The work described in this thesis focused on the use of nuclear magnetic resonance spectroscopy (NMR) to study two classes of metallo enzymes - the Fe(II)- and 2oxoglutarate (2OG)-dependent dioxygenases and the metallo β-lactamases (MBLs). These enzymes are involved in clinically important biological processes, i.e. the hypoxic response and antimicrobial resistance, respectively. Both protein systems are interesting from an NMR perspective because they have dynamic regions involved in catalysis and ligand interactions. The work included mechanistic studies, protein-ligand interaction studies, and method development for inhibitor discovery. NMR was applied to study the human prolyl hydroxylase domain-containing protein 2 (PHD2), which is crucially involved in the chronic hypoxic response. The results reveal that binding of the C- and the N-terminus of the oxygen dependent degradation domains CODD and NODD, respectively, induce different interactions with PHD2. The substitution of a single amino acid, as occurs with PHD2 variants linked to erythrocytosis and breast cancer, can alter the selectivity of PHD2 towards its ODD substrates. Studies with the Trichoplax adhaerens PHD provide insights into the evolutionary substrate preference of the PHDs. Using ¹³C-labelled peptidyl-substrates; NMR was applied to investigate proposed 'alternative' PHD2 substrates/interaction partners. The product release mechanism of PHD2 was investigated using NMR; the results reveal that the presence of 2OG strongly discriminates between the binding of CODD and hydroxylated CODD to PHD2. NMR was also applied to monitor PHD2 kinetics and inhibition. Competition and displacement assays were designed and applied to investigate PHD inhibitor binding modes. Comparative studies on the activities and selectivities of PHD inhibitors in clinical trials should aid in the work on the therapeutic manipulation of the natural hypoxic response. Protein-observe ¹9F-NMR was used to study the São Paolo MBL (SPM-1). The results provide new structural insights into SPM-1 catalysis and the requirements for inhibitor development. They also reveal that the hydrolysed β-amino acid products of MBL catalysis can bind to SPM-1. They illustrate the utility of ¹⁹F-NMR for detecting metal chelation, which is not always readily tractable in studies on metallo enzyme inhibition, new binding modes, and stereoisomer binding/epimerisation in solution. The interaction of a cyclobutanone analogue, a broad-spectrum MBL inhibitor, with SPM-1 was investigated. A combination of ¹H, ¹⁹F, ¹³C-NMR and crystallographic analyses reveal that cyclobutanone binding may mimic formation of the oxyanion tetrahedral intermediate in β-lactam hydrolysis. The susceptibility of avibactam, the first clinically useful non-β-lactam β-lactamase inhibitor, to MBL-catalysed hydrolysis was studied. The results reveal that avibactam is not an MBL inhibitor and a poor substrate of most members of all three clinically relevant subclasses of MBLs. In some cases, avibactam undergoes slow hydrolysis in a process different from that observed with serine β-lactamases. Overall, the results illustrate the utility of NMR for studying dynamic aspects of enzyme catalysis and inhibitor binding.

Nature has evolved an additional level of genetic regulation by-passing direct changes in genetic code through the means of posttranslational modifications (PTMs) of nucleobases and histone proteins. Acetylation, methylation, phosphorylation, O-GlcNAcylation, ubiquitination, sumoylation, and ADP ribosylation are few common examples of various histone modifications. Identification of these modifications and subsequent access to homogeneously modified histone proteins are key for understanding the functional consequence of these PTMs. In this doctoral thesis, the role of PTMs of histone proteins in epigenetics was investigated with emphasis on understanding the role of O-GlcNAcylation in particular. In the second chapter, the functional consequence of O-GlcNAcylation at histone protein, H2B-Ser112 was explored. Homogeneously GlcNAcylated histones and nucleosomes were synthesized using protein chemical reactions. Mass Spectrometry (MS) based quantitative interaction proteomics revealed a direct interaction between GlcNAcylated nucleosomes and the Facilitates Chromatin Transcription (FACT) complex. Preferential binding of FACT to GlcNAcylated nucleosomes provides a molecular mechanism for FACT-driven transcriptional control. In the third chapter, the physical effect of O-GlcNAcylation on the nucleosome structure is described. Homogeneously GlcNAcylated histone protein, H2A-Thr101 was synthesized. The modified protein was used to reconstitute histone sub-complexes and nucleosomes. Various biophysical studies involving circular dichroism and native mass spectrometry revealed that H2A-T101 GlcNAcylation regulates the stability of the nucleosome structure, suggesting a role in transcriptional activation. In the fourth chapter, we discuss an interesting scenario where two PTMs - O-GlcNAcylation and phosphorylation - can compete for the same modification site of histone protein, H2B-Ser36. The resulting outcome is possibly a competitive antagonism or cross-talk, which can modulate the overall control of chromatin regulation. Using a "Tag-and-modify" approach, modified histone proteins bearing both modifications was synthesized, and was later used for nucleosome reconstitution. Quantitative interaction proteomics experiments with the modified nucleosome revealed key interacting protein partners for both the modifications.

