Exploración de los sitios de unión de antitumorales a la tubulina mediante herramientas computacionales y simulaciones de dinámica molecular

El cáncer es sus diferentes manifestaciones es una de las principales enfermedades de nuestros días y, según la Organización Mundial de la Salud, una de las principales causas de muerte en los países desarrollados. El arsenal farmacéutico empleado para tratar los tumores incluye agentes que interfieren en la biosíntesis de ADN y otros que alteran la maquinaria de la célula necesaria para la correcta división celular. A esta familia de agentes terapéuticos pertenecen los fármacos que actúan interfiriendo en la dinámica microtubular. Este grupo fue inicialmente integrado por los alcaloides de la Vinca y posteriormente ampliado con la incorporación del paclitaxel, aislado de la corteza del Tejo del Pacífico y que constituyó un hito en el tratamiento del cáncer de ovario. Aún así, siguen siendo necesarios grandes esfuerzos para obtener nuevas entidades químicas capaces de alterar la dinámica microtubular al unirse a sus dianas con gran afinidad, a la vez que sean capaces de soslayar las diferentes resistencias al tratamiento que se han ido describiendo. Los métodos computacionales se han convertido en herramientas cada vez más útiles para la caracterización de sitios de unión de los ligandos a sus receptores, el estudio del acoplamiento del ligando al receptor y la realización de dinámicas moleculares, todas ellas capaces de aportar información que normalmente va más allá de las capacidades de los métodos experimentales. El objetivo de esta tesis doctoral fue estudiar la unión fármacos estabilizantes y desestabilizantes de microtúbulos a sus diferentes dianas en la tubulina mediante técnicas de modelado molecular y métodos computacionales, y dar una explicación plausible a las relaciones estructura-actividad descritas para ellos. Este trabajo de investigación se realizó en colaboración con los grupos de los profesores Fernando Díaz y José Manuel Andreu del Centro de Investigaciones Biológicas (CSIC), Weishuo Fang del Institute of Materia Medica of the Chinese Academy of Medical Sciences, Dr. Antonio Morreale del Centro de Biología Molecular Severo Ochoa (CSIC) e investigadores de la empresa privada PharmaMar

Exploración de los sitios de unión de antitumorales a la tubulina mediante herramientas computacionales y simulaciones de dinámica molecular

El cáncer es sus diferentes manifestaciones es una de las principales enfermedades de nuestros días y, según la Organización Mundial de la Salud, una de las principales causas de muerte en los países desarrollados. El arsenal farmacéutico empleado para tratar los tumores incluye agentes que interfieren en la biosíntesis de ADN y otros que alteran la maquinaria de la célula necesaria para la correcta división celular. A esta familia de agentes terapéuticos pertenecen los fármacos que actúan interfiriendo en la dinámica microtubular. Este grupo fue inicialmente integrado por los alcaloides de la Vinca y posteriormente ampliado con la incorporación del paclitaxel, aislado de la corteza del Tejo del Pacífico y que constituyó un hito en el tratamiento del cáncer de ovario. Aún así, siguen siendo necesarios grandes esfuerzos para obtener nuevas entidades químicas capaces de alterar la dinámica microtubular al unirse a sus dianas con gran afinidad, a la vez que sean capaces de soslayar las diferentes resistencias al tratamiento que se han ido describiendo. Los métodos computacionales se han convertido en herramientas cada vez más útiles para la caracterización de sitios de unión de los ligandos a sus receptores, el estudio del acoplamiento del ligando al receptor y la realización de dinámicas moleculares, todas ellas capaces de aportar información que normalmente va más allá de las capacidades de los métodos experimentales. El objetivo de esta tesis doctoral fue estudiar la unión fármacos estabilizantes y desestabilizantes de microtúbulos a sus diferentes dianas en la tubulina mediante técnicas de modelado molecular y métodos computacionales, y dar una explicación plausible a las relaciones estructura-actividad descritas para ellos. Este trabajo de investigación se realizó en colaboración con los grupos de los profesores Fernando Díaz y José Manuel Andreu del Centro de Investigaciones Biológicas (CSIC), Weishuo Fang del Institute of Materia Medica of the Chinese Academy of Medical Sciences, Dr. Antonio Morreale del Centro de Biología Molecular Severo Ochoa (CSIC) e investigadores de la empresa privada PharmaMar

The Many Ways to Deal with STING

The stimulator of interferon genes (STING) is an adaptor protein involved in the activation of IFN-β and many other genes associated with the immune response activation in vertebrates. STING induction has gained attention from different angles such as the potential to trigger an early immune response against different signs of infection and cell damage, or to be used as an adjuvant in cancer immune treatments. Pharmacological control of aberrant STING activation can be used to mitigate the pathology of some autoimmune diseases. The STING structure has a well-defined ligand binding site that can harbor natural ligands such as specific purine cyclic di-nucleotides (CDN). In addition to a canonical stimulation by CDNs, other non-canonical stimuli have also been described, whose exact mechanism has not been well defined. Understanding the molecular insights underlying the activation of STING is important to realize the different angles that need to be considered when designing new STING-binding molecules as therapeutic drugs since STING acts as a versatile platform for immune modulators. This review analyzes the different determinants of STING regulation from the structural, molecular, and cell biology points of view.This work was supported by Ministerio de Ciencia e Innovación PID2019-105761RBI00/AEI/10.13039/501100011033. J.A-H. was supported by the PFIS fellowship co-funded by the FEDER/FSE and the ISCIII.S

