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Opioid receptor binding profile of selected dermorphin-like peptides
Peptides. Vol. 7, pp. 755-759, 1986. ~ Ankho InternationalInc. Printed in the U.S.A.
0196-9781/86$3.00 + .00
Opioid Receptor Binding Profile of Selected Dermorphin-Like Peptides A. C. ROSSI, R. DE C A S T I G L I O N E ~ A N D G. P E R S E O C h e m i c a l a n d Biological R e s e a r c h a n d D e v e l o p m e n t , Farmitalia Carlo Erba S . p . A . , Milan, Italy R e c e i v e d 9 D e c e m b e r 1985 ROSSI, A. C., R. DE CASTIGLIONE AND G. PERSEO. Opioid receptor bmdin.~,, pr(ffile ~d selected dermorphin-like peptides. PEPTIDES 7(5) 755-759, 1986.~The receptor binding profile of a selected group of dermorphin-like peptides was determined and correlated with the results of the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays and with the currently used antinociception tests in the rat. For the peptides with the characteristic dermorphin D-Ala~-Phe:~-Gly4 sequence, a linear negative correlation was found between the reciprocal of sodium shift and relative affinity for the/x-type opioid receptor. For the same peptides, a positive correlation was evidenced between relative potency on GPI and MVD and relative affinity for/x- and 8-type receptors, respectively. Analgesia Dermorphins Guinea pig ileum Mouse vas deferens Receptor selectivity Sodium shift Structural correlations
Opioid peptides
Opioid receptors
Amersham; [:~Hl-naloxone (:~H-NLX) from New England Nuclear Corp. Studies on /z- and 8-type opioid receptors were carried out on cerebral membranes prepared from rat brain (minus cerebellum) according to the methods of Pert and Snyder [21] and of Stengaard-Pedersen and Larsson [22], respectively. For studies on K-type opioid receptors the membranes were prepared from guinea pig brain (minus cerebellum) following the method of Kosterlitz et al. [14], with minor modifications. The method of Goodman et al. [ 11], slightly modified, was followed for the binding of '~H-EKC (K-receptors). All binding studies were performed at 4°C. In order to calculate the "sodium shift" (or "'sodium index"), the binding of :~H-NLX was measured according to the method of Pert and Snyder [21], carrying out the experiments in the absence and presence of 100 mM NaCI in the incubation medium. The concentration of drug required to produce 5(F~ of inhibition of specific binding (IC5o) was obtained directly from inhibition curves. Inhibition curves were plotted with 9 different drug concentrations and each data point represented the mean_+SEM of at least 3 different experiments. Dose-response inhibition curves were analyzed by a LOGISTIC computer programme [12]. Relative affinities towards the different types of binding sites (irrespective of the animal species used) were calculated from the equation of McKnight el al. [17]:
DERMORPHIN is a well known opioid peptide originally isolated from the methanol extracts of the skin of the South American frog Phyllomedusa sauvagei [19]. The presence of a D-amino acid residue in its sequence, an occurrence rather unique in a peptide of non-bacterial origin, is posing an embarassing problem about its biosynthesis. The search of the mammalian counterpart, a usual procedure for amphibian skin peptides, has been carried out with immunological methods [2, 20, 23], but unequivocal evidence is still lacking. A number of analogues have been synthesized in several laboratories and assayed for opioid activity both in vitro and in vivo. Structure-activity relatonships have been recently reviewed [6,9]. To get an insight into the mode of action of these peptides at the receptor level, a representative group of dermorphin analogues has been chosen for binding tests. The results of these studies are presented in this paper and analyzed in comparison with the results of the bioassays.
METHOD Dermorphin-like peptides were prepared by chemical syntheses in solution as described elsewhere [7, 8, 18]. Results and methods for in vitro and in vivo bioassays were reported in previous papers [1, 3, 8, 18]. Binding studies on the three generally accepted opioid receptor types were performed by the Battelle Research Centres, Geneva, Switzerland, under a contract with Farmitalia Carlo Erba. [:~H]ID-Ala~,MePhe4-Gly-oPJ enkephalin (:~H-DAGO), [:~HJ-IDAla'-',D-Leu'~lenkephalin (:~H-DADL) and I:~Hl-ethylketocyclazocine I:~H-EKC) were from The Radiochemical Centre,
Relative affinity
(/z, 8 or K)
--
1/Kj (/.t, 8 or K) [I/K~ (/z) + I/K~ (8) + I/K~ (K)]
~Requests for reprints should be addressed to Roberto de Castiglione, Farmitalia Carlo Erba, Ricerca e Sviluppo-Via dei Gracchi 35, 20146 Milano, Italia.
755
756
ROSSI,
TABLE
DECASTIGLIONE
AND
PERSEO
1
R E L A T I V E M O L A R P O T E N C I E S OF D E R M O R P H I N - L I K E PEPTIDES IN VARIOUS I N V I T R O AND I N V I V O TESTS (IN P A R E N T H E S E S . D E R M O R P H I N ' S lC:,0 OR ED:,,,) 18, 9. 181
No. Peptide H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NH2 (dermorphin) 2. 3. 4.
5. 6. 7. 8. 9. 10.
H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-N HN HZ H - T y r - a l a - P h e - G l y - T y r - H y p - S e r - N He H-Tyr-ala-Phe-Gly-Tyr-A3Pro-Ser-NH~ Bzl Bzl
I
H-Tyr-ala-Phe-Gly-Tyr-Hyp-Ser-NHz H - T y r - a l a - G l y - P h e - T y r - P r o - S e r - N Hz H - T y r - A l a - P h e - G l y - T y r - P r o - S e r - N He H-Tyr-ala-Phe-Gly-Tyr- Pro- N H e H-Tyr-ala-Phe-Gly-Tyr-N H ~ H - T y r - a l a - P h e - G l y - N H.,
Hot Plate ICV
TailFlick ICV
Catalepsy ICV
TailPinch SC
I00 (23 pmol/rat) 27 75 15
100 (130 pmol/rat) 100 100 --
100 (1.03 mmol/kg) <25 98 49
---14 12 12
3 <0.5 <0.5 <0.5 0.5-1 <0.5
<25 <2 I <21 < 18 < 16 12
GPI
MVD
100 ( 3 . 3 x 10 '~M)
100 (29×10 'M)
120-160 85-90 90
150 85-90 100
I00 (13.3 pmol/rat) 120 I00 --
12-18 8-9 <0.03 44-52 40-45 2.5-3.5
300-1500 15-20 0.05-0.07 35-40 54-62 3-3.5
10 I <0.02 25 27 22
In t h i s a n d in t h e o t h e r t a b l e s , f o r a b e t t e r a l i g n m e n t D - A I a is r e p r e s e n t e d b y ala.
