Evaluación de compuestos antitumorales dirigidos contra tubulina e implicación en los mecanismos estructurales de control del citoesqueleto

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 28-06-2019Esta tesis tiene embargado el acceso al texto completo hasta el 28-12-2020Los microtúbulos (polímeros de heterodímeros de -tubulina) son uno de los principales componentes del citoesqueleto. Estos polímeros son esenciales para la división celular, así como para otras funciones cruciales de la célula. Para poder realizar dicha función, la tubulina mantiene un ciclo dinámico de polimerización/despolimerización controlado por la actividad GTPasa de la subunidad de -tubulina. Debido al papel de los microtúbulos en el crecimiento y vascularización de los tumores, constituyen una diana para la quimioterapia antitumoral, siendo los ligandos dirigidos frente a tubulina uno de los grupos de fármacos con mejores resultados frente a varios tipos de tumores. Además, debido a la gran cantidad de toxinas anti-tubulina generadas por animales y plantas como mecanismo de defensa, existen hasta seis sitios de unión en tubulina, así como una gran variedad de quimiotipos diferentes. Este trabajo está centrado en la caracterización de dos grupos de fármacos dirigidos contra dos de estos sitios de unión. En la primera parte de este estudio se describe el compuesto Li5, un ligando que se une al sitio de colchicina con la mayor afinidad descrita para este sitio de unión, y que posee un novedoso mecanismo de activación basado en su estado redox, además de una baja toxicidad in vivo. El compuesto fue descubierto como parte de un cribado frente a líneas de linfoma DLBCL. El mecanismo de acción del Li5 ha sido caracterizado a nivel estructural, bioquímico y celular. El compuesto se une a tubulina de forma dependiente de su estado redox, siendo la forma oxidada inactiva. En la forma reducida activa, el ligando puede adoptar dos conformaciones enantioméricas posibles, siendo solo una de ellas capaz de unirse a la diana. Los estudios estructurales han permitido probar que solo la forma enantiomérica S se une al sitio de colchicina. Estos resultados sugieren que el Li5 es un prometedor antimitótico debido a su alta afinidad en tubulina (ideal para generar fármacos conjugados con anticuerpos), su baja toxicidad en ratones y su habilidad para superar mecanismos de resistencia a fármacos por sobreexpresión de transportadores de membrana. La segunda parte de este trabajo se centra en el mecanismo estructural de activación de la tubulina por compuestos de unión covalente (los cuales son interesantes también para conjugarlos con anticuerpos). La estructura a alta resolución del complejo tubulina-ciclostreptina da información sobre los cambios estructurales implicados en la inducción de la estabilización generada por este ligando. El compuesto altera la estructura generando una transición del bucle T5, mimetizando el estado que posee cuando se encuentra GTP unido a la -tubulina, a pesar de tener GDP en el sitio de nucleótido, permitiendo así la activación de la tubulina debido a un efecto alostérico. Este mecanismo es diferente al descrito previamente en otros taxanos, los cuales promueven el ensamblaje al modular la estructuración del bucle M en un plegamiento helicoidal. Finalmente, este trabajo ahonda en el mecanismo de resistencia a fármacos frente a tubulina por sobreexpresión del isotipo III, indicando que este mecanismo se debe a un aumento de la capacidad dinámica de los microtúbulos, el cual contrarresta la estabilidad promovida por los ligandos covalentes del sitio de taxanos

Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity

The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.Agencia Estatal de Investigacion [AEI/10.13039/5011000 ´ 11 033]; Ministerio de Ciencia e Innovacion, and co-´ funded by the European Regional Development Fund(ERDF-UE) [PID2020-114429RB-I00 to O.L., PID2020-112998GB-100 to F.M.-H]; Autonomous Region of Madrid and co-funded by the European Social Fund and the European Regional Development Fund [Y2018/BIO4747 and P2018/NMT4443 to O.L. and F.M.-H.]; National Institute of Health Carlos III to CNIO; J.R.L.O. and O.N. acknowledge support from the Molecular Interactions Facility at the CIB-CSIC; N.G.-R. was supported by a Boehringer Ingelheim Fonds PhD fellowship; N.F.L. is funded by NIH [GM107287]. Funding for open access charge: Agencia Estatal de Investigacion [AEI ´ /10.13039/501100011 033]; Ministerio de Ciencia e Innovacion, co-funded by the Eu-ropean Regional Development Fund (ERDF) [PID2020-114429RB-I00].Peer reviewe

Structure-activity relationships, biological evaluation and structural studies of novel pyrrolonaphthoxazepines as antitumor agents

