Exploring NAG-thiazoline and its derivatives as inhibitors of chitinolytic β-acetylglucosaminidases

• NGT is a poor inhibitor against the insect chitinolytic β-acetylglucosaminidase OfHex1.
• A crystal structure of the enzyme-inhibitor complex reveals that the big active pocket cannot position NGT well.
• A bulky substituted NGT (NMAGT) is obtained as a submicromolar inhibitor of OfHex1.

NAG-thiazoline (NGT) and its derivatives are well-known inhibitors against most β-acetylglucosaminidases (β-GlcNAcases) except for insect and bacterial chitinolytic β-GlcNAcases, including the molting-indispensable OfHex1 from the insect Ostrinia furnacalis. Here, we report the co-crystal structure of OfHex1 in complex with NGT. This structure reveals a large active pocket in OfHex1 that may account for the poor inhibitory activity of NGT. To test this hypothesis, a bulky substituent was designed and synthesized on the thiazoline ring of NGT. The resulting compound (NMAGT) was determined to be a submicromolar inhibitor of OfHex1 with a Ki value of 0.13 μM, which is 600-fold lower than Ki value of NGT. Molecular dynamics simulation analysis supported the good fit of NMAGT to the active pocket.