TABLE
2
M O L A R P O T E N C I E S tiCs0) O F V A R I O U S D E R M O R P H I N - L I K E PEPTIDES A N D S T A N D A R D C O M P O U N D S IN DISPLACING L A B E L L E D L I G A N D S FROM ,a-, 3- AND K-TYPE OPIOtD R E C E P T O R S [:~H]-NLX No.
Compound
1.
H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NHz (dermorphin) 2. H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NHNHZ 3. H-Tyr-ala-Phe-Gly-Tyr-Hyp-Ser-NH~ 4. H-Tyr-ala-Phe-Gly-Tyr-A3Pro-Ser-N H.,, Bz...ll Bz......~l
I
I
5. H-Tyr-ala-Phe-Gly-Tyr-Hyp- Ser- N H.., 6. H-Tyr-ala-Gly-Phe-Tyr-Pro-Ser-N H, 7. H -Tyr-Ala_...._-Phe-Gly-Tyr-Pro- Ser-N H., 8. H-Tyr-ala-Phe-Gly-Tyr-Pro-N H_.....~ 9. H-Tyr-ala-Phe-Gly-Tyr-NH~ 10. H-Tyr-ala-Phe-Gly-NH~ 11. H-Tyr-ala-Gly-MePhe-Gly-ol (DAGO) 12. naloxone (NLX) 13. H-Tyr-Gly-Gly-Phe-Leu-OH (Leu-enkeph.) 14. trifluadom 15. morphine
[:~H]DAGO
- NaCI
1.2±0.11x10 ~'
4.6±0.86x10 '
2 . 1 ± 0 . 2 0 x 1 0 ~' 1.4±0.21x10 " 2.4_+0.39x10 ~'
6.9+0.78x10 4.9_+0.57xl0 1.7±0.17x10 1.6+0.11xl0 9.8+0.87x10 1.5_+0.12x10 3 . 5 + 0 . 2 8 x 10
~' ~ " ' ,o s ~'
Sodium Shift
[:~H]DADL
[:'HIEKC
1.5±0.16x10 7
2.7±0.45x10 "
9.7±1.5 x l 0 " 3 . 4 ± 0 . 3 4 × 1 0 s 3.5 4.3_+0.70xl0 ~ 3.7+_0.57xl0 7 86 8.7±1.5 ×10 ~' 9.9±1.2 x l 0 " 11.4
2.8±0.33xi0 ~ 2.1±0.26×10 7 1.6±0.10×10 ;
5.5±0.64xl0 " 3 . 6 ± 0 . 5 8 x l 0 :' 2 . 4 ± 0 . 3 1 x l 0 :'
1.5±0.23x10 1.5_+0.20x10 4.7±0.60x10 8.7+1.5 ×10 5.9±0.9 x l 0 1.0_+0.12xl0
l.l±0.10xl0 1.7+0.16x10 >10 3.2+0.39×10 8.8-+0.86xl0 1.8+0.18x10
5.4±0.54x10 l.l+0.11xl0 >10 1.5+0.23×10 2.4-+0.30x10 1.0+0.21×10
+ NaCI
s ~ " ~' " 7
3.0±0.31 x 10 '
3 . 5 ± 0 . 6 4 x 1 0 7 76
3.8+0.4 8.9+2.0 6.7+1.0 6.0+1.2 5.6+1.2 7.4±4.0
×10 xl0 xl0 xl0 xl0 xl0
~ 7 ~ 7 : ~
2.5 59.3 14.3 69 95 74
1.8_+0.19x 10 ~'
0.6
~ s ~ 7 ~ '~
7 '; . :'
9.0 + 1.0 x It) " 4.3x10 ~ Ivs. 3H-naloxone)
5.1-+0.67x10 ~
1.5±0.14x10 '; 29.4
6.2×10 ~
7.5± 1.34× 10 "' 7.6x 10 7
L a b e l l e d l i g a n d s : 1.0 n M : H - [ D - A I a 2 , M e P h e ,Gly-oF'] e n k e p h a l i n ( D A G O ) a n d 1.0 n M :~H-naloxone ( N L X ) in the a b s e n c e a n d in the p r e s e n c e o f 100 n M N a C 1 ( ~ - s i t e ) , 1.5 n M S H - [ D - A l a 2 , D - L e u S ] e n k e p h a l i n ( D A D L ) ( & s i t e ) a n d 0 . 6 5 n M Z M - e t h y l k e t o c y c l a z o c i n e ( E K C ) (K-site) in r a t t/x- a n d & s i t e s ) a n d g u i n e a pig (K-site) b r a i n p r e p a r a t i o n s . T h e c o n c e n t r a t i o n o f d r u g r e q u i r e d to p r o d u c e 5 0 % i n h i b i t i o n o f s p e c i f i c b i n d i n g (IC50) is o b t a i n e d d i r e c t l y f r o m t h e i n h i b i t i o n c u r v e s (a f e w e x a m p l e s a r e r e p o r t e d in Fig. 11. T h e s o d i u m shift r e p r e s e n t s t h e r a t i o o f t h e ICs~ v a l u e o f a d r u g f o r t h e d i s p l a c e m e n t o f s p e c i f i c a H - n a l o x o n e b i n d i n g in t h e p r e s e n c e a n d a b s e n c e o f 100 m M N a C I . C o n c e n t r a t i o n s o f t h e r a d i o l i g a n d s h a v e b e e n c h o s e n so as to h a v e t h e s a m e v a l u e s o f t h e i r d i s s o c i a t i o n c o n s t a n t s (KD).
