Addressing the selectivity and toxicity of antiviral nucleosides

Feng, Joy Y

Since the 1950s, mitochondrial respiration has been regarded as the "gold standard" for the measurement of mitochondrial function. 38 It has been used extensively to study mitochondrial toxins that directly impair the electron transport chain but has not been used to study the nucleoside/tide analogs until recently. [39] [40] [41] [42] This assay provides a sensitive functional readout and does not require knowledge of the mechanism of action. 20 A complete loss of mitochondrial respiration capacity can be detected before any change in the cellular ATP level, and this phenomenon has been observed for inhibitors with diverse molecular targets including ddC (inhibitor of mitochondrial DNA synthesis), 4 0 -azido cytidine (inhibitor of mitochondrial RNA synthesis), and chloramphenicol (inhibitor of mitochondrial protein synthesis). For compounds that show both mitochondrial and general toxicity (such as BMS-986094), this method makes it possible to identify whether mitochondria toxicity occurs at a lower drug concentration. 20 For both mitochondrial protein and respiration assays, we found that PC-3 cells offer higher sensitivity and reproducibility than HepG2 cells. 20 An assay with limited predictive value: The glucose-galactose CC 50 assay When faced with mitochondria damage, cells are known to switch to glycolysis to generate ATP; therefore the glucose-galactose CC 50 assay has been widely used to detect mitochondria toxicity. 43 In the absence of glucose, the galactose-adapted cells are forced to rely heavily on mitochondrial OXPHOS and thus would show a !3-fold decrease in CC 50 . 44 However, the predictivity of this assay was challenged by Hyne's 2013 report showing that this assay only detected 2-5% of the 200 potential mitochondrial toxins tested. 44, 45 Even though no nucleoside/tide analogs were included in Hyne's study, it is consistent with our finding from testing >30 clinically relevant nucleoside/tide analogs. None of the known mitochondrial toxins such as ddC, FIAU, or 4 0 -azido C showed enhanced cytotoxicity in galactose-adapted cells.