Cyclopeptides from Sagina japonica (Caryophyllaceae)

Two new cyclic peptides, named sajaponicin C (1) and sajaponicin D (2), were isolated from the whole plants of Sagina japonica (Caryophyllaceae). Their structures were determined as cyclo(Pro(2)-Leu(2)-Tyr-Leu(1)-Phe(1)-Pro(3)-Phe(2)-Pro(1)) (1) and cyclo(Pro(1)-Pro(2)-Pro(3)-Pro(4)-Phe(1)-Gly-Thr-Ser-Phe(2)-Ile-Tyr) (2) on the basis of spectroscopic data, especially by two-dimensional (2D) NMR techniques.     

Unusual thionation of a cyclic hexapeptide. Conformational changes and dynamics.

One carbonyl oxygen of the cyclic hexapeptide cyclo(-Gly1-Pro2-Phe3-Val4-Phe5-Phe6-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=] of Gly1 but that of Phe5 which was substituted and cyclo(-Gly1-Pro2-Phe3-Val4-Phe5 psi [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 beta VIa-turn (minor) and a trans Gly-Pro bond in a beta II'-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 beta I- and beta II-turn about the Val4-Phe5 amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R1 rho) were measured to verify the assumption of this fast beta I/beta II equilibrium within each isomer. Significant contributions to R1 rho-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.     

金铁锁根中的环肽成分

从云南民间重要的药用植物金铁锁（Ｐｓａｍｍｏｓｉｌｅｎｅ ｔｕｎｉｃｏｉｄｅｓ Ｗ．Ｃ．Ｗｕ ｅｔ Ｃ．Ｙ．Ｗｕ）的根中分离得到２个新的天然环二肽以及２个新的环八肽：金铁锁环肽Ａ和Ｂ（ｐｓａｍｍｏｓｉｌｅｎｉｎｓ Ａ ａｎｄ Ｂ）。它们的结构经光谱方法鉴定为ｃｙｃｌｏ（－Ａｌａ－Ａｌａ－）,ｃｙｃｌｏ（－Ｖａｌ－Ａｌａ－）,ｃｙｃｌｏ（－Ｐｒｏ１－Ｐｈｅ１－Ｐｒｏ２－Ｐｈｅ２－Ｐｈｅ３－Ａｌａ－ｐ     

Peptide conformation. 48. Conformation and biological activity of proline containing cyclic retro-analogues of somatostatin

Six cyclic retro-analogues of the peptide hormone somatostatin have been synthesized using the solid phase technique. The peptides cyclo(-Xaa1-Phe2-Thr3-Lys4-Ybb5-Phe6-) and cyclo(-Phe1-Xaa2-Thr3-Lys4-Ybb5-Phe6-) with Xaa = D- or L-Pro and Ybb = D- or L-Trp were cyclized via the azide method. The conformations of the cyclic hexapeptides in DMSO-d6 solution were determined by a number of homo- and heteronuclear two-dimensional n.m.r.-techniques including 2D rotating frame NOE-spectroscopy. Two-step coherence transfers, ROE and chemical exchange, are observed for the first time in ROESY spectra. The backbone conformation of the all-trans cyclopeptides consists of a beta-turn containing the Pro residue in the position i + 1. These retro-analogues of somatostatin exhibit a high activity in the inhibition of cholate and phalloidin uptake by liver cells (cytoprotective effect); however, the hormonal activities of the natural hormone are completely suppressed. The constitutional and conformational requirements for the cytoprotective activity are discussed.     

On the transferability of atomic solvation parameters: Ab initio structural prediction of cyclic heptapeptides in DMSO

