Conformational features responsible for the binding of cyclic analogues of enkephalin to opioid receptors III. Probable binding conformations of μ-agonists with phenylalanine in position 3

Theoretical conformational analysis was carried out for several tetrapeptide analogues of β-casomorphin and dermorphin containing a Phe residue in position 3. Sets of low-energy backbone structures of the μ-selective peptides [N-Me-Phe³, d -Pro⁴]-morphiceptin and Tyr-d -Orn-Phe-Asp-NH₂ were obtained. These sets of structures were compared for geometrical similarity between themselves and with the low-energy conformations found for the δ-selective peptide Tyr-d -Cys-Phe-d -Pen-OH and nonactive peptide Tyr-Orn-Phe-Asp-NH₂. Two pairs of geometrically similar conformations of μ-selective peptides, sharing no similarity with the conformations of peptides showing low affinity to the μ-receptor, were selected as two alternative models of probable μ-receptor-bound backbone conformations. Both models share geometrical similarity with the low-energy structures of the linear μ-selective peptide Tyr-d -Ala-Phe-Phe-NH₂. Putative binding conformations of Tyr¹ and Phe³ side chains are also discussed.     

Single residue modifications of the delta opioid receptor selective peptide, [d-Pen2,d-Pen5]-enkephalin (DPDPE)

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [d -Pen²,d -Pen⁵]enkephalin, Tyr-d -Pen-Gly-Phe-d -PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by ¹H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine¹-DPDPE ([DAT¹]DPDPE) and phenylglycine⁴-DPDPE ([Pgl⁴]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced μ receptor binding affinity of the carboxamide terminal DPDPE-NH₂ appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the α-aminoisobutyric acid substituted analog [Aib³]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the δ opioid receptor.     

Solution conformations of deltorphin-I obtained from combined use of quantitative 2D-NMR and energy calculations: A comparison with dermenkephalin

Deltorphin-I, Tyr-d -Ala-Phe-Asp-Val-Val-Gly-NH₂ and dermenkephalin, Tyr-d -Met-Phe-His-Leu-Met-Asp-NH₂, two highly related opioid peptides from frog skin, display very similar N-termini but strikingly different C-terminal tails. Nevertheless, both peptides are highly potent at, and exquisitely selective for the δ-opioid receptor. To identify common determinants concuring to the remarkably efficient targeting of deltorphin-I and dermenkephalin, combined use of quantitative two-dimensional nuclear magnetic resonance (53 dipolar interactions studied at four temperatures) and energy calculations using simulated annealing generated five groups of deltorphin-I conformers. These groups were pooled into two families whose overall conformation could be described either by a left-handed helix (Family I) or by a big loop (Family II), both stabilized by H-bonds. Proximity of D-Ala²-Phe³-Asp⁴ and Val⁵-Val⁶-Gly⁷ triads is an obvious structural similarity between almost all groups in both families of structures. Whereas differences between the two families originated mostly from a transition at Ψ Asp⁴ backbone dihedral angle, the backbone structures at segment 1–4 are similar and spatial arrangements of Tyr¹ (t) and Phe³ (g^–) are identical in one group of each family. Moreover, these two groups have a N-terminal tetrapeptide whose conformation most closely resembles that of a well-defined group of structures for dermenkephalin. Altogether, these results suggest that conformational attributes that are common to dermenkephalin and deltorphin-I, i.e., the backbone conformation of the N-terminal tetrapeptide and preferential orientations in the side-chain of Tyr¹ (t) and Phe3 (g^–) underlie their ability to bind with high selectivity to the δ-opioid receptor.     

Synthesis,biology, NMR and conformation studies of the topographically constrained δ‐opioid selective peptide analogs of [β‐iPrPhe3]deltorphin I

Abstract: Replacement of Phe³ in the endogenous δ‐opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid β‐isopropylphenylalanine (β‐iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand‐binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [β‐iPrPhe³]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe³ residue in [β‐iPrPhe³]deltorphin I provided the most desirable biological properties with binding affinity (IC₅₀ = 2 n m ), bioassay potency (IC₅₀ = 1.23 n m in MVD assay) and exceptional selectivity for the δ‐opioid receptor over the µ‐opioid receptor (30 000). Further conformational studies based on two‐dimensional NMR and computer‐assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr¹ and (2S,3R)‐β‐iPrPhe³ residues adopt trans side‐chain conformations, and the linear peptide backbone favors a distorted β‐turn conformation.     