RECEPTOR SELECTIVITY
OF DERMORPHINS
757
©
® --
3H-DADL 3H-DAGO
..... ....
lO0"e~"~'~q"t"~
-
-
--
Compound 1
~ 50
{,\ ,,
o
;e
100-
",~ '6i ..
",
7 10
x\~ :\ \
~ 5o
.\
,t~:~ \ ~, \ \
•
QX \ '~
i t
\~("x \,
i0-I0 i0-9 10- 8 I0 -7 I0-6 10-5 10-4 10-3 10-2
"10-10 10-9 ~04 10~? 10-6 10-5 10- 4 10-3 10-2
[DRUG]. M
Trifiuadom Compound 1
....
X
\\
~-
~,
10-4 10-3 10-2
.....
\ \ \\
}
0 10aO 10-9 10-8 10-7 10-6 10.5
........-"'~'eg:k-:~ , % ~:~ \
"', \
,
50
9
100
't,,
{
Compound I ?
.......
DAGO
- -
3H-EKC
[LeuS] Enkephatin
[DRUG].M
[DRUG],M
FIG. 1. Inhibition of 1.0 nM aH-DAGO (A), 1.5 nM aH-DADL (B) and 0.65 nM aH-EKC (C) binding to rat (A and B) and guinea pig (C) brain membrane preparations by 9 different concentrations of selected unlabelled compounds. Results are expressed as percent of total binding. The experiments have been replicated three times.
TABLE 3 COMPARISON BETWEEN SODIUM SHIFT AND PERCENT RELATIVE AFFINITY FOR GPI/MVD PREPARATION AND/x/8/K-TYPE RECEPTOR BINDING
No.
Peptides
1.
H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NHe (dermorphin) 2. H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-N HN HZ 3. H-Tyr-ala-Phe-Gly-Tyr- Hyp-Ser-N He 4. H-Tyr-ala-Phe-Gly-Tyr-$aPrg-Ser-NH2 Bzl Bzl 5. 6. 7. 8. 9. 10.
I
I
H-Tyr-ala-Phe-Gly-Tyr-Hyp-Ser-N H._, H-Tyr-ala-Gly-Phe-Tyr-Pro-Ser-NH2 H-Ty r- Ala- Phe-Gly-Tyr-Pro-Ser-N H_, H-Tyr-ala-Phe-Gly-Tyr-Pro-N H2 H-Tyr-ala-Phe-Gly-Tyr-N He H-Tyr-ala-Phe-Gly-NH2
w h e r e the inhibition c o n s t a n t (KI) could b e c a l c u l a t e d from ICa, v a l u e s using the e q u a t i o n : Ki
-
1C:,,, 1 + c/K,
in w h i c h c was the c o n c e n t r a t i o n o f the r a d i o l i g a n d a n d Kt, its d i s s o c i a t i o n c o n s t a n t . In a similar way, relative p o t e n c i e s in inhibiting t h e electrically s t i m u l a t e d c o n t r a c t i o n s o f t h e g u i n e a pig ileum (GPI) a n d the m o u s e v a s d e f e r e n s ( M V D ) isolated o r g a n p r e p a r a tions were c a l c u l a t e d f r o m t h e formula: l/IC~0 (GPI or MVD) Relative potency (GPI or MVD) = [ l/ICa(, (GP1) + I/IC~o (MVD)]
Sodium Shift
GP1
MVD
/x
8
K
89.9
10.1
99.20
0.794
0.0044
76
89.3 89.9 88.9
10.7 10.1 I 1.1
92.99 99.33 98.51
6.974 0.662 1.48
0.035 0.0039 0.0098
3.5 86 11.4
12.7 81.0 . 87.8 87.0 88.9
87.3 19.0 . 12.2 13.0 11.1
60.97 77.36 . 99.49 98.89 99.16
0.78 0.34
2.5 59.3 14.3 69 95 74
.
38.25 22.30 . 0.497 1.10 0.83
0.01 0.004 0.015
C o r r e l a t i o n c u r v e s b e t w e e n s o d i u m shift a n d r e c e p t o r type selectivity, a n d b e t w e e n relative p o t e n c y o n G P I a n d M V D a n d / , - a n d 8-type r e c e p t o r s , r e s p e c t i v e l y , were o b t a i n e d b y a l i n e a r l e a s t - s q u a r e s c u r v e fitting p r o g r a m m e .
RESULTS AND DISCUSSION T h e t e n p e p t i d e s o f the p r e s e n t s t u d y were c h o s e n e i t h e r b e c a u s e o f t h e i r origin or s t r u c t u r e , or o n the b a s i s of the results of t h e b i o a s s a y s for opioid activity (Table 1). T h e s e p e p t i d e s are the t w o n a t u r a l p r o d u c t s , d e r m o r p h i n (No. 1) a n d [ H y p " ] d e r m o r p h i n (No. 3), a n d the c o r r e s p o n d i n g [A aPro ~] a n a l o g u e (No. 4); the s h o r t e r d e r m o r p h i n h o m o l o g u e s
758
ROSSI, DECASTIGLIONE AND PERSEO 100-
Q "0
•
•
9O I
-'=_= 80.Z-
70
°~ 60
2o
~o
60
Sodium
~
too
shift
FIG. 2. Correlation curve of percent relative affinity for g-type receptor versus sodium shift. (Data are taken from Table 3. The regression line Ix = A + B/SS (SS=sodium shift) with estimates A=101.55 and B= 76.32, has correlation coefficient r=-0.872 (p<0.01).
