Bioactivity-based molecular networking-guided identification of methyl rosmarinate from Orthosiphon aristatus as a PTP1B inhibitor that improves insulin sensitivity in diabetic mice

Background and purpose: Type 2 diabetes (T2D) is a popular chronic metabolic disorder with high morbidity and mortality. Protein tyrosine Phosphatase 1B (PTP1B) acts as a negative regulator of Insulin action, considering as a promising anti-T2D target. The stems and leaves of Orthosiphon aristatus have been widely utilized for treating hyperglycemia. However, the anti-hyperglycemic principles and underlying mechanism remain unclear. Our study aims to identify potential PTP1B inhibitors from O. aristatus and explore the underlying mechanisms.

Methods: The bioactivity-based molecular networking (BMN) was conducted to discover and targeted isolate methyl rosmarinate (MR) from O. aristatus as a PTP1B inhibitor. C2C12 myotubes and high-fat diet-fed plus streptozotocin-injection induced T2D mice were utilized to evaluate the Insulin sensitizing effect of MR.

Results: MR acted as a reversible inhibitor of PTP1B, with competitive inhibition as its dominant mode and an IC₅₀ value of 24.70 ± 0.60 μM. CETSA and DARTS experiments confirmed that MR directly binds to PTP1B in C2C12 myotubes. In C2C12 myotubes, MR (6.25, 12.5 and 25 µM) promoted insulin-stimulated glucose uptake, and enhanced glycogen synthesis. Furthermore, MR (6.25, 25 mg/kg/d) enhanced Insulin sensitivity, improved lipid profile, and rescued skeletal muscle damage in T2D mice.

Conclusions: The BMN strategy was utilized for the quick identification of MR as a PTP1B inhibitor from O. aristatus. MR improves Insulin sensitivity in skeletal muscle of T2D mice by binding to and inhibiting PTP1B activity, which could serve as a promising candidate for treating T2D.

Bioactivity-based molecular networking; Insulin sensitivity; Methyl rosmarinate; Orthosiphon aristatus; Protein tyrosine phosphatase 1B; Type 2 diabetes.