Abstract 4451: Novel small molecule AG311 induces tumor cell death through inhibition of mitochondrial electron transport
Authors: Bastian Anja , Bailey-Downs Lora C. , Thorpe Jessica E. , Devambatla Ravi Kumar Vyas , Gangjee Aleem , Humphries Kenneth M. , Ihnat Michael A. 
Published: August 2015

Mitochondria are emerging as an attractive target for new anticancer therapy. The inhibition of the mitochondrial electron transport chain (ETC) provides an alternate and selective mechanism for killing cancer cells, bypassing upstream apoptosis-inducing pathways. The mechanism is based on the fact that cancer cells have higher mitochondrial membrane potential rendering them inherently susceptible to increased reactive oxygen species (ROS) generation such as superoxide through the respiratory chain. In addition pharmacological inhibition of complex I and III at the ubiquinone-binding site could further increase superoxide production. Taken together, this mitochondrial sensitivity represents a promising approach to selectively kill cancer cells. The purpose of this study was to investigate the mitochondria-associated cell death of AG311, a novel anticancer compound. AG311 (5-[(4-Methylphenyl)thio]-9H-pyrimido[4,5-b]indole-2,4-diamine) is a small molecular weight compound designed by our group. AG311 has been shown to induce rapid mitochondrial depolarization resulting in necrotic cell death in a cancer cell-selective manner. In two mouse orthotopic breast cancer models (MDA-MB-435 and 4T1), AG311 significantly reduced tumor volume by 85% and 81%, respectively (n = 4 - 6), with no apparent systemic toxicity.

In this study, we aimed to identify the mitochondrial protein target(s) of AG311 in cancer cells. First, upregulation of the mitochondrial ETC by culturing MDA-MB-435 cells in galactose media sensitized cells to AG311-induced cell death. Mitochondrial oxygen consumption (XFe96 extracellular flux analyzer) drastically decreased by 62.9% (± 12.9, n = 8) in response to AG311 (7.5 μM). The effect of AG311 on mitochondrial ETC complexes was determined by spectrophotometrically measuring NADH oxidation in the presence of antimycin A (complex I), ubiquinol reduction (complex III-IV) or cytochrome c oxidation (complex IV). It was found that AG311 inhibited complex I and III activity, but not complex IV. Further kinetic assays suggested that AG311 competitively inhibited ubiquinone binding and significantly induced generation of mitochondrial superoxide (estimated with MitoSOX Red). Finally, treatment with antioxidants (e.g. lipoic acid) partially prevented AG311-induced cell death. In summary, the present results indicate mitochondrial ubiquinone as a likely target for AG311-induced selective cancer cell death through generation of mitochondrial superoxide. This together with the previously demonstrated efficacy further substantiates the use of AG311 as an anticancer agent.

Citation Format: Anja Bastian, Lora C. Bailey-Downs, Jessica E. Thorpe, Ravi Kumar Vyas Devambatla, Aleem Gangjee, Kenneth M. Humphries, Michael A. Ihnat. Novel small molecule AG311 induces tumor cell death through inhibition of mitochondrial electron transport. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4451. doi:10.1158/1538-7445.AM2015-4451