44 results on '"Mühlethaler, Tobias"'
Search Results
2. Bridging the maytansine and vinca sites: Cryptophycins target β-tubulin’s T5-loop
- Author
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Abel, Anne-Catherine, Mühlethaler, Tobias, Dessin, Cedric, Schachtsiek, Thomas, Sammet, Benedikt, Sharpe, Timothy, Steinmetz, Michel O., Sewald, Norbert, and Prota, Andrea E.
- Published
- 2024
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3. Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography
- Author
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Wranik, Maximilian, Weinert, Tobias, Slavov, Chavdar, Masini, Tiziana, Furrer, Antonia, Gaillard, Natacha, Gioia, Dario, Ferrarotti, Marco, James, Daniel, Glover, Hannah, Carrillo, Melissa, Kekilli, Demet, Stipp, Robin, Skopintsev, Petr, Brünle, Steffen, Mühlethaler, Tobias, Beale, John, Gashi, Dardan, Nass, Karol, Ozerov, Dmitry, Johnson, Philip J. M., Cirelli, Claudio, Bacellar, Camila, Braun, Markus, Wang, Meitian, Dworkowski, Florian, Milne, Chris, Cavalli, Andrea, Wachtveitl, Josef, Steinmetz, Michel O., and Standfuss, Jörg
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- 2023
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- View/download PDF
4. Filling of a water-free void explains the allosteric regulation of the β1-adrenergic receptor by cholesterol
- Author
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Abiko, Layara Akemi, Dias Teixeira, Raphael, Engilberge, Sylvain, Grahl, Anne, Mühlethaler, Tobias, Sharpe, Timothy, and Grzesiek, Stephan
- Published
- 2022
- Full Text
- View/download PDF
5. Novel fragment-derived colchicine-site binders as microtubule-destabilizing agents
- Author
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de la Roche, Noelia Montel, Mühlethaler, Tobias, Di Martino, Rita Maria Concetta, Ortega, Jose Antonio, Gioia, Dario, Roy, Bibhas, Prota, Andrea E., Steinmetz, Michel O., and Cavalli, Andrea
- Published
- 2022
- Full Text
- View/download PDF
6. Crystal Structure of the Cyclostreptin-Tubulin Adduct: Implications for Tubulin Activation by Taxane-Site Ligands.
- Author
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Balaguer, Francisco, Mühlethaler, Tobias, Estévez-Gallego, Juan, Calvo, Enrique, Giménez-Abián, Juan, Risinger, April, Sorensen, Erik, Vanderwal, Christopher, Altmann, Karl-Heinz, Mooberry, Susan, Steinmetz, Michel, Oliva, María, Prota, Andrea, and Díaz, J
- Subjects
cyclostreptin ,microtubules ,multidrug resistance ,taxanes ,tubulin ,Drug Resistance ,Neoplasm ,Edetic Acid ,HeLa Cells ,Humans ,Mass Spectrometry ,Microtubules ,Polycyclic Compounds ,Taxoids ,Tubulin - Abstract
It has been proposed that one of the mechanisms of taxane-site ligand-mediated tubulin activation is modulation of the structure of a switch element (the M-loop) from a disordered form in dimeric tubulin to a folded helical structure in microtubules. Here, we used covalent taxane-site ligands, including cyclostreptin, to gain further insight into this mechanism. The crystal structure of cyclostreptin-bound tubulin reveals covalent binding to βHis229, but no stabilization of the M-loop. The capacity of cyclostreptin to induce microtubule assembly compared to other covalent taxane-site agents demonstrates that the induction of tubulin assembly is not strictly dependent on M-loop stabilization. We further demonstrate that most covalent taxane-site ligands are able to partially overcome drug resistance mediated by βIII-tubulin (βIII) overexpression in HeLa cells, and compare their activities to pironetin, an interfacial covalent inhibitor of tubulin assembly that displays invariant growth inhibition in these cells. Our findings suggest a relationship between a diminished interaction of taxane-site ligands with βIII-tubulin and βIII tubulin-mediated drug resistance. This supports the idea that overexpression of βIII increases microtubule dynamicity by counteracting the enhanced microtubule stability promoted by covalent taxane-site binding ligands.
- Published
- 2019
7. Strengthening an Intramolecular Non‐Classical Hydrogen Bond to Get in Shape for Binding.
- Author
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Varga, Norbert, Smieško, Martin, Jiang, Xiaohua, Jakob, Roman P., Wagner, Beatrice, Mühlethaler, Tobias, Dätwyler, Philipp, Zihlmann, Pascal, Rabbani, Said, Maier, Timm, Schwardt, Oliver, and Ernst, Beat
- Subjects
ISOTHERMAL titration calorimetry ,HYDROGEN bonding ,PHARMACOKINETICS ,ENTROPY ,MOIETIES (Chemistry) - Abstract
In this research article, we report on the strengthening of a non‐classical hydrogen bond (C−H⋅⋅⋅O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E‐selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre‐organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre‐organization, the stabilization by a non‐classical hydrogen bond between the H−C5 of l‐fucose and the ring oxygen O5 of the neighboring d‐galactose moiety is essential and yields 7.4 kJ mol−1. This effect could be further strengthened by replacing l‐fucose by 6,6,6‐trifluoro‐l‐fucose leading to an improved non‐classical H‐bond of 14.9 kJ mol−1, i.e. an improved pre‐organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR‐shifts of the H−C5Fuc, by X‐ray diffraction analysis of glycomimetics co‐crystallized with E‐selectin as well as by isothermal titration calorimetry. Furthermore, the electron‐withdrawing character of the CF3‐group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid‐stability, a prerequisite for gastrointestinal stability, could be substantially improved. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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8. Crystallization Systems for the High-Resolution Structural Analysis of Tubulin–Ligand Complexes
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Mühlethaler, Tobias, primary, Olieric, Natacha, additional, Ehrhard, Valentin A., additional, Wranik, Maximilian, additional, Standfuss, Jörg, additional, Sharma, Ashwani, additional, Prota, Andrea E., additional, and Steinmetz, Michel O., additional
