39 research outputs found
Estructura tridimensional de un fragmento de 3 Kda del factor derivado de Plaquetas-4 con actividad antiangiogénica e interacción con el factor de crecimiento para fibroblastos
Platelet-Factor-4 ís a protein belonging to the family of ELR negative CXC chemokines which binds to fibroblast growth factor, and inhibits its mitogenic activity.Platelet-factor-4 also inhibits tumor growth by mechanisms involving antiangiogenesis in which the blockage of fibroblast growth factor mitogenic activity seems lo play an essential role. Synthesis of platelet-factor-4 by recombinant technologies is a considerably cumbersome process that does not seen could sustain an industrial production of pharmacological-quoality because of platelet-factor-4 quaternary structure, and because the protein actívation requires an intramonomer Hydrolisys. It has been shown that the proteín's twenty-four residue C-terminal fragment is also antíangtogeníc in vitro and in vivio, forms a specific 1:1 complex with fibroblast growyh factors and decreases their affinity for their cell surface receptor. Thus this fragments seems could constitute an alternative to the use of the whole platelet-factor-4 for several of its uses therapeutic applications. The fragments preferential three-dimensional conformation in solutíon is quite stable and has been found to be composed of two helical sub-domains, which, both, seem required for inhihilion of fíbroblast growth factor dríven mitogenesis. The characterítation, at high resolution, of the three-dimensional structure of this fragment may contribute, to the understanding of the basis of its antiangiogenic activity, and thus, contribute, to the optimitation of their pharmacological properties. At the same time this structural characteritation may also contribute to a deeper perception of the platelet-factor-4 physiology.El factor derivado de plaquetas-a es una proteína perteneciente a la familia de las equemoquinas CXC, carentes de la secuencia ELR• . Esta proteína se une al factor de crecimiemo para Iibroblastos inhibiend o su actividad mitogénica. Así mismo se ha demostrado que inhibe el crecimiento tumoral a través de un mecanismo antiangiogénico en el que debe jugar un pa pel importante el bloqueo de la actividad del factor de crecimiento pura fibroblasros. El factor derivado de plaquetas-4 posee una estructura tridimensional complicada, por lo que es poco probable que puedan obtenerse cantidades industriales del producto, con calidad farmacológica, tanto por síntesis química como mediante ingeniería genética. Se ha demostrado que un fragmento equivalente a los veinticuatro residuos últimos del extremo carboxilo terminal de la proteína completa posee también actividad antíangiogñenica in vitro e in vivo, forma un complejo con estequiomelría 1:1 con el factor de crecimiento para fibroblastos y hace que disminuya su afinidad por su receptor de alta afinidad de la superficie celular. Este fragmento posee numerosas características para constituir un buen sustituto terapéutico de la proteína completa. La estructura tridimensional en solución de este fragmento es relativamente sencilla y notablemente estable. Consta de dos subdominios helicoidales que parece que es necesario que se mantengan unidos, tanto para la inhibición y la formación del complejo, como para que uno de los subdominios siga siendo helicoidal. La caracterización estructural a alta resolución de este fragmento puede contribuir a entender las bases estructurales de su actividad antiangiogéníca y, por tanto, podría permitir mejorar sus propiedad es fa rma co lógica s. A su vez puede ayudar a un a mejor comprensión de las bases estructurales de la fisiología del factor derivado de plaquetas-4
Integrating magnetic capabilities to intracellular chips for cell trapping
Current microtechnologies have shown plenty of room inside a living cell for silicon chips. Microchips as barcodes, biochemical sensors, mechanical sensors and even electrical devices have been internalized into living cells without interfering their cell viability. However, these technologies lack from the ability to trap and preconcentrate cells in a specific region, which are prerequisites for cell separation, purification and posterior studies with enhanced sensitivity. Magnetic manipulation of microobjects, which allows a non-contacting method, has become an attractive and promising technique at small scales. Here, we show intracellular Ni-based chips with magnetic capabilities to allow cell enrichment. As a proof of concept of the potential to integrate multiple functionalities on a single device of this technique, we combine coding and magnetic manipulation capabilities in a single device. Devices were found to be internalized by HeLa cells without interfering in their viability. We demonstrated the tagging of a subpopulation of cells and their subsequent magnetic trapping with internalized barcodes subjected to a force up to 2.57 pN (for magnet-cells distance of 4.9 mm). The work opens the venue for future intracellular chips that integrate multiple functionalities with the magnetic manipulation of cells
