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Rat brain enkephalinase: Characteristion of the active site using mercaptopropanoyl amino acid inhibitors, and comparison with angiotensin-converting enzyme
Life Sciences, Vol. 33, Sup. I, 1983, pp. 113-116 Printed in the U.S.A.
Pergamon Press
RAT BRAIN ENKEPHALINASE: CHARACTERIZATION OF THE ACTIVE SITE USING MERCAPTOPROPANOYL AMINO ACID INHIBITORS, AND COMPARISON WITH ANGIOTENSIN-CONVERTING ENZYME E. M. Gordon,* D. W. Cushman, R. Tung, H. S. Cheung, F. L. Wang and N. G. Delaney Squibb Institute for Medical Research Princeton, New Jersey 08540, USA (Received in final form June 26, 1983) Summary Over fifty mercaptopropanoyl amino acids and related derivatives were synthesized to define the steric, electronic and stereochemical requirements for binding to the active site of enkephalinase (ENKASE), and also for their ability to inhibit angiotensin-converting enzyme (ACE). In this way the character of ENKASE and ACE active sites were compared. Recently it has become increasingly clear that enkephalins are metabolized in brain by at least two types of enzymic processes (1-4): i.) aminopeptidases present primarily in the soluble fraction of brain homogenates cleave the Tyr lGly 2 peptide bond, and 2.) peptidyl dipeptide hydrolases, such as angiotensinconverting enzyme (ACE) (5) and more significantly, a discrete enzyme termed "enkephalinase" (ENKASE) (6-9) cleave the penultimate GlY3-Phe 4 bond to release an intact dipeptide fragment. Our efforts to conceptualize the active site of rat brain ENKASE have been guided by a hypothetical active site model, similar to that used in the design of specific inhibitors of ACE (i0). As a working hypothesis we assumed a mechanistic similarity between these two enzymes. Thus, the active site of ENKASE was expected to contain at least five sites, each of which was anticipated to play a role in the interaction of enzyme with substrate. In addition to the hydrophobic binding interactions of subsites S' I and S'2, a carboxyl binding moiety, most likely an arginine residue, a hydrogen-donor group to participate in binding of the terminal amide linkage, and a tightly bound zinc atom which could serve to polarize the sissile amide carbonyl as a prelude to cleavage were presumed to be present. Similar assumptions were made by Roques, et al., in the design of thiorphan (ii). In the presently described work, we wished to expand on earlier investigations, and focus in depth upon optimization of 3mercaptopropanoyl amino acids as ENKASE inhibitors. In this way, we expected to more clearly define the various subsite specificities of ENKASE and ultimately reveal the differences between this enzyme and ACE (12). Results and Discussion A.)
S' 2 Subsites of ENKASE and ACE
Optimal binding requirements of the ENKASE S' 2 subsite were examined by systematic variation of the C-terminal residue of 3-mercaptopropanoyl amino acids and related C-2 substituted derivatives (See Table I). C-terminal aromatic amino acids and leucine analogs maximized affinity for ENKASE and hence the S'2subsite 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
114
Enkephalinase and ACE Active Sites
Vol. 33, Sup. I, 1983
specificities of ACE and ENKASE for mercaptopropanoyl amino acid inhibitors appear to be quite similar. A major divergence, however, is evident in the very high binding affinity by ACE of mercaptopropanoyl proline analogs, whereas such derivatives were the poorest ENKASE inhibitors tested. B.) S' I Subsites of ENKASE and ACE Table II shows the overall effect of introducing various C-2 side chain substituents into 3-mercaptopropanoyl amino acids. In 3-mercaptopropanoyl glycine, introduction of a 2-benzyl substituent confers a 16,000-fold enhancement in ENKASE inhibition, however, only a modest change is observed for ACE inhibition (5-fold). Inspection of the structure-activity relationships of C-2 variation in 3-mercaptopropanoyl leucine reveals a similar profile for ENKASE inhibition, wherein C-2 benzyl is optimal. Importantly, further homologation of C-2 benzyl (29) to phenethyl (43) causes a sharp drop in activity. In summary, ENKASE inhibition appears to be maximized at subsite S' I by the presence of a C-2 benzyl substituent. Smaller or larger groups are less effective. This substituent occupies an essential binding site of ENKASE and its optimization is critical for manifestation of high inhibitory potency. On the other hand, ACE inhibition is less profoundly affected by the presence of a C-2
side
chain
substituent.
