Structural Basis of Colchicine-Site targeting Acylhydrazones active against Multidrug-Resistant Acute Lymphoblastic Leukemia

Nathália Moreno Cury; Tobias Mühlethaler; Angelo Brunelli Albertoni Laranjeira; Rafael Renatino Canevarolo; Priscila Pini Zenatti; Daniel Lucena-Agell; Isabel Barasoain; Chunhua Song; Dongxiao Sun; Sinisa Dovat; Rosendo Augusto Yunes; Andrea Enrico Prota; Michel Olivier Steinmetz; José Fernando Díaz; José Andrés Yunes

doi:10.1016/j.isci.2019.10.003

Structural Basis of Colchicine-Site targeting Acylhydrazones active against Multidrug-Resistant Acute Lymphoblastic Leukemia

Nathália Moreno Cury, Tobias Mühlethaler, Angelo Brunelli Albertoni Laranjeira, Rafael Renatino Canevarolo, Priscila Pini Zenatti, Daniel Lucena-Agell, Isabel Barasoain, Chunhua Song, Dongxiao Sun, Sinisa Dovat, Rosendo Augusto Yunes, Andrea Enrico Prota, Michel Olivier Steinmetz, José Fernando Díaz, José Andrés Yunes

Research output: Contribution to journal › Article › peer-review

4 Scopus citations

Abstract

Tubulin is one of the best validated anti-cancer targets, but most anti-tubulin agents have unfavorable therapeutic indexes. Here, we characterized the tubulin-binding activity, the mechanism of action, and the in vivo anti-leukemia efficacy of three 3,4,5-trimethoxy-N-acylhydrazones. We show that all compounds target the colchicine-binding site of tubulin and that none is a substrate of ABC transporters. The crystal structure of the tubulin-bound N-(1′-naphthyl)-3,4,5-trimethoxybenzohydrazide (12) revealed steric hindrance on the T7 loop movement of β-tubulin, thereby rendering tubulin assembly incompetent. Using dose escalation and short-term repeated dose studies, we further report that this compound class is well tolerated to >100 mg/kg in mice. We finally observed that intraperitoneally administered compound 12 significantly prolonged the overall survival of mice transplanted with both sensitive and multidrug-resistant acute lymphoblastic leukemia (ALL) cells. Taken together, this work describes promising colchicine-site-targeting tubulin inhibitors featuring favorable therapeutic effects against ALL and multidrug-resistant cells.

Original language	English (US)
Pages (from-to)	95-109
Number of pages	15
Journal	iScience
Volume	21
DOIs	https://doi.org/10.1016/j.isci.2019.10.003
State	Published - Nov 22 2019

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10.1016/j.isci.2019.10.003

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Cury, N. M., Mühlethaler, T., Laranjeira, A. B. A., Canevarolo, R. R., Zenatti, P. P., Lucena-Agell, D., Barasoain, I., Song, C., Sun, D., Dovat, S., Yunes, R. A., Prota, A. E., Steinmetz, M. O., Díaz, J. F., & Yunes, J. A. (2019). Structural Basis of Colchicine-Site targeting Acylhydrazones active against Multidrug-Resistant Acute Lymphoblastic Leukemia. iScience, 21, 95-109. https://doi.org/10.1016/j.isci.2019.10.003

@article{e1f5d06dafe241b8b4e00d88d896e2a8,

title = "Structural Basis of Colchicine-Site targeting Acylhydrazones active against Multidrug-Resistant Acute Lymphoblastic Leukemia",

