A series of new arctigenin and 9-deoxy-arctigenin derivatives bearing different ester and ether side chains at the phenolic hydroxyl positions are designed, synthesized, and evaluated for activating AMPK potency in L6 myoblasts. Initial biological evaluation indicates that some alkyl ester and phenethyl ether arctigenin derivatives display potential activities in AMPK phosphorylation improvement. Further structure–activity relationship analysis shows that arctigenin ester derivatives 3a, 3h and 9-deoxy-arctigenin phenethyl ether derivatives 6a, 6c, 6d activate AMPK more potently than arctigenin. Moreover, the 2-(3,4-dimethoxyphenyl)ethyl ether moiety of 6c has been demonstrated as a potential functional group to improve the effect of AMPK phosphorylation. The structural optimization of arctigenin leads to the identification of 6c as a promising lead compound that exhibits excellent activity in AMPK activation.Figure optionsDownload full-size imageDownload as PowerPoint slide

Improvement of glucose and lipid metabolic dysfunctions is a potent therapeutic strategy against type 2 diabetes mellitus, and identifying new functions for existing drugs may help accelerate the speed of new drug development. Here, we report that latanoprost, a clinical drug for treating primary open-angle glaucoma and intraocular hypertension, effectively ameliorated glucose and lipid disorders in two mouse models of type 2 diabetes. In addition, the glucose-lowering mechanisms of latanoprost were intensively investigated.A binding-affinity assay and enzymatic tests were used to determine the targets of latanoprost. Cell-based assays on 3T3-L1 adipocytes and C2C12 myotubes and animal model-based assays with db/db and ob/ob mice were further performed to clarify the mechanisms underlying latanoprost-regulated glucose and lipid metabolism.Latanoprost functioned as both an indirect activator of AMP-activated protein kinase and a selective retinoid X receptor α (RXRα) antagonist able to selectively antagonise the transcription of a RXRα/peroxisome proliferator-activated receptor γ heterodimer. It promoted glucose uptake, inhibited pre-adipocyte differentiation and regulated the main genes responsible for glucose and lipid metabolism, including Fas, Scd1, Perilipin (also known as Plin1), Lpl and Pdk4. Chronic administration of latanoprost in mice potently decreased the levels of fasting blood glucose, HbA1c, fructosamine (FMN), NEFA and total cholesterol, and effectively improved glucose tolerance and glucose/lipid metabolism-related genes in vivo.Our studies demonstrate that the existing eye drug latanoprost is both an indirect activator of AMP-activated protein kinase and a selective RXRα antagonist. Latanoprost effectively ameliorated glucose and lipid disorders in diabetic mice, which strongly highlights the potential of latanoprost in the treatment of type 2 diabetes mellitus.

Infarct-induced left ventricular (LV) remodeling is a deleterious consequence after acute myocardial infarction (MI) which may further advance to congestive heart failure. Therefore, new therapeutic strategies to attenuate the effects of LV remodeling are urgently needed. Salvianolic acid B (SalB) from Salviae mitiorrhizae, which has been widely used in China for the treatment of cardiovascular diseases, is a potential candidate for therapeutic intervention of LV remodeling targeting matrix metalloproteinase-9 (MMP-9).Molecular modeling and LIGPLOT analysis revealed in silico docking of SalB at the catalytic site of MMP-9. Following this lead, we expressed truncated MMP-9 which contains only the catalytic domain, and used this active protein for in-gel gelatin zymography, enzymatic analysis, and SalB binding by Biacore. Data generated from these assays indicated that SalB functioned as a competitive inhibitor of MMP-9. In our rat model for cardiac remodeling, western blot, echocardiography, hemodynamic measurement and histopathological detection were used to detect the effects and mechanism of SalB on cardio-protection. Our results showed that in MI rat, SalB selectively inhibited MMP-9 activities without affecting MMP-9 expression while no effect of SalB was seen on MMP-2. Moreover, SalB treatment in MI rat could efficiently increase left ventricle wall thickness, improve heart contractility, and decrease heart fibrosis.As a competitive inhibitor of MMP-9, SalB presents significant effects on preventing LV structural damage and preserving cardiac function. Further studies to develop SalB and its analogues for their potential for cardioprotection in clinic are warranted.

The discovery of HpFabZ inhibitors is now of special interest in the treatment of various gastric diseases. In this work, three series of derivatives (compounds 3, 4, and 5) were designed, synthesized, and their biological activities were investigated as potential HpFabZ inhibitors in a two phased manner. First, we designed and synthesized two series of derivatives (3a−r and 4a−u) and evaluated the enzyme-based assay against HpFabZ. Five compounds (3i−k, 3m, and 3q) showed potential inhibitory activity, with IC50 values less than 2 μM. Second, a focused combinatorial library containing 280 molecules was designed employing the LD1.0 program. Twelve compounds (5a−l) were selected and synthesized. The activity of the most potent compound 5h (IC50 = 0.86 μM) was 46 times higher than that of the hit 1. The high hit rate and the potency of the new HpFabZ inhibitors demonstrated the efficiency of the strategy for the focused library design and virtual screening.

Cyclophilin A (CypA) is a member of cyclophilins, a family of the highly homologous peptidyl prolyl cis–trans isomerases (PPIases), which can bind to cyclosporin A (CsA). CypA plays critical roles in various biological processes, including protein folding, assembly, transportation, regulation of neuron growth, and HIV replication. The discovery of CypA inhibitor is now of a great special interest in the treatment of immunological disorders. In this study, a series of novel small molecular CypA inhibitors have been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay. The SPECS_1 database containing 85,000 small molecular compounds was searched by virtual screening against the crystal structure of human CypA. After SPR-based binding affinity assay, 15 compounds were found to show binding affinities to CypA at submicro-molar or micro-molar level (compounds 1–15). Seven compounds were selected as the starting point for the further structure modification in considering binding activity, synthesis difficulty, and structure similarity. We thus synthesized 40 new small molecular compounds (1–6, 15, 16a–q, 17a–d, and 18a–l), and four of which (compounds 16b, 16h, 16k, and 18g) showed high CypA PPIase inhibition activities with IC50s of 2.5–6.2 μM. Pharmacological assay indicated that these four compounds demonstrated somewhat inhibition activities against the proliferation of spleen cells.By using structure-based virtual screening approach in conjunction with chemical synthesis and bioassay, four potent CypA inhibitors have been discovered. Compound 16h is active with very close potency to CsA in inhibiting the proliferation of spleen cells, demonstrating that this compound may be a good lead for discovering new immunosuppressive agents.

The major biochemical and thermodynamic features of nucelocapsid protein of SARS coronavirus (SARS_NP) were characterized by use of non-denatured gel electrophoresis, size-exclusion chromatographic and surface plasmon resonance (SPR) techniques. The results showed that SARS_NP existed in vitro as oligomer, more probably dimer, as the basic functional unit. This protein shows its maximum conformational stability near pH 9.0, and it seems that its oligomer dissociation and protein unfolding occur simultaneously. Thermal-induced unfolding for SARS_NP was totally irreversible. Both the thermal and chemical denaturant-induced denaturation analyses showed that oligomeric SARS_NP unfolds and refolds through a two-state model, and the electrostatic interactions among the charge groups of SARS_NP made a significant contribution to its conformational stability.

•15d-PGJ2 was determined to be an hFXRα antagonist.•15d-PGJ2 effectively regulated the mRNA expressions of hFXRα target genes.•15d-PGJ2 promoted the conversion of cholesterol to bile acids in HepG2 cells.•Key residues of hFXRα responsible for 15d-PGJ2/hFXRα-LBD binding were validated.15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2) is one of the major metabolites from prostaglandin D2 in arachidonic acid (AA) metabolic pathway. It was determined as a ligand of peroxisome proliferator-activated receptor γ (PPARγ) functioning potently in adipocyte development. However, the fact that 15d-PGJ2 exerts also PPARγ-independent biological actions has highly addressed its multi-target behavior. Here, we identified that 15d-PGJ2 was an antagonist of farnesoid X receptor (FXR), as investigated by surface plasmon resonance, fluorescence quenching and homo time-resolved fluorescence based analyses, and the coactivator-recruitment and luciferase-reporter related investigation. Assay of 15d-PGJ2 regulation on hFXRα target genes revealed that treatment of HepG2 cells with 15d-PGJ2 resulted in the stimulation of mRNA expressions of bile-salt export pump (BSEP), and the decrease of cholesterol 7a-hydroxylase (CYP7a1). In addition, functional assays indicated that 15d-PGJ2 promoted the conversion of cholesterol to bile acids in HepG2 cells. Moreover, molecular docking combined with molecular dynamics simulation was applied to develop the possible model of 15d-PGJ2 binding to hFXRα ligand binding domain (LBD) at atomic level, and the responsible residues for 15d-PGJ2/hFXRα-LBD interaction were thereby determined, which were further confirmed by SPR assays against hFXRα-LBD site-directed mutations. Given that hFXRα functions potently in the regulation of hepatic bile acid metabolism and lipid/glucose homeostasis, our current work is expected to help better understand the multi-target features of this PGD2 metabolite in biological pathways, and 15d-PGJ2 as a new discovered FXR antagonist might find its potential application in further anti-hypercholesterol research.

•PGF2α selectively antagonized LXR/RXR or RXR/RXR dimer.•PGF2α antagonized t0901317-stimulated promotion of Aβ clearance in microglia.•PGF2α antagonized t0901317-induced decrease of inflammatory response to LPS or Aβ in microglia.Alzheimer's disease (AD) is characterized by extracellular deposit of β-amyloid (Aβ) and accumulation of intracellular neurofibrillary tangles in the brain. Prostaglandin F2α (PGF2α) is one of the major metabolites of arachidonic acid (AA), and plays essential roles in a series of key physiological processes like luteolysis and parturition. Additionally, PGF2α is also involved in the regulation of chronic and acute inflammation processes. Recent clinical studies have revealed the high content of PGF2α metabolite, 15-keto-dihydro-PGF2α in AD patients, implying the activation of in vivo PGF2α biosynthesis. However, the mechanism underlying the involvement of PGF2α in the progression of AD still remains unclear. Here we discovered that PGF2α selectively antagonized LXR (liver X receptors)/RXR (retinoid X receptor α) and RXR/RXR dimers. Cell based assays indicated that PGF2α effectively antagonized the activation of LXR agonist (t0901317) on Aβ clearance via inhibiting apolipoprotein E (apoE) expression, and cell apoptosis alleviation by accelerating inflammatory response to Aβ or Lipopolysaccharide (LPS) in microglia. Therefore, our current findings have addressed the potential association of PGF2α with AD progression, and highlighted that inhibition of PGF2α biosynthesis might be a useful therapeutic strategy against AD.

Highlights•We firstly reported the protection of PR blockage in islet beta cell survival.•SC51089 as a non-steroid selective PR antagonist promoted Min6 cell survival.•PR antagonist SC51089 has the potential in anti-diabetic research.The progesterone receptor (PR), a member of nuclear receptor superfamily, is closely associated with gestational, type 1 and type 2 diabetes. However, the underlying mechanisms remain obscure. Here we found that PR activation increased the pro-inflammatory cytokines (PIC)-induced injury in Min6 cells, and PR blockage with siRNA interference protected the cells from damage. Moreover, the new discovered PR antagonist SC51089 effectively improved cell survival by reducing the PIC-stimulated cell apoptosis in Min6 cells. Immunoblotting assays indicated that either PR agonist progesterone (P4) or PR-B over-expression promoted the PIC-induced reinforces of extracellular-signal-regulated kinase 1/2 phosphorylation (p-Erk) and protein 53 (p53), and the attenuations of protein kinase B phosphorylation (p-AKT) and tumor necrosis factor receptor-associated factor 2 (TRAF2). SC51089 could reverse all the P4- or PR-B over-expression induced effects. In addition, PR siRNA inference based assay further supported that SC51089 protected pancreatic islet beta cells from the PR activation or PIC-induced injury by targeting PR and this protective action was mediated by AKT signaling pathway. To our knowledge, this current work might be the first report on the regulation of PR in pancreatic islet beta cell survival. It is expected that SC51089, as a non-steroid PR antagonist, might also find its potential in anti-diabetic research.

Flavonoids are polyphenolic compounds that are ubiquitous in nature. They possess varied promising properties for medical use. Quercetin (3,3′,4′,5,7-pentahydroxyflavone) and apigenin (4′,5,7-trihydroxyflavone) are two representative flavonoids, both of which have been reported to possess antibacterial activity by acting on multiple targets. Here, we determined that d-alanine:d-alanine ligase (Ddl) is another new target for quercetin and apigenin. Kinetic analysis indicated that these two flavonoids function as reversible inhibitors that are competitive with the substrate ATP of Ddl, whereas they are non-competitive with the other substrate d-Ala. The fact that quercetin showed lower 50% inhibitory concentration (IC50) and inhibitor binding constant (Ki) values than apigenin against both the Helicobacter pylori Ddl and the Escherichia coli DdlB implies that the two additional hydroxyls on the flavone skeleton of quercetin in structure might facilitate its inhibitory activity and binding affinity to Ddl. This work is expected to help shed more light on the potential antibacterial mechanism of flavonoids.

Estrogen receptors (ERs) belong to nuclear hormone receptor superfamily and can be activated by estrogens and regulate many target genes. Two ER isoforms, ERα and ERβ have been discovered to date and ERβ was indicated to involve in anti-inflammatory and anti-diabetogenic effects. Recently, some studies also demonstrated an association between ERβ and GLUT4 expression. The development of selective ERβ ligand has facilitated probing its novel biological functions and clinical benefits. In this work, a new ERβ selective agonist, butyl 4-(butyryloxy)benzoate (DCW234), was discovered as investigated by surface plasmon resonance (SPR) technology, yeast two-hybrid and cell-based transcription-based assays. SPR results demonstrated that DCW234 has a higher binding affinity against ERβ over ERα and induces a strong and selective stimulation on ERβ/SRC1 interaction, which could be efficiently blocked by Tamoxifen. Meanwhile, the yeast two-hybrid technology-based assay indicated that DCW234 exhibits a higher agonistic activity (∼13-fold) in stimulating ERβ ligand-binding domain (LBD) interaction with SRC1 (EC50 = 2.5 μM) than ERα-LBD/SRC1 interaction (EC50 = 32.7 μM). The cell-based transcriptional assay further proved the potency and selectivity of DCW234. Moreover, DCW234 was found to be able to induce GLUT4 expression in CHO-K1 cell. The discovered DCW234 might be hopefully developed as a potential lead compound for further research.

Estrogen receptors (ERs) belong to nuclear hormone receptor superfamily and can be activated by estrogens and regulate many target genes. Two ER isoforms, ERα and ERβ have been discovered to date and ERβ was indicated to involve in anti-inflammatory and anti-diabetogenic effects. Recently, some studies also demonstrated an association between ERβ and GLUT4 expression. The development of selective ERβ ligand has facilitated probing its novel biological functions and clinical benefits. In this work, a new ERβ selective agonist, butyl 4-(butyryloxy)benzoate (DCW234), was discovered as investigated by surface plasmon resonance (SPR) technology, yeast two-hybrid and cell-based transcription-based assays. SPR results demonstrated that DCW234 has a higher binding affinity against ERβ over ERα and induces a strong and selective stimulation on ERβ/SRC1 interaction, which could be efficiently blocked by Tamoxifen. Meanwhile, the yeast two-hybrid technology-based assay indicated that DCW234 exhibits a higher agonistic activity (∼13-fold) in stimulating ERβ ligand-binding domain (LBD) interaction with SRC1 (EC50 = 2.5 μM) than ERα-LBD/SRC1 interaction (EC50 = 32.7 μM). The cell-based transcriptional assay further proved the potency and selectivity of DCW234. Moreover, DCW234 was found to be able to induce GLUT4 expression in CHO-K1 cell. The discovered DCW234 might be hopefully developed as a potential lead compound for further research.

Acyl carrier protein (ACP) is an essential component in the type II fatty acid biosynthesis (FAS II) process and is responsible for the acyl group transfer within a series of related enzymes. In this work, the ACP from Helicobacter pylori strain SS1 was cloned and the gene sequence of Hpacp was deposited in the GenBank database (Accession No.: AY904356). Two forms of HpACP (apo, holo) were successfully purified and characterized. The thermal stability of these two forms was quantitatively investigated by CD spectral analyses. The results revealed that the holo-HpACP was more stable than apo-HpACP according to the transition midpoint temperature(Tm). Moreover, the interaction of HpACP with the related enzyme (β-hydroxyacyl-ACP dehydratase, HpFabZ) was determined by GST-pull down assay and surface plasmon resonance (SPR) technique in vitro, the results showed that HpACP displays a strong binding affinity to HpFabZ (KD = 1.2 × 10−8 M). This current work is hoped to supply useful information for better understanding the ACP features of Helicobacter pylori SS1 strain.

Hypertension is a common complication of type 2 diabetes mellitus (T2DM), and is the main cause for T2DM-associated mortality. Although the stringent control of blood pressure is known to be beneficial in reducing the cardiovascular mortality of T2DM patients, drugs with both anti-hypertensive and anti-hyperglycemic effects are seldom reported. The traditional Chinese medicine danshen has long been used for lowering both blood pressure and blood glucose in T2DM patients, shedding lights on the development of such medication. However, the molecular mechanism and active component remain unclear. Here, we report that the lipophilic component, 15,16-dihydrotanshinone I (DHTH) from danshen potently antagonized both mineralocorticoid and glucocorticoid receptors, and efficiently inhibited the expression of their target genes like Na+/K+ ATPase, glucose 6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK). In addition, DHTH increased AMPKα phosphorylation and regulated its downstream pathways, including increasing acetyl-CoA carboxylase (ACC) phosphorylation, inhibiting transducer of regulated CREB activity 2 (TORC2) translocation and promoting glucose uptake. Such discovered multi-target effects of DHTH are expected to have provided additional understandings on the molecular basis of the therapeutic effects of danshen against the metabolic syndrome.

Hypertension is a common complication of type 2 diabetes mellitus (T2DM), and is the main cause for T2DM-associated mortality. Although the stringent control of blood pressure is known to be beneficial in reducing the cardiovascular mortality of T2DM patients, drugs with both anti-hypertensive and anti-hyperglycemic effects are seldom reported. The traditional Chinese medicine danshen has long been used for lowering both blood pressure and blood glucose in T2DM patients, shedding lights on the development of such medication. However, the molecular mechanism and active component remain unclear. Here, we report that the lipophilic component, 15,16-dihydrotanshinone I (DHTH) from danshen potently antagonized both mineralocorticoid and glucocorticoid receptors, and efficiently inhibited the expression of their target genes like Na+/K+ ATPase, glucose 6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK). In addition, DHTH increased AMPKα phosphorylation and regulated its downstream pathways, including increasing acetyl-CoA carboxylase (ACC) phosphorylation, inhibiting transducer of regulated CREB activity 2 (TORC2) translocation and promoting glucose uptake. Such discovered multi-target effects of DHTH are expected to have provided additional understandings on the molecular basis of the therapeutic effects of danshen against the metabolic syndrome.

The 3C-like protease of SARS coronavirus (SARS-CoV 3CLpro) is vital for SARS-CoV replication and is a promising drug target. It has been extensively proved that only the dimeric enzyme is active. Here we discovered that two adjacent mutations (Ser139_Ala and Phe140_Ala) on the dimer interface resulted in completely different crystal structures of the enzyme, demonstrating the distinct roles of these two residues in maintaining the active conformation of SARS-CoV 3CLpro. S139A is a monomer that is structurally similar to the two reported monomers G11A and R298A. However, this mutant still retains a small fraction of dimer in solution, which might account for its remaining activity. F140A is a dimer with the most collapsed active pocket discovered so far, well-reflecting the stabilizing role of this residue. Moreover, a plausible dimerization mechanism was also deduced from structural analysis. Our work is expected to provide insight on the dimerization–function relationship of SARS-CoV 3CLpro.

The nuclear receptor retinoid X receptor (RXR) functions potently in the regulation of homeostasis and cell development, while rexinoids as RXR agonists have proved their therapeutic potential in the treatment of metabolic diseases and cancer. Here, the natural product bigelovin was identified as a selective RXRα agonist. Interestingly, this compound could not transactivate RXRα:RXRα homodimer but could enhance the transactivation of RXRα:peroxisome proliferator-activated receptor γ heterodimer and repress that of RXRα:liver X receptor (LXR) α heterodimer, while it had no effects on RXRα:farnesoid X receptor heterodimer. Considering that the effective role of LXR response element involved transactivation of sterol regulatory element-binding protein-1c mediated by RXRα:LXRα in triglyceride elevation, such LXR response element repressing by bigelovin has obviously addressed its potency for further research. Moreover, our determined crystal structure of the bigelovin-activated RXRα ligand-binding domain with the coactivator human steroid receptor coactivator-1 peptide revealed that bigelovin adopted a distinct binding mode. Compared with the known RXR ligands, bigelovin lacks the acidic moiety in structure, which indicated that the acidic moiety rendered little effects on RXR activation. Our results have thereby provided new insights into the structure-based selective rexinoids design with bigelovin as a potential lead compound.