Label-free cell phenotypic profiling of histamine H4R receptor and discovery of non-competitive H4R antagonist from natural products.

Histamine 4 receptor (H4R), the most recently identified subtype of histamine receptor, primarily induces inflammatory reactions upon activation. Several H4R antagonists have been developed for the treatment of inflammatory bowel disease (IBD) and atopic dermatitis (AD), but their use has been limited by adverse side effects, such as a short half-life and toxicity. Natural products, as an important source of anti-inflammatory agents, offer minimal side effects and reduced toxicity. This work aimed to identify novel H4R antagonists from natural products. An H4R target-pathway model deconvoluted downstream Gi and MAPK signaling pathways was established utilizing cellular label-free integrative pharmacology (CLIP), on which 148 natural products were screened. Cryptotanshinone was identified as selective H4R antagonist, with an IC50 value of 11.68 ± 1.30 µM, which was verified with Fluorescence Imaging Plate Reader (FLIPR) and Cellular Thermal Shift (CTS) assays. The kinetic binding profile revealed the noncompetitive antagonistic property of cryptotanshinone. Two allosteric binding sites of H4R were predicted using SiteMap, Fpocket and CavityPlus. Subsequent molecular docking and dynamics simulation indicated that cryptotanshinone interacts with H4R at a pocket formed by the outward interfaces between TM3/4/5, potentially representing a new allosteric binding site for H4R. Overall, this study introduced cryptotanshinone as a novel H4R antagonist, offering promise as a new hit for drug design of H4R antagonist. Additionally, this study provided a novel screening model for the discovery of H4R antagonists.

Discovery and characterization of novel ATP citrate lyase inhibitors from Acanthopanax senticosus (Rupr. & Maxim.) Harms.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism, and abnormally high expression of ACLY occurs in many diseases, including cancers, dyslipidemia and cardiovascular diseases. ACLY inhibitors are prospective treatments for these diseases. However, the scaffolds of ACLY inhibitors are insufficient with weak activity. The discovery of inhibitors with structural novelty and high activity continues to be a research hotpot. Acanthopanax senticosus (Rupr. & Maxim.) Harms is used for cardiovascular disease treatment, from which no ACLY inhibitors have ever been found. In this work, we discovered three novel ACLY inhibitors, and the most potent one was isochlorogenic acid C (ICC) with an IC50 value of 0.14 ± 0.04 µM. We found dicaffeoylquinic acids with ortho-dihydroxyphenyl groups were important features for inhibition by studying ten phenolic acids. We further investigated interactions between the highly active compound ICC and ACLY. Thermal shift assay revealed that ICC could directly bind to ACLY and improve its stability in the heating process. Enzymatic kinetic studies indicated ICC was a noncompetitive inhibitor of ACLY. Our work discovered novel ACLY inhibitors, provided valuable structure-activity patterns and deepened knowledge on the interactions between this targe tand its inhibitors.

Precise Capture and Dynamic Release of Circulating Liver Cancer Cells with Dual-histidine-based Cell Imprinted Hydrogels.

Circulating tumor cells (CTCs) detection presents significant advantages in diagnosing liver cancer due to its non-invasiveness, real-time monitoring, and dynamic tracking. However, the clinical application of CTCs-based diagnosis is largely limited by the challenges of capturing low-abundance CTCs within a complex blood environment while ensuring them alive. Here we design an ultra-strong ligand, L-histidine-L-histidine (HH), specifically targeting sialylated glycans on the surface of CTCs. Further HH is integrated into a cell-imprinted polymer, constructing a hydrogel with precise CTCs imprinting, high elasticity, satisfactory blood-compatibility, and robust anti-interference capacities. These features endow the hydrogel with excellent capture efficiency (>95%) for CTCs in peripheral blood, as well as the ability to release CTCs controllably and alive. Clinical tests substantiate the accurate differentiation between liver cancer, cirrhosis, and healthy groups using this method. The remarkable diagnostic accuracy (94%), lossless release of CTCs, material reversibility, and cost-effectiveness (6.68 dollars per sample) make the HH-based hydrogel a potentially revolutionary technology for liver cancer diagnosis and single-cell analysis. This article is protected by copyright. All rights reserved.

