GS-9620 induces phosphorylation of NF-κB and Akt in pDCs.

<p>PBMC isolated from healthy donors were cultured with GS-9620, resiquimod, or DMSO control. Phosphorylation of NF-κB and Akt was assessed in gated pDC and mDC subsets. (a) Flow cytometric histogram of p-NF-κB (using a phosho NF-κB (S529) specific antibody) following 30min stimulation (left panel) or p-Akt (using a phospho Akt (S473) specific antibody) following 60min stimulation (right panel) with 1μM GS-9620 (blue histogram) or DMSO control (grey histogram). (b) Fold increase in mean fluorescence intensity (MFI) of pDCs for pNF-κB (left panel) or p-Akt (right panel) after stimulation with 1μM GS-9620 normalized to DMSO control treatment. Statistically significant differences relative to DMSO control (p<0.05) are observed with GS-9620 stimulation for all assessed time points (p-NF-κB), and for 10, 15, 30, and 60min time points (p-Akt). (c) Fold increase in MFI upon stimulation with 1μM GS-9620 or resiquimod (R848) normalized to DMSO control treatment in mDCs or pDCs. As expected, phospho-responses to GS-9620 and R848 for both readouts, p-NF-κB and p-Akt, were significantly stronger in pDC compared to donor-matched mDC (p<0.01 for all comparisons). Graphs show data obtained from 6 (p-NF-κB) and 4 (p-Akt) independent healthy donors with mean ±SEM (bars).</p

GS-9620 rapidly distributes to and signals through the endo-lysosomal compartments.

<p>(a-b) Chemical structure of (a) GS-9620 and (b) Resiquimod. (c) Kinetics of intracellular accumulation of ³H-GS-9620 in Daudi cells. (d) Sub-cellular distribution of ³H-GS-9620 in relation to major organelle markers obtained by isopycnic density gradient centrifugation. (e) IFN-α2 secretion by enriched pDCs upon stimulation with GS-9620 or resiquimod (control) in the presence of bafilomycin A1 (BAF) or PBS (mock). Data shown in (c), (d), (e) are representative of 3 independent experiments. (e) Error bars represent mean of triplicate assay conditions and SEM. Abbreviations: Early endosome (Early Endo), endoplasmic reticulum (ER), mitochondria (Mt), lysosome (Lyso).</p

Amino acid changes due to described single nucleotide polymorphisms (SNPs) do not impact GS-9620-dependent TLR7 activation.

<p>Fold increase in NF-kB-luciferase reporter activity upon GS-9620 stimulation in Huh7 cells that were transfected with TLR7 or SNPs rs179008 (Q11L), rs55907843 (V222D), or rs5743781 (A448V). Four 2-fold dose titration curves were performed starting at 5µM for GS-9620 (left to right). Bar graphs show fold change in reporter activity relative to DMSO, and error bars represent the mean of triplicate assay conditions and SEM. AUC calculations were performed as discussed for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146835#pone.0146835.g002" target="_blank">Fig 2</a>, above. By the same AUC criteria none of the three assessed TLR7 SNP variants elicited a significantly altered response, relative to TLR7 WT, after GS-9620 stimulation. Representative data are shown from three independent experiments with similar results.</p

Structure-based mutational analysis identifies residues in TLR7 that are essential for GS-9620 <i>in vitro</i> activity.

<p>(a) Three dimensional molecular model of TLR7 endo-lysosomal domain (left) and magnified view of GS-9620 docked in TLR7 (right). (b,c) Fold increase in NF-κB-luciferase reporter activity upon GS-9620, resiquimod or DMSO control stimulation in Huh7 cells that were transfected with control vector, wild-type TLR7 or point mutants of TLR7. Four 2-fold dose titrations (left to right) were performed starting at 5μM for GS-9620, or 10 μM of resiquimod. Bar graphs show fold change of reporter activity relative to DMSO control, and error bars shown represent the mean of triplicate assay conditions and SEM. Representative data shown from three independent experiments with similar results. Area under the curve (AUC) calculations were performed to quantify reporter activity observed with titrated compound concentrations for each of the TLR7 variants. With GS-9620 stimulation the variants Y356S, F408A, D555A, L557D, and L557Y/T586G, and with R848 stimulation the variants Y356S, F408A, and D555A show a 4-fold or greater reduction in reporter activity compared to TLR7 WT, as assessed by AUC calculation. Therefore, these variants are viewed as significantly compromised in response to the respective compound.</p

TLR7 dimers exist independent of GS-9620 binding.

