Anti-metastatic effects of AGS-30 on breast cancer through the inhibition of M2-like macrophage polarization.

AGS-30, a new andrographolide derivative, showed significant anticancer and anti-angiogenic characteristics. However, its role in controlling macrophage polarization and tumor immune response is unknown. Thus, the main goals of this study are to investigate how AGS-30 regulates macrophage polarization and how it suppresses breast cancer metastasis. AGS-30 inhibited IL-4 and IL-13-induced RAW 264.7 and THP-1 macrophages into M2-like phenotype. However, AGS-30 did not affect the LPS and IFN-γ-induced polarization of M1-like macrophages. AGS-30 reduced the mRNA expressions of CD206, Arg-1, Fizz-1, Ym-1, VEGF, IL-10, MMP2, and MMP9 in M2-like macrophages in a concentration-dependent manner. In contrast, andrographolide treatment at 5 µM did not affect M1-like and M2-like macrophage polarization. The conditioned medium from M2-like macrophages increased 4T1 breast cancer cell migration and invasion, whereas AGS-30 inhibited these effects. In the 4T1 breast tumor xenograft mice, the tumor volume and weight were reduced without affecting body weight after receiving AGS-30. AGS-30 treatment also reduced lung and liver metastasis, with reduced STAT6, CD31, VEGF, and Ki67 protein expressions. Moreover, the tumors had considerably fewer M2-like macrophages and Arg-1 expression, but the proportion of M1-like macrophages and iNOS expression increased after AGS-30 treatment. Same results were found in the tail vein metastasis model. In conclusion, this study shows that AGS-30 inhibits breast cancer growth and metastasis, probably through inhibiting M2-like macrophage polarization. Our findings suggest that AGS-30 may be a potential immunotherapeutic alternative for metastatic breast cancer.

Discovery of a fused bicyclic derivative of 4-hydroxypyrrolidine and imidazolidinone as a new anti-HCV agent.

Hepatitis C virus (HCV) infection causes severe liver diseases and remains a major global public health concern. Current direct-acting antiviral (DAA)-based therapies that target viral proteins involving HCV genome replication are effective, however a minority of patients still fail to cure HCV, rendering a window to develop additional antivirals particularly targeting host functions involving in HCV infection. Here, we utilized the HCV infection cell culture system (HCVcc) to screen in-house compounds bearing host-interacting preferred scaffold for the antiviral activity. Compound HXL-10, a novel fused bicyclic derivative of pyrrolidine and imidazolidinone, was identified as a potent anti-HCV agent with a low cytotoxicity and high specificity. Mechanistic studies showed that HXL-10 neither displayed a virucidal effect nor inhibited HCV genomic RNA replication. Instead, HXL-10 might inhibit HCV assembly by targeting host functions. In summary, we developed a novel anti-HCV agent that may potentially offer additive benefits to the current anti-HCV DDA.

Discovery of ZFD-10 of a pyridazino[4,5-b]indol-4(5H)-one derivative as an anti-ZIKV agent and a ZIKV NS5 RdRp inhibitor.

Zika virus (ZIKV) infection is associated with the birth defect microcephaly and Guillain-Barré syndrome in adults. There is no approved vaccine or specific antiviral agent against ZIKV. ZFD-10, a novel structural skeleton of 1H-pyridazino[4,5-b]indol-4(5H)-one, was firstly synthesized and discovered to be a potent anti-ZIKV inhibitor with very low cytotoxicity. ZFD-10's anti-ZIKV potency is independent of cell lines and ZFD-10 mainly targets the post-entry stages of ZIKV life cycle. Time-of-addition and time-of-withdrawal assays showed that 10 µM ZFD-10 displayed the ability to decrease mainly at the RNA level and weakly the viral progeny particle load. Furthermore, ZFD-10 could protect ZIKV NS5 from thermal unfolding and aggregation and increase the Tagg value of ZIKV NS5 protein from 44.6 to 49.3 °C, while ZFD-10 dose-dependently inhibits ZIKV NS5 RdRp activity using in vitro RNA polymerase assays. Molecular docking study suggests that ZFD-10 affects RdRp enzymatic function through interfering with the fingers and thumb subdomains. These results supported that ZFD-10's cell-based anti-ZIKV activity is related to its anti-RdRp activity of ZIKV NS5. The in vivo anti-ZIKV study shows that the middle-dose (4.77 mg/kg/d) of ZFD-10 protected mice from ZIKV infection and the viral loads of the blood, liver, kidney and brain in the middle-dose and high-dose (9.54 mg/kg/d) were significantly reduced compared to those of the ZIKV control. These results confirm that ZFD-10 has a certain antiviral effect against ZIKV infection in vivo.

