Ribosomally-synthesised cyclic peptides from plants as drug leads and pharmaceutical scaffolds

Owing to their exceptional stability and favourable pharmacokinetic properties, plant-derived cyclic peptides have recently attracted significant attention in the field of peptide-based drug design. This article describes the three major classes of ribosomally-synthesised plant peptides – the cyclotides, the PawS-derived peptides and the orbitides – and reviews their applications as leads or scaffolds in drug design. These ribosomally-produced peptides have a range of biological activities, including anti-HIV, cytotoxic and immunomodulatory activity. In addition, recent interest has focused on their use as scaffolds to stabilise bioactive peptide sequences, thereby enhancing their biopharmaceutical properties. There are now more than 30 published papers on such ‘grafting’ applications, most of which have been reported only in the last few years, and several such studies have reported in vivo activity of orally delivered cyclic peptides. In this article, we describe approaches to the synthesis of cyclic peptides and their pharmaceutically-grafted derivatives as well as outlining their biosynthetic routes. Finally, we describe possible bioproduction routes for pharmaceutically active cyclic peptides, involving plants and plant suspension cultures.     

Novel method for the synthesis of urea backbone cyclic peptides using new Alloc‐protected glycine building units

Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide, disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine functionalized side chains. Here we present urea backbone cyclization as an additional method for the preparation of backbone cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc‐N^α [ω‐amino(Alloc)‐alkyl] glycine building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for protein kinase B inhibition as anticancer leads. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Facile synthesis and evaluation of C-functionalized benzyl-1-oxa-4,7,10-triazacyclododecane-N,N',N″-triacetic acid as chelating agent for ¹¹¹In-labeled polypeptides

A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N,N′,N″-triacetic acid (ODTA), has been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetraacetic acid (DOTA). However, neither synthetic procedure for a C-functionalized ODTA (C-ODTA) nor its chelating ability with a trace amount of radioactive indium-111 (¹¹¹In) has been elucidated. We herein present a facile synthetic procedure for C-ODTA, and estimated its ability as a chelating agent for radiolabeling peptides and proteins with ¹¹¹In. The synthetic procedure involves the synthesis of a linear precursor using a para-substituted phenylalanine derivative as a starting material. The following intramolecular cyclization reaction was best performed (>73% yield) when Boc-protected linear compound and the condensation reagent, HATU, were simultaneously added to the reaction vessel at the same flow rate. The cyclic compound was then reduced with BH₃ and alkylated with tert-butyl bromoacetate. The synthetic procedure was straightforward and some optimization would be required. However, most of the intermediate compounds were obtained easily in good yields, suggesting that the present synthetic procedure would be useful to synthesize C-ODTA derivatives. The intramolecular cyclization reaction might also be applicable to synthesize polyazamacrocycles of different ring sizes and cyclic peptides. In ¹¹¹In radiolabeling reactions, C-ODTA provided ¹¹¹In chelates in higher radiochemical yields at low ligand concentrations when compared with C-DOTA. The ¹¹¹In-labeled C-ODTA remained unchanged in the presence of apo-transferrin. The biodistribution studies also showed that the ¹¹¹In-labeled compound was mainly excreted into urine as intact. These findings indicate that C-ODTA would be useful to prepare ¹¹¹In-labeled peptides of high specific activities in high radiochemical yields.     

