Probing the Pore Drug Binding Site of Microtubules with Fluorescent Taxanes: Evidence of Two Binding Poses

The pore site in microtubules has been studied with the use of Hexaflutax, a fluorescent probe derived from paclitaxel. The compound is active in cells with similar effects to paclitaxel, indicating that the pore may be a target to microtubule stabilizing agents. While other taxanes bind microtubules in a monophasic way, thus indicating a single type of sites, Hexaflutax association is biphasic. Analysis of the phases indicates that two different binding sites are detected, reflecting two different modes of binding, which could arise from different arrangements of the taxane or fluorescein moieties in the pore. Association of the 4-4-20 antifluorescein monoclonal antibody-Hexaflutax complex to microtubules remains biphasic, thus indicating that the two phases observed arise from two different poses of the taxane moiety.This work was supported in part by grant BIO2007-61336 from the Ministry of Science and Innovation to J.F.D., BIPPED-CM from Comunidad de Madrid to J.F.D. and J.M.A., and grant MOST No. 2006DFA31490 to W.S.F

New interfacial microtubule inhibitors of marine origin, PM050489/PM060184, with potent antitumor activity and a distinct mechanism

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αβ-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg2+-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αβ-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to β-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating β-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.We wish to thank J. M. Fernandez Sousa (PharmaMar) for useful discussions and support, E. Hamel (NCI) for providing eribulin, C. Scazzocchio and G. Diallinas for useful advice on mutant screening, H. N. Arst for advice on mutant screening and mapping and for kindly providing strains MAD3688 and MAD4655, T. J. Fitzgerald (A&M University) for MTC and C. Alfonso (CIB) for AUC analysis. We also thank Rhône Poulenc Rorer Aventis for supplying docetaxel and Matadero Municipal Vicente de Lucas de Segovia for providing the calf brains for tubulin purification. B.P. had a contract from Comunidad de Madrid, and A.C. had a Ramon y Cajal contract, J.R.-S. had a fellowship from “Programa de Cooperación Científica entre el Ministerio de Ciencia, Tecnologías y Medio Ambiente de la República de Cuba (CITMA) y el CSIC”. This work was supported by grants BIO2010-16351 (J.F.D.), BQU2009-08536 (J.J.-B.), CAM S2010/BMD-2457 (J.F.D.), CAM S2010/BMD-2353 (J.J.-B., J.M.A.), IPT-2011-0752-900000 and BIO2012-30965 (M.A.P.), BFU2011-23416 (J.M.A.) and PharmaMar-CSIC contracts

Enzyme adaptation to habitat thermal legacy shapes the thermal plasticity of marine microbiomes

Microbial communities respond to temperature with physiological adaptation and compositional turnover. Whether thermal selection of enzymes explains marine microbiome plasticity in response to temperature remains unresolved. By quantifying the thermal behaviour of seven functionally-independent enzyme classes (esterase, extradiol dioxygenase, phosphatase, beta-galactosidase, nuclease, transaminase, and aldo-keto reductase) in native proteomes of marine sediment microbiomes from the Irish Sea to the southern Red Sea, we record a significant effect of the mean annual temperature (MAT) on enzyme response in all cases. Activity and stability profiles of 228 esterases and 5 extradiol dioxygenases from sediment and seawater across 70 locations worldwide validate this thermal pattern. Modelling the esterase phase transition temperature as a measure of structural flexibility confirms the observed relationship with MAT. Furthermore, when considering temperature variability in sites with non-significantly different MATs, the broadest range of enzyme thermal behaviour and the highest growth plasticity of the enriched heterotrophic bacteria occur in samples with the widest annual thermal variability. These results indicate that temperature-driven enzyme selection shapes microbiome thermal plasticity and that thermal variability finely tunes such processes and should be considered alongside MAT in forecasting microbial community thermal response

Identification and characterization of carboxyl esterases of gill chamber-associated microbiota in the deep-sea shrimp rimicaris exoculata by using functional metagenomics

The shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (<52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (<356Umg 1) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities.The European Community project MAMBA (FP7-KBBE-2008-226977), grant BIO2011-25012 from the Spanish Ministry of the Economy and Competitiveness (formerly MICINN). P.N.G. and O.V.G. were supported by EU FP7 project MICROB3 (FP7-OCEAN.2011 287589). This work received support from the Government of Canada through Genome Canada and the Ontario Genomics Institute (grant 2009-OGI-ABC-1405 to A.F.Y. and A.S.) and from the U.S. National Institutes of Health (grants GM074942 and GM094585 to A.S. through the Midwest Center for Structural Genomics).http://aem.asm.orgam201

Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats

The present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040–4908 m depth), moderately warm (14.0–16.5°C) biotopes, characterized by a wide range of salinities (39–348 practical salinity units), were investigated for this purpose. An enzyme from a ‘superficial’ marine hydrothermal habitat (65°C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in saltsaturated deep-sea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deepsea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16°C, was shown to contain halopiezophilic-like enzymes that are most active at 70°C and with denaturing temperatures of 71.4°C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to poly-extremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes.European Community project MAMBA (FP7-KBBE-2008-226977). This grant BIO2011-25012 from the Spanish Ministry of Economy and Competitiveness (formerly MICINN). European Commission for ‘MicroB3’ grant (FP7-OCEAN.2011-2 (contract Nr 287589)). Government of Canada through Genome Canada and the Ontario Genomics Institute (grant 2009-OGI-ABC-1405) and U.S. National Institutes of Health (grants GM074942 and GM094585). Midwest Center for Structural Genomics).http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1462-2920hb2016Biochemistr

<scp>ReSurveyEurope</scp>: A database of resurveyed vegetation plots in Europe

AbstractAimsWe introduce ReSurveyEurope — a new data source of resurveyed vegetation plots in Europe, compiled by a collaborative network of vegetation scientists. We describe the scope of this initiative, provide an overview of currently available data, governance, data contribution rules, and accessibility. In addition, we outline further steps, including potential research questions.ResultsReSurveyEurope includes resurveyed vegetation plots from all habitats. Version 1.0 of ReSurveyEurope contains 283,135 observations (i.e., individual surveys of each plot) from 79,190 plots sampled in 449 independent resurvey projects. Of these, 62,139 (78%) are permanent plots, that is, marked in situ, or located with GPS, which allow for high spatial accuracy in resurvey. The remaining 17,051 (22%) plots are from studies in which plots from the initial survey could not be exactly relocated. Four data sets, which together account for 28,470 (36%) plots, provide only presence/absence information on plant species, while the remaining 50,720 (64%) plots contain abundance information (e.g., percentage cover or cover–abundance classes such as variants of the Braun‐Blanquet scale). The oldest plots were sampled in 1911 in the Swiss Alps, while most plots were sampled between 1950 and 2020.ConclusionsReSurveyEurope is a new resource to address a wide range of research questions on fine‐scale changes in European vegetation. The initiative is devoted to an inclusive and transparent governance and data usage approach, based on slightly adapted rules of the well‐established European Vegetation Archive (EVA). ReSurveyEurope data are ready for use, and proposals for analyses of the data set can be submitted at any time to the coordinators. Still, further data contributions are highly welcome.</jats:sec

Optimización de la interacción microtúbulo-taxol: Diseño de taxanos de alta afinidad

