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Bergofungin D is a helical peptide of the peptaibol family consisting of 14 amino acids, six of which are the helix inducer aminoisobutyric acid (Aib). In the second third of the sequence, a hydroxyproline causes a bending of the helix and a disruption of the hydrogen bond network, and Aib7 is the only amino acid in this region involved in the hydrogen bond network. Therefore, modification of this residue can serve as a probe to monitor the effect of introducing amino acid substitutions on this more fragile helical turn. To validate this approach, we simplified the original bergofungin D by reducing the number of non-classical amino acids, replacing the (R)-isovaleric acid by its enantiomer or an Aib and the hydroxyproline with a proline, respectively, without affecting its secondary structure. Within the modified structure, we replaced Aib7-Aib8 by its 1,2,3-triazolodipeptide equivalent or Aib7 by a serine or a dehydrobutyrine. We have reported and analyzed five crystal structures, three of which are new, demonstrating the usefulness of the modified bergofungin D as a probe for monitoring the introduction of amino acid substitutions within a helical structure., (© 2024 European Peptide Society and John Wiley & Sons Ltd.)

Cone snails are carnivorous marine animals that prey on fish (piscivorous), worms (vermivorous), or other mollusks (molluscivorous). They produce a complex venom mostly made of disulfide-rich conotoxins and conopeptides in a compartmentalized venom gland. The pharmacology of cone snail venom has been increasingly investigated over more than half a century. The rising interest in cone snails was initiated by the surprising high human lethality rate caused by the defensive stings of some species. Although a vast amount of information has been uncovered on their venom composition, pharmacological targets, and mode of action of conotoxins, the venom-ecology relationships are still poorly understood for many lineages. This is especially important given the relatively recent discovery that some species can use different venoms to achieve rapid prey capture and efficient deterrence of aggressors. Indeed, via an unknown mechanism, only a selected subset of conotoxins is injected depending on the intended purpose. Some of these remarkable venom variations have been characterized, often using a combination of mass spectrometry and transcriptomic methods. In this review, we present the current knowledge on such specific predatory and defensive venoms gathered from sixteen different cone snail species that belong to eight subgenera: Pionoconus , Chelyconus , Gastridium , Cylinder , Conus , Stephanoconus , Rhizoconus , and Vituliconus . Further studies are needed to help close the gap in our understanding of the evolved ecological roles of many cone snail venom peptides.

Polymicrobial infections threaten the health of humans and animals but remain understudied in natural systems. We recently described the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease affecting oyster production worldwide. In the French Atlantic coast, the disease involves coinfection with ostreid herpesvirus 1 (OsHV-1) and virulent Vibrio . However, it is unknown whether consistent Vibrio populations are associated with POMS in different regions, how Vibrio contribute to POMS, and how they interact with OsHV-1 during pathogenesis. By connecting field-based approaches in a Mediterranean ecosystem, laboratory infection assays and functional genomics, we uncovered a web of interdependencies that shape the structure and function of the POMS pathobiota. We show that Vibrio harveyi and Vibrio rotiferianus are predominant in OsHV-1-diseased oysters and that OsHV-1 drives the partition of the Vibrio community observed in the field. However only V. harveyi synergizes with OsHV-1 by promoting mutual growth and accelerating oyster death. V. harveyi shows high-virulence potential and dampens oyster cellular defenses through a type 3 secretion system, making oysters a more favorable niche for microbe colonization. In addition, V. harveyi produces a key siderophore called vibrioferrin. This important resource promotes the growth of V. rotiferianus , which cooccurs with V. harveyi in diseased oysters, and behaves as a cheater by benefiting from V. harveyi metabolite sharing. Our data show that cooperative behaviors contribute to synergy between bacterial and viral coinfecting partners. Additional cheating behaviors further shape the polymicrobial consortium. Controlling cooperative behaviors or countering their effects opens avenues for mitigating polymicrobial diseases.

α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii . Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC 50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 β2β3 subtype (IC 50 = 177 nM). Overall, this study contributes to a better understanding of the structure-activity relationships of α-conotoxins, which may help in the design of more selective tools.

The voltage-dependent anion channel (VDAC), the most abundant protein on the outer mitochondrial membrane, is implicated in ATP, ion and metabolite exchange with cell compartments. In particular, the VDAC participates in cytoplasmic and mitochondrial Ca 2+ homeostasis. Notably, the Ca 2+ efflux out of Schwann cell mitochondria is involved in peripheral nerve demyelination that underlies most peripheral neuropathies. Hexokinase (HK) isoforms I and II, the main ligands of the VDAC, possess a hydrophobic N-terminal structured in α-helix (NHKI) that is necessary for the binding to the VDAC. To gain further insight into the molecular basis of HK binding to the VDAC, we developed and optimized peptides based on the NHKI sequence. These modifications lead to an increase of the peptide hydrophobicity and helical content that enhanced their ability to prevent peripheral nerve demyelination. Our results provide new insights into the molecular basis of VDAC/HK interaction that could lead to the development of therapeutic compounds for demyelinating peripheral neuropathies.