From population ecology to metabolism: growth of Trypanosoma evansi,and implications of glucose depletion,in a live host

Little attention has been paid to the potential top-down effects of population ecology on metabolism. Because it is capable of a fast growth that dramatically modifies its blood environment, the parasitic protozoan, Trypanosoma evansi, is a promising model for the study of density-dependent metabolic processes. We assess the in vivo growth rate, doubling time, biomass yield, glycolytic flux, and enzyme expression of T. evansi. Then, we explore the metabolic changes likely occurring during its growth. At low T. evansi densities, most host glucose is used to produce energy, which results in the release of pyruvate. Part of glucose is used for NADPH production through the pentose phosphate pathway. At high T. evansi densities, fructose, mannose, and glycerol become additional energy sources. Part of host glucose is used for biosynthesis and for NADPH production through alternative metabolic pathways, which results in the release of succinate, alanine, and acetate. The ability of organisms to adjust to resource changes is crucial to their survival. Irrespective if the triggering mechanism is direct (nutrient limitation) or indirect (a pheromone-like factor), nutrient availability is necessarily the main evolutionary factor responsible for the density-dependent metabolic properties of trypanosome populations.     

Optimization strategies for metabolic networks

Background  

The increasing availability of models and data for metabolic networks poses new challenges in what concerns optimization for biological systems. Due to the high level of complexity and uncertainty associated to these networks the suggested models often lack detail and liability, required to determine the proper optimization strategies. A possible approach to overcome this limitation is the combination of both kinetic and stoichiometric models. In this paper three control optimization methods, with different levels of complexity and assuming various degrees of process information, are presented and their results compared using a prototype network.     

Light-induced de-epoxidation of violaxanthin in lettuce chloroplasts IV. The effects of electron-transport conditions on violaxanthin availability

1. In isolated chloroplasts of Lactuca sativa var. Manoa, the size of the violaxanthin fraction which is available for de-epoxidation is not directly dependent on electron transport but rather related to the reduced level of some electron carrier between the photosystems. This is concluded from the effects of various electrontransport conditions on violaxanthin availability: Under conditions of electron transport through both photosystems, availability was saturated at a lower electron-transport rate with actinic light at 670 than at 700 nm. Under conditions of electron transport through Photosystem I, availability was smaller for linear electron flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen than for cyclic electron flow mediated by either N-methylphenazonium methosulfate or 2,6-dichlorophenolindophenol; in addition for linear r flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen, availability increased with decreasing light intensity.2. The postulated carrier whose reduced level is related to availability seems to be some carrier between plastoquinone and the primary acceptor of Photosystem II or plastoquinone itself. This conclusion follows from the fact that availability increased with increasing light intensity under conditions of electron flow through both photosystems and that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (≤ μM) had no effect on availability, whereas low levels of 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea resulted in decreased availability (50% decrease at 1 μM). Furthermore, availability in 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts was fully restored by 2-methyl-1,4-naphtoquinone (menadione) which mediates cyclic electron flow through plastoquinone.3. Violaxanthin availability was zero in the dark and increased in the light to a maximum of 67% of the total violaxanthin in chloroplasts. It is proposed that this variable violaxanthin availability reflects conformational changes on the internal surface of the thylakoid membrane which result in variable exposure of violaxanthin to the de-epoxidase. The fact that not all of the violaxanthin was available for de-epoxidation may indicate a heterogenous distribution of violaxanthin in the membrane.     

Interaction of gramicidin S and its aromatic amino-acid analog with phospholipid membranes

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic β-sheet-β-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar ≡ Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic—exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (K_b). The K_b1 and K_b2 values for endothermic two-step binding processes corresponded to high and low binding affinities (K_b1 ≥ 100 K_b2). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.     

Antihypertensive effect of spent brewer yeast peptide

Numerous studies have investigated dietary approaches to prevent chronic lifestyle-related diseases, including hypertension. Spent brewer’s yeast is the second largest byproduct originated by the brewing industry and it deserves considerable attention because of its high nutritional value, ca. 40% of its dry mass is rich in protein which can be hydrolyzed into biologically active peptides. To upgrade this byproduct, the aim of this study was initially in vitro assessment of biological properties, e.g. ACE inhibition and antioxidant activity, and then, the in vivo effect in short-term oral antihypertensive effect of hydrolyzed yeast fractions on a well characterized model to study hypertension - Spontaneously Hypertensive Rats (SHR). Here, it was demonstrated that the fraction with molecular weight below 3 kDa containing tri and tetra- peptides with hydrophobic amino acid residues – SPQW, PWW and RYW, causes the most noticeable decrease in systolic, diastolic and mean blood pressure of SHR and shows highest antioxidant effect. These properties highlight the potential use of yeast extract as nutraceutical or functional food ingredient for the management and treatment of hypertension with antioxidant effect.     

