Deconstruction of Aspartic Protease Inhibitors Enables Fragment-Based Discovery of Plasmepsin V Inhibitors.

Malaria, caused by the Plasmodium parasites, remains a major global health burden, and resistance to current antimalarials drives the need for drugs with new mechanisms of action. Plasmepsin V (PMV), an essential aspartic protease required for PEXEL processing and protein export, is structurally divergent from human aspartic proteases, offering a path to selective inhibition. Here, we applied a structure-informed, deconstruction-based approach to identify non-peptidomimetic PMV inhibitors by mining catalytic dyad-binding motifs from experimentally solved aspartic protease-inhibitor complexes and assembling a focused fragment-like library. Fragment-inspired model compounds based on pyrrolidine, piperidine, and piperazine cores showed measurable PMV inhibition in a Forster resonance energy transfer (FRET)-based assay, and a trans-3,4-disubstituted pyrrolidine hit (7a; IC50 70  µM) was selected for optimization. Guided by PMV structural data and a renin cocrystal structure of a related pyrrolidine inhibitor, we explored SAR around substituents intended to engage the S1 and S2 regions. Optimization identified N-sulfonamide analogs bearing two aromatic substituents as a preferred chemotype, with meta-substitution on the N-aryl group improving potency to the low micromolar range. The most potent compounds obtained displayed ~10 µM potency, establishing a promising non-peptidomimetic scaffold for further development of selective PMV inhibitors.