Identification of the Highly Active, Species Cross-Reactive Complex I Inhibitor BAY-179

Abstract

Mitochondria are key regulators of energy supply and cell death. Generation of ATP within mitochondria occurs through oxidative phosphorylation (OXPHOS), a process which utilizes the four complexes (complex I-IV) of the electron transport chain and ATP synthase. Certain oncogenic mutations (e.g., LKB1 or mIDH) can further enhance the reliance of cancer cells on OXPHOS for their energetic requirements, rendering cells sensitive to complex I inhibition and highlighting the potential value of complex I as a therapeutic target. Herein, we describe the discovery of a potent, selective, and species cross-reactive complex I inhibitor. A high-throughput screen of the Bayer compound library followed by hit triaging and initial hit-to-lead activities led to a lead structure which was further optimized in a comprehensive lead optimization campaign. Focusing on balancing potency and metabolic stability, this program resulted in the identification of BAY-179, an excellent in vivo suitable tool with which to probe the biological relevance of complex I inhibition in cancer indications.

Figure 1

(A) Schematic of the mitochondrial electron transport chain. (B) Representative list of known inhibitors of complex I.

Figure 2

(A) An ATP-dependent luciferase reporter assay was used to determine the effect of electron transport chain inhibitors on ATP synthesis in H1299 cells (left). Luminescent measurements were performed before (M1) and after (M2) addition of complex II substrate succinate. Thereby, this assay can discriminate specific complex I inhibitors (e.g., rotenone, inhibitory effect overridden by succinate) from other mode of actions affecting cellular ATP levels like complex III inhibition by antimycin A or mitochondrial decouplers like FCCP (no rescue effect by succinate). (B) High-throughput screen hit triaging resulted in the identification of one priority cluster represented by screening hit 1.

Figure 3

(A) An ATP-dependent luciferase reporter assay was used to determine complex I inhibition in a variety of cell lines from different species to assess species cross-reactivity. (B) Effects of 8 before and after addition of the complex II substrate succinate to determine specificity. (C) 8 was tested in cells transfected with NDI1, a rotenone-insensitive NADH-quinone oxidoreductase from Saccharomyces cerevisiae to further determine the inhibitor specificity. Reported IC₅₀ values are average values of at least two independent measurements.

Scheme 1. Synthesis of 37