Modulation of human apurinic/apyrimidinic endonuclease 1 (APE1) functions for breast cancer therapy

Tese de doutoramento, Farmácia (Toxicologia), Universidade de Lisboa, Faculdade de Farmácia, 2016DNA repair is required for the maintenance of genome stability. In the last years DNA repair pathways have emerged as important targets for cancer therapy. Since standard anticancer agents are mainly DNA-damaging drugs, its combination with DNA repair inhibitors may contribute to improve treatment outcomes. Among the multiple effectors involved in DNA repair, the multifunctional base excision repair (BER) protein apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most attractive druggable targets in this field. APE1 is the major endonuclease in BER participating in the repair of different DNA lesions including toxic abasic sites. In addition to the DNA repair activity, APE1 also acts independently as a reduction/oxidation signalling protein modulating the activation and DNA binding ability of several transcription factors implicated in cell survival and tumour promotion and progression. In this context, this thesis is focused on the combination of APE1 pharmacological inhibitors with conventional anticancer agents in the highly aggressive human breast cancer MDA-MB-231 cell line. Endonuclease activity has been the most studied function of APE1 in cancer therapy. Methoxyamine (MX), a commercially available indirect inhibitor of APE1 DNA repair function, was evaluated in combination with doxorubicin (Dox) in MDA-MB-231 cells. The chemotherapeutic drug Dox is widely used in the treatment of advanced breast cancer and may act, in part, by inducing oxidative DNA damage. MX had little effects in viability and colony formation of MDA-MB-231 cells. However, a significant increase in the frequency of micronucleated cells and an alteration in the pattern of micronuclei distribution were observed suggesting an increase in Dox genotoxicity. The differential results obtained in terms of cytotoxicity and genotoxicity showed that a therapeutic strategy based on APE1 inhibition is likely to have no relevance for the improvement of outcomes of Dox treatment. Although several putative inhibitors of APE1 endonuclease activity have been reported they still lack potential to be translated to the clinical setting. Therefore, in this thesis a structure-based virtual screening (SBVS) study based on molecular docking analysis of National Cancer Institute (NCI) database of compounds was performed to identify novel small-molecule inhibitors of APE1. The evaluation of SBVS study most promising compounds in a fluorescence-based APE1 endonuclease activity assay revealed three APE1 inhibitors. Compound 22 was a potent APE1 inhibitor showing inhibitory effects at nanomolar concentrations, while compounds 37 and 41 inhibited the enzyme in the micromolar range. These novel scaffolds for the design of more potent APE1 inhibitors did not affect the viability of non-tumourigenic human breast epithelial MCF10A cell line highlighting their promising features. The importance of APE1 modulation is beyond its functions in DNA repair. Therefore, E3330, a commercially available inhibitor of APE1 redox function, was also evaluated as single agent and in combination with the taxane drug docetaxel (DTX) in MDA-MB- 231 cells. DTX has anti-migratory and anti-angiogenic effects and is frequently used in advanced breast cancer refractory to anthracycline-based regimens. Consequently, relevant endpoints of cell migration and invasion were studied in addition to cell viability, proliferation and cell cycle profile assessment. Minor effects were observed in cell proliferation. However, E3330 alone significantly reduced the collective cell migration evaluated by the wound-healing assay without affecting chemotaxis and chemoinvasion. The combination of E3330 with DTX significantly decreased invasion of MDA-MB-231 cells suggesting a potential therapeutic role in metastatic breast cancer. The results described in this work emphasise the importance of preclinical studies of APE1 functions in cancer therapy and highlight the potential of novel drug combinations based on APE1 inhibitors reinforcing the role of targeting DNA repair in cancer treatment

On the development of selective chelators for Cadmium: Synthesis, structure and chelating properties of 3-((5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)amino)benzo[d]isothiazole 1,1-dioxide, a novel Thiadiazolyl Saccharinate

Aquatic contamination by heavy metals is a major concern for the serious negative consequences it has for plants, animals, and humans. Among the most toxic metals, Cd(II) stands out since selective and truly efficient methodologies for its removal are not known. We report a novel multidentate chelating agent comprising the heterocycles thiadiazole and benzisothiazole. 3-((5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)amino)benzo[d]isothiazole 1,1-dioxide (AL14) was synthesized from cheap saccharin and characterized by different techniques, including single crystal X-ray crystallography. Our studies revealed the efficiency and selectivity of AL14 for the chelation of dissolved Cd(II) (as compared to Cu(II) and Fe(II)). Different spectral changes were observed upon the addition of Cd(II) and Cu(II) during UV-Vis titrations, suggesting different complexation interactions with both metals.UIDB/04326/2020, UIDB/04564/2020, ALG-01-0145-FEDER-022121, SFRH/BD/130407/2017info:eu-repo/semantics/publishedVersio

