Oeiras, Portugal
Oeiras, Portugal

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Pena A.C.,University of Lisbon | Penacho N.,Alfama Lda | Mancio-Silva L.,University of Lisbon | Neres R.,University of Lisbon | And 9 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2012

Severe forms of malaria infection, such as cerebral malaria (CM) and acute lung injury (ALI), are mainly caused by the apicomplexan parasite Plasmodium falciparum. Primary therapy with quinine or artemisinin derivatives is generally effective in controlling P. falciparum parasitemia, but mortality from CM and other forms of severe malaria remains unacceptably high. Herein, we report the design and synthesis of a novel carbon monoxide-releasing molecule (CO-RM; ALF492) that fully protects mice against experimental CM (ECM) and ALI. ALF492 enables controlled CO delivery in vivo without affecting oxygen transport by hemoglobin, the major limitation in CO inhalation therapy. The protective effect is CO dependent and induces the expression of heme oxygenase-1, which contributes to the observed protection. Importantly, when used in combination with the antimalarial drug artesunate, ALF492 is an effective adjunctive and adjuvant treatment for ECM, conferring protection after the onset of severe disease. This study paves the way for the potential use of CO-RMs, such as ALF492, as adjunctive/adjuvant treatment in severe forms of malaria infection. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Rodrigues C.A.B.,Alfama Lda | De Matos M.N.,Alfama Lda | Guerreiro B.M.H.,Alfama Lda | Goncalves A.M.L.,Alfama Lda | And 3 more authors.
Tetrahedron Letters | Year: 2011

Decarbonylation of the tertiary aldehydes 4-ethyl-4-formyl-hexanenitrile (2) and 2-methyl-2-phenylpropanal (4) promoted by dioxygen occurs at room temperature only if suspended in water probably via the sequential acyl radical-CO liberation-tertiary radical that is promoted by an 'on water' process originating preferentially from the corresponding tertiary hydroperoxide. © 2011 Elsevier Ltd. All rights reserved.

Kromer L.,New University of Lisbon | Coelho A.C.,New University of Lisbon | Bento I.,New University of Lisbon | Marques A.R.,Alfama Lda. | And 2 more authors.
Journal of Organometallic Chemistry | Year: 2014

A series of cis-[Mo(CO)4(amine)2] complexes (NR 3 = morpholine 1; 4-Me-piperazine, 2; H2NCH 2CH2NH2, 3; H2NCH2CH 2-morpholine (4) R2NCH2CH2- piperazine-4-Me (R = H, 5); R = Me, 6); Me2NCH2CH 2NMe2, 7) was prepared in good yields, in a one-step microwave-assisted synthesis. The X-ray diffraction structures of the complexes 4, 5 and 6 are reported. The stability of the complexes 1-7 in aqueous, aerobic media was studied by UV-Vis spectrophotometry, RP-HPLC and gas chromatography at several pH values. Stability beyond 1 h requires bidentate ligands with at least one tertiary amine ligand and increases in the order 4 < 5 < 6. Stability is approximately the same at pH 7.5 and pH 3.9 for 5 and 6 in solutions acidified with HCl. Acidification with CF3COOH induces decomposition. The order of CO transfer rate to deoxy-Mb and haemoglobin in whole blood is 1 > 2 > 3 > 4 > 5 > 6 >> 7, but it is much faster to whole blood. The haemolytic index of some compounds increases in a similar order: 1 < 2 < 5 < 6; with the exception of 1, the complexes are not toxic to RAW264.7 cells up to a concentration of 100 μM. © 2013 Elsevier B.V. All rights reserved.

Seixas J.D.,Alfama Lda | Mukhopadhyay A.,New University of Lisbon | Santos-Silva T.,New University of Lisbon | Otterbein L.E.,Beth Israel Deaconess Medical Center | And 11 more authors.
Dalton Transactions | Year: 2013

The complex fac-[Mo(CO)3(histidinate)]Na has been reported to be an effective CO-Releasing Molecule in vivo, eliciting therapeutic effects in several animal models of disease. The CO releasing profile of this complex in different settings both in vitro and in vivo reveals that the compound can readily liberate all of its three CO equivalents under biological conditions. The compound has low toxicity and cytotoxicity and is not hemolytic. CO release is accompanied by a decrease in arterial blood pressure following administration in vivo. We studied its behavior in solution and upon the interaction with proteins. Reactive oxygen species (ROS) generation upon exposure to air and polyoxomolybdate formation in soaks with lysozyme crystals were observed as processes ensuing from the decomposition of the complex and the release of CO. © 2013 This journal is The Royal Society of Chemistry.

