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27 results on '"Oliva, María A. [0000-0002-2215-4639]"'

1. Chemical modulation of microtubule structure through the laulimalide/peloruside site

Author

Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, Fundación Tatiana Pérez de Guzmán el Bueno, Swiss National Science Foundation, Ministerio de Universidades (España), Paul Scherrer Institute (Switzerland), European Synchrotron Radiation Facility, Club Escola Hungaresa de Esgrima de Pontevedra, Estévez-Gallego, Juan [0000-0003-3889-8488], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], Menche, Dirk [0000-0002-4724-8383], Lucena-Agell, Daniel [0000-0001-7198-2900], Prota, Andrea E. [0000-0003-0875-5339], Bonato, Francesca [0000-0002-0579-2874], Giménez-Abián, Juan F. [0000-0002-9220-286X], Northcote, Peter [0000-0002-2086-9972], Steinmetz, Michel O. [0000-0001-6157-3687], Kamimura, Shinji [0000-0003-4359-0859], Altmann, Karl-Heinz [0000-0002-0747-9734], Paterson, Ian [0000-0002-8861-9136], Gago, Federico [0000-0002-3071-4878], Van der Eycken, Johan [0000-0002-1623-6750], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Estévez-Gallego, Juan, Álvarez-Bernad, Beatriz, Benet, Pera, Wullschleger, Christoph, Raes, Olivier, Menche, Dirk, Martínez, Juan Carlos, Lucena-Agell, Daniel, Prota, Andrea E., Bonato, Francesca, Bargsten, Katja, Cornelus, Jelle, Giménez-Abián, Juan F., Northcote, Peter, Steinmetz, Michel O., Kamimura, Shinji, Altmann, Karl-Heinz, Paterson, Ian, Gago, Federico, Van der Eycken, Johan, Díaz, José Fernando, Oliva, María A., Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, Fundación Tatiana Pérez de Guzmán el Bueno, Swiss National Science Foundation, Ministerio de Universidades (España), Paul Scherrer Institute (Switzerland), European Synchrotron Radiation Facility, Club Escola Hungaresa de Esgrima de Pontevedra, Estévez-Gallego, Juan [0000-0003-3889-8488], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], Menche, Dirk [0000-0002-4724-8383], Lucena-Agell, Daniel [0000-0001-7198-2900], Prota, Andrea E. [0000-0003-0875-5339], Bonato, Francesca [0000-0002-0579-2874], Giménez-Abián, Juan F. [0000-0002-9220-286X], Northcote, Peter [0000-0002-2086-9972], Steinmetz, Michel O. [0000-0001-6157-3687], Kamimura, Shinji [0000-0003-4359-0859], Altmann, Karl-Heinz [0000-0002-0747-9734], Paterson, Ian [0000-0002-8861-9136], Gago, Federico [0000-0002-3071-4878], Van der Eycken, Johan [0000-0002-1623-6750], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Estévez-Gallego, Juan, Álvarez-Bernad, Beatriz, Benet, Pera, Wullschleger, Christoph, Raes, Olivier, Menche, Dirk, Martínez, Juan Carlos, Lucena-Agell, Daniel, Prota, Andrea E., Bonato, Francesca, Bargsten, Katja, Cornelus, Jelle, Giménez-Abián, Juan F., Northcote, Peter, Steinmetz, Michel O., Kamimura, Shinji, Altmann, Karl-Heinz, Paterson, Ian, Gago, Federico, Van der Eycken, Johan, Díaz, José Fernando, and Oliva, María A.

Abstract

Taxanes are microtubule-stabilizing agents used in the treatment of many solid tumors, but they often involve side effects affecting the peripheral nervous system. It has been proposed that this could be related to structural modifications on the filament upon drug binding. Alternatively, laulimalide and peloruside bind to a different site also inducing stabilization, but they have not been exploited in clinics. Here, we use a combination of the parental natural compounds and derived analogs to unravel the stabilization mechanism through this site. These drugs settle lateral interactions without engaging the M loop, which is part of the key and lock involved in the inter-protofilament contacts. Importantly, these drugs can modulate the angle between protofilaments, producing microtubules of different diameters. Among the compounds studied, we have found some showing low cytotoxicity and able to induce stabilization without compromising microtubule native structure. This opens the window of new applications for microtubule-stabilizing agents beyond cancer treatment.

Published
2023

2. Individually expressed SARS-CoV-2 viroporin ORF3a alters inflammatory responses and cellular structure in lung epithelial cells

Author

European Commission, Consejo Superior de Investigaciones Científicas (España), Junta de Andalucía, Agencia Estatal de Investigación (España), Lucas-Rius, Ana de [0000-0003-1920-2414], Zaldívar-López, Sara [0000-0001-5084-8999], Mendoza-García, Laura [0000-0002-9964-8127], Dies López-Ayllón, Blanca [0000-0003-3889-3644], García-García, Tránsito [0000-0003-3708-0908], Redondo, Natalia [0000-0001-9356-8102], Fernández-Rodríguez, Raúl [0000-0001-8301-0244], Suárez-Cárdenas, José M. [0000-0001-8509-1498], Jiménez-Marín, Ángeles [0000-0001-6546-9174], Pérez-Berna, Ana Joaquina [0000-0003-4145-532X], Pereiro, Eva [0000-0001-7626-5935], Oliva, María A. [0000-0002-2215-4639], Garrido, Juan J. [0000-0001-6592-2231], Montoya, María [0000-0002-5703-7360], Lucas-Rius, Ana de, Zaldívar-López, Sara, Mendoza-García, Laura, Dies López-Ayllón, Blanca, García-García, Tránsito, Redondo, Natalia, Fernández-Rodríguez, Raúl, Suárez-Cárdenas, José M., Jiménez-Marín, Ángeles, Pérez-Berna, Ana Joaquina, Pereiro, Eva, Oliva, María A., Garrido, Juan J., Montoya, María, European Commission, Consejo Superior de Investigaciones Científicas (España), Junta de Andalucía, Agencia Estatal de Investigación (España), Lucas-Rius, Ana de [0000-0003-1920-2414], Zaldívar-López, Sara [0000-0001-5084-8999], Mendoza-García, Laura [0000-0002-9964-8127], Dies López-Ayllón, Blanca [0000-0003-3889-3644], García-García, Tránsito [0000-0003-3708-0908], Redondo, Natalia [0000-0001-9356-8102], Fernández-Rodríguez, Raúl [0000-0001-8301-0244], Suárez-Cárdenas, José M. [0000-0001-8509-1498], Jiménez-Marín, Ángeles [0000-0001-6546-9174], Pérez-Berna, Ana Joaquina [0000-0003-4145-532X], Pereiro, Eva [0000-0001-7626-5935], Oliva, María A. [0000-0002-2215-4639], Garrido, Juan J. [0000-0001-6592-2231], Montoya, María [0000-0002-5703-7360], Lucas-Rius, Ana de, Zaldívar-López, Sara, Mendoza-García, Laura, Dies López-Ayllón, Blanca, García-García, Tránsito, Redondo, Natalia, Fernández-Rodríguez, Raúl, Suárez-Cárdenas, José M., Jiménez-Marín, Ángeles, Pérez-Berna, Ana Joaquina, Pereiro, Eva, Oliva, María A., Garrido, Juan J., and Montoya, María

Abstract

The SARS-CoV-2 coronavirus is the causative agent of the coronavirus disease 2019 (COVID-19). The clinical course of this respiratory disease exhibits a broad spectrum of severity and progression patterns, being potentially fatal. It is known that the underlying cause of severe disease is a cytokine dysregulation and hyperinflammation status. The SARS-CoV-2 genome encodes for eleven accessory proteins which, despite being non-essential for viral replication, may play important roles in viral pathogenesis, and even several variants of concern have mutations among them., ORF3a, the largest accessory protein, is a type III transmembrane protein that forms dimers. It is thought to be a viroporin and function as an ion channel, although this is currently under debate. It has been described to activate the NF-kB pathway and NLRP3 inflammasome, upregulate cytokine expression, induce oxidative stress and cell apoptosis, inhibit interferon-activated JAK/STAT pathway and reduce MHC-I levels among other functions