Elucidation of active components and target mechanism in Jinqiancao granules for the treatment of prostatitis and benign prostatic hyperplasia.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostatitis and benign prostatic hyperplasia (BPH) are inflammations of the prostate gland, which surrounds the urethra in males. Jinqiancao granules are a traditional Chinese medicine used to treat kidney stones and this medicine consists of four herbs: Desmodium styracifolium (Osbeck) Merr., Pyrrosia calvata (Baker) Ching, Plantago asiatica L. and stigma of Zea mays L. AIM OF THE STUDY: We hypothesized that Jinqiancao granules could be a potential therapy for prostatitis and BPH, and this work aimed to elucidate active compounds in Jinqiancao granules and their target mechanisms for the potential treatment of the two diseases. MATERIALS AND METHODS: Jinqiancao granules were commercially available and purchased. Database-driven data mining and networking were utilized to establish a general correlation between Jinqiancao granules and the two diseases above. Ultra-performance liquid chromatography-mass spectrometry was used for compound separation and characterization. The characterized compounds were evaluated on four G-protein coupled receptors (GPCRs: GPR35, muscarinic acetylcholine receptor M3, alpha-1A adrenergic receptor α1A and cannabinoid receptor CB2). A dynamic mass redistribution technique was applied to evaluate compounds on four GPCRs. Nitric acid (NO) inhibition was tested on the macrophage cell line RAW264.7. Molecular docking was conducted on GPR35-active compounds and GPR35 crystal structure. Statistical analysis using GEO datasets was conducted. RESULTS: Seventy compounds were isolated and twelve showed GPCR activity. Three compounds showed potent GPR35 agonistic activity (EC50 < 10 µM) and the GPR35 agonism action of PAL-21 (Scutellarein) was reported for the first time. Docking results revealed that the GPR35-targeting compounds interacted at the key residues for the agonist-initiated activation of GPR35. Five compounds showed weak antagonistic activity on M3, which was confirmed to be a disease target by statistical analysis. Seventeen compounds showed NO inhibitory activity. Several compounds showed multi-target properties. An experiment-based network reflected a pharmacological relationship between Jinqiancao granules and the two diseases. CONCLUSIONS: This study identified active compounds in Jinqiancao granules that have synergistic mechanisms, contributing to anti-inflammatory effects. The findings provide scientific evidence for the potential use of Jinqiancao granules as a treatment for prostatitis and BPH.

An integrated strategy for the discovery of quality marker of Dactylicapnos scandens based on phytochemical analysis, network pharmacology and activity screening.

Dactylicapnos scandens (D. scandens) is an ethnic medicine commonly used for the treatment of analgesia. In this study, an integrated strategy was proposed for the quality evaluation of D. scandens based on "phytochemistry-network pharmacology-effectiveness-specificity" to discover and determine the quality marker (Q-marker) related to analgesia. First, phytochemical analysis was conducted using UPLC-Q-TOF-MS/MS and a self-built compound library, and 19 components were identified in D. scandens extracts. Next, the "compounds-targets" network was constructed to predict the relevant targets and compounds related to analgesia. Then, the analgesic activity of related compounds was verified through dynamic mass redistribution (DMR) assays on D2 and Mu receptors, and 5 components showed D2 antagonistic activity with IC50 values of 39.2 ± 14.7 µM, 5.46 ± 0.37 µM, 17.5 ± 1.61 µM, 7.89 ± 0.79 µM and 3.29 ± 0.73 µM, respectively. Subsequently, nine ingredients were selected as Q-markers in consideration of specificity, effectiveness and measurability, and their content was measured in 12 batches of D. scandens. Furthermore, the hierarchical cluster analysis and heatmap results indicated that the selected Q-marker could be used to discriminate D. scandens and that the content of Q-marker varied greatly in different batches. Our study shows that this strategy provides a useful method to discover the potential Q-markers of traditional Chinese medicine and offers a practical workflow for exploring the quality consistency of medicinal materials.

Profiling CCR3 target pathways for discovering novel antagonists from natural products using label-free cell phenotypic assays.

