Repurposing Fagonia Indica Alkaloids as Allosteric PTP 1B Inhibitors: Computational Profiling and Biochemical Validation.

Protein-tyrosine phosphatase 1B (PTP 1B) is a validated antihyperglycaemic target whose chemical space remains sparsely populated by natural scaffolds. Guided by an explicit repurposing strategy, we screened four quinazolinone and isoquinoline alkaloids isolated from Fagonia indica—harmaline, indicine, protopine and thebaine—against PTP 1B, even though similarity-based target prediction did not nominate the enzyme for any compound. Hierarchical in-silico analysis placed all ligands in the catalytic cleft, where harmaline (–7.1 kcal mol⁻¹) and thebaine (–7.5 kcal mol⁻¹) formed a dual π–π stack with Phe196/Phe280 that dominated docking scores. Two-hundred-nanosecond molecular-dynamics simulations confirmed pose stability, funneled the protein’s free-energy landscape into a single deep basin, and yielded MM/PBSA binding free energies of − 13.3 ± 1.0 and − 13.7 ± 2.3 kcal mol⁻¹, respectively. Per-residue decomposition, hydrogen-bond lifetimes and Coulomb/Lennard-Jones traces consistently highlighted the aromatic clamp as the main energetic hot-spot. SwissADME and pkCSM profiling showed that both alkaloids satisfy Lipinski rules, possess high gastrointestinal absorption, lack hERG and Ames liabilities, and inhibit no more than two cytochrome-P450 isoforms. In vitro, harmaline and thebaine inhibited recombinant PTP 1B with IC₅₀ values of 10.70 ± 1.04 and 11.01 ± 1.00 µM, only 1.5-fold weaker than the reference inhibitor ursolic acid (7.37 ± 0.54 µM), whereas protopine and indicine were four- to ten-fold less potent. Lineweaver–Burk and Michaelis–Menten kinetics revealed a non-competitive mechanism for the top hits, in line with the allosteric WPD-loop trapping predicted computationally. The study uncovers harmaline and thebaine as the first F. indica-derived, low-micromolar, non-competitive inhibitors of PTP 1B, validates an integrated modelling-to-bench workflow for natural-product repurposing, and establishes the Phe196/Phe280 aromatic clamp as a tractable anchor for future optimization toward metabolic-disorder therapeutics.