Conformational preferences of a few enkephalin unsaturated analogs

THEO CHEM ELSEVIER

Journal of Molecular Structure (Theochem) 311 (1994) 255-272

Conformational preferences of a few enkephalin unsaturated analogs G. Alagona, G.M. Ciuffo', C. Ghio* C.N.R. Istituto di Chim ica Quantistica ed Energetica Molecolare, 1-56126 Pisa, Italy

(Received 19 July 1993; accepted 3 December 1993)

Abstract The conformational beha vior of enkephalin analogs containing Q-{3 unsaturated residues was studied employing a recent modification (G. Alagona, C. Ghio and C. Pratesi , J. Comput. Chern., 12 (1991) 934) of an existing force field for nucleic acids and proteins (S.J. Weiner, P.A. Kollm an, D.A. Case, U . Chandra Singh, C. Ghio, G . Alagona, S. Profeta, Jr., and P. Weiner, J. Am. Chern. Soc., 106 (1984) 765) with molecular mechanics and molecular dynamics simulations. On the basis of the structures obtained, the rationale proposed for the morphine-like activity of enkephalins (i.e. the presence of {3-turn of type II', considered important for the binding to opiate receptors) was checked and confirmed on the basis of topological features associated with a compact positioning of the aromatic side chains, tyrosine and phenylalanine or dehydrophenylalanine. The molecular electrostatic potential in the plane perpendicular to the Ct-{3 double bond may account only in part for the enhanced potency often observed in unsaturated compounds, and attributed to the intrinsic reactivity of the double bond toward nucleophilic sites on the opiate receptor or to a stronger binding to receptors. In the presence of the solvent , described as a continuous dielectric medium , most of the least stable conformations in vacuo are greatly stabilized, thus becoming even more favored than the gas-phase minimum-energy structures. Interestingly enough, the solvent stabilization is noticeable not only for the extended conformers, as expected, but also for several {3-tum structures of type II'.

1. Introduction The conformational properties of the linear enkephalins Tyr-Gly-Gly-Phe-Leu and Tyr-GlyGly-Phe-Met, which interact preferentially with the a-opiate receptors, but also bind to the Jt-opiate receptors, have been extensively studied [1-3]. It has been suggested that the low receptor selectivity may ·Corresponding author. (Permanent address: Catedra de Quimica General, Facultad de Quirnica, Bioquimica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina .

be due to the conformational flexibility of these linear peptides; therefore, conformational restrictions have been introduced in order to generate selective analogs. One of the strategies employed for the synthesis of conformationally restricted peptides is the incorporation of structurally constrained amino acid analogs. The substitution in peptide hormones of a certain residue with its Q, {3 unsaturated analog often produces a sharp enhancement in the biological potency of the compound, not completely amenable to an increased resistance to enzymatic degradation [4-7]. Additional explanations concern the greater rigidity [6] induced

0166-1280/94/S07.00 © 1994 Elsevier Science B.V. All rights reserved SSDIOI66-1280(94)03671-7

256

G. Alagona et al.jJ, Mol. Struct, [Theochem} 311 (1994) 255-272

by the presence of the double bond, which is confirmed by the potency observed also in cyclic analogs [8] and the possibility for these compounds of assuming and keeping conformations not at all, or hardly, available to the saturated peptide [9] because of its flexibility. Two types of fi-turn structures have been proposed for enkephalin: namely the Gly2_Gly3 and Gly3_Phe 4 fi-turn structures [10]. Attempts to make these fi-turn structures more rigid have been made by several groups [4,5,1I,12a]. Shimohigashi and coworkers [4,5,13] and Stammer and coworkers [14] have synthesized a series of dehydroenkephalins containing Q, fi dehydroamino acid residues and have shown that the stereo-orientation of the aromatic groups (Tyr and Phe) is crucial for the biological activity. Also, the importance of the aromatic side-chain positions was established for cyclic analogs [11,12b]. Several studies have been carried out which have enabled the basis for 8/J.l selectivity to be determined: the presence of folded structures (active towards the 8-opiate receptor) rather than the presence of extended structures (active towards the J.l receptor) [12a,c-d]. This hypothesis was then confirmed when analogs with a high selectivity for the 8-opiate receptor turned out to have relatively compact structures with a short separation between the aromatic rings [15], whereas a slight preference for the II receptor was found in a highly potent analog of Leu-enkephalin with tyrosine and naphthylalanine side chains rather distant from each other [12d,e]. Recently another study on cyclic analogs of [D-Pen2-D-Pen5]-enkephalin indicated that the Tyr l and Phe4 aromatic side-chain groups were on the same surface [11]. We undertook a complete conformational study by means of molecular mechanics and molecular dynamics simulations to assess whether the activity of the enkephalin analogs containing Q, fi unsaturated residues depends on the possibility they have of keeping selected backbone conformations, other than those available to the corresponding saturated compounds, and on the relative positions of the aromatic rings. The unusual, and possibly favorable, charge distribution produced by the presence of the Q, fi double bond has been explored through the comparison of the molecular electrostatic potential maps of the various conformers. The

solvent effect has also been taken into account because it can simulate alternate intermolecular electrostatic (including those with solvent) and van der Waals interactions occurring in the biological environment. The preferred conformations in solution, however, can be different from those at the receptor site [2,1O,16a], where the environment may be considerably hydrophobic [16b]. Nonetheless, despite the similarity between receptor-bound and gas-phase structures [16c] for these compounds, it is likely that intermolecular interactions, established with polar groups not already involved in intramolecular hydrogen bonds, may compete energetically with intramolecular interactions inferred from vacuum calculations. It is therefore interesting to know which structures are stabilized by these interactions.