Antiviral effects of the fused tricyclic derivatives of indoline and imidazolidinone on ZIKV infection and RdRp activities of ZIKV and DENV.

The prevalence and ravages of Zika virus (ZIKV) seriously endanger human health, especially causing significant neurological defects in both neonates as pediatric microcephaly and adults as Guillain-Barré syndrome. In this work, we studied anti-ZIKV effects of the fused tricyclic derivatives of indoline and imidazolidinone and discovered that some of them are valuable leads for drug discovery of anti-ZIKV agents. The current results show that certain compounds are broad-spectrum inhibitors of ZIKV- and dengue virus (DENV)-infection while distinctive compounds are selective ZIKV inhibitors or selective DENV inhibitors. Compounds of 12, 17 and 28 are more active against Asian ZIKV SZ-VIV01 strain than African ZIKV MR766 strain. It is valued that silylation makes six TBS compounds of 4-nitrophenyl hydrazine series and phenyl hydrazine series more active against ZIKV infection than their phenols. Time-of-addition and withdrawal studies indicate that compound 12 majorly acts on post-infection of RNA synthesis stage of ZIKV life cycle. Moreover, compounds of 12, 17 and 18 are anti-ZIKV agents with the inhibitory activities to ZIKV NS5 RdRp while 12 doesn't inhibit DENV infection even though it is a DENV RdRp inhibitor, 17 is an active agent against DENV infection but is only a weak DENV NS5 RdRp inhibitor, and 28 is inactive against DENV infection and not a DENV NS5 RdRp inhibitor. As a result, a compound's antiviral difference between ZIKV and DENV is not always related to anti-RdRp difference between ZIKV RdRp and DENV RdRp, and structural features of a compound play important roles in executing antiviral and anti-RdRp functions. Further discovery of highly potent broad-spectrum or selective agents against infection by ZIKV and DENV will be facilitated.

Exploration of novel hexahydropyrrolo[1,2-e]imidazol-1-one derivatives as antiviral agents against ZIKV and USUV.

Zika virus (ZIKV) and Usutu virus (USUV) are two emerging flaviviruses mostly transmitted by mosquitos. ZIKV is associated with microcephaly in newborns and the less-known USUV, with its reported neurotropism and its extensive spread in Europe, represents a growing concern for human health. There is still no approved vaccine or specific antiviral against ZIKV and USUV infections. The main goal of this study is to investigate the anti-ZIKV and anti-USUV activity of a new library of compounds and to preliminarily investigate the mechanism of action of the selected hit compounds in vitro. Two potent anti-ZIKV and anti-USUV agents, namely ZDL-115 and ZDL-116, were discovered, both presenting low cytotoxicity, cell-line independent antiviral activity in the low micromolar range and ability of reducing viral progeny production. The analysis of the structure-activity relationship (SAR) revealed that introduction of 2-deoxyribose to 3-arene was fundamental to enhance the solubility and improve the antiviral action. Additionally, we demonstrated that ZDL-115 and ZDL-116 are significantly active against both viruses when added on cells for at least 24 h prior to viral inoculation or immediately post-infection. The docking analysis showed that ZDL-116 could target the host vitamin D receptor (VDR) and viral proteins. Future experiments will be focused on compound modification to discover analogues that are more potent and on the clarification of the mechanism of action and the specific drug target. The discovery and the development of a novel anti-flavivirus drug will have a significant impact in a context where there are no fully effective antiviral drugs or vaccines for most flaviviruses.

Discovery of dehydroandrographolide derivatives with C19 hindered ether as potent anti-ZIKV agents with inhibitory activities to MTase of ZIKV NS5.

