Summary of resistance selection.

<div><p>The <i>Plasmodium falciparum</i> cytoplasmic tyrosine tRNA synthetase (<i>Pf</i>TyrRS) is an attractive drug target that is susceptible to reaction-hijacking by AMP-mimicking nucleoside sulfamates. We previously identified an exemplar pyrazolopyrimidine ribose sulfamate, ML901, as a potent reaction hijacking inhibitor of <i>Pf</i>TyrRS. Here we examined the stage specificity of action of ML901, showing very good activity against the schizont stage, but lower trophozoite stage activity. We explored a series of ML901 analogues and identified ML471, which exhibits improved potency against trophozoites and enhanced selectivity against a human cell line. Additionally, it has no inhibitory activity against human ubiquitin-activating enzyme (UAE) <i>in vitro</i>. ML471 exhibits low nanomolar activity against asexual blood stage <i>P</i>. <i>falciparum</i> and potent activity against liver stage parasites, gametocytes and transmissible gametes. It is fast-acting and exhibits a long <i>in vivo</i> half-life. ML471 is well-tolerated and shows single dose oral efficacy in the SCID mouse model of <i>P</i>. <i>falciparum</i> malaria. We confirm that ML471 is a reaction hijacking inhibitor that is converted into a tight binding Tyr-ML471 conjugate by the <i>Pf</i>TyrRS enzyme. A crystal structure of the <i>Pf</i>TyrRS/ Tyr-ML471 complex offers insights into improved potency, while molecular docking into UAE provides a rationale for improved selectivity.</p></div>