Beyond the four canonical nucleosides as primary building blocks of RNA, posttranscriptional modifications give rise to the epitranscriptome as a second layer of genetic information. In eukaryotic mRNA, the most abundant posttranscriptional modification is N6-methyladenosine (m6A), which is involved in the regulation of cellular processes. Throughout this thesis, the concept of atomic mutagenesis was employed to gain novel mechanistic insights into the substrate recognition by human m6A reader proteins as well as in the oxidative m6A demethylation by human demethylase enzymes. Non-natural m6A atomic mutants featuring distinct steric and electronic properties were synthesized and incorporated into RNA oligonucleotides. Fluorescence anisotropy measurements using these modified oligonucleotides revealed the impact of the atomic mutagenesis on the molecular recognition by the human m6A readers YTHDF2, YTHDC1 and YTHDC2 and allowed to draw conclusions about structural prerequisites for substrate recognition. Furthermore, substrate recognition and demethylation mechanism of the human m6A demethylase enzymes FTO and ALKBH5 were analyzed by HPLC-MS and PAGE-based assays using the modified oligonucleotides synthesized in this work. Modified nucleosides not only expand the genetic alphabet, but are also extensively researched as drug candidates. In this thesis, the antiviral mechanism of the anti-SARS-CoV-2 drug remdesivir was investigated, which causes delayed stalling of the viral RNA-dependent RNA polymerase (RdRp). Novel remdesivir phosphoramidite building blocks were synthesized and used to construct defined RNA-RdRp complexes for subsequent studies by cryogenic electron microscopy (cryo-EM). It was found that the 1'-cyano substituent causes Rem to act as a steric barrier of RdRp translocation. Since this translocation barrier can eventually be overcome by the polymerase, novel derivatives of Rem with potentially improved antiviral properties were designed. / Über die vier kanonischen Nukleoside als primäre RNA-Bausteine hinausgehend bauen posttranskriptionelle Modifikationen eine zweite Informationsebene, das Epitranskriptom, auf. Die häufigste posttranskriptionelle Modifikation in eukaryotischer mRNA ist N6-Methyladenosin (m6A), welches in die Regulierung zellulärer Prozesse involviert ist. In dieser Arbeit wurde das Konzept der atomaren Mutagenese genutzt, um neue Einblicke in die Erkennung von m6A durch menschliche m6A-bindende Proteine sowie in die oxidative Demethylierung von m6A durch menschliche Demethylase-Enzyme zu gewinnen. Es wurden nicht natürlich vorkommende m6A Atommutanten mit unterschiedlichen elektronischen und sterischen Eigenschaften synthetisiert und in RNA-Oligonukleotide eingebaut. Durch Fluoreszenzanisotropie-Messungen mit diesen Oligonukleotiden wurde der Einfluss der Atommutagenese auf die molekulare Erkennung durch die menschlichen m6A-bindenden Proteine YTHDF2, YTHDC1 und YTHDC2 untersucht. Die erhaltenen Ergebnisse ließen Rückschlüsse auf die strukturellen Voraussetzungen für die Erkennung eines Substrates zu. Weiterhin wurden die in dieser Arbeit synthetisierten modifizierten Oligonukleotide zur Untersuchung von Substraterkennung und Demethylierungs-Mechanismus der menschlichen m6A-Demethylasen FTO und ALKBH5 mittels HPLC-MS- und PAGE-basierter Analysen verwendet. Modifizierte Nukleoside dienen nicht nur zur Erweiterung des genetischen Alphabets, sondern werden auch als potentielle Wirkstoff-Kandidaten erforscht. In dieser Arbeit wurde der antivirale Wirkmechanismus des Anti-SARS-CoV-2-Wirkstoffes Remdesivir untersucht, der eine verzögerte Blockade der viralen RNA-abhängigen RNA-Polymerase (RdRp) bewirkt. Neuartige Remdesivir Phosphoramidit-Bausteine wurden synthetisiert und genutzt, um RNA-RdRp-Komplexe mit definierter Struktur zu konstruieren, welche anschließend mittels Cryoelektronenmikroskopie (Cryo-EM) untersucht wurden. Es wurde herausgefunden, dass der 1'-Cyano-Substituent dazu führt, dass Rem als sterische Blockade der RdRp-Translokation agiert. Da diese Tranlokationsbarriere von der Polymerase überwunden werden kann, wurden neuartige Rem-Derivate mit potentiell verbesserten antiviralen Eigenschaften entworfen.

