Tea Bags for Fmoc Solid-Phase Peptide Synthesis: An Example of Circular Economy.

Peptide synthesis is an area with a wide field of application, from biomedicine to nanotechnology, that offers the option of simultaneously synthesizing a large number of sequences for the purpose of preliminary screening, which is a powerful tool. Nevertheless, standard protocols generate large volumes of solvent waste. Here, we present a protocol for the multiple Fmoc solid-phase peptide synthesis in tea bags, where reagent recycling steps are included. Fifty-two peptides with wide amino acid composition and seven to twenty amino acid residues in length were synthesized in less than three weeks. A clustering analysis was performed, grouping the peptides by physicochemical features. Although a relationship between the overall yield and the physicochemical features of the sequences was not established, the process showed good performance despite sequence diversity. The recycling system allowed to reduce N, N-dimethylformamide usage by 25-30% and reduce the deprotection reagent usage by 50%. This protocol has been optimized for the simultaneous synthesis of a large number of peptide sequences. Additionally, a reagent recycling system was included in the procedure, which turns the process into a framework of circular economy, without affecting the quality of the products obtained.

Effects of elderflower extract enriched with polyphenols on antioxidant defense of salmon leukocytes

BACKGROUND: In fish farming, the plant extracts containing antioxidant compounds have been added to the diet for enhancing pathogen resistance. In vitro studies evaluating the antioxidant effect of herbal extracts on fish cell models have focused on ROS production and the respiratory burst mechanism. However, the effects on enzymatic antioxidant defense on salmon leukocytes have not been evaluated. This study aims to evaluate the enzymatic antioxidant defense and ROS-induced cell damage in Salmon Head Kidney-1 (SHK-1) cell line exposed to polyphenol-enriched extract from Sambucus nigra flowers. RESULTS: Firstly, the Total Reactive Antioxidant Power (TRAP) assay of elderflower polyphenol (EP) was evaluated, showing 459 and 489 times more active than gallic acid and butyl hydroxy toluene (BHT), respectively. The toxic effect of EP on salmon cells was not significant at concentrations below 120 mg/ mL and no hemolysis activity was observed between 20 and 400 mg/mL. The treatment of SHK-1 cell line with EP decreased both the lipid peroxidation and protein oxidation induced by H2O2, which could be associated with decreasing oxidative stress in the SHK-1 cells since the GSH/GSSG ratio increased when only EP was added. CONCLUSIONS: These results suggest that plant extracts enriched with polyphenols could improve the enzymatic antioxidant defense of salmon leukocytes and protect the cells against ROS-induced cell damage

The Antiproliferative and Apoptotic Effect of a Novel Synthesized S-Triazine Dipeptide Series, and Toxicity Screening in Zebrafish Embryos.

Several derivatives containing morpholine/piperidine, anilines, and dipeptides as pending moieties were prepared using s-triazine as a scaffold. These compounds were evaluated for their anticancer activity against two human breast cancer cell lines (MCF-7 and MDA-MB-231), a colon cancer cell line (HCT-116), and a non-tumorigenic cell line (HEK 293). Tamoxifen was used as a reference. Animal toxicity tests were carried out in zebrafish embryos. Most of these compounds showed a higher activity against breast cancer than colon cancer. Compound 3a-which contains morpholine, aniline, and glycylglycinate methyl ester-showed a high level of cytotoxicity against MCF-7 cells with IC50 values of less than 1 µM. This compound showed a much lower level of toxicity against the non-tumorigenic HEK-293 cell line, and in the in vivo studies using zebrafish embryos. Furthermore, it induced cell cycle arrest at the G2/M phase, and apoptosis in MCF-7 cells. On the basis of our results, 3a emerges as a potential candidate for further development as a therapeutic drug to treat hormone receptor-positive breast cancer.

Di- and tri-substituted s-triazine derivatives: Synthesis, characterization, anticancer activity in human breast-cancer cell lines, and developmental toxicity in zebrafish embryos.

