## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: IC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: In Vitro Assays for Determining the Inhibition of Janus Kinases: The in vitro inhibition of recombinant human JAKs was determined as follow.Compounds were tested in LANTHASCREEN time-resolved fluorescence energy transfer (TR-FRET) enzymatic assays from Invitrogen. The human Janus kinase 1 (JAK1) used in the assay is the recombinant JAK1 catalytic domain (amino acids 866-1154) expressed and purified from insect cells (Invitrogen, Cat. No. PV4774). The human Janus kinase 2 (JAK2) used in the assay is the recombinant JAK2 catalytic domain (amino acids 808-1132) expressed and purified from insect cells (Invitrogen, Cat. No. PV4210). The human Janus kinase 3 (JAK3) used in the assay is the recombinant JAK3 catalytic domain (amino acids 781-1124) expressed and purified from insect cells (Invitrogen, Cat. No. PV3855). The human tyrosine kinase 2 (TYK2) used in the assay is the recombinant TYK2 catalytic domain (amino acids 833-1187) expressed and purified from insect cells (Invitrogen, Cat. No. PV4790). The substrate is a recombinant STAT1 expressed as a fusion with GFP (Green Fluorescent Protein) to act as a physiological substrate (Invitrogen, Cat. No. PV5211).Test compounds were prepared and diluted in DMSO in 3-fold serial dilutions for 10 doses to 100× of the final testing concentrations. The compounds were then further diluted to 4× by the kinase reaction buffer (Invitrogen, Cat. No. PV3189). The enzymatic reaction for compound testing was performed in a white 384-well polypropylene plate (Packard, Cat. No. 6005214) with a total reaction volume of 10 μL containing 440 ng/mL JAK1, 11 ng/mL JAK2, 400 ng/mL JAK3, or 200 ng/mL TYK2, 100 nM substrate, and 1 mM ATP. The assay started with loading 2.5 μL of JAK1, JAK2, JAK3, or TYK2 diluted in the kinase reaction buffer to wells, followed by addition of an equal volume of 4× compounds for 15-min incubation at room temperature for pre-treatment. The enzymatic reaction was initiated by addition of 5 μL of a mixture of the substrate and ATP prepared in the kinase reaction buffer. After one hour reaction, 10 μL mixture of EDTA (final 10 mM) and terbium-labeled anti-pSTAT1 (pTyr701) antibody (final 2 nM) (Invitrogen, Cat. No. PV4844) prepared in TR-FRET antibody dilution buffer (Invitrogen, Cat. No. PV3574) was added to stop the enzymatic reaction and produce TR-FRET signals. After 30 minutes of incubation at room temperature, the plate was read in Tecan Infinite F200 Pro with the following settings: Excitation 340 nm (30)/Emission1 495 nm (10)/Emission2 520 nm (25). The TR-FRET values were dimensionless numbers that were calculated as the ratio of the acceptor (Green Fluorescent Protein) signal to the donor (Terbium) signal. Percent of inhibition was calculated as (100%−percentage of compound-treated/DMSO vehicle-treated). The dose-response curves were generated and the IC50s were calculated by nonlinear sigmoid curve fitting using GraphPad Prism.
- Assay organism: Homo sapiens
- Target name: Tyrosine-protein kinase JAK1
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: O=C(Nc1cn2c(-c3ccc(CN4CCS(=O)(=O)CC4)cc3)cccc2n1)C1CCC1
- Properties:
- ChEMBL ID: CHEMBL5833136
- Molecular weight: 438.55
- Heavy atoms: 31
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 5
- TPSA: 83.78
- LogP: 2.97
- Rotatable bonds: 5
- Ring count: 5
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.39
- QED: 0.66

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 5.52
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type IC50; relation =; value 3000; units nM; pChEMBL 5.52; comment 935643

## Query Molecule B
- Canonical SMILES: COc1ccc(-c2cccc3nc(NC(=O)C4CC4)cn23)cc1
- Properties:
- ChEMBL ID: CHEMBL5808893
- Molecular weight: 307.35
- Heavy atoms: 23
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 3
- TPSA: 55.63
- LogP: 3.36
- Rotatable bonds: 4
- Ring count: 4
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.22
- QED: 0.8

## Molecule Similarity
- Weighted Tanimoto: 0.58

Answer: 5.52