The current thesis addresses the various facets of the chemistry of photocytotoxic iron complexes including their syntheses, characterization, evaluation of the anti-proliferative activities in various cancer cell lines upon photo-exposure, mechanism of cell death, the cellular uptake, localization inside cells, the interaction with double stranded DNA and their ability to induce DNA photocleavage. Chapter I presents a general introduction to cancer and the anticancer agents. It covers various procedures available for cancer treatment and different aspects of chemotherapy are discussed in details. The mechanism of action of several chemotherapeutic agents, the DNA cleavage pathways and the anticancer activity of bleomycins are delineated. Photo-chemotherapy or photodynamic therapy which has emerged as an alternative treatment modality is described. It also contains a brief description of ideal photosensitizers and the ones that are currently approved. The potential of transition metal complexes as photo-chemotherapeutic agents is discussed based on the recent literature reports on the prospective photocytotoxic metal complexes, the photo-release of cytotoxic molecules from metal complexes, the DNA cleavage activities and their cytotoxicities. The biochemistry of iron and its medical utility which prompted the development of iron based cytotoxins has been presented. The objective of the present investigation is also defined in this chapter. Chapter II describes the syntheses, characterization, evaluation of visible light induced cytotoxicity and interaction with DNA of a series of iron(II) bis-terpyridine complexes. Some interesting redox behaviour observed for two of the complexes has been described in details and rationalized from theoretical calculations. The DNA binding affinities of the complexes and their ability to induce DNA photocleavage in green light are discussed. The importance of this work lies in the remarkable photocytotoxic behaviour of the iron(II) complexes with visible light which was not reported earlier. Chapter III addresses the syntheses of a series of iron(III) catecholate complexes which upon irradiation with red light can initiate photoreactions to generate cytotoxic species and induce death in HeLa, HaCaT, MCF-7 and A549 cells. The mechanisms of cell death, effect of the complexes on the cell cycle under various conditions, the uptake inside cells and the cellular localization of the complexes are studied. The DNA binding affinities of the five complexes and their ability to induce DNA photocleavage in red light are also presented here. These are the first iron based complexes to show red light induced photocytotoxicity. Chapter IV addresses the drawbacks associated with the aforementioned iron(III) catecholates and their modification with a mitochondria targeting triphenylphosphonium unit. The synthesis, characterization, photocytotoxicities in HeLa, HaCaT, MCF-7 and A549, cell death mechanisms and cellular uptake and localization of four iron(III) complexes are discussed. Chapter V describes the syntheses, characterization and the biological activities of carbohydrate appended iron(III) complexes and their non-glucose analogues. The selective and faster internalization of the glyco-conjugated complexes in HeLa cells has been studied using various spectroscopic and microscopic techniques. The red light induced cytotoxicities of the complexes, their effect on the progression of the cell cycle with and without irradiation and the mechanisms of cell death are explored. DNA binding abilities and photocleavage of DNA are also discussed. Chapter VI presents the syntheses, characterization of a series of iron(III) complexes of a pyridoxal derivative and their salicyldehyde analogues for exploring their differential photocytotoxicity and cellular uptake in cancer cells compared to normal cells. The visible light induced cytotoxicities of the complexes in HeLa, HaCaT, MCF-7 A549 cells and HPL1D cells, their effect on the progression of the cell cycle in dark and light, the mechanisms of cell death and the localization of the complexes inside the cells are explored. The references have been compiled at the end of each chapter and given as superscripts in the text. The complexes presented in this thesis are indicated by bold-faced numbers. Crystallography data of the complexes that are structurally characterized by single crystal X-ray crystallography are given in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any unintentional omission that might have happened due to oversight is regretted. INDEX WORDS: Iron complexes • Crystal structure • Red light induced cytotoxicity • Cellular imaging • DNA binding • DNA photocleavage.