TABLE I Effect of Variation of the C-2 Side Chain Substituent and C-Terminal Amino Acids in 3-Mercaptopropanoyl Amino Acids on the Inhibition of Enkephallnase and Angiotensin-Converting Enzyme a
,\\~
Amino Acid
(AA)
150(~M) No.
ENKASE
eeu 3 Phe Trp Dopa Tyr Arg 5 Met E-N-Cbz-Lys Ala 6 Gly 7 Lys 8 Pro 9 Asp i0 Set Glu Val Nle
5.90 3.95
HSA
150(~)
ACE
No.
ENKASE
1.63 .43
ii 12 13
0.21 0.12 0.25
ACE
ENKASE
ACE
0.62 24 0.047i 2-5 0.024 26
0.232 0.24 0.043
1.81 0.116 0.022
27
0.37
0.23
28
0.13
1.59
1.34 0.15 300.
3.0
7.9 6000. 300. >i000. >I000.
0.84 0.27 2.39 0.24 75.
1.41 1.66 12.9
0.37 0.18 0.089
18 19
0.34 22.
0.057 0.36
20 c 21 22 23
1150.
0.023
150(~M)
150(HM) No.
15 16 17
Ph
No.
ENKASE
ACE
29 0.019 30 0.029 31 0.039 32 0.044 33 0.083 34 0.15 35 0.15 36 0.19 37 0.22 38 e 0.36 39 2.62 40 21.8
0.084 0.055 0.024 0.i0 0.019 0.32 0.08 0.58 0.i64 0.86 1.54 0.06
41 d
0.009, 0.297
1.03 0.18 58. 14.4 1.14 2.56 0.52, 1.68
a)All compounds are mixtures of C-2 side chain diastereomers unless otherwise noted. b)Methyl group is of S configuration, c)Methyl group is of S configuration, substance is eaptopril, d)Values are for two separate dlastereomers of undetermined configuration at side chain C-2. ~ S u b s t a n c e is thlorphan.
Vol. 33, Sup. I, 1983
Enkephalinase and ACE Active Sites
TABLE II Effect of C-2 Side Chain Substituent Variation in 3-Mercaptopropanoyl Amino Acids on the Inhibition of Enkephalinase and Angiotensin-Converting Enzyme R
I HSCH2CHCO-AA No. 7 19 38 3 i~ 24 29 43
4 i2 25 30 5 i6 27 34
R
AA
TABLE III Effect of C-2 Side Chain Stereochemistry on the Inhibition of Enkephalinase and Angiotensin-Converting Enzyme by 3-Mercaptopropanoyl Amino Acids a R
150(~M ) ENKASE
-H -CH 3 -CH2Ph -H -CH 3 -CH2CH(CH3) 2 -CH2Ph -CH2CH2Pha
Gly 6000. Gly 22. Gly 0.36 Leu 5.90 Leu 0.21 Leu 0.232 Leu 0.019 Leu (Isomer A) 0.48 (Isomer B) 2.9 -H Phe 3.95 -CH 3 Phe 0.12 -CH2CH(CH3) 2 Phe 0.24 -CH2Ph Phe 0.029 -H Arg 300. -CH 3 Arg 1.66 -CH2CH(CH3) 2 Arg 0.37 -CH2Ph Arg 0.15
115
HSCH2CHCO-AA
ACE 4.5 0.36 0.86 1.63 0.62 1.81 0.084
0.43 0.047 0.116 0.055 3.0 0.18 0.23 0.32
I50(~M) No.
