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Assembly of peptidoglycan fragments: a synthetic challenge
Publication . Queda, Fausto; Covas, Gonçalo; Filipe, Sérgio R.; Marques, M. Manuel B.; LAQV@REQUIMTE; DQ - Departamento de Química; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; Molecular, Structural and Cellular Microbiology (MOSTMICRO); Molecular Diversity Preservation International (MDPI)
Peptidoglycan (PGN) is a major constituent of most bacterial cell walls that is recognized as a primary target of the innate immune system. The availability of pure PGN molecules has become key to different biological studies. This review aims to (1) provide an overview of PGN biosynthesis, focusing on the main biosynthetic intermediates; (2) focus on the challenges for chemical synthesis posed by the unique and complex structure of PGN; and (3) cover the synthetic routes of PGN fragments developed to date. The key difficulties in the synthesis of PGN molecules mainly involve stereoselective glycosylation involving NAG derivatives. The complex synthesis of the carbohydrate backbone commonly involves multistep sequences of chemical reactions to install the lactyl moiety at the O-3 position of NAG derivatives and to control enantioselective glycosylation. Recent advances are presented and synthetic routes are described according to the main strategy used: (i) based on the availability of starting materials such as glucosamine derivatives; (ii) based on a particular orthogonal synthesis; and (iii) based on the use of other natural biopolymers as raw materials.
Neurotoxic effects of MPTP on mouse cerebral cortex: Modulation of neuroinflammation as a neuroprotective strategy
Publication . Mendes, Mariana Oliveira; Rosa, Alexandra Isabel; Carvalho, Andreia Neves; Nunes, Maria João; Dionísio, Pedro; Rodrigues, Elsa; Costa, Daniela; Duarte-Silva, Sara; Maciel, Patrícia; Rodrigues, Cecília Maria Pereira; Gama, Maria João; Castro-Caldas, Margarida; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; Academic Press Inc | Elsevier Science
Parkinson's disease (PD) is a progressive neurological disorder, mainly characterized by the progressive loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc) and by the presence of intracellular inclusions, known as Lewy bodies. Despite SNpc being considered the primary affected region in PD, the neuropathological features are confined solely to the nigro-striatal axis. With disease progression other brain regions are also affected, namely the cerebral cortex, although the spreading of the neurologic damage to this region is still not completely unraveled. Tauroursodeoxycholic acid (TUDCA) is an endogenous bile acid that has been shown to have antioxidant properties and to exhibit a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice model of PD. Moreover, TUDCA anti-inflammatory properties have been reported in glial cells, making it a prominent therapeutic agent in PD. Here, we used C57BL/6 mice injected with MPTP in a sub-acute paradigm aiming to investigate if the neurotoxic effects of MPTP could be extended to the cerebral cortex. In parallel, we evaluated the anti-oxidant, neuroprotective and anti-inflammatory effects of TUDCA. The anti-inflammatory mechanisms elicited by TUDCA were further dissected in microglia cells. Our results show that MPTP leads to a decrease of ATP and activated AMP-activated protein kinase levels in mice cortex, and to a transient increase in the expression of antioxidant downstream targets of nuclear factor erythroid 2 related factor 2 (Nrf-2), and parkin. Notably, MPTP increases pro-inflammatory markers, while down-regulating the expression of the anti-inflammatory protein Annexin-A1 (ANXA1). Importantly, we show that TUDCA treatment prevents the deleterious effects of MPTP, sustains increased levels of antioxidant enzymes and parkin, and most of all negatively modulates neuroinflammation and up-regulates ANXA1 expression. Additionally, results from cellular models using microglia corroborate TUDCA modulation of ANXA1 synthesis, linking inhibition of neuroinflammation and neuroprotection by TUDCA.
Endothelial aquaporins and hypomethylation potential implications for atherosclerosis and cardiovascular disease
Publication . Silva, Inês Vieira da; Barroso, Madalena; Moura, Teresa; Castro, Rita; Soveral, Graça; DQ - Departamento de Química; MDPI - Multidisciplinary Digital Publishing Institute
Aquaporins (AQPs) are transmembrane channels that facilitate water and glycerol permeation through cell membranes. Recently, the water channel AQP1 was suggested to contribute to endothelial homeostasis and cardiovascular health. Less is known about endothelial aquaglyceroporins expression and its implication in cardiovascular disease (CVD). We have previously used cultured human endothelial cells under a hypomethylating environment to study endothelial dysfunction and activation, a phenotype implicated in the establishment of atherosclerosis and CVD. Here, we used the same cell model to investigate aquaporin’s expression and function in healthy or pro-atherogenic phenotype. We first confirmed key features of endothelium dysfunction and activation in our cell model, including an augmented endothelial transmigration under hypomethylation. Subsequently, we found AQP1 and AQP3 to be the most predominant AQPs accounting for water and glycerol fluxes, respectively, in the healthy endothelium. Moreover, endothelial hypomethylation led to decreased levels of AQP1 and impaired water permeability without affecting AQP3 and glycerol permeability. Furthermore, TNF-α treatment-induced AQP1 downregulation suggesting that the inflammatory NF-κB signaling pathway mediates AQP1 transcriptional repression in a pro-atherogenic endothelium, a possibility that warrants further investigation. In conclusion, our results add further support to AQP1 as a candidate player in the setting of endothelial dysfunction and CVD.
