Topographical Requirements for &Selective Opioid Peptides* GREGORY V. NIKIFOROVICH, VICTOR j. HRUBY,+ OM PRAKASH, and CATHERINE A. GEHRIG Department of Chemistry, University of Arizona, Tucson, Arizona 85721

SYNOPSIS

The conformational possibilities of three different &selective opioid peptides, which are DPDPE ( Tyr-D-Pkn-Gly-Phe-D-Ph), DCFPE (Tyr-D-C$s-Phe-D-Pdn), and DRE (TyrD-Met-Phe-His-Leu-Met-Asp-NH2, dermenkephalin) , were explored using energy calculations. Sets of low-energy conformers were obtained for each of these peptides. The sets consisted of 61 structures for DPDPE, 32 for DCFPE, and 38 for DRE, including various types of rotamers of the Tyr and Phe side-chain groups. Comparison of the geometrical shapes of the conformers was performed for these sets using topographical considerations, i.e., examination of the mutual spatial arrangement of the N-terminal a-amino group, and of the Tyr and Phe side-chain groups. The results obtained suggest a model for the 6-receptor-bound conformer ( s ) for opioid peptides. The model suggests the placement of the Phe side chain in a definite position in space corresponding to the g- rotamer of Phe for peptides containing Phe4 and to the t rotamer for peptides containing Phe.3 The position of the Tyr' side chain cannot be specified so precisely. The proposed model is in a good agreement with the results of biological testing of @-Me-Phe4-substituted DPDPE analogues that were not considered in the process of model construction.

INTRODUCTION Efforts to determine the structural basis of receptor selectivity for the opioid peptide-receptor interaction will require careful consideration of the conformational and topographical features of the peptide ligand. T h e suggestion that different conformations are required for recognition and/or binding to p - and 6-opioid receptor types has been strongly supported by the synthesis of conformationally constrained cyclic analogues of opioid peptides showing Hiapolvmers, Vol. 31, 941-955 (1991) 1991 .John Wiley & Sons, Inc.

(c

CCC 0006-3525/91/080941-15$04.00

* Abbreuiations: All amino acids are of the

L variety unless otherwise stated. Abbreviations for amino acid residues follow those recommended by the IUPAC. Other abbreviations include the following: DPDPE, [ D-Pkn2,D-Pdn5]enkephalin, H-Tyr-DPkn-C,ly-Phe-D-Pe'n-OH;Pen, penicillamine or P,P-dimethylcysteine; DCFPE, H-Tyr-D-C4s-Phe-D-PQn-OH;DRE, dermenkephalin, H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2; DPLPE, H-Trr-D-Pkn-Gly-Phe-Pkn-OH;DSLET, H-Tyr-D-Ser-Gly-PheLeu-Thr-OH; DSTBULET, H-Tyr-D-Ser ( 0 % -Gly-Phe-Leu~) Thr-OH; BUBU, H-Tyr-D-Ser( 0%)-Gly-Phe-Leu-Thr(O%u) OH; DAGO, (D-ala2,N-MePhe', Gly'lenkephalin-OL. T o whom reprint requests should be addressed.

'

pronounced receptor selectivity (for reviews, see, e.g., Refs. 1and 2 ) . One of the most &selective cyclic analogues of enkephalin is [ D-Ph2,D-PfhS]enkephalin ( D P D P E ) , a cyclic pentapeptide with the sequence H-Tyr-D-Pe'n-Gly-Phe-D-P&-OH. In the last few years this analogue has been the subject of intensive conformational studies using theoretical 3-8 as well a s e ~ p e r i m e n t a l ~approaches. ~" Models for the solution conformation ( s ) and for the &receptor-bound conformer ( s ) of DPDPE were proposed in all papers3-"; in some of these studies the proposed conformers were compared with the conformational features of other compounds displaying various kinds of receptor ~electivity.~~' T h e theoretical approaches generally were based on energy calculations for DPDPE and other analogues using various computer programs and force fields including ECEPP,3*4,6 AMBER,s and SYBYL (Tripos force field) and a variety of search patterns for finding low-energy conformers. These differences will affect the values of the conformational energy estimations as pointed out ins, but the variety of conformers included in the initial set and selected for further consideration seems to be of more im94 1

942

NIKIFOROVICH ET AL.

