Jiangu formula: A novel osteoclast-osteoblast coupling agent for effective osteoporosis treatment.

BACKGROUND: The discovering of an osteoclast (OC) coupling active agent, capable of suppressing OC-mediated bone resorption while concurrently stimulating osteoblast (OB)-mediated bone formation, presents a promising strategy to overcome limitations associated with existing antiresorptive agents. However, there is a lack of research on active OC coupling agents. PURPOSE: This study aims to investigate the potential of Jiangu Formula (JGF) in inhibiting OCs while maintaining the OCOB coupling function. METHODS: The anti-osteoporosis efficacy of JGF was evaluated in osteoporosis models induced by ovariectomy in C57BL/6 mouse and SD rats. The effect of JGF on OCs was evaluated by detecting its capacity to inhibit OC differentiation and bone resorption in an in vitro osteoclastogenesis model induced by RANKL. The OCOB coupling activity of JGF was evaluated by measuring the secretion levels of OC-derived coupling factors, OB differentiation activity of MC3T3-E1 interfered with conditioned medium, and the effect of JGF on OC inhibition and OB differentiation in a C3H10T1/2-RAW264.7 co-culture system. The mechanism of JGF was studied by network pharmacology and validated using western blot, immunofluorescence (IF), and ELISA. Following that, the active ingredients of JGF were explored through a chemotype-assembly approach, activity evaluation, and LC-MS/MS analysis. RESULTS: JGF inhibited bone resorption in murine osteoporosis without compromising the OCOB coupling effect on bone formation. In vitro assays showed that JGF preserved the coupling effect of OC on OB differentiation by maintaining the secretion of OC-derived coupling factors. Network analysis predicted STAT3 as a key regulation point for JGF to exert anti-osteoporosis effect. Further validation assays confirmed that JGF upregulated p-STAT3(Ser727) and its regulatory factors IL-2 in RANKL-induced RAW264.7 cells. Moreover, 23 components in JGF with anti-OC activity identified by chemotype-assembly approach and verification experiments. Notably, six compounds, including ophiopogonin D, ginsenoside Re, ginsenoside Rf, ginsenoside Rg3, ginsenoside Ro, and ononin were identified as OC-coupling compounds. CONCLUSION: This study first reported JGF as an agent that suppresses bone loss without affecting bone formation. The potential coupling mechanism of JGF involves the upregulation of STAT3 by its regulators IL-2. Additionally, the chemotype-assembly approach elucidated the activity compounds present in JGF, offering a novel strategy for developing an anti-resorption agent that preserves bone formation.

Discovery of Spiro[pyrrolidine-3,3'-oxindole] LXRß Agonists for the Treatment of Osteoporosis.

Osteoclasts have an additional demand for cholesterol compared to normal cells. Liver X receptors (LXRs) are famous for regulation of lipid and cholesterol metabolism. Therefore, we propose that the LXR ß agonist can regulate the cholesterol balance in osteoclasts to inhibit osteoclast differentiation. Here, we designed and synthesized a novel LXRß agonist by introduction of the privileged fragments from anti-osteoporosis agents to the spiro[pyrrolidine-3,3'-oxindole] scaffold which is a novel scaffold of LXR agonists in our previous research. As a result, seven LXRß agonists inhibited osteoclastogenesis with IC50 values ranging from 0.078 to 0.36 µM. Especially, the most potent LXRß agonist B9 significantly inhibited RANKL-induced osteoclast differentiation and bone resorption in vitro and in vivo. Furthermore, B9 selectively activated LXRß to promote intracellular cholesterol exclusion in osteoclasts and reduce extracellular cholesterol uptake and thereby inhibited osteoclast production. This study provides a new strategy to develop LXRß agonists for osteoporosis.

Identification of a binding site on soluble RANKL that can be targeted to inhibit soluble RANK-RANKL interactions and treat osteoporosis.

