The biological activity of the histidine-containing diketopiperazines cyclo(His-Ala) and cyclo(His-Gly)

Two cyclic dipeptides, cyclo(His-Ala) and cyclo(His-Gly,) were synthesized from their linear counterparts and their structures elucidated using standard elucidation techniques. Molecular modeling and predictive NMR results indicated that the majority of energetically favourable conformers adopted a boat conformation with respect to the diketopiperazine ring. Cyclo(His-Ala), at concentrations of 100 microM inhibited the growth, in vitro, of various cancer cell lines, including HT-29, MCF-7 and HeLa carcinoma cells while cyclo(His-Gly) inhibited the growth of MCF-7 cells. While the antibacterial potential of these two compounds was limited, both cyclic dipeptides significantly inhibited the growth of C. albicans. Both compounds at a concentration of 100 microM resulted in a decrease in heart rate, coronary flow rate and left ventricular systolic pressure in the isolated rat heart. Inhibition of thrombin, amounting to a 63.3% and 36.7% reduction in the rate of fibrin formation, was noted for cyclo(His-Ala) and cyclo(His-Gly), respectively. While cyclo(His-Ala) showed no notable effects on platelet aggregation, cyclo(His-Gly) significantly inhibited both pathways tested with greatest effects on thrombin-induced platelet aggregation, yielding an IC(50) of 0.0662 mM (R(2)=0.989). The results of the anticancer and hematological studies indicate that histidine-containing diketopiperazines have potential as a novel group of cytotoxic agents with antithrombotic effects.     

Crystal structure of cyclo(Pro-Gly)3: Li complex: A model for ion transport by cyclo(Pro-Gly)3

The crystal structure of cyclo(Pro-Gly)₃ (PG3) complex with LiSCN (C₂₂H₃₀N₇O₆SLi) has been solved by x-ray diffraction. The crystals belong to the space group R3 in the hexagonal setting with unit cell parameters of a = 12.581(1), c = 29.705(3) Å, V = 4072.0 ų, Z = 6, M_r = 527.53, D_c = 1.23 g/cm³. The crystal structure was solved by direct methods using the program SHELXS-86 and refined to an R value of 5.3% for 1645 reflections (I > 2σI). There are two conformers in the crystal structure. One conformer has three carbonyls on one side and three on the other side of the peptide plane. The other conformer has all six of the carbonyls on the same side of the peptide plane. Both of these conformers bind independently to a Li ion. Based on the conformers of the Li complex and other reported ion complexes formed by PG3, we propose a model for the transport of ions across the lipid membrane. The features of the model are as follows: (1) PG3 forms a hexameric stack in a lipid bilayer when complexing and transporting metal ions. (2) It undergoes a conformational flipping in order pass the ion along the channel. The energy required for the conformational change involved in the flipping of the PG3 molecule may be provided by the applied potential during ion transport. © 1994 John Wiley & Sons, Inc.     

Synthesis and conformation of the cyclic octapeptides cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Cyclic octapeptides, cyclo(X-Pro)₄, where X represents Phe, Leu, or Lys(Z), were synthesized and their conformations investigated. A C₂-symmetric conformer containing two cis peptide bonds was found in all of these cyclic octapeptides. The numbers of available conformations due to the cis–trans isomerization of Pro peptide bonds depended on the nature of the solvent and X residue: they decreased in the following order: cyclo[Lys(Z)-Pro]₄ > cyclo(Leu-Pro)₄ > cyclo(Phe-Pro)₄ in CDCl₃. ¹³C spin-lattice relaxation times (T₁) of these cyclic octapeptides were measured, and the contribution of segmental mobility to T₁ was found to vary with the nature of the X residue.     

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.     

