Optimization of Indole- and Pyrazole-fused Glycyrrhetinic Acid Derivatives as Potent PTP1B Inhibitors: In Silico, In Vitro, In Vivo, and Metabolomic Studies

Protein tyrosine phosphatase 1B (PTP1B) is a crucial enzyme involved in regulating insulin and leptin signaling pathways, making it a promising target for treating type 2 diabetes. In this study, we synthesized 14 derivatives of indole- and pyrazole-fused glycyrrhetinic acid (GA) and evaluated their effects on PTP1B, using both its long (<i>h</i>PTP1B_1-400) and short (<i>h</i>PTP1B_1-285) forms. We analyzed enzymatic kinetics and selectivity over T-cell protein tyrosine phosphatase (TCPTP). Molecular docking and molecular dynamics simulations were performed to understand the binding mode of the compounds within PTP1B. Untargeted metabolomics, using gas chromatography-mass spectrometry, assessed metabolic changes caused by the most potent PTP1B inhibitors in HepG2 cells. We also evaluated these inhibitors <i>in vivo</i> to determine their effects on insulin sensitivity through the insulin tolerance test (ITT) in streptozotocin (STZ)-induced diabetic mice. Two compounds, <b>4b</b> (indole-fused) and <b>5g</b> (pyrazole-fused), showed greater potency against the long form of PTP1B compared to the short form, indicating that both compounds preferentially bind to the disordered C-terminal region of PTP1B. Molecular docking and molecular dynamics studies supported this finding. Furthermore, enzymatic kinetics revealed that compounds <b>4b</b> and <b>5g</b> function as uncompetitive inhibitors of PTP1B, with <i>K</i> _i values of 0.32 and 0.72 μM, respectively. Notably, both GA derivatives exhibited more pronounced inhibition of PTP1B compared to the well-established inhibitors ursolic acid and Ertiprotafib, while also demonstrating selectivity over TCPTP. Metabolomic analysis revealed that these compounds increased pantothenic acid and glycine levels, while decreasing glucose and fatty acid levels in HepG2 cells, suggesting enhanced glycolysis and reduced lipogenesis. Both compounds exhibited low cytotoxicity in HFF-1 cells and significantly reduced glucose levels in the ITT in STZ-induced diabetic mice, outperforming the insulin-sensitizing drug Pioglitazone.