R
AA
,b ENKASE ACE S R
1.34 1.43
0.15 0.70
-CH2Ph L-Leu -CH2Ph L-Leu
S R
0.010 0.048
0.037 1.87
-CH2Ph L-Phe -CH2Ph L-Phe
A B
0.020 0.061
0.029 0.728
14 44
-CH 3 -CH 3
45 46 47 48
L-Dopa L-Dopa
a)Asterisk indicates C-2 side chain stereochemistry. b)A,B indicate pure diastereomers of undetermined configuration.
~)43 was resolved into its 2 diastereomeric components.
TABLE IV Effect of Carboxyl Modification and Stereochemistry on the Inhibition of Enkephalinase by 3-Mercaptopropanoyl Amino Acids
No.
X
, a
29
-C02H (L)
49
Isomer A b -CO2H (D) Isomer B
50
-CH2OH (L)
51
Isomer A c -CONH2 (L)Isomer B
ENKASE (I50LLM) 0.019 5.5 >i000. 145. 0.049 0.640
a)All compounds unless noted are mixtures of R,S C-2 side chain diastereo m ers( * ) b)The values reported are for separate diastereomers of unassigned configuration c)Diastereomer of the S,S configuration, as determined by x-ray crystallography
116
Enkephalinase and ACE Active Sites
Vol. 33, Sup. I, 1983
C.) Stereochemical Demands of ENKASE and ACE S' I Subsites Several mercaptopropanoyl amino acid diastereomeric pairs differing only in C-2 side chain stereochemistry were examined for their effect on ENKASE and ACE inhibition (See Table III). The results suggest that ACE places more stringent stereochemical demands upon P'I inhibitor binding than does ENKASE. A C-2 side chain substituent of the correct (mimicking an L-amino acid residue) configuration is of considerable benefit for ACE inhibition (10-100-fold 150 enhancement vs. the "wrong" isomer). In contrast, the presence of a C-2 substituent is essential for good ENKASE inhibition but its absolute configuration is of secondary and much lesser importance (0-5-fold). That inhibitor sulfhydryl is able to interact with enzymic zinc near equally well in both R and S side chain isomers (in several pairs of derivatives) is somewhat unexpected and indicates a good deal of flexibility exists within the region of the active site of ENKASE encompassing the S' I subsite and zinc ion. (S)-3-mercapto-2benzylpropanoyl-L-leucine (45) (150 = i0 nM), whose absolute configuration mimics a natural L,L-dipeptide, was the most potent ENKASE inhibitor identified in this study (13). D.) Importance of the C-Terminal Carboxylic Acid A number of carboxyl modified 3-mercaptopropanoyl amino acids were synthesized to address the relative significance of this interaction in ENKASE binding (See Table IV). It is well known that the C-terminal carboxylic acid plays a pivotal role in the binding of such inhibitors to ACE (14). Our results indicate that rat brain ENKASE prefers a C-terminal carboxyl group, but to a much lesser extent than ACE, in agreement with the results of Roques, et al (12), and consequently ENKASE is likely not a "pure" exopeptidase (peptidyl dipeptide hydrolase). References i. J.C. SCHWARTZ, B. MALFROY and S. De La BAUME, Life Sci., 29, 1715-1740(1981) 2. C. GORENSTEIN and S.H. SNYDER, Life Sci., 25, 2065-2070 (1979). 3. S. De La BAUME, C. GROS, C.C. YI, P. CHAILLET, H. MARCAIS-COLLAD0, J. COSTENTIN and J.C. SCHWARTZ, Life Sci., 31, 1753-1756 (1982). 4. N. MARKS, M. BENUCK, M.J. BERG and L. SACHS, Ann. N.Y. Acad. Sci., 398, 308-326 (1982). 5. E.G. ERDOS, A.R. JOHNSON and N°T. BOYDEN, Biochem. Pharmacol., 27, 843-848 (1978). 6. J. L. MEEK, H.Y.T. YANG and E. COSTA, Neuropharmacol., 16, 151-154 (1977). 7 J.P. SWERTS, R. PERDRISOT, B. MALFROY and J.C. SCHWARTZ, Eur. J. Pharmacol., 53, 209-210 (1979). 