Thiostrepton (TS: TS; C72H85N19O18S5) is a thiazoline antibiotic that is effective against Gram-positive bacteria and the malarial parasite, Plasmodium falciparum. Tight binding of TS to the bacterial L11-23S ribosomal RNA (rRNA) complex of the large 50S ribosomal unit inhibits protein biosynthesis. The TS producing organism, Streptomyces azureus, biosynthesizes thiostrepton-resistance methyltransferase (TSR), an enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor, to modify the TS target site. Methylation of A1067 (Escherichia coli ribosome numbering) by TSR circumvents TS binding. The S. azureus tsr gene was overexpressed in E. coli and the protein purified for biochemical characterization. Although the recombinant protein was produced in a soluble form, its tendency to aggregate made handling a challenge during the initial stages of establishing a purification protocol. Different purification conditions were screened to generate an isolation protocol that yields milligram quantities of protein with little aggregation and sufficient purity for crystallographic studies. Enzymological characterization of TSR was carried out using an assay to monitor AdoMet-dependent ([methyl-3H]-AdoMet) methylation of the rRNA substrate by liquid scintillation counting. During the optimization of assay, it was found that, although this method is frequently employed, it is very time and labour intensive. A scintillation proximity assay was investigated to evaluate whether it could be a method for collecting kinetic data, and was found that further optimization is required. Comparative sequence analysis of TSR has shown it to be a member of the novel Class IV SpoUT family of AdoMet-dependent MTases. Members of this class possess a non-canonical AdoMet binding site containing a deep trefoil knot. Selected SpoUT family proteins were used as templates to develop a TSR homology model for monomeric and dimeric forms. Validation of the homology models was performed with structural validation servers and the model was then used as the basis of ongoing mutagenesis experiments. The X-ray crystal structure of TSR bound with AdoMet (2.45 Å) was elucidated by our collaborators, Drs. Mark Dunstan and Graeme Conn (University of Manchester). This structure confirms TSR MTase’s membership in the SpoUT MTase family with a deep trefoil knot in the catalytic domain. The AdoMet bound in the crystal structure is in an extended conformation not previously observed in SpoUT MTases. RNA docking simulations revealed some features that may be relevant to binding and recognition of TSR to the L11 binding domain of the RNA substrate. Two structure-activity studies were conducted to investigate the TS-rRNA interaction and TS solubility. Computational analyses of TS conformations, molecular orbitals and dynamics provided insight into the possible modes of TS binding to rRNA. Single-site modification of TS was attempted, targeting the dehydroalanine and dehydrobutyrine residues of the antibiotic. These moieties were modified using the polar thiol, 2-mercaptoethanesulfonic acid (2-MESNA). Similar modifications had been previously used to improve solubility and bioavailability of antibiotics. The resulting analogue was structurally characterized (NMR and mass spectrometry) and showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus.

Thiostrepton (TS: TS; C72H85N19O18S5) is a thiazoline antibiotic that is effective against Gram-positive bacteria and the malarial parasite, Plasmodium falciparum. Tight binding of TS to the bacterial L11-23S ribosomal RNA (rRNA) complex of the large 50S ribosomal unit inhibits protein biosynthesis. The TS producing organism, Streptomyces azureus, biosynthesizes thiostrepton-resistance methyltransferase (TSR), an enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor, to modify the TS target site. Methylation of A1067 (Escherichia coli ribosome numbering) by TSR circumvents TS binding. The S. azureus tsr gene was overexpressed in E. coli and the protein purified for biochemical characterization. Although the recombinant protein was produced in a soluble form, its tendency to aggregate made handling a challenge during the initial stages of establishing a purification protocol. Different purification conditions were screened to generate an isolation protocol that yields milligram quantities of protein with little aggregation and sufficient purity for crystallographic studies. Enzymological characterization of TSR was carried out using an assay to monitor AdoMet-dependent ([methyl-3H]-AdoMet) methylation of the rRNA substrate by liquid scintillation counting. During the optimization of assay, it was found that, although this method is frequently employed, it is very time and labour intensive. A scintillation proximity assay was investigated to evaluate whether it could be a method for collecting kinetic data, and was found that further optimization is required. Comparative sequence analysis of TSR has shown it to be a member of the novel Class IV SpoUT family of AdoMet-dependent MTases. Members of this class possess a non-canonical AdoMet binding site containing a deep trefoil knot. Selected SpoUT family proteins were used as templates to develop a TSR homology model for monomeric and dimeric forms. Validation of the homology models was performed with structural validation servers and the model was then used as the basis of ongoing mutagenesis experiments. The X-ray crystal structure of TSR bound with AdoMet (2.45 Å) was elucidated by our collaborators, Drs. Mark Dunstan and Graeme Conn (University of Manchester). This structure confirms TSR MTase’s membership in the SpoUT MTase family with a deep trefoil knot in the catalytic domain. The AdoMet bound in the crystal structure is in an extended conformation not previously observed in SpoUT MTases. RNA docking simulations revealed some features that may be relevant to binding and recognition of TSR to the L11 binding domain of the RNA substrate. Two structure-activity studies were conducted to investigate the TS-rRNA interaction and TS solubility. Computational analyses of TS conformations, molecular orbitals and dynamics provided insight into the possible modes of TS binding to rRNA. Single-site modification of TS was attempted, targeting the dehydroalanine and dehydrobutyrine residues of the antibiotic. These moieties were modified using the polar thiol, 2-mercaptoethanesulfonic acid (2-MESNA). Similar modifications had been previously used to improve solubility and bioavailability of antibiotics. The resulting analogue was structurally characterized (NMR and mass spectrometry) and showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus.

