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The replication cycle of HIV is a complicated interplay between viral and cellular proteins. Until now antiretroviral therapy mainly targets the viral proteins although the interaction between the viral and cellular proteins could be interesting new drug targets. This strategy is mainly of interest because cellular proteins are much less prone to mutations which gives the virus less opportunity to develop drug resistance. My laboratory has a track record in research on cellular cofactors of the viral enzyme integrase. In 2003 Cherepanov et al. discovered LEDGF/p75 as an interaction partner of integrase and in 2008 Christ et al. found transportin-SR2 in a yeast-two-hybrid screen as an interaction partner of integrase. Transportin-SR2 plays an important role in the nuclear import of the virus and LEDGF/p75 tethers the viral DNA to actively transcribed chromatin where it can integrate. Both proteins form interesting new targets for the treatment of HIV. Next to its role in HIV replication, LEDGF/p75 plays a role in mixed lineage leukemia. This type of cancer is driven by translocations involving the MLL1-gene and mainly affects children. Targeting the chromatin reading function of LEDGF/p75 could therefore be an interesting drug target in the treatment of both HIV and mixed lineage leukemia.In the first chapter of this PhD thesis I focus on the TRN-IN interaction and try to gain more insight into their binding mode. Since nuclear import is generally considered as a bottleneck in the viral replication cycle this could be an interesting new target for antiretroviral therapy. Previous studies have already revealed that mainly the catalytic core domain (CCD) and the C-terminal domain (CTD) of integrase interact with transportin-SR2. The aim of this work was to gain more insight into this protein complex and to pinpoint the important regions in TRN-SR2 that are necessary for integrase binding. In collaboration with the bio-crystallography lab I revealed that one TRN-SR2 molecule can bind a CCD-CTD dimer. Next, I divided transportin-SR2 into small peptides, each corresponding to a single HEAT repeat and used AlphaScreen binding assays to determine if these peptides could still bind integrase. This study revealed that it is mainly the N-terminal region of TRN-SR2 that interacts with integrase, principally through HEAT repeats 4, 10 and 11. Based on these results in combination with small-angle X-ray scattering data for the complex of TRN-SR2 with a truncated integrase, I propose a model of the complex in which the nuclear import of the pre-integration complex can proceed in parallel with nuclear transport of its endogenous cargoes.In the second part of this thesis the aim was to identify novel antivirals that do not suffer from cross-resistance with existing drugs. This chapter describes the development and use of an AlphaScreen-based high-throughput screening cascade for inhibitors of the TRN-IN interaction. In total, 25608 small molecules were tested and after eliminating false positives and nonspecific protein-protein interaction inhibitors two active compound series were discovered. Although the inhibitory effect of these compounds in a multiple and single round antiviral activity assay was only moderate, they significantly reduced the nuclear import of fluorescently labelled HIV particles. This study shows that it is possible to use the AlphaScreen technology as a high throughput platform to screen for a novel class of inhibitors. These results again confirm the important role of the TRN-IN interaction in nuclear import. Our hit compounds represent the first small molecule inhibitors of this step in the viral replication cycle and hold promise for future drug development.The last part of this work focuses on the protein-chromatin interaction between LEDGF/p75 and the trimethylated lysine 36 on histone 3 (H3K36me3). The PWWP domain of LEDGF/p75 plays an important role in tethering the HIV pre-integration complex to the host chromatin. Also in the case of mixed lineage leukemia, LEDGF/p75 is responsible for targeting the leukemogenic MLL-fusion/MENIN complex to actively transcribed genes. Targeting the LEDGF/p75-H3K36me3 interaction could therefore have beneficial therapeutic effects in the treatment of both HIV and mixed lineage leukemia. This part of the thesis describes the development and use of a fragment-based drug discovery campaign. Molecular modelling was used to virtually screen over 4 million compounds for binding to the PWWP domain and the top 525 molecules were selected. An AlphaScreen-based screen was next used to pick initial hit compounds from this small compound library. All compounds were also tested in a nano-DSF set-up in which the intrinsic tryptophan fluorescence of the PWWP domain is monitored upon thermal denaturation in the presence and absence of compounds. Initial results indicate that some of our compounds can indeed inhibit the LEDGF/p75-H3K36me3 interaction, although the effect is only moderate. In parallel we have therefore worked on resolving the crystal structure of the PWWP domain. This structural information could guide further chemical optimization of our compounds.In brief, this work aims to discover and validate novel targets for drug discovery targeting HIV and/or mixed lineage leukemia. Protein-protein and protein-chromatin interactions can be interesting new targets since the risk for cross-resistance with already existing drugs is rather low. However, the main challenge in targeting these interactions is to develop selectivity against the specific interaction while keeping interference with other proteins to a minimum.

