<p dir="ltr">Humans encode approximately 100 deubiquitinating enzymes (DUBs) which are categorized into seven distinct subfamilies. Each family and representative has a unique expression, function and binding topology to ubiquitin. In addition to human DUBs, parasites, bacteria, and viruses contain DUBs with unique structures and functions. One subfamily of DUBs, the ubiquitin C-terminal hydrolases (UCH), has four structurally similar human members and two known members within the <i>Plasmodium falciparum</i> genome. Human UCHL1 and UCHL3 are genetically validated targets in oncology and <i>Plasmodium falciparum</i><i> </i>UCHL3 (PfUCHL3) is a prospective target for antimalarial drug development. Though these three UCH enzymes have potential as therapeutic targets, there is a significant lack of quality small molecule chemical probes to understand the underlying biology and function of the enzymes, pharmacologically validate the targets, and serve as leads for drug development in oncology and malaria.</p><p dir="ltr">The UCH enzymes are cysteine proteases, which our lab has leveraged to identify novel covalent small molecule inhibitors of each enzyme. The workflow for each hit identification and optimization campaign is similar. Covalent fragment screening of electrophilic small molecule libraries against the respective recombinant enzyme was performed to identify chemical space around each enzyme. Subsequent medicinal chemistry hit-to-lead optimization was undertaken to improve upon the moderately potent hit molecules to provide improved small molecule inhibitors for each enzyme. Inhibitor identification and optimization for UCHL1 is described in Chapter 2, revealing a novel scaffold and a cocrystal structure reveals a unique binding pose for UCHL1 inhibitors. These molecules were also characterized in breast cancer cells to validate UCHL1 as a therapeutic target in breast cancer. First-in-class covalent inhibitors of UCHL3 are described in Chapter 3. Medicinal chemistry optimization along with a cocrystal structure of the initial hit has revealed the molecular interactions of this novel inhibitory scaffold. PfUCHL3 inhibitor identification is described in Chapter 4. Characterization of these molecules against Plasmodium falciparum is described along with a comparison to a recently identified reversible PfUCHL3 inhibitor. Finally, conclusions and future directions toward the development of potent, drug-like inhibitors of each UCH enzyme is presented in Chapter 5.</p>

Dans l'introduction, diverses voies de trafic intracellulaire et endocytose seront discutées. Je familiarise le lecteur avec des protéines inactivant les ribosomes, en mettant l'accent sur la structure, l'endocytose, et le trafic intracellulaire de la toxine bactérienne Shiga toxin (STX). STx et la ricine suivent la voie rétrograde pour exercer leur effet toxique sur les cellules. Ils sont respectivement, une menace maladie infectieuse pour la santé humaine et des outils potentiels pour le bioterrorisme pour lequel aucun antidote n’existe actuellement. D'un criblage à haut débit, Retro-1 et Retro-2 avaient déjà été identifiés comme de puissants inhibiteurs de la voie rétrograde à l'interface des endosomes précoces-TGN, et Retro-2 a été démontré pour protéger les souris contre la ricine. Parmi les facteurs de trafic analysés, seule la protéine SNARE syntaxine-5 a été ré- localisée dans les cellules traitées avec Rétro - 2. / In the introduction, various endocytic and intracellular trafficking pathways will be discussed. I acquaint the reader with ribosome-inactivating proteins, with emphasis on the structure, endocytosis, and intracellular trafficking of the bacterial toxin Shiga toxin (STx). STx and ricin follow the retrograde route to exert their toxic effect on cells. They are respectively, an infectious disease threat to human health and potential tools for bioterrorism for which no antidote currently exists. From a high throughput screening, Retro-1 and Retro-2 had previously been identified as potent inhibitors of the retrograde route at the early endosomes-TGN interface, and Retro-2 was demonstrated to protect mice against ricin. Of the trafficking factors analyzed, only the SNARE protein syntaxin-5 was re-localized in Retro-2 treated cells. Yet, whether syntaxin-5 is the direct target of Retro-2 and whether its re-localization was directly responsible for retrograde transport inhibition remained to be established.

