New cyclic peptides with osteoblastic proliferative activity from Dianthus superbus

Two new cyclic peptides, dianthins G-H (1 and 2), together with the known dianthin E (3), were isolated from the traditional Chinese medicinal plant Dianthus superbus. The sequences of cyclic peptides 1 and 2 were elucidated as cyclo (-Gly(1)-Pro(2)-Leu(3)-Thr(4)-Leu(5)-Phe(6)-) and cyclo (-Gly(1)-Pro(2)-Val(3)-Thr(4)-Ile(5)-Phe(6)-), on the basis of ESI tandem mass fragmentation analysis, extensive 2D NMR methods and X-ray diffraction. The isolated three compounds all increase proliferation of MC3T3-E1 cells in vitro using MTT method.     

Design of cyclic peptides with agonist activity at melanocortin receptor-4

A series of cyclic pentapeptides, c(His-D-Phe-Arg-Trp-Z) (Z=omega-amino acid), were prepared and biologically evaluated. The effects of increasing alkyl chain length of omega-amino acid on the functional activities and the receptor binding affinities for human melanocortin receptors (hMC-Rs) were studied. Compound 2 was an agonist for hMC-4R with an EC50 value of 15.4 nM, which was 4.7 times more potent than that of alpha-MSH. Compound 2 also showed a 4.3-fold higher hMC-4R selectivity over hMC-1R, thus providing us with information concerning size and chemical structure of the lactam ring for the development of the agonist with hMC-4R selectivity.     

Cyclic analogs of insect oostatic peptides: synthesis, biological activity, and NMR study

Cyclic peptides 2a-2c, derived from the sequence of the C-terminal shortened analogs of the oostatic decapeptide H-Tyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro-Pro-Pro-OH (1a), were synthesized and assayed on their effect in a reproduction of the flesh fly Neobellieria bullata. The cyclization of the N-terminal linear tetra- and pentapeptides 1b and 1c to the cyclotetra- and cyclopentapeptides 2b and 2c decreased the oostatic activity by one order of magnitude. The cyclodecapeptide 2a, which emerged spontaneously during the pentapeptide cyclization, was quite inactive. Comparative 1H and 13C NMR study on a conformation of the cyclopeptides 2a-2c, and their linear precursors 1b and 1c revealed that a space structure of the cyclic analogues 2b and 2c is too restricted to adopt a biological conformation necessary for receptor binding and therefore only minor oostatic activity is observed after their application. The lack of the oostatic activity in the case of the more flexible dimeric analogue 2a is ascribed to the size of its molecule and its overall shape that is not compatible with a receptor binding.     

CD and NMR structural characterization of ceratotoxins,natural peptides with antimicrobial activity

Antibacterial properties of the secretion from the female reproductive accessory glands of medfly Ceratitis capitata are mostly ascribed to the presence of two peptides, ceratotoxin A and B, which exhibit a strong activity against gram-positive and gram-negative bacterial strains, and show sequence and function homology with cecropins, melittin, and magainins. CD experiments performed in different solvents indicate the presence of a significant content of helical structures in organic solvent. Two-dimensional nmr results for ceratotoxin A in methanol show a helical behavior for the 8–25 region of the peptide. A Ramachandran classification of each residue for the structures obtained from distance geometry calculations lead to the definition of four structural families in which the central segment 10–19 is always helical and differences refer to residues 8–9 and 19–23. A sequence analysis of the two ceratotoxins and a systematic search on the protein data bank revealed the occurrence of a KX-hydrophobic-hydrophobic-P motif that seems to be important for helix stabilization. © 1996 John Wiley & Sons, Inc.     

