Estructura tridimensional de un fragmento de 3 Kda del factor derivado de Plaquetas-4 con actividad antiangiogénica e interacción con el factor de crecimiento para fibroblastos

Platelet-Factor-4 ís a protein belonging to the family of ELR negative CXC chemokines which binds to fibroblast growth factor, and inhibits its mitogenic activity.Platelet-factor-4 also inhibits tumor growth by mechanisms involving antiangiogenesis in which the blockage of fibroblast growth factor mitogenic activity seems lo play an essential role. Synthesis of platelet-factor-4 by recombinant technologies is a considerably cumbersome process that does not seen could sustain an industrial production of pharmacological-quoality because of platelet-factor-4 quaternary structure, and because the protein actívation requires an intramonomer Hydrolisys. It has been shown that the proteín's twenty-four residue C-terminal fragment is also antíangtogeníc in vitro and in vivio, forms a specific 1:1 complex with fibroblast growyh factors and decreases their affinity for their cell surface receptor. Thus this fragments seems could constitute an alternative to the use of the whole platelet-factor-4 for several of its uses therapeutic applications. The fragments preferential three-dimensional conformation in solutíon is quite stable and has been found to be composed of two helical sub-domains, which, both, seem required for inhihilion of fíbroblast growth factor dríven mitogenesis. The characterítation, at high resolution, of the three-dimensional structure of this fragment may contribute, to the understanding of the basis of its antiangiogenic activity, and thus, contribute, to the optimitation of their pharmacological properties. At the same time this structural characteritation may also contribute to a deeper perception of the platelet-factor-4 physiology.El factor derivado de plaquetas-a es una proteína perteneciente a la familia de las equemoquinas CXC, carentes de la secuencia ELR• . Esta proteína se une al factor de crecimiemo para Iibroblastos inhibiend o su actividad mitogénica. Así mismo se ha demostrado que inhibe el crecimiento tumoral a través de un mecanismo antiangiogénico en el que debe jugar un pa pel importante el bloqueo de la actividad del factor de crecimiento pura fibroblasros. El factor derivado de plaquetas-4 posee una estructura tridimensional complicada, por lo que es poco probable que puedan obtenerse cantidades industriales del producto, con calidad farmacológica, tanto por síntesis química como mediante ingeniería genética. Se ha demostrado que un fragmento equivalente a los veinticuatro residuos últimos del extremo carboxilo terminal de la proteína completa posee también actividad antíangiogñenica in vitro e in vivo, forma un complejo con estequiomelría 1:1 con el factor de crecimiento para fibroblastos y hace que disminuya su afinidad por su receptor de alta afinidad de la superficie celular. Este fragmento posee numerosas características para constituir un buen sustituto terapéutico de la proteína completa. La estructura tridimensional en solución de este fragmento es relativamente sencilla y notablemente estable. Consta de dos subdominios helicoidales que parece que es necesario que se mantengan unidos, tanto para la inhibición y la formación del complejo, como para que uno de los subdominios siga siendo helicoidal. La caracterización estructural a alta resolución de este fragmento puede contribuir a entender las bases estructurales de su actividad antiangiogéníca y, por tanto, podría permitir mejorar sus propiedad es fa rma co lógica s. A su vez puede ayudar a un a mejor comprensión de las bases estructurales de la fisiología del factor derivado de plaquetas-4

Synthesis, Microtubule-Binding Affinity, and Antiproliferative Activity of New Epothilone Analogs and of an EGFR-Targeted Epothilone-Peptide Conjugate.

A new simplified, epoxide-free epothilone analog was prepared incorporating an N-(2-hydroxyethyl)-benzimidazole side chain, which binds to microtubules with high affinity and inhibits cancer cell growth in vitro with nM potency. Building on this scaffold, a disulfide-linked conjugate with the purported EGFR-binding (EGFR, epidermal growth factor receptor) peptide GE11 was then prepared. The conjugate retained significant microtubule-binding affinity, in spite of the size of the peptide attached to the benzimidazole side chain. The antiproliferative activity of the conjugate was significantly lower than for the parent scaffold and, surprisingly, was independent of the EGFR expression status of cells. Our data indicate that the disulfide-based conjugation with the GE11 peptide is not a viable approach for effective tumor-targeting of highly potent epothilones and probably not for other cytotoxics

Synthesis and Biological Evaluation As Microtubule-Active Agents of Several Tetrahydrofuran and Spiroacetal Derivatives

The stereoselective preparation of several molecules containing structural fragments of the tetrahydrofuran and spiroacetal type is described. Their degree of cytotoxicity and their interactions with tubulin have been investigated. It has been confirmed that the tetrahydrofuran derivatives are cytotoxic but, in contrast to previous reports, it has been found that the cytoxicity is not due to interactions with the microtubule network. Furthermore, and also in contrast to a previous report on closely related compounds, the spiroacetal derivatives do show interactions with tubulin, even though the precise mechanism and the binding site still remain to be established

