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A new peptide from Crotalus atrox snake venom
Pcptide~, Vol. 6. Suppl. 3. pp. 3a-z-346. 1985. ~ AnkhoInternationalInc. Printed in the U.S.A.
0196-9781 85 $3.tK) + .00
A N e w Peptide From Crotalus atrox Snake Venom V I N C E N Z O P O L I T I , G I O V A N N A D E L U C A , G I O V A N N I DI S T A Z I O , EUGENIA SCHININA'* AND FRANCESCO BOSSA*
Polifarma Research Centre. Via Tor Sapienza, 138-00155 R o m a , Italy *lstituto di Chimica Biologica e Centro di Biologia, Molecolare del C N R Universitfi di R o m a " L a S a p i e n z a , " 00185 Roma, Italy
POLITI. V., G. DE LUCA. G. DI STAZIO, E. SCHININA" AND F. BOSSA. A new peptide/i'om Crotalus atrox snake vcm,n. PEPTIDES 6: Suppl. 3.343-346, 1985.~ The presence of new hypotensive peptides, possibly not related to ACE inhibition, has been investigated on 66 snake venoms from crotalid, viperid and elapid families. Only the venom of Crotalus atr,~x showed a substantial amount of a new decapeptide, called POL-236. with the following aminoacid sequence: PYRLEU-TRP-PRO-ARG-PRO-GLN-1LE-PRO-PRO. Pharmacological assays performed on the synthesized peptide revealed effects on blood pressure, probably derived from vascular and cardiac interferences. Snake venoms
Crotalusatrox small peptides
ACE-inhibitors
Hypertension
Heart
METHOD
S N A K E venoms are very complex mixtures, with absolute predominance of enzymes and peptides, possibly related to two main functions: immobilization of the prey before it can escape, and partial digestion of the swallowed animal through disruption of its tissues. Digestive enzymes are quite common in the venomous secretions of all the well studied snake species, while peptides are apparently aimed to block vital processes in the bitten animal. Elapids and sea snakes, believed to be the oldest poisonous snake families, produce a large amount of curaremimetic neurotoxins that act on cholinergic neuromuscular junctions. So far. more than 40 neurotoxins have been purified and characterized [4]. They are medium-sized peptides, consisting of 62 to 78 aminoacid residues. On the other hand, crotalids and viperids, the other two important venomous snake families, produce many small peptides [5, 8, 9]. In general, these peptides have been named "'Bradykinin potentiating" or "Angiotensin-Coverting Enzyme (ACE) inhibitors'" [3, 6.8, 10]. Essential work on peptido-mimetic synthetic substances derived from ACE-inhibitors a few years ago led to the development of a new class of antihypertensive drugs [7]. Unfortunately, however, the success in chemical modification of peptides stopped a thorough investigation of the possible significance of many analogous peptides found in the same snake venom. We decided therefore to resume the project aimed at the discovery of new hypotensive peptides in snake venoms, possibly not related to ACE inhibition.
Sixty-six dried snake venoms, obtained mainly from Sigma Chemical (sample of Agkistrodon hairs pallas was a gift from Prof. Chi, Shanghai), were suspended in water (10 mg/ml) and ultrafiltered through Amicon YM-10 filters, in order to separate enzymes and neurotoxins from small peptides. Five ml of the filtered solutions were put on a Sephadex G-15 column l x l 0 0 cm, and eluted with water. Pharmacological screenings were performed as follows: male wistar rats, weighing 200-300 g, were anesthetized with ethyl urethane. After incannulation of the trachea, the right carotid artery was isolated and connected by means of a cannula to a transducer for blood pressure. 0.05 ml of the snake venom solution (filtered through YM-10 or recovered from G-15 peaks) was injected into the right femoral vein, and the blood pressure recorded. RESULTS Nine out of 66 filtered snake venoms showed an immediate hypotensive effect when injected in normotensive rats (Table I). Five of them were venoms from viperids (4 Bitis and 1 Echis species), 3 from elapids (Dendroaspis species), I from crotalids (Crotalus atrox). It is interesting to note that only Crotalus atrox venom had some peptide-like components in the chromatographic peak resulted hypotensire. It must be stressed, however, that many other crotalid snake venoms have hypotensive peptide-like materials, even if in smaller amount than Crotalus atrox. Figure 1 shows
~This work was partially supported by Italian National Research Council (CNR).
