Synthesis of novel sulfide-based cyclic peptidomimetic analogues to solonamides

Eight new sulfide-based cyclic peptidomimetic analogues of solonamides A and B have been synthesized via solid-phase peptide synthesis and SN2’ reaction on a Morita–Baylis–Hillman (MBH) residue introduced at the N-terminal of a tetrapeptide. This last step takes advantage of the electrophilic feature of the MBH residue and represents a new cyclization strategy occurring. The analogues were prepared in moderate overall yields and did not show toxic effects on Staphylococcus aureus growth and were not toxic to human fibroblasts. Two of them inhibited the hemolytic activity of S. aureus, suggesting an interfering action in the bacterial quorum sensing similar to the one already reported for solonamides

Potential biological properties of lycopene in a self-emulsifying drug delivery system

In recent years, lycopene has been highlighted due to its antioxidant and anti-inflammatory properties, associated with a beneficial effect on human health. The aim of this study was to advance the studies of antioxidant and anti-inflammatory mechanisms on human keratinocytes cells (HaCaT) of a self-emulsifying drug delivery system (SEDDS) loaded with lycopene purified from red guava (nanoLPG). The characteristics of nanoLPG were a hydrodynamic diameter of 205 nm, a polydispersity index of 0.21 and a zeta potential of −20.57, providing physical stability for the nanosystem. NanoLPG demonstrated antioxidant capacity, as shown using the ORAC methodology, and prevented DNA degradation (DNA agarose). Proinflammatory activity was evaluated by quantifying the cytokines TNF-α, IL-6 and IL-8, with only IL-8 showing a significant increase (p < 0.0001). NanoLPG showed greater inhibition of the tyrosinase and elastase enzymes, involved in the skin aging process, compared to purified lycopene (LPG). In vitro treatment for 24 h with 5.0 µg/mL of nanoLPG did not affect the viability of HaCaT cells. The ultrastructure of HaCaT cells demonstrated the maintenance of morphology. This contrasts with endoplasmic reticulum stresses and autophagic vacuoles when treated with LPG after stimulation or not with LPS. Therefore, the use of lycopene in a nanoemulsion may be beneficial in strategies and products associated with skin health.info:eu-repo/semantics/publishedVersio

Intragenic antimicrobial peptides (IAPs) from human proteins with potent antimicrobial and anti-inflammatory activity

Following the treads of our previous works on the unveiling of bioactive peptides encrypted in plant proteins from diverse species, the present manuscript reports the occurrence of four proof-of-concept intragenic antimicrobial peptides in human proteins, named Hs IAPs. These IAPs were prospected using the software Kamal, synthesized by solid phase chemistry, and had their interactions with model phospholipid vesicles investigated by differential scanning calorimetry and circular dichroism. Their antimicrobial activity against bacteria, yeasts and filamentous fungi was determined, along with their cytotoxicity towards erythrocytes. Our data demonstrates that Hs IAPs are capable to bind model membranes while attaining α-helical structure, and to inhibit the growth of microorganisms at concentrations as low as 1μM. Hs02, a novel sixteen residue long internal peptide (KWAVRIIRKFIKGFISNH2) derived from the unconventional myosin 1h protein, was further investigated in its capacity to inhibit lipopolysaccharide-induced release of TNF-α in murine macrophages. Hs02 presented potent anti-inflammatory activity, inhibiting the release of TNF-α in LPSprimed cells at the lowest assayed concentration, 0.1 μM. A three-dimensional solution structure of Hs02 bound to DPC micelles was determined by Nuclear Magnetic Resonance. Our work exemplifies how the human genome can be mined for molecules with biotechnological potential in human health and demonstrates that IAPs are actual alternatives to antimicrobial peptides as pharmaceutical agents or in their many other putative applications

Lycopene-rich extract from red guava ( Psidium guajava L.) displays cytotoxic effect against human breast adenocarcinoma cell line MCF-7 via an apoptotic-like pathway

