lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc(-c2nnc3n2CCN(C(=O)c2ccc(-c4ccccc4)cc2)[C@@H]3C)cs1
InChIKey: RUDNGJLEKHUVRY-OAHLLOKOSA-N
Target: Transcriptional regulator ERG (SINGLE PROTEIN), Homo sapiens
UniProt: P11308
Assay: hERG Inhibition Assay: The human Ether-a-go-go Related Gene (hERG) encodes the inward rectifying voltage gated potassium channel in the heart (IKr) which is involved in cardiac repolarisation. IKr current inhibition has been shown to elongate the cardiac action potential, a phenomenon associated with increased risk of arrhythmia. IKr current inhibition accounts for the vast majority of known cases of drug-induced QT-prolongation. A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery.The hERG inhibition study aims at quantifying the in vitro effects of compounds of the invention on the potassium-selective IKr current generated in normoxic conditions in stably transfected HEK 293 cells with the human ether-a-go-go-related gene (hERG).Whole-cell currents (acquisition by manual patch-clamp) elicited during a voltage pulse were recorded in baseline conditions and following application of tested compounds (5 minutes of exposure). The concentrations of tested compounds (0.3 μM; 3 μM; 10 μM; 30 μM) reflect a range believed to exceed the concentrations at expected efficacy doses in preclinical models.The pulses protocol applied is described as follow: the holding potential (every 3 seconds) was stepped from −80 mV to a maximum value of +40 mV, starting with −40 mV, in eight increments of +10 mV, for a period of 1 second. The membrane potential was then returned to −55 mV, after each of these incremented steps, for 1 second and finally repolarized to −80 mV for 1 second.The current density recorded were normalized against the baseline conditions and corrected for solvent effect and time-dependent current run-down using experimental design in test compound free conditions.Inhibition curves were obtained for compounds and the concentrations which decreased 50% of the current density determined in the baseline conditions (IC50) were determined. All compounds for which the IC50 value is above 10 M are not considered to be potent inhibitors of the hERG channel whereas compounds with IC50 values below 1 μM are considered potent hERG channel inhibitors.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 415.52, ALogP 4.59, HBA 6, HBD 0, PSA 63.91, QED 0.49
Compound (SMILES): CO[C@]12CC[C@@]3(C[C@@H]1COCc1ccccc1)[C@H]1Cc4ccc(C#N)c5c4[C@@]3(CCN1C)[C@H]2O5
InChIKey: SLLRNVZTIOXEHG-IDMKRTBKSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Measurement: EC50 = 252.11 nM (pChEMBL 6.599999904632568)
Descriptors: MW 456.59, ALogP 4.22, HBA 5, HBD 0, PSA 54.72, QED 0.67
Compound (SMILES): CC(Nc1ncc(F)c(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1cc(-c2ccc(Cl)c(Cl)c2)no1
InChIKey: GBGXXLPKYDLGDN-MVHXDWSJSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 = 1421 nM (pChEMBL 5.849999904632568)
Descriptors: MW 482.3, ALogP 4.46, HBA 8, HBD 2, PSA 113.61, QED 0.53
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3c(C#N)ccc4c(N)n[nH]c34)=CC1=O)c1cc-2cc(OC)n1
InChIKey: BHTHHYTYSKXNGN-CLYVBNDRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki > 457 nM (pChEMBL 6.340000152587891)
Descriptors: MW 634.7, ALogP 5.38, HBA 9, HBD 4, PSA 188.35, QED 0.23
Compound (SMILES): Cc1ncn(-c2ccc(Cl)cc2C2=CC(=O)N([C@H]3CCC[C@@H](C)C(=O)Nc4ccccc4-c4ccnc3c4)CC2)n1
InChIKey: ANAMVZJDWRVIRM-KEEVHDRGSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 40.1 nM (pChEMBL 7.400000095367432)
Descriptors: MW 567.09, ALogP 6.41, HBA 6, HBD 1, PSA 93.01, QED 0.31
Compound (SMILES): CC(F)c1noc(-c2nnc3n2CCN(C(=O)c2ccc(F)cc2)[C@@H]3C)n1
InChIKey: UECKJWCZXJBAJH-QVDQXJPCSA-N
Target: Substance-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21452
Assay: Binding Assay: The affinity of compounds of the invention for the NK-2 receptor was evaluated in CHO recombinant cells which express the human NK-2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [125I]-Neurokinin A (PerkinElmer Cat#NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 μg/ml saponin, at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 3.75 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [125I]-Neurokinin A. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 374.35, ALogP 2.71, HBA 7, HBD 0, PSA 89.94, QED 0.7