Mon projet a porté sur la caractérisation du mécanisme moléculaire des enzymes d'arenavirus (une 3'-5' exoribonucléase et une endonuclease) impliquées dans l'inhibition de la réponse innée IFN de type I et dans le vole de coiffe respectivement, et le développement d'une stratégie thérapeutique basée sur leur structures. Premièrement, j'ai résolu deux structures cristallographiques à haute résolution du domaine exoribonucléases du virus Mopeia (NP-exo MOPV) -un homologue du virus Lassa pathogène- en complexe avec deux ions différents. Ensuite, j'ai effectué une caractérisation fonctionnelle de l’activité exoribonucléase 3'-5' codée par ce domaine. Une corrélation entre la structure et la fonction de NP-exo MOPV démontre que; L’activité exoribonucléase 3'-5' est conservée chez les arenavirus pathogènes ainsi que chez les non-pathogènes. J'ai démontré pour la première fois que l'exoribonucléase est capable d'exciser un ARN misapparié, suggérant ainsi une potentielle activité de correction d'erreur par cette enzyme. Avec la structure de NP-exo MOPV, j'ai développé une stratégie in silico pour identifier des inhibiteurs potentiels spécifiques contre son activité et un inhibiteur a était identifié.En parallèle, nous avons résolu deux structures cristallographiques du domaine de l'endonuclease du virus de la LCMV en complexe avec deux ions catalytiques et deux composés appartenant a la famille des diketo. En résumé, ce travail éclaircit le rôle des exoribonucléases de la famille d'Arenaviridae allant de l’évasion de l'immunité innée à son implication directe dans la réplication. Il ouvre également la voie au développement des inhibiteurs contre ces nucléases. / My PhD work focused on the characterization of the molecular mechanism of two arenavirus enzymes - a 3'-5' exoribonuclease and an endonuclease - implicated in type I IFN suppression and mRNA cap-snatching respectively and the design of a structure based-drug strategy against them. First I solved two high resolution crystal structures of the exoribonuclease domain of Mopeia virus (NP-exo MOPV) -a non pathogenic homologue of the highly pathogenic Lassa virus- in complex with different metal ions. Next I performed an in depth functional characterization of the 3'-5' exoribonuclease activity encoded by this domain. By correlating the structure and function of NP-exo MOPV, I showed that; the 3'-5' exoribonuclease activity is conserved in pathogenic as well as in non-pathogenic arenaviruses. Also, I showed for the first time that this enzyme is able to excise a mismatched RNA suggesting that, arenaviruses might posses a mechanism to limit error incorporation by the RdR polymerase during replication. Using the crystal structure of NP-exo MOPV I designed a structure-based strategy to identify potential inhibitors specific for the 3'-5' exoribonuclease activity and have identified a potential inhibitor.Alongside, we solved two crystal structures of the endonuclease domain of LCMV in complex with two catalytic ions and two compounds belonging to the diketo family.In conclusion, this work has a deep implication extending the role of the Arenaviridae exoribonuclease from innate immunity evasion to direct implication in replication. It also paves the way for the development of inhibitors against these arenavirus nucleases.

