Potent family-18 chitinase inhibitors: x-ray structures, affinities, and binding mechanisms

• Published in: The Journal of biological chemistry Vol. 286; no. 27; pp. 24312 - 24323
• Main Authors: Pantoom, Supansa, Vetter, Ingrid R, Prinz, Heino, Suginta, Wipa
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 08-07-2011
• Subjects: Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Catalytic Domain; Chitinases - antagonists & inhibitors; Chitinases - chemistry; Crystallography, X-Ray; Enzyme Inhibitors - chemistry; Enzymology; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Structure; Protein Binding; Vibrio - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.183376

Six novel inhibitors of Vibrio harveyi chitinase A (VhChiA), a family-18 chitinase homolog, were identified by in vitro screening of a library of pharmacologically active compounds. Unlike the previously identified inhibitors that mimicked the reaction intermediates, crystallographic evidence from 14 VhChiA-inhibitor complexes showed that all of the inhibitor molecules occupied the outer part of the substrate-binding cleft at two hydrophobic areas. The interactions at the aglycone location are well defined and tightly associated with Trp-397 and Trp-275, whereas the interactions at the glycone location are patchy, indicating lower affinity and a loose interaction with two consensus residues, Trp-168 and Val-205. When Trp-275 was substituted with glycine (W275G), the binding affinity toward all of the inhibitors dramatically decreased, and in most structures two inhibitor molecules were found to stack against Trp-397 at the aglycone site. Such results indicate that hydrophobic interactions are important for binding of the newly identified inhibitors by the chitinase. X-ray data and isothermal microcalorimetry showed that the inhibitors occupied the active site of VhChiA in three different binding modes, including single-site binding, independent two-site binding, and sequential two-site binding. The inhibitory effect of dequalinium in the low nanomolar range makes this compound an extremely attractive lead compound for plausible development of therapeutics against human diseases involving chitinase-mediated pathologies.