β‐turn formation by a six‐residue linear peptide in solution

Abstract: A model peptide AAGDYY‐NH₂ (B1), which is found to adopt a β‐turn conformation in the TEM‐1 β‐lactamase inhibitor protein (BLIP) in the TEM‐1/BLIP co‐crystal, was synthesized to elucidate the mechanism of its β‐turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT‐IR and ¹H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT‐IR spectroscopy confirmed the formation of a β‐turn in solution by the model peptide. The dihedral angles [(φ₃, ?₃) (φ₄, ?₄)] of [(?52°, ?32°) (?38°, ?44°)] of Gly‐Asp fragment in the model peptide are consistent with those of a type III β‐turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of β‐turn formation and stability.     

Structure-conformation relationships of synthetic peptide inhibitors of human renin studied by resonance energy transfer and molecular modeling

The structure-conformation relationships of a series of angiotensinogen_6–13 (ANG_6–13, His-Pro-Phe-His-Leu-Val-Ile-His) congeners substituted by Nⁱⁿ-For-Trp (Ftr), d -Ftr or Trp at the N-terminus, Tyr at the C-terminus and PheΨ[CH₂NH]Phe at the P₁-P′₁ cleavage site (i.e. Leu¹⁰-Val¹¹) were studied using resonance energy transfer coupled with molecular modeling of the peptide conformation using macromolecular energy refinement and dynamics simulation. Average end-to-end intramolecular distances (r) of the peptides in solution were determined by fluorescence spectroscopy. For example, Ac-Ftr-Pro-Phe-His-PheΨ[CH₂NH]Phe-Val-Tyr-NH₂ (U-70714E) gave an average intramolecular donor (Tyr)-acceptor (Ftr) distance of 16.3 Å in aqueous solution. This experimental value was consistent with a distance of 17.9 Å determined by molecular modeling of U-70714E to a human renin 3-D structure (developed from known homologous aspartyl protease inhibitor X-ray crystallographic data) followed by simulation of the solution phase conformation of the peptide. An extended backbone secondary structure of U-70714E is suggested from these studies and the relationship(s) of structure-conformation to structure-activity was explored by analysis of several congeners of U-70714E, a potent (IC₅₀= 3.0 × 10^?9m ) inhibitor of human renin in vitro.     

Synthesis and solution structure of an S-glycosylated cyclic hexapeptide

Synthesis and conformational analysis of the S-glycosylated cyclic hexapeptide cyclo(-d -Pro¹-Phe²-Cys³(tetra-O-acetyl-β-d -galactopyranosyl)-Trp⁴-Lys(Z)⁵-Phe⁶-) I was carried out to examine the influence of a saccharide residue in position i of a standard β-turn on the formation of reverse turns and on the biological activity. Synthesis was carried out in the liquid phase employing a galactosylated cysteine building block. The cyclization reagents DPPA/NaHCO₃ avoided high dilution conditions. Spectroscopic data were extracted from homo- and heteronuclear 2D-NMR techniques (TOCSY, NOESY, HMQC, HMQC-TOCSY, HMBCS-270). For structural refinement restrained molecular dynamics (MD) simulations in vacuo and with explicit DMSO as solvent were performed. Finally, simulations in DMSO without experimental restraints provided insight in stability and dynamics of the structural model. A comparison of the S-glycosylated Cys³ peptide with the analogous Thr³ peptide exhibits a similar overall conformation of the hexapeptide [βII’d -Pro-Phe and another β-turn about Trp⁴-Lys⁵(Z)]. However, the latter shows a distinct dynamic flip βI, βII in the glycopeptide, whereas the Thr-analogue only populates βI. This influence is attributed to a βI stabilizing effect of a hydrogen bridge of Thr-O, in position i to the NH of the amino acid in position i+ 2, which is lacking in the glycosylated compound.     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues

The incubation of a solution of the human growth hormone releasing factor analog, [Leu²⁷] hGRF(1-32)NH₂ at pH 7.4 and 37°, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [β-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ and [α-Asp⁸, Leu²⁷] hGRF(1-32)NH₂, produced by deamidation of the Asn⁸ residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [β-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ was estimated to be 400-500 times less potent than the parent Asn⁸ peptide, while [2-Asp⁸, Leu²⁷] hGRF(l-32)NH₂ was calculated to be 25 times less potent than the parent Asn⁸ peptide. Three additional analogs of [Leu²⁷] hGRF(1-32)NH₂ containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37°). Based on disappearance kinetics, [Leu²⁷] hGRF(1-32)NH₂ had a half-life of 202 h while the other analogs had the following half-lives: [Leu²⁷, Asn²⁸] hGRF(1-32)NH_: (150h); [Ser⁸, Leu²⁷, Asn²⁸] hGRF(l-32)NH₂ (746h); and [Ser⁸, Leu²⁷] hGRF(1-32)NH₂ (1550 h). After 14 days, incubated samples of the Asn⁸ analogs lost GH releasing potency, while the Ser⁸ analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.     

Influence of serine in position i on conformation and dynamics of reverse turns

NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cycle(-d -Pro¹-Ala²-Ser³(Bzl)-Trp⁴-Orn⁵(Z)-Tyr⁶-) with and without protecting groups on Ser³ and Orn⁵. This peptide sequence was derived from the active loop sequence of the α-amylase inhibitor Tendamistat (HOE 467). The aim was to investigate the role of serine in position i of a standard β-turn on the conformation and stabilization of this turn. Based on distance and torsion constraints from 2D NMR spectroscopic measurements in DMSO-d₆ solution, structure refinement was accomplished by restrained molecular dynamics (MD) simulations in vacuo and in DMSO. The analysis of both structures in solution reveals a considerable effect of the unprotected serine sidechain on the adjacent β-turn conformation. While in the protected peptide with Ser³(Bzl) a βII-turn is observed between Trp⁴ and Orn⁵, the deprotected compound reveals a βI-turn in this region. The βI-turn is stabilized by a backbone-sidechain hydrogen bond from Orn⁵N_αH to Ser³O_γ. Comparisons with other NMR-derived solution structures of cyclic model peptides and in some protein structures from literature reveal a general structural motif in the stabilization of βI-turns by serine in the i position through backbone-sidechain interactions. © Munksgaard 1995.     

Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal d-Trp or d-Tpi

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the d forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin(6-14) and Leu¹³Ψ(CH₂NH)Phe¹⁴ -bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of ¹²⁵I-Tyr⁴ -bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [³H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist d -Tpi⁶,Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin (6-14)(RC-3095) was slightly more potent in these assays than d -Trp⁶,Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin (6-14)(RC-3125). Nevertheless, d -Trp⁶ Leu¹³Ψ (CH₂NH)Phe¹⁴-bombesin (6-14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC₅₀ value less than 1 nm . Our work indicated that the substitution of d -Trp and d -Tpi at position 6 of the pseudononapeptide bombesin analogs (Ψ^13-14), in which the Met¹⁴ residue is replaced by Leu or Phe, results in potent bombesin/GRP antagonists with improved in vivo activity.     

Comparative conformational analysis and in vitro pharmacological evaluation of three cyclic hexapeptide NK-2 antagonists

The conformational analysis of three cyclic hexapeptides is presented. Cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-d -Met¹¹-) (1) and cyclo-(-Gln⁶-Trp⁷-Phe⁸-Gly⁹-Leu¹⁰-Met¹¹-) (2) are NK-2 antagonists in the hamster trachea assay, whereas cyclo-(-Gln⁶-Trp⁷-Phe⁸-(R)-Gly⁹-[ANC-2]Leu¹⁰-Met¹¹-) (3), where Gly⁹[ANC-2]Leu¹⁰ represents (2S)-2-((3R)-3-amino-2-oxo-1-pyrrolidinyl)-4-methylpentanoyl, is inactive as agonist and antagonist in this assay. In DMSO, the NMR results cannot be interpreted as being consistent with a single conformation. However, the combined interpretation of results from NMR spectroscopy, restrained molecular dynamics simulations with application of proton–proton distance information from ROESY spectra, and pharmacological results leads to a reduced number of conformational domains for each peptide, which can be compared with each other and may be classified as responsible for their biological activity. Trying to match the conformational domains approximately with regular β- and γ-turns, we find a γⁿ-turn at the position of the methionine occuring in all peptides. For the active peptides 1 and 2 we arrive at an inverse γⁱ-turn at Phe⁸, and βI′- or βII-turns with Gly⁹ and Leu¹⁰ at the corner positions, these β-turns having a similar topology with respect to the linking peptide unit. Other conformational domains common to only 1 and 2 support their classification as responsible for the biological activity.     

