Effect of sulfate position on rnyotropic activity of the gastrin/CCK-like insect leucosulfakinins

Leucosulfakinin and leucosulfakinin-II are sulfated insect neuropeptides with homology to human gastrin II and cholecystokinin. Biological evaluation of the analog [Ser(SO₃H)², Tyr⁵]leucosulfakinin-II, two cholecystokinin analogs containing key leucosulfakinin amino acid replacements, and a Tyr(SO₃H) position-analog series has demonstrated that while the presence of a sulfate group is critical to leucosulfakinin myotropic activity, its position is not. Most strikingly, the analog [Tyr (SO₃H)⁵,Phe⁶,Nle⁹]leucosulfakinin, in which the sulfate moiety is shifted one position towards the N-terminus relative to the parent peptide, retains a very significant 38% of the parent's threshold activity. A shift in the location of the sulfate group by two to five positions toward the N-terminus led to reduction of potency to a significant but low plateau of activity, whereas a shift by more than one position towards the C-terminus led to a complete loss of activity. The introduction of a single residue (Arg) in the 7 position of CCK-8 transforms this inactive mammalian hormone into a myotropically-active leucosulfakinin analog on the isolated cockroach hindgut.     

Stabilization of a tyrosine O‐sulfate residue by a cationic functional group: formation of a conjugate acid–base pair

Abstract: Sulfated tyrosine [Tyr(SO₃H)]‐containing peptides showed characteristic peak patterns in their liquid secondary‐ion mass spectrometry (LSIMS) spectra. Protonated molecules were desulfated more easily than their deprotonated counterparts. Therefore, the stabilities of the Tyr(SO₃H) residues were well‐reflected by peak patterns in their positive‐ion spectra. These intrinsic peak patterns were investigated by comparing the behavior of each Tyr(SO₃H) residue in acidic solution. As the peptide chain was lengthened and the number of cationic functional groups increased, the peak representing the [MH]⁺ of a Tyr(SO₃H)‐containing peptide became more prominent than that representing the desulfated [MH–SO₃]⁺. These alterations in peptide structure also increased the stability of the Tyr(SO₃H) residue in acidic solution. Based on the desulfation mechanism of an aryl monosulfate, we predicted that intramolecular cationic functional groups would stabilize Tyr(SO₃H) residues by forming conjugate acid–base pairs (or salt bridges) both in the gaseous phase and in acidic solution. In accordance with this theory, Arg residues would take primary responsibility for this self‐stabilization within Tyr(SO₃H)‐containing peptides. Moreover, a long peptide backbone was expected to have a weak protective effect against desulfation of the [MH]⁺ in the gaseous phase. Tyr(SO₃H) residues were also stabilized by adding an external basic peptide containing multiple Arg residues. Formation of such intermolecular acid–base pairs was demonstrated by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) which detected conjugated peptide ions. The energetically favorable formation of conjugate acid–base pairs prompted by Tyr(SO₃H) residues might be a driving force for protein folding and protein–protein interaction.     

Solid-phase synthesis of cionin,a protochordate-derived octapeptide related to the gastrin/cholecystokinin family of peptides,and its mono-tyrosine-sulfate-containing derivatives