up to the tetrapeptides (Nos. 8-10); the [Gly:~,Phe~]analogue (No. 6) with the characteristic dermorphin Ph&-Gly ~ sequence inverted as in the enkephalins; the inactive [LAla'-']dermorphin (No. 7), initially supposed to be the natural opioid peptide [19]; an analogue (No. 2), which in some tests proved to be as active as or even more active than the parent compound; and finally, a dibenzylated heptapeptide (No. 5) displaying a large dissociation of potency in the GPI and MVD tests. The bioassays for opioid activity reported in Table 1 are the two aforementioned isolated organ preparations and the in vivo tests for analgesia (hot plate, tail-flick, tail-pinch) and for catalepsy in the rat, either by the intracerebroventricular (ICV) or subcutaneous (SC) route of administration. The results of binding assays are reported in Table 2, while a few displacement curves of selected compounds are exemplified in Fig. 1. "Sodium shift" (or "sodium index") is a good indicator of the agonistic or antagonistic properties of an opiate drug. Pure antagonists have sodium shift of 1 or less, pure agonists between 9 and 60. Drugs with agonistic/ antagonistic properties have intermediate sodium shifts 151. To get an insight into the mode of action of these peptides at the opioid receptor level, the experimental results of Table 2 are rearranged in Table 3 as percent relative affinity (selectivity) for/z/6/K binding sites, and compared with the in vitro data of Table l, expressed in the same way. Responses in
GPI and MVD bioassays are, in fact, presumed to be representative for the/.t- and 6-type opioid receptor interactions, respectively (although it is well known that GPI contains also K- and MVD/z- and K-type receptors [13]). For the peptides of Table 3 with the characteristic dermorphin D-Alae-Phe"-Gly ' sequence (Nos. 1-5 and 8-10) a linear correlation is found between percent relative affinity for the different receptor types and the reciprocal of sodium shift. This correlation is negative (level of significativity p<0.01, correlation coefficient r=0.872) for the /x-type receptor, and positive for the 6- and K-type receptors. By plotting percent relative affinity for ~-type receptor against sodium shift (Fig. 2), a steady increase of /z-site selectivity with sodium shift is observed only for sodium shift values <10-15. Higher sodium shift values (>15) appear uniformly linked to maximal percent relative affinity for /x-type receptors. The same compounds show a linear positive correlation (p<0.01, r=0.985) between relative potency on the GPI and MVD isolated organ preparations and /z- and 6-type selectivity, respectively. Antinociception activity is mainly associated with p~-type receptors, and a high ratio of potency in the GPI versus the MVD assay has been suggested as an indication of/x-type receptor specificity [4]. The results reported in the present paper (compare the relative potencies on isolated organ preparations of Table 3 with the antinociceptive activities of Table 1) are fairly in line with these assumptions. The observed discrepancies are probably due to other factors, such as absorption, distribution and enzymatic degradation. Dermorphin, with a sodium shift of 76 (Table 2), is apparently a much "purer" agonist than morphine (sodium shift 29.4 or 37 [4]), and, indeed, it is far more potent and longer lasting as an antinociceptive agent, at least after ICV administration [9]. The two analogues with lower sodium shifts (Nos. 5 and 2) should behave as mixed agonists/antagonists, but we could not yet test this hypothesis. Both displayed also an increased relative affinity for 6- and K-type opioid receptors, and a decrease in GPI/MVD potency ratio, particularly remarkable in the analogue No. 5. Antinociceptive activity (Table 1), on the whole, is also reduced, especially after systemic administration. Surprisingly, the pentapeptide No. 9 with the highest sodium shift (95), is much less analgesic than dermorphin, even after ICV administration, where events in blood and problems governing entry into the CNS should be circumvented. This would mean that analgesic potency is the result of many factors, agonistic properties being only one of them.
REFERENCES
1. Broccardo, M., V. Erspamer, G. Falconieri Erspamer, G. Improta, G. Linari, P. Melchiorri and P. C. Montecucchi. Pharmacological data on dermorphins, a new class of potent opioid peptides from amphibian skin. Br ,I Pharmacol 73: 625-631. 1981. 2. Buffa, R., E. Solcia, E. Magnoni, G. Rindi, L. Negri and P. Melchiorri. lmmunohistochemical demonstration of a dermorphin-like peptide in the brain. Histochemist~3" 76: 273276, 1982. 3. Cervini, M. A.. A. C. Rossi, G. Perseo and R. de Castiglione. Antinociceptive and other opioid effects of a new series of dermorphin analogues after subcutaneous administration in the rat. Peptides 6: 433-438. 1985.
4. Childers, S. R., 1. Creese, A. M. Snowman and S. H. Snyder. Opiate receptor binding affected differentially by opiates and opioid peptides. Eur ,I Pharmacol 55:11-18, 1979. 5. Creese, I. Differentiation of agonists from antagonists using receptor-binding assay. In: Neurotransmitter Receptor Binding, edited by H. J. Yamamura eta/. New York: Raven Press, 1978, pp. 163-166. de Castiglione. R. Structure-activity relationships in dermorphin-like peptides. In: tti~,,hlights in Receptor Chemi.~tO'. edited by C. Melchiorre and M. Giannella. Amsterdam: Elsevier Science Publishers B. V., 1984, pp. 149-168.
