Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRβ inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRβ inhibitors, salvianolic acid B and gomisin D, were screened out with K_D values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl₄-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

As a member of anATP-dependent membrane transport proteins, P-Glycoprotein (P-gp) is known to pump substrates out of cells in the ATP-dependent mechanism. The over-expression of P-gp in tumor cells reduces the intracellular drug concentrations, which decreases the efficacy of extensive antitumor drugs and leads to multidrug resistance (MDR) clinically. The combination of anti-cancer drugs with P-gp inhibitor has been an attractive and promising strategy to reverse MDR in cancer treatment. However, non-specific or non-selective distribution of P-gp inhibitors to non-target organs is one of the most fatal shortcomings in clinical application. Thus, there is an urgent need for effective and non‑toxic MDR reversal agents, particularly in P‑gp‑mediated MDR. Traditional Chinese medicine (TCM) natural products may provide less toxic for use in P-gp inhibition to promote MDR reversal. P-gp modulatory effects have been previously demonstrated using selected TCM, including the flavonoids, alkaloids, terpenoids, coumarins, and quinonoids compounds, and some Chinese medicine extracts. Moreover, the approaches for screening active components from TCM are necessary and facing more challenges. At present, the approaches to study the interaction between TCM and P-gp are divided into in vitro, in vivo and in silico methods. This review will provide an overview and update on the role of TCM in overcoming P-gp mediated MDR and the approaches to study the interaction between TCM and P-gp. Significance Statement This review summarized some traditional Chinese medicine identified to have a modulatory effect towards P-gp, including flavonoids, alkaloids, terpenoids, coumarins, quinonoids compounds and some Chinese medicine extracts, and introduced the possible mechanisms. The approaches to study the interaction between TCM and P-gp are divided into in vitro, in vivo, and in silico methods.