Minimum Inoculum of Resistance Assay for Evaluating Antitoxoplasmosis Compounds That Target Phenylalanine tRNA...

Toxoplasma gondii 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. Herein, we developed a minimum inoculum for resistance assay as a semiquantitative metric for evaluating inhibitors of T. gondii. The resistance assay, adapted from malaria, measures the frequency of pre-existing resistance alleles by exposing different-sized parasite populations to drug pressure. We profiled a series of bicyclic pyrrolidone analogues that inhibit phenylalanine tRNA synthetase. We demonstrate that these inhibitors require higher inocula to lead to parasite resistance (up to >10⁸ parasites) in comparison with an inhibitor of DNA synthesis and that resistance values vary across inhibitors with closely related chemical structures. Clonal analysis of resistant parasites emerging from resistance assays revealed both new and previously identified resistance-conferring mutations in T. gondii phenylalanine tRNA synthetase, and structural modeling revealed their potential impact on the enzyme active site. The minimum inoculum for resistance 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.