## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: EC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: HEK-Blue-hTLR8 Cells Assay: A stable HEK-Blue-hTLR8 cell line was purchased from InvivoGen (Cat. #: HEK-Blue-htlr8, San Diego, Calif., USA). These cells were designed for studying the stimulation of human TLR8 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN-3 minimal promoter fused to five NF-κB and AP-1-binding sites. The SEAP was induced by activating NF-κB and AP-1 via stimulating HEK-Blue-hTLR8 cells with TLR8 ligands. Therefore the reporter expression was regulated by the NF-κB promoter upon stimulation of human TLR8 for 20 hrs. The cell culture supernatant SEAP reporter activity was determined using QUANTI-Blue kit (Cat. #: rep-qb 1, Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple or blue in the presence of alkaline phosphatase.HEK-Blue-hTLR8 cells were incubated at a density of 250,000-450,000 cells/mL in a volume of 180 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50 mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (V/V) heat-inactivated fetal bovine serum for 24 hrs. Then the HEK-Blue-hTLR8 cells were incubated with addition of 20 μL test compound in a serial dilution in the presence of final DMSO at 1% and perform incubation under 37° C. in a CO2 incubator for 20 hrs. Then 20 μL of the supernatant from each well was incubated with 180 μL Quanti-blue substrate solution at 37° C. for 2 hours and the absorbance was read at 620-655 nm using a spectrophotometer. The signalling pathway that TLR8 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was also widely used for evaluating TLR8 agonist (Tsuneyasu Kaisho and Takashi Tanaka, Trends in Immunology, Volume 29, Issue 7, July 2008, Pages 329.sci; Hiroaki Hemmi et al, Nature Immunology 3, 196-200 (2002)).
- Assay organism: Homo sapiens
- Target name: Toll-like receptor 8
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: CCS(=N)(=O)c1nc(N)c2[nH]c(=O)n(Cc3ccc(Cl)cc3)c2n1
- Properties:
- ChEMBL ID: CHEMBL5983543
- Molecular weight: 366.83
- Heavy atoms: 24
- Hydrogen bond donors: 3
- Hydrogen bond acceptors: 6
- TPSA: 130.51
- LogP: 1.83
- Rotatable bonds: 4
- Ring count: 3
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.21
- QED: 0.61

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 4.05
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type EC50; relation =; value 90000; units nM; pChEMBL 4.05; comment 1137177

## Query Molecule B
- Canonical SMILES: CCCCOc1nc(N)c2c(n1)N(Cc1cccc(CN3CCCC3)c1)CC(=O)N2
- Properties:
- ChEMBL ID: CHEMBL2424780
- Preferred name: VESATOLIMOD
- Molecule type: Small molecule
- Max clinical phase: 2
- Molecular weight: 410.52
- Heavy atoms: 30
- Hydrogen bond donors: 2
- Hydrogen bond acceptors: 7
- TPSA: 96.61
- LogP: 2.79
- Rotatable bonds: 8
- Ring count: 4
- Aromatic rings: 2
- Formal charge: 0
- Fraction Csp3: 0.5
- QED: 0.65

## Molecule Similarity
- Weighted Tanimoto: 0.21

Answer: 4.94