lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Fc1ccc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: LYEFGGREPUFIPO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 350.4, ALogP 3.12, HBA 5, HBD 1, PSA 62.11, QED 0.62
Compound (SMILES): Cc1cccc2c(Oc3c(Cl)cccc3Cl)nn(-c3ccc(C(=O)O)cc3)c12
InChIKey: OPMJWULKAWOTPC-UHFFFAOYSA-N
Target: Retinoic acid receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P13631
Assay: Inverse agonist activity at GAL4 DBD-fused RARgamma LBD (unknown origin) expressed in human HG5LN cells after 18 hrs by luciferase reporter gene assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 413.26, ALogP 6.13, HBA 4, HBD 1, PSA 64.35, QED 0.44
Compound (SMILES): O=C(c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1)N1C[C@H]2COC[C@H]2C1
InChIKey: OZWRVBXQKWPWAA-OKILXGFUSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37 °C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30 °C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30 °C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 379.41, ALogP 2.04, HBA 5, HBD 0, PSA 54.9, QED 0.78
Compound (SMILES): O=c1[nH]c2ccc(-c3cccc(OCC(O)CN4CCc5ccccc5C4)c3)cc2[nH]1
InChIKey: YZYBGQZLQHWYCP-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 > 101 nM (pChEMBL 6.995999813079834)
Descriptors: MW 415.49, ALogP 3.32, HBA 4, HBD 3, PSA 81.35, QED 0.45
Compound (SMILES): CC(C)(C)C1=NN(c2ccc(C(=O)O)cc2)C(c2ccccc2Br)C1
InChIKey: OEISQMKGRTXAIX-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23219
Assay: Colorimetric Assay: The activities of COX-1 and COX-2 were measured after the addition of arachidonic acid and incubation at 25° C. for 5 min by absorbance at 590 nm as specified by the manufacturer.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 401.3, ALogP 5.5, HBA 3, HBD 1, PSA 52.9, QED 0.74
Compound (SMILES): O=C(c1ccc(F)cc1)C1CCN(CC(=O)N(CCF)Cc2nc3c(c(=O)[nH]2)COCC3)CC1
InChIKey: NSJLEHJIFVCZLL-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H2K2
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 474.51, ALogP 1.87, HBA 6, HBD 1, PSA 95.6, QED 0.59
Compound (SMILES): CC1(C)COCN1C(=O)c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1
InChIKey: KMMFANKYXNCAKF-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37 °C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30 °C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30 °C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 367.4, ALogP 2.53, HBA 5, HBD 0, PSA 54.9, QED 0.8
Compound (SMILES): CC(C)C(O)c1ccc([C@@H]2[C@@H](CCCCCCC(=O)O)[C@H](Cl)C[C@H]2O)cc1
InChIKey: OPLWXPAXAJYKRO-ISXLOPNHSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Agonist activity at human recombinant EP2 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 396.96, ALogP 4.87, HBA 3, HBD 3, PSA 77.76, QED 0.39
Compound (SMILES): Cc1c(C(=O)N2CCc3c(nnn3-c3ncccn3)C2C)cccc1C(F)(F)F
InChIKey: URMMXPJYAWCRNC-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
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 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 402.38, ALogP 3.14, HBA 6, HBD 0, PSA 76.8, QED 0.66
Compound (SMILES): O=C(Nc1ccc(C2=CCN(C(=O)c3ccccc3)CC2)cc1)N1Cc2ccccc2C1
InChIKey: PSTGFUAJRVBSAV-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 423.52, ALogP 5.16, HBA 2, HBD 1, PSA 52.65, QED 0.63
Compound (SMILES): Cc1cc(Nc2cc(N3CCN(C)CC3)nc(NCc3cc(-c4ccccc4)no3)n2)[nH]n1
InChIKey: BVCJBHICKYJTIS-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 445.53, ALogP 3.27, HBA 9, HBD 3, PSA 111.03, QED 0.39
Compound (SMILES): COc1cc2c(Nc3ccc(F)c(Cl)c3F)ncnc2cc1OC[C@@H]1CN2CCC[C@H]2CO1
InChIKey: AFHNMWQBLLGGSP-KBPBESRZSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 476.91, ALogP 4.56, HBA 7, HBD 1, PSA 68.74, QED 0.52
Compound (SMILES): COCCC(C(=O)Nc1ccc2[nH]ncc2c1)n1cc(OC)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: KHTRQHPDTKFMGV-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of plasma kallikrein from Kordia (0.6 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the substrate H-Pro-Phe-Arg-AMC dissolved in assay buffer (10 μl in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 491.94, ALogP 4.14, HBA 7, HBD 2, PSA 122.03, QED 0.38
