ABSTRACT
Lysine-specific histone demethylase 1 (LSD1) was the first histone demethylase identified in epigenetics and has recently emerged as an attractive therapeutic target for treating tumors. To date, almost all reported LSD1 inhibitors have been chemosynthesized; however, natural products possess pharmacological and biological activity and can be sources for drug development. Here, we established a target separation countercurrent chromatography technique to isolate LSD1 inhibitors from zedoary turmeric oil. Four sesquiterpene-based LSD1 inhibitors were efficiently obtained with an inhibition ratio equal to or less than that of the positive control drug. Compound 2 showed the most potent inhibitory activity, with a half-maximal inhibitory concentration of 3.97 µM, and was further tested to determine its ability to inhibit LSD1 and its antitumor metastatic effects in MDA-MB-231 cells. These four compounds are the first sesquiterpene-based natural LSD1 inhibitors to be characterized. Our findings provide a new molecular framework for studying LSD1 inhibitors and offer a template for designing more sesquiterpene-based LSD1 inhibitors with potential antitumor activity.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Biological Products/pharmacology , Curcuma/chemistry , Enzyme Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Plant Extracts/pharmacology , Sesquiterpenes/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Biological Products/chemistry , Biological Products/isolation & purification , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Histone Demethylases/metabolism , Humans , Molecular Structure , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Oils/chemistry , Sesquiterpenes/chemistry , Sesquiterpenes/isolation & purification , Structure-Activity RelationshipABSTRACT
To develop novel GLS1 inhibitors as effective therapeutic agents for triple-negative breast cancer (TNBC), 25 derivatives were synthesized from the natural inhibitor withangulatin A (IC50 = 18.2 µM). Bioassay optimization identified a novel and selective GLS1 inhibitor 7 (IC50 = 1.08 µM). In MDA-MB-231 cells, 7 diminished cellular glutamate levels by blocking glutaminolysis pathway, further triggering the generation of reactive oxygen species to induce caspase-dependent apoptosis. Molecular docking indicated that 7 interacted with a new reacting site of allosteric binding pocket by forming various interactions in GLS1. The intraperitoneal administration of 7 at a dose of 50 mg/kg exhibited remarkable therapeutic effects and no apparent toxicity in the MDA-MB-231 xenograft model, indicating its potential as a novel GLS1 inhibitor for treatment of TNBC.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Enzyme Inhibitors/pharmacology , Glutaminase/antagonists & inhibitors , Pregnenes/pharmacology , Triple Negative Breast Neoplasms/drug therapy , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Glutaminase/metabolism , Humans , Molecular Structure , Pregnenes/chemical synthesis , Pregnenes/chemistry , Structure-Activity Relationship , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathologyABSTRACT
Eight new germacranolides, minusolides A-H (1-8), along with two known analogues, 9 and 10, were isolated from the whole plant of Carpesium minus. Their structures were elucidated by spectroscopic analysis. Compounds 1 and 2, and 6 and 9 are two pairs of rare epimers with opposite configurations at C-2 of the 2-methylbutyryloxy group. The absolute configurations were determined by X-ray diffraction studies. Compound 7 exhibited cytotoxic activities against MDA-MB-231, A549, and HCT-116 cells with IC50 values of 6.1 ± 0.2, 8.4 ± 0.6, and 3.7 ± 0.6 µM, respectively. Compound 7 induced the apoptosis of HCT-116 cells via suppression of PARP and promoting cleavage of PARP.
Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Asteraceae/chemistry , Sesquiterpenes, Germacrane/isolation & purification , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Sesquiterpenes, Germacrane/chemistry , Sesquiterpenes, Germacrane/pharmacology , Spectrum Analysis/methodsABSTRACT
It is significant to precisely isolate potential active compounds from medicinal herbs containing multiple compounds. Herein, a new strategy for precise separation of lysine-specific demethylase 1 (LSD1) inhibitors from the rhizome of Corydalis yanhusuo (RCY) using counter-current chromatography (CCC) guided by molecular docking and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis was established. First, representative alkaloids from RCY were docked with LSD1 for screening active skeleton compounds. Simultaneously, the crude extract of RCY was preliminarily separated via pH-zone refining CCC. Subsequently, guided by LC-MS/MS analysis of the fragmentation pathways, three potential active fractions were obtained, followed by further online-storage and recycling CCC separation. Finally, three high-purity target quaternary alkaloids compound 3 (dehydrocorydaline), 7 (coptisine), and 8 (columbamine) were successfully isolated as a new class of potential natural LSD1 inhibitors by only one CCC instrument with multiple modes. Compound 3, with the highest LSD1 inhibition ratio of 2.44 µM, was tested for its ability to inhibit tumor invasion and metastasis in U2OS cells. Therefore, the CCC separation guided by virtual screening is a promising method for the targeted isolation of enzyme inhibitors from medicinal herbs.
Subject(s)
Corydalis/chemistry , Countercurrent Distribution/methods , Drug Evaluation, Preclinical , Enzyme Inhibitors/isolation & purification , Histone Demethylases/antagonists & inhibitors , User-Computer Interface , Biological Assay , Cell Line, Tumor , Cell Movement/drug effects , Chromatography, High Pressure Liquid , Enzyme Inhibitors/chemistry , Histone Demethylases/metabolism , Humans , Hydrogen-Ion Concentration , Molecular Docking Simulation , Solvents , Tandem Mass SpectrometryABSTRACT
Six new obovatol trimeric neolignans, houpulignans A-F (1-6) were isolated from the leaves of Magnolia officinalis var. biloba. Their structures were determined on the basis of the interpretation of HRESIMS, NMR data, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are the first examples of neolignans derived from three units of obovatol bearing a rare 1,4-benzodioxepane moiety. Compound 3 possesses a benzodihydropyran ring, meanwhile three units of obovatol in 4-6 are connected by an alkyl chain. Compounds 1-3 inhibited NO production in LPS-stimulated RAW264.7 cells with IC50 values of 8.01, 20.21, and 4.05 µM, respectively.
Subject(s)
Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Lignans/chemistry , Lignans/pharmacology , Magnolia/chemistry , Nitric Oxide/antagonists & inhibitors , Phenyl Ethers/chemistry , Phenyl Ethers/pharmacology , Animals , Macrophages/drug effects , Macrophages/metabolism , Mice , Nitric Oxide/metabolism , Plant Extracts/chemistry , Plant Extracts/pharmacology , Plant Leaves/chemistry , RAW 264.7 CellsABSTRACT
In terms of ligand fishing, the amount and the relative activity recovery of enzymes immobilized on magnetic particles and nanoparticles are not preeminent. Therefore, the metal-organic framework (MOF) UiO-66-NH2 was synthesized to immobilize the porcine pancreatic lipase (PPL) via precipitation-cross-linking, and the resulting novel biological matrices named PPL@MOF manifested high PPL loading capacity (98.31 mg/g) and relative activity recovery (104.4%). Moreover, the novel enzyme-MOF composite was applied to screen lipase inhibitors from Prunella vulgaris L. to enrich and improve the techniques of ligand fishing. As a result, 13 lipase ligands were obtained, and 12 compounds were determined by HPLC-Q-TOF-MS/MS. All of these ligands were further confirmed to be potential inhibitors through the verification of the activity assay and molecular docking. The proposed approach based on PPL@MOF was superior in terms of abundant protein loading capacity, high enzyme catalytic activity and easy preparation process. Taken together, our newly developed method provided a new platform for efficient discovering bioactive molecules from natural herbs.