The molecular basis for antimicrobial activity of pore-forming cyclic peptides

The mechanism of action of antimicrobial peptides is, to our knowledge, still poorly understood. To probe the biophysical characteristics that confer activity, we present here a molecular-dynamics and biophysical study of a cyclic antimicrobial peptide and its inactive linear analog. In the simulations, the cyclic peptide caused large perturbations in the bilayer and cooperatively opened a disordered toroidal pore, 1–2 nm in diameter. Electrophysiology measurements confirm discrete poration events of comparable size. We also show that lysine residues aligning parallel to each other in the cyclic but not linear peptide are crucial for function. By employing dual-color fluorescence burst analysis, we show that both peptides are able to fuse/aggregate liposomes but only the cyclic peptide is able to porate them. The results provide detailed insight on the molecular basis of activity of cyclic antimicrobial peptides.     

Cation-binding cyclic peptides with lipophilic tails

We have synthesized and characterized a series of cation-binding cyclic octapeptides which may function as potential ionophoric substances. The materials contain varying degrees of hydrophobic character, which was controlled systematically through the incorporation of N-alkylglycine residues where N-alkyl = methyl, n-hexyl, cyclohexyl, or n-decyl. The peptides reported include cyclo(Phe-Sar-Gly-Sar)₂, cyclo(Glu(OBzl)-Sar-Gly-Sar-Glu(OBzl)-Sar-Gly-(N-decyl)Gly), cyclo(Glu(OBzl)-Sar-Gly-(N-decyl)Gly)₂, cyclo(Glu(OBzl)-Sar-Gly-(N-hexyl)Gly)₂, cyclo(Glu(OBzl)-Sar-Gly-(N-cyclohexyl)Gly)₂, and the corresponding free diacid forms of the Glu-containing compounds. Using ¹³C- and ¹H-nmr spectra, we demonstrated that the mixture of cis/trans peptide bond-isomer conformers, characteristic of the free-peptide benzyl esters in solution, was converted to unique C₂-symmetric, presumably all-trans conformers on complexation with calcium ions. Cation-transport experiments, using the thick-liquid model of transport in a Pressman cell, established that these compounds transport a variety of cations and that one peptide examined in detail, cyclo(Glu(OBzl)-Sar-Gly-(N-decyl)Gly)₂ (selectivity Ca²⁺ > Na⁺ > K⁺ > Mn²⁺ > Cu²⁺ > Mg²⁺ > Co²⁺ > Zn²⁺), transports calcium about an order of magnitude more efficiently than magnesium.     

Synthesis of aldehydic peptides inhibiting renin

Reduction of peptidyl N, O-dimethyl hydroxamates with lithium aluminium hydride in diethyl ether at 0 degree allowed the preparation of peptidyl aldehydes in excellent yield and optical purity. These aldehydic peptides are able to inhibit renin activity. They are the shortest renin inhibitors known to date.     

Diffusion NMR study of complex formation in membrane-associated peptides

Pulsed-field-gradient nuclear magnetic resonance (PFG-NMR) is used to obtain the true hydrodynamic size of complexes of peptides with sodium dodecyl sulfate SDS micelles. The peptide used in this study is a 19-residue antimicrobial peptide, GAD-2. Two smaller dipeptides, alanine–glycine (Ala–Gly) and tyrosine–leucine (Tyr–Leu), are used for comparison. We use PFG-NMR to simultaneously measure diffusion coefficients of both peptide and surfactant. These two inputs, as a function of SDS concentration, are then fit to a simple two species model that neglects hydrodynamic interactions between complexes. From this we obtain the fraction of free SDS, and the hydrodynamic size of complexes in a GAD-2–SDS system as a function of SDS concentration. These results are compared to those for smaller dipeptides and for peptide-free solutions. At low SDS concentrations ([SDS] ≤ 25 mM), the results self-consistently point to a GAD-2–SDS complex of fixed hydrodynamic size R = (5.5 ± 0.3) nm. At intermediate SDS concentrations (25 mM < [SDS] < 60 mM), the apparent size of a GAD-2–SDS complex shows almost a factor of two increase without a significant change in surfactant-to-peptide ratio within a complex, most likely implying an increase in the number of peptides in a complex. For peptide-free solutions, the self-diffusion coefficients of SDS with and without buffer are significantly different at low SDS concentrations but merge above [SDS] = 60 mM. We find that in order to obtain unambiguous information about the hydrodynamic size of a peptide-surfactant complex from diffusion measurements, experiments must be carried out at or below [SDS] = 25 mM.     

