Vol. 127, No. 2, 1985

March 15, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 649-655

INHIBITION OF ANALGESIA BY C-TERMINAL DELETION ANALOGS OF HUMAN B-ENDORPHIN

Pierre Nicolas and Choh Hao Li

Laboratory of Molecular Endocrinology University of California San Francisco, CA 94143

Received February 5, 1985

SUMMARY: Human B-endorphin (Sh-EP) analogs of variable chain lengths have been investigated for their potency in inhibiting analgesia induced by Bh-EP or by the potent opiate etorphine. It was found that Bh-EP-(I-28) inhibits the analgesic effect of Bh-EP and etorphine when co-injected intracerebroventricularly into mice. Antagonism by competition at same opioid receptor subtypes is suggested from parallel shifts of the dose-response curve of etorphine or Bh-EP in the presence of increasing doses of Bh-EP-(I-28). On a molar basis, Bh-EP-(I-28) is nearly i0 times more potent than naloxone. The reduction of the chain length from residues 1-28 to 1-27 lowered the antagonist potency while further reduction of the peptide chain led to a complete loss of inhibitory activity. From comparison of the opioid- receptor binding affinity, analgesic activity and antagonist potency, it is concluded that the C-terminus of B-EP is critical to the biological efficacy of the molecule and that the antagonist activity of C-terminal deletion analogs is probably mediated through residues 27 and 28. ©1985Acad~c~ ..... I~.

Studies on Bh-EP analogs have shown that systematic

deletion of amino acid residues starting at the C-terminus

results in a stepwise decrease in both the analgesic activity

and the opioid-receptor binding potency (1,2). Recent reports

show Bh-EP analogs with high potency ratio are antagonists to

B-EP induced analgesia (3,4). The potency ratio (B/A) was

defined as the ratio of opioid-receptor binding (B) activity to

analgesic (A) potency. In particular, the naturally occurring

Bh-EP fragment, Bh-EP-(I-27) , with 30% and less than 2%

Abbreviations: Bh-EP, human beta-endorphin; icv, intracerebro- ventricularly; B~A, opioid receptor binding to in vivo analgesic potency ratio; [ H]-Sh-EP, [3H2-Tyr27]-Bh-EP; IC50, fifty per- cent inhibitory concentration; AD50, median antinociceptive dose

649

0006-291X/85 $1.50 Copyright © 1985 by Academic Press, Inc.

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Vol. 127, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

respectively of the binding and analgesic potency of Bh-EP, was

found to inhibit analgesia when co-injected icy into mice with

either Bh-EP (4) or the potent opiate etorphine (5). In order

to explore the contribution of the C-terminal segment of Bh-EP

to the biological efficacy of the molecule, we compared the

ability of synthetic analogs, Bh-EP-(I-28), Bh-EP-(I-27 ) and

Bh-EP-(I-26), in inhibiting analgesia induced by Bh-EP or

etorphine in mice. Results of these studies are herein

presented.

MATERIALS AND METHODS

Bh-EP , 8h-EP-(I-28) , Bh-(l-26) were synthetic products (6-8), Etorphine was a generous gift from Dr. E. L. Way of this university. [3H2~Tyr27],~h-EP (50 Ci/mmol) was prepared as described. (9). [JH]-etorphine (33 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Binding assays were performed as described (i0,Ii) in Tris-Cl, pH 7.5, 0.1% serum albumin, 0.01% bacitracin, with washed rat brain membranes (0.5 mg of membrane proteins per assay) and tritiated Bh-EP (0.3 nM) or eto~phine (0.3 nM) as primary ligand. Analgesic potency was assessed by the tail-flick method (12) using groups of i0 mice per dose as described (3). AD50 values were calculated for each peptide or opiate alone and in combination with various fixed doses of putative antagonist. The ratio of the AD50 value in the presence of antagonist to that in its absence (dose-ratio, x) was calculated for each dose of antagonist. Competitive antag- onism was quantitated by use of the apparent pA 2 for agonist- antagonist interactions (13,14), measured from linear regression of the logarithm [x-l] against the negative logarithm of the molar dose of antagonist injected per animal. We will refer to the corresponding graphs as Schild plots.