Studies on cyclic peptides. IV. Conformation of cyclo(Sar-Sar-Gly)2, cyclo(Sar)6 and cyclo(Sar-Gly-Gly)2 and their conformational change induced by alkali thiocyanates

Conformation of cyclo (Sar-Sar-Gly)₂, cyclo(Sar)₆, and cyclo(Sar-Gly-Gly)₂ was investigated by nmr spectroscopy. cyclo(Sar-Sar-Gly)₂, were shown to assume various conformations in dimethysulfoxide. It was attributed to the distribution of cis as well as trans Gly-Sar or Sar-Sar amide links along the peptide backbone. In particular, cyclo(Sar-Sar-Gly)₂ took five or six different conformations: one or three C₂-symmetric conformations and four or three asymmetric conformations, respectively. Three of nine NH resonance signals were ascribed to the internally hydrogen-bonded glycine residues. cyclo(Sar-Sar-Gly)₂ and cyclo(Sar)₆ showed a spectral change on the addition of alkali thiocyanates, indicating a conformational change induced by a complex formation with the alkali cations. The complex nmr spectrum due to a hybridization of different conformations changed with the salt addition into a simple nmr spectrum, suggesting a preponderence of a new, single conformation. On the basis of the spectral change, the strength for the cations binding the cyclic peptides was found to be in the order of K⁺ > Na⁺ > Rb⁺ > Cs⁺ for cyclo(Sar-Gly-Gly)₂ and K⁺ > Rb⁺ > Cs⁺ for cyclo(Sar)₆. On the other hand, cyclo(Sar-Gly-Gly)₂ in dimethylsulfoxide assumed a single C₂ conformation having two glycyl peptide protons shielded from solvent and the other two exposed to solvent. This conformation did not change with the salt addition. Finally, the conformations of several cyclic peptides containing the sarcosine residue such as cyclo(Sar)₆ cyclo(Sar-Sar-Gly)₂ cyclo(Pro-Sar-Gly)₂, and cyclo (Sar-Gly-Gly)₂ were compared. It appeared that proline and glycine residues reduced the conformational multiplicity of the cyclic peptide backbone, and the ability to bind alkali metal cations decreased in the above order.     

Complex formation with alkali and alkaline earth metal ions of cyclic octapeptides,cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Complex formation with alkali and alkaline earth metal ions of cyclic octapeptides, cyclo(Phe-Pro)₄, cyclo(Leu-Pro)₄, and cyclo[Lys(Z)-Pro]₄ was investigated in relation to conformation. In an alcohol solution, cyclo(Phe-Pro)₄ did not form complexes. However, cyclo(Leu-Pro)₄ and cyclo[Lys(Z)-Pro]₄ formed complexes selectively with Ba²⁺ and Ca²⁺ ions. Changing the solvent from alcohol to acetonitrile, the complexation behavior was very different. In acetonitrile, cyclo(Phe-Pro)₄ was found to form a complex with Ba²⁺, and CD spectra of cyclo(Leu-Pro)₄ and cyclo[Lys(Z)-Pro]₄ changed sharply on complexation with K⁺. Rate constants of the complex formation between the cyclic octapeptides and metal salts were in the range of 0.7–12 L mol^?1 min^?1 in an alcohol solution. One of the two types of complex formation in acetonitrile was much faster than that in an alcohol solution.     

Synthesis and antitumor activity of bis(arylsulfonyl)dihydroimidazolinone derivatives

A series of novel bis(arylsulfonyl)dihydroimidazolinones with different aryl substitution patterns were readily synthesized and evaluated for their antitumor activities. Some of the newly synthesized compounds exhibited cytotoxicity at micromolar range against multiple cancer cell lines, including A549, HepG2, HuCCA-1, and MOLT-3. The most potent analogue contained pentafluorobenzenesulfonyl groups, which could be chemically elaborated to serve as a potential pharmacophore.     

Synthesis and conformational study of a cyclic hexapeptide analogue of somatostatin: cyclo(Phe-D-Trp-Lys-Thr-o-AMPA)

The active sequence Phe7-D-Trp8-Lys9-Thr10 of somatostatin has been cyclized through o-(aminomethyl)phenylacetic acid, a spacer molecule, designed to mimic a Gly-Gly dipeptide containing a cis-constrained peptide bond. The resulting analogue shows no GH-inhibition. A 2D n.m.r. study reveals conformations different from the proposed bio-active one and still sensitive to the medium (solvent).     