CC chemokine receptor 3 (CCR3) plays important roles in atopic dermatitis (AD) and other related allergic diseases. Activation of CCR3 receptor signaling pathways regulates the recruitment of eosinophils to related tissues, releasing inflammatory mediators and causing inflammatory responses. However, none of the known CCR3 antagonists exhibit promising efficacy in clinical trials. In this work, we sought new natural CCR3 antagonists for drug development. To construct a high-throughput screening model, we established a stably transfected CHO-K1-Gα15-CCR3 cell line, and receptor expression was demonstrated by real-time quantitative PCR, confocal detection and flow cytometry analysis. Then, we applied a label-free cell phenotyping technique to profile and deconvolute CCR3 target pathways in CHO-K1-Gα15-CCR3 cells and found that activation of CCR3 triggered the Gq-PLC-Ca2+ and MAPK-P38-ERK pathways. By in vitro and in silico experiments, we discovered a novel CCR3 antagonist emodin, with an IC50 value of 27.28 ± 1.71 µM out of 266 compounds that were identified in 15 traditional Chinese medicines used in the clinical treatment of skin diseases. Molecular docking graphically presented the binding mode of emodin on CCR3. This work reports a new approach for CCR3 antagonist screening and pathway detection and identifies a new antagonist that would benefit future drug development.

The dual-targeting mechanism of an anti-inflammatory diarylheptanoid from Curcuma zedoaria (Christm.) Roscoe with the capacity for ß2-adrenoreceptor agonism and NLRP3 inhibition.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease characterized by symptoms of shortness of breath and chronic inflammation. Curcuma zedoaria (Christm.) Roscoe is a well-documented traditional medical herb that is frequently used in the treatment of COPD. Previously, we identified a diarylheptanoid compound (1-(4-hydroxy-5-methoxyphenyl)-7-(4,5-dihydroxyphenyl)-3,5-dihydroxyheptane; abbreviated as HMDD) from this herb that exhibited potent agonistic activity on ß2-adrenergic receptors (ß2 adrenoreceptor) that are present on airway smooth muscle cells. In this work, we used chemically synthesized HMDD compound, and confirmed its bioactivity on ß2 adrenoreceptors. Then by a proteomics study and anti-inflammatory evaluation detections, we found that HMDD downregulated the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway and suppressed the NLRP3 receptor expression in RAW264.7 macrophages and in a COPD model in A549 lung carcinoma cells. HMDD also decreased nitric oxide production levels, and impacted other interleukins and the phosphorylation of NF-κB and ERK pathways. We performed molecular docking of HMDD on ß2 adrenoreceptor and NLRP3 protein models. This work reports the anti-inflammatory effects of HMDD and suggests a dual-targeting mechanism of ß2-adrenoreceptor agonism and NLRP3 inhibition. Such a mechanism scientifically supports the clinical uses of Curcuma zedoaria (Christm.) Roscoe in treating COPD, as it can simultaneously relieve persistent breathlessness and inflammation. HMDD can be considered as a potential non-steroidal anti-inflammatory drug in novel therapy design for the treatment of COPD and other inflammatory diseases.

Multi-effective components and their target mechanism of Ziziphi Spinosae Semen in the treatment of insomnia.

Insomnia is a common and refractory disease. Since more than 2000 years ago, people have been using Ziziphi Spinosae Semen (ZSS). However, there are lack of molecular mechanisms of sleep promotion effects of ZSS. The purpose of this study is to clarify the active ingredients in ZSS that are used to treat insomnia. Using a method called cellular label-free integrative pharmacology (CLIP), we established five insomnia-related target models, including serotonin (5HT2A and 5HT1A), melatonin (MT1), dopamine (D2) and epinephrine (ß2) receptors. The one-dimensional (1D) fractions of ZSS extract were prepared on a RZC18 column and assayed on five models. Subsequently, the active fraction was further analyzed, fractionated and quantified using a two-dimensional (2D) liquid phase method coupled with a charged aerosol detector (CAD), This CAD-coupled 2D-LC method requires micro-fractions from the 1D separation and thus it greatly saves sample amounts and corresponding preparation time, and quickly conduct activity screening. The composition of the active 2D fractions was then determined using three-dimensional (3D) HPLC-MS, and molecular docking was separately carried out for the described compounds on the targets for activity prediction. Seven compounds were predicted to be active on 5HT2A, and two compounds on D2. We experimentally verified the prediction and found that vitexin exhibited D2 agonistic activity, and nuciferine exhibited 5HT2A antagonistic activity. This study revealed the effective components and their targets of ZSS in the treatment of insomnia, also highlighted the potential of the CLIP technique and bioactivity guided multi-dimensional HPLC-MS in molecular mechanism elucidation for traditional Chinese medicines.