Compound (SMILES): N[C@@H]1CN(c2nc3ccccc3n([C@H]3C[C@H]4CCC[C@@H](C3)N4[C@@H]3C[C@@H]4CCCC[C@@H](C4)C3)c2=O)[C@@H]1C(=O)O
InChIKey: YUONSNDROGRPQU-HBOOAYOWSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: In Vitro Delta-Opioid Receptor Binding Assay: Radioligand dose-displacement assays used 0.2 nM [3H]-Naltrindole (NEN; 33.0 Ci/mmole) with 10-20 μg membrane protein (recombinant delta opioid receptor expressed in CHO-K1 cells; Perkin Elmer) in a final volume of 500 μL binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 μM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 h at a temperature of about 25 °C. Binding reactions were determined by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by five filtration washes with 500 μL ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 1-2 hours. Fifty μL/well scintillation cocktail (MicroScint20, Packard) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: Ki > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 519.69, ALogP 3.91, HBA 7, HBD 2, PSA 104.69, QED 0.63
Compound (SMILES): C[C@]1(c2nc(NC(=O)c3ncc(C#CCO)cc3N)ccc2F)N=C(N)OCC1(F)F
InChIKey: LPHMJKHHQPFBCQ-GOSISDBHSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Inhibition Assay: Chinese hamster ovary cells are transfected with the human gene for amyloid precursor protein. The cells are plated at a density of 8000 cells/well into 96-well microtiter plates and cultivated for 24 hours in DMEM cell culture medium containing 10% FCS. The test compound is added to the cells at various concentrations, and the cells are cultivated for 24 hours in the presence of the test compound. The supernatants are collected, and the concentration of amyloid peptide 1-40 is determined using state of the art immunoassay techniques, for example sandwich ELISA, homogenous time-resolved fluorescence (HTRF) immunoassay, or electro-chemiluminescence immunoassay. The potency of the compound is calculated from the percentage of inhibition of amyloid peptide release as a function of the test compound concentration.
Measurement: IC50 = 62 nM (pChEMBL 7.210000038146973)
Descriptors: MW 434.38, ALogP 0.99, HBA 8, HBD 4, PSA 148.74, QED 0.53
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1noc(-c2ccc(Cl)cc2)n1
InChIKey: HQFBCUBMPLPKFP-MDLDFJCZSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 = 6429 nM (pChEMBL 5.190000057220459)
Descriptors: MW 430.85, ALogP 3.06, HBA 9, HBD 2, PSA 126.5, QED 0.61
Compound (SMILES): COC(=O)c1cc([C@H](Nc2nc3ccc(OC)cc3s2)P(=O)(OC)OC)ccc1O
InChIKey: UFYZZKPXXAXJHI-QGZVFWFLSA-N
Target: 3-hydroxyacyl-CoA dehydrogenase type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99714
Assay: Surface Plasmon Resonance (SPR)Assay: The interactions between compounds and ABAD were performed using the dual flow cell BIACORE 3000 instrument. Surface Plasmon Resonance (SPR) studies were performed on a BIACORE 3000 at 25° C. SPR binding experiments with ABAD were performed in phosphate-buffered saline (PBS, pH 7.4, 0.005% surfactant P20) as the running and the sample buffer. The surface of the sensor chip was first activated with mixtures of N-hydroxysuccinimide (NHS, 115 mg/ml) and N-(3-dimethyl-aminopropyl)-N'-ethyl-carbodiimide-hydrochloride (EDC, 750 mg/ml) for 7 minutes. ABAD was dissolved in PBS buffer (pH 5.0) at a concentration of 10 μg/ml. The protein was immobilized directly and covalently on the hydrophilic carboxymethylated dextran matrix of the CM5 sensor chip (BIACORE) by using the standard primary amine coupling reaction on a CM5 sensor chip according to standard procedures. After the immobilization of the protein, excess activated carboxylic acid groups were quenched with ethanolamine (1 M, pH 8.5). Special care was taken during injection of samples because of carryover effects. Special washing routines were used to clean the system before injection of new samples. In addition, predipping of needles was performed. The sample flow was 40 μl/minute in experiments performed for the determination of the kinetic and equilibrium constants. Regeneration of the surfaces between subsequent binding experiments was achieved by washing the surface extensively (>>1 hour) with buffer solution.
