A crystal structure-guided rational design switching non-carbohydrate inhibitors' specificity between two β-GlcNAcase homologs

Abstract

Selective inhibition of function-specific β-GlcNAcase has great potential in terms of drug design and biological research. The symmetrical bis-naphthalimide M-31850 was previously obtained by screening for specificity against human glycoconjugate-lytic β-GlcNAcase. Using protein-ligand co-crystallization and molecular docking, we designed an unsymmetrical dyad of naphthalimide and thiadiazole, Q2, that changes naphthalimide specificity from against a human glycoconjugate-lytic β-GlcNAcase to against insect and bacterial chitinolytic β-GlcNAcases. The crystallographic and in silico studies reveal that the naphthalimide ring can be utilized to bind different parts of these enzyme homologs, providing a new starting point to design specific inhibitors. Moreover, Q2-induced closure of the substrate binding pocket is the structural basis for its 13-fold increment in inhibitory potency. Q2 is the first non-carbohydrate inhibitor against chitinolytic β-GlcNAcases. This study provides a useful example of structure-based rationally designed inhibitors as potential pharmaceuticals or pesticides.

Figure 1. Structures of naphthalimide-based β-GlcNAcase inhibitors.

Figure 2. Dixon plots of inhibition kinetics of Q1 and Q2 against β-GlcNAcases.

(a). Q1 against OfHex1; (b). Q1 against HsHex; (c). Q2 against OfHex1.

Figure 3. Crystal structure of Q1-complexed OfHex1 and docked structure of Q1-complexed HsHex.

(a). The superimposition of the unliganded-OfHex1 and Q1-complexed OfHex1. Residues of unliganded-OfHex1 and Q1-complexed OfHex1 are shown in white and blue, respectively. Q1 is shown in green. The hydrogen bonds are shown as dashed black lines. (b). The binding mode of Q1 in the substrate-binding pocket of OfHex1. The subsites −1 and +1 are shown in pink and blue, respectively. W483 and V484 are shown in cyan. (c). and (d). The binding mode of Q1 in the substrate binding-pocket of HsHex. The hydrogen bonds are shown as dashed black lines. The subsites -1 and the hydrophobic patch outside the subsite -1 are shown in pink and yellow, respectively.

Figure 4. Crystal structure of Q2-complexed OfHex1.

(a). The superimposition of the unliganded-OfHex1 and Q2-complexed OfHex1. Residues of unliganded-OfHex1 and Q1-complexed OfHex1 are shown in white and cyan, respectively. The hydrogen bonds are shown as dashed black lines. (b). The binding mode of Q2 in the substrate-binding pocket of OfHex1. The subsites −1 and +1 are shown in pink and blue, respectively. W483 and V484 are shown in cyan. (c). The superimposition of the binding modes of Q1 and Q2 in OfHex1. Q1 and Q2 are shown in green and magenta, respectively.