The diverse functions of the MDM2 oncoprotein in growth control and tumourigenesis are managed through coordinated regulation of its discrete domains induced by both extrinsic and intrinsic stimuli. A picture of MDM2 is immerging where structurally discrete but interdependent functional domains are linked through changes in conformation. However compelling insights into how this process is carried out have been hindered by inadequate information on the structure and conformation of the full-length protein. The data presented indicates that the C-terminal RING domain of MDM2, primarily responsible of the E3 ubiquitin ligase activity of the protein, has other intriguing functions. The binding of ATP within the RING domain, triggers conformational changes of MDM2 and its main interaction partner – p53. This in effect promotes efficient binding of the p53 tumour suppressor to specific DNA promoter sequences. Moreover, results presented in this thesis demonstrate a novel role for the RING domain of MDM2 in determining the conformation and activity of its N-terminal hydrophobic cleft, the key target of anticancer drugs designed to activate the function of p53 tumour suppressor protein. Specific modulations within the RING domain, affecting Zinc coordination are synonymous with increased binding affinity of the hydrophobic pocket to the transactivation domain of p53 resulting in a gain of MDM2 transrepressor function thus leading to a decrease in p53-dependant gene expression. ThermoFluor measurements and size exclusion chromatography show that changes in the RING motif lack an effect on the overall integrity of the MDM2 protein. The intrinsic fluorescence measurements manifest that these changes generate long range conformational transitions that are transmitted through the core/central acidic domain of MDM2 resulting in allosteric regulation of the N-terminal hydrophobic pocket. Such RING generated conformational changes result in the relaxation of the hydrophobic pocket. Additionally, it is shown that the cooperation between the RING and the hydrophobic cleft in MDM2 has implications in the efficiency of binding of anticancer drugs such as Nutlin by MDM2. Cooperation between the RING and hydrophobic domain of MDM2 to regulate function demonstrates an allosteric relationship and highlights the need to study MDM2 in a native conformation. In essence the presented data demonstrates that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed towards its hydrophobic pocket.

The tumour suppressor p53 is a key regulatory protein that prevents proliferation of damaged cells. Under unperturbed conditions, the ubiquitin ligase murine double minute 2 (MDM2) mediates p53 ubiquitination and further degradation by the proteasome. In consequence p53 is present at low levels, but becomes rapidly stabilised and activated in response to a variety of stimuli, such as DNA damage or virus infection. P53 responds to these diverse stresses to regulate the expression of many target genes that induce cell cycle arrest, DNA repair, or apoptosis. The attenuation of p53 interaction with MDM2 is maintained by enzymes catalysing p53 post-translational modifications such as phosphorylation. Casein kinase 1 α (CK1α) is one such enzyme; it stimulates p53 after DNA virus infection. Surprisingly depletion of CK1α using small interfering RNA or inhibition using a CK1 kinase inhibitor activated the transcription factor p53, indicating that p53 steady-state level is controlled by CK1α. Disrupting MDM2-p53 interaction using small molecule Nutlin-3 displayed similar pharmacological properties to the CK1 inhibitor on p53, indicating that the MDM2-CK1α complex co-regulates p53 stability. Indeed co-immunoprecipitation of endogenous CK1α with MDM2 occurred in undamaged cells. CK1α was shown in vitro to directly bind to and phosphorylate MDM2. Therefore it appears that CK1α must be recruited into specific complexes under different conditions, which can influence its substrate selectivity and explain its dual role on the p53 pathway. Apart from CK1, there are few other kinases whose action can directly contribute to the inhibition of p53. A novel pyrazolo-pyridine analogue showing dual activity against CK1 and Checkpoint kinase 1 led to increased p53 activation. These data highlighted the potential value of dual kinase inhibitors as therapeutics in cancer. The dominant protein-protein interface that stabilises the MDM2-CK1α complex was mapped using a peptide-based approach. One CK1α peptide bound strongly to MDM2, it specifically disrupted the protein-protein interaction, and its transfection was able to reduce cancer cell growth. A peptide phage display approach was finally combined with Next-Generation Sequencing to define the change in MDM2 binding motifs when the CK1α peptide or Nutlin-3 is bound, compared to ligand-free MDM2, and thus will help to understand protein-protein interaction network re-wirings which led to cell growth inhibition.

