Nambara, Toshihiko

Abstract Alzheimer’s disease (AD) is characterized by diminished capacity to mount adaptive cellular stress responses required to maintain energy homeostasis and proteostasis. An emerging therapeutic strategy is to restore adaptive stress responses by inducing mild energetic stress through inhibition of mitochondrial complex I (mtCI). However, pharmacological inhibition of the respiratory chain has remained challenging, as it can induce bioenergetic failure rather than beneficial signaling. Here, we describe C273, a brain-penetrant small molecule that delivers controlled, weak attenuation of mtCI activity to therapeutically restore endogenous adaptive stress pathways. This work establishes a first-in-class mechanism in which calibrated activation of multifaceted adaptive mechanisms enhances cellular resilience, rather than impairing mitochondrial function. Structure-activity relationship optimization yielded a compound with high potency against Aβ-induced cellular toxicity, strong selectivity for mtCI, and favorable drug-like properties. C273 demonstrated excellent oral bioavailability, metabolic stability in mouse, rat, and human microsomes, minimal CYP liabilities, and a clean ancillary pharmacology profile in the Eurofins CEREP44 panel. In vivo , C273 readily crosses the blood-brain barrier and activates AMP-activated protein kinase (AMPK), initiating a coordinated hormetic response characterized by enhanced antioxidant defenses, suppression of inflammatory signaling, induction of autophagy, and increased mitochondrial biogenesis and turnover. Genetic deletion of AMPKα1/α2 abolished these responses, establishing AMPK as a critical mediator of C273 activity. Pharmacological competition experiments further confirmed the target, as pretreatment with non-toxic concentrations of rotenone blocked C273 interaction with the quinone-binding site of mtCI and eliminated its neuroprotective effects. Repeated oral administration of C273 (20-80 mg/kg/day) to wild-type mice for one month produced no detectable cardiac or hepatic toxicity, indicating a favorable in vivo safety margin. Importantly, C273 activated these mechanisms and reduced Aβ and p-Tau levels in induced pluripotent stem cell-derived cerebral organoids from patients with sporadic AD. Collectively, these results establish controlled mtCI modulation as a therapeutic strategy and position C273 as a promising disease-modifying candidate for AD.

Abstract Despite recent approval of monoclonal antibodies that reduce amyloid (Aβ) accumulation, the development of disease-modifying strategies targeting the underlying mechanisms of Alzheimer’s disease (AD) is urgently needed. We demonstrate that mitochondrial complex I (mtCI) represents a druggable target, where its weak inhibition activates neuroprotective signaling, benefiting AD mouse models with Aβ and p-Tau pathologies. Rational design and structure‒activity relationship studies yielded novel mtCI inhibitors profiled in a drug discovery funnel designed to address their safety, selectivity, and efficacy. The new lead compound C458 is highly protective against Aβ toxicity, has favorable pharmacokinetics, and has minimal off-target effects. C458 exhibited excellent brain penetrance, activating neuroprotective pathways with a single dose. Preclinical studies in APP/PS1 mice were conducted via functional tests, metabolic assessment, in vivo 31 P- NMR spectroscopy, blood cytokine panels, ex vivo electrophysiology, and Western blotting. Chronic oral administration improved long-term potentiation, reduced oxidative stress and inflammation, and enhanced mitochondrial biogenesis, antioxidant signaling, and cellular energetics. These studies provide further evidence that the restoration of mitochondrial function and brain energetics in response to mild energetic stress represents a promising disease- modifying strategy for AD.