CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / A substÃncia 1-(4-Nitrofenil)-3-fenilprop-2-en-1-ona (CG) Ã um derivado de chalcona, sintetizado a partir da reaÃÃo quÃmica entre a acetofenona e para-nitro benzaldeÃdo. Para avaliar o seu potencial anticÃncer foi realizado um estudo farmacolÃgico de suas propriedades antitumorais em vÃrios modelos biolÃgicos in vitro e in vivo. A CG apresentou potente atividade citotÃxica nas 5 linhagens tumorais testadas, inibindo a proliferaÃÃo das cÃlulas tumorais pelo ensaio do MTT e em cÃlulas mononucleares do sangue perifÃrico (PMCB) humano atravÃs do ensaio do Alamar blue. Todas as linhagens mostraram sensibilidade ao tratamento com a CG, e a CI50 variou de 1,18ÂM em HCT-8 a 3,32ÂM em SF-295. O composto apresentou fraca citotoxicidade (CI50 igual a 7,07ÂM) nas cÃlulas PBMC, com exposiÃÃo a CG em 72h, em relaÃÃo Ãs cÃlulas de HL-60, utilizada como modelo nos demais testes biolÃgicos. O tempo de encubaÃÃo com o composto foi de 24h na maioria dos experimentos. Adicionalmente, a CG nÃo induziu efeitos hemolÃticos. O ensaio de exclusÃo por azul de Tripan revelou diminuiÃÃo da viabilidade celular principalmente apÃs 24h na maior concentraÃÃo testada (4ÂM) com 58,4%. Para os testes de atividade antiproliferativa, LA/BE mostrou em sua morfologia cÃlulas em apoptose nas duas maiores concentraÃÃes, enquanto que o BrdU, apresentou incorporaÃÃo do mesmo nas concentraÃÃes testadas. A morfologia analisada por May-Grunwald-Giemsa mostrou reduÃÃo do volume celular, condensaÃÃo da cromatina e fragmentaÃÃo nuclear. Adicionalmente, a CG induziu apoptose em cÃlulas leucÃmicas HL-60, com participaÃÃo das vias intrÃnseca e maior estÃmulo da via extrÃnseca, de maneira concentraÃÃo-dependente, como observado na integridade da membrana citoplasmÃtica, aumento da fragmentaÃÃo do DNA e externalizaÃÃo da fosfatidilserina. Na anÃlise do ciclo celular, foi observado parada na fase G2/M, sendo ativada as caspases 3, 7, 8 e 9 (a Ãltima na maior concentraÃÃo e confirmada pelo teste do Western blot). NÃo houve ativaÃÃo do Citocromo c. A CG nÃo foi capaz de induzir processos genotÃxicos/ mutagÃnicos (testes do cometa e micronÃcleo in vitro). No ensaio de atividade antitumoral in vivo, observou-se inibiÃÃo tumoral nas doses testadas (25 e 50mg/Kg/dia, via oral) de 54,85 e 69,11% respectivamente. As doses de CG causaram tumefaÃÃo celular e o surgimento de focos inflamatÃrios no parÃnquima ou estroma hepÃtico/renal, necrose nefrotÃxica focal, esteatose microvesicular, pigmentos de hemossiderina, hiperplasia das cÃlulas de Kupffer, congestÃo da polpa vermelha e desorganizaÃÃo dos folÃculos linfÃides esplÃnicos. AlÃm disso, os Ãndices bioquÃmicos mostraram aumento do AST e diminuiÃÃo da urÃia (CG 25mg/Kg/dia), diminuiÃÃo do ALT (5-FU e CG 25mg/Kg/dia); as alteraÃÃes hematolÃgicas mostraram leucopenia e plaquetopenia (5-FU), aumento dos leucÃcitos totais (CG 50mg/Kg/dia), aumento de neutrÃfilos e linfÃcitos em todos os grupos tratados. Todos os resultados nos levam a enfatizar que a CG possui grande potencialidade como molÃcula promissora por suas propriedades anticÃncer. / The substance 1- (4-Nitrofenil)-3- fenilprop-2- en-1-ona (CG) is a chalcone derivative, synthesized from a chemical reaction between acetophenone and p-nitro benzaldehyde. To evaluate its anticancer potential a pharmacological study of its antitumor properties in selected biological models in vitro e in vivo. CG presented a powerful cytotoxic activity in the 5 tested tumor lines evaluated, inhibiting cell proliferation of the tumor lines in the MTT assay and human peripheral mononuclear blood cells (PMBC) through the Alamar Blue assay. All cell lines showed sensitivity to the treatment with the CG, and the IC50 varied from 1,18 ÂM in HCT-8 to 3,32 ÂM in SF-295. The sample presented weak cytotoxic effect (IC50 of 7,07 ÂM) in cells PMBC, with 72h exposure to CG, compared to HL-60 cells (leukemic cell line), used in the next biological tests. The sample was incubated with the cells during 24h for the majority of the experiments. Additionally, CG did not induce hemolytic effects. The Tripan Blue assay showed a decrease of the cellular viability especially after 24h of incubation of the higher tested concentration (4 ÂM) with 58,4%. In assays for antiproliferative activity, OA/BE showed in its morphology cells going under apoptosis in the two higher concentrations, whereas the BrdU assay, presented incorporation of the same in the tested concentrations. The morphology analyzed with the May-Grunwald-Giemsa stain showed a decrease of the cellular volume, chromatin condensation and nuclear fragmentation.CG induced apoptosis in HL-60 cells, with participation of the intrinsic pathway and major stimulation of the extrinsic pathway, in a concentration-dependent manner, as observed in the cytoplasmatic membrane integrity, increase of DNA fragmentation and outsourcing of phosphatidylserine. In the cellular cycle analysis, it was observed a stop