Smac-derived Aza-peptide As an Aminopeptidase-resistant XIAP BIR3 Antagonist

The peptidic nature of anti-IAPs N-terminus Smac-derived peptides precludes their utilization as potential therapeutic anticancer agents. Recent advances in the development of novel Smac-derived peptidomimetics and non-peptidic molecules with improved anti-IAPs activity and resistance to proteolytic cleavage have been reported and led to a number of candidates that are currently in clinical trials including LCL-161, SM-406/AT-406, GDC-0512/GDC-0917, and birinapant. As an attempt to improve the proteolytic stability of Smac peptides, we developed the Aza-peptide AzaAla-Val-Pro-Phe-Tyr-NH2 (2). Unlike unmodified peptide Ala-Val-Pro-Phe-Tyr-NH2 (1), analogue (2) exhibited resistance towards proteolytic cleavage by two aminopeptidases; LAP and DPP-IV, while retaining its IAP inhibitory activity. This was due to the altered planar geometry of the P1 residue side chain. Our findings showed that using aza-isosteres of bioactive peptide sequences imbue the residue with imperviousness to proteolysis; underscoring a potential approach for developing a new generation of Smac-derived Aza-peptidomimetics.

Induction of the inflammatory regulator A20 by gibberellic acid in airway epithelial cells

BACKGROUND AND PURPOSE: NF-κB-driven inflammation is negatively regulated by the zinc finger protein A20. Gibberellic acid (GA3 ) is a plant-derived diterpenoid with documented anti-inflammatory activity, which is reported to induce A20-like zinc finger proteins in plants. Here, we sought to investigate the anti-inflammatory effect of GA3 in airway epithelial cells and determine if the anti-inflammatory action relates to A20 induction.

EXPERIMENTAL APPROACH: Primary nasal epithelial cells and a human bronchial epithelial cell line (16HBE14o-) were used. Cells were pre-incubated with GA3 , stimulated with Pseudomonas aeruginosa LPS; IL-6 and IL-8 release, A20, NF-κB and IκBα expression were then evaluated. To determine if any observed anti-inflammatory effect occurred via an A20-dependent mechanism, A20 was silenced using siRNA.

KEY RESULTS: Cells pre-incubated with GA3 had significantly increased levels of A20 mRNA (4 h) and protein (24 h), resulting in a significant reduction in IL-6 and IL-8 release. This effect was mediated via reduced IκBα degradation and reduced NF-κB (p65) expression. Furthermore, the anti-inflammatory action of GA3 was abolished in A20-silenced cells.

CONCLUSIONS AND IMPLICATIONS: We showed that A20 induction by GA3 attenuates inflammation in airway epithelial cells, at least in part through its effect on NF-κB and IκBα. GA3 or gibberellin-derived derivatives could potentially be developed into anti-inflammatory drugs for the treatment of chronic inflammatory diseases associated with A20 dysfunction.

Randomized Phase II trial of nintedanib, afatinib and sequential combination in castration-resistant prostate cancer

AIMS: The aim of this article was to evaluate afatinib (BIBW 2992), an ErbB family blocker, and nintedanib (BIBF 1120), a triple angiokinase inhibitor, in castration-resistant prostate cancer patients.

PATIENTS & METHODS: Patients were randomized to receive nintedanib (250 mg twice daily), afatinib (40 mg once daily [q.d.]), or alternating sequential 7-day nintedanib (250 mg twice daily) and afatinib (70 mg q.d. [Combi70]), which was reduced to 40 mg q.d. (Combi40) due to adverse events. The primary end point was progression-free rate at 12 weeks.

RESULTS: Of the 85 patients treated 46, 20, 16 and three received nintedanib, afatinib, Combi40 and Combi70, respectively. At 12 weeks, the progression-free rate was 26% (seven out of 27 patients) for nintedanib, and 0% for afatinib and Combi40 groups. Two patients had a ≥50% decline in PSA (nintedanib and the Combi40 groups). The most common drug-related adverse events were diarrhea, nausea, vomiting and lethargy.

CONCLUSION: Nintedanib and/or afatinib demonstrated limited anti-tumor activity in unselected advanced castration-resistant prostate cancer patients.

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

Phylloseptin-PBa—A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities.

