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Polymyxins are potent antibiotics that bind to the outer membrane of Gram-negative bacteria, entering the cell and disrupting the inner membrane, resulting in cell death. They were traditionally used as antibiotics of last resort, but the recent surge of multidrug resistant pathogens has renewed interest in these antibiotics. The emergence of polymyxin resistance determinants such as the recently discovered plasmid-mediated phosphoethanolamine transferase MCR-1 may put a strain on the future effectiveness of this antibiotic. One method to combat the rise in antibiotic resistant bacteria is through the identification and development of antibiotic adjuvants. These are small molecules that are able to inhibit the resistance mechanism, allowing previously ineffective antibiotics to once again become effective at treating bacterial infections. In this work, a high throughput cell-based screen was conducted using an in-house library of Actinomycete-derived crude cell extracts in order to search for a natural product inhibitor of an E. coli strain expressing mcr-1. In addition, the development of a new enzyme assay was attempted using purified MCR-1 C-terminal catalytic domain and a chromogenic substrate to test enzymatic activity in vitro, in hopes of establishing a simple means of studying inhibition of MCR-1. The structure-function relationship of MCR-1 was also explored by generating amino acid substitutions and studying their effect on the ability of the enzyme to confer resistance to colistin, as well as the generation of MCR-1 transmembrane truncation mutants to determine if it was possible to generate a shorter variant of MCR-1 that retained its enzymatic activity. This work furthers our understanding of the biochemistry and enzymology of MCR-1, and outlines attempts to identify inhibitors of MCR-1 in order to re-sensitize resistant bacteria to polymyxins. / Thesis / Master of Science (MSc) / Polymyxins are potent antibiotics that are threatened by the spread of multi-drug resistant bacteria. Resistance to these antibiotics is relatively rare, although the recent discovery of a mobile polymyxin resistance enzyme, MCR-1, threatens the future use of this antibiotic for treating infections, as it can readily transfer to other bacteria. The goal of this work was to search for a natural product inhibitor of MCR-1 in order to reverse its ability to confer resistance to polymyxins. A color-changing assay was conducted with MCR-1 in hopes of establishing a method to study the inhibition of MCR-1 in vitro. Additionally, amino acid substitutions were generated in MCR-1 to better understand how key amino acids affect enzyme function, as well as transmembrane domain truncations to determine if it was possible to create a shorter functioning variant of MCR-1.

Interstrand cross-links (ICLs) are a type of DNA damage that prevents strand separation required for basic cellular processes. ICL-based anti-cancer therapies exploit the cytotoxic consequences of replication and transcription inhibition, however, they are limited by the ability of the cell to repair DNA crosslinks. The challenge of ICL repair involves coordinating multiple DNA repair pathways to remove damage occurring on both strands of DNA. Participation of factors that are both exclusive and essential to crosslink repair suggests a pathway requirement to process unique structures and/or intermediates arising only in ICL repair. SNM1A is a nuclease required for survival of human cells in response to ICL exposure, but the specific function and role of SNM1A remain unclear. Here we show that, in addition to known 5’-3’exonuclease activity, SNM1A possesses single-strand specific endonuclease activity. Furthermore, SNM1A exhibits translesion nuclease activity on crosslinks which deform the helical backbone, but not non-distorting stable ICLs. We report the identification and characterization of nine small molecules inhibitors of SNM1A, isolated from an in vitro high-throughput screen of nearly 4,000 bioactive compounds. Finally, we demonstrate that inhibitors of SNM1A potentiate the cytotoxicity of ICL-inducing agent cisplatin in HeLa cells. The work in this thesis expands the possible roles of SNM1A in ICL repair and lays the groundwork for SNM1A inhibition in ICL sensitization efforts. / Thesis / Doctor of Philosophy (PhD)