The past few years have witnessed significant work being directed towards drug delivery systems with layer-by layer (LbL) technique prominently featured as one of the most sought after approach. However, majority of the studies were focused on the fabrication of microcapsules which produced numerous drawbacks resulting in reduced applicability. This has spurred research into nanocapsules which has proved to overcome most of the drawbacks that plagued microcapsules by being able to evade the reticulo-endothelial system, exhibit enhanced permeability and retention in tumours etc. The capsules fabricated by the LbL technique requires a suitable combination of cationic and anionic polyelectrolytes which ensures that it is able to effectively protect the cargo it encapsulates as well as enhance its bio-applications. With numerous advantages such as biocompatibility and biodegradability to name a few, chitosan has proved to be an ideal cationic polyelectrolyte. Thus, this thesis focuses on the various therapeutic applications of LbL fabricated chitosan based nanocapsules. The first work focuses on the targeted delivery of the somatostatin analogue, Octreotide conjugated nanocapsules to over expressed somatostatin receptors. These LbL fabricated nanocapsules composed of chitosan and dextran sulfate (CD) encapsulate the anti cancer drug, doxorubicin and are found to attain site specificity as well as enhanced anti-proliferative activity. The results indicated that the nanocapsules were biocompatible and when conjugated with octreotide was found to have an enhanced internalization into SSTR expressing cells, thereby making it a viable strategy for the treatment of tumors that has an over expression of somatostatin receptors such as pancreatic carcinoma, breast carcinoma etc. The objective of the second work was to develop an efficient drug delivery system such as CD nanocapsules for encapsulation of Ciprofloxacin in order to combat infection by Salmonella, an intracellular and intra-phagosomal pathogen. In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection. The increased retention of ciprofloxacin in tissues delivered by CD nanocapsules as compared to the conventional delivery proved that the same therapeutic effect was obtained with reduced dosage and frequency of Ciprofloxacin administration. The third work deals with the probiotic, Saccharomyces boulardii which is found to be effective against several gastrointestinal diseases but had limited clinical application due to its sensitivity to acidic environment. However, encapsulation of S. boulardii with chitosan and dextran sulfate ensured enhanced viability and selective permeability on exposure to acidic and alkaline conditions experienced during gastro intestinal transit. The final work involves the fabrication of novel pH responsive nanocapsules composed of chitosan-heparin which facilitate the intracellular delivery of a model anti-cancer drug, doxorubicin.

La thèse est divisée en trois chapitres indépendants. Chimie du β-lactames : Synthèse d'α-éthylène et d'α-alkylidène-β-lactames en utilisant la réaction de Kinugasa. Chimie de l'acylsilane : application d'une réaction aldolique intramoléculaire asymétrique sur un dérivé d'acylsilane nouvellement synthétisé. - Chimie médicinale: synthèse de nouvelles molécules à but anticancéreux.Dans le premier chapitre la réaction de Kinugasa a été appliquée pour la première fois à des alcynes vrais, portant en position propargylique un groupe partant permettait d'accéder directement et en une étape aux méthylène- et alkylidene β-lactames recherchés. Dans le second chapitre, la synthèse de molécules originales possédant à la fois une fonction acylsilane et un aldéhyde en position éloignée, et l'aldolisation intramoléculaire asymétrique a été explorée. Dans le dernier chapitre, notre objectif était de restaurer les propriétés apoptotiques au sein des cellules cancéreuses afin d'obtenir de nouveaux composés à activité antitumorale. A partir de données obtenues par modélisation moléculaire, nous avons fait le design de plusieurs séries d'analogues d'un inhibiteur connu(MIM-1) de la protéine anti-apoptotique Mcl-1. Huit composés ont été synthétisés et testés pour trois types de cellules cancéreuses (sein, ovaire et le mélanome). / The thesis is divided into three chapters:- β-lactams chemistry: synthesis of α-methylene and α-alkylidene-β-lactams using the Kinugasa reaction.- Acylsilane chemistry : applying asymmetric intramolecular aldol reaction on a newly synthesized acylsilane derivatives. - Medicinal chemistry: synthesis of new molecules with anticancer aimes. In the first chapter, Kinugasa reaction was applied for the first time with an alkyne bearing a nucleofuge in propargylic position that allowed us to discover a way of synthesis of exoalkylidene β-lactams. In the second chapter, a new acylislane derivatives bearing an aldehyde functional group in a remote position of the molecule were prepared, and asymmetric intramolecular aldolization reaction was performed. In the last chapter, our goal was to reinduce the pro-apoptotic properties in cancer cells in order to obtain new antitumor compounds. Starting from data obtained through molecular modeling studies, we designed and prepared several series of analogs for a known inhibitor (MIM-1) of the anti-apoptotic protein Mcl-1. Eight compounds have been synthetized and screened towards three types of cancer cells (breast, ovarian and melanoma).

