Chemoproteomic Strategy Identifies PfUCHL3 as the Target of Halofuginone

The human malaria parasite Plasmodium falciparum (P. falciparum) continues to pose a significant public health challenge, leading to millions of fatalities globally. Halofuginone (HF) has shown a significant anti‐P. falciparum effect, suggesting its potential as a therapeutic agent for malaria treatment. In this study, we synthesized a photoaffinity labeling probe of HF to identify its direct target in P. falciparum. Our results reveal that ubiquitin carboxyl‐terminal hydrolase 3 (PfUCHL3) acts as a crucial target protein of HF, which modulates parasite growth in the intraerythrocytic cycle. In particular, we discovered that HF potentially forms hydrogen bonds with the Leu10, Glu11, and Arg217 sites of PfUCHL3, thereby inducing an allosteric effect by promoting the embedding of the helix 6’ region on the protein surface. Furthermore, HF disrupts the expression of multiple functional proteins mediated by PfUCHL3, specifically those that play crucial roles in amino acid biosynthesis and metabolism in P. falciparum. Taken together, this study highlights PfUCHL3 as a previously undisclosed druggable target of HF, which contributes to the development of novel anti‐malarial agents in the future. PfUCHL3 was identified as a previously unrecognized anti‐P. falciparum target of halofuginone (HF) through chemical biology strategies. The allosteric modulation of PfUCHL3 stemmed from the potential formation of three hydrogen bonds between HF and PfUCHL3, thereby perturbing the protein‐protein interaction network. PfUCHL3 played a crucial role in regulating the amino acid biosynthesis and metabolism, thereby inhibiting the growth of P. falciparum.