2. Systems considered A wealth of data have been collected on several unsaturated peptide hormones, but we selected enkephalin analogs because enkephalins have elicited our interest in the past [3]. The oligopeptides considered in this study are Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu-OH) analogs, containing dehydrophenylalanine residues, ~Phe, such as Tyr-Gly-Gly-Al'he-Leu-Oll, the corresponding compound with Gly2 substituted with a m-Ala residue (Tyr-n-Ala-Gly-Al'he-Leu-Olf), and the Met-enkephalin amide (Tyr-Gly-Gly-Phe-MetNH 2) analog obtainable from the latter by also substituting the terminal residue (Leu-OH) with Met-NH 2, i.e. Tyr-D-Ala-Gly-~Phe-Met-NH2' Our choice was dictated by the availability for these compounds of experimental studies concerning both their biological activity [5] and their receptor binding affinity [13], data that are useful for a tentative structure-activity correlation. In the following we refer to these oligopeptides as: I, TyrGly-Gly-Phe-Leu-OH; II, Tyr-Gly-Gly-~Phe­ Leu-OH; III, Tyr-n-Ala-Gly-zxl'he-Leu-Ofi; and IV, Tyr-D-Ala-Gly-~Phe-Met-NH2' The list of the compounds taken into account (reported in Table 1, together with their relative biological activity and receptor binding affinity) contains Leu-enkephalin as well because, in order to test

G. Alagona et al.jJ, Mol. Struct. (Theochem} 311 (1994) 255-272

257

Table I Receptor binding activity of enkephalin analogs Compound

Sequence

Binding activity" (nM) Il

I II III

IV

Tyr-Gly-Gly-Phe-Leu-OH Tyr-Gly-Gly-t;,Phe-Leu-OH Tyr-D-Ala-Gly-t;,Phe-Leu-OH Tyr-D-Ala-Gly-t;,Phe-Met-NH 2

2.4

b

(420)" 1.27d 0.61 d

ISb (420)C

io.r' 1.46d

Dose which produces a 50% inhibition of binding. From Ref.13a. C The values in parentheses refer to a combination of 8 and /l receptor binding activity from Ref. 5. d From Ref. l3c.

a

b

the performance of the methods employed and to make the appropriate comparisons, we had to consider also I, the parent compound.

3. Methods employed The structures of the compounds under scrutiny were determined through molecular mechanics (MM) and/or molecular dynamics (MD) simulations using the UNIX VERSION 3.0 REV. A [17a] of the AMBER code [17b], running on the Iris 4D-420 GTXB at ICQEM. The 1984 force field [18a] was employed together with its subsequent modifications developed to deal with non-polar hydrogen atoms [18b] and with dehydroamino acid residues [19], where necessary. As it is impossible to scan systematically all the values of the torsional degrees of freedom with a "brute force" approach, even for the backbone of these oligopeptides, we used as starting conformations for the MM minimizations more than 75 structures, obtained using different techniques. Using a strategy described previously [20], most of the starting conformations were obtained by combining couples of the allowed ¢ and 'l/J values. In this way, all the common structures, such as .a-turns, 3 10 helices and C 7 arrangements, as well as their combinations, were tested. In addition, the ¢ and 'l/J values for .6.Phe from our previous study on dehydroamino acids [19] were used in the combinations. As far as the MD simulations are concerned, we employed both simulations at constant temperature (310K) in order to determine the equilibrium structure (lOps) and the

motions about it (60ps), and simulated annealing techniques (several picoseconds at 1400K, gradually cooling down the structures obtained every 8 ps) to sample the conformational space in an alternative way. More extensive details about the starting conformations and the calculations performed are given in Section 4. The .6.Phe residue consists of two possible stereoisomers with Z and E configurations, corresponding to the phenyl ring placed, respectively, in trans or cis position with respect to the carbonyl group. As the analogs having the Z configuration are the active ones, while those with the E configuration are almost inactive [13b] (and unstable to the usual chemical conditions, converting always into the Z configuration [21]), the force field [19] favors the Z configuration over the E one for dehydrophenylalanine. When .6.Phe residues in the E configuration are to be considered, the specific torsional tenns involving CZ-CM and CM-CA [19] should be cancelled from the parameter set. As several tests have been conducted with different charges for the terminal residues without noticeable variations in the results, only two sets of charges were used throughout. The first (H) (N)=H 2(N) = 0.309, N = -0.434, C = 0.616, 0) = -0.504, O 2 = -0.621, H(02) = 0.226) was obtained by properly adjusting the additional charges (to ensure electroneutrality) with respect to those already present in the NH and CO groups of Tyr and Leu, respectively, while the second (H)(N) = 0.164, H 2(N) = 0.184, N = -0.373, C = 0.352, 0) = -0.339, O2 = -0.329, H(02) = 0.241; the excess charge was placed on C

