Theoretical Conformational Analysis of the Opioid 6 Antagonist H-Tyr-Tic-Phe-OH and the lu. Agonist H-Tyr-D-Tic-Phe-N H2

BRIAN C. WILKES and PETER W. SCHILLER

Laboratory of Chemical Biology and Peptide Research, Clinical RPwarch Institute of Montreal,* 11 0 Pine A v e n u e West, Montreal, Quebec, Canada H 2 W 1 R7

SYNOPSIS

A molecular mechanics study (grid search and energy minimization) of the highly 6 receptor- selective 6 opioid antagonist H-Tyr-Tic-Phe-OH ( TIP; Tic: tetrahydroisoquinoline-3-car- boxylic acid) resulted in four low energy conformers with energies wit,hin 2 kcal/mol of that of the lowest energy structure. These four conformers cont,ain trans peptide bonds only and represent compact structures showing various patterns of aromatic ring stacking. The centrally located Tic residue imposes several conformational constraints on the N- terminal dipeptide segment; however, the results of molecular dynamics simulations in- dicat,ed that this tripeptide still shows some structural flexibility, particularly at the Phe3 residue. Analogous studies performed with the structurally related p receptor-selective p agonist H-Tyr-D-Tic-Phe-NH' resulted in low energy structures that were also compact but showed patterns of ring stacking different from those obtained with TIP. Superim- position of low energy conformers of T IP and H-Tyr-D-Tic-Phe-NH, revealed that the Phe3 residues of the L-'ric- and the u-Tic peptide were always located on opposite sides of the plane defined by the Tic residue, thus providing an explanation for the distinct activity profiles of the two compounds in structural terms. Attempts to demonstrate spatial overlap between the pharmacophoric moieties of low energy conformers of TIP and the nonpeptide 6 antagonist naltrindole were made by superimposing either the Tyr' and Tic' aromatic rings and the N-terminal amino group or the Tyr' and Phe3 aromatic rings arid the N- terminal amino group of the peptide with the corresponding aromatic rings and nitrogen atom in the alkaloid structure. In each case a low energy structure of TIP was found that showed good spatial overlap of all three specified pharmacophoric groups. These two con- formers may represent candidate structures for the 6 receptor-bound conformation of 7'". 0 1994 John Wiley & Sons, Inc.

INTRODUCTION

The development of potent agonists and antagonists with high selectivity for each of the three major opioid receptor types ( p , 6, K ) continues to be an important goal in opioid pharmacology and may lead to new therapeutic agents (for reviews, see Refs. 1 and 2 ) . Receptor-selective opioid antagonists that. have been described so far include both peptide an- alogues ( reviewed in Refs. 1 and 2 ) and nonpeptide

H i q x ~ l y m e r s , Vul. 34, 1213-1219 (1994) (c 19114 J o h n Wilrv & Sons. Inc. CCC OOOfi~3525/94/091213-O7

* Affiliated with the University of Montreal.

compounds (reviewed in Ref. 3 ) . Recently, we dis- covered that the opioid peptide analogue H-Tyr-Tic- Phe-Phe-NH2 (TIPP-NH,; Tic = tetrahydroiso- quinoline-3-carboxylic acid) was a potent. 6 selective 6 antagonist, whereas the diastereoisomeric peptide, H-Tyr-D-Tic-Phe-Phe-NH,, was a p selective p ag- ~ n i s t . ~ The L-Tic-analogue with a free C-terminal carboxyl group, H-Tyr-Tic-Phe-Phe-OH ( TIPP ) , was an even more &selective and more potent 6 an- tagonist than TTPP-NH,. The correspcinding des- Phe4 tripeptide analogues H-Tyr-Tic-Phe-OH ( 6 antagonist ) and H-Tyr-u-Tic-Phe-NH, ( p agonist ) retained an opioid activity profile similar to tha t of their respective parent tetrapeptides but were

1213

1214 WILKES AND SCHILLER

somewhat less potent and less selective. Both TIPP and T I P are more potent and more 6 selective than the enkephalin-derived 6 antagonist N,N-diallyl- Tyr-Aib-Aib-Phe-Leu-OH ( ICI 174,864) .5 The highly potent nonpeptide 6 antagonist naltrindole6 is more potent than TTPP and TIP but considerably less 6-sele~tive.~

The observation that configurational inversion a t the Tic2 residue in T I P ( P ) peptides converts a 6 selective 6 antagonist into a p selective agonist is most interesting and an attempt. should be made to explain the opposite opioid activity profiles of the diastereoisomeric peptides in terms of conforma- tional characteristics. The tripeptides are particu- larly suitable for theoretical conformational analysis because essentially they contain only seven freely rotatable bonds. In this paper, we describe a com- parative conformational analysis of the 6 antagonist H-Tyr-Tic-Phe-OH (TIP) and the p agonist H-Tyr- D-Tic-Phe-NH, by molecular mechanics calcula- tions and molecular dynamics simulations. The re- sults of a conformational comparison of low energy conformers of T I P with the naltrexone-derived 6 antagonist naltrindole are also presented.