in the G2/M phase, activating caspases 3, 7, 8 and 9 (the last one in the highest concentration and confirmed by the Western blot assay). It was not observed activation of Cytochrome c. CG was not capable to induce mutagenic/genotoxic processes (comet assay and micronucleus in vitro). In the in vivo antitumor activity assay, tumor inhibition was observed in the tested doses (25 and 50mg/Kg/day, oral intake) of 54,85 and 69,11%, respectively . The doses of CG caused cellular swelling and the arise of inflammatory focus in the parenchyma or hepatic/renal stroma, focal nephrotoxic necrosis, microvesicular steatosis, hemosiderin pigments, hyperplasia of Kupffer cells, congestion of the red pulp and disorganization of the splenic lymphoid follicles. Furthermore, the biochemical indices had shown increase of AST and reduction of urea (25mg/Kg/day of CG), reduction of ALT (25mg/Kg/day of 5-FU and CG); hematologic alterations showed leukopenia and thrombocytopenia (5-FU), increase of total leukocytes (50mg/Kg/day of CG), increase of neutrophils and lymphocytes in all treated groups. All results led us to emphasize that CG possesses great potential as a promising molecule for its anticancer properties.

Labaditina é um decapeptídeo cíclico, hidrofóbico, extraído da Jatropha Multifida, uma planta da família Euphorbiaceae. É mais resistente à degradação proteolítica que seus respectivos isômeros lineares; e forma pontes de hidrogênio internamente, facilitando sua inserção em membrana biológica. Estudos tem mostrado que a restrição conformacional dos peptídeos cíclicos aumenta sua afinidade e especificidade à membrana. Devido à essas características físicas e às atividades biológicas apresentadas, tais como inibição da via clássica do sistema complemento humano in vitro e atividade antibacteriana para Streptococcus mutans, este peptídeo tem ganhado interesse biológico e farmacológico. Sobretudo, ainda não é conhecido seu mecanismo de ação. Devido à isso, os peptídeos Labaditina (Lo) e o análogo linear (L1), estruturalmente diferentes, foram estudados com o objetivo de obter informações quanto ao mecanismo de ação, interação e possíveis alterações estruturais frente a membranas biológicas. O comportamento do Lo e L1 foi avaliado na presença de diferentes composições de lipídios (DPPC, DPPC:Chol (9:1), DPPC:DPPS (8:2)) e de detergentes (SDS e LPC), utilizando sistemas miméticos de membrana: monocamada, micela e lipossomo. Em monocamada, sistema planar, foi observado um aumento da pressão superficial, provavelmente causado pela presença de peptídeo. Nos sistemas compostos por DPPC:Chol e DPPC:DPPS o efeito foi maior na presença do L1, sugerindo interação eletrostática entre o peptídeo e as monocamadas. Já o peptídeo Lo, por não possuir carga, apresentou maior interação com a monocamada de DPPC, por ser zwitteriônica. Resultados similares foram obtidos através do estudo com lipossomos constituídos por DPPC, DPPC:Chol (9:1) e DPPC:DPPS (8:2). Em todos os meios, através da espectrofotometria de fluorescência, foi observado um blue-shift, ou seja, migração do triptofano para um ambiente mais apolar. Para o Lo, isso foi maior na presença de DPPC; para o L1, na presença de DPPC:Chol e DPPC:DPPS. Através do DSC foi observado um aumento da entalpia e diminuição da cooperatividade (t1/2), causado pela presença de peptídeo na bicamada. Em DPPC:Chol (9:1) e DPPC:DPPS esse efeito foi maior na presença do L1; e em DPPC, na presença do Lo, confirmando os resultados anteriores. Essas interações peptídeo-mimético de membrana foram acompanhadas por mudanças conformacionais, observadas através do CD. O peptídeo Lo, tanto em meio aquoso, quanto na presença dos diferentes lipossomos está não-ordenado, entretanto, possui diferenças conformacionais em cada meio. O peptídeo L1 em meio aquoso apresenta estrutura ao acaso com interação entre os triptofanos, porém em DPPC e em DPPC:Chol (9:1) sofre alteração conformacional, distanciando os triptofanos; em DPPC:DPPS (8:2) sofreu alteração para -folha. Isso demonstra que a composição lipídica induz diferentes conformações nos peptídeos e pode afetar seu mecanismo de ação. No estudo com micelas também foi observado interação de ambos os peptídeos com SDS, e também com LPC. Em SDS os estudos sugerem que o L1 está mais inserido no meio apolar que o Lo; já em LPC, o Lo. Esses peptídeos também apresentaram alteração conformacional na presença das micelas. O peptídeo Lo, tanto em SDS, quanto em LPC, apresentou conformação não-ordenada, porém diferentes. Já o peptídeo L1 apresentou conformação -folha na presença de SDS e LPC, porém também com diferenças. Os resultados demonstram que o peptídeo com estrutura linear (L1) possui maior liberdade conformacional. Portanto, alguns fatores dirigem o processo de interação destes peptídeos: conformação e hidrofobicidade. Devido à diferença estrutural (cíclica e linear), esses peptídeos conferem diferentes hidrofobicidades, e isso interfere na conformação da molécula, além