Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues

Abstract: A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C‐ and N‐terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N‐carboxy alkylated and N‐amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc‐glycine to the N‐alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre‐built in solution. For backbone cyclization at the C‐terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea‐pig ileum (GPI) assays. Additionally, the potentiation of the BK‐induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [d ‐Phe⁷]‐BK. Side chain as well as backbone cyclization in the N‐terminus of [d ‐Phe⁷]‐BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin‐induced contraction of guinea‐pig ileum.     

Synthesis of different types of dipeptide building units containing N‐ or C‐terminal arginine for the assembly of backbone cyclic peptides*

Abstract: Different types of dipeptide building units containing N‐ or C‐terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg). For cyclization in the N‐terminal sequence N‐carboxyalkyl and N‐aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N‐terminal arginine to the N‐alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z‐Arg(Z)₂‐OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C‐terminal arginine was prepared to synthesize BK‐analogues with backbone cyclization in theC‐terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM^? technology was applied using the Boc‐Arg(Alloc)₂‐OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc‐protected amino aldehyde, derived from Boc‐Phe‐OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid‐phase synthesis in the same manner as amino acid derivatives.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.     

Antinociceptive Synergism of MD‐354 and Clonidine. Part II. The α2‐Adrenoceptor Component

Abstract: Previously, we reported that antinociceptive synergism of a 5‐HT₃/α₂‐adrenoceptor ligand MD‐354 (m‐chlorophenylguanidine) and clonidine combination occurs, in part, through a 5‐HT₃ receptor antagonist mechanism. In the present investigation, a possible role for α₂‐adrenoceptors was examined. Mechanistic studies using yohimbine (a subtype non‐selective α₂‐adrenoceptor antagonist), BRL 44408 (a preferential α_2A‐adrenoceptor antagonist) and imiloxan (a preferential α_2B/C‐adrenoceptor antagonist) on the antinociceptive actions of a MD‐354/clonidine combination were conducted. Subcutaneous pre‐treatment with all three antagonists inhibited the antinociceptive synergism of MD‐354 and clonidine in the mouse tail‐flick assay in a dose‐dependent manner (AD₅₀ = 0.33, 2.1, and 0.17 mg/kg, respectively). Enhancement of clonidine antinociception by MD‐354 did not potentiate clonidine’s locomotor suppressant activity in a mouse locomotor assay. When [ethyl‐³H]RS‐79948‐197 was used as radioligand, MD‐354 displayed almost equal affinity to α_2A‐ and α_2B‐adrenoceptors (K_i = 110 and 220 nM) and showed lower affinity at α_2C‐adrenoceptors (K_i = 4,700 nM). MD‐354 had no subtype‐selectivity for the α₂‐adrenoceptor subtypes as an antagonist in functional [³⁵S]GTPγS binding assays. MD‐354 was a weak partial agonist at α_2A‐adrenoceptors. Overall, in addition to the 5‐HT₃ receptor component, the present investigation found MD‐354 to be a weak partial α_2A‐adrenoceptor agonist that enhances clonidine’s thermal antinociceptive actions through an α₂‐adrenoceptor‐mediated mechanism without augmenting sedation.     

Synthesis and modification of dibenzylglycine derivatives via the Suzuki‐Miyaura cross‐coupling reaction

Abstract: The objective of this paper is to describe in details of various available methods to prepare C^α,α‐dibenzylglycines (Db_zg) and then include our work involving the synthesis of side chain Db_zg derivatives. α,α‐Disubstituted amino acids (α,αAAs) are important members in the family of modified amino acids. Replacement of the α‐hydrogens of glycine 1 by alkyl groups leads to α,αAAs. The steric hindrance of the quaternary centre of Aib 2 combined with the helix‐forming capacity has attracted the attention of structural biologists and protein crystallographers. Db_zg 3 is a special structural variant of Aib. The presence of two benzyl groups at C^α‐position not only impart rigidity to the peptide backbone in which it is incorporated, but also acts as a useful vehicle for studying π–π interactions. Although several C^α,α‐disubstituted glycines such as C^α,α‐diethyl glycine (Deg), C^α,α‐dipropyl glycine (Dpg) etc. have been studied in detail, not much has been known about Db_zg because of limited availability of synthetic procedures. Various Db_zg derivatives 19a–f have been prepared using ethyl isocyanoacetate 14 as a glycine equivalent (eq.). A useful and simple methodology has been developed using the Suzuki‐Miyaura cross‐coupling reaction for the modification of Db_zg derivatives 17d , 19d , 22 . Using this ‘Building Block Approach’ (Accounts of Chemical Research 36 , 2003, 342) one can generate a variety of Db_zg derivatives 20a–f and 23a–e , which may find useful applications in combinatorial synthesis and QSAR studies.     