RECEPTOR
SELECTIVITY
OF DERMORPHINS
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759
17. Mc Night, A. T., A. D. Corbett, S. J. Paterson, J. Magnan and H. W. Kosterlitz. Comparison of in vitro potencies in pharmacological and binding assays after inhibition of peptidases reveals that dynorphin (1-9) is a potent ~-agonist. Life Sci 31: 1725-1728, 1982. 18. Melchiorri, P., G. Falconieri Erspamer, V. Erspamer, A. Guglietta, R. de Castiglione, F. Faoro, G. Perseo, S. Piani and F. Santangelo. Synthetic peptides related to the dermorphins, ii. Synthesis and biological activities of new analogues. Peptides 3: 745-748, 1982. 19. Montecucchi, P. C., R. de Castiglione, S. Piani, L. Gozzini and V. Erspamer. Amino acid composition and sequence of dermorphin, a novel opiate-like peptide from the skin of PhylIomedttsa sam'agei, lnt J Pept Protein Res 17: 275-283, 1981. 20. Negri, L., P. Melchiorri, G. Falconieri Erspamer and V. Erspamer. Radioimmunoassay of dermorphin-like peptides in mammalian and non-mammalian tissues. Peptides 2: Suppl 2, 45-49, 1981. 21. Pert, C. B. and S. H. Snyder. Opiate receptor binding. Enhancement by opiate administration itt vivo. Biochem Ph,rmacol 25: 847-853, 1976. 22. Stengaard-Pedersen, K. and L.-J. Larsson. Interaction of putative opioid peptides with opiate receptors. A¢'t~t Pharmacol Toxicol 48: 3%46, 1981. 23. Tsou, K., F.-S. Wang, S.-H. Wang and Y.-Q. Tang. Dermorphin-like immunoreactivity in guinea pig and rat stomach. Neuropeptides 5: 449--452, 1985.
0196-9781/86$3.00 + .00
Opioid Receptor Binding Profile of Selected Dermorphin-Like Peptides A. C. ROSSI, R. DE C A S T I G L I O N E ~ A N D G. P E R S E O C h e m i c a l a n d Biological R e s e a r c h a n d D e v e l o p m e n t , Farmitalia Carlo Erba S . p . A . , Milan, Italy R e c e i v e d 9 D e c e m b e r 1985 ROSSI, A. C., R. DE CASTIGLIONE AND G. PERSEO. Opioid receptor bmdin.~,, pr(ffile ~d selected dermorphin-like peptides. PEPTIDES 7(5) 755-759, 1986.~The receptor binding profile of a selected group of dermorphin-like peptides was determined and correlated with the results of the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays and with the currently used antinociception tests in the rat. For the peptides with the characteristic dermorphin D-Ala~-Phe:~-Gly4 sequence, a linear negative correlation was found between the reciprocal of sodium shift and relative affinity for the/x-type opioid receptor. For the same peptides, a positive correlation was evidenced between relative potency on GPI and MVD and relative affinity for/x- and 8-type receptors, respectively. Analgesia Dermorphins Guinea pig ileum Mouse vas deferens Receptor selectivity Sodium shift Structural correlations
Opioid peptides
Opioid receptors
Amersham; [:~Hl-naloxone (:~H-NLX) from New England Nuclear Corp. Studies on /z- and 8-type opioid receptors were carried out on cerebral membranes prepared from rat brain (minus cerebellum) according to the methods of Pert and Snyder [21] and of Stengaard-Pedersen and Larsson [22], respectively. For studies on K-type opioid receptors the membranes were prepared from guinea pig brain (minus cerebellum) following the method of Kosterlitz et al. [14], with minor modifications. The method of Goodman et al. [ 11], slightly modified, was followed for the binding of '~H-EKC (K-receptors). All binding studies were performed at 4°C. In order to calculate the "sodium shift" (or "'sodium index"), the binding of :~H-NLX was measured according to the method of Pert and Snyder [21], carrying out the experiments in the absence and presence of 100 mM NaCI in the incubation medium. The concentration of drug required to produce 5(F~ of inhibition of specific binding (IC5o) was obtained directly from inhibition curves. Inhibition curves were plotted with 9 different drug concentrations and each data point represented the mean_+SEM of at least 3 different experiments. Dose-response inhibition curves were analyzed by a LOGISTIC computer programme [12]. Relative affinities towards the different types of binding sites (irrespective of the animal species used) were calculated from the equation of McKnight el al. [17]:
DERMORPHIN is a well known opioid peptide originally isolated from the methanol extracts of the skin of the South American frog Phyllomedusa sauvagei [19]. The presence of a D-amino acid residue in its sequence, an occurrence rather unique in a peptide of non-bacterial origin, is posing an embarassing problem about its biosynthesis. The search of the mammalian counterpart, a usual procedure for amphibian skin peptides, has been carried out with immunological methods [2, 20, 23], but unequivocal evidence is still lacking. A number of analogues have been synthesized in several laboratories and assayed for opioid activity both in vitro and in vivo. Structure-activity relatonships have been recently reviewed [6,9]. To get an insight into the mode of action of these peptides at the receptor level, a representative group of dermorphin analogues has been chosen for binding tests. The results of these studies are presented in this paper and analyzed in comparison with the results of the bioassays.
METHOD Dermorphin-like peptides were prepared by chemical syntheses in solution as described elsewhere [7, 8, 18]. Results and methods for in vitro and in vivo bioassays were reported in previous papers [1, 3, 8, 18]. Binding studies on the three generally accepted opioid receptor types were performed by the Battelle Research Centres, Geneva, Switzerland, under a contract with Farmitalia Carlo Erba. [:~H]ID-Ala~,MePhe4-Gly-oPJ enkephalin (:~H-DAGO), [:~HJ-IDAla'-',D-Leu'~lenkephalin (:~H-DADL) and I:~Hl-ethylketocyclazocine I:~H-EKC) were from The Radiochemical Centre,
Relative affinity
(/z, 8 or K)
--
1/Kj (/.t, 8 or K) [I/K~ (/z) + I/K~ (8) + I/K~ (K)]
~Requests for reprints should be addressed to Roberto de Castiglione, Farmitalia Carlo Erba, Ricerca e Sviluppo-Via dei Gracchi 35, 20146 Milano, Italia.
755
756
ROSSI,
TABLE
DECASTIGLIONE
AND
PERSEO
1
R E L A T I V E M O L A R P O T E N C I E S OF D E R M O R P H I N - L I K E PEPTIDES IN VARIOUS I N V I T R O AND I N V I V O TESTS (IN P A R E N T H E S E S . D E R M O R P H I N ' S lC:,0 OR ED:,,,) 18, 9. 181
No. Peptide H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NH2 (dermorphin) 2. 3. 4.
5. 6. 7. 8. 9. 10.
