with the pre- formed symmetrical anhydrides of the Boc amino acids (Hagenmeier & Frank, 1972). The peptide was cleaved from the resin and com- pletely deprotected by reaction with liquid HF. After mild oxidation with potassium ferricy- anide to close the cystine bridges, the highly purified peptide was obtained by chromato- graphy on carboxymethylcellulose and partition chromatography on Sephadex (3-50 (Yamashiro, 1964). Amino acid analyses of acid and enzymic hydrolysates of the two analogs are shown in Table 1.

TABLE 1 Amino acid composition of the synthetic peptides

Peptide I Peptide I1 Amino

acid Acida Enzymeb Acid Enzyme

ASP Thr Ser Glu Pro '/icy s G ~ Y Ala Val Met Ile Leu TY Phe LY s

2.1(2)C 2.9(3) 1.8(2) 2.0(2) 1.0(1) 1.7(2) 4.1(4)

1.0(1) 0.9(1) 1.1(2) 2.0(2) 1.0(1) 2.8(3) 5.0(5)

-

-

8.4d(8)

1.1(1) 0.9( 1) 0.9(2) 4.0(4)

1.2(1) 1.0(1) 1.7(2) 2.2(2) 1.0(1) 2.9(3) 5.0(5)

-

2.1(2) -

2.8(2) 7.4d(8) 1.8(2) -

2.0(2) 1.1(1) 1.1(1) 0.8(1) 1.9(2) 1.3(2) 4.0(4) 4.0(4) 1.0(1) 1.0(1) 1.0(1) 0.9(1) 1.0(1) 1.0(1) 1.2(2) 2.2(2) 1.0(1) l.O(l) l.O(l) 1.0(1) 3.0(3) 3.1(3) 5.1(5) 4.9(5)

aHydrolysis with constant boiling HCI; 22 h at 110'. bDigestion with trypsin/chymotrypsin followed by leucineaminopeptidase; see Blake et al. (1979). 'Numbers in parentheses are the expected values. dCorresponds to sum of Asn + Thr + Ser + Gln.

240

2 5 31 Ala-Ile-Ile-Lys-Asn-Ala-Tyr-LyS-Lys-Lys-Gly-Glu-OH

FIGURE 1 Amino acid sequence of human &endorphin.

Biological assay The peptides were assayed for radioligand binding activity using rat brain membrane pre- parations. The results for the two analogs described here and the previously reported (Blake ef al., 1979) cystine-containing peptides are summarized in Table 2. It is seen that, with the exception of the peptide containing the Cys7-CysZ6 bridge, all of the peptides have a radioligand activity equal to or greater than that of &-EP or the corresponding open chain analog, [Phe", Gly3' ] -&,-EP. Since a covalent linkage between distant neighbors in a peptide chain must restrict the conformational flexi- bility of the peptide, this data indicates that either the conformation of &-EP within the region of residues 11-26 is not critical for radioligand binding activity, or that fortuitously the allowed conformation(s) are compatible with that conformation necessary for efficient interaction with the opiate binding site.

Previous data have shown that the segments of the Ph-EP molecule which are most vital for radioligand binding are the amino terminal pentapeptide (enkephalin) and the carboxyl terminal four to six residues (Ferrara & Li, 1980). Bioassay of peptide analogs which are missing a single amino acid within the central region of the molecule indicates some sensi- tivity for radioligand binding to changes in this region, but clearly less than that to changes at the terminal regions (Li et al., 1980). These results are in agreement with our observations here, although the clearly enhanced binding activity in the peptides containing cystine bridges between positions (14-26), (1 7-26) or (21-26) was not expected from the data on the omission analogs. A similar enhancement of radioligand binding activity has recently been

HUMAN &ENDORPHIN

TABLE 2 Relative potency of synthetic analogs of &ndorphin by radioreceptor binding assay

Peptide G O a

&Endorphin 4.3 x 10-10 [Phe27,Gly31 1 P E P 3.4 x 10-10 [ C y ~ ~ € y s ~ ~ , P h e ~ ~ , G l y ~ ~ 1 #-EP 1.5 x 10-9

[ C y ~ ' ~ € y s ~ ~ , P h e ~ ~ , G l y ~ ~ J P E P 1.2 x 10-10

[Cys" €ys26 ,Phe27 ,Gly31 ] P E P 1.1 x 10-10

[ C y ~ ~ ~ € y s ~ ~ , P h e ~ ~ , G l y ~ ~ ] P E P 3.6 x

[ C ~ s ' ~ € y s ~ ~ ,Phe2' ,Gly3' ] P E P 1.6 x lo-"

inhibiting concentration in M .

observed for Ph-EP analogs with structural extensions at the carboxyl terminus (Yamashiro et al., 1980).