portance than just the absolute values of energies Second, previous studies 3-7 did not consider the toobtained. In this respect some authors selected pographical similarities among the compounds, but rather wide sets of low-energy backbone conformers confined themselves to conformational similarities for further consideration, while others refer to of peptide backbones only. In our view, the model only a few selected conformers5 or mention larger of a receptor-bound peptide conformer should innumbers of low-energy conformers but do not declude the proper positions in space for the functionscribe t,hem.8 ally important side-chain groups. In this way, the Energy calculations also were used to refine peptide backbone can be regarded mainly as a moDPDPE structures constructed in conjunction with lecular scaffold for these functionally significant nmr data in water and DMSO s o l u t i ~ n s , using ~~'~ elements. This concept of topographical similarity the CHARMM and AMBER programs, respectively. appears to be very important for the design of pepThese latter studies start with possible structures tides a n d peptidomimetics.'' In the case of opioid consistent with observed nmr parameters (mostly peptides, the structural elements of primary signifthe values of proton-proton coupling constants, nuicance for &receptor recognition and/or binding are the a-amino group and the aromatic side-chain clear Overhauser effects, and temperature coeffimoieties of the Tyr and Phe residues (see reviews cients of chemical shifts for amide protons) and present models in accord with nmr data. There are in Refs. 1 and especially 2 ) . Other elements that .- ~ ~ appear to be i r n p ~ r t a n t ' , ' ~include the free carsome minor differences in the nmr data that may boxyl group a t the C-terminus and the methyl subbe related to the different solution conformational stituents of the C' atom of D-Pen'. However, since models of DPDPE in Refs. 9 and 10. Molecular dythe C-terminal carboxyl is amidated in DRE and namics simulations also were used for further rethe D-Pen' residue is replaced for D-Cys' in DCFPE finement of the proposed m o d e l ~ . ~With ~ " respect and for D-Met' in DRE, these structural elements t o understanding the "biologically active" confordo not appear to be as crucial. Therefore, we suggest mation ( s ) of 6-opioid ligands, two general limitathat the receptor-bound conformer for opioid peptions are inherent from nmr data: ( a ) The conformtides should take into account the similarity in the ers derived directly from nmr data may represent mutual space arrangement of the N-terminal a n averaged picture of the dynamic equilibrium exa-amino group and the side chains of the Tyr' and isting in solution even for a conformationally rePhe4'3' residues rather than the similarity in peptide stricted peptide like DPDPE; thus the resulting backbone structures as previously ~ t u d i e d . ~T-h~e models might not to be the precise molecular strucpresent paper describes a n examination of &receptor tures present in solution (see also, e.g., Refs. 12 and selectivity based upon all the above considerations. 1 3 ) . ( b ) The solution conformers might not be related in detail to those involved in receptor recognition and/or binding. METHODS AND RESULTS The computational studies also suffer from some limitations of the comparison procedures used pre viously. First, all analogues previously ~ o n s i d e r e d ~ - ~ DPDPE, DCFPE, and DRE were considered in this study as representatives of different sequence types contain a Phe residue in position 4 but not in pothat can give &selective opioid peptides. Both linear sition 3 of the amino acid sequence. More recently, ( D R E ) and cyclic ( D P D P E and DCFPE) comnew cyclic and linear peptides containing Phe3 were pounds are considered, as well as peptides containing discovered that possess good &receptor preference Phe3 (DCFPE and D R E ) or Phe4 ( D P D P E ) suband compete with DPDPE for the same receptor stituents. DRE 1-4 ( Tyr-D-Met-Phe-His-NH,) was site. For example, DCFPE (Tyr-D-C$s-Phe-D-Pdn) used as a n example of a p-selective peptide. and dermenkephalin (DRE, Tyr-D-Met-Phe-HisT h e energy calculations used the ECEPP potenLeu-Met-Asp-NH, ) have been reported (Refs. 14 tial force field22*23 with rigid valence geometry and and 15-17, respectively) to be highly &receptor setrans peptide bonds in all cases. Electrostatic interlective in competitive binding experiments vs the actions were taken into account with the value of p-opioid receptor selective ligands [ 3H] sufentanil, dielectric constant t = 2.0. In accordance with Refs. [ 3 H ]dermorphin, and [ 3 H ]DAGO, and the 6-opioid 22 and 23, only the /3-carboxyl group in the Asp7 receptor selective ligands [ 3H]DPDPE and [ 3 H ]residue in DRE was regarded as negatively charged DSLET. T h e selectivities of DCFPE and DRE are all other ionic groups in these molecules were either similar t o or better t h a n that of DPDPE. Interestdeprotonated (cationic) or protonated (anionic). ingly, the DRE N-terminal tetrapeptide amide There was some difference in valence geometry de( D R E 1-4) shows a preference toward p receptor^.'^ 3,436

943

REQUIREMENTS FOR &-SELECTIVE OPIOID PEPTIDES

scription for the cyclic and the linear compounds. It is natural t o expect some additional sterical hindrance for many conformers of the rigid cycle moieties of DCFPE and DPDPE due to possible atomatom overlappings of closely spaced atomic centers. T o avoid this hindrance the valence geometry of the cyclic compounds should be described with greater accuracy than may be needed in the case of more flexible linear compounds. Thus the o torsional angles were considered as variables only for the cyclic compounds. Also, only the methyl substituents of C~ atoms in u-Pen residues were regarded as united atomic centers for the cyclic peptides DPDPE and DCFPE; all other aliphatic and aromatic hydrogens were taken explicitly. On the other hand, only NH and H " protons were regarded as explicit atoms for the linear compounds DRE and DRE 1-4; all other CH, (aliphatic) or C H (aromatic) groups were represented by united atomic centers. T h e search for low-energy conformers was based on energy calculations for every combination of the backbone conformations that correspond to local minima in the potential maps for the dipeptide units of amino acid residues.24Such potential maps in Ref. 24 include 14 types of local energy minima lettering from A to F in the left half of the Ramachandran map ( i.e., for 4 < 0" ) and from A * to F * in the right half (for 4 > 0" ) . Basically, five points in these maps were considered for every L-amino acid residue as the "starting points" for energy minimization process in this study. These points were 4 = -145O, $ = 140" for E minimum of the map; 4 = -75", $ = 140" for F minimum; 4 = -75", $ = 80" for C / D minima; 4 = -No,$ = -60" for A / B / G minima; and 4 = go", $ = 60" for A * / B * / G * minima. In

Table I

the case of D-amino acid residues, the signs of the dihedral angle values were reversed with minima E *, F*, C*/D*, A*/B*/G*, and A / B / G now considered. Eight minima were assigned to Gly residue, namely E, F, C / D , A / B / G , A * / B * / G * , C*/D*, F*, and E*. For the terminal residues only minima with significant differences in # (N-terminal residue) or 4 (C-terminal) values were taken into account. This confines the Tyr' backbone conformations to E, C, and A / B / G ; the D-Pen475 conformations to E * , C*, and A / B / G for DCFPE and DPDPE; and Asp7 ( His4-NH2)conformations to E, C, and A */ B */ G * for DRE ( DRE 1-4 ) . In all steps of the calculations except the last one, the space arrangement of side-chain groups were optimized by a n algorithm described previously before the energy minimization procedure was started. This algorithm utilizes a stepwise grid search for energy minimum and consists of several steps. First, the 8, dihedral angle chosen from 8,angles ( i = 1 n) is rotated, starting from the 8: value until the value 8;"'"corresponding to local energy minimum U,,, (8,) is reached. Then the 8, angle is fixed in the 8 ; " I n value, instead of 8: value, and the procedure is successively repeated for every 8, angle to 8,,and again starting from 8,.The algorithm stops when all 8: became equal to 8y1", which means that the optimal values of 8, angles are achieved. In this study the algorithm was applied to the x, angles determining the side-chain rotations; the terminal dihedral angles of backbone also were the subject of such a n algorithm. The main calculation steps are described very briefly in Tables I and 11. For the cyclic compounds the first step was to find the low-energy conformers

''

--

Main Calculation Steps for DPDPE and DCFPE

-

No. of Conformers Considered

Energy Criterion (Kcal/mol)

No. of Conformers Selected

Compound

Step

DPDPE

1

Ac-D-Pen-Gly- Ala-D-Pen

360

10

51

2

Ac-D-Pen-Gly-Phe-D-Pen

51

10

45

3

Tyr-D-Pen-Gly-Phe-D-Pen

170

5

14

5

61

DCFPE

Sequence Considered

-

I

4

Tyr-D-P~n-Gly -Phe-D-Pen

1

Ac-D-Cys-Ala-D-Pen

225

10

65

2

Ac-D-Cbs-Phe-D-Pk

65

10

51

51

5

12

5

32

r

3 4

=

126

I

Tyr-D-Cys-Phe-D-Pen I

14 X 9

1

Tyr-D-Cys-Phe-D-Pen

12 x 9

=

108

944

NIKIFOROVICH ET AL.