To improve and diversify the quantification of reactive oxygen species (ROS) in mitochondria of single cells, we connected pyrene derivatives (PB) to a triphenylphosphonium salt (TPP + ) as a mitochondrial vector forming PB-TPP + probes. Two pyrene isomers with the n -butyltriphenylphosphonium moieties attached at their 1- or 2- positions were synthesized and characterized. Using the long fluorescence lifetime of pyrene, it was possible to monitor the variation of cellular free radicals and oxygen and to follow the reversibility of both quenchers in real-time. We compared the behavior of these new probes to the previously published pyrene-probes, functionalized by a mitochondrial-penetrating peptide, allowing their transfer to the mitochondria (Mito-PB) or to the cytosolic membrane for pyrene butyric acid (PBA). The high cellular uptake of the new probes allows cells to be loaded with an initial concentration 40 times lower than that for Mito-PB probes, without inducing perturbations in cell growth. The variation in free radicals and oxygen levels was monitored within cells under different stress conditions through the fluorescence lifetime of the new TPP + -based probes giving comparable results to those obtained for MPP-based probes. However, at a loading concentration as low as 25 nM, our technique allows the detection of increased production of free radicals in the mitochondria in the presence of the TPP + vector, a warning to the user of this well-known vector.

Lipopeptides are a class of compounds generally produced by microorganisms through hybrid biosynthetic pathways involving non-ribosomal peptide synthase and a polyketyl synthase. Cyanobacterial-produced laxaphycins are examples of this family of compounds that have expanded over the past three decades. These compounds benefit from technological advances helping in their synthesis and characterization, as well as in deciphering their biosynthesis. The present article attempts to summarize most of the articles that have been published on laxaphycins. The current knowledge on the ecological role of these complex sets of compounds will also be examined.

The bis-triazole ligand and its corresponding copper complexes were synthesized and characterized for the first time and proposed as new labels for the development of electrochemical aptasensors. The bis-triazole ligand was prepared from methyl 1,6-heptadiyne-4-carboxylate and 2-(azidomethyl)phenol using classical CuAAC in presence of different copper salts. The X-ray structure of bis-triazole showed a symmetry center (C1). UV-Vis and X-band EPR spectra showed that the coordination capacity of the bis-triazole ligand was improved in the presence of triethylamine due to deprotonation of the triazole and phenolate moieties. After complexation with copper, the obtained complex was successfully attached to an anti-estradiol aptamer through thiol-maleimide coupling, and the resulting labelled aptamer was immobilized on a carbon screen-printed electrode by carbodiimide coupling. The electrochemical response of the resulting sensor was shown to decrease in the presence of estradiol, demonstrating that the developed complexes can be applied for the development of aptasensors., (© 2021 Wiley-VCH GmbH.)

In the marine environment, sessile cyanobacteria have developed chemical strategies for protection against grazers. In turn, herbivores have to circumvent these defenses and in certain cases even take advantage of them as shelter from their own predators. This is the case of Stylocheilus striatus , a sea hare that feeds on Anabaena torulosa , a cyanobacterium that produces toxic cyclic lipopeptides of the laxaphycin B family. S. striatus consumes the cyanobacterium without being affected by the toxicity of its compounds and also uses it as an invisibility cloak against predators. In this article, using different substrates analogous to laxaphycin B, we demonstrate the presence of an enzyme in the digestive gland of the mollusk that is able to biotransform laxaphycin B derivatives. The enzyme belongs to the poorly known family of d-peptidases that are suspected to be involved in antibiotic resistance.

Real-time quantification of reactive nitrogen and oxygen species (ROS) in cells is of paramount importance as they are essential for cellular functions. Their excessive formation contributes to the dysfunction of cells and organisms, ultimately leading to cell death. As ROS are mostly produced in the mitochondria, we have synthesized a fluorescent probe able to reach this organelle to detect and quantify, in real time, the variation of ROS by time-resolved microfluorimetry. The new probes are based on the long fluorescence lifetime of pyrene butyric acid (PBA). Two PBA isomers, attached at their 1- or 2-positions to a peptide vector to target mitochondria, were compared and were shown to allow the measurement of free radical species and oxygen, but not non-radical species such as H 2 O 2 ., (© 2020 Wiley-VCH GmbH.)

Laxaphycins are a family of non-ribosomal lipopeptides that have been isolated from several cyanobacteria. Some of these compounds have presented cytotoxic activities, but their mechanism of action is poorly understood. In this work, the already described laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria Anabaena torulosa . Moreover, two new acyclic compounds, [des-(Ala 4 -Hle 5 )] acyclolaxaphycins B and B3, were purified from the herviborous gastropod Stylocheilus striatus , with this being the first description of biotransformed laxaphycins. The structure of these new compounds was elucidated, together with the absolute configuration of acyclolaxaphycins B and B3. The bioactivities of the six peptides were determined in SH-SY5Y human neuroblastoma cells. Laxaphycins B and B3 were cytotoxic (IC 50 : 1.8 and 0.8 µM, respectively) through the induction of apoptosis. In comparison, acyclic laxaphycins did not show cytotoxicity but affected mitochondrial functioning, so their effect on autophagy-related protein expression was analyzed, finding that acyclic peptides affected this process by increasing AMPK phosphorylation and inhibiting mTOR. This work confirms the pro-apoptotic properties of cyclic laxaphycins B and is the first report indicating the effects on autophagy of their acyclic analogs. Moreover, gastropod-derived compounds presented ring opening and amino-acids deletion, a biotransformation that had not been previously described.