G. Alagona et 01./1. Mol. Struct , (Theochem} 311 (1994) 255-272

258

4. Results and discussion

16

f-

-

-64

r

14 12

f-

23

f-

-

f-

-

-

f-

0

ftS 0

~

-

11

8 -

10

E

4.1. Molecular mechanics simulations

55

73

63

-

70

6

4 2 0

--,-

-

-

-... - - 5

71

74

-

-

-

-

51

-

ABC 0 E F G Fig. I. Energies corresponding to the different types of minimumenergy structures obtained for Leu-enkephalin (I) . A: ,B·tums of type I',II. B: A .a-tum of type II'. C: A ,B-tum of type I. D: A seven-membered cycle C 1 . E: Extended conformation. F: One ,B-tum of type III'. G: Non-conventional structures.

(originally = 0.294).) was derived from a MOPAC [22] optimization on Leu-enkephalin (I). To save space we do not report the minimum energy structure distributions or the values of the dihedral angles for both choices, referring to the latter results where needed. The biological environment has been considered, limiting ourselves to study the effect produced by the presence of a continuous dielectric medium [23] (with dielectric constant e = 78.5) surrounding the various minimum energy structures. Due to the system size, the solutes have been described through their point charge distribution contained in the force fields [18,19], making use of an ad hoc enlarged version (MGPIPCM) of MGPIPC [24]. This purely electrostatic approximation was successfully tested in small systems with rotational degrees of freedom [25].

4.1.1. Leu-enkephalin (I) This compound represents the parent compound of the series taken into account in this study. We considered it because it has been (and still is) the object of a number of theoretical studies [2,3,20] aimed at assessing the quality of a variety of methods devised to examine the conformational preferences of small peptides. Due to this wealth of data it is a good reference point for determining the reliability of the sampling as well as to check the response of the conserved parameters. Figure 1 shows the wide variety of conformations available for this peptide, considering only the first choice for the charges of the terminal residues. In any case, different charges (within a reasonable range of variation) do not produce dramatic changes; only the relative stability of the various forms is slightly affected. In Table 2, the backbone dihedral angles of a few of the lowest energy conformations are shown; these are analogous to those reported previously [2], with only small differences in the reciprocal stability, owing to the force fields employed (either AMBER [17,18] or ECEPP [26]). The absolute minimum, SI, is almost perfectly coincident with the lowest energy conformation determined by Scheraga and coworkers [2], who applied several different methods in the search for the minimum. SI is a type ,B-II' turn conformation, with the bend between residues Gly3-Phe4 (G-P), stabilized by five hydrogen bonds, one of which connects the OH group of the tyrosyl side chain to the carbonyl group of Gil. The hydrogen bond between the Tyr phenolic OH proton and the carbonyl oxygen atom of naphthylalanine" is considered a stabilizing factor in cyclic analogs [12e]. The oxygen atom of the Phe 4 carbonyl group participates in two hydrogen bonds (one with the terminal OH group of Leu 5 and one with the NH of Gly2) which give rise together with the other hydrogen bond connecting the NH group of Phe 4 and the CO group of GIl to a ten and a seven membered ring on the backbone. There is another hydrogen bond between the NH group of Tyr l and the CO

G. Alagona et al.lJ, Mol. St ruct, [Theochem} 3 JJ (1 994) 255-272

259

Table 2 9 and V values (deg) for a few selected minimized structures of I and their relative energies with respect to the globa l minimu m (-161.277 kcal rnol'") Conformation

1,\

92

V2

9J

1/;J

94

1/;4

95

1/;5

CJ.E (kcal mol ")

81 34 74

5 71 62 69 7

lSI 184 314 316 181 167 180 162

181 298 288 293 186 283 67 71

67 93 75 84 56 178 67 278

75 81 76 56 67 280 79 279

group of Gly2, as can be seen from the stereo view shown in Fig . 2. Using the MOPAC derived charges for these groups, a hydrogen bond appears between the NH of Tyr' and the oxygen atom belonging to the OH group of Leu 5 • Figure I and the data in Table 2 document the existence of favorable structures (below 4kcalmol- 1 and up to 10kcal mol-I) also of the C 7 and 1',11 types. 4.1.2. [APhe 4 , Leu'] enkephalin (II) The same starting conformations were employed as for I. The presence of the unsaturated residue

289 301 293 52 277 73 303 38

290 294 300 75 279 72 301 58

172 163 132 310 314 298 149 286

284 296 30 1 285 282 288 285 305

74 312 153 71 178 83 77 142

0 1.72 3.68 3.79 3.95 5.22 5.87 6.68

APhe in place of Phe4 stabilizes a structure with a bend II between residues 2 and 3 and a bend I' between residues 3 and 4 (11,1' type (4)), with respect to th e fi-turn type II' (51) by about Skcal mol", thus making it 1.76kcalmol- 1 more stable than 51. While the use of MO PAC deri ved charges for the terminal groups do es not change the resulting structures or relative energies, another choice of point charges make s 51 and 4 exchan ge th eir mutual position, with 51 now being only 0.9 kcal mol- 1 more stable th an 4. The distribution of the minimum energy structures for this an alog is displayed in Fig. 3, while in Table 3

Fi g. 2. St ereo plot of th e lowest energ y structure (81) of I.