Structural Determinants of the Dictyostatin Chemotype for Tubulin Binding Affinity and Antitumor Activity Against Taxane- and Epothilone-Resistant Cancer Cells
A combined biochemical, structural, and cell biology characterization of dictyostatin is described, which enables an improved understanding of the structural determinants responsible for the high-affinity binding of this anticancer agent to the taxane site in microtubules (MTs). The study reveals that this macrolide is highly optimized for MT binding and that only a few of the structural modifications featured in a library of synthetic analogues resulted in small gains in binding affinity. The high efficiency of the dictyostatin chemotype in overcoming various kinds of clinically relevant resistance mechanisms highlights its potential for therapeutic development for the treatment of drug-resistant tumors. A structural explanation is advanced to account for the synergy observed between dictyostatin and taxanes on the basis of their differential effects on the MT lattice. The X-ray crystal structure of a tubulin−dictyostatin complex and additional molecular modeling have allowed the rationalization of the structure−activity relationships for a set of synthetic dictyostatin analogues, including the highly active hybrid 12 with discodermolide. Altogether, the work reported here is anticipated to facilitate the improved design and synthesis of more efficacious dictyostatin analogues and hybrids with other MT-stabilizing agents.We thank Peter T. Northcote for peloruside A, W.-S. Fang for Flutax-2, K. H. Altmann for epothilone D, Dr. Paraskevi Giannakakou (Weill Cornell Medical Center, New York) for the 1A9, PTX10, PTX22, and A8 cell lines, and Prof. Richard Ludueñ a (University of Texas) for the HeLa βIII-transfected cells. We thank Matadero INCOVA (Segovia) for the calf brains for tubulin purification. This work was supported in part by grants BIO2013-42984-R (J.F.D.) and SAF2012-39760-C02-02 (F.G.) from Ministerio de Economia y Competitividad, grant S2010/ ́ BMD-2457 BIPEDD2 from Comunidad Autonoma de Madrid ́ (F.G. and J.F.D.), and the Swiss National Science Foundation grants 310030B_138659 and 31003A_166608 (M.O.S.). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by naturefrom natural products chemistry to drug discovery” and the COST action CM1470. I.P. thanks the EPSRC and AstraZeneca for funding, Dr. John Leonard (AstraZeneca) for useful discussions, Dr. Stuart Mickel (Novartis) for the provision of chemicals, and the EPSRC UK National Mass Spectrometry Facility at Swansea University for mass spectra
Structural Determinants of the Dictyostatin Chemotype for Tubulin Binding Affinity and Antitumor Activity Against Taxane- and Epothilone-Resistant Cancer Cells
A combined biochemical, structural, and cell biology characterization of dictyostatin is described, which enables an improved understanding of the structural determinants responsible for the high-affinity binding of this anticancer agent to the taxane site in microtubules (MTs). The study reveals that this macrolide is highly optimized for MT binding and that only a few of the structural modifications featured in a library of synthetic analogues resulted in small gains in binding affinity. The high efficiency of the dictyostatin chemotype in overcoming various kinds of clinically relevant resistance mechanisms highlights its potential for therapeutic development for the treatment of drug-resistant tumors. A structural explanation is advanced to account for the synergy observed between dictyostatin and taxanes on the basis of their differential effects on the MT lattice. The X-ray crystal structure of a tubulin−dictyostatin complex and additional molecular modeling have allowed the rationalization of the structure−activity relationships for a set of synthetic dictyostatin analogues, including the highly active hybrid 12 with discodermolide. Altogether, the work reported here is anticipated to facilitate the improved design and synthesis of more efficacious dictyostatin analogues and hybrids with other MT-stabilizing agents.We thank Peter T. Northcote for peloruside A, W.