343
344
POL1TI
El
AL.
TABLE 1 HYPOTENSIVE ACTIVITYFOUND IN ULTRAFILTEREDVENOMS Snake Species
A
B
Agkistrodon bilineatus
-
C r o t a l u s viridis helleri
Agkistrodon contortrix contortrix
-
C r o t a l u s viridis ort, gatttls
Agkistrodon contortrix laticinctus Agkistrodon contortrix mokason
-
C r o t a l u s viridis viridis Dendroaspis angusticeps
+
Agkistrodon halys blomhoffii
-
Dendroaspis jamesonii
+
-
Agkistrodon halys pallas
-
Dendroaspis polylepis
+
-
Agkistrodon piscivorus leukostoma
-
Echis carinatus
+
±
Agkistrodon piscivorus piscivorus Agkistrodon rhodostoma
-
Micrurtts fulvitts Naja haje
-
Austrelaps superba Bitis a r i e t a n s
+
-
N a j a h a j e annulifi,ra Naja melanoleuca
-
Bitis c a u d a l i s Bitis g a b o n i c a Bitis n a s i c o r n u s
+ + +
-
Naja mossambica Naja naja N a j a m~ja atra
-
Bothrops alternata Bothrops atrox Bothrops cotiara
-
Naja naja kaouthia N a j a naja o x i a n a Naja naja sputatrix
-
Bothrops jararaca Bothrops jararacussu
-
N a j a nigricollis c r a w s h a w f f N a j a nigricollis nigricol/is
-
Bothrops medusa
-
N a j a nigricollis pallida
-
Bothrops neuwiedi Bothrops nummifer Bungarus fasciatus Causus rhombeatus Crotalus a d a m a n t e u s
-
Naja nivea Notechis ater niger Ophiophagus hannah P s e u d e c h i s austra/is Pseudechis porphyriacus
-
Crotalus atrox Crotalus basiliscus
+ -
Sepedon hemachatus Trhneresurus albolabris
-
Crotalus durissus terrificus Crotalus horridus atricaudatus Crotalus horridus horridus
-
Trimeresurus flavoviridis Trbneresurus okinavensis Trimeresurus popeorum
-
Crotalus molossus molossus Crotalus ruber Crotalus scutulatus
-
Trimeresurus purpureomaculatus Vipera a m m o d y t e s V i p e r a russel/i
-
+
Snake Species
A
B
-
-
-
-
A=Presence (+) or absence ( - ) of hypotensive activity. B=Presence (+) or absence ( - ) of peptide-like materials in hypotensive peaks.
t
TRIMERESURUS ;"-""v' " OKINAVENSIS
," f
f L,'~ ':
CROTALUS DURISSUS
VIPERA RUSSELLI '
::
NAIA
HAlE
T ERRIFICUS
,Y
FIG. I. Sephadex G-15 elution profile of ultrafiltered snake venoms. followed at OD 210 (.... ) and 280 ( ) nm.
typical elution profiles of elapid ( N a j a h a j e ) , viperid (~'ipera and crotalid ( T r i m e r e s u r u s o k i n a v e n s i s and C r o t a / u s t e r r i f i c u s ) snake venoms. Peptide-like compounds are particularly abundant in crotalid and viperid venoms. while they are lacking in elapid venoms. A noteworthy exception is the venom of C r o t a l u s d u r i s s u s t e r r i f i c t t s , a crotalid with an elution profile similar to elapid venoms. Because C r o t a l u s d u r i s s u s t e r r i f i c u s produces some highly toxic neurotoxins, it is possible that small peptides in crotalids and viperids could be evolutive products from bigger peptides acting on membranes. The G-15 elution profile of C r o t a l u s a t r o x ultrafiltered venom shows three main peaks (Fig. 2). Because the hypotensive activity was concentrated in the second peak. this was subjected to HPLC (Fig. 3), using an RP-8 column. A rather pure peptide was obtained, with considerable hypotensive properties. It was methanolized, in order to open the pyroglutamic acid present as amino-terminal residue, and sequenced using the Edman-dansyl manual degrarusse/li) durissus
NE\\
SNAKE VENOM PEPTIDE TABLE
345
2
!fl: ' ,
SUMMARY OF POL-236 PHARMACOLOGICAL ACTIVITIES
Assay ACE inhibition
"'in
Effect vitro"
C~©TALUS ATROX
1C-~,, = 3.3 x 10-" M
ACE inhibition "in vivo'"
Reduction of hypertensive activity of Angiotensin 1
Blood pressure in anesthetized rats
Rapid and short decrease of blood pressure Potentiation of bradykinin effect (abolished by indomethacinl
Isolated rat Heart
negative inotropic and chronotropic activities. Coronary flux reduction
Isolated guinea-pig ileum
Potentiation of Bradykinin activity No effect on acetylcholine
Blood pressure in hypertension: models on freely moving rats
Decrease of blood pressure for more than t~vo hours
Blood pressure follox~ing ICV injection in rats
Slight increase in blood pressure
$ FIG. 2. Sephadex G-15 elution profile o f C r o t a l u s atrox ultra-filtered venom, followed at OD 210 (.... ) and 280 ( J nm. Arrow indicates the most effective hypotensive peak.
