Table 1.
Nucleoside and nucleotide analogs used in this study.
Table 2.
Enzyme kinetics parameter values used for polymerase-γ interaction with each activated analog, in order of decreasing kcat/Km.
Figure 1.
Schematic diagrams of the polymerase-γ reactions in this model.
(A) The four reactions possible following a correct incorporation. DNA Polymerase: polymerase-γ adds one nucleotide to the new DNA strand. This nucleotide may be the correct or incorrect (point mutation) base pair for template strand, or a nucleotide analog. Exonuclease: polymerase-γ removes one nucleotide (correct or incorrect match) from the new DNA strand. This is an error correction mechanism. Disassociation: The DNA polymerase can separate from the DNA molecule. Reassociation: The DNA polymerase re-attaches to the DNA molecule after disassociation. (B) The possible reactions after insertion of analog (Z to represent AZT in this example): exonuclease activity or dissociation. Our model does not allow further polymerization or reassociation once an analog is inserted and not removed by an exonuclease reaction.
Table 3.
Mitochondrial dNTP concentrations and Km values.
Figure 2.
The dose-response curves for incorporation probability of analogs approved for treatment.
Circles are the probability of strand termination calculated from a set of 1000 simulations for each point and the curves are dose-response curves fit to the data points. There is a large difference of incorporation probability between the di-deoxy drugs (ddC, ddI, and d4T) and other approved anti-retrovirals (3TC, TDF, AZT, ABC, and FTC). The anti-herpes drug, acyclovir, falls in the middle of these two extremes, but shows little mitochondrial toxicity in clinical use possibly due to the fact it is dependent upon viral proteins for activation. AZT2001 probabilities were determined using kinetic parameters from reference [25] and AZT2007 probabilities were determined using newly reported kinetic parameters from reference [27].
Table 4.
IC50 values for light strand termination calculated from the simulation.
Table 5.
Reported intracellular concentrations of the activated (tri-phosphate) form of the nucleoside and nucleotide analogs, measured in peripheral blood mononuclear cells in patients.
Figure 3.
The relationship between the IC50 values and the specificity constant, kcat/Km.
The specificity constant is a measurement of direct interaction with polymerase-γ often used as prediction for analog incorporation. The IC50 values are a more direct measure of incorporation probability taking exonuclease activity and other features into account. This relationship shows that the specificity constant is a useful proxy for incorporation probability.
Table 6.
The effect of the exonuclease reaction for each nucleoside or nucleotide analog, in order of increasing Vexo.
Figure 4.
The computed probability of strand termination for an AZT and 3TC combination.
Computed probabilities are shown for AZT-TP alone (at 0.5 µM), 3TC-TP alone (at 0.1 µM) and the combination of both drugs at those concentrations (solid bars). The error bars represent standard deviations from 10 repeated sets of 1000 simulations. Also shown is the predicted probability of strand termination for the combined drugs (hatched bar) with the assumption of no interaction between the two drugs, calculated from the equation given in the text.