Measurement: Ki = 96600 nM (pChEMBL 4.010000228881836)
Descriptors: MW 452.43, ALogP 4.39, HBA 10, HBD 2, PSA 116.21, QED 0.38
Compound (SMILES): Cc1cc(-c2noc(C(C)Nc3nccc(N4C(=O)OC[C@@H]4[C@@H](C)O)n3)n2)ccc1F
InChIKey: DBIUWIKBQRDZEO-AADWAWOOSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 428.42, ALogP 2.85, HBA 9, HBD 2, PSA 126.5, QED 0.61
Compound (SMILES): CCOC(=O)Cc1nnc(Nc2ccc3c(c2)NC(=O)[C@H](C)CCC[C@H](N2CCC(c4c(F)ccc(Cl)c4F)=CC2=O)c2cc-3ccn2)o1
InChIKey: LRJDBUKQKSAXJD-GDJIYFAZSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 0.45 nM (pChEMBL 9.350000381469727)
Descriptors: MW 691.14, ALogP 7.03, HBA 9, HBD 2, PSA 139.55, QED 0.15
Compound (SMILES): O=C(Nc1ccc([C@H]2CNCCO2)cc1)c1cnn(-c2ccc(F)cc2)n1
InChIKey: XKCYWGPISGAXMK-GOSISDBHSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding (rat TAAR1): HEK-293 cells stably expressing rat TAAR1 were maintained at 37Â° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56Â° C.), penicillin/streptomycin (1%), and 375 Î¼g/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1'000 rpm for 5 min at 4Â° C., frozen and stored at â¿¿80Â° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14'000 rpm for 20 s. The homogenate was centrifuged at 48'000Ã¿g for 30 min at 4Â° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14'000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at â¿¿80Â° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 Î¼M unlabeled ligand. All compounds were tested at a broad range of concentrations (10 Î¼M to 10 Î¼M) in duplicates. The test compounds (20 Î¼l/well) were transferred into a 96 deep well plate (TreffLab), and 180 Î¼l of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 Î¼l of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3Ã¿Kd in nM and 500 Î¼l of the membranes (resuspended at 50 Î¼g protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4Â° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 Î¼l of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 367.38, ALogP 2.32, HBA 6, HBD 2, PSA 81.07, QED 0.74
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(Br)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2c(ccc(F)c2F)NC1=O
InChIKey: TXRMRQKQNYNOAW-CMJOXMDJSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 4.46 nM (pChEMBL 8.350000381469727)
Descriptors: MW 618.88, ALogP 7.7, HBA 3, HBD 1, PSA 62.3, QED 0.3
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccc(Nc3cnccn3)cc2NC1=O
InChIKey: KKQBOZNOYSOFLO-GDJIYFAZSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 615.08, ALogP 7.33, HBA 6, HBD 2, PSA 100.11, QED 0.23
Compound (SMILES): O=C(Nc1ccc(C(F)(F)C(F)(F)F)cc1)c1cnc(N2CC[C@@H](O)C2)c(-c2cncnc2)c1
InChIKey: XJCKZYFEKREOHG-QGZVFWFLSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Cellular Proliferation Assay: Test compounds were resuspended in DMSO at a concentration of 10 mM. A serial three-fold dilution of each compound with DMSO was performed in 384-well plates using the Janus Liquid Dispenser (PerkinElmer). Compound was delivered to the assay plates containing 2500 cells in a 50 μL volume via Acoustic delivery from an ATS-100 (EDC). For Ba/F3-BCR-ABL1-WT cell assays, 2 nL of each compound dilution was transferred to the assay plate for final assay concentrations of 0.4 μM, 0.13 μM, 0.044 μM, 0.015 μM, 0.005 μM, 0.001 μM, 0.00033 μM, 0.00011 μM, 0.000037 μM, 0.000012 μM. For Ba/F3-WT and Ba/F3-BCR-ABL1-T315I cell assays, 50 nL of each compound dilution was transferred to the assay plate for final assay concentrations of 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.12 μM, 0.041 μM, 0.014 μM, 0.0046 μM, 0.0015 μM, 0.00051 μM.Cells were incubated at 37° C. in a humidified environment with 5% car
