Targeting Plasmodium falciparum IspD in the Methyl‑d‑erythritol Phosphate Pathway: Urea-Based Compounds with Nanomolar Potency on Target and Low-Micromolar Whole-Cell Activity

The methyl-d-erythritol phosphate (MEP) pathway has emerged as an interesting target in the fight against antimicrobial resistance. The pathway is essential in many human pathogens, including Plasmodium falciparum (Pf), but is absent in human cells. In the present study, we report on the discovery of a new chemical class targeting IspD, the third enzyme in the pathway. Exploration of the structure–activity relationship yielded inhibitors with potency in the low-nanomolar range. Moreover, we investigated the whole-cell activity, mode of inhibition, metabolic, and plasma stability of this compound class, and conducted in vivo pharmacokinetic profiling on selected compounds. Lastly, we disclosed a new mass spectrometry (MS)-based enzymatic assay for direct IspD activity determination, circumventing the need for auxiliary enzymes. In summary, we have identified a readily synthesizable compound class, demonstrating excellent activity and a promising profile, positioning it as a valuable tool compound for advancing research on IspD.