Published
2023

3. Chemical modulation of microtubule structure as a tool to target different diseases

Author

Estévez-Gallego, Juan [0000-0003-3889-8488], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], Lucena-Agell, Daniel [0000-0001-7314-8696], Bonato, Francesca [0000-0002-0579-2874], Giménez-Abián, Juan F. [0000-0002-9220-286X], París, Rebeca [0000-0002-7953-0949], Kamimura, Shinji [0000-0003-4359-0859], Gago, Federico [0000-0002-3071-4878], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Estévez-Gallego, Juan, Álvarez-Bernad, Beatriz, Lucena-Agell, Daniel, Bonato, Francesca, Giménez-Abián, Juan F., Blanco, Óscar, París, Rebeca, Fuertes, L., Martínez, J. C., Kamimura, Shinji, Gago, Federico, Díaz, José Fernando, Oliva, María A., Estévez-Gallego, Juan [0000-0003-3889-8488], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], Lucena-Agell, Daniel [0000-0001-7314-8696], Bonato, Francesca [0000-0002-0579-2874], Giménez-Abián, Juan F. [0000-0002-9220-286X], París, Rebeca [0000-0002-7953-0949], Kamimura, Shinji [0000-0003-4359-0859], Gago, Federico [0000-0002-3071-4878], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Estévez-Gallego, Juan, Álvarez-Bernad, Beatriz, Lucena-Agell, Daniel, Bonato, Francesca, Giménez-Abián, Juan F., Blanco, Óscar, París, Rebeca, Fuertes, L., Martínez, J. C., Kamimura, Shinji, Gago, Federico, Díaz, José Fernando, and Oliva, María A.

Abstract

Microtubules are dynamic filaments involved in many essential cellular functions including those needed for cancer cell growth. Taxanes are microtubule stabilizing agents and the most successful antitumoral drugs targeting these filaments. However, despite their mode of action is the stabilization (i.e., do not destroy the filament structure), they produce a paradoxical neurotoxic effect by inducing axon degeneration. This has been related to microtubule structural modifications upon drug binding. Alternatively, the laulimalide/peloruside binding site also promotes stabilization, but have not been exploits in clinics. We have found that differently to the taxane site, the stabilization mechanism involves exclusively lateral interactions and entails changes on the inter-protofilament angle. Importantly, some compounds do not modify microtubules upon binding when compared to native ones. This feature, together with the low cytotoxicity effect found, open the possibility of exploiting these compounds in neurodegenerative diseases, where microtubule structure and function are compromised due to other primary cellular alterations.

Published
2023

4. Synthesis and biological evaluation of C(13)/C(13′)-Bis(desmethyl)disorazole Z**

Author

Ministerio de Ciencia e Innovación (España), Fundación Tatiana Pérez de Guzmán el Bueno, European Commission, ETH Zurich, Bold, Christian Paul [0000-0003-3810-8407], Lucena-Agell, Daniel [0000-0001-7198-2900], Oliva, María A. [0000-0002-2215-4639], Díaz, José Fernando [0000-0003-2743-3319], Altmann, Karl-Heinz [0000-0002-0747-9734], Bold, Christian Paul, Lucena-Agell, Daniel, Oliva, María A., Díaz, José Fernando, Altmann, Karl-Heinz, Ministerio de Ciencia e Innovación (España), Fundación Tatiana Pérez de Guzmán el Bueno, European Commission, ETH Zurich, Bold, Christian Paul [0000-0003-3810-8407], Lucena-Agell, Daniel [0000-0001-7198-2900], Oliva, María A. [0000-0002-2215-4639], Díaz, José Fernando [0000-0003-2743-3319], Altmann, Karl-Heinz [0000-0002-0747-9734], Bold, Christian Paul, Lucena-Agell, Daniel, Oliva, María A., Díaz, José Fernando, and Altmann, Karl-Heinz

Abstract

We describe the total synthesis of the macrodiolide C(13)/C(13′)-bis(desmethyl)disorazole Z through double inter-/intramolecular Stille cross-coupling of a monomeric vinyl stannane/vinyl iodide precursor to form the macrocycle. The key step in the synthesis of this precursor was a stereoselective aldol reaction of a formal Evans acetate aldol product with crotonaldehyde. As demonstrated by X-ray crystallography, the binding mode of C(13)/C(13′)-bis(desmethyl)disorazole Z to tubulin is virtually identical with that of the natural product disorazole Z. Likewise, C(13)/C(13′)-bis(desmethyl)disorazole Z inhibits tubulin assembly with at least the same potency as disorazole Z and it appears to be a more potent cell growth inhibitor.

Published
2022

5. Targeting the tubulin C-terminal tail by charged small molecules

Author

CAMS Innovation Fund for Medical Sciences, Ministerio de Ciencia e Innovación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundación Tatiana Pérez de Guzmán el Bueno, Voelcker Fund, Natural Science Foundation of Guangdong Province, Li, Shuo [0000-0001-8011-1357], Mori, Mattia [0000-0003-2398-1254], Yang, Mingyan [0000-0001-5141-4594], Elfazazi, Soumia [0000-0001-6242-9900], Hortigüela, Rafael [0000-0002-9621-6441], Chan, Peter [0000-0002-3498-6301], Risinger, April L. [0000-0002-4363-3268], Oliva, María A. [0000-0002-2215-4639], Díaz, José Fernando [0000-0003-2743-3319], Fang, Wei-Shuo [0000-0002-0972-0640], Li, Shuo, Mori, Mattia, Yang, Mingyan, Elfazazi, Soumia, Hortigüela, Rafael, Chan, Peter, Feng, Xinyue, Risinger, April L., Yang, Zhiyou, Oliva, María A., Díaz, José Fernando, Fang, Wei-Shuo, CAMS Innovation Fund for Medical Sciences, Ministerio de Ciencia e Innovación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fundación Tatiana Pérez de Guzmán el Bueno, Voelcker Fund, Natural Science Foundation of Guangdong Province, Li, Shuo [0000-0001-8011-1357], Mori, Mattia [0000-0003-2398-1254], Yang, Mingyan [0000-0001-5141-4594], Elfazazi, Soumia [0000-0001-6242-9900], Hortigüela, Rafael [0000-0002-9621-6441], Chan, Peter [0000-0002-3498-6301], Risinger, April L. [0000-0002-4363-3268], Oliva, María A. [0000-0002-2215-4639], Díaz, José Fernando [0000-0003-2743-3319], Fang, Wei-Shuo [0000-0002-0972-0640], Li, Shuo, Mori, Mattia, Yang, Mingyan, Elfazazi, Soumia, Hortigüela, Rafael, Chan, Peter, Feng, Xinyue, Risinger, April L., Yang, Zhiyou, Oliva, María A., Díaz, José Fernando, and Fang, Wei-Shuo

Abstract

The disordered tubulin C-terminal tail (CTT), which possesses a higher degree of heterogeneity, is the target for the interaction of many proteins and cellular components. Compared to the seven well-described binding sites of microtubule-targeting agents (MTAs) that localize on the globular tubulin core, tubulin CTT is far less explored. Therefore, tubulin CTT can be regarded as a novel site for the development of MTAs with distinct biochemical and cell biological properties. Here, we designed and synthesized linear and cyclic peptides containing multiple arginines (RRR), which are complementary to multiple acidic residues in tubulin CTT. Some of them showed moderate induction and promotion of tubulin polymerization. The most potent macrocyclic compound 1f was found to bind to tubulin CTT and thus exert its bioactivity. Such RRR containing compounds represent a starting point for the discovery of tubulin CTT-targeting agents with therapeutic potential.

