Development of Transmembrane Ion Transport Systems for Biomedical Applications

Abstract

This thesis aims to advance transmembrane ion transport systems as prospective therapeutic agents for the treatment of cancer and bacterial infections by overcoming their limitation of non-specific activity. In this direction, ion transporters formulated in the prodrug form or ‘protransporters’ that are selectively activable in the target via certain stimuli were explored. Initially, a cancer cell targeting protransporter was designed that could be activated by the cell-protecting, quinone-reducing enzyme, NQO1, which is overexpressed in cancer cells. Salicylamide was chosen as the transporter and shown to be capable of transmembrane H+/Cl− symport. Blocking of the salicylamide OH group with NQO1 activable quinones inhibited transport activity, yielding two protransporter systems. NQO1-triggered release of the free transporter from protransporters was verified. Both transporters and protransporters were selectively toxic toward the MCF-7 breast cancer cell line over non-cancerous MEFs. Studies in MCF-7 indicated cytotoxicity to be mediated via ion homeostasis disruption triggered induction of oxidative stress, mitochondrial membrane depolarization, and lysosomal deacidification. Induction of cell death via intrinsic apoptotic pathway was also verified. Next, the utility of the salicylanilide system in the antibacterial role was assessed. Screening of a library of derivatives yielded transporters with significant antibacterial activity, and structure-activity relationships were established. The screened compounds were effective against the Gram-negative bacterial strains: Escherichia coli, Salmonella enterica (serovar Typhimurium), and Klebsiella pneumoniae. Cell surface imaging of E. coli subjected to osmotic shock following salicylanilide treatment showed extensive cell damage, indicating transporter-facilitated H+/Cl− transport across the bacterial OM degrades its cell-protecting ability. The scope of this system was extended as a red light activatable protransporter by conjugating the salicylanilide to the pyrrolidinobenzoquinone group. Recovery of ion transport and antimicrobial activities on irradiation under 655 nm light was verified. To explore the application of a photoactivated protransporter as an anticancer agent, the salicylamide system was extended into a bissalicylamide, and the OH groups conjugated to photocleavable o-nitrobenzyl moieties to cage ion transport. Bissalicylamides were found to be anion antiporters that exhibited cytotoxicity towards MCF-7 breast cancer cells. Protransporters were activated under 405 nm light to recover ion transport activity. The protransporters by themselves were non-toxic towards MCF-7 cells but exhibited significant cytotoxicity on activation under 405 nm light. Due to the limitations of the previous systems in terms of their structural tunability, the little-explored and synthetically versatile pyrrole-2-carboxamide system was assessed as a transmembrane ion transporter. A series of pyrrole-2-carboxamides were studied, which yielded a wide range of ion transport activities and Hill coefficients with minimal structural changes. Mechanistic studies showed pyrroleamides to be anion antiporters and theoretical predictions of ion transport complex structures of derivatives with n = 2 and n = 4 were conducted. This system can be extended as a protransporter by attachment of a labile moiety to either the pyrrole or amide N-H groups. This work would be a significant step forward in the implementation of this novel therapeutic approach for various biomedical applications.