Novel indole-based antimitotic agentsdesign, synthesis and study of the antitumor mechanism of action

Abstract

Cancer is the second leading cause of death, accountable for about 1 in 6 deaths globally. There is massive investment in developing novel therapeutic strategies, although brand-new chemotherapy faces several handicaps such as lack of clinical efficacy, pharmacokinetic problems, or the appearance of resistance. In this work, we focused on designing novel compounds to interact with tubulin, which is a validated target in cancer chemotherapy and arguably one of the most successful ones. The polymerization of α,β-tubulin gives rise to highly dynamic microtubules. Their pivotal roles, as in chromosome segregation, make microtubules an appealing target in cancer chemotherapy since minor disturbance of their dynamics leads to an antimitotic effect and, eventually, cell death. The primary goal of this work is to obtain compounds with advantageous properties to interact with the colchicine domain. Considering that this domain is conceived as an ensemble of three consecutive zones (1-2-3), we have designed compounds directed to zones 1-2, 2-3, and 1-2-3, leveraging the shared zone 2 in the different approaches by using common structural elements for this zone that can be later combined with residues in zones 1 and 3. This work has yielded a series of potent indole-based cytotoxic compounds with two aromatic rings connected by sulfonamide or tetrazole groups that preferentially adopt folded conformations required to interact with the pocket. We have explored different modifications on position 3 of the indole (B ring), on the bridge, or different arrangements for the methoxy groups on the classical trimethoxyphenyl ring (A ring). The in vitro evaluation of the resulting ligands against several human tumor and non-tumorigenic cell lines and mouse dermal fibroblasts reveals antiproliferative potencies in the submicromolar or nanomolar range, with IC50 values as low as 1 nM. The pharmacological inhibition of MDR transport barely influences this effect. Our results indicate that inhibition of cell proliferation can be ascribed to a dose-dependent antimitotic effect, observed by flow cytometry. Here we demonstrate that these compounds bind to the colchicine domain in β-tubulin and preclude the polymerization of microtubules, inducing disorganization of the tubulin cytoskeleton. Additionally, these compounds compromise the tube formation of mouse endothelial 3B-11 cells, suggesting that angiogenesis may also be hindered upon treatment. We have studied the mechanism of action and the induction of cell death in human epithelial cervical carcinoma HeLa cells caused by some representative lead compounds and compared their behavior with other cell lines. These compounds induce caspase-dependent apoptotic cell death with rapid permeabilization of the plasma membrane. Our results point out mitochondria as key players in the cellular response to the treatment. Overexpression of pro-survival Bcl-xL and Bcl-2 proteins in HeLa cells delays the onset of apoptosis but does not entirely prevent it. Despite similar IC50 values, we have shown that non-tumorigenic cell lines challenged with the lead compounds are less prone to undergo apoptosis than tumor cells. The mitotic arrest can be alleviated in drug-free media by re-polymerizing the tubulin cytoskeleton, which depends on protein synthesis. This process turns HeLa cells more sensitive to an additional treatment cycle. This research contributes to further unraveling the molecular processes, both reversible and irreversible, that are involved in the transition from microtubule depolymerization to apoptotic cell death.