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Design and synthesis of small molecule modulators of p53

Publication . Ribeiro, Carlos Jorge Azevedo Costa, 1980-; Santos, Maria Manuel Duque Vieira Marques dos; Moreira, Rui, 1960-; Rodrigues, Cecília M. P., 1968-

Among the tumor suppressor genes, p53 is one of the most studied. It is widely regarded as the “guardian of the genome”, playing a pivotal part in the preservation of genomic integrity by regulating cell cycle, apoptosis, DNA repair, senescence and angiogenesis, and consequently has a major role in carcinogenesis. The function played by p53 in tumor suppression is further highlighted by the fact that direct inactivation of this gene occurs in more than 50% of malignancies. In addition, in tumors that retain wild type p53 status, its function is usually inactivated by overexpression of negative regulators, primarily murine double minute-2 (MDM2), mainly through MDM2 gene amplification or by activity loss of MDM2 inhibitor ARF. Hence, restoring p53 function in cancer cells represents a valuable anticancer approach. Several strategies are being developed, and in particular targeting p53-MDM2 interaction has emerged as a promising viable approach when dealing with cancers that retain wild type p53 function. These two proteins regulate each other through an autoregulatory feedback loop: activation of p53 stimulates the transcription of MDM2, which in turn binds to the Nterminal transactivation domain of p53, disabling its transcriptional function. p53- MDM2 interaction inhibitors share common structural features: a rigid heterocyclic scaffold with three lipophilic groups that mimic the three pivotal p53 Phe19, Trp23 and Leu26 that interact with MDM2. Seven compounds have already entered clinical trials. The main goal of this PhD thesis was to develop new anticancer agents containing a spirooxindole scaffold with different spiro five-membered rings: isoxazoline, oxadiazoline, and triazolines. The spirocycle can potentially function as the rigid heterocyclic scaffold, from which three lipophilic groups are projected to mimic the three pivotal p53 amino acids. This work followed three major strategies: synthesis of spirooxindole derivatives by 1,3 dipolar cycloaddition; biological evaluation of the compounds synthesized; and stability assessment. Overall this PhD thesis contributed with three new families of spirooxindoles with in vitro anti-cancer activity. The most active derivative possessed a GI50 of 1.72 μM in HCT116 p53(+/+) cell line. Furthermore their ability to disrupt the interaction between p53 and MDM2 was confirmed by implementing a cell-base in vitro bimolecular fluorescence complementation assay (BiFC) and the apoptotic outcome verified by immunoblotting analysis and luminescent caspase 3/7 activity assay.

2015Doctoral thesis

Open access

Evaluation of neuroprotective and neurotoxic functions of different microglia phenotypes in Alzheimer’s disease onset and progression, using in vitro and in vivo models

Publication . Caldeira, Cláudia Alexandra Oliveira Lopes, 1973-; Brites, Dora, 1951-; Borralho, Adelaide Maria Afonso Fernandes

