Thesis advisor: Eranthie Weerapana / Functional amino acids that play critical roles in catalysis and regulation are known to display elevated nucleophilicity and can be selectively targeted for covalent modification by reactive electrophiles. Chemical-proteomic platforms, such as activity-based protein profiling (ABPP), exploit this reactivity by utilizing chemical probes to covalently modify active-site residues to inform on the functional state of enzymes within complex proteomes. These and other applications rely on the availability of a diverse array of electrophiles and detailed knowledge of the reactivity and amino-acid specificity of these groups. The sulfonyl fluoride activity-based probe (ABP) DAS1 was discovered to label and inhibit both serine proteases and glutathione S-transferases (GSTs). In the case of GSTs, DAS1 covalently bound to a tyrosine residue, despite predicted serine reactivity. Investigation of potential aryl halide electrophiles for ABPP found that chloronitrobenzene RB2 and dichlorotriazine RB7 covalently modify cysteine and lysine residues in target proteins. Applying an existing ABP, iodoacetamide alkyne (IA-alkyne), demonstrated the ability of ABPP to discover novel reactive residues in short open reading frame (sORF)-encoded peptides, as well as previously unannotated cysteine residues on glycolysis enzymes. These studies illustrate the development and characterization of novel electrophiles and demonstrate the application of ABPs to interrogate biological systems. Looking further ahead, the novel electrophiles also provide new tools for the development of covalent inhibitors for treatment of disease. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Les polyphénols sont des métabolites secondaires d’origine végétale. Ces substances naturelles connues pour leurs pouvoirs antioxydants et anti-radicalaires, contribuent à la protection de la santé humaine notamment contre les maladies cardiovasculaires et neurodégénératives, mais également contre certains cancers et diabètes. Dans certains cas, ces effets biologiques bénéfiques pour la santé pourraient également être liés à une interaction spécifique polyphénol-protéine peu étudiée à ce jour par manque d’outils moléculaires adaptés. Les travaux effectués au cours de cette thèse ont consisté à concevoir, à synthétiser et à évaluer des sondes moléculaires polyvalentes porteuses de polyphénols comme substrats d’affinité pour l’analyse des interactions polyphénol-protéine. Dans ce contexte, de nombreuses sondes arborant différents types de polyphénols ont été synthétisées. Ces différentes sondes pourront être utilisées en protéomique chimique du type "Affinity-Based Protein Profiling" (ABPP) pour identifier au sein d’un mélange complexe de protéines, une protéine ayant une affinité spécifique pour un polyphénol donné. Ces mêmes sondes permettront également d’étudier de manière qualitative les interactions d’un polyphénol avec une protéine donnée en temps réel par la technique de résonance plasmonique de surface (SPR). / Polyphenols are plant secondary metabolites. These natural substances, known for their antioxidant and anti-free radical properties, generally contribute to the protection of human health not only against cardiovascular and neurodegenerative diseases, but also against certain cancers and diabetes. In some cases, these beneficial biological effects could also be related to specific polyphenol-protein interactions. However, studying this type of interactions has suffered from the lack of adequate molecular tools. The work carried out during this thesis has included designing, synthesizing and evaluating modulable polyfunctional molecular probes carrying polyphenols as affinity substrates to analyze polyphenol-protein interactions. In this context, various probes harboring different kinds of polyphenols were synthesized. These probes could be used in chemical proteomics following an “Affinity-Based Protein Profiling” approach (ABPP) to identify a protein within complex protein mixtures, which has a specific affinity with a given polyphenol. These probes will also allow studying the interactions of a polyphenol with a given protein in real time in a qualitative way by surface plasmon resonance (SPR).