Compound (SMILES): COc1cc(OC)cc(N(CCCN)c2ccc3ncc(-c4cnn(CCO)c4)nc3c2)c1
InChIKey: XAJGMXLLRBDBQT-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 448.53, ALogP 2.99, HBA 9, HBD 2, PSA 111.55, QED 0.38
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCN(Cc2cccnc2)CC1
InChIKey: OWPSKDFJWAQALV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF 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 uL of 1.11x enzyme and 1.11x substrate in 1x 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 room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x 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.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 433.49, ALogP 3.16, HBA 7, HBD 2, PSA 112.86, QED 0.59
Compound (SMILES): CCOC(=O)Nc1ccc(-c2nc(N3CC4CCC(C3)O4)nc3c2C(=O)N(CC)CC(C)(C)O3)cc1
InChIKey: LMRJUIZQGMIRPB-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P27986
Assay: Enzyme Assay: The test uses a lucifeurerin/luciferase system to measure the concentration of ATP and its consumption during the enzymatic reaction. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 μl. To 1 μl of inhibitor in 100% DMSO are added (final concentrations) 50 μM of the substrate PIP2 ((L-α-phosphatidyl-D-myoinositol 4,5-bisphosphate, Echelon 117P-4516-0500), 2 μM of ATP and 1.7 μg/ml of PI3Kα, (p110α/p85α, Invitrogen PV4788) in a buffer of Tris/HCl 50 mM pH 7.5, EGTA 1 mM, MgCl2 10 mM, Chaps 0.03%, 1 mM DTT). After 90 minutes, the reaction is quenched by adding 20 μl/well of KinaseGlo reagent (Promega V6713). After 10 minutes in the dark, the luminescence is read on the PHERAStar microplate reader (reading at 0.8 sec/well).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 495.58, ALogP 3.71, HBA 8, HBD 1, PSA 106.12, QED 0.67
Compound (SMILES): O=C(Nc1ccc(C2=CCN(C(=O)c3ccncc3)CC2)cc1)N1Cc2ccccc2C1
InChIKey: AWGHWZRDPKQBTO-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 21.1 nM (pChEMBL 7.679999828338623)
Descriptors: MW 424.5, ALogP 4.56, HBA 3, HBD 1, PSA 65.54, QED 0.67
Compound (SMILES): COCCN1CC(C)(C)Oc2nc(N3CC4CCC(C3)O4)nc(-c3ccc(NC(=O)Nc4ccon4)cc3)c2C1=O
InChIKey: PQGNIMOCUQJMLS-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P27986
Assay: Enzyme Assay: The test uses a lucifeurerin/luciferase system to measure the concentration of ATP and its consumption during the enzymatic reaction. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 μl. To 1 μl of inhibitor in 100% DMSO are added (final concentrations) 50 μM of the substrate PIP2 ((L-α-phosphatidyl-D-myoinositol 4,5-bisphosphate, Echelon 117P-4516-0500), 2 μM of ATP and 1.7 μg/ml of PI3Kα, (p110α/p85α, Invitrogen PV4788) in a buffer of Tris/HCl 50 mM pH 7.5, EGTA 1 mM, MgCl2 10 mM, Chaps 0.03%, 1 mM DTT). After 90 minutes, the reaction is quenched by adding 20 μl/well of KinaseGlo reagent (Promega V6713). After 10 minutes in the dark, the luminescence is read on the PHERAStar microplate reader (reading at 0.8 sec/well).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 563.62, ALogP 3.4, HBA 10, HBD 2, PSA 144.18, QED 0.44
Compound (SMILES): CO[C@]1(C)CC[C@@H](CC(=O)N[C@H]2CC[C@H](CCN3CCC(c4noc5cc(Cl)ccc45)CC3)CC2)CC1
InChIKey: YUMQWMQAMSUQRF-QLVKKYPMSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Radioligand Binding Assay: Aliquots of membrane preparations were thawed at RT, resuspended in assay buffer (D2, D3: 50 mM Tris-HCl, 120 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl2, pH=7.4; 5-HT2A: 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, pH=7.4), homogenized with a Polytron for 20-30 sec at 12,000 rpm and adjusted to a final concentration of approximately 7.5 ug protein/well (D2, D3) and 15 ug protein/well (5-HT2A), respectively.The binding affinity (Ki) of the compounds was determined using radioligand binding. Membranes were incubated in a total volume of 200 ul with a fixed concentration of radioligand (final concentration approximately 0.7 nM [3H]-spiperone for D2, 0.5 nM [3H]-spiperone for D3, and 1.1 nM [3H]-ketanserin for 5-HT2A) and ten concentrations of test compound in ranging between 10 uM-0.1 nM for 1 h at RT. At the end of the incubation, the reaction mixtures were filtered on to unifilter 96-well white microplates with bonded GF/C filters.