One of the major challenges for discovering protein-protein interaction inhibitors is identifying selective and druggable binding sites at the protein surface. Here, we report an approach to identify a small molecular binding site to selectively inhibit the interaction of soluble RANKL and RANK for designing anti-osteoporosis drugs without undesirable immunosuppressive effects. Through molecular dynamic simulations, we discovered a binding site that allows a small molecule to selectively interrupt soluble RANKL-RANK interaction and without interfering with the membrane RANKL-RANK interaction. We describe a highly potent inhibitor, S3-15, and demonstrate its specificity to inhibit the soluble RANKL-RANK interaction with in vitro and in vivo studies. S3-15 exhibits anti-osteoporotic effects without causing immunosuppression. Through in silico and in vitro experiments we further confirm the binding model of S3-15 and soluble RANKL. This work might inspire structure-based drug discovery for targeting protein-protein interactions.

Isochlorogenic Acid C Alleviates High-Fat Diet-Induced Hyperlipemia by Promoting Cholesterol Reverse Transport.

Background: Promoting cholesterol reverse transport (RCT) has been proven to be a promising hyperlipidemia therapy since it is more effective for the treatment of atherosclerosis (AS) caused by hyperlipidemia. Liver X receptor (LXR) agonists can accelerate RCT, but most of them trigger undesirable liver steatosis due to the activation of liver LXRα. Aim: We aim to figure out whether isochlorogenic acid C (ICAC) facilitates RCT without causing hepatic steatosis. Methods: In vitro study, we established foam macrophages and macrophages with loaded NBD-cholesterol models to investigate the competence of RCT promoting ICAC. RT-qPCR and Western blot were used to verify ICAC's regulation of RCT and NF-κB inflammatory pathways. In this in vivo study, male 6-week-old C57BL/6 mice were fed a high-fat diet (HFD) to investigate ICAC's anti-hyperlipidemic effect and its functions in regulating RCT. The anti-hyperlipidemic effect of ICAC was evaluated by blood and liver lipid levels, liver hematoxylin, oil red o staining, and liver coefficient. Finally, mRNA levels of genes involved in RCT and inflammation pathways in the liver and intestine were detected by RT-qPCR. Results: ICAC prevented macrophages from foaming by up-regulating the LXRα mediated RCT pathway and down-regulating expression of the cholesterol absorption genes LDLR and CD36, as well as suppressing iNOS, COX2, and IL-1ß inflammatory factors. In HFD-fed mice, ICAC significantly lowered the lipid level both in the serum and the liver. Mechanistic studies showed that ICAC strengthened the RCT pathway in the liver and intestine but didn't affect liver LXRα. Furthermore, ICAC impeded both adipogenesis and the inflammatory response in the liver. Conclusion: ICAC accelerated RCT without affecting liver LXRα, thus resulting in a lipid-lowering effect without increasing liver adipogenesis. Our results indicated that ICAC could be a new RCT promoter for hyperlipidemia treatment without causing liver steatosis.

Long noncoding RNA PVT1 promotes chondrocyte extracellular matrix degradation by acting as a sponge for miR-140 in IL-1ß-stimulated chondrocytes.

BACKGROUND: Osteoarthritis (OA) is a common degenerative joint disease, and chondrocyte extracellular matrix (ECM) degradation is one vital pathological feature of OA. Long noncoding RNA (lncRNA), a new kind of gene regulator, plays an important role in pathogenesis of many diseases like OA. Recent studies have confirmed that lncRNA plasmacytoma variant translocation 1 (PVT1) expression was upregulated in OA patients; however, its effect on ECM degradation remained unknown. METHODS: Cartilage tissue samples were obtained from 6 OA patients admitted in Guangdong Second Traditional Chinese Medicine Hospital. Chondrocytes were isolated and cultured from the collected cartilage tissue. Plasmid construction, RNA interference, cell transfection, fluorescence in situ hybridization (FISH), and pull-down assay were carried out during the research. RESULTS: In this study, PVT1 expression was significantly increased in chondrocytes stimulated by interleukin-1ß (IL-1ß). In addition, inhibition of PVT1 significantly downregulated the increased expressions of ADAM metallopeptidase with thrombospondin type 1 motif-5 (ADAMTS-5) and matrix metalloproteinase-13 (MMP-13) induced by IL-1ß. Further investigation revealed that PVT1 was an endogenous sponge RNA, which directly bound to miR-140 and inhibited miR-140 expression. CONCLUSION: To sum up, this study showed that PVT1 promoted expressions of ADAMTS-5 and MMP-13 as a competing endogenous RNA (ceRNA) of miR-140 in OA, which eventually led to aggravation of ECM degradation, thus providing a new and promising strategy for the treatment of OA.

