Morphiceptin analogs containing 2-aminocyclopentane carboxylic acid as a peptidomimetic for proline

As part of a program to study the structure-activity relationship of peptide opioids we report the synthesis, conformational characterization and biological activity of four analogs related to morphiceptin in which the proline at position two has been substituted with 2-aminocyclopentane carboxylic acid (beta Ae5c). The beta Ac5c residue is a beta amino acid with two chiral centers resulting in four possible configurations; two configurational cis (R,S and S,R) and two configurational trans (R,R and S,S) forms. Utilizing high resolution n.m.r. at 500 MHz and computer simulations with NOE restraints the chirality of the beta Ac5c residues are assigned. The analog containing the R,S-beta Ac5c is active at both the mu and delta-opioid receptors, with a slight preference for the mu-receptor. The (S,R), (S,S), and (R,R) analogs show minimal activity at the mu-receptor and are inactive at the delta-receptor. A comparison of the findings from the conformational analysis and biological assays lends insight into the structure-activity relationship of this important peptide opiate.     

Topochemical analysis of morphiceptin and dermorphin bioactivities.

To elucidate the topochemical requirements for bioactivities of morphiceptin (Tyr-Pro-Phe-Pro-NH2) and dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), we have designed and synthesized two diastereomers Tyr-(L and D)-(NMe)Ala-Phe-D-Pro-NH2. Both the analogs display high activities in the guinea pig ileum assay. The only difference in the composition of these two diastereomers arises from the chirality at residue 2. The high mu-receptor activities are attributed to structures where the Tyr1-L-(NMe)Ala2 amide bond assumes a cis configuration while the Tyr1-D-(NMe)Ala2 amide bond assumes a trans configuration. Accessible space studied for the second residues of these molecules confirms the fact that the L-(NMe)Ala2 analog belongs to the morphiceptin family of opioids while the D-(NMe)Ala2 analog belongs to the dermorphin class of opioids. The similarity in the spacial array of the analogs explains their high mu-receptor activities and indicates that they are likely recognized at the same opioid receptor.     

Cyclic retro-inverso dipeptides with two aromatic side chains. I. Synthesis.

A series of cyclic retro-inverso dipeptides--2-[(4-hydroxy)benzyl]-5-benzyl-4,6(1H,2H,3H,5H)-pyrimidinedi one (c[mPhe-gTyr]), 2-benzyl-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[mTyr-gPhe]), and 2-benzyl-5-amino-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[(alpha-amino)mTyr-gPhe])--were synthesized in order to define the minimum structural requirements for binding affinity with opiate receptors and biological activity. Although the first two compounds lack a free amine proposed to be necessary for receptor recognition, the c[mPhe-gTyr] and c[mTyr-gPhe] analogues serve as model molecules in conformational studies of the target analogue, c[(alpha-amino)mTyr-gPhe]. The cis- and trans-c[(alpha-amino)mTyr-gPhe] contain all the functional groups such as the amine and phenolic groups in the tyrosine, and the aromatic group in the phenylalanine, necessary for opiate activity. In addition, the c[(alpha-amino)mTyr-gPhe] analogues possess similar geometries to the Tyr-Pro part of morphiceptin (Tyr-Pro-Phe-Pro-NH2) whose high mu-receptor activity is attributed to conformations with the Tyr-Pro amide bond in a cis conformation because the peptide bonds assume a cis conformation. However, both analogues are inactive in the guinea pig ileum and the mouse vas deferens assays. This may result from wrong orientation of the benzyl group of the gPhe residue with respect to the (alpha-amino)mTyr residue. Conformational studies of these molecules using 1H-nmr spectroscopy and molecular mechanics calculations will be reported in the following paper. Results of conformational analysis should provide information about backbone-side-chain interactions in the retro-inverso peptide chains since all the fundamental structural elements of the retro-inverso peptides are included in these model systems even though the peptide bonds must assume a cis conformation.     

Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8

We report the conformational analysis by 1H nmr in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of analogs of the cyclic octapeptide D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys 7]-Thr8-ol (sandostatin, octreotide). The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) contain stereochemical changes in the Thr residues in positions 6 and 8, which allow us to investigate the influence of the stereochemistry within these residues on conformation and binding affinity. The molecular dynamics simulations provide insight into the conformational flexibility of these analogs. The compounds with (S)-configuration at the C(alpha) of residue 6 adopt beta-sheet structures containing a type II' beta-turn with D-Trp in the i+1 position, and these conformations are "folded" about residues 6 and 3. The structures are very similar to those observed for sandostatin, and the disulfide bridge results in a close proximity of the H(alpha) protons of residues 7 and 2, which confirms earlier observations that a disulfide bridge is a good mimic for a cis peptide bond. The compounds with (R)-configuration at the C(alpha) of residue 6 adopt considerably different backbone conformations. The structures observed for these analogs contain either a beta-turn about residue Lys and Xaa6 or a gamma-turn about the Xaa6 residue. These compounds do not exhibit significant binding to the somatostatin receptors, while the compounds with (S) configuration in position 6 bind potently to the sst2, 3, and 5 receptors. The nmr spectra of analogs with (R) or (S) configuration at the C(alpha) of residue 8 are strikingly similar to each other. We have demonstrated that the chemical shifts of protons of residues 3, 4, 5, and 6, which are part of the type II' beta-turn, and especially the effect on the Lys gamma-protons are considerably different in active molecules as compared to inactive analogs. Since the presence of a type II' beta-turn is crucial for the binding to the receptors, the chemical shifts, the amide temperature coefficients of the Thr residue and the medium strength NOE between LysNH and ThrNH can be extremely useful as an initial screening tool to separate the active molecules from inactive analogs.     

Conformational characteristics of receptor-selective opioid peptides. 1H n.m.r. and c.d. spectroscopic studies of delta-kephalin and [Val4]morphiceptin.

An investigation on the conformations of highly receptor-selective opioid peptides was carried out to gain further understanding of the structure-activity relationship of endogenous enkephalins. The preferred conformations of a highly mu-selective [Val4]morphiceptin and a highly delta-selective delta-kephalin have been probed by 1H n.m.r. solvent-, concentration- and temperature-dependences of amide protons to take the folded conformations stabilized by an intramolecular hydrogen bond and the anti-parallely extended dimeric structures respectively. Their possible stereo-conformations were proposed, based on the analyses of the vicinal coupling constants (JHNC alpha H). The conformational difference between the mu- and delta-selective opioid peptides was further ascertained by the c.d. measurements. The c.d. spectra of the mu-selective peptides show negative bands in the range of 210-230 nm, while those of the delta-selective ones show the opposite positive bands. A correlation between c.d. spectra and receptor-selectivity was possible.     

Conformational analysis of morphiceptin by NMR spectroscopy

Three exorphins, beta-casomorphin-5, morphiceptin and its D-Pro4 analog, were studied in DMSO by means of 1H and 13C NMR spectroscopy, with the aim of detecting conformational features of potential biological significance for the mu opioid activity since the presence of two Pro residues restricts the accessible conformational space more than in all other peptides. It is found that the conformational mixtures present in solution contain relevant fractions of folded conformers, a feature that assures the observation of four different Tyr OH signals in the 500 MHz spectrum of morphiceptin. The conformer distribution of (very active) (D-Pro4)-morphiceptin is different from those of its (less active) congeners.     

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

Theoretical conformational analysis was carried out for several tetrapeptide analogues of beta-casomorphin and dermorphin containing a Phe residue in position 3. Sets of low-energy backbone structures of the mu-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 delta-selective peptide Tyr-D-Cys-Phe-D-Pen-OH and nonactive peptide Tyr-Orn-Phe-Asp-NH2. Two pairs of geometrically similar conformations of mu-selective peptides, sharing no similarity with the conformations of peptides showing low affinity to the mu-receptor, were selected as two alternative models of probable mu-receptor-bound backbone conformations. Both models share geometrical similarity with the low-energy structures of the linear mu-selective peptide Tyr-D-Ala-Phe-Phe-NH2. Putative binding conformations of Tyr1 and Phe3 side chains are also discussed.     

14-membered cyclic opioids related to dermorphin and their partially retro-inverso modified analogues. II. Preferred conformations in solution as studied by 1H-NMR spectroscopy.

