Ostroot, Mark

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 Background Despite recent approval of monoclonal antibodies that reduce amyloid (Ab) accumulation, the development of disease‐modifying strategies targeting the underlying mechanisms of Alzheimer’s Disease (AD) are urgently needed. We identified mitochondrial complex I (mtCI) as a small‐molecule druggable target for AD. Using cryo‐EM, we previously demonstrated that the neuroprotective tool compound CP2 directly binds to the Q‐site of mtCI and weakly inhibits its activity. Method Rational design and extensive structure‐activity relationship studies using CP2 as a tool compound allowed the development of novel classes of partial mtCI inhibitors with high selectivity, specificity, safety, and improved drug‐like properties. Target engagement, selectivity, efficacy, safety and the direct binding to mtCI were demonstrated using multiple in vitro assays. Efficacy in vivo was established using APP/PS1 mice. The best new leads, C458 and C273, were evaluated as preclinical candidates. Result Like CP2, C458 and C273 weakly inhibit mtCI, leading to AMPK activation, which triggers a neuroprotective stress response. This response enhances oxidative stress protection, promotes autophagy, and increases mitochondrial biogenesis and turnover, ultimately protecting against Aβ toxicity. Pretreatment with the irreversible mtCI inhibitor rotenone (at non‐toxic concentrations) blocked C458 and C273 from binding to mtCI and abrogated their protection against Aβ toxicity. Both C458 and C273 exhibited excellent bioavailability, blood–brain barrier penetrance, and a clean safety profile in the CEREP 44 safety panel. Chronic administration of C458 (25 mg/kg/day in drinking water ad lib ) to APP/PS1 mice for 7 months preserved cognition, improved long‐term potentiation, reduced oxidative stress and inflammation, and enhanced mitochondrial biogenesis. Similarly, C273 (20, 50, or 80 mg/kg/day) administered to C57BL/6 mice for one month showed no detectable side effects or toxicity in the heart or liver. Conclusion Mechanistic studies revealed that C458 and C273 activate a mitochondrial stress response, augmenting mitochondrial biogenesis, antioxidant signaling, and autophagy, which collectively provided protection against Aβ toxicity in multiple in vitro and in vivo models of AD. These findings demonstrate that weak mtCI inhibition by novel compounds C458 and C273, with improved drug‐like properties, is neuroprotective. This supports the feasibility of developing safe and efficacious mtCI inhibitors as a disease‐modifying strategy for AD.

Abstract Background Although novel treatments targeting amyloid‐beta (Aβ) have been recently approved by the FDA, there remains a critical need for alternative therapeutic strategies to delay the onset and progression of Alzheimer's disease (AD). Mitochondrial dysfunction and disrupted brain energy homeostasis are key hallmarks of early‐stage AD. Developing treatments that address these deficits could offer substantial benefits. We identified mitochondrial complex I (mtCI) as a druggable target and demonstrated that its mild inhibition using the small molecule CP2 improves brain energy homeostasis, restores synaptic and cognitive function, and activates neuroprotective signaling in AD mouse models. However, the mechanistic link between mtCI inhibition, glucose metabolism, and energy homeostasis remains unclear. Methods Cellular energy metabolism was assessed in neuroblastoma SH‐SY5Y cells expressing mutant human APP protein (APPswe) and in control cells. Glycolytic function, mitochondrial energetics, and fatty acid β‐oxidation (FAO) were measured using a Seahorse Analyzer. Glucose transporter (GLUT) translocation to the cell surface was evaluated using flow cytometry and immunofluorescence staining. Protein expression changes were analyzed via Western blot, and glucose uptake was quantified with the Glucose Uptake‐Glo™ assay. Additionally, metabolic flux analysis using 13C‐labeled glucose and targeted metabolomics by mass spectrometry were performed to measure specific metabolites. Results APPswe cells displayed significant reductions in glycolysis and spare respiratory capacity, indicative of impaired mitochondrial energy production under stress. These deficits were accompanied by reduced glucose uptake, which was partially compensated by an increase in FAO. At concentrations relevant to in vivo treatments, CP2 acutely enhanced GLUT translocation to the cell surface, increasing glucose uptake within one hour. Enhanced glucose utilization in OXPHOS machinery was confirmed by inhibition of PDH phosphorylation and increased conversion of pyruvate to acetyl‐CoA, fueling the TCA cycle and generating critical signaling metabolites essential for gene regulation. These mechanisms were mediated by AMPK. Conclusion Our findings suggest that mild inhibition of mtCI activates multiple neuroprotective mechanisms, including improved glucose metabolism and energy homeostasis. These consistent results in human cells and AD mouse models highlight the translational potential of this therapeutic approach for AD.

