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- Decavanadate and metformin-decavanadate effects in human melanoma cellsPublication . de Sousa-Coelho, Ana Luísa; Aureliano, Manuel; Fraqueza, Gil; Serrão, Gisela; Gonçalves, João; Sánchez-Lombardo, Irma; Link, Wolfgang; Ferreira, BibianaDecavanadate is a polyoxometalate (POMs) that has shown extensive biological activities, including antidiabetic and anticancer activity. Importantly, vanadium-based compounds as well as antidiabetic biguanide drugs, such as metformin, have shown to exert therapeutic effects in melanoma. A combination of these agents, the metformin-decavanadate complex, was also recognized for its antidiabetic effects and recently described as a better treatment than the monotherapy with metformin enabling lower dosage in rodent models of diabetes. Herein, we compare the effects of decavanadate and metformin-decavanadate on Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from rabbit skeletal muscles and on cell signaling events and viability in human melanoma cells. We show that unlike the decavanadate-mediated non-competitive mechanism, metformin-decavanadate inhibits Ca2+-ATPase by a mixed-type competitive-non-competitive inhibition with an IC50 value about 6 times higher (87 mu M) than the previously described for decavanadate (15 mu M). We also found that both decavanadate and metformin-decavanadate exert antiproliferative effects on melanoma cells at 10 times lower concentrations than monomeric vanadate. Western blot analysis revealed that both, decavanadate and metformin-decavanadate increased phosphorylation of extracellular signal-regulated kinase (ERK) and serine/ threonine protein kinase AKT signaling proteins upon 24 h drug exposure, suggesting that the anti-proliferative activities of these compounds act independent of growth-factor signaling pathways.
- Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca2+-ATPase: quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibitionPublication . Fraqueza, Gil; Carvalho, Luís A. E. Batista de; Marques, M. Paula M.; Maia, Luisa; Ohlin, C. André; Casey, William H.; Aureliano, M.Recently we demonstrated that the decavanadate (V10) ion is a stronger Ca2+-ATPase inhibitor than other oxometalates, such as the isoelectronic and isostructural decaniobate ion, and the tungstate and molybdate monomer ions, and that it binds to this protein with a 1 : 1 stoichiometry. The V10 interaction is not affected by any of the protein conformations that occur during the process of calcium translocation (i.e. E1, E1P, E2 and E2P) (Fraqueza et al., J. Inorg. Biochem., 2012). In the present study, we further explore this subject, and we can now show that the decaniobate ion, [Nb10 = Nb10O28]6−, is a useful tool in deducing the interaction and the non-competitive Ca2+-ATPase inhibition by the decavanadate ion [V10 = V10O28]6−. Moreover, decavanadate and vanadate induce protein cysteine oxidation whereas no effects were detected for the decaniobate, tungstate or molybdate ions. The presence of the antioxidant quercetin prevents cysteine oxidation, but not ATPase inhibition, by vanadate or decavanadate. Definitive V(IV) EPR spectra were observed for decavanadate in the presence of sarcoplasmic reticulum Ca2+- ATPase, indicating a vanadate reduction at some stage of the protein interaction. Raman spectroscopy clearly shows that the protein conformation changes that are induced by V10, Nb10 and vanadate are different from the ones induced by molybdate and tungstate monomer ions. Here, Mo and W cause changes similar to those by phosphate, yielding changes similar to the E1P protein conformation. The putative reduction of vanadium(V) to vanadium(IV) and the non-competitive binding of the V10 and Nb10 decametalates may explain the differences in the Raman spectra compared to those seen in the presence of molybdate or tungstate. Putting it all together, we suggest that the ability of V10 to inhibit the Ca2+- ATPase may be at least in part due to the process of vanadate reduction and associated protein cysteine oxidation. These results contribute to the understanding and application of these families of mono- and polyoxometalates as effective modulators of many biological processes, particularly those associated with calcium homeostasis.
- Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molyddatePublication . Fraqueza, Gil; Ohlin, C. André; Casey, William H.; Aureliano, M.Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca2+-ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca2+-ATPase, with the following IC50 values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb10), is the strongest P-type enzyme inhibitor, after decavanadate (V10). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca2+-ATPase stoichiometry. Furthermore, V10 binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H2VO4 2−; V1) to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate–Ca2+-ATPase, with a dissociation constant, kd of 1 μM−1. The interaction of decavanadate V10O28 6− (V10) with Ca2+-ATPase is prevented by the isostructural and isoelectronic decaniobate Nb10O28 6− (Nb10), whereas no significant effects were detected with ATP or with heparin, a known competitive ATP binding molecule, suggesting that V10 binds non-competitively, with respect to ATP, to the protein. Finally, it was shown that decaniobate inhibits SR Ca2+-ATPase activity in a non competitive type of inhibition, with respect to ATP. Taken together, these data demonstrate that decameric niobate and vanadate species are stronger inhibitors of the SR calcium ATPase than simple monomeric vanadate, tungstate and molybdate oxometalates, thus affecting calcium homeostasis, cell signalling and cell bioenergetics, as well many other cellular processes. The ability of these oxometalates to act either as phosphate analogues, as a transition-state analogue in enzyme-catalysed phosphoryl group transfer processes and as potentially nucleotide-dependent enzymes modulators or inhibitors, suggests that different oxometalates may reveal different mechanistic preferences in these classes of enzymes.
- Inhibition of SERCA and PMCA Ca2+-ATPase activities by polyoxotungstatesPublication . Aureliano, Manuel; Fraqueza, Gil; Berrocal, Maria; Cordoba-Granados, Juan J.; Gumerova, Nadiia I.; Rompel, Annette; Gutierrez-Merino, Carlos; Mata, Ana M.Plasma membrane calcium ATPases (PMCA) and sarco(endo) reticulum calcium ATPases (SERCA) are key proteins in the maintenance of calcium homeostasis. Herein, we compare for the first time the inhibition of SERCA and PMCA calcium pumps by several polyoxotungstates (POTs), namely by Wells-Dawson phospho-tungstate anions [P2W18O62]6-(intact, {P2W18}), [P2W17O61]10-(monolacunary, {P2W17}), [P2W15O56]12-(trilacunary, {P2W15}), [H2P2W12O48]12-(hexalacunary, {P2W12}), [H3P2W15V3O62]6- (trivanadium-substituted, {P2W15V3}) and by Preyssler-type anion [NaP5W30O110]14-({P5W30}). The speciation in the solu-tions of tested POTs was investigated by 31P and 51V NMR spectroscopy. The tested POTs inhibited SERCA Ca2+- ATPase activity, whereby the Preyssler POT showed the strongest effect, with an IC50 value of 0.37 mu M. For {P2W17} and {P2W15V3} higher IC50 values were determined: 0.72 and 0.95 mu M, respectively. The studied POTs showed to be more potent inhibitors of PMCA Ca2+-ATPase activity, with lower IC50 values for {P2W17}, {P5W30} and {P2W15V3}.
- Polyoxovanadate inhibition of Escherichia coli growth shows a reverse correlation with Ca2+-ATPase inhibitionPublication . Marques-da-Silva, Dorinda; Fraqueza, Gil; Lagoa, Ricardo; Vannathan, Anjana Anandan; Mal, Sib Sankar; Aureliano, ManuelRecently, a global analysis of the structure-activity-relationship of a series of polyoxometalates (POMs) revealed that the most active POMs were ascribed to be polyoxovanadates (POVs), especially decavanadate (V-10), which was very active against certain bacteria (Bijelic et al., Chem. Commun., 2018). The present study explores this observation and compares the effects of three POVs namely MnV11, MnV13 and V-10 against Escherichia coli growth. It was observed that MnV11 presents the lowest growth inhibition (GI(50)) value for Escherichia coli followed by the MnV13 compound, being about 2 times lower than that of V-10 respectively, the values obtained were 0.21, 0.27 and 0.58 mM. All three compounds were more effective than vanadate alone (GI(50) = 1.1 mM) and also than decaniobate, Nb-10 (GI(50) > 10 mM), an isostructural POM of V-10. However, the POVs exhibiting the highest antibacterial activity (MnV11) were shown to have the lowest Ca2+-ATPase inhibitor capacity (IC50 = 58 mM) whereas decavanadate, which was also very active against this membranar ATPase (IC50 = 15 mM), was less active against bacterial growth, suggesting that POV inhibition of ion pumps might not be associated with the inhibition of Escherichia coli growth.