- Published
- 2022
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9. Direct observation of coherent azobenzene photochemistry
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Standfuss, Jörg, primary, Weinert, Tobias, additional, Wranik, Maximillian, additional, Church, Jonathan, additional, Seidel, Hans Peter, additional, Slavov, Chavdar, additional, Masini, Tiziana, additional, James, Daniel, additional, Glover, Hannah, additional, Carrillo, Melissa, additional, Kekilli, Demet, additional, Stipp, Robin, additional, Skopintsev, Petr, additional, Bruenle, Steffen, additional, Gaillard, Natacha, additional, Furrer, Antonia, additional, Gashi, Dardan, additional, Mühlethaler, Tobias, additional, Beale, John, additional, Nass, Karol, additional, Johnson, Philip, additional, Cirelli, Claudio, additional, Ozerov, Dmitry, additional, Dworkowski, Florian, additional, Bacellar, Camila, additional, Milne, Christopher, additional, Steinmetz, Michel, additional, Wachtveitl, Josef, additional, and Schapiro, Igor, additional
- Published
- 2023
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10. Tetra‐ and Hexavalent Siglec‐8 Ligands Modulate Immune Cell Activation
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Conti, Gabriele, primary, Bärenwaldt, Anne, additional, Rabbani, Said, additional, Mühlethaler, Tobias, additional, Sarcevic, Mirza, additional, Jiang, Xiaohua, additional, Schwardt, Oliver, additional, Ricklin, Daniel, additional, Pieters, Roland J., additional, Läubli, Heinz, additional, and Ernst, Beat, additional
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- 2023
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11. Structural Basis of Colchicine-Site targeting Acylhydrazones active against Multidrug-Resistant Acute Lymphoblastic Leukemia
- Author
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Cury, Nathália Moreno, Mühlethaler, Tobias, Laranjeira, Angelo Brunelli Albertoni, Canevarolo, Rafael Renatino, Zenatti, Priscila Pini, Lucena-Agell, Daniel, Barasoain, Isabel, Song, Chunhua, Sun, Dongxiao, Dovat, Sinisa, Yunes, Rosendo Augusto, Prota, Andrea Enrico, Steinmetz, Michel Olivier, Díaz, José Fernando, and Yunes, José Andrés
- Published
- 2019
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12. A Structural-Reporter Group to Determine the Core Conformation of Sialyl Lewisx Mimetics
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Wagner, Beatrice, primary, Binder, Florian P. C., additional, Jiang, Xiaohua, additional, Mühlethaler, Tobias, additional, Preston, Roland C., additional, Rabbani, Said, additional, Smieško, Martin, additional, Schwardt, Oliver, additional, and Ernst, Beat, additional
- Published
- 2023
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13. Structural insight into the stabilization of microtubules by taxanes
- Author
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Prota, Andrea E, primary, Lucena-Agell, Daniel, additional, Ma, Yuntao, additional, Estevez-Gallego, Juan, additional, Li, Shuo, additional, Bargsten, Katja, additional, Josa-Prado, Fernando, additional, Altmann, Karl-Heinz, additional, Gaillard, Natacha, additional, Kamimura, Shinji, additional, Mühlethaler, Tobias, additional, Gago, Federico, additional, Oliva, Maria A, additional, Steinmetz, Michel O, additional, Fang, Wei-Shuo, additional, and Díaz, J Fernando, additional
- Published
- 2023
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14. Tetra- and Hexavalent Siglec-8 Ligands Modulate Immune Cell Activation
- Author
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Chemical Biology and Drug Discovery, Afd Chemical Biology and Drug Discovery, Conti, Gabriele, Bärenwaldt, Anne, Rabbani, Said, Mühlethaler, Tobias, Sarcevic, Mirza, Jiang, Xiaohua, Schwardt, Oliver, Ricklin, Daniel, Pieters, Roland J, Läubli, Heinz, Ernst, Beat, Chemical Biology and Drug Discovery, Afd Chemical Biology and Drug Discovery, Conti, Gabriele, Bärenwaldt, Anne, Rabbani, Said, Mühlethaler, Tobias, Sarcevic, Mirza, Jiang, Xiaohua, Schwardt, Oliver, Ricklin, Daniel, Pieters, Roland J, Läubli, Heinz, and Ernst, Beat
- Published
- 2023
15. Datasets for 'Filling of a water-free void explains the allosteric regulation of the β1-adrenergic receptor by cholesterol'
- Author
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Abiko, Layara Akemi, Dias Teixeira, Raphael, Engilberge, Sylvain, Grahl, Anne, Mühlethaler, Tobias, Sharpe, Timothy, and Grzesiek, Stephan
- Subjects
β1-adrenergic receptor ,cholesterol ,structural biology ,G protein-coupled receptor ,allosteric regulation ,xenon - Abstract
Raw data for the X-ray structure determination of a xenon-derivatized isoprenaline∙β1AR crystal, the xenon atom anomalous maps, NMR spectra, and for the ITC curves presented in the manuscript "Filling of a water-free void explains the allosteric regulation of the β1-adrenergic receptor by cholesterol". The main folder (Xe_isoprenaline_b1AR) contains two subfolders. The coordinate file (.pdb), the hkl file, and the electron density map (.mtz) are placed in the folder "structure". The second folder, “anomalous_map”, contains the anomalous maps (.pha and .map), and the hkl files for the four different crystals evaluated (organized in separate folders for the each crystal 1-4). The images for the 4 crystals are placed in separate folders (images_crystal_1, images_crystal_2, images_crystal_3, and images_crystal_4). The folder “NMR” contains processed 1H-15N TROSY NMR spectra (.DAT files), and full Bruker directories for 1H experiments. The datasets are organized in subfolders named according to the manuscript figures. The folder “ITC” contains the original ITC files (.itc) presented in Figure 4 and supplementary Figure S5 of the manuscript., This work was supported by the Swiss National Science Foundation (Grants CRSK-3_195592 to L.A.A., and 31-149927, 31-173089, 31-201270 to S.G.).