Here we report on a small library based on a 4-aminobenzonitile-s-triazine moiety. We used a straightforward orthogonal synthetic pathway to prepare di- and tri-substituted s-triazine derivatives, whose basic structure was modified. The newly synthesized compounds were fully characterized by 1H NMR, 13C NMR and elemental analysis. They showed strong anticancer activity against two human breast cancer cell lines (MIDA-MB-231 and MCF-7), with IC50 values less than 1 µM. These s-triazine compounds were generally more selective towards hormone receptor-positive breast cancer cell line MCF-7 than the triple negative MDA-MB-231 cell line. Zebrafish embryos were used to test the developmental toxicity of the target compounds in vivo. The phenotype of embryos treated with the derivatives resembled that of those treated with estrogen disruptors. This observation strongly supports the notion that that these compounds induce their anticancer activity in human breast cancer cells via targeting the estrogen and progesterone receptors.

Report of in vitro antileishmanial properties of Iberian macroalgae.

Here is reported the anti Leishmania infantum activity of 48 hexane, CH2Cl2 and MeOH extracts from 16 macroalgae collected on the Iberian Coast. Seven hexane and CH2Cl2 Cystoseira baccata, Cystoseira barbata, Cystoseira tamariscifolia, Cystoseira usneoides, Dictyota spiralis and Plocamium cartilagineum extracts were active towards promastigotes (IC50 29.8-101.8 µg/mL) inducing strong morphological alterations in the parasites. Hexane extracts of C. baccata and C. barbata were also active against intracellular amastigotes (IC50 5.1 and 6.8 µg/mL, respectively). Fatty acids, triacylglycerols, carotenoids, steroids and meroterpenoids were detected by nuclear magnetic resonance (NMR), and gas chromatography in the Cystoseira extracts. These results suggest that Cystoseira macroalgae contain compounds with antileishmanial activity, which could be explored as scaffolds to the development of novel sources of antiparasitic derivatives.

Identification of Peptides in Flowers of Sambucus nigra with Antimicrobial Activity against Aquaculture Pathogens.

The elder (Sambucus spp.) tree has a number of uses in traditional medicine. Previous studies have demonstrated the antimicrobial properties of elderberry liquid extract against human pathogenic bacteria and also influenza viruses. These properties have been mainly attributed to phenolic compounds. However, other plant defense molecules, such as antimicrobial peptides (AMPs), may be present. Here, we studied peptide extracts from flowers of Sambucus nigra L. The mass spectrometry analyses determined peptides of 3 to 3.6 kDa, among them, cysteine-rich peptides were identified with antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens of Chilean aquaculture. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and SYTOX Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that these peptides exert their action by destroying the bacterial membrane.

Natural Snake Venom Inhibitors and their Pharmaceutical Uses: Challenges and Possibilities.

Nowadays, treatment with specific antivenins is considered the only cure for snakebites accidents. However, access to antivenom obstructs the successful implementation of the World Health Organization international guidelines. In the last few years, natural organic compounds, peptides, and proteins with the ability to inhibit snake toxins and obtained from different sources such as plant extracts and animal blood have been proposed as antivenoms. In this work, we will focus on the inhibitors of the main venom toxins, phospholipases A2 and metalloproteinases, and their application as novel antivenoms.

On the Importance of Polyurethane and Polyurea Nanosystems for Future Drug Delivery.

BACKGROUND: During the last decade, polyurethanes and polyureas have emerged as promising alternatives to classical polyacrylate-, polyester- and polyaminoacid-based drug delivery nanosystems. They are not only biocompatible and biodegradable, but also facilitate the manufacture of polymeric nanostructured nanoparticles in quantitative yields. The versatile chemistry reduces the amount of organic solvents used and allows the straightforward multifunctionalization of polymer precursors with the desired targeting molecule at each stage of the process. OBJECTIVES: To highlight the common issues encountered in current drug delivery systems (DDSs) and the state of the art of polyurethane and polyurea polymers that self-assemble in a stratified manner by hydrophobic interactions. Finally, we discuss the importance of taking a holistic view when applying polymer nanotechnologies, in order to enhance their efficiency during preclinical and clinical studies. CONCLUSIONS: Polyurethane-polyurea nanoparticles (PUUa NPs) emerge as suitable platforms to be manufactured in a cost-effective manner at industrial scale and following environmentally friendly synthetic methods. Furthermore, they allow the controlled delivery of a wide range of drugs and can be rapidly adapted to many clinical requirements by means of FDA-approved precursors. Additionally, the ease with which PUUa nanoparticles are biodegraded ensures control over temporal aspects of drug delivery compared to other nanosystems. These advantages make PUUa NPs attractive drug delivery vehicles as long as adequate safety and ethical guidelines for new NP formulations are developed.