Tavares A.F.N.,New University of Lisbon | Teixeira M.,New University of Lisbon | Romao C.C.,New University of Lisbon | Romao C.C.,Alfama Lda. | And 4 more authors.
Journal of Biological Chemistry | Year: 2011

CO-releasing molecules (CO-RMs) were previously shown by us to be more potent bactericides than CO gas. This suggests a mechanism of action for CO-RM, which either potentiates the activity of CO or uses another CO-RM-specific effect. We have also reported that CORM-2 induces the expression of genes related to oxidative stress. In the present study we intend to establish whether the generation of reactive oxygen species by CO-RMs may indeed result in the inhibition of bacterial cellular function. We now report that two CO-RMs (CORM-2 and ALF062) stimulate the production of ROS in Escherichia coli, an effect that is abolished by addition of antioxidants. Furthermore, deletion of genes encoding E. coli systems involved in reactive oxygen species scavenging, namely catalases and superoxide dismutases, potentiates the lethality of CORM-2 due to an increase of intracellular ROS content. CORM-2 also induces the expression of the E. coli DNA repair/SOS system recA, and its inactivation enhances toxicity of CORM-2. Moreover, fluorescence microscopy images reveal that CORM-2 causes DNA lesions to bacterial cells. We also demonstrate that cells treated with CORM-2 contain higher levels of free iron arising from destruction of iron-sulfur proteins. Importantly, we show that CO-RMs generate hydroxyl radicals in a cell-free solution, a process that is abolished by scavenging CO. Altogether, we provide a novel insight into the molecular basis of CO-RMs action by showing that their bactericidal properties are linked to cell damage inflicted by the oxidative stress that they are able to generate. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Santos M.F.A.,New University of Lisbon | Seixas J.D.,Alfama Lda | Seixas J.D.,New University of Lisbon | Coelho A.C.,New University of Lisbon | And 6 more authors.
Journal of Inorganic Biochemistry | Year: 2012

Complexes of the general formula fac-[Ru(CO)3L3] 2 +, namely CORM-2 and CORM-3, have been successfully used as experimental CO releasing molecules (CO-RMs) but their mechanism of action and delivery of CO remain unclear. The well characterized complex [Ru(CO) 3Cl2(1,3-thiazole)] (1) is now studied as a potential model CO-RM of the same family of complexes using LC-MS, FTIR, and UV-vis spectroscopy, together with X-ray crystallography. The chemistry of [Ru(CO) 3Cl2(1,3-thiazole)] is very similar to that of CORM-3: it only releases residual amounts of CO to the headspace of a solution in PBS7.4 and produces marginal increase of COHb after long incubation in whole blood. 1 also reacts with lysozyme to form Ru adducts. The crystallographic model of the lysozyme-Ru adducts shows only mono-carbonyl Ru species. [Ru(H 2O)4(CO)] is found covalently bound to a histidine (His15) and to two aspartates (Asp18 and Asp119) at the protein surface. The CO release silence of both 1 and CORM-3 and their rapid formation of protein-Ru(CO) x(H2O)y (x = 1,2) adducts, support our hypothesis that fac-[Ru(CO)3L3] CO-RMs deliver CO in vivo through the decay of their adducts with plasma proteins. © 2012 Elsevier Inc. All rights reserved.

Fagone P.,University of Catania | Mangano K.,University of Catania | Coco M.,University of Catania | Perciavalle V.,University of Catania | And 3 more authors.
Clinical and Experimental Immunology | Year: 2012

Carbon monoxide (CO) is produced during the catabolism of free haem, catalyzed by haem oxygenase (HO) enzymes, and its physiological roles include vasodilation, neurotransmission, inhibition of platelet aggregation and anti-proliferative effects on smooth muscle. In vivo preclinical studies have shown that exogenously administered quantities of CO may represent an effective treatment for conditions characterized by a dysregulated immune response. The carbon monoxide-releasing molecules (CORMs) represent a group of compounds capable of carrying and liberating controlled quantities of CO in the cellular systems. This review covers the physiological and anti-inflammatory properties of the HO/CO pathway in the central nervous system. It also discusses the effects of CORMs in preclinical models of inflammation. The accumulating data discussed herein support the possibility that CORMs may represent a novel class of drugs with disease-modifying properties in multiple sclerosis. © 2012 The Authors. Clinical and Experimental Immunology © 2012 British Society for Immunology.

