Transcript

Theoretical Conformational Analysis of a p-Selective Cyclic Opioid Peptide Analog

BRIAN C. WILKES and PETER W. SCHILLER,* Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute

of Montreal, Montreal, Quebec, Canada H2W 1R7

Synopsis

The allowed conformations of the p-receptor-selective cyclic opioid peptide analog H-Tyr-D-Om-Phe-Asp-NH, were determined using a grid search through the entire conforma- tional space. Energy mnimization of the 13-membered ring structure lacking the exocyclic Tyr' residue and the Phe3 side chain using the molecular mechanics program Maximin resulted in only four low-energy conformations. These four ring structures served as templates for a further energy minimization study with the Tyr' residue and Phe3 side chain added to the molecule. The results indicated that the Tyr' and Phe3 side chains enjoy considerable orientational freedom, but nevertheless, only a limited number of low-energy side-chain configurations were found. The obtained low-energy conformers are discussed in relation to various proposed models of the bioactive conformation of enkephalins and morphiceptin.

-

INTRODUCTION

During the past decade numerous conformational studies of the opioid peptide enkephalin (H-Tyr-Gly-Gly-Phe-Met[or Leu]-OH) have been car- ried out (for a review, see Ref. 1). Theoretical energy calculations, x-ray diffraction studies, and spectroscopic investigations in solution led to the conclusion that these linear pentapeptides are highly flexible molecules capa- ble of assuming a number of different conformations of comparable low energy. There is no compelling reason to assume that any of these low-energy struetures represents the bioactive (receptor-bound) conformation. The en- kephalins show some preference for opioid receptors of the 8-type, but also bind to p-opioid receptors with somewhat reduced yet still considerable affinity. This low-receptor selectivity may be due to the structural flexibility of these linear peptides which permits adaptation to the conformational requirements of both the p and the 8 receptor.

Biologically active peptide analogs with built-in conformational constraints offer several advantages. First, their enhanced structural rigidity may lead to improved receptor selectivity, since the conformational restriction may only be compatible with one receptor class. Second, conformational studies of such

*To whom correspondence should be addressed.

Biopolymers, Vol. 26, 1431-1444 (1987) 0 1987 John Wiley & Sons, Inc. CCC oooS-3525/87/081431-14$04.00

1432 WILKES AND SCHILLER

analogs are of direct relevance to the bioactive conformation because major conformational changes upon binding to the receptor can no longer occur.

Conformational restriction of enkephalins resulting in active analogs has been achieved through cyclizations via side chains. A cyclic enkephalin analog obtained through substitution of D-a, y-diaminobutyrk acid in po- sition 2 of the peptide sequence followed by amide bond formation be- tween the y-amino group and the C-terminal carboxyl function (H-Tyr-cyclo[-~-A,bu-Gly-Phe-Leu-]),~ was highly potent and showed considerable preference for p receptors over 8 rece~tors .~ Homologs of H-Tyr-cyclo[-D-A,bu-Gly-Phe-Leu-] containing shorter or longer side chains in the 2 p~s i t ion ,~ or related cyclic retro-inverso analog^,^ also were p receptor selective and very stable against enzymatic degradation. Cystine bridged cyclic enkephalin analogs, HzTyr-D-C&-Gly-Phe-D(or L)-C$-X, were synthesized and found to be very potent; peptide amides of this type (X=NH,) were nonselective,6 whereas the corresponding free acids (X=OH) showed about the same moderate &receptor selectivity as [Le~~lenkephalin.~ Structurally related cyclic analogs with a penicillamine (Pen) residue substituted for Cys in position 2,8 or in positions 2 and 5,' displayed greatly improved &receptor selectivity. More recently, H-Tyr-D-OF-Phe-Asp-NH,, a cyclic opioid peptide analog featuring a 13-membered ring structure, has been synthesized.l0*'' This cyclic lactam analog contains a Phe residue in the 3 position of the peptide sequence, and therefore is structurally related to the P-casomorphins and dermorphin. Pharmacologic characterization revealed that H-Tyr-D-Op-Phe-Asjp-NH, ranks among the most selective p agonists known to date.l03"