Cordyceps militaris extract induces apoptosis and pyroptosis via caspase-3/PARP/GSDME pathways in A549 cell line.

Cordyceps militaris (CM) is traditionally used as dietary therapy for lung cancer patients in China. CM extract (CME) is hydrosoluble fraction of CM and extensively investigated. Caspase-3-involved cell death is considered as its major anticancer mechanism but inconclusive. Therefore, we explore its caspase-3-dependent programmed cell death nature (apoptosis and pyroptosis) and validate its caspase-3-dependent property in loss-of-function experiment. Component profile of CME is detected by High Performance Liquid Chromatography- quadrupole time-of-flight mass spectrometry (HPLC-qTOF). Results show that CME causes pyroptosis-featured cell bubbling and cell lysis and inhibits cell proliferation in A549 cell. CME induces chromatin condensing and makes PI+/annexin V+ staining in bubbling cells, indicating genotoxicity, apoptosis, and pyroptosis cell death are caused by CME. High concentration of CME (200 µg/ml) exerts G2/M and G0 cell cycles arresting and suppresses P53-downstream proliferative proteins, including P53, P21, CDC25B, CyclinB1, Bcl-2, and BCL2 associated agonist of cell death (BAD), but 1-100 µg/ml of CME show less effect on proteins above. Correspondingly, caspase-3 activity and caspase-3 downstream proteins including pyroptotic effector gasdermin-E (GSDME) and apoptotic marker cleaved-poly-ADP-ribose polymerase (PARP) are significantly promoted by CME. Moreover, regarding membrane pore formation in pyroptotic cell, expression of membrane GSDME (GSDME antibody conjugated with PE-Cy7 for detection in flow cytometry) is remarkably increased by CME treatment. By contrast, other pyroptosis-related proteins such as P2X7, NLRP3, GSDMD, and Caspase-1 are not affected after CME treatment. Additionally, TET2 is unexpectedly raised by CME. In present of caspase-3 inhibitor Ac-DEVD-CHO (Ac-DC), CME-induced cytotoxicity, cell bubbling, and genotoxicity are reduced, and CME-induced upregulation of apoptosis (cleaved-PARP-1) and pyroptosis (GSDME-NT) proteins are reversed. Lastly, 22 components are identified in HPLC-qTOF experiment, and they are classified into trophism, neoadjuvant component, cytotoxic component, and cancer deterioration promoter according to previous references. Conclusively, CME causes caspase-3-dependent apoptosis and pyroptosis in A549 through caspase-3/PARP and caspase-3/GSDME pathways, and it provides basic insight into clinic application of CME for cancer patients.

Deep learning enables discovery of highly potent anti-osteoporosis natural products.

A pre-trained self-attentive message passing neural network (P-SAMPNN) model was developed based on our anti-osteoclastogenesis dataset for virtual screening purpose. Validation processes proved that P-SAMPNN model was significantly superior to the other base line models. A commercially available natural product library was virtually screened by the P-SAMPNN model and resulted in confirmed 5 hits from 10 selected virtual hits. Among the confirmed hits, compounds AP-123/40765213 and AE-562/43462182 are the nanomolar inhibitors against osteoclastogenesis with a new scaffold. Further studies indicate that AP-123/40765213 and AE-562/43462182 significantly suppress the mRNA expression of RANK and downregulate the expressions of osteoclasts-related genes Ctsk, Nfatc1, and Tracp. Our work demonstrated that P-SAMPNN method can guide phenotype-based drug discovery.