Infection by Zika virus (ZIKV), a mosquito-transmitted arbovirus and a member of Flavivirus, could make pediatric microcephaly and Guillain-Barré syndrome, which remains an ongoing global threat. The efficient antivirals to ZIKV infection are of great medical need. Andrographolide and its analogues were discovered to be active against flaviviral infection. In this study, we discovered some dehydroandrographolide derivatives of 3-oximido- or 3-alcohol-19-hindered ether to be potent anti-ZIKV agents with low cytotoxicities (CC50 > 200 µM). Time of addition assay suggests that compound 5a and its analogues act on inhibition of post-entry stage of ZIKV life cycle. It is discovered by experimental and molecular docking studies that active anti-ZIKV compounds of 3a, 5a, 5b and 5c possess inhibitory activities of ZIKV NS5 MTase (methyl transferase) enzymatic activity. Preliminary SAR reveals that C19-modification with bulky groups is necessary for anti-ZIKV infection and replication, anti-ZIKV activity of 5a comes from itself bearing hindered trityl ether but not from its instability, the backbone of dehydroandrographolide is more effective against ZIKV infection than that of andrographolide, and 3-oxime derivatives are more active against ZIKV infection than 3-alcohol derivatives. To our knowledge, 5a is the first reported MTase inhibitor of andrographolide derivatives. More importantly, discovery of active compound 5b with acid-stable 19-OCHPh2 against ZIKV infection is valued and gives us a clue to design and discover generally acid-stable anti-ZIKV agents.

Discovery of Novel Andrographolide Derivatives as Antiviral Inhibitors against Human Enterovirus A71.

Hand-foot-and-mouth disease (HFMD) caused by human enterovirus A71 (EV-A71) infection has been associated with severe neurological complications. With the lack of an internationally approved antiviral, coupled with a surge in outbreaks globally, EV-A71 has emerged as a neurotropic virus of high clinical importance. Andrographolide has many pharmacological effects including antiviral activity and its derivative, andrographolide sulfonate, has been used in China clinically to treat EV-A71 infections. This study sought to identify novel andrographolide derivatives as EV-A71 inhibitors and elucidate their antiviral mode of action. Using an immunofluorescence-based phenotypic screen, we identified novel EV-A71 inhibitors from a 344-compound library of andrographolide derivatives and validated them with viral plaque assays. Among these hits, ZAF-47, a quinolinoxy-andrographolide, was selected for downstream mechanistic studies. It was found that ZAF-47 acts on EV-A71 post-entry stages and inhibits EV-A71 protein expression. Subsequent luciferase studies confirm that ZAF-47 targets EV-A71 genome RNA replication specifically. Unsuccessful attempts in generating resistant mutants led us to believe a host factor is likely to be involved which coincide with the finding that ZAF-47 exhibits broad-spectrum antiviral activity against other enteroviruses (CV-A16, CV-A6, Echo7, CV-B5, CV-A24 and EV-D68). Furthermore, ZAF-46 and ZAF-47, hits from the screen, were derivatives of the same series containing quinolinoxy and olefin modifications, suggesting that an andrographolide scaffold mounted with these unique moieties could be a potential anti-EV-A71/HFMD strategy.

Design, synthesis, discovery and SAR of the fused tricyclic derivatives of indoline and imidazolidinone against DENV replication and infection.

Dengue virus, belonging to a genus Flavivirus, caused public health problem in recent years. One controversial vaccine of DENV was approved and there is no antiviral for the clinic treatment of DENV, therefore, efficient antivirals to DENV are of great medical significance. In this study, we conducted the design, synthesis, cell-based and target-based activity evaluation of 28 compounds based on indoline structural skeleton against DENV infection. Among them, 13 active compounds against DENV infection were discovered and their structure-activity relationship (SAR) was summarized. In this study, indoline carbohydrazine has derived more active compounds than indoline carboamide. It is discovered that TBS group exhibits a good pharmacophore to enhance anti-DENV activity. Further exploration indicated that post-treatment acts as effective time of addition and compound 15 targeting the post-entry stages of DENV2 viral life cycle. SPR imaging results support there are strong interaction of 13 and 15 with RdRp and compounds 13 and 15 reduce RdRp enzymatic activity, revealing that RdRp of DENV NS5 is the drug target for these series of compounds. Molecular docking deciphered the relationship of the structural feature with the putative binding mode by 13 and 15 with RdRp domain of DENV2 NS5 by hydrogen bonds and hydrophobic interactions to establish the fitted low energy conformation. Future studies will focus on designing more potent inhibitors for the treatment and prevention of dengue virus replication and infection, and understanding the more profound underlying structural features of inhibitors and drug action of the mechanism.