Solution structure of a cathelicidin‐derived antimicrobial peptide,CRAMP as determined by NMR spectroscopy

Abstract: CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin‐derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram‐positive and gram‐negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H₂O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly α‐helical conformation in TFE/H₂O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H₂O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic α‐helices from Leu⁴ to Lys¹⁰ and from Gly¹⁶ to Leu³³. These two helices are connected by a flexible region from Gly¹¹ to Gly¹⁶. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18‐residue fragment with the sequence from Gly¹⁶ to Leu³³ was found to retain antibacterial activity. Therefore, the amphipathic α‐helical region from Gly¹⁶ to Leu³³ of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Conformational study of an Aib‐rich peptide in DMSO by NMR

Abstract: The strong propensity of 2‐amino‐2‐methyl propanoic acid (Aib)‐rich peptides to form stable helical structures is well documented. NMR analysis of the short peptide Z‐(Aib)₅‐L ‐Leu‐(Aib)₂‐OMe indicates the presence of a well‐characterized 3₁₀‐helix even in dimethylsulfoxide (DMSO), a solvent known to disrupt helical structures. The structure remains stable at least up to 348 K. Stereospecific assignment of the diastereotopic methyls of Aib was achieved, with the assumption of a specific helical screw sense. The methyl more eclipsed with respect to the CO vector resonates at a higher field in the carbon dimension. Molecular dynamics simulations successfully predict the ³J_CHNH coupling constant of Leu⁶ and most of the H‐bonding pattern. Discrepancies were found for Aib³ and Aib⁷ amide protons which can be explained by a higher sensitivity of the simulations to the helix fraying at the end of the peptide and by the presence of extended conformations for Leu⁶ during most of the simulations.     

Molecular structures of two crystalline forms of the cyclic heptapeptide antibiotic ternatin,cyclo[-β-OH-d-Leu-d–Ile–(NMe)Ala (NMe)Leu-Leu-(NMe)Ala-d–(NMe)Ala-]

The crystal structures of two solvated forms of ternatin, cyclo[-β-OH-d -Leu-d -Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-d -(NMe)Ala-] are reported. The first crystallizes with two molecules of peptide and one of dioxane in the asymmetric unit: P2₁2₁2₁, a = 11.563(1), b = 21.863(2), c = 36.330(4) Å. The second crystallizes with two molecules of peptide and one of water in the asymmetric unit: P2₁2₁2₁, a = 14.067(2), b = 16.695(1), c = 36.824(6) Å. N-Methylation of four of the seven residues of ternatin appears to reduce the number of low-energy conformations the molecule can assume. The same H-bonded macrocyclic ring conformation is adopted by the backbone of each of the four molecules observed here. All the amino-acid side chains, with the exception of d -Ile², have similar orientations in each of the four conformers. The heptapeptide macrocycle is characterized by: (i) a cis peptide between (NMe)Ala³ and (NMe)Leu⁴, (ii) a type II β-bend, involving residues Leu⁵-(NMe)Ala⁶-d -(NMe)Ala⁷-β-OH-d -Leu², stabilized by two H-bonds, N1′05 and N5′01, between Leu⁵ and β-OH-d -Leu¹ residues, (iii) a third intramolecular H-bond, observed in each of the four molecules, between the hydroxyl group of β-OH-d -Leu¹ and the carbonyl oxygen of d -Ile².     