Measurement: Ki = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 530.15, ALogP 6.71, HBA 5, HBD 1, PSA 67.6, QED 0.41
Compound (SMILES): Fc1ccc([C@@H]2C[C@H]2CN[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: UWOMMJXAXRDMEO-DUPRYPCISA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 404.49, ALogP 4.16, HBA 5, HBD 2, PSA 70.9, QED 0.52
Compound (SMILES): Nc1ncc(-c2csc(CN3CCOCC3)c2)cc1C(=O)Nc1ccncc1
InChIKey: YXNUVQPEHUKSLS-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: IKKepsilon Flashplate Assay: The kinase assay is performed either as 384-well Flashplate assay (for e.g. Topcount measurement). 1 nM IKKepsilon, 800 nM biotinylated IkappaBalpha(19-42) peptide (Biotin-C6-C6-GLKKERLLDDRHDSGLDSMKDEE) SEQ ID NO: 1 and 10 uM ATP (spiked with 0.3 uCi 33P-ATP/well) are incubated in a total volume of 50 ul (10 mM MOPS, 10 mM Mg-acetat, 0.1 mM EGTA, 1 mM Dithiothreitol,0.02% Brij35, 0.1% BSA, 0.1% BioStab, pH 7.5) with or without test compound for 2 hours at 30° C. The reaction is stopped with 25 ul 200 mM EDTA. After 30 Min at room temperature the liquid is removed and each well washed thrice with 100 ul 0.9% sodium chloride solution. Non-specific reaction is determined in presence of 3 uM MSC2119074 (BX-795). Radioactivity is measured with Topcount (PerkinElmer).
Measurement: IC50 = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 395.49, ALogP 2.87, HBA 7, HBD 2, PSA 93.37, QED 0.69
Compound (SMILES): CC(C)c1cnc(N[C@H]2CCC[C@@H]2NC(=O)c2ccccc2-n2nccn2)cn1
InChIKey: JMIWZAWCWVRGMD-IRXDYDNUSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 391.48, ALogP 2.94, HBA 7, HBD 2, PSA 97.62, QED 0.67
Compound (SMILES): CN1CCN(c2ccc(C(=O)Nc3n[nH]c4cc(Oc5cccc(F)c5)ccc34)cc2)CC1
InChIKey: YHSKLCIRXOOVQC-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Assay: The assay has been performed using KIT cytoplasmic domain product and purified in house as GST fused protein. The KIT protein (4.5 microM) was pre activated with 300 microM ATP for 1 hour at 28*C in order to obtain a linear kinetic. Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 5 mM MgCl2, 1 mM MnCl2, 10 mM DTT, 3 microM Na3VO4, and 0.2 mg/mL BSA. The KIT kinase assay was run with a final pre activated enzyme concentration of 4 nM, in the presence of 4.4 microM ATP (residual ATP from KIT pre activation step is negligible), 3.9 nM 33P-γ-ATP and 2.5 microM of substrate BioDB n*138 (Aminoacidic sequence: KWEEINGNNYVYIDPTQLPYDHKWEFPRNR- SEQ ID NO: 2).