Understanding natural defense mechanisms against parasites can be a valuable tool for the development of innovative therapies. We have previously identified a butterflyfish species ( Chaetodon lunulatus ) that avoids gill monogenean parasites while living amongst closely related parasitized species. The metabolome and microbiome of several sympatric butterflyfish species from the island of Moorea (French Polynesia) were previously described. In this study, we used the previously generated datasets in an attempt to identify metabolites and bacteria potentially involved in parasite defense mechanisms. We investigated the interplay between the gill mucus metabolome and microbiome of the non-susceptible C. lunulatus versus sympatric butterflyfish species that were always found parasitized in the Central and Eastern Indo-Pacific. After observing significant differences between the metabolome and bacteria of susceptible versus non-susceptible fish, we obtained the discriminant metabolites and operational taxonomic units (OTUs) using a supervised analysis. Some of the most important discriminant metabolites were identified as peptides, and three new peptides derived from β-subunit hemoglobin from C. lunulatus (CLHbβ-1, CLHbβ-2, and CLHbβ-3) were purified, characterized and synthesized to confirm their structures. We also identified specific bacterial families and OTUs typical from low-oxygen habitats in C. lunulatus gill mucus. By using a correlation network between the two datasets, we found a Fusobacteriaceae strain exclusively present in C. lunulatus and highly correlated to the peptides. Finally, we discuss the possible involvement of these peptides and Fusobacteriaceae in monogenean avoidance by this fish species., Competing Interests: “The authors declare no conflict of interest.”

The growing interest in marine natural substances as potential new drugs has made total synthesis a real asset for structure confirmation. Trichormamide C ( 1 ), a cyclic lipopeptide isolated from the cyanobacteria Oscillatoria sp., is characterized by the presence of nonproteinogenic amino acids in the sequence. Trichormamide C structural confirmation was carried out through the implementation of a flexible synthesis resulting in two new analogs ( 3 and 4 ).

This work presents the synthesis and characterization of salen/salan metal complexes for their future application as electrochemical labels in affinity sensors. Due to its stability and electrochemical properties, an oxovanadium salan complex was selected and coupled to an estradiol-specific aptamer. The response of the resulting aptasensor was shown to decrease with increasing estradiol concentration.

This study investigated the environmental fate of myrigalone A, a light absorbing natural herbicide found on leaves and fruits of Myrica gale. Myrigalone A was irradiated in water and as a dry solid deposit to simulate reactions on leaves, alone and in the presence of the terpenes generated by Myrica gale. The phototransformation was fast ( t 1/2 = 35 min in water). Analyses by liquid chromatography coupled to high resolution orbitrap electrospray mass spectrometry (MS) and gas chromatography-MS revealed the formation of 11 photoproducts in water and solid and 9 in gaseous phase. Some were detected in the leaf glands and oil covering the fruits of Myrica gale, which suggested that photodegradation occurred in the field. Moreover, myrigalone A photoinduced the oxidation of terpenes that in turn protected it against photolysis. This highlights the need for additional research on the effect of terpenes on the photodegradation of pesticides on vegetation.

Brain renin-angiotensin system hyperactivity has been implicated in the development and maintenance of hypertension. We have shown that aminopeptidase A is involved in the formation of brain angiotensin III, which exerts tonic stimulatory control over blood pressure in hypertensive deoxycorticosterone acetate-salt rats and spontaneously hypertensive rats. We have also shown that injection of the specific and selective aminopeptidase A inhibitor, (3S)-3-amino-4-sulfanyl-butane-1-sulfonic acid (EC33), by central route or its prodrug, RB150/firibastat, by oral route inhibited brain aminopeptidase A activity and blocked the formation of brain angiotensin III, normalizing blood pressure in hypertensive rats. These findings identified brain aminopeptidase A as a potential new therapeutic target for hypertension. We report here the development of a new aminopeptidase A inhibitor prodrug, NI956/QGC006, obtained by the disulfide bridge-mediated dimerization of NI929. NI929 is 10× more efficient than EC33 at inhibiting recombinant mouse aminopeptidase A activity in vitro. After oral administration at a dose of 4 mg/kg in conscious deoxycorticosterone acetate-salt rats, NI956/QGC006 normalized brain aminopeptidase A activity and induced a marked decrease in blood pressure of -44±13 mm Hg 4 hours after treatment ( P<0.001), sustained over 10 hours (-21±12 mm Hg; P<0.05). Moreover, NI956/QGC006 decreased plasma arginine-vasopressin levels, and increased diuresis and natriuresis, that may participate to the blood pressure decrease. Finally, NI956/QGC006 did not affect plasma sodium and potassium concentrations. This study shows that NI956/QGC006 is a best-in-class central-acting aminopeptidase A inhibitor prodrug. Our results support the development of hypertension treatments targeting brain aminopeptidase A.