RNA is essential for the transfer of genetic information, as the central dogma of biology dictates. The role of RNA, however, is not limited to serving as an information shuttle between DNA and fully functional protein. Indeed, RNA has experienced a surge of interest in the field of chemical biology for its other critical roles in biology including those in control of transcription, translation, splicing, genetic replication, and catalysis. RNA has proven to be a difficult and complex target for the design of small molecular ligands because of its structural heterogeneity and conformational flexibility. Yet, the highly folded tertiary structures of these oligomers present unique scaffolds which designed ligands should be able to selectively target. To that end, two branched peptide libraries ranging in size from 4,096–46,656 unique sequences were screened for their ability to bind HIV-1 related RNA structures, the transactivation response element (TAR) and the Rev response element (RRE). In addition to discovering a mid-nanomolar branched peptide ligand for TAR, the first branched boronic acid peptide library designed to target RNA was screened for binding to RRE. Each of these efforts resulted in the identification of selective binders to their respective RNA targets, and the unnatural branching of these compounds was demonstrated to provide a multivalent binding interaction with the RNA. Furthermore, these compounds were shown to be cell permeable and displayed little to no cytotoxicity in HeLa and A2780 cells. / Ph. D.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25174号 / 医博第5060号 / 新制||医||1071(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 江藤 浩之, 教授 湊谷 謙司, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

During its intraerythrocytic stages (IE), Plasmodium falciparum, the causative agent of the deadliest human malaria, remodels the host red cell membrane with a poorly defined assortment of parasite-­encoded proteins that undergo antigenic variation. Despite the requirement for immunologic stealth, exported parasite proteins also mediate strain-independent functions such as endothelial sequestration that are critical for parasite survival and pathogenesis. This thesis explores the hypothesis that P. falciparum displays novel structurally conserved proteins on the IE surface and these proteins may serve as useful antigens for a broadly effective anti-­malarial vaccine. In order to test this hypothesis, we developed an in vitro selection technique that sequentially incorporates unique P. falciparum isolates as the targets for Systematic Evolution of Ligands by EXponential enrichment (Serial-SELEX) to generate nucleic acid molecular probes, aptamers, capable of recognizing conserved cell surface determinants. Ten of 11 enriched aptamers were -parasite selective and three of these aptamers demonstrated strain-independent binding to P. falciparum. Aptamer recognition extended beyond the parasites used in Serial-SELEX to other laboratory and recent field isolates. Surprisingly the same three broadly binding aptamer selected against P. falciparum also recognized all laboratory-adapted and clinical isolates of P. vivax and P. knowlesi tested, strongly supporting our hypothesis that structurally conserved molecules are present on the surface IEs. Competition studies showed that the aptamers bound a single target which was confirmed as an IE membrane protein. Aptamer­‐mediated affinity purification and tandem mass spectrometry enabled identification of the aptamer target as parasite-encoded protein. Discovery of a protein conserved between the major human malarias may have implications for vaccine development and validates the Serial‑SELEX technique as a powerful tool for antigen discovery.