FIG. 3. HPLC on RP-8 column of Sephadex G-15 h) potensive peak from C r , , t , l u s atrox ultrafiltered venom, followed at 280 nm OD. Arrow indicates the most effective hypotensive compound(s).
TABLE
3
STRUCTURAL SIMILARITIES BETWEEN POL-236 AND OTHER NATURAL PEPTIDES
POL-236 SQ-20861 SQ-20881 Neurotensin
LH-RH Phyllolitorin Litorin Tryptophyllin
PYR-LEU-TRP - PRO-ARG-PRO-GLN-ILE-PRO-PRO PYR-ASN-TRP - PRO-ARG-PRO-GLN-ILE-PRO-PRO PYR-TRP- PRO-ARG-PRO-GLN-ILE-PRO-PRO PYR-LEU-TRP - GLU-ASN-LYS-PRO-ARG-ARG-PRO ............. PYR-HIS-TRP - SER-TYR-GLY-LEU-ARG-PRO-GLY PYR-LEU-TRP- ALA-VAL-GLY-SER-PHE-MET-NH: PYR-LEU-TRP- ALA-THR-GLY-H1S-PHE-MET-NHz PYR-PRO-TRP - MET-NH:
dation technique. The peptide was identified as a c o m p o u n d with 10 aminoacid residues, and named POL-236: PYRL E U - T R P - P R O - A R G - P R O - G LN-I L E - P R O - P R O . A ,ubslantial amount of POL-236 has been synthesized. in o r d e r to determine its pharmacological activities (Table 2). On A C E . the decapeptide shows an effect similar to o t h e r peptides present in snake v e n o m s 12], but many times losver than ,3nthetic c o m p o u n d s like captopril [I]. This suggests lh~fl probably A C E inhibition is not the most important effect of POL-236. The immediale and short-lasting effect on anesthetized rats. as well as the bradykinin potentiating activitx, are terminated by indomethacin, so that they can be mediated b\ some kind of prostaghmdins. On isolated rat's heart, the decapeptide induces slowing down effects, such as negatixe inotropic and chronotropic activities and c o r o n a o flux reduction. It is the first time that cardiac effects have been obserxed in small peptides fi'om snake v e n o m s and. apparentl,,, they can justify the long-lasting hypotensive efIk'ct shoxved by POL-236 in experimental models of
hypotension (in S p o n t a n e o u s l y H y p e r t e n s i v e rats. in rats made hypertensive by D O C A and in Goldblatt hy pertensive rats). The results obtained by injecting POL-236 directly into the C N S confirm that the hypotension is not centrally mediated. DISCUSSION
Viperid and crotalid snake v e n o m s are extraordinarily rich sources of small peptides, but only a few of them have been characterized until now. It is unfortunate that, due to their supposed activity as "'Bradykinin potentiators'" or " ' A C E inhibitors,'" they were gradually superseded by peplido-mimetic synthetic analogs. In fact. we found that POL-236. a decapeptide newly d i s c o v e r e d in the v e n o m of Crotalu.s u t r o x snake, e v e n if it is structurally very similar to other peptides found in B o t h r o p . s j a r a r a c a snake v e n o m (Table 3). shows a pharmacological profile hardly related to ACE-inhibition alone. On the whole, results obtained in
346
POLITI ET AL.
