with increased activities - PNAS

Proc. Nati. Acad. Sci. USA Vol. 76, No. 7, pp. 3276-3278, July 1979

Biochemistry

3-Endorphin: Synthesis of analogs modified at the carboxyl terminus with increased activities (analgesia/receptor assay/opiate activity/peptide synthesis)

CHOH HAO LI*, DONALD YAMASHIRO*, LIANG-FU TSENGt, WEN-CHANG CHANG*, AND PASCUAL FERRARA* *Hormone Research Laboratory, University of California, San Francisco, California 94143; and tDepartment of Pharmacology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53233

Contributed by Choh Hao Li, April 30, 1979

ABSTRACT Three analogs of human ft-endorphin (#h-EP) have been synthesized: [Gly3l1jWh-EP, [Gly3l1Jth-endorphinamide, and ycine. All are more active than in both the guinea pig ileum bioassay and the opiate reIBh-EP[Gly3f1Bh-endorphiny.g ceptor binding assay. The last two analogs are about twice as active as fth-EP in an assay for analgesia. Modification at position 31 and extension at the COOH terminus may afford a route toward analogs with even greater biological activity. Of all the opioid peptides that correspond to a portion of the structure of f3-lipotropin (1, 2) only the structure corresponding to positions 61-91-namely, human f3-endorphin (13h-EP) (Fig. 1)-has shown potent analgesic activity by the intravenous route (3). Recent studies with synthetic analogs indicate that the complete primary structure of 1h-EP is required for full analgesic activity (4). Although modifications of the pentapeptide [Met]enkephalin (5), representing positions 1-5 of 13h-EP can lead to products with potencies comparable to or even greater than that of 3h-EP (6-8), efforts to make the same modifications in positions 1-5 of 1h-EP have not led to analogs with increased potencies (9-11). Thus far, the only analog of 3h-EP that has exhibited greater analgesic activity than the parent is [Phe27, Gly3l]h-EP whose potency is about 1.48 times that of the parent by the intravenous assay route (11). We have now found that modifications in position 31 and extension at this COOH terminus can lead to even greater biological potencies.

5 10 H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser15 20 Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn25 31 Ala -Ile -Ile.- Lys-Asn-Ala-Tyr- Lys- Lys -Gly-Glu- OH

FIG. 1. Amino acid sequence of 13h-EP. Camel f-endorphin has His-27 and Gln-31.

(13). Final purification by differential hydrophobicity was effected by partition chromatography on Sephadex G-50 (15) with solvent systems used and RF values obtained as follows: I, 1-butanol/pyridine/0.05 M NH4OAc-0.2% HOAc (5:3:10), RF 0.39; II, 1-butanol/HOAc/pyridine/H20 (20:5:1:25), RF 0.37; III, 1-butanol/pyridine/0.6 M NH4OAc (5:3:10), RF 0.61. From 50 Amol of starting Boc-Gly resin there was obtained: I, 65.8 mg; II, 62.2 mg; III, 67.3 mg. The peptides were homogeneous in thin-layer chromatography (silica gel with ninhydrin and C12-tolidine detection) in 1-butanol/pyridine/acetic acid/H20 (5:5:1:4), as follows: I, RF 0.47; II, RF 0.50; III, RF 0.52. They were homogeneous on paper electrophoresis on Whatman 3MM (400 V, 5 hr, ninhydrin detection) at pH 3.7 (I, RF 0.57; II, RF 0.65; III, RF 0.56) and at pH 6.7 (I, RF 0.46; II, RF 0.57; III, RF 0.46) with RF values relative to lysine. Amino acid analyses of 24-hr acid hydrolysates were in agreement with expected values (Table 1). Opiate activity was measured from the depression of electrically stimulated contractions of guinea pig ileum preparations (16, 17). For analgesic assay, male ICR mice weighing 25-30 g (Simonsen Laboratories, Gilroy, CA) were used. Analgesic activity was assessed by the tail-flick method (18) as described

EXPERIMENTAL Solid-phase synthesis (12) was performed on Boc-Gly polymer or brominated polymer (13) for analogs with COOH-terminal glycine residues and on Boc-Gly benzhydrylamine polymer (14) for the analog with a COOH-terminal glycineamide residue. Side-chain protection and coupling were performed as described for the synthesis of 1h-EP (13) except that Z protection was used for the side-chain of Tyr in position 1 (9). Assembly of sequences corresponding to [Gly31]13h-EP (I), [Gly3l]fh-endorphinamide (3h-EP-NH2) (II), and [Gly3l]/3h-endorphinylglycine (fh-EP-Gly-OH) (III) was carried out in a Beckman model 990 peptide synthesizer with a fully automated symmetrical anhydride program (4). After removal of the last Boc group with trifluoroacetic acid and treatment with liquid HF, the peptides were purified by gel filtration on Sephadex G-10 and chromatography on CM-cellulose by described procedures

(4).

