The JmjC-histone lysine demethylases (JmjC-KDMs) are epigenetic regulators responsible for the demethylation of methylated lysine residues on the N-terminal histone tails. As Fe²⁺ and 2-oxoglutarate dependent oxygenases (2OG oxygenases), the JmjC-KDMs possess an absolute requirement for molecular oxygen and are related to the cellular oxygen sensing HIF hydroxylases, PHD2 and FIH. Several JmjC-KDMs are known HIF target genes, hence are upregulated in hypoxia. Moreover, a number of JmjC-KDMs have been shown to have differential oxygen dependences, while aberrant histone methylation has been observed in both hypoxic cells and disease states such as cancer and cardiovascular disease. The work described in this thesis aimed to investigate the impact of hypoxia on the JmjC-KDM, KDM4A. In vitro kinetic analyses revealed a K_m^app(O₂) for recombinant KDM4A of 173 ± 23 μM, which is higher than reported values for the 2OG oxygenases C-P4H, mPAHX and even FIH, and approaching those evaluated for the key oxygen sensor PHD2 (230-1746 μM). These results indicate that KDM4A activity is highly sensitive to oxygen availability, and has the biochemical potential to act as an oxygen sensor in the context of epigenetic regulation. Subsequent investigation of the cellular oxygen dependence of KDM4A, and found that the activity of ectopically expressed KDM4A in U2OS cells demonstrates a graded response to oxygen. Importantly, this trend correlates with the in vitro results, providing further evidence that hypoxia may impact upon epigenetic regulation by the JmjC-KDMs. The various factors that may contribute to the hypoxic inhibition of KDM4A were investigated both in vitro and in cells. The results of these studies suggested that altered concentrations of TCA cycle intermediates, comprising reduced levels of the 2OG oxygenase co-substrate 2OG and increased concentrations of the reported inhibitor 2HG, are likely to only minimally affect the activity of KDM4A in hypoxia. Interestingly, the 2OG oxygenase inhibitor IOX1 possessed increased inhibitory potency against KDM4A under conditions of low oxygen, implying that the use of mixed-mode inhibitors against KDM4A may be of therapeutic benefit in hypoxic disease states. This may be of particular pertinence to cardiac hypertrophy (CH), in which KDM4A activity is reported to have pathophysiological consequences. In a collaboration with Dr Tim McKinsey (University of Colorado, Denver), the KDM4 inhibitor CCT1 was tested in a phenotypic screen of cardiomyocyte hypertrophy, the results of which further support a role for KDM4A in this disease, and suggest that the use of small-molecule inhibitors of KDM4A may be a viable therapeutic strategy in CH. Finally, the effect of reactive oxygen species, levels of which may be increased in hypoxia, on KDM4A activity was explored. Recombinant KDM4A was found to be acutely sensitive to inhibition by hydrogen peroxide (H₂O₂) when compared to the HIF hydroxylases PHD2 and FIH. These results imply that KDM4A may act as a sensor of oxidative stress at the chromatin level, and further investigation in a more biologically relevant context is proposed. Overall, the work described herein demonstrates that the activity of KDM4A is sensitive to oxygen availability, a phenomenon that is likely to have significant implications for epigenetic regulation in hypoxia and the expression of KDM4A-regulated genes in ischaemic disease states.

A notable need exists for noninvasive tools to increase our mechanistic understanding of disease progression at a cellular and molecular level. Studying the functions of proteins in their innate in vivo tissue environment can provide useful information about pathology enabling appropriate treatment and early diagnosis. Chemical exchange saturation transfer MRI contrast provides real-time functional characterization of the biological landscape and can be used to detect multiple enzyme biomarker activities. A dual-enzyme catalyCEST contrast agent was developed as a proof-of-concept to demonstrate the potential of using a salicylic acid scaffold and control the CEST signal through enzyme activation. In addition, a straightforward route was designed to synthesize a diamagnetic catalyCEST MRI agent that is a substrate for β-galactosidase and β-glucuronidase enzymes. The synthesized agents generated two peaks in the CEST spectrum, at 4.25 ppm corresponding to a carbamate moiety and at 9.25 ppm corresponding to the salicylic acid moiety. Chemical exchange rates of liable protons were determined from a QUESP Hanes-Woolf plot. In the presence of the corresponding enzymes, the catalyCEST agent was activated via saccharide hydrolysis followed by a spontaneous disassembly to produce 4-aminosalicylic acid. This reaction converted the carbamate moiety into a free primary amine, and caused a loss of CEST signal at 4.25 ppm. The CEST signal at 9.25 ppm was unaffected by the enzyme catalysis, and therefore used as an internal control signal. Michaelis-Menten enzyme kinetics studies were performed with CEST MRI to verify that catalyCEST MRI could truly detect enzyme activity. The Michaelis-Menten kinetics constants from MRI studies were compared to the kinetics constants measured with UVvis results from the same contrast agent, demonstrating the quantitative potential of catalyCEST MRI with both contrast agents. These findings demonstrate that the newly synthesized modular agents have the potential to become reliable catalyCEST MRI imaging probes. In addition, the modular design of these agents facilitates the conjugation of other enzyme substrates to the carbamate spacer, so that this approach constitutes a platform technology for the detection of enzyme activity.