O-Seco-RA-XXIV,a possible precursor of an antitumor peptide RA-XXIV,from Rubia cordifolia L.

O-Seco-RA-XXIV, a new cyclic peptide, cyclo-(d-alanyl-l-glutaminyl-N,O-dimethyl-l-tyrosyl-l-alanyl-N-methyl-l-tyrosyl-N-methyl-l-tyrosyl), was isolated from the roots of Rubia cordifolia L. along with RA-XXIV. Its structure and relative stereochemistry were determined by interpretation of the spectroscopic data and X-ray crystallography, and its absolute stereochemistry by the Marfey's amino acid analysis of its acid hydrolysate. Isolation of the two peptides from the same plant source may indicate that O-seco-RA-XXIV is a possible precursor of RA-XXIV and that the formation of the diphenyl ether linkage in the cycloisodityrosine moiety is to be formed after the formation of the cyclohexapeptide chain in this series of peptides.     

Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.     

Structure-activity relationship and metabolic stability studies of backbone cyclization and N-methylation of melanocortin peptides

Backbone cyclization (BC) and N-methylation have been shown to enhance the activity and/or selectivity of biologically active peptides and improve metabolic stability and intestinal permeability. In this study, we describe the synthesis, structure-activity relationship (SAR) and intestinal metabolic stability of a backbone cyclic peptide library, BL3020, based on the linear alpha-Melanocyte stimulating hormone analog Phe-D-Phe-Arg-Trp-Gly. The drug lead, BL3020-1, selected from the BL3020 library (compound 1) has been shown to inhibit weight gain in mice following oral administration. Another member of the BL3020 library, BL3020-17, showed improved biological activity towards the mMC4R, in comparison to BL3020-1, although neither were selective for MC4R or MC5R. N-methylation, which restrains conformational freedom while increasing metabolic stability beyond that which is imparted by BC, was used to find analogs with increased selectivity. N-methylated backbone cyclic libraries were synthesized based on the BL3020 library. SAR studies showed that all the N-methylated backbone cyclic peptides demonstrated reduced biological activity and selectivity for all the analyzed receptors. N-methylation of active backbone cyclic peptides destabilized the active conformation or stabilized an inactive conformation, rendering the peptides biologically inactive. N-methylation of backbone cyclic peptides maintained stability to degradation by intestinal enzymes.     

Development of cyclic peptide derivatives from the N-terminal region of LANA for targeting the nucleosome acidic patch

Kaposi’s sarcoma-associated herpesvirus (KSHV) is known to be a carcinogenic agent that causes AIDS-associated Kaposi’s sarcoma (KS). When KSHV infects host’s cells, one of the virus’s proteins, latency-associated nuclear antigen 1 (LANA), binds to the host’s nucleosomes to retain episomes and create latency circumstances. Although the infectious mechanism of KSHV is partly elucidated, the development of drug candidates for targeting KS is ongoing. In this study, we developed cyclic peptides corresponding to an N-terminal LANA sequence that disrupt the LANA–nucleosome interaction. The cyclic peptides showed a different secondary structure compared to their corresponding linear peptide derivatives, which suggests that our cyclization strategy imitates the N-terminal LANA binding conformation on nucleosomes.     