- The Ca2+-ATPase inhibition potential of gold (I, III) compoundsPublication . Fonseca, Custódia; Fraqueza, Gil; Carabineiro, Sónia A.; Aureliano, ManuelThe therapeutic applications of gold are well-known for many centuries. The most used gold compounds contain Au(I). Herein, we report, for the first time, the ability of four Au(I) and Au(III) complexes, namely dichloro (2-pyridinecarboxylate) Au(III) (abbreviated as1), chlorotrimethylphosphine Au(I) (2), 1,3-bis(2,6-diisopropylphenyl) imidazole-2-ylidene Au(I) chloride (3), and chlorotriphenylphosphine Au(I) (4), to affect the sarcoplasmic reticulum (SR) Ca2+-ATPase activity. The tested gold compounds strongly inhibit the Ca2+-ATPase activity with different effects, being Au(I) compounds2and4the strongest, with half maximal inhibitory concentration (IC50) values of 0.8 and 0.9 mu M, respectively. For Au(III) compound1and Au(I) compound3, higher IC(50)values are found (4.5 mu M and 16.3 mu M, respectively). The type of enzymatic inhibition is also different, with gold compounds1and2showing a non-competitive inhibition regarding the native substrate MgATP, whereas for Au compounds3and4, a mixed type of inhibition is observed. Our data reveal, for the first time, Au(I) compounds with powerful inhibitory capacity towards SR Ca(2+)ATPase function. These results also show, unprecedently, that Au (III) and Au(I) compounds can act as P-type ATPase inhibitors, unveiling a potential application of these complexes.
- Repurposing therapeutic drugs complexed to Vanadium in CancerPublication . De Sousa-Coelho, Ana Luísa; Fraqueza, Gil; Aureliano, M.Repurposing drugs by uncovering new indications for approved drugs accelerates the process of establishing new treatments and reduces the high costs of drug discovery and development. Metal complexes with clinically approved drugs allow further opportunities in cancer therapy—many vanadium compounds have previously shown antitumor effects, which makes vanadium a suitable metal to complex with therapeutic drugs, potentially improving their efficacy in cancer treatment. In this review, covering the last 25 years of research in the field, we identified non-oncology-approved drugs suitable as ligands to obtain different vanadium complexes. Metformin-decavanadate, vanadium-bisphosphonates, vanadyl(IV) complexes with non-steroidal anti-inflammatory drugs, and cetirizine and imidazole-based oxidovanadium(IV) complexes, each has a parent drug known to have different medicinal properties and therapeutic indications, and all showed potential as novel anticancer treatments. Nevertheless, the precise mechanisms of action for these vanadium compounds against cancer are still not fully understood.
- Inhibition of Na+/K+- and Ca2+-ATPase activities by phosphotetradecavanadatePublication . Fraqueza, Gil; Fuentes, Juan; Krivosudský, Lukáš; Dutta, Saikat; Mal, Sib Sankar; Roller, Alexander; Giester, Gerald; Rompel, Annette; Aureliano, ManuelPolyoxometalates (POMs) are promising inorganic inhibitors for P-type ATPases. The experimental models used to study the effects of POMs on these ATPases are usually in vitro models using vesicles from several membrane sources. Very recently, some polyoxotungstates, such as the Dawson anion [P2W18O62]6-, were shown to be potent P-type ATPase inhibitors; being active in vitro as well as in ex-vivo. In the present study we broaden the spectrum of highly active inhibitors of Na+/K+-ATPase from basal membrane of epithelial skin to the bi-capped Keggin-type anion phosphotetradecavanadate Cs5.6H3.4PV14O42 (PV14) and we confront the data with activity of other commonly encountered polyoxovanadates, decavanadate (V10) and monovanadate (V1). The X-ray crystal structure of PV14 was solved and contains two trans-bicapped α-Keggin anions HxPV14O42(9-x)-. The anion is built up from the classical Keggin structure [(PO4)@(V12O36)] capped by two [VO] units. PV14 (10 μM) exhibited higher ex-vivo inhibitory effect on Na+/K+-ATPase (78%) than was observed at the same concentrations of V10 (66%) or V1 (33%). Moreover, PV14 is also a potent in vitro inhibitor of the Ca2+-ATPase activity (IC50 5 μM) exhibiting stronger inhibition than the previously reported activities for V10 (15 μM) and V1 (80 μM). Putting it all together, when compared both P-typye ATPases it is suggested that PV14 exibited a high potential to act as an in vivo inhibitor of the Na+/K+-ATPase associated with chloride secretion.