abstract = "Tubulin is one of the best validated anti-cancer targets, but most anti-tubulin agents have unfavorable therapeutic indexes. Here, we characterized the tubulin-binding activity, the mechanism of action, and the in vivo anti-leukemia efficacy of three 3,4,5-trimethoxy-N-acylhydrazones. We show that all compounds target the colchicine-binding site of tubulin and that none is a substrate of ABC transporters. The crystal structure of the tubulin-bound N-(1′-naphthyl)-3,4,5-trimethoxybenzohydrazide (12) revealed steric hindrance on the T7 loop movement of β-tubulin, thereby rendering tubulin assembly incompetent. Using dose escalation and short-term repeated dose studies, we further report that this compound class is well tolerated to >100 mg/kg in mice. We finally observed that intraperitoneally administered compound 12 significantly prolonged the overall survival of mice transplanted with both sensitive and multidrug-resistant acute lymphoblastic leukemia (ALL) cells. Taken together, this work describes promising colchicine-site-targeting tubulin inhibitors featuring favorable therapeutic effects against ALL and multidrug-resistant cells.",

author = "Cury, {Nath{\'a}lia Moreno} and Tobias M{\"u}hlethaler and Laranjeira, {Angelo Brunelli Albertoni} and Canevarolo, {Rafael Renatino} and Zenatti, {Priscila Pini} and Daniel Lucena-Agell and Isabel Barasoain and Chunhua Song and Dongxiao Sun and Sinisa Dovat and Yunes, {Rosendo Augusto} and Prota, {Andrea Enrico} and Steinmetz, {Michel Olivier} and D{\'i}az, {Jos{\'e} Fernando} and Yunes, {Jos{\'e} Andr{\'e}s}",

note = "Funding Information: We thank Ganader{\'i}a Fernando D{\'i}az for calf brains for tubulin purification. The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery.” J.F.D. is a member of the CIB Intramural Program “Molecular Machines for Better Life” (MACBET). N.M.C. was supported by a fellowship from Funda{\c c}{\~a}o de Amparo {\`a} Pesquisa do Estado de S{\~a}o Paulo (FAPESP, 14/08247-8, and 17/14737-6). J.A.Y. received a Productivity fellowship from the Brazilian National Counsel of Technological and Scientific Development (CNPq 305896/2013-0 and 301596/2017-4). This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) (J.F.D.) from Ministerio de Econom{\'i}a y Competitividad. The crystal structure work was supported by grants from the Swiss National Science Foundation (31003A_166608, to M.O.S.) and by the COST action CM1407 (to M.O.S.). Part of the in vivo work was supported by R01CA209829 and R01CA213912, Hyundai Hope On Wheels Scholar Grant, Bear Necessities Pediatric Cancer Foundation, Alex's Lemonade Stand Foundation, the Four Diamonds Fund of the Pennsylvania State University College of Medicine, and the John Wawrynovic Leukemia Research Scholar Endowment (to S.D.). N.M.C. performed the majority of the experiments, contributed to the analyses and interpretation of the results, and drafted the manuscript. T.M. performed the crystal structure work and wrote the manuscript. A.B.A.L. performed the in vivo anti-leukemia experiment. R.R.C. supervised the project and contributed to the interpretation of the results. P.P.Z. performed the in vivo toxicity experiment. D.L.-A. contributed to the standardization and interpretation of biochemical results. I.B. contributed to the standardization and analyses of immunofluorescence assay. C.S. and S.D. supervised the in vivo assays with NALM6 and N6/ADR cell lines. D.S. performed mass spectrometry analyzes. R.A.Y. designed and provided the compounds used in the present work. A.E.P. and M.O.S. supervised the crystal structure work and critically reviewed the manuscript. J.F.D. supervised the biochemical work, contributed to the interpretation of the results, and critically reviewed the manuscript. J.A.Y. was responsible for the conception and design of study. He supervised the entire project and wrote the manuscript. Compounds 7, 12, and 21 are patented under WO 2013/075199 A1. Funding Information: N.M.C. was supported by a fellowship from Funda{\c c}{\~a}o de Amparo {\`a} Pesquisa do Estado de S{\~a}o Paulo (FAPESP, 14/08247-8 , and 17/14737-6 ). J.A.Y. received a Productivity fellowship from the Brazilian National Counsel of Technological and Scientific Development ( CNPq 305896/2013-0 and 301596/2017-4 ). This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) (J.F.D.) from Ministerio de Econom{\'i}a y Competitividad . The crystal structure work was supported by grants from the Swiss National Science Foundation ( 31003A_166608 , to M.O.S.) and by the COST action CM1407 (to M.O.S.). Part of the in vivo work was supported by R01CA209829 and R01CA213912 , Hyundai Hope On Wheels Scholar Grant, Bear Necessities Pediatric Cancer Foundation , Alex{\textquoteright}s Lemonade Stand Foundation , the Four Diamonds Fund of the Pennsylvania State University College of Medicine , and the John Wawrynovic Leukemia Research Scholar Endowment (to S.D.). Publisher Copyright: {\textcopyright} 2019 The Authors",