Cionin, a protochordate-derived octapeptide amide related to the gastrin/cholecystokinin family of peptides, contains two consecutive tyrosine sulfate residues. In order to gain insight into the role of the respective tyrosine sulfate residue in biological activity, cionin and its derivatives in which one of the two tyrosine sulfate residues was replaced by tyrosine, were prepared by two Fmoc-based solid-phase approaches. In approach ( 1 ) Fmoc-Tyr(SO₃Na)-OH was employed as a building block to assemble the Tyr(SO₃Na)-containing peptide-resin, and a global deprotection cleavage was conducted with 90% aqueous TFA in the presence of m-cresol and 2-methylindole at 4°C. In approach ( 2 ) the Tyr(Msib) [Msib =p-(methylsulfinyl)benzyl] derivative was used for the peptide-chain assembly to achieve sulfation on the selective Tyr residue. Partially protected peptide with the Msib Msz protecting groups [Msz =p-(methylsulfinyl)benzyloxycarbonyl], obtained after peptide-resin cleavage, was treated with DMF-SO₃ complex in the presence of ethanedithiol to achieve the sulfation of free Tyr residue and the reduction of the Msib/Msz groups to TFA-labile Mtb/Mtz groups [Mtb =p-(methyithio)benzyl, Mtz =p-(methylthio)benzyloxycarbonyl]. Final deprotection of the Mtb/ Mtz groups with 90° aqueous TFA in the presence of m-cresol and 2-methylindole gave the desired cionin derivative, which contains the tyrosine sulfate residue at the selective position. Yields obtained with approach ( 2 ) were considerably higher than those obtained with approach ( 1 ). Cionin and mono-Tyr(SO³H)-containing derivatives were assayed on exocrine pancreas in dogs.     

Structural and Functional Characterization of Hirudin P6 Derived Novel Bivalent Thrombin Inhibitors – Studying the Effect of Linker Length and Glycosylation on Their Function

HirudinP6 is a glycosylated and sulfated high affinity thrombin inhibitory protein isolated from Hirudineria manillensis. In this study, designing of novel bivalent thrombin inhibitory peptides based on this hirudin isoform is described. The structural and functional impact of varying linker length and glycosylation on their inhibitory potencies and binding kinetics were assessed. The bivalent peptides were obtained by tethering an active site blocking fPRP motif with the carboxy terminal 22 residue segment of hirudin P6 (HP6_42–63) by varying number of glycine residues in the linker region. Among them, analog BiG₁‐HP6 inhibited thrombin with a K_i of 5.12 nm which was comparable to that of glycosylated (disaccharide bearing) and non‐sulfated full length hirudin P6 protein (K_i = 6.38 nm ). Binding kinetics studies revealed increasing linker length can decrease the association rates of peptide─thrombin interactions. Similarly, glycosylation was found to negatively modulate the inhibitory potencies of these peptides by decreasing their rates of association with thrombin. Molecular docking studies revealed that increasing linker length can compromise the electrostatic interactions with the prime subsite residues of thrombin and provided structural explanation for the observed effect of linker length on association rates. These findings thus enhance our understanding of thrombin─(glyco)peptide interactions and provide key insights into the designing of efficient thrombin inhibitors and allosteric modulators of therapeutic potential.     

The receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related δ-opioid antagonists contains all trans peptide bonds

Two different models for the receptor-bound conformation of δ-opioid peptide antagonists containing the N-terminal dipeptide segment H-Tyr-Tic (Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) have been proposed. Both models are based on spatial overlap of the Tyr and Tic² aromatic rings and N-terminal amino group with the corresponding aromatic rings and nitrogen atom of the nonpeptide δ-antagonist naltrindole. However, in one model the peptide bond between the Tyr and Tic² residues assumes the trans conformation, whereas in the other it is in the cis conformation. To distinguish between these two models, we prepared the two peptides H-Tyrψ[CH₂NH]. Tic-Phe-Phe-OH and H-Tyrψ[CH₂NH]. MeTic-Phe-Phe-OH (MeTic = 3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in which a cis peptide bond between the Tyr and Tic (or MeTic) residues is sterically forbidden. Both compounds turned out to be moderately potent δ-opioid antagonists in the mouse vas deferens assay. A molecular mechanics study performed with both peptides resulted in low-energy conformations in which the torsional angle (“ω₁”) of the reduced peptide bond between Tyr and Tic (or MeTic) had a value of 180°(trans conformation) and which were in good agreement with the proposed model with all trans peptide bonds. Furthermore, this study confirmed that neither of these two peptides could assume low-energy conformations in which “ω₁” had a value of 0°(cis conformation). Conformers with that same bond in the gauche- conformation (“ω₁”= -60“) were also identified, but were higher in energy and showed no spatial overlap with naltrindole. On the basis of these results it is concluded that the receptor-bound conformation of δ-peptide antagonists containing an N-terminal H-Tyr-Tic-dipeptide segment must have all trans peptide bonds. © Munksgaard 1998.     