La régulation de la protéolyse est un outil efficace pour le contrôle de la fonction d'une protéine dans des cellules. Nous présentons dans ce travail une stratégie générique permettant d'activer la protéolyse de façon conditionnelle par la lumière, améliorant ainsi la résolution spatio-temporelle. Notre approche repose sur un système de dégradation inductible par l'auxine (AID), mis au point en transposant des composants de la voie de dégradation contrôlée par l'auxine existant chez les plantes dans des cellules de mammifères. Nous présentons une version optimisée du système AID qui a permis de diminuer de façon significative la stabilité de protéines cibles en présence d'auxine. Nous avons en parallèle développé un déclencheur de dégradation photo-activable sous la forme d'une auxine cagée. Une illumination courte et locale permet la libération efficace de l'auxine dans les cellules et induit la dégradation de protéine d'intérêt avec un bon contrôle spatiotemporel. Cette méthode générique a été utilisée dans des contextes nucléaires et cytoplasmiques. / The regulation of proteolysis is an efficient way to control protein function in cells. Here, we present a general strategy enabling to increase the spatiotemporal resolution of conditional proteolysis by using light activation as trigger. Our approach relies on the auxin-inducible degradation (AID) system obtained by transposing components of the plant auxin-dependent degradation pathway in mammalian cells. We developed an optimized version of the AID which enables to significantly destabilize target proteins in presence of auxin. Parallely, we developed a photoactivatable auxin that acts as a photoactivatable inducer of degradation. Upon local and short light illumination, auxin is released in cells and triggers the degradation of a protein of interest with spatiotemporal control. This generic method was implemented in nuclear and cytoplasmic contexts.

Background: Despite multi-modality therapy, the majority of patients with metastatic or recurrent Ewing sarcoma (ES), the second most common pediatric bone malignancy, will die of their disease. ES tumors express aberrantly activated ETS transcription factors through translocations that fuse the EWS gene to ETS family genes FLI1 or ERG. The aberrant activation of ETS transcription factors promotes malignant transformation and proliferation. While, FLI1 or ERG cannot be readily targeted, there is an opportunity to deploy functional genomics screens, to develop novel therapeutic approaches by identifying targetable liabilities in EWS/FLI1 dependent tumors. Materials and Methods: We performed a near whole-genome pooled shRNA screen in a panel of five EWS/FLI1 dependent Ewing sarcoma cell lines and one EWS/ERG cell line to identify essential genes. Essential genes were defined as those genes whose loss resulted in reduced viability selectively in ES cells compared to non-Ewing cancer cell lines. Essential hits were subsequently validated with genomic knockdown and chemical inhibition in vitro, followed by validation of the on-target effect of chemical inhibition. Next, we determined the in vivo effects of small-molecule inhibition on survival and tumor growth in NOD scid gamma (NSG) mice with established subcutaneous ES xenografts. Results: Top hits in our screen that could be readily targeted by small-molecule inhibitors, and thus have potential for rapid clinical validation, were selected for further investigation. These hits included IKBKE, CCND1 and CDK4. IKBKΕ, a non-canonical IKK with an oncogenic role in breast cancer, was one of the top kinase hits in the screen. IKBKΕ shares significant homology to TBK1, another non-canonical IKK that is essential in k-RAS dependent lung cancer. We validated IKBKE through small-molecule inhibition of IKBKE/TBK1 and shRNA based knockdown. Ewing sarcoma cell lines are sensitive to low micromolar concentrations of two IKBKE/TBK1 inhibitors (CYT387 and MRT67307). Additionally, in a panel of ES cell lines, knockdown of IKBKE resulted in decreased growth and impaired colony formation. These observations, paired with impairment of NF-κB nuclear localization following CYT387 treatment suggests that non-canonical IKK mediated signaling may be essential in Ewing sarcoma. We further validated these results through inhibition of IKBKE/TBK1 in in vivo xenograft models treated with 100 mg/kg/day of CYT387. Treatment over the course of twenty-nine days resulted in a significant increase in survival (p-value = 0.0231) and a significant decrease (p-value = 0.036) in tumor size after fifteen days of treatment. CDK4 and CCND1 are highly expressed in Ewing sarcoma as compared to other tumor types. shRNA mediated knockdown of CDK4 and CCND1 resulted in impaired viability and anchorage independent growth. Furthermore, treatment of Ewing sarcoma cell lines with a highly selective CDK4/6 inhibitor, LEE011, resulted in decreased viability (IC50 range of 0.26-18.06 μM), potent G1 arrest in six of eight EWS/FLI1 containing Ewing sarcoma lines tested and apoptosis in a panel of four highly sensitive lines. Administration of 75 mg/kg/day and 250 mg/kg/day of LEE011 in NSG mice with Ewing xenografts resulted in significant impairment of tumor growth, (p-value <0.001 for both treatment arms), as compared to vehicle control. Conclusions: These studies suggest a role for the targeting of IKBKE and CDK 4/6 in Ewing sarcoma, findings with immediate clinical relevance for patients with this malignancy, because small-molecule inhibitors of these proteins have already entered clinical trial for other disease indications.

