One-Step Synthesis and Oriented Immobilization of Strep-Tag II Fused PDGFRβ for Screening Intracellular Domain-Targeted Ligands

Accurate orientations and stable conformations of membrane receptor immobilization are particularly imperative for accurate drug screening and ligand–protein affinity analysis. However, there remain challenges associated with (1) traditional recombination, purification, and immobilization of membrane receptors, which are time-consuming and labor-intensive; (2) the orientations on the stationary phase are not easily controlled. Herein, a novel one-step synthesis and oriented-immobilization membrane-receptor affinity chromatography (oSOMAC) method was developed to realize high-throughput and accurate drug screening targeting specific domains of membrane receptors. We employed Strep-tag II as a noncovalent immobilization tag fused into platelet-derived growth factor receptor β (PDGFRβ) through CFPS, and meanwhile, the Strep–Tactin-modified monolithic columns are prepared in batches. The advantages of oSOMAC are as follows: (1) targeted membrane receptors can be expressed independent of living cell within 1–2 h; (2) orientation of membrane receptors can be flexibly controlled and active sites can expose accurately; and (3) targeted membrane receptors can be synthesized, purified, and orientation-immobilized on monolithic columns in one step. Accordingly, three potential PDGFRβ intracellular domain targeted ligands: tanshinone IIA (Tan IIA), hydroxytanshinone IIA, and dehydrotanshinone IIA were successfully screened out from Salvia miltiorrhiza extract through oSOMAC. Pharmacological experiments and molecular docking further demonstrated that Tan IIA could attenuate hepatic stellate cells activation by targeting the protein kinase domain of PDGFRβ with a K_D value of 9.7 μM. Ultimately, the novel oSOMAC method provides an original insight for accurate drug screening and interaction analysis which can be applied in other membrane receptors.