Five new laxaphycins were isolated and fully characterised from the bloom forming cyanobacteria Anabaena torulosa sampled from Moorea, French Polynesia: three acyclic laxaphycin A-type peptides, acyclolaxaphycin A (1), [des-Gly 11 ]acyclolaxaphycin A (2) and [des-(Leu 10 -Gly 11 )]acyclolaxaphycin A (3), as well as two cyclic ones, [l-Val 8 ]laxaphycin A (4) and [d-Val 9 ]laxaphycin A (5). The absolute configuration of the amino acids, established using advanced Marfey's analysis for compounds 2-5, highlights a conserved stereochemistry at the Cα carbons of the peptide ring that is characteristic of this family. To the best of our knowledge, this is the first report of acyclic analogues within the laxaphycin A-type peptides. Whether these linear laxaphycins with the aliphatic β-amino acid on the N-terminal are biosynthetic precursors or compounds obtained after enzymatic hydrolysis of the macrocycle is discussed. Biological evaluation of the new compounds together with the already known laxaphycin A shows that [l-Val 8 ]laxaphycin A, [d-Val 9 ]laxaphycin A and [des-Gly 11 ]acyclolaxaphycin induce cellular toxicity whereas laxaphycin A and des-[(Leu 10 -Gly 11 )]acyclolaxaphycin A do not affect the cellular viability. An analysis of cellular death shows that the active peptides do not induce apoptosis or necrosis but instead, involve the autophagy pathway., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Peptaibols are linear non ribosomal peptides which have been the object of intense research efforts regarding their synthesis and the elucidation of the mechanism allowing their insertion in biological membranes. Forty years after their discovery they are still considered as model compounds and suitable probes for the investigation of new approaches aiming to test the efficacy of new coupling reagents, to physically and spectroscopically investigate the way by which they interact with the lipid bilayer and to develop artificial membrane pores. The stable helical secondary structure adopted by the peptaibols turn to be an adequate platform for gaining insight on the structural modifications induced by the substitution of the amide bond by 1,2,3-triazoles, but also for monitoring the impact of newly designed α,α-dialkyl glycine with fluorinated and silylated side chains as 2-aminoisobutyric acid mimic. Peptaibols secondary structure dictated by Aib high content has inspired the development of foldamers. Challenges and investigations on the above mentioned topics are discussed in this brief review., (Copyright © 2018 Elsevier Inc. All rights reserved.)

1,4-Disubstituted-1,2,3-triazole (Tz) is widely used in peptides as a trans-amide bond mimic, despite having hazardous effects on the native peptide activity. The impact of amide bond substitution by Tz on peptide secondary structures is scarcely documented. We performed a Tz scan, by systematically replacing peptide bonds following the Aib residues with Tz on two model peptaibols: alamethicin F50/5 and bergofungin D, which adopt stable α- and 310 helices, respectively. We observed that the Tz insertion, whatever its position in the peptide sequences, abolished their antimicrobial activity. The structural consequences of this insertion were further investigated using CD, NMR and X-ray diffraction. Importantly, five crystal structures that were incorporated with Tz were solved, showing various degrees of alteration of the helical structures, from minor structural perturbation of the helix to partial disorder. Together, these results showed that Tz insertions impair helical secondary structures.

A simple and efficient strategy is proposed to significantly improve the antibacterial activity of peptaibols and other antimicrobial peptides by N-terminal capping with 1,2,3-triazole bearing various hydrophobic substituents on C-4. Such N-terminal insertions on alamethicin F50/5 could enhance its antimicrobial activity on Gram-positive bacteria without modification of its overall three-dimensional structure. Although the native peptide and its analogues shared comparable helical contents, the crystal structure of one of the most active derivative showed a local slight distortion of the N-terminal extremity, which was also observed in solution using NMR spectroscopy. Importantly, fluorescence studies showed that the N-capped derivatives had increased affinity for liposomes, which may indicate they interacted more strongly with the bacterial membrane than alamethicin F50/5., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

New chemiluminescence-based immunoassays for sensitive detection of 17-β estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of biotinylated estrogen derivatives. Estrogen derivatives bearing a carboxylic group (E2-COOH and EE2-COOH) on C-3 position were synthesized, covalently bound to aminated biotin and subsequently immobilized on avidin-coated microtiter plates. The assay principle was based on competition between free and immobilized estrogens for their binding to primary antibodies, with subsequent revelation using horseradish peroxidase (HRP)-labeled secondary antibodies. Under optimized conditions, the chemiluminescence immunoassays showed a highly sensitive response to E2 and EE2, with respective detection limits of 0.5 and 1.2 ng L -1 . The LOD achieved using biotinylated E2 was in the same order of magnitude as those obtained using commercially available E2-bovine serum albumin conjugate (E2-BSA). The developed devices were successfully applied to analysis wastewater treatment plants effluents (WWTP) with negligible matrix effect., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Angiogenesis is tightly regulated through the binding of vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). In this context, we showed that human VEGFR1 domain 2 crystallizes in the presence of Zn2+, Co2+ or Cu2+ as a dimer that forms via metal-ion interactions and interlocked hydrophobic surfaces. SAXS, NMR and size exclusion chromatography analyses confirm the formation of this dimer in solution in the presence of Co2+, Cd2+ or Cu2+. Since the metal-induced dimerization masks the VEGFs binding surface, we investigated the ability of metal ions to displace the VEGF-A binding to hVEGFR1: using a competition assay, we evidenced that the metals displaced the VEGF-A binding to hVEGFR1 extracellular domain binding at micromolar level., Competing Interests: The authors have declared that no competing interests exist.

Some terrestrial Trichoderma sp. strains are already used as biological control agents (BCAs). They all produce peptaibols, small antimicrobial peptides which are supposed to play a role in the anti-phytopathogenic activity of Trichoderma sp. Trichoderma strains producing high amounts of peptaibols could represent new potential BCAs. In this context, marine-derived Trichoderma strains from the marine fungal strain collection of the "Mer, Molécules, Santé" (MMS) laboratory were investigated for their peptaibol production. Previously, the quantification of peptaibols was performed using alamethicin, as standard (20-amino acid residues peptaibol). In this study, the development and validation of quantification LC/ESI-TI-MS methods using different standards of peptaibols (11-, 14- and 20-amino acid residues) was performed in order to quantify all of them, in a single analysis, in Trichoderma crude extracts according to their chain length. The developed and validated methods were used to study the peptaibol production kinetic of a marine-derived Trichoderma strain, i.e., Trichoderma longibrachiatum (MMS 151). The results showed the optimal culture time at the 9th day with concentrations reaching 1.4±0.2% and 2.3±0.4% of the fungal biomass respectively for 11- and 20-residue peptaibols. Then, the different peptaibol subgroups produced by 13 Trichoderma strains were quantified. According to their 18-, 19- and 20-residue peptaibol production, three strains referenced as MMS 1541, MMS 639 and MMS 151 seemed to be good candidates as potential new biological control agents with respective production of 0.4, 0.4 and 2.1%., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The laxaphyci's B family constitutes a group of five related cyclic lipopeptides isolated from diverse cyanobacteria from all around the world. This group shares a typical structure of 12 amino acids from the l and d series, some of them hydroxylated at the beta position, and all containing a rare beta-amino decanoic acid. Nevertheless, they can be differentiated due to slight variations in the composition of their amino acids, but the configuration of their alpha carbon remains conserved. Here, we provide the synthesis and characterization of new laxaphycin B-type peptides. In doing so we discuss how the synthesis of laxaphycin B and analogues was developed. We also isolate minor acyclic laxaphycins B, which are considered clues to their biosynthesis.