Human hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and to differentiate into all blood lineages during an adult lifetime. Based on these unique properties, HSCs are used in transplantation procedures to treat various hematological diseases. However, the low number of HSCs in a graft limits the use of this treatment. To overcome this restrain, different approaches were established to expand HSCs ex vivo; yet, the absence of a reliable surface maker that correlates with HSC activity in culture made the assessment labor-intensive and time-consuming. Using a library of small molecules, we were able to identify pyrimidoindole derivative named UM171 as an agonist for HSC self-renewal. UM171 promotes ex vivo expansion of hematopoietic and stem cell progenitors (HSPC) independently of AhR suppression- a pathway reported by Boitano et al. to have the greatest effect in HSC expansion. Unlike AhR suppression that targets a hematopoietic population with limited proliferative potential, UM171 targets the long-term HSCs. Transcriptome analysis showed that UM171 reduces the levels of transcripts associated with lineage differentiation and induces the expression of genes encoding for membrane proteins, one of the best differentially expressed being the endothelial protein c receptor (EPCR). Cell sorting and transplantation experiments of EPCR expressing cells showed a high correlation with HSC activity. We demonstrated EPCR as a first reliable marker to enrich for HSC in culture and that it is required for HSPC function in vivo. These findings provide a valuable tool for clinical and research applications to optimize further HSPC expansion protocols and understand the molecular machinery that governs the HSC self-renewal. / Le terme de cellules souches hématopoïétiques (CSH) désigne une population rare de cellules capables de générer l’ensemble des lignages hématopoïétiques. Cette définition implique une capacité d’auto-renouvèlement, ainsi qu'un potentiel de prolifération et de différenciation important. La greffe de cellules souches hématopoïétiques est aujourd'hui une modalité thérapeutique pour le traitement de diverses maladies hématologiques et représente pour de nombreux patients un traitement de dernier recours. Malheureusement, le nombre limité de ces cellules dans une unité de sang de cordon est à l’origine du faible taux de réussite des greffes de sang de cordon chez l'adulte. Plusieurs stratégies sont actuellement mises en place pour permettre la multiplication de ces CSH ex vivo. Cependant, Il n’y a jusqu’à ce jour aucun critère ou marqueur phénotypique fiable permettant spécifiquement d'identifier ou d'isoler ces CSH amplifiées, et leur caractérisation reste un défi majeur pour les chercheurs. Dans le laboratoire, nous avons effectué un criblage à haut débit afin de tester le potentiel d’un grand nombre de molécules chimiques à multiplier des cellules souches dérivées de sang de cordon ombilical et nous avons ainsi identifié la molécule UM171, un dérivé pyrimido-indole, qui permet de multiplier par 10 le nombre de CSH et par 100 leur descendance. Nous avons démontré qu' UM171 permet de multiplier les CSH sans affecter la voie de signalisation de la protéine AhR, récemment impliquée dans l'auto-renouvèlement des CSH. L'analyse du transcriptome des CSH exposées à la molécule UM171 a permis d'identifier le récepteur endothélial à la protéine C (EPCR), comme marqueur de surface permettant de prédire le nombre et l'activité des CSH en culture et par conséquent de les isoler et de mieux les caractériser. En combinant des techniques de cytométrie de flux et d'ARN interférents avec des expériences de transplantation à long terme dans des souris immuno-déficientes, nous avons pu démontrer qu' EPCR peut être considéré non seulement comme un premier marqueur fiable pour enrichir les CSH en culture mais aussi qu'il est nécessaire pour la fonction de ces CSH in vivo. Les résultats de ces travaux représentent une avancée majeure pour accélérer les recherches et les applications cliniques sur l'expansion des CSH ex vivo et permettra de comprendre les mécanismes moléculaires qui régissent l'auto-renouvèlement des CSH.

