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The article discusses the experiment on the structure-activity relationship of conjugated compounds which were synthesized with imidazole and coumarin derivatives against Hepatitis C virus. Topics mentioned include the results exhibited the values and selective indeces against the virus, the modification of the structure which increased the potency and selectivity, and the provide of the guidelines for development of the compounds as anti-viral agents.

A series of new conjugated compounds with a -SCH2-linkage were synthesized by chemical methods from imidazole and coumarin derivatives. The experimental results indicate that of the twenty newly synthesized imidazole-coumarin conjugates, three of them exhibited appealing EC50 values (5.1-8.4 μM) and selective indices >20 against hepatitis C virus. Their potency and selectivity were increased substantially by modification of their structure with two factors: imidazole nucleus with a hydrogen atom at the N(1) position and coumarin nucleus with a substituent, such as Cl, F, Br, Me, and OMe. These guidelines provide valuable information for further development of conjugated compounds as anti-viral agents. [ABSTRACT FROM AUTHOR]

We previously demonstrated that the epidermal growth factor receptor (EGFR) up-regulated miR-7 to promote tumor growth during lung cancer oncogenesis. Several lines of evidence have suggested that alterations in chromatin remodeling components contribute to cancer initiation and progression. In this study, we identified SMARCD1 (SWI/SNF-related, matrixassociated, actin-dependent regulator of chromatin, subfamily d, member 1) as a novel target gene of miR-7. miR-7 expression reduced SMARCD1 protein expression in lung cancer cell lines. We used luciferase reporters carrying wild type or mutated 3UTR of SMARCD1 and found that miR-7 blocked SMARCD1 expression by binding to two seed regions in the 3UTR of SMARCD1 and down-regulated SMARCD1 mRNA expression. Additionally, upon chemotherapy drug treatment, miR-7 downregulated p53-dependent apoptosis-related gene BAX (BCL2- associatedXprotein) and p21 expression by interfering with the interaction between SMARCD1 and p53, thereby reducing caspase3 cleavage and the downstream apoptosis cascades. We found that although SMARCD1 sensitized lung cancer cells to chemotherapy drug-induced apoptosis, miR-7 enhanced the drug resistance potential of lung cancer cells against chemotherapy drugs. SMARCD1 was down-regulated in patients with non-small cell lung cancer and lung adenocarcinoma cell lines, andSMARCD1and miR-7 expression levels were negatively correlated in clinical samples. Our investigation into the involvement of the EGFR-regulated microRNA pathway in the SWI/ SNF chromatin remodeling complex suggests that EGFRmediated miR-7 suppresses the coupling of the chromatin remodeling factor SMARCD1 with p53, resulting in increased chemo-resistance of lung cancer cells. [ABSTRACT FROM AUTHOR]

Tryptophan metabolism has been recognized as an important mechanism in immune tolerance. Indoleamine 2,3-dioxygenase plays a key role in local tryptophan metabolism via the kynurenine pathway and has emerged as a therapeutic target for cancer immunotherapy. Our prior study identified phenyl benzenesulfonyl hydrazide 2 as a potent in vitro (though not in vivo) inhibitor of indoleamine 2,3-dioxygenase. Further lead optimization to improve in vitro potencies and pharmacokinetic profiles resulted in N'-(4-bromophenyl)-2-oxo-2,3-dihydro-1H-indole-5-sulfonyl hydrazide 40, which demonstrated 59% oral bioavailability and 73% of tumor growth delay without apparent body weight loss in the murine CT26 syngeneic model, after oral administration of 400 mg/kg. Accordingly, 40, is proposed as a potential drug lead worthy of advanced preclinical evaluation. [ABSTRACT FROM AUTHOR]

A structure-basedvirtual screening strategy, comprising homologymodeling, ligand–support binding site optimization, virtualscreening, and structure clustering analysis, was developed and usedto identify novel tryptophan 2,3-dioxygenase (TDO) inhibitors. Compound 1(IC50= 711 nM), selected by virtual screening,showed inhibitory activity toward TDO and was subjected to structuralmodifications and molecular docking studies. This resulted in theidentification of a potent TDO selective inhibitor (11e, IC50= 30 nM), making it a potential compound for furtherinvestigation as a cancer therapeutic and other TDO-related targetedtherapy. [ABSTRACT FROM AUTHOR]

