Fig. 6: Elsulfavirine block SREBP-1 activation through impair ADSL-INSIG1/2 binding upon glucose stimulation.

a The binding complex of INSIG2-Elsulfavirine-ADSL complex was built based on the protein-protein model. b Interaction profile of the INSIG2-Elsulfavirine-ADSL. Residues color such as green, violet, and blue represent hydrophobic, positive charged, and polar residue, respectively. c RMSD of the INSIG2-ADSL in the absence (left) or presence (right) of compound Elsulfavirine from GaMD simulations. d Different frames of binding modes of INSIG2-Elsulfavirine-ADSL complex in 250 ns simulations. e Target engagement assay using Flag-INSIG1 or Flag-INSIG2 purified from Huh7 cells treated with or without indicated drugs (left). The relative distribution of each protein in different fractions was quantified by densitometric analysis of the blots (n = 3 biological replicates) (right), Data are mean ± SD. f The interaction of INSIG2 with Elsulfavirine detected by thermal shift assay. RLU, relative light units. The data were shown as the relative mean values of RLU (n = 3 biological replicates). g, h Huh7 cells were pretreated with or without Tezacaftor, Elsulfavirine, or Pyrithioxin for 1 h before treatment with or without high glucose. Immunoblotting analyses were performed (g). Immunofluorescence analyses were performed (h, left). Scale bars, 20 μm. The relative nucleus intensity was quantified over 30 cells by ImageJ (h, right). n = 10 microscopic fields. i Huh7 cells were treated with or without Tezacaftor, Elsulfavirine, or Pyrithioxin for 1 h and then subjected to a luciferase reporter assay (n = 6 biological replicates). j, k Huh7 cells treated with Elsulfavirine were stimulated with or without high glucose. Immunofluorescence analyses were performed (j). Scale bars, 20 μm. The mRNA expression levels of SREBP-1 target genes were measured using quantitative PCR (k) (n = 3 biological replicates). l The incorporation of ¹⁴C-glucose into TGs (upper) and FAs (lower) was measured (n = 6 biological replicates). m Huh7 cells were pretreated with or without Elsulfavirine in high glucose for 12 h. Fluorescence staining were used with the Bodipy and DAPI (upper). Scale bars, 20 μm. The average number of lipid droplets was quantified by ImageJ (lower). n = 10 microscopic fields. All data are presented as mean ± SD. Statistical significance was determined by a one-way ANOVA (h, i), two-way ANOVA (k), or two-tailed t-test (l, m).