Twin Strep-Tag Modified CPT1A Mitochondrial Membrane Chromatography in Screening Lipid Metabolism Regulators.

Mitochondrial Membrane Chromatography (MMC) is a bioaffinity chromatography technique developed to study the interaction between target proteins embedded in the mitochondrial membrane and their ligand compounds. However, the MMC stationary phases (MMSP) prepared by chemical immobilization are prone to nonspecific binding in candidate agent screening inevitably. To address these challenges, Twin Strep-Tag/Strep Tactin was employed to establish a specific affinity system in the present study. We prepared a carnitine palmitoyltransferase 1A (CPT1A) MMSP by specifically linking Strep-tactin-modified silica gel with the Twin Strep-Tag on the CPT1A-oriented mitochondrial membrane. This Twin Strep-Tag/Strep Tactin modified CPT1A/MMC method exhibited remarkably better retention behavior, longer stationary phase lifespan, and higher screening specificity compared with previous MMC systems with glutaraldehyde immobilization. We adopted the CPT1A-specific MMC system in screening CPT1A ligands from traditional Chinese medicines, and successfully identified novel candidate ligands: ononin, isoliquiritigenin, and aloe-emodin, from Glycyrrhiza uralensis Fisch and Senna tora (L.) Roxb extracts. Biological assessments illustrated that the compounds screened promote CPT1A enzyme activity without affecting CPT1A protein expression, as well as effectively reduce the lipid droplets and triglyceride levels in the high fat induction HepG2 cells. The results suggest that we have developed an MMC system, which is promising for studying the bioaffinity of mitochondrial membrane proteins to candidate compounds. This system provides a platform for a key step in mitochondrial medicine discovery, especially for bioactive molecule screening from complex herbal extracts.

Nebivolol hydrochloride and its impurities induce pseudo-allergic reactions via mast cell activation.

Nebivolol hydrochloride is a third-generation ß-blocker commonly used to treat cardiovascular diseases. However, it has been reported to induce allergic reactions in clinical use which deserves much attention. Therefore, this study focused on the ability of two isomers of nebivolol and chiral isomer impurities to induce allergic reactions. Our findings demonstrate that both nebivolol and two isomeric impurities can activate mast cell degranulation in vitro and show significant retention on Mas-related G-protein-coupled receptor X2 (MRGPRX2)-HEK293 cell membrane chromatography. These effects were further validated in vivo, where nebivolol and impurity IP-3 were observed to cause toe swelling and mast cell degranulation in mice. Molecular docking studies revealed interactions between these compounds and key amino acids of MRGPRX2, suggesting a mechanism for the induced allergic reactions. This work lays the foundation for improving the clinical safety of nebivolol.

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.

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.

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.

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.

MRGPRX2 is critical for clozapine induced pseudo-allergic reactions.

BACKGROUND: Clozapine is one of the most widely used second-generation antipsychotics in clinic. However, allergy-like symptoms such as rash and angioedema have been reported frequently, and the mechanism is still not clear. Mas-related G protein-coupled receptor X2 (MRGPRX2) expressed on mast cells is a crucial receptor for drug induced pseudo-allergic reactions. Therefore, we explored whether the symptoms induced by clozapine were associated with allergic reaction through MRGPRX2. METHODS: The effects of clozapine on pseudo-allergic reactions were evaluated by mast cells degranulation and calcium mobilization assay in vitro, and mice hindpaw swelling, serum histamine detection, avidin and H&E staining assay in vivo. The overexpressed MRGPRX2 cells membrane chromatography (MRGPRX2-HEK293/CMC), MRGPRX2-HEK293 cells calcium mobilization assay and molecular docking were applied to research the correlation between clozapine and MRGPRX2. RESULTS: The study showed that clozapine induced the release of ß-hexosaminidase, histamine and monocyte chemoattractant protein-1 (MCP-1), and trigged calcium mobilization in mast cells. In vivo, clozapine induced paw swelling, degranulation and vasodilation. Furthermore, clozapine could activate the calcium mobilization obviously in MRGPRX2-HEK293 cells, not in NC-HEK293 cells. Clozapine also had a good retention characteristic on MRGPRX2-HEK293/CMC column and the K D value is (2.33 ± 0.21)×10-01M. CONCLUSIONS: Our findings demonstrated that clozapine could induce pseudo-allergic reactions and MRGPRX2 might be the critical receptor for it.