Abstract Background Early mechanisms of neuronal damage in Alzheimer's Disease (AD) include mitochondrial dysfunction and abnormal energy homeostasis. AD‐associated mitochondrial signaling was shown to occur upstream of amyloid precursor protein processing, Ab production, apolipoprotein E (APOE) expression, and tau pathology. However, whether alterations in mitochondrial function are sufficient to instigate AD remains unclear. Mitochondrial complex I (mtCI) is a rate‐limiting enzyme in the oxidative phosphorylation (OXPHOS) machinery. Changes in mtCI activity influence ATP and ROS production, cellular energy and redox homeostasis. We examined how reduced activity of mtCI due to a knockout of one of its accessory subunits Ndufs4 in mice affects transcriptional networks in the brain. We also determined whether reduced activity of mtCI abrogates neuroprotective properties of a small molecule CP2. Method RNA‐sequencing of the brain tissue was performed using Ndufs4 ‐/‐ and control mice ( n = 3–5 per group). The co‐expression modules of differentially expressed genes were compared to the AMP‐AD patient cohort data. Key mechanisms, including mitophagy signaling, were confirmed using ELISA and western blot analyses; the effect on mtCI activity was confirmed using functional assays. Results In Ndufs4 ‐/‐ mice, a residual activity of mtCI was ∼50% of the control. This loss of function led to a global disruption of mitochondrial homeostasis, energy metabolism, synaptic function, significant increase in mitophagy and a reduction in biogenesis. Transcriptomic signatures in brain tissue of male and female Ndufs4 ‐/‐ mice overlapped with changer reported for AD patients and AD mouse models with Ab and p ‐tau pathologies. These changes were partially rescued by a neuroprotective mitochondria‐targeting small molecule CP2. Consistent with studies in AD mice, CP2 treatment in Ndufs4 ‐/‐ mice augmented the expression of genes associated with mitochondrial biogenesis and turnover, synaptic activity, autophagy, redox balance, and reduced expression of genes related to inflammation. Sex‐specific differences included better adaptation to a reduced activity of mtCI and to CP2 treatment in female mice. Conclusion Reduced mtCI activity was sufficient to induce the AD‐like transcriptomic changes in brain. Residual mtCI function was sufficient to mediate neuroprotective effect of CP2, emphasizing a key role of mitochondria in AD development and treatment.

Abstract INTRODUCTION Mitochondrial dysfunction is implicated in Alzheimer's disease (AD), but whether it drives AD‐associated changes is unclear. We assessed transcriptomic alterations in the brains of Ndufs4 −/− mice, a model of mitochondrial complex I (mtCI) deficiency, and evaluated the therapeutic effects of the neuroprotective mtCI inhibitor CP2. METHODS Cortico‐hippocampal tissue from Ndufs4 −/− and wild‐type mice was subjected to transcriptomic analysis, followed by cross‐species comparisons to human late‐onset AD and familial AD mouse datasets. RESULTS Knockout of Ndufs4 ‐mediated mtCI deficiency disrupted mitochondrial homeostasis, energy metabolism, and synaptic gene expression, recapitulating transcriptomic signatures of AD. CP2 treatment partially reversed these changes, with female Ndufs4 −/− mice showing greater compensatory adaptations and treatment responses. DISCUSSION Loss of mtCI activity alone is sufficient to induce AD‐like molecular changes in the brain, independent of amyloid beta or phosphorylated tau. CP2‐mediated rescue highlights the potential of targeting mitochondria as a therapeutic strategy for AD. Sex‐specific responses suggest important considerations for personalized therapeutics. Highlights Activity of mitochondrial complex I (mtCI) affects broad mitochondrial and neuronal transcriptional networks. A reduction of mtCI activity is sufficient to induce transcriptomic changes reminiscent of those observed in late‐onset Alsheimer's disease (AD) patients and familial mouse models of AD. Pharmacological targeting of mtCI mediates neuroprotective signaling. Male and female mice have differential responses to the loss of mtCI activity and to the mitochondria‐targeted therapeutics. Mitochondria play a key role in AD development and treatment.

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.

AbstractBackgroundWe identified small molecule tricyclic pyrone compound CP2 as a mild mitochondrial complex I (MCI) inhibitor that induces neuroprotection in multiple mouse models of AD. One of the major concerns while targeting mitochondria is the production of reactive oxygen species (ROS). CP2 consists of two diastereoisomers, D1 and D2, with distinct activity and toxicity profiles. This study was designed to understand how structure of D1 and D2 affects their binding to MCI and the consequential impact on ROS production.MethodThe X‐ray crystallography and cryo‐electron microscopy (cryo‐EM) at global resolution of 3.25‐3.27Å were employed to identify the molecular structure of D1 and D2 and the D1 binding to the isolated ovine MCI. The assessment of the MCI inhibition and the extent of ROS generation were done in isolated MCI and human neuroblastoma MC65 cells using flow cytometry, a Seahorse extracellular flux analyzer, and the kinetic studies.ResultIn the closed conformation of MCI, D1 selectively binds to the deep Quinone‐site (Qd) but not to the shallow Q‐site (Qs), sharing the same binding pocket as rotenone. In the open MCI state, D1 exclusively binds to the Qs in contrast to rotenone, which binds Qd and Qs in both closed and open states. At the same concentrations, D1 inhibits respiration to a greater extent compared to D2 (5:1 ratio) and produces higher level of ROS.ConclusionCryo‐EM unambiguously identified binding of D1 to both the Qd and Qs sites, contingent upon the conformational state of MCI. In contrast to rotenone, D1 binds Qd only in the closed conformation during catalytic cycle, leading to mild inhibition. Superimposing X‐ray crystallography data of D1 and D2 onto cryo‐EM data suggests that the orientation of the methyl group in D2 induces a flatter conformation, resulting in lower binding affinity to MCI, which correlates with lower inhibition and toxicity compared to D1. At physiologically relevant concentrations, CP2 (D1:D2 = 1:1) demonstrates low MCI inhibition yielding negligible ROS levels. This observation provides new insight into the absence of toxicity associated with CP2 treatment in vivo, further highlighting feasibility for the development of safe and efficacious MCI inhibitors.

