Covalent Design of Cell Membrane Stationary Phase with Enhanced Stability for Fast Screening P-Glycoprotein Inhibitors

Abstract

Cell membrane chromatography (CMC) has been widely used for characterizing the interaction between drugs and membrane receptors to screen target components from herbal medicines. However, the column life, stability, and the efficiency cannot meet the needs of high-throughput screening purpose. In this study, a P-glycoprotein immobilized cell membrane stationary phase (P-gp/CMSP) was prepared with a simple and mild twostep aldehyde modification, realizing the covalent bonding between cell membrane and stationary phase. The column life and stability were significantly enhanced compared with the unmodified columns. The P-gp/CMC column was equipped into a comprehensive 2D P-gp/CMC/Capcell-C18/TOFMS system, which actualizes the automated and high-throughput analytical process and rapid identification of complex chemical samples with no data loss. Five compounds with significant retention were screened out and unambiguously identified by the comprehensive 2D analytical system. Baicalin was confirmed as a P-gp inhibitor with ATP depletion inhibition ratio of 83.4%. Moreover, the reversal index of baicalin on DOX significantly increased to 11.13 when its concentration reached 25 μM, revealing that baicalin could effectively reverse the MDR cell model induced by DOX. The integrated system is a practical drug discovery platform and could be applied to other transmembrane protein models.

Gain deeper insights into traditional Chinese medicines using multidimensional chromatography combined with chemometric approaches

“…Ching based on an on-line 2D-LC system ( Pan et al, 2021 ). Scholars have prepared a P-glycoprotein immobilized cell membrane stationary phase (P-gp/CMSP) and integrated it into a comprehensive 2D P-gp/CMC/Capcell-C18/TOFMS system to screen five compounds in Scutellariae Radix (roots of S. baicalensis ) ( Liu, Wang, Gu, Zhang, & Hong, 2020 ). Experts have loaded a CMC column based on the construction of the hepatocellular carcinoma cell line SK-Hep1-GPC3, screening the anti-tumor components from Scutellariae Radix (roots of S. baicalensis ) ( Chen et al, 2021 a).…”

Multiple mitochondria-targeted components screened from Sini decoction improved cardiac energetics and mitochondrial dysfunction to attenuate doxorubicin-induced cardiomyopathy

“…It should be noted that the CMC models focus on components acting on cell membrane receptors and neglect the active components acting on intracellular organelles from TCM. In addition, the immobilization of biomembrane fragments on stationary phase carriers traditionally utilizes hydrogen bonds and electrostatic forces, which result in the fall-off of biomembrane fragments and further lead to a sharp decline in bioactivity and column life 17 . Consequently, it is urgently needed to develop a stable mitochondrial membrane chromatography method to screen specific active components interacting with intracellular mitochondria from TCM.…”

Site-Specific Covalent Immobilization of SMA-Stabilized ACE2 for SARS-CoV-2 Recognition and Drug Screening

“…Alternatively, chemically bonding MPs can be used to increase the life span of the cell membrane column. For example, 3-aminopropyltriethoxysilane (APTES)-modified silica microspheres can react with glutaraldehyde to generate aldehyde groups on the surface of the microspheres, which can form imine bonds with amine groups on phospholipids to covalently immobilize them . Although these covalent bonding reactions increase the life span of MPs on CMC, these nonspecific reactions result in the inevitable random reactions with numerous active amine groups on the protein and the random orientation of MPs.…”

“…For example, 3-aminopropyltriethoxysilane (APTES)-modified silica microspheres can react with glutaraldehyde to generate aldehyde groups on the surface of the microspheres, which can form imine bonds with amine groups on phospholipids to covalently immobilize them. 10 these covalent bonding reactions increase the life span of MPs on CMC, these nonspecific reactions result in the inevitable random reactions with numerous active amine groups on the protein and the random orientation of MPs. Therefore, on the basis of covalent bonding, people are devoted to optimizing the directional immobilization of proteins.…”