A concise synthetic pathway yielding to hydrophobic α,α-disubstituted disilylated amino acids based on a hydrosilylation reaction is described. As a first example of utilization in solid-phase peptide synthesis, TESDpg was introduced in replacement of Aib in an alamethicin sequence, leading to analogues with modified physicochemical properties and conserved helical structures. This study highlights the potential of these new amino acids as tools for peptide modulation.

Structured peptides gained more attention over a decade because of their biological properties, biocompatibility and ability to act as modulators of protein/protein interactions, antibiotics, analgesics, immunosuppressants or as imaging agents to cite a few relevant applications. However, their poor bioavalability due in part to the susceptibility of the peptide bond to proteolytic cleavages often impaired their development and considerably limited their therapeutic use. To circumvent these problems, many efforts are undertaken to discover stable amide bond mimics resistant to proteolytic degradation. Among them the 1,2,3-triazole emerged as a highly stable analogue of the trans-peptide bond to generate bioactive peptides. Here we report a convenient approach to readily substitute amide bonds by triazole rings in Aib-containing peptides using Aibψ[Tz]-Xaa dipeptide-like units. We defined their application in solid phase synthesis and generated short model peptide sequences to study the impact of the triazole incorporation on their conformations in solution by circular dichroism and nuclear magnetic resonance spectroscopies., (© 2015 Wiley Periodicals, Inc.)

Objectives: We studied whether plasma levels of angiogenic factors VEGF and placental growth factor (PlGF) in coronary artery disease patients or undergoing cardiac surgery are modified, and whether those factors modulate endothelial progenitor's angiogenic potential., Methods and Results: A total of 143 patients' plasmas from two different studies were analyzed (30 coronary artery disease patients, 30 patients with stable angina, coupled with 30 age and sex-matched controls; 53 patients underwent cardiac surgery). Among factors screened, only PlGF was found significantly increased in these pathological populations. PlGF-1 and PlGF-2 were then tested on human endothelial-colony-forming cells (ECFCs). We found that PlGF-1 and PlGF-2 induce VEGFR1 phosphorylation and potentiate ECFCs tubulogenesis in vitro. ECFCs VEGFR1 was further inhibited using a specific small interfering RNA (siRNA) and the chemical compound 4321. We then observed that the VEGFR1-siRNA and the compound 4321 decrease ECFCs tubulogenesis potential in vitro. Finally, we tested the compound 4321 in the preclinical Matrigel(®)-plug model with C57Bl/6J mice as well as in the murine hindlimb ischemia model. We found that 4321 inhibited the plug vascularization, attested by the hemoglobin content and the VE-Cadherin expression level and that 4321 inhibited the post-ischemic revascularization., Conclusion: PlGF plasma levels were found increased in cardiovascular patients. Disrupting PlGF/VEGFR1 pathway could modulate ECFC-induced tubulogenesis, the cell type responsible for newly formed vessels in vivo.

A diisopropylcarbodiimide/Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate) coupling cocktail was successfully incorporated into the automated microwave-assisted synthesis of two peptaibols and one analog, whose previously reported syntheses were complicated by steric hindrance. This method utilizes commercially available reagents and affords alamethicin F50/5 and bergofungin D in high yields and purities along with an appreciable reduction of synthesis time and cost when compared to previously reported methods.

Oxovanadium complexes are presented as new labels for the development of electrochemical immunosensors. The concept was successfully applied to the accurate detection of estradiol, an emerging environmental pollutant, at concentrations ranging from 4 ng L(-1) to 5 μg L(-1).

The first total synthesis of laxaphycin B was accomplished through stepwise automated Solid Phase Peptide Synthesis (SPPS), leading to the structural revision of its stereochemistry especially with regard to the configuration of one of the two 3-hydroxyleucines of this cyclic dodecapeptide of marine origin. The analogous Lyngbyacyclamide A was obtained by an extension of this synthesis.

The design, synthesis, conformational studies and binding affinity for VEGF receptors of a collection of linear and cyclic peptide analogues of the N-terminal α-helix fragments 13-25 of VEGF and 1-13 of Vammin are described. Linear 13(14)-mer peptides were designed with the help of an AGADIR algorithm and prepared following peptide solid-phase synthetic protocols. Cyclic peptide derivatives were prepared on-resin from linear precursors with conveniently located Glu and Lys residues, by the formation of amide linkages. Conformational analysis, CD and NMR, showed that most synthesized peptides have a clear tendency to be structured as α-helices in solution. Some of the peptides were able to bind a VEGFR-1 receptor with moderate affinity. In addition to the described key residues (Phe17, Tyr21 and Tyr25), Val14 and Val20 seem to be relevant for affinity.