- Published
- 2022
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16. Release of a photopharmacological drug from its protein target captured by time-resolved serial crystallography
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Wranik, Maximilian, primary, Weinert, Tobias, additional, Slavov, Chavdar, additional, Masini, Tiziana, additional, Furrer, Antonia, additional, Gaillard, Natacha, additional, Gioia, Dario, additional, Ferrarotti, Marco, additional, James, Daniel, additional, Glover, Hannah, additional, Carrillo, Melissa, additional, Kekilli, Demet, additional, Stipp, Robin, additional, Skopintsev, Petr, additional, Brünle, Steffen, additional, Mühlethaler, Tobias, additional, Beale, John, additional, Gashi, Dardan, additional, Nass, Karol, additional, Ozerov, Dmitry, additional, Johnson, Philip, additional, Cirelli, Claudio, additional, Bacellar, Camila, additional, Braun, Markus, additional, Wang, Meitian, additional, Dworkowski, Florian, additional, Milne, Christopher, additional, Cavalli, Andrea, additional, Wachtveitl, Josef, additional, Steinmetz, Michel, additional, and Standfuss, Jörg, additional
- Published
- 2022
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17. Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol
- Author
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Prota, Andrea E. [0000-0003-0875-5339], Lucena-Agell, Daniel [0000-0001-7198-2900], Estévez-Gallego, Juan [0000-0003-3889-8488], Roca, Carlos [0000-0003-0230-1926], Josa-Prado, Fernando [0000-0002-6162-3231], Giménez-Abián, Juan F. [0000-0002-9220-286X], Canales, Ángeles [0000-0003-0542-3080], Andreu, José Manuel [0000-0001-8064-6933], Altmann, Karl-Heinz [0000-0002-0747-9734], Olieric, Natacha [0000-0002-6273-390X], Kamimura, Shinji [0000-0003-4359-0859], Mühlethaler, Tobias [0000-0001-8437-6619], Oliva, María A. [0000-0002-2215-4639], Steinmetz, Michel O. [0000-0001-6157-3687], Díaz, José Fernando [0000-0003-2743-3319], Prota, Andrea E., Lucena-Agell, Daniel, Ma, Yuntao, Estévez-Gallego, Juan, Roca, Carlos, Josa-Prado, Fernando, Goossens, Kenneth, Giménez-Abián, Juan F., Li, Shuo, Canales, Ángeles, Bargsten, Katja, Andreu, José Manuel, Altmann, Karl-Heinz, Olieric, Natacha, Kamimura, Shinji, Mühlethaler, Tobias, Oliva, María A., Steinmetz, Michel O., Fang, Wei-Shuo, Díaz, José Fernando, Prota, Andrea E. [0000-0003-0875-5339], Lucena-Agell, Daniel [0000-0001-7198-2900], Estévez-Gallego, Juan [0000-0003-3889-8488], Roca, Carlos [0000-0003-0230-1926], Josa-Prado, Fernando [0000-0002-6162-3231], Giménez-Abián, Juan F. [0000-0002-9220-286X], Canales, Ángeles [0000-0003-0542-3080], Andreu, José Manuel [0000-0001-8064-6933], Altmann, Karl-Heinz [0000-0002-0747-9734], Olieric, Natacha [0000-0002-6273-390X], Kamimura, Shinji [0000-0003-4359-0859], Mühlethaler, Tobias [0000-0001-8437-6619], Oliva, María A. [0000-0002-2215-4639], Steinmetz, Michel O. [0000-0001-6157-3687], Díaz, José Fernando [0000-0003-2743-3319], Prota, Andrea E., Lucena-Agell, Daniel, Ma, Yuntao, Estévez-Gallego, Juan, Roca, Carlos, Josa-Prado, Fernando, Goossens, Kenneth, Giménez-Abián, Juan F., Li, Shuo, Canales, Ángeles, Bargsten, Katja, Andreu, José Manuel, Altmann, Karl-Heinz, Olieric, Natacha, Kamimura, Shinji, Mühlethaler, Tobias, Oliva, María A., Steinmetz, Michel O., Fang, Wei-Shuo, and Díaz, José Fernando
- Abstract
Paclitaxel (Taxol®) is a first-line chemotherapeutic drug that promotes the curved-to-straight conformational transition of tubulin, an activation step that is necessary for microtubule formation. Crystallization of Taxol bound to tubulin has been long elusive. We found that baccatin III, the core structure of paclitaxel which lacks the C13 side chain, readily co-crystallizes with curved tubulin. Tailor-made taxanes with alternative side chains also co-crystallized, allowing us to investigate their binding modes. Interestingly, these Taxol derived compounds lost their microtubule stabilizing activity and cytotoxicity but kept their full microtubule binding affinity, and all induced lattice expansion upon binding. Additional nuclear magnetic resonance studies propose that Taxol binds to a small fraction of straight tubulin present in solution. Our results suggest a mode of action of Taxol, where the core structure is responsible for the interacting energy while the bulky hydrophobic C13 side chain enables binding selectively to straight tubulin and promotes stabilization.
- Published
- 2021
18. Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol
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Prota, Andrea E., Lucena-Agell, Daniel, Ma, Yuntao, Estévez-Gallego, Juan, Roca, Carlos, Josa-Prado, Fernando, Goossens, Kenneth, Giménez-Abián, Juan F., Li, Shuo, Canales, Ángeles, Bargsten, Katja, Andreu, José Manuel, Altmann, Karl-Heinz, Olieric, Natacha, Kamimura, Shinji, Mühlethaler, Tobias, Oliva, María A., Steinmetz, Michel O., Fang, Wei-Shuo, Díaz, José Fernando, Prota, Andrea E., Lucena-Agell, Daniel, Estévez-Gallego, Juan, Roca, Carlos, Josa-Prado, Fernando, Giménez-Abián, Juan F., Canales, Ángeles, Andreu, José Manuel, Altmann, Karl-Heinz, Olieric, Natacha, Kamimura, Shinji, Mühlethaler, Tobias, Oliva, María A., Steinmetz, Michel O., Díaz, José Fernando, Prota, Andrea E. [0000-0003-0875-5339], Lucena-Agell, Daniel [0000-0001-7198-2900], Estévez-Gallego, Juan [0000-0003-3889-8488], Roca, Carlos [0000-0003-0230-1926], Josa-Prado, Fernando [0000-0002-6162-3231], Giménez-Abián, Juan F. [0000-0002-9220-286X], Canales, Ángeles [0000-0003-0542-3080], Andreu, José Manuel [0000-0001-8064-6933], Altmann, Karl-Heinz [0000-0002-0747-9734], Olieric, Natacha [0000-0002-6273-390X], Kamimura, Shinji [0000-0003-4359-0859], Mühlethaler, Tobias [0000-0001-8437-6619], Oliva, María A. [0000-0002-2215-4639], Steinmetz, Michel O. [0000-0001-6157-3687], and Díaz, José Fernando [0000-0003-2743-3319]
- Abstract
30 p.-9 fig.-1 tab., Paclitaxel (Taxol®) is a first-line chemotherapeutic drug that promotes the curved-to-straight conformational transition of tubulin, an activation step that is necessary for microtubule formation. Crystallization of Taxol bound to tubulin has been long elusive. We found that baccatin III, the core structure of paclitaxel which lacks the C13 side chain, readily co-crystallizes with curved tubulin. Tailor-made taxanes with alternative side chains also co-crystallized, allowing us to investigate their binding modes. Interestingly, these Taxol derived compounds lost their microtubule stabilizing activity and cytotoxicity but kept their full microtubule binding affinity, and all induced lattice expansion upon binding. Additional nuclear magnetic resonance studies propose that Taxol binds to a small fraction of straight tubulin present in solution. Our results suggest a mode of action of Taxol, where the core structure is responsible for the interacting energy while the bulky hydrophobic C13 side chain enables binding selectively to straight tubulin and promotes stabilization.