RESULTS

Table 1 presents analgesic potency and opioid-receptor

binding activity of Bh-EP, Bh-EP-(I-28), Bh-EP-(I-27), Bh-EP-

(1-26) and etorphine. Comparison of the opioid activities by

either the tail-flick or opioid-receptor binding assays shows

analogs with shorter chain to be less active than 8h-EP. The

reduction of the chain length from residues 1-28 to 1-27 lowered

both the analgesic and binding potency. Further reduction of

the peptide chain to Bh-EP-(I-26) provoked a parallel additional

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Vol. 127, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1. Analgesic potency and opioid-receptor binding affinity of 8h-EP, 8h-EP-(l-28), ~h-EP-(l-27), Bh-EP-(I-26) and etorphine

Compound

Analgesic Activity

AD50 (a) R p. (b)

pmol/mouse

Binding Activity

vs [3H]-Sh-EP

IC50 (a), nM R.P. (b)

vs~ [3H]-Etorphine B/A (a) (b)

IC50 , nM R.P.

8h-EP 31.4 (25.8-37.6

8h-EP-(I-28) 580 (418 - 883)

8h-EP-(I-26) 3200 (2100-4070

Etorphine 20.2 (15.9-25.4

~h-Ep(c) 26.3 (17.2-37.1

~h-EP-(l-27)(c) 1632 (1081-3100

I00 0.96 100 (0.89-1.12)

5.4 1.62 59 (1.49-1.95)

0.98 6.86 14 (6.20-7.12)

155 0.47 205 (0.42-0.51)

I00 i.I0 i00 (1.03-1.25)

1.6 4.11 27 (3.60-5.08)

1.68 70 1.0 (1.52-1.84)

2.56 46 10.9 (2.31-2.63)

ii. 8 i0 14.3 (10.5-13.9)

i. 18 i00 i. 3 (i. I0-i. 27 )

1.66 65 1.0 (i. 50-1.83)

5.85 18.5 16.8 (5.17-7.21)

(a) Numbers in parentheses are 95% confidence limits;

(b) R.P., relative potency;

(e) taken from ref. 5.

lowering of the binding and analgesic potencies. There is a

fair correlation between analgesia and binding potency when

8h-EP (r 2 = 0.94) or etorphine (r 2 = 0.95) are used as primary

ligand. As a consequence, the C-terminal deletion analogs of

8h-EP exhibit nearly similar B/A rations (Table i).

The times for the peak effect of etorphine or ~h-EP either

alone or in the presence of varying doses of 8h-EP-(I-28) are

shown in Figure i. There was a marked decrease in the analgesic

response to etorphine or 8h-EP when small doses of 8h-EP-(I-28)

were co-injected. The percentage of inhibition was almost

constant with time up to 40 min after injection in both cases.

Dose-response curves obtained for the analgesic activity of

etorphine or 8h-EP, either alone or in the presence of 8h-EP-

(1-28) or 8h-EP-(I-26) are shown in Figure 2. Injection of

varying doses of agonists together with an increasing fixed dose

of 8h-EP-(I-28) produced a parallel shift of the dose-response

651

Vol, 127, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

< 8 0

o 60 < z < 40

20 ~u

0

~ D ~ h " EP ( i O O p m o l ) , I •/l h - EP-(1-28), II ( lOOpmol ) 80

0 I + II (SOpmo|) I + II ( lOOpmQI) 6 0

4 0

20

0 __ I ] I I d

0 20 40 60 80 100 TIME (rain)

0 e t o r p h i n e (75pmol) , III • III + II (50pmol)

II ( lOOpmol)

0

I I [ I -J

0 20 40 60 80 100 TIME (rain)

Figure i. (A), Time-course of analgesia produced by 8h-EP alone or in the presence of 8h-EP-(I-28), course of analgesia elicited by 100 pmol of 8h-EP. (B), as in A but etorphine as agonist.

95

8O

60

4O

2O

i,u q < 5 - Z <

95 I - ,z, U

~_ 8o

• p , . r , n A - ~ ;: :.~',:::,~.',', . / / / . / /

o ,+. ( ~ . , ) I / ~5 / / / • 0+,, (ioo~.n) / a / o,. . , , . - , ~ / / / . . /

° " - y / x / ..... / / / / / / / , / -

I I i I I I I I I

• e to r l~ IM, IV a o w + . l ~ m o l ) A iv + ii (5@i0rnol) / . / , i 1

I Y + l l ( la~le l~ l ) 4 1 ~ / / / / o,,+,,,,,,*,-,,/ / / / t / . ~ " /

/ / / 10.0 10.2 10.4 10.6

5 - log (dose}

I I I I I I I I I 10 25 50 100 250 500 1000 2500 5000

DOSE (pmol/mouse)

Figure 2. (A), Log (dose) vs. probit (% analgesia) curves for antinociception produced by icv injection into mice of ~h-EP alone, or in combination with Bh-EP-(I-28) or 8h-EP- (1-26). (B), as in A but etorphine as agonist. (Inset) : Schild plots for antagonism of etorphine or Bh-EP by ~h-EP-(I-28). Abscissa = pA x = -log [mol of antagonist injected per mouse] at which the dose-ratio is x. Ordinate = log [x -I]. pA 2 values were estimated by performing linear regression analysis.