Mechanistic insight into the reaction catalysed by bacterial type II dehydroquinases

DHQ2 (type II dehydroquinase), which is an essential enzyme in Helicobacter pylori and Mycobacterium tuberculosis and does not have any counterpart in humans, is recognized to be an attractive target for the development of new antibacterial agents. Computational and biochemical studies that help understand in atomic detail the catalytic mechanism of these bacterial enzymes are reported in the present paper. A previously unknown key role of certain conserved residues of these enzymes, as well as the structural changes responsible for triggering the release of the product from the active site, were identified. Asp89*/Asp88* from a neighbouring enzyme subunit proved to be the residue responsible for the deprotonation of the essential tyrosine to afford the catalytic tyrosinate, which triggers the enzymatic process. The essentiality of this residue is supported by results from site-directed mutagenesis. For H. pylori DHQ2, this reaction takes place through the assistance of a water molecule, whereas for M. tuberculosis DHQ2, the tyrosine is directly deprotonated by the aspartate residue. The participation of a water molecule in this deprotonation reaction is supported by solvent isotope effects and proton inventory studies. MD simulation studies provide details of the required motions for the catalytic turnover, which provides a complete overview of the catalytic cycle. The product is expelled from the active site by the essential arginine residue and after a large conformational change of a loop containing two conserved arginine residues (Arg109/Arg108 and Arg113/Arg112), which reveals a previously unknown key role for these residues. The present study highlights the key role of the aspartate residue whose blockage could be useful in the rational design of inhibitors and the mechanistic differences between both enzymesFinancial support from the Comunidad de Madrid (S2010-BMD-2457 to F.G.), Xunta de Galicia (10PXIB2200122PR and GRC2010/12 to C.G.-B.) and the Spanish Ministry of Science and Innovation (SAF2009-13914-C02-02 to F.G. and SAF2010-15076 to C.G.-B.) is 5076 to CGB and BFU2008-01588/BMC to MJvR) is gratefully acknowledged. C.C. and A.P. thank the Spanish Ministry of Science and Innovation for their respective FPU fellowshipsS

Tubulin-based structure-affinity relationships for antimitotic Vinca alkaloids

The Vinca alkaloids are a group of widely used anticancer drugs, originally extracted from the Madagascar periwinkle, that disrupt microtubule dynamics in mammalian cells by interfering with proper assembly of α,β-tubulin heterodimers. They favor curved tubulin assemblies that destabilize microtubules and induce formation of spiral aggregates. Their binding energy profiles have been characterized by means of sedimentation velocity assays and the binding site of vinblastine at the interface between two tubulin dimers (α 1β 1-α 2β 2) has been ascertained by X-ray crystallographic studies on a complex of tubulin with the stathmin-like domain of protein RB3, albeit at relatively low resolution. Here we use molecular modeling and simulation techniques to build, refine and perform a comparative analysis of the three-dimensional complexes of vinblastine, vincristine, vinorelbine and vinflunine with a β 1α 2-tubulin interface in explicit water to rationalize the binding affinity differences in structural and energetic terms. Our results shed some more light into the binding determinants and the structure-activity relationships of these clinically useful agents.Peer Reviewe

Comparative Binding Energy (COMBINE) Analysis Supports a Proposal for the Binding Mode of Epothilones to β-Tubulin

The conformational preferences of epothiloneA (EPA) and a 12,13-cyclopropyl C12-epimerized analogue were explored in aqueous solution using molecular dynamics simulations. The simulated conformers that provided an optimal fit in the paclitaxel binding site of mammalian β-tubulin were then selected. The resulting modeled complexes were simulated before and after refinement of the M-loop to improve the fitting and assess ligand stability within the binding pocket. The tubulin-bound conformation of EPA was found to be unlike a previously reported solution obtained through mixed crystallographic/NMR/modeling studies. However, our conformation was in agreement with an NMR-based proposal although the exact binding pose within the site was different. Molecular models were built for the complexes of 14 epothilone derivatives with β-tubulin. A projection to latent structures regression method succeeded in providing a good prediction of the experimentally measured binding enthalpies for the whole set of ligands by assigning weights to a selection of interaction energy terms. These receptor-based, quantitative structure-activity relationships support the proposed binding mode, help confirm and interpret previously acquired experimental data, shed additional light on the effect of several β-tubulin mutations on ligand binding, and can potentially direct further experimental studies. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Comisión Interministerial de Ciencia y Tecnología de España; Comunidad de Madrid; Ministerio de Ciencia e InnovaciónPeer Reviewe

Insights into the interaction of discodermolide and docetaxel with tubulin. Mapping the binding sites of microtubule-stabilizing agents by using an integrated NMR and computational approach

