Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled-receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine-rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the Smo CRD may bind its own endogenous ligand. Here we present the NMR solution structure of the Drosophila Smo CRD, and describe interactions between the glucocorticoid budesonide (Bud) and the Smo CRDs from both Drosophila and human. Our results highlight a function of the Smo CRD, demonstrating its role in binding to small molecule modulators.
In order to investigate the biological activity of novel 1,2,4-triazole compounds, seventeen novel 1,2,4-triazole derivatives containing pyridine moiety were synthesized under microwave assistant condition by multi-step reactions. The structures were characterized by 1H NMR, MS and elemental analyses. The target compounds were evaluated for their fungicidal activities against Stemphylium lycopersici (Enjoji) Yamamoto, Fusarium oxysporum. sp. cucumebrium, and Botrytis cinerea in vivo, and the results indicated that some of the title compounds displayed excellent fungicidal activities. Theoretical calculation of the title compound was carried out with B3LYP/6-31G (d,p). The full geometry optimization was carried out using 6-31G (d,p) basis set, and the frontier orbital energy, atomic net charges were discussed, and the structure-activity relationship was also studied.
1,2,4-triazole; pyridine; synthesis; fungicidal activity; thioether; theoretical calculations
A series of new N,N′-diacylhydrazine derivatives were designed and synthesized. Their structures were verified by 1H-NMR, mass spectra (MS) and elemental analysis. The antifungal activities of these N,N′-diacylhydrazines were evaluated. The bioassay results showed that most of these N,N′-diacylhydrazines showed excellent antifungal activities against Cladosporium cucumerinum, Corynespora cassiicola, Sclerotinia sclerotiorum, Erysiphe cichoracearum, and Colletotrichum orbiculare in vivo. The half maximal effective concentration (EC50) of one of the compounds was also determined, and found to be comparable with a commercial drug. To further investigate the structure–activity relationship, comparative molecular field analysis (CoMFA) was performed on the basis of antifungal activity data. Both the steric and electronic field distributions of CoMFA are in good agreement in this study.
N,N′-diacylhydrazines; antifungal activity; synthesis; three-dimensional quantitative structure-activity relationships (3D-QSAR)
Genomic structural alteration is common in pediatric cancers, and analysis of data generated by the Pediatric Cancer Genome Project reveals such tumor-related alterations in many Wnt signaling–associated genes. Most pediatric cancers are thought to arise within developing tissues that undergo substantial expansion during early organ formation, growth and maturation, and Wnt signaling plays an important role in this development. We examined three pediatric tumors—medullobastoma, early T-cell precursor acute lymphoblastic leukemia, and retinoblastoma—that show multiple genomic structural variations within Wnt signaling pathways. We mathematically modeled this pathway to investigate the effects of cancer-related structural variations on Wnt signaling. Surprisingly, we found that an outcome measure of canonical Wnt signaling was consistently similar in matched cancer cells and normal cells, even in the context of different cancers, different mutations, and different Wnt-related genes. Our results suggest that the cancer cells maintain a normal level of Wnt signaling by developing multiple mutations.
Low-density lipoprotein receptor–related proteins 5 and 6 (LRP5/6) mediate canonical Wnt–β-catenin signaling by forming a complex with the co-receptor Frizzled, which binds to Wnt proteins. Dickkopf (DKK)–related proteins inhibit the Wnt signaling pathway by directly binding to the ectodomains of LRP5/6. However, the mechanism for DKK-mediated antagonism has not been fully understood as of yet. Crystal structures of the LRP6 ectodomain in complex with DKK1, along with mutagenesis studies, provide considerable insights into the molecular basis for DKK-mediated inhibition and Wnt signaling through LRP5/6.
Dishevelled (Dvl) PDZ domains transduce Wnt signals from the membrane-bound receptor Frizzled to the downstream. As abnormal Wnt signaling has been implicated in tumorigenesis, the Dvl PDZ domain is a potential target for small-molecule inhibitors that block Wnt signaling at the Dvl level. We expanded our in silico search to examine the chemical space near previously developed PDZ binders and identified nine additional compounds bind to the Dvl PDZ. We then performed a quantitative structure-activity relationship (QSAR) analysis of these compounds and combined these results with structural studies of the PDZ domain in complex with the compounds to design and synthesize a group of new, further optimized compounds. Two rounds of synthesis and testing yielded a total of six compounds that have greatly improved binding affinity to the Dvl PDZ domain and most potent ones competitively displace Dapper peptide from the PDZ domain. In addition to providing more potent Dvl PDZ domain inhibitors, this study demonstrates that virtual screening and structural studies can be powerful tools in guiding the chemical synthesis hit-to-lead optimization stage during the drug discovery process.
