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In this study, a novel cell membrane chromatography (CMC) model was developed to investigate cluster of differentiation 147 (CD147) targeted anti-tumor drug leads for specific screening and ligand-receptor interaction analysis by SNAP-tagged CD147 fusion protein conjugation and polystyrene microspheres (PS) modification. Traditional Chinese medicines (TCMs) are widely used in the treatment of cancer. CD147 plays important roles in tumor progression and acts as an attractive target for therapeutic intervention; therapeutic drugs for CD147-related cancers are limited to date. Thus, a screening method for active components in TCMs is crucial for the further research and development of CD147 antagonists. However, improvement is still needed to perform specific and accurate drug lead screening using the CMC-based method. Recently, our group developed a covalently immobilized receptor-SNAP-tag/CMC model using silica gel as carrier. Besides the carboxyl group on multi-step modified silica particles, the amino group of benzyl-guanine (BG, substrate of SNAP-tag) also possesses reactivity towards the carboxyl group on available carboxyl-modified PS. Herein, we used PS as carrier and an extended SNAP-tag with CD147 receptor to construct the PS-BG-CD147/CMC model for active compound investigation coupled with HPLC/MS and applied this coupled PS-BG-CD147/CMC-HPLC/MS two-dimensional system to drug lead screening from Nelumbinis Plumula extract (NPE) sample. In addition, to comprehensively verify the pharmacological effects of screened ingredients, a cell proliferation inhibition assay was performed, and the interaction between the ingredients and CD147 was studied by the frontal analysis method. This study developed a high-throughput PS-based CMC screening platform, which could be widely applied and utilized in chromatographic separation and drug lead discovery. [ABSTRACT FROM AUTHOR]

Traditional Chinese medicines played an important role in the treatment of COVID-19 in 2020. Ephedra sinica, one of the major constituent herbs of multi-component herbal formula, has been widely used to treat COVID-19 in China. However, its active components are still unclear. The objectives of this study are to screen and evaluate active components from the traditional Chinese medicine Ephedra sinica for the treatment of COVID-19. In our study, we established an ACE2/CMC bioaffinity chromatography model, and then developed an ACE2/CMC-HPLC-IT-TOF-MS system for the active compounds screening and identification from Ephedra sinica extract. We performed molecular docking and surface plasmon resonance (SPR) assays to assess the binding characteristics (binding mode and KD value). We used CCK-8 staining to assess the toxicity of screened compounds, and also used SARS-CoV-2 pseudovirus to observe the viropexis effect of screened compounds in ACE2h cells. In this current work, one fraction was fished out, separated and identified as ephedrine (EP), pseudoephedrine (PEP), and methylephedrine (MEP). Binding assays showed that the three compounds could bind with ACE2 in a special way to some amino acid residues, similar to the way SARS-CoV-2 bound with ACE2. Additionally, the three compounds, especially EP, can inhibit the entrance of SARS-CoV-2 spike pseudovirus into ACE2h cells because they can reduce the entrance ratio of pseudovirus in the pseudovirus model. Overall, the ACE2/CMC-HPLC-IT-TOF-MS system was established and verified to be suitable for ACE2-targeted bioactive compound screening. EP, PEP, and MEP with ACE2-binding features were screened out from Ephedra sinica, and acted as blockers inhibiting SARS-CoV-2 spike pseudovirus entering ACE2h cells. [ABSTRACT FROM AUTHOR]

Adverse drug reactions of traditional Chinese medicine injection mainly manifested as pseudo‐allergic reactions. In the present study, ginsenoside Rd, Ro, and Rg3 were identified as pseudo‐allergic components in Shengmai injection by a high‐expression Mas‐related G protein‐coupled receptor X2 cell membrane chromatography coupled online with high‐performance liquid chromatography and mass spectrometry. Their pseudo‐allergic activities were evaluated by in vitro and in vivo assay. The three compounds were further found to induce pseudo‐allergic reaction through Mas‐related G protein‐coupled receptor X2. Therefore, we concluded that ginsenoside Rd, Ro and Rg3 may be potential allergens that cause pseudo‐allergic reactions. This study might be helpful for the safe use of Shengmai injection. [ABSTRACT FROM AUTHOR]

A novel stability-enhanced graphene quantum dot (GQD)-decorated epidermal growth factor receptor (EGFR) cell membrane chromatography was constructed to study the potential application of GQDs in bioaffinity chromatography, and to screen active components acting on EGFR from traditional Chinese medicine (TCM). The carboxyl groups on the surface of GQDs reacted with the amino groups of the amino-silica gel (SiO2-NH2) to form a covalent bond, thereby preparing the GQD-decorated silica gel (SiO2-GQDs). The EGFR cell membrane was further immobilized on the SiO2-GQDs through the same covalent binding method to obtain the GQD-decorated cell membrane stationary phase (SiO2-GQDs-CMSP). In this way, the cell membrane was firmly immobilized on the decorated silica carrier. The life span and stability of the GQD-decorated cell membrane chromatographic (SiO2-GQDs-CMC) column were both enhanced, and the optimal immobilization conditions of the EGFR cell membrane were also determined. This model was then verified by establishing a SiO2-GQDs-CMC online liquid chromatography-ion trap-time-of-flight (LC-IT-TOF) system to screen possible active components in Peucedanum praeruptorum Dunn. As a result, praeruptorin B (Pra-B) was screened out, and its inhibitory effect against EGFR cell growth was evaluated by the cell counting kit-8 (CCK-8) assay. Molecular docking assay was also conducted to further estimate the interaction between Pra-B and EGFR. Overall, this research indicated that GQDs may be a promising nanomaterial to be used in prolonging the life span of the CMC column, and Pra-B could be a potential EGFR inhibitor so as to treat cancer. [ABSTRACT FROM AUTHOR]