The 1H-NMR studies were extensively carried out to elucidate preferred conformations of a series of 14-membered cyclic dermorphin analogues containing two phenylalanines at both the third and fourth positions, e.g., Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu], and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu]. The temperature coefficients of the amide proton chemical shifts, vicinal 1H-1H coupling constants for the NH-CH groupings, and nuclear Overhauser effects provided information regarding the preferred conformations of the backbones. The conformational preferences and flexibility of the side chains were also estimated from the vicinal 1H-1H coupling constants around the C-C beta and C beta-C bonds in the articulated side chains. A comparison of the results obtained was made with the results previously obtained for the corresponding enkephalin analogues containing a glycine at the third position. It was found that the replacement of the glycine with the phenylalanine at the third position increases the conformational flexibility of the molecules with an L-, or S-, residue at the fifth position but reduces the flexibility of the molecules with D-, or R-, residue at the same position. The rotating frame nuclear Overhauser experiments gave direct evidence for compact conformations, with the Tyr side chain folding back over the 14-membered ring in Tyr-c[D-Glu-Phe-gPhe-rLeu], which displays relatively high selectivity for the delta-receptor over the mu-receptor. This observation is in agreement with our model proposed for the cyclic enkephalin analogues: folded forms with close aromatic ring placement are required for the activity at the delta-receptor.     

Dermorphin tetra- and heptapeptide analogues containing a [3,4] amide bond replacement by a carbon-carbon double and single bond.

Seven dermorphin hepta- and tetrapeptide analogues containing [3,4] amide bond replacement by a carbon-carbon double and single bond were prepared. 1H NMR studies of the pseudoheptapeptide in DMSO indicate the presence of extended conformations with stacking of the side chains in the N-terminal part and an inverse gamma-turn around Ser7 in the conformational equilibrium. The binding data show that the affinity of the analogues for the mu-receptor is only slightly diminished in the D-Ala2 series and is more affected in the D-Arg2 series. Since the Gly4NH is not present in these compounds we conclude that this NH is not required to stabilize the bioactive conformation nor is it directly involved in binding to the receptor.     

Synthesis, biology, NMR and conformation studies of the topographically constrained delta-opioid selective peptide analogs of [beta-iPrPhe(3)]deltorphin I.

Replacement of Phe3 in the endogenous delta-opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid beta-isopropylphenylalanine (beta-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 [beta-iPrPhe3]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe3 residue in [beta-iPrPhe3]deltorphin I provided the most desirable biological properties with binding affinity (IC50 = 2 nM), bioassay potency (IC50 = 1.23 nM in MVD assay) and exceptional selectivity for the delta-opioid receptor over the mu-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)-beta-iPrPhe3 residues adopt trans side-chain conformations, and the linear peptide backbone favors a distorted beta-turn conformation.     

Rational design of protein stability: effect of (2S,4R)-4-fluoroproline on the stability and folding pathway of ubiquitin

Background

Many strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a C^γ-exo or a C^γ-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying C^γ-exo puckering.

Methodology/Principal Findings

While (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the C^γ-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of −4.71 kJ·mol⁻¹ in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub.

Conclusions/Significance

The results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein.     

(35)S]GTPgammaS binding stimulated by endomorphin-2 and morphiceptin analogs

The ability of several mu-selective opioid peptides to activate G-proteins was measured in rat thalamus membrane preparations. The mu-selective ligands used in this study were three structurally related peptides, endomorphin-1, endomorphin-2 and morphiceptin, and their analogs modified in position 3 or 4 by introducing 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal). The results obtained for these peptides in [(35)S]GTPgammaS binding assay were compared with those obtained for a standard mu-opioid agonist DAMGO. [d-1-Nal(3)]Morphiceptin was more potent in G-protein activation (EC(50) value of 82.5+/-4.5 nM) than DAMGO (EC(50)=105+/-9 nM). [d-2-Nal(3)]Morphiceptin, as well as endomorphin-2 analogs substituted in position 4 by either d-1-Nal or d-2-Nal failed to stimulate [(35)S]GTPgammaS binding and were shown to be potent antagonists against DAMGO. It seems that the topographical location of the aromatic ring of position 3 and 4 amino acid residues can result in a completely different mode of action, producing either agonists or antagonists.     