The development and advancement of prostate cancer is supported by a plethora of genetic and proteomic abnormalities, including events of post-translational modifications. The protein arginine methyltransferase 5 (PRMT5) enzyme regulates epigenetic events of histone modifications and protein post-translational modifications within protein signaling pathways. PRMT5 functions by catalyzing the symmetric dimethylation of terminal arginine residues on target protein substrates. Under abnormal conditions of overexpression and upregulation, PRMT5 methyltransferase activity constitutively drives the growth and proliferation of dysregulated cells. Overexpression or upregulation of PRMT5 correlates with disease progression as observed among numerous cancer types, including breast, colorectal, leukemia, lung, melanoma and prostate cancers. We demonstrated previously that PRMT5 knockdowns attenuated both growth and proliferation of lung and prostatic tumors, in vitro and in vivo. Plants naturally produce chemical toxins as mechanisms of defense against microbial and other biological threats. Human exploitation, consumption and application of agents isolated from plants for therapeutic intervention dates back throughout the millennia. In this study, we extracted, purified and evaluated natural, small, chemical compounds from plant products that antagonize PRMT5 activity in prostate cancer cells. We found that crude and purified extracts of Dendrobium aurantiacum var. denneanum (D. denneanum) plants attenuated prostate tumor growth and proliferation by selective inhibition of PRMT5 methyltransferase activity. These findings establish the first set of natural PRMT5-specific inhibitors reported.

As estruturas cristalinas dos complexos Ln(ClO4)3.6[PONH2(C6H5)2] onde Ln=Eu, La, Cu[NH2(CH3)2CCO2]2, NiBr2.4[AsO(C6H5)3].8H2O (verde) e NiBr2.4[AsO(C6H5)3].1,5(CH3C6H5).H2O (alaranjado) bem como do ligante PONH2(C6H5)2 foram determinadas por difração de raios-X. Os complexos envolvendo íons lantanídeos refinaram a fatores R finais de R(Eu)=0,125, e R(La)=0,133 e foram encontradas a seguintes características principais: a) o sistema cristalino é cúbico; b) a coordenação do cátion é feita por seis átomos de oxigênio dos ligantes em configuração octaédrica (Eu) e antiprismática trigonal (La) com as terras raras em posições de alta simetria (23 para Eu e 3 para La); c) o restante das estruturas apresentam diferentes graus de desordem. À luz da sua configuração geométrica, a presença de uma forte banda 5Do-7F2 no espectro de fluorescência do complexo de Eu, proibida por considerações de simetria, é explicada como decorrente de acoplamentos vibrônicos. O desdobramento da linha &#965;P=0 do espectro de infravermelho do coplxo de La é atribuído à presença de grupos P=0 não equivalentemente ligados a terra rara devido à desordem desta. O composto envolvendo o íon Cu(II) (fator R final 0,053) cristaliza no sistema monoclínico com os complexos se empacotando em camadas paralelas ao plano cristalino (100) com redes de pontes de H intracamada e com francas interações entre camadas consecutivas, o que explica o comportamento magnético quasi-bidimensional observado nestes cristais. O complexo de Ni(II) de coloração verde (fator R final 0,039) apresenta o íon metálico sobre um centro de simetria coordenado por seis moléculas de água numa conformação octaédrica distorcida, as quais estão ligadas aos grupos tfaso[AsO(C6H5)3] e íons brometo por fatores pontes de H. No complexo de coloração alaranjada (fator R final 0,087) o cátion está pentacoordenado com os quatro oxigênios dos ligantes tfaso formando a base de uma pirâmide quadrangular e um ânion Br- ocupando a quinta posição. Como uma conseqüência da resolução e refinamento da estrutura do complexo de Eu, a estrutura cristalina do ligante PONH2(C6H5) puro foi também determinada e refinada a um fator R de 0,033. / The crystal structure of the complexes Ln(ClO4)3.6[PONH2(C6H5)2] where Ln=Eu, La, Cu[NH2(CH3)2CCO2]2, NiBr2.4[AsO(C6H5)3].8H2O (green), NiBr2.4[AsO(C6H5)3.1,5(CH3C6H5).H2O (orange) and of the ligand PONH2(C6H5)2 have been determined by X-ray diffraction. The complexes involving lanthanide ions refined to final R factors of R(Eu)=0.125 and R(La)=0.133 and the following main features were found: a) the crystal system is cubic; b) the cation is coordinated to six ligand oxygens in octahedral earths on position of high symmetry (23 for Eu and 3 for La); c) the rest of the structures shows different degrees of disorder. In the light of the geometrical configuration, the occurrence of a strong band 5Do-7F2 in the fluorescence spectrum of the Eu complex, forbidden on symmetry grounds, is interpreted as a consequence of vibronic coupling. A splitting of the infrared &#965;P=0 band in the La complex is attributed to the presence of P=0 goups non-equivalently bonded to the rare earth due to the disorder of this atom. The compound involving the Cu(II) ion (final R factor of 0.053) crystallizes in the monoclinic system with the complexes packed in layers parallel to the (100) crystal plane, with intralayer nets of H bonds and weak interactions between consecutive layers, which explains the quasi two-dimensional magnetic behavior observed in these crystals. In the green Ni(II) complex (final R=0.039), the metallic ion is sited on a center of symmetry and is octahedrally coordinated to six water molecules which are hydrogen bonded to the tfaso[AsO(C6H5)3] groups and the bromide ions. In the orange complex (final R=0.087) the cation is pentacoordinated with the four oxygens of the tpas ligands forming the basis of a quadrangular pyramid and one Br- anion in the fifth position. As a by product in the solution and refinement of the Eu complex structure, the crystal structure of the pure ligand PONH2(C6H5) was also determined and refined to a R-factor of 0.033.

