Structure basis for the inhibitory mechanism of a novel DNase γ-specific inhibitor, DR396

DNase γ, a member of the DNase I family, has been suggested to cause DNA fragmentation during apoptosis. We recently identified 4-(4,6-dichloro-[1,3,5]-triazine-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396) as a novel specific inhibitor for human DNase γ [Sunaga, S.; Kobayashi, T.; Yoshimori, A.; Shiokawa, D.; Tanuma, S. Biochem. Biophys. Res. Commun. 2004, 325, 1292]. However, the binding mode (coordinate) of DR396 to DNase γ has not yet been defined. Here, we examined the molecular basis for the inhibitory activity of DR396 to DNase γ by structure-based computational docking studies. In the blind-docking study using a human DNase γ homology model, a unique binding site of DR396 was predicted, which is tentatively named the 'DNA trapping site' because of the binding domain of the unhydrolyzed DNA strand, but not the active site. Targeting the DNA trapping site as a hot spot, new human DNase γ inhibitors were obtained from our diverse chemical library in silico. These inhibitors showed high correlations between their predicted binding-free energies (ΔGs) and observed IC50 values in the DNA trapping site but not the active site. The IC50 of a regioisomer of DR396, 5-(4,6-dichloro-[1,3,5]-triazine-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DF365), was 73 μM (ΔG = −9.75 kcal/mol), a 20-fold weaker inhibitory ability than that of DR396 (IC50 = 3.2 μM, ΔG = −11.22 kcal/mol). Fluorescein and triazine derivatives, partial structures of DR396, had little inhibitory activity for DNase γ. Docking analyses of the interaction between DR396 and DNase γ revealed that DR396 binds tightly to three subsites (S1, S2, and S3) in the trapping site of DNase γ by forming six hydrogen bonds, whereas DF365 and the partial structures are unable to form hydrogen bonds at all three subsites. These findings suggest that the specificity and potency of the inhibitory activity of DR396 for DNase γ is due to the specific interaction of DR396 with three subsites in the DNA trapping site of DNase γ.