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第12872号 / 論医科博第1号 / 新制||医科||4(附属図書館) / 31590 / (主査)教授 野田 亮, 教授 楠見 明弘, 教授 瀬原 淳子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18299号 / 工博第3891号 / 新制||工||1597(附属図書館) / 31157 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 梅田 眞郷, 教授 森 泰生 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21668号 / 医博第4474号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 萩原 正敏, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Enzymes are implicated in many diseases including neurodegenerative, cancer, immune deficiency, and inflammatory disorders. There is a constant need to develop novel drug compounds that target enzymes in order to modulate their function, thus treating the disease state. These compounds are typically small molecules with affinity to the enzyme active site or an allosteric site. In order to discover novel compounds for treating disease, the interaction between an enzyme and a small molecule must first be identified and then characterized. With the target enzyme known, it is beneficial to screen libraries of compounds against the target. Immobilizing the enzyme allows for pre-concentration of ligands on the surface and therefore increased signal enhancement, as well as permitting multiple wash steps and enzyme reuse. Immobilized enzyme columns are optimal for coupling to a variety of detection devices by way of liquid chromatography, including absorbance or mass spectrometric detection. Immobilized enzyme reactors (IMERs) were generated and optimized for two target molecules, acetylcholinesterase (AChE) and adenosine deaminase (ADA), for rapid function-based screening of enzyme inhibitors in mixtures. The IMER mode is useful for increasing throughput and facilitating the identification of hit mixtures, but it is slow and tedious to manually deconvolute hit compounds from mixtures and the IMER method is not amenable to natural product extracts, which are good sources of structurally diverse compounds that are more likely to result in a hit compound. Bio-selective solid-phase extraction (BioSPE) is an orthogonal method of isolating and identifying enzyme inhibitors in a single step, and was used to easily deconvolute complex mixtures, rapidly identifying to key compounds EHNA and MAC-0038732 out of mixtures using ADA columns. A data dependent acquisition MS method was developed and used to screen a set of fungal endophyte extracts, identifying two potentially novel inhibitors that were confirmed by IMER-MS/MS. / Thesis / Doctor of Philosophy (PhD) / The discovery of new drug compounds is crucial for the treatment of diseases. Enzymes are proteins that turn a substrate into a product; and in diseases they can often malfunction, overproducing the product. Small molecule compounds can sometimes inhibit enzyme function and can be further developed into therapeutic drugs. This thesis describes a method for detecting small molecule inhibitors that bind to an enzyme that is immobilized in a small column. Once the small molecule is bound to the immobilized enzyme, it can be detected by either showing that enzyme function is inhibited or by removing the compound from the enzyme and identifying the compound by mass spectrometry. These methods can quickly identify compounds at extremely low levels from complex mixtures, such as natural product extracts.

Cyclization reactions play an important role in synthesizing a significant number of small molecule scaffolds for various purposes, including drug discovery. However, the application of cyclization reactions in the modifications of biomolecules in a single step is still limited. This dissertation reports a stereoselective thiomorpholine ring formation reaction to site-selectively modify N-terminal cysteines of unprotected peptides or proteins in a single step. We showed that α-fluoroketone molecules afford the cyclization reaction with the beta amino thiol group of N-terminal cysteine. Both chemo and stereo-selectivity of this reaction have been studied using 2D NMR analyses. Cysteine located at the Nterminal of a short protein (VHP protein) has been modified site-selectively with a fluorescein isothiocyanate (FITC) containing an α-fluoroketone linker to demonstrate the applicability of this reaction in modifying biomolecules. This chemistry has the potential to generate homogeneous and stable antibody-drug conjugates (ADCs) for the treatment of cancer. This dissertation also demonstrates a fluorine-thiol displacement reaction (FTDR) to synthesize various macrocyclic and stapled peptides, which renders medicinally privileged peptides with improved biological properties such as binding affinities and cell membrane permeability. The cyclization of fluoroacetamide containing peptides with benzenedimethane thiol linkers enhances peptides' alpha helicity. These FTDR stapled peptides exhibit better cellular uptake compared to the classic ring-closing metathesis (RCM) stapled peptides. Compared to the proteoglycan-aided cell penetration by peptides stapled with RCM, the preliminary mechanistic studies of our FTDR-stapled peptides revealed that our thiolated linkers allowed peptides to enter cells in multiple pathways. Taken together, our FTDR-based stapling approach may provide a novel class of cell-permeable peptides that might open a new window to probe intracellular targets. This dissertation reports another cyclization reaction between hydrazine and carbamate, synthesized from 7-hdyroxy coumarin derivatives. We demonstrate that the secondary nitrogen of hydrazine is much more reactive than the primary nitrogen for intramolecular cyclization reactions. Hydrazine affords urea bonds upon the substitution of a coumarin moiety of carbamates to generate 6 or 7 membered cyclic scaffolds. The exocyclic free amine might allow facile generation of a library of compounds. In addition, further optimization of this reaction might allow using these hydrazine containing molecules in monitoring the real-time activity of histone deacetylases (HDACs). / Chemistry