Discovery of Inhibitors of Lupin Diadenosine 5′,5′-P1,P4-Tetraphosphate Hydrolase by Virtual Screening

Novel inhibitors of lupin diadenosine 5',5'-P-1,P-4-tetraphosphate (Ap(4)A) hydrolase have been identified by in silico screening of a large virtual chemical library. Compounds were ranked on the basis of a consensus from six scoring functions. From the top 100 ranked compounds six were selected and initially screened for inhibitory activity using a single concentration isothermal titration calorimetry assay. Two of these compounds that showed excellent Solubility properties were further analyzed, but only one [NSC51531; 2-((8-hydroxy-4-(4-methyl-2-sulfoanilino)-9,10-dioxo-9,10-dihydro-1-anthracenyl)amino)5-methylbenzenesulfonic acid] exhibited competitive inhibition with a K-i of 1 mu M. A Structural analogue of this compound also exhibited competitive inhibition with a comparable K-i of 2.9 mu M. H-1, N-15 NMR spectroscopy was used to map the binding site of NSC51531 on lupin Ap(4)A hydrolase and demonstrated that the compound bound specifically in the substrate-binding site, consistent with the competitive inhibition results. Binding of NSC51531 to the human form of Ap(4)A hydrolase is nonspecific, suggesting that this compound may represent a useful lead in the design of specific inhibitors of the plant-like form of Ap(4)A hydrolases.