Martens MC, Won MM, Won HI, Schulze TT, Judge AK, Neville AJ, Vennerstrom JL, Davis PH
In Vitro Selection Implicates ROP1 as a Resistance Gene for an Experimental Therapeutic Benzoquinone Acyl Hydrazone in Toxoplasma gondii.
Antimicrobial agents and chemotherapy. 2021-02-17; 65.3:
is a globally distributed apicomplexan parasite and the causative agent of toxoplasmosis in humans. While pharmaceuticals exist to combat acute infection, they can produce serious adverse reactions, demonstrating a need for enhanced therapies. KG8 is a benzoquinone acyl hydrazone chemotype identified from a previous chemical screen for which we previously showed and efficacy against However, the genetic target and mechanism of action of KG8 remain unknown. To investigate potential targets, we generated resistant lines by chemical mutagenesis followed by selection. Whole-genome sequencing of resistant clones revealed a P207S mutation in the gene encoding rhoptry organelle protein 1 (ROP1) in addition to two lesser resistance-conferring mutations in the genes for rhoptry organelle protein 8 (ROP8) and a putative ADP/ATP carrier protein (TGGT1_237700). Expressing ROP1 in parental parasites was sufficient to confer significant (10.3-fold increased half-maximal effective concentration [EC]) KG8 resistance. After generating a library of mutants carrying hypermutated alleles followed by KG8 pressure, we sequenced the most resistant clonal isolate (>16.9-fold increased EC) and found independent recapitulation of the P207S mutation, along with three additional mutations in the same region. We also demonstrate that a knockout strain is insensitive to KG8. These data implicate ROP1 as a putative resistance gene of KG8. This work further identifies a compound that can be used in future studies to better understand ROP1 function and highlights this novel chemotype as a potential scaffold for the development of improved therapeutics.
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