The tumour suppressor p53 is essential for genome stability and loss of its function can lead to human cancer. The functional roles of p53 are regulated by a variety of mechanisms, some of which are not well understood. However, the murine double minute 2 (MDM2) protein, a major negative regulator of p53, has been found to be overexpressed in many human cancer cell lines in which p53 was not mutated; thus establishing MDM2 as a target for cancer therapeutics. MDM2 is defined as both an oncoprotein and an E3-ubiquitin ligase; its interactions with p53 are controlled through multiple domains, providing different possible pathways to inhibit MDM2 and therefore reactivate p53 function. Previous work undertaken in the Ball laboratory has shown that the MDM2 RING domain plays a critical role in p53 ubiquitination; thus screening for modulation of its activity by small molecules could provide new leads for the inhibition of the E3 ligase activity of MDM2. The MDM2 RING domain was cloned, expressed and purified so that it could be studied using a series of in vitro experiments. The generation of a library of short (12-mer) peptides as potential inhibitors of the MDM2 RING domain was investigated using phage display against His-tagged RING protein to screen the peptide ligands. In order to study the specificity of these peptides towards MDM2 (res. 396-491 and 396-479) compared with MDM4 and BRCA1, the MDM4 RING domain (res. 395-490 and 395-478) and BRCA1 (res. 1-304) domain were expressed and purified for further characterisation. A small selection of peptides was isolated and their binding affinity and activity as MDM2 inhibitors evaluated by in vitro ELISA, affinity chromatography and ubiquitination assays. One peptide in particular, KCCYFETHMPRH, was found to bind to MDM2 and was able to inhibit MDM2-mediated ubiquitination of p53 in vitro. Preliminary optimisation of this peptide by alanine scan revealed a peptide with a 2-fold increased potency. Since peptides provide comparatively weak therapeutic leads due to a combination of poor cellular uptake and susceptibility to cleavage by proteases, cyclic peptidomimetics based upon this lead were developed using side-chain to side-chain cyclisation. These peptidomimetics were successfully generated by the synthesis and incorporation of novel N-propargylated glycine and N-azidoalkyl glycine building blocks into a peptide sequence by Solid Phase Peptide Synthesis (SPPS). Following a Copper-catalysed Azide-Alkyne Cycloaddition (CuAAC) on solid phase or in solution, these peptoid-peptide hybrids were isolated, purified and characterised.

Death domain-associated protein 6 (DAXX) is a multifunctional protein involved in diverse cellular processes. It acts as a histone chaperone or regulator of transcription and apoptosis, in which is its role quite controversial. DAXX also participates in regulation of cell DNA damage response (DDR). DAXX co-creates and stabilizes complex with Mdm2, which negatively regulates the stability of p53, an important tumor suppressor, which is a part of signalling node in the DDR. If DNA damage is not lethal for the cell and unables it to proliferate, the irreversible state of cell cycle called cellular senescence takes place. Under physiological conditions, induction of senescence can prevent the development of tumorigenesis. Therefore, the description of mechanisms involved in the induction of senescence has potential clinical significance. In my thesis, I aimed to determine changes in the level of DAXX protein in senescent cells and to characterize the manner of its regulation. In tumor cells MCF-7 and primary BJ fibroblasts, I observed decrease in DAXX protein level and its regulation. I tested the hypothesis according to which an increase in DAXX level before DNA damage canprevent induction of cellular senescence, or increase in its expression during senescence can cause recovery of cell proliferation....

The MDM2 protein regulates the tumour suppressor protein p53, acting as its chaperone, regulating its translation and targeting p53 for degradation by the 26s proteasome via its E3 ligase activity. The E3 ligase activity of MDM2 is dependent on its C-terminal RING domain. E3 ligases containing a RING domain are traditionally thought to catalyse the transfer of ubiquitin from their conjugating enzyme (E2) partner to the target protein, in the final step of the ubiquitination cascade. Various E2 enzymes have been shown to interact with their partner E3 ligases, yet evidence for the interaction between MDM2 and its partner E2, UbcH5α has not yet been shown. It has been reported that the reason for this lack of evidence is that the interaction between the two is highly unstable. Here I show that MDM2 forms a stable isolatable interaction with UbcH5α, the C-terminal tail of MDM2 is not necessary for this interaction. Although RING E3 ligases were not previously thought to interact with ubiquitin, preliminary evidence is emerging that suggests that this interaction is possible indeed I show that MDM2 and ubiquitin form a stable complex. I demonstrate that UbcH5α and ubiquitin both interact with the RING of MDM2, specifically the 20 most C-terminal amino acids of MDM2. My results show that both these proteins can bind this region of the RING simultaneously. I also highlight specific residues including tyrosine 489 and arginine 479 important for UbcH5α and ubiquitin binding respectively and the negative affect that these mutations have on the E3 ligase activity of MDM2 towards p53. Furthermore I show by limited proteolysis and hydrogen deuterium exchange that UbcH5α can be allosterically activated by MDM2. A novel peptide phage display technique linked to next generation sequencing was developed to further confirm an allosteric change and demonstrates that UbcH5α has different binding specificity for peptides when in a free or ligand bound conformation. MDM2 is a popular target for cancer therapeutics due to its dysregulation throughout many cancer types, including 30% of soft tissue sarcomas. Dissecting the mechanism of MDM2 function is an important step in identifying specific drugable interfaces on MDM2 and its interacting partners so that effective therapeutics can be designed.

