The role of transmembrane proton transport rate in mild mitochondrial uncoupling produced by arylamide substituted fatty acids.

Mitochondrial uncoupling by small molecule protonophores is a promising therapeutic strategy for treatment of cancer and other diseases, however the toxicity associated with uncoupling is a major challenge. A possible solution is the development of mild uncouplers that partially depolarise mitochondria without affecting ATP production. In this study, we prepared a series arylamide substituted fatty acid protonophores and demonstrate that arylamides with appropriate aromatic substitution patterns produce mild uncoupling. Specifically, 3,4-substituted arylamides increased respiration in MDA-MB-231 breast cancer cells and partially collapsed mitochondrial proton gradients without affecting ATP production or cell viability. In contrast, the actions of 3,5-substituted arylamides were similar to those of known full uncouplers DNP and CCCP and decreased intracellular ATP levels. Using a vesicle-based proton transport assay, we found that the rates of transmembrane proton transport by the 3,4-substituted arylamides were slower than those of their 3,5-substituted counterparts, which is attributed to a diminished capacity of 3,4-substituted arylamides to form membrane permeable dimers in the rate limiting step of the protonophoric cycle. This is the first study to assess the role of proton transport rate in protonophore-mediated mitochondrial uncoupling, and the results indicate that development of protonophores with limited proton transport rates may be a viable strategy towards identifying mild uncouplers.