Solid-phase synthesis of peptides with C-terminal asparagine or glutamine

Attempts to anchor Fmoc-asparagine or glutamine as p-alkoxybenzyl esters for solid-phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C-terminal asparagine or glutamine involves quantitative attachment of N^x -Fmoc-C^x -tert.-butyl aspartate or glutamate via the free ω-carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF₃COOH—CH₂Cl₂, 90 min at 25°, releases the desired peptides in >95% yields without side reactions at the C-terminus. Feasibility of the approach has been demonstrated by the syntheses of the C-terminal octapeptide from human proinsulin, H-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gin-OH, and the serum thymic factor pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH.     

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8

Substitution of the -OSO₃H group in the sulfated-tyrosine by the non-hydrolyzable -CH₂SO₃H group was the first described modification of the sulfate ester that does not affect CCK₈ activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH₂SO₃Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH₂SO₃Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK₈ by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK₈ analogues containing the l - or the d -Phe(p-CH₂SO₃Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH₂SO₃Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure l isomer was used. Both CCK₈ analogues displayed high affinities for peripheral and central receptors (KI ～ 1 nm ) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The ?stabilized-CCK₈ peptide”, easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH₂SO₃Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.     

Positional effects of sulfation in hirudin and hirudin PA related anticoagulant peptides

C-Terminal fragment analogues of the leech protein hirudin or the related protein hirudin PA block thrombin's cleavage of fibrinogen. Three series of synthetic peptides were synthesized to study the effects of sulfation in hirudin-derived peptides. Potency of hirudin analogues increased with p-(amino)Phe63, p-(aminosulfonate)Phe63, and p-(sulfate)Tyr63 substitution in place of Tyr63. Sulfation of Tyr56, which in hirudin is normally Phe, resulted in a loss of 1 order of magnitude in potency. The sulfation of Tyr64 of the hirudin PA related analogue resulted in increased potency as for the hirudin analogue. However, in this series the p-(amino)Phe64 and p-(amino-sulfonate)Phe64 did not have increased potency. In addition to these positional effects, replacing all the Glu residues with (O-sulfato)Ser yielded an analogue with full antithrombin potency.     

Histidine-rich human salivary peptides are inhibitors of proprotein convertases furin and PC7 but act as substrates for PC1