Conformational studies on these cyclic opioid peptide analogs have been initiated. Theoretical investigation^'^-'^ and nmr, experiments in conjunction with molecular dynamics and energy minimization studies,15 were carried out in attempts to delineate the conformation(s) of H-Tyr-cyclo[ -D-A ,bu-Gly-Phe-Leu-]. No consensus regarding the ring conformation(s) and transannular hydrogen bonding in this compound has been reached from these studies. Perhaps the most significant result of these efforts is the finding that the 14-membered ring structure in this pep- tide is not entirely rigid but rather exists in a conformational equilibrium involving a few different ~onformers.'~ The results of an nmr study per- formed in [ ,H]-DMSO (dimethyl sulfoxide) indicated the existence of two trans-annular hydrogen bonds in the case of H-Tyr-cyclo[ -~-Om-Gly- Phe-Leu-] and of a conformational equilibrium in the case of H-Tyr-cyclo[-~-Lys-G1y-Phe-Leu-].'~ A theoretical study on H-Tyr-D-CpGly-Phe-D(or L)-C~;~-NH, revealed that the 14-membered ring entity in these peptides is considerably more flexible than that in H-Tyr-cyclo[-~-A,bu-Gly-Phe-Leu].'~ Conformational features of the cystine-containing analogs H-Tyr-D-Cy,s-Gly-Phe-D(or L)-C~S-NH, and the related penicillamine-containing analogs H-Tyr-D-Pen-Gly-Phe-D(or L ) - C ~ N H , were compared in an nmr study performed in D,0.ls The obtained nmr data suggested similar overall conformations; however, in com- parison with the corresponding Cys2 analogs, the Pen2 analogs showed higher rigidity in the C-terminal part of the molecule, which may be related to their more selective activity profiles.

CONFORMATION OF A CYCLIC OPIOID PEPTIDE 1433

In the present paper, we describe a systematic search and subsequent energy minimization by the molecular mechanics method with the highly p-selective cyclic analog H-Tyr-D-OF-Phe-qp-NH 2.

METHODOLOGY

All calculations were performed using the commercial molecular modeling system SYBYL (Tripos Associates, St. Louis, MO) using a VAX 11/750, VMS Version 4.2. Molecules were viewed using an Evans and Sutherland PS 300 computer graphics display terminal. A Hewlett-Packard HP 7475 plotter was used for the preparation of the figures.

A stepwise procedure was employed for the determination of the allowed low-energy conformations of the molecule. The first step involved construct- ing the 13-membered ring along with the atoms directly attached to the ring, including associated hydrogen atoms (Fig. 1, B). The approach of concentrat- ing just on the ring structure is used in order to allow the greatest number of solutions to the conformational search and has been employed by other workers.12* 13, l9 In this manner, possible allowed conformational solutions for the ring structure are not excluded due to the presence of the exocyclic Tyr' residue and the Phe3 side chain. The crude ring is then minimized prior to the conformational search using the versatile energy minimization program called Maximin (J. Labanowski and G. R. Marshall, in preparation). Maximin uses a steepest descent approach to determine the energy minimum of a given

OH

y 2

2 H N-CH-CONH-D-CH-CONH-CH-CONH-CH-CONH - 1 \ 2

2 CH -CH -CH -NHCO-CH 2 2 2

CH 3 1 2 1 3 4

2 H N-D-CH-CONH-CH-CONH-CH-CONH 2 - 1 0 1 9 8 7 6\5

2 CH -CH -CH -NHCO-CH 2 2 2

(B) Fig. 1. Structure of H-Tyr-D-Om-Phe-Asp-NH, and of the parent ring (numbers desig-

nate rotatable bonds).

1434 WILKES AND SCHILLER

structure. The potential energy is calculated from

where the ws represent weight constants and the ES represent energy terms for bond-stretching (str) energy, angle-bending (ang) energy, torsional (tor) energy and Van der Waals (VDW) contact energy (including hydrogen bond- ing). The 13-membered ring was then ready to be searched for allowed conformations.

The second step involved the use of a systematic conformational analysis program called Search,2o which permitted the identification of permissible conformations. The search program checks for VDW contacts among the nonbonded atoms by scanning all possible torsional angles around the rotata- ble bonds. Conformations were eliminated based solely on unfavorable VDW interactions (as well as the requirement for ring closure, see below). A VDW radius factor of 0.80 was used for nonbonded atoms to allow for thermal vibrations of atoms. A VDW radius factor of 0.70 was used for 1,4 interac- tions,21 and a VDW radius factor of 0.40 was used for hydrogen bonds in order to allow for hydrogen-bond formation.

The three amide bonds within the 13-membered ring were held trans and planar. Of the 10 remaining bonds, one was chosen as the ring-closure bond (bond 4, Fig. 1, B) and the remaining 9 bonds were surveyed over a 30" grid over all space. A ring-closure constraint was imposed in that allowed con- formers must allow the tenth bond to form within 0.2 A of the length and within 15 O of the torsion angle of a normal carbon-carbon bond. The program scanned 1O'O possible combinations and required 41.5 h of CPU time. Ten solutions were found. These ten possible ring structures were then minimized using the Maximin program allowing all of the atoms to relax, including the amide bonds. Four of the solutions represented the low-energy structures (within 1.2 Kcal/mol of the minimum), and the remaining six solutions represent the higher energy solutions (greater than 3.5 Kcal/mol above the minimum). The results are shown in Table I.

