TABLE 2

Overview of example assays that can be applied in our proposed tiered evaluation of drug-induced mitochondrial dysfunction, based on methodologies used in the clinical investigation of inherited mitochondrial disease

	Evaluation strategy	Assay examples
Tier 1	Phenotypic observations	Glucose-galactose assay, cellular viability (fluorescence live/dead stain), respirometry assessment (e.g., OCR using Seahorse XF Bioscience), ATP levels (e.g., bioluminescent ATP or CellTiter-Glo), lactic acid (colorimetric assays).
Tier 2	Clinical chemistry	Drug concentration-response curves (IC₅₀/MEC), enzymatic activity (e.g., cytochrome C oxidase, NADH reductase, or succinic dehydrogenase), ATP production rates (using Seahorse XF), OXPHOS complex activity or assembly (e.g., BN-PAGE), MMP (fluorescence, e.g., TMRM or JC-1 or flowcytometry, e.g., MitoTracker Green FM), ROS production (fluorescence, e.g., H₂DCFDA and MitoSOX), network or structure-based model construction (e.g., “omics” or X-ray protein structures/pharmacophore prediction using ProBiS/KRIPO).
Tier 3	Cellular studies	Protein overexpression or knockout (CRISPR/Cas9, RNAi, e.g., shRNA or siRNA) combined with cellular/metabolic parameters (e.g., cellular viability, OXPHOS activity, and ATP production rates).
Tier 4	In vivo studies	In vitro genetic complementation using patient-derived fibroblasts: protein expression (e.g., Western blot), metabolic parameters (e.g., OXPHOS complex activity, MMP) and in vivo evaluation of mitochondrial function (e.g., MITO-Tag mice, MS, ROS, and ATP levels).

BN-PAGE, blue native polyacrylamide gel electrophoresis; H₂DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; KRIPO, key representations of interaction in pockets; MEC, minimal effect concentration; MMP, mitochondrial membrane potential; MS, mass spectrometry; OCR, oxygen consumption rate; OXPHOS, oxidative phosphorylation; RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, small interfering RNA; TMRM, tetramethyl rhodamine methyl ester.