Adenosine is an essential purine nucleoside of particular importance within heart and brain. The widespread and diverse actions of adenosine, driven by activation of cell surface receptors, include regulation of sleep/arousal and neuroprotective properties. The mechanisms involved in regulating adenosine concentrations remain poorly understood but are critical to signaling pathways as they determine the availability of adenosine at corresponding receptors within the extracellular space. The equilibrative nucleoside transporter (ENT) family, bi-directional, Na+-independent nucleoside transporters, are key components in both the release and uptake of adenosine. This study has been conducted to investigate ENT3, a novel member of the ENT family. Our work has demonstrated ENT3 to be expressed throughout brain, located in cortex, cerebellum, striatum and hippocampus, at similar levels. Neurons and astrocytes, but not microglia, showed intracellular ENT3 localisation. This was confirmed by differential centrifugation, of cortex and cerebellum, which suggests ENT3 to be found within the cytoplasm.

Ces dernières années, les analogues nucléosidiques ont eu un rôle majeur dans le traitement de maladies virales infectieuses tels le SIDA, les hépatites, l’herpès, la variole ou la grippe. Cependant, l’apparition de nouveaux virus ou de mutations virales ont renforcé la nécessité de développer des antiviraux plus efficaces et plus résistants. L’intensive recherche de dérivés nucléosidiques cliniquement actifs a permis l’émergence d’une multitude de nouvelles approches pour leurs synthèses. Parmi ce panel, les réactions catalysées par des métaux de transition tardifs font certainement partie des méthodes les plus importantes pour accéder à une large gamme de pharmacomodulations. Lors de ce projet de recherche, nous nous sommes intéressés à la conception, la synthèse et la découverte de nouveaux dérivés nucléosidiques en tant qu’antiviraux contre le virus de la variole. Pour ce faire, nous avons ciblé des 1,2,3-triazolo-carbanucléosides en série racémique ou énantiosélective ainsi que des nucléosides phosphonates acycliques. Dans un effort de développement et/ou d’amélioration de leurs méthodes de synthèse, nous avons également étudié la contribution de complexes au ruthénium et au cuivre contenant un carbène Nhétérocyclique (CNH) dans des réactions de métathèse croisée et de cycloaddition 1,3-dipolaire de Huisgen. Enfin, en vue d’obtenir des outils efficaces utilisables en synthèse nucléosidique, nous avons développés des nouveaux complexes au palladium portant un ligand CNH et testé leurs réactivités dans des réactions de Narylation de Buchwald-Hartwig et d’hydrogénation d’oléfines. / In the last decades, nucleoside analogues have played a major role in the treatment of viral infectious diseases, such as AIDS, hepatitis, herpes, smallpox or influenza. Nevertheless, the apparition of new or mutated viruses highlights the need of more potent and resistant antiviral therapeutics. The intense search for clinically useful nucleoside derivatives has resulted in a wealth of new approaches for their synthesis. Among them, latetransition metal-catalyzed reactions are certainly the most important methods to access variety of appealing pharmacomodulations. In this research project, we were interested in the conception, the synthesis and the discovery of new nucleoside derivatives as antiviral agents against smallpox viruses. Hence, we have targeted racemic or enantiomerically pure 1,2,3-triazolo-carbanucleosides as well as acyclic nucleoside phosphonates. In our effort into the development or/and the improvement of their synthetic methodologies, we have also studied the contribution of ruthenium and copper complexes bearing N-heterocyclic carbene (NHC) ligands in cross-metathesis and Huisgen 1,3-dipolar cycloaddition reactions. Finally, in order to obtain efficient tools useful in nucleoside synthesis, we have developed new NHC-containing palladium complexes and examined their reactivity in Buchwald-Hartwig N-arylation and olefins hydrogenation reactions.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anežka Lalinská Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of interactions of antiretroviral drug tenofovir and its prodrug tenofovir disoproxil fumarate with placental nucleoside transporters Tenofovir (TFV) in the form of ester prodrug tenofovir disoproxil fumarate (TDF) is an essential part of combination antiretroviral therapy. It is often used in the prevention of perinatal HIV transmission. However, precise mechanism(s) involved in transfer of TFV/TDF from mother to fetus are not described in detail. Since these drugs are nucleoside analogues, there is a possibility that the mechanisms of their transplacental passage might include nucleoside transporters (NTs), either equilibrative or concentrative (ENTs/CNTs). The aim of the diploma thesis was to investigate the role of placental NTs in membrane transfer of TFV and TDF. To address this issue, we performed in vitro accumulation in the BeWo cell line derived from placental choriocarcinoma. By evaluating experiments, we found out that both TFV and TDF might not be substrates of NTs, thus the role of these transporters in TFV/TDF placental pharmacokinetics was not confirmed. Therefore, the drug-drug interactions on NTs...

