Revealing receptor-ligand interactions of atypical antipsychotic drugs and screening anti-schizophrenia ingredients in Magnolia officinalis based on 5-HTR2A-SNAP-Tag/CMC and DRD2-SNAP-Tag/CMC models.

Schizophrenia is a serious mental illness with unknown etiology, and shows increasing incidence and high lifetime prevalence rate. The main receptors related to the disease are DRD2 and 5-HTR2A. Thus, a comprehensive understanding of the interaction mode between antipsychotic drugs with relevant receptors is very important for developing more effective drugs. 5-HTR2A-SNAP-Tag/CMC and DRD2-SNAP-Tag/CMC models constructed in this work provided a new method for studying the interaction between atypical antipsychotics and the two receptors. The results of comparative experiments showed that the new models not only met the high selectivity and specificity of the screening requirements but were also more stable and long-lasting than the traditional CMC model. Binding assays showed that the effects of three atypical antipsychotics (including clozapine, olanzapine, and quetiapine) on 5-HTR2A were stronger than their effects on DRD2. Additionally, two potentially active components, magnolol and honokiol, were screened in Magnolia officinalis methanol extract using the 5-HTR2A-SNAP-Tag/CMCHPLC-MS system. Nonlinear chromatographic analysis and molecular docking were conducted to study the interactions between screened compounds and the two receptors. The binding constants (KA) of magnolol and honokiol with 5-HTR2A were 17,854 ± 1,117 M-1 and 38,858 ± 4,964 M-1, respectively, and KA values with DRD2 were 4,872 ± 1,618 M-1 and 20,692 ± 10,267 M-1, respectively. We concluded that the established models are reliable for studying receptor-ligand interactions and screening antagonists of schizophrenia.

Application of SNAP-tag-EGFR cell membrane chromatography model in screening antitumor active components of Silybum marianum (L.) Gaertn.

The SNAP-tag-epidermal growth factor receptor (SNAP-tag-EGFR) cell membrane chromatography (CMC) model is a powerful tool for investigating ligand-receptor interactions and screening active ingredients in traditional Chinese medicine. Most tyrosine kinase inhibitors (TKIs) target epidermal growth factor receptors. However, TKIs associated with significant side effects and drug resistance must be addressed immediately. Therefore, there is an urgent need to develop new TKIs with high efficiency and low toxicity. Because of its low toxicity and side effects, traditional Chinese medicine has been widely employed to treat various diseases, including cancer. Hence, this study aimed to use the SNAP-tag-EGFR/CMC-high-performance liquid chromatography-mass spectrometry (HPLC-MS) two-dimensional system model as the research tool to screen and identify potential EGFR antagonists from the Chinese medicine Silybum marianum (L.) Gaertn. The applicability of the system was verified using the positive control drug osimertinib. Four potential EGFR antagonists were screened from the Chinese medicine Silybum marianum (L.) Gaertn.. They were identified as silydianin, silychristin, silybin, and isosilybin. Additionally, their pharmacological activity was preliminarily verified using a CCK-8 assay. The kinetic parameters of the four active ingredients interacting with EGFR and their binding modes with EGFR were analyzed using nonlinear chromatography (NLC) and molecular docking. This study identified silydianin, silychristin, silybin, and isosilybin from Silybum marianum (L.) Gaertn. and verified their potential antitumor effects on EGFR.

Establishment of angiotensin-converting enzyme 2 and cluster of differentiation 147 dual target cell membrane chromatography based on SNAP-tag technology for screening anti severe acute respiratory syndrome coronavirus 2 active components.

Patients have different responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and these may be life-threatening for critically ill patients. Screening components that act on host cell receptors, especially multi-receptor components, is challenging. The in-line combination of dual-targeted cell membrane chromatography and a liquid chromatography-mass spectroscopy (LC-MS) system for analyzing angiotensin-converting enzyme 2 (ACE2) and cluster of differentiation 147 (CD147) receptors based on SNAP-tag technology provides a comprehensive solution for screening multiple components in complex samples acting on the two receptors. The selectivity and applicability of the system were validated with encouraging results. Under the optimized conditions, this method was used to screen for antiviral components in Citrus aurantium extracts. The results showed that 25 µmol /L of the active ingredient could inhibit virus entry into cells. Hesperidin, neohesperidin, nobiletin, and tangeretin were identified as antiviral components. In vitro pseudovirus assays and macromolecular cell membrane chromatography further verified the interaction of these four components with host-virus receptors, showing good effects on some or all of the pseudoviruses and host receptors. In conclusion, the in-line dual-targeted cell membrane chromatography LC-MS system developed in this study can be used for the comprehensive screening of antiviral components in complex samples. It also provides new insight into small-molecule drug-receptor and macromolecular-protein-receptor interactions.

Methylparaben as a preservative in the development of a multi-dose HPV-2 vaccine.

The human papillomavirus (HPV) vaccine is the simplest, most economical, convenient, and effective method of preventing cervical cancer. However, the current HPV vaccine is supplied as a single-dose vial with a relatively high cost per dose, which hinders its supply to low- and middle-income countries (LMICs), where the demand for HPV vaccine is highest. Therefore, it is necessary to develop a multi-dose HPV vaccine to promote large-scale affordable vaccination in LMICs. Moreover, the addition of preservatives is required to reduce the risk of microbial contamination in multi-dose vaccines within a single vial. In this study, we investigated the effects of six preservatives on HPV 16L1 and 18L1 virus-like particles in solution, as well as the aluminum adsorption status, under normal and high-temperature conditions. Multiple methods were employed, including dynamic light scattering, differential scanning calorimetry, an in vitro relative potency assay, and an in vivo potency assay in mice. Based on the above results, four types of selected preservatives were further studied, and an antimicrobial effectiveness test was performed on the HPV-2 vaccine, which was employed as a model HPV vaccine. Finally, three preservatives were selected based on their performance to evaluate the long-term stability of the HPV-2 vaccine. The results indicated that 0.12% methylparaben is the most suitable preservative for the multi-dose HPV-2 vaccine, guaranteeing the shelf life for at least three years and meeting "B" standards for antimicrobial effectiveness. The formula developed in this study can contribute toward combating cervical cancer in LMICs.

Development of recombinant COVID-19 vaccine based on CHO-produced, prefusion spike trimer and alum/CpG adjuvants.

COVID-19 pandemic has severely impacted the public health and social economy worldwide. A safe, effective, and affordable vaccine against SARS-CoV-2 infections/diseases is urgently needed. We have been developing a recombinant vaccine based on a prefusion-stabilized spike trimer of SARS-CoV-2 and formulated with aluminium hydroxide and CpG 7909. The spike protein was expressed in Chinese hamster ovary (CHO) cells, purified, and prepared as a stable formulation with the dual adjuvant. Immunogenicity studies showed that candidate vaccines elicited robust neutralizing antibody responses and substantial CD4+ T cell responses in both mice and non-human primates. And vaccine-induced neutralizing antibodies persisted at high level for at least 6 months. Challenge studies demonstrated that candidate vaccine reduced the viral loads and inflammation in the lungs of SARS-CoV-2 infected golden Syrian hamsters significantly. In addition, the vaccine-induced antibodies showed cross-neutralization activity against B.1.1.7 and B.1.351 variants. These data suggest candidate vaccine is efficacious in preventing SARS-CoV-2 infections and associated pneumonia, thereby justifying ongoing phase I/II clinical studies in China (NCT04982068 and NCT04990544).