Conformations of cyclo(L-orD-Phe-L-Pro-Aca) and cyclo(L-Pro-L- or D-Phe-Aca). Cyclized dipeptide models for specific types of beta-bends

Conformational analyses on four cyclic model peptides of the beta-bend, cyclo(L- or D-Phe-L-Pro-epsilon-aminocaproyl(Aca] and cyclo(L-Pro-L- or D-Phe-Aca), were carried out both experimentally and theoretically. Cyclo(D-Phe-L-Pro-Aca) was shown to exist as a single conformer taking the type II' beta-bend. The comparison of its CD spectra with those of cyclo(L-Ala-L-Ala-Aca) revealed that type I and II' beta-bends, both with alpha-helix-like CD spectra, can be distinguished. Cyclo(L-Phe-L-Pro-Aca) was shown to exist as a single conformer with a cis L-Phe-L-Pro peptide bond, taking the type VI beta-bend. Its CD spectrum has thus been observed for the first time for the bend containing a cis peptide bond. Cyclo(L-Pro-L-Phe-Aca) was shown to exist as a mixture of two conformers, the major one taking the type I beta-bend with a trans Aca-L-Pro peptide bond and the minor one with a cis Aca-L-Pro peptide bond. Cyclo(L-Pro-D-Phe-Aca) was suggested to exist as a mixture of two conformers, the major one taking the type II beta-bend with a trans Aca-L-Pro peptide bond and the minor one with a cis Aca-L-Pro peptide bond.     

Solution conformation and hydrolytic activity of cyclo(D-leu-L-His)

It has been reported previously that a cyclic dipeptide, cyclo(D -Leu-L -His), showed a high hydrolytic activity toward a hydrophobic ester, p-nitrophenyl laurate. In order to determine the reason for the high catalytic activity, the conformation of cyclo(D -Leu-L -His) in aqueous solution was investigated by nuclear magnetic resonance and circular dichroism spectroscopy and compared with the conformation of cyclo(L -Leu-L -His), which was nearly inactive in otherwise the same conditions for the hydrolysis. It was demonstrated that the spatial arrangement of the hydrophobic isobutyl group of the D -leucyl residue and of the nucleophilic imidazolyl group of the L -histidyl residue in cyclo(D -Leu-L -His) matches very well with the long acyl chain and the active ester function of p-nitrophenyl laurate. On the other hand, in cyclo(L -Leu-L -His) the hydrophobic and the nucleophilic pendant groups are too close with each other to cooperate intramolecularly for the hydrolysis. It was concluded that the different steric structures of the diastereomers can explain the large difference of the catalytic activities.     

Conformational behaviour of A cyclolinopeptide a analogue: Two-dimensional NMR study of cyclo(Pro1-Pro-Phe-Phe-Ac6c-IIe-ala-Val8)

The cyclic octapeptide cyclo[-Pro¹-Pro-Phe-Phe-Ac₆c-Ile-ala-Val⁸-] [C₈-Ac₆c], containing the Pro¹-Pro-Phe-Phe sequence, followed by a bulky helicogenic C^α,α-dialkylated glycine residue Ac₆c (1-aminocyclohexane-1-carboxylic acid), and a _D-Ala residue at position 7 has been synthesized. This cyclic peptide is a deletion analogue of the naturally occurring cyclic nonapeptide cyclolinopeptide A (CLA). It has been designed with the aim of studying the role that the Ac₆c and _D-Ala residues play on the conformational behaviour of the whole molecule and their influence on the conformation of the Pro¹-Pro-Phe-Phe sequence when compared with cyclolinopeptide A. C₈Ac₆c has been investigated in chloroform and acetonitrile solutions by 2D NMR techniques. Only one set of sharp signals is observed in both solvents. This evidence strongly supports the hypothesis that only one conformational state exists in the chosen solvents. The interpretation of the experimental data points to the existence for C₈-Ac₆c of a very rigid structure stabilized by intramolecular hydrogen bonds. The measured NOE effects allow the calculation of internuclear distances, which have been used as restraints in molecular dynamic calculations. The proposed conformation of the molecule shows that the Pro-Pro-Phe segment retains the conformation observed in natural CLA both in solution and in the solid state and that the Ac₆c residue indeed reinforces the ring rigidity not permitting the formation of any appropriate cavity in which inorganic cations could be complexed.     

