Fig 2: ßA1-crystallin regulates PTP1B activity.a Lineweaver-Burk plot showing increasing doses (0, 0.5, 1, and 2 nM) of ßA3/A1-crystallin decreases both Vmax and KM of PTP1B activity for different concentrations of p-Nitrophenyl Phosphate (pNPP). n = 3. *P < 0.05. **P < 0.05. b The N-terminal sequence of Cryba1; ßA1-crystallin KD mice were generated by knocking in 5 base pairs (CCACC, red) before the first start codon to strengthen the Kozak consensus sequence. For generating ßA3-crystallin KO, the first start codon was removed by a single nucleotide mutation in the mouse Cryba1 gene (A > G, red). Another silent mutation (C > G; red) was also introduced to prevent the binding and re-cutting of the sequence by gRNA after homology-directed repair. c Representative western blot and d graph showing densitometry analysis for the expression level of ßA3/A1-crystallin in astrocyte lysates from WT (black bar), ßA3 KO (blue bar), and ßA1 KD (green bar) mice, respectively, showing complete loss of ßA3-crystallin in the ßA3 KO cells and a notable decrease in ßA1-crystallin expression in the ßA1 KD cells, relative to WT astrocytes. In ßA3 KO astrocytes, there is an increase in expression of ßA1-crystallin; n = 4. *P < 0.05, **P < 0.01. e Increased levels of lactate in mouse WT, ßA3 KO and ßA1 KD astrocytes treated with HG (25 or 30 mM for 6 h) relative to untreated cells. Lactate levels in ßA1 KD astrocytes were higher in all experimental conditions, compared to WT and ßA3 KO cells; n = 3. *P < 0.05, **P < 0.01. f Elevated glycolytic flux is evident from increased glycolytic capacity in WT, ßA3 KO and ßA1 KD astrocytes treated with high glucose (HG; 30 mM for 6 h), relative to untreated cells (cultured in 5 mM d-glucose containing medium). Glycolytic capacity in ßA1 KD astrocytes was drastically higher compared to WT and ßA3 KO cells; n = 4. *P < 0.05, **P < 0.01. g Cultured ßA1 astrocytes either untreated or exposed to mannitol (30 mM for 6 h) have elevated PTP1B activity compared to WT and ßA3 KO cells, which increases further with HG (30 mM for 6 h). The elevation in PTP1B activity was rescued by ßA1-crystallin overexpression in untreated or HG-exposed ßA1 KD cells; n = 5. *P < 0.05, **P < 0.01.

Fig 3: 3D molecular docking model for the ligand interactions of compounds 1 (a), 44 (b), and 6 (c); yellow dashed lines indicate H-bonds; the residues of PTP1B are yellow sticks; the compounds are blue sticks. 2D ligand interaction diagrams of compounds 1 (d), 44 (e), and 6 (f) in the PTP1B enzyme.

Fig 4: The histogram of PTP1B-the compound interactions monitored during the whole molecular dynamics simulations. (a) Interaction fraction of PTP1B-compound 1 complex. (b) Interaction fraction of PTP1B-compound 44 complex. (c) Interaction fraction of PTP1B-compound 6 complex.

Fig 5: Lineweaver-Burk plots for PTP1B inhibition of compounds 1 (a), 44 (c), and 6 (e). Dixon plots for PTP1B inhibition of compounds 1 (b), 44 (d), and 6 (f). Each value was presented as mean ± SD, n = 3.