METHODS

All calculations were performed using the molecular modeling software SYBYL ( Tripos Associates, St. Louis, MO) , version 5.5, on a VAXstation 3510. Molecules were viewed with an Evans and Suther- land PS330 computer graphics display terminal. The standard SYBYL force field7 was employed for en- ergy calculations, and a dielectric constant of 78 was used to simulate an aqueous environment.

Molecular mechanics calculations were carried out essentially as previously described.x The two al- lowed side-chain configurations for the cyclic amino acid Tic and its D-isomer are g+ and g - . The two conformers were independently constructed for both Tic and D-Tic using the standard fragment library, and all four striict,ures were minimized. The Tyr' and Phe3 residues were then added to these Tic con- formers to generate the tripeptides to be examined in this study. The two peptides were studied with their N-terminal amino group in protonated form (positively charged), and in the case of TIP, with a negatively charged carboxylate function at the C- terminus. Each of the starting peptide conforma- tions was extensively minimized. Subsequently, a grid search was performed in which the dihedral an- gles of all rotatable bonds were systematically varied

by 30" increments. Both cis and trans peptide bonds were allowed. For each grid search the resulting conformers were grouped into families based on similarity of their dihedral angles ( t -3O0 ) , the num- ber of families ranging from 60 to 120. The lowest energy member of each conformational family was then extensively minimized and the resulting con- formers were ranked in order of increasing energy.

Molecular dynamics simulations were carried out with T I P using the lowest energy conformer ob- tained in the molecular mechanics study as starting conformation. The Verlet method was employed for integrating the equations of motion.' A time step of 1 fs was used and data were recorded for analysis every 50 fs. The simulation was carried out for 300 ps a t 300 K. The dynamics trajectory was analyzed for selected distances between functional groups and all torsional angles were monitored. Conformations were sampled along the trajectory and were com- bined to demonstrate the accessible conformational space for the various residues in TIP (Figure 2 ) .

RESULTS AND DISCUSSION

The systematic conformational analysis resulted in four conformations within 2 kcal/mol (Table I ) and in 13 conformations within 3 kcal/mol of the lowest energy structure found. The four lowest energy con- formers are shown in Figure 1. In the lowest energy structure, the three aromatic rings are stacked on top of each other, with the Tyr' aromatic ring being sandwiched between the Tic2 and Phe3 aromatic rings. The large number of favorable van der Waals contacts between these aromatic rings is the major reason for the low energy of this conformation. In addition, this structure contains an inverse y-turn stabilized by a Tyrl-CO- - -HN-Phe3 hydrogen bond. Other low energy conformers are characterized by various patterns of aromatic ring stacking. In general, in all the low energy conformers observed, the side chains of the three aromatic amino acid residues tended to be clustered together in overall compact structures, whereas more extended confor- mations were found to he higher in energy.

The four lowest energy conformers of T I P con- tained trans peptide bonds only. The lowest energy conformer with a cis peptide bond between the Tyr' and Tic' residues (conformer 861, Table I ) was 2.1 kcal/mol higher in energy than the lowest energy structure with all-trans peptide bonds (conformer 996) and was the fifth lowest energy conformer overall. In the cis peptide bond containing conformer the Tyr' and Tic2 aromatic rings are also in close

Tab

le I

Tor

sion

al A

ngle

s an

d E

nerg

ies

of L

ow E

nerg

y C

onfo

rmer

s of

H-T

yr-T

ic-P

he-O

H an

d H

-Tyr

-D-T

ic-P

he-N

H2

I

996

1070

10

43

927

86 1

1525

68

7 -T

yr-D

-Tic

-Phe

- 12

85

1236

12

07

1224

51

0 50

3 92

6

H-T

yr-T

ic-P

he-O

H

143.

2 70

.6

135.

9 11

2.7

140.

0 13

6.7

170.

6

147.

8 13

8.3

137.

7 13

9.4

86.6

10

3.9

148.

6

W

-177

.8

-176

.7

-175

.2

-168

.2

-7.1

-3

.3

169.

4

179.

2 17

5.4

178.

8 17

9.6

0.6

1.7

-172

.6

-76.

3 -7

8.4

-89.

5 -8

6.1

-65.