do meio lipídico. E finalizando o estudo, foi identificado através da DM que o resíduo de triptofano da posição 2 é o aminoácido mais inserido no meio apolar das micelas, após interação. Assim, um possível mecanismo de interação do peptídeo Lo é baseado, inicialmente, na adsorção do peptídeo na superfície lipídica. Em seguida ocorre a interação hidrofóbica membrana-peptídeo, acompanhada pela inserção do triptofano da posição 2 na região mais profunda da membrana, induzindo alterações conformacionais na molécula mediante a interação, dos outros resíduos, com a membrana. / Labaditin is a cyclic decapeptide with high hydrophobic character, extracted from Jatropha Multifida, a plant from Euphorbiaceae family. It is more resistant to proteolytic degradation than its corresponding linear isomers. Studies have been showed that conformational restriction of cyclic peptide increases its affinity and specificity to the membrane. Due to these physical characteristics and to the biological activities shown, such as inhibition of the classical pathway of human complement system in vitro and antibacterial activity for Streptococcus mutans, this peptide has attracted biological and pharmacological interest. However, neither the target nor the action mechanism are known yet. For this reason, the Labaditin (Lo) and the linear analogue (L1) peptides, different structures, were studied in an attempt to get information regarding the mechanism of action, interaction and possible conformational changes due to the interaction with biological membranes. The behavior of Lo and L1 was studied in the presence of different lipid compositions (DPPC, DPPC:Chol (9:1), DPPC:DPPS (8:2)) and of detergents (SDS and LPC), using membrane mimetic systems: monolayer, micelle and liposome. In monolayer, planar system, it was observed an increase of surface pressure, probably caused by the presence of peptide. In the systems composed by DPPC:Chol and DPPC:DPPS the effect was greater in the presence of L1, implying electrostatic interaction between the peptide and the monolayers. Lo peptide, on the other hand, due to the fact that it does not have charges, presented greater interaction with the DPPC monolayer, a zwitterionic molecule. Similar results were obtained through studies with liposome composed by DPPC, DPPC:Chol (9:1) and DPPC:DPPS (8:2). In all environments, through fluorescence spectroscopy, a blue-shift was observed, which means, migration of the tryptophan to a more non-polar environment. For Lo, it was higher in the presence of DPPC; for L1, in the presence of DPPC:Chol and DPPC:DPPS. Using the DSC technique an increase of enthalpy and a decrease of cooperativity was observed (t1/2), due to the presence of peptide in the bilayers. In DPPC:Chol (9:1) and DPPC:DPPS this effect was greater in the presence of L1; while in DPPC, in the presence of Lo, confirming the previous results. These peptide-membrane mimetic interaction was followed by conformational changes, observed through the CD. The Lo peptide has a unordered conformation in aqueous environment, and in the presence of liposomes also is unordered, although with differences. L1 peptide in aqueous environment presents random coil structure with interaction between tryptophan, but in DPPC and in DPPC:Chol (9:1) it suffers conformational changes, distancing tryptophan; in DPPC:DPPS (8:2) it changes to -sheet. This demonstrates that the lipidic composition induces conformational changes in peptides and it may affect their mechanism of action. In the study with micelles it was also observed interaction between peptides-SDS, and also with peptides-LPC. In SDS, the studies suggest that L1 is more inserted in the non-polar environment than Lo; in LPC, Lo is more inserted. These peptides also presented conformational changes in the presence of micelles. Lo peptide, both in SDS, and in LPC, presented unordered conformation, but differently. L1 peptide presented -sheet conformation in the presence of SDS and LPC, but also with differences. The results show that the peptide with linear structure (L1) has greater conformational liberty. Therefore, some factors are responsible to the interaction process of these peptides: conformation and hydrophobicity. Due to the structural difference (cyclic and linear), these peptides present different hydrophobicity, and it interferes in the conformation of the molecule, as well as the lipidic environment. On the last study it was identified through DM that the tryptophan residue from position 2 is the amino acid most inserted in the micelles, after interaction. Thus, a possible Lo peptide interaction mechanism is based, initially, on the adsorption of the peptide on the lipidic surface. Next, there is a hydrophobic interaction peptide-membrane followed by the tryptophan insertion of the position 2 in the deepest region of the membrane, inducing conformational changes in the molecule, through the interaction of the other residues with the membrane.