260

G. Alagona et al.jl. Mol. Struct. (Theochem) 311 (1994) 255-272

16

r-

-

14

39

13

i-

'ii19_

32

59

--

i-

8

1

i-

10 -

68

i-

2

i-

-

61

-74

35

- -n -

=55

66

-

---r- 67 7 -2-

6 4

54

27-_70

r-

-

ss

o E

-

""TI'i

f-

12

51

-30 7

23

26

60

62

42 50

64

7149 28

63

I-

-

-

51

o

to folded structures, not to extended ones. This compound is, however, less active [5] with respect to compounds III and IV (Table I) because of the rapid degradation of the Tyr 1_Gly2 peptide bond by aminopeptidases [27a,b]. 81 is similar to the minimum energy conformation determined by Scheraga and coworkers [2] for I. It is stabilized by three hydrogen bonds: one connecting the OH group of the tyrosyl side chain to the carbonyl group of ~Phe4; and two between the CO group of Gly2 and Tyr 1 and the NH group of ~Phe4 and Gly3 (see the stereo view in Fig. 4). These hydrogen bonds produce two seven-membered rings on the backbone. Conversely, in structure 4, the stereo view of which is given in Fig. 5, the OH group of the tyrosyl side chain is not engaged in the hydrogen bonds, whereas hydrogen bonds involving the terminal groups are present: the OH group of Leu ' is hydrogen bonded to the NH group of both Gly2 and Tyr 1 and the other hydrogen atom on the NH 2 group of Tyr 1 forms a hydrogen bond with the Ce-O group of ~Phe4. Two ten-membered rings on the backbone are formed due to the hydrogen bonds connecting the NH group of Leu' and 6.Phe4 to the CO group of Gly2 and Tyr 1, respectively. In this case the Tyr residue shows great mobility, in agreement with theoretical and experimental data [27c].

-

4

ABC D E F G Fig. 3. Energies corresponding to the different types of minimumenergy structures obtained for [DoPhe4 , LeusJ enkephalin (II). A: j3-tumsoftype 1',11. B: A j3-tum of type II'_ C: A j3-tum of type I. D: A seven-membered cycle C 7 • E: Extended conformation. F: One j3-tum of type Ill'. G: Non-conventional structures.

4.13. [o-Ala2, ~Phe4, Leu 5] enkeplralin (III) The presence of a n-Ala residue in position 2 produces an enhancement in the selectivity toward the 6-opiate receptor. This fact, coupled with the evidence that relatively extended structures are

the dihedral angles for a few rotamers are reported. Only the 4 and 81 structures are found below 4 kcal mol ". Examination of Fig. 3 shows that all the minimum energy conformations correspond

Table 3 ¢ and '¢ values (deg) for a few selected minimized structures of II and their relative energies with respect to the global minimum (-129.556kcal mol ") Conformation

'¢I

¢2

1/;2

¢3

1/;3

¢4

1/;4

¢S

1/;s

DoE (kcalrnol")

4 SI

63 28 49 71 64

50

308 119 171 286 164 159 168 283

299 278 66 285 73 200 73 285

93 68 271 74 288 74 199 64

52 74 291 68 292 48 71 150

33 291 332 50 142 42 285 160

118 238 136 74 292 125 243 293

321 351 330 326 50 324 346 19

219 199 225 284 214 283 196 200

89 101 95 166 140 174 90 134

0

1.76 4.27 4.69 5.10 5.54 5.79 6.18

G. Alagona 1'1 al.jJ, Mol. Struct. (Theochem) 3JJ (1994) 255-272

261

Fig.4. Stereo plot of the structureSt of II.

responsible for activity at the JL-opiate receptor while folded structures are required for a-opiate receptor activity [I2a,c-e], suggests that folded structures should prevail in this case. The lowest energy structures of this compound, whose distribution as a function of structure type is shown in Fig. 6, do not reveal outstanding differences with respect to those of the previously reported compounds. Just fewer conformers than before are found having an energy within 6 kcal mol-I of the

absolute minimum, with the presence in this range of a conformer of the III' type and of a hardly classifiable type (73). The dihedral angles are reported in Table 4. The lowest energy conformer corresponds to a bend of type II', as in Leuenkephalin, but this molecule is more active than I and II. By comparing the structures of III with the corresponding ones of I, we can observe that the positions of the Q carbon atoms do not greatly

Table4 9 and 1{J values (deg) for a few selected minimized structures of III and their relative energies with respect to the global minimum (-130 .385 kcalmor') Conformation

1{J1

92

tP2

71

t64 319 309 156 28 305 283 170 153

171

30t 303 308 75 306 290 74 167

70 82 89 113 287 109 68 288

4 4F

17 73 61 28 49 51

77

93 73 72

56 81 308 54 68 296 79

tP3

94

tP4

95

1Ps

t!>.E (kcalmol'")

