Exploration of Histone Deacetylase Ligand Binding Modes by Photoaffinity Probes
thesisposted on 22.02.2013, 00:00 by He Bai
Histone deacetylases (HDACs) regulate chromatin structure and function. Since much of the literature has reported aberrant expression and recruitment of HDAC in malignant tissues, the role of HDACs in regulating the genes that are involved in cell cycle progression and arrest makes them attractive therapeutic targets for the treatment of cancer. Even though the pan-HDAC inhibitor showed promising antiproliferative activity, their application may be limited due to their toxicity and negative side-effects. To address this gap, design of isoform-selective HDAC inhibitors might be of significant importance for the treatment of cancer, making the knowledge of how inhibitors interact with HDACs and their binding mode crucial for isoform-specific inhibitor discovery. This study focused on investigating the interaction between HDAC probes and HDACs at the molecular level by using a combination of in vitro bioassays, immunoblotting, fluorescence spectroscopy, and protein mass spectrometry. In this dissertation, a binding ensemble profiling with (f)photoaffinity labeling approach that utilizes photolabeling of HDAC8 or HDAC3/NCoR2-DAD with a probe containing a UV-reactive aromatic azide, mapping of the covalent modification by liquid chromatography-tandem mass spectrometry, and a computational method to characterize the binding poses of the probe is described. 1) Two distinct binding poses of the HDAC8 probe were identified. An “upside-down” pose with the surface binding group of the probe bound in an alternative pocket near the catalytic site may contribute to the binding. 2) Modification sites on HDAC3 and NCoR2-DAD were identified and the interaction between HDAC3 and its corepressor NCoR2-DAD was explored. The results presented in this study showed that our combined approach could not only delineate the regions involved in the interface between two interacting proteins but also lead to structural models where the relative orientation of the proteins and their accessibility by small molecule inhibitors are defined.