Gardiquimod™

TLR7 Agonist - Imidazoquinoline compound

Activation of TLR7 by Gardiquimod™

Gardiquimod™ is an imidazoquinoline compound developed and manufactured by InvivoGen. Gardiquimod™ is a specific agonist for human and mouse Toll-like receptor 7 (TLR7). TLR7 and TLR8 are endosomal pattern recognition receptors that play an important role in the antiviral immune response [1].

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Mode of action

Using our HEK-Blue™ reporter cell lines expressing human or mouse TLR7 or TLR8, we established that Gardiquimod™ is a human and mouse TLR7-specific agonist. Similar to Imiquimod, Gardiquimod™ induces the activation of NF-κB in HEK293 cells expressing human or mouse TLR7 (see figure). It is more potent than Imiquimod. Gardiquimod™ is a TLR7-specific ligand that does not activate TLR8; however, at high concentrations (>10 µg/ml), it might activate human, but not mouse TLR8.

Moreover, Gardiquimod™ is able to activate hTLR7-dependent NF-κB and IRF pathways, as assessed using our monocytic THP1-Dual™ reporter cell lines expressing two reporter genes, for the NF-κB-inducible SEAP and IRF-inducible Lucia luciferase, as well as hTLR7 or hTLR8 (see figure). 

Key features of Gardiquimod™

• Specific activator of human and mouse TLR7
• Does not activate TLR8
• Each lot of Gardiquimod™ is highly pure (≥95%) and functionally tested

References:

1. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31.

Figures

NF-κB response of HEK-Blue™-derived cells to Gardiquimod™. HEK-Blue™ cells expressing human or mouse TLR7 or TLR8 were cultured in HEK-Blue™ Detection reagent and stimulated with increasing concentrations of Gardiquimod™. After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Of note, HEK-Blue™ Null* comprises data from the parental cell lines HEK-Blue Null1, HEK-Blue Null1-v, and HEK-Blue Null2-k.

NF-κB and IRF responses of THP1-Dual™-derived cells to Gardiquimod™. THP1-Dual™, THP1-Dual™ hTLR7, and THP1-Dual™ hTLR8 cells were incubated for 24 hours with increasing concentrations of Gardiquimod™. After 24h incubation, the (A) NF-κB-induced SEAP activity was assessed using QUANTI-Blue™. Data are shown as optical density (OD) at 650 nm (mean ± SEM). (B) The IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown in fold response over non-induced cells (mean ± SEM).

Specifications

Specificity: human/mouse TLR7 agonist

Working concentration: 0.1 - 3 µg/ml

CAS number: 1020412-43-4 (free base)

Solubility: 1 mg/ml in water

Quality control:

• Purity: ≥95% (UHPLC)
• The biological activity of Gardiquimod™ has been validated using cellular assays.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ hTLR2 and HEK-Blue™ hTLR4 cells.

Contents

Gardiquimod™ is available in two quantities:

Catalog code: tlrl-gdqs-1

• 2 x 500 µg Gardiquimod™
• 1.5 ml of sterile endotoxin-free water

Catalog code: tlrl-gdq-10

• 2 x 5 mg Gardiquimod™
• 10 ml of sterile endotoxin-free water

Gardiquimod™ is shipped at room temperature.

 Upon receipt, store at -20°C.

Details

TLR7 and TLR8

TLR7 and TLR8 are endosomal pattern recognition receptors that share structural homology [1]. Both receptors are activated by single-stranded RNA (ssRNA) molecules, however, they exhibit different ligand-binding specificities and cellular expression patterns suggesting that they have nonredundant specialized roles.

TLR7 is essentially expressed by plasmacytoid dendritic cells (pDCs) but is also found in B cells and other myeloid cells [2] while TLR8 is highly expressed by myeloid cells and is absent from pDCs and B cells [2]. 

The endosomal distribution of TLR7 and TLR8 allows them to scan for the presence of microbial RNA in the phagocytic cargo. Their activation leads to NF-κB-, AP1-, and interferon regulatory factor (IRF)-mediated production of type I interferons (IFN-α/β) and pro-inflammatory cytokines [2].

Structural analyses have revealed that both TLR7 and TLR8 possess two binding sites (designated as Site 1 and Site 2) which do not share the same specificities.

Site 1 is highly conserved between TLR7 and TLR8 and binds nucleosides (guanosine (G) for TLR7 and uridine (U) for TLR8) or base analogs. The ligand preference for TLR7 and TLR8 is thus explained by the presence of specific residues in Site 1. Site 1 occupancy allows receptor dimerization and signaling.

Site 2 is less conserved and binds ssRNA with U(U) and U(G) motifs, respectively [3, 4]. Of note, ssRNA-binding to Site 2 is not sufficient for the formation of a signaling-competent TLR dimer but it strongly enhances the binding affinity of Site 1 [3, 4]. Thus, TLR7 and TLR8 appear to sense distinct RNA-degradation products rather than full-length ssRNAs [4].

1. Chuang T.H. & Ulevitch R.J., 2000. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8, and hTLR9. Eur Cytokine Netw, 11:372-8.
2. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31.
3. Zhang Z. et al., 2018. Structural analyses of Toll-like receptor 7 reveal detailed RNA sequence specificity and recognition mechanism of agonistic ligands. Cell Rep. 25:3371.
4. Tanji H. et al., 2015. Toll-like receptor 8 senses degradation products of single-stranded RNA. Nat. Struct. Mol. Biol. 22:109.

Chemical structure of Gardiquimod™