H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-N HN HZ H - T y r - a l a - P h e - G l y - T y r - H y p - S e r - N He H-Tyr-ala-Phe-Gly-Tyr-A3Pro-Ser-NH~ Bzl Bzl
I
H-Tyr-ala-Phe-Gly-Tyr-Hyp-Ser-NHz H - T y r - a l a - G l y - P h e - T y r - P r o - S e r - N Hz H - T y r - A l a - P h e - G l y - T y r - P r o - S e r - N He H-Tyr-ala-Phe-Gly-Tyr- Pro- N H e H-Tyr-ala-Phe-Gly-Tyr-N H ~ H - T y r - a l a - P h e - G l y - N H.,
Hot Plate ICV
TailFlick ICV
Catalepsy ICV
TailPinch SC
I00 (23 pmol/rat) 27 75 15
100 (130 pmol/rat) 100 100 --
100 (1.03 mmol/kg) <25 98 49
---14 12 12
3 <0.5 <0.5 <0.5 0.5-1 <0.5
<25 <2 I <21 < 18 < 16 12
GPI
MVD
100 ( 3 . 3 x 10 '~M)
100 (29×10 'M)
120-160 85-90 90
150 85-90 100
I00 (13.3 pmol/rat) 120 I00 --
12-18 8-9 <0.03 44-52 40-45 2.5-3.5
300-1500 15-20 0.05-0.07 35-40 54-62 3-3.5
10 I <0.02 25 27 22
In t h i s a n d in t h e o t h e r t a b l e s , f o r a b e t t e r a l i g n m e n t D - A I a is r e p r e s e n t e d b y ala.
TABLE
2
M O L A R P O T E N C I E S tiCs0) O F V A R I O U S D E R M O R P H I N - L I K E PEPTIDES A N D S T A N D A R D C O M P O U N D S IN DISPLACING L A B E L L E D L I G A N D S FROM ,a-, 3- AND K-TYPE OPIOtD R E C E P T O R S [:~H]-NLX No.
Compound
1.
H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NHz (dermorphin) 2. H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NHNHZ 3. H-Tyr-ala-Phe-Gly-Tyr-Hyp-Ser-NH~ 4. H-Tyr-ala-Phe-Gly-Tyr-A3Pro-Ser-N H.,, Bz...ll Bz......~l
I
I
5. H-Tyr-ala-Phe-Gly-Tyr-Hyp- Ser- N H.., 6. H-Tyr-ala-Gly-Phe-Tyr-Pro-Ser-N H, 7. H -Tyr-Ala_...._-Phe-Gly-Tyr-Pro- Ser-N H., 8. H-Tyr-ala-Phe-Gly-Tyr-Pro-N H_.....~ 9. H-Tyr-ala-Phe-Gly-Tyr-NH~ 10. H-Tyr-ala-Phe-Gly-NH~ 11. H-Tyr-ala-Gly-MePhe-Gly-ol (DAGO) 12. naloxone (NLX) 13. H-Tyr-Gly-Gly-Phe-Leu-OH (Leu-enkeph.) 14. trifluadom 15. morphine
[:~H]DAGO
- NaCI
1.2±0.11x10 ~'
4.6±0.86x10 '
2 . 1 ± 0 . 2 0 x 1 0 ~' 1.4±0.21x10 " 2.4_+0.39x10 ~'
6.9+0.78x10 4.9_+0.57xl0 1.7±0.17x10 1.6+0.11xl0 9.8+0.87x10 1.5_+0.12x10 3 . 5 + 0 . 2 8 x 10
~' ~ " ' ,o s ~'
Sodium Shift
[:~H]DADL
[:'HIEKC
1.5±0.16x10 7
2.7±0.45x10 "
9.7±1.5 x l 0 " 3 . 4 ± 0 . 3 4 × 1 0 s 3.5 4.3_+0.70xl0 ~ 3.7+_0.57xl0 7 86 8.7±1.5 ×10 ~' 9.9±1.2 x l 0 " 11.4
2.8±0.33xi0 ~ 2.1±0.26×10 7 1.6±0.10×10 ;
5.5±0.64xl0 " 3 . 6 ± 0 . 5 8 x l 0 :' 2 . 4 ± 0 . 3 1 x l 0 :'
1.5±0.23x10 1.5_+0.20x10 4.7±0.60x10 8.7+1.5 ×10 5.9±0.9 x l 0 1.0_+0.12xl0
l.l±0.10xl0 1.7+0.16x10 >10 3.2+0.39×10 8.8-+0.86xl0 1.8+0.18x10
5.4±0.54x10 l.l+0.11xl0 >10 1.5+0.23×10 2.4-+0.30x10 1.0+0.21×10
+ NaCI
s ~ " ~' " 7
3.0±0.31 x 10 '
3 . 5 ± 0 . 6 4 x 1 0 7 76
3.8+0.4 8.9+2.0 6.7+1.0 6.0+1.2 5.6+1.2 7.4±4.0
×10 xl0 xl0 xl0 xl0 xl0
~ 7 ~ 7 : ~
2.5 59.3 14.3 69 95 74
1.8_+0.19x 10 ~'
0.6
~ s ~ 7 ~ '~
7 '; . :'
9.0 + 1.0 x It) " 4.3x10 ~ Ivs. 3H-naloxone)
5.1-+0.67x10 ~
1.5±0.14x10 '; 29.4
6.2×10 ~
7.5± 1.34× 10 "' 7.6x 10 7
L a b e l l e d l i g a n d s : 1.0 n M : H - [ D - A I a 2 , M e P h e ,Gly-oF'] e n k e p h a l i n ( D A G O ) a n d 1.0 n M :~H-naloxone ( N L X ) in the a b s e n c e a n d in the p r e s e n c e o f 100 n M N a C 1 ( ~ - s i t e ) , 1.5 n M S H - [ D - A l a 2 , D - L e u S ] e n k e p h a l i n ( D A D L ) ( & s i t e ) a n d 0 . 6 5 n M Z M - e t h y l k e t o c y c l a z o c i n e ( E K C ) (K-site) in r a t t/x- a n d & s i t e s ) a n d g u i n e a pig (K-site) b r a i n p r e p a r a t i o n s . T h e c o n c e n t r a t i o n o f d r u g r e q u i r e d to p r o d u c e 5 0 % i n h i b i t i o n o f s p e c i f i c b i n d i n g (IC50) is o b t a i n e d d i r e c t l y f r o m t h e i n h i b i t i o n c u r v e s (a f e w e x a m p l e s a r e r e p o r t e d in Fig. 11. T h e s o d i u m shift r e p r e s e n t s t h e r a t i o o f t h e ICs~ v a l u e o f a d r u g f o r t h e d i s p l a c e m e n t o f s p e c i f i c a H - n a l o x o n e b i n d i n g in t h e p r e s e n c e a n d a b s e n c e o f 100 m M N a C I . C o n c e n t r a t i o n s o f t h e r a d i o l i g a n d s h a v e b e e n c h o s e n so as to h a v e t h e s a m e v a l u e s o f t h e i r d i s s o c i a t i o n c o n s t a n t s (KD).