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive cognitive decline, and accumulation of amyloid-β (Aβ) in senile plaques that are associated with inflammatory molecules released by activated glial cells. Among glial cells, microglia that constitute the intrinsic defense system within the central nervous system, may become activated by Aβ aggregates and develop critical neuroprotective and neurotoxic functions with implications in AD onset and progression. Actually,inflammation has been associated with AD, although nonsteroidal anti-inflammatory drugs have not proven efficacy in halting the progression of the disease, reason why they are indicated as beneficial only in the very early stages of AD. Therefore, features of neuroinflammation and associated microglia phenotypes are still open questions in the understanding of AD pathogenesis and neurodegeneration. Another important issue is the association of AD with ageing and the observation of few microglia and accumulation of dystrophic/desensitized microglia in samples from AD old subjects, strongly suggesting their progressive degeneration and diminished replenishment. Investigation of the multiple activated states of microglia when stressed by Aβ, in particular the increased inflammatory status of microglia with aging, referred as primed reactive, or sensitized cell, or in opposite the proposed state of irresponsive aged microglia, are challenged issues once there are no appropriate procedures to isolate degenerative and senescent microglia for experimentation. Therefore, the global aim of this thesis was to explore how different microglia phenotypes and ageing may influence Alzheimer’s disease (AD) pathogenesis and neuroinflammation, by unraveling their associated neuroprotective and neurotoxic functions in in vitro and in vivo models. To achieve these objectives, we first (i) developed an experimental model to naturally age primary microglia, which allowed the evaluation of microglia defensive mechanisms (e.g. migration, phagocytosis, autophagy), of a panoply of inflammatory mediators and of senescence-associated markers, in an acutely isolated and activated microglia [2 days in vitro (2 DIV)] and an aged cell culture (16 DIV). With the characterization of such differently aged microglia, we (ii) progressed towards the assessment of their responsiveness when treated with 1000 nM of Aβ1-42 for 24 h. Finally, in an attempt to translate our in vitro research into the triple transgenic AD (3xTg-AD) mice model, we decided (iii) to explore the inflammatory status of the hippocampus and cortex in animals with 3-, 6- and 9 months-old, by assessing microglia phenotypes, as well as the expression of inflammatory cytokines and microRNAs. We observed in our first study, already published (Caldeira et al Front Cell Neurosci 2014), that (i) in vitro aged microglia switch from a predominant reactive phenotype into cells that although not showing decreased survival, revealed increased dormancy, with morphometric features characteristic of ramified morphology, together with compromised migration, impaired autophagy, reduced phagocytosis, decreased expression of inflamma-miRNAs, and increased presence of senescence-associated markers. In the second study, recently submitted to publication by invitation (Caldeira et al Front Aging Neurosci), using the ageing microglia model and Aβ1-42 treatment, we observed that (ii) Aβ treatment caused soma volume increase and process shortening compatible with activated microglia, in both 2 DIV and 16 DIV cells, together with impairment of neuroprotective functions, namely phagocytosis and migration abilities, as well as autophagy, in in vitro aged microglia. Interestingly, Aβ led to an increased expression of senescence-like associated markers in 2 DIV microglia, similarly to those of the aged cells. Age-dependent changes included the decrease in the expression of inflammatory mediators and surface receptors, together with the reduction of CD11b+ cells and gain of CD86+ microglia and downregulation of miR 155 and miR-124. Lastly, in our in vivo studies at the early-AD stage in the 3xTg-AD at 3 months-old, when Aβ accumulates intraneurally, we observed a downregulation of some activated microglia markers, as well as both typical M1 pro-inflammatory and M2 anti-inflammatory/damage resolution markers. Interestingly, miR-155 revealed to be early upregulated and its increase was sustained at 9 months-old, when extracellular Aβ accumulation is an AD hallmark. At this stage, increase of HMGB1 and decrease of both miR-146a and miR-124 expression is apparent. Curiously, when looking at miR-155 target gene expression we observed new immune-associated molecules that were differently expressed in the 3xTgAD animals by comparison with the wild type mice, both at 3-months and 9-months of age, which will be the subject of study in future works. We may then conclude that the aged in vitro microglia model is very suitable to unravel microglia phenotypic alterations that may explain different cell reactivity in neurodegenerative disorders associated with neuroinflammation and diverse states of disease progression, thus requiring diverse disease-modifying therapies depending on the inflammatory status. We further demonstrate that Aβ induce a heterogeneous population of microglia subtypes instead of only M1 and M2 polarization and that their distribution are age-dependent and influenced by microglia activation state. The increased expression of miR-155 in very early stages of AD in the 3xTg-AD animal model, to be confirmed in AD patients, may additionally reveal as a sensitive biomarker with predictive value if detected in the peripheral blood. The work developed in the present thesis contributed to better define microglial activation phenotypes, in particular the notion of “good” or “bad” states during AD pathogenesis, while identified new targets to be modulated and assessed as predictive biomarkers, with potential relevance for diagnosis and therapeutic tools for developing innovative medicines.

2017Doctoral thesis

Open access