## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: EC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat#AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses @1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat#F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2. A FLEXDROP (Perkin Elmer) was cleaned with ethanol then water, and primed with Buffer 2. A 384 well V bottom polypropylene plate containing d2 labelled cAMP and a second 384 well V bottom polypropylene plate containing anti-cAMP was prepared (50 ul per well). Media as "dumped~ from the cell plate and 30 uL Buffer 1 was added to each well using a Multidrop. The content of the cell plate was again "dumped" and 10 uL Buffer 2 was added to each well using a Flexdrop. 12.5 nL test compound dilutions or control compound dilutions (10 mM to 0.5 uM) were added to the cell plate using an ECHO 555 (Labcyte). The cell plate was mixed (Speed 6, Lab-Line Instruments Titer Plate Shaker) and centrifugated (1000 RPMs, 1 min). Using the Flexdrop, 2 ul additions were made into the cell plate: Buffer 2 was added to Column 24; and, 1.5 uM Forskolin was added to columns 1 through 23. Final volume of the cell plate was 12 ul with 250 nM Forskolin in all wells except column 12, and serial dilutions of test compound or control ranging from 10 uM to 0.5 nM. The cell plate was again mixed (speed 6) and centrifugated (1000 RPMs, 1 min). The cell plate was incubated for 30 minutes at room temperature (~27° C.). The contents of row P were removed and the cAMP standard dilutions were added in duplicate to Row P (P1-12 and P13-24). After incubation, 6 uL d2 labelled cAMP and 6 uL of Anti-cAMP were added to all wells of the cell plate using a BioMek FX (Beckman Coulter). The cell plate was again mixed (speed 6) and centrifugated (1000 RPMs, 1 min) and was incubated for 60 minutes in the dark at room Temp (~27° C.). After this final incubation, the cell plate was read in HTRF mode (fluorescence at 665 nm and 620 nm) on an Envision plate Reader (Perkin Elmer). The Envision reader outputs a ratio of channel 1/channel 2 fluorescenceÃ¿10,000 (Normalized signal (NS)). Amount of cAMP in nM was calculated for each well (based on NS) from a cAMP standard curve located on each plate (at P1-12 and P13-24).
- Assay organism: Homo sapiens
- Target name: Cannabinoid receptor 2
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: O=S(=O)(N1CCC(Nc2nc(O)nc3ccc(C(c4ccc(Cl)cc4)c4ccc(Cl)cc4)cc23)CC1)C(F)(F)F
- Properties:
- ChEMBL ID: CHEMBL4750322
- Molecule type: Unknown
- Molecular weight: 611.47
- Heavy atoms: 40
- Hydrogen bond donors: 2
- Hydrogen bond acceptors: 6
- TPSA: 95.42
- LogP: 6.55
- Rotatable bonds: 6
- Ring count: 5
- Aromatic rings: 4
- Formal charge: 0
- Fraction Csp3: 0.26
- QED: 0.24

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 5.25
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type EC50; relation =; value 5687; units nM; pChEMBL 5.25; comment 465917

## Query Molecule B
- Canonical SMILES: FC(F)(F)c1ccc(N2CCC(Nc3ncnc4ccc(C(c5ccc(Cl)cc5)c5ccc(Cl)cc5)cc34)CC2)cc1
- Properties:
- ChEMBL ID: CHEMBL5833734
- Molecular weight: 607.51
- Heavy atoms: 42
- Hydrogen bond donors: 1
- Hydrogen bond acceptors: 4
- TPSA: 41.05
- LogP: 9.22
- Rotatable bonds: 6
- Ring count: 6
- Aromatic rings: 5
- Formal charge: 0
- Fraction Csp3: 0.21
- QED: 0.2

## Molecule Similarity
- Weighted Tanimoto: 0.51

Answer: 7.14