Cyclopentenones: synthesis and biological evaluation in human cancer cells
Publication . Andrade, Késsia Hapuque Santos de; Afonso, Carlos; Gomes, Rafael
Cyclopentenones (CPs) are cyclic ketones whose structure consists of an α, β-unsaturated carbonyl group attached to a membered ring. Many compounds which contain the cyclopentenone ring are biologically active, for example prostaglandins which have antiviral, anti-inflammatory, antifungal, apoptotic and antitumor properties
It has been shown that most of these properties are due to the Michael acceptor character of the α, β-unsaturated carbonyl, group which can react with various nucleophiles. An example is the reaction with Glutathione, which acts on detoxification mechanisms, contributing positively to combat resistance to antitumor drugs. However, this reactivity of these Michael acceptors makes them promiscuous to unwanted reactions with macromolecules critical to cells.
Based on this promiscuity, our goal was to synthesize a small library of compounds with poor Michael acceptor character and which have cytotoxicity in tumour cells of rectal colon, breast and lung. Thus, we prepared different substituted CPs in positions 2 and 4, basing our reactions on methods already described, including by our group.
From these synthesized CPs, 2-hydroxy-4-substituted CPs (with lower Michael acceptor character) showed considerable cytotoxic activity, while the others, showed no sufficiently cytotoxicity. The CP with a very good Michael acceptor character formed adducts with Glutathione in stability assays, while the 2-morpholine-4-substituted CPs (with lower Michael acceptor character) showed no formation of adducts with Glutathione.
Although the work is still in progress we can infer that CPs can act by a mechanism of action different from those known by alkylation to the α, β-unsaturated carbonyl group, and this is a great advance in the field of medicinal chemistry for the discovery of new drugs with better cancer performance and fewer side effects.
Modulation of HMGB1 in reactive and irresponsive microglia treated with amyloid-β peptide
Publication . Nunes, Maria Carlos Cortegaça da Cruz; Brites, Dora; Vaz, Ana
Neuroinflammation is associated with microglia reactivity in Alzheimer’s disease (AD) in the presence of amyloid-β (Aβ) peptide. Interestingly, expression and activation of the alarmin high mobility group box 1 (HMGB1) were recently associated with neuroinflammation in the aged brain and noticed to contribute to AD pathology. Recent results from our group indicate that Aβ upregulates HMGB1 mRNA in a culture model of reactive microglial cells.
Here we aimed to assess the effects of modulating HMGB1 gene expression in young/reactive and aged/irresponsive microglia, when stimulated by Aβ. Therefore, mixed glial cultures were obtained from CD1 mice pups. Microglia were isolated, maintained for 3 (young) or 16 (aged) days in vitro (DIV) and transiently transfected to silence (si) or overexpress (p) HMGB1, respectively. Cells were then treated (or not) with a mixture of Aβ species (1000 nM). We evaluated: autophagy (LC3II/I and Beclin-1); phagocytosis (MFG-E8); phenotypic markers of pro-inflammatory (TLR2/TLR4/NF-kB signalling pathway, NLRP3-inflammasome/IL-18 complex, TNF-α, iNOS and MHCII) or anti-inflammatory (IL-10 and Arginase-1, CX3CR1) stages; inflamma-miRNAs (miR-155, miR-124 and miR-146a); and senescence.
Aβ induced HMGB1 expression in 3 DIV/young microglia and promoted autophagy, M1 polarization, as well as senescence, while reducing MFG-E8-associated phagocytosis. Efficient siHMGB1 abrogated all these effects, with exception of NLRP3 mRNA upsurge. Contrarily, Aβ couldn’t trigger HMGB1 upregulation in 16DIV/aged microglia, only achieved in pHMGB1-treated samples. pHMGB1 diminished senescence in Aβ-challenged cells and increased the expression of pro- and anti-inflammatory markers, even in the absence of Aβ challenge.
Overall, our data suggest that siHMGB1 protects young microglia from excessive activation without compromising their responsiveness, while pHMGB1 allows aged microglia to regain a reactive profile. Therefore, selective modulation of HMGB1 appears essential to preserve microglia’s key functions, supporting a potential therapeutic application in AD with advantages over conventional broad effect anti-inflammatory agents.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
5876
Número da atribuição
UID/DTP/04138/2013