A statistical mechanics methodology for predicting the solution structures and populations of peptides developed recently is based on a novel method for optimizing implicit solvation models, which was applied initially to a cyclic hexapeptide in DMSO (C. Baysal and H. Meirovitch, Journal of American Chemical Society, 1998, vol. 120, pp. 800-812). Thus, the molecule has been described by the simplified energy function E(tot) = E(GRO) + summation operator(k) sigma(k)A(k), where E(GRO) is the GROMOS force-field energy, sigma(k) and A(k) are the atomic solvation parameter (ASP) and the solvent accessible surface area of atom k, respectively. In a more recent study, these ASPs have been found to be transferable to the cyclic pentapeptide cyclo(D-Pro(1)-Ala(2)-Ala(3)-Ala(4)-Ala(5)) in DMSO (C. Baysal and H. Meirovitch, Biopolymers, 2000, vol. 53, pp. 423-433). In the present paper, our methodology is applied to the cyclic heptapeptides axinastatin 2 [cyclo(Asn(1)-Pro(2)-Phe(3)-Val(4)-Leu(5)-Pro(6)-Val(7))] and axinastatin 3 [cyclo(Asn(1)-Pro(2)-Phe(3)-Ile(4)-Leu(5)-Pro(6)-Val(7))], in DMSO, which were studied by nmr by Mechnich et al. (Helvetica Chimica Acta, 1997, vol. 80, pp. 1338-1354). The calculations for axinastatin 2 show that special ASPs should be optimized for the partially charged side-chain atoms of Asn while the rest of the atoms take their values derived in our previous work; this suggests that similar optimization might be needed for other side chains as well. The solution structures of these peptides are obtained ab initio (i.e., without using experimental restraints) by an extensive conformational search based on E(GRO) alone and E(*)(tot), which consists of the new set of ASPs. For E(*)(tot), the theoretical values of proton-proton distances, (3)J coupling constants, and other properties are found to agree very well with the nmr results, and they are always better than those based on E(GRO).     

Structure investigation of amphiphilic cyclopeptides in isotropic and anisotropic environments-A model study simulating peptide-membrane interactions.

It has been proposed that the membrane allows a much more efficient binding of certain small or medium-sized amphiphilic messenger molecules to their receptor, not only by accumulation of the drug, but also by induction of orientations and conformations that are much more favorable for receptor docking than structures adopted in isotropic phases. A series of eight amphiphilic cyclic peptides containing lipophilic (L-alpha-aminodecanoic acid = Ada, L-alpha-aminohexadecanoic acid = Ahd, Nhdg = N-hexadecylglycine) and hydrophilic (Lys, Asp) amino acids were synthesized and examined by means of NMR spectroscopy and molecular dynamics (MD) simulations in isotropic (CDCl3) and membrane-mimicking anisotropic (SDS/H2O) solvents to study the influence of the environment on their individual conformations. NMR data of cyclo(-Gly1-D-Asp2-Ahd3-Ahd4-Asp5-Gly6+ ++-) (C4), cyclo(-Lys1-D-Pro2-Lys3-Ada4-Pro5-Ada6-) (C5) and cyclo(-Lys1-Pro2-Lys3-Ada4-D-Pro5-Ada6-) (C6) clearly indicate that those compounds are too rigid to perform a conformational change upon transition from an isotropic to an anisotropic environment. On the other hand, the experimental data of cyclo (-Gly1-Asp2-Ahd3-Ahd4-Asp5-Gly6-) (C1), cyclo(-Asp1-Ala2-Nhdg3-Ala4-D-Asp5-) (C7), and cyclo(-D-Asp1-Ala2-Nhdg3-Ala4-Asp5-) (C8) suggest highly flexible unstructured molecules in both environments. However, for cyclo(-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C2) we observed a structure inducing effect of a membrane-like environment. The compound populates three different conformations in SDS/H2O, whereas in CDCI3 no preferred conformation can be detected. cyclo(-D-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C3) clearly exhibits two different conformations with a shifted beta,beta-turn motif in CDCI3 and SDS/H2O solutions. The conformational change could be reproduced in a restraint-free MD simulation using the biphasic membrane mimetic CCl4/H2O. Our results give clear evidence that membrane interactions may not only lead to structure inductions, but can also induce major conformational changes in compounds already exhibiting a defined structure in isotropic solution.     

The importance of the N-terminal beta-turn in bradykinin antagonists

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described. 2. Bk is rapidly inactivated by neuronal perikarya (4.2 +/- 0.6 fmol/min/cell body). 3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages. 4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15. 5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond. 6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases. 7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.     

Strategies for Positioning Fluorescent Probes and Crosslinkers on Formyl Peptide Ligands

Abstract

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1–3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.     

