## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: IC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: 25P Btk Enzyme Activity Assay: Btk enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (lime-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1000 nM to 0.02 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA and 1 mM DTT) containing 26.67 pg/μL (266.7 pM) of 25P Btk enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length Btk; MW=79378 Da). Following a 60 minute compound and enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2), and 100 μM ATP. The final reaction in each well of 10 consists of 200 pM 25P Btk, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.63 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
- Assay organism: Homo sapiens
- Target name: Tyrosine-protein kinase BTK
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: CC(C)(C)c1cc(F)c2c(c1)CCN(c1cccc(-c3cc(C(N)=O)c4[nH]nc(N)c4c3)c1CO)C2=O
- Properties:
- ChEMBL ID: CHEMBL5855747
- Molecular weight: 501.56
- Heavy atoms: 37
- Hydrogen bond donors: 4
- Hydrogen bond acceptors: 5
- TPSA: 138.33
- LogP: 4.04
- Rotatable bonds: 4
- Ring count: 5
- Aromatic rings: 4
- Formal charge: 0
- Fraction Csp3: 0.25
- QED: 0.33

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 9.31
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type IC50; relation =; value 0.49; units nM; pChEMBL 9.31; comment 744468

## Query Molecule B
- Canonical SMILES: CC(C)(C)c1cc(F)c2c(=O)n(-c3cccc(-c4cc(C(=O)N5CCOCC5)c5[nH]nc(N)c5c4)c3CO)ncc2c1
- Properties:
- ChEMBL ID: CHEMBL5872228
- Molecular weight: 570.63
- Heavy atoms: 42
- Hydrogen bond donors: 3
- Hydrogen bond acceptors: 7
- TPSA: 139.36
- LogP: 3.91
- Rotatable bonds: 4
- Ring count: 6
- Aromatic rings: 5
- Formal charge: 0
- Fraction Csp3: 0.29
- QED: 0.3

## Molecule Similarity
- Weighted Tanimoto: 0.54

Answer: 6.76