Potent reaction hijacking inhibitors of Plasmodium falciparum asparagine tRNA synthetase

ABSTRACT 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-aminoquinazoline-based sulfonamides (4AQS), with 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 active against transmissible stages of Plasmodium falciparum and refractory to resistance development. Targeted mass spectrometry reveals that the 4AQS 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 threonine tRNA synthetase ( Pf ThrRS) to cytoplasmic asparagine tRNA synthetase ( Pf AsnRS), a change that is associated with increased potency against P. falciparum . The target preference was confirmed by selective knockdown 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 stabilization assays confirm the susceptibility of Pf AsnRS to reaction hijacking and show that human AsnRS is less susceptible. A molecular model of Asn-tRNA-bound Pf AsnRS reveals that a potent hijacker adopts a pose similar to adenosine 5′-monophosphate (AMP). An AlphaFold model of the native Pf AsnRS dimer helps explain the tolerance-conferring effect of a mutation at the dimer interface. IMPORTANCE Every year, at least 200 million new malaria infections are diagnosed, causing more than 600,000 deaths in Africa and Southeast Asia. Ever-increasing levels of resistance to current antimalarials are leading to an impending crisis. Breakthrough drugs with new modes of action are much needed. Here, we explore a series of inhibitors that hijack critical malaria parasite enzymes. The target enzymes catalyze the formation of a bond between the hijackers and an enzyme substrate, forming a tight-binding compound that disrupts the malaria parasite’s protein translation machinery. The hijacking inhibitors exhibit high-level potency against the disease-causing stage of the malaria parasite and are also active against transmissible stages. They exhibit up to 300-fold selectivity compared with a mammalian cell line, possess drug-like chemical attributes, and are easily synthesized. Importantly, the compounds are refractory to resistance development.