Binding and internalization of an ICAM-1 peptide by the surface receptors of T cells.

The objective of this work was to evaluate the binding characteristics of a cyclic peptide, cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH (cIBR), to Molt-3 T cells. This cIBR peptide is derived from sequence numbers 11-20 of intercellular adhesion molecule-1 (ICAM-1). Binding studies were performed using a fluorescence-labeled peptide (FITC-cIBR) in which the fluorescence marker fluorescein 5-isothiocyanate (FITC) was conjugated to the N-terminal of the cIBR peptide. The binding affinity of the FITC-cIBR peptide to Molt-3 T cells was evaluated using a FACScan flow cytometer. The binding specificity of the FITC-cIBR peptide was also confirmed by inhibition of binding using unlabeled peptide (cIBR). The results show that FITC-cIBR binds to two populations of T cells with different affinities; population 1 has high cell numbers (75%) but low affinity, and population 2 has high binding affinity but low cell numbers (25%). Binding to both populations was saturable and could be inhibited by the unlabeled peptide (cIBR), suggesting a receptor-mediated binding process. In addition to binding, receptor-mediated internalization was also observed for population 2; this was confirmed by confocal microscopy and temperature-dependence studies at 37 degrees C and 4 degrees C. The binding and internalization of this peptide may be carried out by surface receptors on Molt-3 T cells such as LFA-1. In the future, the binding and internalization of cIBR peptide can be utilized as a method of targeted drug delivery to leukocytes for the treatment of leukocyte-related diseases.     

Structural characterization of lipopeptide agonists for the bradykinin B2 receptor

The conformational features of Pam-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PKD) and Pam-Gly(-1)-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PGKD), the Pam-Lys and Pam-Gly-Lys analogues of bradykinin, have been determined by high-resolution NMR in a zwitterionic lipoid environment. Radical-induced relaxation of the (1)H NMR signals was used to probe the topological orientation of the peptides with respect to the zwitterionic lipid interface. The radical-induced relaxation and molecular dynamics (MD) data indicated that the palmitic acid and N-terminal amino acid residues embed into the micelles, while the rest of the polypeptide chain is closely associated with the water-micelle interface. Throughout the entire nuclear Overhauser effect restrained MD simulation, a nonideal type I beta-turn was observed in the C-terminus of PKD between residues 6 and 9, and a gamma-turn was observed in the C-terminus of PGKD between residues 6 and 7. Therefore, the additional glycine has a dramatic effect on the structural preferences of the biologically important C-terminus, an effect brought about by the interaction with the lipid environment. These structural features are correlated to the biological activity at the bradykinin B2 receptor.     

Tetrazole analogues of cyclolinopeptide A: synthesis, conformation, and biology

Linear and cyclic analogues of cyclolinopeptide A (CLA) with two dipeptide segments (Val(5)-Pro(6) and Pro(6)-Pro(7)) replaced by their tetrazole derivatives were synthesized by the SPPS technique and cyclized using TBTU (O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate) reagent. The conformational properties of the c(Leu(1)-Ile(2)-Ile(3)-Leu(4)-Val(5)-Pro(6)-psi[CN(4)]-Ala(7)-Phe(8)-Phe(9)) were investigated by NMR and computational techniques. The overall solution structure of this cyclic peptide resembles that observed for the CLA in the solid state. These studies of cyclic tetrazole CLA analogue confirm that the 1,5-disubstituted tetrazole ring functions as an effective, well-tolerated cis-amide bond mimic in solution. The peptides were examined for their immunosuppressive activity in the humoral response test. For cyclic analogues the immunosuppressive activity, at low doses, is equal in magnitude to the activity presented by cyclosporin A and native CLA. The conformational and biological data seem indicate that the Pro-Pro-Phe-Phe moiety and the preservation of the CLA backbone conformation are important for immunosuppressive activity.     