RECEPTOR SELECTIVITY
OF DERMORPHINS
757
©
® --
3H-DADL 3H-DAGO
..... ....
lO0"e~"~'~q"t"~
-
-
--
Compound 1
~ 50
{,\ ,,
o
;e
100-
",~ '6i ..
",
7 10
x\~ :\ \
~ 5o
.\
,t~:~ \ ~, \ \
•
QX \ '~
i t
\~("x \,
i0-I0 i0-9 10- 8 I0 -7 I0-6 10-5 10-4 10-3 10-2
"10-10 10-9 ~04 10~? 10-6 10-5 10- 4 10-3 10-2
[DRUG]. M
Trifiuadom Compound 1
....
X
\\
~-
~,
10-4 10-3 10-2
.....
\ \ \\
}
0 10aO 10-9 10-8 10-7 10-6 10.5
........-"'~'eg:k-:~ , % ~:~ \
"', \
,
50
9
100
't,,
{
Compound I ?
.......
DAGO
- -
3H-EKC
[LeuS] Enkephatin
[DRUG].M
[DRUG],M
FIG. 1. Inhibition of 1.0 nM aH-DAGO (A), 1.5 nM aH-DADL (B) and 0.65 nM aH-EKC (C) binding to rat (A and B) and guinea pig (C) brain membrane preparations by 9 different concentrations of selected unlabelled compounds. Results are expressed as percent of total binding. The experiments have been replicated three times.
TABLE 3 COMPARISON BETWEEN SODIUM SHIFT AND PERCENT RELATIVE AFFINITY FOR GPI/MVD PREPARATION AND/x/8/K-TYPE RECEPTOR BINDING
No.
Peptides
1.
H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NHe (dermorphin) 2. H-Tyr-ala-Phe-Gly-Tyr-Pro-Ser-N HN HZ 3. H-Tyr-ala-Phe-Gly-Tyr- Hyp-Ser-N He 4. H-Tyr-ala-Phe-Gly-Tyr-$aPrg-Ser-NH2 Bzl Bzl 5. 6. 7. 8. 9. 10.
I
I
H-Tyr-ala-Phe-Gly-Tyr-Hyp-Ser-N H._, H-Tyr-ala-Gly-Phe-Tyr-Pro-Ser-NH2 H-Ty r- Ala- Phe-Gly-Tyr-Pro-Ser-N H_, H-Tyr-ala-Phe-Gly-Tyr-Pro-N H2 H-Tyr-ala-Phe-Gly-Tyr-N He H-Tyr-ala-Phe-Gly-NH2
w h e r e the inhibition c o n s t a n t (KI) could b e c a l c u l a t e d from ICa, v a l u e s using the e q u a t i o n : Ki
-
1C:,,, 1 + c/K,
in w h i c h c was the c o n c e n t r a t i o n o f the r a d i o l i g a n d a n d Kt, its d i s s o c i a t i o n c o n s t a n t . In a similar way, relative p o t e n c i e s in inhibiting t h e electrically s t i m u l a t e d c o n t r a c t i o n s o f t h e g u i n e a pig ileum (GPI) a n d the m o u s e v a s d e f e r e n s ( M V D ) isolated o r g a n p r e p a r a tions were c a l c u l a t e d f r o m t h e formula: l/IC~0 (GPI or MVD) Relative potency (GPI or MVD) = [ l/ICa(, (GP1) + I/IC~o (MVD)]
Sodium Shift
GP1
MVD
/x
8
K
89.9
10.1
99.20
0.794
0.0044
76
89.3 89.9 88.9
10.7 10.1 I 1.1
92.99 99.33 98.51
6.974 0.662 1.48
0.035 0.0039 0.0098
3.5 86 11.4
12.7 81.0 . 87.8 87.0 88.9
87.3 19.0 . 12.2 13.0 11.1
60.97 77.36 . 99.49 98.89 99.16
0.78 0.34
2.5 59.3 14.3 69 95 74
.
38.25 22.30 . 0.497 1.10 0.83
0.01 0.004 0.015
C o r r e l a t i o n c u r v e s b e t w e e n s o d i u m shift a n d r e c e p t o r type selectivity, a n d b e t w e e n relative p o t e n c y o n G P I a n d M V D a n d / , - a n d 8-type r e c e p t o r s , r e s p e c t i v e l y , were o b t a i n e d b y a l i n e a r l e a s t - s q u a r e s c u r v e fitting p r o g r a m m e .