Diseases such as malaria and dengue impose great economic burdens and are a serious threat to public health, with young children being among the worst affected. These diseases are transmitted by mosquitoes, also called disease vectors, which are able to transmit both parasitic and viral infections. One of the most important strategies in the battle against mosquito-borne diseases is vector control by insecticides and the goal is to prevent people from being bitten by mosquitoes. Today’s vector control methods are seriously threatened by the development and spread of insecticide-resistant mosquitos warranting the search for new insecticides. This thesis has investigated the possibilities of vector control using non-covalent inhibitors targeting acetylcholinesterase (AChE); an essential enzyme present in mosquitoes as well as in humans and other mammals. A key requirement for such compounds to be considered safe and suitable for development into new public health insecticides is selectivity towards the mosquito enzyme AChE1. The work presented here is focused on AChE1 from the disease transmitting mosquitoes Anopheles gambiae (AgAChE1) and Aedes aegypti (AaAChE1), and their human (hAChE) and mouse (mAChE) counterparts. By taking a medicinal chemistry approach and utilizing high throughput screening (HTS), new chemical starting points have been identified. Analysis of the combined results of three different HTS campaigns targeting AgAChE1, AaAChE1, and hAChE allowed the identification of several mosquito-selective inhibitors and a number of compound classes were selected for further development. These compounds are non-covalent inhibitors of AChE1 and thereby work via a different mechanism compared to current anti-cholinergic insecticides, whose activity is the result of a covalent modification of the enzyme. The potency and selectivity of two compound classes have been explored in depth using a combination of different tools including design, organic synthesis, biochemical assays, protein X-ray crystallography and homology modeling. Several potent inhibitors with promising selectivity for the mosquito enzymes have been identified and the insecticidal activity of one new compound has been confirmed by in vivo experiments on mosquitoes. The results presented here contribute to the field of public health insecticide discovery by demonstrating the potential of selectively targeting mosquito AChE1 using non-covalent inhibitors. Further, the presented compounds can be used as tools to study mechanisms important in insecticide development, such as exoskeleton penetration and other ADME processes in mosquitoes.

Ces dernières décennies, on a assisté à l’augmentation du nombre de technologies et de concepts permettant l’analyse des interactions intermoléculaires. Dans ce contexte, les puces à fluorescence restent les plus fréquemment utilisées. Cependant, cette technologie bien que très sensible et multiplexée, ne permet pas d’avoir accès aux paramètres cinétiques, indispensables au calcul des constantes d’affinité et la recherche de systèmes alternatifs s’impose. Dans cette optique, la résonance plasmonique de surface par imagerie (SPRi) est considérée comme une véritable option. Cette technologie se caractérise par l’absence de marquage et permet de suivre en temps réel d’infimes variations de masses consécutives à des interactions intermoléculaires sur la surface du prisme. L’obtention de constantes d’affinité est ainsi possible. En revanche, la SPRi présente un certain nombre de limites, principalement au niveau de la sensibilité et du multiplexage. Les objectifs de la thèse ont ainsi consisté à combler en partie ces différentes limites. La chimie de greffage basée sur l’utilisation d’oligonucléotides modifiés par un thiol a permis d’améliorer le multiplexage et de déposer plus de 1000 spots par cm² sur la surface d’or du prisme. Dans le même temps, la modification de la surface avec des colloïdes d’or et des dendrimères a permis pour des interactions ADN/ADN, d’atteindre une limite de détection de 2 nM (d’où un gain de 200%). En parallèle de ces travaux, diverses applications biologiques ont été effectuées. Une première étude a consisté à rechercher des ligands spécifiques des structures G-quadruplex des télomères. Une seconde étude s’est portée sur le complexe de partition bactérien. Par des études de criblage les bases impliquées dans l’interaction avec une protéine indispensable à la partition du plasmide F chez E.coli ont été identifiées. L’ensemble de ces travaux ont montré le fort potentiel de la SPRi et les applications potentielles qui en découlent sont nombreuses. / During the last decades a large number of technologies have been developed to analyze intermolecular interactions. In this context, the fluorescence biochips remain the most frequently used. Although this technology is very sensitive and multiplexed, it does not allow access to the kinetic parameters, essential to the calculation of the constants of affinity. Therefore, the research for alternative systems is essential. In this way, the Surface Plasmon Resonance imaging (SPRi) is considered as an opportunity. It is an optical detection process that can occur when a polarized light hits a prism covered by a thin metal layer. Under certain conditions free electrons at the surface of the biochip absorb