Computational Studies on the Potency and Selectivity of PUGNAc Derivatives Against GH3, GH20, and GH84 β-N-acetyl-D-hexosaminidases

Abstract

β-N-acetyl-D-hexosaminidases have attracted significant attention due to their crucial role in diverse physiological functions including antibacterial synergists, pathogen defense, virus infection, lysosomal storage, and protein glycosylation. In particular, the GH3 β-N-acetyl-D-hexosaminidase of V. cholerae (VcNagZ), human GH20 β-N-acetyl-D-hexosaminidase B (HsHexB), and human GH84 β-N-acetyl-D-hexosaminidase (hOGA) are three important representative glycosidases. These have been found to be implicated in β-lactam resistance (VcNagZ), lysosomal storage disorders (HsHexB) and Alzheimer's disease (hOGA). Considering the profound effects of these three enzymes, many small molecule inhibitors with good potency and selectivity have been reported to regulate the corresponding physiological functions. In this paper, the best-known inhibitors PUGNAc and two of its derivatives (N-valeryl-PUGNAc and EtBuPUG) were selected as model compounds and docked into the active pockets of VcNagZ, HsHexB, and hOGA, respectively. Subsequently, molecular dynamics simulations of the nine systems were performed to systematically compare their binding modes from active pocket architecture and individual interactions. Furthermore, the binding free energy and free energy decomposition are calculated using the MM/GBSA methods to predict the binding affinities of enzyme-inhibitor systems and to quantitatively analyze the contribution of each residue. The results show that PUGNAc is deeply-buried in the active pockets of all three enzymes, which indicates its potency (but not selectivity) against VcNagZ, HsHexB, and hOGA. However, EtBuPUG, bearing branched 2-isobutamido, adopted strained conformations and was only located in the active pocket of VcNagZ. It has completely moved out of the pocket of HsHexB and lacks interactions with HsHexB. This indicates why the selectivity of EtBuPUG to VcNagZ/HsHexB is the largest, reaching 968-fold. In addition, the contributions of the catalytic residue Asp253 (VcNagZ), Asp254 (VcNagZ), Asp175 (hOGA), and Asp354 (HsHexB) are important to distinguish the activity and selectivity of these inhibitors. The results of this study provide a helpful structural guideline to promote the development of novel and selective inhibitors against specific β-N-acetyl-D-hexosaminidases.

Advances in Optical Sensors of N-Acetyl-β-d-hexosaminidase (N-Acetyl-β-d-glucosaminidase)

“…Thus, HEX(NAG) inhibitors are under active investigation as pest control agents or as potential drugs for treating opportunistic infections. 7,65,66 The research often involves computer docking studies of enzyme/inhibitor complexes 67 and standard enzyme inhibition measurements using a commercial chromogenic substrate such as pNP-NAG or fluorogenic substrate such as 4-MU-NAG.…”

“…HEX­(NAG) is a central enzyme in the life cycle of insects and microbial pathogens, because it controls the degradation of chitin, a linear polysaccharide within insect exoskeletons, crustacean shells, and fungal cell walls. Thus, HEX­(NAG) inhibitors are under active investigation as pest control agents or as potential drugs for treating opportunistic infections. ,, The research often involves computer docking studies of enzyme/inhibitor complexes and standard enzyme inhibition measurements using a commercial chromogenic substrate such as pNP-NAG or fluorogenic substrate such as 4-MU-NAG.…”

O‐GlcNAcase: Emerging Mechanism, Substrate Recognition and Small‐Molecule Inhibitors

“…[65] In addition, PUGNAc was reported to inhibit glycoside hydrolase family 3 (GH3). [66,67] GH3 does not use substrate-assisted catalysis, but rather employs double displacement mechanism. [68] Also, PUGNAc inhibits family 89 α-N-acetylglucosaminidase (K i = 6 μM).…”

“…Interestingly, the complexed structure of PUGNAc with Ostriniafurnacalis β‐ N ‐acetyl‐ d ‐hexosaminidase ( Of Hex1) showed a similar planar conformation at the active pocket . In addition, PUGNAc was reported to inhibit glycoside hydrolase family 3 (GH3) . GH3 does not use substrate‐assisted catalysis, but rather employs double displacement mechanism .…”

N‐acetyl‐β‐hexosaminidase activity is important for chitooligosaccharide metabolism and biofilm formation in Burkholderia pseudomallei

“…The N ‐acetyl‐β‐hexosaminidases cleave glycosidically linked terminal non‐reducing N ‐acetyl‐ d ‐hexosamine residues (i.e., GlcNAc, or N ‐acetyl‐ d ‐galactosamine (GalNAc) from both carbohydrate and non‐carbohydrate conjugates. To date, this specific activity is restricted to CAZymes within family GH20 while other homologous glycoside hydrolase (GH) families such as GH3 and GH84 (Dong et al, 2019) are functionally related as they cleave GlcNAc residues, specifically (Macauley et al, 2005; Vocadlo & Withers, 2005).…”