## Endpoint to Predict
- Endpoint scope: assay
- Measurement type: IC50
- Label type: continuous
- Binary semantics: pchembl
- Assay description: In Vitro Enzyme Activity Assay: These compounds according to the afore-mentioned examples were tested for tryptophan hydroxylase (TPH) inhibitory activity in a fluorescence-based in vitro assay, using recombinant human TPH1 (Swiss-Prot: P17752) and TPH2 (Swiss-Prot: Q8IWU9).The full-length coding sequences of human TPH1 and TPH2 were PCR amplified, ligated into a MBP fusion vector (pMalc2x, New England Biolabs, MA, USA) and transformed into SCS1 (Stratagene, CA, USA) to amplify plasmid DNA. For the overexpression of TPH proteins, the constructs were transformed into Rosetta (DE3) (Novagen®/EMD Millipore, MA, USA) and cultivated in terrific broth (TB) medium (AppliChem, Darmstadt, Germany) at 37° C. When the bacterial cultures reached an ≈2, expression was induced with 0.5 mM IPTG (AppliChem, Darmstadt, Germany) over night at 17° C. The purification of soluble proteins started with sonication-mediated cell disruption in lysis buffer (1×PBS pH 7.4, 0.5 M NaCl, 5% Glycerol+CHAPS, DTT, PMSF, benzonase), followed by affinity purification (MBPTrap, GE Healthcare, UK) and gel filtration (26/60 Superdex 200 prep grade, GE Healthcare, UK), according to the manufacturer's protocol. The quality of protein expression and solubility was controlled by SDS-PAGE and Coomassie blue staining.The enzymatic reaction was carried out in black 96-well flat bottom plates (Corning GmbH, Wiesbaden). TPH1 and TPH2 activities were measured in a reaction mixture containing 50 mM 4-Morpholineethanesulfonic acid (MES), pH 7, 40 μM tryptophan, 200 mM ammonium sulfate, 25 μM ferrous ammonium sulfate, 50 μM tetrahydrobiopterin, 25 μg/ml catalase, and 7 mM DTT. The reactions were initiated by adding TPH1 or TPH2 to a final concentration of 5 μg/ml. Initial velocity of the reactions was determined by following the change of fluorescence at 330 nm (excitation wavelength=300 nm) (Infinite M200, Tecan, Crailsheim).TPH1 and TPH2 inhibition was determined by measuring a compound dose response, using a serial dilution of a 5 mM DMSO stock solution. The potency of a given compound was calculated in GraphPad PRISM 6 software (San Diego, USA) with a Nonlinear Regression fit (log(inhibitor) vs. response-variable slope) using the relative fluorescence units (RFU) of the sample triplicates.
- Assay organism: Homo sapiens
- Target name: Tryptophan 5-hydroxylase 2
- Target type: SINGLE PROTEIN
- Target organism: Homo sapiens

## Reference Molecule A
- Canonical SMILES: Cn1c(=O)[nH]c(=O)c2c1nc(Cc1nc3cc(NC(=O)CC[C@H](N)C(=O)O)ccc3[nH]1)n2Cc1ccccc1
- Properties:
- ChEMBL ID: CHEMBL5172824
- Molecular weight: 530.55
- Heavy atoms: 39
- Hydrogen bond donors: 5
- Hydrogen bond acceptors: 7
- TPSA: 193.78
- LogP: 1.07
- Rotatable bonds: 9
- Ring count: 5
- Aromatic rings: 5
- Formal charge: 0
- Fraction Csp3: 0.23
- QED: 0.19

## Known Reference Result on This Endpoint
- Molecule A clean result: median pChEMBL = 6.77
- This result is provided as context for predicting Molecule B on the same endpoint.

## Reference Molecule A Other ChEMBL Evidence
- type IC50; relation =; value 170; units nM; pChEMBL 6.77; comment 707070

## Query Molecule B
- Canonical SMILES: Cc1ccc2[nH]c(Sc3nc4c(c(=O)[nH]c(=O)n4C)n3Cc3ccccc3)nc2c1
- Properties:
- ChEMBL ID: CHEMBL5850224
- Molecular weight: 418.48
- Heavy atoms: 30
- Hydrogen bond donors: 2
- Hydrogen bond acceptors: 5
- TPSA: 101.36
- LogP: 2.81
- Rotatable bonds: 4
- Ring count: 5
- Aromatic rings: 5
- Formal charge: 0
- Fraction Csp3: 0.14
- QED: 0.47

## Molecule Similarity
- Weighted Tanimoto: 0.44

Answer: 6.24