Philosophiae Doctor - PhD / Glioblastoma multiforme (GBM) remains the most malignant of all primary adult brain tumours. It is a highly invasive and vascularized neoplasm with limited treatment options and very low survival rate. GBM tumours are heterogeneous in nature with cellular hierarchy and at the apex of this hierarchy are the glioblastoma stem cells, known to promote tumourigenesis and resistance to chemotherapeutic agents and tumour recurrence. Currently, the standard care for GBM involves surgical resection, radiation, and chemotherapy treatment with temozolomide. Unfortunately, median survival after treatment is still daunting and tumour relapse is very frequent. Indeed, patients with recurrent glioblastoma have less than a year survival. To address this, novel therapies need to be developed with the early introduction of promising agents into clinical trials and subsequent approval for use. Importantly, for these novel therapies to be approved for GBM, they need to be safe, effective as well as being able to penetrate the blood-brain barrier (BBB). Due to the high cost and process time for the development of new drugs, existing approved drugs are currently being repurposed for new indications and this is gaining significance in clinical pharmacology as it allows rapid delivery of useful drugs from bench to bedside. Drugs of the antipsychotic class are well known to cross the BBB due to their neuroleptic action. To this end, the aim of this study was to identify and characterize the anti-cancer activities of novel phenothiazine-derivatives belonging to the antipsychotic class of drugs in glioblastoma. To achieve this, several novel phenothiazine-derivatives were initially screened for possible anti-cancer activity in the U87 and U251 malignant GBM cells. Two lead compounds, DS00326 and DS00329, were identified and their anti-cancer activities were determined in U87 and U251 cells as well as in primary patient-derived xenograft (PDX) glioblastoma cultures. DS00326 and DS00329 significantly inhibited glioblastoma cell viability, with minimal effects observed in the non-cancerous FG0 fibroblasts. The IC50 values of DS00326 and DS00329 for U251, U87 and PDX cells ranged from 1.61 to 12.53μM. Flow cytometry analyses showed that DS00326 and DS00329 treatment led to an increase in the G1 population of cells. Additionally, DS00326 and DS00329 induced double-strand DNA breaks, which lead to activation of the canonical DNA damage response pathway. Furthermore, DS00326 and DS00329 induced apoptosis as shown by morphological markers, flow cytometry with annexin V-FITC/propidium iodide staining, as well as western blotting with an antibody to detect levels of cleaved PARP. Interestingly, treatment with DS00326 and DS00329 also induced autophagy as evident by the increase of acidic vesicular organelles in cells following staining with acridine orange as well as an increase in levels of the autophagy marker LC3-II. Autophagy was seen as a pro-death pathway in the U87 and U251 cells as inhibition of autophagy led to a reversal of cytotoxicity and consequently increased cell survival. Moreover, it was demonstrated that DS00326 and DS00329 inhibited the PI3/Akt pathway while modulating the mitogen-activated protein kinases p38, ERK1/2 and JNK signalling pathways. Importantly DS00326 and DS00329 displayed anti-cancer stem cell activities by the inhibition of neurosphere formation and regulation of stem cell markers SOX2 and GFAP in PDX cells. Together, the findings from this study suggest that DS00326 and DS00329 may be effective in the treatment of glioblastoma and provide a strong rationale for further clinical studies exploiting phenothiazines and their derivatives as treatments for glioblastoma. / 2021-09-01

No description available.

