Targeting Plasmodium falciparum IspD in the Methyl-D-Erythritol Phosphate Pathway: Urea-Based Compounds with Nanomolar Potency on target with 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, reducing the risk of off-target side effects. In the present study, we conducted a high-throughput screening on the third enzyme of the pathway, IspD, and discovered a new chemical class for which we ran a structure–activity relationship investigation, resulting in low-nanomolar inhibitors of PfIspD. In addition, we determined the whole-cell activity (PfNF54 IC50 = 3.4 ± 1.0 μM), mode of inhibition, metabolic, and plasma stability of the new compound class. In vivo pharmacokinetic profiling of a selection of compounds demonstrated promising behavior for future development. Lastly, we disclosed a new MS-based enzymatic assay for direct IspD activity determination, circumventing the need for auxiliary enzymes. We used this assay to investigate the mode of inhibition of our new compound class. 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.