A versatile and automatic on-line screening method: Transverse diffusion of laminar flow profiles-based capillary...

Natural products (NPs) play an important role in drug discovery and drug development due to their diverse chemical properties and biological activities. In the present work, an on-line capillary electrophoresis (CE) method was developed and applied to screen protein tyrosine phosphatase 1B (PTP1B) inhibitors in NPs. As a generic technique, transverse diffusion of laminar flow profiles (TDLFP) was utilized to mix all reactants in the capillary for on-line enzymatic reaction. The procedures of TDLFP were optimized in terms of the concentration of PTP1B, injection order of the plugs, PTP1B injection time, overall plugs' length, and temperature set point. After validation, this developed on-line method was used for kinetic constant determination and inhibitor screening. As a result, the proposed method was validated with good repeatability and a short analysis time of 3 min. The obtained K_m value was as 3.66 ± 0.97 mM. The IC₅₀ value of the phosphatase inhibitor Na₃VO₄ was determined as 98.50 ± 24.82 µM. An enhanced inhibition effect was found by the combined extract of Morus alba L. and quercetin/caffeic acid which could be a synergistic effect on PTP1B. Afterwards, molecular docking was performed for mechanism clarification. According to the docking results, it is speculated that the compounds in the extract of M. alba L. contribute to the enhanced effect through occupation of the catalytic region and substrate recognition site. In conclusion, a universal and automatic on-line method using CE was developed and applied for inhibitor screening. Owing to the merits of automation and low consumption of samples, this method can be an alternative for inhibitor screening of other dephosphorylating enzymes, especially some valuable enzymes.