The new andrographolide derivative AGS-30 induces apoptosis in human colon cancer cells by activating a ROS-dependent JNK signalling pathway.

BACKGROUND: The anti-cancer activity of andrographolide (Andro) has been extensively demonstrated in recent years. It is supposed that modifying the chemical structure of Andro can improve its efficacy and reduce its toxicity. PURPOSE: In this study, the anti-cancer effect of a 14ß-(2'-chlorophenoxy) derivative of andrographolide known as AGS-30 was investigated, and its underlying mechanisms were also explored. STUDY DESIGN/METHODS: Different cancer cells were used to evaluate and compare the in vitro anti-cancer effects of Andro and AGS-30. Human colon cancer cells HT-29 and HCT-116 were used to study the underlying anti-cancer mechanisms of AGS-30. HT-29 cells xenografted in nude mouse model was used to compare the in vivo anti-tumour efficacies of Andro and AGS-30. RESULT: In vitro studies showed that AGS-30 possessed an anti-cancer effect by inhibiting the viability, colony formation and migration of cancer cells. It significantly induced the generation of reactive oxygen species (ROS), caused the loss of mitochondrial membrane potential and triggered the apoptosis in colon cancer cells. These effects of AGS-30 were more potent than those of Andro. In addition, the expression levels of proteins associated with apoptosis, including phospho-JNK1/2 as well as cleaved caspase 9, caspase 3, and poly(ADP ribose) polymerase, were elevated in AGS-30-treated colon cancer cells. Moreover, these elevated levels of the proteins were inhibited by the antioxidant N-acetylcysteine and the JNK inhibitor SP600125, suggesting the involvement of ROS/JNK-dependent mechanisms in AGS-30-induced apoptosis. The in vitro anti-cancer effect could be reproduced in an HT-29 colon cancer cell xenografted nude mouse model. CONCLUSION: The anti-cancer effect of AGS-30 is stronger than that of Andro. AGS-30 induces apoptosis of colon cancer cells through ROS/JNK-dependent pathway. Our findings may provide insights for the future development of derivatives of Andro as novel chemotherapeutic agents.

Synthesis and Characterization of Andrographolide Derivatives as Regulators of ßAPP Processing in Human Cells.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, one of the main characteristics of which is the abnormal accumulation of amyloid peptide (Aß) in the brain. Whereas ß-secretase supports Aß formation along the amyloidogenic processing of the ß-amyloid precursor protein (ßAPP), α-secretase counterbalances this pathway by both preventing Aß production and triggering the release of the neuroprotective sAPPα metabolite. Therefore, stimulating α-secretase and/or inhibiting ß-secretase can be considered a promising anti-AD therapeutic track. In this context, we tested andrographolide, a labdane diterpene derived from the plant Andrographis paniculata, as well as 24 synthesized derivatives, for their ability to induce sAPPα production in cultured SH-SY5Y human neuroblastoma cells. Following several rounds of screening, we identified three hits that were subjected to full characterization. Interestingly, andrographolide (8,17-olefinic) and its close derivative 14α-(5',7'-dichloro-8'-quinolyloxy)-3,19-acetonylidene (compound 9) behave as moderate α-secretase activators, while 14α-(2'-methyl-5',7'-dichloro-8'-quinolyloxy)-8,9-olefinic compounds 31 (3,19-acetonylidene) and 37 (3,19-diol), whose two structures are quite similar although distant from that of andrographolide and 9, stand as ß-secretase inhibitors. Importantly, these results were confirmed in human HEK293 cells and these compounds do not trigger toxicity in either cell line. Altogether, these findings may represent an encouraging starting point for the future development of andrographolide-based compounds aimed at both activating α-secretase and inhibiting ß-secretase that could prove useful in our quest for the therapeutic treatment of AD.