No description available.

This thesis describes the design, synthesis and reactivity of bimetallic complexes of doubly-pillared Schiff-base calixpyrrole ligands. Chapter One introduces the oxygen reduction reaction in light of the global energy scenario at present and in the future. Compounds and materials known to catalyse this reaction are discussed, with particular focus on transition metal complexes of pyrrole-containing macrocycles and the ability of these compounds to act as catalysts in redox reactions. Chapter Two describes the design and synthesis of several of the macrocyclic ligands developed during this project. The wide range of metals and geometries supported by one of the ligands, H4L, are outlined and include complexes of alkali-metals (Li, K), a rare earth metal (Mg), transition metals (Pd, Fe) and an actinide (UO2 2+). Chapter Three presents the use of [Co2(L)] for the reduction of dioxygen to water. The redox behaviour of the complex and its ability to reversibly bind oxygen were evaluated. The catalytic activity of [Co2(L)] was investigated in solution by UV-Vis spectrophotometry and electrochemically by rotating ring-disk electrochemistry. In Chapter Four, the ability of [Zn2(L)] to bind anions is described. Isothermal microcalorimetry, NMR, UV-Visible spectrophotometry, and fluorophotometry were used to study the de-aggregation of the anion free complex and the subsequent anion binding event. The stability of the complexes was estimated by DFT calculations. Chapter Five outlines the synthesis of complexes of L for other transition metals relevant to small molecule activation. Chapter Six contains a conclusion and suggestions on further investigations to carry out. Chapter Seven presents the full experimental details and analytical data for this work.