Naturally-occurring TGR5 agonists modulating glucagon-like peptide-1 biosynthesis and secretion

Selective GLP-1 secretagogues represent a novel potential therapy for type 2 diabetes mellitus. This study examined the GLP-1 secretory activity of the ethnomedicinal plant, Fagonia cretica, which is postulated to possess anti-diabetic activity. After extraction and fractionation extracts and purified compounds were tested for GLP-1 and GIP secretory activity in STC-1 pGIP/neo cells. Intracellular levels of incretin hormones and their gene expression were also determined. Crude F. cretica extracts stimulated both GLP-1 and GIP secretion, increased cellular hormone content, and upregulated gene expression of proglucagon, GIP and prohormone convertase. However, ethyl acetate partitioning significantly enriched GLP-1 secretory activity and this fraction underwent bioactivity-guided fractionation. Three isolated compounds were potent and selective GLP-1 secretagogues: quinovic acid (QA) and two QA derivatives, QA-3β-O-β-D-glycopyranoside and QA-3β-O-β-D-glucopyranosyl-(28→1)-β-D-glucopyranosyl ester. All QA compounds activated the TGR5 receptor and increased intracellular incretin levels and gene expression. QA derivatives were more potent GLP-1 secretagogues than QA. This is the first time that QA and its naturally-occurring derivatives have been shown to activate TGR5 and stimulate GLP-1 secretion. These data provide a plausible mechanism for the ethnomedicinal use of F. cretica and may assist in the ongoing development of selective GLP-1 agonists.

Caracterização química e actividade biológica dos óleos essenciais e extractos aquosos de algumas Lavandula spp. do Sul de Portugal

No presente trabalho estudaram-se três espécies do género Lavandula, do sul de Portugal: L. luisieri e L. pedunculata (Alentejo) e L.viridis (Algarve), com vista caraterizar a composição química dos óleos essenciais das diferentes espécies, parte vegetativa (folha) e espiga floral, e dos respetivos extratos de hidrodestilação, e avaliar as propriedades antioxidante, antimicrobiana, toxicológica, analgésica e antiinflamatória de alguns dos seus óleos essenciais e extratos aquosos, mais promissores. Os resultados mostraram importantes diferenças na composição química dos óleos essenciais, quanto à diversidade e à proporção dos seus constituintes. Os óleos essenciais e hidrolatos, em estudo, apresentaram importantes propriedades antioxidantes e antimicrobianas. Os estudos toxicológicos e farmacológicos mostraram que os óleos apresentaram citotoxicidade em Artemia salina, apresentaram valores de DL50 muito superiores a 2000 mg/kg em ratinhos Swiss, e que possuem importantes propriedades analgésicas e anti-inflamatória. Estes resultados sugerem o seu potencial uso para aplicações farmacológicas como agentes nutracêuticos e/ou fitoterapêuticos; ABSTRACT:The aim of present work was to develop a set of studies of three species of the genera Lavandula, at the South of Portugal: L. luisieri e L. pedunculata (Alentejo) e L.viridis (Algarve), in order to characterize the chemical composition of the essential oils (leaves and flowers) and to evaluate the antioxidant, antimicrobial, toxicological and pharmacological properties of selected essential oils and aqueous extracts. Results show important differences in chemical composition of essential oils, both in diversity as the proportion of their constituents. Essential oils and aqueous extracts of different Lavandula spp. showed important antimicrobial and antioxidant properties. Pharmacological studies have shown that essential oils showed cytotoxicity against Artemia salina, low acute toxicity, with LD50 >> 2000 mg/kg for mice, and important analgesic and anti-inflammatory properties. These results suggest their potential use for pharmacological applications as nutraceutical and/or phytotherapeutic agents.