-S. Fang for Flutax-2, K. H. Altmann for epothilone D, Dr. Paraskevi Giannakakou (Weill Cornell Medical Center, New York) for the 1A9, PTX10, PTX22, and A8 cell lines, and Prof. Richard Ludueñ a (University of Texas) for the HeLa βIII-transfected cells. We thank Matadero INCOVA (Segovia) for the calf brains for tubulin purification. This work was supported in part by grants BIO2013-42984-R (J.F.D.) and SAF2012-39760-C02-02 (F.G.) from Ministerio de Economia y Competitividad, grant S2010/ ́ BMD-2457 BIPEDD2 from Comunidad Autonoma de Madrid ́ (F.G. and J.F.D.), and the Swiss National Science Foundation grants 310030B_138659 and 31003A_166608 (M.O.S.). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by naturefrom natural products chemistry to drug discovery” and the COST action CM1470. I.P. thanks the EPSRC and AstraZeneca for funding, Dr. John Leonard (AstraZeneca) for useful discussions, Dr. Stuart Mickel (Novartis) for the provision of chemicals, and the EPSRC UK National Mass Spectrometry Facility at Swansea University for mass spectra
Structural Determinants of the Dictyostatin Chemotype for Tubulin Binding Affinity and Antitumor Activity Against Taxane- and Epothilone-Resistant Cancer Cells
13 p.-5 fig.-2 tab.-1 graph.abst.A combined biochemical, structural, and cell biology characterization of dictyostatin is described, which enables an improved understanding of the structural determinants responsible for the high-affinity binding of this anticancer agent to the taxane site in microtubules (MTs). The study reveals that this macrolide is highly optimized for MT binding and that only a few of the structural modifications featured in a library of synthetic analogues resulted in small gains in binding affinity. The high efficiency of the dictyostatin chemotype in overcoming various kinds of clinically relevant resistance mechanisms highlights its potential for therapeutic development for the treatment of drug-resistant tumors. A structural explanation is advanced to account for the synergy observed between dictyostatin and taxanes on the basis of their differential effects on the MT lattice. The X-ray crystal structure of a tubulin–dictyostatin complex and additional molecular modeling have allowed the rationalization of the structure–activity relationships for a set of synthetic dictyostatin analogues, including the highly active hybrid 12 with discodermolide. Altogether, the work reported here is anticipated to facilitate the improved design and synthesis of more efficacious dictyostatin analogues and hybrids with other MT-stabilizing agents.This work was supported in part by grants BIO2013-42984-R (J.F.D.) and SAF2012-39760-C02-02 (F.G.) from Ministerio de Economía y Competitividad, grant S2010/BMD-2457 BIPEDD2 from Comunidad Autónoma de Madrid (F.G. and J.F.D.), and the Swiss National Science Foundation grants 310030B_138659 and 31003A_166608 (M.O.S.). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature—from natural products chemistry to drug discovery” and the COST action CM1470. I.P. thanks the EPSRC and AstraZeneca for funding, Dr. John Leonard (AstraZeneca) for useful discussions, Dr. Stuart Mickel (Novartis) for the provision of chemicals, and the EPSRC UK National Mass Spectrometry Facility at Swansea University for mass spectra
Synthesis and structure-activity relationship studies of C(13)-desmethylene-(−)-zampanolide analogs
14 p.-4 fig.-2 tab.We describe the synthesis and biochemical and cellular profiling of five partially reduced or demethylated analogs of the marine macrolide (−)-zampanolide (ZMP). These analogs were derived from 13-desmethylene-(−)-zampanolide (DM-ZMP), which is an equally potent cancer cell growth inhibitor as ZMP. Key steps in the synthesis of all compounds were the formation of the dioxabicyclo[15.3.1]heneicosane core by an intramolecular HWE reaction (67–95 % yield) and a stereoselective aza-aldol reaction with an (S)-BINOL-derived sorbamide transfer complex, to establish the C(20) stereocenter (24–71 % yield). As the sole exception, for the 5-desmethyl macrocycle, ring-closure relied on macrolactonization; however, elaboration of the macrocyclization product into the corresponding zampanolide analog was