Upregulated expression of MMP family genes is associated with poor survival in patients with esophageal squamous cell carcinoma via regulation of proliferation and epithelial­mesenchymal transition.

Matrix metalloproteinases (MMPs) are involved in the cleavage of several components of the extracellular matrix and serve important roles in tumor growth, metastasis and invasion. Previous studies have focused on the expression of one or several MMPs in esophageal squamous cell carcinoma (ESCC); however, in the present study, the transcriptomics of all 23 MMPs were systematically investigated with a focus on the prognostic value of the combination of MMPs. In this study, 8 overlapping differentially expressed genes of the MMP family were identified based on data obtained from Gene Expression Omnibus and The Cancer Genome Atlas. The prognostic value of these MMPs were investigated; the receiver operating characteristic curves, survival curves and nomograms showed that the combination of 6 selected MMPs possessed a good predictive ability, which was more accurate than the prediction model based on Tumor­Node­Metastasis stage. Gene set enrichment analysis and gene co­expression analysis were performed to investigate the potential mechanism of action of MMPs in ESCC. The MMP family was associated with several signaling pathways, such as epithelial­mesenchymal transition (EMT), Notch, TGF­ß, mTOR and P53. Cell Counting Kit­8, colony formation, wound healing assays and western blotting were used to determine the effect of BB­94, a pan­MMP inhibitor, on proliferation and migration of ESCC cells. BB­94 treatment decreased ESCC cell growth, migration and EMT. Therefore, MMPs may serve both as diagnostic and prognostic biomarkers of ESCC, and MMP inhibition may be a promising preventive and therapeutic strategy for patients with ESCC.

Tanshinol A Ameliorates Triton-1339W-Induced Hyperlipidemia and Liver Injury in C57BL/6J Mice by Regulating mRNA Expression of Lipemic-Oxidative Injury Genes.

Tanshinol A, which is derived from a traditional Chinese herbal Radix Salviae Miltiorrhizae is indicative of a hypolipidemic candidate. Therefore, we aim to validate its hypolipidemic activity of tanshinol A and explore its mechanism in triton-1339W-induced hyperlipidemic mice model, which possess multiply pathogenesis for endogenous lipid metabolism disorder. Experimental hyperlipidemia mice are treated with or without tanshinol A (i.g. 40, 20, 10 mg/kg), and blood and liver tissue were collected for validating its hypolipidemic and hepatic protective effect, and hepatic mRNA expression profile, which was associated with lipid metabolism dysfunction and liver injury, was detected by RT-qPCR. As results show, triton-1339W-induced abnormal of serum TC, TAG, HDL-C, LDL-C, SOD, MDA, GOT, and GPT is remarkably attenuated by tanshinol A. In pathological experiment, triton-1339W-induced hepatocellular ballooning degeneration, irregular central vein congestion, and inflammation infiltration are alleviated by tanshinol A. Correspondingly, hepatic mRNA expression of Atf4, Fgf21, Vldlr, Nqo1, Pdk4, and Angptl4, which are genes regulating lipemic-oxidative injury, are significantly increased by tanshinol A by 2~6 fold. Abcg5, Cd36, and Apob, which are responsible for cholesterol metabolism, are mildly upregulated. Noticeably, triton-1339W-suppressed expressions of Ptgs2/Il10, which are genes responsible for acute inflammation resolution in liver injury, are remarkably increased by tanshinol A. Conclusively, tanshinol A exerted hypolipidemic effect and hepatoprotective effect through restoring triton-1339W-suppressed mRNA expression, which may be involved in Atf4/Fgf21/Vldlr and Ptgs2/Il-10 signaling pathways.

Diterpenoids from the aerial parts of Flueggea acicularis and their activity against RANKL-induced osteoclastogenesis.

