In silico design, synthesis and in vitro evaluation of antimitotic agents

Abstract

One of the most successful group of chemotherapeutic compounds currently in clinical use for anticancer treatment are those that interfere with normal progression of mitosis through the interference of microtubule dynamics. 2-Methoxyestradiol (2ME) is an endogenous metabolite of 17â-estradiol exerting both antiangiogenic and antimitogenic effects in vitro and in vivo Abrogation of microtubule dynamics is one of the mechanisms of action of 2ME and it is proposed that 2ME interacts with the colchicine binding site of microtubules. 2ME has a short half life and has been shown to be a target for 17â-hydroxysteroid dehydrogenase-mediated metabolism. The aim of this study was to utilize non-commercial bioinformatics software to identify compounds with improved bioavailability and potency. Academically available bioinformatics software was used to develop an in silico protocol in order to identify potential inhibitors of microtubule dynamics that are capable of selectively inhibiting carbonic anhydrase IX (CAIX) activity. Over expression of CAIX contributes to the acidification of the extracellular microenvironment. Acidic extracellular pH in turn contributes to the breakdown of the basement membrane, as well as the induction of the expression of proteinases that facilitate invasion and metastasis. Therefore, it was decided to identify selective inhibitors of CAIX as it provides a valuable strategy for curtailing the development of metastatic processes associated with acidotic microenvironmental conditions in tumors. Three new and novel antimitotic compounds, ESE-15-one, ESE-15-ol and ESE-16, with nanomolar anticarbonic anhydrase activity were synthesized. Of the three new compounds, ESE- 15-ol and ESE-16 were more selective towards inhibiting a mimic of CAIX over wild-type CAII. These compounds reduced cell proliferation in both the non-tumorigenic MCF-12A and the tumorigenic MCF-7 cell line in a dose-dependent manner. The compounds are 5 to 20 times more potent than 2ME. The tumorigenic MCF-7 cells and metastatic MDA-MB-231 cells were more susceptible to ESE-15-ol and ESE-16 treatment when compared to the non-tumorigenic MCF-12A cells. Morphological investigations using confocal microscopy revealed that the compounds interfere with microtubule dynamics in actively dividing cells. Flow cytometry confirmed that the compounds are antimitotic compounds since they block cells in the G2/M phase with subsequent induction of apoptosis via mitochondrial membrane depolarization. In ESE-16-exposed cells, JNK and p38 stress-activated protein kinases are differently affected in each cell line with the JNK pathway playing an important role in mediating mitochondrial membrane depolarization in MCF-7 cells. In MDA-MB-231 and MCF- 12A cells, the p38 pathway plays a role in inducing apoptosis. Gene and proteins expression studies provided evidence for the selectivity. In MCF-7 cells, DAB2 up regulation is a likely candidate for contributing towards activating the JNK stress pathway and subsequent Bcl-2 phosphorylation and apoptosis induction. Reactive oxygen species induction due to ESE-16 exposure is argued to play an important role in inducing cell death. Several testable hypotheses regarding the mechanism of action of ESE-16 were generated from the data. These include focusing on polyamine metabolism and its causal role in inducing apoptosis,the role that PLK2 up regulation plays in inducing apoptosis and autophagy, the role that the UPR plays in inducing cell death and lastly, the phosphorylation status of Mcl-1 in response to ESE-16 exposure. Finally, additional targets that may be used for combination treatment were identified. Altogether, the study provides a basis for future research projects to develop the newly synthesized compounds into clinically usable anticancer agents.