Orientadores: João Ernesto de Carvalho, Mary Ann Foglio / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T00:15:44Z (GMT). No. of bitstreams: 1 Longato_GiovannaBarbarini_D.pdf: 11931836 bytes, checksum: 6a9508b3296a68bdd39eb4630f1e89de (MD5) Previous issue date: 2014 / Resumo: O câncer é a segunda causa de morte da população nos países desenvolvidos e em desenvolvimento. Caracteriza-se pelo desenvolvimento de células instáveis que tendem a não responder aos estímulos internos e externos que controlam a proliferação, diferenciação e morte celular. O crescente entendimento da biologia tumoral tem fornecido alvos moleculares para a triagem orientada de quimioterápicos e de agentes quimiopreventivos, geralmente de origem natural ou sintetizados com base em produtos naturais. A partir do extrato bruto diclorometânico das folhas de Piper regnellii foram isolados dois compostos, o eupomatenóide-5 e o apiol. O eupomatenóide-5 é uma neolignana cuja potencial atividade anticâncer foi demonstrada in vitro (cultura de células tumorais humanas) e in vivo (tumor sólido de Ehrlich) em trabalhos anteriores publicados pelo nosso grupo. Dentre as linhagens mais sensíveis, MCF-7 (mama) e 786-0 (rim) foram selecionadas para dar continuidade aos estudos de mecanismo de ação deste composto. Os resultados obtidos no presente estudo sugerem que o eupomatenóide-5 possui ação multialvo, uma vez que demonstraram claramente que existe um mecanismo de ação diferente para cada linhagem celular. Sugere-se que o efeito antiproliferativo do eupomatenóide-5 esteja associado com indução de apoptose em linhagem MCF-7 e necrose programada em linhagem 786-0. O apiol, outro composto isolado, é um fenilpropanóide com conhecida ação bloqueadora dos canais de cálcio. Sabe-se que a inibição de canais de cálcio tem relação com a reversão da resistência a múltiplas drogas (MDR) e, por este motivo, o efeito do apiol sobre a glicoproteína-P, uma das principais proteínas moduladoras dessa resistência, foi avaliado; entretanto o apiol não inibiu sua ação. Por outro lado, neste estudo foi possível evidenciar que embora tenha apresentado atividade citostática baixa para as linhagens tumorais, ele potencializou a atividade específica dos quimioterápicos doxorrubicina e vincristina quando associado a estes em linhagens tumorais com e sem fenótipo de resistência, sugerindo um efeito sinérgico. Além disso, o apiol potencializou a atividade antiproliferativa do eupomatenóide-5 e essa associação reduziu a quantidade de células tumorais de Ehrlich em modelo in vivo, sem sinais de toxicidade evidentes. Os resultados encorajam o aprofundamento desses estudos para a determinação dos mecanismos de ação envolvidos nas atividades anticâncer do eupomatenóide-5 e de reversão da MDR do apiol / Abstract: Cancer is the second leading cause of mortality worldwide. The loss of normal cell growth control is the main event in the development of cancer and includes specific steps known as cell proliferation, differentiation and apoptosis. Natural compounds are good source of new anticancer agents, being the investigation into cell death mechanisms involved in this activity a significant endeavor. From the crude dichloromethane extract of Piper regnellii leaves two compounds were obtained: eupomatenoid-5 and apiole. Eupomatenoid-5 is a neolignan whose in vitro (tumor cell line culture) and in vivo (Ehrlich solid tumor) anticancer activity was previously reported by our group. Among the cancer cell lines most sensible to eupomatenoid-5 treatment, MCF-7 (breast) and 786-0 (kidney) were chosen to continue the cell death mechanism of action studies. The results obtained suggest that eupomatenoid-5 is a multiple-target compound. Our findings support the possibility that eupomatenoid-5 might be acting in both cell death via: apoptosis in MCF-7 cell line and programmed necroptosis in 786-0 cell line. Apiole, the other compound isolated, is a phenylpropanoid and a calcium channel blocker. Recent pharmacological studies have reported the ability of several calcium channel blockers to reverse the multidrug resistance (MDR) and, therefore, the effect of apiole on P-glycoprotein (P-gp), the main protein involved in MDR, was evaluated. Apiole did not inhibit P-gp and, moreover, presented low cytotoxic activity for the most tumor cell lines. Nonetheless, the combined therapy with chemotherapies (doxorubicin and vincristine) was efficient in potentializing their antiproliferative action in tumor cell lines with or without resistance phenotype, suggesting synergic effect. Furthermore, apiole potentiated the antiproliferative activity of eupomatenoid-5 and this association reduced the in vivo tumor cell quantity with no side effects. The results encourage further studies to be developed with the aim to determine the mechanisms of action involved in the anticancer activity of eupomatenoid-5 and in the MDR reversion by apiole / Doutorado

Bactérias multirresistentes (MRs) pertencentes ao grupo ESKAPE (i.e., Enterococcus faecium resistente à vancomicina, VRE; Staphylococcus aureus resistente à meticilina, MRSA; Klebsiella spp., e Escherichia coli produtoras de β-lactamases de amplo espectro; Acinetobacter baumannii, Pseudomonas aeruginosa e Enterobacter spp. resistentes aos carbapenêmicos) são importantes patógenos de infecções relacionadas à assistência à saúde (IRAS), onde a sua endemicidade e prevalência tem sido decorrente da seleção de linhagens clonais. Embora, o fenótipo MR decorra da expressão de mecanismos mediados por genes intrínsecos e/ou adquiridos, o crescimento bacteriano na forma de biofilme contribui para um importante fenômeno fisiológico de resistência, o qual é inespecífico quanto ao substrato antimicrobiano. O presente estudo teve como objetivo avaliar a eficácia de agentes físicos e químicos contra biofilmes produzidos por clones de