Novel modulators of mitochondrial complex I ROS production as potential AD therapeutics

Abstract Background Among the indicated pathogenic factors of Alzheimer’s disease (AD), mitochondrial dysfunction has been recognized as an essential contributor, especially for the late onset AD (LOAD). That this observed dysfunction is the cause or consequences of other AD‐related injuries has been debated, results from both sides are in agreement that the roles of mitochondria in connection with other AD pathologies are convergent. This strongly advocates that targeting mitochondrial dysfunction represents a promising approach to develop more effective AD therapeutics. Method Chemical probes based on a novel lead structure that showed promising neuroprotective activities both in vitro and in vivo AD models were designed and synthesized to identify protein targets. Mouse brain mitochondria were purified from C57BL/6 mice and complex I was further isolated. OXPHOS studies were conducted in purified mitochondria upon treatment with our lead compound. Photo‐affinity labeling studies were conducted upon incubation of chemical probes with complex I. Colocalization was studied using neuronal MC65 cells and chemical probes. Result OXPHOS studies established that the lead compound only modulated the state 3 respiratory of pyruvate, the complex I substrate, but not succinate, the complex II substrate. Enzymatic studies of ETC also confirmed its selective modulation of complex I. The immunocytochemistry studies in MC65 cells indicated that the designed chemical probe localized to mitochondria. Protein labeling studies confirmed the direct interaction of the chemical probe with mitochondria complex I. Further analysis suggested that our chemical probe binds to the FMN binding site of complex I. Competition studies using the lead compound and NADH supported the selective labeling of complex I sub‐units. Conclusion Studies from in vitro, ex vivo, and in vivo models established that the lead compound selectively modulates the production of mitochondria complex I ROS without interfering with mitochondria membrane potential and bioenergetics, representing a novel mechanism of action. Chemical biology studies employing photo‐affinity labeling probes also confirmed the interaction of the probes with complex I and suggested the FMN binding site as the interaction site. Collectively, the results strongly encourage developing mitochondria complex I ROS modulators based on this novel chemical scaffold as potential AD therapeutics.