A second search of the ring was performed by choosing a different ring- closure bond (bond 9, Fig. 1, B), leaving the remaining parameters unaltered. This program required 54.2 h of central processing unit (CPU) time and resulted in five solutions. Of these five solutions, four corresponded to the four lowest energy solutions obtained in the original search, and the fifth solution corresponded to one of the higher energy solutions.

The third step involved taking the four low-energy ring solutions and adding the exocyclic Tyr' residue and the Phe3 side chain (Fig. 1, A). The two exocyclic amide bonds were held trans and planar, and the remaining seven bonds were surveyed over a 30 grid over all space. In each case, the program scanned approximately lo6 possible conformations requiring between 30 and 60 min of CPU time. For each grid search there were 3000-7000 possible solutions. The energies of these conformers were calculated and the resulting solutions were grouped into low-energy families. For ring conformer 1 there were five low-energy families. For ring conformer 2 there were eight low- energy families, for ring number 7 there were five low-energy families, and for ring number 10 there were two-low energy families.

0

0 2 3 E

TA

BL

E I

Tor

sion

al A

ngle

s and

Ene

rgie

s of

the

13-M

embe

red R

ings

8 E

nerg

y Z

N

o.

(Kca

l/mol

) q2

*2

(P3

$3

*3

(P4

x41

x42

*4

x24

x23

x22

x21

1 -0

.325

-1

11.4

-1

74.6

53

.2

65.7

18

0.0

-137

.9

37.9

-1

47.5

18

0.0

-69.

5 58

.4

146.

6 13

7.1

2 0.

533

-113

.2

-176

.6

55.7

82

.4

177.

7 17

0.3

64.5

-5

6.9

178.

4 -1

59.1

-8

8.2

115.

8 16

7.0

0

5.81

2 -1

39.7

-1

77.3

87

.1

-139

.2

177.

8 79

.9

-17.

9 12

4.7

180.

0 11

3.7

-148

.6

115.

8 -1

63.5

4

fl

3 4 3.

250

141.

8 -1

67.7

-1

76.2

-8

8.6

180.

0 20

.8

-31.

2 72

.1

178.

2 13

7.2

87.9

-1

52.4

-7

2.2

5 4.

428

71.3

- 1

78.9

- 15

8.4

101.

4 18

0.0

-169

.5

2.2

45.2

18

0.0

162.

6 - 1

49.8

11

4.3

-135

.6

0

6 7.

073

13.9

- 1

77.1

-1

34.0

40

.1

180.

0 -5

9.1

7.3

-84.

1 17

9.0

-71.

5 - 1

17.1

11

3.7

-102

.9

7 0.

689

10.0

- 1

73.5

-4

2.2

-74.

2 18

0.0

55.1

52

.5

123.

4 17

5.5

142.

5 -8

6.3

116.

0 16

4.9

8 3.

488

-1.7

-1

78.7

-5

4.3

94.8

18

0.0

-96.

2 -5

1.4

-26.

8 11

7.9

-156

.9

122.

1 -9

4.6

-104

.3

9 4.

384

-16.

4 -1

75.0

-1

4.3

-64.

7 17

8.1

-38.

0 72

.7

-58.

2 17

8.9

-130

.5

-81.

9 11

1.8

-166

.6

E: 2 10

0.

838

-44.

3 -1

75.5

-4

4.3

-48.

6 17

8.4

-30.

0 70

.7

-29.

1 17

6.1

114.

6 91

.5

-92.

9 -4

7.7

q

M 3

TA

BL

E I1

Various

Para

met

ers o

f L

ow-E

nerg

y Con

form

ers o

f H

-Tyr

-D-O

m-P

he-

Asp

-NH

, -

Tor

siona

l ang

les (

degr

ees)

In

term

olec

ular

dis

tanc

es (A

) E

nerg

y T

yrri

ng

Tyr

ring

T

yr-O

H

No.

(KcW

mol

) x1

6 x12

x11

#1

01

'pz

x31

x3,

$4

toph

erin

g to

NH

, to

NH

,

1

- 12

.096

2

- 9.

936

3 - 7.

317

4 - 10

.930

5

- 9.

184

6 - 7.