bactérias MRs de importância clínica e epidemiológica no Brasil. Cerdas de poliamida foram utilizadas como modelo de superfície de adesão para o crescimento de biofilmes, os quais foram monitorados por microscopia eletrônica de varredura (MEV). In vivo, o modelo de biofilme foi avaliado pela inserção das cerdas na proleg de larvas de Galleria mellonella, enquanto que, diferentes tratamentos foram aplicados para inibir a formação do biofilme. Adicionalmente, mediante ao ensaio de bioluminescência, o modelo de biofilme produzido pela cepa de P. aeruginosa PAO1/lecA::lux foi avaliado na presença de soluções hipertônicas de cloreto de sódio (NaCl). In vitro, soluções hipertônicas de cloreto de sódio (> 6%) utilizadas de maneira profilática, apresentaram efeito bacteriostático (CIM90= 1,7 M) contra biofilmes produzidos por todos os isolados analisados. Além disso, através do uso profilático de soluções hipertônicas de NaCl, foi possível visualizar a inibição da motilidade dos isolados. Por outro lado, os compostos quaternários de amônio (CQAs) cloreto de benzalcônio (CBA) e cloreto de cetilpiridínio (CCP) apresentaram efeito bactericida (CBM90= 256 µg/mL) contra biofilmes previamente formados em 24h. A atividade de ambos os CQAs foi potencializada na presença de soluções salinas hipertônicas, como avaliado pela metodologia de checkerboard, tendo um efeito sinérgico contra E. coli (ST10, ST101) MCR-1 (∑FIC= 0,5); parcialmente sinérgico contra A. baumannii OXA-23 (ST79), E. cloacae CTX-M-8 (ST131), E. faecium VRE (ST478) e K. pneumoniae KPC-2 (ST340) (∑FIC= 0,75); e indiferente contra cepas de P. aeruginosa SPM-1 (ST277) e S. aureus MRSA (ST5). Adicionalmente, a CIM de carbapenêmicos, fluoroquinolonas e aminoglicosídeos contra biofilmes de bactérias Gram-negativas MRs foi potencializada na presença de solução salina hipertônica resultando em uma queda da CIM >=2. Finalmente, in vivo, para todas as espécies MRs estudadas, biofilmes formados em 08, 12 e 24h resultaram em 100% de morte das larvas de G. mellonella em até 96 horas pós-infecção. O mesmo comportamento foi observado para a cepa PAO1/lecA::lux, sendo possível detectar sinais intensos de bioluminescência nas larvas infectadas com os biofilmes. Entretanto, para os biofilmes previamente tratados com solução salina hipertônica, observou-se a diminuição dos sinais de bioluminescência em até 60%. Já para biofilmes formados em 24, 12 e 08h, o tratamento prévio em solução salina hipertônica e posteriormente com antibióticos resultou em um aumento de até 40, 70 e 80% da sobrevida de G. mellonella, respectivamente. / ESKAPE pathogens (ie, vancomycin-resistant (VRE) Enterococcus faecium; methicillin-resistant (MRSA) Staphylococcus aureus; extended spectrum β-lactamase-producing Klebsiella spp., and Escherichia coli; Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. Resistant to carbapenems), represents an important group of multidrug-resistant (MDR) bacteria related to healthcare-associated infections (HAIs), whereas endemicity have been associated with selection and predominance of clones. Although the MR phenotype derives from the expression of mechanisms mediated by intrinsic and/or acquired genes, bacterial growth in the biofilm form contributes to an important physiological phenomenon of resistance, which is non-specific to the antimicrobial substrate. The present study aimed to evaluate the efficacy of physical and chemical agents against biofilms produced by clones of MDR bacteria of clinical and epidemiological importance, in Brazil. Polyamide bristles were used as adhesion surface model for the growth of biofilms, which were monitored by scanning electron microscopy (SEM). In vivo, the biofilm model was evaluated by the insertion of the bristles into the proleg of larvae of Galleria mellonella, while different treatments and physicochemical conditions were applied to inhibit biofilm formation. Additionally, the biofilm model produced by the P. aeruginosa PAO1/lecA::lux strain was evaluated in the presence of hypertonic solutions of sodium chloride (NaCl). In vitro, hypertonic solutions of sodium chloride presented a bacteriostatic effect (MIC90 = 1.7 M) against biofilm formation of all the isolates analyzed. Moreover, through the prophylactic use of hypertonic solutions of NaCl, it was possible to observe the inhibition of the motility of the isolates. On the other hand, the ammonium quaternary compounds (QACs) benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) had a bactericidal effect (CBM90 = 256 µg / mL) against previously formed biofilms in 24h. The activity of both QACs was potentiated in the presence of hypertonic saline solutions, as evaluated by the checkerboard methodology, having a synergistic effect against E. coli (ST10, ST101) MCR-1 (∑FIC = 0.5); (ST340) (∑FIC = 0.75), E. faecium VRE (ST478) and K. pneumoniae KPC-2 (ST340), E. cloacae CTX-M-8 (ST131); and indifferent effect against strains of P. aeruginosa SPM-1 (ST277) and S. aureus MRSA (ST5). Furthermore, the MIC of carbapenems, fluoroquinolones and aminoglycosides against biofilms of MDR Gram-negative bacteria was potentiated in the presence of hypertonic saline solution resulting in a decrease in MIC >=2-fold. Finally, for all MDR species studied, biofilms formed at 08, 12 and 24h resulted in 100% death of G. mellonella larvae within 96h post-infection. In fact, the same behavior was observed for the strain PAO1/lecA::lux, and it is possible to detect intense bioluminescence signals in the larvae infected with biofilms. However, for biofilms previously treated with hypertonic saline solution, bioluminescence signs decreased by up to 60%. As for biofilms formed at 24, 12 and 8h, pretreatment in hypertonic saline solution and later with antibiotics resulted in an increase of up to 40, 70 and 80% of the survival of G. mellonella, respectively.

