Potent tetrapeptide enkephalins

Life Sciences, Vol. 28, pp. 1517-1522 Printed in the U.S.A.

Pergamon Press

POTENT TETRAPEPTIDE ENKEPHALINS Richard E. Chipkin*, Dan H. Morris*, Michael G. English**, James D. Rosamond***, Charles H. Stammer**, Eunice J. York*, and John M. Stewart* *Department of Biochemistry, University of Colorado School of Medicine, Denver, Colorado, 80262 **University of Georgia, Athens, Georgia, 30602 ***Pennwalt Pharmaceutical Division, Rochester, New York,

14603

(Received in final form January 26, 1981) SUMMARY Small peptides with opiate-like activity have generally had structures closely resembling that of the opioid pentapeptide enkephalin: Tyr-Gly-Gly-Phe-Met-COOH. Single deletions of any one of the amino acids has been demonstrated to reduce opiate activity drastically. In this work we show that the potency losses resulting from the removal of glycine3 can be fully attenuated by substitution of D-alanine in position two and derivatization of the acid to the amide. This tetrapeptide (Tyr-DAla-Phe-Met-NH 2) has narcotic activity similar to the parent pentapeptide in the guinea pig ileum and mouse tail-flick tests. This enhanced potency, relative to the unaltered tetrapeptide, is theorized to arise from increased resistance to enzymatic destruction. The data presented show that a five amino acid sequence is not mandatory for the expression of opiate activity in enkephalin analogs. The structure of enkephalin, as defined by Hughes et al. (I) is Tyr-GlyGly-Phe-Met-COOH (Enk), with the leucine 5 pentapeptide also occurring naturally In the years since its discovery, there has been much work on the synthesis of novel analogs based on these structures (2) as well as conformational studies (3) all assuming that a five amino acid sequence is necessary for significant biological activity. This position was based on evidence that removal of either the C-terminal or N-terminal amino acids of Enk resulted in peptides with severely diminished activity (4). However, McGregor et al. (5) have reported that the tetrapeptide Tyr-DAIa-GIy-Phe-NH 2 is a potent opiate agonist. This activity was thought to be due to increased resistance to enzymatic degradation conferred upon the molecule by the D-alanine2 substitution. Others have extended this finding by showing the activity of C-terminal derivatized des-methionine 5 enkepha!ins both in vitro (6) and in vivo (7). Thus, a pentapeptide sequence does not appear mandatory. Agarwal, et al. (8) showed that the tetrapeptide Tyr-Gly-Phe-Leu-COOH was not active in the opiate receptor binding assay. We wished to see if the potency of tetrapeptides of this class could be increased by modifications similar to those used by McGregor et al. We report here the synthesis and biological activities of enkephalin tetrapeptides in which glycine 3 has been 0024-3205/81/131517-06502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

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deleted. The potencies of some of these peptides are comparable the parent pentapeptides from which they are derived.