pharmacological assays suggest that POL-236 can bind to vascular receptors (possibly associated to bradykinin binding sites), stimulating a phospholipase A2 and the release of prostaglandins. The resulting vasodilation is potentiated by a long-lasting depressive effect on the cardiac muscle, so that a profound hypotension might ensue, sometimes leading to animal death (hypotension is observed frequently in animals bitten by crotalid snakes). Of course, the decapeptide is also able to inhibit ACE, but tile activity is not very strong, and therefore one can speculate that it is a secondary effect due to -PRO-PRO C-terminal dipeptide. Interestingly enough, POL-236 has structure analogies not only with other snake venoms, but also with frog skin
and some mammalian endogenous peptides (Table 3). This suggests that the decapeptide from Crotalus atrox snake venom could have other hitherto undiscovered pharmacological effects. In fact, in Prof. Melchiorri's Laboratory in Rome it has been demonstrated that POL-236 can inhibit gastric secretion (unpublished results). In conclusion, snake venoms are a huge factory of small. pharmacologically active peptides. Contrary to endogenous neuro-gastrointestinal hormones, they have to be injected from outside, in order to have a strong effect on the host's physiological responses. It is possible, therefore, that they could become very useful pharmacological tools, and possibly new drugs.
REFERENCES
1. Antonaccio, M. J. Angiotensin Converting Enzyme (ACE) inhibitors. Annu Rev Pharmacol 22: 57-87, 1982. 2. Cheung, H. S. and D. W. Cushman. Inhibition of homogeneous angiotensin-converting enzyme of rabbit lung by synthetic venom peptides of Bothrops Jararaca. Biochim Biophys Acta 293: 451-463, 1973. 3. Ferreira, S. H., D. C. Bartelt and L. J. Greene. Isolation of bradykinin-potentiating peptides from Bothrops Jararaca venom. Biochemiso T 9: 2583-2593, 1970. 4. Karlsson, E. Chemistry of protein toxins in snake venom. In: Handbook ofExptl Pharmacol, Vol 52. Berlin: Springer-Verlag, 1979, pp. 15%212. 5. Kato, H., S. lwanaga and T. Suzuki. The isolation and amino acid sequences of new pyroglutamylpeptides from snake venoms. Experientia 22: 4%50, 1966. 6. Kato, H. and T. Suzuki. Bradykinin potentiating peptides from the venom of Agkistrodon halys blomhoffii. Biochemistry 10: 972-980, 1971.
7. Ondetti, M. A., B. Rubin and D. W. Cushman. Design of specific inhibitors of angiotensin-converting enzyme: New class of orally active antihypertensive agents. Science 196: 441-444. 1977. 8. Ondetti, M. A., N. J. Williams, E. F. Sabo et a/. Angiotensinconverting enzyme inhibitors from the venom of Bothrops jararac'a. Isolation, elucidation of structure, and synthesL,. Biochemistry 10: 4033-4039, 1971. 9. Shipolini, R., C. P. lvanov, G. Dimitrov and B. V. Alexiex. Composition of the low molecular fraction of the Bulgarian viper venom. Biochim Biophys Acta 104: 292-295, 1965. 10. Wang. M., S. Luo and Z. Qi. Studies on bradykinin potentiating peptide (BPP) from venom of Zhejiang pit viper. Sci Sin [B] 26: 716--724, 1983.