Table I1 Main Calculation Steps for Linear Compounds

Compound DRE

DRE 1-4

Step

Sequence Considered Ac-His-Leu-Met-Asp-NH,

No. of Conformers Considered

Energy Criterion (Kcal/mol)

Conformers Selected

375 1240 1495 375 135

10 10 10 10 7 7

248 299 125 44 15 38 68 28

252

7 5 5

1 2 3 4

Ac-D-Met-Phe-His-Leu-Met-Asp-NH, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH,

5

Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 15 X 9

1

Tyr-D-Met-Phe-His-NH2

2

Tyr-D-Met-Phe-His-NH,

Ac-Phe-His-Leu-Met-Asp-NH,

of the model ring where the Phe residue was substituted for Ala. The disulfide bond closing was achieved using the system of parabolic potential functions of Uo(r - ro) type previously proposed22”,‘3 to maintain the proper ro distances for S-S valence bond and C@-S-Svalence angles ( CLI-S distances). In the very first step of the calculations rather “soft” U, values were used ( 100 k c a l / m o l - A 2 ) for the S-S bond and the valence angles (10 kcal/mol-A2) to avoid situations where disulfide bond closing would be achieved despite significant steric hindrance in the peptide backbone. Such situations could result in conformers with relatively high total energies that would be discarded by subsequent selection. Then the Uo values of 1000 and 100 kcal/ mol A’ respectively were applied in accordance with Refs. 22 and 23. The “soft” cycle closing step was not used in previous4” calculations for DPDPE. This is the main reason why the lists of possible lowenergy backbone conformations obtained in Refs. 4 and 6 are generally shorter than the same lists in the present study. T h e low-energy conformers were selected accordafter each energy ing to the criterion A E = E Emin calculation step. The calculation pattern utilized the successive “growing” of the molecule toward the N-terminus so that a t every step the dihedral angle 4 values considered for a second N-terminal residue were not only those found a t the previous step using the algorithm, 2 5 but also others consistent with already found Ic, values. Generally, a t each step AE values were 10 kcal/mol, except for the last two steps, where they were 5 kcal/mol for DPDPE, DCFPE, and DRE 1-4, and 7 kcal/mol for DRE. In the last step all combinations of X I rotamers (i.e., g i , t , and 6 - ) for Tyr and Phe residues were explored; sometimes this step gained conformations ~

=

225 28 X 9

=

No. of

69

with energies lower than those obtained a t the previous step. On the other hand, the additional calculations for DPDPE that explored the possibilities of disulfide bond closing other than those achieved by the algorithmz5 found that the algorithm had found the closing options with lowest energies for all DPDPE conformers. It is interesting to compare the conformers considered for DPDPE in the present study and those in previous studies.“-’ The reasons for the differences between the present calculation results and those described in Refs. 4 and 6 were discussed above. The authors of Ref. 3 used the well-known Go-Scheraga procedure2fito close the model ring accurately. They found 104 potentially low-energy conformers (before the energy minimization s t e p ) , which resulted in 31 structures of the entire molecule including various 7.5 Tyr and Phe rotamers characterized by AE I kcal/mol ( D P L P E but not DPDPE was most thoroughly examined in Ref. 3 ) . In Ref. 5, 40 conformations of DPDPE were generated as starting points for energy minimization. In Ref. 8,23 conformations of the model ring were considered after energy minimization, which in turn were used for constructing more than 130 conformers of the entire molecule for further calculation steps; it appeared that 12 of them 2 kcal/mol (Tripos possessed energies within AE I force field). In the present study we are examining a more comprehensive range of DPDPE conformations than previously:’.”’ considered. The entire lists of low-energy conformers including various Tyr and Phe rotamers contain 61 structures for DPDPE, 32 for DCFPE, 38 for DRE, and 69 for DRE 1-4, respectively (Tables I and 11). Tables 111-VI list those backbone conformers in which the differences exceed 40” (cyclic compounds) and 60” (linear compounds) for a t least one of the

REQUIREMENTS FOR &SELECTIVE OPIOID PEPTIDES

945

Table 111 Different Types of Low-Energy Backbone Conformers for DPDPE Angle

Residue $1

d I L

x:, x.1

Y,,I

ic ., U'L I XI2 4

$ 3

U' I.,

9

4

$4

"115 Xl4

x 24 &> $ -,

x li CSSC"

mol 1

R

c

D

E

F

c:

H

I

J

141 -176 -179 59 30 129 - 143 173 175 63 33 178 -167 -57 180 178 61 127 -150 -67 -144

141 180 -180 60 -30 80 -143 174 173 64 34 177 -167

-39 176 60 102 -30 133 - 140 171 176 61 3' 180 -167 -57 -179 173 -117 125 -150 -68 -144

137 -177 -178 -117 30 138 -126 -171 -167 116 -66 171 -152 -56 -175 174 64 126 -145 68 -112

139 -179 -179 -120 30 139 -123 -177 -166 138 -79 I77 -89 -37 179 -179 89 95 -150 -69 98

140 -178 -179 -119 30 136 -106 -178 -170 118 -20 178 -159 -57 I77 176 -118 122 -150 -71 100

-55 180 178 -121 30 80 -142 174 173 64 33 178 -166 -58 180 178 -119 127 150 -67 144

-40 180 61 99 -30 140 -126 -172 -166 115 -65 172 -152 -56 -175 174 --I15 126 -145 68

-52 -178 178 -93 30 139 -124 -176 -166 138 -80 177 -90 -37 179 -59 -62 96 -150 -69 99

142 180 180 -119 30 79 -133 -176 -178 145 -68 179 -96 -56 176 179 60 86 -150 48 -144

-

92

9

A

-58 180 180 -120 127 -150 -67 -144

17.2

17.9

18.0

18.2

18.4

-

-

18.4

18.4

-111

20.0

20.4

20.7

K

L

1-11

144 180 180 -119 30 79 -124 -168 -176 118 -65 170 -153 -58 -175 178 61 124 -121 71 -112

180

1x0 -119 :10 XO -l:j3

178 178 I27 -1.50 ~

179 65 .'$,'I 175 212 140

-1'1 49 I60 21.0

21.2

' ' ('SSC is the C-S-S-C dihedral angle

backbone dihedral angles. These conformers can be regarded as various types of three-dimensional shape for the peptide backbone. The combinations of Tyr

Table IV Residue

l)-CyS

Phe

and Phe side-chain rotamers in Tables 111-VI correspond to the lowest energy for a given type of backbone.