to those of

Methods Synthesis: Peptides were synthesized by the solid phase method (9), using standard techniques (I0) with the aid of a Beckman 990 synthesizer. Some peptides were also synthesized by solution methods. Peptides having free carboxyl groups were synthesized on standard Merrifield resins and removed by cleavage with anhydrous HF in the presence of anisole. Peptide alcohols were synthesized by reductive cleavage of the corresponding peptide-resins with LiBH 4 in tetrahydrofuran or by reduction of the peptide methyl ester in tetrahydrofuran solution. The tetrapeptide alcohol sulfoxide was prepared by oxidation of the peptide alcohol in glacial acetic acid solution with hydrogen peroxide. Unsubstituted peptlde amides were synthesized on the methylbenzhydrylamine resin which yields peptide amides directly upon HF cleavage. Substituted amides were synthesized by aminolysis of the corresponding peptide methyl esters. Peptides were purfied by countercurrent distribution or partition chromatography on Sephadex. Homogeneity of the purified peptides was demonstrated by paper electrophoresis and thin layer chromatography, and composition was verified by amino acid analysis after hydrolysis with HCI. Methionine sulfoxide was determined on hydrolysates made with 3_N methanesulfonic acid (II). Boc-DAla-A-Phe azlactone was synthesized by dehydration and azlactonization of Boc-DAla-DL-8-phenylserine with acetic anhydride (12). The azlactone was coupled to Met-methylbenzhydrylamine resin in a 24-hr reaction in2dimeth[l formamide. The remainder of the synthesis was straightforward. DAla -A-Phe~Enk-NH 2 was synthesized by English and Stammer (13). Stimulated Guinea Pig Ileum: Male Hartley guinea pigs (225-275 g) were sacrificed ~nd strips of intact ileum (2-4 cm) were placed in a I0 ml tissue bath at 37 ~ I-C containing Tyrode's solution bubbled with 98% 02 - 2% CO 2 (pH 7.4). Isometric contractions, recorded via a Grass force transducer on a Grass polygraph, were induced by a Grass stimulator using the following parameters: 0.2 Hz, 0.4 msec, 100 volts. ED50"s were determined from the mean of at least three independent observations for each drug. See Chipkin et al. (14) for additional details. Mouse Tall Flick: Male Swiss Webster mice (Charles River, Inc.) weighing 23-32 g were used. Analgesic testing using a radiant heat tail-fllck technique was conducted according t o Dewey and Harris (15). Briefly, initial control latenties for all mice were first determined. The intensity of the stimulus was adjusted such that all mice had control latencles of 2-3 seconds; all other animals were discarded (<5%). Following determination of pre-drug latencles, the mice were injected intracerebrally (ic) according to the method of Haley and McCormick (16) using a Hamilton syringe with a collared 27 gauge needle that permitted a penetration of only 3 mm. A volume of I0 B1 was injected in all experiments. At I0, 30, 45, 60 and 90 minutes following injection, the tail-fllck latencies were redetermined. A cut-off of 6 sec was used to prevent tissue damage in instances of maximal drug effectiveness. N = six mice/dose. For experiments examining the in vlvo reversibility of the effect, naloxone was given subcutaneously (0.I ml/10 g body wt) ten minutes before the ic injection of the peptide. All drugs were dissolved in distilled water.

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Enkephalin Tetrapeptides

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Table I Potencies Of Enkephalins On Guines Pig Ileum COMPOUND

I II III IV V VI

ED50(MxI0 -8)