Structural and ICAM-1-docking properties of a cyclic peptide from the I-domain of LFA-1: an inhibitor of ICAM-1/LFA- 1-mediated T-cell adhesion

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)-Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7- Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 alpha-subunit. We found that cyclic peptide 1 can bind to the D1-domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1-domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable betaII -turn at Asp4- Gly5-Glu6-Ala7 and a betaI-turn at Pen1-Ile2-Thr3-Asp4; a less stable betaV-turn is found at the C-terminal region. The beta-turn at Asp4- Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1-domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

Isolation and characterization of ornitho-kininogen

Ornitho-kininogen was purified from chicken blood plasma by a two-stage method using chromatography on columns of S-alkylated papain-Cellulofine and DEAE-5PW. The yield was 1.7 mg from 44 ml plasma. The isolated preparation gave a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) with or without 2-mercaptoethanol and on disc/polyacrylamide gel electrophoresis. The relative molecular mass, Mr, of ornitho-kininogen was estimated as 74,000 on SDS-PAGE using the Ferguson plot method. Ornitho-kininogen was found to have the similar properties to those of mammalian high-Mr kininogen, in terms of the amino acid composition, molecular mass, and susceptibility to plasma kallikrein. No kininogen corresponding to mammalian low-Mr kininogen and rat T-kininogen could be detected in chicken plasma. In fact, ornitho-kininogen was degraded rapidly by bovine plasma kallikrein, liberating a kinin. This kinin was isolated from the digest by reversed-phase HPLC. The primary structure of the isolated kinin was determined as Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-Pro7-Leu8-Arg9. The sequence of this peptide, named ornitho-kinin, was similar to that of bradykinin except for the substitution of Thr6 and Leu8 for Ser6 and Phe8. The isolated ornitho-kinin induced a contraction of chicken smooth muscle and had a strong hypotensive effect in the chicken. However, it did not contract the isolated rat uterus. It is suggested that this specificity difference is due to the replacement of Phe8 by Leu8. The sequence of residues 1-30 of ornitho-kininogen exhibited 43% identity with that of bovine kininogen.     

Structural and ICAM-1-Docking Properties of a Cyclic Peptide from the I-domain of LFA-1: An inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion

Abstract

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)- Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7-Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 α-subunit. We found that cyclic peptide 1 can bind to the D1- domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1- domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable βII'-turn at Asp4-Gly5-Glu6-Ala7 and a βI-turn at Pen1-Ile2-Thr3-Asp4; a less stable βV-turn is found at the C-terminal region. The β-turn at Asp4-Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1- domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

Nonpeptide antagonists for kinin receptors

Kinins are a family of small peptides acting as mediators of inflammation and pain in the peripheral and central nervous system. The two main 'kinins' in mammals are the nonapeptide bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and the decapeptide kallidin (KD, [Lys0]-BK, Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8-Phe9- Arg10). Their biological actions are mediated by two distinct receptors, termed B1 and B2. Kinin B and B2 receptor antagonists may be useful drugs endowed with analgesic and anti-inflammatory properties, with potential use in asthma, allergic rhinitis and other diseases. The first nonpeptide kinin B2 receptor antagonist, WIN 64338, was reported in 1993. Despite its low selectivity, the compound provided a reference for pharmacological and modeling studies. Several quinoline and imidazo[1,2-a]pyridine derivatives have been shown by Fujisawa to possess high affinity and selectivity for kinin B2 receptors. Among them, FR 173657 displayed excellent in vitro and in vivo antagonistic activity, while FR 190997 emerged as the first nonpeptide agonist for B2 receptor. Two structurally related Fournier compounds were recently published. Other kinin B2 receptor ligands were obtained by rational design, through library screening or from natural sources. The only example of a nonpeptide kinin B1 receptor ligand has been reported in a patent by Sanofi.     

Solution conformations of two flexible cyclic pentapeptides: cyclo(Gly-Pro-D-Phe-Gly-Ala) and cyclo(Gly-Pro-D-Phe-Gly-Val).