This study investigated a lycopene-rich extract from red guava (LEG) for its chemical composition using spectrophotometry, mass spectrometry, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), and computational studies. The cytotoxic activity of LEG and the underlying mechanism was studied in human breast adenocarcinoma cells (MCF-7), murine fibroblast cells (NIH-3T3), BALB/c murine peritoneal macrophages, and sheep blood erythrocytes by evaluating the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and flow cytometry. Spectrophotometry analysis showed that LEG contained 20% of lycopene per extract dry weight. Experimental and theoretical ATR-FTIR suggests the presence of lycopene, whereas MS/MS spectra obtained after fragmentation of the molecular ion [M]+• of 536.4364 show fragment ions at m/z 269.2259, 375.3034, 444.3788, and 467.3658, corroborating the presence of lycopene mostly related to all-trans configuration. Treatment with LEG (1600 to 6.25μg/mL) for 24 and 72h significantly affected the viability of MCF-7 cells (mean half maximal inhibitory concentration [IC50]=29.85 and 5.964μg/mL, respectively) but not NIH-3T3 cells (IC50=1579 and 911.5μg/mL, respectively). Furthermore LEG at concentrations from 800 to 6.25μg/mL presented low cytotoxicity against BALB/c peritoneal macrophages (IC50≥800μg/mL) and no hemolytic activity. LEG (400 and 800μg/mL) caused reduction in the cell proliferation and induced cell cycle arrest, DNA fragmentation, modifications in the mitochondrial membrane potential, and morphologic changes related to granularity and size in MCF-7 cells; however, it failed to cause any significant damage to the cell membrane or display necrosis or traditional apoptosis. In conclusion, LEG was able to induce cytostatic and cytotoxic effects on breast cancer cells probably via induction of an apoptotic-like pathway.The authors acknowledge the computational time provided by CENAPAD/SP on the project proj697. Alexandra Plácido is gratefully to FCT by her grant SFRH/BD/97995/2013, financed by POPH–QREN–Tipologia 4.1–Formação Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologia e Ensino Superior. The work at REQUIMTE/LAQV received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT) through project UID/QUI/50006/2013. Adriany das G. N. Amorim is grateful to CAPES by for the doctoral fellowship process no. 99999.004236/2014-09 in Federal University of Piauí (UFPI). Eder A. Barbosa is grateful to PNPD/CAPES for its post-doctoral fellowship.info:eu-repo/semantics/publishedVersio

In Silico, In Vitro and In Vivo Toxicological Assessment of BPP-BrachyNH2, A Vasoactive Proline-Rich Oligopeptide from Brachycephalus ephippium

BPP-BrachyNH2 is a proline-rich oligopeptide (PRO) firstly identified in skin secretion of the frog Brachycephalus ephippium, which possess in vitro inhibitory activity of angiotensin-I converting enzyme (ACE) and endothelium-dependent vasorelaxant activity. Considering its potential application in the treatment of cardiovascular diseases, the present work assessed the toxicological profile of the BPP-BrachyNH2. The in silico toxicity prediction was performed from the best model obtained through the optimization of the FASTA query peptide. This prediction study revealed that BPP-BrachyNH2 induced high predicted LD50 values for both humans and rats, and then is well-tolerated in the recommended range. The MTT assay was applied for the in vitro cytotoxic evaluation in murine macrophages. In this assay, a decrease of cell viability was not observed. The in vivo acute toxicological study was performed after the intraperitoneal administration of BPP-BrachyNH2 at doses of 5 and 50 mg/kg. After intraperitoneal administration, no death, alterations in behavioral parameters or weight gain curve was observed, as well as none in the serum biochemical parameters, and gross pathological and histopathological analyses. These observations demonstrates an acceptable safety profile for BPP-BrachyNH2, leading towards further studies focused on investigation of pharmacological and therapeutical applications for this peptide.info:eu-repo/semantics/publishedVersio

Promising self-emulsifying drug delivery system loaded with lycopene from red guava (Psidium guajava L.): in vivo toxicity, biodistribution and cytotoxicity on DU-145 prostate cancer cells