Compounds 1-11 were isolated from the aerial parts of Flueggea acicularis Croizz Webster, including three new rearranged clesistanthane diterpenoids fluacinoids A-C (1-3) and five new norditerpenoid fluacinoids D-H (4-8). The new compounds were identified from spectroscopic data combined with single crystal X-ray diffraction analysis, modified Mosher's methods, and ECD data analyses. All the isolated compounds were evaluated for their activities on RANKL-induced osteoclastogenesis in bone marrow monocytes (BMMs). Compound 6 showed the most potent inhibition against osteoclast differentiation (IC50, 0.7 µM) and decreased the expression level of osteoclast-related genes. Moreover, compound 6 prompted the apoptosis of osteoclasts. Compound 6 also suppressed RANKL-induced NF-κB activation. This study reveals that norditerpenoids may be resource for anti-osteoporosis agents.

Identifying Novel Anti-Osteoporosis Leads with a Chemotype-Assembly Approach.

In this paper, we applied a chemotype-assembly approach for ligand-based drug discovery (LBDD) to discover novel anti-osteoporosis leads. With this new approach, we identified 12 chemotypes and derived 18 major chemotype assembly rules from 245 known anti-osteoporosis compounds. Then, we selected 19 compounds from an in-house compound library using chemotype-assembly approach for anti-osteoporosis assays, which resulted in 13 hits. Based on structural features in these 13 compounds, we synthesized 50 possible anti-osteoporosis compounds from the anti-osteoporosis chemotypes by means of click chemistry techniques and discovered a compound (10a, IC50 = 2 nM) with nanomolar activity. Compound 10a was then proved to be an anti-osteoporosis lead since it can prevent bone loss in vivo.

Discovery of new inhibitors against both NF-κB and osteoclastogenesis from in-house library with α, ß-unsaturated-enone fragment.

The α,ß-unsaturated-enone contained natural products have been reported showing NF-κB inhibition effect. It is well known that NF-κB inhibitors can also be used to inhibit osteoclastogenesis. In a continual discovery new agents for anti-osteoclastogenesis, 8 different type compounds with α,ß-unsaturated-enone fragments from our in-house library were evaluated for NF-κB inhibition and anti-osteoclastogenesis. Experimental results indicated five compounds exhibited inhibition of NF-κB signal pathway. Among them, one compound ((E)-2-(4-fluorobenzylidene)-3,4-dihydronaphthalen-1(2H)-one, 6a) simultaneously inhibits both osteoclastogenesis and NF-κB signal pathway. Furthermore, 12 compounds with similar scaffold with 6a were tested for anti-osteoclastogenesis. As a result, 9 compounds inhibited both NF-κB and osteoclastogenesis. Among them, compound 6b is the most potent inhibitor against NF-κB (IC50 = 2.09 µM) and osteoclast differentiation (IC50 = 0.86 µM). Further studies show that compound 6b blocks the phosphorylation of both p65 and IκBα, and suppresses NF-κB targeted gene expression without interfering MAPKs and PI3K/Akt signal transduction pathways. This study demonstrates that we can identify promising synthesized compounds with new scaffolds as therapeutic solutions against osteoclastogenesis inspired by the privileged fragment derived from natural leads.

Mechanism of YLTZ on glycolipid metabolism based on UPLC/TOF/MS metabolomics.

Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by dysfunction of glycolipid metabolism. YLTZ is used to treat hyperlipidemia, yet its hypolipidemic and hypoglycemic mechanism on T2DM are unknown. Thus, UPLC/TOF/MS was applied in this study to identify the potential bio-markers, and deduce the possible metabolic pathways. According to bio-indexes, the increased blood lipid levels, including TC, TG, LDL and FA, and the decreased HDL, the elevated glucose, reduced insulin level and impaired OGTT were observed in diabetic rat model. While YLTZ can decrease the lipid levels and glucose content, as well as increased insulin standards and improve OGTT. After data from UPLC/TOF/MS processed, 17 metabolites were obtained, including phospholipids (LPCs, PCs and PGP (18:1)), beta-oxidation production (HAA, VAG and CNE) and precursors (THA), bile acid (CA, CDCA and IDCA), hydrolysate of TG (MG (22:4)), glycometabolism (G6P), cholesterol-driven synthetics (ADO) and production of arachidonate acid (THETA). As a result, YLTZ was able to reduce LPCs, PCs, PGP (18:1), HAA, VAG, CNE, CA, ADO and THETA, as well as enhance MG (22:4) and G6P. After analyzing results, several metabolic pathways were deduced, which containing, cholesterol synthesis and elimination, FA beta-oxidation, TG hydrolysis, phospholipids synthesis, glycolysis, gluconeogenesis and inflammation. Consequently, YLTZ performed to prohibit the FA beta-oxidation, synthesis of cholesterol and phospholipids, gluconeogenesis and inflammation level, as well as promote TG hydrolysis, glycolysis and blood circulation. Hence, applying metabonomics in TCM research can uncover its pharmacological edges, elucidating comprehensively that YLTZ has capacity of hypolipidemic, hypoglycemic and promoting blood circulation, matching the effect of removing blood stasis, eliminating phlegm and dampness.

Evaluation of type 2 diabetic mellitus animal models via interactions between insulin and mitogen­activated protein kinase signaling pathways induced by a high fat and sugar diet and streptozotocin.

Type 2 diabetic mellitus (T2DM), which is characterized by insulin resistance (IR), hyperglycemia and hyperlipidemia, is a comprehensive dysfunction of metabolism. The insulin receptor (INSR)/phosphoinositide 3­kinase (PI3K)/AKT signaling pathway is well acknowledged as a predominant pathway associated with glucose uptake; however, the effect of streptozotocin (STZ) plus a high fat and sugar diet (HFSD) on the proteins associated with this pathway requires further elucidation. In order to explore this effect, a T2DM rat model was constructed to investigate T2DM pathogenesis and potential therapeutic advantages. Rats were randomly divided into control and model groups, including normal diet (ND) and HFSD types. ND types were administered intraperitoneal (IP) injections of STZ (35 mg/kg) or a combination of STZ and alloxan monohydrate (AON) (40 mg/kg), whereas HFSD types were composed of HFSD pre­given, post­given and simul­given groups, and were modeled as follows: IP or intramuscular (IM) injection of STZ (35 mg/kg) or a combination of STZ and AON (40 mg/kg). Results indicated that, compared with controls, blood glucose, insulin, homeostatic model assessment­insulin resistance and total triglyceride were significantly elevated in groups with HFSD and modeling agents (P<0.05 or P<0.01), whereas total cholesterol and low­density lipoprotein levels were significantly elevated in groups simultaneously administered HFSD and modeling agents (P<0.05 or P<0.01), in addition to downregulation of the expression of insulin signaling pathway proteins in the liver, including INSR, PI3K, AKT1, phosphatidylinositol-5-phosphate 4­kinase type­2α (PIP5Kα) and glucose transporter (GLUT)2, and increased expression of inflammatory factors, including p38, tumor necrosis factor (TNF)α and interleukin (IL)6. Furthermore, compared with other two HFSD types including pre­given and post­given group, the simul­given group that received IM injection with STZ exhibited decreased expression levels of major insulin signal pathway proteins INSR, PI3K, AKT1, PIP5Kα, GLUT2 or GLUT4 in the liver and pancreas (P<0.05 or P<0.01), whereas the opposite was observed in the skeletal muscle. In addition, the protein expression levels of phosphorylated­p38, p38, IL6 and TNFα in the simul­given group that received IM injection with STZ were increased (P<0.05 or P<0.01), and histopathology also indicated inflammation in pancreas and liver. The present findings suggest that a low dose of STZ may partially impair the ß cells of the pancreas, whereas long­term excess intake of HFSD may increase lipid metabolites, inhibit the insulin signaling pathway and activate the mitogen­activated protein kinase p38 signaling pathway. The combined action of STZ and AON may result in insulin resistance, which ultimately results in abnormalities in glucose and lipid metabolism. The present model, analogue to T2DM onset of humans, evaluated the medical effect on metabolic dysfunction and provides an insight into the underlining mechanism of IR.

Selective reduction in the expression of UGTs and SULTs, a novel mechanism by which piperine enhances the bioavailability of curcumin in rat.