unsuccessful. All modifications led to reduced cellular activity and lowered microtubule-binding affinity compared to DM-ZMP, albeit to a different extent. For compounds incorporating the reactive enone moiety of ZMP, IC50 values for cancer cell growth inhibition varied between 5 and 133 nM, compared to 1–12 nM for DM-ZMP. Reduction of the enone double bond led to a several hundred-fold loss in growth inhibition. The cellular potency of 2,3-dihydro-13-desmethylene zampanolide, as the most potent analog identified, remained within a ninefold range of that of DM-ZMP.This workwas supported by the Swiss National Science Foundation (KHA,project200021_149253). Institutional support by the ETH Zurich is also gratefully acknowledged(KHA).Funding was also received from Ministerio de Ciencia e Innovación(Spain) (JFD,Project PID2019-104545RB-I00/AEI/10.13039/501100011033),the European Commission-NextGenerations EU(RegulationEU 2020/2094),through CSIC’s Global Health Platform(PTI Salud Global) and Proyecto de Investigación en Neurociencia Fundación Tatiana Pérez de Guzmán el Bueno 2020 (JFD).Peer reviewe
Manganese Superoxide Dismutase: Guardian of the Powerhouse
The mitochondrion is vital for many metabolic pathways in the cell, contributing all or important constituent enzymes for diverse functions such as β-oxidation of fatty acids, the urea cycle, the citric acid cycle, and ATP synthesis. The mitochondrion is also a major site of reactive oxygen species (ROS) production in the cell. Aberrant production of mitochondrial ROS can have dramatic effects on cellular function, in part, due to oxidative modification of key metabolic proteins localized in the mitochondrion. The cell is equipped with myriad antioxidant enzyme systems to combat deleterious ROS production in mitochondria, with the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) acting as the chief ROS scavenging enzyme in the cell. Factors that affect the expression and/or the activity of MnSOD, resulting in diminished antioxidant capacity of the cell, can have extraordinary consequences on the overall health of the cell by altering mitochondrial metabolic function, leading to the development and progression of numerous diseases. A better understanding of the mechanisms by which MnSOD protects cells from the harmful effects of overproduction of ROS, in particular, the effects of ROS on mitochondrial metabolic enzymes, may contribute to the development of novel treatments for various diseases in which ROS are an important component
Fatty acids revert the inhibition of respiration caused by the antidiabetic drug metformin to facilitate their mitochondrial β-oxidation
8 páginas, 4 figuras -- PAGS nros. 1768-1775While metformin has been widely used to treat type 2 diabetes for the last fifty years, its mode of action remains unclear. Hence, we investigated the short-term alterations in energy metabolism caused by metformin administration in 3T3-L1 adipocytes. We found that metformin inhibited mitochondrial respiration, although ATP levels remained constant as the decrease in mitochondrial production was compensated by an increase in glycolysis. While AMP/ATP ratios were unaffected by metformin, phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase augmented. The inhibition of respiration provoked a rapid and sustained increase in superoxide levels, despite the increase in UCP2 and superoxide dismutase activity. The inhibition of respiration was rapidly reversed by fatty acids and thus respiration was lower in treated cells in the presence of pyruvate and glucose while rates were identical to control cells when palmitate was the substrate. We conclude that metformin reversibly inhibits mitochondrial respiration, it rapidly activates AMPK without altering the energy charge, and it inhibits fatty acid synthesis. Mitochondrial β-oxidation is facilitated by reversing the inhibition of complex I and, presumably, by releasing the inhibition of carnitine palmitoyltransferase. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012)This work was supported by project grants from the Spanish Ministry of Science and Innovation (BFU2006-08182, SAF2009-07126, SAF2010-20256 and Consolider-Ingenio CSD2007-00020) and the Comunidad de Madrid (S2010/BMD-2402). A.A. was supported by a predoctoral fellowship from the “Master and Back” programme of the autonomous region of Sardinia (Italy). M.M.G.-B. was supported by the “Ramón y Cajal” programme of the Spanish Ministry of Science and InnovationPeer reviewe