Measurement: IC50 = 564 nM (pChEMBL 6.25)
Descriptors: MW 445.5, ALogP 4.5, HBA 5, HBD 2, PSA 73.49, QED 0.47
Compound (SMILES): CC1([C@@H]2CC(=O)N(CCOc3ccc(F)c(C#N)c3)C2)CCS(=O)(=O)CC1
InChIKey: SYEMWCXBADSSGE-OAHLLOKOSA-N
Target: Platelet-activating factor acetylhydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q13093
Assay: Inhibition of recombinant human Lp-PLA2 pre-incubated for 30 mins before PED6 fluorogenic substrate
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 394.47, ALogP 2.14, HBA 5, HBD 0, PSA 87.47, QED 0.76
Compound (SMILES): CNC(=O)c1c(C2CC2(C)C)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CO4)cc12
InChIKey: SIFXXVBVKHGGSE-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhbition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 588.66, ALogP 5.87, HBA 7, HBD 1, PSA 106.67, QED 0.28
Compound (SMILES): COc1ccc2[nH]cc(CCn3cc(NC(=O)c4ncoc4-c4cccc(C)c4)cn3)c2n1
InChIKey: OWUGHOGLSOEBHT-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Antagonist activity at human OX1R expressed in CHO cells assessed as orexin A-induced intracellular Ca2+ mobilization preincubated for 120 mins followed by orexin A addition by FLIPR assay
Measurement: IC50 = 619 nM (pChEMBL 6.210000038146973)
Descriptors: MW 442.48, ALogP 4.23, HBA 7, HBD 2, PSA 110.86, QED 0.39
Compound (SMILES): CCCCCC(=O)N[C@H]1CCC(=O)NCC[C@@H](C(=O)N[C@@H](Cc2c[nH]c3ccccc23)C(N)=O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC1=O
InChIKey: ZOFPYCCSYGWTCN-WRQFUQGOSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
Assay: Competitive Inhibition Binding Assay: A competitive inhibition binding assay was performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMCR-1a or hMCR-4 (in each instance where the h prefix refers to human), or alternatively membrane homogenates from B16-F10 mouse melanoma cells containing endogenous murine MCR-1. In the examples that follow, all MCR-1 and MCR-4 values are for human recombinant receptors, unless otherwise noted. Assays were performed in 96 well polypropylene round-bottom plates (VWR catalog number 12777-030). Membrane homogenates were incubated with 0.1 nM [I125]-NDP-α-MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.3 mM 1,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for 90 minutes at 37° C., the assay mixture was filtered onto GF/B Unifilter plates (Perkin-Elmer catalog number 6005177) and washed with 3 mL of ice-cold buffer per well. Filters were air dried and 35 uL of scintillation cocktail added to each well. Plates were counted in a Microbeta counter for bound radioactivity. Non-specific binding was measured by inhibition of binding of [I125]-NDP-α-MSH in the presence of 1 uM NDP-α-MSH. Maximal specific binding (100%) was defined as the difference in radioactivity (cpm) bound to cell membranes in the absence and presence of 1 uM NDP-α-MSH. Each assay was conducted in duplicate and the actual mean values are described, with results less than 0% reported as 0%. Ki values for peptides of the present invention were determined using Graph-Pad Prism curve-fitting software.
Measurement: Ki = 0.025 nM (pChEMBL 10.600000381469727)
Descriptors: MW 953.12, ALogP -0.54, HBA 10, HBD 13, PSA 353.16, QED 0.03
Compound (SMILES): CCCC1(C(O)c2cccc([C@@H]3[C@@H](CCCCCCC(=O)O)[C@H](Cl)C[C@H]3O)c2)CCC1
InChIKey: QJVZVQODDFMWMY-BBPSQCEISA-N
Target: Prostaglandin E2 receptor EP3 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43115
Assay: Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Measurement: EC50 = 3118 nM (pChEMBL 5.510000228881836)
Descriptors: MW 451.05, ALogP 6.19, HBA 3, HBD 3, PSA 77.76, QED 0.26
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1cc(N(Cc2cccnc2)C(=O)Nc2c(Cl)c(OC)cc(OC)c2Cl)ncn1
InChIKey: IOCUPPAUQZXBRS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Kinase HotSpotâ¿¢ Assay: Compounds were profiled for FGFR inhibition activity at Reaction Biology Corporation (Malvern, Pa.) with their Kinase HotSpot assay. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.Recombinant FGFR1 (2.5 nM), FGFR2 (1 nM), FGFR3 (5 nM), or FGFR4 (12 nM) (Invitrogen) was prepared as a mixture with substrate KKKSPGEYVNIEFG (SEQ ID NO:1) (20 uM, FGFR1 substrate); and Poly [E,Y]4:1 (0.2 mg/ml, FGFR2,3,4 substrate)] in kinase reaction buffer (20 mM HEPES-HCl, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij35, 0.1 mM Na3VO4, 0.02 mg/ml BSA, 2 mM DTT, and 1% DMSO). Compound was added to the enzyme/substrate mixture using acoustic technology (Labcyte Echo 550, Sunnyvale, Calif.) (see, Olechno et al., 2006, Improving IC50 results with acoustic droplet ejection. JALA 11, 240-246) and pre-incubated for 0, 15, or 60 minutes at room temperature. After compound pre-incubation, a mixture of ATP (Sigma-Aldrich) and 33P-γ-ATP (PerkinElmer) was added to a final concentration of 10 uM to initiate kinase reactions. Reactions were incubated for 120 minutes at room temperature and then spotted onto Whatman P81 ion exchange filter paper. Unbound phosphate was removed by extensively washing filters in 0.75% phosphoric acid. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.
