Browsing by Author "Figueiredo, Joana Patrícia Rodrigues"
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- Desenvolvimento de heterociclos pirimidínicos derivados de ácidos barbitúricos, como potenciais inibidores da xantina oxidasePublication . Figueiredo, Joana Patrícia Rodrigues; Silvestre, Samuel Martins; Almeida, Paulo Jorge da SilvaO desenvolvimento da biologia molecular e da química computacional entre outras áreas levou ao isolamento e caraterização de diversos alvos biológicos, sendo a xantina oxidase um destes casos. Esta enzima chamou a atenção dos químicos medicinais, uma vez que o aumento da sua atividade conduz a estados patológicos graves. A xantina oxidase é uma importante e versátil molibdoflavoproteina, envolvida no metabolismo de purinas, que catalisa a transformação da hipoxantina em xantina e da xantina em ácido úrico, com concomitante produção de espécies reativas de oxigénio. Além disso, níveis elevados de ácido úrico podem levar à gota, uma doença geralmente controlada utilizando o fármaco alopurinol, que é considerado o inibidor protótipo desta enzima. No entanto, a utilização deste fármaco na clínica está associada a efeitos adversos relevantes, o que levou ao desenvolvimento de novos inibidores. Os barbituratos constituíram, ao longo dos anos uma estrutura base importante para o desenvolvimento de novos fármacos, sendo as modificações no anel pirimidínico, em especial, alterações em C-5, as mais relevantes na descoberta de novas moléculas bioativas. Com este trabalho de dissertação, e com o objetivo de se obterem análogos ao alopurinol, pretendia-se a síntese de novos barbituratos ciclizados em C-5-C-6 do anel pirimidínico. Não se confirmando a ciclização pretendida, optou-se por realizar modificações ao anel pirimídico em C-5, obtendo-se derivados de hidrazinilpirimidinas, descritos com diversas atividades biológicas. Após a caraterização estrutural dos compostos sintetizados foram testadas as suas atividades biológicas, avaliando-se as suas capacidades de inibição da atividade da enzima xantina oxidase, e o seu potenciai anti-oxidante, anti-bacteriano e anti-proliferativo, numa linha celular do cancro da mama e numa linha celular normal. Adicionalmente, foram realizados estudos complementares in silico, nomeadamente estudos de docking molecular e de previsão de propriedades farmacocinéticas. Dos resultados obtidos, destaca-se o potencial de inibição da atividade da enzima xantina oxidase de duas hidrazinilpirimidinas, tal como a capacidade de outros destes compostos como anti-oxidantes e anti-bacterianos, sendo estes últimos resultados bastantes promissores.
- G-quadruplex targeting by ligands as a lung cancer therapeutic strategyPublication . Figueiredo, Joana Patrícia Rodrigues; Cruz, Carla Patrícia Freire Madeira Alves da; Mergny, Jean-LouisG-quadruplexes (G4s) are non-canonical four-stranded nucleic acid secondary structures that can form in guanine-rich regions in the human genome and transcriptome. DNAs G4s are found in critical regulatory regions of the human genome such as the end of telomers and the promoter regions of several oncogenes. These structures have been implicated in the control of key cellular events including replication, transcription, genome stability, and epigenetic regulation. On the other hand, RNAs G4 are found within non-coding regions and are implicated in crucial RNA metabolism events, including the regulation of RNA processing and translation. Due to their biological relevance and structural features, G4s are considered suitable therapeutic targets. The prevalence of G4 folding in the cancer context and its stabilization can be used as an anti-tumor treatment strategy in different cancer types. Several synthetic small molecules with high specificity for G4s DNAs and RNAs relative to double-stranded DNA (G4 ligands) have been developed and evaluated for their therapeutic potential. Over the years, thousands of small molecules have been reported as G4 ligands with in vitro and/or in vivo anti-tumor activity. Often, but not always, G4 ligands have a planar aromatic core for π-π stacking with G-quartet and a positively charged or basic group(s) to interact with the phosphate backbone of nucleic acid. Different scaffolds containing these chemical features, such as quindolines, acridines, naphthalene diimides, and phenanthrolines have proved to have G4 binding properties and anti-tumor activities. These small organic molecules interacting with G4s may affect cancer cell growth in different ways, including inhibition of telomerase or interference with telomere function, modulation of oncogenes expression by stabilizing G4 in their promoter and regulating microRNA biogenesis (miRNAs) by stabilizing