- Published
- 2021
19. Potent neutralization by monoclonal human IgM against SARS‐CoV‐2 is impaired by class switch
- Author
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Callegari, Ilaria, primary, Schneider, Mika, additional, Berloffa, Giuliano, additional, Mühlethaler, Tobias, additional, Holdermann, Sebastian, additional, Galli, Edoardo, additional, Roloff, Tim, additional, Boss, Renate, additional, Infanti, Laura, additional, Khanna, Nina, additional, Egli, Adrian, additional, Buser, Andreas, additional, Zimmer, Gert, additional, Derfuss, Tobias, additional, and Sanderson, Nicholas S R, additional
- Published
- 2022
- Full Text
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20. A Structural-Reporter Group to Determine the Core Conformation of Sialyl Lewis x Mimetics.
- Author
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Wagner, Beatrice, Binder, Florian P. C., Jiang, Xiaohua, Mühlethaler, Tobias, Preston, Roland C., Rabbani, Said, Smieško, Martin, Schwardt, Oliver, and Ernst, Beat
- Subjects
SPATIAL orientation ,FUCOSE ,MOIETIES (Chemistry) ,GALACTOSE - Abstract
The d-GlcNAc moiety in sialyl Lewis
x (sLex , 1) acts predominantly as a linker to position the d-Gal and the l-Fuc moieties in the bioactive spatial orientation. The hypothesis has been made that the NHAc group of GlcNAc pushes the fucose underneath the galactose and, thus, contributes to the stabilization of the bioactive conformation of the core of sLex (1). To test this hypothesis, GlcNAc mimetics consisting of (R,R)-1,2-cyclohexanediols substituted with alkyl and aryl substituents adjacent to the linking position of the fucose moiety were synthesized. To explore a broad range of extended and spatially demanding R-groups, an enzymatic approach for the synthesis of 3-alkyl/aryl-1,2-cyclohexanediols (3b-n) was applied. These cyclohexanediol derivatives were incorporated into the sLex mimetics 2b-n. For analyzing the relationship of affinity and core conformation, a1 H NMR structural-reporter-group concept was applied. Thus, the chemical shift of H-C5Fuc proved to be a sensitive indicator for the degree of pre-organization of the core of this class of sLex mimetics and therefore could be used to quantify the contribution of the R-groups. [ABSTRACT FROM AUTHOR]- Published
- 2023
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21. Rational Design of a Novel Tubulin Inhibitor with a Unique Mechanism of Action
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Mühlethaler, Tobias, primary, Milanos, Lampros, additional, Ortega, Jose Antonio, additional, Blum, Thorsten B., additional, Gioia, Dario, additional, Roy, Bibhas, additional, Prota, Andrea E., additional, Cavalli, Andrea, additional, and Steinmetz, Michel O., additional
- Published
- 2022
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22. Molecular snapshots of drug release from tubulin over eleven orders of magnitude in time
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Wranik, Maximillian, primary, Weinert, Tobias, additional, Slavov, Chavdar, additional, Masini, Tiziana, additional, Furrer, Antonia, additional, Gaillard, Natacha, additional, Gioia, Dario, additional, Ferrarotti, Marco, additional, James, Daniel, additional, Glover, Hannah, additional, Carrillo, Melissa, additional, Kekilli, Demet, additional, Stipp, Robin, additional, Skopintsev, Petr, additional, Brünle, Steffen, additional, Mühlethaler, Tobias, additional, Beale, John, additional, Gashi, Dardan, additional, Nass, Karol, additional, Ozerov, Dmitry, additional, Johnson, Philip J.M., additional, Cirelli, Claudio, additional, Bacellar, Camila, additional, Braun, Markus, additional, Wang, Meitian, additional, Dworkowski, Florian, additional, Milne, Chris, additional, Cavalli, Andrea, additional, Wachtveitl, Josef, additional, Steinmetz, Michel O., additional, and Standfuss, Jörg, additional
- Published
- 2022
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23. Comprehensive analysis of binding sites in tubulin to develop small molecule ligands
- Author
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Mühlethaler, Tobias, Steinmetz, Michel, Maier, Timm, and Glockshuber, Rudolf
- Abstract
In eukaryotic cells, microtubules are highly dynamic filaments of the cytoskeleton where they are involved in vital tasks like cell division, intracellular transport, and cell polarization. Microtubules are assembled from their building block tubulin, and both microtubules and tubulin are known to bind a multitude of regulators. In cells, protein partners bind to microtubules to either modulate their behavior or to use them to fulfill various cellular functions. In addition to proteins, several dozen small molecule ligands are known to influence microtubule dynamics and they target six distinct binding sites on tubulin. As tumor cells exhibit faster rates of cell division than healthy cells, it is not surprising that mitotic inhibition is one of the many strategies to treat cancer. Indeed, five of these ligands are currently used in the clinic as monotherapeutic antitubulin drugs. While their mechanism of action differ, all original ligands are derived from natural sources and have complex chemical structures. Based on these observations, we aimed in the present thesis to address the two outstanding open questions whether there are additional binding sites present in tubulin and if it is possible to develop a small molecule ligand targeting microtubules. To tackle these two questions, we first performed an X-ray crystallography based fragment screen. Using this approach, we found 56 chemically diverse fragments that bound to 10 distinct binding sites on tubulin. Moreover, we combined a computational binding-site search with the results from our fragment screen to comprehensively map all binding sites in tubulin. Indeed, we found 18 novel sites that are not targeted by any of the known ligands. Out of these, 11 sites are also not targeted by any known tubulin protein partner. These findings demonstrate that our combined computational