Atovaquone Tolerance in Plasmodium falciparum Parasites Selected for High-Level Resistance to a Dihydroorotate Dehydrogenase Inhibitor

Abstract

Atovaquone is a component of Malarone, a widely prescribed antimalarial combination, that targets malaria respiration. Here we show that parasites with high-level resistance to an inhibitor of dihydroorotate dehydrogenase demonstrate unexpected atovaquone tolerance. Fortunately, the tolerance is diminished with proguanil, the second partner in Malarone. It is important to understand such "genetic cross talk" between respiration and pyrimidine biosynthesis since many antimalarial drug development programs target these two seemingly independent pathways. Malarone is an antimalarial drug combination, often prescribed for travelers as a prophylactic. but its high cost so far has limited its general use in countries where malaria is endemic. The patent for this drug expired in 2013, and its global usage could soon surge. Atovaquone and proguanil are the synergistic pair of antimalarials that make up this effective, well-tolerated cocktail. While the mechanism of action for proguanil remains uncertain (1, 2), it is well know that atovaquone targets the cytochrome bc 1 complex of the Plasmodium falciparum respiratory chain (3, 4). One function of this mitochondrial pathway is to generate a proton gradient that is required for maintaining the membrane potential for important processes such as protein synthesis, heme biogenesis, and the citric acid cycle (reviewed in reference 2). Importantly, the P. falciparum cytochrome bc 1 complex has been implicated in the direct regeneration of oxidized ubiquinone for dihydroorotate dehydrogenase (DHODH), a key enzyme in the biosynthesis of pyrimidine nucleotides (5-7). Thus, DHODH is physically and functionally tied to the respiratory chain.Recently, P. falciparum parasites resistant to DSM1, a potent inhibitor of DHODH (8, 9), were generated at two levels (333 nM for round 1 and 3 or 10 M for round 2) (10). Amplification of the genome segments that encompassed the DHODH gene were responsible for the observed phenotype. Whole-genome sequence methods ruled out resistance-conferring mutations elsewhere, and no alterations in the sensitivities to several unrelated antimalarials were detected. However, here we report the unexpected development of tolerance to atovaquone in parasites resistant to higher levels of the DHODH inhibitor DSM1 (round 2).Following DSM1 selection and subcloning (10), atovaquone sensitivity was tested. Round 1 parasites (ϳ3-fold DSM1 resistance) exhibited wild-type sensitivity to atovaquone in a 72-h assay (Fig. 1A and Table 1). Long-term exposure to low levels of atovaquone confirmed the sensitivity of these parasites: 10 nM atovaquone completely prevented the proliferation of parental Dd2, as well as round 1 parasites within 2 days, and continued to do so over an extended period of 8 days (Fig. 1C and D, red lines). We further assessed the survival of Dd2 and round 1 clone C by exposing small clonal populations of parasites to atovaquone before extensive washing and replating in the absence of drug and following the number of viable colonies over 60 days (11)...

Selection of Plasmodium falciparum cytochrome B mutants by putative PfNDH2 inhibitors

“…Results showed similar nanomolar EC 50 sensitivities of the Dd2, 106/1, 3D7, and HB3 control parasites to ATQ, RYL-552, and CK-2-68. Activities of the Q i antagonist AMA, chloroquine (CQ), and the PfDHODH inhibitor DSM1 were also in the expected ranges for these four controls (31)(32)(33)(34)(35). The clones from the ETC inhibitor-selected lines retained the same CQ responses as those of the original Dd2 (CQ-resistant) and 106/1 (CQ-sensitive) clones ( Table 2).…”

“…The nuclear pfdhod gene is normally single-copy in P. falciparum but amplification has been found with increases in ATQ EC 50 levels upon DSM1 selection (28,35,38). We tested for pfdhod CNV in the clones listed in Table 2.…”

Identification of inhibitors that dually target the new permeability pathway and dihydroorotate dehydrogenase in the blood stage of Plasmodium falciparum

“…It is well established that proguanil (whose metabolised form cycloguanil, inhibits dihydrofolate reductase) synergises with cytochrome bc1 inhibitors both increasing their potency and abrogating yDHODH-conferred resistance2526. To determine whether our MMV compounds were inhibiting Pf DHODH directly or indirectly by targeting bc1 all the inhibitors were reanalysed in the presence or absence of 1 μM proguanil and the results are summarised in Tables 3 and 4.…”

Ellagic acid microspheres restrict the growth of Babesia and Theileria in vitro and Babesia microti in vivo

“…The preferable treatment of babesiosis in humans is the combination of atovaquone (AQ) with azithromycin due to their low side effects [4]. Guler et al [5] reported that Plasmodium falciparum rapidly developed resistance to AQ when used as a single drug. Another report showed the relapse of Babesia gibsoni due to the change of amino acid in the mitochondrial cytochrome B that led to a reduction in the efficacy of AQ [6].…”