Thymidylate kinase (TMK) is a key enzyme for DNA synthesis. It occurs at the junction of the de novo and salvage pathways for the synthesis of deoxythymidine triphosphate (dTTP). Its inhibition affects cell viability, thereby making it an important target for the development of anticancer, antibacterial and antiparasitic drugs. This thesis describes the analyses of the sequence, structure and dynamics of thymidylate kinase to obtain insights into its function. Two thermophilic variants of the enzyme were chosen for our studies. The studies provide valuable insights about the active site residues and the mechanism of catalysis, which have implications in protein engineering and design of specific inhibitors. Following is a chapter-wise description of the overall layout of the thesis. Chapter 1 | Introduction: This chapter provides a brief survey of the literature on TMKs and the scope of the work presented in the thesis. TMK belongs to the nucleoside monophosphate kinase (NMPK) family of enzymes, which includes adenylate kinase (AMK), guanylate kinase (GMK), uridylate kinase (UMK) and cytidylate kinase (CMK). The NMPK family of enzymes is associated with the reversible transfer of the terminal phosphoryl group from a nucleoside triphosphate (NTP) (usually adenosine triphosphate, i.e., ATP) to a nucleoside monophosphate (NMP). The identity of the NMP substrate varies among different enzymes. NMPKs share a common Rossmann fold and are comprised of a conserved P-loop, Lid region, CORE and NMP domains. The enzymes in the NMPK family also contain structurally similar active site architecture. Besides the three signature motifs, there are other conserved residues at the active site of TMK which are involved in interactions with the substrates ATP and dTMP. Despite the overall similarity, TMKs exhibit significant variations in sequence, residue conformation, substrate specificity and oligomerization mode. However, the residues responsible for these differences have not been studied. This thesis describes a comprehensive analysis of the sequence space of TMKs to detect the residues involved in such diversity. Subsequently, TMKs from a thermophilic archaeon (Sulfolobus tokodaii) and a hyperthermophilic bacterium (Aquifex aeolicus) were chosen for biochemical characterization and structural studies. Of these, the Sulfolobus tokodaii TMK (StTMK) has low sequence identity to the other TMKs of known three dimensional (3D) structures. Crystal structure analyses depicted the presence of some novel structural features and provided insights into the role of a conserved Arginine residue in function, which was verified through computational studies and mutagenesis experiments. Finally, the study on Aquifex aeolicus TMK resulted in multiple crystal structures of the apo form and different holo forms. These helped us to understand the mechanistic details of TMK-mediated catalysis, namely, the order of substrate binding and the reaction mechanism for phosphate transfer. Chapter 2 | Materials and Methods: This chapter provides a brief description of the procedures used to carry out the thesis work. The protein samples were purified to a high degree using column chromatography, and the purity was assessed using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis. Circular dichroism (CD) spectroscopy was employed to assess if the purified protein was well-folded. The pure and properly folded protein samples were used in further experiments. Differential scanning fluorimetry (DSF) was performed to determine the melting temperature of the thermophilic protein. MicroScale Thermophoresis (MST) and Surface Plasmon Resonance (SPR) were carried out to detect protein-substrate interactions. The protein samples were crystallized using the hanging drop vapour diffusion and microbatch under-oil techniques using commercially available crystallization screens, and the conditions which gave crystals were further optimized. Diffraction data, collected at either the home source or the synchrotron, were processed and scaled. Subsequently, phase information was obtained using the molecular replacement (MR) calculations. The MR solution was refined till convergence and its geometry was validated using different softwares. Finally, molecular dynamics (MD) simulations were performed to study the functionally important motions in the protein. Chapter 3 Insights into substrate specificity and oligomerization mode of Thymidylate Kinases from sequence evolution and conformational dynamics: Thymidylate kinase homologs exhibit significant variations in sequence, residue conformation, substrate specificity and oligomerization mode. However, the influence of sequence evolution and conformational dynamics on its quaternary structure and function has not been studied before. Based on extensive sequence and structure analyses, our study detected several non-conserved residues which are linked by co-evolution and are implicated in the observed variations in flexibility, oligomeric assembly and substrate specificity among the homologs. These lead to differences in the pattern of interactions at the active site in TMKs of different specificity. The method was further tested on thymidylate kinase from Sulfolobus tokodaii (StTMK) which has substantial differences in sequence and structure compared to other TMKs. Our sequence analyses pointed to a more flexible dTMP-binding site in StTMK compared to the other homologs, which was also indicated in MD simulations on the protein 3D structure. Binding assays proved that the protein can accommodate both purine and pyrimidine nucleotides at the dTMP binding site with comparable affinity. Additionally, the residues responsible for the narrow specificity of Brugia malayi TMK, whose three dimensional structure is unavailable, were detected. Our study provides a residue-level understanding of the differences observed among