Effect of secondary structure on the rate of deamidation of several growth hormone releasing factor analogs

The objective of this study was to determine whether the rates of deamidation of Asn⁸ in selected growth hormone releasing factor (GRF) analogs were related to the peptide's secondary structures in solution. Bovine or human [Leu²⁷]GRF(1–32)NH₂ (both having Gly at position 15), [Ala¹⁵ Leu²⁷]bGRF(1–32)NH₂ and [Pro¹⁵ Leu²⁷]bGRF(1–32)NH₂ were used as model peptides. The peptide helical content (assessed by CD) increased with the increasing methanol concentration and was as follows: 7, 12 and 18% in 0% MeOH; 24, 48 and 52% in 40% MeOH; and 41, 77 and 81% in 80% MeOH for Pro¹⁵ Leu²⁷ bGRF(1–32)NH₂, [Leu²⁷]hGRF(1–32)NH₂, and Ala¹⁵ Leu²⁷ bGRF(1–32)NH₂, respectively. 2D NMR studies done in the presence of 40% CD₃OH indicated more helical structure for the Ala¹⁵ analog as compared to [Len²⁷]hGRF(1–32)NH₂. In both these peptides Asn⁸ was included in the helical region. In contrast, the lack of conformational information for the Pro¹⁵ analog indicated little helical structure around Asn⁸. The peptides’ deamidation rates decreased and their half-lives increased with increasing MeOH concentrations. At 40% MeOH, the least helical Pro¹⁵ bGRF analog (t_1/2= 10.78 h) deamidated 1.5 and 2 times faster than its Gly¹⁵ (t_1/2= 15.74 h) and Ala¹⁵ (t_1/2= 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn⁸ in GRF makes this residue less prone to deamidation.     

Importance of the C‐terminal phenylalanine of gastrin for binding to the human CCK2 receptor

Abstract: The importance of the C‐terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK₂ receptor were assessed using analogs of [Leu¹⁵]G(11?17). The following peptides were synthesized, Ac[Leu¹⁵]G(11?17), Ac[Leu¹⁵]G(11?16)NH₂, [Leu¹⁵]G(11?17), [Leu¹⁵,Ala¹⁷]G(11?17), [Leu¹⁵,Abu¹⁷]G(11?17), [Leu¹⁵,Val¹⁷]G(11?17), [Leu¹⁵,Leu¹⁷]G(11?17), [Leu¹⁵,Cha¹⁷]G(11?17), [Leu¹⁵,Trp¹⁷]G(11?17), [Leu¹⁵,Tic¹⁷]G(11?17), [Leu¹⁵, d ‐Phe¹⁷]G(11?17) and [Leu¹⁵,p‐X‐Phe¹⁷]G(11?17), where X = F, Cl, Br, I, OH, CH₃, NH₂ and NO_2. Competition binding experiments with [³H]CCK‐8 were performed using human CCK₂ receptors stably expressed in CHO cells. Phe¹⁷ was shown to be important for binding. A hydrophobic side‐chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side‐chain either in the g(+) or g(–) conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d ‐Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des‐Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK₂ receptor.     

Fluorescence study on the solution conformation of dynorphin in comparison to enkephalin

Conformational parameters of the opioid peptides dynorphin and [Leu⁵] enkephalin in dilute aqueous solution (3 times 10^-5 M) were investigated by performing singlet-singlet energy transfer experiments with dynorphin and with the biologically active 4-tryptophan analogs of dynorphin-(1–13) and [Leu⁵] enkephalin at pH 5.5 and 8.0. Efficiencies of transfer of excitation energy from the phenol ring of tyrosine (donor) to the indole moiety of tryptophan (acceptor) were determined and average intramolecular Tyr-Trp distances were calculated on the basis of the Förster equation. The observed absence of energy transfer between Tyr¹ and Trp¹⁴ of dynorphin indicates that the two fluorophores are at least 20 Å apart and rules out a close proximity between the N- and C-terminal segments of the peptide. Evaluation of energy transfer in [Trp⁴] dynorphin-(1–13) resulted in an average intramolecular Tyr¹-Trp⁴ distance of at least 15 Å whereas the corresponding average distance in [Trp⁴, Leu⁵] enkephalin was found to be much shorter (10 Å). It thus appears that in [Trp⁴] dynorphin-(1–13) the predominant conformation of the N-terminal tetrapeptide segment is almost completely extended, whereas in [Trp⁴, Leu⁵] enkephalin folded conformations of that same segment occur in a major proportion. This drastic conformational difference is of interest with regard to the different preferences of dynorphin and [Leu⁵] enkephalin for the various opiate receptor subclasses.     