0196-9781 85 $3.tK) + .00
A N e w Peptide From Crotalus atrox Snake Venom V I N C E N Z O P O L I T I , G I O V A N N A D E L U C A , G I O V A N N I DI S T A Z I O , EUGENIA SCHININA'* AND FRANCESCO BOSSA*
Polifarma Research Centre. Via Tor Sapienza, 138-00155 R o m a , Italy *lstituto di Chimica Biologica e Centro di Biologia, Molecolare del C N R Universitfi di R o m a " L a S a p i e n z a , " 00185 Roma, Italy
POLITI. V., G. DE LUCA. G. DI STAZIO, E. SCHININA" AND F. BOSSA. A new peptide/i'om Crotalus atrox snake vcm,n. PEPTIDES 6: Suppl. 3.343-346, 1985.~ The presence of new hypotensive peptides, possibly not related to ACE inhibition, has been investigated on 66 snake venoms from crotalid, viperid and elapid families. Only the venom of Crotalus atr,~x showed a substantial amount of a new decapeptide, called POL-236. with the following aminoacid sequence: PYRLEU-TRP-PRO-ARG-PRO-GLN-1LE-PRO-PRO. Pharmacological assays performed on the synthesized peptide revealed effects on blood pressure, probably derived from vascular and cardiac interferences. Snake venoms
Crotalusatrox small peptides
ACE-inhibitors
Hypertension
Heart
METHOD
S N A K E venoms are very complex mixtures, with absolute predominance of enzymes and peptides, possibly related to two main functions: immobilization of the prey before it can escape, and partial digestion of the swallowed animal through disruption of its tissues. Digestive enzymes are quite common in the venomous secretions of all the well studied snake species, while peptides are apparently aimed to block vital processes in the bitten animal. Elapids and sea snakes, believed to be the oldest poisonous snake families, produce a large amount of curaremimetic neurotoxins that act on cholinergic neuromuscular junctions. So far. more than 40 neurotoxins have been purified and characterized [4]. They are medium-sized peptides, consisting of 62 to 78 aminoacid residues. On the other hand, crotalids and viperids, the other two important venomous snake families, produce many small peptides [5, 8, 9]. In general, these peptides have been named "'Bradykinin potentiating" or "Angiotensin-Coverting Enzyme (ACE) inhibitors'" [3, 6.8, 10]. Essential work on peptido-mimetic synthetic substances derived from ACE-inhibitors a few years ago led to the development of a new class of antihypertensive drugs [7]. Unfortunately, however, the success in chemical modification of peptides stopped a thorough investigation of the possible significance of many analogous peptides found in the same snake venom. We decided therefore to resume the project aimed at the discovery of new hypotensive peptides in snake venoms, possibly not related to ACE inhibition.
Sixty-six dried snake venoms, obtained mainly from Sigma Chemical (sample of Agkistrodon hairs pallas was a gift from Prof. Chi, Shanghai), were suspended in water (10 mg/ml) and ultrafiltered through Amicon YM-10 filters, in order to separate enzymes and neurotoxins from small peptides. Five ml of the filtered solutions were put on a Sephadex G-15 column l x l 0 0 cm, and eluted with water. Pharmacological screenings were performed as follows: male wistar rats, weighing 200-300 g, were anesthetized with ethyl urethane. After incannulation of the trachea, the right carotid artery was isolated and connected by means of a cannula to a transducer for blood pressure. 0.05 ml of the snake venom solution (filtered through YM-10 or recovered from G-15 peaks) was injected into the right femoral vein, and the blood pressure recorded. RESULTS Nine out of 66 filtered snake venoms showed an immediate hypotensive effect when injected in normotensive rats (Table I). Five of them were venoms from viperids (4 Bitis and 1 Echis species), 3 from elapids (Dendroaspis species), I from crotalids (Crotalus atrox). It is interesting to note that only Crotalus atrox venom had some peptide-like components in the chromatographic peak resulted hypotensire. It must be stressed, however, that many other crotalid snake venoms have hypotensive peptide-like materials, even if in smaller amount than Crotalus atrox. Figure 1 shows
~This work was partially supported by Italian National Research Council (CNR).
343
344
POL1TI
El
AL.