Discovery of 14S-(2'-chloro-4'-nitrophenoxy)-8R/S,17-epoxy andrographolide as EV-A71 infection inhibitor.

Human enterovirus A71 (EV-A71) is a major etiological agent of hand-foot-and-mouth disease (HFMD) and there is presently no internationally approved antiviral against EV-A71. In this study, it is disclosed that 14S-(2'-chloro-4'-nitrophenoxy)-8R/S,17-epoxy andrographolide (2) was discovered to be an effective inhibitor against EV-A71 infection showing significant reduction of viral titre. In addition to EV-A71, compound 2 exerts broad-spectrum antiviral effects against other enteroviruses. It is revealed that compound 2 inhibits the post-entry stages of EV-A71 viral replication cycle and significantly reduces viral protein expression of structural proteins such as VP0 and VP2 via inhibiting EV-A71 RNA replication. Moreover, the inhibitory property of compound 2 is specific to viral RNA replication. Furthermore, compound 2 is more likely to target a host factor in EV-A71 RNA replication. As a result, introduction of epoxide at positions 8 and 17 of andrographolide is effective for anti-EV-A71 infection and is a potential anti-EV-A71 strategy. Further work to discover more potent andrographolide derivatives and elucidate comprehensive SAR is under way.

Andrographolide derivative as antagonist of vitamin D receptor to induce lipidation of microtubule associate protein 1 light chain 3 (LC3).

Lipidation of microtubule associated protein 1 light chain 3 (LC3) is the critical step in autophagosome formation, numerous efforts have been made to design and develop small molecules that trigger LC3 lipidation to activate autophagy. In this study, we discovered a series of andrographolide derivatives as potent antagonists of vitamin D receptor (VDR) by luciferase reporter assay. Structure-activity-relationship study revealed that andrographolide derivative ZAV-12 specifically inhibited VDR signaling but not NF-κB or STAT3 activation. Western blot analysis indicates that ZAV-12 markedly triggered lipidation of LC3 in MPP+-induced Parkinsonism in vitro in an mTOR-independent manner. The ZAV-12 triggered lipidation was mediated through SREBP2 activation instead of changing expression levels of lipid synthesis genes. Furthermore, ZAV-12 treatment increased the ratio of LC3-II/LC3-I and oligomerization of A53T α-synuclein (SNCA) in SNCA triggered neurotoxicity. Taken together, these results demonstrate the therapeutic potential of VDR antagonist as novel drug candidate for neurodegenerative diseases.

Andrographolide and Its 14-Aryloxy Analogues Inhibit Zika and Dengue Virus Infection.

Andrographolide is a labdene diterpenoid with potential applications against a number of viruses, including the mosquito-transmitted dengue virus (DENV). In this study, we evaluated the anti-viral activity of three 14-aryloxy analogues (ZAD-1 to ZAD-3) of andrographolide against Zika virus (ZIKV) and DENV. Interestingly, one analogue, ZAD-1, showed better activity against both ZIKV and DENV than the parental andrographolide. A two-dimension (2D) proteomic analysis of human A549 cells treated with ZAD-1 compared to cells treated with andrographolide identified four differentially expressed proteins (heat shock 70 kDa protein 1 (HSPA1A), phosphoglycerate kinase 1 (PGK1), transketolase (TKT) and GTP-binding nuclear protein Ran (Ran)). Western blot analysis confirmed that ZAD-1 treatment downregulated expression of HSPA1A and upregulated expression of PGK1 as compared to andrographolide treatment. These results suggest that 14-aryloxy analogues of andrographolide have the potential for further development as anti-DENV and anti-ZIKV agents.

Inhibition of zika virus infection by fused tricyclic derivatives of 1,2,4,5-tetrahydroimidazo[1,5-a]quinolin-3(3aH)-one.