4 -7

0.0

-53.

6

109.

4 91

.2

94.6

97

.8

103.

8 68

.1

55.7

83.1

77

.7

163.

3 74

.0

170.

5 -3

2.0

-45.

6

-167

.9

-66.

0 -6

8.0

-69.

1 -4

1.0

-162

.1

-93.

5

178.

0 17

8.9

177.

0

176.

1 17

7.3

178.

7

174.

4

-179

.8

-177

.4

-178

.2

-176

.2

-177

.6

179.

6 17

2.7

-67.

0 -1

50.2

-1

37.4

-1

50.8

-1

47.5

-4

6.9

-70.

0

-142

.5

59.9

-7

0.3

-150

.7

-145

.6

-148

.4

-54.

7

-55.

5 -4

7.6

-52.

8 -5

2.8

173.

5 -4

0.4

-14.

0

169.

0 17

0.1

163.

2 16

9.9

178.

4 -6

2.2

-36.

6

174.

6 -1

75.6

-1

75.5

-1

76.8

-1

79.9

17

9.5

-58.

1

178.

5 -1

79.9

17

9.9

-179

.8

-175

.8

-175

.2

64.4

98.7

53

.6

70.8

55

.1

89.9

58

.6

61.8

56

.7

93.8

-4

2.2

94.5

51

.1

95.9

-4

7.4

93.9

-4

4.0

91.7

-5

8.1

92.6

-5

6.3

93.3

-5

6.6

78.5

-6

1.1

89.9

41

.6

84.9

46

.9

-46.

6 -4

6.9

-44.

3 -4

6.2

48.1

46.3

16.5

40

.6

36.0

35

.0

38.1

-46.

5

-47.

1 -4

7.4

-61.

4 10

4.5

-57.

0 98

.7

-62.

0 92

.5

-63.

4 90

.8

64.0

92

.2

61.0

86

.9

62.4

81

.3

-54.

1 11

4.4

-48.

2 11

3.6

59.8

79

.7

-55.

0 93

.6

-53.

6 96

.3

-60.

3 10

2.0

58.4

80

.9

0.02

4 1.

028

1.11

9 1.

213

2.04

5 2.

084

2.68

2

2.01

1 2.

444

2.47

2 2.

627

2.81

4 3.

851

4.79

6

a T

he d

esig

natio

n of

the

low

est e

nerg

y co

nfor

mer

s is

bas

ed o

n th

e nu

mbe

ring

of

the

conf

orm

atio

ns u

sed

in t

he c

onfo

rmat

iona

l se

arch

pro

cedu

re.

1216 WILKES AND SCHILLER

Figure 1. The four lowest energy structures of H-Tyr- Tic-Phe-OH ( T I P ) . Clockwise from upper left: 996,1070, 104% 927.

proximity to one another (Figure 6 ) . However, in contrast to the compact structures seen with the all- trans lowest energy conformers, the Phe3 residue in conformer 861 assumes an orientation that prevents van der Waals interactions of its aromatic ring with the rest of the molecule.

The presence of the Tic2 residue in the tripeptide TIP gives rise to several local conformational con- straints around the 2 position of the peptide se- quence. Whereas the side chains of most proteino- genic amino acids can assume three different con- formations characterized by the torsional angle ( X l ) values f 6 0 " ( g + ) , 180" ( t ) , and -60" ( g - ) , only the g+ and g configurations are possible in the case of Tic. In either configuration the heterocyclic six- membered ring assumes a twisted-boat conforma- tion. In all four lowest energy conformers of TIP the Tic residue was found to have the g + configu- ration. An all-trans conformer (687) with Tic in the g- configuration was about 2.7 kcal/mol higher in energy than the lowest energy structure found (con- former 996). Interestingly, in the lowest energy czs peptide bond containing conformer (861) the Tic' side-chain conformation is g- . However, another conformer with a Tyr1-Tic2 cis peptide bond and the Tic' residue in the g ' configuration (conformer 1525 ) was only slightly higher in energy. Torsional angles and energies of conformers 1525 and 687 are also listed in Table I.

The & dihedral angle at the Tic' residue is limited to narrow ranges of values because the N-C" bond is part of the tetrahydropyridine ring. Allowed values

for this angle are around -80" (-65" to -100") when the side chain is in the g+ configuration and around -60" ( -40" to -75" ) with the side chain in the 6- configuration. Indeed, in the four lowest en- ergy conformers of T I P with Tic' in the g+ config- uration, & was around -80". The lowest energy conformers of T I P with Tic2 in the g - configuration showed 4' values of -65" and -54" for the cis pep- tide containing conformer (861 ) and for the trans peptide bond containing one ( 687 ) , respectively. A further conformational constraint due to the pres- ence of an N-alkylated amino acid in a peptide is the limitation of the + dihedral angle a t the preced- ing amino acid residue to positive values." In the molecular dynamics simulations performed with H- Tyr-Tic-Phe-OH, the + angle a t Tyr' (g1) was in- deed limited to positive values ranging from f60" to +180° (data not shown).