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 479.41, ALogP 4.02, HBA 6, HBD 2, PSA 91.24, QED 0.54
Compound (SMILES): CS(=O)(=O)N1C(=O)N(c2cccc(C(F)(F)F)c2)C2=C(C(=O)CC2)[C@H]1c1ccc(C#N)cc1
InChIKey: ZSGUFCFIQTTZBC-HXUWFJFHSA-N
Target: Cytochrome P450 2C19 (SINGLE PROTEIN), Homo sapiens
UniProt: P33261
Assay: Cytochrome P450 2C9 Inhibition Assay (Mephenytoin): The inhibition of cytochrome P450 2C19-isoenzyme catalysed hydroxylation of Mephenytoin by the test compound is assayed at 37° C. with human liver microsomes. All assays are carried out on a robotic system in 96 well plates. The final incubation volume contains TRIS buffer (0.1 M), MgCl2 (5 mM), human liver microsomes (0.5 mg/ml), (S)-Mephenytoin (70 uM) and the test compound at five different concentrations or no compound (high control) in duplicate (e.g. highest concentration 10-50 uM with subsequent serial 1:4 dilutions). Following a short preincubation period, reactions are started with the cofactor (NADPH, 1 mM) and stopped by cooling the incubation down to 8° C. and subsequently by addition of one volume of acetonitrile.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 475.45, ALogP 4.14, HBA 5, HBD 0, PSA 98.55, QED 0.66
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1nc(-c2ccc(Cl)cc2C(F)(F)F)no1
InChIKey: UXGRUBLQGMQKGE-WUMONGPASA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 = 1145 nM (pChEMBL 5.940000057220459)
Descriptors: MW 498.85, ALogP 4.08, HBA 9, HBD 2, PSA 126.5, QED 0.52
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccc(Nc3nnc(CC#N)o3)cc2NC1=O
InChIKey: KJPOONPDKYPIKE-CLYVBNDRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 1.17 nM (pChEMBL 8.930000305175781)
Descriptors: MW 644.08, ALogP 6.99, HBA 8, HBD 2, PSA 137.04, QED 0.22
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-n2nc(C3CC3)cc2NC1=O
InChIKey: ZSIJVOIVCNQFGT-GYCJOSAFSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 302 nM (pChEMBL 6.519999980926514)
Descriptors: MW 552.03, ALogP 6.19, HBA 5, HBD 1, PSA 80.12, QED 0.39
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4CSc4nc6ccccc6s4)[C@@H](C2)N(C)CC[C@]315
InChIKey: YFKQYHWQDZXKGP-RIWDBKMTSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Measurement: EC50 = 502.68 nM (pChEMBL 6.300000190734863)
Descriptors: MW 520.72, ALogP 5.54, HBA 7, HBD 0, PSA 43.82, QED 0.41
Compound (SMILES): C[C@@H]1Cc2c(nnn2-c2ccn[nH]2)[C@@H](C)N1C(=O)c1cccc(C(F)(F)F)c1Cl
InChIKey: IQRKGPSJDGZJEV-NXEZZACHSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 92.3 nM (pChEMBL 7.039999961853027)
Descriptors: MW 424.81, ALogP 3.81, HBA 5, HBD 1, PSA 79.7, QED 0.68
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(cc[n+]2[O-])-c2ccc(N)cc2NC1=O
InChIKey: SKANAQYYDPFGRP-GYCJOSAFSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 8.02 nM (pChEMBL 8.100000381469727)
Descriptors: MW 553.01, ALogP 5.62, HBA 4, HBD 2, PSA 102.37, QED 0.19
Compound (SMILES): CCCc1cc(COC[C@H]2C[C@@]34CC[C@]2(OC)[C@@H]2Oc5c(OC)ccc6c5[C@@]23CCN(C)[C@@H]4C6)on1
InChIKey: VGPKXDDDJZDRDW-NWCUSBOBSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Measurement: EC50 = 650.11 nM (pChEMBL 6.190000057220459)