INTRODUÇÃO: O aumento da incidência de infecções causadas por bactérias resistentes a múltiplos antimicrobianos limita cada vez mais as opções terapêuticas, dificultando o tratamento e aumentando os índices de morbidade e mortalidade, além dos gastos em saúde. Ao longo dos últimos cinco anos, essa limitação tem levado ao reestabelecimento do uso de antimicrobianos consideradas ultrapassados, como as polimixinas. Este grupo passou a ser utilizado com cada vez mais frequência no tratamento de infecções causadas por microrganismos gram-negativos resistentes aos carbapenêmicos. As enterobactérias, em particular a espécie Klebsiella pneumoniae, tem apresentado frequentemente esse perfil, porém, a resistência à polimixinas têm sido relatada, eliminando essa importante alternativa terapêutica. Apesar da importância do tema, são escassas as publicações sobre frequência de resistência às polimixinas em K. pneumoniae e a relação clonal entre isolados resistentes à polimixina B no Brasil. OBJETIVOS: Avaliar a relação genética, perfil de sensibilidade antimicrobiana e mecanismos de resistência às polimixinas em K. pneumoniae. MATERIAIS E MÉTODOS: A execução deste trabalho dividiu-se em duas partes principais: (i) levantamento de dados de culturas positivas para K. pneumoniae da rotina de pacientes hospitalizados em instituições atendidas pelo serviço de análises clínicas do Fleury Medicina e Saúde; (ii) confirmação das concentrações inibitórias mínimas (CIM) para polimixina B, avaliação da relação clonal por eletroforese em campos pulsados (PFGE),e sequenciamento de múltiplos loci (MLST), avaliação da integridade do gene mgrB e da presença do gene mcr-1 por PCR entre isolados resistentes à polimixina B e aos carbapenêmicos (CPRKp). RESULTADOS e CONCLUSÕES: Na análise de 3.085 isolados de K. pneumoniae obtidos de pacientes internados em 11 hospitais da Grande São Paulo entre os anos de 2011 e 2015, foi evidenciado um aumento estatisticamente significativo na resistência aos carbapenêmicos de 6,8% em 2011 para 35,5% em 2015. Em 2015, KPC foi detectada em 96,2% dos isolados resistentes aos carbapenêmicos. A distribuição das concentrações inibitórias mínimas de polimixina B entre todos os isolados de K. pneumoniae evidenciou uma distribuição bimodal com a CIM de 2 mg/L como o valor de ponto de corte para a susceptibilidade à polimixina B; assim, 3,6% do número total de isolados sensíveis aos carbapenêmicos foram interpretados como resistentes enquanto essa proporção foi de 22,5% entre as resistentes aos carbapenêmicos (CRKp). Entre esses últimos isolados também houve um aumento estatisticamente significativo na tendência anual de resistência à polimixina B, de 0% em 2011 para 27,1% em 2015. Estas taxas variaram de 0,7% em 2011 para 3,9% até junho de 2014 entre os sensíveis aos carbapenêmicos. Entre os antimicrobianos alternativos, a amicacina e a tigeciclina foram os compostos mais ativos. A análise por PFGE de 60 isolados de CPRKp obtidos de pacientes distintos nos anos de 2014 e 2015 evidenciou dois grandes grupos clonais: CPRKp1 e CPRKp2, os quais segundo a análise por MLST pertencem, respectivamente, aos grupos ST11 e ST437, ambos do complexo clonal 258. Foi observado o mesmo grupo ST entre isolados obtidos dentro de um mesmo hospital e também entre diferentes hospitais, públicos e privados. O mecanismo de resistência mais comum entre os isolados de CPRKp foi a presença de sequências de inserção interrompendo o gene mgrB. O gene mcr-1 não foi detectado em nenhum dos isolados. / INTRODUCTION: The increasing incidence of infections caused by bacteria resistant to multiple antimicrobials increasingly limits therapeutic options, making treatment difficult and increasing the morbidity and mortality and health spending. Over the past five years, this limitation has led to the reestablishment of the use of antimicrobials deemed outdated, such as polymyxins. This group is now used with increasing frequency to treat infections caused by carbapenem-resistant gram-negative microorganisms. Enterobacteria, especially Klebsiella pneumoniae, have often presented this profile, however, resistance to polymyxins have been also reported, eliminating this important therapeutic alternative. Despite the importance of this issue, the publications are scarce on the polymyxins resistance frequency in K. pneumoniae and clonal relationship among isolates resistant to polymyxin B in Brazil. OBJECTIVES: To evaluate the genetic relationship, antimicrobial susceptibility profile and polymyxin B resistance mechanisms in K. pneumoniae. MATERIALS AND METHODS: The execution of this work was divided into two main parts: (i) survey data on routine cultures positive for K. pneumoniae from patients hospitalized in institutions attended by the clinical analysis service of Fleury Health and Medicine; (ii) confirmation of to polymyxin B minimum inhibitory concentrations (MIC), evaluation of clonal relationship by electrophoresis pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), evaluation of the integrity of the mgrB gene and the presence of mcr-1 gene by PCR among isolates resistant to polymyxin B and carbapenems (CPRKp). RESULTS AND CONCLUSIONS: The analysis of 3,085 K. pneumoniae isolates obtained from inpatients from 11 hospitals in the São Paulo urban area between 2011 and 2015, has shown a statistically significant increase in carbapenem resistance from 6.8% in 2011 to 35.5% in 2015. In 2015, KPC was detected in 96.2% of isolates resistant to carbapenems. The polymyxin B MIC distribution of all Klebsiella pneumoniae showed a bimodal distribution with the MIC of 2 mg/L as the cutoff value for polymyxin B susceptibility; thus, 3.6% of the total number of isolates susceptible to carbapenems were interpreted as resistant while this proportion was 22.5% among carbapenem-resistant isolates (CRKp). Among these isolates there was also a statistically significant increase in the annual trend of polymyxin B resistance, from 0% in 2011 to 27.1% in 2015. These rates ranged from 0.7% in 2011 to 3.9% by June 2014 between carbapenem-susceptible isolates. Among alternative antimicrobials, amikacin and tigecycline were the most active compounds. The analysis by PFGE of 60 CPRKp isolates obtained from different patients in the years 2014 and 2015 showed two major clonal groups: CPRKp1 and CPRKp2, which according to the analysis by MLST belong respectively to ST11 and ST437 groups, both from clonal complex 258. We observed the same ST group of isolates obtained within a hospital and between different public and private hospitals. The most common mechanism of polymyxin B resistance among CPRKp isolates was the presence of insertion sequences interrupting the mgrB gene. The mcr-1 gene was not detected in any of the isolates.