Despite these various conformational constraints, the tripeptide still has some structural flexibility, as indicated by the number of low energy structures that resulted from the molecular mechanics calcu- lations and the molecular dynamics simulations. Examination of 20 conformations sampled at 15 ps intervals along the dynamics trajectory (Figure 2 ) demonstrated that in particular the Phe3 residue enjoys extensive orientational freedom. As expected, the Tyr' residue was found to be somewhat confor- mationally restricted and the centrally located Tic residue showed very limited movement during the entire 300 ps simulation period. The Tic' residue

r'igure 2. 'l'wenty snapshots of H-Tyr-Tic-Phe-OH taken at 15 ps intervals along the dynamics trajectory. The C" of Tyr' and the N and C* of Tic2 were superim- posed, and hydrogen atoms were omitted.

ANALYSIS OF THE OPIOID IS ANTAGONIST AND u AGONIST 1217

did assume both t,he g+ and the g- side-chain con- formation in this molecular dynamics simulation, but spent most of the time (80% ) in the g+ config- uration.

A systematic grid search and energy minimization study of the p opioid receptor-selective p agonist H- Tyr-D-Tic-Phe-NH2 resulted in 11 conformations with energies within 2 kcal/mol and in 18 confor- mations with energies within 3 kcal/mol of that of the lowest energy conformer. The four lowest energy structures are shown in Figure 3, and their structural parameters and energies are listed in Table I. All four contain trans peptide bonds only. As i t was the case with the lowest energy structure of H-Tyr-Tic- Phe-OH, the three aromatic rings in the lowest en- ergy conformer (1285) of the D-Tic2-tripeptide were also stacked one upon another in a sandwich-type configuration. However, in contrast to TIP, the D- Tic2 side chain rather than the Tyr' side chain was located in the middle of the sandwich structure. The lowest energy structure of H-Tyr-D-Tic-Phe-NH, with a cis peptide bond between the Tyr' and Tic2 residues (conformer 510) is 0.8 kcal/mol higher in energy than the lowest energy conformer with all- trans peptide bonds.

In the four lowest energy all-trans structures the n-Tic2 residue assumes the g - conformation. Inter- estingly, in the cis peptide bond containing con- former (510) the Tic2 residue is also in the g- con- figuration. It is of interest to note that in recently determined crystal structures of D-Tic containing model peptides the side-chain configuration of D-

Figure 3. The four lowest energy structures of H-Tyr- D-Tic-Phe-NH,. Clockwise from upper left: 1285, 1236, 1207, 1224.

Figure 4. Superimposition of lowest energy conformers of H-Tyr-Tic-Phe-OH (conformer 996, drawn in heavy lines) and H-Tyr-D-Tic-Phe-NH, (conformer 1285, drawn in light lines). An inverse y-turn stabilized by a Tyr 'CO - - * HNPhe3 hydrogen bond is observed with the L-Tic' analogue.

Tic was also g-'I. The lowest energy conformer with the D-Tic2 residue in the g+ conformation (con- former 503) also contained a Tyr'-D-Tic* cis peptide bond and was about 1.8 kcal/mol higher in energy than the overall lowest energy structure (1285), whereas the energy of an all-trans conformer (926) with D-Tic' in the gt configuration was 2.8 kcal/ mol higher. Again, the range of accessible & values was very limited. Observed values for & in conform- ers with D-Tic' in the g- configuration fell into the range from 65" to 110", whereas those seen in con- formers with D-Tic2 in the g+ configuration ranged from 40" to 75".

The lowest energy conformations of H-Tyr-Tic- Phe-OH and H-Tyr-D-Tic-Phe-NH, were compared by superimposing the peptide backbone atoms of the centrally located Tic residue contained in these two compounds (Figure 4). The most striking difference between the L-Tic2- and the D-Tic2-tripeptide re- vealed by this superimposition is the location of the Phe3 residue of these peptides on opposite sides of the plane defined by the Tic residue. This dramat- ically different spatial location of the Phe" residue due to the conformational constraints imposed by the Tic residue is seen in spatial comparisons of all the low energy conformers (including the cis peptide bond containing ones) of these two compounds and may explain the fact that configurational inversion from D to L a t the Tic2 residue converts a p selective agonist into a 6 selective antagonist. Finally, it is

1218 WILKES AND SCHILLER

interesting to point out that the tyrosine residue shows the same conformational preferences xI1, and Xl2 angles) in the lowest energy conformers of both peptides.