RESULTS AND DISCUSSION T h e t e n p e p t i d e s o f the p r e s e n t s t u d y were c h o s e n e i t h e r b e c a u s e o f t h e i r origin or s t r u c t u r e , or o n the b a s i s of the results of t h e b i o a s s a y s for opioid activity (Table 1). T h e s e p e p t i d e s are the t w o n a t u r a l p r o d u c t s , d e r m o r p h i n (No. 1) a n d [ H y p " ] d e r m o r p h i n (No. 3), a n d the c o r r e s p o n d i n g [A aPro ~] a n a l o g u e (No. 4); the s h o r t e r d e r m o r p h i n h o m o l o g u e s
758
ROSSI, DECASTIGLIONE AND PERSEO 100-
Q "0
•
•
9O I
-'=_= 80.Z-
70
°~ 60
2o
~o
60
Sodium
~
too
shift
FIG. 2. Correlation curve of percent relative affinity for g-type receptor versus sodium shift. (Data are taken from Table 3. The regression line Ix = A + B/SS (SS=sodium shift) with estimates A=101.55 and B= 76.32, has correlation coefficient r=-0.872 (p<0.01).
up to the tetrapeptides (Nos. 8-10); the [Gly:~,Phe~]analogue (No. 6) with the characteristic dermorphin Ph&-Gly ~ sequence inverted as in the enkephalins; the inactive [LAla'-']dermorphin (No. 7), initially supposed to be the natural opioid peptide [19]; an analogue (No. 2), which in some tests proved to be as active as or even more active than the parent compound; and finally, a dibenzylated heptapeptide (No. 5) displaying a large dissociation of potency in the GPI and MVD tests. The bioassays for opioid activity reported in Table 1 are the two aforementioned isolated organ preparations and the in vivo tests for analgesia (hot plate, tail-flick, tail-pinch) and for catalepsy in the rat, either by the intracerebroventricular (ICV) or subcutaneous (SC) route of administration. The results of binding assays are reported in Table 2, while a few displacement curves of selected compounds are exemplified in Fig. 1. "Sodium shift" (or "sodium index") is a good indicator of the agonistic or antagonistic properties of an opiate drug. Pure antagonists have sodium shift of 1 or less, pure agonists between 9 and 60. Drugs with agonistic/ antagonistic properties have intermediate sodium shifts 151. To get an insight into the mode of action of these peptides at the opioid receptor level, the experimental results of Table 2 are rearranged in Table 3 as percent relative affinity (selectivity) for/z/6/K binding sites, and compared with the in vitro data of Table l, expressed in the same way. Responses in
GPI and MVD bioassays are, in fact, presumed to be representative for the/.t- and 6-type opioid receptor interactions, respectively (although it is well known that GPI contains also K- and MVD/z- and K-type receptors [13]). For the peptides of Table 3 with the characteristic dermorphin D-Alae-Phe"-Gly ' sequence (Nos. 1-5 and 8-10) a linear correlation is found between percent relative affinity for the different receptor types and the reciprocal of sodium shift. This correlation is negative (level of significativity p<0.01, correlation coefficient r=0.872) for the /x-type receptor, and positive for the 6- and K-type receptors. By plotting percent relative affinity for ~-type receptor against sodium shift (Fig. 2), a steady increase of /z-site selectivity with sodium shift is observed only for sodium shift values <10-15. Higher sodium shift values (>15) appear uniformly linked to maximal percent relative affinity for /x-type receptors. The same compounds show a linear positive correlation (p<0.01, r=0.985) between relative potency on the GPI and MVD isolated organ preparations and /z- and 6-type selectivity, respectively. Antinociception activity is mainly associated with p~-type receptors, and a high ratio of potency in the GPI versus the MVD assay has been suggested as an indication of/x-type receptor specificity [4]. The results reported in the present paper (compare the relative potencies on isolated organ preparations of Table 3 with the antinociceptive activities of Table 1) are fairly in line with these assumptions. The observed discrepancies are probably due to other factors, such as absorption, distribution and enzymatic degradation. Dermorphin, with a sodium shift of 76 (Table 2), is apparently a much "purer" agonist than morphine (sodium shift 29.4 or 37 [4]), and, indeed, it is far more potent and longer lasting as an antinociceptive agent, at least after ICV administration [9]. The two analogues with lower sodium shifts (Nos. 5 and 2) should behave as mixed agonists/antagonists, but we could not yet test this hypothesis. Both displayed also an increased relative affinity for 6- and K-type opioid receptors, and a decrease in GPI/MVD potency ratio, particularly remarkable in the analogue No. 5. Antinociceptive activity (Table 1), on the whole, is also reduced, especially after systemic administration. Surprisingly, the pentapeptide No. 9 with the highest sodium shift (95), is much less analgesic than dermorphin, even after ICV administration, where events in blood and problems governing entry into the CNS should be circumvented. This would mean that analgesic potency is the result of many factors, agonistic properties being only one of them.
REFERENCES
1. Broccardo, M., V. Erspamer, G. Falconieri Erspamer, G. Improta, G. Linari, P. Melchiorri and P. C. Montecucchi. Pharmacological data on dermorphins, a new class of potent opioid peptides from amphibian skin. Br ,I Pharmacol 73: 625-631. 1981. 2. Buffa, R., E. Solcia, E. Magnoni, G. Rindi, L. Negri and P. Melchiorri. lmmunohistochemical demonstration of a dermorphin-like peptide in the brain. Histochemist~3" 76: 273276, 1982. 3. Cervini, M. A.. A. C. Rossi, G. Perseo and R. de Castiglione. Antinociceptive and other opioid effects of a new series of dermorphin analogues after subcutaneous administration in the rat. Peptides 6: 433-438. 1985.
4. Childers, S. R., 1. Creese, A. M. Snowman and S. H. Snyder. Opiate receptor binding affected differentially by opiates and opioid peptides. Eur ,I Pharmacol 55:11-18, 1979. 5. Creese, I. Differentiation of agonists from antagonists using receptor-binding assay. In: Neurotransmitter Receptor Binding, edited by H. J. Yamamura eta/. New York: Raven Press, 1978, pp. 163-166. de Castiglione. R. Structure-activity relationships in dermorphin-like peptides. In: tti~,,hlights in Receptor Chemi.~tO'. edited by C. Melchiorre and M. Giannella. Amsterdam: Elsevier Science Publishers B. V., 1984, pp. 149-168.
RECEPTOR
SELECTIVITY
OF DERMORPHINS
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759
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