Pyrimethamine upregulates BNIP3 to interfere SNARE-mediated autophagosome-lysosomal fusion in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common tumor types and remains a major clinical challenge. Increasing evidence has revealed that mitophagy inhibitors can enhance the effect of chemotherapy on HCC. However, few mitophagy inhibitors have been approved for clinical use in humans. Pyrimethamine (Pyr) is used to treat infections caused by protozoan parasites. Recent studies have reported that Pyr may be beneficial in the treatment of various tumors. However, its mechanism of action is still not clearly defined. Here, we found that blocking mitophagy sensitized cells to Pyr-induced apoptosis. Mechanistically, Pyr potently induced the accumulation of autophagosomes by inhibiting autophagosome-lysosome fusion in human HCC cells. In vitro and in vivo studies revealed that Pyr blocked autophagosome-lysosome fusion by upregulating BNIP3 to inhibit synaptosomal-associated protein 29 (SNAP29)-vesicle-associated membrane protein 8 (VAMP8) interaction. Moreover, Pyr acted synergistically with sorafenib (Sora) to induce apoptosis and inhibit HCC proliferation in vitro and in vivo. Pyr enhances the sensitivity of HCC cells to Sora, a common chemotherapeutic, by inhibiting mitophagy. Thus, these results provide new insights into the mechanism of action of Pyr and imply that Pyr could potentially be further developed as a novel mitophagy inhibitor. Notably, Pyr and Sora combination therapy could be a promising treatment for malignant HCC.

Screening of CPT1A-Targeting Lipid Metabolism Modulators Using Mitochondrial Membrane Chromatography.

Carnitine palmitoyltransferase 1A (CPT1A), which resides on the mitochondrial outer membrane, serves as the rate-limiting enzyme of fatty acid ß-oxidation. Identifying the compounds targeting CPT1A warrants a promising candidate for modulating lipid metabolism. In this study, we developed a CPT1A-overexpressed mitochondrial membrane chromatography (MMC) to screen the compounds with affinity for CPT1A. Cells overexpressing CPT1A were cultured, and subsequently, their mitochondrial membrane was isolated and immobilized on amino-silica gel cross-linked by glutaraldehyde. After packing the mitochondrial membrane column, retention components of MMC were performed with LC/MS, whose analytic peaks provided structural information on compounds that might interact with mitochondrial membrane proteins. With the newly developed MMC-LC/MS approach, several Chinese traditional medicine extracts, such as Scutellariae Radix and Polygoni Cuspidati Rhizoma et Radix (PCRR), were analyzed. Five noteworthy compounds, baicalin, baicalein, wogonoside, wogonin, and resveratrol, were identified as enhancers of CPT1A enzyme activity, with resveratrol being a new agonist for CPT1A. The study suggests that MMC serves as a reliable screening system for efficiently identifying modulators targeting CPT1A from complex extracts.

Development of an environmentally sensitive fluorescent peptide probe for MrgX2 and application in ligand screening of peptide antibiotics.

Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.

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

Membrane protein (MP)-based biomaterials have a wide range of applications in drug screening, antigen detection, and ligand-receptor interaction analysis. Traditional MP immobilization methods have the disadvantage of disordered protein immobilization orientation, leading to the shielded binding domain and unreliable binding pattern. Herein, we describe a site-specific covalent immobilization of MPs, which utilizes the styrene maleic acid (SMA) detergent-free extraction method of MPs as well as the covalent reaction between His-tag and divinyl sulfone (DVS). As an example, we covalently immobilized angiotensin-converting enzyme 2 (ACE2) on a cell membrane chromatography system (ACE2-His-SMALPs/CMC) in a site-specific manner and verified the specificity and stability of this system. This technique significantly improves the service life compared to the physisorption CMC column. The improved protein immobilization strategies of the ACE2-His-SMALPs/CMC system enable it to effectively recognize SARS-CoV-2 pseudoviral particles as well as detect viral particles in ambient air once combined with an aerosol collector; as a powerful ligand biosensor, the ACE2-His-SMALPs/CMC system was used to screen for compounds with anti-SARS-CoV-2 pseudovirus activity. In conclusion, the optimized MP immobilization strategy has been successfully applied to CMC technology, showing enhanced stability and sensitivity, which can provide an efficient and convenient membrane protein immobilization method for biomaterials.

Harringtonine: A more effective antagonist for Omicron variant.