The recently discovered nonpeptide 6 antagonist naltrindole6 contains an indole moiety fused to the C6-7 position of the morphinan skeleton, and it has been postulated that the six-membered ring of the indole group in this compound may play the same role in the interaction with the 6 receptor as the Phe4 aromatic ring in the enkephalins or the Phe' aromatic ring in the deltorphins. However, a T IPP analogue containing pipecolic acid in place of Tic2 has recently been shown to be a potent p agonist, indicating that the Tic2 aromatic ring in TIPP pep- tides plays a crucial role for 6 antagonist behavior.12 Therefore, two different attempts were made to demonstrate spatial overlap between either one of two different proposed sets of pharmacophoric moi- eties in low energy conformers of T I P and corre- sponding functional groups in naltrindole. In the first case, the phenolic ring of Tyr ' , the phenyl moiety of Tic *, and the N-terminal amino group of the peptide were superimposed with the corre- sponding aromatic rings and nitrogen atom in the alkaloid structure. Best spatial overlap was achieved with the fourth lowest energy conformer (927), which is 1.2 kcal/mol higher in energy than the low- est energy conformer (Figure 5). In this conformer the intramolecular distance between the Tyr and Tic2 aromatic rings (6.5 A ) was close to that ob- served between the aromatic moieties in naltrindole (6.4 A) and the spatial overlap of the three proposed functional groups with those in naltrindole was ex- cellent, the rms deviation being 0.6 A. In an alter-

Figure 5. Superimposition of the fourth lowest energy all-trans structure of T IP (conformer 927, drawn in heavy lines) with the minimized structure of naltrindole (drawn in light lines). The N-terminal amino group and the Tyrl and Tic2 aromatic rings of the peptide are superimposed with the corresponding moieties in the alkaloid structure. Hydrogen atoms are omitted and the superimposed mol- ecules are shown in two different orientations.

Figure 6. Superimposition of the lowest energy struc- ture of TIP containing a Tyr '-Tic2 cis peptide bond (con- former 861, drawn in heavy lines) with the minimized structure of naltrindole (drawn in light lines). The N- terminal amino group and the Tyr' and Phe:' aromatic rings of the peptide are superimposed with the corre- sponding moieties in the alkaloid structure. Hydrogen at- oms were omitted and the superimposed molecules are shown in two different orientations.

native attempt to demonstrate spatial overlap be- tween TIP and naltrindole, the Tyr' and Phe3 ar- omatic rings and the N-terminal amino group of the peptide were superimposed with the corresponding functional groups in naltrindole. In this case, best results were obtained with the lowest energy con- former (861 ) containing a cis peptide bond between the Tyr and Tic2 residues (Figure 6) . In this con- formation the intramolecular distance between the Tyr' and Phe3 aromatic rings was 6.3 A and t,he overall spatial overlap of the three proposed func- tional groups was also excellent (rms deviation of 0.6 A ) . In both superpositions the respective aro- matic rings of T IP conformers 927 and 861 and the corresponding aromatic moieties of naltrindole were nearly coplanar. While it, was also possible to su- perimpose the N-terminal amino group and the Tyr' and Phe3 aromatic rings of the second lowest energy, all-trans conformer (1070) of T IP with the corre- sponding moieties of naltrindole, the obtained spa- tial overlap of the three pharmacophoric moieties was less satisfactory, insofar as the rms deviation was higher (0.75 A ) and the Phe3 aromatic ring was oriented perpendicular to the corresponding aro- matic ring in naltrindole.

Since conformers 927 and 861 both have energies less than 2.1 kcal/mol higher than that of the lowest energy structure, either one of them may represent the 6 receptor-bound conformation, albeit conformer 927 appears the more likely candidate structure in view of the demonstrated importance of the Tic2 aromatic ring for 6 antagonism."

ANALYSIS OF T H E OPIOID 6 ANTAGONIST AND fi AGONIST 1219

This work was supported by a maintenance grant from the Medical Research Council of Canada (MT-10131). We thank Dr. Ralf Schmidt for helpful discussions and CBcile Gravel for the preparation of the manuscript.

6. Portoghese, P. S., Sultana, M. & Takemori, A. E.

7. Clark, M., Cramer, R. D., I11 & Opdenbosch, N. V.

8. Wilkes, B. C. & Schiller, P. W. (1990) Biopolymers

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Received December 6, 1993 Accepted March 28, 1994