The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds’ effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

Phone number +33(0)4 24 76 54 95 71 laurence.lafanechere@univ-grenoble-alpes.fr 25 2 Running title: A new paclitaxel sensitizer that targets tubulin 26 Scientific Research (NWO) CW ECHO grant (711.015.005) to AA. 32 33 The authors declare no potential conflicts of interest 34 35 Word count: 36 Abstract: 158 words 37 Main manuscript, including acknowledgments: 4667 words 38 Figure legends: 1115 words 39 Number of references: 34 40 Abstract 43Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy 44 inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched 45 for drugs which could potentiate its therapeutic effect. We have screened a chemical library 46 3 and selected Carba1, a carbazolone, which exerts synergistic cytotoxic effects on tumor cells 47 grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the 48 colchicine binding-site of tubulin and is a microtubule-destabilizing agent. The Carba1-49 induced modulation of microtubule dynamics increases the accumulation of fluorescent 50 paclitaxel inside microtubules, providing a mechanistic explanation of the observed synergy 51 between Carba1 and paclitaxel. The synergistic effect of Carba1 with paclitaxel on tumor cell 52 viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring 53 paclitaxel accumulation in microtubules can be transposed to in vivo mouse cancer treatments, 54 paving the way for new therapeutic strategies combining low doses of microtubule targeting 55 agents with opposite mechanisms of action. 56 Introduction 57 Microtubules (MTs), dynamic polymeric filaments composed of -tubulin and -tubulin 58 heterodimers, are key components of the cytoskeleton of eukaryotic cells. Their crucial roles 59 in cell division and physiology mainly rely on their ability to rapidly polymerize or 60 depolymerize. Targeted perturbation of this finely tuned process constitutes a major 61 therapeutic strategy. Drugs interfering with MTs are major constituents of chemotherapies for 62 the treatment of carcinomas. A number of compounds bind to the tubulin-MT system. They 63 can be roughly classified into MT-stabilizers such as taxanes or epothilones, and MT-64 destabilizers such as vinca alkaloids, combretastatin and colchicine [1]. It has been 65 demonstrated that binding of vinca alkaloids or colchicine prevents the curved-to-straight 66 conformational change of tubulin at the tip of the growing MT, necessary for proper 67 incorporation of new tubulin dimers into the MT lattice (see reviews [1,2]). 68 Paclitaxel (PTX) binds to the taxane-site of -tubulin and stabilizes the MT lattice by 69 strengthening lateral and/or longitudinal tubulin contacts within the MT [1]. At stoichiometric 70 concentrations, it promotes MT assembly. At low and clinically relevant concentrations, PTX 71