Conformational features responsible for binding of cyclic analogues of enkephalin to opioid receptors

Low-energy peptide backbone conformers were found by means of energy calculation for several cyclic analogues of enkephalin in an attempt to assess models for receptor-bound conformations for opioid receptors of the μ- and §-types. They included [D-Cys², L-Cys⁵]- and [D-Cys², D-Cys⁵]-enkephalinamides showing moderate preference for preceptors, the selective compounds [D-Pen², L-pen⁵] and [D-Pen², D-Pen⁵]-enkephalins and Tyr-D-Lys-Gly-Phe analogue possessing very high affinity to receptors of the μ-type. The low-energy conformers obtained for these analogues were in good agreement with the results of calculations by other authors and with experimental evidence. All of the analogues contain a Phe residue in position 4 of the peptide chain which facilitates the eventual search for geometrical similarity between the low-energy backbone conformers of different analogues in question.     

[d-Pen2,d-Pen5]enkephalin,the standard delta opioid agonist,induces morphine-like behaviors in mice

[d-Pen²,d-Pen⁵]enkephalin (DPDPE; 3–30 µg) and morphine (10 µg) both caused Straub tails, increased locomotion, and circling after ICV administration to ICR mice. DPDPE-induced tail stiffening was reduced when mice were pretreated with naloxone (0.5 mg/kg SC) or-funaltrexamine (10 µg ICV), but not with ICI 174864 (2 mg/kg SC), the selective antagonist at delta opioid receptors. These results point to (a) mu receptors mediating the tail stiffening and (b) the loss of delta receptor selectivity after 10 and 30 µg DPDPE.     

Theoretical conformational analysis of enkephalin analogues related to fluorescence and n.m.r. measurements in aqueous solution

The properties related to non-radiative energy transfer of a number of enkephalin analogues with tryptophan substituted for phenylalanine in position 4 and n.m.r. ³JNH-C^αH coupling constants of corresponding [Phe⁴]-enkephalin analogues are being derived from semi-empirical conformational energy. The molecules considered contain a glycyl, a D-alanyl or an L-alanyl as second residue; two of the compounds are N-methylated at position 4 or 5. The [Trp⁴]- enkephalin analogues and the corresponding [Phe⁴]-enkephalin analogues display nearly parallel affinities in the opiate receptor binding assay (Schiller et al.(1)). The comparison of computed and experimental properties shows that an ensemble of conformers is a satisfactory representation of the state of these molecules in water.     

Crystal structure,conformation, and potential energy calculations the chemotactic peptide N- formyl-l-Met-l-Leu-l-Phe-OMe

The tripeptide N-formyl-L-Met-l -Leu-l -Phe-OMe (FMLP-OMe) crystallizes in the orthorhombic system, space group P 2₁2_l2₁, with the following unit-cell parameters: a = 21.727, b = 21.836, c = 5.133Å, Z = 4. The structure has been solved and refined to a final R of 0.068 for 1838 independent reflexions with I > 2σ(I). The peptide backbone is folded at the Leu residue (φ_L=?67.7,Ψ_L=?49.1°) without intramolecular hydrogen bonds. Considering each peptide plane, the Leu side-chain is oriented on the same side of that of the Phe residue and on the opposite side of that of the Met residue, respectively. The crystal conformation differs from all the other conformations proposed for FMLP-OMe and the anionic form of N-formyl-l -Met-l -Leu-l -Phe-OH (FMLP) in solution accounts for the amphiphilic character of the peptide, giving rise, through intermolecular hydrogen bonds, to a stacking of molecules which could be maintained in the aggregation states experimentally observed in solvents of low polarity. Intramolecular potential energy calculations have ben carried out in order to compare the energies of the various backbone conformers.     

Conformationally stabilized gramicidin S analog containing dehydrophenylalanine in place of d-phenylalanine4,4′

Unsaturated gramicidin S analog, [ΔPhe^4,4′]gramicidin S, was synthesized by conventional solution method in order to evaluate the role of the dehydrophenylalanine residues replacing d -phenylalanine^4,4′ in stabilizing the bioactive β-shect conformation. The dehydrophenylalanine (ΔPhe) moiety was introduced by dehydroazlactonization of the β-phenylserine residue. The [ΔPhe^4,4′]gramicidin S prepared by this method showed very strong antimicrobial activities against Gram-positive bacteria, but not against Gram-negative ones. Several lines of spectroscopic evidence indicated that [ΔPhe^4,4′] gramicidin S has a reinforced β-sheet backbone conformation necessary for a full biological activity of gramicidin S. These results suggested that :α,β-dehydrogenation of the amino acid residue in a cyclic peptide can stabilize the turn structure.     