Aim. The aim of the study was to evaluate the efficacy, safety and possible mechanism of action of potassium humate loaded with selenium Objectives. The objectives of the study were to evaluate the possible in vitro cytotoxic effect of potassium humate loaded with selenium (Phse) on the growth of primary cell cultures (lymphocytes), to evaluate the in vitro antioxidant activity of Phse, to evaluate the in vitro effect of Phse on CR3 expression using mixed leukocytes, to evaluate the anti-inflammatory properties of Phse using the carrageenan-induced paw oedema rat model and finally to evaluate the effect of Phse on acute phase proteins in the rat model Methods. For the cytotoxicity effects on lymphocytes, the MTT assay was used where lymphocytes were isolated and divided in to two groups, one group was stimulated with PHA and the other not, then the cells were treated with different concentrations of the test compounds. The evaluation of the antioxidant activity was done using the ORAC and DCFH-DA assays with the DCFH-DA assay also done using the HepG2 cell line. The expression of CR3 by mixed leukocytes was quantified by flow cytometry. The evaluation of the anti-inflammatory properties of Phse was done using the carrageenan-induced paw oedema rat model. The rats were randomly assigned to six groups, the negative control, positive control, experimental group 1, 2, 3 and sham group. A once daily dose by gavage for five consecutive days of their respective treatment was administered. Prior to the assay the rats were dosed according to the experimental group to which they were assigned. On the fifth day of the experiment, 50 µl of λ- Carrageenan was injected subplanter into the right hind paw of the rats and 50 µl of saline into the left hindpaw. The right hind paw volume of each rat was measured hourly from the time of injection for seven consecutive hours with a water displacement plethysmometer. At the end of the 7 hours the rats were anaesthetised and approximately 5 ml of blood was collected via cardiac puncture. The blood was centrifuged, the plasma removed and frozen at -80°C until assayed. For evaluating the effects of the test compounds on acute phase proteins ELISA was used according to the manufacturer’s protocols. Results. None of the test compounds were toxic to lymphocytes but rather caused cell proliferation. The test compounds demonstrated no antioxidant activity, with Phse showing pro-oxidant activity. All the test compounds inhibited the expression of CR3 significantly with selenium free Ph being the most potent inhibitor. Ph reduced the carrageenan induced paw oedema volumes in a similar manner to indomethacin and Phse had an insignificant effect. Ph decreased SP slightly but the results were not statistically significant whereas Se and Phse had no effect. All the test compounds statistically significantly decreased plasma CRP levels with Se showing the greatest effect. Conclusion. Phse is safe but not more effective than Ph as an anti-inflammatory agent. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

Antimicrobial peptides (AMPs) have been identified as important therapeutic agents that can be developed as new multifunctional antibiotic compounds, which may address antibiotic resistance. AMPs have a wide range of bioactivities, including antimicrobial, antioxidant, anti-inflammatory and anticancer properties. Os and Os-C (a derivative of Os, lacking cysteine residues) are two synthetic AMPs derived from the tick defensin OsDef2 which have been shown to have antibacterial, antioxidant and anti-inflammatory activity. Differences in bacterial killing times between these peptides indicate differences in the modes of bacterial killing. For the further development of Os and Os-C for therapeutic application, the aim of this study was to establish the mode of bacterial killing, to determine if these peptides are cytotoxic to human erythrocytes and leukocytes. Lastly, to determine if these peptides have additional beneficial cellular effects such as antioxidant activity. Ultrastructural analysis with electron microscopy techniques revealed that both peptides adversely affected the membranes and intracellular structures of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis bacteria. Effects included membrane ruffling, cytoplasmic retraction, intracellular granulation and the formation of dense fibres. At the minimum bactericidal concentrations (MBCs) of 0.77 μM for Os and 1.74 μM for Os-C membrane permeabilisation measured with the SYTOX green assay was found not to be the principle mode of action. In stationary phase bacteria, fluorescent triple staining showed that both peptides caused permeabilisation. Studies using fluorescently labelled peptides revealed that the membrane penetrating activities of Os and Os-C were similar to buforin II, a cell-penetrating peptide. Os was able to enter stationary phase E. coli and B. subtilis while Os-C was unable to enter E. coli cells and accumulated on B. subtilis septa. Using plasmid binding and fluorescence displacement assays both peptides could bind DNA, while a dosage effect was only observed for Os. Evaluation of cytotoxicity revealed that Os and Os-C caused no erythrocyte haemolysis or changes to erythrocyte morphology. Only the highest concentration of Os (100 μM), which is 130 fold greater than the MBC for E. coli and B. subtilis, caused cellular damage to peripheral mononuclear (MN) and polymorphonuclear (PMN) cells. In contrast, Os-C caused leukocyte activation identified by associated morphological features and reactive oxygen species (ROS) formation. Chemical and erythrocyte antioxidant assays indicated that both Os and Os-C had antioxidant activity. Both peptides provided extracellular protection of erythrocytes against 2,2'-azobis(2-amidinopropane) dihydrochloride induced oxidative damage. In MN and PMN cells Os showed low levels of antioxidant activity while Os-C had minimal activity. In conclusion, both peptides showed a dual mechanism of bacterial killing, targeting both the membrane and intracellular elements. Os had a predominant membrane effect while Os-C targeted the septa of B. subtilis and had a higher affinity for DNA. Cytotoxicity in erythrocytes and leukocytes was minimal. In addition, Os exhibited antioxidant properties while Os-C caused leukocyte activation. Both peptides have been identified as promising therapeutic agents although activity in plasma and the effect on coagulation must still be determined. / Thesis (PhD)--University of Pretoria, 2017. / Anatomy / PhD / Unrestricted