incident light photons and convert them into surface plasmon waves. Perturbations at the surface of the biochip, such as an interaction between probes immobilized on the chip and targets, induce a modification of resonance conditions which can be measured. It is a label free technology which allows intermolecular interactions in real time and gives access to the kinetics parameters. However, SPRi is limited in sensitivity and multiplexing. The objectives of my PhD were to circumvent these various limits. Thus, we validated the immobilization of DNA probes on gold surface using thiol-modified oligonucleotide probes. Deposition carried out on non-modified gold surface, does not require electrical stimulation and expensive specific robotic devices. The thiol modification of the probes was shown to be very stable at room temperature, contrary to pyrrole and diazonium probes that need to be prepared just prior to their spotting. We demonstrate that thiol-modified oligonucleotide probes spotted on a gold surface of the SPRi-prisms are very robust and reproducible. We also demonstrated that this simple chemistry is compatible with high density arrays fabrication bearing more than 1000 spots using a classical spotter. Furthermore, the modification of the prism surface with gold colloids and dendrimers allowed for DNA/DNA interactions, to reach a detection limit of 2 nM. In parallel of this work, various biological applications were carried out and validate our previous developments. A first study was to screen G-quadruplex specific ligands to inhibit telomerase activity. We demonstrated that SPRi technology is particularly well adapted to the screening of interaction of small molecules with DNA probes and is sensitive enough to permit distinction between interactions with different DNA structures. The second study was on the bacterial partition complex. We study the DNA binding requirement involved in SopB-sopC specific interactions and analysed at the nucleotide level the bases involved in the binding efficiency and essential for the partition All this PhD work improved the SPRi technology and demonstrated its great potential in biological applications.

With the continued need for drug discovery and the quest to understand disease and treatment, there remains a requirement for improved methods to study protein-protein interactions and to identify potential drug leads for protein targets. We sought to develop a new approach to directly link genotype and phenotype to use as a probe for the identification of binding partners of proteins. The method creates millions of water-in-oil emulsions, each of which functions as a micro-environment for the amplification of a library of random peptide-encoding DNA molecules, which covalently bind to a bead. Subsequently the emulsions are broken and the bead-DNA complexes are recovered, which subsequently form another emulsion with in vitro transcription and translation components and incubated under suitable protein synthesis conditions. The synthetic peptide is designed with tags that link to the same bead which it is translated from. In chapter 3, the detailed design and optimisation of the method will be discussed. Cross-clade neutralising antibodies specific to HIV-1 are rare, partly because glycosylation restricts access to conserved backbone residues of gp120. In chapter 4, we hypothesized that peptides may have greater access than relatively large antibody structures, and so used our method to display random peptides on beads using a protein domain scaffold. Using a single round of selection, we identified 22 gp120-binding peptides, 4 of which were able to inhibit HIV-1 replication in vitro. One of the inhibitory peptides was found to bind the CCR5/CXCR4-binding site of gp120 and was able to inhibit clade B and C CCR5-tropic isolates of HIV-1. We have identified HIV-1 cross-clade neutralising peptides using a novel in vitro bead display library. Comprehensive antigenic characterization of a T cell population of unknown specificity is challenging. Existing MHC class I expression systems are limited by the practical difficulty of probing cell populations with an MHC class I peptide library and the cross-reactivity of T cells that are able to recognise many variants of an index peptide. We reasoned that a bead-based display high-throughput approach may overcome these challenges. Using emulsion PCR and emulsion in vitro transcription/translation of a random library of peptides conjugated to β-2-microglobulin on beads, we refolded with exogenous wild-type HLA-A*0201 or CD8-null A*0201 (domains 1 and 2 of HLA-A*0201 and domain 3 of Kb with mutated residues 226A/227L). The HLA bead libraries were used to probe HLA-A*0201-restricted T cells with specificity for influenza, CMV and EBV. High-throughput sequencing was used to rank sequences of identified peptides. Compared to pre-selection sequences, we observed significant enrichment for sequences containing HLA-A2 anchors and correct viral fragments for all T cell populations. HLA bead display provides a novel approach to identify the specificity of T cells. In summary, we combine the convenient handling of beads, the homogeneity of micro-environment in emulsion, and next-generation sequencing to create an attractive alternative to identify ligands of protein targets and antigenic peptides.