A 32 amino acid peptide called histatin-3 (H3; 22% His) and its TV-terminal 24 amino acid fragment histatin-5 (H5, 33% His), are found in human saliva and possess powerful antimicrobial properties. These His-rich peptides have been synthesized by Fmoc-based solid-phase chemistry. Their sequences are: DSHAKRHHGYKRKFHEKHHSHRGYRSNYLYDN (H3) and DSHAKRHHGYKRKFHEKHHSHRGY (H5). In addition, we also prepared two H5 and one H3 mutants. The H5 mutants were: DH5 (all amino acids in D configuration) and H5F (where all His are replaced by Phe at positions 3, 7, 8, 15, 18, 19, 21). The 9–24 segment of H3 with all the His at positions 15, 18, 19, 21 replaced by Tyr was also prepared (Δ_1–8H3Y). The behavior of these five peptides was examined with three proprotein convertases (PC's) which possess cleavage specificity directed towards single and pairs of basic residues. These were: human (h)PCl, an endocrine and neural convertase, hfurin and rat (r)PC7, two widely expressed enzymes. All are serine endoproteases belonging to the kexin/subtilisin family. Our in vitro study revealed that H3 behaves as a substrate for PCI, being cleaved by this endoprotease primarily at a site carboxy terminal to the single Arg²⁵ residue (HRGYR↓SN). On prolonged incubation some minor cleavage was also observed C-terminal to the first Lys Arg⁶ pairs of basic amino acids namely at: HAKR↓HH, which contains a P4 as well as P'l and P'2 His residues. The second potential site YKRK¹²-FH which does not have a P4 basic residues is not cleaved, even upon incubation with excess protease. PCI only poorly cleaves H5 at the same site mentioned above for H3, i.e., at HAKR↓HH. As expected, neither the D-amino acid analogue (DH5), nor the Phe and Tyr mutant analogues of the long and short histatins, respectively, are cleaved at all. In contrast to the above findings for hPCl, the convertase hfurin did not cleave any of the five synthetic peptides. Instead, H3 and H5 were found to be moderately potent inhibitors of the furin-mediated cleavage of the pentapeptide pGlu-Arg-Thr-Lys-Arg-MCA fluorogenic substrate. This inhibition was reversible and competitive, with an estimated inhibition constant K_i of 1.98 μM for H3 and 2.98 μM for H5. The other analogs exhibited only a moderate to weak inhibition of furin, suggesting that substitution of all His by aromatic residues (Phe or Tyr) drastically reduces their inhibitory potency. When tested against rPC7, H3 exhibited almost identical inhibition profile with a measured K_i of 2.4 μM. The partial sequence identity of H3 to the inhibitory pro-peptide of furin and PC7 provides a rationale for our observation. © Munksgaard 1997.     

Acyl,pseudotetra-, tri- and dipeptide active-core analogs of insect neuropeptides

Pseudopeptides of the achetakinin insect neuropeptide family were synthesized by replacing the amino acid blocks Phe-, Phe-Tyr-, and Phe-Tyr-Pro- of the active-core pentapeptide Phe-Tyr-Pro-Trp-GIy-NH₂ with hydrocinnamic acid, 6-phenylhexanoic acid, and both 9-phenylnonanoic and 6-phenylhexanoic acid, respectively. All four of these analogs retained myotropic activity, demonstrating that the active core could be reduced from a pentapeptide to a modified dipeptide. Most notable of these was the pseudotetrapeptide hydrocinnamyl-Tyr-Pro-Trp-Gly-NH₂, which retained 70% of the potency and over 85% of the maximal activity of the parent pentapeptide. The N-terminal amino group, the phenol ring of the Tyr residue, the sulfate moiety and the Gly residue of the insect sulfakinin active core Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂ were all replaced by dodecanedioic acid. The resulting pseudotetrapeptide, dodecandioyl-His-Nle-Arg-Phe-NH₂, elicited myostimulatory activity. Conversely, the related acyl pseudopentapeptide azelayl-Gly-His-Nle-Arg-Phe-NH₂ proved myoinhibitory. A possible explanation for these disparate biological responses is discussed. These acyl pseudopeptides are important advances towards the eventual development of stable, potent mimetic agonists and antagonists of insect neuropeptides.     

Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method

A segment condensation method was developed for the chemical synthesis of large (>90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the non-phosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO₃(Bzl)₂)-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifu-gation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the α-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53. © Munksgaard 1996.     

Replacement of Tyr-SO3H by a p-carboxymethyl-phenylalanine in a CCK8-derivative preserves its high affinity for CCK-B receptor