Wnt signaling pathway; Dishevelled; PDZ domain
Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or “snatched” from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.
Seasonal and pandemic influenza have enormous impacts on global public health. The rapid emergence of influenza virus strains that are resistant to current antiviral therapies highlights the urgent need to develop new therapeutic options. A promising target for drug discovery is the influenza virus PA protein, whose endonuclease enzymatic activity is essential for the “cap-snatching” step of viral mRNA transcription that allows transcripts to be processed by the host ribosome. Here, we describe a structure-based analysis of the mechanism of inhibition of the influenza virus PA endonuclease by small molecules. Our X-ray crystallographic studies have resolved the modes of binding of known and predicted inhibitors, and revealed that they directly block the PA endonuclease active site. We also report a number of molecular interactions that contribute to binding affinity and specificity. Our structural results are supported by biochemical analyses of the inhibition of enzymatic activity and computational docking experiments. Overall, our data reveal exciting strategies for the design and optimization of novel influenza virus inhibitors that target the PA protein.
Mesenchymal stem cells (MSCs) are used in many of the current stem cell-based clinical trials and their therapeutic efficacy has increasingly been attributed to secretion of paracrine factors. We have previously demonstrated that a therapeutic constituent of this secretion is exosome, a secreted bilipid membrane vesicle of ~50–100 nm with a complex cargo that is readily internalized by H9C2 cardiomyocytes. It reduces infarct size in a mouse model of myocardial ischemia/reperfusion (MI/R) injury. We postulate that this therapeutic efficacy is derived from the synergy of a select permutation of individual exosome components. To identify protein candidates in this permutation, the proteome was profiled and here we identified 20S proteasome as a protein candidate. Mass spectrometry analysis detected all seven α and seven β chains of the 20S proteasome, and also the three beta subunits of “immunoproteasome” with a very high confidence level. We demonstrated that a functional proteasome copurified with MSC exosomes with a density of 1.10–1.18 g/mL, and its presence correlated with a modest but significant reduction in oligomerized protein in a mouse model of myocardial infarction. Circulating proteasomes in human blood also copurified with exosomes. Therefore, 20S proteasome is a candidate exosome protein that could synergize with other constituents to ameliorate tissue damage.
Wnt/β-catenin-mediated gene transcription plays important roles in a wide range of biological and pathophysiological processes including tumorigenesis where β-catenin-mediated transcription activity frequently elevates. TRABID, a deubiquitinase, was shown to have a positive Wnt/β-catenin-mediated gene transcription and hence holds a promise as a putative anti-cancer target.
In this study, we used a combination of structure based virtual screening and an in vitro deubiquitinase (DUB) assay to identify several small molecules that inhibit TRABID DUB activity. However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription. In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.
TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.
Cell growth can be suppressed by stressful environments, but the role of stress pathways in this process is largely unknown. Here we show that a cascade of p38β mitogen activated protein kinase and p38 regulated/activated kinase (PRAK) plays a role in energy starvation-induced suppression of mammalian target of rapamycin (mTOR), that energy starvation activates the p38β-PRAK cascade, and that p38β- or PRAK-deletion diminishes energy depletion-induced suppression of mTORC1 and reduction of cell size. We show that p38β-PRAK operates independent from the known mTORC1 inactivation pathways – phosphorylation of tuberous sclerosis protein 2 (TSC2) and raptor by AMP activated protein kinase (AMPK), and surprisingly, PRAK directly regulates Ras homolog enriched in brain (Rheb), a key component of the mTORC1 pathway by phosphorylation. Phosphorylation of Rheb at serine 130 by PRAK impairs Rheb’s nucleotide-binding ability and inhibits Rheb-mediated mTORC1 activation. The direct regulation of Rheb by PRAK integrates a stress pathway with the mTORC1 pathway in response to energy depletion.
AIM: To investigate the expression of programmed death (PD)-1, PD ligand 1 (PD-L1) and PD-L2 in liver tissues in the context of chronic hepatitis and hepatocellular carcinoma (HCC).
METHODS: Liver biopsies and HCC specimens from patients were collected and histologically examined. The expression of PD-1, PD-L1, and PD-L2 in biopsy specimens of chronic hepatitis and HCC specimens was evaluated by immunohistochemical staining. The association between the expression level of PD-1, PD-L1, and PD-L2 and clinical and pathological variables was analyzed statistically.
