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Cortes-Ciriano I.,University of Zaragoza | Koutsoukas A.,University of Cambridge | Abian O.,University of Zaragoza | Abian O.,Aragon Health science Institute ICS | And 3 more authors.
MedChemComm | Year: 2013

Two relatively recent trends have become apparent in current early stage drug discovery settings: firstly, a revival of phenotypic screening strategies and secondly, the increasing acceptance that some drugs work by modulating multiple targets in parallel ('multi-target drugs'). The work presented here combines both those aspects by integrating experimental phenotypic screening for cytotoxic compounds with an experimental validation of individual protein targets predicted in silico. In this first step of this work, in silico target predictions for a dataset comprising cytotoxic compounds showed an enrichment of enzymes involved in cell cycle progression (such as Topoisomerase I, Bcl-X and Protein Kinase C alpha) as well as in the defense against xenobiotic compounds (such as P-gp 1 and the CYPs). Ten compounds predicted to be active on each of two of the enriched targets, P-glycoprotein 1 and Topoisomerase I, were tested in vitro to validate (or invalidate) the predicted mode of action. Hoechst 33342 dye uptake, P-gp ATPase activity and Topoisomerase I DNA relaxation assays were able to identify two inhibitors of P-gp with IC50 values of 37 ± 5 and 28 ± 2 μM, respectively (comparable to the activity of Verapamil of 12 μM measured with the same assay) as well as five moderate inhibitors of Topoisomerase I. Furthermore, we also screened combinations of compounds with different modes of action to evaluate possible synergistic effects. When evaluating compound synergies, four of the five compounds exhibit synergistic effects in HeLa cell cultures in the presence of the two P-gp inhibitors identified (two independent samples t-test, p < 0.01). Hence, this appears to be one of the first studies where multiple aspects of compound action as predicted by in silico models are prospectively validated, namely phenotypic effect as well as on-target activities, and where synergies between compound combinations could also be experimentally confirmed. © The Royal Society of Chemistry 2013. Source

Conesa C.,Aragon Health science Institute ICS | Conesa C.,University of Zaragoza | Doss M.X.,Stem Cell Center | Antzelevitch C.,Stem Cell Center | And 4 more authors.
Stem Cell Reviews and Reports | Year: 2012

A potential application of embryonic and inducible pluripotent stem cells for the therapy of degenerative diseases involves pure somatic cells, free of tumorigenic undifferentiated embryonic and inducible pluripotent stem cells. In complex collections of chemicals with pharmacological potential we expect to find molecules able to induce specific pluripotent stem cell death, which could be used in some cell therapy settings to eliminate undifferentiated cells. Therefore, we have screened a chemical library of 1120 small chemicals to identify compounds that induce specifically apoptotic cell death in undifferentiated mouse embryonic stem cells (ESCs). Interestingly, three compounds currently used as clinically approved drugs, nortriptyline, benzethonium chloride and methylbenzethonium chloride, induced differential effects in cell viability in ESCs versus mouse embryonic fibroblasts (MEFs). Nortriptyline induced apoptotic cell death in MEFs but not in ESCs, whereas benzethonium and methylbenzethonium chloride showed the opposite effect. Nortriptyline, a tricyclic antidepressant, has also been described as a potent inhibitor of mitochondrial permeability transition, one of two major mechanisms involved in mitochondrial membrane permeabilization during apoptosis. Benzethonium chloride and methylbenzethonium chloride are quaternary ammonium salts used as antimicrobial agents with broad spectrum and have also been described as anticancer agents. A similar effect of benzethonium chloride was observed in human induced pluripotent stem cells (hiPSCs) when compared to both primary human skin fibroblasts and an established human fibroblast cell line. Human fibroblasts and hiPSCs were similarly resistant to nortriptyline, although with a different behavior. Our results indicate differential sensitivity of ESCs, hiPSCs and fibroblasts to certain chemical compounds, which might have important applications in the stem cell-based therapy by eliminating undifferentiated pluripotent stem cells from stem cell-derived somatic cells to prevent tumor formation after transplantation for therapy of degenerative diseases. © 2011 Springer Science+Business Media, LLC. Source

Abian O.,University of Zaragoza | Abian O.,Unidad University | Abian O.,Aragon Health science Institute ICS | Vega S.,University of Zaragoza | And 3 more authors.
Biophysical Journal | Year: 2010

