Ye, X.; Esherick, L. Y.; Ketprasit, N.; Narwal, S. K.; Godoy, L. C.; Boonyalai, N.; Dogovski, C.; Morton, C. J.; Rabie, T.; Famodimu, M.; Tai, C.-W.; Yeo, T.; Le, L. H. M.; Leeming, M. G.; Laureano De Souza, M.; Chenu, E.; Creek, D. J.; Delves, M.; Birkholtz, L.-M.; Duffy, J.; Lobb, K.; Durst, G.; Lee, M. C. S.; Fidock, D.; Niles, J.; Siegel, M. G.; Tilley, L.; Xie, S. C.

Malaria remains one of the major threats to human health. Breakthrough drugs with high potency and low resistance risk are needed to combat the ever-increasing resistance to currently deployed antimalarials. Here, we explore a series of 4-amino-quinazoline-based sulfonamides, with drug-like physicochemical parameters and a synthetically accessible scaffold. Exemplars exhibit nanomolar potency against blood stage Plasmodium cultures, with up to 300-fold selectivity compared with a mammalian cell line. The compounds are also active against transmissible stages of P. falciparum and are refractory to resistance development. Targeted mass spectrometry reveals that the compounds act as reaction hijacking inhibitors targeting P. falciparum aminoacyl tRNA synthetases (aaRSs). Subtle changes to the chemical structure switch the main target from cytoplasmic tRNA threonine synthetase (PfThrRS) to cytoplasmic asparagine synthetase (PfAsnRS), a change that is associated with increased potency and selectivity. The target preference was confirmed by selective knock-down of different P. falciparum aaRSs and by tolerance selection in a mutator line. Consistent with aaRS targets, exemplar compounds activate the amino acid starvation response. Recombinant enzyme inhibition and thermal stabilisation assays confirm the susceptibility of PfAsnRS to reaction hijacking and show that human AsnRS is less susceptible. A molecular model of Asn-tRNA-bound PfAsnRS reveals that a potent hijacker adopts a pose similar to adenosine 5-monophosphate (AMP). An AlphaFold model of the native PfAsnRS dimer helps explain the tolerance-conferring effect of a mutation at the dimer interface.