Descriptors: MW 494.63, ALogP 4.3, HBA 7, HBD 0, PSA 66.19, QED 0.54
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4[C@](O)(CC(O)CO)c4ccccc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: CNTAUNWPRVDTGK-RQFDAPRJSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: GTPgammaS Functional Assay (kappa): Membranes from recombinant HEK-293 cells, CHO cells expressing the recombinant human kappa opioid receptor (kappa) were prepared by lysing cells in ice cold hypotonic buffer (2.5 mM MgCl2, 50 mM HEPES, pH 7.4) (10 mL/10 cm dish) followed by homogenization with a tissue grinder/Teflon pestle. Functional [35S]GTPgammaS binding assays were conducted as follows. kappa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/ul kappa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2,20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B.
Measurement: EC50 = 122.29 nM (pChEMBL 6.909999847412109)
Descriptors: MW 561.72, ALogP 3.55, HBA 7, HBD 3, PSA 91.62, QED 0.43
Compound (SMILES): C[C@H](Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)F)n1)c1cn(-c2ccc(Cl)cc2)cn1
InChIKey: JJRFKCRKJUIUTG-IIYDPXPESA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 Biochemical Assay: LC-MS Detection of 2-HG: Mutant IDH1 R132H catalytic activity was monitored using the quantitative liquid chromatography/mass spectrometry (LC-MS) detection of 2-HG, a product of the NADPH-dependent alpha-KG reduction reaction.More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG.The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, 20 minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below).LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively. The relative responses (2-HG/13C-2-HG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC50 values (normalized IC50 regression curves).
Measurement: IC50 < 23 nM (pChEMBL 7.638000011444092)
Descriptors: MW 430.87, ALogP 4.17, HBA 7, HBD 1, PSA 85.17, QED 0.63
Compound (SMILES): Cc1cnc2c(c1)nnn2-c1ccc(C(=O)N(c2ncccc2Cl)[C@@H]2CCCNC2)nc1
InChIKey: UVVXRMZCPKQLAO-OAHLLOKOSA-N
Target: Proprotein convertase subtilisin/kexin type 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NBP7
Assay: Inhibition of full length human PCSK9 expressed in human Huh-7 cells by ELISA analysis
Measurement: IC50 = 1460 nM (pChEMBL 5.840000152587891)
Descriptors: MW 448.92, ALogP 2.97, HBA 8, HBD 1, PSA 101.72, QED 0.51
Compound (SMILES): N#C[C@@H]1CS(=O)(=O)CC[C@H]1n1cc(C(N)=O)c(Nc2ccc(C(F)(F)F)cc2)n1
InChIKey: VONAFDGGTACLKB-QMTHXVAHSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 146 nM (pChEMBL 6.840000152587891)
Descriptors: MW 427.41, ALogP 2.24, HBA 7, HBD 2, PSA 130.87, QED 0.77
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(C#N)c3F)=CC2=O)c2cc(ccn2)-c2cc(C#N)ccc2NC1=O
InChIKey: MFZYQWVLTUKKTJ-CLYVBNDRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki > 408 nM (pChEMBL 6.388999938964844)
Descriptors: MW 537.57, ALogP 5.89, HBA 5, HBD 1, PSA 109.88, QED 0.44
Compound (SMILES): COc1ccc2c3c1O[C@H]1C4(OC)CC[C@@]5(C[C@@H]4COCC#N)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: JCCODHWQLAIKEU-XYBUWLCTSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Binding Assay: Binding assay of certain compounds of this invention to the opioid receptor was determine using radioligand binding assay (screening and dose-displacement).
