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.     

Analogues of cyclolinopeptide A containing alpha-hydroxymethyl amino acid residues

Linear and cyclic cyclolinopeptide A (CLA) analogues containing alpha-hydroxymethylleucine (HmL) in positions 1, 4, and 1&4, and alpha-hydroxymethylvaline (HmV) in position 5, were synthesized by the solid-phase peptide strategy and cyclized with the 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide/1-hydroxy-7-azabenzotriazole (EDC/HOAt) reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation assays (LPA). Only HmL-containing peptides demonstrated at about 25% lower immunosuppressive activity, but they are four times more soluble in water solutions than the native CLA. It seems from the LPA results that peptide [(HmL4)CLA] is the most promising for further studies. This peptide was characterized in solution, at room temperature in CDCl3, and the conformation compared with that observed for CLA in the solid state.     

Antamanide, a derivative of Amanita phalloides, is a novel inhibitor of the mitochondrial permeability transition pore

Antamanide is a cyclic decapeptide derived from the fungus Amanita phalloides. Here we show that antamanide inhibits the mitochondrial permeability transition pore, a central effector of cell death induction, by targeting the pore regulator cyclophilin D. Indeed, (i) permeability transition pore inhibition by antamanide is not additive with the cyclophilin D-binding drug cyclosporin A, (ii) the inhibitory action of antamanide on the pore requires phosphate, as previously shown for cyclosporin A; (iii) antamanide is ineffective in mitochondria or cells derived from cyclophilin D null animals, and (iv) abolishes CyP-D peptidyl-prolyl cis-trans isomerase activity. Permeability transition pore inhibition by antamanide needs two critical residues in the peptide ring, Phe6 and Phe9, and is additive with ubiquinone 0, which acts on the pore in a cyclophilin D-independent fashion. Antamanide also abrogates mitochondrial depolarization and the ensuing cell death caused by two well-characterized pore inducers, clotrimazole and a hexokinase II N-terminal peptide. Our findings have implications for the comprehension of cyclophilin D activity on the permeability transition pore and for the development of novel pore-targeting drugs exploitable as cell death inhibitors.     

Cyclopeptides of Linum usitatissimum.

Cyclolinopeptide A (CLA), a cyclic nonapeptide from linseed, possesses strong immunosuppressive and antimalarial activity along with the ability to inhibit cholate uptake into hepatocytes. The structure of the peptide was studied extensively in solution as well as in the solid state. It is postulated that both the Pro-Pro cis-amide bond and an 'edge-to-face' interaction between the aromatic rings of two adjacent Phe residues are important for biological activity. Structure-activity relationship studies of many linear and cyclic analogues of CLA suggest that the Pro-Xxx-Phe sequence and the flexibility of the peptide are important for the immunosuppressive activity.     

Synthesis and immunosuppressive activity of new cyclolinopeptide A analogs modified with beta-prolines

Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases. As a continuation of our previous work searching for new, effective suppressors devoid of toxicity, we present the synthesis, conformational analysis, and biological activity of nonapeptides 1-6, analogs of naturally existing immunomodulatory peptide CLA. New CLA analogs were modified with (S)-beta(2)-iso-proline 7 or (S)-beta(3)-homo-proline 8, respectively. The conformational influence of the beta-iso-proline and beta-homo-proline building blocks was analyzed by NMR spectroscopy. Peptides 1-6 exist as a mixture of four isomers due to cis/trans isomerization of the Xxx-Pro peptide bond. The major isomers of peptides 1, 3, and 4 contain all peptide bonds of the trans geometry. The geometry of the proline-proline bond of the second populated isomer of peptides 3 and 4 is cis. The proline-proline peptide bond is cis for the major isomers of peptides 2, 5, and 6. The peptides were tested for their ability to suppress the proliferative response of mouse splenocytes to T- and B-cell mitogens and the secondary humoral immune response to sheep erythrocytes in vitro in parallel with a reference drug-cyclosporine A. The immunoregulatory actions of the peptides depended on the position and content of proline isomers and were, with some exceptions, strongly inhibitory at the highest dose tested (100 microg/ml). In addition, the peptides were practically devoid of toxicity at that dose. In conclusion, the replacement of Pro by beta-Pro may be useful for fine-tuning CLA immunosuppressive potency and undesirable toxicity.     