Synthesis and biological evaluation of novel biphenyl-furocoumarin derivatives as vasodilator agents.

A series of novel biphenyl-furocoumarin derivatives were synthesized based on the nuclear structure of imperatorin and identified by IR, 1H NMR, 13C NMR and MS, and evaluated for their ability to relax vessel on isolated rat mesenteric artery, basilar artery and renal artery, respectively. The majority of compounds demonstrated potent vasodilatation, and compound 8e expressed the highest activity (EC50 = 0.56 µM) in MA. Compounds with fluorine at 2-position of 5-phenyl get better activity than others with chlorine or bromine, and the compounds containing a bulky structure had relatively low activity, such as 8c (EC50 = 22.39 µM) in MA. As a follow-up, 8e, 10e, and 8c were docked into L-calcium channel (PDB code: 3G43) to explain the difference in the activity of the compounds.

Design, synthesis, and evaluation of new series of Imperatorin analogs with potential vasodilatory activity.

Two series of imperatorin analogs were synthesized based on our previous research and evaluated for their vasodilatation activities on in vitro rat mesenteric artery, basilar artery, and renal artery ring models. Target compounds were characterized by infrared, 1H NMR, and mass spectra. Most derivatives possessed significant vasodilatory activity on the mesenteric artery, and compound 3a exhibited favorable and broad vasodilatation activities on three kinds of rat artery ring models. The pharmacological results indicated that introducing nitrogen-contained ring in side chain or large steric hindrance at the distal end could increase the vasodilatory activity. Further, replacement of oxygen atom (-O-) in the skeleton of furocoumarin derivatives with nitrogen (-NH-) could cause the decrease of vasodilatory activity. The molecular docking also indicated that compound 3a showed a best affinity with α-1C receptor (PDB ID: 3G43). All these results suggested compound 3a would be a potential vasodilatory agent for hypertension.

Refined assessment of heavy metal-associated health risk due to the consumption of traditional animal medicines in humans.

Little is known about the extent of heavy metal accumulation in traditional Chinese medicines (TCMs). In this study, the levels of lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg) in traditional animal medicines were monitored using inductively coupled plasma mass spectroscopy (ICP-MS). Additionally, for the first time, a heavy metal risk assessment strategy was used to evaluate the potential risks of traditional animal medicines by calculating estimated daily intake (EDI), target hazard quotient (THQ), and cancer risk (CR). To obtain a refined risk assessment, the frequency of exposure to traditional animal medicines was determined from questionnaire data, and the safe factor for TCM was applied. Based on the standard levels for leech, it was found that earthworm, hive, scorpion, and leech accumulated high levels of heavy metals. The combined THQ (cTHQ) values indicated that ingestion of most traditional animal medicines would not pose a risk to the health of either male or female human beings. However, it was indicated that attention should be paid to the potential risk associated with cicada slough, earthworm, scorpion, turtle shells, and hive. Among heavy metals, As and Hg contributed to a major extent to the risk to human health. The CR assessment for Pb and As indicated that, with the exception of earthworm, the cancer risk was less than the acceptable lifetime risk for both males and females. Owing to the higher body weight, both THQ and CR were generally lower for males than for females.

Simultaneous identification of three pseudoallergic components in Danshen injection by using high-expression Mas-related G protein coupled receptor X2 cell membrane chromatography coupled online to HPLC-ESI-MS/MS.

Adverse drug reactions of Danshen injection mainly manifested as pseudoallergic reactions. In the present study, salvianolic acid A and a pair of geometric isomers (isosalvianolic acid C and salvianolic acid C) were identified as pseudoallergic components in Danshen injection by a high-expression Mas-related G protein coupled receptor X2 cell membrane chromatography coupled online with high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Their pseudoallergic activities were evaluated by in vitro assay, which were consistent with the retention times on the cell membrane chromatography column. Salvianolic acid C, the most outstanding compound, was further found to induce pseudoallergic reaction through Mas-related G protein coupled receptor X2. All the results above indicated that the system developed in this study is an effective method for simultaneously analyzing pseudoallergic components, even those with similar structures and the microcomponents in complex samples (salvianolic acid C in Danshen injection).

5-Phenylcoumarin Derivatives: Design, Synthesis, and Vasodilatory Activity.

