Effects of atovaquone and diospyrin-based drugs on the cellular ATP of Pneumocystis carinii f. sp. carinii

Abstract

Atovaquone (also called Mepron, or 566C80) is a napthoquinone used for the treatment of infections caused by pathogens such as Plasmodium spp. and Pneumocystis carinii. The mechanism of action against the malarial parasite is the inhibition of dihydroorotate dehydrogenase (DHOD), a consequence of blocking electron transport by the drug. As an analog of ubiquinone (coenzyme Q [CoQ]), atovaquone irreversibly binds to the mitochondrial cytochrome bc(1) complex; thus, electrons are not able to pass from dehydrogenase enzymes via CoQ to cytochrome c. Since DHOD is a critical enzyme in pyrimidine biosynthesis, and because the parasite cannot scavenge host pyrimidines, the drug is lethal to the organism. Oxygen consumption in P. carinii is inhibited by the drug; thus, electron transport has also been identified as the drug target in P. carinii. However, unlike Plasmodium DHOD, P. carinii DHOD is inhibited only at high atovaquone concentrations, suggesting that the organism may salvage host pyrimidines and that atovaquone exerts its primary effects on ATP biosynthesis. In the present study, the effect of atovaquone on ATP levels in P. carinii was measured directly from 1 to 6 h and then after 24, 48, and 72 h of exposure. The average 50% inhibitory concentration after 24 to 72 h of exposure was 1.5 microgram/ml (4.2 microM). The kinetics of ATP depletion were in contrast to those of another family of naphthoquinone compounds, diospyrin and two of its derivatives. Whereas atovaquone reduced ATP levels within 1 h of exposure, the diospyrins required at least 48 h. After 72 h, the diospyrins were able to decrease ATP levels of P. carinii at nanomolar concentrations. These data indicate that although naphthoquinones inhibit the electron transport chain, the molecular targets in a given organism are likely to be distinct among members of this class of compounds.

FIG. 1

Structures of naphthoquinone drugs tested. (A) Atovaquone; (B) diospyrin; (C) diospyrin dimethylether; (D) diospyrin dimethylether hydroquinone.

FIG. 2

Effects of atovaquone on the cellular ATP contents of P. carinii f. sp. carinii populations in vitro, expressed as percent inhibition compared to ATP levels in untreated controls. Bars represent the averages of 12 separate experiments using P. carinii isolated from different individual rats ± standard errors of the means. The three concentrations of atovaquone tested and expressed as 0.1 to 10.0 μg are equivalent to 0.27, 2.72, and 27.2 μM, respectively.

FIG. 3

ATP levels of P. carinii populations exposed to varying concentrations of atovaquone over a 6-h period. “Medium control” represents the ATP levels from P. carinii organisms unexposed to experimental compounds. Data are expressed as relative light units and are averages of nine separate readings ± standard errors of the means. The atovaquone concentrations expressed as 10.0 to 0.5 μg are equivalent to 27.2, 13.6, 2.72, and 1.36 μM, respectively.

FIG. 4

Effects of diospyrin compounds on the cellular ATP contents of P. carinii f. sp. carinii populations in vitro, expressed as percent inhibition compared to ATP levels in untreated controls. Bars represent the average results from a representative experiment performed in triplicate. The micromolar equivalents are 0.37, 3.74, and 37.4 μM for diospyrin; 0.40, 4.02, and 40.2 μM for diospyrin dimethylether; and 0.41, 4.06, and 40.6 μM for diospyrin dimethylether hydroxyquinone.

FIG. 5

Effects of pentamidine on the cellular ATP contents of P. carinii f. sp. carinii populations in vitro, expressed as percent inhibition compared to ATP levels in untreated controls. Bars represent the averages ± standard errors of the means of three to nine separate experiments using different P. carinii populations. The micromolar equivalents of the concentrations of pentamidine evaluated are 0.169, 1.69, and 16.87 μM.