The opiate receptor binding assay was performed by the method of Pasternak et al. (19) with modifications (unpublished results) by using a membrane fraction from rat brain homogenate. [3H-Tyr27h h-EP (20) was used as primary ligand and synthetic Oh-EP (13) was used as standard competing ligand. RESULTS AND DISCUSSION The synthesis of [Gly3l A]f-EP, [Gly3l ]fh-EP-NH2 and [Gly31 ]Bh-EP-Gly-OH was accomplished by the solid-phase method (12) according to procedures used for the synthesis of 13h-EP (13). Purification of the analogs was effected by chromatog-

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Abbreviations:

fh-EP, human f-endorphin;

Ohb-EP-NH2,

human

f3-endorphinamide; 13h-EP-Gly-OH, human f3-endorphinylglycine.

this fact.

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Li et al.

Proc. Natl. Acad. Sci. USA 76 (1979)

Table 1. Amino acid analyses of synthetic 13h-EP analogs Amino [Gly31][Gly3t][Gly3t]acid 1h-EP 3h-EP-Gly-OH fh-EP-NH2 Lys 4.98 (5) 5.02 (5) 4.91 (5) Asp 1.98 (2) 2.01 (2) 2.01 (2) Thr 2.74 (3) 2.72 (3) 2.79 (3) Ser 1.76 (2) 1.75 (2) 1.76 (2) Glu * 2.23 (2) 2.20 (2) 2.15 (2) Pro 1.04(1) 1.03(1) 1.04(1) Gly 3.84 (4) 3.78 (4) 4.82 (5) Ala 2.09 (2) 2.07 (2) 2.10 (2) Val 1.01 (1) 1.00(1) 0.99(1) Met 0.98 (1) 0.95 (1) 0.98 (1) Ile* 1.44 (2) 1.39 (2) 1.37 (2) Leu 2.08 (2) 2.05 (2) 2.09 (2) Tyr 1.93 (2) 1.94 (2) 1.93 (2) Phe 1.99 (2) 1.99 (2) 1.93 (2) The analyses were done on 24-hr 6 M HCl hydrolysates (theoretical values in parentheses). * Low values are accounted for by the presence of the acid-resistant Ile-Ile moiety.

raphy on CM-cellulose and by partition chromatography on Sephadex G-50. The latter method has been shown to be useful for the separation of deletion sequences (15). The highly purified peptides were characterized by thin-layer chromatography, paper electrophoresis, and amino acid analysis. The biological activities of the analogs were measured by in vitro and in vivo procedures as summarized in Tables 2, 3, and Table 2. Opiate activity of synthetic fh-EP analogs substituted at position 31 and extended at the COOH terminus

IC50,* Synthetic peptides

nM

Relative potency

Oh-EP

100 128 76 168 64 200 97 132 *Fifty percent inhibitory concentration in the guinea pig ileum assay.

[Gly3l] 3h-EP [Gly3l] h-EP-NH2 [Gly3llfh-EP-Gly-OH

4. For comparison, the relative potencies (fBh-EP = 100) previously obtained for [Phe27, Gly3t]3h-EP were 128 in the guinea pig ileum assay and 119 in the in vivo assay used here. Thus, the double substitutions in positions 27 and 31, which are the two variable residues when camel and human f3-endorphins are compared (21), did not substantially change either activity. However, the single replacement of Glu-31 in 1h-EP by Gly appears to substantially raise the in vitro activity (Table 2) but

3277

Table 4. Receptor binding assay of synthetic fh-EP analogs substituted at position 31 and extended at the COOH terminus

IC50,* Peptide

Relative potency

nM

0.75 100 3h-EP 0.59 127 [Gly3l] 3h-EP 0.49 153 [Gly3l]3h-EP-Gly-OH 0.30 [Gly31hlh-EP-NH2 250 * Fifty percent inhibitory concentration (see Fig. 2) in the opiate receptor binding assay.

not significantly alter the analgesic potency (Table 3). Such results would be consistent with earlier observations that the structural requirements for the two activities differ (9, 10). The opiate activities of the synthetic analogs as measured by the rat brain receptor assay are summarized in Fig. 2 and Table 4. All the analogs exhibited greater potency than fh-EP in the receptor and guinea pig ileum assays. Interestingly, the analog with a COOH1-terminal carboxamide appears to be the most active. In the receptor assay (Table 4), [Gly3113h-EP-NH2 is almost 3 times more potent in comparison with the parent peptide. Similar increases in activity in the myenteric plexus bioassay has been observed in going from [Met]enkephalin to [Metlenkephalinamide and from f-lipotropin-(61-76) to the

amide form (22). The analgesic potency of 13h-EP appears to be practically unchanged by the replacement of Glu-31 by Gly, indicating that the side-chain of Glu is not necessary for this activity. Conversion of the COOH-terminal carboxyl group of [Gly3l]/h-EP to a carboxamide or extension by an additional Gly residue results in increases in analgesic potency. In view of the fact that the entire chain length of 1h-EP is required for full analgesic activity (4), it is evident that even limited enzymatic attack at the COOH terminus could rapidly destroy its activity. Thus, modification of position 31 and extension at the COOH terminus may be one approach toward obtaining analogs with greater biological activity than 3-endorphin. 100