Synthesis of new Aβ-ligands useful in diagnosis of Alzheimer's disease

Alzheimer’s disease is a chronic progressive neurodegenerative disease and is the most common form of dementia (estimated 50−60% of all cases), associated with loss of memory (in particular episodic memory), cognitive decline, and behavioural and physical disability, ultimately leading to death. Alzheimer’s disease is a complex disease, mostly occurring sporadically with no apparent inheritance and being the age the main risk factor. The production and accumulation of amyloid-beta peptide in the central nervous system is a key event in the development of Alzheimer’s disease. This project is devoted to the synthesis of amyloid-beta ligands, fluorophores and blood brain barrier-transporters for diagnosis and therapy of Alzheimer’s disease. Different amyloid-beta ligands will be synthesized and their ability to interact with amyloid-beta plaques will be studied with nuclear magnetic resonance techniques and a process of lead optimization will be performed. Many natural and synthetic compounds able to interact as amyloid-beta ligands have been identified. Among them, a set of small molecules in which aromatic moieties seem to play a key role to inhibit amyloid-beta aggregation, in particular heteroaromatic polycyclic compounds such as tetracyclines. Nevertheless tetracyclines suffer from chemical instability, low water solubility and possess, in this contest, undesired anti-bacterial activity. In order to overcome these limitations, one of our goals is to synthesize tetracyclines analogues bearing a polycyclic structure with improved chemical stability and water solubility, possibly lacking antibacterial activity but conserving the ability to interact with amyloid-beta peptides. Known tetracyclines have in common a fourth cycle without an aromatic character and with different functionalisations. We aim to synthesize derivatives in which this cycle is represented by a sugar moiety, thus bearing different derivatisable positions or create derivatives in which we will increase or decrease the number of fused rings. In order to generate a potential drug-tool candidate, these molecules should also possess the correct chemical-physical characteristics. The glycidic moiety, not being directly involved in the binding, it assures further possible derivatizations, such as conjugation to others molecular entities (nanoparticles, polymeric supports, etc.), and functionalization with chemical groups able to modulate the hydro/lipophilicity. In order to be useful such compounds should perform their action within the brain, therefore they have to be able to cross the blood brain barrier, and to be somehow detected for diagnostic purposes.

Evaluation of the in vitro biological activities of extracts from carob tree and mediterranean oaks

Dissertação mest., Engenharia Biológica, Universidade do Algarve, 2009

Searching biocompounds in marine natural extracts with potential use in tumor prevention and treatment

Cancer is a multistage process characterized by three stages: initiation, promotion and progression; and is one of the major killers worldwide. Oxidative stress acts as initiator in tumorigenesis; chronic inflammation promotes cancer; and apoptosis inactivation is an issue in cancer progression. In this study, it was investigated the antioxidant, antiinflammatory and antitumor properties of hexane, ether, chloroform, methanol and water extracts of five species of halophytes: A. macrostachyum, P. coronopus, J. acutus, C. edulis and A. halimus. Antioxidant activity was assessed by DPPH• and ABTS•+ methods, and the total phenolics content (TPC) was evaluated by the Folin-Ciocalteau method. The anti-inflammatory activity of the extracts was determined by the Griess method, and by evaluating the inhibition of NO production in LPS-stimulated RAW- 264.7 macrophages. The cytotoxic activity of the extracts against HepG2 and THP1 cell lines was estimated by the MTT assay, and the results obtained were further compared with the S17 non-tumor cell line. The induction of apoptosis of J. acutus ether extract was assessed by DAPI staining. The highest antioxidant activities was observed in C. edulis methanol and the J. acutus ether extracts against the DPPH• radical; and J. acutus ether and A. halimus ether extracts against the ABTS•+ radical. The methanol extracts of C. edulis and P. coronopus, and the ether extract of J. acutus revealed a high TPC. Generally the antioxidant activity had no correlation with the TPC. The A. halimus chloroform and P. coronopus hexane extracts demonstrated ability to reduce NO production in macrophages (> 50%), revealing their anti-inflammatory capacity. The ether extract of J. acutus showed high cytotoxicity against HepG2 cancer cells, with reduced cellular viability even at the lowest concentrations. This outcome was significantly lower than the obtained with the non-tumor cells (S17). This result was complemented by the induction of apoptosis.