41 p.-7 fig.-3 fig.-supl.The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/β-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/β-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/β-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubulesThis work was supported in part by grant BIO2010-16351 and BQU2009-08536 from MICINN (to J.F.D. and J.J.B., respectively), BIPPED-CM from Comunidad de Madrid (J.F.D., J.J.B., and J.M.A.), and EPSRC (I.P.). We also thank CESGA and the CAI NMR of Universidad Complutense for providing access to their facilitiesPeer reviewe

Optimization of Taxane Binding to Microtubules: Binding Affinity Dissection and Incremental Construction of a High-Affinity Analog of Paclitaxel

13 páginas, 5 figuras, 2 tablas -- PAGS nro.s 573-585The microtubule binding affinities of a series of synthetic taxanes have been measured with the aims of dissecting individual group contributions and obtaining a rationale for the design of novel compounds with the ability to overcome drug resistance. As previously observed for epothilones, the positive and negative contributions of the different substituents to the binding free energies are cumulative. By combining the most favorable substitutions we increased the binding affinity of paclitaxel 500-fold. Insight into the structural basis for this improvement was gained with molecular modeling and NMR data obtained for microtubule-bound docetaxel. Taxanes with affinities for microtubules well above their affinities for P-glycoprotein are shown not to be affected by multidrug resistance. This finding strongly indicates that optimization of the ligand-target interaction is a good strategy to overcome multidrug resistance mediated by efflux pumpsThis work was supported in part by grant BIO2007-61336 from M.E.C. to J.F.D., BIPPED-CM from Comunidad de Madrid to F.G., J.F.D., J.J.B., and J.M.A., and grants NSFC 20572135 and MOST 2006DFA31490 to W.S.FPeer reviewe

Molecular Imaging Probes Based on Matrix Metalloproteinase Inhibitors (MMPIs)

Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases which are secreted or anchored in the cell membrane and are capable of degrading the multiple components of the extracellular matrix (ECM). MMPs are frequently overexpressed or highly activated in numerous human diseases. Owing to the important role of MMPs in human diseases, many MMP inhibitors (MMPIs) have been developed as novel therapeutics, and some of them have entered clinical trials. However, so far, only one MMPI (doxycycline) has been approved by the FDA. Therefore, the evaluation of the activity of a specific subset of MMPs in human diseases using clinically relevant imaging techniques would be a powerful tool for the early diagnosis and assessment of the efficacy of therapy. In recent years, numerous MMPIs labeled imaging agents have emerged. This article begins by providing an overview of the MMP subfamily and its structure and function. The latest advances in the design of subtype selective MMPIs and their biological evaluation are then summarized. Subsequently, the potential use of MMPI-labeled diagnostic agents in clinical imaging techniques are discussed, including positron emission tomography (PET), single-photon emission computed tomography (SPECT) and optical imaging (OI). Finally, this article concludes with future perspectives and clinical utility

Identification of Aldo-keto reductase AKR1B10 as a selective target for modification and inhibition by prostaglandin A1: Implications for anti-tumoral activity

11 páginas, 6 figuras -- PAGS nros. 4161-4171Cyclopentenone prostaglandins (cyPG) are reactive eicosanoids that may display anti-inflammatory and antiproliferative actions, possibly offering therapeutic potential. Here we report the identification of members of the aldo-keto reductase (AKR) family as selective targets of the cyPG prostaglandin A1 (PGA1). AKR enzymes metabolize aldehydes and drugs containing carbonyl groups and are involved in inflammation and tumorigenesis. Thus, these enzymes represent a class of targets to develop small molecule inhibitors with therapeutic activity. Molecular modeling studies pointed to the covalent binding of PGA1 to Cys299, close to the active site of AKR, with His111 and Tyr49, which are highly conserved in the AKR family, playing a role in PGA1 orientation. Among AKR enzymes, AKR1B10 is considered as a tumor marker and contributes to tumor development and chemoresistance. We validated the direct modification of AKR1B10 by biotinylated PGA1 (PGA1-B) in cells, and confirmed that mutation of Cys299 abolishes PGA1-B incorporation, whereas substitution of His111 or Tyr49 reduced the interaction. Modification of AKR1B10 by PGA1 correlated with loss of enzymatic activity and both effects were increased by depletion of cellular glutathione. Moreover, in lung cancer cells PGA1 reduced tumorigenic potential and increased accumulation of the AKR substrate doxorubicin, potentiating cell-cycle arrest induced by this hemotherapeutic agent. Our findings define PGA1 as a new AKR inhibitor and they offer a framework to develop compounds that could counteract cancer chemoresistanceThis work was supported by grants SAF2009-11642 from MiCInn and RETICS RD07/0064/0007 from ISCIII to D. Pérez-Sala and SAF2006-12713-C02-02 (CICYT) and S-BIO/0214/2006 (CAM) to F. Gago. J. Gayarre was the recipient of fellowships from CSIC (I3P), EMBO, and FEBS (short term fellowships). C. Coderch was supported by a MiCInn FPU (AP2007-01225) fellowship. Part of this work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme. Feedback from COST Action CM1001 is appreciated. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this factPeer reviewe