Ce travail porte sur la synthèse et l’évaluation de systèmes pi-conjugués en tant matériaux actifs pour des dispositifs opto(électroniques). Un premier chapitre décrit une série d’oligothiophènes cruciformes et leur évaluation dans des dispositifs électrochromes. Le second chapitre décrit la synthèse d’accepteurs moléculaires à base de benzodithiophène et l’analyse de leur potentialités comme matériaux accepteurs dans des cellules solaires organiques. La plus large part du travail porte sur l’analyse des relations structure-propriétés d’une série de petites molécules push-pull comportant un groupe donneur triphénylamine (TPA) relié à un groupe accepteur par un espaceur thiényl. Une première étape a consisté à remplacer l’un des cycles phényles de la TPA par des groupes aromatiques tels que p-fluorophényle, anthryle et naphtyle. Ces modifications ont peu d’influence sur les propriétés électroniques de la molécule mais induisent de large variations des propriétés de transport de charge et de conversion photovoltaïque des matériaux correspondants. Au cours d’une seconde étape l’un des groupes phényles de la TPA a été remplacé par des chaînes alkyle, perfluoroalkyle et oligo(oxyethylene). Les résultats de diffraction X, spectroscopie d’absorption et de fluorescence, génération de second harmonique et électrochimie démontrent que certaines de ces molécules présentent des propriétés d’émission contrôlées par agrégation tandis que les matériaux correspondants se réorganisent spontanément à l’état solide sous forme d’agrégats H ou J dotés de propriétés de transport de charges et de conversion photovoltaïque fortement améliorées et de propriétés d’absorption, d’émission et de génération de second harmonique mécaniquement modulables. / This work deals with the design, synthesis and evaluation of molecular pi-conjugated systems as active materials for (opto)electronics devices. A short first chapter describes three X-shaped oligothiophenes, thecharacterization of their structure and properties and a first evaluation of their performances in electrochromic devices. The second chapter describes the synthesis of molecular acceptors based on a benzodithiophene and the analysis of their potentialities when combined with molecular donors in organic solar cells.The major part of the work is focused on the analysis of structure-properties relationships of a series of smallpush-pull molecules involving di- or tri-arylamine donorblocks linked to an acceptor group by a thienyl bridge. In a first step, a phenyl ring of triphenylamine (TPA) is replaced by p-fluorophenyl, anthryl and naphtyl groups.Optical and electrochemical results show that substitution has little effect at the molecular level but can markedly affect solid-state properties with in particular an improvement of charge-transport and short-circuit current density of solar cells based on these donor materials.In a second step, a phenyl ring of TPA is replaced by alkyl, perfluoroalkyl and oligo(oxyethylene) chains. Results of X-ray diffraction, absorption and photoluminescence spectroscopies, second harmonic generation, and electrochemistry demonstrate that some of these molecules under go aggregation controlled photoluminescence emission wave length while the corresponding materials spontaneous lyre organize in the solid-state to form either H or Jaggregates with enhanced charge mobility, photovoltaic conversion efficiency and mechanically-induced chromism, fluorochromism and NLO-chromism.

Chemical biology encompasses the study and manipulation of biological system using chemistry, often by virtue of small molecules or unnatural amino acids. Much insight has been gained into the mechanisms of biological processes with regards to protein structure and function, metabolic processes and changes between healthy and diseased states. As an ever expanding field, developing new tools to interact with and impact biological systems is an extremely valuable goal. Herein, work is described towards the synthesis of a small library of heterocyclic-containing small molecules and the mechanistic details regarding the interesting and unexpected chemical compounds that arose; an alternative set of non-toxic copper catalyzed azide-alkyne click conditions for in vivo metabolic labelling; and the synthesis of an unnatural amino acid for further chemical modification via [3+2] cycloadditions with nitrones upon incorporation into a peptide of interest. Altogether, these projects strive to supplement pre-existing methodology for the synthesis of small molecule libraries and tools for metabolic labelling, and thus provide further small molecules for understanding biological systems.

Chemical biology encompasses the study and manipulation of biological system using chemistry, often by virtue of small molecules or unnatural amino acids. Much insight has been gained into the mechanisms of biological processes with regards to protein structure and function, metabolic processes and changes between healthy and diseased states. As an ever expanding field, developing new tools to interact with and impact biological systems is an extremely valuable goal. Herein, work is described towards the synthesis of a small library of heterocyclic-containing small molecules and the mechanistic details regarding the interesting and unexpected chemical compounds that arose; an alternative set of non-toxic copper catalyzed azide-alkyne click conditions for in vivo metabolic labelling; and the synthesis of an unnatural amino acid for further chemical modification via [3+2] cycloadditions with nitrones upon incorporation into a peptide of interest. Altogether, these projects strive to supplement pre-existing methodology for the synthesis of small molecule libraries and tools for metabolic labelling, and thus provide further small molecules for understanding biological systems.