Les méthyltransférases de l'ADN (DNMT) catalysent la méthylation de l'ADN, l'une des marques épigénétiques les plus étudiées. Dans les cancers, on observe une hyperméthylation spécifique de promoteurs de gènes suppresseurs de tumeurs (GST) conduisant à leur extinction génique, ce qui participe au maintien et à la progression de tumeurs. A ce jour, les mécanismes responsables de cette hyperméthylation spécifique des promoteurs de GST dans les cancers sont indéterminés. Ces travaux de thèse sont consacrés à l'inhibition des DNMT dans les cancers afin de restaurer l'expression des GST mais également à l'utilisation d'une approche innovante de chemobiologie pour l'identification de partenaires des DNMT potentiellement responsables de leur adressage vers les régions promotrices des GST. Les partenaires ainsi identifiés peuvent constituer de nouvelles cibles épigénétiques pour le ciblage indirect de la méthylation de l'ADN dans les cancers. Deux séries d'inhibiteurs de DNMT ont été étudiées. La première est la famille des chloronitro-flavanones, précédemment identifiée par criblage, pour laquelle de nouveaux dérivés de type bromonitro-flavanones ont été synthétisés afin d'améliorer la stabilité en conditions physiologiques. J'ai réalisé l'étude des effets pharmacologiques de cette famille de molécules. J'ai également entrepris la synthèse et la caractérisation pharmacologique de nouveaux inhibiteurs de type bi-substrats, analogues de l'adénosine et de la désoxycytidine, conçus par une approche rationnelle. Ces deux études ont permis respectivement d'identifier un dérivé flavanone plus stable et plus actif que le composé de référence et deux dérivés quinazoline-quinoléine très prometteurs, actifs sur les DNMT et dans les lignées cellulaires, à la fois pour la réexpression d'un gène rapporteur mais surtout dans l'induction de la déméthylation du GST CDKN2A et de sa réexpression. Pour identifier les partenaires de DNMT, nous avons employé une approche de chemobiologie (" Activity-Based Protein Profiling - ABPP ") basée sur la conception de sondes chimiques comportant un inhibiteur de DNMT. Ces sondes, utilisées sur des cellules vivantes, permettent, grâce à une étape de fonctionnalisation par chimie bioorthogonale, de purifier les protéines partenaires des DNMT. Vingt sondes ont été synthétisées et leurs activités ont été évaluées sur des modèles enzymatiques et cellulaires. Les sondes sélectionnées ont été utilisées dans des lignées cellulaires cancéreuses pour purifier les protéines partenaires qui ont ensuite été identifiées par analyse protéomique. Suite à leur validation, ces protéines pourront constituer de nouvelles cibles de la méthylation aberrante de l'ADN dans les cancers. / DNA methyltransferases (DNMTs) catalyse DNA methylation, one of the most studied epigenetic marks. In cancers, a specific hypermethylation of the promoters of the tumour suppressor genes (TSGs) is observed, which leads to their silencing. This abnormal DNA methylation pattern participates to the maintenance and the progression of the tumour. Today, the mechanisms that direct this specific hypermethylation of TSG promoters and their transcriptional repression in cancers are still unknown. The aim of my PhD is to identify DNMT inhibitors that are able to reactivated TSGs in cancer cells but also to identify the DNMT partners that address specifically these enzymes to TSG promoter regions. Such partners can constitute new anticancer "epitargets" to indirectly target DNA methylation specifically in cancer cells. Two families of DNMT inhibitors were studied. The first one starts from the chloronitro-flavanones previously identified by screening. New derivatives including bromonitro-flavanones were synthesised aiming at improving compound stability. I pharmacologically characterised these compounds and show for one of them an increased stability and activities compared to reference compound. In parallel, I synthesised and pharmacologically characterised new bi-substrate analogue inhibitors, mimicking the adenosine and the deoxycytidine. Two very promising quinazoline-quinoline derivatives were identified. They are active against DNMT and in cell lines, both for reexpression of a reporter gene but mostly in CDKN2A TSG demethylation inducing its reexpression. To identify DNMT partners we adopted a chemical biology approach (Activity-Based Protein Profiling (ABPP)) based on the use of chemical probes including in-house non- nucleoside DNMT inhibitors as bait to trap the DNMT partners. We designed and synthesised twenty chemical probes and evaluate them using enzymatic and cellular-based assays. Selected probes were used to carry out ABPP directly in living cells. After functionalization by bioorthogonal chemistry, DNMT protein partners were purified and identified by proteomic analysis. Target validation would enable to determine new targets for the aberran

Identification of differential expressions of proteins in proteomic profiles of biological samples shows great potential as a valuable technique for the early diagnosis of various diseases. An important challenge in modern protein profiling approaches is to reduce the complexity of the samples by limiting the number of proteins that need to be evaluated for distinction in the expression between normal and deceased cells. In this research, an affinity based approach for the enrichment of nucleotide and nucleoside binding proteins from a complex cell proteome has been developed. To achieve this goal, new N6-biotinylated-8-azido-adenosine probes (AdoRs) have been designed and synthesized to photolabel the nucleotide and nucleoside binding proteins. These probes contain a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Further, a mass spectrometric protein profiling approach is employed to quantitatively identify small variations in expression of nucleoside and nucleotide binding proteins in samples of interest. Mouse neuroblastoma N18TG2 cell proteome has been used as a model system for the development of the LC-MS/MS based proteomic analysis of these affinity enriched protein fractions. Upon enrichment, the photolabeled proteome exhibited an approximately four-fold abundance of nucleoside and nucleotide binding proteins over nonlabeled proteome. The approach was extended to compare the proteomic profiles of nucleotide and nucleoside binding proteins in cancerous (Hey) and non-cancerous (T-80) human ovarian cell proteome. Certain proteins that were not detected in cell lysate were also identified in labeled proteome, thereby demonstrating the strength of our approach in enriching low abundant proteins. To substantiate the qualitative analysis, we have employed the Stable Isotope Labeling in Amino Acid Cell Culture (SILAC) for the quantitative study of the protein expression in cancerous and non-cancerous human ovarian cells. A modest panel of proteins with differential expressions in these cell lines was identified, a few of which have been correlated to various forms of cancer. Vimentin, stress induced phosphoprotein-1, and heat shock protein 90 that were identified to have altered expressions in these cell lines are among some of the proteins associated with ovarian cancer.

