As an important posttranslational modification, protein acetylation plays critical roles in many biological processes such as gene transcription, DNA damage repair, apoptosis and metabolism. The acetylation occurs on the ε-amino group of specific lysine residues, and is catalyzed by histone acetyltransferases (HATs). In cellular contexts, HATs are found to target hundreds and thousands of substrates including histone and nonhistone proteins. Lysine acetylation changes the microenvironment of protein and may potentially alter protein activity and protein-protein interaction. The goal of this dissertation project is to investigate the impact of lysine acetylation on the catalysis of MYST HATs, and to establish the strategy for labeling substrates of the MYST HATs at cellular level. To understand the regulatory mechanism of MYST HATs, a detailed study was carried out to investigate the active site lysine acetylation of two MYST HATs (MOF and Tip60). Autoradiography and immunoblotting data shows that mutation of active site lysine differentially affects the enzyme autoacetylation activity and the cognate substrate acetylation activity. In addition, deacetylated MOF and Tip60 were prepared by using the nonspecific lysine deacetylase Sirt1. Kinetic study demonstrated that the acetylation of the active site lysine on MYST HATs marginally modulates the HAT catalysis. This work provides new insights into the regulatory mechanism of MYST catalysis. In the second part of my work, we designed and synthesized a series of Ac-CoA analogs conjugated with alkynyl or azido functional groups. Meanwhile, the active site of the MOF was engineered to expand the cofactor binding capability. Fluorescence screening was carried out to characterize the enzyme activity to Ac-CoA analogs. MOF-I317A with all analogs and MOF-I317A/H273A–5HYCoA were identified and further applied in the labeling of the cognate histone H4 protein and HAT substrates in 293T cell lysate. Visualizing of the labeled substrate was achieved using the alkynyl or azido-tagged fluorescent reporters through the copper-catalyzed azide−alkyne cycloaddition. As expected, the histone H4 protein was successfully labeled by the active enzyme-cofactor pairs. More intriguingly, multiple protein bands in cell lysate were labeled and observed. This work provides a new versatile strategy in exploring the substrates of MYST HATs at the proteomic level.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23215号 / 工博第4859号 / 新制||工||1758(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 大江 浩一, 教授 近藤 輝幸, 教授 深澤 愛子 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Chemical probes are necessary for elucidating the biochemical roles of proteins. Bromodomains are protein-interaction modules found in a family of proteins implicated in the epigenetic regulation of transcription; however, the individual roles remain unknown for many bromodomain proteins, without potent and selective ligands available to assist in their study. From lead compounds, a structure-based drug discovery program was to be explored with the use of biophysical assays and appropriate chemical methods to expediate development of probes against a number of these proteins. A fragment lead against BRD4 was developed into PNZ5, a potent (K_D 5 nM) BRD4 probe with a high ligand efficiency. Although enantioselective syntheses and the use of an alternative synthetic route were unsuccessful, PNZ5 showed cytotoxic activity against gastric cancer cell lines that had proved resilient to existing anticancer agents. Optimisation of a lead compound against BRD9 resulted in the development of LP99, the first reported BRD7/9 probe, that was potent (BRD9 K_D 99 nM, BRD7 K_D 909 nM), selective amongst bromodomain proteins and active in cells. An enantioselective synthesis was performed using chiral organocatalyts and LP99 was used to identify a previously unknown role of BRD7/9 in the regulation of inflammatory processes. Research is ongoing to assess further biochemical roles of these proteins with LP99. Arising from a more potent lead against BRD9, a series of structurally related compounds were synthesised to explore SAR around this ligand, however no improvement on the affinity of the lead was realised. Finally, based on disclosed lead structures against PCAF, a series of compounds were synthesised to replicate their activity. A number of important binding interactions were assessed and a lead structure was identified (K_D 1 μM). Development is ongoing to progress this lead into the first reported PCAF probe.

As depicted in Chapter I, 2-oxoglutarate- (2OG) dependent oxygenases are ubiquitous in living systems and display a wide range of cellular functions, spanning metabolism, transcription, and translation. Although functionally diverse, the 2OG oxygenases share a high degree of structural similarities between their catalytic sites. From a medicinal chemistry point of view, the combination of biological diversity and structural similarity presents a rather challenging task for the development of selective small molecules for functional studies in vivo. The non-selective metal chelator 8-hydroxyquinoline (8HQ) was used as a template for the generation of tool compound <b>I</b> for the KDM4 subfamily of histone demethylases via application of the Betti reaction. Structural analogue <b>II</b> was used as the corresponding negative control (Figure A). These compounds were characterised in vitro against a range of 2OG oxygenases and subsequently used for studies in cells. <b>I</b> displays selectivity for KDM4 and increases the level of the H3K9me3 histone mark in cells. It has an effect on the post-translational modification pattern of histone H3, but not other histones, and reduces the viability of lung cancer cells, but not normal lung cells, derived from the same patient. <b>I</b> also stabilises hypoxia-inducable factor HIF in cells via a mechanism which seems to be independent from prolyl hydroxylase inhibition. This work is described in Chapters II and III. The chemical biology research in epigenetics is complemented by qualitative analysis conducted in the social sciences at Said Business School. With a global view on how innovation occurs and may actively be fostered, Chapter IV focuses on the potential of epigenetics in drug discovery and how this process may actively be promoted within the framework of open innovation. Areas of focus include considerations of incremental and disruptive technology; how to claim, demarcate, and control the market; how knowledge brokering occurs; and insights about process, management, organisation, and culture of open innovation. In contrast to the open-skies approach adopted for the development of a tool compound in Chapters II and III, a focused-library approach was taken for the generation of a tool compound for the OGFOD1 ribosomal prolyl hydroxylase. The development of a suitable in vitro activity assay for OGFOD1 in Chapter V enabled the development of lead compound <b>III</b> in Chapter VI. <b>III</b> is selective for OGFOD1 against the structurally closely related prolyl hydroxylase PHD2.