279 287 31 304 309 36 58 142 294

247 241 108 316 253 61 71 288 259

349 332 330 334 143 8 320 46 204

266 219 229 82 324 281 285 214 288

182 98 85 306 309 76 174 146 154

0 0.93 1.90 3.32 4.75 4.96 7.14 7.35 7.36

G. Alagona et al.jJ. Mol. Struct, (Theochem) 311 (1994) 255-272

262

Fig. 5. Stereo plot of the lowest energy structure (4) of II.

of the Co of71 and 4, respectively. The phenyl ring of .6.Phe4 , however, is almost perpendicular to the one in II, while the positions of the other side chains do not change appreciably. For the 81 structures there is considerable difference in the positions of Leu and .6.Phe between III and II. For the structure 49, despite the very low Lm.S. deviation (0.09) of the 0: carbon atoms, the

differ: their root mean square (r.m.s.) deviation is 0.35 and 0.31 for the structures of the 81 type and those of the 71 type, respectively. The main differences are found in the position of the .6.Phe group with respect to those occupied by the Phe one. Carrying out the same kind of comparison for the corresponding structures of II and III we obtain r.m.s. deviations of 0.22 and 0.26 for the positions

Table 5 9 and !/J values (deg) for a few selected minimized structures of IV and their relative energies with respect to the global minimum (-151.787 kcal mol ") Conformation

!/J.

92

81 71 61

354 174 301 290 166 182 158

291 177 300 289 312 296 172

28

54 60 66

!/J2

67 71 82 65 97 73 84

93

74 75 65 68 53 77 80

!/J3

94

!/J4

95

!/J5

1::..£ (kcalrnol")

288 284 51 59 28 287 296

237 244 73 71 46 42 238

346 349 327 320 22 219 204

289 277 262 285 275 248 199

70 152 293 174 57 145 307

0 4.96 6.10 6.44 7.24 7.46 8.59

G. Alagona et al.fJ. Mol. Struct, (Theochem) 311 (1994) 255-272

16

-

-

14

-

M

27

62

-

f-

12 10

I

-

8

ro

67 21

f-

6

28

S1

f-

f-

73

17

f-

4F f-

0

r-

-

-

-

f-

61

2

-

49

f-

4

35

65 _ _ 26 50_ 42 (;6

-

f-

o

~

f-

54

2-23-

f-

0

E

n-:±.-=- 5 _

ff-

-

-

4

71

-

-

ABC D E F G Fig. 6.Energies corresponding to the different types of minimumenergy structures obtained for [D-Alaz, L).Phe4 , LeuSj enkephalin (III). A: ,a-turns of type I',II. B: A ,a·turn of type II'. C: A ,a·turn of type I. 0: A seven-membered cycle C 7 • E: Extended conformation. F: One ,a-turn of type III': G: Non-eonventional structures.

263

4.1.4. [D-Ala2 , t.Phe4 , Mer'] enkephalin amide (IV) The substitution in III of a methionine amide residue in place of the terminal Leu residue makes a terminal amide group to appear instead of the -COOH group. The lowest energy conformation obtained shows a bend of the II' type between residues Gly3 and t.Phe4 • Again only few conformations (see Fig.8 and Table 5) are found within 6 kcal mol"! of the most stable conformer. Structure 72 has been omitted from the table, because its dihedral angles are close to those of structure 71; only the relative positions of the aromatic rings differ. It seems that this molecule has a lower flexibility as compared with Leu-enkephalin. The lowest energy conformation (Fig.9) is stabilized by six hydrogen bonds: two of them between the NH groups of n-Ala 2 and Tyr' and the CO group of Met 5 ; two of them between the NH groups of Mer' and t.Phe4 and the CO group of n-Ala 2 ; one between the NH group of D-Alaz and the Tyr' carbonyl group; and one between a hydrogen atom of the terminal NH z group (Mer' -NH z) and the CO group of t.Phe4 • This conformation, though more compact, resembles in part the lowest energy conformation obtained for Leuenkephalin (Fig. 2), to which several final conformations are closely related, but it differs from those obtained for compound III.

4.2. Molecular dynamics simulations positions of the Tyr rings are almost specular with a very large angle between them (the tyrosyl OH groups, conversely, point in the same direction): the t.Phe residues are also almost specular, but they are very close to one another. In structure 73, the stereo plot for which is displayed in Fig.7, the two rings are very close (much closer even than in structure 71). This is a feature observed in molecular dynamics simulations of a series of cyclic analogs, showing high selectivity toward the <5-opiate receptor [15]. Therefore, because of the relevance of the distance between the aromatic rings in opiate activity [12], the correlation between energy and separation of the aromatic rings for systems I-IV will be examined in detail at the end of the MD section.