Conformational stability of antamanide and analogs. Crystal structure of perhydrosymmetric antamanide, cyclic (-Val-Pro-Pro-Cha-Cha-Val-Pro-Pro-Cha-Cha-)

The synthetic perhydrogenated symmetric analog of the cyclic decapeptide antamanide is biologically inactive, although the conformation of the molecule and the crystal packing are very similar to that of the active symmetric analog of antamanide. In fact, the same conformation for the molecule has now been found in six polymorphs of uncomplexed antamanide and its analogs. The differences between the active and inactive antamanide analogs are displayed dramatically in the conformations of their metal ion (Na+ or Li+) complexes, thus suggesting strongly that for physiological activity antamanide is not in the conformation assumed by the uncomplexed molecule, but rather in the conformation assumed by the complexed state of natural antamanide. The present structure crystallizes in space group P2(1)2(1)2(1) with a = 20.515(14) A, b = 21.316(16) A, c = 17.128(16) A and four peptide molecules in the unit cell. There are three cocrystallized water molecules at full occupancy intrinsic to the peptide, and several more water molecules or other solvent molecules at partial occupancy. The formula of the peptide is C66H106N10O10.4-H2O.2X.     

A new cyclolinopeptide containing nonproteinaceous amino acid N-methyl-4-aminoproline

We have found that besides the known cyclolinopeptides A (CLA) and B (CLB), there is a new cyclic peptide in linseed mill cake that we have named CLX. Its composition is very similar to that of CLA, a cyclic peptide with a distinct immunosuppressive activity. The sequence of this peptide has been established as cyclo(PPFFILLX), where X is a non-proteinaceous amino acid, N-methyl-4-aminoproline. This amino acid substitutes for two amino acid residues of CLA, mimicking a dipeptide moiety with a nonplanar cis amide bond. The non-proteinaceous amino acid X may mimic a transition state of the peptide bond which exists in such processes as, e.g., PPIase-catalysed cis/trans amide-Pro bond isomerisation.     

Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability.

The aim of this study was to determine whether phalloidin (1 microM) or antamanide (1 microM), cyclic peptides that stabilize dense peripheral band and stress fiber F-actin in endothelium, would attenuate the increase in microvascular permeability induced by 4 h of ischemia and 30 min of reperfusion (I/R) in the isolated canine gracilis muscle. Changes in microvascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in muscles subjected to 4.5 h of continuous perfusion (nonischemic controls), I/R alone, I/R + phalloidin, or I/R + antamanide. Muscle neutrophil content was assessed by determination of myeloperoxidase (MPO) activity in tissue samples obtained at the end of the experiments. Fluorescent detection of nitrobenzoxadiazole-phallicidin in endothelial cell monolayers confirmed that phalloidin enters these cells. I/R was associated with marked increases in microvascular permeability and muscle neutrophil content (1 - sigma = 0.45 +/- 0.07; MPO = 8.9 +/- 0.5 units/g) relative to control (4.5 h continuous perfusion) preparations (1 - sigma = 0.12 +/- 0.03; MPO = 0.5 +/- 0.8 unit/g). These I/R-induced changes were largely prevented by administration of phalloidin (1 - sigma = 0.19 +/- 0.02; MPO = 0.8 +/- 0.4 U/g) or antamanide (1 - sigma = 0.07 +/- 0.11; MPO = 0.9 +/- 0.3 unit/g) at reperfusion. Similar results were obtained when phalloidin was administered before ischemia (1 - sigma = 0.24 +/- 0.04; MPO = 1.2 +/- 1.0 units/g). Although antamanide decreased superoxide production (by approximately 60%) and adherence to plastic (by approximately 75%) by activated neutrophils in vitro, phalloidin failed to alter these aspects of granulocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new cyclolinopeptide containing nonproteinaceous amino acid N-methyl-4-aminoproline

Summary We have found that besides the known cyclolinopeptides A (CLA) and B (CLB), there is a new cyclic peptide in linseed mill cake that we have named CLX. Its composition is very similar to that of CLA, a cyclic peptide with a distinct immunosuppressive activity. The sequence of this peptide has been established ascyclo(PPFFILLX), where X is a non-proteinaceous amino acid,N-methyl-4-aminoproline. this amino acid substitutes for two amino acid residues of CLA, mimicking a dipeptide moiety with a nonplanarcis amide bond. The non-proteinaceous amino acid X may mimic a transition state of the peptide bond which exists in such processes as, e.g., PPIase-catalysedcis/trans amide-Pro bond isomerisation.     