Fusion with host cell membrane is the main mechanism of infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we propose that a new strategy to screen small-molecule antagonists blocking SARS-CoV-2 membrane fusion. Using cell membrane chromatography (CMC), we found that harringtonine (HT) simultaneously targeted SARS-CoV-2 S protein and host cell surface TMPRSS2 expressed by the host cell, and subsequently confirmed that HT can inhibit membrane fusion. HT effectively blocked SARS-CoV-2 original strain entry with the IC50 of 0.217 µM, while the IC50 in delta variant decreased to 0.101 µM, the IC50 in Omicron BA.1 variant was 0.042 µM. Due to high transmissibility and immune escape, Omicron subvariant BA.5 has become the dominant strain of the SARS-CoV-2 virus and led to escalating COVID-19 cases, however, against BA.5, HT showed a surprising effectiveness. The IC50 in Omicron BA.5 was even lower than 0.0019 µM. The above results revealed the effect of HT on Omicron is very significant. In summary, we characterize HT as a small-molecule antagonist by direct targeting on the Spike protein and TMPRSS2.

5-Hydroxymethylfurfural multimers induce pseudo-allergic reaction through FcεRI at high doses.

Under acidic and high temperature conditions, 5-hydroxymethylfurfural (5-HMF) converted from sugar further produces dimers (Compound II) and trimers (Compound III). The polymers were less reported, and sensitization effect of them was reported in this study. Compounds II and III induced the local and systemic anaphylaxis effect in passive cutaneous anaphylaxis mice model and activated RBL-2H3 cell inducing [Ca2+ ] mobilization, resulting in the release of ß-hexosaminidase and histamine in vitro. The gene knockdown assay figured out that Compounds II and III induced degranulation through FcεRI. Further, Compounds II and III had a certain affinity with FcεRI by cell membrane chromatography and may combine on the "proline sandwich" structure indicated by molecular docking. All above suggested Compounds II and III can induce pseudo-allergic reaction through FcεRI in vivo and in vitro. Our work provides basic research to prove that the newly discovered 5-HMF transformants, Compounds II and III, induce pseudo-allergic reaction in vitro and in vivo through FcεRI, which is different pathway from 5-HMF. In foods with high sugar content, the sensitization of Compounds II and III needs more attention. In high-sugar foods and medicines, especially traditional Chinese medicine injections, the content of transformants needs to be detected.

Screening potential anaphylactoid components in vinpocetine injection using a high expression Mas-related G-protein-coupled receptor X2 cell membrane chromatography.

Vinpocetine injection is often used in clinical treatment of acute cardiovascular and cerebrovascular diseases. However, it was reported that vinpocetine injection caused allergic reactions in clinical use; therefore, its safety needs urgent attention. Until now, research on its sensitization is rarely reported. Here, the components contained in three vinpocetine injections were examined. It was found that besides vinpocetine, the synthetic raw material vincamine, the excipients benzyl alcohol and ethyl p-toluenesulfonate, and the impurities A, B, C, and D, which are excipients specified in the European Pharmacopoeia, were also present in them. Then the Mas-related G-protein-coupled receptor X2 (MRGPRX2)-HEK293 cell membrane chromatography was used to investigate the affinity of them with MRGPRX2 and found that vinpocetine, vincamine, and impurities A, B, C, and D bind to MRGPRX2. Afterwards, these compounds were further used to investigate the local sensitization ability in vivo. The results showed that vinpocetine, vincamine, and impurity C could induce swelling of the paw and decrease body temperature in mice, but only impurity C could cause local skin mast cell degranulation and serum histamine release increase. In vitro, the results also indicated that impurity C could increase intracellular [Ca2+ ] in MRGPRX2-HEK293 cells, whereas vinpocetine and vincamine did not. Therefore, the impurity C was the potential anaphylactoid component in vinpocetine injection, which may be one of the reasons for the occurrence of allergic reactions in the clinical use of vinpocetine injection. This work provides evidence on the sensitization of impurity C and also contributes to promoting the clinical safety of vinpocetine injection.

Convenient Diaryl Ureas as Promising Anti-pseudo-allergic Agents.