Published
2022

6. Pharmacological modulation of the interaction between tubulin and a SARS-CoV-2 protein

Author

Ministerio de Ciencia e Innovación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fuertes-Monge, Laura [0000-0002-9764-3111], Schaerlaekens, Sofie [0000-0002-4410-2311], Lucena-Agell, Daniel [0000-0001-7314-8696], García-Dorival, Isabel [0000-0002-5654-5662], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Alonso, Covandoga [0000-0002-0862-6177], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Fuertes-Monge, Laura, Schaerlaekens, Sofie, Lucena-Agell, Daniel, García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Inmaculada, Alonso, Covadonga, Díaz, José Fernando, Oliva, María A., Ministerio de Ciencia e Innovación (España), European Commission, Consejo Superior de Investigaciones Científicas (España), Fuertes-Monge, Laura [0000-0002-9764-3111], Schaerlaekens, Sofie [0000-0002-4410-2311], Lucena-Agell, Daniel [0000-0001-7314-8696], García-Dorival, Isabel [0000-0002-5654-5662], Cuesta-Geijo, Miguel Ángel [0000-0003-4694-1968], Barrado-Gil, Lucía [0000-0002-1053-2997], Galindo, Immaculada [0000-0001-8257-9797], Alonso, Covandoga [0000-0002-0862-6177], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Fuertes-Monge, Laura, Schaerlaekens, Sofie, Lucena-Agell, Daniel, García-Dorival, Isabel, Cuesta-Geijo, Miguel Ángel, Barrado-Gil, Lucía, Galindo, Inmaculada, Alonso, Covadonga, Díaz, José Fernando, and Oliva, María A.

Abstract

The cytoskeleton is the main communication/transport route within cells and many viruses abuse on this cellular machine to fulfil their cycle. We initially identified the interaction of a SARS-CoV-2 protein with tubulin using a proteomic analysis. We next confirmed the interaction and identified the viral protein domain involved through in vitro co-immunoprecipitation assays and analytical ultracentrifugation experiments. Then, we focused on unveiling the molecular mechanism of the interaction to determine if the SARS-CoV-2 protein promote a stable microtubule assembly (as roads for motors) or induce microtubule dynamics (as main force generation for transport). We have combined biochemical, biophysical and structural studies to determine the ratio of protein-protein interaction and the resulting effect on tubulin assembly. We have found that this protein domain is able to promote microtubule depolymerization into rings and tubulin assembly into non-functional filaments likely because a preference for tubulin curved-conformation. Importantly, this effect is not dependent on nucleotide or nucleotide hydrolysis. Finally, tubulin is a well-known target in cancer diseases and there are four of the seven tubulin druggable sites exploited on chemotherapy. Hence, we have analyzed the ability of microtubule stabilizing (MSA) and destabilizing agents (MDA) on disrupting the interaction of SARS-CoV-2 protein with tubulin. We have found that MSAs keep microtubule structures even in the presence of the viral protein, while the effect of MDAs varies depending on their mechanism of action.

Published
2022

7. Effect of Clinically Used Microtubule Targeting Drugs on Viral Infection and Transport Function

Author

Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, La Caixa, Club Escola Hungaresa de Esgrima de Pontevedra, Oliva, María A. [0000-0002-2215-4639], Tosat-Bitrian, Carlota [0000-0002-0525-3512], Barrado-Gil, Lucía [0000-0002-1053-2997], Bonato, Francesca [0000-0002-0579-2874], Galindo, Inmaculada [0000-0001-8257-9797], Garaigorta, Urtzi [0000-0002-0683-5725], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], París-Ogáyar, Rebeca [0000-0002-7953-0949], Lucena-Agell, Daniel [0000-0001-7314-8696], Giménez-Abián, Juan F. [0000-0002-9220-286X], Urquiza, Jesús [0000-0002-7870-4580], Díaz, José Fernando [0000-0003-2743-3319], Palomo, Valle [0000-0002-1473-4086], Alonso, Covadonga [0000-0002-0862-6177], Oliva, María A., Tosat-Bitrian, Carlota, Barrado-Gil, Lucía, Bonato, Francesca, Galindo Barreales, Inmaculada, Garaigorta, Urtzi, Álvarez-Bernad, Beatriz, París-Ogáyar, Rebeca, Lucena-Agell, Daniel, Giménez-Abián, Juan F., García-Dorival, Isabel, Urquiza, Jesús, Gastaminza, Pablo, Díaz, José Fernando, Palomo, Valle, Alonso Martí, Covadonga, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, La Caixa, Club Escola Hungaresa de Esgrima de Pontevedra, Oliva, María A. [0000-0002-2215-4639], Tosat-Bitrian, Carlota [0000-0002-0525-3512], Barrado-Gil, Lucía [0000-0002-1053-2997], Bonato, Francesca [0000-0002-0579-2874], Galindo, Inmaculada [0000-0001-8257-9797], Garaigorta, Urtzi [0000-0002-0683-5725], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], París-Ogáyar, Rebeca [0000-0002-7953-0949], Lucena-Agell, Daniel [0000-0001-7314-8696], Giménez-Abián, Juan F. [0000-0002-9220-286X], Urquiza, Jesús [0000-0002-7870-4580], Díaz, José Fernando [0000-0003-2743-3319], Palomo, Valle [0000-0002-1473-4086], Alonso, Covadonga [0000-0002-0862-6177], Oliva, María A., Tosat-Bitrian, Carlota, Barrado-Gil, Lucía, Bonato, Francesca, Galindo Barreales, Inmaculada, Garaigorta, Urtzi, Álvarez-Bernad, Beatriz, París-Ogáyar, Rebeca, Lucena-Agell, Daniel, Giménez-Abián, Juan F., García-Dorival, Isabel, Urquiza, Jesús, Gastaminza, Pablo, Díaz, José Fernando, Palomo, Valle, and Alonso Martí, Covadonga

Abstract

Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds. In order to understand the reasons for the observed antiviral activity, we studied the impact of these compounds in motor proteins-mediated intracellular transport. To do so, we used labeled peptide tools, finding that clinically available MTAs impaired the movement linked to MT motors in living cells. However, their effect on viral infection lacked a clear correlation to their effect in motor-mediated transport, denoting the complex use of the cytoskeleton by viruses. Finally, we further delved into the molecular mechanism of action of Mebendazole by combining biochemical and structural studies to obtain crystallographic high-resolution information of the Mebendazole-tubulin complex, which provided insights into the mechanisms of differential toxicity between helminths and mammalians.

Published
2022

9. Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol

Author

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

10. Identification of novel anti-cancer agents by the synthesis and cellular screening of a noscapine-based library

Author

Iranian National Science Foundation, Shahid Beheshti University, Consejo Superior de Investigaciones Científicas (España), Bischoff-Kont, Iris [0000-0002-2057-0604], Salehi, Peyman [0000-0001-5774-3372], Nejad-Ebrahimi, Samad [0000-0003-2167-8032], Bararjanian, Morteza [0000-0003-2235-9877], Schaerlaekens, Sofie [0000-0002-4410-2311], Lucena-Agell, Daniel [0000-0001-7314-8696], Oliva, María A. [0000-0002-2215-4639], Fürst, Robert [0000-0002-9926-7578], Nemati, Faezeh, Bischoff-Kont, Iris, Salehi, Peyman, Nejad-Ebrahimi, Samad, Mohebbi, Maryam, Bararjanian, Morteza, Hadian, Nasim, Hassanpour, Zahra, Jung, Yvonne, Schaerlaekens, Sofie, Lucena-Agell, Daniel, Oliva, María A., Fürst, Robert, Nasiri, Hamid R., Iranian National Science Foundation, Shahid Beheshti University, Consejo Superior de Investigaciones Científicas (España), Bischoff-Kont, Iris [0000-0002-2057-0604], Salehi, Peyman [0000-0001-5774-3372], Nejad-Ebrahimi, Samad [0000-0003-2167-8032], Bararjanian, Morteza [0000-0003-2235-9877], Schaerlaekens, Sofie [0000-0002-4410-2311], Lucena-Agell, Daniel [0000-0001-7314-8696], Oliva, María A. [0000-0002-2215-4639], Fürst, Robert [0000-0002-9926-7578], Nemati, Faezeh, Bischoff-Kont, Iris, Salehi, Peyman, Nejad-Ebrahimi, Samad, Mohebbi, Maryam, Bararjanian, Morteza, Hadian, Nasim, Hassanpour, Zahra, Jung, Yvonne, Schaerlaekens, Sofie, Lucena-Agell, Daniel, Oliva, María A., Fürst, Robert, and Nasiri, Hamid R.

