Design and synthesis of multifunctional microtubule targeting agents endowed with dual pro-apoptotic and anti-autophagic efficacy

96 p.-20 fig.-7 tab.Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion.This research was funded by the European Union's Horizon 2020 (EU) Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No.721906-TRACT (TK, SM, MG, NMcC, GO’S, RT, VK, SG, DZ, GC, SB); Tuscany strategic project POR-FSE 2014-2020, ‘Medicina di Precisione e Malattie Rare’(MePreMaRe), (ACE-ESCC) (NR, GC, SB), and by “Epigenetic Hallmarks of Multiple Sclerosis” (acronym Epi-MS) (id:415, Merit Ranking Area ERC LS) in VALERE 2019 Program; V:ALERE 2020 – Progetto competitivo “CIRCE” in risposta al bando D.R. n. 138 del February 17, 2020 Program; Blueprint 282510; EPICHEMBIO CM1406; AIRC-17217; VALERE: Vanvitelli per la Ricerca; Campania Regional Government Technology Platform Lotta alle Patologie Oncologiche: iCURE; Campania Regional Government FASE2: IDEAL. MIUR, Proof of Concept POC01_00043. POR Campania FSE 2014-2020 ASSE III.Peer reviewe