Solid-phase synthesis of protected peptides using new cobalt(III) ammine linkers†

Cobalt(III) ammine complexes of the type cis-[CoL₄(4-AMB)O-AA-Boc](CF₃SO₃)₂, where L₄= bisethylenediamine (en)₂ or tetraammine (NH₃)₄, and 4-AMB = 4-(aminomethyl)benzoic acid, have been synthesized and used as linkers to polystyrene resins for solid-phase synthesis of protected peptides. Boc/t-Bu-protected [Leu⁵]enkephalin was assembled on the two different Co(III) resins, and then cleaved from the resins by reduction of the Co(III) center in 93–96%; yield. HPLC-purified protected [Leu⁵]enkephalin was obtained in 67–69% overall yield and characterized by amino acid analysis and ¹H NMR. Stepwise synthesis on the Co(en)₂-resin was also used in the assembly of Boc-Asp(OcHex)-Arg(Mts)-Gly-Asp(OcHex)-Ala-Pro-Lys(2Cl-Z)-Gly-OH, a sequence from collagen α1 Type 1. The protected peptide was cleaved from the Co(III) resin in 74% yield, and the HPLC-purified nonapeptide was characterized by amino acid analysis, ¹H NMR and liquid secondary-ion mass spectrometry (LSIMS). New routes are described for the synthesis of isomerically pure Co(III) anchor complexes. The Co(III) resins were found to be compatible with both the tert-butyloxycarbonyl (Boc) and the 9-fluorenylmethoxycarbonyl (Fmoc) N^α-protecting group strategies used in solid-phase peptide synthesis.     

Immunosuppressive analogues of hexapeptide Tyr-Val-Pro-Leu-Phe-Pro,an immune system stimulant

It was found that substitution of Val² and/or Leu⁴ residue in a hexapeptide Tyr-Val-Pro-Leu-Phe-Pro (I) transforms this peptide immunostimulant into analogues possessing the immunosuppressor activity – Tyr-Gly-Pro-Leu-Phe-Pro (II), Tyr-Val-Pro-Gly-Phe-Pro (III), and Tyr-Gly-Pro-Gly-Phe-Pro (IV). Biological effects of peptides I-IV were studied using PFC (plaque forming cell) test, GvH (graft vs host) reaction in mice, and ARFC (autologous rosette forming cell) test. The strongest immunosuppressor activity was observed for III – in this case the immunosuppressor effect was observed even in PFC test in vitro in which II and IV showed no such activity. These results suggest that simultaneous presence of both Val² and Leu⁴ residues is necessary for the generation of immunostimulation. The CD study of I-IV in methanol solution suggests that the conformational preferences of II-IV change towards stabilization of the β-turn structure, whereas in the case of I the γ-turn on Leu⁴ and cis orientation of the Pro³ carbonyl (distorted β-turn of type I) was found (1) as the preferred conformation. Competition in biological effects observed for I and III in PFC in vitro test suggests that these analogues may interact with the same cellular receptor. Drastic changes in the activity which accompany changes in the sequence are discussed in the terms of our stereochemical hypothesis (1).     

Solution conformation of Leu27 hGRF(1-32)NH2 and its deamidation products by 2D NMR