Allergic diseases are a group of allergen-induced unfavorable immune responses initiating various symptoms in different organs. Mas-related G protein-coupled receptor X2 (MRGPRX2) on mast cells has been reported to be responsible for immunoglobulin E (IgE)-independent immune diseases and allergic drug reactions and has therefore been a crucial drug target for the development of anti-pseudo-allergic agents. Considering the active structural features of MRGPRX2, we designed and synthesized a series of diaryl ureas (DPUs). DPUs exert promising potency for inhibiting ß-hexosaminidase release in LAD2 cells with half-maximal inhibitory concentrations (IC50) values of 2.51-0.62 µM, as well as favorable antilocal and systemic anaphylaxis in mice at a dosage of 10 mg/kg. MRGPRX2 is further revealed to participate in the anti-pseudo-allergic activity of DPUs by binding with electrophilic urea and trifluoromethyl substituents. In brief, these results highlight entities with powerful electrophilic substituents as a prospective therapeutic strategy for the treatment of IgE-independent disorders.

Synthetic imperatorin derivatives alleviate allergic reactions via mast cells.

Anaphylaxis is a severe systemic allergic reaction that exhibits multiple clinical symptoms. The Mas-related G protein-coupled receptor X2 (MRGPRX2) is recognized as a key cell receptor mediating allergic diseases and drug-induced anaphylactoid reactions. Thus, it has been a promising target for preventing and treating these reactions. Based on the potential activity of imperatorin and active structural feature of MRGPRX2, we first demonstrated that the synthetic imperatorin derivatives (IDs) could significantly inhibit MRGPRX2 agonist-induced degranulation and cytokine release in LAD2 cells, as well as alleviate local and systemic anaphylaxis in mice. The IC50 value of the most promising compound is an order of magnitude lower than that of imperatorin. IDs were further identified to display anti-pseudo-allergic activity by binding MRGPRX2 with the tertiary nitrogen substructures, just liking the reported MRGPRX2-ligand. These results would propose evidence for discovery of agents for treating MCs-dependent allergic disorders.

Synthesis and evaluation of new potential anti-pseudo-allergic agents.

Pseudo-allergic reactions frequently occur following clinical drug use and sometimes even cause mortal danger. Mas-related G-protein-coupled receptor member X2 (MRGPRX2) is a novel receptor that mediates pseudo-allergy and is an important target in the treatment of allergies. However, to date, there are no synthetic small-molecule inhibitors that prevent anaphylactoid reactions through this pathway. Our preliminary research suggested that B10-S and mubritinib effectively inhibited LAD2 cells. Therefore, two novel derivatives were synthesized by integrating the active substructures of B10-S and mubritinib, according to the molecular docking results. The antiallergic inhibitory effects of the two compounds were preliminarily evaluated in vitro using ß-hexosaminidase release, histamine release, and intracellular Ca2+ mobilization assays, and their binding sites on MRGPRX2 were analyzed by molecular docking. Both substances inhibited ß-hexosaminidase and histamine release in LAD2 cells and decreased intracellular Ca2+ by inhibiting MRGPRX2 in MRGPRX2-HEK293 cells treated with C48/80 in a dose-dependent manner. The docking results suggested that the molecules could competitively bind to the active site on MRGPRX2 and Glu141, which were combined by C48/80. Our study indicated that the two compounds have potential anti-allergic properties, which may provide evidence that will facilitate the development of synthetic molecules with anti-pseudo-allergic activity for clinical use in the future.

Design and synthesis of first environment-sensitive coumarin fluorescent agonists for MrgX2.

Mas related G-protein-coupled receptor member X2 (MrgX2) has been identified as the crucial receptor in drug induced pseudo-allergic reactions and allergic diseases. In this research, the first type of fluorescent agonists (ZX1, ZX2 and ZX3) for MrgX2 were developed by conjugating environment-sensitive fluorophore coumarin to MrgX2 selective agonists (R)-ZINC-3573. Their environment-sensitive property was confirmed by the dramatically increase of fluorescent intensity after binding to the hydrophobic ligand binding domain MrgX2, which help to overcome the high background signal. Based on these characteristics, they can be used for selective visualization of MrgX2 in living cells even with their own background interference. Among these fluorescent agonists, compound ZX2 possessed splendid spectroscopic properties, outstanding pharmacological activities (EC50 = 0.93 µM, KD = 1.97 µM). And a competitive binding assay was established with ZX2 to analysis the binding affinity of MrgX2 agonists, which shown high coherence with the results of cell membrane chromatography. To our knowledge, these probes are the first fluorescent ligands of MrgX2 with agonistic activity and environment-sensitive property, which is expected to use for the development of MrgX2 molecular pharmacology and serve as a convenient high-throughput screening tool for the drug candidates targeting MrgX2.

