Structure-Activity Relationship Study Identifies a Novel Lipophilic Amiloride Derivative that Efficiently Kills Chemoresistant Breast Cancer Cells

Abstract

Amiloride and its derivatives have long attracted attention as potential anticancer therapeutic agents. Several original studies characterized amilorides as inhibitors of sodium-proton antiporter tumor growth and urokinase plasminogen activator-mediated metastasis. However, more recent observations indicate that amiloride derivatives are specifically cytotoxic toward tumor cells relative to normal cells and have the capacity to target tumor cell populations resistant to currently-employed therapies. A major barrier to clinical translation of the amilorides is their modest cytotoxic potency, with EC50 values in the high micromolar to low millimolar range. Here we report structure-activity relationship observations that underscore the importance of the guanidinium group and the presence of lipophilic substituents at the C(5) position of the amiloride pharmacophore in promoting cytotoxicity. Moreover, we demonstrate that our most potent derivative called LLC1 is cytotoxic toward mouse mammary tumor organoids and drug-resistant populations of various breast cancer cell lines, and induces lysosomal membrane permeabilization as a prelude to lysosome-dependent cell death. Our observations offer a roadmap for the future development of amiloride-based cationic amphiphilic drugs that engage the lysosome to specifically kill breast tumor cells.

Saponin and Ribosome-Inactivating Protein Synergistically Trigger Lysosome-Dependent Apoptosis by Inhibiting Lysophagy: Potential to Become a New Antitumor Strategy

“…For instance, hexamethylene amiloride significantly inhibits mouse breast tumor tissue only at concentrations higher than 40 μM, whereas in an in vivo model of metastatic breast tumor in mice, it needs to be injected at a dose of 30 mg/kg . LLC1, a structurally optimized amiloride derivative, still exhibits a high EC 50 of nearly 20 μM against MCF-7 chemoresistant cells . Our study also showed that QS-21 below 10 μM induced LMP but was not cytotoxic to tumor cells.…”