RELATIVE POTENCY

Morphine

1.4

1.6

Tyr-Gly-Gly-Phe-Met-COOH

2.3

1.0

Tyr-Gly-Gly-Phe-Met-NH 2

1.4

1.6

Tyr-DAla-Gly-Phe-Met-NH 2

1.7

1.4

Tyr-DAla-Gly-Phe-Met-ol

0.64

3.6

Tyr- Gly-Phe-Met-COOH

6300

0.004

VII

Tyr- Gly-Phe-Met-NH 2

I00

0.02

VIII

Tyr-DAla-Phe-Met-COOH

140

0.02

IX

Tyr-DAla-Phe-Met-NH 2

2.0

1.2

X

Tyr-DAla-Phe-Met-OCH 3

6.0

0.4

Tyr-DAla-Phe-Met-ol

4.2

0.5

Tyr-DAla-Phe-Met(0)-ol

14.0

0.2

Tyr-DAIa-Phe-Met-NH-CH2CH 3

12.0

0.2

XI XII XIII XIV XV XVI XVII XVIII

Tyr-DAIa-Phe-Met-NHCH2CH2OH

19.0

0.i

Tyr-DAla-Phe-Leu-NH 2

2.0

0.2

Acetyl-Tyr-DAla-Phe-Met-NH 2 Tyr-DAIa-APhe-Met-NH 2

6300 115.0

Tyr-DAIa-GIy-APhe-Met-NH 2

0.33

0.004 0.02 7.0

Results Table I shows the structures of the peptldes, their potencies on stimulated guinea pig ileum, and the relative potency of the compounds as compared to enkephalin. As can be seen, Enk (II) is slightly less active than morphine (I) but derlvatlzatlon to the amide, Enk-NH 2 (III), confers additional potency on the pentapeptide to make it equal to morphine. Substitution of D-alanine in position two of the pentapeptide amide does not have a significant effect (III vs IV) whereas reduction of the carboxyl of DAla2-Enk to the corresponding alcohol (V) enhances potency to about four times that of Enk. On the other hand, removal of Gly 3 from Enk dramatically reduced the efficacy of the peptide (VI); however, this can be somewhat offset by making the tetrapeptide amide (VII) or by substitution of Gly 2 in VI with D-alanine to give VIII. Simultaneous incorporation of both changes yields DAla2-desGly3-Enk-NH2 (IX). This peptide (IX) is of essentially equal potency with Enk and only slightly less active than morphine. Interestingly, derlvatizatlon of the methlonine in VIII

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rq-E]

DAla 2 - M e t 5 - E n k

O O

D A l a 2 - d e s G l y 3 -Enk ( 10 pg/mouse I

(10~Jg/mouse}

N=6 N=6

A--A

DAla 2 -desGly 3 - E n k

N=6

?--? ~-~, x--x

DAla2-desGly3-Enk {100pg/mouse ) Saline {10 ~JI/mouse } DAla 2 -desGly 3 -Enk ( 50 {Jg / mouse ) + Naloxone (5 m g / K g , sc )

[ 0

I 10

(50 Pg/mouse)

N=4 ITwo died) N= 6 N=6

6

5

o .E

4

>- 3

2 E_ ! p0

I 30

I 45

Time After lntracraniallnjection

FIG. Analgesia produced by flick test.

I 60

F 90

} i n minutes}

1

DAla2-Enk-NH2 and DAla2-desGly3-Enk-NH2 in

the mouse tail-

to give peptides X-XIV results in reduced potency compared to the amide (IX); although peptides X-XIV are considerably more potent than the parent acid (VIII). Replacement of Met 5 in IX with leucine to give DAla2-desGly3-Leu5-Enk NH 2 (XV) does not change the potency. Finally, acetylation of the alpha-nitrogen of DAla2-desGly3-Enk-NH 2 to give XVI reduced potency to less than 1/1000th that of Enk. Surprisingly, the potency of the dehydrophenylalanine tetrapeptide amide (XVII) was only 2% that of Enk. All the inhibitor~ effects of these peptides on guinea pig ileum were reversed by naloxone (5x10- 8M). 2 3 2 The effects of DAIa -desGly -Enk-NH~ and DAIa -Enk-NH 2 on the tail-flick latencies of mice are shown in Fig. I. D~Ia2-Enk-NH2 (10 ~g/mouse) injected intracranially showed pronounced analgesia that peaked within ten minutes after administration. Similarly, DAla2-desGly3-Enk-NH2 showed a dose-related effect on the tail-flick response, with 10 ~g causing only slight analgesia and 50 ~g being virtually identical to i0 ~g of DAIa2-Enk-NH 2. Animals treated with the tetrapeptide displayed typical narcotic behavioral signs such as hyperactivity and Straub tail after injection. A dose of 100 ~g/mouse of the tetrapeptide was lethal to two of six mice tested; death was apparently due to respiratory depression. These analgesic effects were naloxone reversible. No analgesia was produced by intraperitoneal injection of 20 mg/kg of DAla2-desGly3-Enk-NH 2.