In continuation of our previous efforts towards the development of coumarin derivatives with potential vasodilatory activity, 5-phenylcoumarin derivatives were designed and synthesized. Target compounds and their precursors exhibited moderately vasodilatory ability with EC50 at 2.5-49.0 µm. Docking studies also revealed the good binding mode of 7-methoxy-8-[2-(pyrrolidin-1-yl)ethoxy]-5-[2-(trifluoromethyl)phenyl]-2H-chromen-2-one (8c) with the target protein. Moreover, intermediates and the final products exhibited different fluorescent properties due to their substituent effect. These results may provide new ideas for the synthesis and application of 5-substituted coumarins.

The human mast cell line-1 cell membrane chromatography coupled with HPLC-ESI-MS/MS method for screening potentical anaphylactic components from chuanxinlian injection.

Chuanxinlian injection is a traditional Chinese medicine injection widely used in China to treat sore throat, cough and dysentery, although a high occurrence of severe adverse reactions has been reported in clinical practice in recent years. In the present study, a human mast cell line-1 cell membrane chromatography coupled with HPLC-ESI-MS/MS method was established to screen and identify potentical anaphylactic components in chuanxinlian injection, and the dehydroandrographolide was identified as a potential anaphylactic component. In vitro anaphylactic assay showed that intracellular Ca2+ concentration clearly increased under dehydroandrographolide (100 µm) treatment. ß-Hexosaminidase and histamine release in human mast cell line-1 cells were both markedly enhanced with increased concentrations of dehydroandrographolide, confirming the anaphylactic activity of dehydroandrographolide. The application for chuanxinlian injection in this study suggested that the developed human mast cell line-1 cell membrane chromatography coupled with HPLC-ESI-MS/MS system may be effective and rapid for screening the potentical anaphylactic components from complex samples.

Synthesis and fluorescent study of 5-phenyl furocoumarin derivatives as vasodilatory agents.

Two series of 5-phenyl furocoumarin derivatives were designed and prepared based on our previous research. All new compounds were characterized by (1)H NMR, (13)C NMR and mass spectra. Furthermore, they were screened for their vasodilatory activity on the mesenteric artery of Sprague-Dawley rats, and they all presented with moderate vasodilatory activity. Fluorescent properties of the target compounds were tested in methanol. The fluorescence variation of 4a was investigated in different solvents, various pH and the migration time was determined. All results indicated that this type of fluorescent compound can be used as vasodilatory agents and probes simultaneously after further structural modifications.

The imperatorin derivative OW1, a new vasoactive compound, inhibits VSMC proliferation and extracellular matrix hyperplasia.

Chronic hypertension induces vascular remodeling. The most important factor for hypertension treatment is reducing the risk of cardiovascular disease. OW1 is a novel imperatorin derivative that exhibits vasodilative activity and antihypertensive effects in two-kidney one-clip (2K1C) renovascular hypertensive rats. It also inhibited vascular remodeling of the thoracic aorta in a previous study. Here, the inhibitory effects and mechanisms of OW1 on arterial vascular remodeling were investigated in vitro and in 2K1C hypertensive rats in vivo. OW1 (20µM, 10µM, 5µM) inhibited Ang II-induced vascular smooth muscle cells (VSMCs) proliferation and ROS generation in vitro. OW1 also reversed the Ang II-mediated inhibition of α-SMA levels and stimulation of OPN levels. Histology results showed that treatment of 2K1C hypertensive rats with OW1 (20, 40, and 80mg/kg per day, respectively for 5weeks) in vivo significantly decreased the number of VSMCs, the aortic cross-sectional area (CSA), the media to lumen (M/L) ratio, and the content of collagen I and III in the mesenteric artery. Western blot results also revealed that OW1 stimulated the expression of α-SMA and inhibited the expression of collagen I and III on the thoracic aorta of 2K1C hypertensive rats. In mechanistic studies, OW1 acted as an ACE inhibitor and affected calcium channels. The suppression of MMP expression and the MAPK pathway may account for the effects of OW1 on vascular remodeling. OW1 attenuated vascular remodeling in vitro and in vivo. It could be a novel candidate for hypertension intervention.

Antihypertensive and vascular remodelling effects of the imperatorin derivative OW1 in renovascular hypertension rats.