Predicting the Solubility of the Anti-Cancer Agent Docetaxel in Small Molecule Excipients using Computational Methods

Purpose To develop an in silico model that provides an accurate prediction of the relative solubility of the lipophilic anticancer agent docetaxel in various excipients. Materials and Methods The in silico solubility of docetaxel in the excipients was estimated by means of the solubility (δ) and Flory-Huggins interaction (χ _FH) parameters. The δ values of docetaxel and excipients were calculated using semi-empirical methods and molecular dynamics (MD) simulations. Cerius² software and COMPASS force-field were employed for the MD simulations. The χ _FH values for the binary mixtures of docetaxel and excipient were also estimated by MD simulations. Results The values obtained from the MD simulations for the solubility of docetaxel in the various excipients were in good agreement with the experimentally determined values. The simulated values for solubility of docetaxel in tributyrin, tricaproin and vitamin E were within 2 to 6% of the experimental values. MD simulations predicted docetaxel to be insoluble in β-caryophyllene and this result correlated well with experimental studies. Conclusions The MD model proved to be a reliable tool for selecting suitable excipients for the solubilization of docetaxel. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Synthesis,biological activity,and solution structures of a cyclic dodecapeptide from the EGF‐2 domain of blood coagulation factor VII

Abstract: The cyclic dodecapeptide, disulfide‐cyclo‐[H‐Cys‐Val‐Asn‐Glu‐Asn‐Gly‐Gly‐Cys(Acm)‐Glu‐Gln‐Tyr‐Cys‐OH], which corresponds to the 91–102 sequence of the second epidermal growth factor domain of human blood coagulation factor VII, was synthesized using solid‐phase procedures. It was shown to be an inhibitor at the key step in the induction of coagulation by the extrinsic pathway, i.e. the factor VII/tissue factor‐catalyzed activation of coagulation factor X. The solution structure of this peptide was investigated by NMR spectroscopy and was computer‐modeled via molecular mechanics. Structures were calculated based on 112 distance and nine dihedral angle constraints. The resulting backbone structures were classified into two structural subsets: one which exhibited a twisted ‘8’‐shaped folding and another describing an open, circular ‘O’ outline. The local backbone structures of segments Asn³‐Glu⁴‐Asn⁵, Gly⁷‐Cys⁸ and Gln¹⁰‐Tyr¹¹ were well preserved among the two subsets. Apart from the unrestrained N‐ and C‐termini, Gly⁶ and Glu⁹ sites exhibited marked local disorder between the two subsets, suggesting localized flexible hinges likely to govern tertiary structure interconversion between the two subsets. Two transient hydrogen bonds were identified from pH chemical shift titrations by matching the pK_a values of NH and carboxylate groups, which supported the occurrence of the ‘8’ structure, and agreed with temperature coefficients of peptidyl NH resonances. Structure?function relationships of the peptide were discussed in terms of the likely physiological function of the disulfide‐bonded loop in factor VII which the peptide represents.     

Conformational comparison of µ‐selective endomorphin‐2 with its C‐terminal free acid in DMSO solution,by 1H NMR spectroscopy and molecular modeling calculation

Abstract: In order to make clear the structural role of the C‐terminal amide group of endomorphin‐2 (EM2, H‐Tyr‐Pro‐Phe‐Phe‐NH₂), an endogenous µ‐receptor ligand, in the biological function, the solution conformations of endomorphin‐2 and its C‐terminal free acid (EM2OH, H‐Tyr‐Pro‐Phe‐Phe‐OH), studied using two‐dimensional ¹H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr‐Pro ω bond in a population ratio of ≈ 1 : 2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton?proton distance constraints derived from the ROE cross‐peaks. These energy‐minimized conformers, which were all in the φ torsion angles estimated from J_NHCαH coupling constants within ± 30°, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro²–Phe³ moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the ‘bow’‐shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C‐ and N‐terminal groups. These results indicate clearly that the substitution of carboxyl group for C‐terminal amide group makes the peptide flexible. The conformational requirement for µ‐receptor activation has been discussed based on the active form proposed for endomorphin‐1 and by comparing conformational features of EM2 and EM2OH.     

