Abstract 306 - Mechanistic Studies of Dioxolane Guanosine 5´- Triphosphate: Implications for Efficacy, Lack of Cross- Resistance and Selectivity of DAPD.

306 Mechanistic Studies of Dioxolane Guanosine 5´- Triphosphate: Implications for Efficacy, Lack of Cross- Resistance and Selectivity of DAPD.

J. Feng, J. Jeffrey, K. Anderson, W. Copeland, and P. Furman.
Triangle Pharmaceuticals, Inc., Durham, NC.

Background:DAPD, a prodrug of dioxolane guanosine (DXG), is a nucleoside analog currently in Phase I/II clinical trials for the treatment of HIV infection. DAPD is deaminated by adenosine deaminase to DXG, which is subsequently phosphorylated to the corresponding 5´-triphosphate, DXG-TP.

Methods:Steady-state and pre-steady-state kinetics were used to evaluate the activity of DXG-TP against wild- type and resistant HIV-1 RT and human cellular DNA polymerasesα;,bandg.

Results:DXG-TP was shown to be a potent alternative substrate inhibitor of the HIV RT, with a K_Ivalue of 0.019 + 0.002mM. Pre-steady-state kinetic studies demonstrated that the binding affinity of DXG-TP for the RT was similar to that determined for dGTP with either a DNA or RNA template. However, dGTP was incorporated 11 and 23 times faster than DXG-TP when the template was DNA or RNA, respectively. Comparing the overall efficiency of incorporation of dGTP with that of DXG-TP showed that the RT incorporated dGTP 17 times more efficiently than DXG-TP. Furthermore, the overall efficiency of incorporation of DXG-TP was not affected by mutations conferring resistance to zidovudine or lamivudine. To give further insight into the origin of the selective activity of DAPD/DXG, steady-state kinetic studies with human DNA polymerasesα;,bandgwere performed. DXG-TP was a relatively weak inhibitor of human DNA polymerasesα;andb; however, DNA polymerasegproved to be somewhat sensitive to inhibition by DXG-TP. Nevertheless, mitochondrial toxicity studies performed using HepG2 cells showed that DXG was not toxic to mitochondria at concentrations up to and including 50mM. A pre-steady-state kinetic study showed that DXG-TP was a poor substrate for DNA polymerasegcompared with dideoxy-CTP, a potent inhibitor of mitochondrial DNA synthesis. Excision of the terminally incorporated DXG-MP by thegpolymerase-associated exonuclease was not observed during a 12- hour incubation.

Conclusions:These data provide a mechanistic explanation for the overall activity and lack of toxicity for DAPD/DXG and the lack of cross-resistance to viruses resistant to lamivudine and zidovudine.