The impact of COVID-19 pandemic on hand hygiene compliance of healthcare workers in a tertiary hospital in East China.

Introduction: Hand hygiene is a cost-effective measure to reduce healthcare-associated infections (HAIs) in healthcare facilities. The impact of the coronavirus disease 2019 (COVID-19) pandemic on hand hygiene performance (HHP) provided evidence for targeted hand hygiene intervention measures. Methods: This study evaluated the HHP rate in a tertiary hospital before and after the COVID-19 outbreak. HHP was checked by infection control doctors or nurses every day, and they inputted the HHP rate to the full-time infection control staff every week. A random examination of HHP was conducted by a confidential worker every month. The HHP of healthcare workers (HCWs) was monitored in the outpatient department, inpatient ward, and operating room from January 2017 to October 2022. The influence of COVID-19 prevention and control strategies on HHP was elucidated by analyzing the results of HHP during the study period. Results: The average HHP rate of HCWs was 86.11% from January 2017 to October 2022. The HHP rate of HCWs after the COVID-19 pandemic was statistically significantly higher than that before the pandemic (P < 0.001). The HHP rate was the highest (93.01%) in September 2022 when the local epidemic occurred. Among the different occupation categories, medical technicians showed the highest HHP rate (89.10%). The HHP rate was the highest after contact with body fluids or blood of patients (94.47%). Conclusion: The HHP rate of HCWs in our hospital showed an increasing trend in the recent 6 years, especially during the COVID-19 pandemic, and the increase was most obvious during the local epidemic.

The effect of meropenem versus piperacillin-tazobactam in critically ill patients with sepsis and septic shock.

Background: Antibiotics are a popular and efficient treatment for sepsis and septic shock. However, there is presently little proof of Meropenem with piperacillin-therapeutic tazobactam's benefits. Methods: From January 1, 2010 to January 1, 2021, we treated a total of 1244 patients with sepsis and septic shock using either Meropenem (n = 622, 1 g every 8 h) or piperacillin-tazobactam (n = 622, 3.375 g or 4.5 g every 8 h). The intervention was administered for 7 days following randomization and continued for up to 14 days thereafter, or until the patient was discharged from the critical care unit or passed away, whichever occurred first. Results: First, we discovered that there were no significant changes in the duration of stay in ICU, Cardiovascular in SOFA, Coagulation in SOFA, Hepatic in SOFA, or Central Nervous System in SOFA between the meropenem alone group and the piperacillin-tazobactam group. In addition, WBC beyond the standard limit was 68.00% in the meropenem alone group against 61.89% in the piperacillin-tazobactam group (P = 0.03). However, Meropenem had a lower mortality rate on ventilator-free days, vasopressor-free days, and hospital-free days. Conclusion: This procedure may offer clinical evidence for the safety and efficacy of meropenem with piperacillin-tazobactam in critically sick patients with sepsis and septic shock.

Development and Characterization of Fluorescent Probes for the G Protein-Coupled Receptor 35.

The orphan G protein-coupled receptor 35 (GPR35) is a potential target for the treatment of pain, inflammation, and metabolic diseases. Although many GPR35 agonists have been discovered, research on functional GPR35 ligands, such as fluorescent probes, is still limited. Herein, we developed a series of GPR35 fluorescent probes by conjugating a BODIPY fluorophore to DQDA, a known GPR35 agonist. All probes exhibited excellent GPR35 agonistic activity and desired spectroscopic properties, as determined by the DMR assay, bioluminescence resonance energy transfer (BRET)-based saturation, and kinetic binding experiments. Notably, compound 15 showed the highest binding potency and the weakest nonspecific BRET binding signal (K d = 3.9 nM). A BRET-based competition binding assay with 15 was also established and used to determine the binding constants and kinetics of unlabeled GPR35 ligands.

