La voie biosynthétique du mevalonate (MVA) est indispensable à la production de molécules qui sont essentielles dans grand nombre de processus physiologiques mais la plupart des études effectuées sur le rôle physiologique de cet important processus métabolique dans le système nerveux central restent simplement en corrélation. Pour autant, le but des études présentées dans cette thèse a été: évaluer les protéines de la voie du MVA dans différentes régions du cerveaux de rat en prenant en considération l’éventuelle modulation selon le sexe et l’âge; analyser l’impact de la voie sur le développement des neurones set sur le comportement des rats; explorer l’impact de la voie dans une pathologie du neurodéveloppement comme l’autisme. Les résultats démontrent que la voie biosynthétique du MVA est régulée différemment selon la région du cerveau analysée, en fonction de l’âge et du sexe. La voie biosynthétique du MVA revêt un rôle critique dans la modulation physiologique du comportement animal et du développement des neurones Pour finir, mes données démontrent, dans un modèle expérimental d’autisme, que la voie biosynthétique du MVA est modulée dans plusieurs région du SNC selon l’âge. / The mevalonate (MVA) pathway is an essential metabolic pathway that leads to the production of molecules important in several physiological processes and play pivotal roles in the brain. An imbalance of this pathway in CNS is accompanied by the onset of several neuropathological descriptions. Despite these observations, the physiological importance of this metabolic process in the brain has remained unclear. My aim was to study the presence and the regulation of the proteins involved in the MVA pathway in different rat brain areas in a sex- and age-dependent manner, to analyze the impact of the key enzymes on neuronal development and on rat behavior, and to explore whether the MVA pathway is affected in a neurodevelopmental disease such as autism. My results demonstrate that this metabolic process is expressed and modulated in a highly region-dependent manner and that age and sex induce physiological differences. Notably, it impact on behavior and neuronal development suggesting that this pathway may be considered as potential molecular target when designing novel therapeutic approaches for the treatment of these pathologies.

The objective of this work is to investigate the structures of two nucleotide binding proteins: mevalonate kinase (MVK) and FtsZ. MVK is the key enzyme involved in terpenoid biosynthesis. In this study, we solved the crystal structures of the M. jannaschii MVK apoprotein, as well as the protein in complex with ligands. Its fold was analyzed and firmly established within the GHMP kinase family, in which homoserine kinase (HSK), phosphomevalonate kinase and galactokinase also belong. Structural analysis in combination with enzyme kinetics studies revealed the mechanism of this enzyme upon substrate binding, catalysis and inhibition. In particular, the phosphate-binding loop was found to be critically involved in the binding of nucleotides and terpenoids, via the interaction with a di-phosphate moiety from the ligand. An enzymatic reaction mechanism was constructed based on our structural data and it is consistent with kinetics studies from the literature. In this mechanism, the invariant residue Asp 155 functions as a general base that increases the nucleophilicity of the phosphoryl acceptor. Finally, a virtual screening study has been performed to explore the ligand binding potential of MVK. Compounds predicted to bind MVK were tested and analyzed. FtsZ is a prokaryotic homologue of tubulin that forms the apparatus for bacterial cell division. The structure of a crystal filament of the M. tuberculosis FtsZ complexed with GDP was described in this study. It shows an anti-parallel, left-handed double helical architecture. Compared with the straight crystal filament revealed earlier by other groups, the catalytic T7 loop in our structure is found to be outside the nucleotide binding site, indicating the GTPase is inactive. Furthermore, the buried surface area in our crystal filament is less, probably suggesting the helical FtsZ filament is less stable. We therefore proposed that the hydrolysis of GTP and the releasing of the γ-phosphate group will trigger the rearrangement of the FtsZ fibler, characterized by the exclusion of the T7 loop, which might lead to a less stable helical filament and would be the first step for the disassembly of FtsZ polymer.

