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

Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance oforganismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancertherapy attracted considerable attention in the past as stress-induced autophagy has been demonstratedto 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 fordesigning innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of themost 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 thatcombine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combinationof computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyseswe identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and alsodemonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, weinvestigated their effects on basal and starvation-mediated autophagic flux by quantifying the expressionof LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through westernblotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues5i 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.