Aminosalicylates target GPR35, partly contributing to the prevention of DSS-induced colitis.

GPR35, a class A G-protein-coupled receptor, is considered an orphan receptor; the endogenous ligand and precise physiological function of GPR35 remain obscure. GPR35 is expressed relatively highly in the gastrointestinal tract and immune cells. It plays a role in colorectal diseases like inflammatory bowel diseases (IBDs) and colon cancer. More recently, the development of GPR35 targeting anti-IBD drugs is in solid request. Nevertheless, the development process is in stagnation due to the lack of a highly potent GPR35 agonist that is also active comparably in both human and mouse orthologs. Therefore, we proposed to find compounds for GPR35 agonist development, especially for the human ortholog of GPR35. As an efficient way to pick up a safe and effective GPR35 targeting anti-IBD drug, we screened Food and Drug Administration (FDA)-approved 1850 drugs using a two-step DMR assay. Interestingly, we found aminosalicylates, first-line medicine for IBDs whose precise target remains unknown, exhibited activity on both human and mouse GPR35. Among these, pro-drug olsalazine showed the most potency on GPR35 agonism, inducing ERK phosphorylation and ß-arrestin2 translocation. In dextran sodium sulfate (DSS)-induced colitis, the protective effect on disease progression and inhibitory effect on TNFα mRNA expression, NF-κB and JAK-STAT3 pathway of olsalazine are compromised in GPR35 knock-out mice. The present study identified a target for first-line medicine aminosalicylates, highlighted that uncleaved pro-drug olsalazine is effective, and provided a new concept for the design of aminosalicylic GPR35 targeting anti-IBD drug.

Chemical profiling of Ziziphi spinosae semen using on-line comprehensive two-dimensional liquid chromatography-mass spectrometry based on a novel phthalic anhydride bonded stationary phase.

Ziziphi spinosae semen has been widely used to treat insomnia and anxiety. To profile its chemical components, an online comprehensive two-dimensional liquid chromatography-mass spectrometry was developed. In this two-dimensional liquid chromatography system, a novel phthalic anhydride-bonded stationary phase column was combined with a C18 column. As a result, this new stationary phase exhibited remarkable differences in separation selectivity from C18, achieving a good orthogonality of 83.3%. Moreover, this new stationary phase with weaker hydrophobicity than C18 realized solvent compatibility in the online configuration. Coupled with tandem MS, 154 compounds were identified, including 51 unreported compounds. Compared with one-dimensional liquid chromatography-mass spectrometry, this online two-dimensional liquid chromatography-mass spectrometry system exhibited a much higher resolving power in isomer separation. This work provided an effective separation and characterization method for the material basis of Ziziphi spinosae semen. This strategy provides ideas for the material basis research of other traditional Chinese medicines.

Design and synthesis of 2-(2,2-diarylethyl)-cyclamine derivatives as M3 receptor antagonists and functional evaluation on COPD.

Muscarine acetylcholine receptors (mAChRs) regulate a variety of central and peripheral physiological functions and emerge as important therapeutic targets for a number of diseases including chronic obstructive pulmonary disease (COPD). Inspired by two active natural products, we designed and synthesized a series of 2-(2,2-diarylethyl)-cyclamine derivatives for screening M3 mAChR antagonists. On this skeleton, the structural units including N heterocycle, aryl groups and its substituents on aryl were examined and resulted in a clear structure-activity relationships on the M3 mAChR. In general, these 2-(2,2-diarylethyl)-cyclamine derivatives exhibited good to excellent M3 antagonistic potency and receptor selectivity. The most active 5b-C1 had an IC50 value of 3 nM and the most of compound 6 displayed inactivity against histamine H1 receptor closely related to M3. In in vitro and in vivo evaluations of tracheo-relaxation function, some compounds even showed comparable activity to tiotropium bromide, a known blockbuster drug for COPD. Such excellent properties made these novel compounds potential candidates for COPD drug development.

Uncariphyllin A-J, indole alkaloids from Uncaria rhynchophylla as antagonists of dopamine D2 and Mu opioid receptors.