8 J.P. SWERTS, R. PERDRISOT, G. PATEY, S. De La BAUME and J.C. SCHWARTZ, Eur. J. Pharmacol., 57, 279-281 (1979). 9 A. ARREQUI, C.M. LEE, P.C. EMSON and L.L. IVERSEN, Eur. J. Pharmacol., 59, 141-144 (1979). i0. D.W. CUSHMAN, H.S° CHEUNG, E.F. SABO and M.A. ONDETTI, Biochem., 16, 54845491 (1977). ii B.P. ROQUES, M.C. FOURNIE-ZALUSKI, E. SOROCA, J.M. LeCOMTE, B. MALFROY, C. LLORENS and J.C. SCHWARTZ, Nature, 288, 286-288 (1980). 12 B.P. ROQUES, M.C. FOURNIE-ZALUSKI, D. FLORENTIN, G. WAKSMAN, A. SASSI, P. CHAILLET, H. COLLADO and J. COSTENTIN, Life Sci., 31, 1749-1752 (1982). 13 Previously reported as a mixture of diastereomers: M.C. FOURNIE-ZALUSKI, C. LLORENS, G. GACEL, B. MALFROY, J.P. SWERTS, J.M. LeCOMTE, J.C. SCHWARTZ and B.P. ROQUES, in Proceedings of the Sixteenth Eur. Pept. Sympo., pp. 476-481, K. Brunfeldt, Ed., Scriptor, Copenhagen (1981). 14. E.W. PETRILLO, JR., and M.A. ONDETTI, Medicinal Res. Reviews, 2, 1-41 (1982).
Pergamon Press
RAT BRAIN ENKEPHALINASE: CHARACTERIZATION OF THE ACTIVE SITE USING MERCAPTOPROPANOYL AMINO ACID INHIBITORS, AND COMPARISON WITH ANGIOTENSIN-CONVERTING ENZYME E. M. Gordon,* D. W. Cushman, R. Tung, H. S. Cheung, F. L. Wang and N. G. Delaney Squibb Institute for Medical Research Princeton, New Jersey 08540, USA (Received in final form June 26, 1983) Summary Over fifty mercaptopropanoyl amino acids and related derivatives were synthesized to define the steric, electronic and stereochemical requirements for binding to the active site of enkephalinase (ENKASE), and also for their ability to inhibit angiotensin-converting enzyme (ACE). In this way the character of ENKASE and ACE active sites were compared. Recently it has become increasingly clear that enkephalins are metabolized in brain by at least two types of enzymic processes (1-4): i.) aminopeptidases present primarily in the soluble fraction of brain homogenates cleave the Tyr lGly 2 peptide bond, and 2.) peptidyl dipeptide hydrolases, such as angiotensinconverting enzyme (ACE) (5) and more significantly, a discrete enzyme termed "enkephalinase" (ENKASE) (6-9) cleave the penultimate GlY3-Phe 4 bond to release an intact dipeptide fragment. Our efforts to conceptualize the active site of rat brain ENKASE have been guided by a hypothetical active site model, similar to that used in the design of specific inhibitors of ACE (i0). As a working hypothesis we assumed a mechanistic similarity between these two enzymes. Thus, the active site of ENKASE was expected to contain at least five sites, each of which was anticipated to play a role in the interaction of enzyme with substrate. In addition to the hydrophobic binding interactions of subsites S' I and S'2, a carboxyl binding moiety, most likely an arginine residue, a hydrogen-donor group to participate in binding of the terminal amide linkage, and a tightly bound zinc atom which could serve to polarize the sissile amide carbonyl as a prelude to cleavage were presumed to be present. Similar assumptions were made by Roques, et al., in the design of thiorphan (ii). In the presently described work, we wished to expand on earlier investigations, and focus in depth upon optimization of 3mercaptopropanoyl amino acids as ENKASE inhibitors. In this way, we expected to more clearly define the various subsite specificities of ENKASE and ultimately reveal the differences between this enzyme and ACE (12). Results and Discussion A.)
