Reverse Fosmidomycin Analogs as Bisubstrate Inhibitors: Binding Mode Elucidation and Mechanistic Insights

Inhibitors of the 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the nonmevalonate pathway, represent a promising class of antiplasmodial compounds, as DXR is essential for human pathogens but absent in their host. The natural product fosmidomycin was the first clinical DXR inhibitor, however high rates of recrudescence attributed to the polar phosphonic acid group have prevented progression beyond phase II clinical trials. Herein, lipophilic N-benzamidoalkyl and N-phthalimidoalkyl substituents were introduced into reverse α-phenyl fosmidomycin derivatives to mimic the nicotinamide moiety of the NADPH cofactor and enhance antiplasmodial activity. Elongation of the alkyl linker markedly improved enzymatic inhibition (15e PfDXR IC₅₀ = 4.3 nM) and antiplasmodial activity (16e PfDd2 IC₅₀ = 0.28 μM). Mode of inhibition studies showed competitive inhibition with the DXR substrate and the cofactor NADPH (16e K_i = 0.068 μM). Co-crystallization with the PfDXR enzyme revealed that the introduced residues bind within the NADPH binding site.