Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target

Abstract

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial monotopic membrane protein that plays an essential role in the pyrimidine de novo biosynthesis and electron transport chain pathways. In Eimeria tenella, an intracellular apicomplexan parasite that causes the most severe form of chicken coccidiosis, the activity of pyrimidine salvage pathway at the intracellular stage is negligible and it relies on the pyrimidine de novo biosynthesis pathway. Therefore, the enzymes of the de novo pathway are considered potential drug target candidates for the design of compounds with activity against this parasite. Although, DHODHs from E. tenella (EtDHODH), Plasmodium falciparum (PfDHODH), and human (HsDHODH) show distinct sensitivities to classical DHODH inhibitors, in this paper, we identify ferulenol as a potent inhibitor of both EtDHODH and HsDHODH. Additionally, we report the crystal structures of EtDHODH and HsDHODH in the absence and presence of ferulenol. Comparison of these enzymes showed that despite similar overall structures, the EtDHODH has a long insertion in the N-terminal helix region that assumes a disordered configuration. In addition, the crystal structures revealed that the ferulenol binding pocket of EtDHODH is larger than that of HsDHODH. These differences can be explored to accelerate structure-based design of inhibitors specifically targeting EtDHODH.

Plasmodium Parasite Malate-Quinone Oxidoreductase Functionally Complements a Yeast Deletion Mutant of Mitochondrial Malate Dehydrogenase

“…4 ), because of the additional target (i.e., PfMQO) present in the strain, confirming that PfMQO contributes to both the energy metabolism of the TCA cycle and the ETC in yeast mitochondria. It is of interest that ferulenol is known to inhibit several ETC dehydrogenases, including MQO and succinate dehydrogenase, but the degree of inhibition in this class of enzyme differs among species ( 41 ). Similarly, the reported sensitivity to ferulenol in rat succinate dehydrogenase (IC 50 of 17 μM and no apparent inhibition at 1.5 μM) was much weaker than our observation for yeast succinate dehydrogenase ( 42 ).…”

Biochemical Studies of Mitochondrial Malate: Quinone Oxidoreductase from Toxoplasma gondii

“…Ferulenol is a sesquiterpene prenylated coumarin isolated from Ferula communis [79] that is reported to have antibacterial [80], anti-coagulant [81][82][83], and anti-cancer [84] activities. Several targets of ferulenol have been identified so far, such as vitamin K epoxide reductase of rat [85] and bacteria [86], complex II [79,87], PfMQO [23], Eimeria tenella DHODH [88], TAO, trypanosomal glycerol kinase [34,89,90], and human DHODH [88]. In this study, we showed that ferulenol is also an inhibitor of TgMQO; however, its IC 50 was 14 times higher (0.822 ± 0.151 µM) than that of PfMQO (0.057 µM) (Figure 5a) [23].…”

“…The electrophoresis was carried out at 25 mA for 90 min at room temperature, and the gel was washed three times for 5 min with purified water and stained with GelCode TM Blue Safe Protein stain (Thermo Fisher Scientific, Waltham, MA, USA) according to the provider's manual. hrCNE was performed by adapting the reported protocol for DHODH [88]. Briefly, purified TgMQO was diluted to 0.3, 0.1, and 0.03 mg/mL in 50 mM MOPS pH 8.0, 0.05% (v/v) n-dodecyl β-D-maltoside (DDM, Sigma), 0.05% (v/v) sodium deoxycholate (DOC, Nacalai Tesque, Kyoto, Japan), 0.0001% (w/v) ponceau S (MP Biomedicals LLC, Illkirch, France), and 5% (v/v) glycerol (final concentrations).…”

Distinct non-synonymous mutations in cytochrome b highly correlate with decoquinate resistance in apicomplexan parasite Eimeria tenella

“…The hydrophobic subunit of cytochrome b is an essential component of the cyt bc 1 complex, which functions as a proton-motive ubiquinol [ 44 ] and plays a crucial role in oxidizing ubiquinol (QH 2 ) to ubiquinone (Q) and donating electrons to cytochrome c oxidase. In parasitic protozoa, including E. tenella , dihydroorotate dehydrogenase (DHODH) also acts as an essential enzyme for the de novo synthesis of pyrimidines, which is the exclusive pathway to synthesize pyrimidines [ 45 , 46 ]. However, in one study, the addition of uracil to the medium did not have a substantial effect on the in vitro anti-parasite activity of decoquinate, suggesting that de novo pyrimidine synthesis was not the primary biochemical target of decoquinate in T. gondii [ 36 ].…”