Martin Luther University Halle-Wittenberg

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Histone modifying enzymes

Structure-based drug design and virutal screening on histon-modifying enzymes - methyltransferases, acetylases and deacetylases

I) Structure-based design and development of novel HDAC and HAT inhibitors

Imbalance in histone acetylation can lead to changes in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and/or apoptosis. Histone acetyltransferases (HATs) and histone deacetylases (HDACs), are two classes of enzymes regulating histone acetylation and whose altered activity has been identified in several cancers. HATs and HDACs enzymes also target non histone protein substrates, including transcription factors, nuclear import factors, cytoskeleton and chaperon proteins. HDAC inhibitors are a novel class of anticancer agents which have been recently shown to induce growth arrest and apoptosis in a variety of human cancer cells by mechanism that cannot be solely attributed to the level of histone acetylation. Several clinical studies with HDAC inhibitors are ongoing, however the molecular basis for their tumour selectivity remains unknown and represent a challenge for the cancer research community.
Information on HDAC subtype selectivity of available inhibitors is limited and the consequences of such selectivity are mostly unclear. Also only limited information is available on the three-dimensional structure of HDACs. The X-ray structure of a bacterial homologue, with yet unknown function, has been resolved and was used by us to construct a homology model of human HDAC1 and HDAC6. These models showed a potential link between certain biological activities and the different binding modes of structurally similar inhibitors, thus implying subtype selectivity. Very recently, the X-ray structure of one human subtype (HDAC8) has been resolved and showed that some inhibitors are able to influence the conformation of that HDAC subtype. The structural information of X-ray data and homology models will now be used to analyze the mode of interaction of different chemical types of HDAC inhibitors.Further, novel modular HDAC inhibitors containing structural elements that allow for conformational control in the different modules shall be developed. They will be tested for the enzyme inhibition as well as for anticancer activities. Analyses of conformations that lead to stronger or more selective inhibition and docking studies using homology models of HDAC subtypes should drive further inhibitor optimization. This in turn should lead to valuable tools for gene regulation studies and the analysis of the therapeutic potential of that class of compounds.
Our investigations aim at the analysis of conformational control synthetic chemistry can achieve on different levels in drug modulated chromatin acetylation. The first level we will address is the possibility of conformational control in the inhibitor molecules and systematic variations in each of the three inhibitor modules will be performed. They will be guided by theoretical analysis of the resulting conformations. The second level will deal with the ability of certain inhibitors to induce conformational changes in the target histone deacetylases. The third level will be the consequences of histone deacetylase inhibition in terms of chromatin conformation and hence transcriptional activity and cellular function. The synthesis of ideas resulting from structure based design is carried out in the lab.

Further information: A-PARADDISE website (    ). German press release.http://pressemitteilungen

Collaborators: Prof. Dr. M. Jung, Department of Pharmaceutical Sciences; University of Freiburg, Germany (Analysis of the in-vitro enzyme-inhibition, synthesis of novel bioactive molecules). Dr. Chris Romier, IGBMC, Universite Strasbourg, France (X-ray crystallography).

II) Analysis of subtype selectivity of Sirtuin histone deacetylases inhibitors

Strongly related to the before mentioned project is the analysis of histone deacetylases from the Sir2 family and histone arginine methylases. Both enzymes have been implicated in transcription silencing and suppression of recombination. The Sir2 complex represses transcription at telomeres, mating-type loci, and ribosomal DNA. The Sir2 gene is highly conserved in organisms ranging from archaea to humans. Interestingly, Sir2 is active as an NAD+-dependent deacetylase (in contrast to the HDAC histone deacetylases, which are Zinc-dependent enzymes), which is broadly conserved from bacteria to higher eukaryotes.
To further analyze the function of human Sir2 enzymes, for which several subtypes have been detected so far, potent and subtype-selective inhibitors are needed. For the human Sirtuin subtype II the crystal structure has been recently described which will now be used for structure-based design of inhibitors. The docking of the lead compound splitomycin resulted in a hypothesis for the binding mechanism of this molecule. On the basis of a variety of different theoretical approaches we have designed modified splitomycin derivatives which have been synthesized and tested by our collaboration partner Prof. Manfred Jung in Freiburg. The novel compounds are active in the sub-micromolar range on Sirtuin subtype II with a moderate selectivity towards subtype I and III. In the ongoing projects we are interested in further optimizing the inhibitors (activity and selectivity) using theoretical and analytical methods. To investigate the selectivity profile of the compounds we have generated in addition a homology model for the human subtype I and III. The aim is to derive potent ligands to analyze the therapeutic potential of this new drug family in detail.

Collaborators: Prof. Dr. M. Jung, Department of Pharmaceutical Sciences; University of Freiburg, Germany (Analysis of the in-vitro enzyme-inhibition, synthesis of novel bioactive molecules). Prof. Mike Schutkowski, Institute of Biochemistry, MLU Halle-Wittenberg

III) Virtual screening and experimental testing of histone methyltransferase inhibitors

The third part of the project considers the histone methylases – a novel and so far therapeutically not analyzed enzyme. Although the number of enzymes involved in protein methylation is increasing, the function of this modification is not cleared up. Within the family of histone arginine methylases a broad spectrum of different substrates has been reported, indicating a high level of complexity. Therefore, the study of the responsible enzymes and the analysis of substrate specificities represent important steps for understanding the functions of these proteins in cellular regulation. Several x-ray structures of arginine methylases from different species have been solved recently which will be used a starting point for the structure-based design of potential inhibitors. Until now no histone arginine methylase inhibitor has been described in the literature. To investigate this novel target class we have initiated combined virtual and in vitro screenings to find inhibiting compounds. A variety of different compound databases, ranging from natural product libraries (provided by the Hans-Knöll-Institute, Jena), the NCI-Diversity Dataset, up to commercially available libraries from Maybridge and Leadquest were virtually screened by different protein-based docking methods. Due to the limited testing capacity a subset of about 100 compounds was selected out of the 100.000 database molecules and tested within a fluorescence-assay by the cooperation partner. Out of the selected compounds more than 70% of the compounds are active in the micromolar range. Beside the inhibition on arginine methylases also the selectivity profile of the compounds towards lysine methylases was investigated by theoretical approaches and experimental testing. The most promising compounds were selected and were tested in addition on an in vivo reporter gene assay. The bioactive molecules represent a novel class of pharmacological tools to study the various effects of histone methylation. In addition biophysical studies of the binding characteristics of these novel inhibitors are planned.

Prof. Dr. M. Jung, Department of Pharmaceutical Sciences; University of Freiburg, Germany (Analysis of the in-vitro enzyme-inhibition, synthesis of novel bioactive molecules)