RESULTS: Expression of PD-1 was found in liver-infiltrating lymphocytes. In contrast, PD-L1 and PD-L2 were expressed in non-parenchyma liver cells and tumor cells. The expression of PD-L1 was significantly correlated with hepatitis B virus infection (1.42 ± 1.165 vs 0.50 ± 0.756, P = 0.047) and with the stage of HCC (7.50 ± 2.121 vs 1.75 ± 1.500 vs 3.00 ± 0.001, P = 0.018). PD-1 and PD-Ls were significantly up-regulated in HCC specimens (1.40 ± 1.536 vs 5.71 ± 4.051, P = 0.000; 1.05 ± 1.099 vs 4.29 ± 3.885, P = 0.004; 1.80 ± 1.473 vs 3.81 ± 3.400, P = 0.020).
CONCLUSION: PD-L1 may contribute to negative regulation of the immune response in chronic hepatitis B. PD-1 and PD-Ls may play a role in immune evasion of tumors.
Hepatitis B virus; Programmed death-1; Programmed death ligands; Hepatitis; Hepatocellular carcinoma
We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose dependent inhibition on HIV-1 fusion core formation with EC50s of 66–58 nM, with no detectable toxicity. Our findings on effects on HIV-1 integrase are reported separately.
HIV-1; AIDS; natural product; small molecule HIV-1 inhibitors; HIV-1 entry inhibitor; Olive Leaf Extract (OLE); Oleuropein (Ole); Hydroxytyrosol (HT); structure-function; molecular modeling
AIM: To establish a cell model harboring replicative clinical hepatitis B virus (HBV) isolates and evaluate its application in individualized selection of anti-HBV agents for chronic hepatitis B (CHB) patients.
METHODS: The full-length HBV genomic DNA from 8 CHB patients was amplified by polymerase chain reaction (PCR). All the patients were treated with lamivudine for at least seven months and finally became resistant to lamivudine. The amplified HBV DNA fragments were inserted into pHY106 vectors by Sap I digestion. The recombinant plasmids containing 1.1 copies of HBV genome were transiently transfected into Huh7 cell line, and the levels of HBsAg, HBeAg and intercellular HBV replicative intermediates were determined by ELISA and Southern blot analysis, respectively, with or without lamivudine and adefovir treatment. The antiviral treatment with adefovir was administered to the patients and analyzed in parallel.
RESULTS: A total of 25 independent HBV isolates were obtained from the sera of 8 patients, each patient had at least two isolates. One isolate from each individual was selected and subcloned into pHY106 vector, including 5 isolates with YVDD mutation and 3 isolates with YIDD mutation. All recombinant plasmids harboring HBV isolates were transfected into Huh7 cells. The results indicated that HBV genome carried in HBV replicons of clinical HBV isolates could effectively replicate and express in Huh7 cells. Adefovir, but not lamivudine, inhibited HBV replication both in vitro and in vivo, and in vitro inhibition was dose-dependent.
CONCLUSION: The novel method described herein enables individualized selection of anti-HBV agents in clinic and is useful in future studies of antiviral therapy for CHB.
Hepatitis B virus; Chronic hepatitis B; Hepatitis B virus isolate; Antiviral agents
We report molecular modeling and functional confirmation of Ole and HT binding to HIV-1 integrase. Docking simulations identified two binding regions for Ole within the integrase active site. Region I encompasses the conserved D64-D116-E152 motif, while region II involves the flexible loop region formed by amino acid residues 140–149. HT, on the other hand, binds to region II. Both Ole and HT exhibit favorable interactions with important amino acid residues through strong H-bonding and van der Waals contacts, predicting integrase inhibition. To test and confirm modeling predictions, we examined the effect of Ole and HT on HIV-1 integrase activities including 3′-processing, strand transfer and disintegration. Ole and HT exhibit dose-dependent inhibition on all three activities, with EC50s in the nM range. These studies demonstrate that molecular modeling of target–ligand interaction coupled with structural-activity analysis should facilitate the design and identification of innovative integrase inhibitors and other therapeutics.
HIV-1; AIDS; natural product; small molecule HIV-1 inhibitors; HIV-1 integrase inhibitor; Olive Leaf Extract (OLE); Oleuropein (Ole); Hydroxytyrosol (HT); structure-function; molecular modeling
AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo.
METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively.
RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls.
CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.
Cytosine deaminase domain; Apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G; Hepatitis B virus; Antiviral therapy
AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model.
METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively.
RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with in vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups.
CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.
APOBEC3G; Hepatitis B virus; Antiviral therapy