TMK homologs in previous experiments. It also illustrates the correlation among sequence evolution, conformational dynamics, oligomerization mode and substrate recognition in thymidylate kinases and detects co-evolving residues that affect binding, which should be taken into account while designing novel inhibitors. Chapter 4 | Biochemical and Structural characterization of a thermophilic variant of thymidylate kinase: This chapter reports the biochemical characterization and crystal structure determination of thymidylate kinase from the hyperthermophilic organism Sulfolobus tokodaii (StTMK) in its apo and ADP-bound forms. Our study describes the first three-dimensional structure of an archaeal TMK. The different structures had resolution ranging between 1.60 Å and 2.40 Å. StTMK is a thermostable enzyme with a melting temperature of 85.3 °C, as observed from thermal unfolding studies. The protein exists as a dimer in solution. A coupled enzyme assay, performed using thermo-stable lactate dehydrogenase (TtLdh) and pyruvate kinase (TtPk) from Thermus thermophilus, showed that StTMK has optimum activity at 80 °C. Despite the overall similarity to homologous TMKs, StTMK structures revealed several residue substitutions at the active site. However, enzyme assays demonstrated specificity to its natural substrates ATP and dTMP. A novel insertion (9 residues long) is observed in the C-terminal stretch of the Lid region. However, it is relatively rigid, which may be attributed to the presence of two proline residues and a hydrogen bond with an arginine residue in the α4/α5 loop. The C-terminus of the α2 helix points away from the Lid region in StTMK to avoid steric clashes with the Lid insertion. The main chain dihedral angles of the conserved Arg in the DRX motif are in the disallowed region of the Ramachandran plot in all holo TMK structures, wherein it forms several conserved hydrogen bonds with residues in the P-loop, α4 helix and α7 helix, as well as with the phosphate groups of both the substrates. A similar feature is observed in some of the StTMK structures. However, torsion angles in the allowed region of the Ramachandran plot are observed in one chain each in two of the apo structures. Further, conformational rearrangements of this Arg and its neighboring residues at the binding site of the second substrate are observed. The functional implication of this variation is described in the next chapter (chapter 5). Chapter 5 | Role of a conserved active site Arginine residue in Thymidylate kinase: Analysis of the structures of StTMK revealed multiple conformational states of Arg93 which is located at the reaction centre and is a part of the highly conserved DRX motif. Conformational heterogeneity of Arg can also be observed in some structures of Staphylococcus aureus and human TMK. However, the functional implication of this feature has not been probed before. The rearrangements of Arg93 are accompanied by related changes in the conformations of its neighbouring residues at the active site. This leads to three distinct conformational states in the dTMP-binding site, namely ‘Arg in’, ‘Arg intermediate’ and ‘Arg out’. Only the ‘Arg in’ state was found to be suitable for the proper positioning of the α-phosphate group of dTMP at the active site. This is hindered in the ‘Arg out’ and ‘Arg intermediate’ states. MD simulations showed that the torsion angles of the DRX Arg can sample between allowed and disallowed values in the apo-protein, with a preference for the catalytically suitable disallowed conformation in the holo-protein. Computational alanine scanning and MM/PBSA binding energy calculation further revealed the importance of Arg93 side chain in substrate binding. Subsequent site directed mutagenesis at this position to an Ala resulted in the loss of activity. Our work provides the first experimental evidence for the functional importance of Arg93 and gives insight into its regulatory role in the catalytically competent placement of dTMP. Our study also has implications for the development of potent inhibitors to lock the enzyme in the catalytically non-productive state. Chapter 6 | Characterizing active site dynamics from structural studies on the Intermediates along the reaction coordinate of a hyperthermophilic Thymidylate Kinase: TMK belongs to the family of nucleoside monophosphate kinases (NMPKs), several of which undergo structure-encoded conformational changes to perform their function. However, the absence of three dimensional structures for all the different reaction intermediates of a single TMK homolog hinders a clear understanding of its functional mechanism. We herein report the different conformational states along the reaction coordinate of a hyperthermophilic TMK from Aquifex aeolicus, determined via X-ray diffraction and further validated through normal mode studies. The analyses implicate an arginine residue in the Lid region in catalysis, which was confirmed through site-directed mutagenesis and subsequent enzyme assays on the wild type protein and mutants. Further, the enzyme was found to exhibit broad specificity towards phosphate group acceptor nucleotides. Our comprehensive analyses of the conformational landscape of TMK, together with associated biochemical experiments, provide insights into the mechanistic details of TMK-driven catalysis, for example, the order of substrate binding and the reaction mechanism for phosphate transfer. Such a study has utility in the design of potent inhibitors for these enzymes. Finally, the implications of the work described in this thesis and its future applications have been discussed in the section titled ‘Future prospects’. The work described in chapters 3 – 6 have been published in peer reviewed journals. Additionally, the author was involved in several collaborative projects which also resulted in publications (reprints attached in appendix).