Antibiotics are an important resource in modern medicine used to treat serious infections and enable a wide array of vital medical interventions including surgery and cancer chemotherapy. However, due to the increasing prevalence of antibiotic resistant pathogens, many clinically useful antibiotics are being rendered ineffective with too few new antibiotics in development to combat them. With highly diverse chemistry and bioactivity exquisitely shaped by evolution, natural products provide an unrivaled source of antibiotic compounds that is impossible to reproduce instinctively in the laboratory. The armeniaspirols are polyketide natural products with a unique spiro-[4.4]non-8-ene core that were isolated from Streptomyces armeniacus and were shown to be active against drug-resistant Gram-positive bacteria. Promisingly, in vitro resistant Staphylococcus aureus strains could not be readily obtained even after thirty serial passages under sub-lethal doses. Herein, we decipher the mechanism of action for this structurally unprecedented natural product antibiotic in the Gram-positive model organism Bacillus subtilis. Through chemical proteomics with an armeniaspirol-inspired activity-based probe, quantitative proteomics, biochemical assays, and microscopy, we show that armeniaspirol is a competitive inhibitor of the AAA+ proteases ClpXP and ClpYQ. Armeniaspirol represents the first known natural product inhibitor of ClpP, a highly coveted target due to its prominent role in bacterial virulence. Using overlapping proteomic fingerprints of armeniaspirol-treatment with ΔclpQ and ΔclpP deletions in B. subtilis, inhibition or deletion of these proteases appears to dysregulate key proteins involved in cell division, including FtsZ, DivIVA, and MreB. The dual ClpXP and ClpYQ inhibition is responsible for armeniaspirol’s potent antibiotic activity and this unique pharmacology makes it a promising candidate for antibiotic development. Several armeniaspirol-inspired analogs were generated as part of a medicinal chemistry study and evaluated for antibiotic activity towards a panel of clinically relevant Gram-positive pathogens. As a result, we identify three exciting armeniaspirol analogs with improved antibiotic activity. Lastly, the foundation for elucidating the ClpYQ degradome is developed. Our proteomic fingerprint of the B. subtilis ΔclpQ deletion strain generated some of the first insights into potential substrates of the ClpYQ protease. As a largely uncharacterized AAA+ protease implicated in the mechanism of action of armeniaspirol, we pursued a previously established acyl-intermediate covalent trapping strategy to characterize the ClpYQ-substrate complexes in B. subtilis cell lysate. Through unnatural amino acid incorporation using an evolved tRNA/aminoacyl-tRNA synthetase pair, the N-terminal active site serine of ClpQ is substituted with a photocleavable precursor that generates 2,3-diaminopropionic acid. While we were successful in synthesizing the photocleavable precursor, initial experiments to incorporate this unnatural amino acid in ClpQ expression proved unsuccessful, leading us to outline necessary control experiments for future endeavours. Ultimately, the covalently trapped substrates will be identified by LC-MS/MS, where we expect to identify key divisome and elongasome proteins in corroboration with the armeniaspirol mechanism of action study.

Hydroxychloroquine, initially used as an antimalarial, is used as an immunomodulatory and anti-inflammatory agent for the management of autoimmune and rheumatic diseases such as systemic lupus erythematosus. Lately, there has been interest in its potential efficacy against severe acute respiratory syndrome coronavirus 2, with several speculated mechanisms. The purpose of this review is to elaborate on the mechanisms surrounding hydroxychloroquine. The review is an in-depth analysis of the antimalarial, immunomodulatory, and antiviral mechanisms of hydroxychloroquine, with detailed and novel pictorial explanations. The mechanisms of hydroxychloroquine are related to potential cardiotoxic manifestations and demonstrate potential adverse effects when used for coronavirus disease 2019 (COVID-19). Finally, current literature associated with hydroxychloroquine and COVID-19 has been analyzed to interrelate the mechanisms, adverse effects, and use of hydroxychloroquine in the current pandemic. Currently, there is insufficient evidence about the efficacy and safety of hydroxychloroquine in COVID-19.KEY MESSAGES HCQ, initially an antimalarial agent, is used as an immunomodulatory agent for managing several autoimmune diseases, for which its efficacy is linked to inhibiting lysosomal antigen processing, MHC-II antigen presentation, and TLR functions. HCQ is generally well-tolerated although severe life-threatening adverse effects including cardiomyopathy and conduction defects have been reported. HCQ use in COVID-19 should be discouraged outside clinical trials under strict medical supervision.

