La estructura del dominio de Colchicina como base para el diseño y síntesis de compuestos con propiedades antimitóticas y antivasculares

El desarrollo de redes de vasos sanguíneos que puedan aportar oxígeno y nutrienteses una de las características esenciales para el crecimiento de tumores sólidos. Dadala importancia de un adecuado flujo sanguíneo en el entorno tumoral para la expansióncelular y la metástasis tumoral, la búsqueda de compuestos capaces de inhibir dichavascularización centra grandes expectativas en el estudio actual de nuevas estrategiasantineoplásicas. Dentro de la terapia antivascular, los agentes interruptores de lavascularización tumoral (VDAs) ocupan un papel primordial debido a su mecanismo deacción, complementario a las terapias ya existentes. Los VDAs actúan directa yselectivamente sobre el endotelio tumoral, induciendo cambios morfológicos yfuncionales que provocan el descenso rápido y dramático del flujo sanguíneo en el senodel tumor, el cual acaba necrosándose. Dentro de los VDAs, los compuestos másestudiados se encuadran dentro del grupo de ligandos del sitio de colchicina en tubulina,que inhiben la polimerización de los microtúbulos celulares, actuando así como agentesantimitóticos.La acción antitumoral dual de los VDAs basados en colchicina (antivascular yantimitótico) explica el enorme potencial terapéutico de esta clase de compuestos. Entreestos VDAs cabe destacar la combretastatina A4 (CA-4), compuesto de referencia delque además existen varios derivados que actualmente se encuentran en fases clínicasavanzadas. Ahora bien, estos candidatos, junto a otros VDAs que también están siendoobjeto de investigación clínica, todavía presentan notables limitaciones que obstaculizanel desarrollo farmacéutico de los mismos, tales como la baja estabilidad química y/o lalimitada solubilidad. Por ello, el objetivo central de esta Tesis ha consistido en labúsqueda de nuevos VDAs ligandos del sitio de colchicina de mejor perfil farmacológico,que solventen los problemas de los compuestos desarrollados hasta el momento..

Protein-ligand complex for structure-based design: impact on the affinity and antitumor activity of new tubulin ligands

Resumen del trabajo presentado en el XVIII Congreso de la Sociedad Española de Química Terapéutica, celebrado en Salamanca (España), del 23 al 26 de enero de 2018Microtubules, made of ¿ß¿tubulin heterodimers, are the key components of the cytoskeleton and play a crucial role in many cellular processes, such as cell motility, morphogenesis and mitosis.[1] Interference with microtubule dynamics induces cell cycle arrest during mitosis and triggers cell death. Compounds that interact with tubulin, especially those binding at the colchicine domain, have been deeply investigated as anticancer drugs due to their dual mechanism of action as antimitotics and as vascular disrupting agents.[2,3] Our research group has recently described a new family of colchicine¿domain binders, based on a cyclohexanedione skeleton, with potent antiproliferative activity against tumor and endothelial cells.[4] Moreover, to gain insight into the binding mode of these cyclohexanediones, we have determined the crystal structure of ¿ß¿tubulin in complex with our hit compound (TUB075). Based on this detailed information and by applying the affinity maps program cGRILL, a structurebased synthesis of new cyclohexanedione derivatives has been accomplished with the objective of improving their affinity for tubulin and their antitumor activity. Following this approach, we have obtained new compounds with potent antiproliferative activity against tumor and endothelial cells (IC50=8¿31 nM) and with the highest Kb value reported for compounds binding at the colchicine site in tubulin. Additional studies have shown that they arrest cell cycle at G2/M and disrupt a network of endothelial cells. Moreover they keep antiproliferative activity against cell lines overexpressing P¿gp, further supporting the potential of these compounds.The financial support of the Spanish MINECO (SAF2012‐39760‐C02‐01 and SAF 2015‐64629‐C2‐1‐R), Comunidad de Madrid (BIPEDD2; ref P2010/BMD‐2457) and the COST action CM1407 (to M J. P.P., S.L., M.O.S. and J.F.D.) is sincerely acknowledge