A biodiversidade de plantas brasileiras é uma fonte muito rica de moléculas bioativas, dentro da proposta da busca por novas drogas antitumorais, avaliamos neste estudo o potencial antiproliferativo do composto 7-epi-clusianona. Foram utilizadas duas linhagens celulares derivadas de tumor de mama humana, Hs 578T e MCF-7, cultivadas em monocamada e como esferoides. O IC50 após 48 horas de tratamento das células é de 20 μM para Hs 578T e 6 μM para MCF-7. A análise do ciclo celular mostrou que o composto é capaz de reter as células em fase G1/G0 em ambas as linhagens em 2D, mas não em 3D. O composto é capaz de induzir as células a senescência celular, como mostrado pelo ensaio de detecção de β-galactosidase. Esses dados indicam que o composto 7-epi-clusianona é uma molécula promissora, que demonstrou potencial antitumoral em células de tumor de mama. A cultura tridimensional se mostrou mais resistente ao tratamento com 7-epi-clusianona, portanto estudos mais abrangentes são necessários para melhor entendimento dos efeitos do composto sobre esse tipo de cultura. / Brazilian flora is considered one of the most diverse in the world and natural products are some of the important sources of new antitumoral compounds. The aim of this study was to evaluate the antiproliferative potential of 7-epi-clusianone. Two cell lines derived from human breast tumor were used, Hs 578T and MCF-7, cultured in monolayer and as spheroids. The IC50 after 48 hours of treatment is 20 μM to Hs 578T cells and 6 μM to MCF-7 cells. Cell cycle analysis showed induction of cell cycle arrest in G1/S phase in cells cultured in monolayers, but not in spheroids. The amount of cells in senescence after the treatment with 7-epi-clusianone is higher than the control group, as seen by the senescence β-galactosidase staining assay. These data suggest that 7-epi-clusianone is a promising molecule against breast cancer cells. We show that 3D culture was more resistant to treatment than 2D culture, therefore more comprehensive studies are needed to better understand the effects of 7-epi-clusianone on this kind of culture.

Les glycosides cardiotoniques sont des inhibiteurs des pompes sodium / potassium utilisés pour le traitements des insuffisances cardiaques, qui détiennent également des activités anticancéreuses et épigénétiques récemment caractérisées. Toutefois, dans l’objectif de repositionner ces médicaments comme traitement anticancéreux, les mécanismes sousjacents aux activités anticancéreuses et épigénétiques des glycosides cardiotoniques restent à être déterminés. Dans nos travaux, nous révélons que la proscillaridine A est le glycoside cardiotonique qui détient des activités anticancéreuses et épigénétiques les plus puissantes dans des lignées de cancer du côlon, de leucémies et de sarcomes pédiatrique. De plus, nous avons identifié que l’activité anticancéreuse de la proscillaridine A corrèle positivement avec le niveau d’expression protéique du proto-oncogène MYC dans un panel de 14 lignées cellulaires cancéreuses. Dans les lignées cellulaires exprimants un haut niveau de MYC telles que les lignées leucémiques, la proscillaridine A agit comme un inhibiteur de MYC et module sa stabilité protéique ainsi que la régulation transcriptionnelle et translationnelle de ces cibles. Cette inhibition est induite par la baisse significative de l’expression des enzymes épigénétiques les lysines acétyltransférases (KATs), qui contrôlent l’ajout des résidus d’acétylcoenzyme A sur les histones et sur d'autres protéines dont MYC. La baisse d’expression des KATs résultent à une baisse de l’acétylation des résidus de l’histone 3 et à une reprogrammation de l’acétylome des cellules cancéreuses surexprimant