<p>Immunoblot analysis of whole cell lysates of Huh7 cells transfected with V5-tagged TLR7 (TLR7-V5) or HA-tagged TLR7 (TLR7-HA) or HA-tagged TLR9 (TLR9-HA) untreated or treated with increasing amounts of GS-9620 (0.1μM, 1μM and 5μM) for 1 hour before preparation of cell lysates and immunoprecipitation (IP) analysis. Lysates were immunoprecipitated with anti-V5 agarose (panel C,D) and immunoblot probed with anti-V5 mAb (panel A,C) or anti-HA mAb (panel B,D). Total cell lysates used in panels A and B were probed with anti-V5 mAb and anti-HA mAb respectively, to control for protein expression and loading.</p

Molecular Determinants of GS-9620-Dependent TLR7 Activation

<div><p>GS-9620 is an orally administered agonist of Toll-like receptor (TLR)7 currently being evaluated in clinical studies for the treatment of chronic HBV and HIV patients. GS-9620 has shown antiviral efficacy in preclinical models of chronic hepadnavirus infection in woodchuck as well as chimpanzee. However, the molecular determinants of GS-9620-dependent activation of TLR7 are not well defined. The studies presented here elucidate GS-9620 subcellular distribution and characterize its molecular interactions with human TLR7 using structure-guided mutational analysis. Based on our results we present a molecular model of TLR7 bound to GS-9620. We also determine that several coding SNPs had no effect on GS-9620-dependent TLR7 activation. In addition, our studies provide evidence that TLR7 exists in a ligand-independent oligomeric state and that, TLR7 activation by GS-9620 is likely associated with compound-induced conformational changes. Finally, we demonstrate that activation of NF-κB and Akt pathways in primary plasmacytoid dendritic cells occur as immediate downstream cellular responses to GS-9620 stimulation. The data presented here further our understanding of the molecular parameters governing TLR7 activation by GS-9620, and more generally by nucleos/tide-related ligands.</p></div

Synthetic Stimulator of Interferon Genes (STING) Agonists Induce a Cytokine-Mediated Anti-Hepatitis B Virus Response in Nonparenchymal Liver Cells

Chronic hepatitis B (CHB) remains a major public health problem worldwide, with limited treatment options, but inducing an antiviral response by innate immunity activation may provide a therapeutic alternative. We assessed the cytokine-mediated anti-hepatitis B virus (HBV) potential for stimulating the cyclic GMP–AMP synthase–stimulator of interferon genes (STING) pathway using STING agonists in primary human hepatocytes (PHH) and nonparenchymal liver cells (NPCs). The natural STING agonist, 2′,3′-cyclic GMP–AMP, the synthetic analogue 3′,3′-c-di(2′F,2′dAMP), and its bis(pivaloyloxymethyl) prodrug had strong indirect cytokine-mediated anti-HBV effects in PHH regardless of HBV genotype. Furthermore, STING agonists induced anti-HBV cytokine secretion in vitro, in both human and mouse NPCs, and triggered hepatic T cell activation. Cytokine secretion and lymphocyte activation were equally stimulated in NPCs isolated from control and HBV-persistent mice. Therefore, STING agonists modulate immune activation regardless of HBV persistence, paving the way toward a CHB therapy

Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors

<div><p>Influenza polymerase is a heterotrimer composed of polymerase acidic protein A (PA) and basic proteins 1 (PB1) and 2 (PB2). The endonuclease active site, located in the PA subunit, cleaves host mRNA to prime viral mRNA transcription, and is essential for viral replication. To date, the human influenza A endonuclease activity has only been studied on the truncated active-site containing N-terminal domain of PA (PA_N) or full-length PA in the absence of PB1 or PB2. In this study, we characterized the endonuclease activity of recombinant proteins of influenza A/PR8 containing full length PA, PA/PB1 dimer, and PA/PB1/PB2 trimer, observing 8.3-, 265-, and 142-fold higher activity than PA_N, respectively. Using the PA/PB1/PB2 trimer, we developed a robust endonuclease assay with a synthetic fluorogenic RNA substrate. The observed <i>K</i>_m (150 ± 11 nM) and <i>k</i>_<i>cat</i> [(1.4 ± 0.2) x 10^-3s^-1] values were consistent with previous reports using virion-derived replication complex. Two known influenza endonuclease phenylbutanoic acid inhibitors showed IC₅₀ values of 10–20 nM, demonstrating the utility of this system for future high throughput screening.</p></div

Determination of apparent binding affinity (<i>K</i>_app) of GTP, T-1106-TP, and 4’vinyl-FdGTP during single nucleotide incorporation by the recombinant influenza RdRp.

<p>(A) Diagram illustrating formation of RNA 9-mer product from ApG primer and further elongation to RNA 14-mer product; (B) Summary of <i>K</i>_app and selectivity for GTP and analogs; Incorporation of GTP (panel C), T-1106-TP (panel D), and 4’vinyl-FdGTP (panel E) as a function of NTP analog concentration. The product formation is plotted as a function of compound concentration for GTP (panel F), T-1106-TP (panel G), and 4’vinyl-FdGTP (panel H), respectively. The calculated <i>K</i>_<i>app</i> values for GTP, T-1106-TP, and 4’vinyl-FdGTP are 9 nM, 27 nM, and 29 μM respectively.</p

Inhibition of influenza virus and crude replication complex.

<p>Inhibition of influenza virus and crude replication complex.</p