TABLE 1 HYPOTENSIVE ACTIVITYFOUND IN ULTRAFILTEREDVENOMS Snake Species
A
B
Agkistrodon bilineatus
-
C r o t a l u s viridis helleri
Agkistrodon contortrix contortrix
-
C r o t a l u s viridis ort, gatttls
Agkistrodon contortrix laticinctus Agkistrodon contortrix mokason
-
C r o t a l u s viridis viridis Dendroaspis angusticeps
+
Agkistrodon halys blomhoffii
-
Dendroaspis jamesonii
+
-
Agkistrodon halys pallas
-
Dendroaspis polylepis
+
-
Agkistrodon piscivorus leukostoma
-
Echis carinatus
+
±
Agkistrodon piscivorus piscivorus Agkistrodon rhodostoma
-
Micrurtts fulvitts Naja haje
-
Austrelaps superba Bitis a r i e t a n s
+
-
N a j a h a j e annulifi,ra Naja melanoleuca
-
Bitis c a u d a l i s Bitis g a b o n i c a Bitis n a s i c o r n u s
+ + +
-
Naja mossambica Naja naja N a j a m~ja atra
-
Bothrops alternata Bothrops atrox Bothrops cotiara
-
Naja naja kaouthia N a j a naja o x i a n a Naja naja sputatrix
-
Bothrops jararaca Bothrops jararacussu
-
N a j a nigricollis c r a w s h a w f f N a j a nigricollis nigricol/is
-
Bothrops medusa
-
N a j a nigricollis pallida
-
Bothrops neuwiedi Bothrops nummifer Bungarus fasciatus Causus rhombeatus Crotalus a d a m a n t e u s
-
Naja nivea Notechis ater niger Ophiophagus hannah P s e u d e c h i s austra/is Pseudechis porphyriacus
-
Crotalus atrox Crotalus basiliscus
+ -
Sepedon hemachatus Trhneresurus albolabris
-
Crotalus durissus terrificus Crotalus horridus atricaudatus Crotalus horridus horridus
-
Trimeresurus flavoviridis Trbneresurus okinavensis Trimeresurus popeorum
-
Crotalus molossus molossus Crotalus ruber Crotalus scutulatus
-
Trimeresurus purpureomaculatus Vipera a m m o d y t e s V i p e r a russel/i
-
+
Snake Species
A
B
-
-
-
-
A=Presence (+) or absence ( - ) of hypotensive activity. B=Presence (+) or absence ( - ) of peptide-like materials in hypotensive peaks.
t
TRIMERESURUS ;"-""v' " OKINAVENSIS
," f
f L,'~ ':
CROTALUS DURISSUS
VIPERA RUSSELLI '
::
NAIA
HAlE
T ERRIFICUS
,Y
FIG. I. Sephadex G-15 elution profile of ultrafiltered snake venoms. followed at OD 210 (.... ) and 280 ( ) nm.
typical elution profiles of elapid ( N a j a h a j e ) , viperid (~'ipera and crotalid ( T r i m e r e s u r u s o k i n a v e n s i s and C r o t a / u s t e r r i f i c u s ) snake venoms. Peptide-like compounds are particularly abundant in crotalid and viperid venoms. while they are lacking in elapid venoms. A noteworthy exception is the venom of C r o t a l u s d u r i s s u s t e r r i f i c t t s , a crotalid with an elution profile similar to elapid venoms. Because C r o t a l u s d u r i s s u s t e r r i f i c u s produces some highly toxic neurotoxins, it is possible that small peptides in crotalids and viperids could be evolutive products from bigger peptides acting on membranes. The G-15 elution profile of C r o t a l u s a t r o x ultrafiltered venom shows three main peaks (Fig. 2). Because the hypotensive activity was concentrated in the second peak. this was subjected to HPLC (Fig. 3), using an RP-8 column. A rather pure peptide was obtained, with considerable hypotensive properties. It was methanolized, in order to open the pyroglutamic acid present as amino-terminal residue, and sequenced using the Edman-dansyl manual degrarusse/li) durissus
NE\\
SNAKE VENOM PEPTIDE TABLE
345
2
!fl: ' ,
SUMMARY OF POL-236 PHARMACOLOGICAL ACTIVITIES
Assay ACE inhibition
"'in
Effect vitro"
C~©TALUS ATROX
1C-~,, = 3.3 x 10-" M
ACE inhibition "in vivo'"
Reduction of hypertensive activity of Angiotensin 1
Blood pressure in anesthetized rats
Rapid and short decrease of blood pressure Potentiation of bradykinin effect (abolished by indomethacinl
Isolated rat Heart
negative inotropic and chronotropic activities. Coronary flux reduction
Isolated guinea-pig ileum
Potentiation of Bradykinin activity No effect on acetylcholine
Blood pressure in hypertension: models on freely moving rats
Decrease of blood pressure for more than t~vo hours
Blood pressure follox~ing ICV injection in rats
Slight increase in blood pressure
$ FIG. 2. Sephadex G-15 elution profile o f C r o t a l u s atrox ultra-filtered venom, followed at OD 210 (.... ) and 280 ( J nm. Arrow indicates the most effective hypotensive peak.
FIG. 3. HPLC on RP-8 column of Sephadex G-15 h) potensive peak from C r , , t , l u s atrox ultrafiltered venom, followed at 280 nm OD. Arrow indicates the most effective hypotensive compound(s).
