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. 2023 Nov 24;28(23):7758.
doi: 10.3390/molecules28237758.

A Natural Compound Containing a Disaccharide Structure of Glucose and Rhamnose Identified as Potential N-Glycanase 1 (NGLY1) Inhibitors

Ruijie Liu  1 , Jingjing Gu  2 , Yilin Ye  1 , Yuxin Zhang  1 , Shaoxing Zhang  1 , Qiange Lin  1 , Shuying Yuan  3 , Yanwen Chen  4 , Xinrong Lu  5 , Yongliang Tong  5 , Shaoxian Lv  5 , Li Chen  5 , Guiqin Sun  1
Affiliations

A Natural Compound Containing a Disaccharide Structure of Glucose and Rhamnose Identified as Potential N-Glycanase 1 (NGLY1) Inhibitors

Ruijie Liu et al. Molecules. .

Abstract

N-glycanase 1 (NGLY1) is an essential enzyme involved in the deglycosylation of misfolded glycoproteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, which could hydrolyze N-glycan from N-glycoprotein or N-glycopeptide in the cytosol. Recent studies indicated that NGLY1 inhibition is a potential novel drug target for antiviral therapy. In this study, structure-based virtual analysis was applied to screen candidate NGLY1 inhibitors from 2960 natural compounds. Three natural compounds, Poliumoside, Soyasaponin Bb, and Saikosaponin B2 showed significantly inhibitory activity of NGLY1, isolated from traditional heat-clearing and detoxifying Chinese herbs. Furthermore, the core structural motif of the three NGLY1 inhibitors was a disaccharide structure with glucose and rhamnose, which might exert its action by binding to important active sites of NGLY1, such as Lys238 and Trp244. In traditional Chinese medicine, many compounds containing this disaccharide structure probably targeted NGLY1. This study unveiled the leading compound of NGLY1 inhibitors with its core structure, which could guide future drug development.

Keywords: N-glycanase 1 (NGLY1); NGLY1 inhibitor; natural compound; structure-based virtual screening.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic diagram of antiviral effect by downregulating NGLY1 expression. NGLY1 is an essential enzyme involved in deglycosylation of misfolded glycoproteins. Knocking out the expression of NGLY1 leads to severe mitochondrial fragmentation, causing the release of mitochondrial DNA into the cytoplasm. Innate immune detection of self-DNA by the DNA sensor cGAS activates downstream STING-TBK1-IRF3 signaling cascade, inducing the expression of type I interferons (IFN) and IFN-stimulated genes (ISGs), effectively promoting an antiviral response [10]. mtDNA: mitochondrial DNA; cGAS: cyclic GMP–AMP synthase; cGAMP: 2′3′ cyclic GMP–AMP; STING: stimulator of interferon genes; COP-II: coatomer protein complex II; TBK1: TANK-binding kinase 1; IRF3: interferon regulatory factor 3; type I IFN: type I interferons; ISGs: IFN-stimulated genes.
Figure 2
Figure 2
The screening method for targeting NGLY1 lead compounds. Red circle: Location of the receptor pocket. Red arrow: Location of the NGLY1. RNase B (g): N-glycosylated RNase B (NGLY1 is inhibited); RNase B (dg): N-glycosylated RNase B (NGLY1 is not inhibited).
Figure 3
Figure 3
Analysis of interaction mode between NGLY1 inhibitors and NGLY1 protein, and NGLY1 inhibitors’ mediated inhibition of NGLY1 activity were tested at various concentrations using the electrophoretic mobility shift assay. (A) Sites of Z-VAD-FMK binding to NGLY1 and Z-VAD-FMK mediated inhibition of NGLY1 activity. (B) Sites of Poliumoside binding to NGLY1 and Poliumoside Bb mediated inhibition of NGLY1 activity. (C) Sites of Soyasaponin Bb binding to NGLY1 and Soyasaponin Bb mediated inhibition of NGLY1 activity. (D) Sites of Saikosaponin B2 binding to NGLY1 and Saikosaponin B2 mediated inhibition of NGLY1 activity. RNase B (g): N-glycosylated RNase B (NGLY1 is inhibited); RNase B (dg): N-glycosylated RNase B (NGLY1 is not inhibited).
Figure 4
Figure 4
The inhibitory effect of Rutinose as a core structure of Poliumoside, Soyasaponin Bb, and Saikosaponin B2 on NGLY1. (A) Chemical structure of Poliumoside, Soyasaponin Bb, Saikosaponin, Rutinose. (B) Rutinose mediated inhibition of NGLY1 activity. Further quantification of the enzyme cleavage results was performed using Image J (Version 2.1.0) software. (C) Sites of Rutinose binding to NGLY1. (D) The activity of NGLY1 mutants. RNase B (g): N-glycosylated RNase B (NGLY1 is inhibited); RNase B (dg): N-glycosylated RNase B (NGLY1 is not inhibited). Red box: The glycan structure of the compound.
Figure 5
Figure 5
Doismin and Rutin mediated inhibition of NGLY1 activity were tested at various concentrations using the electrophoretic mobility shift assay. (A,C) Chemical structure diagram of Diosmin and Rutin. (B) Diosmin and (D) Rutin mediated inhibition of NGLY1 activity were tested at various concentrations using the electrophoretic mobility shift assay. RNase B (g): N-glycosylated RNase B (NGLY1 is inhibited); RNase B (dg): N-glycosylated RNase B (NGLY1 is not inhibited). Red box: The glycan structure of the compound.
Figure 6
Figure 6
CCK8 analysis of HEK 293T cells treated with NGLY1 inhibitors for 24 h.
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