521

7 - 10

.663

8

- 9.

632

9 - 9.

761

10

- 11

.226

11

- 11

.043

12

- 11

.465

13

- 11

.481

14

- 7.

577

15

- 7.

181

16

- 6.

851

17

- 6.

589

18

- 7.

253

19

- 10

.362

20

- 10

.642

0.0

159.

0 97

.0

0.0

17.5

79

.0 0.0

144.

8 21

.1

13.0

18

0.0

0.0 1.2

6.2

9.7

178.

7 18

0.0

16.3

0.

0 17

8.8

63.2

- 1

53.3

87

.7

-58.

3 94

.1

-60.

9 89

.2

64.2

62

.8

-178

.3

90.8

62

.7

120.

0 85

.1

121.

8 -1

55.5

65

.4

-178

.8

63.9

18

0.0

64.5

17

8.6

88.5

-6

0.3

90.9

-1

76.6

94

.3

57.7

58

.0

178.

3 82

.8

157.

9 94

.9

60.0

60

.8

-149

.7

5.2

177.

5 33

.8

173.

0

- 59

.2

- 62

.4

84.3

17

5.9

149.

4 17

7.4

- 17

6.9

- 58

.2

154.

0 - 58

.1

- 62

.2

- 62

.0

154.

7 28

.8

- 63

.2

139.

3 16

5.5

- 16

8.2

- 88

.9

- 83

.1

173.

8 87

.0

176.

7 87

.4

174.

8 89

.4

175.

5 91

.3

176.

7 14

6.3

178.

4 93

.4

174.

6 15

2.8

176.

7 15

0.9

177.

7 15

0.0

178.

8 86

.9

178.

2 62

.9

180.

0 91

.1

177.

7 83

.5

173.

5 12

0.8

170.

7 -1

20.5

17

3.3

125.

0 17

6.6

130.

1 17

6.5

133.

1 17

6.8

147.

0 17

3.9

144.

2

- 59

.2

- 58

.7

- 58

.8

- 58

.7

- 59

.4

- 56

.5

- 56

.6

- 56

.7

- 57

.0

- 17

6.1

- 83

.3

- 56

.6

- 56

.6

- 86

.4

120.

5 - 87

.6

- 87

.7

- 87

.6

- 87

.8

180.

0

89.9

61

.5

91.0

62

.1

90.3

61

.9

90.5

61

.4

90.4

62

.3

92.4

-46.

4 92

.1

-46.

8 93

.3

-46.

3 91

.8

-46.

5 -6

1.5

-46.

8 11

9.3

-46.

3 92

.6

-46.

6 11

8.7

-46.

5 11

4.6

55.2

11

7.8

56.2

86

.4

55.4

86

.5

55.4

85

.7

55.0

30

.0

127.

2 86

.5

126.

0

4.17

0 7.

527

7.74

9 7.

869

8.38

6 7.

819

8.61

4 5.

054

7.96

2 6.

022

5.31

0 7.

027

8.23

7 4.

475

5.22

9 9.

183

10.1

21

5.10

2 4.

400

6.39

2

5.03

9 3.

878

3.92

3 3.

959

5.12

6 3.

920

4.27

2 5.

077

5.11

4 5.

114

5.11

1 3.

856

5.13

6 3.

727

5.09

9 5.

073

3.82

1 4.

999

5.18

9 5.

155

8 x 3

6.27

4 * w P

7.65

7 6.

262

6.28

4 6.

332

7.78

1

U

6.68

9 7.

694

X

7.73

9 7.

733

7.74

9 6.

182

M

7.78

7 z

5.99

1 7.

727

7.68

8 6.

101

7.60

4 7.

853

7.80

7

CONFORMATION OF A CYCLIC OPIOID PEPTIDE 1437

In the fourth step the lowest energy member of each family (20 families total) was taken and subjected to extensive energy minimization as described above. Each program required 1.5 h of CPU time. The results are shown in Table 11. This entire procedure required more than 120 h of CPU time.

RESULTS

Conformation of the 13-Membered Ring

A conformational grid search of the parent ring resulted in a set of ten discrete allowed ring conformations (Table I). A VDW contour map of the lowest energy conformer (ring 1) is shown in Fig. 2 and in Fig. 3 ring conformer 1 is depicted together with the three other lowest energy ring conformers. A second independent grid search of the parent ring resulted in five allowed conformations, four of which were identical with the four lowest energy conformers obtained in the original search. Relaxing the parameters of the search procedure (VDW radii and ring closure constraints) resulted in more allowed conformations, but these were found to be higher in energy than the conformations described above (data not shown).

