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Tetraoxane-based tumor-activated prodrug strategy to target intracellular labile ferrous iron
Publication . Magalhães e Silva, Diogo; Moreira, Rui Ferreira Alves; Lopes, Francisca da Conceição; Rodrigues, Cecília Maria Pereira
Iron is a critical component in many cellular functions including DNA replication and repair, essential for cell vitality. However, iron metabolism dysregulation is associated with infectious diseases and cancer. Despite clinical precedent for iron-dependent pharmacology in antimalarial therapy with endoperoxides, tumour targeting strategies designed to exploit changes in iron homeostasis remain poorly studied. In this work, two tetraoxane-based Fe(II)-activable drug conjugate (FeADC) systems were developed as targeted therapy. Type I FeADCs spontaneously releases the cytotoxic payload after reaction with Fe(II). Ten type I FeADCs were obtained with different cytotoxic payloads for malaria and cancer. Type I FeADCs coupled with antimalarials were potent on the nanomolar range in chloroquine-sensitive strain. Type I FeADCs conjugated with doxorubicin showed high efficiency in reducing cellular viability in Fe(II)-rich cancer cell lines. Importantly, this FeADC was significantly less toxic to non-tumorigenic cells than the parent drug. Type I FeADC activity against Fe(II)-rich cancer cell lines was abrogated when incubated with the iron chelator desferoxamine (DFO). Type II FeADCs, in addition to the Fe(II)-activable tetraoxane scaffold shared with the type I system, also contains an unique structural feature that enables reaction with another metabolite, which is elevated in cancer. This feature only becomes available after endoperoxide activation by Fe(II), thus increasing the potential for improved selectivity. We have prepared a type II FeADC that showed high efficiency in reducing cellular viability in Fe(II)-rich cancer cell lines. Type II FeADC selectivity was evaluated in AML12 cells, being 200-fold less toxic than the parent drug. Importantly, our results also demonstrate that type II FeADC induced cell death is iron-dependent. In summary, the tetraoxane scaffold was validated as a drug delivery system for diseases with iron metabolism dysregulation.
Facile access to structurally diverse antimalarial indoles using a one‐pot A3 coupling and domino cyclization approach
Publication . Silva, Gustavo Da; Luz, André F. S.; Duarte, Denise; Fontinha, Diana; Silva, Vera L. M.; Almeida Paz, Filipe A.; Madureira, Ana Margarida; Simões, Sandra; Prudêncio, Miguel; Nogueira, Fátima; Silva, Artur M. S.; Moreira, Rui
A multistep and diversity-oriented synthetic route aiming at the A3 coupling/domino cyclization of o-ethynyl anilines, aldehydes and s-amines is described. The preparation of the corresponding precursors included a series of transformations, such as haloperoxidation and Sonogashira cross-coupling reactions, amine protection, desilylation and amine reduction. Some products of the multicomponent reaction underwent further detosylation and Suzuki coupling. The resulting library of structurally diverse compounds was evaluated against blood and liver stage malaria parasites, which revealed a promising lead with sub-micromolar activity against intra-erythrocytic forms of Plasmodium falciparum. The results from this hit-to-lead optimization are hereby reported for the first time.
Recovery of depleted miR-146a in ALS cortical astrocytes reverts cell aberrancies and prevents paracrine pathogenicity on microglia and motor neurons
Publication . Barbosa, Marta; Gomes, Cátia; Sequeira, Catarina; Gonçalves-Ribeiro, Joana; Pina, Carolina Campos; Carvalho, Luís A.; Moreira, Rui; Vaz, Sandra H.; Vaz, Ana Rita; Brites, Dora
Reactive astrocytes in Amyotrophic Lateral Sclerosis (ALS) change their molecular expression pattern and release toxic factors that contribute to neurodegeneration and microglial activation. We and others identified a dysregulated inflammatory miRNA profile in ALS patients and in mice models suggesting that they represent potential targets for therapeutic intervention. Such cellular miRNAs are known to be released into the secretome and to be carried by small extracellular vesicles (sEVs), which may be harmful to recipient cells. Thus, ALS astrocyte secretome may disrupt cell homeostasis and impact on ALS pathogenesis. Previously, we identified a specific aberrant signature in the cortical brain of symptomatic SOD1-G93A (mSOD1) mice, as well as in astrocytes isolated from the same region of 7-day-old mSOD1 mice, with upregulated S100B/HMGB1/Cx43/vimentin and downregulated GFAP. The presence of downregulated miR-146a on both cases suggests that it can be a promising target for modulation in ALS. Here, we upregulated miR-146a with pre-miR-146a, and tested glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS) for their immunoregulatory properties. VS was more effective in restoring astrocytic miR-146a, GFAP, S100B, HMGB1, Cx43, and vimentin levels than GUDCA, which only recovered Cx43 and vimentin mRNA. The miR-146a inhibitor generated typical ALS aberrancies in wild type astrocytes that were abolished by VS. Similarly, pre-miR-146a transfection into the mSOD1 astrocytes abrogated aberrant markers and intracellular Ca2+ overload. Such treatment counteracted miR-146a depletion in sEVs and led to secretome-mediated miR-146a enhancement in NSC-34-motor neurons (MNs) and N9-microglia. Secretome from mSOD1 astrocytes increased early/late apoptosis and FGFR3 mRNA in MNs and microglia, but not when derived from pre-miR-146a or VS-treated cells. These last strategies prevented the impairment of axonal transport and synaptic dynamics by the pathological secretome, while also averted microglia activation through either secretome, or their isolated sEVs. Proteomic analysis of the target cells indicated that pre-miR-146a regulates mitochondria and inflammation via paracrine signaling. We demonstrate that replenishment of miR-146a in mSOD1 cortical astrocytes with pre-miR-146a or by VS abrogates their phenotypic aberrancies and paracrine deleterious consequences to MNs and microglia. These results propose miR-146a as a new causal and emerging therapeutic target for astrocyte pathogenic processes in ALS.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/MED-QUI/30021/2017