A 1H-NMR study of the solution conformation of cyclo(GRGDSPA): conformational effects on the physiological activity.

Solution conformations of cyclo(GRGDSPA) have been analyzed by the use of two-dimensional proton nuclear magnetic resonance spectroscopy and the dynamical simulated annealing calculation. It has been shown that the RGDS segment in cyclo(GRGDSPA) takes a beta-turn conformation. We have concluded that this beta-turn conformation is essential for the physiological activity of cyclo(GRGDSPA).     

Conformation and complexation with metal ions of cyclic hexapeptides: cyclo (L-Leu-L-Phe-L-Pro)2 and cyclo [L-Cys(Acm)-L-Phe-L-Pro]2

Cyclic hexapeptides, cyclo (L-Leu-L-Phe-L-Pro)2 and cyclo[L-Cys(Acm)-L-Phe-L-Pro]2, in which Acm represents an acetoamide-methyl group, were synthesized, and the conformation and complexation with metal ions were investigated. Cooperation of the carbonyl groups of the Cys(Acm) side chains with those of the cyclic skeleton in complexation was especially examined. Cyclo(L-Leu-L-Phe-L-Pro)2, which possesses no functional groups on side chains, was taken as the reference compound. 13C- and two-dimensional n.m.r. measurements revealed that cyclo(L-Leu-L-Phe-L-Pro)2 and cyclo[L-Cys(Acm)-L-Phe-L-Pro]2 took a C2-symmetric conformation containing cis L-Phe-L-Pro bonds in chloroform and acetonitrile. Both cyclic hexapeptides were found to complex selectively with Ba2+ and Ca2+ in acetonitrile. On complexation the conformation of either cyclic hexapeptide changed into a similar one. However, the binding constant of cyclo[L-Cys(Acm)-L-Phe-L-Pro]2 was higher than that of cyclo(L-Leu-L-Phe-L-Pro)2. The n.m.r. measurements showed that the amide carbonyl groups of Cys(Acm) side chains as well as those of cyclic skeleton in cyclo[L-Cys(Acm)-L-Phe-L-Pro]2 cooperatively bound the cations.     

Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid

We have isolated a Lactobacillus plantarum strain (MiLAB 393) from grass silage that produces broad-spectrum antifungal compounds, active against food- and feed-borne filamentous fungi and yeasts in a dual-culture agar plate assay. Fusarium sporotrichioides and Aspergillus fumigatus were the most sensitive among the molds, and Kluyveromyces marxianus was the most sensitive yeast species. No inhibitory activity could be detected against the mold Penicillium roqueforti or the yeast Zygosaccharomyces bailii. An isolation procedure, employing a microtiter well spore germination bioassay, was devised to isolate active compounds from culture filtrate. Cell-free supernatant was fractionated on a C(18) SPE column, and the 95% aqueous acetonitrile fraction was further separated on a preparative HPLC C(18) column. Fractions active in the bioassay were then fractionated on a porous graphitic carbon column. The structures of the antifungal compounds cyclo(L-Phe-L-Pro), cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid (L/D isomer ratio, 9:1), were determined by nuclear magnetic resonance spectroscopy, mass spectrometry, and gas chromatography. MIC values against A. fumigatus and P. roqueforti were 20 mg ml(-1) for cyclo(L-Phe-L-Pro) and 7.5 mg ml(-1) for phenyllactic acid. Combinations of the antifungal compounds revealed weak synergistic effects. The production of the antifungal cyclic dipeptides cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) by lactic acid bacteria is reported here for the first time.     

Conformational study of cyclo(1,5)-Ac-Pen-Arg-Gly-Asp-Cys-NH2 in water by NMR and molecular dynamics.