Different Types of Low-Energy Backbone Conformers of DCFPE Angle

A

B

C

150 180 180 87 0 76 50 171 168 -77 -38 167 -63 100 124 27 46 79 18.3

155 -179 -173 79 -1 106 -6 156 -89 -91 -20 175 68 -99 144 - 143 -52 -152 20.3

155 -175 178 86 0 78 74 164 147 - 74 -47 171 -67 109 118 29 50 88 20.6 -

D 88 176 - 175 76

0 80 53 171 160 -72 -32 170 -65 102 111 -152 48 81 21.4

E

F

119 180 -180 85 0 151 -31 170 -42 -73 -26 163 67 85 108 38 54 136 22.1

153 177 180 88

0 81 53 165 175 -95 38 -169 -71 - 104 66 - 148 -68 -118 22.6

946

NIKIFOROVICH ET AL

Table V Different Types of Low-Energy Backbone Conformers of DRE Residue TYr

Angle *1 XI1

x21 x 61

D-Met

$2 *2 XI2 X22

X 32

Phe

d3 $3 XI3 x23

His

d4 $4 XI4 x24

Leu

45

*S

x1s x25

Met

d6 $6 XI6 x26 x36

Asp-NH2

47 *7

XI7

x27

E (kcal/mol)

A

B

C

D

E

F

G

G*

H

154 -173 82

154 -164 79 -9 102 40 121 180 -179 -91 -17 -64 101 62 43 -57 87 -127 -67 -67 159 -133 25 -67 179 179 -75 -29 -49 -100 15.6

129 -175 73 -6 92 30 67 -175 -165 -99 -36 - 79 95 -96 -4 -57 96 66

140 -177 90 1 88 40 174 -178 -179 -101 -28 -77 97 -139 -65 -54 93 165 99 -177 86 46 59 -62 179 - 180 -72 91 -52 -117 17.5

162 54 90 -1 118 -164 70 179 179 100 -22 -60 98 -139 -59 -54 95 -164 88 -173 82 49 59 -60 179 -179 -69 148 -55 54 19.2

-48 177 81 -1 95 -141 65 71 -178 -101 -28 -60 95 -138 -62 -54 94 -165 93 -174 83 47 59 -63 179 -180 -68 149 -54 -118 20.1

156 180 86 -2 97 33 152 180 -180 -93 - 16 -66 93 - 140 151 -177 -119 -50 -45 - 180 89 -112 67 -61 180 -180 -89 150 -151 151 20.5

153 -177 77 -28 92 37 172 176 -175 -91 -20 -58 -58 -158 159 -178 -85 -57 119 -177 75 56 45 -65 176 -178 -71 -34 57 -26 17.3

152 -171 78 -8 101 34 171 179 -179 -100 -32 -65 98 -133 -176 -52 112 -56 100 -176 82 55 36 -64 178 -179 -140 155 174 -123 20.8

0

102 44 151 -179 178 -86 -11 -65 96 65 36 -59 88 -125 55 -64 155 57 26 -64 179 -180 -94 34 57 -56 14.8

21

-78 76 -161 32 58 -178 -180 -88 150 -58 108 17.4

The most characteristic feature of all DPDPE conformers in Table I11 is the distorted inverse y-turn (or a y-like t u r n ) centered a t the Gly residue. Some conformers are characterized by the same geometrical shape of the cycle backbone, namely conformers A , B , C, and G; D , H , and K ; E , F , I , and J; and L . Conformer pairs A / B and D / K could be distinguished by the 4 angle value of the D-Pen' residue, while conformers pairs A / C, B / G , D / H , and E / I differ mainly in the positive/negative value of the 1c, dihedral angle of the Tyr residue. A complete list of the 61 low-energy structures of DPDPE contains all low-energy structures found previously6 using the same E C E P P potential field except one (structure 6, Table 5 in Ref. 6 ) . The conformational freedom of DCFPE is even more limited. T h e six conformers listed in Table IV contain two different geometrical shapes of the cyclic

-

-

backbone: A , C, D , and F ; and B and E . The A I D conformers differ in the positive/ negative value of the angle for Tyr residue. The very similar conformers B and E differ in values of the C'-S-S-Co torsional angle only. Again, only one conformer from those found earlier (structure 2, Table 5 from Ref. 27) is not present in the complete list of 32 lowenergy conformations found in the present study. As might be expected, the greatest variety of three-dimensional backbone conformer is found for DRE (Table V ) ; the only common feature of these conformers is the a-helix-like backbone structure ( A type) for the Phe3 residue. DRE conformers have intramolecular hydrogen bonds not found in the cyclic peptides. Conformers A and B are quite similar, both containing two hydrogen bonds between the oxygens of the Asp7 P-carboxyl and the amide protons of the D-Met' and Leu5 residues. One of

REQUIREMENTS FOR &SELECTIVE OPIOID PEPTIDES

Table VI

Different Types of Low-Energy Backbone Conformers of DRE 1-4

Residue TYr

Angle $1

XI1

xz1 XSI

II-Met

42 $2 XlZ x22

X 32

Phe

43 $3

x 1:i X28

His-NH,

4 4

$4

x14 x24

E (kcal/mol)

