Masters of Science / Retinoblastoma Binding Protein 6 (RBBP6) is a RING finger-containing protein which plays a critical role in the 3'-end processing of mRNA transcripts. It is a constituent of the human pre-mRNA processing complex but also interacts directly with core splicing-associated proteins. RBBP6 also interacts with both major tumour suppressor proteins p53 and pRb and is known to play a critical role in suppression of p53 during development, in cooperation with MDM2. Through its RING finger it interacts with the C-terminus of the oncogenic protein Y-Box Binding Protein 1 (YB-1) both in vitro and in vivo, catalysing its ubiquitination and degradation in the proteasome. YB-1 is closely associated with tumour progression, poor patient prognosis and chemotherapeutic resistance, making it a promising target for therapeutic intervention. Unpublished data from our laboratory suggests that RBBP6 is able to poly-ubiquitinate YB-1 in vitro, using UbcH1 as the ubiquitin- conjugating enzyme (E2). This study aims to identify RBBP6 RING protein-protein interactions involved in the down regulation of YB-1 by RBBP6. These interactions include the C-terminal fragment of YB-1 (substrate), MDM2 (E3) and UbcH1 (E2). The C-terminal fragment of YB-1, denoted YB-1₂₂₀₋₃₂₄, was successfully cloned and expressed in bacteria and demonstrated to interact directly with the RBBP6 RING finger domain in in vitro affinity pull down assays. This is in good agreement with our unpublished data that RBBP6 is able to ubiquitinate full length YB-1 as well as the YB-1₂₂₀₋₃₂₄ fragment. UbcH1 was successfully expressed and shown to interact directly with RBBP6 RING in in vitro affinity pull down assays. This is also in agreement with our data showing that RBBP6 is able to ubiquitinate YB-1 using UbcH1 as E2. ¹⁵N-labelled samples of RBBP6 RING was successfully expressed in bacteria and used to investigate the putative interaction with UbcH1 in NMR-based chemical shift perturbation assays. However no interaction was observed, possibly because the sample of UbcH1 was subsequently found using mass spectrometery to be partially degraded. GST-tagged RBBP6 RING was able to precipitate MDM2 from HeLa lysate. This extends previous reports that full length RBBP6 and MDM2 interact directly and play a role in the suppression of p53 during development. The result was validated by showing that GST-MDM2 was able to precipitate RBBP6 RING in in vitro. This study includes a side project which involved the cloning and expression of DWNN-GG. GST-HADWNN-GG was successfully cloned and expressed in bacteria. An HA tag was included immediately upstream of DWNN-GG for immunodetection using anti-HA antibodies; the construct was designed in such as way that it could be re-used to generate HA-tagged versions of existing constructs cloned into pGEX-6P-2. The above findings lay the foundation for future structural and functional studies of the involvement of RBBP6 in regulation of the cancer-related proteins p53 and YB-1, which may have far-reaching consequences in the fight against cancer. / National Research Foundation (NRF)