Antivascular and antitumor properties of the tubulin-binding chalcone TUB091

We investigated the microtubule-destabilizing, vascular-targeting, anti-tumor and anti-metastatic activities of a new series of chalcones, whose prototype compound is (E)-3-(3’’-amino-4’’-methoxyphenyl)-1-(5’-methoxy-3’,4’-methylendioxyphenyl)- 2-methylprop-2-en-1-one (TUB091). X-ray crystallography showed that these chalcones bind to the colchicine site of tubulin and therefore prevent the curved-tostraight structural transition of tubulin, which is required for microtubule formation. Accordingly, TUB091 inhibited cancer and endothelial cell growth, induced G2/M phase arrest and apoptosis at 1-10 nM. In addition, TUB091 displayed vascular disrupting effects in vitro and in the chicken chorioallantoic membrane (CAM) assay at low nanomolar concentrations. A water-soluble L-Lys-L-Pro derivative of TUB091 (i.e. TUB099) showed potent antitumor activity in melanoma and breast cancer xenograft models by causing rapid intratumoral vascular shutdown and massive tumor necrosis. TUB099 also displayed anti-metastatic activity similar to that of combretastatin A4-phosphate. Our data indicate that this novel class of chalcones represents interesting lead molecules for the design of vascular disrupting agents (VDAs). Moreover, we provide evidence that our prodrug approach may be valuable for the development of anti-cancer drugs.M-DC thanks the Fondo Social Europeo (FSE) and the JAE Predoc Programme for a predoctoral fellowship. This work has received the Ramón Madroñero award for young researchers (to M-DC and OB) in the XVII call www.impactjournals.com/oncotarget 17 Oncotarget sponsored by the Spanish Society of Medicinal Chemistry (SEQT). This project has been supported by the Spanish Ministerio de Economia y Competitividad (SAF2012- 39760-C02-01 to M-JC, M-JP-P, SV and E-MP; and BIO2013-42984-R to JFD), Comunidad de Madrid (BIPEDD2; ref. P2010/BMD-2457 to M-JC and J-FD), the Swiss National Science Foundation (310030B_138659 and 31003A_166608; to MOS). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery” and COST action CM1470.Peer reviewe

Blocking blood flow to solid tumors by destabilizing tubulin: An approach to targeting tumor growth

The unique characteristics of the tumor vasculature offer the possibility to selectively target tumor growth and vascularization using tubulin-destabilizing agents. Evidence accumulated with combretastatin A-4 (CA-4) and its prodrug CA-4P support the therapeutic value of compounds sharing this mechanism of action. However, the chemical instability and poor solubility of CA-4 demand alternative compounds that are able to surmount these limitations. This Perspective illustrates the different classes of compounds that behave similar to CA-4, analyzes their binding mode to αβ-tubulin according to recently available structural complexes, and includes described approaches to improve their delivery. In addition, dissecting the mechanism of action of CA-4 and analogues allows a closer insight into the advantages and drawbacks associated with these tubulin-destabilizing agents that behave as vascular disrupting agents (VDAs).status: publishe

Blocking Blood Flow to Solid Tumors by Destabilizing Tubulin : An Approach to Targeting Tumor Growth (Perspective)

The unique characteristics of the tumor vasculature offer the possibility to selectively target tumor growth and vascularization using tubulin-destabilizing agents. Evidence accumulated with combretastatin A-4 (CA-4) and its prodrug CA-4P support the therapeutic value of compounds sharing this mechanism of action. However, the chemical instability and poor solubility of CA-4 demand alternative compounds that are able to surmount these limitations. This Perspective illustrates the different classes of compounds that behave similar to CA-4, analyzes their binding mode to αβ- tubulin according to recently available structural complexes, and includes described approaches to improve their delivery. In addition, dissecting the mechanism of action of CA-4 and analogues allows a closer insight into the advantages and drawbacks associated with these tubulin-destabilizing agents that behave as vascular disrupting agents (VDAs).Due to the amount of original research articles and reviews on this subject, we were unable to cite all of them; any omissions were unintentional. Author’s research in this subject has been financed by the Spanish Ministerio de Economıá y Competitividad (SAF2012-39760-C02-01 and SAF2015-64629-C2- 1-R (MINECO-FEDER) to M.-J.P.-P. and E.-M.P.) and Comunidad de Madrid (BIPEDD2; ref P2010/BMD-2457). S.L. and M.-J.P.-P. acknowledge networking contribution by COST Action CM1407 “Challenging organic synthesis inspired by nature-from natural products chemistry to drug discovery”. M.-D.C. thanks the Fondo Social Europeo (FSE) and the JAE Predoc Programme for a predoctoral fellowship.Peer reviewe