S' 2 Subsites of ENKASE and ACE
Optimal binding requirements of the ENKASE S' 2 subsite were examined by systematic variation of the C-terminal residue of 3-mercaptopropanoyl amino acids and related C-2 substituted derivatives (See Table I). C-terminal aromatic amino acids and leucine analogs maximized affinity for ENKASE and hence the S'2subsite 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
114
Enkephalinase and ACE Active Sites
Vol. 33, Sup. I, 1983
specificities of ACE and ENKASE for mercaptopropanoyl amino acid inhibitors appear to be quite similar. A major divergence, however, is evident in the very high binding affinity by ACE of mercaptopropanoyl proline analogs, whereas such derivatives were the poorest ENKASE inhibitors tested. B.) S' I Subsites of ENKASE and ACE Table II shows the overall effect of introducing various C-2 side chain substituents into 3-mercaptopropanoyl amino acids. In 3-mercaptopropanoyl glycine, introduction of a 2-benzyl substituent confers a 16,000-fold enhancement in ENKASE inhibition, however, only a modest change is observed for ACE inhibition (5-fold). Inspection of the structure-activity relationships of C-2 variation in 3-mercaptopropanoyl leucine reveals a similar profile for ENKASE inhibition, wherein C-2 benzyl is optimal. Importantly, further homologation of C-2 benzyl (29) to phenethyl (43) causes a sharp drop in activity. In summary, ENKASE inhibition appears to be maximized at subsite S' I by the presence of a C-2 benzyl substituent. Smaller or larger groups are less effective. This substituent occupies an essential binding site of ENKASE and its optimization is critical for manifestation of high inhibitory potency. On the other hand, ACE inhibition is less profoundly affected by the presence of a C-2
side
chain
substituent.
TABLE I Effect of Variation of the C-2 Side Chain Substituent and C-Terminal Amino Acids in 3-Mercaptopropanoyl Amino Acids on the Inhibition of Enkephallnase and Angiotensin-Converting Enzyme a
,\\~
Amino Acid
(AA)
150(~M) No.
ENKASE
eeu 3 Phe Trp Dopa Tyr Arg 5 Met E-N-Cbz-Lys Ala 6 Gly 7 Lys 8 Pro 9 Asp i0 Set Glu Val Nle
5.90 3.95
HSA
150(~)
ACE
No.
ENKASE
1.63 .43
ii 12 13
0.21 0.12 0.25
ACE
ENKASE
ACE
0.62 24 0.047i 2-5 0.024 26
0.232 0.24 0.043
1.81 0.116 0.022
27
0.37
0.23
28
0.13
1.59
1.34 0.15 300.
3.0
7.9 6000. 300. >i000. >I000.
0.84 0.27 2.39 0.24 75.
1.41 1.66 12.9
0.37 0.18 0.089
18 19
0.34 22.
0.057 0.36
20 c 21 22 23
1150.
0.023
150(~M)
150(HM) No.
15 16 17
Ph
No.