Leída en Universidad Autónoma de Madrid. Facultad de Ciencias el 01-13-2009; 214 págs.Developed resistance to drugs is one of the causes of cancer hemotherapy failure. The effect of drug-site interaction optimization to fight against resistance have been studied in cells resistant to taxanes by two different mechanisms, P-glycoprotein ediated multidrug resistance and mutations in the taxane site. To design drugs with high affinity for the taxane site microtubule binding affinities of a series of synthetic taxanes have been measured. From these measurements we could dissect individual group contributions and obtain a rationale for the design of novel compounds with the ability to vercome P-glycoprotein-mediated drug resistance. As previously observed for epothilones, contributions of the different substituents to the binding free energy are cumulative. Combination of the most favourable substitutions in one single analogue increased the binding affinity 500-fold over that of taxol. Structural models built in this study assigned an important role to the interactions of C2 and C13 substituents with the protonated side chain of His229 in β-tubulin. The relative orientation of these groups was found to be in agreement with NMR data obtained for microtubules-bound docetaxel. The cytotoxicities of the compounds in A2780 cells correlate with their affinities with an apparent cytotoxicity limit in the nanomolar range. Taxane resistance index plotted versus binding affinity fits a bell-shaped curve showing that the P-gp-overexpressing cells (A2780AD) are sensitive to the highest and lowest affinity compounds, whereas resistance indexes in the range of 100 to 1000 were obtained for those ligands whose binding affinities for tubulin and Pgp are similar. This finding strongly indicates that for a series of compounds with similar pharmacokinetic andioavailability properties, optimization of the ligand-target interaction is a good strategy to overcome multidrug resistance mediated by efflux pumps. Moreover same high-affinity compounds seem to avoid resistance in cells with mutated tubulins as probed with their cytotoxicities in cells PTX10 and PTX22 when compared with the parental 1A9 cell line wich contains wild type tubulin.Peer reviewe

Thermodynamics as a Tool for the Optimization of Drug Binding, Thermodynamics - Interaction Studies - Solids, Liquids and Gases

20 páginas, 5 figuras, 2 tablas -- PAGS nros. 777-796A non-covalent interaction is a kind of chemical bond, typically between macromolecules, that involves dispersed variations of electromagnetic interactions (Alberts et al. 1994; Connors & Mecozzi 2010). Non-covalent interactions are individually weak as compared with covalent bonds, but their net strength is higher than the sum of that of the individual interactions. There are few drugs that bind irreversibly to their targets, in pharmacology, most drugs establish non-covalent interactions with their target molecules (usually proteins). From a chemical point of view, the affinity constant (Ka) is a very useful measurement for the study of binding reactions as it provides much information about the mechanism. In many cases some chemical or physical properties of ligand or target change with the interaction between them, these changes might help to measure binding constants. It is important to establish the stoichiometry of the complex to be sure that the constants are accurately calculated. From the affinity constants measured it is possible to calculate the standard thermodynamic quantities for the binding reaction: free-energy (ΔG), enthalpy (ΔH) and entropy (ΔS). Our group has already demonstrated that, in some cases, binding affinity measurements are very helpful for the optimization of ligand binding as it can be determined the contribution of every single chemical modification of the ligand to the binding affinity (Buey et al. 2004; Matesanz et al. 2008) One of the objectives of drug development is the search of new or modified compounds with improved properties such as better potency, higher selectivity, better pharmacokinetics or superior drug resistance profiles. An important goal in this objective is the optimization of drugs binding affinity towards their targets, as binding affinity is directly related to potency (Ruben et al. 2006). Moreover, it has been shown that extremely high affinity drugs reflect as well changes in other properties like selectivity (Ohtaka et al. 2004; Ohtaka & Freire 2005) or resistance overcoming ability (Matesanz et al. 2008). Examples of the importance of ligand affinity in drug optimization can be observed in the development of HIV-1 protease inhibitors and statins (cholesterol lowering drugs) over the years as remarked in (Freire 2008). In this chapter we will study the nature of non-covalent interations and the concept of binding constant for these interactions. Examples of methodologies to measure binding constants of small ligands to macromolecules will be introduced and we will emphasize theneed to determine the stoichiometry of the studied system to calculate accurately the constants. Once the thermodynamic concepts were introduced, we will show the use of these kind of studies for the optimization of drug binding to its target. We will detail the role of single chemical modifications in the molecule of study to modulate its binding affinity, and the way to quantify these changes. We will finally further discuss how the selection of the best sustituents can result in the optimization of bindinPeer reviewe