Abstract

Noscapine is a natural product first isolated from the opium poppy (Papaver somniferum L.) with anticancer properties. In this work, we report the synthesis and cellular screening of a noscapine-based library. A library of novel noscapine derivatives was synthesized with modifications in the isoquinoline and phthalide scaffolds. The so generated library, consisting of fifty-seven derivatives of the natural product noscapine, was tested against MDA-MB-231 breast cancer cells in a cellular proliferation assay (with a Z' > 0.7). The screening resulted in the identification of two novel noscapine derivatives as inhibitors of MDA cell growth with IC50 values of 5 µM and 1.5 µM, respectively. Both hit molecules have a five-fold and seventeen-fold higher potency, compared with that of lead compound noscapine (IC50 26 µM). The identified active derivatives retain the tubulin-binding ability of noscapine. Further testing of both hit molecules, alongside the natural product against additional cancer cell lines (HepG2, HeLa and PC3 cells) confirmed our initial findings. Both molecules have improved anti-proliferative properties when compared to the initial natural product, noscapine.

Published
2021

11. N‑alkylisatin‑based microtubule destabilizers bind to the colchicine site on tubulin and retain efficacy in drug resistant acute lymphoblastic leukemia cell lines with less in vitro neurotoxicity

Author

European Commission, Keenan, Bryce [0000-0002-5543-1495], Finol-Urdaneta, Rocío [0000-0003-2157-4532], Hope, Ashleigh [0000-0001-7182-083X], Bremner, John B. [0000-0002-7701-0034], Kavallaris, Maria [0000-0003-2309-898X], Lucena-Agell, Daniel [0000-0001-7314-8696], Oliva, María A. [0000-0002-2215-4639], Díaz, José Fernando [0000-0003-2743-3319], Vine, Kara L. [0000-0001-6871-1149], Keenan, Bryce, Finol-Urdaneta, Rocío, Hope, Ashleigh, Bremner, John B., Kavallaris, Maria, Lucena-Agell, Daniel, Oliva, María A., Díaz, José Fernando, Vine, Kara L., European Commission, Keenan, Bryce [0000-0002-5543-1495], Finol-Urdaneta, Rocío [0000-0003-2157-4532], Hope, Ashleigh [0000-0001-7182-083X], Bremner, John B. [0000-0002-7701-0034], Kavallaris, Maria [0000-0003-2309-898X], Lucena-Agell, Daniel [0000-0001-7314-8696], Oliva, María A. [0000-0002-2215-4639], Díaz, José Fernando [0000-0003-2743-3319], Vine, Kara L. [0000-0001-6871-1149], Keenan, Bryce, Finol-Urdaneta, Rocío, Hope, Ashleigh, Bremner, John B., Kavallaris, Maria, Lucena-Agell, Daniel, Oliva, María A., Díaz, José Fernando, and Vine, Kara L.

Abstract

Background:Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment., Methods:Vincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey’s post hoc test or a two-tailed paired t test was used to determine statistical significance., Results: CEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing., Conclusions:The N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted.

Published
2020

12. Structural model for differential cap maturation at growing microtubule ends

Author

Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), European Commission, Swiss National Science Foundation, Ministerio de Educación, Cultura y Deporte (España), Estévez-Gallego, Juan [0000-0003-3889-8488 ], Josa-Prado, Fernando [0000-0002-6162-3231], Martínez Buey, Rubén [0000-0003-1263-0221], Prota, Andrea E. [0000-0003-0875-5339], Lucena-Agell, Daniel [0000-0001-7314-8696], Kamma-Lorger, C.S. [0000-0001-6686-9161], Yagi, Toshiki[0000-0002-1379-460X], Barasoain, Isabel [0000-0003-2013-085X], Steinmetz, Michel O. [0000-0001-6157-3687], Kamimura, Shinji [0000-0003-4359-0859], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Estévez-Gallego, Juan, Josa-Prado, Fernando, Ku, Siou, Martínez Buey, Rubén, Balaguer, Francisco de Asís, Prota, Andrea E., Lucena-Agell, Daniel, Kamma-Lorger, C.S., Yagi, Toshiki, Iwamoto, Hiroyuki, Duchesne, Laurence, Barasoain, Isabel, Steinmetz, Michel O., Chrétien, Denis, Kamimura, Shinji, Díaz, José Fernando, Oliva, María A., Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), European Commission, Swiss National Science Foundation, Ministerio de Educación, Cultura y Deporte (España), Estévez-Gallego, Juan [0000-0003-3889-8488 ], Josa-Prado, Fernando [0000-0002-6162-3231], Martínez Buey, Rubén [0000-0003-1263-0221], Prota, Andrea E. [0000-0003-0875-5339], Lucena-Agell, Daniel [0000-0001-7314-8696], Kamma-Lorger, C.S. [0000-0001-6686-9161], Yagi, Toshiki[0000-0002-1379-460X], Barasoain, Isabel [0000-0003-2013-085X], Steinmetz, Michel O. [0000-0001-6157-3687], Kamimura, Shinji [0000-0003-4359-0859], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Estévez-Gallego, Juan, Josa-Prado, Fernando, Ku, Siou, Martínez Buey, Rubén, Balaguer, Francisco de Asís, Prota, Andrea E., Lucena-Agell, Daniel, Kamma-Lorger, C.S., Yagi, Toshiki, Iwamoto, Hiroyuki, Duchesne, Laurence, Barasoain, Isabel, Steinmetz, Michel O., Chrétien, Denis, Kamimura, Shinji, Díaz, José Fernando, and Oliva, María A.

Abstract

Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDP•Pi transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in Pi release.

Published
2020

13. Chapter 17-Purification and assembly of bacterial tubulin BtubA/B and constructs bearing eukaryotic tubulin sequences

Author

Ministerio de Ciencia e Innovación (España), Andreu, José Manuel [0000-0001-8064-6933], Oliva, María A. [0000-0002-2215-4639], Andreu, José Manuel, Oliva, María A., Ministerio de Ciencia e Innovación (España), Andreu, José Manuel [0000-0001-8064-6933], Oliva, María A. [0000-0002-2215-4639], Andreu, José Manuel, and Oliva, María A.

Abstract

Bacterial tubulin BtubA/B is a close structural homolog of eukaryotic ab-tubulin, thought to have originated by transfer of ancestral tubulin genes from a primitive eukaryotic cell to a bacterium, followed by divergent evolution. BtubA and BtubB aremeasily expressed homogeneous polypeptides that fold spontaneously without eukaryotic chaperone requirements, associate into weak BtubA/B heterodimers and assemble forming tubulin-like protofilaments. These protofilaments coalesce into pairs and bundles, or form five-protofilament tubules proposed to share the architecture of microtubules. Bacterial tubulin is an attractive framework for tubulin engineering. Potential applications include humanizing different sections of bacterial tubulin with the aims of creating recombinant binding sites for anti-tumor drugs, obtaining well defined substrates for the enzymes responsible for tubulin posttranslational modification, or bacterial microtubule-like polymeric trails for motor proteins. Several divergent sequences from the surface loops of bacterial tubulin have already been replaced by the corresponding eukaryotic sequences, yielding soluble folded chimeras. We describe the purification protocol of untagged bacterial tubulin BtubA/B by means of ion exchange, size exclusion chromatography and an assembly-disassembly cycle. This is followed by methods and examples to characterize its assembly, employing light scattering, sedimentation and electron microscopy.