Background Self-emulsifying drug delivery systems (SEDDSs) have attracted attention because of their effects on solubility and bioavailability of lipophilic compounds. Herein, a SEDDS loaded with lycopene purified from red guava (nanoLPG) was produced. The nanoemulsion was characterized using dynamic light scattering (DLS), zeta potential measurement, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), lycopene content quantification, radical scavenging activity and colloidal stability in cell culture medium. Then, in vivo toxicity and tissue distribution in orally treated mice and cytotoxicity on human prostate carcinoma cells (DU-145) and human peripheral blood mononuclear cells (PBMC) were evaluated. Results NanoLPG exhibited physicochemical properties with a size around 200 nm, negative zeta-potential, and spherical morphology. The size, polydispersity index, and zeta potential parameters suffered insignificant alterations during the 12 month storage at 5 °C, which were associated with lycopene stability at 5 °C for 10 months. The nanoemulsion showed partial aggregation in cell culture medium at 37 °C after 24 h. NanoLPG at 0.10 mg/mL exhibited radical scavenging activity equivalent to 0.043 ± 0.002 mg Trolox/mL. The in vivo studies did not reveal any significant changes in clinical, behavioral, hematological, biochemical, and histopathological parameters in mice orally treated with nanoLPG at 10 mg/kg for 28 days. In addition, nanoLPG successfully delivered lycopene to the liver, kidney and prostate in mice, improved its cytotoxicity against DU-145 prostate cancer cells—probably by pathway independent on classical necrosis and apoptosis—and did not affect PBMC viability. Conclusions Thus, nanoLPG stands as a promising and biosafe lycopene delivery system for further development of nanotechnology-based health products

Layer-by-layer films containing peptides of the Cry1Ab16 toxin from Bacillus thuringiensis for potential biotechnological applications

Cry1Ab16 is a toxin of crystalline insecticidal proteins that has been widely used in genetically modified organisms (GMOs) to gain resistance to pests. For the first time, in this study, peptides derived fromthe immunogenic Cry1Ab16 toxin (from Bacillus thuringiensis) were immobilized as layer-by-layer (LbL) films. Given the concern about food and environmental safety, a peptide with immunogenic potential, PcL342–354C, was selected for characterization of the electrochemical, optical, and morphological properties. The results obtained by cyclic voltammetry (CV) showed that the peptide have an irreversible oxidation process in electrolyte of 0.1 mol·L−1 potassiumphosphate buffer (PBS) at pH7.2. Itwas also observed that the electrochemical response of the peptide is governed mainly by charge transfer. In an attempt to maximize the electrochemical signal of peptide, it was intercalated with natural (agar, alginate and chitosan) or synthetic polymers (polyethylenimine (PEI) and poly(sodium 4-styrenesulfonate (PSS)). The presence of synthetic polymers on the film increased the electrochemical signal of PcL342–354C up to 100 times. Images by Atomic Force Microscopy (AFM) showed that the immobilized PcL342–354C formed self-assembled nanofibers with diameters ranging from 100 to 200 nm on the polymeric film. By UV–Visible spectroscopy (UV–Vis) it was observed that the ITO/PE/PSS/PcL342–354C film grows linearly up to the fifth layer, thereafter tending to saturation. X-ray diffraction confirmed the presence on the films of crystalline ITO and amorphous polypeptide phases. In general, the ITO/PEI/PSS/PcL342–354C film characterization proved that this systemis an excellent candidate for applications in electrochemical sensors and other biotechnological applications for GMOs and environmental indicators.info:eu-repo/semantics/publishedVersio

Structure-function studies of BPP-BrachyNH2 and synthetic analogues thereof with angiotensin I-converting enzyme