Zika virus (ZIKV) infection represents a significant threat to the global health system, and the search for efficient antivirals to ZIKV remains necessary and urgent. In this study, we extended the exploration of our previously discovered scaffold of 1H-pyrrolo[1,2-c]imidazol-1-one and revealed that two trans isomers of compounds 2 and 7 and one mixture with major trans isomer of compound 3 as novel tetrahydroquinoline-fused imidazolone derivatives are active against ZIKV infection but they are not virucidal. Western Blot and ELISA analyses of ZIKV NS5 and NS1 further demonstrate that compounds of (±)-2, (±)-3 and (±)-7 act as effective agents against ZIKV infection. We show that the N10's basicity is not the basic requirement for these compounds' antiviral activity in the current work. Importantly, tuning of some pharmacophores including substituents at arene can generate promising candidates for anti-ZIKV agents.

Activity of vitamin D receptor agonists against dengue virus.

Infections with the mosquito-transmitted dengue virus (DENV) are a pressing public health problem in many parts of the world. The recently released commercial vaccine for DENV has encountered some problems, and there is still no effective drug to treat infections. Vitamin D has a well characterized role in calcium and phosphorus homeostasis, but additionally has a role in the immune response to bacterial and viral pathogens. In this study a number of fused bicyclic derivatives of 1H-pyrrolo[1,2]imidazol-1-one with vitamin D receptor (VDR) agonist activity were evaluated for possible anti-DENV activity. The results showed that five of the compounds were able to significantly inhibit DENV infection. The most effective compound, ZD-3, had an EC50 value of 7.47 µM and a selective index of 52.75. The compounds were only effective when used as a post-infection treatment and treatment significantly reduced levels of infection, virus output, DENV protein expression and genome copy number. These results suggest that these VDR agonists have the potential for future development as effective anti-DENV agents.

Design, synthesis and discovery of andrographolide derivatives against Zika virus infection.

The Zika endemic established by imported and local transmission is of significant concern and effective anti-ZIKV drugs remain an urgent unmet need. As andrographolide was identified to be an inhibitor of DENV and CHIKV and the importance of quinoline structure against infectious diseases was considered, we are interested in studying its andrographolide derivatives with quinoline moiety against Zika virus infection. In addition to screening eight in-house derivatives of andrographolide, sixteen new derivatives were designed, synthesized and tested against Zika virus infection. Among these compounds, two most potent anti-Zika compounds of 19-acetylated 14α-(5',7'-dichloro-8'-quinolyloxy) derivative 17b and 14ß-(8'-quinolyloxy)-3,19- diol derivative 3 with the highest selectivity were discovered. The SAR analysis indicates that rational and optimal combined modification/s at 3-, 14-, or 19-positions can make derivatives less toxic and more potent against Zika infection, and both of 3 and 17b are suitable as leads for designing new generation of andrographolide derivatives with quinoline or its structure- and property-related moieties against Zika virus and other arboviruses.

AGS-30, an andrographolide derivative, suppresses tumor angiogenesis and growth in vitro and in vivo.

Poor bioavailability and limited efficacy are challenges associated with using andrographolide as a therapeutic agent. We recently synthesized AGS-30, a new andrographolide derivative, in our laboratory. In this study we investigated the potential anti-tumor effect of AGS-30 and the underlying mechanisms, particularly those related to angiogenesis. Results from our in vitro experiments showed that AGS-30 exerted anti-angiogenic effects by inhibiting endothelial cell proliferation, migration, invasion, and tube formation. Phosphorylation and activation of angiogenesis-related signaling molecules (e.g., vascular endothelial growth factor [VEGF] receptor 2, mitogen-activated protein kinase kinase 1/2, extracellular signal-regulated kinase 1/2, mechanistic target of rapamycin [mTOR], protein kinase B [Akt], and p38) were markedly reduced by AGS-30. Meanwhile, AGS-30 potently inhibited cell proliferation and phosphorylation of cell survival-related proteins (e.g., Akt, mTOR, and ERK1/2) and decreased the expression of VEGF in HT-29 colon cancer cells. AGS-30 blocked microvessel sprouting in a rat aortic ring model and blood vessel formation in zebrafish embryos and a mouse Matrigel plug model. Additionally, AGS-30 suppressed tumor growth and angiogenesis in HT-29 colon cancer cell xenografts in nude mice. These effects were not observed when same concentration of andrographolide, the parent compound of AGS-30, was used. Thus, AGS-30 exerted a strong antitumor effect by inhibiting tumor cell growth and angiogenesis and is a candidate compound for the treatment of cancer.