Novel bioavailability-based risk assessment of Cd in earthworms and leeches utilizing in vitro digestion/Caco-2 and MDCK cells.

In the present study, the oral bioavailability of cadmium (Cd) in earthworms and leeches was investigated through in vitro physiologically based extraction test (PBET) digestion/Caco2 and MDKC cell models. We are the first to create an innovative assessment strategy which has capacity to offer a more precise evaluation of Cd-associated health risks in traditional animal medicines (TAMs), by combinational usage of bioavailable Cd levels, the duration and frequency of the exposure to TAMs obtained by questionnaire data, as well as safety factor of TAMs. Our data showed that the percentage of bioavailability for Caco-2 cells in earthworms and leeches ranged from 3.29 to 14.17% and 4.32 to 12.61%, respectively. The percentage of bioavailability of MDCK cells in earthworms and leeches ranged from 4.83 to 15.74% and 6.53 to 15.04%, respectively. After adjusting by the bioavailability of Cd to target hazard quotient (THQ), excitingly, our findings manifested that the health risks induced by the ingestion of earthworms and leeches were acceptable in the clinic. Our key findings suggest that bioavailability characterization cannot be ruled out and health risks should be assessed on the basis of the bioavailable Cd levels rather than total levels. Our novel strategy provides insight into the bio-accumulation of Cd in organisms as well as a more realistic and accurate assessment of Cd-associated health risks in TAMs, with the main purpose of improving public health by scientifically using TAMs.

Establishment of substance P modified affinity chromatography for specific detection and enrichment of Mas-related G protein-coupled receptor X2.

Mas-related G protein-coupled receptor X2 (MrgX2) has been identified to be critical in drug-induced pseudo-allergic reactions and allergic diseases. Herein, an affinity high-performance liquid chromatography was established for the specific detection and enrichment of MrgX2. Substance P was used as an affinity ligand and immobilized on a glutaraldehyde-modified amino silica gel. The successful grafting of substance P was characterized by infrared spectroscopy, elemental analysis, X-ray photoelectron spectroscopy, thermogravimetric analysis, and nitrogen adsorption and desorption analyzes. The prepared materials were then used as the stationary phase to investigate the retention behavior of MrgX2 recombinant protein on the affinity column. The results obtained with the analytical techniques show the specificity and selectivity of the MrgX2 recombinant protein on the affinity column. The repeatability and reproducibility for the analysis of MrgX2 on the NH2-Silico@GD@SP column show relative standard deviation (RSD) values lower than the acceptance criteria of 2 and 5% of retention time, and RSD of peak areas < 7%. The RSD value of the results obtained for the control of the activity of the prepared columns respond to the acceptance criteria of 5% and proves that the NH2-Silico@GD@SP column are stable until 48 h. The suitability of the NH2-Silico@GD@SP column offline SEC system has been tested by using MrgX2 as positive control. The results of this experiment indicate that the offline system may be used to analyze the retention fraction. MrgX2 extracted from human mast cells LAD2 was also verified. An obvious retention can be observed and the natural MrgX2 was concentrated 114.6 times compared with the original complex components by using the affinity column. These results may provide a new approach for the specific detection and enrichment of G-protein-coupled receptors.

Astemizole as a drug to inhibit the effect of SARS-COV-2 in vitro.

Since the beginning of December 2019, a novel Coronavirus severe respiratory disease, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which also been termed 2019-new CoV (2019-nCoV), has continued to spread worldwide. As of August 27, 2020, a total of 24,232,429 people have been infected and 826,518 people have died. In our study, we found that astemizole can antagonize ACE2 and inhibit the entry of SARS-COV-2 spike pseudovirus into ACE2-expressed HEK293T cells (ACE2hi cells). We analysied the binding character of astemizole to ACE2 by molecular docking and surface plasmon resonance (SPR) assays and molecule docking, SARS-COV-2 spike pseudotype virus was also taken to investigate the suppression viropexis effect of astemizole. The results showed that astemizole can bind to the ACE2 receptor and inhibit the invasion of SARS-COV-2 Spike pseudoviruses. Thus astemizole represent potential drug candidates that can be re-used in anti-coronavirus therapies.

Azelastine inhibits viropexis of SARS-CoV-2 spike pseudovirus by binding to SARS-CoV-2 entry receptor ACE2.

