Journal: bioRxiv

Article Title: Discovery and Preclinical Validation of a Clinically Optimized Mitochondrial Complex I Modulator for Alzheimer’s Disease

doi: 10.64898/2026.04.10.717554

Figure Lengend Snippet: a A drug discovery funnel used for the identification of C273. b C273 protects MC65 Tet-Off cells from Aβ toxicity (red line). # indicates cell death in Tet-Off cells treated with vehicle. No toxicity was observed in MC65 Tet-On cells (Aβ not expressed, black line). EC 50 values were calculated using nonlinear regression in GraphPad Prism 10. c Treatment with C273 for 24 h does not induce cytotoxicity in HepG2 human hepatic cells at concentrations up to 100 μM. d C273 increases NADH levels in MC65 Tet-On cells, consistent with inhibition of mtCI. NADH levels were measured over 24 h. e Inhibition of mtCI activity in isolated mouse brain mitochondria by C273 measured using an NADH oxidation assay. f Activities of mitochondrial ETC complexes I–IV were assessed in permeabilized MC65 cells using an electron flow assay in the Seahorse XFe96 Extracellular Flux Analyzer in the presence of C273 (25 and 50 μM). g Injection of rotenone or C273 to monitor OCR kinetics in MC65 Tet-On cells. h Pretreatment with rotenone blocks the protective effect of C273 in MC65 Tet-Off cells (Aβ expressed). Open orange squares: Tet-On cells, no Aβ expression, treated with vehicle (positive control, 100% viability). Blue triangles: Tet-Off cells (Aβ expressed), which undergo extensive cell death after 3 days. Tet-Off cells treated with C273 (2 nM - 5 μM; blue line) show complete protection from Aβ toxicity. Solid red triangles: Tet-On cells treated with 32 nM rotenone, which show no toxicity. Open red triangles: Tet-Off cells treated with 32 nM rotenone alone, resulting in complete cell death. Pretreatment with 32 nM rotenone blocks the protective effect of C273 in Tet-Off cells (red line, grey box), supporting a competitive interaction at mtCI. Partial rescue at higher C273 concentrations reflects incomplete occupancy of the mtCI binding site by rotenone, which inhibits ∼50% of mtCI activity at this dose. The grey box indicates the EC 50 concentration range for C273. P values were calculated using unpaired two-tailed Student’s t -tests; ** P < 0.01. Data are presented as mean ± SD ( n = 4 technical replicates). All experiments were replicated at least twice.

Article Snippet: NF-κB Reporter (Luc) HEK293 cell line and the antioxidant response element (ARE) Luciferase Reporter HepG2 hepatic cell line were purchased from BPS Bioscience (CA, USA) and were cultured in accordance of manufacturer instructions.

Techniques: Drug discovery, Inhibition, Activity Assay, Isolation, Oxidation Assay, Injection, Expressing, Positive Control, Binding Assay, Concentration Assay, Two Tailed Test