Fig. 2. Contour map of the VDW radii around ring 1.

1438 WILKES AND SCHILLER

w Fig. 3. Four lowest energy ring structures. Clockwise from upper left: Ring 1, ring 2, ring 10,

ring 7.

A display of the calculated VDW distances for ring 1 results in a “dough- nut”-like structure with multiple VDW contacts all the way around the ring (Fig. 2). In addition, it can be seen that the “central cavity” in the lowest energy conformer is smaller than the size of a hydrogen atom. The three internal amide bonds are perpendicular to the plane of the ring, with two of the amide carbonyl groups (w2 and us) pointing in the same direction and the third amide carbonyl ( w 4 ) pointing in the opposite direction.

Ring number 2 is 0.85 Kcal/mol higher in energy than ring number 1. The major difference between this ring conformation and the conformation of ring number 1 is that, in this case, the w, amide group is no longer perpendicular to the plane of the ring. The w4 amide proton of this ring is now pointing at an angle of approximately 60 O toward the center of the ring. Calculated VDW radii show that this proton now completely fills the central cavity observed in ring 1 and therefore makes VDW contacts across the ring. Due to the physical constraints imposed by the 13-membered ring, the amide proton is not able to rotate further toward the center of the ring (see also below). There was no apparent hydrogen bonding observed with this ring structure.

Ring number 7 is just over 1.01 Kcal/mol higher in energy than ring number 1 and very close in energy to ring 2. There are two major features that distinguish this structure from that observed in ring 1. The w3 amide bond is

CONFORMATION OF A CYCLIC OPIOID PEPTIDE 1439

rotated 180" with respect to its orientation in ring 1, and therefore the carbonyl group of this amide bond is pointing in the same direction as the w4 amide bond carbonyl. In addition, both the w3 and w4 amide groups are perpendicular to the plane of the ring. On the other hand, the w2 amide bond carbonyl group is bent into the plane of the ring at an angle of about 45". This again results in a filling of the central cavity observed in ring 1. Thus, this carbonyl group is within the VDW contact range of the atoms directly across the ring.

Ring number 10 is 1.16 Kcal/mol higher in energy than ring 1 and represents a hybrid between ring structures 2 and 7. The w3 amide group is rotated 180" relative to ring 1, and the w2 amide bond carbonyl is pointing toward the center of the ring, as it is the case in ring 7. In comparison with ring conformation 2, the w4 amide proton is also pointing toward the center o,f the ring but is located on the opposite side of the ring. The distance of 2.71 A observed between this carbonyl group and the amide nitrogen would be compatible with hydrogen-bond formation; however, the observed valence angle of 130 " indicates that this nonlinear hydrogen bond must be very weak a t best. The higher energy observed for this ring relative to rings 1, 2, and 7, which lack any apparent hydrogen bonds, indicates that hydrogen bonding cannot have any real influence on the overall conformation of this constrained ring system.

The next higher energy ring conformer is more than 3.5 Kcal/mol greater in energy than ring conformation 1. Therefore, further work on these rings was not done. It was noted that, within these higher energy ring conformations, the greatest flexibility was observed around the w4 amide group, the x24 and x23 (~ -0 rn side chain), and the ~ 4 2 and x4' (Asp side chain). No strong hydrogen bonds were observed with any of these ring conformations.

Conformation of the Side Chains

The next step involved placing the Tyr' residue and the Phe3 side chain on the four lowest energy ring conformers and performing a grid search on each of these completed structures. Using a 30" grid on each rotatable bond resulted in between 3000 and 7000 allowed conformations for each ring studied. Clearly, there is a considerable amount of conformational freedom for these seven rotatable bonds defining the conformations of the Tyr' residue and the Phe3 side chain. However, when the energies were calculated for these conformations it was found that one could map out regions of low energy and regions of high energy within the allowed conformational space. Structures of a family of conformers located in a particular low-energy region were gener- ally within a 30"-120" range for each rotatable bond and within these regions an energy well could be located as the lowest energy member of each family of conformations. Rotation about each bond can be summarized as follows:

1. The #4 bond was found to have the greatest freedom of movement, generally showing one long continuous region of low energy. Rotation about this bond appeared independent of (not influenced by) the other rotatable bonds.

1440 WILKES AND SCHILLER

Fig. 4. Lowest energy structure found for H-Tyr-D-Qrn-Phe-bp-NH, (two orientations).

2. The phenylalanine side chain (xS1, x32 bonds) generally showed one or two regions of low energy. These low energy regions were small (30-60 O ) and were influenced by rotation about other rotatable bonds.