AbstractBackgroundWhile disease‐modifying treatments that reduce Aβ have been recently approved by the FDA, the identification of novel therapeutic targets and strategies that target underlying mechanisms to delay the AD development are still needed. Abnormal brain energy homeostasis and mitochondria dysfunction are observed early in AD. Therefore, the development of treatments to restore these defects could be beneficial. We identified small molecule (code name CP2) as a mild and specific mitochondrial complex I (MCI) inhibitor. Application of CP2 improved brain energy homeostasis, restored synaptic and cognitive function in APP/PS1 and 3xTgAD mice. However, mechanistic relationship between MCI inhibition, glucose uptake/utilization and mitochondrial function remains to be determined.MethodCellular energy metabolism was assessed in the neuroblastoma SH‐SY5Y cells that express either mutant human APP protein (APPswe) or empty vector (control) treated with vehicle or CP2. A Seahorse Extracellular Flux Analyzer was used to measure glycolysis, oxygen consumption rate, and fatty acids β‐oxidation (FAO). Flow cytometry allowed determining the translocation of glucose transporters to the cell surface. Changes in protein expression in response to treatment were assessed using Western Blot analysis. Changes in mitochondrial morphology were monitored using electron microscopy. The non‐radioactive Glucose Uptake‐Glo™ assay was utilized for measuring glucose uptake in cells. Metabolic flux analysis was done using 13C D‐Glucose stable isotope‐labeling.ResultsAPPswe cells have a significant decrease in glycolysis and spare respiratory capacity, an indicator of the mitochondrial ability to produce energy under stress conditions. These observations were consistent with decreased glucose uptake, which was compensated by an increased FAO to provide axillary fuel for ATP production. Mechanistically, at the concentrations relevant to in vivo treatment in APP/PS1 and 3xTgAD mice, acute CP2 treatment increased glucose uptake and utilization through the translocation of glucose transporters to the plasma membrane while prolong CP2 treatment activated metabolic sensors and mitochondrial morphofunctional pathways (e.g., fission, fusion, biogenesis, and turnover) consistent with the improved cellular bioenergetics in the AMPK‐dependent pathway.ConclusionData suggested that mild MCI inhibition activates multiple neuroprotective mechanisms improving cellular energy homeostasis and mitochondrial function in vivo and in vitro, representing promising strategy that target early AD mechanisms.

Background: Accumulation of hyperphosphorylated tau (pTau) protein is associated with synaptic dysfunction in Alzheimer’s disease (AD). We previously demonstrated that neuroprotection in familial mouse models of AD could be achieved by targeting mitochondria complex I (MCI) and activating the adaptive stress response. Efficacy of this strategy on pTau-related pathology remained unknown. Objective: To investigate the effect of specific MCI inhibitor tricyclic pyrone compound CP2 on levels of human pTau, memory function, long term potentiation (LTP), and energy homeostasis in 18-month-old 3xTg-AD mice and explore the potential mechanisms. Methods: CP2 was administered to male and female 3xTg-AD mice from 3.5–18 months of age. Cognitive function was assessed using the Morris water maze. Glucose metabolism was measured in periphery using a glucose tolerance test and in the brain using fluorodeoxyglucose F18 positron-emission tomography (FDG-PET). LTP was evaluated using electrophysiology in the hippocampus. The expression of key proteins associated with neuroprotective mechanisms were assessed by western blotting. Results: Chronic CP2 treatment restored synaptic activity in female 3xTg-AD mice; cognitive function, levels of synaptic proteins, glucose metabolism, and energy homeostasis were improved in male and female 3xTg-AD mice. Significant reduction of human pTau in the brain was associated with increased activity of protein phosphatase of type 2A (PP2A), and reduced activity of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β). Conclusion: CP2 treatment protected against synaptic dysfunction and memory impairment in symptomatic 3xTg-AD mice, and reduced levels of human pTau, indicating that targeting mitochondria with small molecule specific MCI inhibitors represents a promising strategy for treating AD.