La recherche de nouvelles molécules à visée thérapeutique est un enjeu majeur pour la communauté scientifique et actuellement, 60% des médicaments utilisés cliniquement sont des produits naturels ou leurs analogues. Dans ce contexte, afin d’être capable de générer rapidement une bibliothèque de composés, le développement d’outils de synthèse efficaces est fondamental et c’est dans cette optique que s’inscrit le travail mené au cours de cette thèse. Les produits d’origine marine sont une source d’inspiration considérable dans le domaine de la création de nouveaux médicaments. Lors de ce travail, une attention particulière a été accordée à la ningaline B et à ses analogues avec pour objectif de mettre au point une voie d’accès simple et efficace. En effet, si la capacité de la forme hexaméthyléther à reverser la résistance aux anticancéreux par inhibition de la glycoprotéine-P a déjà été rapportée dans la littérature, l’étude d’analogues n’a été que peu exploitée.Dans ce contexte, une voie de synthèse reposant sur une étape clé de double couplage de Suzuki-Miyaura sur un dérivé (pseudo)dihalogéné symétrique a été proposée. Cette étape a fait l’objet d’une étude approfondie en envisageant deux approches. La première approche, basée sur des travaux antérieurs réalisés au laboratoire, a été de type simultané. Ainsi, à la manière d’une réaction multicomposante, tous les réactifs sont introduits dès le départ dans le milieu réactionnel. Cette méthode a montré des résultats intéressants dans le cas où deux dérivés borés électroniquement différents sont employés. En revanche, il a été montré que l’utilisation d’espèces borés électroniquement similaires était peu viable avec l’obtention d’un mélange statistique en produits de dicouplage. Toutefois, la préparation des cibles visées requérant l’utilisation d’aryles borés riches en électrons, une autre approche, séquentielle, a été envisagée. Après un travail d’optimisation sur des substrats simplifiés, avec pour objectif de disposer de conditions efficaces, adaptables, faciles avec des réactifs standards, une méthode de monoarylation de dérivés (pseudo)dihalogénés symétriques a été mise en place. Le champ d’application a ensuite été élargi en incluant les noyaux pyrroles nécessaires à la synthèse de la ningaline B et de ses dérivés. Ces travaux ont permis d’accéder aux molécules ciblées. En effet, une fois, les teraryles préparés, quelques étapes de manipulations fonctionnelles permettront d’avoir rapidement accès à de nombreux analogues. D’autre part, les motifs pouvant être obtenus par ce type de séquence réactionnelle pourraient être facilement dérivatisables afin de générer une bibliothèque d’analogues. / New therapeutic targets inversigation is one of the most serious challenges for the scientific community. Nowadays, 60% of clinically used drugs are natural products or their analogs. Thus, the development of new efficient synthetic tools to easily access such library of compounds is crucial. Marin natural products are an important source of inspiration for original drug design. Here we focused on development of an easy and efficient synthesis of ningalin B and its analogs. Indeed, while the literature reports the potential of the hexamethyl ether form to reverse multidrug resistance inhibing P-glycoprotein, only few studies have been done on analogs.Thereby, here we suggest a synthesis based on double Suzuki-Miyaura couplings on symmetrical dihaloarenes as a key step. This is a step to deal with studies in depth and two approaches have been envisaged. The first one relies on a previous simultaneous work in the laboratory. Thus, like a multicomponent reaction, all the starting materials are introduced in the mixture at the beginning of the reaction. This method show promising results when opposed electronic aryl boron derivatives are employ. Nevertheless, using electronic similar boron derivatives, a statistical mixture of dicoupling products is obtained. Since the preparation of our targets only needs the introduction of electron rich aryl moieties, a sequential approach has been envisaged. Preliminary optimizations afford optimal monocoupling conditions on symmetrical dihaloarenes. The scope of the reaction has then been studied including pyrrol nucleuses that are required for the synthesis of ningalins.This work led to desired molecules and analogs can be easily access in a few steps to fulfil a library of compounds.

Breast cancer resistance protein (BCRP/ ABCG2), an ATP-binding cassette half transporter, confers multidrug resistance (MDR) to a series of antitumor agents such as mitoxantrone, daunorubicin, SN-38, and topotecan, and often limits the efficacy of chemotherapy. Recent studies have indicated that a putative estrogen response element (ERE) is located in the promoter region of the BCRP gene. However, whether and how BCRP is regulated transcriptionally by toremifene (TOR) remains unknown. In the present study, two plasmid vectors have been designed to express the wild-Type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a constitutive cytomegalovirus (CMV) promoter as control, respectively, which were transfected into estrogenresponsive MCF-7 and estrogen-independent MDA-MB-231 human breast adenocarcinoma cell lines. We showed that toremifene alone significantly downregulated BCRP mRNA and protein levels in estrogen receptor a (ERa)-positiveMCF- 7 cells in a dose-dependent manner, and the inhibitory effect was partially reversed by estrone (E1). Furthermore, gel shift assays demonstrated that specific binding of ERa to the ERE in the BCRP promoter is essential for transcriptional inhibition of BCRP by toremifene. Interestingly, toremifene alone increased the cellular accumulation of mitoxantrone inBCRPtransfected cells, suggesting that TOR indeed inhibits BCRPmediated drug efflux and overcome drug resistance. To the best of our knowledge, this is the first report describing a direct effect of toremifene on BCRP. Our results thus indicate that toremifene by itself downregulates BCRP expression to reverse BCRP-mediated atypical multidrug resistance via a novel transcriptionally mechanism, which might be involved inTOR-ERcomplexes binding to theEREofBCRP promoter to repress transcription of BCRP gene.

