Human Toll-like receptor 8 (TLR8) (IC50: picomolar range) [1]

CU-CPT9b is a selective TLR8 antagonist, with an IC50 of 0.7±0.2 nM. ITC studies have demonstrated the robust binding of CU-CPT9b with a Kd of 21 nM. It is proven that CU-CPT-9b attaches to the inactive TLR8 dimer in a similar way to CU-CPT8m. CU-CPT9b exploits hydrogen bonds with G351 and V520*, which are conserved among TLR8/antagonist structures. Additionally, CU-CPT9b develops water-mediated contacts with S516* and Q519*, which are not detected in TLR8/CU-CPT8m structure, suggesting that the higher potency of CU-CPT9b arises from the novel interactions with these polar residues. The orientation of Y567* also alters to enhance van der Waals interactions with CU-CPT9b as compared to TLR8 /CU-CPT8m[1].

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1. Isothermal Titration Calorimetry (ITC) experiments: When CU-CPT-9b was pre-incubated with purified TLR8 protein, TLR8 agonists (e.g., R848) failed to bind to TLR8, confirming that CU-CPT-9b competitively blocks the interaction between TLR8 and its agonists [1]
2. Proinflammatory cytokine inhibition: In cultured cell lines (e.g., HEK-Blue 293 cells overexpressing TLR8), human peripheral blood mononuclear cells (PBMCs), and splenocytes, treatment with CU-CPT-9b significantly reduced the expression and secretion of TLR8 agonist-induced proinflammatory cytokines, including TNF-α and IL-8. The reduction was concentration-dependent and consistent across different cell types [1]
3. NF-κB signaling pathway suppression: In NF-κB reporter gene assays, CU-CPT-9b dose-dependently inhibited R848-induced activation of the NF-κB pathway (a key downstream signaling cascade of TLR8). This was evidenced by decreased activity of the NF-κB-responsive reporter gene (e.g., secreted alkaline phosphatase or luciferase) [1]
4. Structural and binding mechanism: X-ray crystallographic analysis revealed that CU-CPT-9b binds to a unique pocket at the protein-protein interface of the TLR8 homodimer. This pocket is formed by leucine-rich repeats (LRR) 11–13 of one protomer and LRR 15–16 of the other protomer. CU-CPT-9b forms multiple interactions with surrounding residues: hydrogen bonds with Gln 519 and Val 520, stacking interactions with Tyr 348 and Phe 495, and hydrophobic interactions with Phe 261, Phe 346, Val 378, Ile 403, Phe 405, Phe 494, Ala 518, and Tyr 567. These interactions stabilize TLR8 in its resting (inactivated) dimeric state, preventing agonist-induced conformational changes required for TLR8 activation [1]

1. Efficacy in TLR8-transgenic mice: Splenocytes isolated from human TLR8-transgenic mice were treated with CU-CPT-9b followed by stimulation with a TLR8 agonist. CU-CPT-9b significantly reduced the production of proinflammatory cytokines (e.g., TNF-α, IL-8) in these splenocytes, consistent with in vitro results. This indicated that CU-CPT-9b retains TLR8 inhibitory activity in a physiological context relevant to human TLR8 function [1] 2. Therapeutic potential in autoimmune disease models: Although direct in vivo efficacy in autoimmune disease models was not fully detailed, the high efficacy of CU-CPT-9b in TLR8-transgenic mouse splenocytes and human primary cells suggested its potential for treating autoimmune disorders associated with excessive TLR8 activation (e.g., rheumatoid arthritis) [1]

1. TLR8 protein preparation: Recombinant human TLR8 protein was expressed in a suitable expression system (e.g., insect cells or mammalian cells) and purified using standard biochemical techniques (e.g., affinity chromatography) to obtain active, soluble TLR8 protein [1]
2. ITC reaction setup: Purified TLR8 protein was diluted in a buffer matching the experimental conditions. A solution of CU-CPT-9b was prepared at an appropriate concentration and mixed with the TLR8 protein. The mixture was incubated at a constant temperature (e.g., 25°C) to allow sufficient binding between CU-CPT-9b and TLR8 [1]
3. Agonist addition and data collection: After incubation, a TLR8 agonist (e.g., R848) was titrated into the TLR8-CU-CPT-9b mixture using an ITC instrument. The heat changes associated with molecular interactions were recorded in real time. A lack of heat signals corresponding to agonist-TLR8 binding indicated that CU-CPT-9b blocked agonist access to TLR8 [1]
4. Data analysis: The ITC data were processed to calculate binding parameters (e.g., binding affinity). The absence of detectable agonist-TLR8 binding confirmed the antagonistic activity of CU-CPT-9b [1]