Published
2013

14. Tubulin homolog TubZ in a phage-encodedpartition system

Author

Oliva, María A. [0000-0002-2215-4639], Martín-Galiano, Antonio J.[0000-0002-6662-329X], Andreu, José Manuel [0000-0001-8064-6933], Oliva, María A., Martín-Galiano, Antonio J., Shakaguchi, Yoshihiko, Andreu, José Manuel, Oliva, María A. [0000-0002-2215-4639], Martín-Galiano, Antonio J.[0000-0002-6662-329X], Andreu, José Manuel [0000-0001-8064-6933], Oliva, María A., Martín-Galiano, Antonio J., Shakaguchi, Yoshihiko, and Andreu, José Manuel

Abstract

Partition systems are responsible for the process whereby large and essential plasmids are accurately positioned to daughter cells during bacterial division. They are typically made of three components: a centromere-like DNA zone, an adaptor protein, and an assembling protein that is either a Walker-box ATPase (type I) or an actin-like ATPase (type II). A recently described type III segregation system has a tubulin/FtsZ-like protein, called TubZ, for plasmid movement. Here, we present the 2.3 Å structure and dynamic assembly of a TubZ tubulin homolog from a bacteriophage and unravel the Clostridium botulinum phage c-st type III partition system. Using biochemical and biophysical approaches, we prove that a gene upstream from tubZ encodes the partner TubR and localize the centromeric region (tubS), both of which are essential for anchoring phage DNA to the motile TubZ filaments. Finally, we describe a conserved fourth component, TubY, which modulates the TubZ-R-S complex interaction.

Published
2012

15. Features critical for membrane binding revealedby DivIVA crystal structure

Author

Oliva, María A. [0000-0002-2215-4639], Halbedel, Sven [0000-0002-5575-8973], Veprintsev, Dmitry B. [0000-0002-3583-5409], Löwe, Jan [0000-0002-5218-6615], Oliva, María A., Halbedel, Sven, Dutow, Pavel, Leonard, Thomas A., Veprintsev, Dmitry B., Hamoen, L. W., Löwe, Jan, Oliva, María A. [0000-0002-2215-4639], Halbedel, Sven [0000-0002-5575-8973], Veprintsev, Dmitry B. [0000-0002-3583-5409], Löwe, Jan [0000-0002-5218-6615], Oliva, María A., Halbedel, Sven, Dutow, Pavel, Leonard, Thomas A., Veprintsev, Dmitry B., Hamoen, L. W., and Löwe, Jan

Abstract

DivIVA is a conserved protein in Gram-positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N-terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled-coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low-resolution crystal structure of the C-terminal (Ct) domain displays a curved tetramer made from two parallel coiled-coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.

Published
2010

16. Structural insights into the conformational variability of FtsZ

Author

Oliva, María A. [0000-0002-2215-4639], Trambaiolo, Daniel [0000-0002-7242-3948], Löwe, Jan [0000-0002-5218-6615], Oliva, María A., Trambaiolo, Daniel, Löwe, Jan, Oliva, María A. [0000-0002-2215-4639], Trambaiolo, Daniel [0000-0002-7242-3948], Löwe, Jan [0000-0002-5218-6615], Oliva, María A., Trambaiolo, Daniel, and Löwe, Jan

Abstract

FtsZ is a prokaryotic homologue of the eukaryotic cytoskeletal protein tubulin and plays a central role in prokaryotic cell division. Both FtsZ and tubulin are known to pass through cycles of polymerization and depolymerization, but the structural mechanisms underlying this cycle remain to be determined. Comparison of tubulin structures obtained in different states has led to a model in which the tubulin monomer undergoes a conformational switch between a "straight" form found in the walls of microtubules and a "curved" form associated with depolymerization, and it was proposed recently that this model may apply also to FtsZ. Here, we present new structures of FtsZ from47 Aquifex aeolicus,47 Bacillus subtilis, Methanococcus jannaschii and Pseudomonas aeruginosa that provide strong constraints on any proposed role for a conformational switch in the FtsZ monomer. By comparing the full range of FtsZ structures determined in different crystal forms and nucleotide states, and in the presence or in the absence of regulatory proteins, we find no evidence of a conformational change involving domain movement. Our new structural data make it clear that the previously proposed straight and curved conformations of FtsZ were related to inter-species differences in domain orientation rather than two interconvertible conformations. We propose a new model in which lateral interactions help determine the curvature of protofilaments.

Published
2007

17. Structural insights into FtsZ protofilament formation

Author

Ministerio de Ciencia y Tecnología (España), Oliva, María A. [0000-0002-2215-4639], Löwe, Jan [0000-0002-5218-6615], Oliva, María A., Cordell, Suzanne C., Löwe, Jan, Ministerio de Ciencia y Tecnología (España), Oliva, María A. [0000-0002-2215-4639], Löwe, Jan [0000-0002-5218-6615], Oliva, María A., Cordell, Suzanne C., and Löwe, Jan

Abstract

The prokaryotic tubulin homolog FtsZ polymerizes into a ring structure essential for bacterial cell division. We have used refolded FtsZ to crystallize a tubulin-like protofilament. The N- and C-terminal domains of two consecutive subunits in the filament assemble to form the GTPase site, with the C-terminal domain providing water-polarizing residues. A domain-swapped structure of FtsZ and biochemical data on purified N- and C-terminal domains show that they are independent. This leads to a model of how FtsZ and tubulin polymerization evolved by fusing two domains. In polymerized tubulin, the nucleotide-binding pocket is occluded, which leads to nucleotide exchange being the rate-limiting step and to dynamic instability. In our FtsZ filament structure the nucleotide is exchangeable, explaining why, in this filament, nucleotide hydrolysis is the rate-limiting step during FtsZ polymerization. Furthermore, crystal structures of FtsZ in different nucleotide states reveal notably few differences.

Published
2004

18. Reversible unfolding of FtsZ cell division proteins from archaea and bacteria. Comparison with eukaryotic tubulin folding and assembly

Author

Comunidad de Madrid, Fondo Nacional de Desarrollo Científico y Tecnológico (Chile), Andreu, José Manuel [0000-0001-8064-6933], Oliva, María A. [0000-0002-2215-4639], Andreu, José Manuel, Oliva, María A., Monasterio, Octavio, Comunidad de Madrid, Fondo Nacional de Desarrollo Científico y Tecnológico (Chile), Andreu, José Manuel [0000-0001-8064-6933], Oliva, María A. [0000-0002-2215-4639], Andreu, José Manuel, Oliva, María A., and Monasterio, Octavio

Abstract

The stability, refolding, and assembly properties of FtsZ cell division proteins from Methanococcus jannaschii and Escherichia coli have been investigated. Their guanidinium chloride unfolding has been studied by circular dichroism spectroscopy. FtsZ from E. coli and tubulin released the bound guanine nucleotide, coinciding with an initial unfolding stage at low denaturant concentrations, followed by unfolding of the apoprotein. FtsZ from M. jannaschii released its nucleotide without any detectable secondary structural change. It unfolded in an apparently two-state transition at larger denaturant concentrations. Isolated FtsZ polypeptide chains were capable of spontaneous refolding and GTP-dependent assembly. The homologous eukaryotic tubulin monomers misfold in solution, but fold within the cytosolic chaperonin CCT. Analysis of the extensive tubulin loop insertions in the FtsZ/tubulin common core and of the intermolecular contacts in model microtubules and tubulin-CCT complexes shows a loop insertion present at every element of lateral protofilament contact and at every contact of tubulin with CCT (except at loop T7). The polymers formed by purified FtsZ have a distinct limited protofilament association in comparison with microtubules. We propose that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for extended two-dimensional polymerization into microtubule polymers.