Measurement: Ki = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 450.58, ALogP 3.46, HBA 6, HBD 0, PSA 63.95, QED 0.59
Compound (SMILES): CC(C)(C)[C@@]1(C)OCO[C@]23CC[C@@]4(C[C@@H]21)[C@H]1Cc2ccc(O)c5c2[C@@]4(CCN1CC1CC1)[C@H]3O5
InChIKey: OWNPADNXDQCYAS-IHFGGWKQSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: κ-Opioid Receptor Functional Assay: Functional [35S]GTPγS binding assays were conducted as follows. κ opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 μg/μl κ membrane protein (in-house), 10 μg/mL saponin, 3 μM GDP and 0.20 nM [35S]GTPγS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Packard) and followed by three filtration washes with 200 μl ice-cold binding buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. Fifty μl/well scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: EC50 = 22.66 nM (pChEMBL 7.639999866485596)
Descriptors: MW 465.63, ALogP 4.78, HBA 5, HBD 1, PSA 51.16, QED 0.68
Compound (SMILES): O=C(O)[C@H]1CCN1c1nc2ccccc2n([C@H]2C[C@H]3CCC[C@@H](C2)N3[C@@H]2C[C@@H]3CCCC[C@@H](C3)C2)c1=O
InChIKey: ZELGCSSWSLNFRN-UTZFDERYSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: In Vitro Delta-Opioid Receptor Binding Assay: Radioligand dose-displacement assays used 0.2 nM [3H]-Naltrindole (NEN; 33.0 Ci/mmole) with 10-20 μg membrane protein (recombinant delta opioid receptor expressed in CHO-K1 cells; Perkin Elmer) in a final volume of 500 μL binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 μM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 h at a temperature of about 25 °C. Binding reactions were determined by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by five filtration washes with 500 μL ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 1-2 hours. Fifty μL/well scintillation cocktail (MicroScint20, Packard) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: Ki = 6410 nM (pChEMBL 5.190000057220459)
Descriptors: MW 504.68, ALogP 4.98, HBA 6, HBD 1, PSA 78.67, QED 0.63
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4C(N[C@@H](C(N)=O)c4ccccc4)c4ccccc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: MGYBPVKOXFORNZ-KFGYFWJXSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: GTPgammaS Functional Assay (kappa): Membranes from recombinant HEK-293 cells, CHO cells expressing the recombinant human kappa opioid receptor (kappa) were prepared by lysing cells in ice cold hypotonic buffer (2.5 mM MgCl2, 50 mM HEPES, pH 7.4) (10 mL/10 cm dish) followed by homogenization with a tissue grinder/Teflon pestle. Functional [35S]GTPgammaS binding assays were conducted as follows. kappa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/ul kappa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2,20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B.
Measurement: EC50 = 771.49 nM (pChEMBL 6.110000133514404)
Descriptors: MW 619.81, ALogP 5.48, HBA 6, HBD 2, PSA 86.05, QED 0.31