1. Proinflammatory cytokine detection assay:
- Cell seeding: Target cells (e.g., HEK-Blue 293/TLR8 cells, human PBMCs, or splenocytes) were seeded into 96-well or 24-well culture plates at a density of 1×10⁵–5×10⁵ cells/well and incubated overnight in a 37°C, 5% CO₂ incubator to allow cell adhesion or adaptation [1]
- Drug pretreatment: CU-CPT-9b was serially diluted in culture medium to generate multiple concentrations. The cells were treated with each concentration of CU-CPT-9b and incubated for 1–2 hours at 37°C [1]
- Agonist stimulation: After pretreatment, a TLR8 agonist (e.g., R848) was added to the cells at a final concentration that induces maximal proinflammatory response. The cells were further incubated for 24–48 hours [1]
- Cytokine measurement: The cell culture supernatant was collected by centrifugation. The levels of proinflammatory cytokines (TNF-α, IL-8) were measured using enzyme-linked immunosorbent assay (ELISA) kits. For mRNA expression analysis, total RNA was extracted from cells, reverse-transcribed into cDNA, and quantified by quantitative real-time polymerase chain reaction (qPCR) [1]
2. NF-κB reporter gene assay:
- Cell preparation: HEK-Blue 293 cells stably transfected with both human TLR8 and an NF-κB-responsive reporter gene (e.g., secreted alkaline phosphatase) were seeded into 96-well plates at 2×10⁴ cells/well and cultured overnight [1]
- Drug and agonist treatment: CU-CPT-9b (serial concentrations) was added to the cells for 1 hour of pretreatment, followed by addition of R848. The cells were incubated for 16–24 hours [1]
- Reporter activity detection: The culture supernatant was mixed with a reporter substrate (e.g., p-nitrophenyl phosphate for alkaline phosphatase). The absorbance (at 405 nm) or luminescence (for luciferase reporter) was measured using a microplate reader. The percentage of NF-κB activation inhibition was calculated relative to the agonist-only control group [1]

[1]. Small-molecule inhibition of TLR8 through stabilization of its resting state. Nat Chem Biol. 2018 Jan;14(1):58-64.

1. Background: Toll-like receptor 8 (TLR8) is an endogenous immune receptor that recognizes single-stranded RNA. TLR8 overactivation is associated with the pathogenesis of autoimmune diseases such as rheumatoid arthritis, but selective small molecule TLR8 antagonists were scarce before the discovery of CU-CPT-9b. [1] 2. Design principle: CU-CPT-9b was developed based on rational structural design to enhance its binding affinity to TLR8. Compared with other CU-CPT derivatives, CU-CPT-9b is genetically engineered to form more hydrogen bonds with residues in the TLR8 binding pocket, thereby enhancing its potency [1]. 3. Selectivity and tool value: CU-CPT-9b is a highly selective TLR8 antagonist (inactive against other TLRs, such as TLR3, TLR7 and TLR9) and is an important tool for studying the TLR8 signaling pathway in vitro and in TLR8 transgenic models [1]. 4. Therapeutic potential: Due to its strong TLR8 inhibitory activity and efficacy in human primary cells and TLR8 transgenic mouse cells, CU-CPT-9b has good therapeutic potential in treating autoimmune diseases driven by TLR8 overactivation [1].

C16H13NO2

251.279924154282

251.094

2162962-69-6

135567366

White to yellow solid powder

3.5

2

3

1

19

309

0

OC1C=CC(=CC=1C)C1C=CN=C2C=C(C=CC=12)O

QXFYDRYRLOHSBD-UHFFFAOYSA-N

InChI=1S/C16H13NO2/c1-10-8-11(2-5-16(10)19)13-6-7-17-15-9-12(18)3-4-14(13)15/h2-9,18-19H,1H3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.28 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.28 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.28 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)