Dynamic post-translational histone modifications studied by NMR spectroscopy

DNA molecules of human cells are many times longer than the diameter of the cell and consequently the DNA is packed into a compact structure called the chromatin. The chromatin consists of DNA molecules coiled around histone proteins (sticky pulleys) in a very systematic manner. The cell utilises several mechanisms to control exactly what inheritable information from the DNA molecule that is turned into functional product (cellular machines). One mechanism that the cell exploits is to change the charge of certain histone proteins (weaken or strengthen the stickiness of the pulleys) and thus expose or restrict a specific part of the DNA to the cells gene production apparatus. HDAC, an enzyme that is responsible for changing the charge of histone proteins (a stickiness enhancer) will be the focus of the proposed research project. The HDAC enzyme works as a scissors that strips a negative charge off the histone proteins, thereby rendering the histone tails positively charged which strengthen the interaction with the negatively charged DNA. In particular, the focus of the proposed project is the dynamics and molecular motions of the HDAC enzyme (how does the scissors cut) and the dynamics will be studied primarily with nuclear magnetic resonance (NMR) spectroscopy. Thus, one of the key objectives of the research is to characterise, at atomic resolution, the mechanism by which the HDAC enzyme alter the histone charges. The goal is also to characterise how HDAC enzymes interact with inhibitors (drugs) and histones. HDAC enzymes are involved in cancers where they are believed to suppress the production of tumour suppressors. Inhibitors of HDAC enzymes have shown anti-tumour activity and it is therefore likely that the outcome of the proposed research will lead to the design of specific inhibitors of HDAC enzymes ultimately resulting in more efficient cancer therapy. An understanding of histone modifications requires a detailed picture of the three-dimensional structure of the involved enzymes and an appreciation of how these structures vary and fluctuate with time (a scissors cuts due to its opening and closing motions). Static structures of HDAC enzymes have been determined over the last decade, however, very few studies on the flexibility and dynamics of these regulatory molecules have been published. The proposed project focuses on the use of NMR spectroscopy as the primary biophysical tool to elucidate molecular flexibility and interactions since NMR has the potential to provide a description of the dynamics and interactions at atomic resolution. It is the goal that the NMR measurements together with other experimental techniques and computer simulations will create a coherent characterisation of the enzyme function. Another major objective of the proposed research is to develop new NMR methods to characterise molecular dynamics and flexibility in general. These developments aim at a time-resolved description of enzyme motions, that is, a visualisation of the enzyme motions over time - as a movie - as opposed to previous methods that primarily provides the amplitudes of protein motions. The research will be carried out at the Institute of Structural and Molecular Biology (ISMB), a joint venture between Birkbeck and University College London (UCL). UCL and ISMB provide a state-of-the-art and stimulating research environment with dedicated NMR machines suitable for the proposed project. Also, the highly collaborative environment and world-class expertise at ISMB and UCL open up the possibility for fruitful collaborations. For example, to increase the likelihood of success in the development of new HDAC inhibitors, I have initiated a collaboration with Prof Charles Marson, UCL, who is an expert on the productions of HDAC inhibitors. In my opinion this collaboration will allow the results about the HDAC enzyme dynamics, obtained by NMR spectroscopy, to be taken one important step further towards the design of new medicine.