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Human Immunodeficiency Virus type-1 (HIV-1) hijacks the host replication machinery for efficient replication and completion of its viral life cycle. To ensure that the viral RNA genome is transcribed, it retrotranscribes its RNA into DNA that is integrated into the host genome. During integration, HIV integrase (HIV-IN) interacts with the viral cDNA and the human protein lens epithelium-derived growth factor p75 (LEDGF/p75). LEDGF/p75 tethers binding partners in the nucleus to the chromatin. The N-terminus of LEDGF/p75 (PWWP) recognizes actively transcribed genes whereby HIV-IN is tethered to these genes. Consequently the cDNA is integrated in the human genome catalyzed by HIV-IN. Due to an interaction with human proteins during the viral life cycle it is necessary to first understand the cellular function of LEDGF/p75. Earlier studies published the ability of LEDGF/p75 dimerization. NMR experiments revealed amino acids involved in the dimerization, located at the IBD and CTD. Independently, the IBD and CTD of LEDGF/p75 was examined in other studies as well. Long-term nonprogressor HIV infected patients carry mutations (clinical mutations) in CTD. LEDGF/p75 analysed during the presence of autoimmunity diseases was cleaved in the CTD by caspase-3 and -7. In this thesis I examined whether NMR based mutations, clinical mutations or cleaved LEDGF/p75 analogues affect LEDGF/p75 dimerization. By the use of Alphascreens it was revealed that NMR based mutants still show dimerization but less compared to WT LEDGF/p75. Cleaved LEDGF/p75 analogues show better dimerization while dimerization of clinical mutants is abolished. Before variations in dimerization can be correlated with the development of long- term nonprogressors or autoimmunity diseases, the cellular functions of dimerization first need to be clarified.

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Mucoviscidose is de meest voorkomende autosomaal recessieve aandoening en treft voornamelijk het Kaukasische ras. Wereldwijd wordt het aantal patiënten op 70.000 geschat en de ziekte leidt tot een gemiddelde levensverwachting van 37 jaar. Mucoviscidose, ook gekend als taaislijmziekte of cystische fibrose, wordt veroorzaakt door een mutatie in één gen, het Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gen dat codeert voor het CFTR proteïne. Dit eiwit fungeert als anionenkanaal en mutaties in het CFTR gen leiden tot een verhoogde morbiditeit en mortaliteit voornamelijk van respiratoire oorsprong. Voor de zeldzame mutaties, N1303K (3%) en G85E (0,7%), is tot op heden nog maar weinig onderzoek gedaan naar het beter begrijpen van de defecten en het ontwikkelen van nieuwe therapieën. Bovendien vertonen beiden geen CFTR activiteit na behandeling met de recent ontwikkelde lumacaftor en/of ivacaftor therapie. Het onderzoek, waar deze masterproef deel van uitmaakt, beklemtoont het belang van het creëren van meer algemene kennis naar de basisdefecten van twee minder onderzochte vouwingsmutanten. Bovendien heeft de optimalisatie van de hoge doorvoer testen, op basis van high-content analysis van plasmamembraan gelokaliseerd CFTR, enkele essentiële voordelen ten opzichte van hoge doorvoer- programma’s, die de functie van het ionenkanaal analyseren. Naast een verruiming van de algemene kennis over het opstellen van corrector-screeningsprogramma’s, kan dit onderzoek in de toekomst eventueel bijdragen aan een ontdekking van een therapie voor de G85E en/of N1303K mutant. Dit academisch onderzoek vult de beperkte kennis over deze mutanten verder aan en kan op die manier aanvullend zijn aan de farmaceutische industrie om samen te slagen in één opzet: het tegemoet komen aan de hoge noden van deze patiënten door nieuwe, inventieve therapieën.

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HIV remains a global health concern. Currently, HIV can only be suppressed through lifelong combination antiretroviral therapy (cART), because of a latent HIV reservoir where the virus can persist and from which it can rebound after cART interruption. In the quest for a functional or sterilizing cure, research on this reservoir was mainly conducted in blood. However, research in tissues is important, since less than 2% of body lymphocytes actually reside in blood and there is still much that is unknown about the penetrance of cART in different tissues. We thoroughly evaluated the current studies on the cellular and anatomical latent HIV reservoir and on its replication-competency and latency. We found that these reservoirs were primarily situated in secondary lymphoid organs and promising results identified the brain and adipose tissue as possible reservoirs too. Memory CD4+ T cells make up the largest cellular latent HIV reservoir. Several subsets of these cells were identified as major contributors, including CXCR5+/-PD-1+ cells in B follicles, CCR6+ in colon, lymph nodes and blood. To date, it has not been shown that macrophages contain a stable latent HIV reservoir. Similarly, the extent to which ongoing-replication contributes to the persistence of the latent HIV reservoir is still unclear. Further research on HIV latency should be carried out by identifying specific tissue macrophages and subsets of memory CD4+ T-cells to provide more insights into the specific latent HIV reservoirs in tissues, as well as into subset-targeted eradication strategies.

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Gene therapy has the potential to provide a mutation-independent and curative therapy for cystic fibrosis airway disease. To date, 27 clinical trials have been completed, of which only one achieved a mildly improved clinical outcome. Failure of these early trials is partially attributed to limitations in preclinical evaluation of potential gene therapy. In recent years, the development of CF animal models that mimic CF airway disease have helped to further elucidate CF pathophysiology. Moreover, several new read-out assays allow efficacy evaluation of potential therapies on a number of different lung disease parameters. In this review, we provide an overview of the advances in animal models and read-out assays, propose a timeline for gene therapy evaluation and discuss future perspectives for CF gene therapy.