ENKASE
ACE
29 0.019 30 0.029 31 0.039 32 0.044 33 0.083 34 0.15 35 0.15 36 0.19 37 0.22 38 e 0.36 39 2.62 40 21.8
0.084 0.055 0.024 0.i0 0.019 0.32 0.08 0.58 0.i64 0.86 1.54 0.06
41 d
0.009, 0.297
1.03 0.18 58. 14.4 1.14 2.56 0.52, 1.68
a)All compounds are mixtures of C-2 side chain diastereomers unless otherwise noted. b)Methyl group is of S configuration, c)Methyl group is of S configuration, substance is eaptopril, d)Values are for two separate dlastereomers of undetermined configuration at side chain C-2. ~ S u b s t a n c e is thlorphan.
Vol. 33, Sup. I, 1983
Enkephalinase and ACE Active Sites
TABLE II Effect of C-2 Side Chain Substituent Variation in 3-Mercaptopropanoyl Amino Acids on the Inhibition of Enkephalinase and Angiotensin-Converting Enzyme R
I HSCH2CHCO-AA No. 7 19 38 3 i~ 24 29 43
4 i2 25 30 5 i6 27 34
R
AA
TABLE III Effect of C-2 Side Chain Stereochemistry on the Inhibition of Enkephalinase and Angiotensin-Converting Enzyme by 3-Mercaptopropanoyl Amino Acids a R
150(~M ) ENKASE
-H -CH 3 -CH2Ph -H -CH 3 -CH2CH(CH3) 2 -CH2Ph -CH2CH2Pha
Gly 6000. Gly 22. Gly 0.36 Leu 5.90 Leu 0.21 Leu 0.232 Leu 0.019 Leu (Isomer A) 0.48 (Isomer B) 2.9 -H Phe 3.95 -CH 3 Phe 0.12 -CH2CH(CH3) 2 Phe 0.24 -CH2Ph Phe 0.029 -H Arg 300. -CH 3 Arg 1.66 -CH2CH(CH3) 2 Arg 0.37 -CH2Ph Arg 0.15
115
HSCH2CHCO-AA
ACE 4.5 0.36 0.86 1.63 0.62 1.81 0.084
0.43 0.047 0.116 0.055 3.0 0.18 0.23 0.32
I50(~M) No.
R
AA
,b ENKASE ACE S R
1.34 1.43
0.15 0.70
-CH2Ph L-Leu -CH2Ph L-Leu
S R
0.010 0.048
0.037 1.87
-CH2Ph L-Phe -CH2Ph L-Phe
A B
0.020 0.061
0.029 0.728
14 44
-CH 3 -CH 3
45 46 47 48
L-Dopa L-Dopa
a)Asterisk indicates C-2 side chain stereochemistry. b)A,B indicate pure diastereomers of undetermined configuration.
~)43 was resolved into its 2 diastereomeric components.
TABLE IV Effect of Carboxyl Modification and Stereochemistry on the Inhibition of Enkephalinase by 3-Mercaptopropanoyl Amino Acids
No.