Acylpeptide hydrolase inhibition as targeted strategy to induce proteasomal down-regulation

Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy.Here, we illustrate a new pathway modulating UPS and proteasome activity through inhibition of APEH. To find novel molecules able to down-regulate APEH activity, we screened a set of synthetic peptides, reproducing the reactive-site loop of a known archaeal inhibitor of APEH (SsCEI), and the conjugated linoleic acid (CLA) isomers. A 12-mer SsCEI peptide and the trans10-cis12 isomer of CLA, were identified as specific APEH inhibitors and their effects on cell-based assays were paralleled by a dose-dependent reduction of proteasome activity and the activation of the pro-apoptotic caspase cascade. Moreover, cell treatment with the individual compounds increased the cytoplasm levels of several classic hallmarks of proteasome inhibition, such as NFkappaB, p21, and misfolded or polyubiquitinylated proteins, and additive effects were observed in cells exposed to a combination of both inhibitors without any cytotoxicity. Remarkably, transfection of human bronchial epithelial cells with APEH siRNA, promoted a marked accumulation of a mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), herein used as a model of misfolded protein typically degraded by UPS. Finally, molecular modeling studies, to gain insights into the APEH inhibition by the trans10-cis12 CLA isomer, were performed.Our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of strategies aimed at modulation of cancer progression.     

Influence of conjugated linoleic acid isomers on the metastasis of colon cancer cells in vitro and in vivo

This study investigated the isomer-specific effects of cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) conjugated linoleic acid (CLA) on the metastasis of colon cancer cells in vitro and in vivo. Cell migration was examined by a Boyden chamber assay in SW480 cells. MMP-9 activity was monitored by gelatin zymography, and MMP-9 protein and mRNA levels were determined by Western blot and RT-PCR analysis, respectively, in SW480 cells. For the experimental metastasis, BALB/c mice were injected intravenously with CT-26 cells in the tail vein. Mice were fed a diet containing either no CLA or 0.1% c9,t11 or t10,c12 CLA for 4 weeks. In experimental metastasis, the numbers of pulmonary nodules were significantly lower in mice fed CLA isomers than in mice fed a control diet (P<.05). Results from the Boyden chamber assay revealed that c9,t11 CLA significantly inhibited cell migration (P<.05), whereas t10,c12 CLA had no effect on cell migration. The activity of MMP-9 was significantly inhibited by c9,t11 CLA (P<.05) but not by t10,c12 CLA. However, neither MMP-9 protein nor mRNA levels were altered by either of these CLA isomers. We have demonstrated that diets containing 0.1% c9,t11 and t10,c12 CLA were equally effective in inhibiting colon cancer cell metastasis in vivo. However, in vitro, only c9,t11 but not t10,c12 inhibited colon cancer cell migration and MMP-9 activity.     

A new strain of Butyrivibrio fibrisolvens that has high ability to isomerize linoleic acid to conjugated linoleic acid

A new strain of Butyrivibrio fibrisolvens (TH1) that has high potential to produce conjugated linoleic acid (CLA) was isolated. Strain TH1 had higher LA isomerase (LA-I) activity, and was much more tolerant to linoleic acid (LA) than other strains examined. However, high CLA reductase (CLA-R) activity resulted in the temporary accumulation of CLA and subsequent conversion to trans-vaccenic acid (t-VA). When LA was added to growing TH1 cultures in a solution with dimethylsulfoxide (LA/DMSO), CLA produced was greater than when LA was added in a mixture with bovine serum albumin (BSA). The number of viable cells decreased upon addition of LA/DMSO, but then increased as the CLA decreased upon its conversion to t-VA. This result suggests that B. fibrisolvens can resume growing by the removal of CLA from the cells. Most CLA was released from B. fibrisolvens cells by gentle washing with BSA, suggesting that CLA bound to the cells might be removed in the rumen and large intestine. Thus, CLA production by B. fibrisolvens in the digestive tract could be increased by a reduction in CLA-R activity without accompanying an overall decrease in the cell number of B. fibrisolvens. Fatty acids (FAs) with 18 carbon backbone inducted LA-I activity, whereas unsaturated FAs induced CLA-R activity, suggesting that FAs stimulate the synthesis of LA-I and CLA-R. Providing a diet with a low ratio of unsaturated to saturated FAs may favor CLA production.     

