Tyrosine Amide and Triazaspirocyclic Antimalarials Are Resistant to the Mutant Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL)

Tyrosine amide and triazaspirocyclic chemotypes were identified via high-throughput screening against P. falciparum parasites. Chemical synthesis confirmed potently active stereoisomers for both hit classes. Whole-genome sequencing of resistant lines, combined with pooled resistance barcoding, revealed distinct mutations in the cyclic amine resistance locus (PfCARL) and the UDP-galactose transporter (PfUDP), two ER transmembrane proteins previously associated with resistance to the clinical candidate KAF156. Live-cell thermal proteome integral solubility assays suggest that these compounds target proteins connected with ER-mediated trafficking, supporting the role of PfCARL and PfUDP as multidrug resistance loci rather than direct targets. Both chemotypes disrupted protein export, arrested parasites from late ring-to-early trophozoite stages, and inhibited liver schizont and gamete development. In addition, both tyrosine amide and triazaspirocyclic analogs exhibited high solubility and metabolic stability, highlighting their promise for future development as next-generation multistage antimalarials.