Synthesis and biological evaluation as microtubule-active agents of several tetrahydrofuran and spiroacetal derivatives

The stereoselective preparation of several molecules containing structural fragments of the tetrahydrofuran and spiroacetal type is described. Their degree of cytotoxicity and their interactions with tubulin have been investigated. It has been confirmed that the tetrahydrofuran derivatives are cytotoxic but, in contrast to previous reports, it has been found that the cytoxicity is not due to interactions with the microtubule network. Furthermore, and also in contrast to a previous report on closely related compounds, the spiroacetal derivatives do show interactions with tubulin, even though the precise mechanism and the binding site still remain to be established.Financial support has been granted by the Spanish Ministry of Education and Science (CTQ2008-02800), by the Consellería d´Empresa, Universitat i Ciencia de la Generalitat Valenciana (ACOMP09/113) and by the BANCAJA-UJI Foundation (P1-1B2002-06 and P1-1B-2008-14). The biological work has been supported in part by grants from the Spanish Ministry of Education and Science (BIO2007- 61336) and from the Comunidad de Madrid (grants S2010/ BMD-2457 and BIPEDD2-CM) (both to J.F.D.). We further thank the Matadero Municipal Vicente de Lucas in Segovia for providing the calf brains which were the source of tubuli

Synthesis and biological evaluation of truncated α-tubulin-binding pironetin analogues lacking alkyl pendants in the side chain or the dihydropyrone ring

The preparation of several new truncated analogues of the natural dihydropyrone pironetin is described. They differ from the natural product mainly in the suppression of some of the alkyl pendants in either the side chain or the dihydropyrone ring. Their cytotoxic activity and their interactions with tubulin have been investigated. It has been found that all analogues are cytotoxic towards two either sensitive or resistant tumoral cell lines with similar IC50 values in each case, thus strongly suggesting that, like natural pironetin, they also display a covalent mechanism of action. Their cytotoxicity is, however, lower than that of the parent compound. This indicates that all alkyl pendants are necessary for the full biological activity, with the ethyl group at C-4 seemingly being particularly relevant. Most likely, the alkyl groups cause a restriction in the conformational mobility of the molecule and reduce the number of available conformations. This makes it more probable that the molecule preferentially adopts a shape which fits better into the binding point in α-tubulin

Structural Determinants of the Dictyostatin Chemotype for Tubulin Binding Affinity and Antitumor Activity Against Taxane- and Epothilone-Resistant Cancer Cells

13 p.-5 fig.-2 tab.-1 graph.abst.A combined biochemical, structural, and cell biology characterization of dictyostatin is described, which enables an improved understanding of the structural determinants responsible for the high-affinity binding of this anticancer agent to the taxane site in microtubules (MTs). The study reveals that this macrolide is highly optimized for MT binding and that only a few of the structural modifications featured in a library of synthetic analogues resulted in small gains in binding affinity. The high efficiency of the dictyostatin chemotype in overcoming various kinds of clinically relevant resistance mechanisms highlights its potential for therapeutic development for the treatment of drug-resistant tumors. A structural explanation is advanced to account for the synergy observed between dictyostatin and taxanes on the basis of their differential effects on the MT lattice. The X-ray crystal structure of a tubulin–dictyostatin complex and additional molecular modeling have allowed the rationalization of the structure–activity relationships for a set of synthetic dictyostatin analogues, including the highly active hybrid 12 with discodermolide. Altogether, the work reported here is anticipated to facilitate the improved design and synthesis of more efficacious dictyostatin analogues and hybrids with other MT-stabilizing agents.This work was supported in part by grants BIO2013-42984-R (J.F.D.) and SAF2012-39760-C02-02 (F.G.) from Ministerio de Economía y Competitividad, grant S2010/BMD-2457 BIPEDD2 from Comunidad Autónoma de Madrid (F.G. and J.F.D.), and the Swiss National Science Foundation grants 310030B_138659 and 31003A_166608 (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” and the COST action CM1470. I.P. thanks the EPSRC and AstraZeneca for funding, Dr. John Leonard (AstraZeneca) for useful discussions, Dr. Stuart Mickel (Novartis) for the provision of chemicals, and the EPSRC UK National Mass Spectrometry Facility at Swansea University for mass spectra

Synthesis and structure-activity relationship studies of C(13)-desmethylene-(−)-zampanolide analogs

