| dc.description.abstracteng | The intracellular distribution of proteins and ribonucleoproteins is a fundamental process in all eukaryotes that is critically vital for the normal functionality of the eukaryotic cell. In the last decades, it has been discovered that cancer cells utilize the transport machinery to stimulate tumor growth and to effectively evade apoptotic mechanisms. Chromosome region maintenance 1 (CRM1) is a major nuclear export receptor that was found to mediate the mislocalization of several tumor suppressor proteins such as Rb, APC, p53, p21 and p27 as well as cancer drug targets like topoisomerase II α. In addition, elevated CRM1 expression has been observed in several cancers and was correlated with poor patient prognosis. This renders CRM1 a particularly interesting target for therapeutic intervention in diverse cancer diseases. In the recent years, structural characterization of CRM1 inhibitor complexes has been performed exclusively using CRM1 from the yeast Saccharomyces cerevisiae. The yeast protein was genetically modified to mimic its human homolog by the incorporation of a cysteine residue that is required for inhibitor binding. This approach has been used as a model system for the structural characterization of several natural and synthetic inhibitors bound to yeast CRM1. The performed studies defined a typical mechanism of CRM1 inhibition by the covalent modification of a reactive cysteine residue that is located in the nuclear export signal (NES)-binding cleft (Cys528 in human). The aim of this work was to gain structural and biochemical insight into the Inhibition of human CRM1, the actual protein targeted for anti-tumor drug development. This study focused on the biochemical and structural characterization of four novel CRM1 inhibitor candidates: the compounds C3, C6, C10 and DP2392-E10. In addition, during this work Leptomycin B (LMB), a well-studied CRM1 inhibitor, was implemented for the development of a successful crystallization approach of human CRM1 – inhibitor complexes. Genetic modifications coupled with intensive screening for potential crystallization conditions succeeded to gain the crystal structure of LMB bound to human CRM1-RanGTP complex. The obtained structure revealed that LMB interactions with human CRM1 are rather similar but not identical to those with the yeast variant. Moreover, binding assays showed that some of the tested novel compounds as well as a NES peptide exhibit no binding or lower binding affinity towards yeast CRM1 when compared to the human protein. Introducing the reactive cysteine to yeast CRM1 did not lead to the binding of some of the tested compounds, indicating that the used model system is limited to a certain class of human CRM1 inhibitors. Furthermore, during this work several experimental and computational methods were applied using human CRM1 to elucidate the inhibitory mechanisms of C3, C6, C10 and DP2392-E10. Quantitative binding assays revealed that all four compounds bind directly to CRM1 in a concentration dependent manner. Further analysis unveiled that CRM1 inhibition by C3, C6 and C10 is mediated by the reactive cysteine (Cys528), which indicated they act by the direct blocking of the NES-binding cleft. In contrast, DP2392-E10 exhibited unique binding properties that are not dependent on Cys528. Computational docking, using ‘ligand free’ human CRM1-LMB complex structure as a template, defined the binding of DP2392-E10 outside the NES-binding cleft, at the base of the acidic loop. The identified binding mode suggests a novel mechanism of CRM1 inhibition by the allosteric modulation of the NES-binding cleft. Moreover, computational analysis on human CRM1 identified several potential ligand binding sites outside the NES-binding cleft, which indicates for potential alternative mechanisms for the inhibition of CRM1-mediated nuclear export.
The work presented here provides new insights into human CRM1 inhibition and it emphasizes the importance of using the human protein for inhibitor studies. Furthermore, the crystal structure of CRM1-RanGTP-LMB complex obtained during this study represents a valuable framework for different experimental and computational methods that can be applied for CRM1-targeting drug design. | de |