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...

Therapeutic resistance of tumours represents an important clinical issue. We can classify the therapeutic tumour resistance in two ways. According to the clinical course, tumours can behave either as primary resistant, i.e. from the very beginning not responsive, or they can display a secondary (also called acquired) resistance, whereby an initial clinical response is lost and the tumour develops into chemo-, radio- or immunoresistant disease. An alternative classification distinguishes cell autonomous resistance mechanisms from resistance that relies on complex interactions within the context of tumour microenvironment. From the research perspective, modelling therapeutic resistance frequently involves experimental treatment of sensitive cancer cells and selection of daughter resistant cell lines. The Ph.D. thesis includes derivation of two unique models of urothelial bladder carcinoma therapeutic resistance. The first model involves newly established urothelial carcinoma cell lines BC44 and BC44DoxoR, which resulted from a prolonged doxorubicin exposure of the mother cell line. The daughter chemoresistant cell line exhibits multidrug resistant phenotype, which extends beyond the selecting drug - doxorubicin - to four additional chemotherapeutic drugs (cisplatin, methotrexate, vinblastine, and...

One of the main reasons for the treatment failure of infections caused by pathogenic microorganisms is the overexpressing of efflux membrane proteins, which actively remove drugs from cells, leading to a phenomenon called multidrug resistance MDR. In this work, we focused on the functional characterization of the MDR pump Pdr5p in the yeast Saccharomyces cerevisiae. We have verified that diS-C3(3) fluorescence method can be used to determine the binding sites where the substrates bind in the binding pocket of the pump ScPdr5p. We focused on the study of the ScPdr5p binding pocket using triazole derivatives: ravuconazole, voriconazole and fluconazole. Using disc diffusion assay, we showed that all three studied triazoles are substrates of the pump ScPdr5p. We have found that these structural analogs have a significantly different effect on the inhibition of the potentiometric fluorescent probe diS-C3(3) transport by the pump ScPdr5p, and also that ravuconazole and voriconazole compete with each other for transport by the pump ScPdr5p. We have used a fluorescent approach to study the binding of azoles to the binding pocket of pump ScPdr5p using benchmark substrates, that bind selectively to only one binding site in the binding pocket of the pump ScPdr5p, and we have supported the hypothesis that ravuconazole...

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