## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: IC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: 100P 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 & 1 mM DTT) containing 13.3 pg/μL (133.3 pM) of 100P 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) (SEQ.ID.NO.: 1) and 100 μM ATP. The final reaction in each well of 10 consists of 100 pM 100P Btk, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 45 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(=O)n(-c3cccc(-c4cc(C(F)(F)F)c5[nH]nc(N)c5c4)c3CO)ncc2c1
- Properties:
- ChEMBL ID: CHEMBL5870967
- Molecular weight: 525.51
- Heavy atoms: 38
- Hydrogen bond donors: 3
- Hydrogen bond acceptors: 5
- TPSA: 109.82
- LogP: 5.46
- Rotatable bonds: 3
- Ring count: 5
- Aromatic rings: 5
- Formal charge: 0
- Fraction Csp3: 0.22
- QED: 0.27

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 6
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type IC50; relation =; value 1000; units nM; pChEMBL 6; comment 744445

## Query Molecule B
- Canonical SMILES: Cc1nn(C)cc1-c1cc(-c2cccc(-n3ncc4cc(C(C)(C)C)cc(F)c4c3=O)c2CO)cc2c(N)n[nH]c12
- Properties:
- ChEMBL ID: CHEMBL5968326
- Molecular weight: 551.63
- Heavy atoms: 41
- Hydrogen bond donors: 3
- Hydrogen bond acceptors: 6
- TPSA: 127.64
- LogP: 5.15
- Rotatable bonds: 4
- Ring count: 6
- Aromatic rings: 6
- Formal charge: 0
- Fraction Csp3: 0.23
- QED: 0.28

## Molecule Similarity
- Weighted Tanimoto: 0.75

Answer: 7.16