In order not to leave important conformers out of the subsequent analysis, the structures obtained were checked through molecular dynamics simulations, employing several strategies. The first one, successfully tested by various authors [28], consists in starting from an extended structure of the compound under study, minimized with MM, and equilibrating it with a short dynamics run (lOps in this instance). During this period a conformational change usually occurs, due to the formation of intramolecular hydrogen bonds that stabilize folded structures. Once these hydrogen bonds are established, only small motions and displacements with respect to the folded conformation may occur, involving, at most, one of them: it is unlikely in fact that two or more

264

G. Alagona et al.fJ. Mol. Struct, [Theochem} 311 (1994) 255-272

Fig. 7. Stereo plot of the structure 73 of III.

hydrogen bonds are broken and formed again, even if a long run (60ps) is carried out. During the dynamics only limited fluctuations around the structure reached at the end of the equilibration step are obtained, with no guarantee of efficient sampling, even though several backbone transitions have been observed in a lucky case [29a], during a very short simulation at room temperature. The second strategy is the so-called "simulated annealing" approach [29], which consists of a short MD [30] or Monte Carlo [31] simulation at high temperature, followed by a slow cooling down of the system, which should eventually settle into a lower minimum. A more effective sampling could be obtained, for our purposes, by carrying out high temperature dynamics and cooling down or minimizing with MM the structures obtained at regular time intervals, even though this method does not necessarily provide the lowest minimum structure. A recently proposed method [32]that can be used to efficiently locate the global minimum by making use of protein-folding potentials would produce the a-carbon positions, but no information about

the side chains whose separation, at least as far as the aromatic rings are concerned, seems to be essential in these analogs. The final structure of compound II, once minimized with MM, after a 10ps equilibration (10 pseq) and a 60 ps simulation (60 ps-av), both 'performed at physiological constant temperature (310K), is similar to the conformation obtained using MM alone, when starting from the Scheraga conformation. Although simulated annealing starting from the extended conformation at a temperature of 1400K was performed, we were unable to find a structure similar to the minimum energy one obtained employing MM alone. On the contrary, an MD simulation (10 ps-eq + 20 ps-av) followed by a minimization on the structure obtained after the simulated annealing produced a conformation that was 5 kcal mor ' more stable than the initial one. It was even more stable than the conformation previously obtained starting from the extended conformation (-134.7 vs -130.8kcalmol- I ) . Interestingly enough, the backbone dihedral angles ("pI = 351°, ¢2 = 297°, "p2 = 107°, ¢3 = 55°, "p3 = 36°, ¢4 = 67°, "p4 = 359°, ¢s = 265°,

G. Alagona et al.jJ. Mol. Struct, (Theochem} 311 (1994) 255-272

16 -

30- 16- -

7 _ 34

-

14 12 -

_

4 -

55

27

6-

36 21

62 -10

--

23 32

10

-

26

-

-

65

8 ff-

4

f-

2

f-

o

f-

-

f-

6

-

249,56

63

o E

-

66

54 61

-

-

=72 71

ABC D E F G Fig. 8. Energies corresponding to the different types of minimumenergy structures obtained for [o-A1a2 , ~Phe4, MetSj enkephalin amide (IV). A: ,S-turns of type I',II. B: A ,S-turn of type II'. C: A ,S-turn of type 1. D: A seven-membered cycle C 7 • E: Extended conformation. F: One ,S-turn of type III'. G: Non-conventional structures.

265

'l/Js = 312°) are fairly similar to those in conformation 4, whereas the relative ring positions are similar to those found in the Scheraga conformation. Several structures, obtained at high temperature (1400K), were minimized with the aim of obtaining different low energy conformers, but unsuccessfully. In any case, with a good starting point a lower minimum energy conformer was found employing MD. For compound III the MD simulation starting from the extended conformation did not lead to the most stable conformer previously obtained (71), though the final energy (-126.6kcalmol- l ) is in the range of the previous MM values; neither were remarkably different low energy rotamers obtained. Starting again from the minimum energy structure, after 10 ps-eq and 20 ps-av, and minimizing the resulting structure, we arrived at an energy of -128.2 kcal mol", which is lower than that obtained from the extended conformation, but higher than the result of the purely MM simulation. The final conformation ('l/JI = 88°, ¢2 = 290°, 'l/J2 = 55°, ¢3 = 64°, 'l/J3 = 280°, ¢4 = 279°, 'l/J4 = 338°, ¢s = 234°, 'l/Js = 40°), however, is fairly similar to 71 in Table 4. In the case of compound IV, the conformation obtained following the first strategy is somewhat higher in energy (-138.1 kcalrnol") than the lowest energy minimum obtained with MM, but its structure is similar to that of conformers 17

Fig. 9. Stereo plot of the structure 81 of IV.

266

G. Alagona et al.jJ, Mol. Struct, (Theochem) 311 (1994) 255-272

16

I

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(b)

"II'

" r,I1

e

Ui

"II'

8 6 (kcal/mol)

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0

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2 4 Relative energy

"c7 6 8 (kcal/mol)

Fig. 10. Aromatic ring separation as a function of the relative energy of the various rotamers and of their structure type: (a) I; (b) II; (c) III; (d) IV.

and 10 (not reported in Table 5 because of their energy difference with respect to the lowest minimum). Examining the intermediate structures along the dynamics simulation it is obvious that they all belong to the same family, as observed in general using the first strategy. If we use as a starting conformation the MM lowest minimum and we minimize the structure obtained after IOps-eq and 20ps-av, a decidedly lower energy structure (-156.2 kcal mol-I) is obtained, although still corresponding to a conformation ('l/J. = 353°, (h = 285°, 'l/J2 = 56°, ¢3 = 75°, 'l/J3 = 294°, ¢4 = 242°, 'l/J4 = 344°, ¢s = 292°, 'l/Js = 70°) quite similar to the initial one. 4.3. Correlation between energy and aromatic ring separation