14 p.-4 fig.-2 tab.We describe the synthesis and biochemical and cellular profiling of five partially reduced or demethylated analogs of the marine macrolide (−)-zampanolide (ZMP). These analogs were derived from 13-desmethylene-(−)-zampanolide (DM-ZMP), which is an equally potent cancer cell growth inhibitor as ZMP. Key steps in the synthesis of all compounds were the formation of the dioxabicyclo[15.3.1]heneicosane core by an intramolecular HWE reaction (67–95 % yield) and a stereoselective aza-aldol reaction with an (S)-BINOL-derived sorbamide transfer complex, to establish the C(20) stereocenter (24–71 % yield). As the sole exception, for the 5-desmethyl macrocycle, ring-closure relied on macrolactonization; however, elaboration of the macrocyclization product into the corresponding zampanolide analog was unsuccessful. All modifications led to reduced cellular activity and lowered microtubule-binding affinity compared to DM-ZMP, albeit to a different extent. For compounds incorporating the reactive enone moiety of ZMP, IC50 values for cancer cell growth inhibition varied between 5 and 133 nM, compared to 1–12 nM for DM-ZMP. Reduction of the enone double bond led to a several hundred-fold loss in growth inhibition. The cellular potency of 2,3-dihydro-13-desmethylene zampanolide, as the most potent analog identified, remained within a ninefold range of that of DM-ZMP.This workwas supported by the Swiss National Science Foundation (KHA,project200021_149253). Institutional support by the ETH Zurich is also gratefully acknowledged(KHA).Funding was also received from Ministerio de Ciencia e Innovación(Spain) (JFD,Project PID2019-104545RB-I00/AEI/10.13039/501100011033),the European Commission-NextGenerations EU(RegulationEU 2020/2094),through CSIC’s Global Health Platform(PTI Salud Global) and Proyecto de Investigación en Neurociencia Fundación Tatiana Pérez de Guzmán el Bueno 2020 (JFD).Peer reviewe

Intracellular Mechanical Drugs Induce Cell-Cycle Altering and Cell Death

Current advances in materials science have demonstrated that extracellular mechanical cues can define cell function and cell fate. However, a fundamental understanding of the manner in which intracellular mechanical cues affect cell mechanics remains elusive. How intracellular mechanical hindrance, reinforcement, and supports interfere with the cell cycle and promote cell death is described here. Reproducible devices with highly controlled size, shape, and with a broad range of stiffness are internalized in HeLa cells. Once inside, they induce characteristic cell-cycle deviations and promote cell death. Device shape and stiffness are the dominant determinants of mechanical impairment. Device structural support to the cell membrane and centering during mitosis maximize their effects, preventing spindle centering, and correct chromosome alignment. Nanodevices reveal that the spindle generates forces larger than 114 nN which overcomes intracellular confinement by relocating the device to a less damaging position. By using intracellular mechanical drugs, this work provides a foundation to defining the role of intracellular constraints on cell function and fate, with relevance to fundamental cell mechanics and nanomedicine.Peer reviewe

Estructura tridimensional de un fragmento de 3Kda del factor derivado de Plaquetas-4 con actividad antiangiogénica e interacción con el factor de crecimiento para fibroblastos

El factor derivado de plaquetas-a es una proteína perteneciente a la familia de las equemoquinas CXC, carentes de la secuencia ELR� . Esta proteína se une al factor de crecimiemo para Iibroblastos inhibiend o su actividad mitogénica. Así mismo se ha demostrado que inhibe el crecimiento tumoral a través de un mecanismo antiangiogénico en el que debe jugar un pa pel importante el bloqueo de la actividad del factor de crecimiento pura fibroblasros. El factor derivado de plaquetas-4 posee una estructura tridimensional complicada, por lo que es poco probable que puedan obtenerse cantidades industriales del producto, con calidad farmacológica, tanto por síntesis química como mediante ingeniería genética. Se ha demostrado que un fragmento equivalente a los veinticuatro residuos últimos del extremo carboxilo terminal de la proteína completa posee también actividad antíangiogñenica in vitro e in vivo, forma un complejo con estequiomelría 1:1 con el factor de crecimiento para fibroblastos y hace que disminuya su afinidad por su receptor de alta afinidad de la superficie celular. Este fragmento posee numerosas características para constituir un buen sustituto terapéutico de la proteína completa. La estructura tridimensional en solución de este fragmento es relativamente sencilla y notablemente estable. Consta de dos subdominios helicoidales que parece que es necesario que se mantengan unidos, tanto para la inhibición y la formación del complejo, como para que uno de los subdominios siga siendo helicoidal. La caracterización estructural a alta resolución de este fragmento puede contribuir a entender las bases estructurales de su actividad antiangiogéníca y, por tanto, podría permitir mejorar sus propiedad es fa rma co lógica s. A su vez puede ayudar a un a mejor comprensión de las bases estructurales de la fisiología del factor derivado de plaquetas-4