Planten als bron voor inhibitoren van proteïne tyrosine fosfatase 1B

Natural products (NPs) have been a significant resource for providing potential drug candidates because of their documented therapeutic effects over decades. The recognition and identification of biologically active compounds plays a crucial role in drug discovery and drug development. In this project, multi-techniques were employed to develop efficient methods to screen protein tyrosine phosphatase 1B (PTP1B) inhibitors from plant extracts. In chapter 1, a brief introduction on the history and application of NPs in drug discovery and development, as well as technologies of target identification in NPs and plants was presented. The text summarizes the principles, advantages and disadvantages of various screening methods classified by phenotype-based assays and target-based approaches. In addition, the structure and functions of PTP1B were illustrated. In chapter 2, the main aims of the study were introduced. The objectives and methodologies were outlined for the subsequent works. In chapter 3, a rapid and efficient inhibitor screening method of PTP1B was established based on capillary electrophoresis (CE). The results indicated that the extracts of Astragalus membranaceus (Fisch) Bunge and Morus alba L. induced a prominent decrease of the activity of PTP1B. In addition, some advantages of the method were illustrated: (1) short analysis time of less than 2 min; (2) selective product detection due to excellent separation by CE; (3) accurate determination of kinetic parameters; (4) direct evaluation of inhibitory effect of plant extracts on PTP1B. Thereby, it was concluded that this rapid and efficient screening method could be a useful tool for inhibitor screening from natural plant extracts. In chapter 4, an on-line CE method was developed and applied to screen PTP1B inhibitors in plant extracts. The established on-line method was evaluated by a statistical factor Z'. The latter was determined as 0.67, hence located in the standard range of 0.5-1.0, demonstrating the reliability of the method for screening. What's more, an enhanced inhibition effect was found by the combined extract of Morus alba L. and quercetin/caffeic acid. According to the docking results, it is assumed 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. All in all, owing to the merits of automation and low consumption of samples, this method can be an alternative for inhibitor screening of other dephosphorylating enzymes. In chapter 5, an immobilized PTP1B affinity column was constructed to discover and characterize the compounds in M. alba L., binding on PTP1B. Five compounds, chlorogenic acid, rutin, mulberrofuran G, sanggenon C, and astragalin were found and identified. Notably, sanggenon C and mulberrofuran G showed explicit inhibition effects on PTP1B with IC50 values of 13.38 ± 0.22 µM and 17.17 ± 0.30 µM, respectively, pointing towards useful PTP1B inhibition. Thereby, these findings proved the efficiency of the constructed PTP1B affinity column for drug discovery, offering an alternative for future exploration. Chapter 6 presents a general discussion and future perspectives related to the current work. Initially, the merits and limitations of the instrumental CE technique were discussed. Then, the key factors influencing the separation performance of CE were explained. In addition, the rationale for the use of the on-line TDLFP mixing method was also interpreted, as well as the difference in Km value between the two methods. Afterwards, several important points for the construction of the PTP1B affinity column were discussed. Finally, perspectives for future study were proposed.