The vasoactive proline-rich oligopeptide termed BPP-BrachyNH2 (H-WPPPKVSP-NH2) induces in vitro inhibitory activity of angiotensin I-converting enzyme (ACE) in rat blood serum. In the present study, the removal of N-terminal tryptophan or C-terminal proline from BPP-BrachyNH2 was investigated in order to predict which structural components are important or required for interaction with ACE. Furthermore, the toxicological profile was assessed by in silico prediction and in vitro MTT assay. Two BPP-BrachyNH2 analogues (des-Trp(1)-BPP-BrachyNH2 and des-Pro(8)-BPP-BrachyNH2) were synthesized, and in vitro and in silico ACE inhibitory activity and toxicological profile were assessed. The des-Trp(1)-BPP-BrachyNH2 and des-Pro(8)-BPP-BrachyNH2 were respectively 3.2- and 29.5-fold less active than the BPP-BrachyNH2-induced ACE inhibitory activity. Molecular Dynamic and Molecular Mechanics Poisson-Boltzmann Surface Area simulations (MM-PBSA) demonstrated that the ACE/BBP-BrachyNH2 complex showed lower binding and van der Wall energies than the ACE/des-Pro(8)-BPP-BrachyNH2 complex, therefore having better stability. The removal of the N-terminal tryptophan increased the in silico predicted toxicological effects and cytotoxicity when compared with BPP-BrachyNH2 or des-Pro(8)-BPP-BrachyNH2. Otherwise, des-Pro(8)-BPP-BrachyNH2 was 190-fold less cytotoxic than BPP-BrachyNH2. Thus, the removal of C-terminal proline residue was able to markedly decrease both the BPP-BrachyNH2-induced ACE inhibitory and cytotoxic effects assessed by in vitro and in silico approaches. In conclusion, the aminoacid sequence of BPP-BrachyNH2 is essential for its ACE inhibitory activity and associated with an acceptable toxicological profile. The perspective of the interactions of BPP-BrachyNH2 with ACE found in the present study can be used for development of drugs with differential therapeutic profile than current ACE inhibitors

Antiviral Action against SARS-CoV-2 of a Synthetic Peptide Based on a Novel Defensin Present in the Transcriptome of the Fire Salamander (<i>Salamandra salamandra</i>)

The potential emergence of zoonotic diseases has raised significant concerns, particularly in light of the recent pandemic, emphasizing the urgent need for scientific preparedness. The bioprospection and characterization of new molecules are strategically relevant to the research and development of innovative drugs for viral and bacterial treatment and disease management. Amphibian species possess a diverse array of compounds, including antimicrobial peptides. This study identified the first bioactive peptide from Salamandra salamandra in a transcriptome analysis. The synthetic peptide sequence, which belongs to the defensin family, was characterized through MALDI TOF/TOF mass spectrometry. Molecular docking assays hypothesized the interaction between the identified peptide and the active binding site of the spike WT RBD/hACE2 complex. Although additional studies are required, the preliminary evaluation of the antiviral potential of synthetic SS-I was conducted through an in vitro cell-based SARS-CoV-2 infection assay. Additionally, the cytotoxic and hemolytic effects of the synthesized peptide were assessed. These preliminary findings highlighted the potential of SS-I as a chemical scaffold for drug development against COVID-19, hindering viral infection. The peptide demonstrated hemolytic activity while not exhibiting cytotoxicity at the antiviral concentration

Antioxidant and Neuroprotective Effects of the First Tryptophyllin Found in Snake Venom (Bothrops moojeni)

In this study, we report the isolation, characterization, and synthesis of the peptide BmT-2 belonging to the tryptophyllins family, isolated from the venom of the snake Bothrops moojeni. This is the first time a tryptophyllin is identified in snake venom. We tested whether BmT-2 had cytotoxic effects and antioxidant activity in a set of experiments that included both in vitro and cell-based assays. BmT-2 presented a radical scavenging activity toward ABTS• and AAPH-derived radicals. BmT-2 protected fluorescein, DNA molecules, and human red blood cells (RBCs) from free radicals generated by the thermal decomposition of AAPH. The novel tryptophyllin was not toxic in cell viability tests, where it (up to 0.4 mg/mL) did not cause hemolysis of human RBCs and did not cause significant loss of cell viability, showing a CC50 > 1.5 mM for cytotoxic effects against SK-N-BE(2) neuroblastoma cells. BmT-2 prevented the arsenite-induced upregulation of Nrf2 in Neuro-2a neuroblasts and the phorbol myristate acetate-induced overgeneration of reactive oxygen species and reactive nitrogen species in SK-N-BE(2) neuroblastoma cells. Electronic structure calculations and full atomistic reactive molecular dynamics simulations revealed the relevant contribution of aromatic residues in BmT-2 to its antioxidant properties. Our study presents a novel peptide classified into the family of the tryptophyllins, which has been reported exclusively in amphibians. Despite the promising results on its antioxidant activity and low cytotoxicity, the mechanisms of action of BmT-2 still need to be further elucidated