The Major Facilitator Superfamily groups a vast number of secondary transporters that import or export distinct substrates. Among these, multidrug antiporters constitute a peculiar class of transporters, both because of their multispecificity, recognizing structurally very diverse substrates, and because of their transport mechanism, that relies on bilayer-mediated extrusion of cytotoxic compounds. An accurate and detailed description of the conformational changes that underlie the transport cycle is still lacking and the structural basis for energetic coupling in these transporters has not been elucidated, with so far only limited crystallographic evidence available. We investigate the molecular basis of secondary multidrug transport with biochemical and biophysical studies on LmrP, a Major Facilitator Superfamily multidrug transporter from Lactococcus lactis. We used extensive continuous-wave electron paramagnetic resonance and double electron-electron resonance measurements on a library of spin-labeled LmrP mutants to uncover the conformational states involved in transport and to investigate how protons and ligands shift the equilibrium between conformers to enable transport. We find that the transporter switches between outward-open and outward-closed conformations depending on the protonation states of specific acidic residues forming a transmembrane protonation relay. We observe that substrate binding restricts the conformational freedom of LmrP and induces localized conformational changes. Our data allows to build a model of secondary multidrug transport wherein substrate binding initiates the transport cycle by opening the extracellular side to protons. Subsequent protonation of membrane-embedded acidic residues induces substrate release to the extracellular side and triggers a cascade of conformational changes that culminates in a proton release to the intracellular side. Parallel to this, we have optimized our purification and expression protocol in order to set up crystallization trials on LmrP. Through extensive screening and optimization of the lipidation state of LmrP, using ad hoc methods for sample preparation, we were able to obtain low-resolution diffracting crystals. By improving our lipidation technique and modifying the lipid composition we further improved crystal quality. Other factors such as ligand addition, the presence of secondary detergent and additives for controlling phase separation and nucleation were tested, paving the way to high resolution structure determination of LmrP. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

La diffusion des entérobactéries multirésistantes aux antibiotiques (MDR) à l’échelle mondiale constitue une menace de santé publique majeure. Résistantes à au moins trois classes d’antibiotiques, les entérobactéries MDR entrainent des infections échappant aux traitements de première intention. La première partie de ce travail s’intéresse à l’épidémiologie moléculaire des souches d’entérobactéries MDR isolées dans les infections et les colonisations des patients hospitalisés en Languedoc-Roussillon, en France, et dans un pays où cette épidémiologie est encore peu connue, l’Algérie. Nous avons montré, dans notre région et au niveau national, que la résistance aux carbapénèmes était essentiellement liée à des modifications de la perméabilité membranaire (87,4% des entérobactéries résistantes, au niveau national). Dans la deuxième partie de ce travail, nous avons étudié les modulations de la perméabilité membranaire et de l’efflux chez Escherichia coli ST131, l’exemple-type d’un clone MDR. Nous avons montré que ce clone mondial présentait une remarquable adaptabilité à la pression antibiotique. Cette adaptabilité avait un impact significatif sur la virulence et le fitness de E. coli. Les capacités de formation de biofilm et la virulence chez Caenorhabditis elegans étaient augmentées chez les souches de phénotypes « efflux ». Inversement, les souches de phénotypes « imperméabilité » présentaient un faible potentiel de virulence, associé à une diminution significative de la formation de biofilm et de la mobilité par swimming. / The spread of multidrug-resistant (MDR) Enterobacteriaceae is a major public health threat worldwide. Resistant to at least three classes of antibiotics, MDR Enterobacteriaceae cause infections for which first-line treatments are inefficient. The first part of this work focused on the molecular epidemiology of MDR Enterobacteriaceae strains isolated in infections and colonizations of patients hospitalized in Languedoc-Roussillon, in France and in Algeria, a country where few data are currently available. We showed in our region and nationally, that resistance to carbapenems was mainly due to changes in membrane permeability (87.4% of resistant Enterobacteriaceae, nationally).In the second part of this work, we studied the modulation of membrane efflux and permeability in the quintessential example of an international MDR high-risk clone, Escherichia coli ST131. We showed that this global clone had a remarkable adaptability to antibiotic pressure. This adaptability had a significant impact on the virulence and the fitness of E. coli. The biofilm formation and virulence capacities in Caenorhabditis elegans model were increased in strains overexpressing an efflux system. Conversely, the strains with altered porins expression had a low potential virulence, associated with a significant reduction in biofilm formation and swimming mobility.