Non-communicable diseases (NCD) such as cancer, heart disease and cerebrovascular injury are dependent on or aggravated by inflammation. Their prevention and treatment is arguably one of the greatest challenges to medicine in the 21st century. The pleiotropic, proinflammatory cytokine; interleukin-l beta (IL-l~) is a primary, causative messenger of inflammation. Lipopolysaccharide (LPS) induction ofIL-l~ expression via toll-like receptor 4 (TLR4) in myeloid cells is a robust experimental model of inflammation and is driven in large part via p38-MAPK and NF-KB signaling networks. The control of signaling networks involved in IL-l~ expression is distributed and highly complex, so to perturb intracellular networks effectively it is often necessary to modulate several steps simultaneously. However, the number of possible permutations for intervention leads to a combinatorial explosion in the experiments that would have to be performed in a complete analysis. We used a multi-objective evolutionary algorithm (EA) to optimise reagent combinations from a dynamic chemical library of 33 compounds with established or predicted targets in the regulatory network controlling IL-l ~ expression. The EA converged on excellent solutions within 11 generations during which we studied just 550 combinations out of the potential search space of - 9 billion. The top five reagents with the greatest contribution to combinatorial effects throughout the EA were then optimised pair- wise with respect to their concentrations, using an adaptive, dose matrix search protocol. A p38a MAPK inhibitor (30 ± 10% inhibition alone) with either an inhibitor of IKB kinase (12 ± 9 % inhibition alone) or a chelator of poorly liganded iron (19 ± 8 % inhibition alone) yielded synergistic inhibition (59 ± 5 % and 59 ± 4 % respectively, n=7, p≥O.04 for both combinations, tested by one way ANOVA with Tukey's multiple test correction) of macrophage IL-l~ expression. Utilising the above data, in conjunction with the literature, an LPS-directed transcriptional map of IL-l ~ expression was constructed. Transcription factors (TF) targeted by the signaling networks coalesce at precise nucleotide binding elements within the IL-l~ regulatory DNA. Constitutive binding of PU.l and C/EBr-~ TF's are obligate for IL-l~ expression. The findings in this thesis suggest that PU.l and C/EBP-~ TF's form scaffolds facilitating dynamic control exerted by other TF's, as exemplified by c-Jun. Similarly, evidence is emerging that epigenetic factors, such as the hetero-euchromatin balance, are also important in the relative transcriptional efficacy in different cell types. Evolutionary searches provide a powerful and general approach to the discovery of novel combinations of pharmacological agents with potentially greater therapeutic indices than those of single drugs. Similarly, construction of signaling network maps aid the elucidation of pharmacological mechanism and are mandatory precursors to the development of dynamic models. The symbiosis of both approaches has provided further insight into the mechanisms responsible for IL-lβ expression, and reported here provide a - platform for further developments in understanding NCD's dependent on or aggravated by inflammation.