Normal blood pressure and plasma renin activity in mice lacking the renin-binding protein, a cellular renin inhibitor

In renal extracts, some renin is present as "high molecular weight renin," a heterodimeric complex of renin with the 46-kDa renin-binding protein (RnBP), also known as N-acyl-D-glucosamine 2-epimerase. Because RnBP specifically inhibits renin activity, the protein was proposed to play an important role in the regulation of the renin-angiotensin system (RAS). Using gene targeting, we have generated mice lacking RnBP and tested this hypothesis in vivo. In particular, we analyzed biosynthesis, secretion, and activity of renin and other components of the RAS in mice lacking RnBP. Despite extensive investigations, we were unable to detect any major effects of RnBP deficiency on the plasma and renal RAS or on blood pressure regulation. Contrary to previous hypotheses, we conclude that RnBP does not play a significant role in the regulation of renin activity in plasma or kidney. However, RnBP knockout mice excrete an abnormal pattern of carbohydrates in the urine, indicating a role of the protein in renal carbohydrate metabolism.     

Production and characterization of human renin antibodies with region-oriented synthetic peptides

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.     

Computational study of the conformational profiles of model bis-cystine cyclic peptides

Bis-cystine cyclic peptides are a new kind of molecules with potential use as cavitands, transporters or antagonists of target ligands. Studies aimed at establishing their conformational profiles may prove useful in understanding their characteristics and potentiate their use in molecular design. The present investigation reports the results of a computational study devoted to establishing the conformational preferences of model bis-cystine cyclic peptides and the properties in common with their linear analogs. For this purpose a study of four model compounds: (Ac-Cys-X-Cys-NHMe)₂ and (Ac-Cys-X-X-Cys-NHMe)₂ with X = Ala, Val, was performed. The goal of the study was to explore the importance of the conformational nature of the central residues, the effect of the number of them, and the loss of conformational freedom after cyclization on model molecules. Accordingly, the conformational space and the dynamic behaviour of the four cyclic peptides as well as the corresponding linear analogs was carefully explored. The results indicate the existence of structural patterns that might be useful for the use of this kind of molecule in de novo molecular design     

Synthesis of cyclic tryptathionine peptides

The helicity of the tryptathionine moiety of the phallotoxins has been recognized by comparison with cyclic tryptathionine tripeptides. In order to investigate the influence of the configuration of the component amino acids on the conformation of the cyclic peptides, six analogue thioether tripeptides containing L- and D-alanine and L- and D-cysteine, respectively, have been synthesized. The CD spectra of the peptides are very similar to each other, showing mirror images of the CD of phalloidin and, therefore, negative helicity. The spectra of the D-cysteine containing compounds differ from the L-cysteine containing compounds by their weakly positive ellipticity values around 270 nm. The cyclization reaction of Boc-Hpi-D-Ala-D-Cys(STrt)OCH3, along with the cyclic tripeptide, afforded a cyclic hexapeptide by dimerization. The CD spectrum of the dimer is very similar to that of phalloidin, thus pointing to a positive helicity of its two tryptathionine moieties. The dimeric thioether peptide forms a rather strong complex with Cu2+ ions.     

NMR study of the structure of short-chain peptides in solution.

