Theoretical conformational analysis of a μ-selective cyclic opioid peptide analog

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

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

    of Montreal, Montreal, Quebec, Canada H2W 1R7

    Synopsis

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

    -

    INTRODUCTION

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

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

    *To whom correspondence should be addressed.

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

  • 1432 WILKES AND SCHILLER

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

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

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

  • CONFORMATION OF A CYCLIC OPIOID PEPTIDE 1433

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

    METHODOLOGY

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

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

    OH

    y 2

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

    CH 3 1 2 1 3 4

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

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

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

    nate rotatable bonds).

  • 1434 WILKES AND SCHILLER

    structure. The potential energy is calculated from

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

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

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

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

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

  • 0

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  • CONFORMATION OF A CYCLIC OPIOID PEPTIDE 1437

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

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