Minimum inoculum of resistance assay for evaluating for anti-toxoplasmosis compounds that target phenyl alanine tRNA synthetase

<i>Toxoplasma gondii</i> is a globally important intracellular parasite, and treatment regimens are limited by the failure of drugs to target latent tissue cysts. Developing new candidates for treatment also needs to address the potential for resistance to arise. Here, we developed a Minimum Inoculum for Resistance (MIR) assay as a quantitative metric for evaluating inhibitors of <i>T. gondii</i> . The MIR assay, adapted from assays used in malaria drug discovery, measures the frequency for pre-existing resistance alleles by exposing different sized parasite populations to drug pressure. We profiled a series of bicyclic pyrrolidone analogs that inhibit phenylalanine tRNA synthetase (PheRS). We demonstrate that these inhibitors require higher inocula to lead to parasite resistance (up to > 10 ⁸ parasites) in comparison with an inhibitor of DNA synthesis, and that MIR values vary across inhibitors with closely related chemical structures. Clonal analysis of resistant parasites emerging from MIR assays revealed both new and previously identified resistance conferring mutations in <i>Tg</i> PheRS, and structural modeling revealed their potential impact the enzyme active site. The MIR assay provides a functional benchmark to compare new and existing inhibitors, allowing for rational prioritization of lead compounds with a high genetic barrier to resistance.