A

B

C

D

E

F

G

H

I

J

144 176 75 0 62 -103 74 -179 179 -136 28 -57 103 -141 151 50 -89 15.2

-51 -173 92 0 68 -94 167 179 -180 -143 24 52 89 -146 145 57 -82 17.2

142 178 73 0 66 -116 73 -178 179 -88 -14 -177 69 -81 -29 -44 90 18.2

146 -178 70 0 66 -98 73 -179 -178 -80 -20 179 70 58 43 -64 82 18.4

138 -180 72 0 74 -91 72 -179 -178 -87 144 178 75 -141 153 -61 85 19.1

155 -174 70 -1 161 -122 169 179 180 -86 130 -64 107 -137 151 -50 -64 19.6

-53 178 76 0 81 - 105 69 -179 179 -154 143 179 74 -140 154 -74 78 19.7

140 -178 70 0 74 29 73 -180 -176 -149 148 180 75 140 153 -55 86 19.9

153 -179 74 1 86 42 170 -178 -174 -87 -24 -60 117 -153 146 53 77 20.0

140 -178 70 0 74 30 74 179 -176 -83 147 179 75 -138 153 -65 82 20.1

these oxygens can also be involved in hydrogen bonding with the amide proton of either the His4 (conformers C, D , F ) or the Phe3 (conformers C, G ) residues. Conformers A , B , C, D , E , and F contain Lj-like turns in the region of His4-Leu5-Met6Asp ?, but without stabilizing hydrogen bonds. In addition to this structural feature, conformers C, D , and E contain another P-like turn in the D-Met2Phe:'-His4-Leu5region. A hydrogen bond between the ( T y r ) CO and the H N (Met') is found in conformers E and F . A well-defined @-turnis located in the His4-Leu5-Met6-Asp7 sequence of conformer G; this turn is stabilized by a ( H i s 4 ) C 0 HN ( Asp') hydrogen bond. The same type of 6-turn with the same hydrogen bond also occurs in conformer H ; the turn extends in this case into a kind of @-hairpinadditionally stabilized by ( His4)N H OC(Aspi) and ( H i s 4 ) N 6 H * * POOC(AspT) hydrogen bonds. The low-energy conformers of DRE 1-4 listed in Table VI can be divided into three groups. The first one is formed by the @-turnor P-turn-like structures A , B , C, D , and E . This p-turn is stabilized by the hydrogen bond between ( Tyr ) CO and H N ( His ) in conformers A , B , and D with a n additional ( T y r 1 NH2 to OC ( H i s ) hydrogen bond in the case of A conformer. The H * atom of the His side chain can also be involved in hydrogen bonding with the OC ( T y r ) for conformer C and with the OC ( D-Met) for conformers E and J . In all other conformers of

---

---

947

this group intramolecular hydrogen bonding is lacking. Backbone conformers F , G , and H form the second group; they are somewhat L shaped with a reversal turn ca. 90" a t the D-Met residue. And conformers I and J represent a Z-like backbone structures with the two 90" turns in opposite directions centered at the D-Met2and Phe3 residues. Interestingly, backbone conformer I of DRE 1-4 is very similar to conformer G of DRE. T h e calculated data for DRE 1-4 were also used to validate the build-up procedure for DRE presented in Table 11. Thus, we have performed additional calculations by building the peptide chain toward the C-terminus from DRE 1-4 to DRE 1-5 (Tyr-D-Met-Phe-His-Leu-NMe)and then to DRE. The AE values were 7 kcal/mol a t each step of the build-up procedure. Only one type of DRE peptide backbone conformer found by this procedure meets the requirement of 5 7 kcal/mol comparing with DRE conformers A - H listed in Table V. The geometrical shape of the conformer is quite similar to that of conformer G in Table V, the only difference being a rotation of the Leu-Met peptide bond plane a t ca. 160". This conformer is given in Table V as conformer G*. This result once more indicates the importance of the C-terminal Asp residue for stabilization of the low-energy structures of DRE. All final low-energy conformers in Tables I and I1 can be regarded as energetically equivalent for the purpose of geometrical comparison. The inac-

948

NIKIFOROVICH ET AL.

Table VII Models of 6-Receptor-Bound Conformers for DPDPE

Residue

TYr

Angle

*I w12

XI1 X2l

Xti1 D-Pen

d2 *2

w2:l x21

GlY

43 *3

w34

Phe

*4 *4 a45 x14 x24

D-Pen

45 *5 XIS

cssc

E (kcal/mol)

Table VIII

Angle *I w12

XI1 XP1 xti1

D-CYS

d2 $2 w23

XI2

Phe

43 *3 w34 x13 x23

D-Pen

44 *4

x 14

cssc

E (kcal/mol)

2

3

142 -179 -180 62 -30 80 -145 174 173 66 27 175 -157 -57 179 -75 -64 126 -149 -66 -146 18.4

146 180 61 92 -30 81 -145 173 172 66 27 175 -157 -57 180 -75 -64 126 -149 -66 -143 19.6

142 180 -61 91 30 81 - 145 174 172 66 28 175 -157 -58 179 - 74 -64 126 - 150 66 -146 20.5 -

Models of 6-Receptor-Bound Conformers for DCFPE

Residue TYr

1

curacy in energy calculations makes moot the distinction between conformers differing by 2 or 3 kcall mol in energy. It is noteworthy that generally the use of different force field parameters could change the ordering of energies for particular conformers, but it is likely that the variety of geometrical shapes for conformers that make up the set of low-energy structures would be basically the same. For comparison of DPDPE, DCFPE, DRE, and DRE 1-4 structures, six atomic centers were selected as important: the nitrogen atom of the a-amino group; the C7 and C r - atoms of the Tyr and the Phe aromatic rings representing their mutual arrangement; and the C ” atom of the residue in position 2. T h e latter center was selected to prevent the “enantiomeric” situations when, e.g., aromatic rings of the Tyr and Phe residues in the pair of conformations compared would overlap properly, but the backbones of this pair would look like “mirror images” of each other. T h e comparison procedure included the assessment of the best fit (according to Ref. 28) of the space arrangement of the six atomic centers for each pair of conformations belonging to the entire lists of low-energy conformers found for the different molecules (e.g., 61 X 32 = 1952 pairs of conformers were compared in the case of DPDPE vs DCFPE).