year = "2019",

month = nov,

day = "22",

doi = "10.1016/j.isci.2019.10.003",

language = "English (US)",

volume = "21",

pages = "95--109",

journal = "iScience",

issn = "2589-0042",

publisher = "Elsevier Inc.",

}

Cury, NM, Mühlethaler, T, Laranjeira, ABA, Canevarolo, RR, Zenatti, PP, Lucena-Agell, D, Barasoain, I, Song, C, Sun, D , Dovat, S, Yunes, RA, Prota, AE, Steinmetz, MO, Díaz, JF & Yunes, JA 2019, 'Structural Basis of Colchicine-Site targeting Acylhydrazones active against Multidrug-Resistant Acute Lymphoblastic Leukemia', iScience, vol. 21, pp. 95-109. https://doi.org/10.1016/j.isci.2019.10.003

TY - JOUR

T1 - Structural Basis of Colchicine-Site targeting Acylhydrazones active against Multidrug-Resistant Acute Lymphoblastic Leukemia

AU - Cury, Nathália Moreno

AU - Mühlethaler, Tobias

AU - Laranjeira, Angelo Brunelli Albertoni

AU - Canevarolo, Rafael Renatino

AU - Zenatti, Priscila Pini

AU - Lucena-Agell, Daniel

AU - Barasoain, Isabel

AU - Song, Chunhua

AU - Sun, Dongxiao

AU - Dovat, Sinisa

AU - Yunes, Rosendo Augusto

AU - Prota, Andrea Enrico

AU - Steinmetz, Michel Olivier

AU - Díaz, José Fernando

AU - Yunes, José Andrés

N1 - Funding Information: We thank Ganadería Fernando Díaz for calf brains for tubulin purification. The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery.” J.F.D. is a member of the CIB Intramural Program “Molecular Machines for Better Life” (MACBET). N.M.C. was supported by a fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 14/08247-8, and 17/14737-6). J.A.Y. received a Productivity fellowship from the Brazilian National Counsel of Technological and Scientific Development (CNPq 305896/2013-0 and 301596/2017-4). This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) (J.F.D.) from Ministerio de Economía y Competitividad. The crystal structure work was supported by grants from the Swiss National Science Foundation (31003A_166608, to M.O.S.) and by the COST action CM1407 (to M.O.S.). Part of the in vivo work was supported by R01CA209829 and R01CA213912, Hyundai Hope On Wheels Scholar Grant, Bear Necessities Pediatric Cancer Foundation, Alex's Lemonade Stand Foundation, the Four Diamonds Fund of the Pennsylvania State University College of Medicine, and the John Wawrynovic Leukemia Research Scholar Endowment (to S.D.). N.M.C. performed the majority of the experiments, contributed to the analyses and interpretation of the results, and drafted the manuscript. T.M. performed the crystal structure work and wrote the manuscript. A.B.A.L. performed the in vivo anti-leukemia experiment. R.R.C. supervised the project and contributed to the interpretation of the results. P.P.Z. performed the in vivo toxicity experiment. D.L.-A. contributed to the standardization and interpretation of biochemical results. I.B. contributed to the standardization and analyses of immunofluorescence assay. C.S. and S.D. supervised the in vivo assays with NALM6 and N6/ADR cell lines. D.S. performed mass spectrometry analyzes. R.A.Y. designed and provided the compounds used in the present work. A.E.P. and M.O.S. supervised the crystal structure work and critically reviewed the manuscript. J.F.D. supervised the biochemical work, contributed to the interpretation of the results, and critically reviewed the manuscript. J.A.Y. was responsible for the conception and design of study. He supervised the entire project and wrote the manuscript. Compounds 7, 12, and 21 are patented under WO 2013/075199 A1. Funding Information: N.M.C. was supported by a fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 14/08247-8 , and 17/14737-6 ). J.A.Y. received a Productivity fellowship from the Brazilian National Counsel of Technological and Scientific Development ( CNPq 305896/2013-0 and 301596/2017-4 ). This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) (J.F.D.) from Ministerio de Economía y Competitividad . The crystal structure work was supported by grants from the Swiss National Science Foundation ( 31003A_166608 , to M.O.S.) and by the COST action CM1407 (to M.O.S.). Part of the in vivo work was supported by R01CA209829 and R01CA213912 , Hyundai Hope On Wheels Scholar Grant, Bear Necessities Pediatric Cancer Foundation , Alex’s Lemonade Stand Foundation , the Four Diamonds Fund of the Pennsylvania State University College of Medicine , and the John Wawrynovic Leukemia Research Scholar Endowment (to S.D.). Publisher Copyright: © 2019 The Authors