Conformational mimetics of the α-methyl chalcone TUB091 binding tubulin: Design, synthesis and antiproliferative activity

Based on the conformation of the α-methyl chalcone TUB091 in its complex with tubulin, a series of conformational mimetics have been designed and synthesized where the methyl group of the chalcone has been fused to phenyl ring B resulting in 1,2,3,4-tetrahydronaphthalen-2-yl aryl ketones. Among the synthesized compounds, the 5-amino-6-methoxy derivative, with a similar substitution pattern to that of TUB091, showed antiproliferative activity around 20 nM against tumor and endothelial cells. Tubulin binding experiments confirmed its binding to tubulin at the colchicine site with a Kb of 2.4 × 10 M resulting in the inhibition of the in vitro assembly of purified tubulin. Moreover, based on the recently reported complex of combretastatin A4 (CA4) with tubulin, a comparative analysis of the binding mode of CA4 and the α-methyl chalcone to tubulin has been performed.This project has been supported by MINECO (SAF2012-39760- C02-01) and MINECO/FEDER (SAF2015-64629-C2-1-R), and to M.- J.P.-P., E.-M.P. E. Q. and M. J. C. and MINECO/FEDER BFU2016- 75319-R to J.F.D. 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-J. P.-P. S.L. and J.-F.D.) We also would like to acknowledge Eef Meyen and Lizette Van Berckelaer for excellent technical assistance.Peer Reviewe

Antiviral activity of [1,2,3]triazolo[4,5- d ]pyrimidin-7(6 H )-ones against chikungunya virus targeting the viral capping nsP1

International audienceChikungunya virus (CHIKV) is a re-emerging alphavirus transmitted to humans by Aedes mosquitoes. Since 2005, CHIKV has been spreading worldwide resulting in epidemics in Africa, the Indian Ocean islands, Asia and more recently in the Americas. CHIKV is thus considered as a global health concern. There is no specific vaccine or drug available for the treatment of this incapacitating viral infection. We previously identified 3-aryl-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones as selective inhibitors of CHIKV replication and proposed the viral capping enzyme nsP1 as a target. This work describes the synthesis of novel series of related compounds carrying at the aryl moiety a methylketone and related oximes combined with an ethyl or an ethyl-mimic at 5-position of the triazolopyrimidinone. These compounds have shown antiviral activity against different CHIKV isolates in the very low μM range based on both virus yield reduction and virus-induced cell-killing inhibition assays. Moreover, these antivirals inhibit the in vitro guanylylation of alphavirus nsP1, as determined by Western blot using an anti-cap antibody. Thus, the data obtained seem to indicate that the anti-CHIKV activity might be related to the inhibition of this crucial step in the viral RNA capping machinery

High-affinity ligands of the colchicine domain in tubulin based on a structure-guided design