Curcumin (CUR) is known to exert numerous health-promoting effects in pharmacological studies, but its low bioavailability hinders the development of curcumin as a feasible therapeutic agent. Piperine (PIP) has been reported to enhance the bioavailability of curcumin, but the underlying mechanism remains poorly understood. In an attempt to find the mechanism by which piperine enhances the bioavailability of curcumin, the dosage ratio (CUR: PIP) and pre-treatment with piperine were hypothesized as key factors for improving the bioavailability in this combination. Therefore, combining curcumin with piperine at various dose ratios (1:1 to 100:1) and pre-dosing with piperine (0.5-8 h prior to curcumin) were designed to investigate their contributions to the pharmacokinetic parameters of curcumin in rats and their effects on the expression of UGT and SULT isoforms. It was shown that the Cmax and AUC0-t of curcumin were slightly increased by 1.29 and 1.67 fold at a ratio of 20:1, while curcumin exposure was enhanced significantly in all the piperine pre-treated rats (0.5-8 h), peaking at 6 h (a 6.09-fold and 5.97-fold increase in Cmax and AUC0-t , p < 0.01), regardless of the unchanged t1/2 and Tmax . Also observed was a time-dependent inhibition of the hepatic expression of UGT1A6, 1A8, SULT1A1, 1A3, and the colonic expression of UGT1A6 that occurred within 6 h of piperine pre-treatment but was reversed at 8 h, which correlated with the changes in curcumin exposure. Similarly, the inhibitory effect of piperine on most of the UGTs and SULTs are time-dependent in Caco-2 and HepG2 cells. It is concluded that piperine pre-treatment time-dependently improves the bioavailability of curcumin through the reversible and selective inhibition of UGTs and SULTs. Copyright © 2016 John Wiley & Sons, Ltd.

Euphomilones A and B, ent-Rosane Diterpenoids with 7/5/6 and 5/7/6 Skeletons from Euphorbia milii.

Three highly modified ent-rosane diterpenoids, euphomilones A (1) and B (2) and euphomianol A (3), were isolated from the aerial parts of Euphorbia milii. The structures were elucidated from physical, spectroscopic, and X-ray diffraction data, as well as experimental and calculated electronic circular dichroism (ECD) analysis. Plausible biogenetic pathways to 1-3 are proposed. Also, compound 1 exhibited inhibition of receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclast formation (IC50 = 12.6 µM).

Comparative effects of SNX-7081 and SNX-2112 on cell cycle, apoptosis and Hsp90 client proteins in human cancer cells.

SNX-2112, a novel 2-aminobenzamide inhibitor of Hsp90, previously showed a broad spectrum of anticancer activity. However, subsequent development has been discontinued due to ocular toxicity as identified in a phase I study. SNX-7081, another closely related Hsp90 inhibitor with a side chain of indole instead of indazole, has recently attracted attention. The aim of the present study was to investigate the anticancer effects of SNX-7081 in eleven cell lines, as well as the mechanisms involved, with SNX-2112 serving as a reference. The cytotoxic effects were determined using an MTT assay and apoptosis was measured using flow cytometry. The results showed that SNX-7081 exerted better inhibitory effects than SNX-2112 in six eighths of the human cancer cell lines, with an average IC50 of 1 µM. The two inhibitors exerted low cytotoxicity in L-02, HDF and MRC5 normal human cells (IC50 >50 µM), and arrested cancer cells at the G2/M phase in a similar manner to normal cells. Compared with SNX-2112, SNX-7081 exhibited more potent effects on cell apoptosis in four sixths of the human cancer cell lines, and was more active in the downregulation of Hsp90 client proteins. In addition, SNX-7081 exhibited a stronger binding affinity to Hsp90 than SNX-2112 in molecular docking experiments. Considering the superior effects against Hsp90 affinity, cell growth, apoptosis, and Hsp90 client proteins in a majority of human cancer cells, the novel SNX-7081 may be a promising alternative to SNX-2112, which merits further evaluation.

Piperine potentiates the hypocholesterolemic effect of curcumin in rats fed on a high fat diet.