Short AntiMicrobial Peptides (SAMPs) as a class of extraordinary promising therapeutic agents.

The emergence of multidrug resistant bacteria has a direct impact on global public health because of the reduced potency of existing antibiotics against pathogens. Hence, there is a pressing need for new drugs with different modes of action that can kill microorganisms. Antimicrobial peptides (AMPs) can be regarded as an alternative tool for this purpose because they are proven to have therapeutic effects with broad-spectrum activities. There are some hurdles in using AMPs as clinical candidates such as toxicity, lack of stability and high budgets required for manufacturing. This can be overcome by developing shorter and more easily accessible AMPs, the so-called Short AntiMicrobial Peptides (SAMPs) that contain between two and ten amino acid residues. These are emerging as an attractive class of therapeutic agents with high potential for clinical use and possessing multifunctional activities. In this review we attempted to compile those SAMPs that have exhibited biological properties which are believed to hold promise for the future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Isololiolide, a carotenoid metabolite isolated from the brown alga Cystoseira tamariscifolia, is cytotoxic and able to induce apoptosis in hepatocarcinoma cells through caspase-3 activation, decreased Bcl-2 levels, increased p53 expression and PARP cleavage.

BACKGROUND: Brown macroalgae have attracted attention because they display a wide range of biological activities, including antitumoral properties. Inthis study we isolated isololiolide from Cystoseira tamariscifolia for the first time. PURPOSE: To examine the therapeutical potential of isololiolide against tumor cell lines. METHODS/STUDY DESIGN: The structure of the compound was established and confirmed by 1D and 2D NMR as well as HRMS spectral analysis. The in vitro cytotoxicity was analyzed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in tumoral as well as in non-tumoral cell lines. Cell cycle arrest and induction of apoptosis were assessed by flow cytometry. Alteration of expression levels in proteins important in the apoptotic cascade was analyzed by western blotting. RESULTS: Isololiolidewas isolated for the first time from the brown macroalga C.tamariscifolia. Isololiolide exhibited significant cytotoxic activity against three human tumoral cell lines, namely hepatocarcinoma HepG2 cells, whereas no cytotoxicity was found in non-malignant MRC-5 and HFF-1 human fibroblasts. Isololiolide completely disrupted the HepG2 normal cell cycle and induced significant apoptosis. Moreover, western blot analysis showed that isololiolide altered the expression of proteins that are important in the apoptotic cascade, increasing PARP cleavage and p53 expression while decreasing procaspase-3 and Bcl-2 levels. CONCLUSION: Isololiolide isolated from C. tamariscifolia is able to exert a selective cytotoxic activity on hepatocarcinoma HepG2 cells as well as induce apoptosis through the modulation of apoptosis-related proteins.

A comparative evaluation of biological activities and bioactive compounds of the seagrasses Zostera marina and Zostera noltei from southern Portugal.

This work assessed the antioxidant potential, acetylcholinesterase (AChE) inhibition and the in vitro cytotoxic activity of extracts of the seagrasses Zostera marina and Zostera noltei collected from southern Portugal. The total phenolic contents (TPCs), the rosmarinic acid (RA) concentration (HPLC/DAD) and the fatty acid (FA) profile (GC/MS) are also described. Z. marina had the highest TPC, radical scavenging activity against DPPH radicals and copper chelating activity. Z. noltei had metal chelation capacity to copper and iron ions. None of the species was able to inhibit AChE. Both seagrasses had high levels of polyunsaturated FAs. Z. marina significantly and selectively reduced the viability of tumorous neuronal cells. Z. noltei was highly toxic for the three cell lines tested and was selective against hepatocarcinoma cells at the concentration of 100 µg/mL. RA was the main compound identified in Z. marina, but not in Z. noltei.