EXPERIMENTAL PROCEDURES

Protected peptide resins corresponding to peptides I and IZ Boc-glycyl resin (1.20 g, 0.58 mmol) was treated exactly as described for the previous synthesis of &,-EP analogs (Blake et al., 1979). After the coupling of Ile-22, the peptide resin was divided into two portions and synthesis of the two peptide analogs proceeded. The final peptide resin was deblocked in 55% trifluoroacetic acid/ methylene chloride, washed, and dried in vacuo .

/Cys2 -Cy~~~,Phe~',Gly~' J -oh endorphin (I ) A portion (402 mg, 0.064 mol) of the protected peptide resin was treated with 0.95 ml anisole and 10 ml liquid HF for 1 h at 0". The HF gas evaporated at 0", and the residue was washed with ethyl acetate. The peptide product was dissolved in 10 ml of 0.5 N acetic acid. After filtration to remove resin, the peptide solution was diluted with 60ml water and 1 N ammoni- um hydroxide was added until pH 8.2 was attained. Analysis of 100 pl aliquot with the Ellman reagent (Ellman, 1959) indicated the presence of 0.074mmol of -SH group. Then 0.01 M K3Fe(CN)6 was added until a slight yellow color persisted. Acetic acid was added to give pH 4.5 and the solution was desalted on IRC-50 column which was washed with 0.2N acetic acid and water; the peptide was eluted

Relative potency

100 126

28 120 358 269 390

with pyridine :acetic acid :water (30 : 4 : 66). The lyophilized crude peptide was chromato- graphed on carboxymethylcellulose as previ- ously described (Li et al., 1976) to give 31.6 mg peptide. Partition chromatography on Sephadex G-50 column (1.75 X 43.5cm) in the system n-butanol : pyridine : 0.1% acetic acid (5 : 3 : 1 1) gave 22.7mg peptide (10% yield based on starting Boc-glycyl resin) at Rf 0.27.

Paper electrophoresis at pH 3.7 and 6.7 (400 V, 2.5 h) gave single ninhydrin, chlorine positive spots at RkYs 0.59 and 0.47, respec- tively. Thin-layer chromatography in the BPAW system consisting of n-butanol : pyridine : acetic acid :water (5 : 5 : 1 : 4) gave a single ninhydrin, chlorine positive spot at Rf 0.44. Amino acid analyses (Spackman et al., 1958) of acid and enzyme hydrolysates are shown in Table 1.

[ C y ~ ' ~ - C y s ~ ~ , p h e ~ ~ , G l y ~ ~ ] - ~ h ~ n d o r p h i n (II) A portion (401 mg, 0.063 mmol) of the pro- tected peptide resin was treated with HF/ anisole as described above for peptide I. Chromatography on carboxymethylcellulosee gave 47mg of peptide 11. Partition chromato- graphy on Sephadex G-50 in the system n- butanol : pyridine : 0.1 M ammonium acetate (5 : 3 : 10) gave 34.1 mg (16% yield) of peptide I1 at Rf 0.35.

Paper electrophoresis at pH 3.7 and 6.7 gave single ninhydrin, chlorine positive spots at RkYs 0.61 and 0.50, respectively. Thin-layer chromatography in the BPAW system gave a single spot at Rf0.43. Amino acid analyses of acid and enzyme hydrolysates are summarized in Table 1.

24 1

J . BLAKE ET AL.

Bioassay for opiate activity Opiate activity was measured in the radioligand binding assay in which tritiated fih-EP (Houghten & Li, 1978) is used as the primary ligand (Ferrara ef al., 1979). The results are indicated in Table 2.

ACKNOWLEDGMENTS

We thank W. Hain and K. Hoey for technical assistance. This work was supported in part by the National Institute of Mental Health (MH-30245) and National Institute of Health (GM-2907).

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Address: Dr. C.H. Li Hormone Research Laboratory 1088 HSW University of California San Francisco, CA 94143 U.S.A.

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