GCM1 (glial cell missing 1) is a short-lived transcription factor essential for placental development. The F-box protein, FBW2 (F-box and WD-repeat domain-containing 2), which contains five WD (tryptophan-aspartate) repeats, recognizes GCM1 and mediates its ubiquitination via the SCFFBW2 E3 ligase complex. Although the interaction between GCM1 and FBW2 is facilitated by GCM1 phosphorylation, it is possible that this interaction might be regulated by additional cellular factors. In the present study, we perform tandem-affinity purification coupled with MS analysis identifying RACK1 (receptor for activated C-kinase 1) as an FBW2-interacting protein. RACK1 is a multifaceted scaffold protein containing seven WD repeats. We demonstrate that the WD repeats in both RACK1 and FBW2 are required for the interaction of RACK1 and FBW2. Furthermore, RACK1 competes with GCM1 for FBW2 and thereby preventsGCM1ubiquitination, which is also supported by the observation that GCM1 is destabilized in RACK1-knockdown BeWo placental cells. Importantly, RACK1 knockdown leads to decreased expression of the GCM1 target gene HTRA4 (hightemperature requirement protein A4), which encodes a serine protease crucial for cell migration and invasion. As a result, migration and invasion activities are down-regulated in RACK1- knockdown BeWo cells. The present study reveals a novel function for RACK1 to regulate GCM1 activity and placental cell migration and invasion. [ABSTRACT FROM AUTHOR]

Autotrophic plants have evolved distinctive mechanisms for maintaining a range of homeostatic states for sugars. The on/off switch of reversible gene expression by sugar starvation/provision represents one of the major mechanisms by which sugar levels are maintained, but the details remain unclear. α-Amylase (αAmy) is the key enzyme for hydrolyzing starch into sugars for plant growth, and it is induced by sugar starvation and repressed by sugar provision. αAmy can also be induced by various other stresses, but the physiological significance is unclear. Here, we reveal that the on/off switch of αAmy expression is regulated by 2 MYB transcription factors competing for the same promoter element. MYBS1 promotes αAmy expression under sugar starvation, whereas MYBS2 represses it. Sugar starvation promotes nuclear import of MYBS1 and nuclear export of MYBS2, whereas sugar provision has the opposite effects. Phosphorylation of MYBS2 at distinct serine residues plays important roles in regulating its sugar-dependent nucleocytoplasmic shuttling and maintenance in cytoplasm by 14-3-3 proteins. Moreover, dehydration, heat, and osmotic stress repress MYBS2 expression, thereby inducing αAmy3. Importantly, activation of αAmy3 and suppression of MYBS2 enhances plant growth, stress tolerance, and total grain weight per plant in rice. Our findings reveal insights into a unique regulatory mechanism for an on/off switch of reversible gene expression in maintaining sugar homeostatic states, which tightly regulates plant growth and development, and also highlight MYBS2 and αAmy3 as potential targets for crop improvement. [ABSTRACT FROM AUTHOR]

The BH3-only pro-apoptotic protein BIK is regulated by the ubiquitin–proteasome system. However, the mechanism of this regulation and its physiological functions remain elusive. Here, we identify Cul5-ASB11 as the E3 ligase targeting BIK for ubiquitination and degradation. ER stress leads to the activation of ASB11 by XBP1s during the adaptive phase of the unfolded protein response, which stimulates BIK ubiquitination, interaction with p97/VCP, and proteolysis. This mechanism of BIK degradation contributes to ER stress adaptation by promoting cell survival. Conversely, genotoxic agents down-regulate this IRE1α–XBP1s–ASB11 axis and stabilize BIK, which contributes in part to the apoptotic response to DNA damage. We show that blockade of this BIK degradation pathway by an IRE1α inhibitor can stabilize a BIK active mutant and increase its antitumor activity. Our study reveals that different cellular stresses regulate BIK ubiquitination by ASB11 in opposing directions, which determines whether or not cells survive, and that blocking BIK degradation has the potential to be used as an anticancer strategy. [ABSTRACT FROM AUTHOR]

Indoleamine 2,3-dioxygenase 1 (IDO1), promoting immune escape of tumors, is a therapeutic target for the cancer immunotherapy. A number of IDO1 inhibitors have been identified, but only limited structural biology studies of IDO1 inhibitors are available to provide insights on the binding mechanism of IDO1. In this study, we present the structure of IDO1 in complex with 24, a NLG919 analogue with potent activity. The complex structure revealed the imidazole nitrogen atom of 24 to coordinate with the heme iron, and the imidazoleisoindole core situated in pocket A with the 1-cyclohexylethanol moiety extended to pocket B to interact with the surrounding residues. Most interestingly, 24 formed an extensive hydrogen bond network with IDO1, which is a distinct feature of IDO1/24 complex structure and is not observed in the other IDO1 complex structures. Further structure-activity relationship, UV spectra, and structural biology studies of several analogues of 24 demonstrated that extensive hydrophobic interactions and the unique hydrogen bonding network contribute to the great potency of imidazoleisoindole derivatives. These results are expected to facilitate the structure-based drug design of new IDO inhibitors. [ABSTRACT FROM AUTHOR]