X
, a
29
-C02H (L)
49
Isomer A b -CO2H (D) Isomer B
50
-CH2OH (L)
51
Isomer A c -CONH2 (L)Isomer B
ENKASE (I50LLM) 0.019 5.5 >i000. 145. 0.049 0.640
a)All compounds unless noted are mixtures of R,S C-2 side chain diastereo m ers( * ) b)The values reported are for separate diastereomers of unassigned configuration c)Diastereomer of the S,S configuration, as determined by x-ray crystallography
116
Enkephalinase and ACE Active Sites
Vol. 33, Sup. I, 1983
C.) Stereochemical Demands of ENKASE and ACE S' I Subsites Several mercaptopropanoyl amino acid diastereomeric pairs differing only in C-2 side chain stereochemistry were examined for their effect on ENKASE and ACE inhibition (See Table III). The results suggest that ACE places more stringent stereochemical demands upon P'I inhibitor binding than does ENKASE. A C-2 side chain substituent of the correct (mimicking an L-amino acid residue) configuration is of considerable benefit for ACE inhibition (10-100-fold 150 enhancement vs. the "wrong" isomer). In contrast, the presence of a C-2 substituent is essential for good ENKASE inhibition but its absolute configuration is of secondary and much lesser importance (0-5-fold). That inhibitor sulfhydryl is able to interact with enzymic zinc near equally well in both R and S side chain isomers (in several pairs of derivatives) is somewhat unexpected and indicates a good deal of flexibility exists within the region of the active site of ENKASE encompassing the S' I subsite and zinc ion. (S)-3-mercapto-2benzylpropanoyl-L-leucine (45) (150 = i0 nM), whose absolute configuration mimics a natural L,L-dipeptide, was the most potent ENKASE inhibitor identified in this study (13). D.) Importance of the C-Terminal Carboxylic Acid A number of carboxyl modified 3-mercaptopropanoyl amino acids were synthesized to address the relative significance of this interaction in ENKASE binding (See Table IV). It is well known that the C-terminal carboxylic acid plays a pivotal role in the binding of such inhibitors to ACE (14). Our results indicate that rat brain ENKASE prefers a C-terminal carboxyl group, but to a much lesser extent than ACE, in agreement with the results of Roques, et al (12), and consequently ENKASE is likely not a "pure" exopeptidase (peptidyl dipeptide hydrolase). References i. J.C. SCHWARTZ, B. MALFROY and S. De La BAUME, Life Sci., 29, 1715-1740(1981) 2. C. GORENSTEIN and S.H. SNYDER, Life Sci., 25, 2065-2070 (1979). 3. S. De La BAUME, C. GROS, C.C. YI, P. CHAILLET, H. MARCAIS-COLLAD0, J. COSTENTIN and J.C. SCHWARTZ, Life Sci., 31, 1753-1756 (1982). 4. N. MARKS, M. BENUCK, M.J. BERG and L. SACHS, Ann. N.Y. Acad. Sci., 398, 308-326 (1982). 5. E.G. ERDOS, A.R. JOHNSON and N°T. BOYDEN, Biochem. Pharmacol., 27, 843-848 (1978). 6. J. L. MEEK, H.Y.T. YANG and E. COSTA, Neuropharmacol., 16, 151-154 (1977). 7 J.P. SWERTS, R. PERDRISOT, B. MALFROY and J.C. SCHWARTZ, Eur. J. Pharmacol., 53, 209-210 (1979). 8 J.P. SWERTS, R. PERDRISOT, G. PATEY, S. De La BAUME and J.C. SCHWARTZ, Eur. J. Pharmacol., 57, 279-281 (1979). 9 A. ARREQUI, C.M. LEE, P.C. EMSON and L.L. IVERSEN, Eur. J. Pharmacol., 59, 141-144 (1979). i0. D.W. CUSHMAN, H.S° CHEUNG, E.F. SABO and M.A. ONDETTI, Biochem., 16, 54845491 (1977). ii B.P. ROQUES, M.C. FOURNIE-ZALUSKI, E. SOROCA, J.M. LeCOMTE, B. MALFROY, C. LLORENS and J.C. SCHWARTZ, Nature, 288, 286-288 (1980). 12 B.P. ROQUES, M.C. FOURNIE-ZALUSKI, D. FLORENTIN, G. WAKSMAN, A. SASSI, P. CHAILLET, H. COLLADO and J. COSTENTIN, Life Sci., 31, 1749-1752 (1982). 13 Previously reported as a mixture of diastereomers: M.C. FOURNIE-ZALUSKI, C. LLORENS, G. GACEL, B. MALFROY, J.P. SWERTS, J.M. LeCOMTE, J.C. SCHWARTZ and B.P. ROQUES, in Proceedings of the Sixteenth Eur. Pept. Sympo., pp. 476-481, K. Brunfeldt, Ed., Scriptor, Copenhagen (1981). 14. E.W. PETRILLO, JR., and M.A. ONDETTI, Medicinal Res. Reviews, 2, 1-41 (1982).