Preparação de lectinas de Moringa oleifera com atividade coagulante

info:eu-repo/semantics/publishedVersio

Understanding the clinical spectrum of complicated Plasmodium vivax malaria: a systematic review on the contributions of the Brazilian literature

The resurgence of the malaria eradication agenda and the increasing number of severe manifestation reports has contributed to a renewed interested in the Plasmodium vivax infection. It is the most geographically widespread parasite causing human malaria, with around 2.85 billion people living under risk of infection. The Brazilian Amazon region reports more than 50% of the malaria cases in Latin America and since 1990 there is a marked predominance of this species, responsible for 85% of cases in 2009. However, only a few complicated cases of P. vivax have been reported from this region. A systematic review of the Brazilian indexed and non-indexed literature on complicated cases of vivax malaria was performed including published articles, masters' dissertations, doctoral theses and national congresses' abstracts. The following information was retrieved: patient characteristics (demographic, presence of co-morbidities and, whenever possible, associated genetic disorders); description of each major clinical manifestation. As a result, 27 articles, 28 abstracts from scientific events' annals and 13 theses/dissertations were found, only after 1987. Most of the reported information was described in small case series and case reports of patients from all the Amazonian states, and also in travellers from Brazilian non-endemic areas. The more relevant clinical complications were anaemia, thrombocytopaenia, jaundice and acute respiratory distress syndrome, present in all age groups, in addition to other more rare clinical pictures. Complications in pregnant women were also reported. Acute and chronic co-morbidities were frequent, however death was occasional. Clinical atypical cases of malaria are more frequent than published in the indexed literature, probably due to a publication bias. In the Brazilian Amazon (considered to be a low to moderate intensity area of transmission), clinical data are in accordance with the recent findings of severity described in diverse P. vivax endemic areas (especially anaemia in Southeast Asia), however in this region both children and adults are affected. Finally, gaps of knowledge and areas for future research are opportunely pointed out

Analysis of shared heritability in common disorders of the brain

ience, this issue p. eaap8757 Structured Abstract INTRODUCTION Brain disorders may exhibit shared symptoms and substantial epidemiological comorbidity, inciting debate about their etiologic overlap. However, detailed study of phenotypes with different ages of onset, severity, and presentation poses a considerable challenge. Recently developed heritability methods allow us to accurately measure correlation of genome-wide common variant risk between two phenotypes from pools of different individuals and assess how connected they, or at least their genetic risks, are on the genomic level. We used genome-wide association data for 265,218 patients and 784,643 control participants, as well as 17 phenotypes from a total of 1,191,588 individuals, to quantify the degree of overlap for genetic risk factors of 25 common brain disorders. RATIONALE Over the past century, the classification of brain disorders has evolved to reflect the medical and scientific communities' assessments of the presumed root causes of clinical phenomena such as behavioral change, loss of motor function, or alterations of consciousness. Directly observable phenomena (such as the presence of emboli, protein tangles, or unusual electrical activity patterns) generally define and separate neurological disorders from psychiatric disorders. Understanding the genetic underpinnings and categorical distinctions for brain disorders and related phenotypes may inform the search for their biological mechanisms. RESULTS Common variant risk for psychiatric disorders was shown to correlate significantly, especially among attention deficit hyperactivity disorder (ADHD), bipolar disorder, major depressive disorder (MDD), and schizophrenia. By contrast, neurological disorders appear more distinct from one another and from the psychiatric disorders, except for migraine, which was significantly correlated to ADHD, MDD, and Tourette syndrome. We demonstrate that, in the general population, the personality trait neuroticism is significantly correlated with almost every psychiatric disorder and migraine. We also identify significant genetic sharing between disorders and early life cognitive measures (e.g., years of education and college attainment) in the general population, demonstrating positive correlation with several psychiatric disorders (e.g., anorexia nervosa and bipolar disorder) and negative correlation with several neurological phenotypes (e.g., Alzheimer's disease and ischemic stroke), even though the latter are considered to result from specific processes that occur later in life. Extensive simulations were also performed to inform how statistical power, diagnostic misclassification, and phenotypic heterogeneity influence genetic correlations. CONCLUSION The high degree of genetic correlation among many of the psychiatric disorders adds further evidence that their current clinical boundaries do not reflect distinct underlying pathogenic processes, at least on the genetic level. This suggests a deeply interconnected nature for psychiatric disorders, in contrast to neurological disorders, and underscores the need to refine psychiatric diagnostics. Genetically informed analyses may provide important "scaffolding" to support such restructuring of psychiatric nosology, which likely requires incorporating many levels of information. By contrast, we find limited evidence for widespread common genetic risk sharing among neurological disorders or across neurological and psychiatric disorders. We show that both psychiatric and neurological disorders have robust correlations with cognitive and personality measures. Further study is needed to evaluate whether overlapping genetic contributions to psychiatric pathology may influence treatment choices. Ultimately, such developments may pave the way toward reduced heterogeneity and improved diagnosis and treatment of psychiatric disorders

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo