Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase

Abstract

Incorporation of nucleoside analogues by the mitochondrial DNA polymerase has been implicated as the primary cause underlying many of the toxic side effects of these drugs in HIV therapy. Recent success in reconstituting recombinant human enzyme has afforded a detailed mechanistic analysis of the reactions governing nucleotide selectivity of the polymerase and the proofreading exonuclease. The toxic side effects of nucleoside analogues are correlated with the kinetics of incorporation by the mitochondrial DNA polymerase, varying over 6 orders of magnitude in the sequence zalcitabine (ddC) > didanosine (ddI metabolized to ddA) > stavudine (d4T) >> lamivudine (3TC) > tenofovir (PMPA) > zidovudine (AZT) > abacavir (metabolized to carbovir, CBV). In this review, we summarize our current efforts to examine the mechanistic basis for nucleotide selectivity by the mitochondrial DNA polymerase and its role in mitochondrial toxicity of nucleoside analogues used to treat AIDS and other viral infections. We will also discuss the promise and underlying challenges for the development of new analogues with lower toxicity.

Figure 1:

Toxicity and structures of nucleoside analogues. The structures of various nucleoside analogues are shown over a bar graph showing mitochondrial toxicity on a log scale spanning 6 orders of magnitude. There are currently seven FDA-approved nucleoside/nucleotide analogues, zalcitabine (ddC), lamivudine ((−)3TC), didanosine (ddI which is metabolized to ddA), zidovudine (AZT), stavudine (D4T), abacavir (prodrug form of CBV), and tenofovir (PMPA). We also show (+) 3TC (β-d-(+)-2′,3′-dideoxy-3′-thiacytadine), ddA (2′,3′-dideoxyadenosine), and 1-(2-deoxy-2-fluoro-γ-d-arabinofuranosyl)-5-iodoracil (FIAU) (see inset), which led to the deaths of five patients in a hepatitis B clinical trial; unlike other analogues, FIAU is not a chain terminator, and it is extended rather than removed 60% of the time, leading to severe toxicity probably due to mutagenesis of the mitochondrial genome (5). The calculation of the toxicity index is described in the text. Note that the definition given here differs slightly from that used previously (5) in that we have removed the value of 1 so that now the index refers to a fractional increase in time rather than the fold increase in time required to replicate the mitochondrial genome. For example, the 1.2-fold increase for (−)3TC is now reported as 0.2-fold (20%) increase in time. We also have introduced the results of a more accurate measurement of the rate of exonuclease removal of ddC (0.0004 s⁻¹, J. Hanes and K. A. Johnson, unpublished results), and corrected minor math errors in the previous report of the toxicity index (5). The values for the toxicity index in order from CBV to ddC are 0.03, 0.20, 0.40, 1.5, 2.6, 6.8, 2000, 5600, and 9000.

Figure 2:

Therapeutic index of nucleoside analogues. The theoretical, enzyme-based therapeutic index is presented on a log scale for the seven nucleoside analogues approved by the FDA. The therapeutic index is calculated as the ratio of the discrimination by RT divided by the discrimination by Pol γ as described in the text and in ref . The year in which each analogue was approved for AIDS treatment by the FDA is shown under each bar.

Figure 3:

Contributions of K_d and k_pol to discrimination. The ratio of K_d values for each analogue triphosphate divided by the K_d for the normal dNTP $K_{\mathrm{d}}^{\text{AnaTP}}∕K_{\mathrm{d}}^{\text{dNTP}}$, and the corresponding ratios of k_pol values $k_{\text{pol}}^{\text{dNTP}}∕k_{\text{pol}}^{\text{AnaTP}}$ are shown. The values are displayed on a log scale to represent the contributions of ground-state binding (K_d, darker color) and rate of polymerization (k_pol, lighter color) to the net discrimination (k_pol/K_d), represented by the sum of the two. For those analogues that bind more tightly than the normal nucleotide, the bar for the K_d contribution is negative and therefore is superimposed on the selectivity factor resulting from k_pol to get the net discrimination. On the right-hand axis, the same information is displayed on a free energy scale where ΔΔG = RT ln(R), where R is the ratio of K_d or k_pol values. Formally, the free energy scale for the ratio of rates equates to ΔΔG^‡, differences in the free energy of activation. Note that on this scale, a positive ΔΔG equals the free energy difference favoring the normal nucleotide over the analogue, whereas a negative value that the analogue is favored over the normal nucleotide. Values of K_d and k_pol ratios are from ref .