and crystallographic approach is a powerful tool to map binding sites in any protein for which a well diffracting crystal system is available. With the structural information at hands on a plethora of new fragments bound to tubulin, we went on to develop novel small molecule ligands targeting tubulin. As a first step and proof of principle, we chose a fragment that binds to the well-known colchicine site and has structural resemblance to nocodazole, a microtubule-destabilizing agent that was proven to be too toxic to use in the clinic. This fragment establishes an intricate interaction network with tubulin, consisting of four hydrogen bonds. Indeed, when probing for the importance of these hydrogen bonds by removing them individually we learned that all of them are necessary to retain binding. While leaving the interaction network unchanged, we still had the option to grow our fragment by attaching continuously larger substitutions and in this way, we managed to improve our fragment from an IC50 of 54 μM to a final IC50 of 0.94 μM in our best compound. These results demonstrate that already a small fragment can exhibit optimal interactions to its target and that indeed our fragment hits can be used to obtain potent tubulin inhibitors. As our pilot project showed that our fragments can be improved, we went on to apply our freshly gained insight to a novel tubulin-binding site. This time we chose a fragment linking strategy, in which several fragments that bind close to each other are combined. First, we took a fragment that contained a common binding motif as a starting point to gain specificity. Second, we looked for additional fragments binding in close proximity that could be chemically linked to our starting fragment. The two additional fragments that we selected suggested that expanding our starting fragment with a phenyl moiety should increase its affinity. Indeed, with our linking effort we obtained a first ligand with an IC50 of 48 μM, whereas none of the starting fragments showed any effect on cells. Additional rounds of modification of this first ligand brought us our most potent compound, which we named Todalam. Todalam has an IC50 value of 8.8 μM and showed microtubule depolymerizing effects in vitro as well as in cells. Furthermore, we proposed a novel, twofold mechanism of action for Todalam: First, Todalam acts like a “plug” locking tubulin in its curved conformation and second, it links free tubulin to ring like structures that are incompatible with microtubule assembly. This is to the best of our knowledge the first rationally designed tubulin-targeting agent with a novel binding site and a novel molecular mechanism of action. Overall, we demonstrated that crystallographic fragment screening is a very powerful tool both for finding novel binding sites as well as for developing small molecule ligands. Our results set a basis for future ligand design efforts by providing a diverse set of novel tubulin-binding sites and ligands. They further offer two lead-like compounds that are ready to be developed into small lead molecules.
- Published
- 2022
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24. Prodrugs of E‐selectin Antagonists with Enhanced Pharmacokinetic Properties
- Author
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Dätwyler, Philipp, primary, Jiang, Xiaohua, additional, Wagner, Beatrice, additional, Varga, Norbert, additional, Mühlethaler, Tobias, additional, Hostettler, Katja, additional, Rabbani, Said, additional, Schwardt, Oliver, additional, and Ernst, Beat, additional
- Published
- 2021
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25. Preclinical and Early Clinical Development of PTC596, a Novel Small-Molecule Tubulin-Binding Agent
- Author
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Jernigan, Finith, primary, Branstrom, Arthur, additional, Baird, John D., additional, Cao, Liangxian, additional, Dali, Mandar, additional, Furia, Bansri, additional, Kim, Min Jung, additional, O'Keefe, Kylie, additional, Kong, Ronald, additional, Laskin, Oscar L., additional, Colacino, Joseph M., additional, Pykett, Mark, additional, Mollin, Anna, additional, Sheedy, Josephine, additional, Dumble, Melissa, additional, Moon, Young-Choon, additional, Sheridan, Richard, additional, Mühlethaler, Tobias, additional, Spiegel, Robert J., additional, Prota, Andrea E., additional, Steinmetz, Michel O., additional, and Weetall, Marla, additional
- Published
- 2021
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26. Structural insight into the stabilization of microtubules by taxanes
- Author
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Prota, Andrea E., primary, Lucena-Agell, Daniel, additional, Ma, Yuntao, additional, Estévez-Gallego, Juan, additional, Li, Shuo, additional, Bargsten, Katja, additional, Josa-Prado, Fernando, additional, Altmann, Karl-Heinz, additional, Gaillard, Natacha, additional, Kamimura, Shinji, additional, Mühlethaler, Tobias, additional, Gago, Federico, additional, Oliva, María A., additional, Steinmetz, Michel O., additional, Fang, Wei-Shuo, additional, and Díaz, J. Fernando, additional
- Published
- 2021
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- View/download PDF
27. Comprehensive Analysis of Binding Sites in Tubulin
- Author
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Mühlethaler, Tobias, primary, Gioia, Dario, additional, Prota, Andrea E., additional, Sharpe, May E., additional, Cavalli, Andrea, additional, and Steinmetz, Michel O., additional
- Published
- 2021
- Full Text
- View/download PDF
28. Structural Refinement of the Tubulin Ligand (+)-Discodermolide to Attenuate Chemotherapy-Mediated Senescence
- Author
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Guo, Boying, primary, Rodriguez-Gabin, Alicia, additional, Prota, Andrea E., additional, Mühlethaler, Tobias, additional, Zhang, Nan, additional, Ye, Kenny, additional, Steinmetz, Michel O., additional, Horwitz, Susan Band, additional, Smith, Amos B., additional, and McDaid, Hayley M., additional