Fluorine‐18 labelling of PNAs functionalized at their pseudo‐peptidic backbone for imaging studies with PET

Peptide nucleic acids (PNAs) form a unique class of synthetic macromolecules, originally designed as ligands for the recognition of double‐stranded DNA, where the deoxyribose phosphate backbone of original DNA is replaced by a pseudo‐peptide N‐(2‐aminoethyl)glycyl backbone, while retaining the nucleobases of DNA. We have previously developed an original method to label oligonucleotide‐based macromolecules with the short‐lived positron‐emitter fluorine‐18 (t_1/2: 109.8 min) using the N‐(4‐[¹⁸F]fluorobenzyl)‐2‐bromoacetamide reagent. Using this method, we herein report the fluorine‐18‐labelling of 13 decameric PNAs ( OLP_1‐13 ), of the same sequence (CTCATACTCT), but presenting selected modification of the pseudo‐peptidic backbone at two or three of the thymine residues (positions 2, 5 and 8). Structural characteristics of these backbone modifications include either an amino acid side chain (L ‐Lys, L ‐Glu, L ‐Leu and L ‐Arg) or a glycosyl moiety (mannose, galactose, fucose, N‐Ac‐galactosamine and N‐Ac‐glucosamine) attached via an appropriate spacer. N‐(4‐[¹⁸F]fluorobenzyl)‐2‐bromoacetamide was synthesized in three radiochemical steps from 4‐cyano‐N,N,N‐trimethylanilinium trifluoromethanesulfonate and HPLC‐purified in 85–90 min (typical production: 3.7–4.8 GBq starting from a batch of 29.6–31.4 GBq of [¹⁸F]fluoride). Conjugation of the fluorine‐18‐labelled bromoacetamide reagent with the PNAs was performed in a mixture of acetonitrile and HEPES buffer (0.1 M, pH 7.9) for 10 min at 60°C and gave the corresponding pure labelled conjugated PNAs ([¹⁸F] c‐OLP_1‐13 ) after RP‐HPLC purification. The whole synthetic procedure, including the preparation of the fluorine‐18‐labelled reagent, provides up to 0.9 GBq (25 mCi) of HPLC‐purified [¹⁸F] c‐OLP_1‐13 in 160 min with a specific radioactivity of 45–65 GBq/µmol (1.2–1.7 Ci/µmol) at the end of synthesis starting from 29.6 to 31.4 GBq (800–850 mCi) of [¹⁸F]fluoride. Copyright © 2004 John Wiley & Sons, Ltd.     

Characterization of a novel binding site for 125I-Tyr-D-Arg-[Hyp3,D-Phe7,Leu8]bradykinin on epithelial membranes of guinea pig ileum

We have recently shown that (a) [^125]I-Tyr8]bradykinin (BK) recognized bradykinin binding sites in guinea pig epithelium membranes with a K_d value of 1.6 nM and a B_max of 156 fmol/mg protein, and (b) B₂ agonists and some B₂ antagonists, such as D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK, inhibited this specific binding with a K_i value of 32 nM. In the present study, we have radioiodinated the B₂ antagonists Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK and have performed a full characterization of the binding properties of this tracer in the same membrane preparation. Equilibrium experiments performed in the absence or presence of an excess of BK (10⁻⁵ M) showed that ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK specifically labelled two different sites. One of these is the same as the site labelled by [¹²⁵I-Tyr⁸]BK, and this indicates that ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK interacts specifically with kinin B₂ receptors. Equilibrium experiment performed in the presence of an excess of BK (10⁻⁵ M) indicated that specific binding of ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK to the second site is also saturable and Scatchard analysis showed that the site is of high affinity with a K_d of 16.8 nM and a B_max of 2.08 pmol/mg protein. Surprisingly, unlabelled B₂ agonists such as bradykinin, [Tyr⁸]BK,[Leu⁸]BK,[Hyp³,Tyr⁸(OMe)]BK,D-Arg-[Hyp³]BK and kallidin were found to be inactive on this second site. A series of B₂ receptor antagonists, Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK, D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK, D-Arg-[Hyp³,Leu^5.8,D-Phe⁷]BK, D-Arg-[Hyp³,Gly⁶,D-Phe⁷,Leu⁸]BK and D-Arg-[Hyp³,Thi^5.8,D-Phe⁷]BK inhibited ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu Leu⁸]BK binding with K_i values of 25.0, 20.9, 15.8, 64.6 and 6606.9 nM respectively. On the other hand, [Thi^5.8,D-Phe⁷]BK did not interfere with ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK but was found to be a potent inhibitor of [¹²⁵I-Tyr⁸]BK binding (K_i = 53.7 nM). As expected, B₁ receptor agonists, antagonists and peptides non-related to BK such as substance P, neurokinin A, neurokinin B, angiotensin II, bombesin, vasopressin and the calcitonin gene related peptide were unable to compete with ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK. The results show that ¹²⁵I-Tyr-D-Arg-[Hyp³,D-Phe⁷,Leu⁸]BK is interacting with two distinct binding sites in the guinea pig epithelium: one is the well known bradykinin B₂ receptor and the other is a new, non-characterized binding site that interacts exclusively with some bradykinin receptor antagonists.     

