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HIV nuclear import and chromatin reading as novel drug targets

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.

Date:1 Sep 2013 →  30 Nov 2018
Keywords:drug discovery, HIV, MLL
Disciplines:Microbiology, Systems biology, Laboratory medicine, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering
Project type:PhD project