A recent study have reported that pre-use of azelastine is associated with a decrease in COVID-19 positive test results among susceptible elderly people. Besides, it has been reported that antihistamine drugs could prevent viruses from entering cells. The purpose of this study is to investigate whether azelastine have antiviral activity against SARS-CoV-2 in vitro and the possible mechanism. Here, we discovered antihistamine azelastine has an affinity to ACE2 by cell membrane chromatography (CMC); Then we determined the equilibrium dissociation constant (KD) of azelastine-ACE2 as (2.58 ± 0.48) × 10-7 M by surface plasmon resonance (SPR). The results of molecular docking showed that azelastine could form an obvious hydrogen bond with Lys353. The pseudovirus infection experiments showed that azelastine effectively inhibited viral entry (EC50 = 3.834 µM). Our work provides a new perspective for the screening method of drug repositioning for COVID-19, and an attractive and promising drug candidate for anti-SARS-CoV-2.

Testing of the inhibitory effects of loratadine and desloratadine on SARS-CoV-2 spike pseudotyped virus viropexis.

Currently, there is an urgent need to find a treatment for the highly infectious coronavirus disease (COVID-19). However, the development of a new, effective, and safe vaccine or drug often requires years and poses great risks. At this critical stage, there is an advantage in using existing clinically approved drugs to treat COVID-19. In this study, in vitro severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike pseudotyped viral infection experiments indicated that histamine H1 antagonists loratadine (LOR) and desloratadine (DES) could prevent entry of the pseudotyped virus into ACE2-overexpressing HEK293T cells and showed that DES was more effective. Further binding experiments using cell membrane chromatography and surface plasmon resonance demonstrated that both antagonists could bind to ACE2 and that the binding affinity of DES was much stronger than that of LOR. Molecular docking results elucidated that LOR and DES could bind to ACE2 on the interface of the SARS-CoV-2-binding area. Additionally, DES could form one hydrogen bond with LYS31 but LOR binding relied on non-hydrogen bonds. To our knowledge, this study is the first to demonstrate the inhibitory effect of LOR and DES on SARS-CoV-2 spike pseudotyped virus viropexis by blocking spike protein-ACE2 interaction. This study may provide a new strategy for finding an effective therapeutic option for COVID-19.

Repositioning of histamine H1 receptor antagonist: Doxepin inhibits viropexis of SARS-CoV-2 Spike pseudovirus by blocking ACE2.

The spread of the corona virus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been intensifying in the past year, posing a huge threat to global health. There is an urgent need for effective drugs and vaccines to fight the COVID-19, but their advent may not be quite fast. Drug repurposing is a feasible strategy in the current situation, which could greatly shorten drug development time and help to response quickly to the novel virus outbreak. It has been reported that histamine H1 receptor antagonists have broad-spectrum antiviral effects. Therefore, in this study, we aim to screen potential drugs among histamine H1 receptor antagonists that may inhibit SARS-CoV-2 infection. Based on the model of angiotensin-converting enzyme 2 (ACE2) overexpressing HEK293T cell membrane chromatography (CMC), five FDA-approved histamine H1 receptor antagonists were found to have bioaffinity to ACE2. Then we determined the interaction between these drugs and ACE2 by frontal analysis and surface plasmon resonance (SPR), which consistently demonstrated that these hits bind to ACE2 at micromolar levels of affinity. Through the pseudovirus assay, we finally identified that doxepin could inhibit SARS-CoV-2 spike pseudovirus from entering the ACE2-expressing cell, reducing the infection rate to 25.82%. These preliminary results indicate that the histamine H1 receptor antagonist, doxepin, is a viable drug candidate for clinical trials. Therefore, we hope the work timely provides rational help for developing anti-SARS-CoV-2 drugs to control the rapid spread of SARS-CoV-2.

Fingerprint analysis of phenolic acid extract of Salvia miltiorrhiza by digital reference standard analyzer with one or two reference standards.

BACKGROUND: Fingerprint analysis and simultaneous multi-components determination are crucial for the holistic quality control of traditional Chinese medicines (TCMs). Yet, reference standards (RS) are often commercially unavailable and with other shortages, which severely impede the application of these technologies. METHODS: A digital reference standard (DRS) strategy and the corresponding software called DRS analyzer, which supports chromatographic algorithms, spectrum algorithms, and the combination of these algorithms, was developed. The extensive function also enabled the DRS analyzer to recommend the chromatographic column based on big data. RESULTS: Various quality control methods of fingerprints of 11 compounds in polyphenolic acid extract of Salvia miltiorrhiza (S. miltiorrhiza) were developed based on DRS analyzer, involving relative retention time (RRT) method, linear calibration using two reference substances (LCTRS) technique, RRT combined with Photon Diode Array (PDA) method, LCTRS combined with PDA method. Additionally, the column database of samples was established. Finally, our data demonstrated that the DRS analyzer could accurately identify 11 compounds of the samples, using only one or two physical RSs. CONCLUSIONS: The DRS strategy is an automated, intelligent, objective, accurate, eco-friendly, universal, sharing, and promising method for overall quality control of TCMs that requires the usage of fewer RSs.