Ten new indole alkaloids (1-10) as well as eleven known analogs (11-21) were isolated from the stems and hooks of Uncaria rhynchophylla. Their structure elucidation was based on extensive NMR studies, MS and ECD data, with the essential aid of DFT prediction of ECD spectra. Compound 1 was determined as a 17,19-seco-cadambine-type alkaloid, and compound 3 was confirmed to be a 3,4-seco-tricyclic monoterpene indole alkaloid, which are the first seco-alkaloids possessing such cleavage positions from U. rhynchophylla. All the isolated compounds were evaluated for their bioactivities on dopamine D2 and Mu opioid receptors for discovering natural therapeutic drugs targeting central nervous system (CNS) diseases. Compounds 1, 2, 4, 5, 20 and 21 showed antagonistic bioactivities on the D2 receptor (IC50 0.678-15.200 µM), and compounds 1, 3, 6, 9, 10, 13, 18, 19 and 21 exhibited antagonistic effects on the Mu receptor (IC50 2.243-32.200 µM). Among them, compounds 1 and 21 displayed dual-target activities. Compound 1 showed conspicuous antagonistic activity on D2 and Mu receptors with the IC50 values of 0.678 ± 0.182 µM and 13.520 ± 2.480 µM, respectively. Compound 21 displayed moderate antagonistic activity on the two receptors with the IC50 values at 15.200 ± 1.764 µM and 32.200 ± 5.695 µM, respectively.

Integration of Two-Dimensional Liquid Chromatography-Mass Spectrometry and Molecular Docking to Characterize and Predict Polar Active Compounds in Curcuma kwangsiensis.

Curcuma kwangsiensis, one species of Curcumae zedoaria Ros. c, is a commonly used traditional Chinese medicine (TCM) for treating cardiovascular disease, cancer, asthma and inflammation. Polar compounds are abundant in water decoction, which would be responsible for critical pharmacological effects. However, current research on polar compounds in Curcumae zedoaria Ros. c remains scarce. In this study, the polar fraction from Curcuma kwangsiensis was firstly profiled on G protein-coupled receptor 109A (GPR109A), ß2-adrenergic receptor (ß2-AR), neurotensin receptor (NTSR), muscarinic-3 acetylcholine receptor (M3) and G protein-coupled receptor 35 (GPR35), which were involved in its clinical indications and exhibited excellent ß2-AR and GPR109A receptor activities. Then, an offline two-dimensional reversed-phase liquid chromatography (RPLC) coupled with the hydrophilic interaction chromatography (HILIC) method was developed to separate polar compounds. By the combination of a polar-copolymerized XAqua C18 column and an amide-bonded XAmide column, an orthogonality of 47.6% was achieved. As a result of coupling with the mass spectrometry (MS), a four-dimensional data plot was presented in which 373 mass peaks were detected and 22 polar compounds tentatively identified, including the GPR109A agonist niacin. Finally, molecular docking of these 22 identified compounds to ß2-AR, M3, GPR35 and GPR109A receptors was performed to predict potential active ingredients, and compound 9 was predicted to have a similar interaction to the ß2-AR partial agonist salmeterol. These results were supplementary to the material basis of Curcuma kwangsiensis and facilitated the bioactivity research of polar compounds. The integration of RPLC×HILIC-MS and molecular docking can be a powerful tool for characterizing and predicting polar active components in TCM.

Discovery of Flavonoids as Novel Inhibitors of ATP Citrate Lyase: Structure-Activity Relationship and Inhibition Profiles.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism and its aberrantly high expression is closely associated with various cancers, hyperlipemia and atherosclerotic cardiovascular diseases. Prospects of ACLY inhibitors as treatments of these diseases are excellent. To date, flavonoids have not been extensively reported as ACLY inhibitors. In our study, 138 flavonoids were screened and 21 of them were subjected to concentration-response curves. A remarkable structure-activity relationship (SAR) trend was found: ortho-dihydroxyphenyl and a conjugated system maintained by a pyrone ring were critical for inhibitory activity. Among these flavonoids, herbacetin had a typical structure and showed a non-aggregated state in solution and a high inhibition potency (IC50 = 0.50 ± 0.08 µM), and therefore was selected as a representative for the ligand-protein interaction study. In thermal shift assays, herbacetin improved the thermal stability of ACLY, suggesting a direct interaction with ACLY. Kinetic studies determined that herbacetin was a noncompetitive inhibitor of ACLY, as illustrated by molecular docking and dynamics simulation. Together, this work demonstrated flavonoids as novel and potent ACLY inhibitors with a remarkable SAR trend, which may help design high-potency ACLY inhibitors. In-depth studies of herbacetin deepened our understanding of the interactions between flavonoids and ACLY.

