## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: IC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: TR-FRET Assay for Retinol-Induced RBP4-TTR Interaction: Binding of a desired RBP4 antagonist displaces retinol and induces hindrance for RBP4-TTR interaction resulting in the decreased FRET signal (FIG. 7 ). Bacterially expressed MBP-RBP4 and untagged TTR were used in this assay. For the use in the TR-FRET assay the maltose binding protein (MBP)-tagged human RBP4 fragment (amino acids 19-201) was expressed in the Gold(DE3)pLysS E. coli strain (Stratagene) using the pMAL-c4x vector. Following cell lysis, recombinant RBP4 was purified from the soluble fraction using the ACTA FPLC system (GE Healthcare) equipped with the 5-ml the MBP Trap HP column. Human untagged TTR was purchased from Calbiochem. Untagged TTR was labeled directly with Eu3+ Cryptate-NHS using the HTRF Cryptate Labeling kit from CisBio following the manufacturer's recommendations. HTRF assay was performed in white low volume 384 well plates (Greiner-Bio) in a final assay volume of 16 μl per well. The reaction buffer contained 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% NP-40, 0.05% Prionex, 6% glycerol, and 400 mM KF. Each reaction contained 60 nM MBP-RBP4 and 2 nM TTR-Eu along with 26.7 nM of anti-MBP antibody conjugated with d2 (Cisbio). Titration of test compounds in this assay was conducted in the presence of 1 μM retinol. All reactions were assembled in the dark under dim red light and incubated overnight at +4° C. wrapped in aluminum foil. TR-FRET signal was measured in the SpectraMax M5e Multimode Plate Reader (Molecular Device). Fluorescence was excited at 337 nm and two readings per well were taken: Reading 1 for time-gated energy transfer from Eu(K) to d2 (337 nm excitation, 668 nm emission, counting delay 75 microseconds, counting window 100 microseconds) and Reading 2 for Eu(K) time-gated fluorescence (337 nm excitation, 620 nm emission, counting delay 400 microseconds, counting window 400 microseconds). The TR-FRET signal was expressed as the ratio of fluorescence intensity: Flu665/Flu620×10,000.
- Assay organism: Homo sapiens
- Target name: Retinol-binding protein 4
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: N#Cc1ccc2nnc(C(=O)N3CCC(c4cccc(Cl)c4C(F)(F)F)CC3)n2c1
- Properties:
- ChEMBL ID: CHEMBL3960081
- Molecule type: Small molecule
- Molecular weight: 433.82
- Heavy atoms: 30
- Hydrogen bond donors: 0
- Hydrogen bond acceptors: 4
- TPSA: 74.29
- LogP: 4.29
- Rotatable bonds: 2
- Ring count: 4
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.3
- QED: 0.6

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 7.97
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type IC50; relation =; value 10.7; units nM; pChEMBL 7.97; comment 1278979

## Query Molecule B
- Canonical SMILES: O=C(c1n[nH]c2c1CN(CC(F)(F)F)C2)N1CCC(c2ccc(F)c(F)c2C(F)(F)F)CC1
- Properties:
- ChEMBL ID: CHEMBL3920509
- Molecule type: Small molecule
- Molecular weight: 482.38
- Heavy atoms: 33
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 3
- TPSA: 52.23
- LogP: 4.6
- Rotatable bonds: 3
- Ring count: 4
- Aromatic rings: 2
- Formal charge: 0
- Fraction Csp3: 0.5
- QED: 0.65

## Molecule Similarity
- Weighted Tanimoto: 0.34

Answer: 7.11