- Interação de oxometalatos de vanádio, nióbio, tungsténio e molibdénio com a Ca2+-ATPase de retículo sarcoplasmático: um alvo de ação de fármacoPublication . Fraqueza, Gil; Aureliano, M.Os oxometalatos são compostos que têm sido descritos por apresentarem propriedades insulino-miméticas, antitumorais, antibióticas, além de serem inibidores das ATPases, nomeadamente a Ca2+-ATPase. Contudo, os efeitos da interação dos oxometalatos com a Ca2+-ATPase, não é um assunto completamente esclarecido. Este estudo teve como objetivo, esclarecer o modo de atuação de oxometalatos de vanádio, nióbio, tungsténio e molibdénio na função e estrutura da Ca2+-ATPase de RS, combinando-se estudos cinéticos com técnicas espetroscópicas. Verificou-se que os oxometalatos estudados inibem a atividade da Ca2+-ATPase, sendo o inibidor mais potente o decavanadato com um IC50 de 15 µM. Foi observado que decavanadato e decaniobato inibem a Ca2+-ATPase de modo não competitivo. Por EAA, observou-se que V10 liga-se às conformações E1, E1P, E2 e E2P, enquanto foi confirmado que V1 liga-se preferencialmente às conformações E2 e E2P. Por outro lado, o vanadato e o decavanadato induzem a oxidação de cisteínas na proteína. A presença do antioxidante quercetina previne a oxidação das cisteínas, mas não a inibição da Ca2+-ATPase por vanadato ou decavanadato. O sinal de V(IV), observado no espetro de RPE de decavanadato na presença Ca2+-ATPase de retículo sarcoplasmático e ATP, sugere uma redução de vanadato, devido à interação de V10 com a proteína. Estes resultados sugerem que a maior capacidade do V10 para inibir a Ca2+-ATPase pode estar, pelo menos em parte, relacionada com o processo de redução do vanadato associada à oxidação das cisteínas na proteína. Finalmente estes resultados contribuem para a compreensão e aplicação de oxometalatos e polioxometalatos como moduladores eficazes de muitos processos biológicos, particularmente aqueles associados com homeostasia do cálcio. De facto os oxometalatos, em especial os polioxometalatos apresentam capacidade inibitória sobre as bombas iónicas, em especial na Ca2+-ATPase, semelhante a fármacos que têm como alvo de ação terapêutica estas bombas iónicas. Sendo por isso de considerar em estudos, a inclusão deste tipo de oxometalatos em fármacos com aplicação terapêutica.
- The P-type ATPase inhibiting potential of polyoxotungstates.Publication . Gumerova, Nadiia; Krivosudský, Lukáš; Fraqueza, Gil; Breibeck, Joscha; Al-Sayed, Emir; Tanuhadi, Elias; Bijelic, Aleksandar; Fuentes, Juan; Aureliano, M.; Rompel, AnnettePolyoxometalates (POMs) are transition metal complexes that exhibit a broad diversity of structures and properties rendering them promising for biological purposes. POMs are able to inhibit a series of biologically important enzymes, including phosphatases, and thus are able to affect many biochemical processes. In the present study, we analyzed and compared the inhibitory effects of nine different polyoxotungstates (POTs) on two P-type ATPases, Ca2+-ATPase from skeletal muscle and Na+/K+-ATPase from basal membrane of skin epithelia. For Ca2+-ATPase inhibition, an in vitro study was performed and the strongest inhibitors were determined to be the large heteropolytungstate K9(C2H8N)5[H10Se2W29O103] (Se2W29) and the Dawson-type POT K6[α-P2W18O62] (P2W18) exhibiting IC50 values of 0.3 and 0.6 μM, respectively. Promising results were also shown for the Keggin-based POTs K6H2[CoW11TiO40] (CoW11Ti, IC50 = 4 μM) and Na10[α-SiW9O34] (SiW9, IC50 = 16 μM), K14[As2W19O67(H2O)] (As2W19, IC50 = 28 μM) and the lacunary Dawson K12[α-H2P2W12O48] (P2W12, IC50 = 11 μM), whereas low inhibitory potencies were observed for the isopolytungstate Na12[H4W22O74] (W22, IC50 = 68 μM) and the Anderson-type Na6[TeW6O24] (TeW6, IC50 = 200 μM). Regarding the inhibition of Na+/K+-ATPase activity, for the first time an ex vivo study was conducted using the opercular epithelium of killifish in order to investigate the effects of POTs on the epithelial chloride secretion. Interestingly, 1 μM of the most potent Ca2+-ATPase inhibitor, Se2W29, showed only a minor inhibitory effect (14% inhibition) on Na+/K+-ATPase activity, whereas almost total inhibition (99% inhibition) was achieved using P2W18. The remaining POTs exhibited similar inhibition rates on both ATPases. These results reveal the high potential of some POTs to act as P-type ATPase inhibitors, with Se2W29 showing high selectivity towards Ca2+-ATPase.