1

2

3

4

5

6

149 179 180 90 0 78 49 170 167 -74 -41 169 180 88 125 28 47 77 19.5

150 180 180 90 0 77 44 172 166 -74 -25 168 -180 -91 109 30 47 75 20.5

149 179 60 90 0 78 49 170 167 - 74 -41 169 180 88 125 28 47 77 20.6

150 180 60 90 0 77 44 172 166 - 74 -25 168 -180 -91 109 30 47 75 21.5

149 179 -60 90 0 78 49 170 167 -74 -41 169 180 88 125 28 47 77 21.2

150 180 -60 90 0 77 44 172 166 - 74 -25 168 - 180 -91 109 30 47 75 22.2

REQUIREMENTS FOR &SELECTIVE OPIOID PEPTIDES

Conformers were regarded as geometrically similar when the rms value was less or equal to 1.0 A for the six atoms in question. On the basis of these assumptions, three types of structures were found to be geometrically similar for all &selective compounds (DPDPE, DCFPE, and D R E ) , and a t the same time nonsimilar for all conformers of the p-selective DRE 1-4 compound. These types are described in Tables VII-IX. The first type is composed of structures 1 for DPDPE, and 1 and 2 of DCFPE and DRE (numbering of structures according to Tables VII-IX) . The second type consists of structures 2 for DPDPE, and 3 and 4 for DCFPE and DRE, and the third type is represented by structures 3 for DPDPE, and 5 and 6 for DCFPE and DRE. (There are two DRE 1-4 conformers that possess rms values less than 1.0 A when

Table IX

949

comparing them with DCFPE structures 1 and 2; it would be more reasonable not to reject these DCFPE structures, keeping in mind all the assumptions made.) The similarity for different types of structures is illustrated by Figure la-c. The main difference between these three structure types is in the Tyr side-chain rotamers being t , g+ , and g in Figure l a , b, and c, respectively. It is interesting to note that when the same comparison procedure with the same criterion of rms I1.0 A was applied to the same conformers, but comparing the space arrangement of atomic centers that characterize the backbone rather than side chains (i.e., the nitrogen of the a-amino group and C" and Cd atoms of the Tyr and Phe residues, the centers being selected in Ref. 7 ) , quite different results were obtained. For example, it was found that DCFPE and DRE backbone

Models of 6-Receptor-Bound Conformers for DRE

Residue TYr

Angle

80 -6 98 38 124 180 -179 -88 - 15 -155 86 60 48 -58 87 -134 -61 172 103 -139 26 -80 178 -179

78 -6 96 38 126 179 -179 -86 -15 -156 85 61 49 -60 85 -134 -63 -175 109 -140 27 -152 -180 -180 -76 -31 -47 -101 20.4

42

43

X1:r x2:r

44 $4

XI4 x24 4 5 *5

XIS X2.5

4fi *fi XI6

XPfi x36

Asp-NH,

4i *7

XI7 x27

E (kcal/rnol)

163 67 96 -1 90 32 68 -177 179 -84 -16 -155 84 58 55 -63 79 -141 -63 174 80 -132 21 -68 176 -177 -79 -34 -41 -116 20.9

X2l

$3

Met

158 67 96 0 97 25 64 -176 180 -97 -37 -176 81 -95 -4 -58 96 67 20 -80 73 -160 32 57 -179. - 180 -88 148 -57 110 20.3

156 - 166

X 22 X 32

Leu

4

156

XI2

His

3

- 166

*2

Phe

2

XI I

*I

X6l

LI-Met

1

-79 -31 -48 -99 20.2

5

6

139

163

- 70

- 70

98 0 97 24 62 -175 179 -98 -37 -176 81 -95 -4 -58 96 67 20 -80 73 -160 32 57 -179 -180 -89 149 -57 109 20.4

101 0 89 30 67 -177 179 -86 -16 -155 84 59 54 -62 80 -139 -64 -174 80 -131 21 -68 176 -177 -78 -34 -41 -114 21.1

950

NIKIFOROVICH ET AL.

Figure 1. Stereoviews of topographically equivalent conformers of DPDPE (solid line), DCFPE (solid dashed line), and DRE (thin line). The Phe3 side-chain rotamers are of the t type while the Phe4 side-chain rotamer is of the g- type. The T y r side-chain rotamers are t ( a ) , g + ( b ), and g- ( c ) . All hydrogen atoms are omitted for clarity.

conformers were similar t o some DRE 1-4 conformers. At the same time, DPDPE and DCFPE molecules had no similar backbone conformers. In other words, it appeared impossible by this comparison to elucidate any structures as models for “ 6 conformers” by comparing backbone structures only.

The DPDPE structures listed in Table VII belong to the same backbone type B described in Table 111. They possess one more common feature: the g - rotamer of the x1 dihedral angle for the Phe4 side chain. At the same time, the X I rotamers for the Tyr side chain are different in these structures, as

REQUIREMENTS FOR &SELECTIVE OPIOID PEPTIDES

Figure 1.

951

(Continued from the previous page. )

mentioned previously. These DPDPE structures are superimposed in Figure 2. The same differences for the Tyr' side chain and similarity for the Phe sidechain rotamers are characteristic of the DCFPE and

DRE conformers in Tables VIII and IX. The Phe rotamer is t ( b u t not g- ) for these two peptides; nonetheless the Phe3 aromatic moiety can occupy the same location in space as the Phe4 moiety in

Figure 2. Stereoview of overlapped models for DPDPE possible 6-receptor-bound conformers with different rotamers of the Tyr side chain. The omissions are the same as in Fimre 1.

952

NIKIFOROVICH ET AL.

DPDPE (Figure 1) . Two slightly different backbone types of DCFPE are represented in Table VIII, both belonging to the conformer type A of Table IV. And three backbone types of DRE are listed in Table IX, two of them (structures 1, 2, 4, and 6 ) being somewhat different but belonging to the same conformer type B in Table V, and structures 3 and 5 belonging to type C in Table V.