Published
2002

19. Effect of Clinically Used Microtubule Targeting Drugs on Viral Infection and Transport Function

Author

María Ángela Oliva, Carlota Tosat-Bitrián, Lucía Barrado-Gil, Francesca Bonato, Inmaculada Galindo, Urtzi Garaigorta, Beatriz Álvarez-Bernad, Rebeca París-Ogáyar, Daniel Lucena-Agell, Juan Francisco Giménez-Abián, Isabel García-Dorival, Jesús Urquiza, Pablo Gastaminza, José Fernando Díaz, Valle Palomo, Covadonga Alonso, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, La Caixa, Club Escola Hungaresa de Esgrima de Pontevedra, Oliva, María A., Tosat-Bitrian, Carlota, Barrado-Gil, Lucía, Bonato, Francesca, Galindo, Inmaculada, Garaigorta, Urtzi, Álvarez-Bernad, Beatriz, París-Ogáyar, Rebeca, Lucena-Agell, Daniel, Giménez-Abián, Juan F., Urquiza, Jesús, Díaz, José Fernando, Palomo, Valle, Alonso, Covadonga, Oliva, María A. [0000-0002-2215-4639], Tosat-Bitrian, Carlota [0000-0002-0525-3512], Barrado-Gil, Lucía [0000-0002-1053-2997], Bonato, Francesca [0000-0002-0579-2874], Galindo, Inmaculada [0000-0001-8257-9797], Garaigorta, Urtzi [0000-0002-0683-5725], Álvarez-Bernad, Beatriz [0000-0002-2492-9215], París-Ogáyar, Rebeca [0000-0002-7953-0949], Lucena-Agell, Daniel [0000-0001-7314-8696], Giménez-Abián, Juan F. [0000-0002-9220-286X], Urquiza, Jesús [0000-0002-7870-4580], Díaz, José Fernando [0000-0003-2743-3319], Palomo, Valle [0000-0002-1473-4086], and Alonso, Covadonga [0000-0002-0862-6177]

Subjects
Mammals, antivirals, microtubule targeting drugs, SARS-CoV-2, mebendazole-tubulin complex crystal structure, Organic Chemistry, Mebendazole-tubulin complex crystal structure, General Medicine, Antivirals, Antiviral Agents, Microtubules, Catalysis, COVID-19 Drug Treatment, Computer Science Applications, Inorganic Chemistry, Mebendazole, Tubulin, Animals, Microtubule targeting drugs, Physical and Theoretical Chemistry, Molecular Biology, Spectroscopy
Abstract

Microtubule targeting agents (MTAs) have been exploited mainly as anti-cancer drugs because of their impact on cellular division and angiogenesis. Additionally, microtubules (MTs) are key structures for intracellular transport, which is frequently hijacked during viral infection. We have analyzed the antiviral activity of clinically used MTAs in the infection of DNA and RNA viruses, including SARS-CoV-2, to find that MT destabilizer agents show a higher impact than stabilizers in the viral infections tested, and FDA-approved anti-helminthic benzimidazoles were among the most active compounds. In order to understand the reasons for the observed antiviral activity, we studied the impact of these compounds in motor proteins-mediated intracellular transport. To do so, we used labeled peptide tools, finding that clinically available MTAs impaired the movement linked to MT motors in living cells. However, their effect on viral infection lacked a clear correlation to their effect in motor-mediated transport, denoting the complex use of the cytoskeleton by viruses. Finally, we further delved into the molecular mechanism of action of Mebendazole by combining biochemical and structural studies to obtain crystallographic high-resolution information of the Mebendazole-tubulin complex, which provided insights into the mechanisms of differential toxicity between helminths and mammalians., This work was supported by Ministerio de Ciencia e Innovación PID2019-104545RBI00/ AEI/10.13039/501100011033 to JFD and RTI2018-097305-R-I00 to CA, Consejo Superior de Investigaciones Científicas PIE 201920E111 to JFD, PIE 202020E301 and the European Commission– NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global) to JFD and CA, H2020-MSCA-ITN-2019 860070 TUBINTRAIN to JFD, “La Caixa” Banking Foundation (LCF/PR/HR19/52160012) to CA, Postdoctoral Junior Leader Fellowship Programme from ‘La Caixa’ Banking Foundation (LCF/BQ/PR18/11640007) to VP and Ministerio de Ciencia e Innovacion FPI program (PRE2020-094384) to BAB and Ministerio de Educación FPU program (FPU18/06310) to CTB. We also acknowledge a generous donation from Club Deportivo Escuela Hungaresa de Pontevedra.

Published
2022
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21. Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol

Author

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

22. Identification of novel anti-cancer agents by the synthesis and cellular screening of a noscapine-based library

Author

Zahra Hassanpour, Sofie Schaerlaekens, Hamid R. Nasiri, Samad Nejad-Ebrahimi, María A. Oliva, Morteza Bararjanian, Faezeh Nemati, Maryam Mohebbi, Yvonne Jung, Robert Fürst, Daniel Lucena-Agell, Nasim Hadian, Peyman Salehi, Iris Bischoff-Kont, Iranian National Science Foundation, Shahid Beheshti University, Consejo Superior de Investigaciones Científicas (España), Bischoff-Kont, Iris [0000-0002-2057-0604], Salehi, Peyman [0000-0001-5774-3372], Nejad-Ebrahimi, Samad [0000-0003-2167-8032], Bararjanian, Morteza [0000-0003-2235-9877], Schaerlaekens, Sofie [0000-0002-4410-2311], Lucena-Agell, Daniel [0000-0001-7314-8696], Oliva, María A. [0000-0002-2215-4639], Fürst, Robert [0000-0002-9926-7578], Bischoff-Kont, Iris, Salehi, Peyman, Nejad-Ebrahimi, Samad, Bararjanian, Morteza, Schaerlaekens, Sofie, Lucena-Agell, Daniel, Oliva, María A., and Fürst, Robert

Subjects
Noscapine, Drug repurposing, Antineoplastic Agents, Huisgen reaction, Opium Poppy, 01 natural sciences, Biochemistry, Natural product, Phthalide, Tubulin binding, Small Molecule Libraries, HeLa, chemistry.chemical_compound, Tubulin, Cell Line, Tumor, Drug Discovery, medicine, Humans, Papaver, Isoquinoline, Molecular Biology, Benzofurans, Cell Proliferation, biology, 010405 organic chemistry, Cell growth, Tubulin-binding, Organic Chemistry, Isoquinolines, biology.organism_classification, 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, chemistry, Drug Design, Anticancer agents, Protein Binding, medicine.drug
Abstract

53 p.-7 fig.-1 tab.-1 schem.-1 graph. abst., Noscapine is a natural product first isolated from the opium poppy (Papaver somniferum L.) with anticancer properties. In this work, we report the synthesis and cellular screening of a noscapine-based library. A library of novel noscapine derivatives was synthesized with modifications in the isoquinoline and phthalide scaffolds. The so generated library, consisting of fifty-seven derivatives of the natural product noscapine, was tested against MDA-MB-231 breast cancer cells in a cellular proliferation assay (with a Z' > 0.7). The screening resulted in the identification of two novel noscapine derivatives as inhibitors of MDA cell growth with IC50 values of 5 µM and 1.5 µM, respectively. Both hit molecules have a five-fold and seventeen-fold higher potency, compared with that of lead compound noscapine (IC50 26 µM). The identified active derivatives retain the tubulin-binding ability of noscapine. Further testing of both hit molecules, alongside the natural product against additional cancer cell lines (HepG2, HeLa and PC3 cells) confirmed our initial findings. Both molecules have improved anti-proliferative properties when compared to the initial natural product, noscapine., We are also grateful to the Iran National Science Foundation (INSF, grant number 98026465) for financial support of this project and Shahid Beheshti University Research Council for providing facilities of to conduct this study. This work was supported by CSIC PIE 201920E111 (MAO).