Microtubule-targeting agents (MTAs) are a class of clinically successful anti-cancer drugs. The emergence of multidrug resistance to MTAs imposes the need for developing new MTAs endowed with diverse mechanistic properties. Benzoxazepines were recently identified as a novel class of MTAs. These anticancer agents were thoroughly characterized for their antitumor activity, although, their exact mechanism of action remained elusive. Combining chemical, biochemical, cellular, bioinformatics and structural efforts we developed improved pyrrolonaphthoxazepines antitumor agents and their mode of action at the molecular level was elucidated. Compound 6j, one of the most potent analogues, was confirmed by X-ray as a colchicine-site MTA. A comprehensive structural investigation was performed for a complete elucidation of the structure-activity relationships. Selected pyrrolonaphthoxazepines were evaluated for their effects on cell cycle, apoptosis and differentiation in a variety of cancer cells, including multidrug resistant cell lines. Our results define compound 6j as a potentially useful optimized hit for the development of effective compounds for treating drug-resistant tumors.This work was supported in part by a grant from the Swiss National Science Foundation (31003A_166608; to M.O.S), grant BFU2016-75319-R (AEI/FEDER, EU) from Ministerio de Economia y Competitividad, Blueprint 282510, AIRC-17217. The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery” (to M.O.S. and J.F.D.) and the COST Action EPICHEMBIO CM-1406 (to L.A. and G.C.). This work has also received partial funding from the European Union’s Horizon 2020 (EU) research and innovation programme under the Marie Sklodowska-Curie grant agreement No 721906. Finally, this work was partially funded by MIUR-PRIN project n. 2015Y3C5KP (to L.M.)

Quinolin-6-yloxyacetamides are microtubule destabilizing agents that bind to the colchicine site of tubulin

Quinolin-6-yloxyacetamides (QAs) are a chemical class of tubulin polymerization inhibitors that were initially identified as fungicides. Here, we report that QAs are potent anti-proliferative agents against human cancer cells including ones that are drug-resistant. QAs act by disrupting the microtubule cytoskeleton and by causing severe mitotic defects. We further demonstrate that QAs inhibit tubulin polymerization in vitro. The high resolution crystal structure of the tubulin-QA complex revealed that QAs bind to the colchicine site on tubulin, which is targeted by microtubule-destabilizing agents such as colchicine and nocodazole. Together, our data establish QAs as colchicine-site ligands and explain the molecular mechanism of microtubule destabilization by this class of compounds. They further extend our structural knowledge on antitubulin agents and thus should aid in the development of new strategies for the rational design of ligands against multidrug-resistant cancer cells.This work was supported by grants from Spanish Ministerio de Economía y Competitividad (BFU2016-75319-R (AEI/FEDER, UE) to J. Fernando Díaz) and from the Swiss National Science Foundation (31003A_166608 to Michel O. Steinmetz). The authors acknowledge networking contribution by the COST (European Cooperation in Science and Technology) Action CM1407 “Challenging organic syntheses inspired by nature—from natural products chemistry to drug discovery”. X-ray data were collected at beamline X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland). Ashwani Sharma was supported by an EMBO (European Molecular Biology Organization) Long Term Fellowship, J. Fernando Díaz is member of the CIB Intramural Program “Molecular Machines for Better Life” (MACBET)

Quinolin-6-Yloxyacetamides Are Microtubule Destabilizing Agents That Bind to the Colchicine Site of Tubulin

Quinolin-6-yloxyacetamides (QAs) are a chemical class of tubulin polymerization inhibitors that were initially identified as fungicides. Here, we report that QAs are potent anti-proliferative agents against human cancer cells including ones that are drug-resistant. QAs act by disrupting the microtubule cytoskeleton and by causing severe mitotic defects. We further demonstrate that QAs inhibit tubulin polymerization in vitro. The high resolution crystal structure of the tubulin-QA complex revealed that QAs bind to the colchicine site on tubulin, which is targeted by microtubule-destabilizing agents such as colchicine and nocodazole. Together, our data establish QAs as colchicine-site ligands and explain the molecular mechanism of microtubule destabilization by this class of compounds. They further extend our structural knowledge on antitubulin agents and thus should aid in the development of new strategies for the rational design of ligands against multidrug-resistant cancer cells

Molecular architecture and oligomerization of C. glabrata Cdc13 underpin its telomeric DNA binding and unfolding activity