The sulfated tyrosine present in the sequence of CCK₈ Asp²⁶-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-PheNH₂, seems to play a critical role in the recognition of CCK-A binding sites. In this work, we have investigated whether the presence of an anionic charge on the tyrosine moiety is strictly necessary and whether the sulfate moiety interacts with a divalent cation in the receptor subsite. For this purpose, the novel amino acids (l,d ) Phe(p-CH₂CO₂H) and (l,d ) Phe(p-CH₂CONHOH), as well as their l -resolved forms were introduced into the sequence of Ac[X²⁷, Nle²⁸, Nle³¹]-CCK_27-33 by solid phase method. The biological activities of these new derivatives were compared to two almost equiactive analogues of CCK₈, Ac[Phe(p-CH2SO₃H)²⁷, Nle²⁸, Nle³¹]-CCK_27-33 and Boc[Nle²⁸, Nle³¹]-CCK₂₇-3₃ (BDNL) and to the nonsulfated analogue of the latter peptide (BDNL NS). All these new CCK-related analogues behave as agonists in stimulating pancreatic amylase release and display high affinity for brain binding sites (K₁～ 3-11 nm ) but the only peptides which retain affinity for CCK-A receptors (K₁～ 20 nm ) are those containing a p-caiboxymethyl phenylalanine. Thus, introduction of this amino acid under an esterified form on the side chain, into specific and potent CCK-B agonists could allow compounds endowed with good bioavailabilities to be obtained.     

Synthesis,metabolic stability and chemotactic activity of peptide T and its analogues

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD₄ present on T₄ helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure-activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3-8)-OH and T(4–8)-OH displayed potent bioactivity (maximal chemotactic activity in the range 10^-11-10^-10M). The C-terminal heptapeptide showed a reduction of potency, while further truncations at N-terminus of T(4–8)-OH abolished the biological action. In the octapeptide series, whereas the α-amino butyric acid (Abu) substitution for Thr⁴ was well tolerated, the same “slight” structural change at Thr⁵ or Thr⁸ was very detrimental. Finally, [d -Asn⁶]T(1-8)-OH analogue has low chemotactic activity. All these results indicate that i) the C-terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr⁴ does not interfere with biological characteristics of peptides. With regard to the enzymic degradation, the in vitro experiments showed the susceptibility of peptide T to rapid metabolism by human or rat plasma (T1/2 = 5.2 min), rat brain (T1/2 = 2.1 min) and kidney (T1/2 = 0.4 min) enzymes. The main metabolic product appears to be the C-terminal heptapeptide, which retains, in turn, a low enzymatic stability.     

Single diastereomeric desaminotyrosylalanyl tetra- and heptapeptides with opioid antagonistic activity

The N-terminal dipeptide Tyr-d -Ala of a p-selective agonist, dermorphin tetrapeptide (DT, H-Tyr-d -Ala-Phe-Gly-NH₂) and δ-selective agonist deltorphin C (DEL-C, H-Tyr-d -Ala-Phe-Asp-Val-Val-Gly-NH₂) was changed into an aminodiacyl moiety. The relevant synthetic step is a nucleophilic substitution of bromine from a chiral 2-bromopropanamide by the amino group of tyrosine, with overall retention of configuration. The resulting pseudo tetra- and heptapeptides I-VI were characterized for μ and δ-opioid receptor binding properties using [³H]DAGO and [³H]DPDPE, respectively, and in a bioassay using guinea pig ileum (GPI) and mouse vas deferens (MVD). As a result of chemical alteration of N-terminal dipeptide moiety, all synthesized analogs showed considerable reduction in opioid receptor affinity compared to μ and δ-prototypes (500-fold on the μ-site, analog I , and 125-fold on the δ-site, analog IV ). Interestingly, analogs I and IV showed moderate antagonist activity, respectively, on GPI and MVD, with pA₂ values of 6.05 and 6.82. Analog IV did not exhibit the δ-antagonist potency and δ-selectivity of TIPP peptides. © Munksgaard 1995.     