- Published
- 2020
- Full Text
- View/download PDF
29. Prodrugs of E‐selectin Antagonists with Enhanced Pharmacokinetic Properties.
- Author
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Dätwyler, Philipp, Jiang, Xiaohua, Wagner, Beatrice, Varga, Norbert, Mühlethaler, Tobias, Hostettler, Katja, Rabbani, Said, Schwardt, Oliver, and Ernst, Beat
- Published
- 2022
- Full Text
- View/download PDF
30. MAP7 family proteins regulate kinesin-1 recruitment and activation
- Author
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Hooikaas, Peter Jan, Martin, Maud, Mühlethaler, Tobias, Kuijntjes, Gert Jan, Peeters, Cathelijn A.E., Katrukha, Eugene A., Ferrari, Luca, Stucchi, Riccardo, Verhagen, Daan G.F., Van Riel, Wilhelmina E., Grigoriev, Ilya, Altelaar, A. F.Maarten, Hoogenraad, Casper C., Rüdiger, Stefan G.D., Steinmetz, Michel O., Kapitein, Lukas C., Akhmanova, Anna, Sub Cell Biology, Sub Cellular Protein Chemistry, Afd Biomol.Mass Spect. and Proteomics, Celbiologie, Biomolecular Mass Spectrometry and Proteomics, Cellular Protein Chemistry, Sub Cell Biology, Sub Cellular Protein Chemistry, Afd Biomol.Mass Spect. and Proteomics, Celbiologie, Biomolecular Mass Spectrometry and Proteomics, and Cellular Protein Chemistry
- Subjects
Allosteric effect ,Kinesins ,Diketopiperazines ,macromolecular substances ,Microtubules ,Article ,HeLa ,03 medical and health sciences ,0302 clinical medicine ,Microtubule ,Chlorocebus aethiops ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Research Articles ,030304 developmental biology ,0303 health sciences ,biology ,Chemistry ,Processivity ,Cell Biology ,biology.organism_classification ,In vitro ,Mitochondria ,3. Good health ,Cell biology ,Enzyme Activation ,Protein Transport ,Lower affinity ,HEK293 Cells ,Benzamides ,COS Cells ,Kinesin ,Microtubule-Associated Proteins ,Linker ,030217 neurology & neurosurgery ,HeLa Cells ,Protein Binding - Abstract
Hooikaas et al. show that mammalian MAP7 family proteins act redundantly to activate the kinesin-1 motor protein. Using experiments in cells and in vitro reconstitution assays, they demonstrate that MAP7 proteins promote microtubule recruitment and processivity of kinesin-1 by transiently associating with the stalk region of the motor., Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1, and MAP7D3 act redundantly to enable kinesin-1–dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared with MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be cotransported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.
- Published
- 2019
31. MAP7 family proteins regulate kinesin-1 recruitment and activation
- Author
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Sub Cell Biology, Sub Cellular Protein Chemistry, Afd Biomol.Mass Spect. and Proteomics, Celbiologie, Biomolecular Mass Spectrometry and Proteomics, Cellular Protein Chemistry, Hooikaas, Peter Jan, Martin, Maud, Mühlethaler, Tobias, Kuijntjes, Gert Jan, Peeters, Cathelijn A.E., Katrukha, Eugene A., Ferrari, Luca, Stucchi, Riccardo, Verhagen, Daan G.F., Van Riel, Wilhelmina E., Grigoriev, Ilya, Altelaar, A. F.Maarten, Hoogenraad, Casper C., Rüdiger, Stefan G.D., Steinmetz, Michel O., Kapitein, Lukas C., Akhmanova, Anna, Sub Cell Biology, Sub Cellular Protein Chemistry, Afd Biomol.Mass Spect. and Proteomics, Celbiologie, Biomolecular Mass Spectrometry and Proteomics, Cellular Protein Chemistry, Hooikaas, Peter Jan, Martin, Maud, Mühlethaler, Tobias, Kuijntjes, Gert Jan, Peeters, Cathelijn A.E., Katrukha, Eugene A., Ferrari, Luca, Stucchi, Riccardo, Verhagen, Daan G.F., Van Riel, Wilhelmina E., Grigoriev, Ilya, Altelaar, A. F.Maarten, Hoogenraad, Casper C., Rüdiger, Stefan G.D., Steinmetz, Michel O., Kapitein, Lukas C., and Akhmanova, Anna
- Published
- 2019
32. VISAGE Reveals a Targetable Mitotic Spindle Vulnerability in Cancer Cells
- Author
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Patterson, Jesse C., primary, Joughin, Brian A., additional, Prota, Andrea E., additional, Mühlethaler, Tobias, additional, Jonas, Oliver H., additional, Whitman, Matthew A., additional, Varmeh, Shohreh, additional, Chen, Sen, additional, Balk, Steven P., additional, Steinmetz, Michel O., additional, Lauffenburger, Douglas A., additional, and Yaffe, Michael B., additional
- Published
- 2019
- Full Text
- View/download PDF
33. Front Cover: Improvement of Aglycone π‐Stacking Yields Nanomolar to Sub‐nanomolar FimH Antagonists (ChemMedChem 7/2019)
- Author
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Schönemann, Wojciech, primary, Cramer, Jonathan, additional, Mühlethaler, Tobias, additional, Fiege, Brigitte, additional, Silbermann, Marleen, additional, Rabbani, Said, additional, Dätwyler, Philipp, additional, Zihlmann, Pascal, additional, Jakob, Roman P., additional, Sager, Christoph P., additional, Smieško, Martin, additional, Schwardt, Oliver, additional, Maier, Timm, additional, and Ernst, Beat, additional
- Published
- 2019
- Full Text
- View/download PDF
34. Improvement of Aglycone π‐Stacking Yields Nanomolar to Sub‐nanomolar FimH Antagonists
- Author
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Schönemann, Wojciech, primary, Cramer, Jonathan, additional, Mühlethaler, Tobias, additional, Fiege, Brigitte, additional, Silbermann, Marleen, additional, Rabbani, Said, additional, Dätwyler, Philipp, additional, Zihlmann, Pascal, additional, Jakob, Roman P., additional, Sager, Christoph P., additional, Smieško, Martin, additional, Schwardt, Oliver, additional, Maier, Timm, additional, and Ernst, Beat, additional
- Published
- 2019
- Full Text
- View/download PDF
35. MAP7 family proteins regulate kinesin-1 recruitment and activation
- Author
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Hooikaas, Peter Jan, primary, Martin, Maud, additional, Mühlethaler, Tobias, additional, Kuijntjes, Gert-Jan, additional, Peeters, Cathelijn A.E., additional, Katrukha, Eugene A., additional, Ferrari, Luca, additional, Stucchi, Riccardo, additional, Verhagen, Daan G.F., additional, Riel, Wilhelmina E. van, additional, Grigoriev, Ilya, additional, Altelaar, A.F. Maarten, additional, Hoogenraad, Casper C., additional, Rüdiger, Stefan G.D., additional, Steinmetz, Michel O., additional, Kapitein, Lukas C., additional, and Akhmanova, Anna, additional
- Published
- 2018
- Full Text
- View/download PDF
36. KinITC-One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists
- Author
