Methods: ELQ-316 resistant parasites were isolated by exposing the RH ∆uprt strain to the mutagen, ENU, followed by continuous ELQ-316 pressure. We obtained a clonal population of parasites through limiting dilution. mRNA was isolated from two different resistant clones ER1 and ER2, the parental RH ∆uprt strain, and the ME49 strain. The cytochrome b gene was amplified from mRNA by reverse transcriptase PCR and sequenced. In vitro growth inhibition of T. gondii was quantified by a crystal violet cell lysis assay. Cytochrome bc1 inhibition was determined by measuring the rate of cytochrome c reduction by T. gondii mitochondrial fragments across a range of inhibitor concentrations.
Results: The cytochrome b gene of both ELQ-316 resistant clones showed an A -> C point mutation at position 664, resulting in a Thr -> Pro substitution at position 222 in the transcribed protein. Measurement of growth inhibition by the cell lysis assay showed that the T222P mutation was associated with resistance to ELQ-316 (3.4nM vs 155 nM), ELQ-271 (6.4nM vs 17nM) and antimycin A (47nM vs 525 nM). Likewise, the bc1 with the T222P mutation displayed elevated EC50s to ELQ-316 compared to the parental strain (41pM vs 9.0nM) and antimycin A (439pM vs 72nM).
Conclusion: The association of the T222P mutation with resistance to ELQ-316, ELQ-271 and antimycin A suggests that ELQ-316 inhibits the Qi site of the T gondii cytochrome bc1. This finding provides insight into the structural interactions of Qi site inhibitors that will aid in the development of cytochrome bc1 inhibitors for clinical use.
P. H. Alday Jr.,
S. Pou, None
A. Nilsen, None
M. K. Riscoe, None
J. Doggett, None