Fatty acid profile of different species of algae of the Cystoseira genus: a nutraceutical perspective.

The fatty acid (FA) composition of six macroalgae from the Cystoseira genus, namely Cystoseira compressa, Cystoseira humilis, Cystoseira tamariscifolia, Cystoseira nodicaulis, Cystoseira baccata and Cystoseira barbata, was determined. Polyunsaturated fatty acids (PUFA) corresponded to 29-46% of the total FA detected. C. compressa, C. tamariscifolia and C. nodicaulis stood out for their low PUFA/saturated fatty acid, low n-6 PUFA/n-3 PUFA ratios as well as favourable unsaturation, atherogenicity and thrombogenicity indices, suggesting a high nutritional value with potential applications in the nutraceutical industry.

In vitro antioxidant and inhibitory activity of water decoctions of carob tree (Ceratonia siliqua L.) on cholinesterases, α-amylase and α-glucosidase.

This work reports the in vitro inhibitory activity of water decoctions of leaves, germ flour, pulp, locust bean gum and stem bark of carob tree on α-amylase, α-glucosidase, acetylcholinesterase and butyrylcholinesterase. The antioxidant activity and the chemical characterisation of the extracts made by spectrophotometric assays and by high-performance liquid chromatography are also reported. Leaves and stem bark decoctions strongly inhibited all the enzymes tested, had significant antioxidant activity and the highest total phenolics content. The major compounds were identified as gallic acid in the leaves and gentisic acid in the stem bark.

Semi-synthesis of acylated triterpenes from olive-oil industry wastes for the development of anticancer and anti-HIV agents.

A broad set of potential bioactive conjugate compounds has been semi-synthesized through solution- and solid-phase organic procedures, coupling two natural pentacyclic triterpene acids, oleanolic (OA) and maslinic acids (MA), at the hydroxyl groups of the A-ring of the triterpene skeleton, with 10 different acyl groups. These acyl OA and MA derivatives have been tested for their anti-proliferative (against the b16f10 murine melanoma cancer cells) and antiviral (as inhibitors of the HIV-1-protease) effects. Several derivatives have shown high levels of early and total apoptosis (up to 90%). Most of the compounds that exhibited anti-proliferative effects also generated ROS, probably involving the activation of an intrinsic apoptotic route. The only four compounds that did not cause the release of ROS could be related to the participation of a probable extrinsic activation of the apoptosis mechanism. A great number of these acyl OA and MA derivatives have proved to be potent inhibitors of the HIV-1-protease, the most active inhibitors having IC50 values between 0.31 and 15.6 µM, these values being between 4 and 186 times lower than their non-acylated precursors. The potent activities exhibited in the apoptosis-activation processes and in the inhibition of the HIV-1-protease by some OA and MA acylated derivatives imply that these compounds could be used as new, safe, and effective anticancer and/or antiviral drugs.

Synthesis and in vivo evaluation of the biodistribution of a 18F-labeled conjugate gold-nanoparticle-peptide with potential biomedical application.

Gold nanoparticles (AuNPs) have been extensively used in biological applications because of their biocompatibility, size, and ease of characterization, as well as an extensive knowledge of their surface chemistry. These features make AuNPs readily exploitable for biomedical applications, including drug delivery and novel diagnostic and therapeutic approaches. In a previous work, we studied ex vivo distribution of the conjugate C(AuNP)-LPFFD for its potential uses in the treatment of Alzheimer's disease. In this study, we covalently labeled the conjugate with [(18)F]-fluorobenzoate to study the in vivo distribution of the AuNP by positron emission tomography (PET). After intravenous administration in rat, the highest concentration of the radiolabeled conjugate was found in the bladder and urine with a lower proportion in the intestine, demonstrating progressive accumulation compatible with biliary excretion of the conjugate. The conjugate also accumulated in the liver and spleen. PET imaging allowed us to study the in vivo biodistribution of the AuNPs in a noninvasive and sensitive way using a reduced number of animals. Our results show that AuNPs can be covalently and radioactively labeled for PET biodistribution studies.