Published
2021

23. Gatorbulin-1, a distinct cyclodepsipeptide chemotype, targets a seventh tubulin pharmacological site

Author

María A. Oliva, Susan Matthew, Ranjala Ratnayake, Hendrik Luesch, Francesca Bonato, April L. Risinger, Andrea E. Prota, Daniel Lucena-Agell, Seok Ting Lim, Qi-Yin Chen, J. Fernando Díaz, Charles S. Fermaintt, Xiaomeng Wang, Valerie J. Paul, National Cancer Institute (US), National Institute of General Medical Sciences (US), University of Florida, Ministerio de Ciencia e Innovación (España), Consejo Superior de Investigaciones Científicas (España), European Commission, Matthew, S. [0000-0003-4943-4777], Chen, Qi-Yin [0000-0002-4364-1057], Ratnayake, R. [0000-0002-5980-2070], Fermaintt, Charles S. [0000-0001-8140-5461], Lucena-Agell, Daniel [0000-0001-7198-2900], Bonato, Francesca [0000-0002-0579-2874], Prota, Andrea E. [0000-0003-0875-5339], Lim, Seok Ting [0000-0002-5173-5715], Wang, Xiaomeng [0000-0002-1036-2764], Díaz, José Fernando [0000-0003-2743-3319], Risinger, April L. [0000-0002-4363-3268], Paul, Valerie J. [0000-0002-4691-1569], Oliva, María A. [0000-0002-2215-4639], Luesch, H. [0000-0002-4091-7492], Matthew, S., Chen, Qi-Yin, Ratnayake, R., Fermaintt, Charles S., Lucena-Agell, Daniel, Bonato, Francesca, Prota, Andrea E., Lim, Seok Ting, Wang, Xiaomeng, Díaz, José Fernando, Risinger, April L., Paul, Valerie J., Oliva, María A., and Luesch, H.

Subjects
Models, Molecular, Protein Conformation, alpha-Helical, Marine natural product, Antineoplastic Agents, Apoptosis, Total synthesis, Crystallography, X-Ray, Cyanobacteria, 01 natural sciences, Pentapeptide repeat, Microtubules, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Microtubule, Tubulin, Cell Line, Tumor, Depsipeptides, Drug Discovery, Humans, Maytansine, Protein Interaction Domains and Motifs, Binding site, Vinca Alkaloids, 030304 developmental biology, 0303 health sciences, Biological Products, Multidisciplinary, Natural product, Binding Sites, biology, Chemotype, 010405 organic chemistry, Biological Sciences, HCT116 Cells, In vitro, Tubulin Modulators, 3. Good health, 0104 chemical sciences, chemistry, Biochemistry, Pyrones, biology.protein, Protein Conformation, beta-Strand, Taxoids, Colchicine, Protein Binding
Abstract

35 p.-5 fig.-1 tab., Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/β-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/β-tubulin−GB1 complex., This research was supported by the NIH, National Cancer Institute Grants R01CA172310 (to H.L.) and R50CA211487 (to R.R.) and National Institute of General Medical Sciences Grant P41GM086210 (to H.L. and V.J.P.), Commercialization Fund Award from University of Florida (UF) Innovate (UF Office of Technology Licensing), and Debbie and Sylvia DeSantis Chair professorship (H.L.). The biochemical and the crystal structure work was supported by grants Ministerio de Ciencia e Innovación PID2019-10454RB-I00/AEI/10.13039/501100011033, Fondo de Investigaciones Sanitarias and COV20/01007E Proyecto Intramural Especial 201920E111 from Consejo Superior de Investigaciones Científicas (to J.F.D.) and European Union H2020-MSCA-ITN-2019 860070 TUBINTRAIN grant (to J.F.D. and A.E.P.).

Published
2021

24. N-alkylisatin-based microtubule destabilizers bind to the colchicine site on tubulin and retain efficacy in drug resistant acute lymphoblastic leukemia cell lines with less in vitro neurotoxicity

Author

Kara L. Vine, John B. Bremner, Rocio K. Finol-Urdaneta, Bryce Keenan, José Fernando Díaz, Daniel Lucena-Agell, Ashleigh Hope, María A. Oliva, Maria Kavallaris, European Commission, Keenan, Bryce, Finol-Urdaneta, Rocío, Hope, Ashleigh, Bremner, John B., Kavallaris, Maria, Lucena-Agell, Daniel, Oliva, María A., Díaz, José Fernando, Vine, Kara L., Keenan, Bryce [0000-0002-5543-1495], Finol-Urdaneta, Rocío [0000-0003-2157-4532], Hope, Ashleigh [0000-0001-7182-083X], Bremner, John B. [0000-0002-7701-0034], Kavallaris, Maria [0000-0003-2309-898X], Lucena-Agell, Daniel [0000-0001-7314-8696], Oliva, María A. [0000-0002-2215-4639], Díaz, José Fernando [0000-0003-2743-3319], and Vine, Kara L. [0000-0001-6871-1149]

Subjects
Cancer Research, Vincristine, Pharmacology, P-glycoprotein, Acute lymphoblastic leukemia, lcsh:RC254-282, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, In vivo, Genetics, medicine, Neurotoxicity, Colchicine, Chemotherapy, lcsh:QH573-671, Cytotoxicity, 030304 developmental biology, 0303 health sciences, biology, lcsh:Cytology, Chemistry, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, N-alkylisatin, Vinblastine, Multi-drug resistance, Tubulin, Oncology, Paclitaxel, 030220 oncology & carcinogenesis, biology.protein, Primary Research, medicine.drug
Abstract

16 p.-7 fig.-1 tab., Background:Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment., Methods:Vincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey’s post hoc test or a two-tailed paired t test was used to determine statistical significance., Results: CEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing., Conclusions:The N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted., European Union H2020-MSCA-ITN-ETN/0582 ITN TubInTrain to J.F. Diaz is gratefully acknowledged.

Published
2020

25. Structural model for differential cap maturation at growing microtubule ends

Author

Isabel Barasoain, Andrea E. Prota, J. Fernando Díaz, Francisco de Asís Balaguer, Christina Kamma-Lorger, Siou Ku, María A. Oliva, Rubén M. Buey, Toshiki Yagi, Laurence Duchesne, Denis Chrétien, Daniel Lucena-Agell, Shinji Kamimura, Juan Estévez-Gallego, Michel O. Steinmetz, Hiroyuki Iwamoto, Fernando Josa-Prado, Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Department of Structural Engineering - University of Naples 'Federico II', Microbiologia Molecular, Centro Investigaciones Biologicas. CSIC, Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Biomolecular Research, Structural Biology, Paul Scherrer Institute (PSI), Interactions cellulaires et moléculaires (ICM), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS), BFU2013-47014P, Ministerio de Economía y Competitividad, H2020-MSCA-ITN-EJD-860070, H2020 European Research Council, 31003A_166608, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, KAKENHI 16K07328/17H03668, Japan Society for the Promotion of Science, ANR-16-C11-0017-01, Agence Nationale de la Recherche, 2019B1365, SPring-8 Proposal, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), European Commission, Swiss National Science Foundation, Ministerio de Educación, Cultura y Deporte (España), Estévez-Gallego, Juan, Josa-Prado, Fernando, Martínez Buey, Rubén, Prota, Andrea E., Lucena-Agell, Daniel, Kamma-Lorger, C.S., Yagi, Toshiki, Barasoain, Isabel, Steinmetz, Michel O., Kamimura, Shinji, Díaz, José Fernando, Oliva, María A., Estévez-Gallego, Juan [0000-0003-3889-8488 ], Josa-Prado, Fernando [0000-0002-6162-3231], Martínez Buey, Rubén [0000-0003-1263-0221], Prota, Andrea E. [0000-0003-0875-5339], Lucena-Agell, Daniel [0000-0001-7314-8696], Kamma-Lorger, C.S. [0000-0001-6686-9161], Yagi, Toshiki[0000-0002-1379-460X], Barasoain, Isabel [0000-0003-2013-085X], Steinmetz, Michel O. [0000-0001-6157-3687], Kamimura, Shinji [0000-0003-4359-0859], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A. [0000-0002-2215-4639], Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), University of Naples Federico II = Università degli studi di Napoli Federico II, Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), and ANR-16-CE11-0017,Tubulin_GTP,Mécanisme d'hydrolyse du GTP lors de l'assemblage des microtubules(2016)