Microtubule-targeting agents that bind at the colchicine-site of tubulin are of particular interest in antitumoral therapy due to their dual mechanism of action as antimitotics and vascular disrupting agents. Cyclohexanediones derivatives have been described as a new family of colchicine-domain binders with an association constant to tubulin similar to that of colchicine. Here, the highresolution structures of tubulin in complex with cyclohexanediones TUB015 and TUB075 were solved by X-ray crystallography. A detailed analysis of the tubulin-TUB075 interaction by means of computational affinity maps allowed the identification of two additional regions at the binding site that were addressed with the design and synthesis of a new series of cyclohexanediones with a distal 2-substituted benzofurane. These new compounds showed potent antiproliferative activity with IC50 values in the nM range, arrested cell cycle progression at the G2/M phase and induced apoptosis at sub μM concentrations. Moreover, they caused the destruction of a preformed vascular network in vitro and inhibited the migration of endothelial cells at non-toxic concentrations. Finally, these compounds displayed high affinity for tubulin as substantiated by a Kb value of 2.87 × 108 M−1 which, to the best of our knowledge, represents the highest binding constant measured to date for a colchicine-domain ligand.This project has been supported by the Spanish Ministerio de Economía y Competitividad (SAF2015–64629- C2-1-R to EMP, MJPP. and MJC and SAF2015-64629-C2-2 to F.G.), by the Swiss National Science Foundation (31003A_166608, to MOS) and COST action CM1407 (to MJPP, SL, AP, MS, and JFD). We acknowledge Lizette Van Berckelaer, Eef Meyen and Sam Noppen for excellent technical assistance. We thank V. Olieric and M. Wang for excellent technical assistance with the collection of X-ray data at beamline X06SA of the Swiss Light Source (Paul Scherrer Institut, Villigen PSI, Switzerland). We thank José Fernando Escolar for his help with electron microscopy. We thank ganadería Fernando Díaz for calf brains for tubulin purification. This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) (JFD.) JFD. is a member of the CIB Intramural Program “Molecular Machines for Better Life” (MACBET). We also thank SEQT for the “Ramón Madroñero Award” to O.B.Peer Reviewe

Antiviral activity of [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones against chikungunya virus targeting the viral capping nsP1

Chikungunya virus (CHIKV) is a re-emerging alphavirus transmitted to humans by Aedes mosquitoes. Since 2005, CHIKV has been spreading worldwide resulting in epidemics in Africa, the Indian Ocean islands, Asia and more recently in the Americas. CHIKV is thus considered as a global health concern. There is no specific vaccine or drug available for the treatment of this incapacitating viral infection. We previously identified 3-aryl-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones as selective inhibitors of CHIKV replication and proposed the viral capping enzyme nsP1 as a target. This work describes the synthesis of novel series of related compounds carrying at the aryl moiety a methylketone and related oximes combined with an ethyl or an ethyl-mimic at 5-position of the triazolopyrimidinone. These compounds have shown antiviral activity against different CHIKV isolates in the very low μM range based on both virus yield reduction and virus-induced cell-killing inhibition assays. Moreover, these antivirals inhibit the in vitro guanylylation of alphavirus nsP1, as determined by Western blot using an anti-cap antibody. Thus, the data obtained seem to indicate that the anti-CHIKV activity might be related to the inhibition of this crucial step in the viral RNA capping machinery.A. G. has received a JAE-predoctoral fellowship financed by the CSIC and the FSE (Fondo Social Europeo).We thank Caroline Collard and Kim Donckers for their excellent technical assistance in the acquisition of the antiviral data. This work has been supported by grants of the MINECO/FEDER (SAF2015-64629-C2-1-R), BIPEDD-2- CM (S2010/BMD-2457) and by EU F7 projects SILVER and EUVIRNA (grant numbers 260644 and 264286, respectively).Peer Reviewe

Antiviral activity of [1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones against chikungunya virus targeting the viral capping nsP1

Chikungunya virus (CHIKV) is a re-emerging alphavirus transmitted to humans by Aedes mosquitoes. Since 2005, CHIKV has been spreading worldwide resulting in epidemics in Africa, the Indian Ocean islands, Asia and more recently in the Americas. CHIKV is thus considered as a global health concern. There is no specific vaccine or drug available for the treatment of this incapacitating viral infection. We previously identified 3-aryl-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones as selective inhibitors of CHIKV replication and proposed the viral capping enzyme nsP1 as a target. This work describes the synthesis of novel series of related compounds carrying at the aryl moiety a methylketone and related oximes combined with an ethyl or an ethyl-mimic at 5-position of the triazolopyrimidinone. These compounds have shown antiviral activity against different CHIKV isolates in the very low μM range based on both virus yield reduction and virus-induced cell-killing inhibition assays. Moreover, these antivirals inhibit the in vitro guanylyl-transfer activity of alphavirus nsP1, as determined by Western blot using an anti-cap antibody. Thus, the data obtained seem to indicate that the anti-CHIKV activity might be related to the inhibition of this crucial step in the viral RNA capping machinery.status: publishe