Ecosystem constraints to symbiotic nitrogen fixers: a simple model and its implications

The widespread occurrence of N limitation to net primary production (NPP) and other ecosystem processes, despite the ubiquitous occurrence of N-fixing symbioses, remains a significant puzzle in terrestrial ecology. We describe a simple simulation model for an ecosystem containing a generic nonfixer and a symbiotic N fixer, based on: (1) a higher cost for N acquisition by N fixers than nonfixers; (2) growth of fixers and fixation of N only when low N availability limits the growth of nonfixers, and other resources are available; and (3) losses of fixed N from the system only when the quantity of available N exceeds plant and microbial demands. Despite the disadvantages faced by the N fixer under these conditions, N fixation and loss adjust N availability close to the availability of other resources, and biomass and NPP in this simple model can be substantially but only transiently N limited. We then modify the model by adding: (1) losses of N in forms other than excess available N (e.g., dissolved organic N, trace gases produced by nitrification); and (2) constraints to the growth and activity of N fixers imposed by differential effects of shading, P limitation, and grazing. The combination of these processes is sufficient to describe an open system, with input from both precipitation and N fixation, that is nevertheless strongly N-limited at equilibrium. This model is useful for exploring causes and consequences of constraints to N fixation, and hence of N limitation, and we believe it will also be useful for evaluating how N fixation and limitation interact with elevated CO₂ and other components of global enviromental change.     

Priming and microbial nutrient limitation in lowland tropical forest soils of contrasting fertility

Priming is an increase in soil organic carbon decomposition following input of labile organic carbon. In temperate soils where biological activity is limited commonly by nitrogen availability, priming is expected to occur through microbial acquisition of nitrogen from organic matter or stimulated activity of recalcitrant-carbon degrading microorganisms. However, these priming mechanisms have not yet been assessed in strongly weathered tropical forest soils where biological activity is often limited by the availability of phosphorus. We examined whether microbial nutrient limitation or community dynamics drive priming in three lowland tropical forest soils of contrasting fertility (‘low’, ‘mid’ and ‘high’) by applying C₄-sucrose (alone or in combination with nutrients; nitrogen, phosphorus and potassium) and measuring (1) the δ¹³C-signatures in respired CO₂ and in phospholipid fatty acid (PLFA) biomarkers, and (2) the activities of enzymes involved in nitrogen (N-acetyl β-glucosaminidase), phosphorus (phosphomonoesterase) and carbon (β-glucosidase, cellobiohydrolase, xylanase, phenol oxidase) acquisition from organic compounds. Priming was constrained in part by nutrient availability, because priming was greater when sucrose was added alone compared to when added with nutrients. However, the greatest priming with sucrose addition alone was detected in the medium fertility soil. Priming occurred in parallel with stimulated activity of phosphomonoesterase and phenol oxidase (but not N-acetyl β-glucosaminidase); when sucrose was added with nutrients there were lower activities of phosphomonoesterase and phenol oxidase. There was no evidence according to PLFA δ¹³C-incorporation that priming was caused by specific groups of recalcitrant-carbon degrading microorganisms. We conclude that priming occurred in the intermediate fertility soil following microbial mineralization of organic nutrients (phosphorus in particular) and suggest that priming was constrained in the high fertility soil by high nutrient availability and in the low fertility soil by the low concentration of soil organic matter amenable to priming. This first study of priming mechanisms in tropical forest soils indicates that input of labile carbon can result in priming by microbial mineralization of organic nutrients, which has important implications for understanding the fate of organic carbon in tropical forest soils.     

Conformational aspects of N-glycosylation of proteins. Studies with linear and cyclic peptides as probes