Novel electrochemical immunosensors for sensitive detection of 17-β estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of magnetic beads (MBs) as solid support and screen-printed electrodes as sensing platforms. Four synthetic estrogen derivatives containing either a carboxylic group or an amine group at the C-3 position were synthesized and covalently bound to MBs functionalized with amine or carboxyl groups, respectively. The assay was based on competition between the free and immobilized estrogen for the binding sites of the primary antibody, with subsequent revelation using alkaline phosphatase-labeled secondary antibody. Preliminary colorimetric tests were performed in order to validate the applicability of the synthetic estrogens to immuno-recognition and to optimize different experimental parameters. In a second step, electrochemical detection was carried out by square wave voltammetry (SWV). Under the optimized working conditions, the electrochemical immunosensors showed a highly sensitive response to E2 and EE2, with respective detection limits of 1 and 10 ng/L. Cross-reactivity evaluated against other hormones demonstrated an excellent selectivity. The developed devices were successfully applied to analysis of spiked and natural water samples. These new immunosensors offer the advantages of being highly sensitive, easy, and rapid to prepare, with a short assay time.

The present study was conducted to assess the structures of the main unknown oxygenated metabolites of EAPB0203. The first step was to assign all the (1)H and (13)C NMR of both EAPB0203 and its demethylated metabolite (EAPB0202) to the corresponding atoms in their molecular structures and to elucidate the fragmentation pathways for the [M + H](+) ions of these compounds using high-resolution mass spectrometry (MS). MS/MS spectra showed that both protonated molecules possessing an even number of electrons were unexpectedly losing radicals such as H(•), CH(3)(•), or even C(7)H(7)(•) giving stable radical cations. In vitro metabolism studies were investigated in rat and dog liver microsomes and in the filamentous fungus Cunninghamella elegans. Structural elucidation of six oxygenated metabolites was performed based on the following: (i) their fragmentation pathways in liquid chromatography-MS/MS (LC-MS/MS) analyses; (ii) comparison of their changes in their molecular masses and fragment ions with those of the parent drugs; and (iii) the results of online H/D exchange experiments that provided additional evidence in differentiating hydoxylated metabolites from N-oxides. Structures of the metabolites were elucidated by LC-MS/MS and comparison with synthetic standards; structures of these standards were confirmed using one- and two-dimensional (1)H NMR spectroscopies.

Protein-protein interactions play a central role in medicine, and their modulation with small organic compounds remains an enormous challenge. Because it has been noted that the macromolecular complexes modulated to date have a relatively pronounced binding cavity at the interface, we decided to perform screening experiments over the vascular endothelial growth factor receptor (VEGFR), a validated target for antiangiogenic treatments with a very flat interface. We focused the study on the VEGFR-1 D2 domain, and 20 active compounds were identified. These small compounds contained a (3-carboxy-2-ureido)thiophen unit and had IC(50) values in the low micromolar range. The most potent compound inhibited the VEGF-induced VEGFR-1 transduction pathways. Our findings suggest that our best hit may be a promising scaffold to probe this macromolecular complex and for the development of treatments of VEGFR-1-dependent diseases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The design, synthesis, conformational studies and binding affinity for VEGFR-1 receptors of a collection of linear and cyclic peptide analogues of the β-hairpin fragment VEGF(81-91) are described. Cyclic 11-mer peptide derivatives were prepared from linear precursors with conveniently located Cys, Asp or Dap residues, by the formation of disulfide and amide bridges, using solid-phase synthesis. Molecular modelling studies indicated a tendency to be structured around the central β-turn of the VEGF(81-91) β-hairpin in most synthesized cyclic compounds. This structural behavior was confirmed by NMR conformational analysis. The NHCO cyclic derivative 7 showed significant affinity for VEGFR-1, slightly higher than the native linear fragment, thus supporting the design of mimics of this fragment as a valid approach to disrupt the VEGF/VEGFR-1 interaction., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: The application (WO2010075475) is involved in the diagnosis of angiogenesis-dependent diseases related with the soluble FMS-like tyrosine kinase-1 (sFlt-1) biomarker., Objective: It aims to identify, characterize and produce antibodies raised against sFlt-1 for the diagnosis of preeclampsia as well as other cardiovascular diseases., Methods: The diagnostic kit is based on a double mAb sandwich assay, comprising of a capture anti-sFlt-1 mAb, grafted onto paramagnetic particles that are able to bind sFlt-1 both in bound and free forms. An acridinium conjugated anti-sFlt-1 antibody, which binds to s-Flt1 on another epitope, serves as a chemioluminescent label., Results: Using this assay, the total amount of sFlt-1 could be estimated in various biological samples., Conclusion: Antibodies against the free and bound forms of sFlt-1 offer new opportunities in the diagnostics of preeclampsia and other angiogenesis-dependent disorders. Furthermore, as demonstrated in this patent, the immunoassay could be automated and is fast and reliable.