Les analogues de choline constituent une nouvelle classe d'antipaludiques qui inhibent la biosynthèse de la phosphatidylcholine (PC) de Plasmodium, parasite responsable du paludisme. Les études conduites ont mis en relief des particularités uniques de ces composés. Nous avons élucidé le mécanisme d'action biochimique de l'albitiazolium, actuel candidat clinique, caractérisant chacune des 5 étapes conduisant à la biosynthèse de PC. L'albitiazolium affecte en premier lieu l'entrée de choline dans le parasite intraerythrocytaire, choline et albitiazolium utilisant le même transporteur et affecte de façon différentielle les autres étapes de synthèse. L'activité antipaludique est fortement antagonisée par la choline indiquant que le mécanisme d'action primaire est bien l'inhibition de la synthèse de PC. L'accumulation des analogues de choline dans le parasite intracellulaire leur permet de restreindre leur toxicité aux seuls érythrocytes infectés. Des études comparatives réalisées chez Plasmodium et Babesia montrent une double compartimentation de l'albitiazolium uniquement chez Plasmodium, l'une d'elles correspondant à la vacuole digestive. L'accumulation chez Plasmodium est glucose-dépendante et exige aussi le maintien des gradients ioniques dans la cellule. Bien que les analogues de choline exercent leur effet antiparasitaire dès les premières heures de contact, l'effet dit « cheval de Troie » exige des conditions particulières pour les mesures d'activités pharmacologiques, nous amenant à comparer différents tests d'activité. Seuls les tests isotopiques basés sur l'incorporation d'hypoxanthine ou d'éthanolamine après un cycle parasitaire entier et le test fluorescent au SYBR green appliqué après 72h obtiennent des résultats fiables quel que soit le mécanisme d'action des antipaludiques. Enfin, des études de pharmacocinétique / pharmacodynamie montrent une exposition plasmatique supérieure chez les souris infectées par Plasmodium, due au recyclage de l'albitiazolium après son accumulation dans l'érythrocyte infecté. / Choline analogues form a new class of antimalarial drugs that inhibit the biosynthesis of phosphatidylcholine (PC) in Plasmodium, the malaria-causing parasite. The studies presented here highlighted the unique features of these compounds. We elucidated the biochemical mechanism of action of albitiazolium, the current clinical candidate, characterizing each of the 5 steps leading to the biosynthesis of PC. Albitiazolium primarily affects the entry of choline into the intraerythrocytic parasite and choline and albitiazolium use the same carrier. The other steps of synthesis are differentially affected. Antimalarial activity is strongly antagonized by choline indicating that the primary mechanism of action is the inhibition of PC synthesis Accumulation of choline analogs in the intracellular parasite allows them to restrict their toxicity to infected erythrocytes. Comparative studies in Plasmodium and Babesia show a double compartmentalization of albitiazolium only in Plasmodium, one of them corresponding to the food vacuole. Accumulation in Plasmodium is glucose-dependent and requires maintaining ionic gradients in the cell.Although choline analogues exert their antiparasitic effect in the first hours of contact, the “Trojan horse effect” requires specific conditions for the determination of pharmacological activity, leading us to evaluate various tests of activity. Only the isotopic tests based on hypoxanthine or ethanolamine incorporation after one parasite cycle and the fluorescent SYBR green assay applied after 72 hours give reliable results regardless of the mode of action of the tested antimalarials. Finally, pharmacokinetics/pharmacodynamics studies in Plasmodium-infected mice revealed that albitiazolium is recycled after its accumulation in the infected erythrocyte leading to increased plasma levels.

HIV-1 protease plays a crucial role in the late state of the life cycle of HIV virus when it cleaves the viral polyprotein precursors into the structural and functional proteins. If it is effectively inhibited, HIV particles remain immature and noninfectious. The application of highly active antiretroviral therapy (HAART) including protease inhibitors can reduce plasma HIV-1 levels below the detection limit in adherent patients and thus dramatically change their life expectancy. The clinical utility of the first inhibitors was limited by severe side effects, low bioavailability, high pill burdens, and rapid development of viral resistance under the selection pressure of HIV antiretrovirals. To overcome these difficulties, second-generation inhibitors were developed. Despite an indisputable improvement they brought to antiretroviral therapy, the development of new highly active HIV-1 protease inhibitors with optimal pharmacokinetic properties, higher metabolic stability, little off-target activity, and particularly, more favorable resistance profiles is still of high importance. This thesis provides an overview of anti-HIV- drugs including development of substituted metallacarboranes, a new class of potent, unusual, nonpeptidic HIV protease inhibitors with therapeutic potential. Next, the impact of...