Synthesis of novel protected Nα(ω‐thioalkyl) amino acid building units and their incorporation in backbone cyclic disulfide and thioetheric bridged peptides

Abstract: General methods for the preparation of protected N^α(ω‐thioalkyl) amino acids building units for backbone cyclization using reductive alkylation and on‐resin preparation are described. The synthesis of non‐Gly Fmoc‐protected S‐functionalized N‐alkylated amino acids is based on the reaction of readily prepared protected ω‐thio aldehyde with the appropriate amino acid. Preparation of Fmoc‐protected S‐functionalized N‐alkylated Gly building units was carried out using two methods: reaction of glyoxylic acid with Acm‐thioalkylamine and an on‐resin reaction of bromoacetyl resin with Trt‐thioalkylamines. Three model peptides were prepared using these building units. The GlyS2 building unit was incorporated into a backbone cyclic analog of somatostatin that contains a disulfide bridge. Formation of the disulfide bridge was performed by on‐resin oxidation using I₂ or Tl(CF₃COO^–)₃. Both methods resulted in the desired product in a high degree of purity in the crude. The AspS3 building unit was also successfully incorporated into a model peptide. In addition, the in situ generation of sulfur containing Gly building units was demonstrated on a Substance P backbone cyclic analog containing a thioether bridge.     

Influence of hydrophobic interactions on the conformational adaptability of the β‐Ala residue

Abstract: The chemical synthesis and X‐ray crystal structure analysis of a model peptide incorporating a conformationally flexible β‐Ala residue: Boc‐β‐Ala‐Pda, 1 (C₂₃H₄₆N₂O₃: molecular weight = 398.62) have been described. The peptide crystallized in the crystal system triclinic with space group P2₁: a = 5.116(3) Å, b = 5.6770(10) Å, c = 21.744(5) Å; α = 87.45°, β = 86.87°, γ = 90.0°; Z = 1. An attractive feature of the crystal molecular structure of 1 is the induction of a reasonably extended backbone conformation of the β‐Ala moiety, i.e. the torsion angles φ ≈ ?115°, µ ≈ 173° and ψ ≈ 122°, correspond to skew^?, trans and skew⁺ conformation, respectively, by an unbranched hydrophobic alkyl chain, Pda, which prefers an all‐anti orientation (θ₁ ≈ ?153°, θ₂ ≈ … θ₁₄ ≈ ±178°). The observation is remarkable because, systematic conformational investigations of short linear β‐Ala peptides of the type Boc‐β‐Ala‐Xaa‐OCH₃ (Xaa = Aib or Acc⁶) have shown that the chemical and stereochemical characters of the neighboring moieties may be critical in dictating the overall folded and/or unfolded conformational features of the β‐Ala residue. The overall conformation of 1 is typical of a ‘bar’. It appears convincing that, in addition to a number of hydrophobic contacts between the parallel arranged molecules, an array of conventional N‐H…O=C intermolecular H‐bonding interactions stabilize the crystal molecular structure. Moreover, the resulting 14‐membered pseudo‐ring motif, generated by the amide–amide interactions between the adjacent molecules, is completely devoid of nonconventional C?H…O interaction. The potentials of the conformational adaptation of the β‐Ala residue, to influence and stabilize different structural characteristics have been highlighted.     

