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

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

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

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

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

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

C17 protein is monomeric.

<p>(A) Recombinant purified human C17-V5H8 was analyzed by SDS-PAGE under reducing (1) and non-reducing (2) loading conditions and subsequent Coomassi staining. A protein marker is shown in the right column (3). (B) Baseline drift-corrected A280 nm elution profile of size exclusion chromatography after loading affinity-purified human C17-V5H8. Retention times of marker proteins (in kDa) are indicated above.</p

Sustained over-expression of C17 does not result in an inflammatory response <i>in vivo</i>.

<p>Age-and sex-matched mice received a hydrodynamic injection of C17 or GFP (control) minicircle, or were kept naïve. (A) After five weeks, animals were euthanized and hepatic over-expression of C17 mRNA was verified. In addition, (B) hepatic expression of C17 protein was confirmed by IHC using an anti-C17 mAb (top row) or isotype control Ab (bottom row); arrowheads highlight areas of positive C17 staining in C17-transfected, but not naïve or GFP-transfected liver; images were taken at 20× magnification. (C) Systemic exposure to C17 was verified by measuring serum levels of tagged C17 protein. Data shown represent a typical result from at least two independent experiments with five or more animals per group.</p

Reduced expression of inflammatory markers and genes associated with joint destruction in paws of C17-treated animals.

<p>One hind paw per animal was harvested at the time of euthanasia and quantitative RT-PCR was performed to measure mRNA levels of cytokines associated with joint inflammation (A), and genes associated with joint remodeling and arthritic tissue destruction (B). (C) mRNA expression of selected genes from paws with severe swelling and matched clinical disease score 3. (D) C17 expression in paws from naïve animals and animals that received arthrogen with GFP control or C17 minicircle. Values from naïve animals are shown as filled triangles, from GFP mice as filled squares, from C17 mice as open circles, throughout the figure. Not significant: ns; p<0.05: *; p<0.01: **. Similar data were obtained in at least two independent experiments with five or more mice per group.</p

C17 mRNA and protein are expressed in cartilage-rich tissues and chondrocytes, respectively.

<p>(A) A variety of tissues were harvested and pooled from three C57BL/6 mice and then analyzed for C17 mRNA expression using qPCR (n.d., not detected). (B) Immunohistochemical staining for C17 on mouse sternal cartilage, using a mAb anti-C17, demonstrates protein expression by chondrocytes. Scale bars represent 0.1 mm.</p