Subjects
Models, Molecular, none, Structural Biology and Molecular Biophysics, [SDV]Life Sciences [q-bio], Molecular Conformation, GTPase, Microtubules, 0302 clinical medicine, Tubulin, structural biology, Nucleotide, Biology (General), ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, 0303 health sciences, taxol, biology, Chemistry, General Neuroscience, dynamic instability, General Medicine, Molecular biophysics, Medicine, Guanosine Triphosphate, Structural biology, Research Article, microtubule, QH301-705.5, Taxol, Science, Microtubule, Dynamic instability, macromolecular substances, Guanosine Diphosphate, General Biochemistry, Genetics and Molecular Biology, Structure-Activity Relationship, 03 medical and health sciences, None, Pi, molecular biophysics, 030304 developmental biology, GTP-cap, General Immunology and Microbiology, Cell growth, Cryoelectron Microscopy, Hydrogen Bonding, biology.protein, Biophysics, 030217 neurology & neurosurgery
Abstract

26 p.-5 fig.-5 tab., Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDP•Pi transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in Pi release., This work was supported by Ministerio de Economia y Competitividad grants BFU2013-47014-P to MAO and BFU2016-75319-R to FDP (both AEI/FEDER, UE); Ministerio de Ciencia e Innovación RYC-2011–07900 to MAO; European Union H2020-MSCA-ITN-ETN/0582 ITN TubInTrain to FDP and AEP; Swiss National Science Foundation (31003A_166608) to MOS; JSPS KAKENHI (16K07328/17H03668) to SK; (FSE) ANR-16-CE11-0017-01 to DC and LD. JE-G was supported by Ministerio de Educación, Cultura y Deporte FPU15-03140 and SK by French Ministry of Higher Education of Research and Innovation (IGDR). Cryo-EM data were adquired on the MRic platform (Univ. Rennes, CNRS, Inserm, BIOSIT-UMS 3480, US_S 018, F-35000 Rennes, France). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery"

Published
2020
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26. Structural basis of noscapine activation for tubulin binding

Author

Jesús Jiménez-Barbero, Ángeles Canales, Katja Bargsten, María A. Oliva, Michel O. Steinmetz, Andrea E. Prota, Javier Rodríguez-Salarichs, J. Fernando Díaz, Youssef L. Bennani, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Swiss National Science Foundation, European Commission, Club Escola Hungaresa de Esgrima de Pontevedra, Oliva, María A. [0000-0002-2215-4639], Prota, Andrea E. [0000-0003-0875-5339], Rodríguez-Salarichs, Javier [0000-0002-2932-0142], Jiménez-Barbero, Jesús [0000-0001-5421-8513], Canales, Ángeles [0000-0003-0542-3080], Steinmetz, Michel O. [0000-0001-6157-3687], Díaz, José Fernando [0000-0003-2743-3319], Oliva, María A., Prota, Andrea E., Rodríguez-Salarichs, Javier, Jiménez-Barbero, Jesús, Canales, Ángeles, Steinmetz, Michel O., and Díaz, José Fernando

Subjects
Steric effects, Noscapine, Antineoplastic Agents, macromolecular substances, Crystallography, X-Ray, 01 natural sciences, Microtubules, Anticancer drugs, Tubulin binding, 03 medical and health sciences, chemistry.chemical_compound, In vivo, Tubulin, Drug Discovery, medicine, Animals, Humans, Colchicine, Cytotoxicity, 030304 developmental biology, 0303 health sciences, Binding Sites, biology, Alkaloid, Tubulin Modulators, 0104 chemical sciences, 3. Good health, Molecular Docking Simulation, 010404 medicinal & biomolecular chemistry, chemistry, Drug Design, biology.protein, Biophysics, Molecular Medicine, Protein Binding, medicine.drug
Abstract

Noscapine is a natural alkaloid that is used as an antitussive medicine. However, it also acts as a weak anticancer agent in certain in vivo models through a mechanism that is largely unknown. Here, we performed structural studies and show that the cytotoxic agent 7A-O-demethoxy-amino-noscapine (7A-aminonoscapine) binds to the colchicine site of tubulin. We suggest that the 7A-methoxy group of noscapine prevents binding to tubulin due to a steric clash of the compound with the T5-loop of α-tubulin. We further propose that the anticancer activity of noscapine arises from a bioactive metabolite that binds to the colchicine site of tubulin to induce mitotic arrest through a microtubule cytoskeleton-based mechanism., This work was supported by Ministerio de Ciencia e Innovación PID2019-104545RB-I00 and Fondo de Investigaciones Sanitarias COV20-01007 to JFD; CTQ2016-76263-P to AC; Swiss National Science Foundation (31003A_166608) to MOS; H2020 MSCA-ITN-2019 860070 TUBINTRAIN to AEP and JFD and a donation from Club deportivo Escuela Hungaresa de Pontevedra.

Published
2020

27. Reversible unfolding of FtsZ cell division proteins from archaea and bacteria. Comparison with eukaryotic tubulin folding and assembly

Author

Andreu, José Manuel, Oliva, María A., Monasterio, Octavio, Comunidad de Madrid, Fondo Nacional de Desarrollo Científico y Tecnológico (Chile), Andreu, José Manuel, Oliva, María A., Andreu, José Manuel [0000-0001-8064-6933], and Oliva, María A. [0000-0002-2215-4639]

Subjects
Ring, Self-association, Methanococcus-jannaschii, bacteria, Alpha-beta-tubulin, Microtubule, macromolecular substances, Escherichia-coli, Binding, gtp hydrolysis, actin, Chaperonin
Abstract

10 p.-7 fig.1 tab., The stability, refolding, and assembly properties of FtsZ cell division proteins from Methanococcus jannaschii and Escherichia coli have been investigated. Their guanidinium chloride unfolding has been studied by circular dichroism spectroscopy. FtsZ from E. coli and tubulin released the bound guanine nucleotide, coinciding with an initial unfolding stage at low denaturant concentrations, followed by unfolding of the apoprotein. FtsZ from M. jannaschii released its nucleotide without any detectable secondary structural change. It unfolded in an apparently two-state transition at larger denaturant concentrations. Isolated FtsZ polypeptide chains were capable of spontaneous refolding and GTP-dependent assembly. The homologous eukaryotic tubulin monomers misfold in solution, but fold within the cytosolic chaperonin CCT. Analysis of the extensive tubulin loop insertions in the FtsZ/tubulin common core and of the intermolecular contacts in model microtubules and tubulin-CCT complexes shows a loop insertion present at every element of lateral protofilament contact and at every contact of tubulin with CCT (except at loop T7). The polymers formed by purified FtsZ have a distinct limited protofilament association in comparison with microtubules. We propose that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for extended two-dimensional polymerization into microtubule polymers., This work was supported in part by MCyT Grant BIO99-0859-C03-02/BIO2000-0748, the Programa de Grupos Estratégicos de la Comunidad de Madrid (to J. M. A.), an FPI predoctoral fellowship (to M. A. O.), and FONDECYT Grant 1010848 (to O. M.).

Published
2002

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