Kinin B1 and B2 receptors in pig vessels: characterization of two monoreceptor systems

The coronary artery and renal vein of the adult pig are sensitive and reliable monoreceptor systems for studying kinin receptors. The pig coronary artery with intact endothelium is highly sensitive to bradykinin (BK, pEC₅₀ 8.6), while being insensitive to the B₁ receptor agonist, LysdesArg⁹BK. The tissue responds to BK with concentration-dependent relaxation, which is prevented by B₂ receptor antagonists, particularly dArg[Hyp³, Thi⁵, dTic⁷, Oic⁸]BK (HOE 140, pK_B 9.3), (E)-3-(6-acetoamido-3-pyridyl)-N-(N-{2, 4-dichloro-3-[(2-methyl-8-quinolinyl)oxy-methyl]phenyl}-N-methylaminocarbonylmethyl)acrylamide (FR 173657), a new non peptide compound (pK _B 9.3), while B₁ receptor antagonists (e.g. Lys[Leu⁸]desArg⁹BK) are inactive. The order of potency of kinin-related peptides in this vessel is: LysBK ≥ BK > [Hyp³]BK>[Aib⁷]BK, a sequence typical of a B₂ receptor system. Antagonists such as HOE 140 and FR 173657, at high concentrations reduce the maximum effect of BK and thus behave as noncompetitive antagonists. The kinin B₁ receptor was studied in the pig renal vein without endothelium and incubated for several hours in order to allow for the de novo formation of this functional site. After 7–8 h in vitro incubation, the vessel shows high sensitivity to LysdesArg⁹BK (pEC₅₀ 8.3) and is insensitive to BK. The pig renal vein responds to B₁ receptor agonists with concentration-dependent contraction which maintains a stable plateau and is prevented by selective B₁ receptor antagonists such as Lys[Leu⁸]desArg⁹BK (pK_B 6.7). The most active antagonist has been found to be desArg⁹HOE 140 (pA₂ 7.6) which acts as competitive antagonist in this preparation. Some B₂ antagonists (e.g. HOE 140) show weak (pK _B 6.1) anti-B₁ receptor activity while the non-peptide compound FR 173657 is inactive on the B₁ receptor and therefore acts as a potent and selective kinin B₂ receptor antagonist in the pig. The data obtained in this study allow us to compare the porcine B₂ and B₁ receptors with those of other species including man, and underline some interesting features that are unique to the porcine functional sites. Received: 9 April / Accepted: 20 June 1997     

Conformational analysis of endomorphin‐1 by molecular dynamics methods

Abstract: Endomorphin‐1 (EM1, H‐Tyr‐Pro‐Trp‐Phe‐NH₂) is a highly potent and selective agonist for the μ‐opioid receptor. A conformational analysis of this tetrapeptide was carried out by simulated annealing and molecular dynamics methods. EM1 was modeled in the neutral (NH₂‐) and cationic (NH‐) forms of the N‐terminal amino group. The results of NMR measurements were utilized to perform simulations with restrained cis and trans Tyr¹‐Pro² peptide bonds. Preferred conformational regions in the Φ₂–Ψ₂, Φ₃–Ψ₃ and Φ₄–Ψ₄ Ramachandran plots were identified. The g(+), g(?) and trans rotamer populations of the side‐chains of the Tyr¹, Trp³ and Phe⁴ residues were determined in χ₁ space. The distances between the N‐terminal N atom and the other backbone N and O atoms, and the distances between the centers of the aromatic side‐chain rings and the Pro² ring were measured. The preferred secondary structures were determined as different types of β‐turns and γ‐turns. In the conformers of trans‐EM1, an inverse γ‐turn can be formed in the N‐terminal region, but in the conformers of cis‐EM1 the N‐terminal inverse γ‐turn is absent. Regular and inverse γ‐turns were observed in the C‐terminal region in both isomers. These β‐ and γ‐turns were stabilized by intramolecular H‐bonds and bifurcated H‐bonds.     

Biological and conformational study of β‐substituted prolines in MT‐II template: steric effects leading to human MC5 receptor selectivity*

