Thèse d'exercice : Pharmacie : Nantes : 2007. / Bibliogr.

Thesis (doctoral)--Uppsala University. / Description based on print version record.

Thesis (M.Sc.)--University of Alberta, 1997. / Submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Master of Science, Department of Medicine. Also available online.

Since Bortezomib®, a proteasome inhibitor, was approved by US FDA for the treatment of multiple myeloma in 2003, proteasome is recognized as one of the most promising targets for cancer therapeutics. The proteasomes play a critical role in regulating the level of cellular proteins and recycling damaged and misfolded proteins. Although the activity of the proteasome is essential for normal cells, it is especially critical for the proliferation and survival of cancer cells. In an attempt to develop effective and safe proteasome inhibitor-based anticancer drugs, the Lee laboratory created a chemical library by a hybrid approach using a 4-piperazinylquinoline scaffold and a sulfonyl phamarcophore. It is known that the chloroquine scaffold possesses a weak proteasome inhibition activity, and chloroquine itself preferentially kills malignant cells over non-cancer cells, alone or in combination with other therapeutics. To identify compounds with desirable anticancer activities, I have screened the aforementioned chemical library. The screening yielded several hits with substantial efficacy and selectivity against malignant cells. In this thesis, I describe the functional mechanism of VR23, one of the most promising compounds identified from my screening, as it kills cancer cells up to 17 fold more effectively than non-cancer cells. Molecular docking and substrate competition studies revealed that VR23 binds to the β2 peptide of the 20S proteasome catalytic subunit. The IC50 value of VR23 in inhibiting trypsin-like proteasome activity is 1.0 nM. VR23 is also substantially effective in inhibiting chymotrypsin-like proteasome activity (IC50, 50-100 nM). The inhibition of proteasome activity by VR23 led to the accumulation of ubiquitinated cyclin E at centrosomes. This, in turn, induces abnormal centrosome amplification by a de novo centrosome synthesis pathway in cancer cells, but not in non-cancer cells. The presence of multiple centrosomes in single cancer cells results in cell cycle arrest at prometaphase and, eventually, cell death by apoptosis. Thus, VR23 possesses a very desirable property as a safe anticancer drug.

No description available.

The aim of this study was to elucidate the oxidation products of a candidate antimalarial drug, PL69, using a porphyrin system and to determine the accuracy of the oxidation products produced, as compared to what is expected in metabolism. PL69 is a reversed chloroquine (RCQ) that is active against chloroquine resistant malaria. Porphyrin oxidation systems have been shown to mimic in vitro enzymatic metabolism reactions. PL69 and its known metabolite, PL16, were incubated with the porphyrin system, and then the oxidation products were collected and separated by HPLC. The oxidation products were characterized by NMR and mass spectrometry and compared to previous metabolism studies of PL69 with liver microsomes. The results of this research show that this porphyrin system is an acceptable mimic of in vitro metabolism methods for RCQs and provides a good framework for understanding the types of metabolism that will occur in vivo for RCQs.

Tuberous sclerosis complex (TSC) is a multisystem genetic disease that is caused by a germ line mutation in the genes TSC1 and TSC2. Patients with the disease tend to suffer from benign tumors of the brain, heart, kidneys, skin, and other organs that contain giant cells. Although mTORC1 inhibitors (rapamycin and rapalogs) are often used to treat TSC because of their efficacy in promoting tumor shrinkage, clinical studies in the past have shown that when treatment is taken away, the tumor size returns to its original state. The objective of this study was to identify compounds that selectively inhibit proliferation of TSC2-deficient cells. A high-throughput screen of about 4000 compounds was performed using a lysosomal inhibitor (chloroquine [CQ], 5 μM) and a “repurposing” library of compounds. Through some yet to be determined mechanism, the combination of ritanserin (a selective serotonin reuptake inhibitor [SSRI]) and chloroquine was found to synergize to selectively inhibit the cell viability of DJK MEFs and TSC2-/-KO cells (TFFs) starting at 48 hours after treatment. The effects of this combination treatment were confirmed in a second cell line (TFFs) exhibiting similar reduction in proliferation. Interestingly, treatment with CQ (5 μM) and ritanserin (20 μM) showed synergistic action (combination index [CI] = 0.6) against TSC2-deficient cells. This combination treatment induced apoptosis (41%) in TSC2-deficient cells but not in TSC2-expressing cells. These results suggest a novel treatment approach in tuberous sclerosis complex and provide an incentive for further investigation of the mechanisms contributing to the vulnerability of TSC2-deficient cells. Moreover, the use of an already Food and Drug Administration (FDA)-approved compound can lead to a more rapid pharmacologic approach for TSC patients.