Cyclo(1,5)-Ac-Pen-Arg-Gly-Asp-Cys-NH2 (3) is a potent inhibitor of platelet aggregation. Nuclear magnetic resonance (NMR) and restrained molecular dynamics were used to study the conformations of 3. Elucidation of RGD conformations in 3 will increase the understanding of interaction between the RGD-sequence with GPIIb/IIIa.     

Ten-membered cyclotripeptides. III. Synthesis and conformation of cyclo(-Me beta Ala-Phe-Pro-) and cyclo(-Me beta Ala-Phe-DPro-).

The 10-membered cyclotripeptide cyclo(-Me beta Ala-Phe-Pro) 3 and its diastereoisomer cyclo(-Me beta Ala-Phe-DPro-) 4 have been synthesized under mild cyclization conditions starting from linear precursors containing C-terminal proline. The crystal and molecular structure of the two models has been determined by X-ray crystallography. Analysis of the NMR spectra supported by NOE data clearly indicates that the conformations found in the crystals are retained in solution. Both cyclotripeptides exhibit a cis-cis-trans backbone conformation. The two tertiary peptide bonds, at the proline and Me beta Ala nitrogen atoms, adopt a cis conformation whereas the CO-NH junctions are trans in both the models. The deviations from planarity of the peptide units vary from delta omega values of ca. 18 degrees for the Pro-Me beta Ala and DPro-Me beta Ala bonds to ca. 7 degrees for Phe-Pro and Phe-DPro bonds. Relevant conformational details of 3 and 4, as revealed by X-ray and NMR analysis, are reported. Crystals of 3 are monoclinic: P2(1), a = 5.317(2), b = 17.059(6), c = 9.514(3) A, beta = 99.18(3), Z = 2. The final R and Rw are 0.054 and 0.071 respectively. Crystals of 4 are orthorhombic: P2(1)2(1)2(1), a = 8.797(2), b = 19.440(9), c = 21.605(10) A, Z = 8. The final R and Rw are 0.069 and 0.104 respectively.     

Radioprotective effect of cyclo(L-phenylalanyl-L-prolyl) on irradiated rat lung

In the present study, we investigated the radioprotective effect of cyclo(L-phenylalanyl-L-prolyl) on irradiated rat lungs to determine its potential as a radioprotective agent. We found that early lung damage induced by irradiation was reduced by treatment with 40 mg/kg of cyclo(Lphenylalanyl- L-prolyl) in the latent and early pneumonitis phases. Expression of TNF-alpha and TGF-beta1 at 2 and TGF-beta1 at 8 weeks post-irradiation was decreased in animals that received both radiation and cyclo(L-phenylalanyl-L-prolyl) compared with animals that received radiation alone. Evidence indicated that the proinflammatory cytokine TNF-alpha and the fibrogenic cytokine TGF-beta1 likely play a role in the radioprotective effect of cyclo(L-phenylalanyl-L-prolyl). However, besides TNF-alpha and TGF-beta1 expressions, the precise mechanism by which cyclo(L-phenylalanyl-L-prolyl) ameliorates the induced radiation damage is not clear.     

Is all cyclo(His-Pro) derived from thyrotropin-releasing hormone?

Cyclo(His-Pro), or histidyl-proline diketopiperazine, is an endogenous cyclic dipeptide that is ubiquitously distributed in tissues and body fluids of both man and animals. This cyclic dipeptide is not only structurally related to thyrotropin-releasing hormone (TRH, pGlu-His-ProNH₂), but it can also arise from TRH by the action of the enzyme pyroglutamate amino-peptidase (pGlu-peptidase). The data on the distribution of TRH, cyclo(His-Pro), and pGlu-peptidase under normal and abnormal conditions are summarized and potential relationships analyzed. We conclude that all of the cyclo(His-Pro) cannot be derived from TRH. Two additional sources of cyclo(His-Pro) are suggested. It is proposed that 29,247 molecular weight TRH prohormone, prepro TRH, which contains 5 copies of TRH sequence, can be processed to yield cyclo(His-Pro). Thus, both TRH and cyclo(His-Pro) share a common precursor, prepro[TRH/Cyclo(His-Pro)].