Antioxidant activities of eight Algerian plant extracts and two essential oils

Total phenol, hydroxycinnamic acid derivatives, flavone/flavonol and flavanones/dihydroflavonol contents of hydro-alcoholic extracts, obtained by sonication, from the aerial parts of Artemisia campestris L., Anthemis arvensis L., Haloxylon scoparium Pomel, Juniperus phoenicea L., Arbutus unedo L., Cytisus monspessulanus L., Thymus algeriensis Boiss et Reut, Zizyphus lotus L (Desf.) collected in Djebel Amour (Sahara Atlas, Algeria) were quantified by spectrophotometric methods. The chemical composition of the essential oils obtained by hydrodistillation from Artemisia campestris L. and Juniperus phoenicea I aerial parts were also evaluated by gas chromatography (GC) and gas chromatography coupled to mass spectrometry (GC-MS). The antioxidant activity of the extracts and essential oils was assessed measuring the capacity for preventing lipid peroxidation using two lipidic substrates (egg yolk and liposomes), the capacity for scavenging DPPH, ABTS, superoxide anion radicals, hydroxyl radicals and peroxyl radicals. Anti-inflammatory activity was assessed by measuring the capacity for inhibiting lipoxygenase. Reducing power and chelating capacity were also assayed. The results showed different amounts of total phenols depending on the method used: A. campestris extract had the highest levels of total phenols when the measurement was made at lambda = 280 nm, whereas H. scoparium and A. unedo extracts showed the highest levels of total phenols with Folin-Ciocalteau. C. monspessulanus had the highest levels of flavones/flavonols and flavanones/dihydroflavonols. The essential oils of A. campestris and J. phoenicea were mainly constituted by alpha-pinene, beta-pinene and sabinene; and a-pinene, respectively. The methods used for assaying the capacity for preventing lipid peroxidation revealed to be inadequate for extracts due to the great interferences detected. The essential oils were more active than the generality of extracts for scavenging peroxyl radicals and for inhibiting lipoxygenase, whereas A. unedo extract was the most active for scavenging ABTS, DPPH, superoxide anion radicals and it also had the best reducing capacity. In a general way, the great majority of the antioxidant activities correlated well with the phenol content although such correlation was not so clear with the flavonoid content. (c) 2013 Elsevier B.V. All rights reserved.

Activities of an aqueous plant extract against Herpes simplex virus type 2 (HSV 2): role in virus replication and progeny yield

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2015

In-situ development of self-defensive antibacterial biomaterials: phenol-g-keratin-EC based bio-composites with characteristics for biomedical applications

Recently, the development of highly inspired biomaterials with multi-functional characteristics has gained considerable attention, especially in biomedical, and other health-related areas of the modern world. It is well-known that the lack of antibacterial potential has significantly limited biomaterials for many challenging applications such as infection free wound healing and/or tissue engineering etc. In this perspective, herein, a series of novel bio-composites with natural phenols as functional entities and keratin-EC as a base material were synthesised by laccase-assisted grafting. Subsequently, the resulting composites were removed from their respective casting surfaces, critically evaluated for their antibacterial and biocompatibility features and information is also given on their soil burial degradation profile. In-situ synthesised phenol-g-keratin-EC bio-composites possess strong anti-bacterial activity against Gram-positive and Gram-negative bacterial strains i.e., B. subtilis NCTC 3610, P. aeruginosa NCTC 10662, E. coli NTCT 10418 and S. aureus NCTC 6571. More specifically, 10HBA-g-keratin-EC and 20T-g-keratin-EC composites were 100% resistant to colonisation against all of the aforementioned bacterial strains, whereas, 15CA-g-keratin-EC and 15GA-g-keratin-EC showed almost negligible colonisation up to a variable extent. Moreover, at various phenolic concentrations used, the newly synthesised composites remained cytocompatible with human keratinocyte-like HaCaT, as an obvious cell ingrowth tendency was observed and indicated by the neutral red dye uptake assay. From the degradation point of view, an increase in the degradation rate was recorded during their soil burial analyses. Our investigations could encourage greater utilisation of natural materials to develop bio-composites with novel and sophisticated characteristics for potential applications.

Blacklisting and its legacy in the UK construction industry: employment relations in the aftermath of exposure of the Consulting Association

This paper explores responses to the exposure of blacklisting in the UK construction industry in the period following the closure of the Consulting Association (CA) in 2009. It asks whether employer collusion to blacklist in this way has been terminated and concludes that it is now largely of historical interest although other forms of anti-union activity continue. It highlights particularly the historic and continuing importance of ‘double breasting’ and reports on divergent employer paths in the aftermath of the exposure and subsequent closure of the activities of the CA.

Efeito da combinação de antibióticos e sinvastatina sobre microrganismos de interesse endodôntico e na expressão de marcadores odontoblásticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)