The design, synthesis and binding affinity for VEGFR-1 receptors of a small library of linear and cyclic analogues of the VEGF(81-91) fragment are described. Cyclic 11- and 10-mer peptide derivatives were prepared using parallel solid-phase protocols. The formation of hydrocarbon alkene-bridged cyclic peptides was achieved through optimized ring-closing metathesis reactions from linear derivatives with conveniently located allylGly residues. Alkane-bridged analogues were successfully obtained by ulterior on-resin hydrogenation. Binding assays showed that some of these compounds were able to compete with labeled VEGF for interaction with the VEGFR-1 receptor. Several peptide derivatives, 2, 7 and 8, showed modest but significant binding affinity, indicating that the designed peptide could mimic the VEGF(81-91) fragment and therefore disrupt the VEGF/VEGFR-1 interaction. This fact opens the way for using these peptides as the starting point for biological/pharmacological tools to deeply investigate this protein-protein system., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cyclic peptide antagonist c[YYDEGLEE]-NH(2), which disrupts the interaction between vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), represents a promising tool in the fight against cancer and age-related macular degeneration. Furthermore, coupled to a cyclen derivative, this ligand could be used as a medicinal imaging agent. Nevertheless, before generating such molecular probes, some preliminary studies need to be undertaken in order to define the more suitable positions for introduction of the cyclen macrocycle. Through an Ala-scan study on this peptide, we identified its binding motif, and an NMR study highlights its binding sites on the VEGFR-1D2 Ig-like domain. Guided by the structural relationship results deduced from the effect of the peptides on endothelial cells, new peptides were synthesized and grafted on beads. Used in a pull-down assay, these new peptides trap the VEGFRs, thus confirming that the identified amino acid positions are suitable for further derivatization.

The interaction of the vascular endothelial growth factor (VEGF) with its cellular receptors exerts a central role in the regulation of angiogenesis. Among these receptors, the VEGF receptor 1 may be implicated in pathological angiogenesis. Here, we report the first total chemical synthesis of the VEGF-binding domain of the VEGF receptor 1. Aggregation issues were overcome by the use of a low-substituted resin and the stepwise introduction of pseudoproline dipeptides and Dmb-glycines. The folding of the protein was achieved by air oxidation and its biological activity was verified on ELISA-based assays.

APA (aminopeptidase A; EC 3.4.11.7) is a membrane-bound zinc metallopeptidase, also activated by Ca(2+), involved in the formation of brain angiotensin III, which exerts a tonic stimulatory action on the central control of blood pressure in hypertensive animals. In the present study, in the three-dimensional model of the ectodomain of mouse APA, we docked the specific APA inhibitor glutamate phosphonate, in the presence of Ca(2+). The model showed the presence of one Ca(2+) atom in an hydrophilic pocket corresponding to the S1 subsite in which the lateral chain of the inhibitor is pointing. In this pocket, the Ca(2+) atom was hexaco-ordinated with the acidic side chains of Asp(213) and Asp(218), the carbonyl group of Glu(215) and three water molecules, one of them being engaged in a hydrogen bond with the negatively charged carboxylate side chain of the inhibitor. Mutagenic replacement of Asp(213) and Asp(218) with a conservative residue maintained the ability of mutated APAs to be activated by Ca(2+). However, the replacement by a non-conservative residue abolished this property, demonstrating the crucial role of these residues in Ca(2+) binding. We also showed the involvement of these residues in the strict specificity of APA in the presence of Ca(2+) for N-terminal acidic residues from substrates or inhibitors, since mutagenic replacement of Asp(213) and Asp(218) induced a decrease of the inhibitory potencies of inhibitors homologous with acidic residues. Finally, this led to the rational design of a new potent APA inhibitor, NI926 (K(i)=70 nM), which allowed us to precisely localize Asp(213) at the entrance and Asp(218) at the bottom of the S1 subsite. Taken together, these data provide new insight into the organization and functional role of the APA S1 subsite and will allow the design of pharmacophore of the inhibitor, helpful for the development of a new generation of APA inhibitors as central-acting antihypertensive agents.

Dysregulated angiogenesis is implicated in several pathologies, including cancer and age-related macular degeneration. A potential antiangiogenic strategy consists in developing VEGF receptor ligands capable of preventing VEGF binding and the subsequent activation of these receptors. Herein, we describe the structure-based design of a VEGF-mimicking peptide, VG3F. This 25-mer peptide was doubly cyclized, on-resin, by formation of both a disulfide bridge and an intramolecular amide bond to constrain it to adopt a bioactive conformation. Tested on in vitro assays, VG3F was able to prevent VEGF binding to VEGF receptor 1 and inhibit both VEGF-induced signal transduction and cell migration.

Overactivity of the brain renin-angiotensin system has been implicated in the development and maintenance of hypertension. We reported previously that angiotensin II is converted to angiotensin III by aminopeptidase A in the mouse brain. We then used specific and selective aminopeptidase A inhibitors to show that angiotensin III is one of the main effector peptides of the brain renin-angiotensin system, exerting tonic stimulatory control over blood pressure in hypertensive rats. Aminopeptidase A, the enzyme generating brain angiotensin III, thus represents a potential candidate central nervous system target for the treatment of hypertension. Given this possible clinical use of aminopeptidase A inhibitors, it was, therefore, important to investigate their pharmacological activity after oral administration. We investigated RB150, a dimer of the selective aminopeptidase A inhibitor, EC33, generated by creating a disulfide bond. This chemical modification allows prodrug to cross the blood-brain barrier when administered by systemic route. Oral administration of RB150 in conscious DOCA-salt rats inhibited brain aminopeptidase A activity, resulting in values similar to those obtained with the brains of normotensive rats, demonstrating the central bioavailability of RB150. Oral RB150 treatment resulted in a marked dose-dependent reduction in blood pressure in DOCA-salt but not in normotensive rats, with an ED(50) in the 1-mg/kg range, achieved in <2 hours and lasting for several hours. This treatment also significantly decreased plasma arginine-vasopressin levels and increased diuresis, which may participate to the blood pressure decrease by reducing the size of fluid compartment. Thus, RB150 may be the prototype of a new class of centrally active antihypertensive agents.