Conformation of sequential peptides containing proline residues

The conformational properties of Boc-Leu₃-(Pro₂GlyLeu₃)_n-Pro₂Gly-OH(n = 0, 1, 3, and 5) and Boc-Leu₃-(Pro₂GlyLeu₃)_n-Pro₂Gly-OBzl (n = 7, 9, and 11) were investigated in various organic solvents of high polarity using molar rotation and CD measurements. The peptides examined are assembled by the N-terminal, internal, and C-terminal segments having the sequences of Boc-Leu₃, Pro₂Gly-Leu₃, and Pro₂Gly-OX (X = H or Bzl), respectively. The conformational studies using the molar rotation of the oligopeptides and polypeptides in MeOH, DMSO, DMF, DMA, and NMP have made it clear that all the peptides have a randomly coiled structure. CD spectral patterns of the peptides in MeOH were essentially the same as each other and indicative of the randomly coiled structure. The additive nature of the total molar rotation and total molar ellipticity in Boc-Leu₃-(Pro₂GlyLeu₃)_n-Pro₂Gly-OX (X = H or Bzl) indicates that the “peptide segment separation” is indeed in operation in organic solvents of high polarity and that the peptide molecule can be considered to be assembled with the small peptide segments having a randomly coiled structure.     

Folded conformations of the δ-selective opioid dermenkephalin with head-to-tail interactions. A simulated annealing study through NMR restraints

Despite similar tripeptide N-termini, dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) and dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH₂), naturally occuring opioid peptides from frog skin, exhibit high affinity but contrasting selectivity for the μ- and δ-opioid receptors, respectively. Structure-activity relationship studies have shown that the N-terminal tripeptide, Tyr-D-Xaa-Phe (where Xaa is either Ala or Met), is necessary for binding with both the μ- and δ-receptors while the nature and/or the conformation of the C-terminus His-Leu-Met-Asp-NH₂ of dermenkephalin are responsible for addressing the peptide to the δ-receptor. In order to examine the conformational characteristics that are related to the selectivity of dermenkephalin towards the δ-receptor, 50 NOE restraints (10 between nonadjacent residues), and 7 dihedral angles, derived from a two-dimensional ¹H-NMR study of dermenkephalin in dimethyl sulfoxide, were used in simulated annealing and energy minimization procedures. Twenty-four resulting conformers (60% of the generated structures) with no severe distance restraint violation were pooled into seven groups and three related families. These 24 conformers show close proximity between the two methionine residues, S-shaped structures, mean planes of N-terminal and C-terminal moieties almost at right angles to each other, a C-terminus region above the plane of the N-terminal region and g^? as preferential orientation in the side chain of the. Aside these similarities, families of conformers differ by the preferential orientation in the side chain of Tyr (t or g^?) and proximity between Tyr and Asp, or Tyr and the C-terminus. In contrast to previous models, practically no β-turn structures exist for dermenkephalin, most of the NH hydrogen bonds participating to γ-turns. The possible relationship between the conformational characteristics of dermenkephalin and the δ-opioid receptor selectivity is discussed. © Munksgaard 1996.     

Analgesic,receptor binding,and peripheral opioid activities of synthetic dermorphin-dynorphin hybrid peptides

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin-dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin^1–5 sequence caused a remarkable increase in analgesic potency, and a 3-6 fold increase in potency of binding against [³H]-dihydromorphine. The potency of receptor binding against [³H]-EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100mM), the effect of enhancing binding against [³H]-EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid μreceptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decrease in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that the N-terminal dermorphin moiety, but not the C-terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.     

Amidation of growth hormone releasing factor (1–29) by serine carboxypeptidase catalysed transpeptidation