The electron-mediated spin–spin coupling constant J between the amide NH and the α-CH protons in the dipeptide fragment C^α? CO(NH? C^αH)R? C′ONH? C^α is dependent on the dihedral angle of rotation (Φ) around the N? C bond. Measurement of J in a series of zwitterionic dipeptides H₃N⁺? CHR₁? CONH? CHR₂? CO₂^? (which is conformationally similar to the dipeptide fragment) in TFA solution shows that J is independent of R₁, but dependent on the steric bulk of R₂. The data are interpreted in terms of a model that assumes that what we measure is an average value of J? a thermal average over all the possible rotamers. The groups R₁ and R₂ are, in most cases, sterically kept apart by the trans and planar amide bonds, and hence the independence of J of R₁. This model is consistent with the theoretical calculations done on the dipeptide fragment. The effect of the structural characteristics of the side chains (e.g., the effect of lengthening and branching the side chains) on the J values in dipeptides is discussed in the light of the existing results of theoretical calculations. Study of 〈J〉 values in tripeptides (C₆H₅CH₂OCONH? CHR₁? CONH? CHR₂? CO₂CH₃, essentially three linked peptide units) shows that electrostatic interaction between the two amide bonds modifies the potential energy surface and the 〈J〉 value of a dipeptide subunit in the tripeptides. Also in some cases, direct steric interaction between the two side chains in the two adjacent dipeptide subunits in the tripeptide affects the potential energy surfaces of the individual dipeptide subunits and hence the 〈J〉 values. The influence of the structural characteristics of the side chains of individual amino acids on structure formation at or beyond the dipeptide level is discussed at various points. The J(NH? ^αCH) values of CH₃CONH? CHR? CONH₂ and CH₃CONH? CHR? CO₂CH₃ with the same R are quite different for R = valine, leucine, phenylalanine, methionine, but equal for R = glycine. This, coupled with the fact that one of the carboxamide NH resonances has a chemical shift different from its counterpart in simple amides like CH₃CONH₂ and the other carboxamide NH has the same chemical shift as its counterpart in CH₃CONH₂, suggest the presence of a hydrogen bond in dipeptide CH₃CONH? CHR? CONH₂ with carboxamide NH as the donor. Theoretical evidence for two seven-membered hydrogen-bonded rings with the carboxamide NH as donor and the acetyl oxygen as acceptor is summarized. Our data cannot suggest the number of such hydrogen-bonded rings, nor can they conclude the relative proportion of these rings in a particular dipeptide. A discussion of the difficulty of interpretation is presented and the data are discussed under certain simplifying assumptions.     

Effect of propranolol on cyclic AMP excretion and plasma renin activity in labile essential hypertension

Labile hypertension is often associated with elevated cardiac output, increased plasma renin activity (PRA) and urinary cyclic AMP excretion in response to upright posture and to isoproterenol. The β-blocking agent propranolol was demonstrated to be an effective therapeutic agent in this condition. The effect of posture on cyclic AMP, PRA, pulse rate and blood pressure was therefore studied during the administration of propranolol and a placebo in patients with labile hypertension. With the patient on placebo, upright posture induced an increase in pulse rate, cyclic AMP excretion and PRA. Propranolol administration decreased the recumbent and upright blood pressures, pulse rate and PRA. Cyclic AMP excretion remained unchanged in the recumbent position but the postural increase was abolished. No appreciable changes in catecholamine excretion occurred during propranolol administration. Propranolol normalizes some humoral as well as hemodynamic abnormalities of labile hypertension and therefore may be of benefit in long-term treatment and possibly also in the prevention of stable hypertension.     

Studies on cyclic peptides. II. Synthesis of cyclic peptides containing sarcosine.

Cyclic peptides containing sarcosine, cyclo-(Pro-Sar-Gly)₂, cyclo-(Sar-Sar-Gly)₂, cyclo-(Sar₄), and cyclo-(Sar₆) have been synthesized by the cyclization of the p-nitrophenyl ester of linear peptides. The tert-butoxycarbonyl group was used as the N^α-protecting group, which was removed by acid. Benzyl ester was used to protect the C-terminal. tert-butoxycarbonylpeptide was obtained by the stepwise elongation of the peptide bond by the carbodiimide method. Deblocking and cyclization of the linear peptides gave the cyclic peptides.     

