## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: IC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: Inhibitory Activity Assay: Essentially, the enzyme activity is measured by quantification of the Coumberol from Coumberone (Halim, M., Yee, D. J., and Sames, D., J. AM. CHEM. SOC. 130, 14123-14128 (2008) and Yee, D. J., Balsanek, V., Bauman, D. R., Penning, T. M., and Sames, D., Proc. Natl. Acad. Sci. USA 103, 13304-13309 (2006)). In this test, the increase of the highly fluorescent Coumberol by NADPHâ¿¿(nicotinamide adenine dinucleotide phosphate)-dependent reduction of the non-fluorescent Coumberone by AKR1C3 can be determined.The enzyme used was recombinant human AKR1C3 (Aldo-keto reductase family 1 member C3) (GenBank Accession No. NM_003739). This was expressed in E. coli as GST (glutathione S transferase) fusion protein and purified by glutathione Sepharose affinity chromatography. The GST was removed by digestion with thrombin and subsequent size exclusion chromatography (Dufort, I., Rheault, P., Huang, X F., Soucy, P., and Luu-The, V., Endocrinology 140, 568-574 (1999)). For the assay, 50 nl of a 100-fold concentrated solution of the test substance in DMSO were pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 ul of a solution of AKR1C3 in assay buffer [50 mM potassium phosphate buffer pH 7, 1 mM DTT, 0% (w/v) Pluronic F-127, 0.01% BSA (w/v) and protease inhibitor cocktail (Complete, EDTA-free Protease Inhibitor Cocktail from Roche)] were added and the mixture was incubated for 15 min to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. The enzyme reaction was then started by addition of 3 ul of a solution of NADPH (16.7 uM final concentration in 5 ul of assay volume is 10 uM) and Coumberone (0.5 uM final concentration in 5 ul of assay volume is 0.3 uM) in assay buffer, and the resulting mixture was incubated at 22Â° C. for the reaction time of 90 min. The concentration of the AKR1C3 was adapted to the respective activity of the enzyme preparation and adjusted such that the assay was carried out in the linear range. Typical concentrations were in the region of 1 nM. The reaction was stopped by addition of 5 ul of a stop solution consisting of the inhibitor EM-1404 [F. Labrie et al. U.S. Pat. No. 6,541,463, 2003] (2 uM final concentration in 5 ul of assay volume is 1 uM). The fluorescence of the Coumberole was then measured at 520 nm (excitation at 380 nm) using a suitable measuring instrument (Pherastar from BMG Labtechnologies). The intensity of the fluorescence was used as a measure of the amount of Coumberole formed and thus of the enzyme activity of AKR1C3. The data were normalized (enzyme reaction without inhibitor=0% inhibition; all other assay components, but no enzyme=100% inhibition). Usually, the test substances were tested on the same microtiter plate at 11 different concentrations in the range from 20 uM to 96.8 pM (20 uM, 5.9 uM, 1.7 uM, 0.5 uM, 0.15 uM, 44 nM, 12.9 nM, 3.8 nM, 1.1 nM, 0.3 nM and 96.8 pM, the dilution series were prepared prior to the assay on the level of the 100-fold concentrated solution by serial 1:3 dilutions with 100% DMSO) in double for each concentration, and the IC50 values were calculated using a 4-parameter fit.
- Assay organism: Homo sapiens
- Target name: Aldo-keto reductase family 1 member C3
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: CN(CCC(=O)O)C(=O)c1ccc2c(c1)CC[C@@H]1[C@@H]2CC[C@]2(C)C(c3cncc(C(F)(F)F)c3)=CC[C@@H]12
- Properties:
- ChEMBL ID: CHEMBL4112723
- Molecule type: Small molecule
- Molecular weight: 512.57
- Heavy atoms: 37
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 3
- TPSA: 70.5
- LogP: 6.2
- Rotatable bonds: 5
- Ring count: 5
- Aromatic rings: 2
- Formal charge: 0
- Fraction Csp3: 0.48
- QED: 0.52

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 8.38
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type IC50; relation =; value 4.2; units nM; pChEMBL 8.38; comment 443610

## Query Molecule B
- Canonical SMILES: C[C@]12CC[C@@H]3c4ccc(C(=O)N[C@@H]5CCC[C@@H]5C(=O)O)cc4CC[C@H]3[C@@H]1CC=C2c1cncc(F)c1
- Properties:
- ChEMBL ID: CHEMBL4114888
- Molecule type: Small molecule
- Molecular weight: 488.6
- Heavy atoms: 36
- Hydrogen bond donors: 2
- Hydrogen bond acceptors: 3
- TPSA: 79.29
- LogP: 5.75
- Rotatable bonds: 4
- Ring count: 6
- Aromatic rings: 2
- Formal charge: 0
- Fraction Csp3: 0.5
- QED: 0.57

## Molecule Similarity
- Weighted Tanimoto: 0.56

Answer: 8.7