1 p.-7 fig.The CST complex, composed of Cdc13, Stn1 and Ten1 in yeast, mediates the replication and stability of telomeric DNA. Cdc13, the least evolutionarily conserved component, features four concatenated OB-fold domains, whose architecture and functions remain poorly understood. We dissected the molecular architecture of Candida glabrata Cdc13 and showed how each of its OB folds contributes to its self-association and binding to telomeric DNA sequences. Using a combination of biochemical and biophysical tools, we concluded that all individual domains contribute to DNA binding despite not being directly implicated in the binding itself. Analyzing Cdc13 mutants lacking one or more OB-fold domains, we observed that Cdc13 forms dimers primarily through the interaction between OB-fold 2 (OB2) domains, stimulating the binding of OB3 to telomeric sequences. Furthermore, we showed that C. glabrata Cdc13 and CST form higher-order complexes via oligomerization through OB1. Our results reveal the molecular organization of C. glabrata Cdc13, how this regulates DNA binding, and imply that the distinct architectures of yeast Cdc13 share common principles.Peer reviewe

Structure, Thermodynamics, and Kinetics of Plinabulin Binding to Two Tubulin Isotypes

25 p.-5 fig.-1 tab. + 6 fig. supl.+ 1 tab. supl.αβ-Tubulin is a validated target for anticancer drug discovery, and molecules binding to this protein are used to treat several types of tumors. Here, we report on a combined X-ray crystallography and molecular dynamics approach to study drug binding within the colchicine site of αβ-tubulin, focusing on plinabulin, an agent currently in phase 3 clinical testing for the treatment of cancer and chemotherapy-induced neutropenia. We found that plinabulin is more persistently bound to the colchicine site of βII- compared to βIII-tubulin, allowing for a prediction of isotype-expression-dependent drug sensitivity. Additionally, computational residence time and exit paths from the βII-tubulin were compared between plinabulin and two other compounds, colchicine and combretastatin-A4. The former displayed the highest residence time, followed by plinabulin and then distantly by combretastatin-A4. Our combined experimental and computational protocol could help to investigate anti-tubulin drugs, improving our understanding of their mechanism of action, residence time, and tubulin isotype selectivity.This work was financially supported by BeyondSpring Pharmaceuticals Inc. (to M.O.S. and A.C.) and by grants from the Ministerio de Ciencia Innovacion y Universidades (BFU2016-75319-R; to J.F.D.), from the Swiss National Science Foundation (31003A_166608, to M.O.S.) and from the Regione Lombardia (Accordo per la Ricerca e l’Innovazione).Peer reviewe

Triazolopyrimidines stabilize microtubules by binding to the vinca inhibitor site of tubulin

53 p.-7 fig.+ 7 p.-1 tab.supl.-6 fig.supl. Saez Calvo, Gonzalo et al.Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells.This work was supported in part by grants BFU2016‐75319‐R (AEI/FEDER, UE) (JFD), BFU2014‐51823‐R (to JMA) from Ministerio de Economia y Competitividad and 31003A_166608 from the Swiss National Science Foundation (M.O.S.). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature ‐ from natural products chemistry to drug discovery”. The SAXS experiments were performed at BL11‐NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff. X‐ray data were collected at beamline X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland).Peer reviewe

Crystal Structure of the Cyclostreptin-Tubulin Adduct: Implications for Tubulin Activation by Taxane-Site Ligands

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.This research was funded by Ministerio de Economia y Competitividad grant BFU2016-75319-R to JFDP (both AEI/FEDER, UE); Ministerio de Ciencia e Innovación RYC-2011-07900 to MAO; Swiss National Science Foundation grant (31003A_166608) to MOS and CA121138 to SLM. The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature”—from natural products chemistry to drug discoveryWe acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Structure-activity relationships, biological evaluation and structural studies of novel pyrrolonaphthoxazepines as antitumor agents

Microtubule-targeting agents (MTAs) are a class of clinically successful anti-cancer drugs. The emergence of multidrug resistance to MTAs imposes the need for developing new MTAs endowed with diverse mechanistic properties. Benzoxazepines were recently identified as a novel class of MTAs. These anticancer agents were thoroughly characterized for their antitumor activity, although, their exact mechanism of action remained elusive. Combining chemical, biochemical, cellular, bioinformatics and structural efforts we developed improved pyrrolonaphthoxazepines antitumor agents and their mode of action at the molecular level was elucidated. Compound 6j, one of the most potent analogues, was confirmed by X-ray as a colchicine-site MTA. A comprehensive structural investigation was performed for a complete elucidation of the structure-activity relationships. Selected pyrrolonaphthoxazepines were evaluated for their effects on cell cycle, apoptosis and differentiation in a variety of cancer cells, including multidrug resistant cell lines. Our results define compound 6j as a potentially useful optimized hit for the development of effective compounds for treating drug-resistant tumors