DISCUSSION The present study proposes a model for the 6-receptor-bound conformations ( ‘‘6 conformations”) for opioid peptides based on topographical considerations concerning the mutual space arrangement of the functionally important structural moieties, the N-terminal a-amino group and the aromatic moieties of the Tyr and Phe residues. T h e model is described in Tables VII-IX and is depicted in Figure 1 for DPDPE, DCFPE, and DRE. The most characteristic feature of the model is the placement of Phe side chain in a more or less definite position in the space corresponding to a X I rotamer, which is g- for peptides containing Phe4 and t for peptides with Phe3. These findings are consistent with the importance of the Phe aromatic moiety for interaction with the &receptor pointed out in Ref. 2 . The position in space for the Tyr’ side chain cannot be specified so precisely using the peptides under consideration. ( Very recent energy calculations performed for the “hybrid” analogues of DRE and dermorphin suggest a g- rotamer for the Tyr’ side chain2’.) A more distant spacing of the Tyr’ and P h e 4 side chains is found in the proposed model, which is different than the more or less close spacing proposed for DPDPE from nmr and some ca1cu1ations”’~“’(but not in other calculations [ I t should be noted that using the ECEPP force field all previously proposed &conformer models for DPDPE appeared to be higher in energy compared t o structure 1 in Table VII by 20.44,310.45 and 9.83 (conformers 2a and 2b; Ref. 5 ) , 1.63 and 3.86 (conformers 4 and 8; Ref. 7 ) , 11.57,’ and 12.10 kcal/ mol, lo respectively.] A large amount of synthetic work was performed recently to insert topographical constrains in DPDPE by substitution of Phe4 with P-Me-Phe4 and by various modifications of the aromatic ring."^:" In particular, four DPDPE analogues with all possible isomers of P-Me-Phe (erythro-L-, S,S; threoL-, S,R; erythro-D-, R,R; and threo-D-, R,S) were synthesized, and tested for their binding potencies and biological activities using isolated organs 3 3 7 ) .

(guinea pig ileum and mouse vas deferens). T h e substitution of one P hydrogen by a methyl group should bias the conformational possibilities for the Phe side chain. Hence conformational analysis of P-Me-Phe-substituted DPDPE analogues followed by comparison of low-energy conformers with the proposed model in the present study should provide further insights. The search procedure for the sets of low-energy conformers for P-Me-substituted DPDPE analogues was exactly the same as outlined in Table I for DPDPE itself. It resulted in 55 (45; 60; 72) structures possessing AE I 5 kcal/mole taking into account various rotamers of Tyr and Phe side chains for DPDPE analogues for the (S,S) -P-Me-Phe4 (S,R-; R,S-; R,R-, respectively) analogues. A more detailed description of these conformers will be presented elsewhere along with the corresponding nmr data; it is noteworthy only to point out the strong preference of the /3-Me-Phe4 side-chain rotamer of g - found for (S,S)-P-Me-Phe, t for ( S , R ) - and (R,S)-@-Me-Phe, and g t for (R,R) -P-Me-Phe4containing analogues. Comparison of peptide backbones for these structures with those of DPDPE (atomic centers for fitting were the C” and CO-atoms) revealed that almost every type of backbone conformer for [ (S,S)] - or [ (S,R)-/3-Me-Phe4]DPDPE is similar to some type of DPDPE backbone. However, the same is true only for a few conformers of [ (R,S)]- or [ ( R , R )-P-Me-Phe4]DPDPE. On the other hand, comparison of Tyr and Phe sidechain space arrangement performed with the same six atomic centers mentioned before and with the same similarity criterion shows very close similarity of every DPDPE structure from Table VIII to some low-energy structures of [ ( S , S )-P-Me-Phe4]DPDPE ( t h e lowest rms = 0.14 A,see Figure 3 ) . There is reasonable similarity of [ ( R , R )-P-MeP h e 4 ]DPDPE ( t h e lowest rms = 0.85 A ) , poor similarity of [ ( S , R ) -P-Me-Phe4]DPDPE ( r m s 2 1.32 A ) , and very poor similarity of [ ( R,S) $-MeP h e 4 ] D P D P E (rms > 3.25 A ) to DPDPE. Thus, the 6-receptor-bound conformer model proposed in this study is most consistent with low-energy conformers of [ (S,S) -P-Me-Phe4]DPDPE, which possesses a space arrangement of the Phe aromatic moiety predicted by this model. A t the same time this analogue is the most potent and apparently the most &selective compound among the four 8-MePhe4-substitutedanalogues of DPDPE in both binding studies and bioassays (data from Ref. 3 1 ) . In a recent paper32Mosberg et al. considered the possibility for the existence of two different subsites a t the 6 receptor, interacting differently with the

REQUIREMENTS FOR 6-SELECTIVE OPIOID PEPTIDES

953

Figure 3. Stereoview of overlapped models for 6-receptor-bound conformers of D P D P E an d [ ( S , S ) - & M e - P h e 4 ] D P D P E .T h e omissions are the same a s in Figure 1.

P h e 4 in D P D P E and the Phe" in DCFPE. This possibility was based primarily on the slightly different binding and activity profiles found in Ref. 32 for the

[ p F P h e 4 ]DPDPE and [pFPhe"]DCFPE analogues compared t o their parent compounds. It seems, however, t h a t the use of the model proposed in our

Figure 4. Stereoview of overlapped models for 6-receptor-bound conformers of D PD PE (solid line) a n d D S L E T (thin line). T h e omissions are the same as in Figure 1.

954

NIKIFOROVICH ET AL.

study could explain the above-mentioned observation in the frame of the hypothesis of the same &receptor site interacting both with Phe4 in DPDPE and Phe3 in DCFPE. Figure 1clearly shows the different orientation of the vectors connecting atomic centers CO, C y , and Cc, and the would be para-F atom in DPDPE a s compared to that in DCFPE. This difference might be sufficient for the 6 receptor t o distinguish these two analogues. Finally, it is interesting to compare the models for the 6-receptor-bound conformer proposed in the present study for DPDPE, and in Ref. 33 for the 6-selective linear peptides DSTBULET [ H-Tyr-DSer ( OtBu) -Gly-Phe-Leu-ThrJ and BUBU [ H-TyrD-Ser ( O t B u ) -Gly-Phe-Leu-Thr ( O t B u ) 1 . T h e backbone conformation C from Table IV in Ref. 33 was proposed as a model for the 6-receptor-bound conformer. T h e energy of this conformation was recalculated by us employing the procedure described in this study (all rotamers of the Tyr and Phe4 side chains were considered for energy minimization). Figure 4 shows the overlap of one of the recalculated conformers of DSLET from Ref. 33 and one of the possible 6-receptor-bound conformations found in our study. There is good agreement between the models proposed in these studies in terms of the mutual and spatial arrangement of the a-amino group and the aromatic moieties of the Tyr' and Phe4 residues. Thus the model proposed in the present study for the 6-receptor-bound conformation for opioid peptides appears t o be consistent in its main features with the assay data for compounds not involved in the process of model constructing. We would suggest that this model, based mainly upon the topographical considerations involving the space arrangement of aromatic moieties in relation to each other and t o the N-terminal a-amino group as the most important factors for 6 selectivity, might be successfully applied t o the problems of conformationally directed drug design of 6-opioid receptor ligands. This work was supported by the U S . Public Health Service Grants DA 06284 and NS 19972, and by a grant from the National Science Foundation (DMB-8712133).