No description available.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide and is recognized as the second leading cause of cancer-related deaths in North America. CRC follows a hierarchal tumor organization, the root being a small population of self-renewing and highly tumorigenic colon cancer stem cells (CSC). There is an epigenetic signature that exists within these colon CSCs contributing to their maintenance and dynamic plasticity. A key hallmark of this colon CSC epigenetic signature is the Histone-3 Lysine-9 di-methylation (H3K9me2) histone mark which is overrepresented in many types of cancer. Pharmacological modulation of this epigenetic signature (i.e through H3K9me2 modulation) serves as a novel way to selectively target and eliminate human CSCs while sparing normal progenitor cells. Direct inhibitors of key methyltransferases such as G9a, have been identified to have high specificity, however none of these inhibitors have shown success during early stages of clinical trials, leaving us to question the clinical relevance. My research has shown that the overexpression of histone methyltransferase G9a in colon cancer serves as a risk factor for patients and is associated with shorter-relapse free survival. G9a activity has been shown to be essential for the maintenance of embryonic-like transcriptional signature which promotes self-renewal, tumorigenicity and an undifferentiated state. This work provides insights into the role of G9a as a driver of a cancer stem cell phenotype. To combat the toxicity and issues associated with targeting the catalytic activity of G9a, I utilized a phenotypic screening pipeline consisting of thousands of clinically-approved compounds, and identified CSC-bioactive epigenetic inhibitors showing promise in CSC-like models. RNA-seq profiling, dose response treatments and molecular techniques were used to confirm the selectivity of these candidates to colon-CSC like cells with minimal impact on normal progenitors. The lead candidate compound, vanoxerine (VXN) restricts organoid-initiating capacity of patient derived colon CSCs in serial 3D organoid formation assays that I developed throughout my research project. Using two murine syngeneic models resembling microsatellite instability and stability in CRC, I found this compound to be successful in decreasing primary tumor volumes compared to vehicle control mice, through epigenetic reprogramming and infiltration of immune cells. Drug treated tumors that were harvested, dissociated and re-injected into secondary mice showed diminished tumor initiating capacity compared to vehicle controls. Furthermore, the target of vanoxerine, SLC6A3, was investigated and the expression pattern characterized revealed a new potential biomarker for colon cancer stem cells. This SLC6A3-G9a axis discovered for the first time in colon cancer and serves as a novel and important pathway to block H3K9me2 deposition in CRC, rewiring the CSC epigenome and suppressing neoplastic self-renewal and tumor-initiating functions. Together, the identified repurposed compound selectively targets and modulates the epigenetic signature of CSCs which diminishes the tumor initiating function of these cells. This hints at an interesting interaction between CRC stemness and tumor immunology, a promising future therapeutic avenue.

Several Fungal species have the potential to cause a broad spectrum of diseases in humans, ranging from mild superficial to disseminated invasive infections that involve the bloodstream and vital organs. Invasive fungal infections are severe, life-threatening diseases that result in the deaths of 1.5 million patients each year. The most common fungal species responsible for the majority of invasive fungal infections include Candida, Cryptococcus, and Aspergillus. The current treatment options for invasive fungal infections are restricted to three classes of antifungals: Azoles, polyenes, and echinocandins. The emergence of new fungal species, especially C. auris, marked by high resistance profiles and increased mortality rates (30-60%), has further exacerbated the limitations in its therapeutic options. This emphasizes the urgent need for effective alternatives to combat these deadly pathogens. C. auris isolates exhibited high resistance capability especially against azole (fluconazole) and polyene (amphotericin B) antifungals. Here, we utilized the combinatorial strategy to screen ~3400 FDA-approved drugs and clinical compounds to identify hits that can enhance/restore the antifungal activity of azoles and amphotericin B against resistant C. auris. The HIV protease inhibitors (lopinavir and ritonavir) were identified as potent enhancers to the antifungal activity of azole drugs (fluconazole, voriconazole and itraconazole). We confirmed that lopinavir and ritonavir have the capability to interfere with fungal efflux pump machinery. The in vivo efficacy of the combination of azole antifungals and HIV protease inhibitors was also evaluated to discover the best combination of itraconazole, lopinavir and ritonavir. Three drugs (lansoprazole, rolapitant and idebenone) were identified to effectively enhance the antifungal effects of amphotericin B and overcome its resistance in C. auris. Furthermore, the synergistic interactions of these combinations were applied on other medically important Candida, Cryptococcus, and Aspergillus species. In