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Zihlmann, Pascal, primary, Silbermann, Marleen, additional, Sharpe, Timothy, additional, Jiang, Xiaohua, additional, Mühlethaler, Tobias, additional, Jakob, Roman P., additional, Rabbani, Said, additional, Sager, Christoph P., additional, Frei, Priska, additional, Pang, Lijuan, additional, Maier, Timm, additional, and Ernst, Beat, additional
- Published
- 2018
- Full Text
- View/download PDF
37. Target‐directed Dynamic Combinatorial Chemistry: A Study on Potentials and Pitfalls as Exemplified on a Bacterial Target
- Author
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Frei, Priska, primary, Pang, Lijuan, additional, Silbermann, Marleen, additional, Eriş, Deniz, additional, Mühlethaler, Tobias, additional, Schwardt, Oliver, additional, and Ernst, Beat, additional
- Published
- 2017
- Full Text
- View/download PDF
38. Structural insight into the stabilization of microtubules by taxanes
- Author
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Andrea E Prota, Daniel Lucena-Agell, Yuntao Ma, Juan Estevez-Gallego, Shuo Li, Katja Bargsten, Fernando Josa-Prado, Karl-Heinz Altmann, Natacha Gaillard, Shinji Kamimura, Tobias Mühlethaler, Federico Gago, Maria A Oliva, Michel O Steinmetz, Wei-Shuo Fang, J Fernando Díaz, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), Fundación Tatiana Pérez de Guzmán el Bueno, European Commission, Swiss National Science Foundation, Japan Society for the Promotion of Science, National Natural Science Foundation of China, Chinese Academy of Medical Sciences, Prota, Andrea E., Lucena-Agell, Daniel, Estévez-Gallego, Juan, Josa-Prado, Fernando, Altmann, Karl-Heinz, Gaillard, Natacha, Kamimura, Shinji, Mühlethaler, Tobias, Gago, Federico, Oliva, María A., Steinmetz, Michel O., and Díaz, José Fernando
- Subjects
General Immunology and Microbiology ,General Neuroscience ,General Medicine ,General Biochemistry, Genetics and Molecular Biology - Abstract
35 p.-12 fig.-2 tab.-3 animat.-1 schem.This article is dedicated to the memory of Dr Linda Amos, a dear friend and pioneer in the study of microtubules and the mechanism of action of paclitaxel (Amos and Löwe, 1999), who passed away while we were assembling this manuscript., Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules’ diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a βM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding., Ministerio de Ciencia e Innovación (PID2019-104545RB-I00 (AEI/10.13039/501100011033)) J Fernando Díaz Consejo Superior de Investigaciones Científicas (PIE 201920E111) J Fernando Díaz Fundación Tatiana Pérez de Guzmán el Bueno (Proyecto de Investigación en Neurociencia 2020) J Fernando Díaz European Union NextGenerationEU (H2020-MSCA-ITN-2019 860070 TUBINTRAIN) Andrea E Prota J Fernando Díaz Swiss National Science Foundation (310030_192566) Michel O Steinmetz JSPS KAKENHI (16K07328/17H03668) Shinji Kamimura National Natural Science Foundation of China (30930108) Wei-Shuo Fang Chinese Academy of Medical Sciences (2016-I2M-1-010) Wei-Shuo Fang Ministerio de Ciencia e Innovación (PID2019-104070RB-C22 (AEI/10.13039/501100011033)) Federico Gago We thank Ganadería Fernando Díaz for calf brains supply and staff of beamlines X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen PSI, Switzerland) and BL11-NDC-SWEET (ALBA, Cerdanyola del Vallès, Spain) for their support. We also thank Mr Pedro Gascón Blanco for his private donation to the project to support a month of a student salary.
- Published
- 2023
- Full Text
- View/download PDF
39. Tetra- and Hexavalent Siglec-8 Ligands Modulate Immune Cell Activation.
- Author
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Conti G, Bärenwaldt A, Rabbani S, Mühlethaler T, Sarcevic M, Jiang X, Schwardt O, Ricklin D, Pieters RJ, Läubli H, and Ernst B
- Subjects
- Ligands, Sialic Acid Binding Immunoglobulin-like Lectins metabolism, Polysaccharides chemistry, Eosinophils metabolism
- Abstract
Carbohydrate-binding proteins are generally characterized by poor affinities for their natural glycan ligands, predominantly due to the shallow and solvent-exposed binding sites. To overcome this drawback, nature has exploited multivalency to strengthen the binding by establishing multiple interactions simultaneously. The development of oligovalent structures frequently proved to be successful, not only for proteins with multiple binding sites, but also for proteins that possess a single recognition domain. Herein we present the syntheses of a number of oligovalent ligands for Siglec-8, a monomeric I-type lectin found on eosinophils and mast cells, alongside the thermodynamic characterization of their binding. While the enthalpic contribution of each binding epitope was within a narrow range to that of the monomeric ligand, the entropy penalty increased steadily with growing valency. Additionally, we observed a successful agonistic binding of the tetra- and hexavalent and, to an even larger extent, multivalent ligands to Siglec-8 on immune cells and modulation of immune cell activation. Thus, triggering a biological effect is not restricted to multivalent ligands but could be induced by low oligovalent ligands as well, whereas a monovalent ligand, despite binding with similar affinity, showed an antagonistic effect., (© 2023 Wiley-VCH GmbH.)
- Published
- 2023
- Full Text
- View/download PDF
40. Rational Design of a Novel Tubulin Inhibitor with a Unique Mechanism of Action.
- Author
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Mühlethaler T, Milanos L, Ortega JA, Blum TB, Gioia D, Roy B, Prota AE, Cavalli A, and Steinmetz MO
- Subjects
- Cell Death, Microtubules metabolism, Protein Binding, Tubulin chemistry, Tubulin Modulators chemistry
- Abstract
In this study, we capitalized on our previously performed crystallographic fragment screen and developed the antitubulin small molecule Todalam with only two rounds of straightforward chemical synthesis. Todalam binds to a novel tubulin site, disrupts microtubule networks in cells, arrests cells in G2/M, induces cell death, and synergizes with vinblastine. The compound destabilizes microtubules by acting as a molecular plug that sterically inhibits the curved-to-straight conformational switch in the α-tubulin subunit, and by sequestering tubulin dimers into assembly incompetent oligomers. Our results describe for the first time the generation of a fully rationally designed small molecule tubulin inhibitor from a fragment, which displays a unique molecular mechanism of action. They thus demonstrate the usefulness of tubulin-binding fragments as valuable starting points for innovative antitubulin drug and chemical probe discovery campaigns., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)