The solution structure and helical content of a human growth hormone releasing factor analog, Leu²⁷ hGRF(1–32)NH₂ (hGRF), and its deamidation products Asp⁸ Leu²⁷ hGRF(l-32)NH₂ and isoAsp⁸ Leu²⁷ hGRF(1-32)NH₂, were determined by CD and 2D NMR. Chemical-shift assignments of ¹H NMR resonances were made from DQFCOSY, HOHAHA and NOESY spectra, and qualitative secondary structure was determined from NOESY spectra. 2D NMR studies in aqueous MeOH showed the Asn⁸, Asp⁸ and isoAsp⁸ hGRF analogs to have significant α-helical character. However, the β-linked isoAsp⁸ analog did not retain helical structure in the N-terminal region, most likely because of disruption of the hydrogen bonding pattern upon substitution of the extra methylene into the peptide backbone. The helical content, as determined by CD, was ~ 12% in 0% MeOH for all three peptides, and 77, 72 and 69% in 80% MeOH for the Asn⁸, Asp⁸ and isoAsp⁸ hGRF analogs, respectively. However, 2D NMR solution structure data indicated a decrease in helicity in the N-terminal region for the isoAsp⁸ analog when compared with the other two analogs. In the Asn⁸ and Asp⁸ hGRF analogs, the helix began at Asp³ or Ala⁴, while the isoAsp⁸ analog helix was disrupted until Arg. The higher helicity value for the Asn⁸ peptide over the isoAsp⁸ analog may be associated with reported biological activity, where the in vitro activity decreased from 100 to 4 and < 1% for Asn⁸, Asp⁸ and isoAsp⁸ hGRF, respectively.     

Potent pseudopeptide bombesin-like agonists and antagonists

Bombesin-like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13–14 and 8–9, respectively, with ψ[CH₂S] and ψ[CH₂N(CH₃)]. [Phe¹³ψ[CH₂S]Leu¹⁴]bombesin and [Phe⁸ψ[CH₂S]-Leu⁹]litorin bound to the murine pancreatic bombesin gastrin releasing peptide receptor with similar dissociation constants (K_d= 3.9 and 3.4 nM. respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, K_d= 1.6 nM and isomer II, K_d= 0.89nM. Further oxidation to the sulfone decreased potency ([Phe⁸ψ[CH₂SO₂]Leu⁹]litoin, K_d= 9.9nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a ψCH₂N(CH₃)] at the 8–9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (K_d= 0.1 nM) and [Leu⁹]litorin (K_d= 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH-dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pK_a= 6.3 and 8.5). The lower pK_a is found in the agonist analogs but not in the ψ[CH₂S]-containing antagonist.     

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.     

Esterase-Sensitive Cyclic Prodrugs of Peptides: Evaluation of a Phenylpropionic Acid Promoiety in a Model Hexapeptide

Purpose. To evaluate a cyclic phenylpropionic acid prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of a peptide and stabilize it to metabolism. Methods. Conversion to the linear hexapeptide was studied at 37°C in HBSS, pH 7.4, and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model. Results. In aqueous buffered solution, pH 7.4, the cyclic prodrug degraded quantitatively (t _1/2 = 1795 ± 289 min) to the linear hexapeptide and the lactone. Substantially faster degradation of the cyclic prodrug was observed in 90% human plasma (t _1/2 = 508 ± 24 min), and in homogenates of Caco-2 cells (t _1/2 = 940 ± 13 min), the rat intestinal mucosa (t _1/2 = 1286 ± 32 min), and rat liver (t _1/2 = 840 ± 42 min). Pretreatment of these biological media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug was significantly more stable than the hexapeptide and at least 71-fold more able to permeate (P_app = 1.21 ± 0.12 × 10^–7 cm/s) than was the parent peptide (P_app 0.17 × 10^–8 cm/s). In the presence of 0.1 mM palmitoyl-DL-carnitine, the transport rate of the cyclic prodrug (P_app = 2.19 × 10^–6 cm/s) was 1250-fold greater than that of the linear hexapeptide. Conclusions. Preparation of a cyclic peptide using a phenylpropionic acid promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.     

Unusual thionation of a cyclic hexapeptide

One carbonyl oxygen of the cyclic hexapeptide cycle(-Gly¹-Pro²-Phe³-Val⁴-Phe⁵-Phe⁶-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=O of Gly¹ but that of Phe⁵ which was substituted and cyclo(-Gly¹-Pro62-Phe³-Va1⁴-Phe⁵ψ[CS-NH]Phe⁶-) (B)was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe⁵ and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a CIS Gly¹-Pro² bond in a βVIa-turn (minor) and a trans Gly-Pro bond in a βII′-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOES, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a βI- and βII-turn about the Val⁴-Phe⁵ amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R_1p) were measured to verify the assumption of this fast βI/βII equilibrium within each isomer. Significant contributions to R_1p-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations.