HIV‑1 X4 Activities of Polycationic “Viologen” Based Dendrimers by Interaction with the Chemokine Receptor CXCR4: Study of Structure–Activity Relationship

A series of “viologen” based dendrimers with polycationic scaffold carrying 10, 18, 26, 42, and 90 charges per molecule were used to determine the structure–activity relationship (SAR) with regard to HIV-1 inhibitory activity. The studies involved five compounds with a high activity against HIV-1 already utilized in our previous study and five new dendrimers. Such dendrimers block HIV-1 entry into the cell, indicating that they bind to HIV-1 surface proteins and/or on the host cell receptors required for entry. The increasing positive character of dendrimers leads to more cytotoxicity. The 10 charges dendrimers (<b>1</b>, <b>6</b>) have less influence on the cell viability but low inhibition of the binding of the CXCR4 mAb clone 1D9. Thus, dendrimers with 18 charges (<b>2</b>, <b>7</b>) are the most promising CXCR4 imaging probes. We report the design, synthesis, and biological activity of new HIV-1 inhibitors that are conceptually distinct from those of the existing HIV-1 inhibitors

Nucleoside Diphosphate Prodrugs: Nonsymmetric Di<i>PP</i>ro-Nucleotides

Nonsymmetric Di<i>PP</i>ro-nucleotides are described as nucleoside diphosphate (NDP) delivery systems. The concept is to attach different bis­(acyloxybenzyl) moieties at the β-phosphate moiety of a NDP. Di<i>PP</i>ro compounds bearing two alkanoylbenzyl residues and Di<i>PP</i>ro compounds bearing an alkanoylbenzyl or a benzoylbenzyl group as bioreversible prodrug moieties were studied. Compounds bearing short chain alkanoyl esters led to a fast hydrolysis by chemical or enzymatic means. The ester group in the second prodrug group comprised a long lipophilic aliphatic or an aromatic residue. The lipophilicity of this group enabled the prodrug to penetrate the cell membrane. The introduction of two different groups allowed a controlled stepwise removal of the prodrug moieties to achieve a highly selective delivery of the NDP in CEM cell extracts. The compounds were highly active against HIV even in thymidine kinase-deficient CEM cells. Thus, the compounds, although charged at the α-phosphate group, were taken up by the cells and released NDPs

Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors

<div><p>The chemokine receptor CXCR4 is activated by its unique chemokine ligand CXCL12 and regulates many physiological and developmental processes such as hematopoietic cell trafficking. CXCR4 is also one of the main co-receptors for human immunodeficiency virus (HIV) entry. Dysfunction of the CXCL12/CXCR4 axis contributes to several human pathologies, including cancer and inflammatory diseases. Consequently, inhibition of CXCR4 activation is recognized as an attractive target for therapeutic intervention. In this regard, numerous agents modifying CXCR4 activity have been evaluated in <i>in vitro</i> experimental studies and pre-clinical models. Here, we evaluated a CXCL12 competition binding assay for its potential as a valuable initial screen for functional and competitive CXCR4 inhibitors. In total, 11 structurally diverse compounds were included in a side-by-side comparison of <i>in vitro</i> CXCR4 cell-based assays, such as CXCL12 competition binding, CXCL12-induced calcium signaling, CXCR4 internalization, CXCL12-guided cell migration and CXCR4-specific HIV-1 replication experiments. Our data indicated that agents that inhibit CXCL12 binding, <i>i</i>.<i>e</i>. the anti-CXCR4 peptide analogs T22, T140 and TC14012 and the small molecule antagonists AMD3100, AMD3465, AMD11070 and IT1t showed inhibitory activity with consistent relative potencies in all further applied CXCR4-related assays. Accordingly, agents exerting no or very weak receptor binding (i.e., CTCE-9908, WZ811, Me6TREN and gambogic acid) showed no or very poor anti-CXCR4 inhibitory activity. Thus, CXCL12 competition binding studies were proven to be highly valuable as an initial screening assay and indicative for the pharmacological and functional profile of competitive CXCR4 antagonists, which will help the design of new potent CXCR4 inhibitors.</p></div

Antiviral activity of heparin and heparan sulfate analogs against DENV-2 in HMEC-1 cells.

<p>EC₅₀: effective concentration or drug concentration required to inhibit DENV-2 infection in HMEC-1 cells by 50% as measured by flow cytometry. The means ± standard deviations for three independent experiments are shown.</p

Anti-DENV-2 activity of sulfated GAGs.

<p>HMEC-1 cells were infected with DENV-2 at a MOI 1. Viral infectivity was quantified 24 h after infection by flow cytometry using an anti-DENV-2 specific antibody. Heparin (A), heparan sulfate (B), chondroitin sulfate A (C) and dermatan sulfate (D) dose-dependently inhibited DENV-2 infection in HMEC-1 cells. Data represent the % of infected cells relative to the positive control (DENV-2 infected cells). The means and standard deviations of three independent experiments are shown.</p

Expression of surface molecules on HMEC-1 and HMVEC-d cells as determined by flow cytometry.

<p>HMEC-1 and HMVEC-d cells were stained with antibodies against different DENV receptors or endothelial cell markers. The mean fluorescence intensities for DC-SIGN, L-SIGN, mannose receptor (MR), heparan sulfate (HS), vascular endothelial growth factor receptor (VEGFR)-2 and CD31 (PECAM-1) are shown (after background reduction in the presence of labeled secondary antibody only or corresponding FITC-/PE-conjugated isotypic control antibody).</p