Conformational aspects of N-glycosylation of glycoproteins have been studied by using a series of peptides which contained, in addition to the `marker sequence' Asn-Gly-Thr, two cysteine residues in various positions of the peptide chain. The presence of two cysteines permitted a partial fixation of the above triplet sequence in cyclic structures of various size by intramolecular disulphide bond formation. Comparison of the glycosyl acceptor properties of the linear peptides and their corresponding cyclic analogues allows the following statements. The considerably lower acceptor capabilities of the cyclic derivatives indicate that the restriction of rotational degrees of freedom imposed by disulphide bonding results in a conformation which hinders a favourable interaction of the peptide substrate with the N-glycosyltransferase. On the other hand, the glycosylation rate of linear peptides increases with increasing chain length, suggesting that the amino acids on both the N- and C-terminal side of the `marker sequence' may contribute to a considerable extent to the induction of an `active' conformation. Realization of a potential sugar attachment site requires a hydrogen bond interaction within the `marker sequence' between the oxygen of threonine (serine) as the hydrogen bond acceptor and the β-amide of asparagine as the donor [Bause & Legler (1981) Biochem. J. 195, 639–644]. This interaction is obviously facilitated when the peptide chain can adopt a conformation which resembles a β-turn or other loop structure. The available experimental and statistical data are discussed in terms of possible structural features for N-glycosylation, with the aid of space-filling models.     

Rhizobium leguminosarum Biovar viciae Symbiotic Hydrogenase Activity and Processing Are Limited by the Level of Nickel in Agricultural Soils

Analysis of levels of hydrogenase processing and activity in Rhizobium leguminosarum biovar viciae bacteroids from pea (Pisum sativum) plants showed that the oxidation of nitrogenase-evolved hydrogen is limited by the availability of nickel in agricultural soils. This limitation was overcome by using an inoculant strain engineered for higher hydrogenase expression.     

Studies on the inhibition of hepatic lipogenesis by N6,O2′-dibutyryl adenosine 3′,5′-monophosphate

Fatty acid synthesis by isolated liver cells is dependent upon the availability of lactate and pyruvate. A lag in fatty acid synthesis is explained by time being required for lactate and pyruvate to accumulate to maximum concentrations in the incubation medium. The initial rate of fatty acid synthesis is not linear with cell concentration, being disproportionately greater at higher cell concentrations because optimal lactate and pyruvate concentrations are established in the medium more rapidly. The accumulation of lactate and pyruvate is inhibited markedly by N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate. This accounts in part for the inhibition of fatty acid synthesis caused by this cyclic nucleotide. Other sites of action are apparent, however, because exogenous lactate plus pyruvate only partially relieves the inhibition. The profile of metabolic intermediates suggests that N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate inhibits the conversion of glycogen to pyruvate and lactate by decreasing the effectiveness of phosphofructokinase and pyruvate kinase.     

Developing novel hCT derived cell-penetrating peptides with improved metabolic stability

Many promising therapeutics are currently awaiting their clinical application. Due to their low capability of cell membrane crossing, these compounds do not reach their site of action. One way to overcome this problem might be the fusion of these agents to cell-penetrating peptides (CPP), which are able to shuttle various cargoes across cellular membranes. One disadvantage in using CPP in drug delivery is their low metabolic stability. The aim of our work was to increase the proteolytic resistance of the CPP hCT(9-32), a truncated C-terminal fragment of human calcitonin. Thus, we synthesised six modified N-terminally carboxyfluorescein labelled hCT(9-32) derivatives by replacing positions 12 and/or 16 of hCT(9-32) with either N-methylphenylalanine or d-phenylalanine, respectively. By using confocal laser scanning microscopy we showed that the modifications did neither affect the peptide internalisation efficiency in HeLa nor HEK 293T cells. The metabolic stability of the peptides was investigated in human blood plasma and HEK 293T cell culture supernatant. To analyse the degradation patterns, we used RP-HPLC and MALDI-TOF mass spectrometry. However, we found for all of the new derivatives high metabolic stabilities. In blood plasma, the half-lives for five of the six peptides increased compared to unmodified hCT(9-32). The degradation patterns showed a distinct stabilisation in the N-terminal part of the modified peptides, in the C-terminal part, we found some cleavage to a minor extent. Furthermore, we studied the conformation of the peptides by CD spectroscopy and demonstrated that they possess no cell toxicity. Since our metabolically more stable compounds are still able to pass the cell membrane they provide powerful tools as drug delivery vectors.