0\ 0

21 80 4#--

0

0 It

- 60

a-

w

Table 3. Analgesic potency of synthetic Oh-EP analogs substituted at position 31 and extended at the COOH terminus Synthetic peptides Relative potency AD50,* nmol 13h-EP 0.064 (0.026-0.17) 100 0.077 (0.038-0.17) 83 [Gly3lf~h-EP 3h-EP

[Gly3l]fh-EP-NH2 13h-EP

0.036 (0.019-0.068) 0.016 (0.008-0.032)

100 225

0.092 J0.061-0.14) 100 0.043 (0.031-0.057) 217 *AD50 is the mean analgesic dose (by intracerebroventricular injection). The numbers in parentheses refer to the 95% confidence limit.

[Gly3lt]fh-EP-Gly-OH

0

A r"

§_40 0

I> 0 C c

0~ ~

mo 20

1

~

10

~

~

--

100

Picomoles per tube

FIG. 2. Opiate receptor binding assay. *, 3h-EP; D, [Gly3l]3h-EP; 0, [Gly3l] Oh-EP-Gly-OH; A, [Gly3l]Oh-EP-NH2.

3278

Biochemistry:

Li et al.

We thank K. Hoey and W. F. Hain for technical assistance. This work was supported in part by grants from the National Institute of Mental Health (MH-30245 to C.H.L.), National Institute of Drug Abuse (DA-02352 to L-F.T.), and the Hormone Research Foundation. 1. Li, C. H., Barnafi, L., Chretien, M. & Chung, D. (1965) Nature (London) 208, 1093-1094. 2. Li, C. H. & Chung, D. (1976) Nature (London) 260,622-624. 3. Tseng, L-F., Loh, H. H. & Li, C. H. (1976) Nature (London) 263, 239-240. 4. Li, C. H., Tseng, L-F. & Yamashiro, D. (1978) Biochem. Biophys. Res. Commun. 85, 795-800. 5. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. & Morris, H. R. (1975) Nature (London) 258,

577-579. 6. Bajusz, S., Ronai, A. Z., Szekely, J. I., Graf, L., Dunai-Kovacs, Zs. & Berzetei, I. (1977) FEBS Lett. 76,91-92. 7. Roemer, D., Buescher, H. H., Hill, R. C., Pless, J., Bauer, W., Cardinaux, F., Closse, A., Hauser, D. & Huguenin, R. (1977)

Nature (London) 268,547-549. 8. Yamashiro, D., Tseng, L-F. & Li, C. H. (1977) Biochem. Biophys. Res. Commun. 78, 1124-1129. 9. Yamashiro, D., Tseng, L-F., Doneen, B. A., Loh, H. H. & Li, C. H. (1977) Int. J. Peptide Protein Res. 10, 159-166.

Proc. Natl. Acad. Sci. USA 76 (1979) 10. Yamashiro, D., Li, C. H., Tseng, L-F. & Loh, H. H. (1978) Int. J. Pept. Protein Res. 11, 251-257. 11. Blake, J., Tseng, L-F., Chang, W-C. & Li, C. H. (1978) Int. J. Pept. Protein Res. 11, 323-328. 12. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85,2149-2154. 13. Li, C. H., Yamashiro, D., Tseng, L-F. & Loh, H. H. (1977) J. Med. Chem. 20, 325-328. 14. Pietta, P. G. & Marshall, G. R. (1970) J. Chem. Soc. D, 650651. 15. Yamashiro, D. (1979) Int. J. Pept. Protein Res. 13,5-11. 16. Kosterlitz, H. W., Lydon, R. T. & Watt, A. F. (1970) J. Pharmacol. 39, 398-413. 17. Doneen, B. A., Chung, D., Yamashiro, D., Law, P. Y., Loh, H. H. & Li, C. H. (1977) Biochem. Biophys. Res. Commun. 74, 656-662. 18. D'Amour, F. E. & Smith, D. L. (1941) J. Pharmacol. Exp. Ther. 72,74-79. 19. Pasternak, G. W., Wilson, H. A. & Snyder, S. H. (1975) Mol. Pharmacol. 11,340-351. 20. Houghten, R. A. & Li, C. H. (1978) Int. J. Pept. Protein Res. 12, 325-326. 21. Li, C. H. (1977) Arch. Biochem. Biophys. 183,592-604. 22. Ling, N. & Guillemin, R. (1976) Proc. NatI. Acad. Sci. USA 73, 3308-3310.