The hepatitis C virus NS3 protease is responsible for the processing of the nonstructural region of viral precursor polyprotein in infected hepatic cells. NS3 has been considered a target for drug discovery for a long time. NS3 is a zinc-dependent serine protease. However, the zinc ion is not involved in the catalytic mechanism, because it is bound far away from the active site. Thus, zinc is essential for the structural integrity of the protein and it is considered to have a structural role. The first thermodynamic study on the conformational equilibrium and stability of NS3 and the effect of zinc on such equilibrium is presented here. In agreement with a previous calorimetric study on the binding of zinc to NS3, the global unfolding heat capacity is dominated by the zinc dissociation step, suggesting that the binding of zinc induces a significant structural rearrangement of the protein. In addition, contrary to other homologous zinc-dependent proteases, the zinc-free NS3 protease is not completely unstructured. It is apparent that the conformational landscape of hepatitis C virus NS3 protease is fairly complex due to its intrinsic plasticity, and to the interactions with its different effectors (zinc and the accessory viral protein NS4A) and their modulation of the population of the different conformational states. © 2010 by the Biophysical Society. Source

Ayuso-Tejedor S.,University of Zaragoza | Abian O.,University of Zaragoza | Abian O.,Aragon Health science Institute ICS | Abian O.,CIBER ISCIII | And 2 more authors.
Biochemistry | Year: 2011

Flavodoxins are bacterial electron transport proteins whose redox competence is due to the presence of a tightly but noncovalently bound FMN molecule. While the thermodynamics of the complex are understood, the mechanism of association between the apoflavodoxin and the redox cofactor is not so clear. We investigate here the mechanism of FMN binding to the apoflavodoxin from Helicobacter pylori, an essential protein that is being used as a target to develop antimicrobials. This flavodoxin is structurally peculiar as it lacks the typical bulky residue interacting with the FMN re face but bears instead a small alanine. FMN binding is biphasic, regardless of the presence of phosphate molecules in solution, while riboflavin binding takes place in a single step, the rate constant of which coincides with the fast phase of FMN binding. A mutational study at the isoalloxazine and phosphate subsites for FMN binding clearly indicates that FMN association is always limited by interaction with the isoalloxazine subsite because mutating residues that interact with the phosphate moiety of FMN in the native complex hardly changes the observed rate constants and amplitudes. In contrast, replacing tyr92, which interacts with the isoalloxazine, greatly lowers the rate constants. Our analysis indicates that the two FMN binding phases observed are related neither with alternative or sequential interaction with the two binding subsites nor with the presence of bound phosphate. It is possible that they reflect the intrinsic conformational heterogeneity of the apoflavodoxin ensemble. © 2011 American Chemical Society. Source

Luesma M.J.,University of Zaragoza | Luesma M.J.,Aragon Health science Institute ICS | Cantarero I.,University of Cordoba, Spain | Cantarero I.,Maimonides Institute for Biomedical Research of Cordoba IMIBIC | And 4 more authors.
BioMed Research International | Year: 2014

The receptor tyrosine kinase Ret (c-Ret) transduces the glial cell line-derived neurotrophic factor (GDNF) signal, one of the neurotrophic factors related to the degeneration process or the regeneration activity of motor neurons in amyotrophic lateral sclerosis (ALS). The phosphorylation of several tyrosine residues of c-Ret seems to be altered in ALS. c-Ret is expressed in motor neurons and in the enteric nervous system (ENS) during the embryonic period. The characteristics of the ENS allow using it as model for central nervous system (CNS) study and being potentially useful for the research of human neurological diseases such as ALS. The aim of the present study was to investigate the cellular localization and quantitative evaluation of marker c-Ret in the adult human gut. To assess the nature of c-Ret positive cells, we performed colocalization with specific markers of cells that typically are located in the enteric ganglia. The colocalization of PGP9.5 and c-Ret was preferentially intense in enteric neurons with oval morphology and mostly peripherally localized in the ganglion, so we concluded that the c-Ret receptor is expressed by a specific subtype of enteric neurons in the mature human ENS of the gut. The functional significance of these c-Ret positive neurons is discussed. © 2014 M. J. Luesma et al. Source

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