Presence of antibodies against cyclic citrullinated peptides in patients with 'rhupus': a cross-sectional study

'Rhupus' is a rare condition sharing features of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). If rhupus is a distinctive entity, an overlap between RA and SLE or a subset of SLE is currently debated. This study was performed to explore the prevalence of antibodies against cyclic citrullinated peptides (anti-CCP antibodies) in rhupus. Patients meeting American College of Rheumatology criteria for RA, SLE, or both were included. Clinical and radiographic features were recorded and sera were searched for anti-CCP antibodies, rheumatoid factor, antinuclear antibodies, anti-extractable nuclear antigens, and antibodies against double-stranded DNA (anti-dsDNA antibodies). Seven patients for each group were included. Clinical and serological features for RA or SLE were similar between rhupus and RA patients, and between rhupus and SLE patients, respectively. Values for anti-CCP antibodies obtained were significantly (p < 0.05) higher in RA (6/7) and rhupus (4/7) than in SLE patients (0/7) and healthy subjects (0/7). Our data support the possibility that rhupus is an overlap between RA and SLE, because highly specific autoantibodies for RA (anti-CCP) and for SLE (anti-dsDNA and anti-Sm) are detected in coexistence.     

1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides

The recent crystal structure of Pin1 protein bound to a doubly phosphorylated peptide from the C-terminal domain of RNA polymerase II revealed that binding interactions between Pin1 and its substrate take place through its Trp-Trp (WW) domain at the level of the loop Ser(11)-Arg(12) and the aromatic pair Tyr(18)-Trp(29), and showed a trans conformation for both pSer-Pro peptide bonds. However, the orientation of the ligand in the aromatic recognition groove still could be sequence-specific, as previously observed in SH3 domains complexed by peptide ligands or for different class of WW domains (Zarrinpar, A., and Lim, W. A. (2000) Nat. Struct. Biol. 7, 611-613). Because the bound peptide conformation could also differ as observed for peptide ligands bound to the 14-3-3 domain, ligand orientation and conformation for two other biologically relevant monophosphate substrates, one derived from the Cdc25 phosphatase of Xenopus laevis (EQPLpTPVTDL) and another from the human tau protein (KVSVVRpTPPKSPS) in complex with the WW domain are here studied by solution NMR methods. First, the proton resonance perturbations on the WW domain upon complexation with both peptide ligands were determined to be essentially located in the positively charged beta-hairpin Ser(11)-Gly(15) and around the aromatic Trp(29). Dissociation equilibrium constants of 117 and 230 microm for Cdc25 and tau peptides, respectively, were found. Several intermolecular nuclear Overhauser effects between WW domain and substrates were obtained from a ligand-saturated solution and were used to determine the structures of the complexes in solution. We found a similar N to C orientation as the one observed in the crystal complex structure of Pin1 and a trans conformation for the pThr-Pro peptidic bond in both peptide ligands, thereby indicating a unique binding scheme for the Pin1 WW domain to its multiple substrates.     

Activity and NMR structure of synthetic peptides of the bovine ATPase inhibitor protein,IF1

The protein IF₁ is a natural inhibitor of the mitochondrial F_oF₁-ATPase. Many investigators have been prompted to identify the shortest segment of IF₁, retaining its native activity, for use in biomedical applications. Here, the activity of the synthetic peptides IF₁-(42–58) and IF₁-(22–46) is correlated to their structure and conformational plasticity determined by CD and [¹H]-NMR spectroscopy. Among all the IF₁ segments tested, IF₁-(42–58) exerts the most potent, pH and temperature dependent activity on the F_oF₁ complex. The results suggest that, due to its flexible structure, it can fold in helical and/or β-spiral arrangements that favor the binding to the F_oF₁ complex, where the native IF₁ binds. IF₁-(22–46), instead, as it adopts a rigid -helical conformation, it inhibits ATP hydrolysis only in the soluble F₁ moiety.