Biologically active conformation of tuftsin

The main features of the assumed biologically active conformation of the tuftsin molecule have been described using the energy calculations as well as the n.m.r. and CD-spectroscopy measurements performed for the conformationally rigid active cycloanalogue, Thr-L-ys-Pro-Arg. The biologically active conformation has been shown to be of BBRB or BLRB backbone type with the trans-conformation of proline residue.     

Comparative conformational studies on cyclic hexapeptides corresponding to message sequence His-Phe-Arg-Trp of α-Melanotropin by NMR

Solution conformation of cyclo(Gly¹-His²-Phe³-Arg⁴-Trp⁵-Gly⁶) and its d -Phe analog corresponding to the message sequence [Gly-α-MSH_5-10] of α-MSH has been studied by 1D and 2D proton magnetic resonance spectroscopy in dimethyl sulfoxide (DMSO)-d₆ solution and in a DMSO-d₆/H₂O cryoprotective mixture. The NMR data for both the analogs in solution at 300 K cannot be interpreted based on a single ordered conformation, as evidenced by the broadening of only -NH resonances as well as the temperature coefficients of the amide protons. An analysis of the nuclear Overhauser effect (NOE) cross-peaks in conjunction with temperature coefficient data indicates an equilibrium of multiple conformers with a substantial population of particular conformational states at least in the d -analog. The molecular dynamics simulations without and with NOE constraints also reveal numerous low-energy conformers with two γ-turns, a γ-turn and a β-turn, two β-turns, etc. for both the analogs. The observed NMR spectra can be rationalized by a dynamic equilibrium of conformers characterized by a γ-bend at Gly⁶, two γ-bends at Phe³ and Gly⁶ and a conformer with a single β-turn and a γ-bend for the l -Phe analog. On the other hand, a conformation with two fused β-turns around the two tetrads His²-d -Phe³-Arg⁴-Trp⁵ and Trp⁵-Gly⁶-Gly¹-His² dominates the equilibrium mixture for the d -Phe analog. For the d -Phe analog, the experimentally observed average conformation is corroborated by molecular dynamics simulations as well as by studies in cryoprotective solvent.     

Dehydro-enkephalins

Two kinds of dehydropeptide analogs of enkephalin containing a ΔTyr unit at the N-terminus have been synthesized by coupling Boc-ΔTyr-(Cl₂Bzl)-OH with amino acid amides and tetrapeptide esters using the water soluble carbodiimide-HOBt method. Pentapeptides consisting of ΔTyr¹, and ΔPhe⁴ or ΔLeu⁵ were also prepared. Ultraviolet difference spectroscopy was important in the characterization of the dehydro moieties, ΔTyr, ΔPhe and ΔLeu. Attempts to liberate ΔTyr¹-enkephalins have been unsuccessful because of the instability of an N-terminal ΔTyr residue having p-phenolic group in the side chain.     

Synthesis and biological properties of β-MePhe3 analogues of deltorphin I and dermenkephalin: influence of biased X1 of Phe3 residues on peptide recognition for δ-opioid receptors

Using the method of conformational constraint, we have designed and synthesized analogues of deltorphin I and dermenkephalin containing each of the four stereoisomers (2S, 3S: 2S, 3R; 2R, 3S; 2R, 3R) of the unusual amino acid ß-methylphenylalanine in position three. The potency and selectivity of these analogues were evaluated by radioreceptor binding assays in the rat brain using [³H]CTOP (δ-ligand) and [³H][p-CβPhe⁴]DPDPE (β-ligand), and by bioassay using the mouse vas deferens (β-receptor assay) and guinea pig ileum (β-receptor assay) assays. The substitution of a ß-MePhe for Phe³ in deltorphin I and dermenkephalin has a large and variable effect on the bioactivities of the synthesized analogues. The synthesized analogues are somewhat less potent than the native peptides. Both [(2S, 3R)-ß-MePhe³]deltorphin and [(2S, 3R)-ß-MePhe³]dermenkephalin are more selective, however, and interact essentially specifically with the receptor in the binding assays and bioassays. The bioassay data in vitro of the synthesized analogues of deltorphin I and dermenkephalin follow the same general trends as the receptor binding data. These results demonstrate that topographical modifications of the side-chain conformation of critical structural moieties in a ligand can significantly modulate both the potency and receptor selectivity for ligands that have multiple sites of biological activity, and they illustrate that this approach has general application to peptide and peptidomimetic ligand design.     

