9-Arylpurines as a Novel Class of Enterovirus Inhibitors

Here we report on a novel class of enterovirus inhibitors that can be structurally described as 9-arylpurines. These compounds elicit activity against a variety of enteroviruses in the low μM range including Coxsackie virus A16, A21, A24, Coxsackie virus B3, and echovirus 9. Structure−activity relationship (SAR) studies indicate that a chlorine or bromine atom is required at position 6 of the purine ring for antiviral activity. The most selective compounds in this series inhibited Coxsackie virus B3 replication in a dose-dependent manner with EC50 values around 5−8 μM. No toxicity on different cell lines was observed at concentrations up to 250 μM. Moreover, no cross-resistance to TBZE-029 and TTP-8307 CVB3 resistant strains was detected

Intramolecular Cation−π Interactions As the Driving Force To Restrict the Conformation of Certain Nucleosides

Despite the well-established importance of intermolecular cation−π interactions in molecular recognition, intramolecular cation−π interactions have been less studied. Here we describe how the simultaneous presence of an aromatic ring at the 5′-position of an inosine derivative and a positively charged imidazolium ring in the purine base drive the conformation of the nucleoside toward a very major conformer in solution that is stabilized by an intramolecular cation−π interaction. Therefore, the cation−π interaction between imidazolium ions and aromatic rings can also be proposed in the design of small molecules where this type of interaction is desirable. The imidazolium ion can be obtained by a simple acidification of the pH of the media. So a simple change in pH can shift the conformational equilibrium from a random to a restricted conformation stabilized by an intramolecular cation−π interaction. Thus the here described nucleosides can be considered as a new class of pH-dependent conformationally switchable molecules

Targeting HIV Entry through Interaction with Envelope Glycoprotein 120 (gp120): Synthesis and Antiviral Evaluation of 1,3,5-Triazines with Aromatic Amino Acids

On the basis of the interesting inhibitory properties that lectins show against HIV-replication through their interaction with glycoprotein 120 (gp120), we here describe the design, synthesis, and anti-HIV evaluation of three series of 1,3,5-triazine derivatives (monomers, dimers, and trimers) functionalized with aromatic amino acids meant to mimic interactions that lectins establish with gp120. While monomers were inactive against HIV replication, dimers showed limited anti-HIV activity that is, however, considerably more significant in the trimers series, with EC50 values in the lower μM range. These findings most likely reflect the requirement of multivalency of the 1,3,5-triazine derivatives to display anti-HIV activity, as lectins do. The pronounced anti-HIV activity (EC50 ∼ 20 μM) is accompanied by the absence of toxicity in CEM T-cell line (CC50 > 250 μM). Moreover, SPR experiments revealed that the prototype trimers with a central core of 2,4,6-triethylbenzene and six l-Trp or six l-Tyr residues at the periphery were efficient binders of CXCR4- and CCR5-tropic HIV-1 gp120 (estimated KD: lower micromolar range). The collected data support the interest of this novel family of anti-HIV agents and qualify them as potential novel microbicide lead compounds

GSH depletion interferes with the interaction of VP1 and 2C.

<p>BGM cells were infected with wt or T₇₇M CVB3 at a MOI of 10, in the absence or the presence of 50 µM TP219. Cells were fixed with saponin 0.5% at 5 h p.i. and costained with antibodies targeting 2C (and Alexa Fluor 568-conjugated secondary antibody (red color)) and VP1 (and Alexa Fluor 488-conjugated secondary antibody (green color)). Regions of colocalization are indicated by arrows.</p

Antiviral activity of TP219 against a selected panel of enteroviruses.

<p>Data are mean values ± SD from at least three independent experiments. The cytotoxicity values (CC₅₀) were for all cell lines >100 µM.</p>a<p>50% effective concentration.</p

Resistance to GSH depletion maps to the interface between 2 protomers.