a comprehensive mechanistic study, we discovered that lansoprazole interferes with an essential target in the fungal mitochondrial cytochrome system, cytochrome bc1. This interference induces oxidative stress in fungal cells and subsequently enhances the antifungal activity of amphotericin B. For rolapitant, a transcriptomic analysis along with ATP luminescence assays confirmed that rolapitant at sub-inhibitory concentrations significantly interferes with ATP production in C. auris. For idebenone, checkerboard assays confirmed the synergistic interactions between amphotericin B and idebenone against a diversity of medically important fungal species. This combination exhibited a rapid fungicidal activity within 4 hours. Additionally, the cytotoxicity of this combination was assessed in a cell line model of kidney cells. Based on the potent in vitro and in vivo synergistic relationships observed for the identified combinations, it can be concluded that our approach offers a new hope to restore the antifungal activity of the existing antifungal drugs, even against resistant fungal infections. Additionally, it provides valuable insights into identifying novel targets to overcome resistance in multidrug-resistant fungal pathogens. / Doctor of Philosophy / Fungi comprise a diverse group of organisms that interact with humans in many good and bad aspects. Candida auris, a recently identified fungus, poses a significant threat to patients with weak immune systems. Infections with C. auris can be associated with mortality rates of up to 60%. Notably, this fungus is characterized by its powerful spreading capability and displays extraordinary resistance to antifungal agents, rendering many existing antifungal drugs ineffective. As a result, there is an unmet need to find efficient treatments for such deadly fungal infections. In this study, several drugs were identified with the potential to restore the activity of traditional antifungal drugs. The study identified four promising drugs (lopinavir, lansoprazole, rolapitant, and idebenone) with the potential to enhance the activity of the antifungal drugs against C. auris. lopinavir showed great potential to enhance the activity of azole antifungals, including fluconazole, voriconazole, and itraconazole. Furthermore, three other drugs (lansoprazole, rolapitant, and idebenone) were identified for their potential to enhance the activity of amphotericin B, which is considered a last-line antifungal therapy. We clarified the mechanisms by which these drugs could restore the activity of antifungal agents. Finally, we confirmed the effectiveness of these combinations in animal models, providing valuable insights into their potential for clinical applications. In summary, our research has opened promising avenues to overcome resistance and develop new treatments for hard-to-treat fungal infections.

Investigating novel treatment approaches to combat Clostridioides difficile Rusha Pal ABSTRACT Clostridioides difficile is the leading cause of antibiotic-induced diarrhea and colitis in hospitals and communities worldwide. The enteric pathogen, classified to be an "urgent threat" by the United States Center for Disease Control and Prevention (CDC), capitalizes on disrupted intestinal microbiome to establish infection with disease symptoms ranging from mild diarrhea to potentially fatal conditions. Disruption of the intestinal microbiome, caused mostly by antibiotic use, enables C. difficile to colonize and proliferate within the host. Paradoxically, antibiotics are used to treat C. difficile infection. These antibiotics decimate the gut microbial community further, thus priming the gastrointestinal tract to become more prone to recurrence of infection. To tackle this clinical setback, we utilized a combination of traditional and non-traditional drug discovery approaches and identified chemical entities and targeted treatment options effective against this toxin-producing intestinal pathogen. Herein, we exploited the strategy of high-throughput screening to identify leads that harbor anticlostridial activity. Our primary phenotypic screen of FDA-approved drugs and natural product libraries led to the identification of novel molecules that were further characterized for their anticlostridial efficacy both in vitro and in vivo. The most potent scaffolds identified were those of mitomycin C, mithramycin A, aureomycin, NP-003875, NAT13-338148, NAT18-355531, and NAT18-355768. Of these, mithramycin A, aureomycin, and NP-003875 were also found to harbor anti-virulence properties as they inhibited toxin production by the pathogen. Furthermore, natural product NP-003875 could confer protection to 100% of the infected mice from clinical manifestations of the disease in a primary infection model of C. difficile. Our final approach has been to develop targeted therapeutics called peptide nucleic acids (PNAs). PNAs are antisense agents capable of inhibiting gene expression in bacteria. In this study, antisense inhibition of the RNA polymerase  subunit gene (rpoA) of C. difficile was found to be bactericidal for the pathogen and could also inhibit the expression of its virulence factors. Additionally, antisense inhibition of the C. difficile rpoA gene was found to be non-deleterious for the tested commensal microflora strains. Given their intriguing anticlostridial properties, it can be concluded