Development of Artificial Anion Transport Systems and Evaluation of Their Biological Activity

Abstract

Several cellular physiological functions rely on naturally occurring ion channels, membrane- embedded proteins that permit ions to flow across cell membranes. This controlled flow of ions across the membranes balances the osmolality within and outside of the cell. However, cystic fibrosis (CF) and other fatal disorders can result from mutations or structural rearrangement in the genes that encode crucial membrane transport proteins. The advent of artificial ion transport systems has opened up a way to replace dysfunctional natural ion channels in channel replacement therapy. However, these cannot be used to treat CF in most cases since they harm healthy cells. In an effort to address these concerns, we introduced artificial channels developed from isophthalic acid-based small organic molecules to transport chloride ions across epithelial cells while being non-toxic even up to a loading concentration of 100 μM. These compounds have the realistic potential for treating diseases related to the dysfunctions in Cl ‒ channels in the near future. Recent reports have shown that membrane-active synthetic ionophores can induce apoptosis in various cancer cell lines. In an effort to fight against cancer, these findings have rekindled interest among scientists to further explore the field. Along these veins, we developed benzoylbenzohydrazide-based ion channel systems that induce apoptosis and disrupt autophagy, a combination seldom seen in targeting cancers. However, ion transport activity and IC50 against cancer cells were only moderate. We anticipated that the effectiveness of the anticancer activity might greatly improve if we could build a system with extremely high HCl transport activity, as they possess an advantage of working more effectively in acidic microenvironments of cancer cells. Along these lines, we developed a class of pyridyl-linked benzimidazolyl hydrazone, the highest active synthetic HCl transporters hitherto reported. Viability tests showed that the transporters cause high cytotoxicity in human breast cancer MCF-7 cells but are comparatively harmless to non-cancerous HEK293T cells. Before diving into the in-depth mechanistic details of cell death by these HCl carriers, we contemplated the possibility of rendering these systems into an HCl channel, a system that is very rarely reported in the literature. We replaced the pyridyl moiety with the pyrrole ring, and as an outcome, the compound was found to self-assemble in the presence of HCl to construct a nanochannel assembly filled with chloride ions. The anticancer activity improved significantly with the IC 50 value of only 0.1 uM in human osteosarcoma U2OS cell lines while being nearly non-toxic toward healthy cells. The detailed mechanistic study revealed that the compound induces apoptosis by disrupting intracellular ion homeostasis, producing oxidative stress, and depolarizing mitochondrial membrane potential leading to the release of cytochrome c to the cytosol. Overall, these systems have realistic potential to combat cancers in due course.