Multidrug resistance (MDR) is a major obstacle of current chemotherapy. In this study, we investigated the effects ofpolyamines, acetylated polyamines and APAH, a polyamine deacetylase inhibitor, on LS180 cells and the derived MDR LS180 Ad-50 cells. L.ethal concentrations for 20% of the cells (LC20) for N8 -acetylspermidine (N8 - AcSPD) and SPD were 12.5 and 7 times greater for LS180 Ad-50 cells than for LS180 cells, respectively. However, there was no difference in LC20 levels for N1-AcSPD in LS180 and LS180 Ad-50 cells. Addition of 100 nM APAH to N8-AcSPD increased its LC20 to 17 foldgreater in MDR compared to non:·MDR cells. and resulted in increased cell growth at 5 μM N8-AcSPD. Furthermore, exposure to the LC20 concentrations of N8 -AcSPD for 48 hrs caused no significant change of intracellular Rhodamine (Rh) 123 concentrations. Adding 100 nM APAH to this N8 -AcSPD experiment led to a decrease of intracellular Rh123 concentration of 15-20% in both LS 180 and LS 180 Ad-50 cells. These Rh 123 concentrations further decreased in the presence of higher concentrations of APAH. Pretreatment of the cells with APAH did not affect these results. Kinetic studies suggest that APAH may be a non-competitive transport inhibitor of Rh123 transporter.

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Multidrug resistance (MDR) in breast cancer treatment is the major cause leading to the failure of chemotherapy. P-glycoprotein (P-gp), the product of the human MDR1 gene, plays a key role in resistance to chemotherapy and confers cross-resistance to many structurally unrelated anticancer drugs. We have previously reported that integrin αvβ6 plays a critical role in breast cancer invasion and metastasis. However, whether and how αvβ6 is associated with P-gp and regulated by potential genetic mechanisms in breast cancer remains unclear. In the present study, we further investigated the reversal effect and underlying mechanisms of MDR in breast cancer. Two small interfering RNA constructs (pSUPER-β6shRNAs) targeting two different regions of the β6 gene have been designed to inhibit αvβ6 expression by transfecting them into adriamycin-resistant MCF-7/ADR cell lines. Suppression of αvβ6 dramatically downregulated the levels of MDR1 gene mRNA and P-gp. In particular, β6shRNA-mediated silencing of αvβ6 gene increased significantly the cellular accumulation of Rhodamine 123 and markedly decreased drug efflux ability, suggesting that β6shRNAs indeed inhibit P-gp mediated drug efflux and effectively overcome drug resistance. In addition, inhibition of integrin αvβ6 suppressed the expression of ERK1/2. Interestingly, our data demonstrate that suppression of integrin αvβ6 caused significant downregulation of Bcl-2, Bcl-xL and upregulation of caspase 3, Bad, accompanied by increasing activity of cytochrome C. A possible connection between αvβ6 and P-gp in drug resistance biology is suggested. Taken together, β6shRNA could efficiently inhibit αvβ6 and MDR1 expression in vitro and these findings may offer specifically useful means to reverse MDR in breast cancer therapy.

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Yes / Recent studies portend a rising global spread and adaptation of human- or healthcare- associated pathogens. Here, we analyse an international collection of the emerging, multi-drug-resistant, opportunistic pathogen Stenotrophomonas maltophilia from 22 countries to infer population structure and clonality at a global level. We show that the S. maltophilia complex is divided into 23 monophyletic lineages, most of which harbour strains of all degrees of human virulence. Lineage Sm6 comprises the highest rate of human-associated strains, linked to key virulence and resistance genes. Transmission analysis identifies potential outbreak events of genetically closely related strains isolated within days or weeks in the same hospitals.

No description available.