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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.

41 p.-8 fig.-2 tab. Field, Jessica J. et al., The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 105 M-1 in the case of unmodified tubulin to 1.4 ± 0.3 × 105 M-1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin., This work was supported in part by grants BFU2016-75319-R (AEI/FEDER, UE) from Ministerio de Economia y Competitividad and the Cancer Society of New Zealand, and the Wellington Medical Research Foundation (JJF & JHM). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery”. The CNIC is supported by the Ministerio de Ciencia e Innovación and the Fundación Pro CNIC.

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).

Cyclohexanedione derivatives represent a new family of colchicine-site binders that were identified through a ligand-based virtual screening approach. Structural modifications have now been performed at both distal sites of our identified hit [2-(1-((2-methoxyphenyl)amino)ethylidene)-5-phenylcyclohexane-1,3-dione (4)] in order to improve tubulin binding affinity, anti-proliferative activity and/or aqueous solubility. The results obtained indicate that the 2-methoxyphenyl ring, the fragment located closer to the αβ-tubulin interface according to docking studies, is the one that allows structural variation in order to improve the K value against tubulin (as in compound 20a with a K = 1.3 × 10 M, analogous to colchicine) or to improve aqueous solubility, as in compound 22c, being more than 10 times more soluble than the previous hit 4., M.-D. C. thanks the Fondo Social Europeo (FSE) and the JAE Predoc Programme for a predoctoral fellowship. This project has been supported by the Spanish Ministerio de Econom´ıa y Competitividad (SAF2012-39760-C02-01 to E.-M. P, M.-J. P.-P. and M.-J. C and BIO2013-42984-R to J. F. D.), and Comunidad de Madrid (BIPEDD2; ref. P2010/BMD-2457 to M.-J. C and J. F. D.). J. F. D. acknowledges networking contribution by the COST Action CM1407 “Challenging organic synthesis inspired by nature-from natural products chemistry to drug discovery” and the COST action CM1470. We also wish to thank Eef Meyen and Lizette van Berckelaer for excellent technical assistance

4 p.-1 fig.-1 tab.-6 schem., To probe the exact role of the oxetane D ring in both tubulin binding and cytotoxicity of taxanes, novel D-seco taxanes bearing a C4 ether substituent have been prepared from paclitaxel 1a. Among them, 20-hydroxymethyl-4-allyloxy D-seco taxane 5e is the most active in both tubulin and cytotoxicity assays. It is only slightly less potent than 1a on tubulin polymerization promotion in vitro and the most cytotoxic among all D-seco taxanes known to date. The reason for the loss and restoration of bioactivity for these D-seco taxanes is also discussed with the assistance of NMR and molecular modeling studies. From these results, we draw a conclusion that the intact D ring of taxanes is not strictly necessary for their binding to tubulin and cytotoxic effects., S.R.W. and W.S.F. thank the National Natural Science Foundation of China (Grant Nos. 30930108 and 81202434)for financial support. F.G. is grateful to the Spanish Ministerio de Economıa y Competitividad (SAF2012-39760-C02-02) and Comunidad Autónoma de Madrid (S2010-BMD-2457).

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).

Vascular disrupting agents (VDAs) constitute an innovative anticancer therapy that targets the tumor endothelium, leading to tumor necrosis. Our approach for the identification of new VDAs has relied on a ligand 3-D shape similarity virtual screening (VS) approach using the ROCS program as the VS tool and as query colchicine and TN-16, which both bind the α,β-tubulin dimer. One of the hits identified, using TN-16 as query, has been explored by the synthesis of its structural analogues, leading to 2-(1-((2-methoxyphenyl) amino)ethylidene)-5-phenylcyclohexane-1,3-dione (compound 16c) with an IC 50 = 0.09 ± 0.01 μM in HMEC-1 and BAEC, being 100-fold more potent than the initial hit. Compound 16c caused cell cycle arrest in the G2/M phase and interacted with the colchicine-binding site in tubulin, as confirmed by a competition assay with N,N′-ethylenebis(iodoacetamide) and by fluorescence spectroscopy. Moreover, 16c destroyed an established endothelial tubular network at 1 μM and inhibited the migration and invasion of human breast carcinoma cells at 0.4 μM. In conclusion, our approach has led to a new chemotype of promising antiproliferative compounds with antimitotic and potential VDA properties.

Due to its central role in growth, proliferation, survival and migration, phosphoinositide 3-kinase (PI3K) is considered as an important drug target in oncology (1). BKM120 is one of the clinically most advanced PI3K inhibitors (PI3Ki), and is currently listed in more than 80 clinical studies aimed at attenuating tumour progression. As an off-target effect, BKM120 was reported to disrupt microtubules (MT) at concentrations around 1 μM (2). Here, we elucidate in detail the structural factors defining PI3K- and tubulin-binding of BKM120, and present a pure PI3K inhibitor (PQR309) and a potent MT disruptor (MTD147) differing from BKM120 by only 1 Dalton. Separation of PI3Ki and MT disruption activities of BKM120 allowed profiling of BKM120 against PQR309 and MTD147: cellular growth profiles of PQR309 clustered with other PI3Ki such as GDC0941/GDC0980, while BKM120 matched MTD147. Both yielded a G2/M cell cycle arrest with typical histone3 phosphorylation. Accumulation of G2/M arrested cells was already evident at concentrations yielding 50% growth inhibition. Interestingly, BKM120 concentrations for 50% cell growth inhibition (with evident G2/M arrest) ranged below or within its reported AUC0-24 levels at day 8 in patient plasma (3,4). This result implies that the two activities of BKM120 cannot be separated, thus complicating the understanding of drug action and impacting on the rational of combination therapies at relevant drug doses. Using X-ray crystallography we found that BKM120 binds to the colchicine pocket on β-tubulin. This study further highlights the importance of the pyrimidine core orientation for tight tubulin binding. Interestingly, activities of regio-isomers of the pyrimidine core are inversed for PI3Ki and tubulin association, and modulate binding by a factor of >30x. Finally, a combination of biochemical, cellular and structural data suggests an inverted orientation of BKM120 in the catalytic cleft of PI3K as previously proposed (6). In summary, the dissection of BKM120 functions allows reassessment of its dominant activity, to increase drug safety, and to flexibly control PI3K and/or MT targeting in combination therapy. 1. M. P. Wymann, R. Schneiter, Nat Rev Mol Cell Biol 9, 162 (2008).; 2. S. M. Brachmann et al., Mol Cancer Ther 11, 1747 (2012). 3. J. C. Bendell et al., J Clin Oncol 30, 282 (2012). 4. C. Saura et al., Clin Cancer Res 20, 1935 (2014). 5. A. E. Prota et al., Science 339, 587 (2013). 6. S. M. Maira et al., Mol Cancer Ther 11, 317 (2012). Citation Format: Thomas Bohnacker, Florent Beaufils, Andrea E. Prota, John E. Burke, Anna Melone, Alison J. Inglis, Ludovico Fusco, Vladimir Cmiljanovic, Natasa Cmiljanovic, Denise Rageot, Katja Bargsten, Gonzalo Saez-Calvo, Olivier Pertz, Amol B. Aher, Anna Akhmanova, Fernando J. Diaz, Doriano Fabbro, Marketa Zvelebil, Roger L. Williams, Michel O. Steinmetz, Matthias P. Wymann. BKM120-mediated G2 arrest: Structural and functional segregation of off-target action and PI3K inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 671. doi:10.1158/1538-7445.AM2015-671