Phosphonates represent an important class of organophosphorus compounds. Their use as reagents in organic synthesis is prevalent, and there is a plethora of examples of biologically active compounds possessing the phosphonate moiety. To further our exploration of phosphonates as both reagents and biologically active compounds we have developed a one-flask protocol for the direct synthesis of phosphonates from benzylic and allylic alcohols. This transformation is unprecedented and is applicable to a range of substrates. Both electron rich and electron deficient benzylic alcohols react under the conditions developed. Furthermore, good yields are achieved when converting allylic alcohols to the corresponding allylic phosphonates. In at least one case, the one-flask protocol allows for phosphonate formation that was not achievable under the standard conditions. Bisphosphonates represent a significant subclass of phosphonates. Several nitrogenous bisphosphonates have found use in the clinic as treatments for bone-related disease including osteoporosis, and there is speculation that bisphosphonates that are enzyme-specific inhibitors may be used as cancer therapies. To develop our understanding of isoprenoid metabolism, we have prepared a range of bisphosphonates as potential inhibitors of geranylgeranyl pyrophosphate synthase. After much experimentation, an α-amino analog of a potent inhibitor of GGDPS has been synthesized and biological data is forthcoming. Furthermore, a new class of aromatic bisphosphonates, analogs of digeranyl bisphosphonic acid, has been synthesized and assayed. The bioassay results indicate that this series of compounds retains its specificity for the GGDPS enzyme, and that the dialkyl analogues retain much of their potency in the assays in spite of the increased steric bulk of the aromatic substructure. We have also begun the design and synthesis of compounds as potential inhibitors of Rab geranylgeranyl transferase (RGGTase). The lead compound, 3-PEHPC, is documented to inhibit RGGTase selectively, albeit at less than desirable concentrations. Using 3-PEHPC as the model compound we have elected to probe the impact of modifications on the hydrophilic "head" portion of the molecule. Using the phosphonophosphinate functionality as a surrogate for the phosphonocarboxylate moiety we have successfully synthesized digeranyl phosphonophosphinate. Initial assay data indicates that this novel phosphonophosphinate does not act upon GGDPS as does the analogous bisphosphonate substructure. The bioassay data to probe this compound's impact on RGGTase is forthcoming. Given the worldwide impact of tuberculosis infection and the emergence of drug-resistant strains of tuberculosis-causing pathogens, new and potent treatments for tuberculosis are necessary. We have engaged in the synthesis of several compounds as inhibitors of Rv2361c, an enzyme key to cell wall biosynthesis in Mycobacterium tuberculosis, the principle causative agent of tuberculosis in humans. To probe the impact of modifications at the C-9-position of the most potent of our Rv2361c inhibitors, we have made several analogues having phenyl and indole substituents. The in vitro enzyme assay data for the set of compounds has clarified understanding of the essential components of the pharmacophore, and helped to establish the direction for future efforts.

Lung cancer is the most common cause of cancer-related mortality worldwide with current treatments providing limited therapeutic benefit in most cases. TP53 (Trp53, p53) mutations occur in approximately 50% of lung adenocarcinoma cases and are associated with poor prognosis and so novel therapies that target these p53 mutant lung tumours are urgently needed. Despite the high frequency of p53 mutations in lung tumours, the impact these mutations have on response to therapy remains unclear in this cancer type. The aim of my project is to characterise the gain-of-function and dominant-negative effects of p53 mutations in lung tumours and to identify ways of therapeutically targeting these p53 mutant tumours based on dependencies and susceptibilities that our analysis uncovers. To characterise the gain-of-function and dominant-negative effects of p53 mutations I compared p53 mutant murine lung tumour cells that endogenously express either a contact (R270H, equivalent to R273H in humans) or conformational (R172H, equivalent to R175H in humans) p53 mutant protein and p53 null lung tumour cell lines; both in the presence and absence of wild-type p53. Interestingly, transcriptional and functional analysis uncovered metabolic gain-of-functions that are specific to the type of p53 mutation. Upregulation of mevalonate pathway expression was observed only in R270H lung tumours and consequently R172H and R270H lung tumours displayed distinct sensitivities to simvastatin, a mevalonate pathway inhibitor widely used in the clinic. Furthermore, the transcriptional signature underlying this sensitivity to simvastatin was also present in human lung tumours with contact p53 mutations, indicating that these findings may be clinically relevant. On the other hand, our analysis of the potential dominant-negative effects of the p53 mutants on wild-type p53 demonstrated that wild-type p53 was able to induce typical p53 target genes to a similar level in p53 null and mutant cells. Furthermore, wild-type p53 restoration resulted in comparable tumour suppressive responses in p53 mutant and null tumours and thus, p53-restoration therapy will likely be of benefit to patients with p53 mutations in lung cancer. Hence, I have demonstrated that lung tumours harbouring contact and conformational p53 mutations display common and distinct therapeutic susceptibilities.

Malaria is a parasitic infectious disease that results in millions of clinical cases per year and accounts for approximately 1 million deaths annually. Because the parasite has developed resistance to all current antimalarials, new therapies are urgently needed. Purine and pyrimidine biosynthesis for DNA and RNA synthesis has been recognized as a source of therapeutic targets. Targeted metabolite profiling has aided in the understanding of several biological processes in the parasite besides drug discovery. Therefore, having a robust analytical platform to quantify the purines and pyrimidines is of a great value. For this purpose an ion pair reversed phase ultra-performance liquid chromatography in tandem with mass spectrometry method was developed and validated. In addition, the apicoplast is an organelle present in the malaria parasite and other apicomplexan parasites. It was demonstrated that the apicoplast is essential for parasite's survival. The supply of isopentenyl diphosphate and dimethylallyl diphosphate for isoprenoid biosynthesis is the sole function of this organelle in the asexual intraerythrocytic stages. Isoprenoid precursors are synthesized through the methylerythritol phosphate (MEP) pathway in the malaria parasite while humans utilize the mevalonate pathway. Therefore, the MEP pathway is a source of drug targets for drug development. Our group has identified MMV008138 as anti-apicoplast inhibitor through phenotypic screening. Preliminary data suggest that the molecular target of MMV008138 may be within the MEP pathway. We used proteomic and metabolomic approaches to identify the molecular target of MMV008138 to aid future medicinal chemistry to improve the efficacy of this inhibitor. / Master of Science