Imperatorin ameliorates mast cell-mediated allergic airway inflammation by inhibiting MRGPRX2 and CamKII/ERK signaling pathway.

BACKGROUND: Allergic asthma is a common inflammatory lung disease associated with complex pathogenesis. Mast cell (MC) is one of the key drivers of allergic asthma, Mas-related G protein-coupled receptor X2 (MRGPRX2) on the MC could mediate MC activation and trigger a pseudo-allergic reaction. Imperatorin (IMP), the main active compound of Radix Angelicae Dahuricae, has been reported to exert various pharmacological effects. In this study, we focused on the therapeutical mechanism of IMP on MRGPRX2-induced pseudo-allergy and allergic asthma. METHODS: We examined the effect of IMP on MRGPRX2 related mast cell activation in mouse peritoneal MC (MPMC), Human Laboratory of Allergic Disease 2 MCs (LAD2 cells) and Mrgprx2-expressing HEK293 cells. Molecular docking and Surface plasmon resonance (SPR) were taken to reveal the binding character between IMP and MRPGRX2. MRGPRX2 downstream proteins were also detected by western blotting. IgE-independent responses was evaluated by using passive cutaneous anaphylaxis (PCA) and active systemic anaphylaxis (ASA) models. The therapeutic effect of IMP on asthma was evaluated by a lung inflammation mouse model which was induced by ovalbumin (OVA). RESULTS: IMP was found to reduce substance P (SP) induced calcium flux and suppressed degranulation of MC. SP can promote the phosphorylation of ERK and CamKII, which regulates the synthesis of inflammatory factors such as MIP-2 and TNF-α in MC. In vivo assays revealed that IMP can mitigate SP-induced mouse PCA and ASA. IMP could also mitigate lung inflammation in an OVA induced mice model by inhibiting MC activation in the lung tissue. Furthermore, IMP binds well to MRGPRX2 protein. The binding constant (KD) is 4.48 ± 0.49 × 10-7 M. The data suggeste that IMP is a novel inhibitor of MRGPRX2 to treat allergic asthma.

Screened antipsychotic drugs inhibit SARS-CoV-2 binding with ACE2 in vitro.

AIM: The coronavirus disease 2019 (COVID-19) pandemic has swept the globe and no specific effective drug has been identified. Drug repurposing is a well-known method to address the crisis in a time-critical fashion. Antipsychotic drugs (APDs) have been reported to inhibit DNA replication of hepatitis B virus, measles virus germination, and HIV infection, along with replication of SARS-CoV and MERS-CoV, both of which interact with host cells as SARS-CoV-2. METHODS: Nineteen APDs were screened using ACE2-HEK293T cell membrane chromatography (ACE2-HEK293T/CMC). Cytotoxicity assay, coronavirus spike pseudotype virus entry assay, surface plasmon resonance, and virtual molecular docking were applied to detect affinity between ACE2 protein and drugs and a potential antiviral property of the screened compounds. KEY FINDINGS: After the CMC screening, 8 of the 19 APDs were well-retained on ACE2-HEK293T/CMC column and showed significant antiviral activities in vitro. Three quarters of them belong to phenothiazine and could significantly inhibit the entrance of coronavirus into ACE2-HEK293T cells. Aother two drugs, aripiprazole and tiapride, exhibited weaker inhibition. We selected five of the drugs for subsequent evaluation. All five showed similar affinity to ACE2 and virtual molecular docking demonstrated they bound with different amino acids respectively on ACE2 which SARS-CoV-2 binds to. SIGNIFICANCE: Eight APDs were screened for binding with ACE2, five of which demonstrated potential protective effects against SARS-CoV-2 through acting on ACE2. Although the five drugs have a weak ability to block SARS-CoV-2 with a single binding site, they may provide a synergistic effect in adjuvant therapy of COVID-19 infection.