## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: IC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: OPR Mu Antagonist Assay: The purpose of this assay is to confirm the potency and selectivity of compounds synthesized to be OPRK1 Antagonists. This assay monitors the OPRMu1 activation, in membrane recruitment of β-arrestin. The assay monitors GPCR-β-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). It employs U20S cells which express OPRMu1 fused to the complementary beta-gal fragment (enzyme acceptor). As designed, compounds that act as antagonists will prevent receptor activation resulting in reduce well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 10 micromolar.The Discover X OPRMu1-U20S cell line was routinely cultured in T175 flasks at 37° C., 5% CO2 and 95% relative humidity (RH). The growth media consisted of DMEM/F12 1:1 Media supplemented with 10% v/v heat inactivated fetal bovine serum, 25 mM HEPES, Non-essential amino acids, 1 mM Sodium Pyruvate, 1× antibiotic mix (penicillin streptomycin) plus 500 ug/mL Geneticin and 300 ug/mL Hygromycin (selection antibiotics).On Day 1 of the assay, 5000 cells in 20 ul of assay buffer (Discover X's Cell Plating Reagent 5) were seeded into each well of a 384 Corning 3570 standard white plate, and incubated 16-24 hours at 37° C., 5% CO2 and 95% (RH). On Day 2, 100 nl of test compound in DMSO was added to the appropriate wells, 100 nl of DMSO added to control wells and plates were incubated for 30 min at 37° C., 5% CO2 and 95% (RH). Next 100 nl of DAMGO OPRMu1 or DMSO in assay media (EC80 Challenge consists of 100 nl of 50 uM DAMGO, final assay concentration=250 nM, 100% Response wells receive 100 nl 200 uM DAMGO). After incubation for 3 hours at 37° C., 5% CO2 and 95% (RH), 10 ul of Path Hunter Detection Mix is added to each well, plate placed on a plate rotator/mixer for 10 minutes and then incubated at room temperature, in the dark for 1 hour. Well luminescence was measured on Perkin Elmer's Envision.The Percent Inhibition was calculated from the median ratio as follows:% ⁢ ⁢ Inhibition = 100 - ( 1 ⁢ 0 ⁢ 0 ⁢ ( Compound ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well EC ⁢ ⁢ 80 ⁢ ⁢ Control ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well ) )where: COMPOUND WELL is defined as the well containing test compound; EC80 CONTROL WELL is defined as wells containing DAMGO challenge (250 nM final)=0% inhibition; and 0% RESPONSE WELL is defined as wells containing DMSO=100% inhibition.IC50 is defined as the concentration of compound required to achieve 50% inhibition.
- Assay organism: Homo sapiens
- Target name: Mu-type opioid receptor
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: Cc1noc(-c2c(N3CCC(N[C@@H]4CCCOC4)CC3)nc3c(C#N)cc(CC(F)(F)F)cc3c2C)n1
- Properties:
- ChEMBL ID: CHEMBL5961770
- Molecular weight: 514.55
- Heavy atoms: 37
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 8
- TPSA: 100.1
- LogP: 4.62
- Rotatable bonds: 5
- Ring count: 5
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.54
- QED: 0.53

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 6.26
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type IC50; relation =; value 550; units nM; pChEMBL 6.26; comment 1094761

## Query Molecule B
- Canonical SMILES: CCc1cc(F)c2nc(N3CCC(N[C@H]4CCCOC4)CC3)c(-c3nc(C)no3)c(C)c2c1
- Properties:
- ChEMBL ID: CHEMBL4440683
- Molecule type: Unknown
- Molecular weight: 453.56
- Heavy atoms: 33
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 7
- TPSA: 76.31
- LogP: 4.34
- Rotatable bonds: 5
- Ring count: 5
- Aromatic rings: 3
- Formal charge: 0
- Fraction Csp3: 0.56
- QED: 0.62

## Molecule Similarity
- Weighted Tanimoto: 0.72

Answer: 6.26