It has previously been demonstrated that curcumin possesses a hypocholesterolemic effect and potentiates numerous pharmacological effects of curcumin, however, the mechanisms underlying this hypocholesterolemic effect and the interaction between curcumin and piperine remain to be elucidated. In the present study, male Sprague-Dawley rats were fed on a high-fat diet (HFD) to establish a hyperlipidemia (HLP) model. Co-administration of curcumin plus piperine was found to decrease the levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol in the serum and liver, as well as increase the levels of fecal TC, TG and total bile acid, compared with administration of curcumin alone. Curcumin plus piperine also markedly increased the levels of high-density lipoprotein cholesterol. Furthermore, compared with administration of curcumin alone, administration of curcumin plus piperine resulted in a significant upregulation of the activity and gene expression of apolipoprotein AI (ApoAI), lecithin cholesterol acyltransferase (LCAT), cholesterol 7α-hydroxylase (CYP7A1) and low-density lipoprotein receptor (LDLR). In conclusion, these results indicated that co-administration of curcumin plus piperine potentiates the hypocholesterolemic effects of curcumin by increasing the activity and gene expression of ApoAI, CYP7A1, LCAT and LDLR, providing a promising combination for the treatment of HLP.

SNX-25a, a novel Hsp90 inhibitor, inhibited human cancer growth more potently than 17-AAG.

17-Allylamino-17-demethoxygeldanamycin (17-AAG), a typical Hsp90 inhibitor derived from geldanamycin (GA), has entered Phase III clinical trials for cancer therapy. However, it has several significant limitations such as poor solubility, limited bioavailability and unacceptable hepatotoxicity. In this study, the anticancer activity and mechanism of SNX-25a, a novel Hsp90 inhibitor, was investigated comparing with that of 17-AAG. We showed that SNX-25a triggered growth inhibition more sensitively than 17-AAG against many human cancer cells, including K562, SW-620, A375, Hep-2, MCF-7, HepG2, HeLa, and A549 cell lines, especially at low concentrations (<1 µM). It showed low cytotoxicity in L-02, HDF and MRC5 normal human cells. Compared with 17-AAG, SNX-25a was more potent in arresting the cell cycle at G2 phase, and displayed more potent effects on human cancer cell apoptosis and Hsp90 client proteins. It also exhibited a stronger binding affinity to Hsp90 than 17-AAG using molecular docking. Considering the superiority effects on Hsp90 affinity, cell growth, cell cycle, apoptosis, and Hsp90 client proteins, SNX-25a is supposed as a potential anticancer agent that needs to be explored in detail.

Linker-extended native cyanovirin-N facilitates PEGylation and potently inhibits HIV-1 by targeting the glycan ligand.

Cyanovirin-N (CVN) potently inhibits human immunodeficiency virus type 1 (HIV-1) infection, but both cytotoxicity and immunogenicity have hindered the translation of this protein into a viable therapeutic. A molecular docking analysis suggested that up to 12 residues were involved in the interaction of the reverse parallel CVN dimer with the oligosaccharide targets, among which Leu-1 was the most prominent hot spot residue. This finding provided a possible explanation for the lack of anti-HIV-1 activity observed with N-terminal PEGylated CVN. Therefore, linker-CVN (LCVN) was designed as a CVN derivative with a flexible and hydrophilic linker (Gly4Ser)3 at the N-terminus. The N-terminal α-amine of LCVN was PEGylated to create 10 K PEG-aldehyde (ALD)-LCVN. LCVN and 10 K PEG-ALD-LCVN retained the specificity and affinity of CVN for high mannose N-glycans. Moreover, LCVN exhibited significant anti-HIV-1 activity with attenuated cytotoxicity in the HaCaT keratinocyte cell line and MT-4 T lymphocyte cell lines. 10 K PEG-ALD-LCVN also efficiently inactivated HIV-1 with remarkably decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. The linker-extended CVN and the mono-PEGylated derivative were determined to be promising candidates for the development of an anti-HIV-1 agent. This derivatization approach provided a model for the PEGylation of biologic candidates without introducing point mutations.