Conjugated linoleic acid and hepatic lipogenesis in mouse: role of the mitochondrial citrate carrier

Conjugated linoleic acid (CLA) is able to reduce adiposity by affecting lipid metabolism. In particular, CLA administration to mice reduces body fat mass with a concomitant lipid accumulation in the liver. We investigated the effects of CLA on the activity of the mitochondrial citrate carrier (CIC), which is implicated in hepatic lipogenesis. The transport activity of the CIC, measured both in intact mitochondria and in the proteoliposomes, progressively increased with the duration of CLA feeding. An increase in the CIC activity of approximately 1.7-fold was found in 16 week CLA-treated mice with respect to control animals. A kinetic analysis showed a 1.6-fold increase in the V(max) of citrate transport but no change in the K(m) value. Western blot experiments revealed an increase of approximately 1.7-fold in the expression of CIC after CLA treatment. A strict correlation between the increase in CIC activity and the stimulation of the cytosolic lipogenic enzymes was also found. These data indicate that the CIC may play a role in the onset of hepatic steatosis in CLA-fed mice by supplying the carbon source for de novo fatty acid synthesis.     

Modulation of Serotonin Transporter Activity by a Protein Kinase C Activator and an Inhibitor of Type 1 and 2A Serine/Threonine Phosphatases

Abstract: We studied the effects of 12- O -tetradecanoylphorbol 13-acetate (TPA), a protein kinase C (PKC) activator, and calyculin A (CLA), an inhibitor of type 1 and 2A serine/threonine phosphatases, on serotonin uptake by a human placenta choriocarcinoma cell line (BeWo) and COS-7 cells expressing recombinant serotonin transporter (SET). In BeWo cells, treatment with TPA decreased imipramine-sensitive serotonin uptake with a reduction in V _max without affecting K _m. CLA also decreased imipramine-sensitive serotonin uptake in a manner similar to that of TPA. TPA and CLA also decreased the uptake activity of recombinant SET expressed in COS-7 cells as seen in BeWo cells. These effects of TPA and CLA were reversed by staurosporine, a protein kinase inhibitor. To elucidate whether the inhibitory effects of TPA and CLA were due to direct phosphorylation of SET by PKC, site-directed mutagenesis of five putative PKC phosphorylation sites in SET was performed. Serotonin uptake was also down-regulated by TPA and CLA in all nine mutants, suggesting that these inhibitory modulation of SET activity did not act via direct phosphorylation of SET by PKC.     

Down-regulation of PPARgamma2-induced adipogenesis by PEGylated conjugated linoleic acid as the pro-drug: Attenuation of lipid accumulation and reduction of apoptosis

This study is designed to evaluate whether the PEGylated conjugated linoleic acid (PCLA) as the pro-drug can have favorable stability, bioavailability, and anti-adipogenic activity in 3T3-L1 cells for anti-obesity when compared with conjugated linoleic acid (CLA) itself. The CLA was simply coupled to poly(ethylene glycol) (PEG) at the melting state without solvents or catalysts through ester linkages between the carboxylic group of CLA and the hydroxyl group of PEG. To confirm of PCLA as the pro-drug, CLA release from PCLA was investigated by using high-performance liquid chromatographic (HPLC), showing that CLA release from PCLA was almost 90% in a nearly continuous fashion over the next 75h. Apoptosis was promoted by both CLA- and PCLA-treatments with increasing concentrations. However, the level of cell apoptosis induced by PCLA was lower than that induced by CLA owing to the biocompatible and hydrophilic properties of PEG. Moreover, the PCLA decreased glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 cells by acting upon major adipocyte marker proteins such as PPARgamma2, C/EBPalpha, and aP2 modulators. Furthermore, either CLA or PCLA stimulated basal, but not isoproterenol-sensitive, lipolysis in our cell model, suggesting that both CLA and PCLA may stimulate lipolysis via hormone sensitive lipase (HSL)-independent mechanisms. These results suggest that the PCLA may prove to be a stable pro-drug to control the deposition of fat in the human body, and that the anti-adipogenic effect of the PCLA on 3T3-L1 cells will offer a challenging approach for anti-obesity.     