that our research opened exciting possibilities that can be further evaluated to uncover new treatments for CDI. / Doctor of Philosophy / Investigating novel treatment approaches to combat Clostridioides difficile Rusha Pal LAYMAN LANGUAGE ABSTRACT Clostridioides difficile is a prominent human pathogen that can colonize the gut and cause fatal infections. C. difficile is the most common cause of microbial healthcare-associated infection and results in substantial morbidity and mortality. The "most urgent worldwide public health threat" label has been assigned to C. difficile by the United States Centers for Disease Control and Prevention (CDC). There is a pressing need to develop new classes of antibiotics with improved efficacy to treat C. difficile infections (CDI). To address the need for novel strategies to combat the growing problem of CDI, we screened FDA-approved drugs and natural products library in search of novel drugs that possess potent and specific anticlostridial activity. Several promising hits were identified and evaluated successfully both in vitro and in vivo. The most potent and novel hits that displayed exceptional activity were mitomycin C, mithramycin A, aureomycin, NP-003875, NAT13-338148, NAT18-355531, and NAT18-355768. Furthermore, a murine model of C. difficile infection revealed that compound NP-003875 conferred 100% protection to the infected mice from clinical manifestations of the disease. Interestingly, these compounds were non-toxic to the gut microflora and human cells. Our final approach has been to develop non-traditional therapeutics to target specific genes in C. difficile. These novel therapeutics are called peptide nucleic acids (PNA). Herein, we designed a PNA targeting RNA polymerase  subunit gene (rpoA) of C. difficile. The designed PNA could successfully inhibit the growth of the pathogen and expression of its virulence factors. In conclusion, our research opened exciting possibilities that can be further evaluated to uncover new treatments for CDI.

No description available.

No description available.

Résumé : Les méthodes de détection de similarités de sites de liaison servent entre autres à la prédiction de fonction et à la prédiction de cibles croisées. Ces méthodes peuvent aider à prévenir les effets secondaires, suggérer le repositionnement de médicament existants, identifier des cibles polypharmacologiques et des remplacements bio-isostériques. La plupart des méthodes utilisent des représentations basées sur les atomes, même si les champs d’interaction moléculaire (MIFs) représentent plus directement ce qui cherche à être identifié. Nous avons développé une méthode bio-informatique, IsoMif, qui détecte les similarités de MIF entre différents sites de liaisons et qui ne nécessite aucun alignement de séquence ou de structure. Sa performance a été comparée à d’autres méthodes avec des bancs d’essais, ce qui n’a jamais été fait pour une méthode basée sur les MIFs. IsoMif performe mieux en moyenne et est plus robuste. Nous avons noté des limites intrinsèques à la méthodologie et d’autres qui proviennent de la nature. L’impact de choix de conception sur la performance est discuté. Nous avons développé une interface en ligne qui permet la détection de similarités entre une protéine et différents ensembles de MIFs précalculés ou à des MIFs choisis par l’utilisateur. Des sessions PyMOL peuvent être téléchargées afin de visualiser les similarités identifiées pour différentes interactions intermoléculaires. Nous avons appliqué IsoMif pour identifier des cibles croisées potentielles de drogues lors d’une analyse à large échelle (5,6 millions de comparaisons). Des simulations d’arrimage moléculaire ont également été effectuées pour les prédictions significatives. L’objectif est de générer des hypothèses de repositionnement et de mécanismes d’effets secondaires observés. Plusieurs exemples sont présentés à cet égard. / Abstract : Methods that detect binding site similarities between proteins serve for the prediction of function and the identification of potential off-targets. These methods can help prevent side-effects, suggest drug repurposing and polypharmacological strategies and suggest bioisosteric replacements. Most methods use atom-based representations despite the fact that molecular interaction fields (MIFs) represents more closely the nature of what is meant to be identified. We developped a computational algorithm, IsoMif, that detects MIF similarities between binding sites. We benchmark IsoMif to other methods which has not been previously done for a MIF-based method. IsoMif performed best in average and more consistently accross datasets. We highlight limitations intrinsic to the methodology or to nature. The impact of design choices on performance is discussed. We built a freely available web interface that allows the detection of similarities between a protein and pre-calculated MIFs or user defined MIFs. PyMOL sessions can be downloaded to visualize similarities for the different intermolecular interactions. IsoMif was applied for a large-scale analysis (5,6 millions of comparisons) to predict offtargets of drugs. Docking simulations of the drugs in the binding site of their top hits were performed. The primary objective is to generate hypotheses that can be further investigated and validated regarding drug repurposing opportunities and side-effect mechanisms.