Histone deacetylases (HDACs) are involved in the regulation of gene expression by catalysing the deacetylation of histone proteins, which in turn leads to a condensed chromatin at point of modification. The physiological importance of HDACs is stressed by the fact that aberrant recruitment of HDACs has been linked to lymphomas and inhibitors of HDACs have shown anti-tumour activity in preclinical trials. The X-ray structures of HDAC:inhibitor complexes are available, however, very few reports on the molecular dynamics of HDACs have been published. It is suggested that structural flexibility of the HDACs is one of the factors that control the formations of HDAC:substrate and HDAC:inhibitor complexes, thus, experimental determinations of such flexibilities become important. A major goal of the proposed research is to characterise the molecular dynamics and interactions of the HDAC isoform HDAC8. This includes a characterisation of the formation of HDAC8:substrate and HDAC8:inhibitor complexes, including those interactions that are transient. Experimental determinations of the dynamics and structural changes of regions that surround the entrance to the active site (inhibitor binding site) will likely facilitate the design of isoform-specific inhibitors. Another goal of the proposed research is to develop new NMR methods to characterise molecular dynamics. Methods will be developed to determine the dynamics of active sites of metalloproteins by exploiting the strong interactions between unpaired electrons and nuclear spins. Moreover, methods to determine the dynamics of flexible proteins (histone tails and C-terminal domains of HDACs) will be developed. The new methods will together with theoretical molecular dynamics simulations allow for a time-resolved description of the molecular motions including models that visualize the molecular motions as a function of time.

I strongly believe that the outcome of the proposed research will be beneficial for both the private sector (commercial drug discovery), the public sector, and in general beneficial for enhancing the health of society. The description of the histone deacetylase (HDAC) structural dynamics can be used readily by commercial drug discovery for molecular docking purposes and the new NMR methods that will be developed during the project will be useful for the general research community to characterise protein dynamics. Moreover, the outcome of the research will most likely facilitate the design of HDAC isoform specific inhibitors that will be beneficial for therapy of certain cancers. A major objective of the proposed research is to provide a description of the HDAC structures that encompasses dynamics and heterogeneity. Such a representation of the structure that surrounds the entrance to the active site will provide an accurate and veracious template to facilitate computer-assisted molecular docking and screenings for new HDAC inhibitors relevant for cancer and lymphoma therapy. In general, the development of methodology to provide a time-resolved description of protein dynamics will greatly assist in silico predictions of drugs and inhibitor binding, which in turn reduces the number of tests to perform in the laboratory. Consequently drug design will be faster and cheaper, which is of considerable economic and societal relevance. I therefore believe that both the new methods that will be developed and the characterisations of the HDAC dynamics will be beneficial for commercial drug design. The scientific results will be made available by publications in peer-reviewed journals and by presentations at national and international conferences. Furthermore, the software that will be developed during the project will be made available online, as I have done previously during my postdoctoral and graduate studies. I expect that cancer patients will benefit ultimately from the outcome of the proposed research. One of the goals of the proposed project is to facilitate the design of HDAC isoform specific inhibitors to improve therapy of certain cancers, possibly as suggested previously by using HDAC inhibitors as single agents or in combination with a chemotherapeutic agent (e.g. dexamethasone) or radiation therapy. In order to increase the likelihood of success in the development of isoform specific inhibitors, I have initiated a collaboration with Prof C Marson, UCL, who is an expert on the synthesis of HDAC inhibitors. In this future collaboration, I will provide the group of Prof Marson with dynamical descriptions of the HDAC structure and characterisations of binding interfaces obtained via NMR spectroscopy. New inhibitors will thereafter be designed collaboratively and synthesised in the lab of Prof Marson for further investigations. In my opinion this collaboration with a synthetic organic chemist will allow the results obtained by NMR spectroscopy to be taken one important step further towards the design of new drugs. One of the first HDAC inhibitors (pan-inhibitor) took ca. one decade from discovery to FDA approval, thus giving an approximate timeline. Overall the outcome of the proposed project will likely impact the research fields of chromatin remodelling and proteins dynamics. For example, the design of isoform specific HDAC inhibitors will provide a valuable tool to elucidate the individual functions of the HDAC isoforms. Moreover, the proposed research will provide the growing research community that utilises NMR spectroscopy with several new NMR methods and tools to investigate macromolecular dynamics and interactions. The new methods will be generally applicable to study molecular dynamics and interactions, i.e., not specific to HDAC proteins. I expect that the new methods will be particularly beneficial to the growing group of researchers that are studying intrinsically disordered proteins.