<p>(<b>A</b>) Surface rendered view of a single pentameric subunit (colored) in a whole capsid of CVB3 strain M (PDB 1COV). The remaining surface of the virion is colored grey. Viral proteins within a single pentameric unit are colored denoting VP1 (yellow), VP2 (green) and VP3 (aquamarine). A pentameric unit showing the location of the different mutations conferring GSH-insensitivity identified from the revertant screen (red) at the interface of 2 protomers. One protomer within the pentameric structure is outlined and an enlarged view on the antiparallel β-sheets in VP1 (colored yellow) and the location of the identified amino acids (colored red) on the interface between two protomers is provided as well. Comparison of pentamers of various GSH-insensitive (<b>B</b>) [echo11 (1h8t) and EV71 (4aed)] and GSH-sensitive (<b>C</b>) [CVB3 (1cov), echo 1 (1ev1), CVA21 (1z7s) and RV14 (1ro8)] enteroviruses. Surface representation of different protomers within a pentamer. Surface exposed methionines are indicated in red. An enlarged view on the location of the identified surface exposed methionines (colored red) on the interface between two protomers is also provided. Molecular graphics and analyses were performed with the UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004039#ppat.1004039-Pettersen1" target="_blank">[58]</a>.</p

Resistance pattern of recombinant CVB3 viruses carrying mutations in VP1 and VP3, either alone or combined.

<p><i>Recombinant viruses were evaluated for inhibition by TP219, enviroxime, TBZE-029 and BSO</i>.</p><p><i>Data represent EC₅₀ values [µM] ± SD</i>.</p><p><i>ND, not determined</i>.</p

Growth phenotype of TP219-resistant CVB3 recombinants.

<p>The genomic organization of the structural proteins of CVB3 recombinants are illustrated on the left. Mutations in the genomic structure of P1 are shown by black rectangles. Virus infectious virus yields were calculated by endpoint titration and expressed as the tissue culture 50% infectious dose per ml (Log₁₀ TCID₅₀/ml). The plaque phenotype was determined using a plaque assay.</p

Direct interaction between VP1 and GSH.

<p>(<b>A</b>) Heat inactivation of 3×10⁷ TCID₅₀/ml of wt, T₇₇M and T₇₇M/A₁₈₀T CVB3 in the absence or presence of various concentrations of GSH. Following 30 minutes incubation at 46°C infectious virus titers were determined and expressed as Log₁₀ TCID₅₀/ml. (<b>B</b>) GSH pull down assay using glutathione-sepharose beads of CVB3-infected BGM cells. Labeling was done with Methionine ^3S[S] and the lysate was analyzed by SDS-PAGE for the presence of viral proteins (input, lane 1). The lysate was loaded onto glutathione-sepharose beads and analyzed by SDS-PAGE (GSH pull down, lane 2). Lane 2′ shows a shorter exposure time of lane 2. (<b>C</b>) GSH pull down of PV Mahoney or PV Sabin1 infected Vero cells that were treated with TP219 or left untreated. Lysates were analyzed by SDS-PAGE (input, lanes 1–4) and loaded onto GSH Sepharose beads after which the pulled down material was analyzed by SDS-PAGE (pull down, lanes 5–8).</p

TP219 inhibits virus morphogenesis.

<p>(<b>A</b>) Structural formula of TP219. (<b>B</b>) Effect of TP219 on cell viability and CVB3-induced cytopathic effect in BGM cells. Toxicity (black circles) and CPE (white squares) was quantified by MTS assay at 3 d p.i. and expressed as percentage of untreated controls. Data are average values ± SD. (<b>C</b>) Analysis of the effect of TP219 on RNA replication and infectious virus titers. BGM cells were infected with RLuc-CVB3. The indicated compounds were added immediately after infection at the indicated concentrations. The enterovirus inhibitors GuaHCl and geldanamycin were included as controls. At 8 h p.i. intracellular viral RNA replication in the absence or presence of the indicated molecules was quantified by measuring luciferase activity (<b>C, left panel</b>). Lysates were used to determine infectious virus yields calculated by endpoint titration and expressed as the tissue culture 50% infectious dose per ml (TCID₅₀) (<b>C, right panel</b>). Experiments were performed in triplicate and mean values ± SD are depicted. (<b>D</b>) Effect of TP219 on polyprotein processing. Cells were infected with CVB3 at MOI 50 and pulse-labeled with Methionine ^3S[S] in the absence or presence of TP219. Subsequently, proteins were analyzed by SDS-PAGE.</p