L’instabilité d’ADN mitochondrial (ADNmt) peut être quantitative avec la déplétion de l’ADNmt ou qualitative avec des délétions de l’ADNmt. Ces anomalies sont une des causes les plus commmunes des maladies mitochondriales. Un des gènes qui contrôle la stabilité et le maintien de l’ADNmt est POLG. Ce gène code pour la polymerase gamma mitochondriale. Chez l’homme, les mutations dans le gène POLG sont liées aux maladies mitochondriales telle que; l’insuffisance hépatique, le syndrome d’Alpers, le PEO ou Progressive External Ophtalmoplegia, la neuropathie sensorielle et l’ataxie. Des mutations dans le gène POLG sont aussi associées au syndrome de Parkinson. Aujourd’hui, il n’existe aucune thérapie pour ces maladies. Compte tenu de la conservation évolutive de la fonction mitochondriale de la levure à l’homme, nous avons utilisé deux organismes modèles, Saccharomyces cerevisiae et Caenorhabditis elegans, pour identifier des molecules chimiques capables de compenser l’instabilité de l’ADNmt liée à des mutations du gène POLG dans des fibroblastes d’un patient. Nous avons trouvé trois molécules candidates potentielles: MRS2, MRS3 et MRS4, à partir d’un criblage primaire chez la levure, en utilisant une chimiothèque d’environ 2000 molécules chimiques. MRS3 est la molécule candidate la plus efficace pour la stabilization d’ADNmt chez des mutants POLG de la levure, du champignon filamenteux, du nématode et sur des fibroblastes de patients. MRS3, ou clofilium tosylate (CLO), est un agent antiarrhytmique, médicament pour soigner les troubles du rythme cardiaque. Dans cette étude, nous avons aussi montré que deux autres antiarrhythmiques appartenant à la même classe que CLO avaient un effet positive chez un mutant POLG de C. elegans. En utilisant une approche de chemogénomique chez la levure, nous avons identifié Fis1, un acteur de la fission mitochondriale qui pourrait être impliqué dans la mode d’action de CLO. Fis1 est requise pour la viabilité cellulaire en concentration légèrement toxique de CLO et nécesaire pour la stabilization de l’ADNmt par CLO. L’ensemble de ces résultats ont montré que CLO pourrait être la première molécule chimique qui stimule la réplication de l’ADNmt et qui pourrait être développée pour le traitement des maladies liées à des mutations dans le gène POLG. Ces résultats ont aussi permis de mettre en évidence une nouvelle connexion entre replication de l’ADNmt et la fission mitochondriale. / The instability of mitochondrial DNA (mtDNA) in form of mtDNA depletion (quantitative instability) or large deletion (qualitative instability) is one of the most common cause of mitochondrial diseases.. One of the genes responsible for human mtDNA stability, POLG, is exploited in this study. POLG encodes the human mitochondrial polymerase gamma. In human, POLG mutations are a major cause of mitochondrial disorders including hepatic insufficiency; Alpers syndrome, progressive external ophthalmoplegia, sensory neuropathy and ataxia. They are also associated with Parkinsonism. Currently, there is no effective and disease-specific therapy for these diseases. Based on the conservation of mitochondrial function from yeast to human, we used Saccharomyces cerevisiae and Caenorhabditis elegans as first pass filters to identify chemical compounds that suppresses mtDNA instability in cultured fibroblasts of a POLG-deficient patient. We