A general and modular synthetic approach to 4-substituted phenyl, 2-substituted pyridin- 5-yl and 5-substituted pyridin-2-yl 2′-C-methyl-C-ribonucleosides as potential anti-HCV agents was developed. Addition of halo(het)aryllithium reagents to benzylated 2-C-methyl-D- ribonolactone gave the corresponding hemiketals, which were subsequently converted to the β-anomeric benzyl-protected bromo(het)aryl-C-nucleosides via either direct reduction (in the case of phenyl derivative) or acetylation followed by reduction of the resulting hemiketal acetates (in the case of pyridyl derivatives). The key halogenated (het)aryl-C-nucleoside intermediates were further transformed by Pd-catalyzed cross-coupling, hydroxylation and amination reactions affording series of protected C-nucleosides with small hydrophilic and hydrophobic substituents. The final protecting group removal was rather problematic, and different debenzylation methods, such as hydrogenation on Pd/C or treatment with BCl3, had to be optimized for each derivative to minimize the formation of side-products. The final C- nucleosides were also converted into their 5′-O-triphosphates, and biological activity screenings revealed that none of the free C-nucleosides possesses any antiviral activity in the HCV replicon assay, and none of their NTPs...

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19188号 / 工博第4065号 / 新制||工||1627(附属図書館) / 32180 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation. As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The physiological relevance of this is explained.

Nucleoside (S)-cEt-LNA a été synthétisé par trois voies différentes à partir de la 5- méthyluridine qui est commercialement disponible. Le chemin le plus court comprend une méthylation diastéréosélective d'un aldéhyde, et une cyclisation 5-exo-tet d'un éther par déplacement SN2. / (S)-cEt-LNA nucleoside was synthesized by three different routes starting from commercially available 5-methyluridine. The shortest route includes a diastereoselective methylation of an aldehyde, and a 5-exo-tet cylization of an ether via SN2 displacement.

Les analogues de nucléosides représentent une famille thérapeutique très largement utilisée en cancérologie dans le traitement des hémopathies malignes et autres cancers. Leur implication sur les variations des pools des nucléotides endogènes a été largement décrite et a constitué le point de départ de ce travail. C’est dans le but d’étudier ces variations que nous avons développé une méthode de dosage sensible et spécifique des ribonucléotides et désoxyribonucléotides physiologiques intracellulaires. Dans la première partie de ce mémoire nous avons réalisé une revue de la bibliographie sur les méthodes de dosage décrites dans la littérature puis nous avons détaillé le métabolisme des nucléotides endogènes et de leurs analogues. Dans la deuxième partie, nous présentons la validation de notre méthode de dosage et l’application à l’étude des variations des pools endogènes de ribonucléotides et désoxyribonucléotides dans différents modèles cellulaires : lignées résistantes versus lignées parentales et lignées traitées versus lignées non traitées. Les résultats obtenus sur les cellules montrent que notre méthode de dosage permet d’étudier l’impact des analogues nucleosidiques sur les pools endogènes de nucléotides mais aussi les dosages des dérivés phosphates de ces nucléosides / Nucleoside analogues constitute family of drugs widely used in cancer therapeutic for the treatment of malignancy hemopathies and several type of cancer. Numerous studies described their action on the variation of endogen nucleotide pools. This constitutes the point of starting of the present work. A sensible and specific assay for the quantification of intracellular ribonucleotides and désoxyribonucleotides was developed. In the first part, a review concerning the analytical methods published is presented. We also focused on the metabolism of endogen nucleotides and their analogues. In the second part, our method for the quantification of endogen nucleotides and désoxynucleotides is presented from different cellular models: resistant cell lines versus wild type lines, treated cells with analogues versus untreated cells. Results obtain from the cells lines studied show that our analytical method allows to study the impact of nucleoside analogues on endogenous nucleotides, but also the quantification of the phosphate derivatives of these analogues