Conformational characteristics of peptides and unanticipated results from crystal structure analyses

Preferred conformation and types of molecular folding are some of the topics that can be addressed by structure analysis using x-ray diffraction of single crystals. The conformations of small linear peptide molecules with 2-6 residues are affected by polarity of solvent, presence of water molecules, hydrogen bonding with neighboring molecules, and other packing forces. Larger peptides, both cyclic and linear, have many intramolecular hydrogen bonds, the effect of which outweighs any intermolecular attractions. Numerous polymorphs of decapeptides grown from a variety of solvents, with different cocrystallized solvents, show a constant conformation for each peptide. Large conformational changes occur, however, upon complexation with metal ions. A new form of free valinomycin grown from DMSO exhibits near three-fold symmetry with only three intramolecular hydrogen bonds. The peptide is in the form of a shallow bowl with a hydrophobic exterior. Near the bottom of the interior of the bowl are three carbonyl oxygens, spaced and directed so that they are in position to form three ligands to a K+, e.g., complexation can be completed by the three lobes containing the beta-bends closing over and encapsulating the K+ ion. In another example, free antamanide and the biologically inactive perhydro analogue, in which four phenyl groups become cyclic hexyl groups, have essentially the same folding of backbone and side chains. The conformation changes drastically upon complexation with Li+ or Na+. However, the metal ion complex of natural antamanide has a hydrophobic globlar form whereas the metal ion complex of the inactive perhydro analogue has a polar band around the middle. The structure results indicate that the antamanide molecule is in a complexed form during its biological activity. Single crystal x-ray diffraction structure analyses have identified the manner in which water molecules are essential to creating minipolar areas on apolar helices. Completely apolar peptides, such as membrane-active peptides, can acquire amphiphilic character by insertion of a water molecule into the helical backbone of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib-Ala-Leu-Aib-OMe, for example. The C-terminal half assumes an alpha-helix conformation, whereas the N-terminal half is distorted by an insertion of a water molecule W(1) between N(Ala5) and O(Ala2), forming hydrogen bonds N(5)H...W(1) and W(1)...O(2). The distortion of the helix exposes C = O(Aib1) and C = O(Aib4) to the outside environment with the consequence of attracting additional water molecules. The leucyl side chains are on the other side of the molecule. Thus a helix with an apolar sequence can mimic an amphiphilic helix.     

The effect of conjugated linoleic acid on the viability and metabolism of human osteoblast-like cells

Studies in experimental animals and murine osteoblast cells in culture have produced conflicting findings on the effect of conjugated linoleic acid (CLA) on bone formation. The present study investigated the influence of CLA on viability and metabolism of two human osteoblast-like cell lines (SaOS2 and MG63). Both cell lines were exposed to increasing concentrations (0-50 microM) of CLA either as pure cis (c) 9: trans (t) 11 and t10:c12 CLA isomers or a blend of isomers, or linoleic acid (C18:2). Cell cytotoxicity and degree of DNA fragmentation were unaffected by any fatty acid treatment. PGE2 biosynthesis by both cell lines was variably reduced by CLA isomer blend and t10:c12 CLA, but not c9:t11 CLA. Alkaline phosphatase activity was variably increased by all CLA treatments. These results suggest a lack of cytotoxic effect of CLA on human osteoblast-like cells and tentatively suggest a possible beneficial effect on bone formation in humans.     

Metabolism and short-term metabolic effects of conjugated linoleic acids in rat hepatocytes

Metabolic fate and short-term effects of a 1:1 mixture of cis-9,trans-11 and trans-10,cis-12-conjugated linoleic acids (CLA), compared to linoleic acid (LA), on lipid metabolism was investigated in rat liver. In isolated mitochondria CLA-CoA were poorer substrates than LA-CoA for carnitine palmitoyltransferase-I (CPT-I) activity. However, in digitonin-permeabilized hepatocytes, where interactions among different metabolic pathways can be simultaneously investigated, CLA induced a remarkable stimulatory effect on CPT-I activity. This stimulation can be ascribed to a reduced malonyl-CoA level in turn due to inhibition of acetyl-CoA carboxylase (ACC) activity. The ACC/malonyl-CoA/CPT-I system can therefore represent a coordinate control by which CLA may exert effects on the partitioning of fatty acids between esterification and oxidation. Moreover, the rate of oxidation to CO2 and ketone bodies was significantly higher from CLA; peroxisomes rather than mitochondria were responsible for this difference. Interestingly, peroxisomal acyl-CoA oxidase (AOX) activity strongly increased by CLA-CoA compared to LA-CoA. CLA, metabolized by hepatocytes at a higher rate than LA, were poorer substrates for cellular and VLDL-triacylglycerol (TAG) synthesis. Overall, our results suggest that increased fatty acid oxidation with consequent decreased fatty acid availability for TAG synthesis is a potential mechanism by which CLA reduce TAG level in rat liver.