3. The remaining four bonds (a2, +1, xll, x12) tended to show considerable free rotation. The a2 bond had 1 or 2 low-energy regions in a 30-60" range each, and the remaining bonds showed larger low-energy regions (60-90° range).

Two to eight low-energy families were found for each ring conformation chosen and a total of 20 low-energy families were found altogether (Table 11). The lowest energy conformation (1) is shown in Fig. 4, and the next four lowest energy conformations are shown in Fig. 5. It should be noted that in some cases there was considerable similarity in the side-chain configurations found between structures with different ring conformations (cf. conformers 1 and 11).

DISCUSSION It is apparent that formation of the cyclic 13-membered ring limits the

conformational possibilities for that portion of the molecule. In addition, it should be noted that there is considerable similarity between the 10 ring conformations allowed by the search procedure used. All 10 conformations are fairly symmetrical in that they are round, and when viewed from the side they are flat and show little boat- or chairlike formation. The three amide bonds within the ring are generally either perpendicular to the plane of the ring or tilted slightly toward the center of the ring.

Extension of the analysis to include the exo-cyclic Tyrl residue and the Phe3 side chain indicates that the orientational freedom of the 1- and 3-position side chains is not highly restricted by the ring, and that many

CONFORMATION OF A CYCLIC OPIOID PEPTIDE 14-41

Fig. 5. Low-energy conformations of H-Tyr-D-?rn-Phe-Asp-NH2: Clockwise from upper left: 13, 12, 11, 10 (Table 11).

low-energy configurations of the two aromatic side chains are possible. In particular, the Tyr' residue enjoys a high degree of conformational freedom. This amino acid residue has four preferred low-energy regions (Figs. 4 and 5). The Phe3 side chain, on the other hand, has only two preferred low-energy regions and its lowest energy configuration is dependent upon the torsional angles of the T Y ~ ' residue. There are, however, only 9 conformations within 2 Kcal/mol of the lowest energy structure.

In morphine, the distance between the center of the aromatic A ring and the nitrogen atom is 4.55 A, and the distance between the hydroxyl oxygen of the A ring and the nitrogen atom is 7.08 A.22 It is generally believed the tyramine moiety of morphine and the tyramine portion of the enkephalins interact with the same region of opioid receptors.23 None of the low-energy solutions of the cyclic opioid peptide analog have interatomic distances that would correspond to the interatomic distances found in morphine (Table 11). The results of theoretical energy calculations performed with another cyclic

1442 WILKES AND SCHILLER

enkephalin analog had also shown that the interatomic distances in the tyramine portion of the obtained lowest energy conformers were different from the corresponding distances in morphine.12 Furthermore, a theoretical conformational analysis of the linear peptide [Met'lenkephalin indicated that the tyramine segment in the resulting lowest energy conformer did not show spatial overlap with the tyramine portion present in morphine or ~ r ipav ine .~~ However, a peptide conformer only 3.5 Kcal/mol higher in energy than the lowest energy conformer was obtained that did show this spatial overlap with morphine in the tyramine region. I t is thus conceivable that both linear and cyclic enkephalin analogs could undergo a conformational change when bind- ing to the receptor to produce a conformation of their tyramine segments identical to that of the tyramine moiety in morphine. Since such a conforma- tional change could occur at an energy expenditure of only a few Kcal/mol, it could be paid for by part of the binding interaction energy.

Another important conformational parameter is the intramolecular distance between the aromatic rings of Tyr' and Phe3. Several proposals concerning the structural correspondence between the Phe4 aromatic ring in enkephalin and various moieties in morphinelike opiates have been made. According to one hypothesis2' the aromatic ring in the 4 position of enkephalin would interact with the same receptor subsite as does the phenethyl substituent on carbon-19 of 7-a-[1-(R)-hydroxy-l-methyl-3-phenylpropyl]-6,14-e~- ethenotetrahydrooripavine (PEO). A [Met'lenkephalin conformer of rela- tively low energy which shows spatial overlap of the Tyr' tyramine segment and Phe4 aromatic ring with the corresponding moieties in PEO has indeed been found.24 The approximate distance between the two aromatic rings in PEO is about 10 and fluorescence energy transfer experiments performed with linear Trp4 enkephalin analogs in solution had indicated that the average intramolecular distance between the aromatic rings in the 1 and 4 position is between 9 and 11 However, it should be realized that linear enkephalins in solution exist in a conformational equilibrium, and that only one of the many conformers may interact with the receptor-or that the bioactive conformation may not be present at all in solution and may only be assumed in the receptor-bound state. Thus, the average intramolecular distance be- tween the two aromatic rings of enkephalin determined in solution may be quite different from that in the peptide in its receptor-bound conformation. In a second proposal the meta and para positions of the Phe4 aromatic ring in enkephalin are considered as functional correlates of atoms C-5 and C-6 contained in the C-ring of m~rph ine .~ '*~~ A conformer of the enkephalin tetrapeptide H-Tyr-D-Ala-Gly-Phe-OH based on this proposal and ob- tained by an extensive computer search is characterized by a close proximity of the two aromatic rings (- 5 A). The intramolecular distances between the aromatic rings of the cyclic peptide determined in the present study would thus appear more compatible with the latter model of the bioactive conforma- tion.

