β-N-Acetylhexosaminidases are widely distributed exoglycosidases and have attracted significant attention due to their important roles in the field of pesticide and drug discovery. Remarkably, human O-GlcNAcase (hOGA) and human β-N-acetylhexosaminidase (HsHex) possess the same catalytic mechanism but play different physiological actions in vivo. In this Letter, we aim to improve the inhibitory potency and selectivity of previously reported thioglycosyl−naphthalimides against hOGA. The rational compound design led to the synthesis of 13r bearing a 4-piperidylnaphthalimide moiety as a highly potent hOGA inhibitor (K i = 0.6 μM against hOGA) with good selectivity (K i > 100 μM against HsHexB). Furthermore, to investigate the basis for the potency and selectivity of 13r against hOGA, the possible inhibitory mechanisms of selected inhibitors (15b, 13b, and 13r) against hOGA and HsHexB were studied using molecular docking and MD simulations. These 4-substituted naphthalimide thioglycosides may potentially serve as useful tools for the further study of the function of hOGA.

β-N-Acetylhexosaminidases have emerged as promising targets for drug and pesticide discovery due to their critical physiological functions in various cellular processes. In particular, human O-GlcNAcase (hOGA) from the glycoside hydrolase family 84 (GH84) has gained significant attention. This enzyme was found to be linked to various diseases such as diabetes, cancer, and Alzheimer's disease (AD). In this study, to develop novel hOGA inhibitors with suitable pharmaceutical properties, virtual screening of the Drugbank database was performed using a docking-based approach targeting hOGA. Chlorhexidine (4, K i = 4.0 μM) was identified as a potent hOGA inhibitor with excellent selectivity (K i > 200 μM against human β-N-acetylhexosaminidase B) and subjected to structural modifications and SAR studies. Furthermore, molecular dynamics simulations as well as binding free energy and free energy decomposition calculations were carried out to investigate the basis for the efficiency of potent inhibitors against hOGA. This present work revealed the new application of the disinfectant chlorhexidine and provided useful information for the future design of hOGA inhibitors.

Insect chitinolytic β-N-acetylhexosaminidase OfHex1, from the agricultural pest Ostrinia furnacalis (Gueneé), is considered as a potential target for green pesticide design. In this study, rational molecular design and optimization led to the synthesis of compounds 15r (K i = 5.3 μM) and 15y (K i = 2.7 μM) that had superior activity against OfHex1 than previously reported lead compounds. Both compounds 15r and 15y had high selectivity toward OfHex1 over human β-Nacetylhexosaminidase B (HsHexB) and human O-GlcNAcase (hOGA). In addition, to investigate the basis for the potency of glycosylated naphthalimides against OfHex1, molecular docking and molecular dynamics simulations were performed to study possible binding modes. Furthermore, the in vivo biological activity of target compounds with efficient OfHex1 inhibitory potency was assayed against Myzus persicae, Plutella xylostella, and O. furnacalis. This present work indicates that glycosylated naphthalimides can be further developed as potential pest control and management agents targeting OfHex1.

The development of effective detection methods for hexosaminidase is of great importance for the rapid screening of potential inhibitors in vitro and for the early diagnosis of related diseases ex vivo. In this study, the activatable fluorescent probes that are based on naphthalimide decorated with ethylene glycol units were synthesized using N-acetyl-β-d-glucosaminide as a hexosaminidase-responsive group. When exposed to this enzyme, the glucoside-linked naphthalimide moiety of 1c can be cleaved quickly with significant changes in both color (from colorless to yellow) and fluorescence (from blue to green). Probe 1c shows better water-solubility and fluorescence properties than common substrate 4-methylumbelliferyl N-acetyl-β-d-glucosaminide. Furthermore, the response mechanism of 1c to hexosaminidase was evaluated using HPLC analysis and TD-DFT calculations. Molecular docking was performed to investigate the interaction mode. In addition, 1c has successfully achieved the straightforward rapid discovery of effective hexosaminidase inhibitors. Fluorescence imaging experiments indicate that 1c has good cell safety and can be employed as a useful tool for detecting intracellular hexosaminidase activity.