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2. Hruby, V. J. & Gehrig, C. A. (1989) Med. Res. Rev. 9, 343-401. 3. Keys, C., Payne, P., Amsterdam, P., Toll, L. & Loew, G. ( 1988) Mol. Pharmacol. 33, 528-536. 4. Nikiforovich, G. V. & Balodis, J. (1988) F E B S Lett. 227, 127-130. 5. Froimowitz, M. & Hruby, V. J. ( 1989) Znt. J . Peptide Protein Res. 34, 88-96. 6. Nikiforovich, G. V., Balodis, J., Shenderovich, M. D. & Golbraikh, A. A. ( 1990) Int. J. Peptide Protein Res. 36,67-78. 7. Nikiforovich, G. V., Golbraikh, A. A., Shenderovich, M. D. & Balodis, J. ( 1990) Int. J. Peptide Protein Res. 36, 209-218. 8. Wilkes, B. C. & Schiller, P. W. (1990) in Peptides. Chemistry, Structure and Biology, Proceedings of the Eleventh American Peptide Symposium, Rivier, J. E. & Marshall, G. R., Eds., ESCOM, Leiden, pp. 341343. 9. Hruby, V. J., Kao, L.-F., Pettitt, B. M. & Karplus, M. ( 1988) J. A m . Chem. SOC.110, 3351-3359. 10. Mosberg, H. I., Sobczyk-Kojiro, K., Subramanian, P., Crippen, G. M., Ramalingam, K. & Woodward, R. W. ( 1990) J. Am. Chem. SOC.112,822-829. 11. Pettitt, B. M., Gehrig, C., Matsunaga, T. M., Karplus, M. & Hruby, V. J. (1990) Biophys. J., submitted. 12. Jardetsky, 0. (1980) Biochim. Biophys. Acta 621, 227-232. 13. Nikiforovich, G. V., Vesterman, B., Betins, J. & Podins, L. (1987) J . Biomol. Struct. Dynam. 4, 11191135. 14. Mosberg, H. I., Omnaas, J. R., Medzihradsky, F. & Smith, C. B. (1988) Life Sci. 4 3 , 1013-1020. 15. Sagan, S., Amiche, M., Delfour, A., Mor, A., Camus, A. & Nicolas, P. (1989) J . Biol. Chem. 264, 1710017106. 16. Amiche, M., Sagan, S., Mor, A., Delfour, A. & Nicolas, P. (1989) Mol. Pharmacol. 35, 774-779. 17. Kreil, G., Barra, D., Simmaco, M., Erspamer, G. F., Negri, L., Severini, C., Corsi, R. & Melchiorri, P. (1989) Eur. J. Pharmacol. 162,123-128. 18, Hruby, V. J., Al-Obeidi, F. & Kazmierski, W. (1990) Biochem. J. 268, 249-262. 19. Mosberg, H. I., Haaseth, R. C., Ramalingam, K., Mansour, A., Akil, H. & Woodward, R. ( 1988) Znt. J . Peptide Protein Res. 32, 1-8. 20. Toth, G., Kramer, T. H., Knapp, R., Lui, G., Davies, P., Burks, T. F., Yamamura, H. I. & Hruby, V. J. (1990) J . Med. Chem. 33, 249-253. 21. Hruby, V. J., Kao, L.-F., Hirning, L. D. & Burks, T. F. ( 1986) in Peptides: Structure and Function, Deber, C. M., Hruby, V. J. & Kopple, K. D., Eds., Pierce Chemical Co., Rockford, IL, pp. 487-490. 22. Nemethy, G., Pottle, M. S. & Scheraga, H. A. (1983) J. Phys. Chem. 8 7 , 1883-1887. 23. Dunfield, L. G., Burgess, A. W. & Scheraga, H. A. (1978) J . Phys. Chem. 82, 2609-2616.

REQUIREMENTS FOR &SELECTIVE OPIOID PEPTIDES

24. Zimmerman, S. S. & Scheraga, H. A. (1977) Biopolymr’rs 16, 811-843. 25. Nikiforovich, G. V., Shenderovich, M. D. & Balodis, J. ( 1981 ) Bioorgan. Khimia 7,179-188 ( i n Russian). 26. Go, N. & Scheraga, H. A. (1970) Macromolecules 3, 178-187. 27. Shenderovich, M. D., Nikiforovich, G. V. & Golbraikh, A. A. (1991) Znt. J. Peptide Protein Res. 37,241-251. 28. Nyburg, S. C. (1974) Acta Crystal. B 30 (part I ) , 25 1- 253. 29. Nikiforovich, G. V. & Hruby, V. J. (1990) Biochern. Biophys. Res. Commun. 173, 521-527. 30. Hruby, V. J., Toth, G., Prakash, O., Davis, P. & Burks, T. F. ( 1989) in Peptides 1988, Proceedings of the 20th

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EPS, Jung, G. & Bayer, E., Eds., Walter de Gruyter, Berlin, pp. 616-618. 31. Hruby, V. J., Gehrig, C., Toth, G., Kao, L.-F., Knapp, R., Yamamura, H. I., Kramer, T., Davis, P. & Burks, T. F. (1991) J. Med. Chem., in press. 32. Mosberg, H. I., Heyl, D. L., Haaseth, R. C., Omnaas, J. R., Medzihradsky, F. & Smith, C. B. (1990) Mol. Pharmacol. 38, 924-928. 33. Belleney, J., Gacel, G., Fournie-Zaluski, M. C., Maigret, B. & Roques, B. P. (1989) Biochemistry 28, 7392-7400. Received November 4, 1990 Accepted March 27, 1991