Structure-Activity Relationship and Mechanistic Studies of Bisaryl Urea Anticancer Agents Indicate Mitochondrial Uncoupling by a Fatty Acid-Activated Mechanism.

Targeting the cancer cell mitochondrion is a promising approach for developing novel anticancer agents. The experimental anticancer agent ,'-bis(3,5-dichlorophenyl)urea () induces apoptotic cell death in several cancer cell lines by uncoupling mitochondrial oxidative phosphorylation (OxPhos) using a protein-free mechanism. However, the precise mechanism by which depolarizes mitochondria is unclear because lacks an acidic functional group typically found in protein-independent uncouplers. Recently, it was shown that structurally related thioureas can facilitate proton transport across lipid bilayers by a fatty acid-activated mechanism, in which the fatty acid acts as the site of protonation/deprotonation and the thiourea acts as an anion transporter that shuttles deprotonated fatty acids across the phospholipid bilayer to enable proton leak. In this paper, we show that mediated proton transport is enhanced by the presence of free fatty acids in the lipid bilayer, indicating that uncouples mitochondria through the fatty acid-activated mechanism. This mechanistic insight was used to develop a library of substituted bisaryl ureas for structure-activity relationship studies and subsequent cell testing. It was found that lipophilic electron-withdrawing groups on bisaryl ureas enhanced electrogenic proton transport via the fatty acid-activated mechanism and had the capacity to depolarize mitochondria and reduce the viability of MDA-MB-231 breast cancer cells. The most active compound in the series reduced cell viability with greater potency than and was more effective at inhibiting adenosine triphosphate production.