<p><a>The processive plant cellulose synthase (CESA) synthesizes (1→4)-β-D-glucans. CESAs assemble into a six-fold symmetrical cellulose synthase complex (CSC), with an unknown symmetry and number of CESA isomers. The CSC synthesizes a cellulose microfibril as the fundamental scaffolding unit of the plant cell wall. CESAs are approximately 110 kDa glycosyltransferases with an N-terminal RING-type zinc finger domain (ZnF), seven transmembrane α-helices (TMHs) and a cytoplasmic catalytic domain (CatD). In the CatD, the uridine diphosphate glucose (UDP-Glc) substrate is synthesized into</a> (1→4)-β-D-glucans. The ZnF is likely to facilitate dimers in the CSC. Recombinant class-specific region (CSR), a plant specific insertion to the C-terminal end of the CatD is also known to form dimers<i> in vitro</i>. The CSR sequence is the primary source of distinction between CESA isoforms and class structure. Also within the CESA CatD is a 125-amino acid insertion known as the plant-conserved region (P-CR), whose molecular structure was unknown. The function of the P-CR is still unclear, especially in the context of complete CESA and CSC structures. Thus, one major knowledge gap is understanding how multimeric CSCs synthesize multiple chains of (1→4)-β-D-glucans that coalesce to form microfibrils. The specific number of CESAs in a CSC and how interactions of individual CESA isoforms contribute to the CSC are not known. Elucidating the structure-function relationships of the P-CR domain, and with the consideration of the ability of CSR and ZnF domains to dimerize, it is possible to more completely model the structure of the CSC.</p> <p>Recombinantly expressed rice (<i>Oryza sativa</i>) secondary cell wall OsCESA8 P-CR domain purifies as a monomer and shows distinct α-helical secondary structure by circular dichroism analysis. A molecular envelope of the P-CR was derived by small angle X-ray scattering (SAXS). The P-CR was crystallized and structure solved to 2.4 Å resolution revealing an anti-parallel coiled-coiled domain. Connecting the coiled-coil α-helices is an ordered loop that bends back towards the coiled-coils. The P-CR crystal structure fits the molecular envelope derived by SAXS, which in turn fits into the CatD molecular envelope. The best fit places the P-CR between the membrane and substrate entry portal. In depth analysis of structural similarity to other proteins, and 3D-surface structure of the P-CR, leads to hypotheses that it could function in protein-protein interactions as a dimer, trimer or tetramer in the CSC, that it could form protein-protein interactions with CESA-interacting proteins, and/or modulate substrate entry through its N- and/or C-terminus. From modeling, hypothetically important residues within the P-CR or related to the P-CR through potential protein contacts were mutated in <i>Arabidopsis thaliana</i> <i>AtCESA1</i> constructs. These constructs were expressed in the temperature-sensitive <i>radial swelling</i> (<i>rsw</i>)<i> rsw1-1</i> mutant of <i>AtCESA1 </i>to test for complementation of growth phenotypes at restrictive temperatures. Preliminary experiments indicate that some mutated CESA1 sequences fail to complement the <i>rsw1-1</i> phenotype, suggesting that specific functions of individual amino can be tested using this system.</p>

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Text in English / Specially prepared 2-amino-5-bromo-3-iodoacetophenone and 5-bromo-2-hydroxy-3 iodoacetophenone were subjected to Claisen-Schmidt aldol condensation with benzaldehyde derivatives followed by sequential and/or one-pot palladium catalyzed Sonogashira cross coupling and heteroannulation of the 3-alkynylated intermediates to afford indole-chalcones and benzofuran-chalcones, respectively. The indole-chalcones derivatives were, in turn, subjected to trifluoroacetic anhydride in tetrahydrofuran under reflux to afford the corresponding 3-trifluoroacetyl substituted indole-chalcone derivatives. The coupling constant values (Jtrans) of about 16.0 Hz for the chalcone derivatives corresponding to the vinylic protons confirmed the trans geometry of the α,β-unsaturated carbonyl framework in all the cases. Their trans geometry of the chalcone derivatives was further confirmed by single crystal X-ray diffraction (XRD) analyses. Further structural elaboration of the ambident electrophilic α,β unsaturated carbonyl (chalcone) moiety of the indole-chalcones and the analogous benzofuran chalcones with 2-aminothiophenol afforded novel benzothiezapine-appended indole and benzofuran hybrids, respectively. Sonogashira cross-coupling of 5-bromo-2-hydroxy-3 iodoacetophenone with terminal acetylenes followed by heteroannulation of the intermediate 3-alkynylated 5-bromo-2-hydroxyacetophenones afforded the corresponding 7-acetyl-2-aryl-5-bromobenzofurans in a single-pot operation. The oximes derived from the 7-acetyl–substituted 2-aryl-5-bromobenzofurans were subjected to Beckmann rearrangement with