1. The anti-HIV agents 2',3'-dideoxyadenosine (ddAdo) and (2'-beta- fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo) are rapidly converted both in vitro and in vivo to the corresponding inosine analogs by the widely distributed enzyme adenosine deaminase (EC 3.5.4.4.4). We have determined the effects of the potent adenosine deaminase inhibitor 2'-deoxycoformycin (2'-dCF) on ddAdo and 2'-beta-F-ddAdo metabolism in MOLT-4 cells and on ddAdo antiviral activity in the ATH8 test system. At levels as low as 5 nM in the incubation medium, 2'-dCF effectively blocks the extracellular deamination of both agents, thus permitting their rapid cellular uptake as the unchanged parent compounds. In anti-HIV assays, an increase of 2.2- fold was seen in ddAdo antiviral potency at 2'-dCF levels of 20 and 50 nM. 2. The anti-HIV thymidine analogue 2',3'-dideoxythymidinene (D4T) has been the subject of extensive clinical evaluation, but there are several aspects of D4T pharmacology which are still unexplored. Since the 5'- phosphorylation of D4T is catalyzed by thymidine kinase, we have examined the effects of agents which alter the activity of this enzyme on D4T phosphorylation. Agents which lower dTTP (e.g., deoxyguanosine) stimulate D4T phosphorylation. Conversely, agents which cause an increase in dTTP pools (e.g. ribavirin, mycophenolic acid) inhibit D4T phosphorylation because of feedback inhibition of the kinase. 3. Competitive PCR assay for mitochondrial DNA. Several dideoxynucleoside anti-HIV agents (particularly ddCyd and its analogues) are thought to exert their toxic effects by depleting mitochondrial DNA, presumably by inhibition of DNA polymerase gamma. We have developed a rapid and sensitive competitive PCR assay for mitochondrial DNA content which can be used to quantitate mtDNA in total cellular DNA samples of as little as 25 ng. The method should be of value in preclinical and Phase I/II studies of new antiviral and antitumor agents with possible mitochondrial toxicity.