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Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2016-11-17T16:34:28Z No. of bitstreams: 2 Dissertação - Sandro Antônio Gomes - 2014.pdf: 4737361 bytes, checksum: f0e85ab14dec862e8af685d9be88c2db (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-11-21T20:36:03Z (GMT) No. of bitstreams: 2 Dissertação - Sandro Antônio Gomes - 2014.pdf: 4737361 bytes, checksum: f0e85ab14dec862e8af685d9be88c2db (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-11-21T20:36:03Z (GMT). No. of bitstreams: 2 Dissertação - Sandro Antônio Gomes - 2014.pdf: 4737361 bytes, checksum: f0e85ab14dec862e8af685d9be88c2db (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-11-05 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / A rapid, sensitive and selective liquid chromatography tandem mass spectrometry (LC/MS-MS) bioanalytical method was developed and validated to quantify LQFM018 and LQFM030 prototypes obtained by molecular simplification nutlins (inhibitors of MDM2-p53 interaction) in mouse, rat and human plasma. The prototypes and the internal standard domperidone were extracted from the plasma samples by liquid-liquid extraction using methyl tert-butyl ether. The chromatographic analysis was performed using ACE ® C18 analytical column (5µm 100 x 4.6 mm) for a total time of 4 minutes. In MRM method transitions (m/z) were used: 349.138/191.10, 319.162/191.20 and 426.032/175.20 Daltons to LQFM018; LQFM030 and internal standard, respectively. The mobile phase consists of methanol-2 mM ammonium acetate containing 0.025% formic acid. The calibration curve was linear over the concentration range examined 10- 15000 ng/mL with r> 0.99 and the limit of quantitation of 10 ng/mL for both prototypes. For LQFM018, intra-assay precision was 0.8 to 7.3% and accuracy from 96.8 to 105.8%. And inter-assay to the precision was 2.3 and 6.6% and accuracy was 99.3 to 104.3%. The average recovery was 65.0% and the matrix effect between -11.2 to 2.1%. To LQFM030, intra-assay precision was 0.6 to 5.5% and accuracy from 95.5 to 111.3%. Inter-assay precision was 1.8 to 6.7%, and accuracy was 99.0 to 107.0%. The average recovery was 74.1% and the matrix effect from -7.9 to 1.5%. Recovery for internal standard was 61.3%. The prototypes were considered stable in the biological matrix and the solution proposed tests. Thus, the analytical method was developed and validated is capable to quantify traces of concentration in plasma. / Uma técnica bioanalítica rápida, sensível e seletiva que utiliza cromatografia líquida de alta eficiência acoplada à espectrometria de massas em sequência foi desenvolvida e validada para quantificar os protótipos LQFM018 e LQFM030 obtidos por simplificação molecular de nutlins (inibidores da interação MDM2-p53) em plasma de camundongo, rato e humano. Os protótipos e o padrão interno (PI) domperidona foram extraídos das amostras de plasma por meio de extração líquido-líquido utilizando éter metil-tercbutílico. A análise cromatográfica foi executada utilizando-se coluna analítica ACE® C18 (5 µm, 100 x 4,6 mm) num tempo total de 4 minutos. Na técnica MRM foram utilizadas as transições (m/z) 349,138/191,10; 319,162/191,20; 426,032/175,20 Daltons para LQFM018; LQFM030 e padrão interno, respectivamente. A fase móvel consistiu-se da mistura de metanol e acetato de amônio 2 mM contendo 0,025% de ácido fórmico. A curva de calibração foi linear em toda a faixa de concentração analisada 10-15000 ng/mL com r > 0,99 e o limite de quantificação de 10 ng/mL para ambos os protótipos. Para o LQFM018, a precisão intraensaio, foi de 0,8 a 7,3% e exatidão de 96,8 a 107,6% e a precisão interensaio foi de 2,3 a 6,6% e a exatidão foi de 99,3 a 104,3%. A recuperação média foi 65,0% e o efeito matriz entre -11,2 a 2,1%. Para LQFM030, a precisão intraensaio foi de 0,6 a 5,5% e exatidão de 95,5 a 111,3%. A precisão interensaio foi de 1,8 a 6,7% e exatidão foi de 99,0 a 107,0%. A recuperação média foi 74,1% e o efeito matriz entre -7,9 a 1,5%. A recuperação para o padrão interno foi 61,3%. Os protótipos se mostraram estáveis na matriz biológica e em solução nos ensaios propostos. Sendo assim, a técnica bioanalítica desenvolvida e validada é capaz de quantificar traços de concentrações dos protótipos LQFM018 e LQFM030 em plasma.

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