OW1 is a novel imperatorin derivative that exhibits vasodilator activity. In the present study, the antihypertensive effect of and inhibition of vascular remodelling by OW1 were investigated in two-kidney, one-clip (2K1C) renovascular hypertensive rats. Rats were subjected to the 2K1C procedure and treated with OW1 (40 or 80 mg/kg per day) for 8 weeks. Blood pressure was measured in conscious rats. Microalbumin (mALB) and total protein (U-TP) concentrations were determined in the urine, as were plasma concentrations of angiotensin (Ang) II, calcitonin gene-related peptide (CGRP) and angiotensin-converting enzyme 1 (ACE). The unclipped kidney was stained with haematoxylin and eosin and Masson trichrome, whereas aortic sections were stained with Masson trichrome. In addition, OW1-induced vasodilatation was evaluated in vitro in rat mesenteric and renal arteries. Immunohistochemical analysis was used to quantify collagen I and III expression. OW1 relaxed rat mesenteric and renal arterial rings in vitro. Treatment of 2K1C hypertensive rats with OW1 (40 and 80 mg/kg per day) for 8 weeks significantly decreased blood pressure. In addition, OW1 reduced plasma AngII and ACE concentrations and increased plasma CGRP concentrations. At 80 mg/kg per day, OW1 decreased blood urea nitrogen, mALB and U-TP levels. Histological analysis revealed that OW1 reduced renal arteriolar thickness and relieved the structural hypertrophic arteries. Moreover, OW1 had an inhibitory effect on vascular remodelling and renal lesions in hypertensive rats. In conclusion, the results suggest that OW1 could potentially be a novel candidate for hypertension intervention.

Design, synthesis and evaluation of 9-hydroxy-7H-furo[3,2-g]chromen-7-one derivatives as new potential vasodilatory agents.

Two new 9-hydroxy-7H-furo[3,2-g]chromen-7-one derivatives were designed, synthesized and evaluated for their in vitro vasodilatory activity. The structures of two compounds were elucidated by infrared, ¹H NMR, and mass spectral data. The in vitro pharmacological evaluation indicated that both of them possessed well vasodilatory activity compared with imperatorin. The molecule docking also showed two target compounds docked well with L-calcium channel (PDB code: 3G43). The result suggested that they would be potential vasodilatory agents for hypertension.

Synthesis and anti-SARS-CoV-2 activity of amino acid modified cephalotaxine derivatives.

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic has triggered a significant impact on global public health security, it is urgent to develop effective antiviral drugs. Previous studies have found that binding to ACE2 is a key step in the invasion of SARS-CoV-2 into host cells, so virus invasion can be inhibited by blocking ACE2, but there are few reports on this kind of specific inhibitor. Our previous study found that Harringtonine (HT) can inhibit the entry of SARS-CoV-2 spike pseudovirus into ACE2h cells, but its relatively high cytotoxicity limits its further development. Amino acid modification of the active components can increase their solubility and reduce their cytotoxicity. Therefore, in this study, seven new derivatives were synthesized by amino acid modification of its core structure Cephalotaxine. The target compounds were evaluated by cell viability assay and the SARS-CoV-2 spike pseudovirus entry assay. Compound CET-1 significantly inhibited the entry of pseudovirus into ACE2h cells and showed less cytotoxicity than HT. Molecular docking results showed that CET-1 could bind TYR83, an important residue of ACE2, just like HT. In conclusion, our study provided a novel compound with more potential activity and lower toxicity than HT on inhibiting the SARS-CoV-2 spike pseudovirus infection, which makes it possible to be a lead compound as an antiviral drug in the future.

Construction and application of H1R ligand screening materials based on SMA stabilization and his-tag covalent immobilization of membrane proteins.

The histamine H1 receptor (H1R) plays a pivotal role in allergy initiation and undergoes the necessity of devising a high-throughput screening approach centered on H1R to screen novel ligands effectively. This study suggests a method employing styrene maleic acid (SMA) extraction and His-tag covalent bonding to immobilize H1R membrane proteins, minimizing the interference of nonspecific proteins interference while preserving native protein structure and maximizing target exposure. This approach was utilized to develop a novel material for high-throughput ligand screening and implemented in cell membrane chromatography (CMC). An H1R-His-SMALPs/CMC model was established and its chromatographic performance (selectivity, specificity and lifespan) validated, demonstrating a significant enhancement in lifespan compared to previous CMC models. Subsequently, this model facilitated high-throughput screening of H1R ligands in the compound library and preliminary activity verification of potential H1R antagonists. Identification of a novel H1R antagonist laid the foundation for further development in this area.

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.