found three potential candidates, MRS2, MRS3 and MRS4, from a chemical screening of nearly 2000 compounds in yeast. MRS3 is the most efficacious in stabilizing mtDNA in yeast, filamentous fungi, worm and patient fibroblasts. This unsuspected compound, clofilium tosylate (CLO), belongs to a class of antiarrhythmic agents for cardiovascular disease. Two other antiarrhythmic agents (FDA-approved) sharing common pharmacological properties and chemical structure with CLO also show potential benefit for POLG deficiency in C. elegans. Using a chemogenomic approach in yeast, we also discovered that a mitochondrial fission actor Fis1 is implicated in the mechanism of action of CLO. Fis1 is important for cellular viability in a slightly toxic concentration of CLO and is required for the mtDNA stabilizing potency of CLO. Our findings provide evidence of the first mtDNA-stabilizing compound that may be an effective pharmacological alternative for the treatment of POLG-related diseases and uncover a new connection between the mitochondrial fission process and mtDNA replication. / Ketidakstabilan DNA mitokondria (mtDNA) dalam bentuk pengurangan kopi mtDNA di dalam sel (ketidakstabilan kuantitatif), atau pun dalam bentuk delesi pada sekuens mtDNA (ketidakstabilan kualitatif) merupakan salah satu penyebab penyakit mitokondria. Salah satu gen yang bertanggung jawab dalam menjamin kestabilan mtDNA adalah POLG. Gen POLG mengkode protein polimerase gamma pada manusia, yang mereplikasi dan mereparasi mtDNA di dalam mitokondria. Mutasi pada gen POLG dapat menyebabkan penyakit kelainan mitokondria pada manusia, seperti gagal ginjal, sindrom Alpers, Progressive External Ophtalmoplegia, neuropati sensorial, ataxia dan bisa dikaitkan dalam beberapa gejala Parkinsonisme. Saat ini, belum ada terapi obat yang dapat mengatasi penyakit – penyakit tersebut. Berdasarkan kesamaan evolutif dari ragi hingga manusia, pada studi ini kami menggunakan Saccharomyces cerevisiae dan Caenorhabditis elegans untuk mengidentifikasi molekul obat yang berpotensi mengatasi ketidakstabilan mtDNA dari fibroblas pasien manusia yang memiliki mutasi gen POLG. Kami mengidentifikasi tiga kandidat potensial, yakni MRS2, MRS3 dan MRS4 dari penapisan kurang lebih 2000 molekul obat dengan menggunakan ragi. MRS3 adalah kandidat yang paling berkhasiat dan mampu mengatasi ketidakstabilan mtDNA pada ragi, Podospora, cacing dan fibroblas manusia. MRS3 adalah alias bagi clofilium tosylate (CLO), sebuah molekul antiaritmia untuk penyakit kardiovaskuler. Pada studi ini, kami juga menguji aktifitas dua molekul antiaritmia lain yang tergabung dalam kelas yang sama dengan CLO, dan menemukan bahwa kedua molekul ini juga berpotensi mengatasi defisit POLG pada cacing C. elegans. Dengan menggunakan metode kemogenomik pada ragi, kami juga mengidentifikasi sebuah aktor prosesus pembelahan mitokondria, Fis1, yang berpotensi terlibat dalam mekanisme seluler CLO. Fis1 dibutuhkan untuk: (1) kelangsungan hidup ragi pada konsentrasi toksik CLO dan (2) efek CLO dalam menstabilkan mtDNA pada ragi. Keseluruhan studi ini membuktikan potensi CLO sebagai molekul penstabil mtDNA yang pertama, yang dapat dikembangkan sebagai salah satu alternatif terapi obat untuk penyakit – penyakit mitokondria terkait mutasi POLG. Melalui studi ini, juga diungkap adanya hubungan antara kestabilan mtDNA dan prosesus pembelahan mitokondria.