In an effort to explore the residue preferences in three-residue reverse turns (so-called gamma-turns), two cyclic pentapeptides--cyclo(Gly1-Pro2-D-Phe3-Gly4-Ala5) (I) and cyclo(Gly1-Pro2-D-Phe3-Gly4-Val5) (II)--have been synthesized and analyzed by nmr. It was anticipated that the Gly-Pro-D-Phe-Gly portions of these molecules would favor a beta-turn conformation, leaving the remainder of the molecule to adopt a gamma turn, as seen in several previously studied model cyclic pentapeptides. The nmr data for both peptides in CDCl3 (5% DMSO-d6) and in neat DMSO-d6 indicate that the most populated conformation contains a distorted beta turn around Pro2-D-Phe3, which includes a gamma turn around D-Phe3. The distortion in the beta turn does not impede the formation of an inverse gamma turn around residue 5, and indeed, this conformation is observed in both peptides. Both the alanine and the bulkier valine residues are therefore found to be compatible with an inverse gamma turn. Molecular dynamics simulations on the title peptides are reported in the following paper. These simulations indicate that there is conformational flexibility around the D-Phe3-Gly4 peptide bond, which enables the formation of the gamma turn around D-Phe3. The third paper in this series explores the impact of a micellar environment on conformational equilibria in II.     

Ortho-aminobenzoic acid-labeled bradykinins in interaction with lipid vesicles: fluorescence study

The peptide hormone bradykinin (BK) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) and its shorter homolog BK(1-5) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)) were labeled with the extrinsic fluorescent probe ortho-aminobenzoic acid (Abz) bound to the N-terminal and amidated in the C-terminal carboxyl group (Abz-BK-NH(2) and Abz-BK(1-5)-NH(2)). The fragment des-Arg(9)-BK was synthesized with the Abz fluorescent probe attached to the 3-amino group of 2,3-amino propionic acid (DAP), which positioned the Abz group at the C-terminal side of BK sequence, constituting the peptide des-Arg(9)-BK-DAP(Abz)-NH(2). The spectral characteristics of the probe were similar in the three peptides, and their fluorescent properties were monitored to study the interaction of the peptides with anionic vesicles of dimyristoylphosphatidylglycerol (DMPG). Time-resolved fluorescence experiments showed that the fluorescence decay of the peptides was best described by double-exponential kinetics, with mean lifetimes values around 8.0 ns in buffer pH 7.4 that increased about 10% in the presence of DMPG vesicles. About a 10-fold increase, compared with the values in aqueous solution, was observed in the steady-state anisotropy in the presence of vesicles. A similar increase was also observed for the rotational correlation times obtained from time-resolved anisotropy decay profiles, and related to the overall tumbling of the peptides. Equilibrium binding constants for the peptide-lipid interaction were examined monitoring anisotropy values in titration experiments and the electrostatic effects were evaluated through Gouy-Chapman potential calculations. Without corrections for electrostatic effects, the labeled fragment Abz-BK(1-5)-NH(2) presented the major affinity for DMPG vesicles. Corrections for the changes in peptide concentration due to electrostatic interactions suggested higher affinity of the BK fragments to the hydrophobic phase of the bilayer.     

Impact of a micellar environment on the conformations of two cyclic pentapeptides.

In an effort to explore the influence of interfacial environments on reverse turns, we have performed a detailed analysis by nmr of the solution conformations of two cyclic pentapeptides in sodium dodecyl sulfate (SDS) micelles. The first peptide, cyclo (D-Phe1-Pro2-Gly3-D-Ala4-Pro5), adopts a single rigid conformation in solution (either chloroform or dimethylsulfoxide) and in crystals, whereas the second, cyclo (Gly1-Pro2-D-Phe3-Gly4-Val5), is much more flexible and adopts different conformations in the crystal and in solution. Both of these peptides are solubilized by SDS micelles, and nmr relaxation rates indicate that they are both partially immobilized by interaction with the micelles. Furthermore, some amide protons in both peptides participate in hydrogen bonds with water. In the presence of micelles, the former peptide retains a conformation essentially the same as that found in crystals and in solution, which consists of a beta turn and an inverse gamma turn. However, the micellar environment has a significant effect on the latter peptide. In particular, the population of a conformer containing a cis Gly-Pro peptide bond is increased significantly. The most likely conformation of the cis isomer, determined by a combination of nmr and restrained molecular dynamics, contains a Gly1-Pro2 delta turn and a gamma turn about D-Phe3. The nmr data on the trans isomer indicate that this isomer is averaging between two conformations that differ mainly in the orientation of the D-Phe3-Gly4 peptide bond.