O chumbo (Pb) é um metal tóxico e promove diversos efeitos adversos no organismo que incluem distúrbios neurológicos, hematopoiéticos, esqueléticos, renais e cardiovasculares. A análise proteômica têm se tornado cada vez mais uma ferramenta importante na elucidação de mecanismos de toxicidade, bem como na identificação de potenciais biomarcadores. Nesse sentido, o objetivo deste estudo foi comparar diferenças de abundância de proteínas plasmáticas em amostras de sangue coletadas de trabalhadores de uma fábrica de baterias automotivas, e de um grupo sem histórico de exposição ocupacional ao Pb. Pb no sangue total (Pb-S) foi determinado por espectrometria de massas com plasma acoplado indutivamente (ICP-MS). Para a análise proteômica, o plasma foi separado do sangue total, seguido de depleção de albumina e imunoglobulina G (IgG) através de cromatografia de imunoafinidade. Em seguida, foi realizada digestão das proteínas com tripsina e os peptídeos foram analisados no sistema nanoACQUITY UPLC acoplado ao espectrômetro de massas em tandem SYNAPT® G2-Si HDMS. A identificação das proteínas foi realizada através do banco de dados SwissProt e as proteínas identificadas com pelo menos 3 peptídeos foram classificadas de acordo com o banco de dados do Gene Ontology (GO) e ranqueadas através de algoritmos de classificação utilizando a mineração de dados. A quantificação das proteínas foi realizada por label-free e foram consideradas proteínas com diferença de abundância aquelas com fold-change >=1,5 e p>0,05 através do teste t de Student. A média de Pb-S para os grupos expostos ocupacionalmente e sem histórico de exposição ocupacional foi de 43,7?g/dL e 0,386 ?g/dL, respectivamente. No total, 82 proteínas foram identificadas no plasma e apenas 4 proteínas apresentaram fold change >= 1,5, sendo elas: Apolipoproteína B-100 (APOB) e ?-II-antiplasmina (A2AP2), além de plasminogênio (PLMN) e gelsolina (GELS) cuja diferença de abundância entre os grupos foi significativa (p=0,036 e <0,01, respectivamente). As proteínas com diferença de abundância também foram classificadas entre as mais importantes para a diferenciação dos grupos através da mineração de dados. Assim, o presente estudo foi capaz de demonstrar diferenças de abundância em proteínas plasmáticas entre indivíduos com elevadas concentrações de Pb-S e com baixas concentrações de Pb-S, evidenciando possíveis novos mecanismos de toxicidade do metal. / Lead (Pb) is a toxic metal and may cause several adverse effects including neurological, hematopoietic, skeletal, renal and cardiovascular disorders. Proteomic analyses have become an important tool for the discovery of new mechanism of toxicity as well to identify potential new biomarkers of metal exposure, which may help in early detection of toxic effects. This study aimed to evaluate the differences in the plasma proteome of two groups: i) Pb-exposed workers and ii) a group with no occupational lead exposure history. Blood samples were collected for blood lead levels (BLL) determination by inductively coupled plasma mass spectrometry (ICP-MS) and for proteomic analyses in plasma fraction. Plasma samples were separated from whole blood using centrifugation. Antibody-based affinity columns were used to deplete plasma fractions from albumin and immunoglobulin G (IgG). Thereafter, proteins were digest with trypsin solution and peptide analyses were conducted using a nanoACQUITY UPLC System coupled to a SYNAPT® G2-Si tandem mass spectrometer. Identification of proteins were performed against SwissProt database and proteins identified by at least three peptides were classified according to the Gene Ontology terms (GO). In addition, identified proteins were ranked through classification algorithms by data mining analyses. Proteins quantitation was performed by label-free and only proteins with fold-change >=1.5 were considered differentially expressed. Mean BLL were 43.7?g/dL and 0.386 ?g/dL for occupational and non-occupational lead exposed groups, respectively. Eighty-two proteins were identified in the plasma samples out of which only four - plasminogen (PLMN), gelsolin (GELS), ?-II-anti-plasmin (A2AP2) and apolipoprotein B-100 (APOB) showed >=1.5 folds differential abundance. Differential abundances were confirmed for PLMN and GELS proteins (p=0.036 and <0.01, respectively). In conclusion, the present study indicated that the plasma proteome was a function of BLL in the individuals

O aumento na incidência de infecções fúngicas e a alta toxicidade ou elevado índice de resistência associado aos antimicóticos comerciais, criou um mercado carente de novas drogas. Neste contexto, os peptídeos antimicrobianos (AMPs) surgem como uma alternativa promissora ou fonte de conhecimento por desempenhar ação inibidora de crescimento e/ ou letal contra bactérias Gram-positivas e Gram-negativas, fungos, parasitas e/ ou vírus, além de atividade antitumoral e efeito imunomodulador. Como os mecanismos pelos quais eles o fazem são diferentes daqueles das drogas não peptídicas, os AMPs estão pouco associados ao desenvolvimento de resistência microbiana. A hemoglobina (Hb) é uma fonte de peptídeos com funções biológicas diversas. O fragmento 33-61 (Hb33-61) da cadeia &#945; da Hb bovina foi o primeiro AMP descrito a ser gerado in vivo no trato gastrointestinal do carrapato Boophilus microplus. Nossos estudos posteriores usando CD e H1-RMN revelaram que a amidação C-terminal deste fragmento o tornava ainda mais ativo que o primeiro e que em presença de micelas de SDS o Hb33-61a apresenta uma dobra &#946; na porção N-terminal (Lys40-Phe43) e outra (Ser49- Ser52) seguida de &#945;-hélice no C-terminal (Ala53-Ala60), bem como um segmento Pro44-Leu48 capaz de mover-se independentemente e agir como uma dobradiça. Nossas investigações usando análogos sintéticos truncados do Hb33-61a mostraram que o Hb40-61a poderia ser sua porção mínima ativa por apresentar comportamento conformacional idêntico. Nossos estudos subsequentes enfocando as suas propriedades evidenciaram a sua capacidade de causar morte rápida de cepas de Candida, incluindo C. albicans resistentes ao fluconazol e extravasamento de conteúdo e formação de poros em LUVs, revelando sua ação permeabilizante de membrana. Em continuidade ao estudo do Hb40-61a, investigamos no presente trabalho as suas propriedades e o seu mecanismo de ação contra C. albicans. Para isso, sintetizamos, purificamos e caracterizamos esta hemocidina, o seu análogo inteiro composto por D aminoácidos (ent-Hb40-61a) e o seu análogo marcado com 5 (6) carboxifluoresceína (FAM-Hb40-61a). Ensaios com eritrócitos humanos confirmaram a baixa atividade hemolítica