Killing of Anopheles stephensi mosquitoes by selective triketone inhibitors of 4-hydroxyphenylpyruvate dioxygenase depends on a high protein meal.

The malaria vector Anopheles stephensi has expanded from Asia into Eastern Africa, posing a growing global health threat due to its adaptive biology and increasing resistance to conventional control methods. Here, we characterise 4-hydroxyphenylpyruvate dioxygenase (HPPD), a crucial enzyme in the tyrosine degradation pathway, and demonstrate its potential as a novel drug target in An. stephensi. Homology modeling combined with molecular dynamics simulations confirmed that key inhibitor-binding residues are highly conserved across mosquito HPPDs and predicted potent inhibition by triketone-based compounds. Using cell-based assay with codon-optimized recombinant expression in Escherichia coli, we screened several triketone and diketonitrile HPPD inhibitors and identified nitisinone as the most potent inhibitor, displaying nanomolar-range IC50 values. Membrane feeding assays showed that nitisinone's insecticidal activity relies on ingestion of a high-protein meal, with haemoglobin identified as the potent dietary factor driving toxicity. These results highlight HPPD inhibition as a promising blood-meal-dependent vector control strategy specifically targeting haematophagous mosquitoes.