Fig. 6: Development of a high-throughput screening system that can quantify MGAT1 enzyme activity with luminance signal to develop specific MGAT1 inhibitors.

a Schematic of an MGAT1 function-based drug screening assay converting enzymatic activity to a luminance signal. Key components include MGAT1, mannose-5-AEAD (substrate), UDP-GlcNAc (sugar donor), and a compound library. MGAT1 inhibition reduces free UDP, decreasing luminance. Created in BioRender. Zhu, Y. (2025) https://BioRender.com/t24b827. b Normalized MGAT1 enzymatic function in high-throughput screening with an anti-cancer compound library (~ 3500 compounds). c AutoDock Vina-predicted most energy-favorable binding of TW-37 (pink) to MGAT1, with interacting residues (within 3.5 Å) shown. d Pharmacophore model 1 (PM1) based on binding of TW-37 to MGAT1. e Pharmit-predicted binding of the top 10 compounds (colored differently) screened against PM_1. f Normalized MGAT1 enzyme function upon treatment with 15 screened compounds was measured by function-based screening assay. g Flow cytometry analysis of membrane CD73 in MDA-MB468 cells treated with 15 compounds. h, i The membrane-fractionated protein levels of CD73 in MDA-MD468 cells upon treatment with screened compounds were determined by (h) immunostaining and (i) immunoblotting. j The chemical structures of two top-ranked compounds (No.8 and No.9). k Dose-dependent MGAT1 inhibition by No.2, No.8, and No.9 in the function-based assay. l–o MDA-MB468 cells were cocultured with PBMC cells and the indicated compounds. Percentages of TNFα⁺ (l), IFNγ⁺ (m), Ki-67⁺ (n), and Granzyme B⁺ (o) in CD8 + T cells were measured using flow cytometry. p, q Time-lapse quantification of MDA-MB468 survival in PBMC coculture treated with No.2/No.8(W-GTF01)/No.9 (p) or No.8 in MDA-MB468-WT/CD73-KD cells (q). r Binding of W-GTF01 (MolPort-004-851-686) to MGAT1, highlighting π-stacking interaction between F324 (yellow) and W-GTF01 (magenta). Data (mean ± SEM), images, western blot, and flow cytometry are representative of at least three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (i, l–o) or two-way ANOVAs followed by Tukey’s multiple comparison tests (p, q). Source data are provided as a Source Data file.