G4 in microRNA precursors (pre-miRNAs). Lung cancer (LC) is the leading cause of cancer-related death worldwide and is divided into two major subtypes, small-cell lung cancer (SCLC), and non-small cell lung cancer (NSCLC). Although therapeutic options such as surgery and chemoradiotherapy prove efficacy in the early stage of the disease, their effectiveness is limited in advanced LC where most patients are diagnosed. Although immune checkpoint inhibitors have shown promising clinical outcomes, the high level of molecular heterogeneity in LC makes treatment difficult and new therapeutic strategies are needed. The prevalence of G4 formation in important regulatory systems in LC including proto-oncogenes (e.g. MYC, BCL-2, KRAS, KIT, and VEGF) and telomere regions make then attractive targets. Similarly to other cancer types, G4 ligand-induced stabilization promotes changes in telomere maintenance and decreases oncogene expression levels. Ongoing efforts have been made to harness G4 ligands for inducing G4 stabilization in the LC context. Different classes of G4s ligands may lead to in vitro antiproliferative activity and, in some cases, in vivo anti-tumor effects. Overall, this thesis aims to develop novel G4 ligands derivatived from 1,10-phenanthroline, which can bind/stabilize G4 structures present in important regulatory regions in LC acting as anti-tumor agents. The chemical synthesis and screening of heterocyclic compounds featuring phenyl, quinoline, naphthalene, acridines, and phenanthroline scaffolds were assessed to explore their stabilization effect towards G4 structures in c-MYC, KRAS and VEGF promoters, human telomeric motif, and pre-MIR150 RNA G4. The acridine moiety exhibited the highest G4 stabilization, followed by phenanthroline. Additionally, most compounds proved greater anti-tumor efficacy in LC cells than in non-malignant cells. Subsequently, a novel class of phenanthroline derivatives was synthesized and structurally characterized. In addition, to assess their potential to bind/stabilize G4-forming sequences, several biophysical techniques, including förster resonance energy transfer (FRET) melting assay, circular dichroism (CD) studies and fluorescence titrations were employed. The in vitro anti-tumor activity of these compounds has been evaluated against LC cell lines via MTT assay. Sixteen derivatives of 1,10-phenanthroline were synthesized, featuring substituents at positions 2 and 9 with either amine or amide side chains, by direct condensation reactions. Among these derivatives, those bearing methoxyaniline and ethane-diaminium side chains have been shown to stabilize various G4 topologies derived from human telomere sequences. These included hybrid, parallel, and antiparallel G4 structures, with a marked preference for the hybrid topology of the F21T sequence (ΔTm =7.2 – 12 °C, by FRET melting). Additionally, the ligands displayed promising cytotoxic profiles and exhibited greater efficacy against A549 than H1299 LC cells. Additionally, novel ten 1,10-phenanthroline-2,9-bistriazoles derivatives were synthesized via cooper catalyze azide/alkyne cycloaddition reactions. Through biophysical assessment, three promising G4 ligands against KRAS G4 sequences were identified, exhibiting increases in melting temperature (ΔTm =4.7 – 11.2 °C, by FRET melting) and binding affinities ranging between 10-6 and 10-9 M. Evaluation of anti-tumor activity revealed that the compound bearing a phenyl ring linked to the triazole moiety exhibited a potent inhibitory effect on A549 and H1299 cancer cell growth (IC50 = 14.6 and 10.9 μM, respectively). Overall, the experimental results demonstrated that the synthesized compounds have the potential to bind/stabilize different G4-forming sequences and may serve as promising scaffolds for the development of G4 ligands. Additionally, this study provides invaluable insights into the structure-activity relationships for this class of compounds to maximize their activity. Finally, to the best of our knowledge, we have identified, for the first time, a G4-forming region within the human precursor of MIR150 (pre-MIR150). The G4-forming region folds into a parallel RNA G4 and has the potential to interact with nucleolin (NCL), which is a protein overexpressed on the cell surface of cancer cells involved in several cellular processes, including tumorigenesis, angiogenesis, and signaling pathways. The thermal stability of G4 increases in the presence of a commercial phenanthroline ligand (PhenDC3), and the formation of ternary complex G4/PhenDC3/NCL was observed. Moreover, the G4 structure in pre-MIR150 recognized NCL-positive LC cancer cells, and liquid biopsies when fluorescently labeled, can be used as a probe.