TABLE
3
STRUCTURAL SIMILARITIES BETWEEN POL-236 AND OTHER NATURAL PEPTIDES
POL-236 SQ-20861 SQ-20881 Neurotensin
LH-RH Phyllolitorin Litorin Tryptophyllin
PYR-LEU-TRP - PRO-ARG-PRO-GLN-ILE-PRO-PRO PYR-ASN-TRP - PRO-ARG-PRO-GLN-ILE-PRO-PRO PYR-TRP- PRO-ARG-PRO-GLN-ILE-PRO-PRO PYR-LEU-TRP - GLU-ASN-LYS-PRO-ARG-ARG-PRO ............. PYR-HIS-TRP - SER-TYR-GLY-LEU-ARG-PRO-GLY PYR-LEU-TRP- ALA-VAL-GLY-SER-PHE-MET-NH: PYR-LEU-TRP- ALA-THR-GLY-H1S-PHE-MET-NHz PYR-PRO-TRP - MET-NH:
dation technique. The peptide was identified as a c o m p o u n d with 10 aminoacid residues, and named POL-236: PYRL E U - T R P - P R O - A R G - P R O - G LN-I L E - P R O - P R O . A ,ubslantial amount of POL-236 has been synthesized. in o r d e r to determine its pharmacological activities (Table 2). On A C E . the decapeptide shows an effect similar to o t h e r peptides present in snake v e n o m s 12], but many times losver than ,3nthetic c o m p o u n d s like captopril [I]. This suggests lh~fl probably A C E inhibition is not the most important effect of POL-236. The immediale and short-lasting effect on anesthetized rats. as well as the bradykinin potentiating activitx, are terminated by indomethacin, so that they can be mediated b\ some kind of prostaghmdins. On isolated rat's heart, the decapeptide induces slowing down effects, such as negatixe inotropic and chronotropic activities and c o r o n a o flux reduction. It is the first time that cardiac effects have been obserxed in small peptides fi'om snake v e n o m s and. apparentl,,, they can justify the long-lasting hypotensive efIk'ct shoxved by POL-236 in experimental models of
hypotension (in S p o n t a n e o u s l y H y p e r t e n s i v e rats. in rats made hypertensive by D O C A and in Goldblatt hy pertensive rats). The results obtained by injecting POL-236 directly into the C N S confirm that the hypotension is not centrally mediated. DISCUSSION
Viperid and crotalid snake v e n o m s are extraordinarily rich sources of small peptides, but only a few of them have been characterized until now. It is unfortunate that, due to their supposed activity as "'Bradykinin potentiators'" or " ' A C E inhibitors,'" they were gradually superseded by peplido-mimetic synthetic analogs. In fact. we found that POL-236. a decapeptide newly d i s c o v e r e d in the v e n o m of Crotalu.s u t r o x snake, e v e n if it is structurally very similar to other peptides found in B o t h r o p . s j a r a r a c a snake v e n o m (Table 3). shows a pharmacological profile hardly related to ACE-inhibition alone. On the whole, results obtained in
346
POLITI ET AL.
pharmacological assays suggest that POL-236 can bind to vascular receptors (possibly associated to bradykinin binding sites), stimulating a phospholipase A2 and the release of prostaglandins. The resulting vasodilation is potentiated by a long-lasting depressive effect on the cardiac muscle, so that a profound hypotension might ensue, sometimes leading to animal death (hypotension is observed frequently in animals bitten by crotalid snakes). Of course, the decapeptide is also able to inhibit ACE, but tile activity is not very strong, and therefore one can speculate that it is a secondary effect due to -PRO-PRO C-terminal dipeptide. Interestingly enough, POL-236 has structure analogies not only with other snake venoms, but also with frog skin
and some mammalian endogenous peptides (Table 3). This suggests that the decapeptide from Crotalus atrox snake venom could have other hitherto undiscovered pharmacological effects. In fact, in Prof. Melchiorri's Laboratory in Rome it has been demonstrated that POL-236 can inhibit gastric secretion (unpublished results). In conclusion, snake venoms are a huge factory of small. pharmacologically active peptides. Contrary to endogenous neuro-gastrointestinal hormones, they have to be injected from outside, in order to have a strong effect on the host's physiological responses. It is possible, therefore, that they could become very useful pharmacological tools, and possibly new drugs.
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