La surexpression des pompes d’efflux (PE) appartenant à la famille Resistance-Nodulation-Division (RND) est l’un des contributeurs majeurs de la multirésistance (MDR) et la pathogénicité des bactéries Gram-négatives. Ces transporteurs sont capables d'expulser à l’extérieur de la cellule bactérienne différentes classes d'antibiotiques, ce qui contribue de manière significative à l'échec thérapeutique du traitement des maladies infectieuses. Dans ce contexte, les PEs sont des cibles intéressantes pour la découverte de nouveaux antimicrobiens. Afin de combattre ce mécanisme de résistance, des inhibiteurs des pompes d’efflux (EPIs) sont développés comme adjuvants d'antibiotiques dans le but de restaurer ou d'améliorer leur activité. L'archétype AcrAB-TolC est particulièrement répandu chez les espèces d’Enterobacter pertinentes en clinique (pathogènes « ESKAPE »). Cette étude décrit une stratégie basée sur des analogues des fluoroquinolones pour le drug design des EPIs, contre la pompe AcrB chez E. aerogenes. Ainsi, la synthèse et l'évaluation microbiologique des dérivés de quinazoline-4(3H)-one ont été effectuées. Les propriétés structurales et moléculaires des composés testés (i.e. rigidité et flexibilité) ont également été étudiées. Pour cela, de nouveaux scaffolds ont été évaluées. Plusieurs molécules ont montré une augmentation de la sensibilité des bactéries à la norfloxacine et au chloramphénicol. Les résultats obtenus, appuyés par la modélisation moléculaire, suggèrent que la flexibilité moléculaire et la nature des fonctions chimiques des EPIs jouent un rôle essentiel dans l'amélioration de l'activité et la sélectivité vis-à-vis des fluoroquinolones. / Overexpression of Resistance-Nodulation-Division (RND) efflux pumps (EP) is a major contributor in multidrug resistance (MDR) and pathogenicity in Gram-negative bacteria. These transporters are able to expel out of the bacterial cell clinically important antibiotic classes, contributing in a significant manner to the treatment failure of infectious diseases. With the worrying levels of bacterial resistance reported worldwide and the continuous spreading of MDR pathogens, EPs are interesting targets for the discovery of new antimicrobial drugs. Therefore, to overcome this mechanism, efflux pump inhibitors (EPIs) are being developed as adjuvants in order to restore or improve the activity of usual antibiotics. The AcrAB-TolC archetype is particularly widespread in Enterobacter spp. presenting clinical relevance (ESKAPE pathogens). In this study, we described the drug design strategy based on fluoroquinolone antibiotic analogs, against the AcrB pump of E. aerogenes. Thus, synthesis and microbiological evaluation of quinazolin-4(3H)-one derivatives were performed. The structural and molecular properties of the tested compounds (i. e. rigidity and flexibility) were also investigated. In this purpose, a scaffold hopping of the quinazolinone core to homologous benzoquinazolinones and precursors benzamides were carried out. Several molecules increased the bacterial susceptibility towards norfloxacin and chloramphenicol. The obtained results, supported by molecular modeling, suggest that molecular flexibility and the nature of chemical functions play a critical role to improve activity and selectivity on fluoroquinolone potentiation targeting AcrB efflux pump.