Conformation-function relationships in LHRH analogs

A systematic conformational build-up procedure was performed for the LHRH molecule, pGlu¹-His²-Trp³-Ser⁴-Tyr⁵-Gly⁶-Leu⁷-Arg⁸-Pro⁹-Gly¹⁰-NH₂. The results showed a very high flexibility of the LHRH backbone, with 300 conformers being regarded as having low energy. At the same time, the conformational flexibility of LHRH differs among the fragments of the molecule. The most rigid fragments of LHRH are the Ser⁴-Tyr⁵-Gly⁶-Leu⁷ and Tyr⁵-Gly⁶-Leu⁷-Arg⁸ central tetrapeptides, the latter possessing only eight different types of low-energy backbone conformers. These eight conformer types belong to different kinds of chain reversals which are stabilized by different systems of intramolecular hydrogen bonds. Some of them resemble the β-II′ turn, which was derived as the LHRH structure from energy calculations by others. The results obtained are in good agreement with the experimental data on LHRH flexibility in solution.     

Conformational analyses by 1H NMR and computer simulations of cyclic hexapeptides related to somatostatin containing acidic and basic peptoid residues

We report the conformational analysis by ¹H NMR in DMSO and computer simulations involving distance geometry and molecular dynamics simulations at 300K of peptoid analogs of the cyclic hexapeptide c-[Phe¹¹-Pro⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰]. The analogs c-[Phe¹¹-Nasp⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰] ( 1 ), c-[Phe¹¹-Ndab⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰] ( 2 ) and c-[Phe¹¹-Nlys⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰] ( 3 ) where Nasp denotes N-(2-carboxyethyl) glycine, Ndab N-(2-aminoethyl) glycine and Nlys N-(4-aminobutyl) glycine are subject to conformational studies. The results of free and restrained molecular dynamics simulations at 300K are reported and give insight into the conformational behaviour of these analogs. The compounds show two sets of nuclear magnetic resonance signals corresponding to the cis and trans orientations of the peptide bond between residues 11 and 6. The backbone conformation of the cis isomers that we believe are the bioactive isomers of the three compounds are very similar to each other while there are larger variations amongst the trans isomers. The binding data to the isolated receptors show that the introduction of the Nlys residue in analog 3 leads to an enhancement of binding potency to the hsst5 receptor compared with analog 2 while maintaining identical binding potency to the hsst2 receptor. The Nasp⁶ analog 1 binds weakly to the hsst2 and is essentially inactive towards the other receptors. Comparison of the conformations and binding activities of these three analogs indicates that the Nlys residue extends sufficiently far to allow binding to a negatively charged binding domain on the hsst5 receptor. According to this model, the Ndab analog 2 cannot extend far enough to allow for binding to the receptor pocket. The loss of activity observed for the Nasp⁶ compound 1 indicates that the presence of a negatively charged residue in position 6 is unfavorable for binding to the hsst receptors.     

Proton n.m.r. investigation of conformational influence of penicillamine residues on the disulfide ring system of opioid receptor selective Somatostatin derivatives

Three cyclic disulfide analogs related to somatostatin, d -Phe¹ -Cys²-Tyr³-d -Trp⁴-Lys⁵-Thr⁶-Xxx⁷-Thr⁸NH₂ (where Xxx =l -Pen 1; l -Cys 3; or d -Pen 4) were examined in DMSO-d₆ by one- and two-dimensional proton n.m.r. spectroscopy in order to analyze the conformational influence of the position-7 residue on the 20-membered disulfide ring. From these studies it was concluded that all three analogs maintain a β II turn solution conformation for the core tetrapeptide-Tyr³-d -Trp⁴-Lys⁵-Thr⁶-. However, the disulfide conformation differs in the analogs, with 1 and 3 having a left-handed and 4 a right-handed disulfide chirality.     