triflic acid in acetonitrile under reflux. We isolated the corresponding 7-amino-2-aryl-5 bromobenzofuran derivatives formed from hydrolysis in situ of the intermediate 7-acetamide 2-aryl-5-bromobenzofurans. Amino-dechlorination of the 4-chloroquinazoline derivatives with the 7-aminobenzofurans afforded novel benzofuran 4-aminoquinazoline hybrids. The prepared compounds were characterized using a combination of nuclear magnetic resonance (1H-NMR & 13C-NMR including 19F-NMR), infrared (IR) and mass spectroscopic techniques complemented with single crystal X-ray diffraction (XRD) analyses and/or density functional (DFT) method. The benzofuran-chalcone 203a–y derivatives were evaluated for anti-growth effect against the breast cancer (MCF-7) cell line by the MTT cell viability assay. Their mode of cancer cell death (apoptosis versus necrosis) was detected by Annexin V-Cy3 SYTOX staining and caspase-3 activation. The most cytotoxic compounds 203i and 203o were also evaluated for potential to inhibit tubulin polymerization and/or epidermal growth factor receptor-tyrosine kinase (EGFR-TK) phosphorylation. The experimental results were complemented with theoretical data from molecular docking into ATP binding site of the EGFR and colchicine binding site of tubulin, respectively. The benzofuran–4-aminoquinazoline hybrids 215a–j, on the other hand, were evaluated for antiproliferative propeties in vitro against the human lung cancer (A549), epithelial colorectal adenocarcinoma (Caco-2) and hepatocellular carcinoma (C3A) cell lines. The benzofuran-aminoquinazoline hybrids were also evaluated for potential to induce apoptosis and for their capability to inhibit EGFR-TK phosphorylation complemented with molecular docking (in silico) into the ATP binding site of EGFR. Mechanistic studies demonstrated that the benzofuran-appended aminoquinazoline hybrids 215d and 215j induced apoptosis via activation of caspase-3 pathway. Moreover, compounds 215d and 215j exhibited significant and moderate inhibitory effects against EGFR (IC50 = 29.3 nM and 61.5 nM, respectively) when compared to Gefitinib (IC50 = 33.1 nM). Molecular docking of compounds 215 into EGFR-TK active site suggested that they bind to the region of EGFR like Gefitinib does. / Chemistry / D. Phil. (Chemistry)

To study the functions of glycoconjugates in biological systems reliable and efficient protocols for glycoconjugate synthesis are needed. To reach this goal we have developed methods for solid-phase synthesis of glycoconjugates that can be monitored with gel-phase 19F spectroscopy using fluorinated linkers, building blocks, and protecting groups. We have developed a new fluorine containing linker suitable for solid-phase synthesis of glycoconjugates. The linker was more acid-labile than similar linkers in order to enable cleavage under mild conditions of the target compound from the linker resin.  A carbamate-based strategy has been applied to attach a spacer carrying an amino group to a fluorinated Wang linker for synthesis of amino-functionalized glycoconjugates using thioglycoside donors with fluorinated protective groups. Cleavage from the solid support was performed with trifluoroacetic acid and subsequent protecting group removal gave the target compound. The terminal amine was conjugated with didecyl squarate and this derivative can be attached to various proteins and solid surfaces carrying primary or secondary amines. To evaluate this methodology we have immobilized glycoconjugates in amino-functionalized microtiter plates and successfully probed them with lectin. In addition, a novel fluorine containing protecting group has been designed, synthesized and evaluated. The protecting group was used for protection of the unreactive 4-OH in a galactose building block that was applied in the synthesis of 6-aminohexyl galabioside and was removed with TBAF in THF. Adenovirus serotype 8 (Ad8), Ad19, and Ad37 cause the severe ocular infection, epidemic keratoconjunctivities (EKC). During infection, the adenoviruses interact with sialic acid containing glycoconjugates on the epithelial cells via fiber structures extending from the viral particles. The virus particle most likely binds to the host cell in a multivalent way by simultaneously using multiple fiber proteins and binding sites. Multivalent sialic acid containing conjugates could efficiently inhibit Ad37 cell attachment and subsequent infection of human corneal epithelial (HCE) cells. Three compact tri- and tetravalent sialic acid conjugates were prepared and evaluated as inhibitors of adenoviral host cell attachment and subsequent infection and all conjugates were potent as anti-adenoviral agents. The conjugates can readily be synthesized from accessible starting materials. A crystal structure of the Ad37 fiber knob protein and the trivalent sialic acid conjugate showed that the three binding sites were all occupied by one sialic acid residue each.

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