Nous avons évalué les différents aspects de la synergie entre des antibiotiques classiques et les huiles essentielles de certaines plantes, endémiques du Maroc, sur des bactéries résistantes impliquées dans les infections nosocomiales. Il s’agit de Thymus maroccanus, T. broussonetii, T. pallidus, T. riataraum et Rosmarinus officinalis. La combinaison de ces huiles essentielles avec des antibiotiques a révélé que parmi 80 combinaisons testées, 71% des combinaisons ont montré une synergie totale, 20% un effet synergique partiel et 9% des combinaisons n’ont pas d’effet synergique. De plus, les huiles essentielles de T. maroccanus et T. broussonetii ont pu augmenter considérablement la sensibilité de plusieurs isolats résistant au chloramphénicol. Elles peuvent restaurer la sensibilité au chloramphénicol en produisant une compétition vis-à-vis de son extrusion en bloquant les pompes impliquées dans son efflux. Par ailleurs, l’huile essentielle de T. maroccanus augmentait la perméabilité de la membrane des bactéries étudiées, et favorisait la libération des protéines intracellulaires dans le milieu extérieur. Elle perméabilise à la fois la membrane interne et la membrane externe des bactéries, mais sans causer une dégradation détectable des constituants cellulaires. Des altérations protéiques ont été associées à l'exposition prolongée aux composés naturels testés. Ces altérations se sont traduites par un efflux accru dont l'effet synergique est renforcé par la baisse du niveau de porines qui peuvent être impliquées dans la diffusion passive de ces antimicrobiens naturels, ce qui confère aux bactéries testées une tolérance accrue à ces composés. / We evaluated different aspects of the synergy between conventional antibiotics and essential oils of some Moroccan endemic plants, on resistant bacteria involved in nosocomial infections. These plants were: Thymus maroccanus, T. broussonetii, T. pallidus, T. riataraum and Rosmarinus officinalis. Out of 80 combinations tested between these two essential oils and antibiotics, 71% showed total synergism, 20% had partial synergistic interaction and 9% showed no effect. Combination with carvacrol, the major constituent of T. maroccanus and T. broussonetii, showed also an interesting synergistic effect in combination with ciprofloxacin. Moreover, T. maroccanus and T. broussonetii essential oils increased chloramphenicol susceptibility of several resistant isolates. they could restore sensitivity to chloramphenicol producing a competition to its extrusion by blocking the efflux pumps expressed in the isolates and involved in its efflux. Furthermore, T. maroccanus essential oil increased permeability of studied bacteria membrane, and favoured intracellular proteins release into the external medium. Thymus maroccanus essential oil permeabilized both outer and inner membranes of tested bacteria, but without causing detectable degradation of cellular constituents. Protein alterations have been associated with prolonged exposure to natural compounds tested, an increased efflux associated with a lower level of porins that may be involved in the passive diffusion of these natural antimicrobials, conferring a native protection to bacteria towards these natural compounds.

A reemergência de algumas bactérias Gram-positivas, em particular, do gênero Staphylococcus, como principal foco causador de infecções hospitalares, tem se intensificado nas últimas décadas, e, apesar da existência de potentes fármacos voltados para o tratamento de infecções causadas por este gênero de bactéria, as taxas de morbidade e mortalidade prevalecem com perfil crescente. Além disso, um grande problema associado a cepas de MRSA (Methicillin-Resistant Staphylococcus aureus) é o fenótipo de multi-resistência, característica que confere a este microrganismo resistência não apenas à meticilina como também a uma série de outros fármacos, exceto frente à vancomicina e à teicoplanina. Muito tem se feito, mas ainda são poucos os resultados efetivamente aplicáveis no tratamento de infecções com caráter de multi-resistência, justificando, desta forma, a necessidade de desenvolvimento de sucedâneos que sejam consideravelmente mais efetivos para a solução deste problema. Baseado nestes fatos, a proposta deste estudo envolveu o planejamento, síntese, identificação e estudos de QSAR (Quantitative Structure-Activity Relationships) em duas e três dimensões de derivados 5-nitro-2-tiofilidênicos com atividade antimicrobiana frente a cepas padrão e multi-resistente de Staphylococcus aureus. A escolha dos grupos substituintes foi realizada em duas etapas. Na primeira delas seguiu-se metodologia de substituição em anéis aromáticos proposta por Topliss para a otimização da bioatividade de compostos. Em uma segunda etapa, predominantemente quantitativa, foram selecionados mais alguns derivados baseando-se em faixa de hidrofobicidade ótima pré-determinada experimentalmente e na variação de efeito estérico dos grupos substituintes. Quatorze derivados 5-nitro-2-tiofilidênicos foram sintetizados, estruturalmente identificados e avaliados quanto à atividade antimicrobiana frente às cepas padrão (ATCC 25923) e multi-resistente (3SP/R33) de Staphylococcus aureus por determinação da concentração inibitória mínima empregando-se método de macrodiluição sucessiva em tubos. Salienta-se que a cepa 3SP/R33 se mostra resistente a dezenove antibióticos empregados na prática médica e apresenta suscetibilidade apenas à vancomicina. As concentrações inibitória e bactericida mínimas apresentadas