However, it should be realized that the cyclic opioid peptide analog de- scribed in the present paper contains the Phe residue in the 3 position of the peptide sequence as it is in the case with the p-casomorphins (morphiceptin) and the dermorphins, whereas in the enkephalins the Phe residue is in the 4 position. Structure-activity studies with analogs of morphiceptin, dermorphin,

CONFORMATION OF A CYCLIC OPIOID PEPTIDE 1443

and H - T ~ ~ - D - O F - P ~ ~ - A ~ ~ - N H , containing a p-nitrophenylalanine re- sidue in place of the Phe r&idue indicated that the Phe3 aromatic ring in these peptides may interact with a p-opioid receptor subsite different from that with which the Phe4 aromatic ring in the enkephalins i n t e r a ~ t s . ~ ~ . ~ ~ Therefore, a structural comparison of H-Tyr-~-OpPhe-hp-NH, con- formers with conformational models of morphiceptin- and dermorphin-related peptides may be more relevant. A receptor-bound conformation of morphi- ceptin was proposed based on a theoretical energy calculation performed with various active and inactive morphiceptin Inspection of the latter conformational model reveals that the two aromatic rings are not in close proximity. The distance between the Tyr'-Phe3 aromatic rings in another receptor-bound conformation obtained from theoretical conformational analy- sis of various conformationally restricted morphiceptin analogs also appears larger (9-12 A)32 than the corresponding distances observed with the low- energy conformers of the cyclic analog in the present study. Furthermore, the two aromatic rings in a proposed bioactive conformation of the dermorphin- related tetrapeptide Tyr-D-Ala-Phe-Gly-NH 233 are also separated by a distance much larger than that seen in the lowest energy structure of H-Tyr-D-Orn-Phe-Asp-NH,. In a proposed model of the opioid receptor, the Tyrl and $he4 aromatic rings of enkephalin are assumed to interact with recepter subsites (T and P) that are well separated from one another,34 and it is obvious that the lowest energy conformer of the cyclic opioid peptide analog described in this paper would not fit into this receptor topography if one assumes interaction of the Tyr' and Phe3 aromatic rings with the T and P subsites, respectively. However, it should be pointed out again in this context that the Phe3 aromatic ring in H-Tyr-D-O~-Phe-~p-NH, may bind to a receptor subsite different from that with which the Phe4 aromatic ring in enkephalin interacts (see above). An extension of the conformational analysis described in this paper to various active and inactive cyclic analogs of H-Tyr-~-Ov-Phe-hp-NH, can be expected to provide more insight into the pharmacophoric conformation at the p receptor.

This work was supported by operating grants from the Medical Research Council of Canada (MT-5655) and the Quebec Heart Foundation. The authors would like to thank Mamdouh Mikhail, Pierre Pa&, and Pierre Chbnier for the excellent upkeep of the VAX 750. We also thank Dr. Jacques Drouin for the use of his Tektronics terminal at need.

References 1. Schiller, P. W. (1984) in The Peptides: Analysis, Synthesis, Biology, Vol. 6, Udenfriend, S.

2. DiMaio, J. & Schiller, P. W. (1980) Proc. Nutl. Acad. Scz. USA 77, 7162-7166. 3. Schiller, P. W. & DiMaio, J. (1982) Nature (London) 297,74-76. 4. DiMaio, J., Nguyen, T. M.-D., Lemieux, C. & Schiller, P. W. (1982) J. Med. C h m . 25,

5. Berman, J. M., Goodman, M., Nguyen, T. M.-D. & Schiller, P. W. (1983) Biochem. Biophys. Res. Commun. 115,864-870.

6. Schiller, P. W., Eggimann, B., DiMaio, J., Lemieux, C. & Nguyen, T. M.-D. (1981) Biochem. Bwphys. Res. Commun. 101,337-343.