Antibiotics are an important resource in modern medicine used to treat serious infections and enable a wide array of vital medical interventions including surgery and cancer chemotherapy. However, due to the increasing prevalence of antibiotic resistant pathogens, many clinically useful antibiotics are being rendered ineffective with too few new antibiotics in development to combat them. With highly diverse chemistry and bioactivity exquisitely shaped by evolution, natural products provide an unrivaled source of antibiotic compounds that is impossible to reproduce instinctively in the laboratory. The armeniaspirols are polyketide natural products with a unique spiro-[4.4]non-8-ene core that were isolated from Streptomyces armeniacus and were shown to be active against drug-resistant Gram-positive bacteria. Promisingly, in vitro resistant Staphylococcus aureus strains could not be readily obtained even after thirty serial passages under sub-lethal doses. Herein, we decipher the mechanism of action for this structurally unprecedented natural product antibiotic in the Gram-positive model organism Bacillus subtilis. Through chemical proteomics with an armeniaspirol-inspired activity-based probe, quantitative proteomics, biochemical assays, and microscopy, we show that armeniaspirol is a competitive inhibitor of the AAA+ proteases ClpXP and ClpYQ. Armeniaspirol represents the first known natural product inhibitor of ClpP, a highly coveted target due to its prominent role in bacterial virulence. Using overlapping proteomic fingerprints of armeniaspirol-treatment with ΔclpQ and ΔclpP deletions in B. subtilis, inhibition or deletion of these proteases appears to dysregulate key proteins involved in cell division, including FtsZ, DivIVA, and MreB. The dual ClpXP and ClpYQ inhibition is responsible for armeniaspirol’s potent antibiotic activity and this unique pharmacology makes it a promising candidate for antibiotic development. Several armeniaspirol-inspired analogs were generated as part of a medicinal chemistry study and evaluated for antibiotic activity towards a panel of clinically relevant Gram-positive pathogens. As a result, we identify three exciting armeniaspirol analogs with improved antibiotic activity. Lastly, the foundation for elucidating the ClpYQ degradome is developed. Our proteomic fingerprint of the B. subtilis ΔclpQ deletion strain generated some of the first insights into potential substrates of the ClpYQ protease. As a largely uncharacterized AAA+ protease implicated in the mechanism of action of armeniaspirol, we pursued a previously established acyl-intermediate covalent trapping strategy to characterize the ClpYQ-substrate complexes in B. subtilis cell lysate. Through unnatural amino acid incorporation using an evolved tRNA/aminoacyl-tRNA synthetase pair, the N-terminal active site serine of ClpQ is substituted with a photocleavable precursor that generates 2,3-diaminopropionic acid. While we were successful in synthesizing the photocleavable precursor, initial experiments to incorporate this unnatural amino acid in ClpQ expression proved unsuccessful, leading us to outline necessary control experiments for future endeavours. Ultimately, the covalently trapped substrates will be identified by LC-MS/MS, where we expect to identify key divisome and elongasome proteins in corroboration with the armeniaspirol mechanism of action study.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18902号 / 医科博第58号 / 新制||医科||4(附属図書館) / 31853 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 山下 潤, 教授 楠見 明弘, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright ©American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/jacs.5b10162. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19611号 / 医博第4118号 / 新制||医||1015(附属図書館) / 32647 / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 渡邊 直樹, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20213号 / 理博第4298号 / 新制||理||1617(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)講師 嶋田 知生, 教授 長谷 あきら, 教授 鹿内 利治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23247号 / 農博第2454号 / 新制||農||1084(附属図書館) / 学位論文||R3||N5337(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 宮川 恒, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23955号 / 農博第2504号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5390(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 宮川 恒, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Thesis advisor: Jia Niu / High-Throughput Particle Display Screening of RNA-Protein Interactions RNA-protein binding interactions have essential roles in many biological processes including transcriptional and translational control; thus, it is important to quantify the binding affinities of these biological complexes through functional binding assays. Although conventional binding assays have provided significant insight to these dynamic networks, they generally provide a relatively low throughput for a limited number of samples. To overcome the limitations of these conventional binding assays to study RNA-protein binding interactions, we propose to develop an in vitro, high-throughput particle display-based for RNA aptamer screening of RNA-protein complexes for the subsequent identification and characterization of novel RNA aptamers that influence protein binding. With this technique, we will be able to profile large numbers of binding events based on binding-induced fluorescence-enhancement for a more holistic understanding of the corresponding RNA-protein network. So far, we have confirmed that this particle display-based technique can be used to estimate the binding affinity of the well-characterized MS2-MCP model system, and plan to advance this technique to screen a library of MS2 variants for mutational analysis. Prime Editor-Mediated Programmable Insertion of UAAs into Endogenous Proteins The introduction of unnatural amino acids (UAAs) to endogenous cell surface proteins for site-specific bioconjugation reactions allows for the incorporation of clickable, fluorescent handles in vivo; however, the transient expression of proteins harboring UAAs is limited by its transfection efficiency. Thus, we propose to employ prime editors and tRNA/aminoacyl-tRNA synthetase technologies to introduce an UAA to endogenous proteins for downstream bioconjugation applications. Briefly, we propose to stably incorporate a stop codon into mammalian cells by prime editing which will be confirmed with a reporter system, such that this stop codon can mediate the introduction of an UAA through the associated tRNA/aminoacyl-tRNA synthetase technology. By permanently introducing a bioorthogonal, clickable handle onto an endogenous protein, its cellular signaling and localization patters can be monitored in vivo for further classification of the behaviors of these proteins. So far, we identified a promising fluorescent reporter construct to validate the introduction of a stop codon into the mammalian genome by prime editing. / Thesis (MS) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.