pelos compostos sintetizados mostraram sofrer influência significativa da hidrofobicidade sobre as referidas atividades de acordo com os estudos de QSAR-2D e QSAR-3D, sendo os resultados obtidos para a cepa multi-resistente absolutamente compatíveis com os anteriormente determinados para a cepa padrão. Os estudos de QSAR-2D indicaram que a atividade antimicrobiana das 5nitro-2-tiofilideno benzidrazidas substituídas sofre influência significativa de duas propriedades físico-químicas que são a hidrofobicidade e a distribuição eletrônica. A relevância dos descritores estruturais σ e efe na determinação da atividade antimicrobiana, sinalizam que a distribuição eletrônica influencia fortemente o aumento da potência antimicrobiana dos compostos em estudo tanto pela influência dos efeitos indutivo e de ressonância na estrutura química do ligante, como também pelos campos moleculares gerados ao redor de grupos substituintes, sugerindo uma possível interação dos mesmos com uma área específica do sítio receptor. Nos estudos de QSAR-3D, foi evidenciado, em concordância com o estudo clássico anteriormente realizado, que a hidrofobicidade prevalece como propriedade de fundamental importância no estabelecimento da atividade antimicrobiana. Foi observada a importância da presença de regiões hidrofílicas pontuais nos compostos de forma a propiciar processos de solvatação e dessolvatação que são críticos na difusão através de membranas biológicas. Pode-se afirmar que a análise de QSAR, considerando os aspectos tridimensionais ligantes, ressaltou a necessidade de um balanço lipofílico-hidrofílico para um bom desempenho das 5-nitro-2-tiofilideno benzidrazidas ρ-substituídas como agentes antimicrobianos. A partir dos resultados obtidos evidenciou-se, neste estudo, o forte potencial de derivados 5-nitro-2-tiofilidênicos como possível alternativa para o desenvolvimento racional, em nível molecular, de fármacos voltados para o tratamento de infecções causadas por cepas multi-resistentes de Staphylococcus aureus. / In the last decade, there has been a reemergence of Gram-positive bacteria, in particular Staphylococcus, which isconsidered one of the. most causing of nosocomial infections. Although potent antistaphylococcal drugs are available, this infection continues presenting increasing morbidity and mortality rates. Besides, a serious problem associated with MRSA (Methicillin-Resistant Staphylococcus aureus) is the phenotype of multidrug resistance, which is, resistance not only to methicillin but also to many other drugs, except to vancomycin and teicoplanin. Many efforts have been made in a tentative to reduce this problem, nevertheless there is only a few number of alternatives to combat Staphylococcus aureus multidrug-resistant strains, justifying the necessity of development of more effective compounds to the treatment of these infections. Based in these facts, the purpose of this study was the design, synthesis, structural identification and 2D-QSAR and 3D-QSAR (Quantitative Structure-Activity Relationships) studies of 5-nitro-2-thiophylidene derivatives with antimicrobial activity against multidrug-resistant strains of Staphylococcus aureus. The choice of substituent groups was made in two stages. The first stage comprises on application of Topliss operational scheme for aromatic substitution. In a second quantitative stage, more derivatives were selected according by hydrophobicity range previously determined. Other standard considered at the selection of substituent groups was the variation of steric effect. Fourteen 5-nitro-2-thiophylidene derivatives were synthesized, structural identified and tested against standard (A TCC 25923) and multidrug-resistant (3SP/R33) strains of Staphylococcus aureus. The Minimal Inhibitory Concentration, MIC, was determined using the serial dilution tests in two sequential stages. The 3SP/R33 strain is resistant to nineteen antimicrobial agents in use, except to vancomycin. The minimal inhibitory and bactericidal concentrations of synthesized compounds showed, according by 2D-QSAR and 3D-QSAR studies, a significant influence of hydrophobic properties on antimicrobial activity determination and the results obtained for multidrug-resistant strain were consistent with those determined for A TCC 25923 strain. 2D-QSAR studies showed that antimicrobial activity are mainly influenced by two physico-chemical properties: hydrophobicity and electronic distribution. The relevance of σ e ephe parameters on antimicrobial activity determination, denotes the contribution of inductive and resonance effects for the polar performed by the substituent groups, probably suggesting an interaction between them and specific receptor site. 3D-QSAR studies showed that hydrophobicity is a essential property to antimicrobial activity determination, sustained the same conclusions previously obtained by Hansch Analysis. It was observed a great concern of small hydrophilic regions distributed on derivatives in order to promote solvation and desolvation process, that have critical importance on diffusion process through the biological membranes. QSAR studies considering three-dimensional properties of ligands indicated the necessity of accurate hydrophilic-hydrophobic balance on nitrothiophene derivatives for their good performance as antimicrobial agents. The results obtained in this preliminary study have shown the potential of synthesized compounds as alternatives to the treatment of infections caused by multidrug-resistant strains of Staphylococcus aureus.