Subject(s)
Biological Products/pharmacology , Drugs, Chinese Herbal/pharmacology , Enzymes, Immobilized/antagonists & inhibitors , Lipase/antagonists & inhibitors , Metal-Organic Frameworks/pharmacology , Prunella/chemistry , Animals , Biological Products/chemistry , Biological Products/metabolism , Drug Discovery , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/metabolism , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Ligands , Lipase/chemistry , Lipase/metabolism , Medicine, Chinese Traditional , Metal-Organic Frameworks/chemistry , Metal-Organic Frameworks/metabolism , Molecular Docking Simulation , Pancreas/enzymology , SwineABSTRACT
The ability to separate bioactive compounds from herbal medicines, which contain abundant components, is crucial for drug discovery. Conventional Countercurrent chromatography (CCC) methods for separating bioactive compounds are labor intensive and show low efficiency. Here, we present a novel integrative CCC method for separating lysine-specific demethylase 1 (LSD1) inhibitors from the roots of Salvia miltiorrhiza (RSM). The methanol extracts of RSM were separated into hydrosoluble and liposoluble fractions, which were online stored in coils. Subsequently, the targeting LSD1 constituents were isolated using isocratic, gradient, or recycling elution mode. All separation processes could be accomplished using one CCC apparatus. Using our separation strategy, two phenylpropanoids and four tanshinones were isolated, which were determined to be new classes of natural LSD1 inhibitors. Salvianolic acid B, which showed the most potent inhibitory activity with an IC50 of 0.11⯵M, exhibiting a considerable potential as an anticancer agent. Promisingly, the integrative CCC could be a crucial tool for the target separation of enzyme inhibitors from herbal medicines.
Subject(s)
Enzyme Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Plant Roots/chemistry , Salvia miltiorrhiza/chemistry , Benzofurans/isolation & purification , Benzofurans/metabolism , Benzofurans/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cinnamates/isolation & purification , Cinnamates/metabolism , Cinnamates/pharmacology , Countercurrent Distribution/methods , Depsides/isolation & purification , Depsides/metabolism , Depsides/pharmacology , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/metabolism , Histone Demethylases/metabolism , Humans , Molecular Docking Simulation , Phenanthrenes/isolation & purification , Phenanthrenes/metabolism , Phenanthrenes/pharmacology , Protein Binding , Rosmarinic AcidABSTRACT
Synephrine, a chiral adrenergic agonist, exists as R- (more active) and S-enantiomers (less active) that are difficult to separate because of their alkalinity and high polarity. Herein, we show that although this enantioseparation can hardly be achieved by high-performance liquid chromatography, acceptable preparative separation can be realized using high-speed counter-current chromatography (HSCCC) and pH-zone-refining counter-current chromatography (CCC) under optimal conditions. Specifically, 23.0 mg of S-synephrine and 25.0 mg of R-synephrine were isolated from 60.0 mg of racemic synephrine by conventional HSCCC, while 70.0 mg of S-synephrine and 69.0 mg of R-synephrine were obtained from 200.0 mg of racemic synephrine by pH-zone-refining CCC. The pH-zone-refining CCC was identified as the most efficient enantioseparation technique for synephrine and can be applied for the preparative enantioseparation of other ß-amino alcohols.
Subject(s)
Chemistry Techniques, Analytical/methods , Countercurrent Distribution , Synephrine/isolation & purification , Chemistry Techniques, Analytical/instrumentation , Hydrogen-Ion Concentration , StereoisomerismABSTRACT
Countercurrent chromatography (CCC) has gradually become a widely used method for preparative separation of bioactive natural molecules. These molecules generally contain distinct scaffolds and characteristics that cannot be readily isolated from plants. While one-dimensional CCC is typically used for the initial purification with insufficiently resolved peaks after locating bioactive components, two-dimensional (2D) or multi-dimensional CCC strategies are employed to improve the resolution of peaks. However, these methods usually present certain disadvantages, such as complicated procedures and increased time consumption, experimental costs, and equipment requirements. Here, a bioactivity-guided cut CCC strategy was established to isolate lysine-specific demethylase 1 (LSD1) inhibitors from Scutellaria baicalensis Georgi. Gradient-elution CCC coupled with real-time detection of LSD1 inhibition by the collected fractions was developed. Next, an online-storage recycling CCC mode was designed to enable the active fractions to be stored in coils, and these active fractions were further separated to obtain pure compounds by using sequential recycling elution. In this strategy, active fractions are first identified, and then pure LSD1 inhibitors are isolated in the 2D CCC mode through continuous separation on a single instrument. By using our bioactivity-guided cut CCC strategy, we successfully isolated six natural LSD1 inhibitors from S. baicalensis Georgi, five of which were identified for the first time as natural LSD1 inhibitors.