MYC. Ces changements au niveau de la chromatine induisent une reprogrammation transcriptionnelle et phénotypique des cellules surexprimant MYC, qui se traduit par une perte de la transcription des programmes oncogéniques et l’induction des programmes associés à la différenciation cellulaire. Pour finir, nous avons évalué le potentiel synergique anticancéreux et épigénétique de la proscillaridine A avec le médicament épigénétique la décitabine dans des lignées cancéreuses exprimants des niveaux différentiels de MYC. Dans une lignée résistante à la proscillaridine A et exprimant de faible niveau de MYC (lignée de cancer de côlon), la décitabine et la proscillaridine A démontrent des activités épigénétiques synergiques tandis que dans une lignée sensible à la proscillaridine A et surexprimant MYC (lignée de sarcome pédiatrique), la décitabine et la proscillaridine A démontrent des activités antiprolifératives synergiques. Dans ces travaux, nous avons donc démontré le potentiel de repositionner la proscillaridine A dans les cancers surexprimant MYC. Également, nous démontrons le potentiel synergique anticancéreux et épigénétique de la proscillaridine A avec la décitabine et nous suggérons d’étudier cette combinaison de médicaments dans les cancers plus résistants à la proscillaridine A. / Cardiac glycosides are sodium/potassium pomps’ inhibitors used for the treatment of heart failure, and whose anticancer and epigenetic activities have been recently characterized. However, in order to repurpose cardiac glycosides as anticancer drugs, mechanistic studies are required to identify the anticancer and epigenetic mechanism of actions. In our experiments, proscillaridin A exhibited the most powerful anticancer and epigenetic activities in colon cancer, leukemia, and sarcoma cell lines. Moreover, we demonstrated that in a panel of 14 cancer cell lines, proscillaridin A anticancer activities positively correlated with MYC protooncogene expression level. In high MYC expressing cell lines such as leukemia, proscillaridin A inhibited MYC expression through protein destabilization and through transcriptomic and translational regulation of MYC targets. Theses inhibitions are induced by the loss of lysine acetylatransferase (KAT) expressions, which are epigenetic enzymes controlling the addition of acetyl-coenzyme A on histones and other proteins such as MYC. KAT inhibitions are responsible for the global loss of histone 3 acetylation and acetylome reprogrammation in high MYC expressing cancer cells. These chromatin changes induced transcriptomic and phenotypic reprogrammation, defined by a loss of the transcription of oncogenic programs and the induction of cell differentiation. To finish, we evaluated the anticancer and epigenetic synergic potential of proscillaridin A in combination with the epigenetic drug the decitabine in cancer cell lines expressing different MYC levels. In a cancer cell line resistant to proscillaridin A treatments and expressing low MYC level (colon cancer cell line), the combination of decitabine and proscillaridin A demonstrated synergistic epigenetic activity although, in a cell line sensitive to proscillaridin A treatments and expressing high MYC level (sarcoma cell line), the combination of decitabine and proscillaridin A exhibited synergistic anti-proliferative activity. To conclude, we highlighted the potential of repurposing proscillaridin A as an anticancer treatment in high MYC expressing cells. Furthermore, we demonstrated the anticancer and epigenetic synergistic potential of proscillaridin A in combination with decitabine and we propose to study the drug combination in cancers that are resistant to proscillaridin A treatment.