La métastase et la chimiorésistance sont les principales causes de mortalité chez les patients atteints d’un cancer. La compréhension des mécanismes moléculaires régissant ces deux processus devient donc un domaine de recherche important pour la conception de nouvelles stratégies thérapeutiques. Dans ma thèse, je me concentre sur la compréhension du rôle du facteur d’initiation de la traduction chez les eucaryotes 4E (eIF4E) dans l’invasion du cancer, et je décris un nouveau mécanisme de résistance que nous avons découvert en étudiant le développement de la résistance à un inhibiteur connu d’eIF4E, la ribavirine. eIF4E est un puissant oncogène qui est connu pour être élevé dans une multitude de cancers comprenant entre autres les sous-types M4 / M5 de la leucémie myéloïde aiguë (AML). Il fonctionne dans la traduction et l'exportation nucléocytoplasmique d'ARNm en se liant à la coiffe m7G des ARNm possédant des codes USER spécifiques dans leur région UTR 5' et/ou 3'. En reconnaissant ces codes USER, le complexe dans lequel se trouve eIF4E régule de manière coordonnée l'expression de gènes essentiels à la croissance, à la prolifération et à la survie, et ainsi, eIF4E a été placée en tant que nœud central d'un régulon d'ARN régissant la prolifération. En analysant les voies dans lesquelles l’export est régulé de façon coordonnée par eIF4E et les effets physiologiques qui en découlent, j'ai trouvé un enrichissement de la voie biosynthétique de l'acide hyaluronique (HA) et de son principal récepteur CD44 qui sont des médiateurs clés connus des métastases cancéreuses. J’ai également démontré que l'élévation d’eIF4E modifie la surface des cellules cancéreuses en les recouvrant de protrusions riches en HA de type microvillus et enrichies d'armes de destruction métastatique. Heureusement, en dégradant le manteau HA ou en utilisant des inhibiteurs de CD44 en combinaison avec la ribavirine, nous pouvons alors nous défendre. Compte tenu de l'avantage prolifératif que confère la surexpression d’eIF4E, il est devenu un talon d'Achille attrayant pour le traitement de cancers ayant un niveau élevé d'eIF4E. En effet, lors d'un essai clinique de phase II parmi des patients atteints de leucémie myéloïde aiguë M4 / M5 réfractaire et récidivante, la ribavirine a conduit au ciblage d'eIF4E et a donné lieu à des réponses cliniques significatives, incluant des réponses complètes ou partielles. Cependant, tel qu’attendu lors d’un traitement monothérapique, les patients ayant répondu finissent par développer une résistance au médicament. Mon analyse a révélé que cette résistance est due à un mécanisme nouveau caractérisé par l'élévation du facteur de transcription Sonic Hedgehog GLI1 qui conduit à la glucuronidation du médicament et donc à la perte de l'interaction entre la drogue et sa cible. Heureusement, ce mécanisme peut être inversé en utilisant des inhibiteurs de la voie Hedgehog. En conclusion, ces découvertes fournissent de nouvelles cibles thérapeutiques pour le traitement des cellules cancéreuses agressives et résistantes. / Metastasis and chemoresistance are the leading cause of mortality among cancer patients. The discovery of molecular mechanisms governing these two processes is becoming an important area of research for the design of novel therapeutic strategies. In my thesis, I focus on understanding the role of the eukaryotic translation initiation factor 4E (eIF4E) in cancer invasion and describe a novel mechanism of resistance that we discovered while studying the development of resistance to a known eIF4E inhibitor, ribavirin. eIF4E is a potent oncogene that is known to be elevated in a multitude of cancers including M4/M5 subtypes of acute myeloid leukemia (AML). It functions in mRNA translation and nucleocytoplasmic export by binding to the m7G cap of mRNAs possessing specific USER codes in their 5’ and/or 3’ UTRs. By recognizing these USER codes, eIF4E complex coordinately regulates the expression of genes essential for growth, proliferation and survival and as such has been placed as a central node of an RNA regulon governing proliferation. When analyzing which pathways have their export coordinately regulated by eIF4E and what physiological effects arise from it, I found an enrichment in the hyaluronic acid (HA) biosynthetic pathway as well as its major receptor CD44 which are known key mediators of cancer metastasis. I demonstrate that eIF4E elevation changes the surface of cancer cells sugar-coating them with HA-rich microvillus-like protrusions that are enriched with weapons of metastatic destruction. Luckily, through degrading the HA-coat or using inhibitors of CD44 in combination with ribavirin we can strike back. Given the proliferative advantage that eIF4E overexpression conveys, this rendered it as an attractive Achilles heel for the treatment of cancers where eIF4E levels are high. Indeed, in a phase II clinical trial in refractory and relapsed poor prognosis M4/M5 AML patients, ribavirin led to eIF4E targeting and resulted in significant clinical responses including complete and partial remissions. However, as it is expected for monotherapy treatment, all responding patients eventually developed resistance to the drug. My analysis revealed that resistance is due to a novel mechanism characterized by elevation of the Sonic Hedgehog transcription factor GLI1 which leads to drug glucuronidation and the subsequent loss of drug-to-target interaction. Fortunately, this mechanism can be reversed using Hedgehog pathway inhibitors. Taken together, these findings provide novel therapeutic venues for the treatment of aggressive and resistant cancer cells.

Yeast plasma membrane transporters play crucial roles in many cellular processes, including detoxification and build-up and maintenance of the plasma membrane potential (ΔΨ). The former development of the diS-C3(3) fluorescence assay by the Biophysics Group of the Institute of Physics, Charles University, enabled us to conveniently study both, including their changes, using a simple fluorescent probe diS-C3(3). Many studies carried out on both animal and yeast cells have revealed that ethanol and other alcohols inhibit the functions of various membrane channels, receptors and solute transport proteins, and a direct interaction of alcohols with these membrane proteins has been proposed. Using the diS- C3(3) assay for multidrug-resistance pump inhibitors in a set of isogenic yeast pdr5 and snq2 deletion mutants we found that n-alcohols (from ethanol to hexanol) exhibit an inhibitory effect on both pumps, increasing with the length of the alcohol carbon chain. The inhibition is not connected with loss of plasma membrane structural or functional integrity and is fully reversible. This supports a notion that the inhibitory action does not necessarily involve only changes in the lipid matrix of the membrane but may entail a direct interaction of the alcohols with the pump proteins. Tok1p is a highly specific...