Hepatitis C virus (HCV) is a hepatotropic positive-sense RNA virus of the Flaviviridae virus family and is a major cause of chronic liver disease worldwide. Like all obligate parasites, HCV relies on host pathways to enable its pathogenesis. HCV, in particular, has a clear link with hepatic lipid metabolism, promoting a lipid-rich environment for its proliferation. This manifests as liver steatosis in many patients harboring chronic HCV infection. Based on our recent findings regarding an immunometabolic and HCV antiviral microRNA (miRNA), miRNA-185 targeting and down regulating serine hydrolases (SH) involved in lipid and endocannabinoid metabolism, here we investigate HCV and its dependency on certain metabolic serine hydrolases involved in lipid and endocannabinoid metabolism. Serine hydrolases are one of the largest and most diverse enzyme families. This enzyme family has emerged as a center of therapeutic potential due to its implications in many metabolic roles. Here, we demonstrate that pharmacological inhibition of metabolic serine hydrolases alpha-beta hydrolyzing domain 6 (ABHD6), carboxylesterase 1 (CES1), and monoacylglycerol lipase (MGLL), enzymes involved in the hydrolysis of the endogenous cannabinoid receptor 1 (CB1) agonist 2-arachidonoyl glycerol (2-AG) are potently antiviral against HCV. Serine hydrolase inhibition with the MGLL inhibitor MJN110 paired with endocannabinoid signaling antagonization led to additive antiviral effects against HCV and has revealed modulation of the viral pathogenic phenotype to be its key course of action. MGLL inhibitor MJN110 transcriptomic characterization revealed modulations in humoral immunity and phagocytosis and acts antiviraly against HCV independent of CB1 antagonization. This provides an avenue for future investigation, assessing the viability of CB1 antagonization, and MGLL as a key host targeted antiviral factor in affecting HCV viral life cycle.

Herkömmliche Zytostatika greifen vornehmlich in den Zellzyklus ein und somit werden Zellen mit hoher Proliferationsrate geschädigt. Allerdings fallen hierunter nicht ausschließlich Krebszellen, sondern auch gesunde, schnell proliferierende Gewebearten. Auf Grund dessen verursacht eine klassische Chemotherapie schwerwiegende Nebenwirkungen. Neuere Therapieansätze greifen daher geno- sowie phänotypischer Unterschiede zwischen malignen und gesunden Zellpopulationen auf und können selektiv den zytotoxischen Wirkstoff in die Tumorpopulation einbringen. Dazu werden sogenannte Prodrug-Konzepte verfolgt, bei denen ein möglichst „untoxisches” Prodrug gezielt im entarteten Gewebe enzymatisch zum zytotoxischen Wirkstoff (Drug) aktiviert wird. In diesem Rahmen werden Substrate für die Antibody-Directed Enzyme Prodrug Therapy (ADEPT) hergestellt. Bei diesem Konzept wird eine hohe Tumorspezifität durch Konjugate aus Enzymen und Antikörpern erlangt, indem das Immunglobulin selektiv an tumorassoziierte Antigene bindet und durch das konjugierte Enzym die Drugfreisetzung ermöglicht wird. Die natürlichen zytotoxischen Antibiotika (+)-CC 1065 und (+)-Duocarmycin SA dienen hierbei als Leitstrukturen für die Synthese entsprechender Prodrugs. Im Rahmen der vorliegenden Arbeit wurden insgesamt zwei neue Duocarmycin-analoge Prodrugs sowie neun neue seco-Drugs synthetisiert, wobei vier Vertreter eine terminale Alkinfunktion aufweisen. Für die Darstellung der Prodrugs wurden auf die Galaktose als Glykosideinheit zurückgegriffen. Zudem wurde ein neuartiges dimeres seco Drug hergestellt, das aus zwei pharmakophoren Einheiten sowie einem verbrückenden Linker mit Alkineinheit besteht. Die jeweiligen Substanzen wurden auf ihre In-vitro-Zytotoxizitäten sowie die Eignung für eine Anwendung im ADEPT-Ansatz evaluiert. Neun der neuen Duocarmycin-Analoga wurden in Form von seco- und Prodrugs wurden im Rahmen des aktivitätsbasierten Protein-Profilings untersucht. Hierbei konnte die Aldehyddehydrogenase 1 als wichtiges Angriffsziel der Duocarmycin-Familie verifiziert werden.