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Enkephalin Tetrapeptides

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Discussion The data presented herein show that des-glycine 3 enkephalins can be modified to give highly active compounds in both in vitro and in vivo tests. A five amino acid sequence is therefore not necessary for opiate activity, the prototype analog- DAla2-desGly3-Met5-enkephalinamlde (IX)- is as active on stimulated guinea pig ileum as the parent pentapeptide. Furthermore, it shows dose related naloxone-reversible analgesic efficacy in vivo after intracerebral injections in mice. Thus, it is apparent that a pentapeptide sequence is not critical for opioid-like activity. 2 3 3 The reasons for DAIa -desGly -Enk-NH2"s high potency compared to desGly Enk may be related to the rapid enzymatic degradation of the underivatized tetrapeptide. In the ileum assay, an EDb0 dose of Tyr-Gly-Phe-Met-COOH (VI) has a shorter duration of action than does that of the pentapeptide (II) (Chipkin, unpublished observations). Whereas previously synthesized tetrapepz tides may have been degraded very quickly, DAIa -desGly -Enk-NH2"s stability makes measurement of its biological efficacy possible. This hypothesis would explain why Agarwal et al. (8) failed to detect significant activity of TyrGly-Phe-Leu-COOH in the opiate receptor binding assay. The activity of DAIa2-desGIy3-EnkTNH2 implies that a precise intramolecular distance between Tyr I and Phe 4 in the amino acid sequence of enkephalin is not critical for opioid activity. Bradbury et al. (17) have suggested there must be two glycines in the molecule for Tyr I and Phe 4 of enkephalin to correspond spatially with the aromatic rings3of oripavine. However, the data presented here show that the removal of Gly and the subsequent reorientation of the tyrosine and phenylalanine closer together does not compromise potency. Thus, it appears that the molecule is rather flexible or the receptor site does not have a rigid conformation on the membrane. In either ease, it seems that a change in peptide lengt h can be accomodated via peptidereceptor interactions or altered peptide folding. The two peptides containing e,8-dehydrophenylalanine (XVII & XVlII) provide further information on conformational requirements for opioid activity. As previously reported (18), the A-Phe 4 pentapeptide (XVlII) is five times as potent as the corresponding phenylalanine pentapeptide. This increased potency might be due either to an increased resistance of the peptide to enzymatic degradation or to an enhanced fit of the peptide to receptors (increased affinity). Since the potency increase was equivalent both in vitro and in vivo, it was suggested that increased affinity was responsible for the increased potency (18). In these peptides the A-Phe is in the Z conformation, in which the phenyl ring is held rigidly toward the amino end of the peptide. In the pentapeptide this conformational restriction apparently favors peptide-receptor interaction over that of the peptide containing the more flexible phenylalanine residue. In the tetrapeptide, on the other hand, the phenyl ring is apparently held rigidly in a position which does not allow it to interact with the aromatic-binding site on the receptor. The phenylalanine tetrapeptides, being more flexible than the A-Phe analog, can evidently assume a conformation which will allow this essential interaction to take place. It appears that the structure-activity relationships that exist for the tetrapeptide may be different than those for the parent peptide. For example, DAla2-Enk-ol is more potent than its corresponding amide (IV vs V), whereas the same relationship is not true in the tetrapeptide (IX vs XI). Furthermore, it has been reported (19) that derivatization of the pentapeptide to the ethylamide increases activity on the ileum, while the same is not true of the tetrapeptlde3(IX vs XIII). Therefore, it seems as if increasing the length of desglycine tetrapeptides with lipophilic moieties decreases activity, whereas the

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opposite is the case in the pentapeptide. However, both penta- and tetrapeptides have a strict requirement for a tyrosine at position one with a free amino group in order for opiate-like activity to be observed (2). In summary, this work shows that the des-~lycine 3 derivatiea may be suitably modified to give active analogs. DAlaZ-desGly3-Enk-NH 2 is a potent opioid with naloxone-reversible actions both in vitro and in vivo. The activity of these tetrapeptides implies a flexible receptor that can adapt itself to opiate peptide analogs with differing chemical characteristics. Acknowledgements Grateful appreciation is extended to Ms. Virginia Sweeney for her help in the amino acid analyses of these peptides. REC was supported by USPHS Grant No. DA07043. DHM was supported by a grant from Pennwalt Corp. Dr. Chipkln's present address is c/o Schering Corp., 60 Orange Street, Bloomfield, NJ, 07003. References i. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12.

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