Growth-associated protein 43 (GAP43) is known to regulate axon growth, but whether it also plays a role in synaptogenesis remains unclear. Here, we found that GAP43 regulates the aggregation of gephyrin, a pivotal protein for clustering postsynaptic GABAA receptors (GABAARs), in developing cortical neurons. Pharmacological blockade of either protein kinase C (PKC) or neuronal activity increased both GAP43-gephyrin association and gephyrin misfolding-induced aggregation, suggesting the importance of PKC-dependent regulation of GABAergic synapses. Furthermore, we found that PKC phosphorylation-resistant GAP43S41A, but not PKC phosphorylation-mimicking GAP43S41D, interacted with cytosolic gephyrin to trigger gephyrin misfolding and its sequestration into aggresomes. In contrast, GAP43S41D, but not GAP43S41A, inhibited the physiological aggregation/clustering of gephyrin, reduced surface GABAARs under physiological conditions, and attenuated gephyrin misfolding under transient oxygen-glucose deprivation (tOGD) that mimics pathological neonatal hypoxia. Calcineurin-mediated GAP43 dephosphorylation that accompanied tOGD also led to GAP43-gephyrin association and gephyrin misfolding. Thus, PKC-dependent phosphorylation of GAP43 plays a critical role in regulating postsynaptic gephyrin aggregation in developing GABAergic synapses. [ABSTRACT FROM AUTHOR]

Acetaminophen (APAP), is a safe analgesic and antipyretic drug at therapeutic dose, and is widely used in the clinic. However, high doses of APAP can induce hepatotoxicity and nephrotoxicity. Most studies have focused on high-dose APAP-induced acute liver and kidney injury. So far, few studies have investigated the effects of the therapeutic dose (1/10 of the high dose) or of the low dose (1/100 of the high dose) of APAP on the cells. The aim of this study was to investigate the cellular effects of therapeutic- or low-dose APAP treatment on hepatoma cells and kidney fibroblasts. As expected, high-dose APAP treatment inhibited while therapeutic and low-dose treatment did not inhibit cell survival of kidney tubular epithelial cells. In addition, therapeutic-dose treatment induced an increase in the H2O2 level, activated the caspase-9/-3 cascade, and induced cell apoptosis of hepatoma cells. Notably, APAP promoted fibroblast proliferation, even at low doses. This study demonstrates that different cellular effects are exerted upon treatment with different APAP concentrations. Our results indicate that treatment with the therapeutic dose of APAP may exert an antitumor activity on hepatoma, while low-dose treatment may be harmful for patients with fibrosis, since it may cause proliferation of fibroblasts. [ABSTRACT FROM AUTHOR]

In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressininduced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin- 2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase. [ABSTRACT FROM AUTHOR]

Background: Mammalian M6A, a member of the proteolipid protein (PLP/DM20) family expressed in neurons, was first isolated by expression cloning with a monoclonal antibody. Overexpression of M6A was shown to induce filopodium formation in neuronal cells; however, the underlying mechanism of is largely unknown. Possibly due to gene duplication, there are two M6A paralogs, M6Aa and M6Ab, in the zebrafish genome. In the present study, we used the zebrafish as a model system to investigate the role of zebrafish M6Ab in filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos. Methodology/Principal Findings: We demonstrated that zebrafish M6Ab promoted extensive filopodium formation in NGF-treated PC12 cells, which is similar to the function of mammalian M6A. Phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Transfection of the S263A mutant protein greatly reduced filopodium formation in PC12 cells. In zebrafish embryos, only S263D could induce neurite outgrowth. Conclusions/Significance: Our results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical for its role in regulating filopodium formation and neurite outgrowth. [ABSTRACT FROM AUTHOR]

The role of general acid–base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr91 and Lys162 residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in kcat values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the kcat values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(kcat) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pKa value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys162 as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr91 residue could form a hydrogen bond with Lys162 to act as a catalytic dyad that contributes a proton to complete the enol–keto tautomerization. [ABSTRACT FROM AUTHOR]