A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer

Structural basis of colchicine-site targeting acylhydrazones active against multidrug-resistant acute lymphoblastic leukemia

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 cell2195109CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP305896/2013-0; 301596/2017-414/08247-8; 17/14737-6We 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.

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

Ultrafast photochemistry produces superbright short-wave infrared dots for low-dose in vivo imaging

12 p.-5 fig.Optical probes operating in the second near-infrared window (NIR-II, 1,000-1,700 nm), where tissues are highly transparent, have expanded the applicability of fluorescence in the biomedical field. NIR-II fluorescence enables deep-tissue imaging with micrometric resolution in animal models, but is limited by the low brightness of NIR-II probes, which prevents imaging at low excitation intensities and fluorophore concentrations. Here, we present a new generation of probes (Ag2S superdots) derived from chemically synthesized Ag2S dots, on which a protective shell is grown by femtosecond laser irradiation. This shell reduces the structural defects, causing an 80-fold enhancement of the quantum yield. PEGylated Ag2S superdots enable deep-tissue in vivo imaging at low excitation intensities (<10 mW cm-2) and doses (<0.5 mg kg-1), emerging as unrivaled contrast agents for NIR-II preclinical bioimaging. These results establish an approach for developing superbright NIR-II contrast agents based on the synergy between chemical synthesis and ultrafast laser processing.Authors thank Dr A. Benayas (CICECO, U. Aveiro, Portugal), Prof G. Lifante and Prof J. García Sole (UAM) for helpful discussions. This work has been founded by Ministerio de Economı́a y Competitividad-MINECO (MAT2017-83111R and MAT2016-75362-C3-1-R) and the Comunidad de Madrid (B2017/BMD-3867 RENIM-CM) co-financed by European Structural and Investment Fund. D.M.-G. thanks UCM-Santander for a predoctoral contract (CT17/17-CT18/17). We thank the staff at the ICTS-National Centre for Electron Microscopy at the UCM for the help in the electron microscopy studies and C.M. at the beamline BL22-CLAESS of the Spanish synchrotron ALBA for his help in the XANES experiments. We also thank J.G.I at the Ultrafast Laser Laboratory at UCM for his help and fruitful discussion. Y.S. acknowledges the support from the China Scholarship Council (CSC File No. 201806870023). Additional funding was provided by the European Commission Horizon 2020 project NanoTBTech, the Fundación para la Investigación Biomédica del Hospital Universitario Ramón y Cajal project IMP18_38 (2018/0265). Ajoy K. Kar and Mark D. Mackenzie acknowledge support from the UK Engineering and Physical Sciences Research Council (Project CHAMP, EP/M015130/1). C. Jacinto thanks the financial support of the Brazilian agencies: CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) through the grants: Projeto Universal Nr. 431736/2018-9 and Scholarship in Research Productivity 1C under the Nr. 304967/20181; FINEP (Financiadora de Estudos e Projetos) through the grants INFRAPESQ-11 and INFRAPESQ-12; FAPEAL (Fundação de Amparo à Pesquisa do Estado de Alagoas) grant Nr. 1209/2016. H. D. A. Santos was supported by a graduate studentship from CNPq and by a sandwich doctoral program (PDSE-CAPES) developed at Universidad Autonoma de Madrid, Spain, Project Nr. 88881/2016-01.Peer reviewe

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

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).Peer reviewe

Maytansinol functionalization:towards useful probes for studying microtubule dynamics

11 p.-7 fig.-4 tab. 7 schem.Maytansinoids are a successful class of natural and semisynthetic tubulin binders, known for their potent cytotoxic activity. Their wider application as cytotoxins and chemical probes to study tubulin dynamics has been held back by the complexity of natural product chemistry. Here we report the synthesis of long-chain derivatives and maytansinoid conjugates. We confirmed that bulky substituents do not impact their high activity or the scaffold's binding mode. These encouraging results open new avenues for the design of new maytansine-based probes.This work was supported by the H2020-MSCA-ITN-2019 (860070 TUBINTRAIN), Ministerio de Ciencia e Innovación PID2019-104545RB−I00 (JFD), Proyecto de Investigación en Neurociencia Fundación Tatiana Pérez de Guzmán el Bueno 2020, and by the European Union NextGenerationEU (J.F.D.). Open Access Funding provided by $INSTITUTION within the CRUI-CARE Agreement.Peer reviewe

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

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.).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.)

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

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.N