La recherche de nouvelles réactions est un des enjeux fondamentaux de la chimie organique. En dehors de l’approche classique basée sur la conception d’une réaction en s’appuyant sur les propriétés chimiques des substrats, une nouvelle approche utilisant le criblage systématique de combinaisons aléatoires de fonctions réactives a été récemment adoptée par plusieurs groupes. Cette stratégie nécessite un outil analytique permettant de cribler un très grand nombre de réactions par jour et d’identifier les meilleures combinaisons conduisant à la formation de produits intéressants. Les travaux de thèse présentés dans ce mémoire s’inscrivent dans le contexte de l’utilisation des techniques de dosages immuno-enzymatiques (ELISA) comme outil de criblage pour la recherche de nouvelles réactions de couplage. Dans un premier temps le criblage de 2688 combinaisons de fonctions réactives et de catalyseurs choisies au hasard a été effectué. Ce criblage a permit de mettre en évidence deux nouveaux couplages en présence de sels de cuivre : une réaction entre les thiourées et les phénols conduisant à la formation des isourées et une réaction entre les N-hydroxythiourées et les alcynes conduisant à la formation des thiazole-2-imines. Dans un second temps le criblage de 2816 combinaisons de fonctions sélectionnées, cette fois-ci, de façon rationnelle a été effectué. Ce criblage a visé la découverte de nouvelles cycloadditions [3+2] répondant aux critères de la chimie « click ». Ainsi l’utilisation de dosage immuno-enzymatique a été étendue à l’optimisation des nouvelles réactions découvertes ainsi qu’à l’évaluation de leurs cinétique, chimiosélectivité et biocompatibilité. Près de 3000 tests complémentaires effectuées sur les « hits » issus du criblage primaire ont ainsi permit de mettre en évidence 4 nouvelles réactions de couplage dont une nouvelle réaction « click » : la cycloaddition sydnone-alcyne catalysée au cuivre (CuSAC). Dans la dernière partie de ce manuscrit les études plus détaillées sur la réaction CuSAC ont été effectuées, notamment l’identification de la structure du produit de couplage et l’étendue du champ d’application de cette réaction. Enfin, l’aspect « click » de la réaction CuSAC a été illustré par l’application de cette réaction au marquage d’une protéine. / Discovery of new reactions is one of the fundamental goals in organic chemistry. In addition to the traditional approach to reaction discovery, consisting in designing a reaction on the basis of known chemical properties of reagents, new approaches based on the screening of random combinations of reactive functions and catalysts have been recently developed. The main prerequisite of this strategy is an analytical tool allowing screening of a big number of reactions per day and identifying combinations leading to the formation of unanticipated products. In the work presented herein a high-throughput immunoassay screening has been used for the discovery of new coupling reactions. In the first part of this work a screening of 2688 combinations of randomly chosen reactive functions and catalysts was carried out. This screening led to the discovery of two copper-promoted coupling reactions: a reaction between thioureas and phenols leading to the formation of isoureas through desulfurization; and a reaction between N-hydroxythioureas and alkynes leading to the formation of thiazole-2-imines. In the second part of the work a screening of 2816 combinations of rationally designed chemical functions and catalysts was carried out. This screening was focused on the discovery of catalytic [3+2] cycloadditions that comply with the standards of “click” chemistry. In this study, the use of immunoassay screening was extended to optimize new reactions and to evaluate their kinetics, chemoselectivity and biocompatibility. Therefore, around 3000 complementary tests were carried out on the hits, identified in the primary screening. This allowed the discovery of 3 new coupling reactions and one new “click” reaction: a copper-catalyzed sydnone-alkyne cycloaddition (CuSAC). The last part of the work was focused on detailed studies of the CuSAC reaction. Identification of the structure of the coupling product and substrate scope of this reaction was carried out. Finally, the applicability of the CuSAC reaction for bioconjugation was demonstrated by an example of protein labeling.