The applicability of serine carboxypeptidase catalysed transpeptidation reactions, using amino acid amides as nucleophiles, for C-terminal amidation of peptides has been investigated. With the aim of converting an unamidated precursor into GRF(1–29)-NH₂, an interesting biologically active derivative of growth hormone releasing factor, a number of model reactions were initially investigated. In such a transpeptidation reaction, where the C-terminal amino acid is replaced by the amino acid amide, used as nucleophile, the C-terminal amino acid residue of the substrate can be chosen freely since it functions as leaving group and does not constitute part of the product. Since the C-terminal sequence of GRF(1–29)-NH₂ is -Met-Ser-Arg-NH₂ the model reactions Bz-Met-Ser-X-OH (X = Ala, Leu, Arg) + H-Arg-NH₂→ Bz-Met-Ser-Arg-NH₂+ H-X-OH were first studied. With carboxypeptidase Y and X = Ala or Leu the amidated product could be obtained of 98% and 41%, respectively. With carboxypeptidase W-II and X = Arg a yield of no more than 72 % could be obtained. The choice of Ala as leaving group in combination with carboxypeptidase Y therefore appeared optimal. With the longer peptide Bz-Leu-Gln-Asp-Ile-Met-Ser-Ala-OH the amidated product could be obtained in a yield of 78%, using carboxypeptidase Y, the only other product being Bz-Leu-Gln-Asp-Ile-Met-Ser-OH, formed due to the competing hydrolysis reaction. The full length peptide GRF(1–28)-Ala-OH was synthesized by the continuous flow polyamide solid-phase method. With this peptide the yield of amidation was 87 % and the remaining products were GRF (1–28)-OH and GRF(1–28)-Arg-Arg-NH₂. However, these can be separated from the desired product by ion exchange chromatography. The results demonstrate that it is feasible to use the generally stable and easily accessible serine carboxy-peptidase for C-terminal amidation of peptides, e.g. produced by fermentation of genetically manipulated microorganisms.     

Comparison of conformational properties of linear and cyclic δ selective opioid ligands DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) and DPLPE (Tyr-c[D-Pen-Gly-Phe-Pen]) by 1H n.m.r. spectroscopy

The preferential conformations of the δ selective opioid peptides DPLPE (Tyr-c[D-Pen-Gly-Phe-Pen]) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) were studied by 400 MHz ¹H n.m.r. spectroscopy in DMSO-d₆ solution. In neutral conditions, the weak NH temperature coefficients of the C-terminal residue (Pen⁵ or Thr⁶), associated with interproton NH-NH and α-NH NOE's (ROESY experiments), indicated large analogies between the backbone folding tendency of both the linear and cyclic peptides. Various γ and/or β turns may account for these experimental data. A similar orientation of the N-terminal tyrosine related to the folded backbones is observed for the two agonists, with a probable γ turn around the amino acid in position 2. Finally, a short distance, about 10 Å, between Tyr and Phe side chains and identical structural roles for threonyl and penicillamino residues are proposed for both peptides. These results suggest the occurrence of similar conformers in solution for the constrained peptide DPLPE and the flexible hexapeptide DTLET. Therefore, it may be hypothesized that the enhanced δ selectivity of DPLPE is related to a very large conformational expense of energy needed to interact with the μ opioid receptor, a feature not encountered in the case of DTLET. These findings might allow peptides to be designed retaining a high affinity for δ opioid receptors associated with a very low cross-reactivity with μ binding sites.     

Design of chimeric peptide ligands to galanin receptors and substance P receptors

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (K_D(1)<0.1 nM and K_D(2)∼ 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (K_D∼ 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD ∼ 1 nm ) or its N-terminal fragment galanin-(1-13) (K_D∼ 1,nm ), which constitutes the A′-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (K_D= 0.4μm ), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp²], in the galanin portion of the chimeric peptide or substitution of ([Phe⁷] or [Met¹¹]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors. In line with this suggestion is the finding that the binding of the chimeric ligands to the galanin receptor is strongly influenced by the presence of SP (1 μm ) or spantide (1 μm ). We have performed the synthesis and binding studies with 11 chimeric peptides, all composed of the N-terminal galanin-(1-13) fragment or of its analogs, linked to the C-terminal portion of SP or its peptide antagonist, spantide. Our results, similar to earlier reports on chimeric peptides, suggest that high-affinity ligands to peptide receptors can be produced by linking biologically active N-terminal and C-terminal portions of peptides via linkers, enabling a) independent recognition of the chimeric peptide by the relevant receptors and b) intramolecular interactions between the joined N- and C-terminal peptide fragments. These two phenomena may also explain why some of the chimeric peptides have higher affinity than the endogenous peptide(s) and why galantide, and some of its analogs presented here, behave(s) as a galanin receptor antagonist(s).