A novel interaction between dengue virus nonstructural protein 1 and the NS4A-2K-4B precursor is required for viral RNA replication but not for formation of the membranous replication organelle

<div><p>Dengue virus (DENV) has emerged as major human pathogen. Despite the serious socio-economic impact of DENV-associated diseases, antiviral therapy is missing. DENV replicates in the cytoplasm of infected cells and induces a membranous replication organelle, formed by invaginations of the endoplasmic reticulum membrane and designated vesicle packets (VPs). Nonstructural protein 1 (NS1) of DENV is a multifunctional protein. It is secreted from cells to counteract antiviral immune responses, but also critically contributes to the severe clinical manifestations of dengue. In addition, NS1 is indispensable for viral RNA replication, but the underlying molecular mechanism remains elusive. In this study, we employed a combination of genetic, biochemical and imaging approaches to dissect the determinants in NS1 contributing to its various functions in the viral replication cycle. Several important observations were made. First, we identified a cluster of amino acid residues in the exposed region of the <i>β-ladder</i> domain of NS1 that are essential for NS1 secretion. Second, we revealed a novel interaction of NS1 with the NS4A-2K-4B cleavage intermediate, but not with mature NS4A or NS4B. This interaction is required for RNA replication, with two residues within the connector region of the NS1 “<i>Wing</i>” domain being crucial for binding of the NS4A-2K-4B precursor. By using a polyprotein expression system allowing the formation of VPs in the absence of viral RNA replication, we show that the NS1 –NS4A-2K-4B interaction is not required for VP formation, arguing that the association between these two proteins plays a more direct role in the RNA amplification process. Third, through analysis of polyproteins containing deletions in NS1, and employing a <i>trans</i>-complementation assay, we show that both <i>cis</i> and <i>trans</i> acting elements within NS1 contribute to VP formation, with the capability of NS1 mutants to form VPs correlating with their capability to support RNA replication. In conclusion, these results reveal a direct role of NS1 in VP formation that is independent from RNA replication, and argue for a critical function of a previously unrecognized NS4A-2K-NS4B precursor specifically interacting with NS1 and promoting viral RNA replication.</p></div

Synthesis and Biological Evaluation of 2‑(Alkoxycarbonyl)-3-Anilinobenzo[<i>b</i>]thiophenes and Thieno[2,3‑<i>b</i>]pyridines as New Potent Anticancer Agents

Two new series of inhibitors of tubulin polymerization based on the 2-(alkoxycarbonyl)-3-(3′,4′,5′-trimethoxyanilino)­benzo­[<i>b</i>]­thiophene and thieno­[2,3-<i>b</i>]­pyridine molecular skeletons were synthesized and evaluated for antiproliferative activity on a panel of cancer cell lines, inhibition of tubulin polymerization, cell cycle effects, and in vivo potency. Antiproliferative activity was strongly dependent on the position of the methyl group on the benzene portion of the benzo­[<i>b</i>]­thiophene nucleus, with the greatest activity observed when the methyl was located at the C-6 position. Also, in the smaller thieno­[2,3-<i>b</i>]­pyridine series, the introduction of the methyl group at the C-6 position resulted in improvement of antiproliferative activity to the nanomolar level. The most active compounds (<b>4i</b> and <b>4n</b>) did not induce cell death in normal human lymphocytes, suggesting that the compounds may be selective against cancer cells. Compound <b>4i</b> significantly inhibited in vivo the growth of a syngeneic hepatocellular carcinoma in Balb/c mice

Synthesis, Antimitotic and Antivascular Activity of 1‑(3′,4′,5′-Trimethoxybenzoyl)-3-arylamino-5-amino-1,2,4-triazoles

A new class of compounds that incorporated the structural motif of the 1-(3′,4′,5′-trimethoxtbenzoyl)-3-arylamino-5-amino-1,2,4-triazole molecular skeleton was synthesized and evaluated for their antiproliferative activity in vitro, interactions with tubulin, and cell cycle effects. The most active agent, <b>3c</b>, was evaluated for antitumor activity in vivo. Structure–activity relationships were elucidated with various substituents on the phenyl ring of the anilino moiety at the C-3 position of the 1,2,4-triazole ring. The best results for inhibition of cancer cell growth were obtained with the <i>p</i>-Me, <i>m</i>,<i>p</i>-diMe, and <i>p</i>-Et phenyl derivatives <b>3c</b>, <b>3e</b>, and <b>3f</b>, respectively, and overall, these compounds were more or less as active as CA-4. Their vascular disrupting activity was evaluated in HUVEC cells, with compound <b>3c</b> showing activity comparable with that of CA-4. Compound <b>3c</b> almost eliminated the growth of syngeneic hepatocellular carcinoma in Balb/c mice, suggesting that <b>3c</b> could be a new antimitotic agent with clinical potential

New Nitrogen Containing Substituents at the Indole-2-carboxamide Yield High Potent and Broad Spectrum Indolylarylsulfone HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors

New indolylarylsulfone (IAS) derivatives bearing nitrogen containing substituents at the indole-2-carboxamide inhibited the HIV-1 WT in MT-4 cells at low nanomolar concentrations. In particular, compound <b>9</b> was uniformly effective against the mutant Y181C, Y188L, and K103N HIV-1 strains; it was highly active against the multidrug resistant mutant IRLL98 HIV-1 strain bearing the K101Q, Y181C, and G190A mutations conferring resistance to NVP, DLV, and EFV and several HIV-1 clades A in PBMC

Potent, Long-Acting Cyclopentane-1,3-Dione Thromboxane (A₂)‑Receptor Antagonists

A series of derivatives of the known thromboxane A₂ prostanoid (TP) receptor antagonists, 3-(6-((4-chlorophenyl)­sulfonamido)-5,6,7,8-tetrahydronaphthalen-1-yl)­propanoic acid and 3-(3-(2-((4-chlorophenyl)­sulfonamido)­ethyl)­phenyl) propanoic acid, were synthesized in which the carboxylic acid functional group was replaced with substituted cyclopentane-1,3-dione (CPD) bioisosteres. Characterization of these molecules led to the discovery of remarkably potent new analogues, some of which were considerably more active than the corresponding parent carboxylic acid compounds. Depending on the choice of the C2 substituent of the CPD unit, these new derivatives can produce either a reversible or an apparent irreversible inhibition of the human TP receptor. Given the potency and the long-lasting inhibition of TP receptor signaling, these novel antagonists may comprise promising leads for the development of antithromboxane therapies

Concise Synthesis and Biological Evaluation of 2‑Aroyl-5-Amino Benzo[<i>b</i>]thiophene Derivatives As a Novel Class of Potent Antimitotic Agents

The biological importance of microtubules make them an interesting target for the synthesis of antitumor agents. The 2-(3′,4′,5′-trimethoxybenzoyl)-5-aminobenzo­[<i>b</i>]­thiophene moiety was identified as a novel scaffold for the preparation of potent inhibitors of microtubule polymerization acting through the colchicine site of tubulin. The position of the methoxy group on the benzo­[<i>b</i>]­thiophene was important for maximal antiproliferative activity. Structure–activity relationship analysis established that the best activities were obtained with amino and methoxy groups placed at the C-5 and C-7 positions, respectively. Compounds <b>3c</b>–<b>e</b> showed more potent inhibition of tubulin polymerization than combretastatin A-4 and strong binding to the colchicine site. These compounds also demonstrated substantial antiproliferative activity, with IC₅₀ values ranging from 2.6 to 18 nM in a variety of cancer cell lines. Importantly, compound <b>3c</b> (50 mg/kg), significantly inhibited the growth of the human osteosarcoma MNNG/HOS xenograft in nude mice

Indolylarylsulfones Carrying a Heterocyclic Tail as Very Potent and Broad Spectrum HIV‑1 Non-nucleoside Reverse Transcriptase Inhibitors

We synthesized new indolylarylsulfone (IAS) derivatives carrying a heterocyclic tail at the indole-2-carboxamide nitrogen as potential anti-HIV/AIDS agents. Several new IASs yielded EC₅₀ values <1.0 nM against HIV-1 WT and mutant strains in MT-4 cells. The (<i>R</i>)-<b>11</b> enantiomer proved to be exceptionally potent against the whole viral panel; in the reverse transcriptase (RT) screening assay, it was remarkably superior to NVP and EFV and comparable to ETV. The binding poses were consistent with the one previously described for the IAS non-nucleoside reverse transcriptase inhibitors. Docking studies showed that the methyl group of (<i>R</i>)-<b>11</b> points toward the cleft created by the K103N mutation, different from the corresponding group of (<i>S</i>)-<b>11</b>. By calculating the solvent-accessible surface, we observed that the exposed area of RT in complex with (<i>S</i>)-<b>11</b> was larger than the area of the (<i>R</i>)-<b>11</b> complex. Compounds <b>6</b> and <b>16</b> and enantiomer (<i>R</i>)-<b>11</b> represent novel robust lead compounds of the IAS class

Toward Highly Potent Cancer Agents by Modulating the C‑2 Group of the Arylthioindole Class of Tubulin Polymerization Inhibitors

New arylthioindole derivatives having different cyclic substituents at position 2 of the indole were synthesized as anticancer agents. Several compounds inhibited tubulin polymerization at submicromolar concentration and inhibited cell growth at low nanomolar concentrations. Compounds <b>18</b> and <b>57</b> were superior to the previously synthesized <b>5</b>. Compound <b>18</b> was exceptionally potent as an inhibitor of cell growth: it showed IC₅₀ = 1.0 nM in MCF-7 cells, and it was uniformly active in the whole panel of cancer cells and superior to colchicine and combretastatin A-4. Compounds <b>18</b>, <b>20</b>, <b>55</b>, and <b>57</b> were notably more potent than vinorelbine, vinblastine, and paclitaxel in the NCI/ADR-RES and Messa/Dx5 cell lines, which overexpress P-glycoprotein. Compounds <b>18</b> and <b>57</b> showed initial vascular disrupting effects in a tumor model of liver rhabdomyosarcomas at 15 mg/kg intravenous dosage. Derivative <b>18</b> showed water solubility and higher metabolic stability than <b>5</b> in human liver microsomes