Measurement: IC50 = 720 nM (pChEMBL 6.139999866485596)
Descriptors: MW 594.46, ALogP 6.3, HBA 8, HBD 3, PSA 130.6, QED 0.18
Compound (SMILES): CC(C)(C)NC(=O)C1(c2ccccc2)CCN(c2ncnc3c2nc(-c2ccccc2Cl)n3-c2ccc(Cl)cc2)CC1
InChIKey: FXJQCXUIMZHDEI-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Radioligand Displacement Assay (CP55940): Further characterization of select compounds was performed using radioligand displacement of [3H]1 and equilibrium dissociation constant (Ki) values were determined. Selectivity of these compounds at CB1 versus CB2 was also determined by obtaining Ki values at either receptor using displacement of [3H]CP55940 in membranes of CHO-K1 cells over-expressing either receptor. Data reported are average values from 3-6 measurements.
Measurement: Ki = 20000 nM (pChEMBL 4.699999809265137)
Descriptors: MW 599.57, ALogP 7.24, HBA 6, HBD 1, PSA 75.94, QED 0.23
Compound (SMILES): CNC(=O)[C@H](CC(C)C)NC(=O)c1cccc(-c2cccc(F)c2)n1
InChIKey: BWIKOKDINCKYTF-KRWDZBQOSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 343.4, ALogP 2.78, HBA 3, HBD 2, PSA 71.09, QED 0.85
Compound (SMILES): Cn1cc(-c2cc(N3CCCS3(=O)=O)ccc2Oc2ccc(F)cc2F)c2cc[nH]c2c1=O
InChIKey: LBXQLGRAZRCPAY-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 471.49, ALogP 4.14, HBA 5, HBD 1, PSA 84.4, QED 0.48
Compound (SMILES): C[C@H](Oc1cc(C(N)=O)nc(-c2ccc(Oc3ccc(Cl)cc3F)cc2)n1)C(N)=O
InChIKey: TZZDBEDTIJHZJP-JTQLQIEISA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR Assay: Assay Buffer: The assay buffer was formulated by removing 120 mL from a 1 L bottle of fresh, sterile dH2O (Mediatech, Herndon, Va.) and adding 100 mL of 10×HBSS that does not contain Ca++ or Mg++ (Gibco, Invitrogen, Grand Island, N.Y.) followed by 20 mL of 1.0 M Hepes, pH 7.3 (Fisher Scientific, BP299-100). The final buffer consisted of 20 mM Hepes, pH 7.3, 1.261 mM CaCl2, 0.493 mM MgCl2, 0.407 mM Mg(SO)4, 5.33 mM KCl, 0.441 mM KH2PO4, 137 mM NaCl, 0.336 mM Na2HPO4 and 0.556 mM D-glucose (Hanks et al., Proc. Soc. Exp. Biol. Med. 71:196 (1949)), and the simple formulation was typically the basic buffer throughout the assay (i.e., all wash and addition steps).