Conformation-activity relationship of peptide T and new pseudocyclic hexapeptide analogs.

Peptide T (ASTTTNYT), a segment corresponding to residues 185-192 of gp120, the coat protein of HIV, has several important biological properties in vitro that have stimulated the search for simpler and possibly more active analogs. We have previously shown that pseudocyclic hexapeptide analogs containing the central residues of peptide T retain considerable chemotactic activity. We have now extended the design of this type of analogs to peptides containing different aromatic residues and/or Ser in lieu of Thr. The complex conformation-activity relationship of these analogs called for a reexamination of the basic conformational tendencies of peptide T itself. Here, we present an exhaustive NMR conformational study of peptide T in different media. Peptide T assumes a gamma-turn in aqueous mixtures of ethylene glycol, a type-IV beta-turn conformation in aqueous mixtures of DMF, and a type-II beta-turn conformation in aqueous mixtures of DMSO. The preferred conformations for the analogs were derived from modeling, starting from the preferred conformations of peptide T. The best models derived from the gamma-turn conformation of peptide T are those of peptides XII (DSNYSR), XIII (ETNYTK) and XVI (ESNYSR). The best models derived from the type-IV beta-turn conformation of peptide T are those of peptides XIV (KTTNYE) and XV (DSSNYR). No low-energy models could be derived starting from the type-II beta-turn conformation of peptide T. The analogs with the most favored conformations are also the most active in the chemotactic test.     

Synthesis,biological evaluation and structural analysis of novel peripherally active morphiceptin analogs

Morphiceptin (Tyr-Pro-Phe-Pro-NH₂), a tetrapeptide amide, is a selective ligand of the μ-opioid receptor (MOR). This study reports the synthesis and biological evaluation of a series of novel morphiceptin analogs modified in positions 2 or/and 4 by introduction of 4,4-difluoroproline (F₂Pro) in l or d configuration. Depending on the fluorinated amino acid configuration and its position in the sequence, new analogs behaved as selective full MOR agonists showing high, moderate, or relatively low potency. The most potent analog, Tyr-F₂Pro-Phe-d-F₂Pro-NH₂, was also able to activate the κ-opioid receptor (KOR), although with low potency. Docking studies and the comparison of results with the high resolution crystallographic structure of a MOR-agonist complex revealed possible structure–activity relationships of this compound family.     

Comparative structure-function studies with analogs of dynorphin-(1-13) and [Leu5]enkephalin

Analogs of dynorphin-(1-13) with modifications in the enkephalin segment were compared with correspondingly modified analogs of [Leu5]enkephalin in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assay as well as in mu- and delta-receptor selective binding assays. The obtained results indicate that a) the enkephalin binding domain of the dynorphin (kappa) receptor has structural requirements which are distinct from those of the enkephalin binding site at the mu-receptor and b) the introduction of an identical conformational constraint in [Leu5]enkephalin and in the enkephalin segment of dynorphin-(1-13) produces a superpotent agonist in both cases. Fluorescence energy transfer measurements with the active [4-tryptophan]analogs of dynorphin-(1-13) and [Leu5]enkephalin and with dynorphin-(1-17) demonstrated a more extended conformation of the N-terminal tetrapeptide segment in [Trp4]dynorphin-(1-13) than in [Trp4, Leu5]enkephalin as well as the absence of an interaction between the N- and C-terminal segments of dynorphin-(1-17).     

Impact of cis-proline analogs on peptide conformation

The beta-turn is a common motif in both proteins and peptides and often a recognition site in protein interactions. A beta-turn of four sequential residues reverses the direction of the peptide chain and is classified by the phi and psi backbone torsional angles of residues i + 1 and i + 2. The type VI turn usually contains a proline with a cis-amide bond at residue i + 2. Cis-proline analogs that constrain the peptide to adopt a type VI turn led to peptidomimetics with enhanced activity or metabolic stability. To compare the impact of different analogs on amide cis-trans isomerism and peptide conformation, the conformational preference for the cis-amide bond and the type VI turn was investigated at the MP2/6-31+G** level of theory in water (polarizable continuum water model). Analogs stabilize the cis-amide conformations through different mechanisms: (1) 5-alkylproline, with bulky hydrocarbon substituent on the C(delta) of proline, increases the cis-amide population through steric hindrance between the alkyl substituent and the N-terminal residues; (2) oxaproline or thioproline, the oxazolidine- or thiazolidine-derived proline analog, favors interactions between the dipole of the heterocyclic ring and the preceding carbonyl oxygen; and (3) azaproline, containing a nitrogen atom in place of the C(alpha) of proline, prefers the cis-amide bond by lone-pair repulsion between the alpha-nitrogen and the preceding carbonyl oxygen. Preference for the cis conformation was augmented by combining different modifications within a single proline. Azaproline and its derivatives are most effective in stabilizing cis-amide bonds without introducing additional steric bulk to compromise receptor interactions.     