Les produits naturels (PNs) englobent une énorme diversité chimique qui a conduit à la découverte de médicaments révolutionnaires contre le cancer, contre les maladies infectieuses et contre d'autres maladies. La majorité des médicaments actuellement approuvés sont des dérivés de PNs, où nombre d’entre eux engagent des cibles considérées comme non thérapeutiques. Malgré ces avantages, les PNs posent des problèmes au niveau de l’isolement, de la déréplication, du réapprovisionnement et de la traçabilité chimique. Compte tenu du besoin urgent de découvrir de nouvelles molécules bioactives contre de nouvelles cibles pour tous les types de maladies, des stratégies innovantes sont nécessaires pour revigorer la découverte de médicaments à partir des PNs. Nous avons développé une plateforme utilisant Saccharomyces cerevisiae pour la production hétérologue de molécules similaire aux PNs, appelée « produits naturels synthétiques » (PNSs). Nous avons synthétisé une vaste bibliothèque de gènes impliqués dans la biosynthèse de PNs (GBSs) provenant de plantes, de champignons et de bactéries, pour lesquels leur contenu en GC et leurs codons ont été optimisés pour l’expression dans S. cerevisiae. Ces gènes sont assemblés en chromosomes artificiels de levure pour générer de vastes bibliothèques combinatoires de BSG pour la production de molécules similaires aux PNs. Les bibliothèques de PNSs peuvent être directement criblées contre des microorganismes ou des cibles spécifiques dans des essais à haut débit. J'ai effectué le criblage de bibliothèques de PNSs contre une variété de cibles bactériennes et humaines. L'un de ces criblages a conduit à la découverte de PNSs ayant une activité antimicrobienne contre un groupe de pathogènes cliniquement pertinents. Récemment, certaines équipes scientifiques, dont la nôtre, ont découvert que l'hyperactivation de la protéase mitochondriale humaine CLPP par les composés anticancéreux ONC201 et ONC212, qui sont présentement en phase préclinique, provoque la mort cellulaire par protéolyse mitochondriale incontrôlée. Cependant, j'ai trouvé que ONC201/212 activent également la version bactérienne de ClpP et ils pourraient donc perturber le microbiome. J'ai donc développé des essais génétiques de substitution dans la levure pour les protéases ClpP afin de cribler pour des activateurs plus spécifiques. Ensuite, j'ai adapté mon approche dans la levure pour le criblage d’inhibiteurs de la protéase principale (Mpro) et de l'endoribonucléase (NendoU) de SRAS-CoV-2, afin de répondre au besoin pour des thérapies antivirales efficaces afin de traiter les personnes atteintes de la forme grave de la COVID-19. Enfin, une autre variante de mon approche dans la levure a également été développée pour le criblage de stabilisateurs de l'interaction entre FKBP12 et calcineurine dans le but d'identifier de nouveaux immunosuppresseurs qui présentent moins d'effets secondaires. Le criblage de ces différents essais m’a permis d’identifier des candidats potentiels pour chaque cible. Bien que les tests faits dans la levure soient utilisés dans le contexte de criblages traditionnels, l’utilisation de la plateforme PNS permet d’explorer un espace chimique inaccessible auparavant afin de favoriser la découverte de médicaments, le tout de manières économique, modulable et durable. / Natural products (NPs) encompass enormous chemical diversity, leading to revolutionary medicines in cancer, infectious disease, and other indications. The majority of currently approved drugs are derived from NPs, with many of them engage targets otherwise viewed as undruggable. Despite these advantages, NPs pose problems in isolation, dereplication, resupply and chemical tractability. Given the pressing need to discover bioactive chemical matter against new targets in all disease areas, innovative strategies are required to reinvigorate NP-based drug discovery. We have developed a Saccharomyces cerevisiae platform for heterologous production of NP-like chemical matter, termed Synthetic Natural Products (SynNPs). We synthesized an extensive library of codon- and GC-content optimized NP biosynthetic genes (BSGs) from plants, fungi and bacteria. These genes are then assembled into programmable yeast artificial chromosomes (YAC) to generate vast combinatorial BSG libraries that produce NP-like molecules. SynNP libraries can be directly screened in high-throughput in either cell- or target-based assays. I constructed and screened SynNP libraries in yeast-based surrogate genetic assays against a variety of bacterial and human targets. One of these screens led to the discovery of SynNPs with antimicrobial activity against a panel of clinically relevant pathogens. Recently, we and others discovered that hyperactivation of the human mitochondrial caseinolytic protease proteolytic subunit (CLPP) by the preclinical anti-cancer compounds ONC201 and ONC212 causes cell death by rampant mitochondrial proteolysis. However, I found that ONC201/212 also activates bacterial ClpP and could therefore disrupt the microbiome. I thus developed yeast-based surrogate genetic assays for ClpP proteases to screen for more specific activators. Then, I adapted my yeast-based approach to screen for inhibitors of SARS-CoV-2 main protease (Mpro) and endoribonuclease (NendoU) to address the need for efficacious antiviral therapies to mitigate the COVID-19 pandemic. Finally, I developed another variant of my yeast-based approach to screen for stabilizers of the interaction between FKBP12 and calcineurin to identify novel candidate immunosuppressants. Screens with these various assay formats allowed me to identify candidate hits for each target. In summary, the SynNP platform allows the exploration of new-to-nature NP-like chemical space for drug discovery in a cost-effective, scalable and sustainable manner, and yeast-based surrogate genetic assays can be used to screen both existing chemical libraries and SynNP libraries.