Discovery of fused bicyclic derivatives of 1H-pyrrolo[1,2-c]imidazol-1-one as VDR signaling regulators.

The modulation of VDR signaling is important in regulating tumor-related signal transduction and protecting from microorganismal infection. In this study we discovered by luciferase reporter assay that several fused bicyclic derivatives of 1H-pyrrolo[1,2-c]imidazol-1-one with the assistance of calcitriol result in up to three-fold increases of VDR promoter activity. Preliminary SAR results from 20 compounds disclose that ideal VDR signaling regulators of these compounds are built up by the optimal combination of multiple factors. Western blot analysis indicates that compounds of ZD-3, ZD-4 and ZD-5 not only significantly upregulate p62 and LC3-II but also elevate the ratio of LC3-II/LC3-I, which possibly leads to activated autophagy. All of five compounds also significantly downregulate p65 and upregulate p-p65 and ZD-3 is the most active one to NF-κB signaling, suggesting a possible induction of apoptosis through the regulation of NF-κB signal transduction mediated by VDR signaling. Compounds of ZD-3, ZD-4 and ZD-5 significantly counteract the interference by VDR shRNA, in which ZD-3 gets the highest compensation of VDR expression and the highest ratio of LC3-II/LC3-I, indicating that ZD-3 very likely activates VDR-mediated autophagy. Taken together, these 1H-pyrrolo[1,2-c]imidazol-1-one derivatives can modulate VDR signaling, possibly resulting in the regulation of some signal pathways to induce autophagy and apoptosis.

Andrographolide derivative ameliorates dextran sulfate sodium-induced experimental colitis in mice.

The therapeutic efficacy of immunosuppressive agents has been intensively studied for colitis management. We synthesized a series of andrographolide derivatives and reported their structure-activity-relationship and anti-inflammatory activity in our previous studies. Among these derivatives, compound 3b exhibited the most potent immunosuppressive activity. In the present study, we assessed the efficacy of 3b in dextran sulfate sodium (DSS)-induced model of acute colitis. Compound 3b was administered intragastrically. The therapeutic effect of 3b was evaluated using disease score and immune cell infiltration. The effect of 3b on Toll-like receptor 4/NF-κB and ß-catenin signaling was primarily determined by using immunohistochemistry staining and quantitative real-time PCR. The crosstalk between NF-κB and ß-catenin signaling was then assessed in HCT-116 cells. Treatment with 3b significantly downregulated the disease activity index and suppressed the histologic evidence of inflammation in DSS-induced model of acute colitis. Compound 3b inhibited proinflammatory cytokine expression at both the serum and transcription levels. Treatment with 3b also upregulated the number of PCNA-positive and goblet cells in the intestinal crypt and the intestinal expression of mRNA levels of ß-catenin target genes. ß-Catenin level regulation affected the antiinflammation and anti-apoptotic activities of 3b. This study demonstrated that 3b, a novel andrographolide derivative, suppressed inflammation and significantly reversed colitis pathology. The outcome of colitis treatment with an immunosuppressive agent depends upon the intestinal expression and mutation status of ß-catenin.

Andrographolide derivative as STAT3 inhibitor that protects acute liver damage in mice.

Sustained activation of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway contributed to the progression of cancer and liver diseases. STAT3 signaling inhibitor has been extensively investigated for pharmacological use. We synthesized a series of andrographolide derivatives, and characterized their activity against STAT3 signaling pathway both in vitro and in the CCl4-induced acute liver damage mice model. Among these derivatives, compound 24 effectively inhibited phosphorylation and dimerization of STAT3 but not its DNA binding activity. Compound 24 significantly ameliorated carbon tetrachloride-induced acute liver damage in vivo without changing mice body weight. Treatment with 24 attenuated hepatic pathologic damage and promoted hepatic proliferation and activation of STAT3. Compound 24 inhibited elevated expression of α-smooth muscle actin and serum pro-inflammatory cytokines downstream of STAT3 but not those factors that are regulated by NF-κB or SMADs. In summary, our results suggest that compound 24 may serve as a potential therapeutic agent for the treatment of hepatic damage or a liver protection agent via regulating STAT3 activation.