Pseudo-allergic reactions (PARs) are IgE-independent hypersensitivity reactions. Mas-related G protein-coupled receptor-X2 (MrgX2) was proved the key receptor of PAR. The anti-pseudo-allergic compound discovery based on MrgX2 was of great value. Cell membrane chromatography (CMC) based on MrgX2 provides a convenient and effective tool in anti-pseudo-allergic compound screening and discovery, and further improvements of this method are still needed. In this work, SNAP-tag was introduced at C-terminal of Mas-related G protein-coupled receptor (MrgX2-SNAP-tag), and an MrgX2-SNAP-tag/CMC model was then conducted using CMC technique. Comparative experiments showed that the new model not only satisfied the good selectivity and specificity of screening but also exhibited more stable and longer life span than traditional MrgX2/CMC model. By coupling with HPLC–MS, two compounds were screened out from Arnebiae Radix and identified as shikonin and acetylshikonin. Nonlinear chromatography was performed to study the interactions between two screened compounds and MrgX2, and binding constant (KA) of shikonin and acetylshikonin with MrgX2 were 2075.67 ± 0.34 M−1 and 32201.36 ± 0.35 M−1, respectively. Furthermore, β-hexosaminidase and histamine release assay in vitro demonstrated that shikonin (1–5 μM) and acetylshikonin (2.5–10 μM) could both antagonize C48/80-induced allergic reaction. In conclusion, the MrgX2-SNAP-tag/CMC could be a reliable model for screening pseudo-allergy-related components from complex systems. [ABSTRACT FROM AUTHOR]

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride‐decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride‐decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride‐decorated epidermal growth factor receptor/cell membrane chromatography online HPLC‐IT‐TOF‐MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography. [ABSTRACT FROM AUTHOR]

Chloroquine and hydroxychloroquine have been studied since the early clinical treatment of SARS‐CoV‐2 outbreak. Considering these two chiral drugs are currently in use as the racemate, high‐expression angiotensin‐converting enzyme 2 cell membrane chromatography was established for investigating the differences of two paired enantiomers binding to angiotensin‐converting enzyme 2 receptor. Molecular docking assay and detection of SARS‐CoV‐2 spike pseudotyped virus entry into angiotensin‐converting enzyme 2‐HEK293T cells were also conducted for further investigation. Results showed that each single enantiomer could bind well to angiotensin‐converting enzyme 2, but there were differences between the paired enantiomers and corresponding racemate in frontal analysis. R‐Chloroquine showed better angiotensin‐converting enzyme 2 receptor binding ability compared to S‐chloroquine/chloroquine (racemate). S‐Hydroxychloroquine showed better angiotensin‐converting enzyme 2 receptor binding ability than R‐hydroxychloroquine/hydroxychloroquine. Moreover, each single enantiomer was proved effective compared with the control group; compared with S‐chloroquine or the racemate, R‐chloroquine showed better inhibitory effects at the same concentration. As for hydroxychloroquine, R‐hydroxychloroquine showed better inhibitory effects than S‐hydroxychloroquine, but it slightly worse than the racemate. In conclusion, R‐chloroquine showed better angiotensin‐converting enzyme 2 receptor binding ability and inhibitory effects compared to S‐chloroquine/chloroquine (racemate). S‐Hydroxychloroquine showed better angiotensin‐converting enzyme 2 receptor binding ability than R‐hydroxychloroquine/hydroxychloroquine (racemate), while the effect of preventing SARS‐CoV‐2 pseudovirus from entering cells was weaker than R‐hydroxychloroquine/hydroxychloroquine (racemate). [ABSTRACT FROM AUTHOR]

• CMC was used to study the interaction of CQ and HCQ with ACE2 receptors. • The article provides new insights on the use of CQ and HCQ in treating with COVID-19. • ACE2/CMC can be used to separate bioactive ingredients active towards ACE2 receptor. The recent emergence of the novel pathogenic coronavirus disease 2019 (COVID-19) is responsible for a worldwide pandemic. In sight of this, there has been growing interest in the use of chloroquine (CQ) and hydroxychloroquine (HCQ) as potential treatments. In this study, we use angiotensin converting enzyme 2 (ACE2) over-expressed cell membrane chromatography (CMC) to study the interaction of CQ and HCQ with ACE2 receptor. Both CQ and HCQ were retained on the ACE2/CMC column. Then we analyzed the binding character of CQ and HCQ to ACE2 by CMC frontal analysis, ionic force investigation and competitive binding experiment. Results showed that CQ and HCQ KD values obtained from the CMC frontal analysis method were 8.22(±0.61) × 10−7 M and 11.70(±2.44) × 10−7 M. Compare to CQ, HCQ has the weaker affinity with ACE2. The action force of CQ, HCQ and ACE2 is mainly ionic force. CQ and HCQ have different degrees of competitive binding relationship with ACE2. Our study revealed the interaction of CQ and HCQ with ACE2 receptor, which provides new insights for the use of CQ and HCQ in the treatment of COVID-19. Moreover, this biomimetic drug screening method is expected to open the door for rapid targeting and separating bioactive ingredients active towards ACE2 receptor. [ABSTRACT FROM AUTHOR]