Conformational studies of irreversible HIV-1 protease inhibitors containing cis-epoxide as an amide isostere.

We have carried out NMR and molecular modeling studies of peptidomimetic HIV-1 protease inhibitors, LB71116: Qc-Asn-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isopropyl)2 where Qc stands for quinaldic acid and LB71148: Qc-(SMe)Pen(O)2-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isoprop yl)2 where (SMe)Pen(O)2 stands for S-methyl-S-dioxo-penicillamine. Through conformational calculations and NMR data analysis, we have obtained preferred conformations of the two inhibitors in solution. To our knowledge, this work is one of the first extensive conformational studies of peptidomimetics containing cis-epoxide amide isostere. The resulting preferred conformations contain extended structures. In these conformations, the psi of Phe(cep) is maintained about 130 degrees and the phi angle of (cep)Gly prefers +/- 150 degrees [where Phe(cep) and (cep)Gly are the residues generated by the replacement of the Phe-Gly peptide bond with cis-epoxide]. Two conformations were commonly observed in the preferred conformations of each inhibitor. Through restrained molecular dynamics simulating the hydrogen bond formation between our inhibitor and a water molecule ('flap water'), one of the conformations is assumed as the conformation which can bind to the enzyme without large conformational changes. Recently, we had the opportunity to compare the selected preferred conformation with the binding conformation of LB71116 observed from the X-ray studies of the complex between LB71116 and HIV-1 protease. These two conformations are surprisingly similar to each other. Thus, we can explain high activity and selectivity of our inhibitors to the HIV-1 protease by the similarity between the preferred conformations in solution and the binding conformation.     

Key analogs of the tetrapeptide subunit of RA-VII and deoxybouvardin

The synthesis and evaluation of two key analogs 3 and 4 of the potent antitumor antibiotics deoxybouvardin (1) and RA-VII (2) which contain fundamental modifications in the tetrapeptide subunit are described. Unlike the natural products, these agents 3 and 4, which substitute (Gly)₄ and (Gly)₃ for the -Ala-Ala-NMe-Tyr(OMe)-Ala tetrapeptide subunit, adopt conformations in which the central amide in the cycloisodityrosine subunit adopts its inherently preferred trans stereochemistry and both were found to be biologically inactive.     

Influence of conformational flexibility on biological activity in cyclic astin analogues

The astins, a family of natural antitumor cyclopeptides, from the roots of Aster tataricus, consist of a 16-membered ring system containing uncoded amino acid residues. The backbone conformation, with a cis-3,4-dichlorinated proline residue, plays an important role in antineoplastic activity. The acyclic astins, on the other hand, do not show antitumor activity, suggesting that the cyclic nature of astins may be a key role in their biological properties. Although the antineoplastic activity of natural astins has been screened in vitro and in vivo, the mechanism of action has never been investigated. With the aim at elucidating the influence of conformational flexibility on biological activity, we have designed and synthesized several astin analogues containing either Aib and the nonproteinogenic Abu and (S)beta3-hPhe residues, able to modify the peptide backbone structure, or the peptide bond surrogate -SO2-NH-. Tested for their antitumor effect, our astin-related cyclopeptides are able to inhibit the growth of tumor cell lines, while the acyclic astins are inefficacious. The present work reports on the structure-activity study of a selected synthetic cyclotetrapeptide corresponding to the sequence c[Thr-Aib-(S)beta3-hPhePsi(CH2-SO2-NH)-Abu], synthesized by classical methods and characterized conformationally by two-dimensional NMR and molecular dynamics analyses.