PY - 2019/11/22

Y1 - 2019/11/22

N2 - Tubulin is one of the best validated anti-cancer targets, but most anti-tubulin agents have unfavorable therapeutic indexes. Here, we characterized the tubulin-binding activity, the mechanism of action, and the in vivo anti-leukemia efficacy of three 3,4,5-trimethoxy-N-acylhydrazones. We show that all compounds target the colchicine-binding site of tubulin and that none is a substrate of ABC transporters. The crystal structure of the tubulin-bound N-(1′-naphthyl)-3,4,5-trimethoxybenzohydrazide (12) revealed steric hindrance on the T7 loop movement of β-tubulin, thereby rendering tubulin assembly incompetent. Using dose escalation and short-term repeated dose studies, we further report that this compound class is well tolerated to >100 mg/kg in mice. We finally observed that intraperitoneally administered compound 12 significantly prolonged the overall survival of mice transplanted with both sensitive and multidrug-resistant acute lymphoblastic leukemia (ALL) cells. Taken together, this work describes promising colchicine-site-targeting tubulin inhibitors featuring favorable therapeutic effects against ALL and multidrug-resistant cells.

AB - Tubulin is one of the best validated anti-cancer targets, but most anti-tubulin agents have unfavorable therapeutic indexes. Here, we characterized the tubulin-binding activity, the mechanism of action, and the in vivo anti-leukemia efficacy of three 3,4,5-trimethoxy-N-acylhydrazones. We show that all compounds target the colchicine-binding site of tubulin and that none is a substrate of ABC transporters. The crystal structure of the tubulin-bound N-(1′-naphthyl)-3,4,5-trimethoxybenzohydrazide (12) revealed steric hindrance on the T7 loop movement of β-tubulin, thereby rendering tubulin assembly incompetent. Using dose escalation and short-term repeated dose studies, we further report that this compound class is well tolerated to >100 mg/kg in mice. We finally observed that intraperitoneally administered compound 12 significantly prolonged the overall survival of mice transplanted with both sensitive and multidrug-resistant acute lymphoblastic leukemia (ALL) cells. Taken together, this work describes promising colchicine-site-targeting tubulin inhibitors featuring favorable therapeutic effects against ALL and multidrug-resistant cells.

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UR - http://www.scopus.com/inward/citedby.url?scp=85073606426&partnerID=8YFLogxK

U2 - 10.1016/j.isci.2019.10.003

DO - 10.1016/j.isci.2019.10.003

M3 - Article

C2 - 31655259

AN - SCOPUS:85073606426

SN - 2589-0042

VL - 21

SP - 95

EP - 109

JO - iScience

JF - iScience

ER -

Structural Basis of Colchicine-Site targeting Acylhydrazones active against Multidrug-Resistant Acute Lymphoblastic Leukemia

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