No description available.

La química bioinorgánica se ocupa de estudiar la función de los metales en los sistemas biológicos y de la síntesis de modelos para estudiar sistemas más complejos. Enmarcados en esta, se han estudiado los modos de interacción y los patrones de reconocimiento presentes en los complejos ternarios metal - base nitrogenada - derivado de aminoácido. En primer lugar, se han obtenido un serie de complejos binarios de 5-halouracilatos con cobre(II), zinc(II) y níquel(II) que presentan un reconocimiento por tándem de enlaces de hidrógeno entre uracilos. Con ácido orto-iodohipúrico se han preparado una serie de complejos binarios con cobalto(II), níquel(II), cobre(II), zinc(II) y plata(I). Con estos y aciclovir se ha conseguido la formación de los complejos ternarios donde los de cobalto, níquel y zinc son isoestructurales y, debido a la presencia de un apilamiento entre el aciclovir y un anillo aromático de o-iodohipúrico, se generan en la estructura los dos enantiómeros δ (delta minúscula) y λ (lambda minúscula) si consideramos los dos ligandos como un quelato gracias a la interacción por apilamiento. Con cobre(II), ácidos hipúricos y 1,10-fenantrolina o 2,2'-bipiridilo se han preparado una serie de complejos ternarios capaces de generar la especie de cobre(I) [Cu(phen)2]+ y que presentan actividad anticancerosa frente a células A549. En todos los complejos con ácidos hipúricos se ha observado que la presencia del anillo aromático perpendicular al plano amídico genera estructuras bidimensionales, mientras que con ligandos planares del mismo tipo las estructuras son tridimensionales. Finalmente se han preparado análogos de nucleótidos tipo N1-alquiluracilos y de dinucleótidos tipo N1,N1'-polimetilen-bis-uracilos. / Bioinorganic chemistry deals with the study of the role of metal ions in biologic systems and the synthesis of model compounds to study more complex systems. In line with it, we have studied the interactions modes and recognition patterns that are present in ternary complexes of the type: metal ion - nucleobase - amino acid derivative. First of all, we have obtained a series of binary compounds with 5-halouracils and copper(II), zinc(II) and nickel(II) with a recognition pattern formed by a tandem of hydrogen bonds between uracils. With orto-iodohippuric acid, we have prepared some binary complexes with cobalt(II), nickel(II), copper(II), zinc(II) and silver(I). With these binary derivatives and acyclovir we have been capable to obtain ternary complexes were, the cobalt, nickel and zinc ones are isostructural and, due to the presence of a stacking between the acyclovir and one aromatic ring of o-iodohippuric acid, they generate in the crystal structure the two enantiomers δ (lower case delta) and λ (lower case lambda) if we consider the two ligands as a chelate by means of the stacking interaction. In the case of copper, we have prepared a series of ternary derivatives with different hippuric acids and 1,10-phenanthroline and 2,2'-bipyridyl. These complexes are capable to generate the copper(I) species [Cu(phen)2]+ and they present anticancer activity against A549 cell line. An important fact in all the coordination compounds with hippuric acids is that the presence of the aromatic ring normal to the amide bond promotes the formation of bi-dimensional structures, whereas with planar ligands of the same kind we obtain tri-dimensional structures. Last but not least, we have prepared nucleotide (N1-alkiluracils) and dinucleotide (N1,N1'-polymethilen-bis-uracils) type ligands.