2249. Mechanism of Action of ELQ-316 Against Toxoplasma gondii: Evidence for Qi Site Inhibition of Cytochrome bc1.
Session: Poster Abstract Session: New Antibiotics in Development
Saturday, October 29, 2016
Room: Poster Hall
Background: The endochin-like quinolones (ELQs) are a series of preclinical compounds with broad activity against apicomplexan parasites, including PlasmodiumBabesia and Toxoplasma. ELQ-316 is highly effective in a murine model of acute and latent toxoplasmosis and has an in vitro IC50 in the picomolar range. Prior experiments in S. cerevisiae and P. falciparum have suggested that the mechanism of action of ELQs is cytochrome bc1 inhibition. Using chemical mutagenesis and ELQ-316 exposure, we selected a T gondii clone with resistance to ELQ-316, ELQ-271 and antimycin A, a well-characterized inhibitor of the cytochrome bc1 Qi site.

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. Holland Alday Jr., MD, PhD1,2, Igor Bruzal, PhD1,2, Sovitj Pou, PharmD2, Aaron Nilsen, PhD2,3, Michael K Riscoe, PhD2,3 and Joseph Doggett, MD1,2, (1)Division of Infectious Diseases, Oregon Health and Science University, Portland, OR, (2)Portland VA Medical Center, Portland, OR, (3)Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR

Disclosures:

P. H. Alday Jr., None

I. Bruzal, None

S. Pou, None

A. Nilsen, None

M. K. Riscoe, None

J. Doggett, None

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