The separation between the aromatic rings (as

measured from the distance of the oxygen atom on the tyrosyl ring and the carbon atom of the 6Phe phenyl ring opposite to that bearing the connection to the backbone) versus the relative energy of the conformation is plotted in Fig. 10 for each compound. As far as I is concerned (Fig. lO(a)), there are five structures, two 13-11' turns and three C 7 , with a separation about or below 12 A, which is the limiting distance used to discriminate between extended (average separation about 14 A [12a,c-e]) and compact structures, considered to be highly selective for the 8 receptor [15b]. None of them, however, should be very active, due to the fairly large separation (at the top of the interval) between the aromatic rings; neither would they be very prevalent, due to this energy gap. Five structures fall about or below 12 A for II (Fig. lOeb)) within a reasonable range of energy:

G. Alagona et al.j.I, Mol. Struct. (Theochem) 311 (1994) 255-272

the II' conformation (though it is not the most stable one), with a distance slightly above 6.5 A, should be much more prevalent than the C 7 conformer of I. The other four structures should at most contribute as much as the last two of I. From the view point of aromatic ring separation (~8 A) III and IV are more favorable, the plots for which (Fig. IO(c,d)) present a rather sharp distinction into two parts, indicating that there is a strong preference for either short (below 7.4 A) or long distances (above 10.7 A) between the aromatic rings, whereas the distributions for I and II show more spread. The most stable structures for both III and IV are type ,8-11' turns, which are also very compact. A little higher in energy is the second compact structure for III, again of the same type, followed by two hardly classifiable types and by a III' turn, of course not in this order. One of the outstanding features of the three most stable structures of Iv (even though the last two are

267

about 5kcalmol- 1 higher in energy than the first one) is that they are the only Il'-type conformers with an energy below IOkcalmol- l . The short separation between the aromatic rings has been already noted. This distance for the II' conformers turns out to be only 5.4-6.5 A. In addition, in the case of analog IV, there is a noticeably strong preference for short separations; five out of eight structures are below 7 A. The two analogs III and IV should thus be more active than I and II. As can be seen (Fig. 10), a short separation does not always correspond to a II' conformation. Moreover, II' conformations can often give rise to large distances between the aromatic rings, with the only exception of IV. Thus, the presence of the unsaturated residue seems to introduce into the structure a sort of conformational constraint, such as those considered helpful to enhance the activity toward the 8-receptor [33].

(b)

8

S1

4

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-4

Fig. 11.Molecular electrostatic potential maps along the C" -Cf3 bond in the plane perpendicular to that containing the carbonyl carbon atom, C" and Cp for a few rotarners of I (a) and II (b).

268

G. Alagolla et al.fJ. Mol. Struct, [Theochem} 3JJ (1994) 255-272

Fig. 12. Molecular electrostatic potential maps in the same plane as in Fig. II for a few rotamers of III.

4.4. Molecular electrostatic potential

Fig. 13. Molecular electrostatic potential maps in the same plane as in Fig. I I for a few rotamers of IV.

Among the several factors supposed to be responsible for the enhanced activity and potency observed in the unsaturated analogs, two can be studied and evaluated by examining the molecular electrostatic potential (MEP) in the region surrounding the C=C double bond. Both the intrinsic reactivity of the double bond [34] toward nucleophilic sites on the receptors [35] and the strengthening of binding to receptors [36] should be brought out, in fact, by an increase in the MEP in the plane perpendicular to the double bond. The value of the MEP on the molecular surface, although pictorially impressive, is less adequate for showing small differences in the MEP depending either on the distance from the binding site or on the orientation with respect to it. From a perusal of the maps shown in Figs. 11-13 it is evident that, as an almost general rule, the differences arise more from the assumed conformations than from the residues making up the peptide. This is particularly true for structures SI, 28 and 61. The MEP maps for SI, which is the

G. Alagona et al.ll. Mol. Struct, (Theochem) 3Il (1994) 255-272 16 (d)

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somewhat higher in energy than 81 (compounds I, II and IV), are again fairly similar, despite the fact that they correspond to different kinds of conformation (C 7 for 28 and ,B-turns of type III' or I' ,II for 61). It is, however, possible to detect preferential trends in the MEP for each compound, although related to different conformational types. The MEP for structures 71, 17, and 73 of III in the region of the double bond is similar to the MEP for 81. In addition, the MEP of structure 4F of III is very close to that obtained for structures 71 and 4 of II, which are different from the MEP computed for structure 4 of III, despite the analogous separation between the aromatic rings in all the structures of type 4. In any case, there is no clear-cut evidence that the MEP for at least compounds III and IV (which are more active than I and II) is stronger or more positive in the outer space region than that of I or II.

