Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase

To examine the role of the mitochondrial polymerase (Pol gamma) in clinical ly observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol gamma for each Food and Drug Administration-approved analog plus 1-(2-deoxy2-fluoro-beta -D-arabinofura nosyl)-5-iodouracil (FIAU), beta -L-(-)-2',3'-dideoxy-3'-thiacytidine (-)3T C, and (R)-9-(2-phosphonylmethoxypropyl) adenine (PMPA). We used recombinan t exonuclease-deficient (E200A), reconstituted human Poly holoenzyme in sin gle turnover kinetic studies to measure K-d (K-m) and k(pol) (k(cat)) to es timate the specificity constant (k(cat)/K-m) for each nucleoside analog tri phosphate. The specificity constants vary more than 500,000-fold for the se ries ddC > ddA (ddI) much greater than 2',3'-didehydro-2',3'-dideoxythymidi ne (d4T) much greater than (+)3TC (-)3TC > PMPA > azidothymidine (AZT) Carb ovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are e xpected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT . We also examined the rate of exonuclease removal of each analog after inc orporation. The rates varied from 0.06 to 0.0004 s(-1) for the series FIAU > (+)3TC (-)3TC > CBV > AZT > PMPA similar to d4T much greater than ddA (dd I) much greater than ddC. Removal of ddC was too slow to measure (<0.00002 s(-1)). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (-)3TC, CBV , and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s(-1)) faster than it is removed (0.06 s(-1)) and, therefore, is stably incorporated and highly mutagenic. We define a t oxicity index for chain terminators to account for relative rates of incorp oration versus removal. These results provide a method to rapidly screen ne w analogs for potential toxicity.