Discovery and characterization of novel ATP citrate lyase inhibitors from natural products by a luminescence-based assay.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism with therapeutic prospect for treating hyperlipidemia and various cancers. Much effort has been put into discovering ACLY inhibitors. However, current screening approaches have limitations in sensitivity, portability and high-throughput. To develop a general screening assay, we investigated series of conditions affecting the enzymatic reaction based on the ADP-Glo luminescence assay. Bovine serum albumin (0.001%) added triggered strong and stable fluorescence signal. The optimized assay was validated and applied to screen our natural product library. Two novel inhibitors were identified with IC50 values of 3.86 ± 0.62 µM (2) and 15.48 ± 2.51 µM (4). Their aggregations and target specificities were also examined. 2 was characterized as a noncompetitive inhibitor of ACLY, while 4 was a competitive inhibitor of CoA, which was also elucidated by docking studies. In anticancer activity evaluation, 2 with higher inhibition potency did not exhibit anticancer effect, probably owing to its insufficient cell-permeability. 4 showed moderate inhibition in the proliferation of A549 and PC3 cells. This study not only developed a general approach for ACLY inhibitor discovery, but also identified a new scaffold ACLY inhibitor, which could be served as a hit compound in drug design.

Menisperdaurines A-W, structurally diverse isoquinoline alkaloids from Menispermum dauricum and their dopamine D1 receptor activities.

A total of 33 structurally diverse isoquinoline alkaloids were isolated from the rhizomes of Menispermum dauricum, including seventeen benzylisoquinoline analogues (menisperdaurines A-Q, 1-17), five protoberberine analogues (menisperdaurines R-V, 18-22), a quaternary phenanthrene alkaloid (menisperdaurine W, 23) and ten known compounds (24-33). Compound structures, including absolute configurations, were determined by extensive spectroscopic methods, quantum chemical calculations of chemical shifts, and calculated and experimental electronic circular dichroism (ECD) data. Compounds 1-5 were glycosidic benzylisoquinolines with glucose moieties attached at the C-12 position. Compound 8 was the first example that was isolated from the rhizomes of Menispermum dauricum, benzylisoquinoline and an aromatic unit connected by a sugar bridge. Compounds were evaluated for their inhibitory effects on the dopamine D1 receptor. Compounds 1, 8, 21, 24 and 29 showed potent D1 antagonistic activities, with IC50 values ranging from 1.0 to 4.5 µM. Compound 1 exhibited the highest antagonistic activity with an IC50 value of 1.0 ± 0.2 µM.

Corybungines A-K: Isoquinoline alkaloids from Corydalis bungeana with dopamine D2 receptor activity.

Eleven undescribed isoquinoline alkaloids corybungines A-K including a protoberberine-type alkaloid, an isoquinoline alkaloid with a unique 6-norprotoberberine skeleton, one 13,14-seco-protoberberine-type alkaloid, two 1a,14-seco-protoberberine-type alkaloids with a 4-(hydroxymethyl)phenoxy moiety and six aporphine alkaloids, together with seven known alkaloids, have been isolated from the whole herb extract of Corydalis bungeana Turcz. Their structures and absolute configurations were elucidated based on an analysis of spectroscopic data and electronic circular dichroism (ECD) spectra. (R)-stephanine displayed high antagonistic activity against the dopamine D2 receptor with an IC50 value of 0.85 ± 0.09 µM in CHO-D2 cells. Additionally, corybungines D, F, H, (R)-roemerine, (R)-vireakine and (R)-tuduranine showed moderate D2 antagonism (IC50 5.20-26.07 µM). The preliminary structure-activity relationships (SARs) of aporphine alkaloids were discussed.