7. Schiller, P. W., DiMaio, J. & Nguyen, T. M.-D. (1985) in Proceedings of the 16th FEBS Congress, Ovchinnikov, Y. A., Ed., VNU Science Press, Utrecht, The Netherlands, pp. 457-462.

& Meienhofer, J., Eds., Academic Press, Orlando, FL, pp. 219-268.

1432-1438.

WILKES AND SCHILLER

8. Mosberg, H. I., Hurst, R., Hruby, V., Galligan, J. J., Burks, T. F., Gee, K. & Yamamura,

9. Masberg, H. I., Hurst, R., Hruby, V. J., Gee, K., Yamamura, H. I., Galligan, J. J. & Burks,

10. Schiller, P. W., Nguyen, T. M.-D., Maziak, L. A. & Lemieux, C. (1985) Biochem. Biophys.

11. Schiller, P. W., Nguyen, T. M.-D., Lemieux, C. & Maziak, L. A. (1985) J. Med. Chem. 28,

12. Hall, D. & Pavitt, N. (1984) BiopoZymers 23, 144-1455. 13. Hall, D. & Pavitt, N. (1985) BiopoZymers 24,935-945. 14. Maigret, B., FouniB-Zaluski, M.-C., Roques, B. & Premilat, S. (1986) Mol. Phurmucol. 29,

15. Mammi, N. J., Hassan, M. &Goodman, M. (1985) J. Am. Chem. Soc. 107,4008-4013. 16. Kessler, H., Holzemann, G. & Zechel, C. (1985) Znt. J. Pept. Protein Res. 25, 267-279. 17. Hall, D. & Pavitt, N. (1984) BiopoZymers 23,2325-2334. 18. Mosberg, H. I. & Schiller, P. W. (1984) Znt. J. Pept. Protein Res. 23, 462-466. 19. Smith, G. M. & Veber, D. F. (1986) Biochem. Biophys. Res. Commun. 134,907-914. 20. K h a n , B. L. (1982) Technical Memorandum, No. 49, Department of Computer Science,

21. Motoc, I. & Marshall, G. R. (1985) Chem. Phys. Lett. 116, 415-419. 22. Mackay, M. & Hodgkin, D. C. (1955) J. Chem. Soc., 3261-3267. 23. Horn, A. S. & Rodgers, J. R. (1976) Nature 260, 795-797. 24. Loew, G. H. & Burt, S. K. (1978) Proc. Natl. Acad. Sci. USA 75, 7-11. 25. Bradbury, A. F., Smyth, D. G. & Snell, C. R. (1976) Nature (London) 260, 165-166.

27. Gorin, F. A. & Marshall, G. R. (1977) Proc. Natl. A d . Sci. USA 74, 5179-5183. 28. GoM, F. A., Balasubramanian, T. M., Barry, C. D. & Marshall, G. R. (1978) J. Supramol.

29. Schiller, P. W., Nguyen, T. M.-D., DiMaio, J. & Lemieux, C. (1983) Life Sci. 33 (suppl.),

30. Schiller, P. W., Nguyen, T. M.-D., Maziak, L. A., Lemieux, C. & Wilkes, B. C. (1987) in

31. Luew, G., Keys, C., Luke, B., Polgar, W. & Toll, L. (1986) Mol. Phurmucol. 29, 546-553. 32. Nelson, R. D., Gottlieb, D. I., Balasubramanian, T. M. & Marshall, G. R. (1986) in NIDA

Research Momgraph 69, Rapaka, R. S., Barnett, G. & Hawks, R. L., Eds., US. Government Printing Office, Washington, D.C., pp. 204-230.

33. Pastore, A., Tancredi, T. & Temussi, P. A. (1985) in Peptides: Structure and Function, Proceedings of the Ninth American Peptide Symposium, Deber, C. M., Hruby, V. J. & Kopple, K. D., Eds., Pierce Chemical Company, Rockford, IL., pp. 529-532.

H. I. (1983) Life Sci. 32, 2565-2569.

T. F. (1983) P ~ o c . Natl. A ~ a d . SCi. 80, 5871-5874.

Res. Commun. 127, 558-564.

1766-1771.

314-320.

Washington University, St. Louis.

26. Schillm, P. W., Yam, C. F. & L~s, M. (1977) Bio~hemisQ 16, 1831-1838.

StrLcct. 1, 27-39.

319-322.

Peptides 1986, Thdoropoulas, D., Ed., W. DeGruyter, Berlin, in press.

34. Portoghese, P. S., Alreja, B. D. & Larson, D. L. (1981) J. Med. Chem. 24, 782-787.

Received December 3, 1986 Accepted March 20, 1987


Top Related