desses AMPs em meio de alta e baixa força iônica. O análogo ent-Hb40-61a apresentou a mesma atividade antifúngica que o análogo L, evidenciando um mecanismo de ação não-estereoespecífico. Análises de células de Candida tratadas com FAM-Hb40-61a por microscopia confocal mostraram que em ½ MIC e MIC o peptídeo deposita-se na membrana plasmática e é internalizado, respectivamente. Citometria de fluxo demonstrou que na MIC cerca de 97% das células encontram-se marcadas pelo peptídeo, confirmou a influência negativa da alta força iônica em sua atividade, mostrou que a internalização celular na MIC é independente da temperatura e que a alteração no metabolismo energético da célula afeta de maneira negativa a internalização do peptídeo. Ensaios de permeabilidade celular com Syto 09 e iodeto de propídeo confirmaram danos progressivos à membrana plasmática de C. albicans com o aumento da concentração do Hb40-61a. Experimentos usando DiBAC4(5) e de DPH revelaram que o Hb40-61a altera o potencial de membrana e afeta sua fluidez, respectivamente. Imagens preliminares das células tratadas e não tratadas com Hb40-61a por microscopia de força atômica (AFM) sugeriram alterações nas células de C. albicans após tratamento com a hemocidina. Medidas preliminares do diâmetro médio das células de C. albicans revelaram que elas diminuem após o tratamento com o peptídeo, o que pode ser mais um indício de dano à membrana plasmática por formação de poros e extravasamento de conteúdo intracelular. Assim, obtivemos fortes indícios de que o alvo do Hb40-61a é, de fato, a membrana plasmática das células de Candida, de que ele apresenta potencial de uso tópico para tratamento de candidíase e pode servir como modelo para o desenho de novas drogas antimicrobianas, peptídicas ou não, com propriedades ainda mais valiosas e índices terapêuticos mais elevados. Testes preliminares mostraram que é possível a adsorção do Hb40-58a à nanopartículas de PSS e que, em relação ao peptídeo livre, este arranjo mantém a atividade antifúngica com MIC superior e apresenta menor atividade hemolítica. / The increased incidence of fungal infections and the high toxicity or high level of resistance associated with conventional antimycotics created a demand for new drugs. Antimicrobial peptides (AMPs) constitute a promising alternative and/or an important source of knowledge due to their growth inhibitory action and/or lethality against Gram-positive and Gram-negative bacteria, fungi, parasites and/or viruses. Besides, AMPs display antitumoral and immunomodulator effects. As their mechanisms of action are different from those of non-peptide drugs, AMPs are less associated with the development of antimicrobial resistance. Hemoglobin (Hb) is a source of peptides with diverse biological functions. The fragment 33-61 (Hb33-61) of bovine Hb &#945; chain was the first AMP reported to be generated in vivo in gastrointestinal tract of Boophilus microplus. Our studies of Hb33-61 using CD and H1-NMR showed that amidation of its C-terminal (Hb33-61a) increases its activity; in the presence of SDS micelles, Hb33-61a is characterized by a central hinge joining the C-terminal region (containing a &#946; turn followed by a helical element) to the N-terminal region (that presents only a &#946; turn). Our previous investigations using synthetic truncated analogues of Hb33-61a suggested that Hb40-61a could be its minimal active portion as it presented equal biological and structural properties. Our subsequent studies focusing on its properties showed its ability to quickly kill Candida albicans strains (including those resistant to fluconazole) and to cause leakage of the contents of LUVs and pore formation in GUVs, revealing its membrane permeabilizing action. We further investigated the properties of Hb40-61a and its possible mechanism of action against C. albicans. To do it, we synthesized, purified and chemically characterized it, its all-D analogue (ent-Hb40-61a) and its analogue labeled with 5 (6) carboxyfluorescein (FAM-Hb40-61a). Tests using human erythrocytes confirmed the low toxicity of these hemocidins at high or low ionic strength. The ent-analogue was as active as the all-L compound suggesting a non stereospecific mechanism of action. Confocal microscopy analysis of Candida cells treated with FAM-Hb40-61a showed that at ½ MIC and MIC, the peptide deposits on the plasma membrane and is internalized, respectively. Flow cytometry results showed that at the MIC about 97% of the cells are marked by the peptide, confirmed the negative influence of high ionic strength on its antifungal activity and showed that the cellular internalization at the MIC is partially dependent on ATP, but independent on the temperature. Cell permeabilization assays using Syto 09 and propidium iodide confirmed progressive damage of the membrane as a function of Hb40-61a concentration. Experiments employing DiBAC4 (5) and DPH revealed that the Hb40- 61a alters the membrane potential and affects its fluidity, respectively. Preliminary atomic force microscopy (AFM) images of C. albicans cells before and after treatment with Hb40-61a suggested morphological changes in the plasma membrane. Preliminary measurements of the average diameters of the fungal cells indicated size reduction after treatment with the Hb40-61a probably resulting from pore formation and leakage of cell contents. Thus, we obtained strong evidences that the target of this peptide is indeed the plasma membrane of Candida cells. Thus, this hemocidin have the potential to be used topically for treating candidiasis and/or serve as model for the design of new antimicrobial drugs, peptide or non-peptide, with even more valuable properties and improved therapeutic indexes. Preliminary tests confirmed the possibility of adsorbing Hb40-58a to nanoparticles of polystyrene sulfate (PSS) and that resulting assembly is still active and less hemolytic than the free peptide.