8

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269

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Conformers

Fig. 14. Energy differences between various conformers of (a) I, (b) II, (c) III and (d) IV in vacuo (- - -) and in solution ( - ) . The solvent effect on the conformers taken as zero is -17.3, -14.3, -9.9 and -11.4kcalmol- 1 for I, II, III and IV, respectively.

lowest energy structure for most of the systems considered, present a unique large lobe of negative potential for these compounds (with the exception ofIII which, however, assumes a conformation not amenable to usual types, see Table 4), while the MEP maps for structures 28 and 61, which are

The inclusion of the solvent in the calculations produces a stabilizing effect of the order of 10-22 kcal mol-I , depending on the system and the geometry (Fig. 14); overall this causes a generalized decrease in the energy differences between the various conformers. The solvent stabilizing effect is larger for the conformers that are less stable in vacuo, either because of the reduced number of intramolecular hydrogen bonds with respect to the lowest energy structures, or because of a backbone arrangement which is particularly accessible to the solvent, or because of the cooperative effect of the carbonyl oxygen lone pairs. An example of this feature is shown in Fig. 14(a) where structure 5 of I is somewhat stabilized, despite the presence of six intramolecular hydrogen bonds, due to the favorable positions of two of its carbonyl oxygen atoms which point toward the same region of the cavity surface. This arrangement produces a strong solvent reaction field that affects the free energy of solution. The solvent stabilizing effect can also be considerable when the backbone is noticeably exposed to the solvent (see, for instance, 65 in Fig. 14(b), 50 in Fig. 14(c), and 49 in Fig.14(d), despite the

270

G. Alagona el al.l.I, Mol. Struct. (Theochem) 311 (1994) 255-272

non-negligible number of hydrogen bonds (about four) still present in the aforementioned structures. The largest effects are however observed for the structures that present several polar groups available to interact with the solvent,due to the very limited number of intramolecular hydrogen bonds. The structures with only two intramolecular hydrogen bonds (namely: 70 (Fig. 14(a); 70 and 67 (Fig.14(b); 36 and 67 (Fig. 14(c); and 36 (Fig. 14(d)) have most of their NH and carbonyl groups exposed to the solvent, as well as both the phenolic and the carboxyl OH groups. The most interesting outcome of these calculations is that several ,B-turn structures of type II', presenting a relatively short separation between the aromatic rings, are greatly stabilized by the solvent (see Fig. lO). Moreover, structures that in vacuo fell outside of the plot because of their high energy even though they have a relatively short separation (below 12A) between the aromatic rings, turn out to be among the most stable in solution and thus the most probable structures for I (64, IO.2A), II (70, lO.oA), III (17, 5.4A; 2, 11.6 A) and IV (63, I LoA; 32,5.7 A; 55,11.7 A).

5. Conclusions The lower conformational flexibility summed up to the similarity to the Leu-enkephalin conformational behavior seems to be a necessary, but not sufficient, condition to explain the higher specificity toward the 8-receptor of these analogs. The presence of conformational constraints seems to be responsible for the enhanced activity towards the 8-receptor [33], more than folded, but flexible, conformations, because these constraints help the molecule maintain the aromatic rings at short separation (~12A). The aromatic ring separation seems to be an index that can successfully be employed to explain the activity toward the 8-receptor. The present results suggest that it is not only the backbone dihedral angles that determine the energy and the activity of the compounds, but also the relative position of the aromatic side chains. As for almost all the compounds studied similar backbone conformations were obtained,

but with a quite different spatial orientation of the side chains (see, for instance, Figs,2, 4, 5, 7 and 9), it is evident that the biological activity is the result of a suitable mixture of all these elements. Interestingly enough, the most active compound in this series (compound IV) presents not only a ,B-I1' bent structure for the backbone, but also the closest distance between the aromatic rings. It is attractive to propose that a combination of both features could be a good rationale for the requirements of receptor binding. Our results are in good agreement with experimental data for 8-selective analogs [1l,15c] of rigid compounds with a very short aromatic ring separation. The distributions of the minimum-energy conformations of these compounds are remarkably different in vacuo and in solution; a generalized flattening is observed in solution. The conformers that, at first sight, show the largest effect are the extended ones but, as expected, in the gas phase these in general have unfavorable energies. The ,B-turns of type II', however, remain the most stable conformations, even though they correspond to a different geometry. The ,B-turn conformers favored in vacuo show about 5-6 intramolecular hydrogen bonds, whereas the ,B-turn conformers favored in solution have 2-3 (or 4 at most) such hydrogen bonds, with an energy gain with respect to the gas phase as large as 22 kcal mol-I. Short distances between the aromatic rings are found also in the ,B-turn rotamers most stable in solution. The effect of the solvent field, which can act as a model of the biological environment as well, is to reduce the energy difference between the various rotamers, thus allowing a larger number of rotamers with favorable topological features to approach the receptor. Several ,B-tum II' structures having a close distance between the aromatic rings are in fact now accessible, and compound IV is favored in this respect because of the number of possible conformations that fulfil both requirements. The short relative separation between the aromatic rings appears to be the main factor responsible for the 8 receptor activity and specificity, which is therefore related to the increased rigidity of the peptide backbone, rather than to a direct electronic reactivity of the double bond towards the receptor.

G. Alagona et alp. Mol. Struct , [Theochem ) 311 (1994) 255-272

Acknowledgments G.M.C. is grateful to the Italian CNR for a fellowship that allowed her to carry out this study. The drawings from the Drexel University Molecular Editor on a Macintosh SE/30 have been used as hard copy for the MOGLI pictures produced by the calligraphic display of the Evans and Sutherland PS330 at ICQEM (Pisa).

[13]

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