Conformational analysis of two somatostatin analogues by 1H 500 MHz n.m.r. spectroscopy

A series of nine closely related somatostatin analogues, containing the hexapeptide H-Cys² -Phe³ -D-Trp⁴-Lys⁵ -Thr⁶ -Cys⁷-NH₂ sequence have been synthesized by Bauer et al. The conformational properties of two of them, showing intermediate activities between those of SMS 201–995 and somatostatin, have been studied by high field n.m.r. spectroscopy in DMSO. Assignments were made using 2D-n.m.r. methods, in particular NOESY experiments and detection of long-range connectivities in aromatic residues. In all the compounds of this series, the biologically active ones as well as the inactive ones, the n.m.r. parameters are in favour of a predominant conformation with a type II' β turn involving amino acids Phe³ to Thr⁶. A clearcut correlation exists between the predominant conformation at the cystine bridge side and the activity. The presence of the exocyclic amino acids Phe¹ and Thr⁸ (ol) plays a major role in stabilization of the active conformation.     

Dual effect of [ -Pen, -Pen]enkephalin on ion transport in guinea pig colon

Effects of the δ-opioid receptor ligand, [ -Pen², -Pen⁵]enkephalin (DPDPE) on basal and endothelin-1-induced ion secretion in guinea pig colon were investigated. Muscle-stripped segments of guinea pig colon were mounted in Ussing flux chambers and changes in the short-circuit current (I_sc) were monitored continuously. DPDPE significantly reduced baseline I_sc at a low dose, 1 nM; however DPDPE increased I_sc at 10 and 100 μM. Endothelin-1 stimulated ion secretion that was unaltered in tissues pretreated with DPDPE. In guinea pig colon, δ-opioid receptor activation evoked both a proabsorptive and prosecretory response.     

Selective ligands for opioid receptors: β-Cyclopropylalanyl containing analogs of enkephalin

The synthesis and resolution of the amino acid β-cyclopropylalanine (Cpr) and its incorporation into four enkephalin analogs is reported. The analogs prepared were: Tyr - l - Cpr - Gly - Phe - Pen (des - COOH - Nle = n - pentylamide = Pen) (l -Cpr²-Pen⁵-ENK), Tyr-d -Cpr-Gly-Phe-Pen (d -Cpr²-Pen⁵-ENK), l -Cpr-Tyr-d -Ala-Gly-Phe-Pen (l -Cpr⁰-d -Ala²-Pen⁵-ENK) and d -Cpr-Tyr-d -Ala-Gly-Phe-Pen (d -Cpr⁰-d -Ala²-Pen⁵-ENK). Each was tested for its ability to inhibit the field stimulated guinea pig ileum (GPI) and rat vas deferens (RVD) and the results compared to the effect d -Ala²-d -Leu⁵-enkephalin (DADLE) has on the same preparations. The results show that at concentrations up to 10^-5 m all four analogs, as well as DADLE, are full agonists on the GPI preparation. The concentrations necessary to produce a 50% inhibition of the twitch response were, DADLE, 3.5 °× 10^-8 m ; l - Cpr⁰-d -Ala²-Pen⁵-ENK, 6.0 × 10^-8 m ; d -Cpr²-Pen⁵-ENK, 1.1 × 10^-7 m ; l -Cpr²-Pen⁵-ENK, 1.2 × 10^-6 m and d -Cpr⁰-d -Ala²-Pen⁵-ENK, > 10^-5 m . On RVD a different result was observed with only DADLE (1.3 × 10^-6 m ) and l -Cpr⁰-Pen⁵-enkephalin (1.8 × 10^-6 m ) showing full agonist activity. d -Cpr²-Pen⁵-ENK was a partial agonist (29 · 5% inhibition of the twitch at 10^-5 m ) while d -Cpr⁰-d -Ala²-Pen⁵-ENK and l -Cpr²-Pen⁵-ENK did not inhibit the twitch at concentrations up to 10^-5 m . These compounds which were inactive or of low potency on each preparation were also tested as antagonists. Only d -Cpr²-Pen⁵-ENK was an antagonist (pA₂ = 6.09) versus DADLE on RVD while d -Cpr⁰-d -Ala²-Pen⁵-ENK was inactive as an antagonist on both GPI and RVD. d -Cpr²-Pen⁵-ENK, therefore, represents the first enkephalin analog to be categorized as a mixed agonist-antagonist.     

Conformational energy calculations on Substance P

The conformational and spacial configurations of the biologically active undecapeptide, Substance P, were studied using conformational energy calculations. Low energy conformers of residues 1–5 and 6–11 were found by energy minimization and the two fragments were then combined to find low-energy structures for Substance P. Several configurational classes were found with different backbone conformations. A comparison of the final low-energy structures with data from biological tests on analogs of Substance P gives some insight into the conformation required for interaction at the biological receptor.