Malaria is an infectious, often fatal disease that afflicts nearly 200 million people every year. The disease, characterized by recurring and extreme flu-like symptoms, is caused by the protozoan parasite Plasmodium falciparum. Victims usually contract the disease through a mosquito vector. Chloroquine is a chemotherapeutic that was introduced in the 1940s. For many years the drug was the foremost treatment of malaria, being effective and producing few side effects. Unfortunately, tolerance to chloroquine developed when the parasite evolved a resistance mechanism. Newer drugs have been developed and implemented, but these medicines also show a decreasing effect with continued administration. It is imperative that a new pipeline of drugs be developed in order to combat the disease and anticipated resistance. Reversed chloroquines are a new class of multiple-ligand compounds that are active against chloroquine-sensitive and chloroquine-resistance malaria species. This thesis describes research targeted at the modification of lead reversed chloroquine molecules to discover new and effective moieties, as well as to improve pharmacokinetic-related properties. An especial emphasis of this project is the addition of a sulfonamide functional group to a reversed chloroquine. Preliminary evidence indicates that this is a promising direction for this line of research. Brief discussions of some reversed chloroquine characterization studies are included in appendices.

Les gliomes malins prolifèrent d’une manière très anarchique et elles s’infiltrent dans le parenchyme cérébral. Plusieurs facteurs de croissance, tels que le Transforming Growth Factor Beta (TGF-ß), sont surexprimés dans les gliomes malins. La voie de signalisation du TGF-ß joue un rôle clé dans la régulation des mécanismes liés au métabolisme et à la prolifération cellulaire. L’objectif principal de cette étude est d’évaluer les effets de l’inhibition de la voie de signalisation du TGF-ß sur le phénotype des gliomes malins. Deux méthodes ont été utilisées pour inhiber la voie du TGF-ß. La première est un traitement à la chloroquine, un agent alcalinisant lysosomotropique, qui module à la hausse le pH du Trans Golgi Network (TGN )/endosome. Cet agent inhibe la furine, une protéase nécessaire à la maturation du TGF-ß, ainsi que la métalloprotéases-2 (MMP-2) qui permet la libération du TGF-ß de la matrice extracellulaire (MEC). La deuxième méthode est un traitement avec l’Avasimibe™, un agent qui inhibe l'Acyl-CoA-Acétyltransférase (ACAT) démontrée comme étant activé par le TGF-ß. Les effets de ces deux agents ont été testés sur la sécrétion de TGF-ß, sur le métabolisme et la prolifération cellulaire de la lignée de gliomes murins F98. Par l’immunobuvardage de type Western blot, notre étude a révélé la présence de la forme immature de TGF-ß1 et de Latency Associated Peptide (LAP) dans le surnageant de la lignée de gliomes murins F98. Par le test Enzyme Linked Immunosorbent Assay (ELISA), l’étude a indiqué que la chloroquine et l’Avasimibe™ diminuent de 85 ± 3 % et 65 ± 1 % la concentration de TGF-ß1 mature sécrétée dans le surnageant cellulaire. Les essais WST-1 et BrdU ont montré que des expositions de 24 h et 48 h à la chloroquine et l’Avasimibe™ diminuent significativement de plus de 74 ± 9 % et 86 ± 2 % % le métabolisme et la prolifération cellulaire. La zymographie pousse à croire que les effets de chloroquine étaient associés à une inhibition de 75 ± 1 % de l’activité de la MMP-2 en 48 h. L’ajout de la chloroquine et de l’Avasimibe™ pourrait être bénéfique au traitement standard des gliomes malins en réduisant leur phénotype.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 6 uppsatser.