Abstract: To investigate the molecular basis for the interaction of the χ‐constrained conformation of melanotropin peptide with the human melanocortin receptors, a series of β‐substituted proline analogs were synthesized and incorporated into the Ac‐Nle‐c[Asp‐His‐d ‐Phe‐Arg‐Trp‐Lys]‐NH₂ (MT‐II) template at the His⁶ and d ‐Phe⁷ positions. It was found that the binding affinities generally diminished as the steric bulk of the p‐substituents of the 3‐phenylproline residues increased. From (2S, 3R)‐3‐phenyl‐Pro⁶ to (2S, 3R)‐3‐(p‐methoxyphenyl)‐Pro⁶ analogs the binding affinity decreased 23‐fold at the human melanocortin‐3 receptor (hMC3R), 17‐fold at the hMC4R, and eight‐fold at the hMC5R, but selectivity for the hMC5R increased. In addition, the substitution of the d ‐Phe⁷ residue with a (2R, 3S)‐3‐phenyl‐Pro resulted in greatly reduced binding affinity (10³–10⁵) at these melanocortin receptors. Macromodel's Large Scale Low Mode (LLMOD) with OPLS‐AA force field simulations revealed that both MT‐II and SHU‐9119 share a similar backbone conformation and topography with the exception of the orientation of the side chains of d ‐Phe⁷/d ‐Nal (2′)⁷ in χ space. Introduction of the dihedrally constrained phenylproline analogs into the His⁶ position (analogs 2 – 6 ) caused topographical changes that might be responsible for the lower binding affinities. Our findings indicate that hMC3 and hMC4 receptors are more sensitive to steric effects and conformational constraints than the hMC5 receptor. This is the first example for melanocortin receptor selectivity where the propensity of steric interactions in χ space of β‐modified Pro⁶ analogs of MT‐II has been shown to play a critical role for binding as well as bioefficacy of melanotropins at hMC3 and hMC4 receptors, but not at the hMC5 receptor.     

Three‐dimensional structure of the α‐MSH‐derived candidacidal peptide [Ac‐CKPV]2

Abstract: Previous research has shown that the immunomodulatory peptide α‐melanocyte‐stimulating hormone (α‐MSH) and its carboxy‐terminal tripeptide KPV (Lys‐Pro‐Val α‐MSH_11?13) have antimicrobial influences. By inserting a Cys‐Cys linker between two units of KPV, we designed the dimer [Ac‐CKPV]₂ that showed excellent candidacidal effects in pilot tests and was the subject of further investigations. [Ac‐CKPV]₂ was active against azole‐resistant Candida spp. Therefore, the molecule appeared a promising candidate for therapy of fungal infections and was the subject of a structural study. ¹H‐NMR and restrained mechanic and dynamic calculations suggest that the peptide adopts an extended backbone structure with a β‐turn‐like structure. These results open a pathway to development of additional novel compounds that have candidacidal effects potentially useful against clinical infections.     

NMR solution structure of a potent cyclic nonapeptide inhibitor of ICAM‐1‐mediated leukocyte adhesion produced by homologous amino acid substitution

Abstract: We have previously described a disulfide‐linked cyclic nonapeptide (inhibitory peptide‐01, IP01), with the sequence CLLRMRSIC, which binds to intercellular adhesion molecule‐1 (ICAM‐1), and blocks binding to its counter‐structure, the integrin α_Lβ₂ (leukocyte functional antigen‐1, LFA‐1) (Sillerud et al., J. Peptide Res. 62, 2003: 97). We now report the optimization of this peptide by means of single homologous amino acid substitutions to yield a new peptide (IP02‐K6; CLLRMKSAC) which shows an approximately sixfold improvement in inhibitory activity of multivalent leukocyte binding (inhibition constant for 50% inhibition, IC₅₀ = 90 μm ) compared with IP01 (IC₅₀ = 580 μm ). This improvement in activity gives IP02‐K6 potent in vivo activity in a murine model of ischemia reperfusion injury (Merchant et al., Am. J. Physiol. Heart Circ. 284, 2003: H1260). In order to determine the structural features relevant to ICAM‐1‐binding, we have determined the structure of IP02‐K6 using proton nuclear magnetic resonance (NMR) spectroscopy and restrained molecular modeling. In our previously reported study of solution models of IP01, we observed three interconverting conformations during low‐temperature molecular dynamics simulation. In the present study, we find a single conformation of IP02‐K6 similar to one of the previously found conformations of IP01 (family C). In particular, an R4‐S7 β‐turn is present in similar proportions in both conformation C of IP01 and in IP02‐K6; this motif is important in binding to ICAM‐1 because this turn enables the IP02‐K6 backbone to drape over proline‐36 on ICAM‐1. The NMR‐derived solution model of IP02‐K6 was found to dock at the α_Lβ₂‐binding site on ICAM‐1 with no changes in peptide backbone conformation. This docking model displaced five of the 15 α_Lβ₂ residues at the ICAM‐1‐binding site and provided a rationale for understanding the quantitative relationship between IP02‐K6 structure and biologic activity.