## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: IC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: Btk Enzyme Activity Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-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 1 μM 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 & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & 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) (SEQ. ID. NO.: 1), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 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: CCOc1cc(C(=O)Nc2cc(C(F)(F)F)ccn2)ccc1-c1nc([C@]2(C)CC[C@H]3CCC(=O)N3C2)n2ccnc(N)c12
- Properties:
- ChEMBL ID: CHEMBL6063741
- Molecular weight: 593.61
- Heavy atoms: 43
- Hydrogen bond donors: 2
- Hydrogen bond acceptors: 7
- TPSA: 127.74
- LogP: 5.09
- Rotatable bonds: 6
- Ring count: 6
- Aromatic rings: 4
- Formal charge: 0
- Fraction Csp3: 0.37
- QED: 0.32

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 9.36
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type IC50; relation =; value 0.44; units nM; pChEMBL 9.36; comment 530467

## Query Molecule B
- Canonical SMILES: CCOc1cc(C(=O)Nc2cc(C(F)(F)F)ccn2)ccc1-c1nc([C@]2(C)CC[C@H]3CN(C(C)C)CC(=O)N3C2)n2ccnc(N)c12
- Properties:
- ChEMBL ID: CHEMBL5983869
- Molecular weight: 650.71
- Heavy atoms: 47
- Hydrogen bond donors: 2
- Hydrogen bond acceptors: 8
- TPSA: 130.98
- LogP: 5.02
- Rotatable bonds: 7
- Ring count: 6
- Aromatic rings: 4
- Formal charge: 0
- Fraction Csp3: 0.42
- QED: 0.29

## Molecule Similarity
- Weighted Tanimoto: 0.83

Answer: 9.38