Santos-Silva T.,New University of Lisbon | Mukhopadhyay A.,New University of Lisbon | Seixas J.D.,New University of Lisbon | Seixas J.D.,Alfama Lda. | And 5 more authors.
Current Medicinal Chemistry | Year: 2011

The biological role of carbon monoxide (CO) has completely changed in the last decade. Beyond its widely feared toxicity, CO has revealed a very important biological activity as a signaling molecule with marked protective actions namely against inflammation, apoptosis and endothelial oxidative damage. Its direct use as a therapeutic gas showed significant and consistent positive results but also intrinsic severe limitations. The possibility of replacing the gas by pro-drugs acting as CO-Releasing Molecules (CO-RMs) has clearly been demonstrated with several experimental compounds. Transition metal carbonyls complexes have proven to be the most versatile experimental CO-RMs so far. Presently, the challenge is to equip them with drug-like properties to turn them into useful pharmaceuticals. This requires studying their interactions with biological molecules namely those that control their pharmacokinetic and ADME profiles like the plasma proteins. In this account we analyze these questions and review the existing interactions between Metal Carbonyls and proteins. The recently explored case of CORM-3 is revisited to exemplify the methodologies involved and the importance of the results for the understanding of the mode of action of such pro-drugs. © 2011 Bentham Science Publishers Ltd.

Marazioti A.,University of Patras | Bucci M.,University of Naples Federico II | Coletta C.,University of Texas Medical Branch | Vellecco V.,University of Naples Federico II | And 11 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2011

Objective-: Carbon monoxide (CO) is a weak soluble guanylyl cyclase stimulator, leading to transient increases in cGMP and vasodilation. The aim of the present work was to measure the effect of CO-releasing molecules (CORMs) on the cGMP/nitric oxide (NO) pathway and to evaluate how selected CORMs affect NO-induced vasorelaxation. Methods and Results-: Incubation of smooth muscle cells with some but not all of the CORMs caused a minor increase in cGMP levels. Concentration-response curves were bell-shaped, with higher CORMs concentrations producing lower increases in cGMP levels. Although exposure of cells to CORM-2 enhanced cGMP formation, we observed that the compound inhibited NO-stimulated cGMP accumulation in cells and NO-stimulated soluble guanylyl cyclase activity that could be reversed by superoxide anion scavengers. Reactive oxygen species generation from CORMs was confirmed using luminol-induced chemiluminescence and electron spin resonance. Furthermore, we observed that NO is scavenged by CORM-2. When used alone CORM-2 relaxed vessels through a cGMP-mediated pathway but attenuated NO donor-stimulated vasorelaxation. Conclusion-: We conclude that the CORMs examined have context-dependent effects on vessel tone, as they can directly dilate blood vessels, but also block NO-induced vasorelaxation. © 2011 American Heart Association. All rights reserved.

Santos-Silva T.,New University of Lisbon | Mukhopadhyay A.,New University of Lisbon | Seixas J.D.,New University of Lisbon | Seixas J.D.,Alfama Lda. | And 4 more authors.
Journal of the American Chemical Society | Year: 2011

CORM-3, [fac-Ru(CO)3Cl(κ2-H2NCH 2CO2)], is a well-known carbon monoxide releasing molecule (CORM) capable of delivering CO in vivo. Herein we show for the first time that the interactions of CORM-3 with proteins result in the loss of a chloride ion, glycinate, and one CO ligand. The rapid formation of stable adducts between the protein and the remaining cis-RuII(CO)2 fragments was confirmed by Inductively Coupled Plasma-Atomic Emission Spectrocopy (ICP-AES), Liquid-Chromatography Mass Spectrometry (LC-MS), Infrared Spectroscopy (IR), and X-ray crystallography. Three Ru coordination sites are observed in the structure of hen egg white lysozyme crystals soaked with CORM-3. The site with highest Ru occupancy (80%) shows a fac-[(His15)Ru(CO)2(H 2O)3] structure. © 2011 American Chemical Society.

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