Phytochemical profile, antioxidant and cytotoxic activities of the carob tree (Ceratonia siliqua L.) germ flour extracts.

This work aimed to evaluate the phytochemical content and to determine the antioxidant and cytotoxic activities of methanol extracts of the carob tree (Ceratonia siliqua L.) germ flour. The extracts were rich in phenolic compounds, had considerable antioxidant activity, and reduced the viability of cervical (HeLa) cancer cells. The chemical content and the biological activities of the extracts were significantly affected by gender and cultivar. Female cultivar Galhosa had the highest levels of phenolic compounds, and the highest antioxidant activity. Extracts from the hermaphrodite trees and from the female cultivars Galhosa and Costela/Canela exhibited the highest cytotoxic activity. The most abundant compound was theophylline. The phenolic content was correlated to both antioxidant and cytotoxic activities. Our findings provide new knowledge about the health implications of consuming food supplemented with carob germ flour.

Screening of one-bead-one-peptide combinatorial library using red fluorescent dyes. Presence of positive and false positive beads.

To screen one-bead-one-compound (OBOC) combinatorial libraries, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with this molecule are then identified and isolated for compound structure determination. Here we describe an OBOC peptide library screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using the COPAS BIO-BEAD flow sorting equipment to separate fluorescent from nonfluorescent beads. The red dyes used were ATTO 590 and Texas Red. After incubating the library with the SA-red fluorescent dye conjugate, we isolated positive beads caused by peptide-SA interaction and false positive beads produced by peptide fluorescent dye interaction. These false positives were a drawback when sorting beads by COPAS. However,an in depth analysis of both kinds of beads allowed the differentiation of positives from false positives. The false positive beads showed bright homogeneous fluorescence, while positive beads had a heterogeneous fluorescence, exhibiting a characteristic halo appearance, with fluorescence intensity greatest at the bead surface and lowest in the core. The difference was more evident when using Texas Red instead of ATTO 590. Thus, positive beads could be manually separated from false positive ones. The beads were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Most of the sequences obtained from positive beads had the His-Pro-Gln motif. Peptides from false positive beads were rich in Leu/Ileu, His, Phe, and Tyr.

Conformationally restricted hydantoin-based peptidomimetics as inhibitors of caspase-3 with basic groups allowed at the S3 enzyme subsite.

By using a combination of molecular modeling, combinatorial chemistry, and biological essays, novel scaffold molecules for the inhibition of caspase-3 have been developed. These compounds have an overall attenuated negative charge and show similar IC(50) values for both recombinant and human endogenous caspase-3. This might provide the basis for a novel strategy for the discovery of potent and more druglike inhibitors of caspase-3.

Solid-phase combinatorial synthesis of a lysyl-tRNA synthetase (LysRS) inhibitory library.

The solid-phase combinatorial synthesis of a new library with potential inhibitory activity against the cytoplasmic lysyl-tRNA synthetase (LysRS) isoform of Trypanosoma brucei is described. The library has been specifically designed to mimic the lysyl adenylate complex. The design was carried out by dividing the complex into four modular parts. Proline derivatives (cis-gamma-amino-L-proline or trans-gamma-hydroxy-L-proline) were chosen as central scaffolds. After primary screening, three compounds of the library caused in vitro inhibition of the tRNA aminoacylation reaction in the low micromolar range.

Synthesis of oligonucleotide derivatives using ChemMatrix supports.

The synthesis of oligonucleotides on poly(ethylene glycol)-based (ChemMatrix) supports was studied. Results show that oligonucleotides can be indeed prepared in good yields using slightly modified synthesis cycles and automated DNA synthesizers. The use of these supports for the synthesis of oligonucleotide-peptide conjugates and for the ligation of oligonucleotides using Cu(+)-catalyzed cycloadition reactions is reported. Moreover, these supports can be used for the preparation of oligonucleotides in anhydrous solvents, followed by hybridization of the complementary sequences in aqueous buffers.