Angiogenesis is the development of a novel vascular network from a pre-existing structure. Blocking angiogenesis is an attractive strategy to inhibit tumor growth and metastasis formation. Based on structural and mutagenesis data, we have developed novel cyclic peptides that mimic, simultaneously, two regions of the VEGF crucial for the interaction with the VEGF receptors. The peptides, displaying the best affinity for VEGF receptor 1 on a competition assay, inhibited endothelial cell transduction pathway, migration, and capillary-like tubes formation. The specificity of these peptides for VEGF receptors was demonstrated by microscopy using a fluorescent peptide derivative. The resolution of the structure of some cyclic peptides by NMR and molecular modeling has allowed the identification of various factors accounting for their inhibitory activity. Taken together, these results validate the selection of these two regions as targets to develop molecules able to disturb the development of cancer and angiogenesis-associated diseases.

Cyclic peptides were obtained, on-resin, by the copper (I) catalysed 1,3-dipolar cycloaddition of azides and alkynes. The reaction led exclusively to the formation of the expected cyclomonomeric products which acted as ligands of the Vascular Endothelial Growth Factor receptor 1.

Aminopeptidase A (APA) is involved in the maturation of angiotensin III, a peptide which seems to be implicated in blood pressure regulation at the brain level. Therefore APA inhibitors are potential new antihypertensive agents with possible novel applications. With the aim of enhancing the bioavailability and potency of EC 33, the APA inhibitor (Ki = 300 nM) initially used in the earlier studies, we have synthesized new non-peptidic inhibitors able to interact with the S1 and S'1 subsites of the targeted enzyme. Compound 10a, (3S,4S)-3-amino-4-mercapto-6-phenyl-hexane-1-sulfonic acid was obtained using an asymmetric synthesis. Inhibitor 10a exhibits a Ki value of 30 nm.

Three zinc metallopeptidases are implicated in the regulation of fluid homeostasis and vascular tone and represent interesting targets for the treatment of chronic heart failure. We have previously reported the synthesis of a triple inhibitor able to simultaneously inhibit neprilysin (NEP, EC 3.4.24.11), angiotensin-converting enzyme (ACE, EC 3.4.15.1) and endothelin-converting enzyme (ECE-1, EC 3.4.24.71) with nanomolar potency towards NEP and ACE and a lesser affinity for ECE. Here, we report the optimization and biological activities of analogs derived from lead compound 1 (2S)-2-[(2R)-2-((1S)-5-bromo-indan-1-yl)-3-mercapto-propionylamino]-3- (1H-indol-3-yl)-propionic acid by a structural approach. Among several inhibitors, compound 21, (2S)-2-[(2R)-2-((1S)-5-bromo-indan-1-yl)-3-mercapto-propionylamino]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)-propionic acid was selected by taking into account its good molecular adaptation with the recently published structures of the three vasopeptidases. This optimization procedure led to an improved pharmacologic activity when compared with 1.

We have previously reported the design of a lead compound 1a for the joint inhibition of neprilysin (NEP, EC 3.4.24.11), angiotensin converting enzyme (ACE, EC 3.4.15.1) and endothelin converting enzyme (ECE-1, EC 3.4.24.71), three metallopeptidases which are implicated in the regulation of fluid homeostasis and vascular tone. We report here the synthesis and biological activities of analogues derived from this lead with inhibitory potencies in the nanomolar range for the three enzymes. Compounds 8b and 15c are the most potent triple inhibitors described to date.

The formation of vasoconstrictors (e.g., angiotensin II and endothelin) and the inactivation of vasodilators (e.g., bradykinin and atrial natriuretic) by membrane-bound zinc metallopeptidases are key mechanisms in the control of blood pressure and fluid homeostasis. The way in which these peptides modulate physiological functions has been intensively studied. With the aim to develop compounds that can jointly block the three metallopeptidases-neutral endopeptidase (NEP, neprilysin), angiotensin-converting enzyme (ACE), and endothelin-converting enzyme (ECE-1)-we studied the common structural specificity of the S1' subsites of these peptidases. Various mercaptoacyl amino acids of the general formula HS-CH2-CH(R1')CO-Trp-OH, possessing more or less constrained R1' side chains, were designed. The mercapto-acyl synthons contain one or two asymmetrical centers. The K(i) values of the separated stereoisomers of the most efficient inhibitors were used to determine the stereochemical preference of each enzyme. A guideline for the joint inhibition of the three peptidases was obtained with the (2R,3R) isomer of compound 13b. Its K(i) values on NEP, ACE, and ECE were 0.7, 43, and 26 nM, respectively.

Based on the recently described three-dimensional model of the 507-749 region of neprilysin, which contains the catalytic site of the enzyme, experiments were performed to improve the proposed topology of its large and hydrophobic S(')(1) subsite. Docking studies, site-directed mutagenesis, and biochemical studies were combined. The mutations of various residues proposed to be part of the S(')(1) subsite (F563A, F564A, M579A, F716A, and I718A) did not induce major structural reorganization of the active site as demonstrated by the slight modification of the enzyme activity. The mutations were also analyzed by measuring the inhibitory potencies of thiol inhibitors containing P(')(1) moieties of increasing sizes. These results combined with molecular modeling studies support the proposed topology of the S(')(1) subsite. This, and the critical role of F563 and M579 in inhibitor binding, could facilitate the synthesis of new potent and selective inhibitors.