All animal cells maintain cell size homeostasis, where cell growth (increase in size) is balanced with proliferation (reduction in size via cell division). Yet, different cell types have different sizes and there are physiologically relevant situations where animal cells undergo major cell size changes. So how is cell size regulated? And why is cell size regulated? Are there specific cellular processes that have different functionality in different sized cells? This thesis investigates these questions from the perspective of cellular metabolism. Using a Cyclin dependent kinase 1 knockout mouse model with different degrees of hepatocytes enlargement, gene expression levels were correlated with cell size in vivo. This revealed that the relative expression of mitochondrial and lipid biosynthesis genes are downregulated with increasing cell size. However, mitochondrial content of the liver samples was not decreased, suggesting that cell functions and cell contents scale differently with cell size. To better investigate how mitochondrial functions scale with cell size in non-mutant cells, a novel and high throughput flow cytometry based single-cell analysis method called CoSRA was developed. Using fluorescence mitochondrial probes CoSRA revealed that, while mitochondrial content increases linearly with cell size, mitochondrial membrane potential is decreased in the very smallest and the largest cells. These effects were independent of cell cycle and all animal cell types examined displayed similar effects. Similar nonlinearity was observed in mitochondrial respiration. Furthermore, cell-to-cell variability in mitochondrial membrane potential was minimised in cells which are close to the median cell size of the whole population. The cell size dependence of mitochondrial functions was regulated by mitochondrial dynamics. It was also investigated if mitochondrial functions or lipid biosynthesis are capable of regulating cell size in human cell culture models. Various mitochondrial inhibitions increased cell size by reducing proliferation. Similar results were seen with inhibitions on lipid biosynthesis and especially with inhibitions of mevalonate pathway. Systematic dissection of the mevalonate pathway revealed that protein geranylgeranylation is required for maintaining normal cell size and proliferation ratio. Geranylgeranylation of the recycling endosome regulating protein RAB11 was identified to be at least partially responsible for the cell size regulation by the mevalonate pathway. Furthermore, the link from the mevalonate pathway to RAB11 was found to regulate basal autophagic flux, thus providing a novel connection from lipid biosynthesis to other growth regulating processes. In conclusion, this thesis provides evidence for cell size dependent metabolism, where mitochondrial functions do not increase linearly with cell size. This provides conceptual insights into organelle scaling with cell size and a potential mechanism for maintenance of cell size homeostasis. In addition, mitochondria and lipid synthesis are identified as critical processes for normal cell size homeostasis.

Schweinfurthins are a family of natural products with significant anti-cancer activities. They were originally identified in the National Cancer Institute (NCI) human 60 cancer cell line screening. The growth inhibition profile of schweinfurthins is distinct from other clinically used anti-cancer agents, indicating that they have a novel mechanism of action or have a previously unrecognized protein target. Previous studies showed that schweinfurthins affect multiple cellular processes in cancer cells. For example, schweinfurthins can alter cytoskeleton organization, induce ER stress and apoptosis, and inhibit the mevalonate pathway. The mevalonate pathway is responsible for the production of isoprenoids and cholesterol, which have been shown to play regulatory roles in the Hedgehog (Hh) signaling pathway. In this study, we found that the Hh signaling pathway in NIH-3T3 and SF-295 cells was inhibited by schweinfurthins. The supplementation of mevalonate and cholesterol partially restored Hh signaling, indicating that schweinfurthins inhibit Hh signaling partially by down-regulating the products from the mevalonate pathway. Interestingly, schweinfurthins in combination with cyclopamine, an inhibitor of the Hh singaling pathway, synergistically decreased cell viability. In order to better understand the underlying mechanism of the anti-cancer action of schweinfurthins, we attempted to identify the protein target of schweifnurthins. Affinity chromatography was performed to pull down the protein target. We found that schweinfurhtins bound to the M2 isoform of pyruvate kinase (PKM2) and inhibit its pyruvate kinase activity. Knockdown of PKM2 by siRNA increased the sensitivity of SF-295 cells to schweinfurthins. The inhibition of PKM2 by schweinfurthins led to a reduction in the rate of glycolysis in cancer cells. Fructose 1,6-bisphosphate (FBP), an activator of PKM2, could alleviate schweinfurthin-mediated inhibition on PKM2 and glycolysis. Notably, FBP could also partially reverse the reduction of cell viability in the presence of schweinfurthins. Taken together, these studies revealed the mechanism by which schweinfurthins inhibit Hh signaling. In addition, we uncovered PKM2 as a schwienfurthin target and highlighted the importance of glycolysis suppression as a mechanism of the anti-cancer action of schweinfurthins.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.