- Published
- 2022
- Full Text
- View/download PDF
41. Comprehensive Analysis of Binding Sites in Tubulin.
- Author
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Mühlethaler T, Gioia D, Prota AE, Sharpe ME, Cavalli A, and Steinmetz MO
- Subjects
- Allosteric Regulation, Binding Sites, Crystallography, X-Ray, Molecular Dynamics Simulation, Protein Binding, Tubulin chemistry, Tubulin Modulators chemistry, Tubulin Modulators metabolism, Ligands, Tubulin metabolism
- Abstract
Tubulin plays essential roles in vital cellular activities and is the target of a wide range of proteins and ligands. Here, using a combined computational and crystallographic fragment screening approach, we addressed the question of how many binding sites exist in tubulin. We identified 27 distinct sites, of which 11 have not been described previously, and analyzed their relationship to known tubulin-protein and tubulin-ligand interactions. We further observed an intricate pocket communication network and identified 56 chemically diverse fragments that bound to 10 distinct tubulin sites. Our results offer a unique structural basis for the development of novel small molecules for use as tubulin modulators in basic research applications or as drugs. Furthermore, our method lays down a framework that may help to discover new pockets in other pharmaceutically important targets and characterize them in terms of chemical tractability and allosteric modulation., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)
- Published
- 2021
- Full Text
- View/download PDF
42. MAP7 family proteins regulate kinesin-1 recruitment and activation.
- Author
-
Hooikaas PJ, Martin M, Mühlethaler T, Kuijntjes GJ, Peeters CAE, Katrukha EA, Ferrari L, Stucchi R, Verhagen DGF, van Riel WE, Grigoriev I, Altelaar AFM, Hoogenraad CC, Rüdiger SGD, Steinmetz MO, Kapitein LC, and Akhmanova A
- Subjects
- Animals, Benzamides pharmacology, COS Cells, Chlorocebus aethiops, Diketopiperazines pharmacology, Enzyme Activation, HEK293 Cells, HeLa Cells, Humans, Kinesins genetics, Microtubule-Associated Proteins genetics, Microtubules drug effects, Microtubules genetics, Mitochondria genetics, Protein Binding, Protein Interaction Domains and Motifs, Protein Transport, Kinesins metabolism, Microtubule-Associated Proteins metabolism, Microtubules enzymology, Mitochondria enzymology
- Abstract
Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1, and MAP7D3 act redundantly to enable kinesin-1-dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared with MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be cotransported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules., (© 2019 Hooikaas et al.)
- Published
- 2019
- Full Text
- View/download PDF
43. KinITC-One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists.
- Author
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Zihlmann P, Silbermann M, Sharpe T, Jiang X, Mühlethaler T, Jakob RP, Rabbani S, Sager CP, Frei P, Pang L, Maier T, and Ernst B
- Subjects
- Calorimetry methods, Drug Discovery, Fimbriae Proteins antagonists & inhibitors, Hydrogen Bonding, Kinetics, Ligands, Models, Molecular, Protein Binding, Protein Conformation, Protein Domains, Structure-Activity Relationship, Thermodynamics, Adhesins, Escherichia coli chemistry, Fimbriae Proteins chemistry, Mannosides chemistry
- Abstract
Affinity data, such as dissociation constants (K
D ) or inhibitory concentrations (IC50 ), are widely used in drug discovery. However, these parameters describe an equilibrium state, which is often not established in vivo due to pharmacokinetic effects and they are therefore not necessarily sufficient for evaluating drug efficacy. More accurate indicators for pharmacological activity are the kinetics of binding processes, as they shed light on the rate of formation of protein-ligand complexes and their half-life. Nonetheless, although highly desirable for medicinal chemistry programs, studies on structure-kinetic relationships (SKR) are still rare. With the recently introduced analytical tool kinITC this situation may change, since not only thermodynamic but also kinetic information of the binding process can be deduced from isothermal titration calorimetry (ITC) experiments. Using kinITC, ITC data of 29 mannosides binding to the bacterial adhesin FimH were re-analyzed to make their binding kinetics accessible. To validate these kinetic data, surface plasmon resonance (SPR) experiments were conducted. The kinetic analysis by kinITC revealed that the nanomolar affinities of the FimH antagonists arise from both (i) an optimized interaction between protein and ligand in the bound state (reduced off-rate constant koff ) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on-rate constant kon ). Based on congeneric ligand modifications and structural input from co-crystal structures, a strong relationship between the formed hydrogen-bond network and koff could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on kon ., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2018
- Full Text
- View/download PDF
44. Target-directed Dynamic Combinatorial Chemistry: A Study on Potentials and Pitfalls as Exemplified on a Bacterial Target.
- Author
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Frei P, Pang L, Silbermann M, Eriş D, Mühlethaler T, Schwardt O, and Ernst B
- Subjects
- Adhesins, Escherichia coli genetics, Adhesins, Escherichia coli metabolism, Aldehydes chemistry, Aniline Compounds chemistry, Catalysis, Combinatorial Chemistry Techniques, Escherichia coli metabolism, Fimbriae Proteins genetics, Fimbriae Proteins metabolism, Hydrazones chemical synthesis, Hydrazones metabolism, Hydrogen-Ion Concentration, Small Molecule Libraries chemistry, Small Molecule Libraries metabolism, Surface Plasmon Resonance, Fimbriae Proteins antagonists & inhibitors, Hydrazones chemistry
- Abstract
Target-directed dynamic combinatorial chemistry (DCC) is an emerging technique for the efficient identification of inhibitors of pharmacologically relevant targets. In this contribution, we present an application for a bacterial target, the lectin FimH, a crucial virulence factor of uropathogenic E. coli being the main cause of urinary tract infections. A small dynamic library of acylhydrazones was formed from aldehydes and hydrazides and equilibrated at neutral pH in presence of aniline as nucleophilic catalyst. The major success factors turned out to be an accordingly adjusted ratio of scaffolds and fragments, an adequate sample preparation prior to HPLC analysis, and the data processing. Only then did the ranking of the dynamic library constituents correlate well with affinity data. Furthermore, as a support of DCC applications especially to larger libraries, a new protocol for improved hit identification was established., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2017
- Full Text
- View/download PDF
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