© 2017 American Chemical Society. SmNACE is a NAD catabolizing enzyme expressed on the outer tegument of S. mansoni, a human parasite that is one of the major agents of the neglected tropical disease schistosomiasis. Recently, we identified aroylhydrazone derivatives capable of inhibiting the recombinant form of the enzyme with variable potency (IC50 ranging from 88 μM to 33 nM). In the present study, we investigated the mechanism of action of the least potent micromolar inhibitor (compound 1) and the most potent nanomolar inhibitor (compound 2) in the series on both the recombinant and native SmNACE enzymes. Using mass spectroscopy, spectrophotometry, and activity assays under different experimental conditions, we demonstrated that the >3 log gain in potency against recombinant SmNACE by this class of compounds is dependent on the formation of a coordination complex with metal cations, such as Ni(II), Zn(II), and Fe(II), that are loaded on the protein surface. Testing the compounds on live parasites, we observed that only the weak micromolar compound 1 was active on the native enzyme. We showed that S. mansoni effectively sequesters the metal from the coordination complex, resulting in the loss of inhibitory activity of the potent nanomolar compound 2. Importantly, the modeling of the transition complex between Zn(II) and compound 2 enabled the discovery of a new metal-independent aroylhydrazone analogue, which is now the most potent and selective inhibitor of native SmNACE known.