Measurement: IC50 = 83 nM (pChEMBL 7.079999923706055)
Descriptors: MW 430.82, ALogP 3.08, HBA 6, HBD 2, PSA 130.42, QED 0.59
Compound (SMILES): O=C(Oc1ccc2c(c1)CCC2)N(Nc1ccccc1)S(=O)(=O)c1ccc(Cl)cc1
InChIKey: DEFZOWRYMRTWHT-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 1 (SINGLE PROTEIN), Ovis aries
UniProt: P05979
Assay: Inhibition of ovine COX1 by enzyme immunoassay
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 442.92, ALogP 5.05, HBA 5, HBD 1, PSA 75.71, QED 0.56
Compound (SMILES): O=C1NC(=S)S/C1=C\c1ccc2c(c1)N(Cc1ccc(F)c(Cl)c1)C(=O)CO2
InChIKey: KWDKVVZOYYHCRG-APSNUPSMSA-N
Target: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00750
Assay: Inhibition of PI3KC2beta (unknown origin) expressed in HEK293 cells using phosphatidylinositol and gamma-32P-ATP incubated for 20 mins by TLC assay or high-throughput membrane capture assay
Measurement: IC50 = 370 nM (pChEMBL 6.429999828338623)
Descriptors: MW 434.9, ALogP 3.89, HBA 5, HBD 1, PSA 58.64, QED 0.59
Compound (SMILES): CC(C)(C)Nc1cnc2cccc(-c3cc4n(c3)CCNC4=O)c2n1
InChIKey: GIZDEODKRGICNS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 335.41, ALogP 3.05, HBA 5, HBD 2, PSA 71.84, QED 0.75
Compound (SMILES): Cc1nn(C)c(C)c1N(C)S(=O)(=O)c1ccc(CCCN(C)C)cc1
InChIKey: OXWRQYIDBMXMPC-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30419
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 364.52, ALogP 2.36, HBA 5, HBD 0, PSA 58.44, QED 0.76
Compound (SMILES): Cc1cnc(O)c(-c2cc3c(cn2)cnn3-c2cncc(N3CCCNCC3)n2)c1
InChIKey: ZLBGPWHOXABCIP-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 527 nM (pChEMBL 6.28000020980835)
Descriptors: MW 402.46, ALogP 2.09, HBA 9, HBD 2, PSA 104.88, QED 0.54
Compound (SMILES): Cn1nc(-c2ccc(F)cc2)c(-c2ccc3nc(NC(=O)c4ccnc(N5CCOCC5)c4)cn3n2)c1N1CCN(C(=O)CC(F)(F)F)CC1
InChIKey: MHRHPSXNYRHWIU-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 692.68, ALogP 4.02, HBA 11, HBD 1, PSA 126.02, QED 0.25
Compound (SMILES): CNC(=O)c1c(-c2ccc(OC3CC3)nc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CC4)cc12
InChIKey: ZEHBQEBUDKOCMZ-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 2.299 nM (pChEMBL 8.640000343322754)
Descriptors: MW 651.72, ALogP 6.17, HBA 8, HBD 1, PSA 119.56, QED 0.22
Compound (SMILES): Cc1ccc(Cl)cc1NC(=O)N1CCC(N2CC(CC#N)(n3cc(-c4ncnc5[nH]ccc45)cn3)C2)CC1
InChIKey: ITRRPSKNDSTOKN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of human N-terminal His-tagged JAK2 catalytic domain (828 to 1132 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 10 nM (pChEMBL 8)
Descriptors: MW 530.04, ALogP 4.4, HBA 7, HBD 2, PSA 118.76, QED 0.4
Compound (SMILES): Cc1c(CC(=O)O)c2cccnc2n1Cc1ccc(S(=O)(=O)C(C)C)cc1
InChIKey: XMCRMQJEYLGRKF-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Antagonist activity against human CRTh2 expressed in CHO-K1 cells assessed as inhibition of PGD2-mediated attenuation of forskolin-induced cAMP accumulation incubated for 60 mins by scintillation counting method
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 386.47, ALogP 3.2, HBA 5, HBD 1, PSA 89.26, QED 0.7
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ccc(F)cn1)c1nc2c(F)ccc(F)c2c(=O)n1-c1cc(F)cc(F)c1
InChIKey: GYLKQJYQGVMLDU-ZDUSSCGKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 549.46, ALogP 4.42, HBA 8, HBD 2, PSA 111.61, QED 0.25