652

Vol. 127° No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 2. Quantitative antagonism of Sh-EP and etorphine by ~h-EP-(I-28), ~h-EP-(I-27), Bh-EP-(I-26) and naloxone

Agonist Antagonist pA 2 Slope R.A.P. (a)

8h-EP

Etorphine

8h-EP-(I-28) 10.56 -I.06 226

8h-EP-(I-27) (b) 10.22 -0.96 I00

~h-EP-(I-26) .......... nil

Naloxone (b) 9.55 -1.06 23

Bh-EP-(I-28) 10.59 -1.13 230

8h-EP-(l-27) (b) 10.09 -1.12 74

8h-EP-(I-26) .......... nil

Naloxone (b) 9.47 -1.16 18

(a) antagonist potency relative to the interaction of 8h-EP with ~h-EP-(I-27)

are calculated from the respective apparent association constants K 2 by the

relation logK 2 = pA 2

(b) taken from ref. 4,5.

curve of the agonist to the right. As shown in Figure 2, Bh-EP-

(1-26) failed to demonstrate any inhibitory effect even in high

doses. From dose-response obtained for etorphine and 8h-EP in

the presence of 8h-EP-(I-28), apparent AD50 and dose-ratio (x)

were calculated. Quantitation of the inhibitory effect was

assessed by Schild plots (inset of Figure 2). Linear

relationships resulted in both cases. The correspondent appar-

ent pA 2 values (Table 2) give the apparent antagonist potency

assuming the potency of Bh-EP-(I-27) against Bh-EP to be i00.

DISCUSSION

This study demonstrates that Bh-EP-(I-28) inhibits the

analgesic effect of 8h-EP and etorphine in a dose-related

fashion when co-injected into mice. This inhibition is

effective during the entire time-course of analgesia induced by

these agonists. Linear Schild plots were obtained in both cases

with slope very close to that expected (-i.0) for competitive

antagonism at similar receptors (13,14). Moreover, pA 2 values

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Vol. 127, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for antagonism of etorphine or 8h-EP by 8h-EP-(I-28) were found

not significantly different (p <0.01 by the t-test). This

argues for a close similarity within the opioid receptor

subtypes that mediate analgesia induced by etorphine or Bh-EP.

On a molar basis, 8h-EP-(I-28) is 9.6 times more potent than

naloxone and 2.2 times more potent than 8h-EP-(I-27) in

inhibiting analgesia elicited by Bh-EP or etorphine (Table 2).

In similar experiments, Bh-EP-(I-26) is inactive as antagonist

against 8h-EP or etorphine. The reduction of the chain length

starting from residues 1-28 to residues 1-27 and 1-26 led to a 2

times decrease and then to a complete loss of the antagonist

potency upon removal of residue Tyr-27 (Table 2). This was

further reinforced by the lack of ability of 8h-EP-(I-21) and

8h-EP-(I-17) to antagonize Sh-EP analgesia (results not shown).

Although Bh-EP-(I-28) , ~h-EP-(l-27) and Bh-EP-(I-26)

possess a similar B/A ratio (Table I), they do exhibit great

differences in antagonist potency. It thus appears that even

though a high B/A is required for effective antagonism,

structural features at the C-terminus of the molecule are

additionally needed. In that regard, residue Tyr-27 appears to

be of utmost importance since its deletion fully abolished the

antagonist potency while affecting only slightly both the

analgesic and binding potency. Previous reports on structure-

activity studies of 8-EP analogs have emphasized the importance

of that particular residue in affecting the biological efficacy

of 8-EP (15-17) while conformational studies (18) have shown

Lys-28 to be involved in maintaining an active conformation of

~EP in solution.

ACKNOWLEDGMENTS: This work was supported in part by the National Institute of Drug Abuse (DA-03434), National Institutes of Health (GM02907) and the Hormone Research Foundation. Permanent address of P.N.: Groupe de Neurobiochimie, Universite P. et M. Curie, 96 Bd Raspail, 75006 Paris, France.

654

Vol. 127, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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