lovingscience/chembl-v37-raw-activities huggingface dataset:
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 JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 > 1000 nM (pChEMBL 6)
Descriptors: MW 404.49, ALogP 4.16, HBA 5, HBD 2, PSA 70.9, QED 0.52
Compound (SMILES): CC1([C@@H]2CC(=O)N(CCOc3ccc(F)c(C#N)c3)C2)CCS(=O)(=O)CC1
InChIKey: SYEMWCXBADSSGE-OAHLLOKOSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of CYP3A4 (unknown origin) expressed in Escherichia coli co-expressing human NADPH reductase using both 7-benzyloxyquinolone and diethoxyfluorescein substrate in presence of NADPH regenerating system incubated for 10 mins by spectrofluorometry
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 394.47, ALogP 2.14, HBA 5, HBD 0, PSA 87.47, QED 0.76
Compound (SMILES): COc1ccc2[nH]cc(CCn3cc(NC(=O)c4ncoc4-c4cccc(C)c4)cn3)c2n1
InChIKey: OWUGHOGLSOEBHT-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20'000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 °C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37 °C. in 5% CO2 followed by equilibration at RT for 30 min before measurement. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μl/well, incubated for 120 min and finally 10 μl/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 442.48, ALogP 4.23, HBA 7, HBD 2, PSA 110.86, QED 0.39
Compound (SMILES): O=C(Oc1ccc2c(c1)CCC2)N(Nc1ccccc1)S(=O)(=O)c1ccc(Cl)cc1
InChIKey: DEFZOWRYMRTWHT-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35354
Assay: Inhibition of recombinant human COX2 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: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of PI3Kalpha/p85alpha (unknown origin) using phosphatidylinositol and gamma-32P-ATP incubated for 20 mins by TLC assay or high-throughput membrane capture assay
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 434.9, ALogP 3.89, HBA 5, HBD 1, PSA 58.64, QED 0.59
Compound (SMILES): Cc1cnc(O)c(-c2cc3c(cn2)cnn3-c2cncc(N3CCCNCC3)n2)c1
InChIKey: ZLBGPWHOXABCIP-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
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 = 14.8 nM (pChEMBL 7.829999923706055)
Descriptors: MW 402.46, ALogP 2.09, HBA 9, HBD 2, PSA 104.88, QED 0.54
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 JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
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: Displacement of [3H]PGD2 from human DP2 receptor expressed in CHO cell membranes after 60 mins by scintillation proximity assay
Measurement: Ki = 86 nM (pChEMBL 7.070000171661377)
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 delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
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 < 100 nM (pChEMBL 7)
Descriptors: MW 549.46, ALogP 4.42, HBA 8, HBD 2, PSA 111.61, QED 0.25
Compound (SMILES): N#Cc1ccc(N[C@H]2CC[C@@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: FRJMJEGDBSCIBR-FZNQNYSPSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 = 1000 nM (pChEMBL 6)
Descriptors: MW 357.42, ALogP 3.62, HBA 6, HBD 2, PSA 94.69, QED 0.58
Compound (SMILES): O=C(COc1cccc(F)c1)NCC(O)CN1CCc2ccccc2C1
InChIKey: DYLGANMZPUAKDT-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay: Molecular Biology: Full-length human PRMT5 (NM_006109.3) transcript variant 1 clone was amplified from a fetal brain cDNA library, incorporating flanking 5′ sequence encoding a FLAG tag (MDYKDDDDK) (SEQ ID NO.:4) fused directly to Ala 2 of PRMT5. Full-length human MEP50 (NM_024102) clone was amplified from a human testis cDNA library incorporating a 5′ sequence encoding a 6-histidine tag (MHHHHHH) (SEQ ID NO.:5) fused directly to Arg 2 of MEP50. The amplified genes were sublconed into pENTR/D/TEV (Life Technologies) and subsequently transferred by Gateway attL×attR recombination to pDEST8 baculvirus expression vector (Life Technologies).Protein Expression. Recombinant baculovirus and Baculovirus-Infected Insect Cells (BIIC) were generated according to Bac-to-Bac kit instructions (Life Technologies) and Wasilko, 2006, respectively. Protein over-expression was accomplished by infecting exponentially growing Spodoptera frugiperda (SF9) cell culture at 1.2×106 cell/ml with a 5000 fold dilution of BIIC stock. Infections were carried out at 27° C. for 72 hours, harvested by centrifugation, and stored at −80° C. for purification.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 358.41, ALogP 1.74, HBA 4, HBD 2, PSA 61.8, QED 0.79
Compound (SMILES): O=S(=O)(Nc1ncns1)c1ccc2c(c1)OCCN2c1ccc(C(F)(F)F)cc1C1=CCNCC1
InChIKey: BHJSCRNVDBKWGW-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 349 nM (pChEMBL 6.460000038146973)
Descriptors: MW 523.56, ALogP 4.26, HBA 8, HBD 2, PSA 96.45, QED 0.52
Compound (SMILES): CC(C)CS(=O)(=O)NCc1cccc(-n2c([C@H](C)Nc3ncnc(N)c3C#Cc3ccc(F)cn3)nc3cccc(Cl)c3c2=O)c1
InChIKey: PPLLJHIOPMUCFQ-FQEVSTJZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
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 < 100 nM (pChEMBL 7)
Descriptors: MW 661.16, ALogP 4.59, HBA 10, HBD 3, PSA 157.78, QED 0.19
Compound (SMILES): Nc1nc(Nc2ccccc2)nc2sc(-c3ccco3)nc12
InChIKey: YEXYVRKJKQADJF-UHFFFAOYSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Displacement of [125I]AB-MECA from human adenosine A3 receptor expressed in CHO cell membranes measured after 120 mins by scintillation counting method
Measurement: Ki = 321 nM (pChEMBL 6.489999771118164)
Descriptors: MW 309.35, ALogP 3.67, HBA 7, HBD 2, PSA 89.86, QED 0.6
Compound (SMILES): CN(C)CCOc1ccc(Nc2cc(-n3ccnc3)ncn2)nc1
InChIKey: PKZBARFMXVRZEC-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: Inhibition of recombinant full-length human C-terminal His6-tagged CDK9/full-length human cyclin T1 expressed in baculovirus infected insect Sf21 cells using KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC as substrate in presence of [gamma-33P]-ATP after 40 mins by scintillation counting analysis
Measurement: IC50 = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 325.38, ALogP 1.74, HBA 8, HBD 1, PSA 80.99, QED 0.71
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4cc(O)cc(F)c4)c4c(N)ncnc43)n(-c3ccccc3O)c(=O)c12
InChIKey: XOZCRBUNRWGVCR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 508.51, ALogP 4.29, HBA 9, HBD 3, PSA 132.08, QED 0.32
Compound (SMILES): CCN(c1ccc2ncc(-c3cnn(C)c3)nc2c1)c1cc(OC)cc(OC)c1Cl
InChIKey: RKRDVEBFGMJINX-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of TEL-fused FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 72 hrs by Alamar blue assay
Measurement: IC50 = 12.88 nM (pChEMBL 7.889999866485596)
Descriptors: MW 423.9, ALogP 4.86, HBA 7, HBD 0, PSA 65.3, QED 0.44
Compound (SMILES): COc1cc(N2CCC(N3CCSCC3)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: XRMQCRQMFWASRC-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 732.86, ALogP 7.76, HBA 10, HBD 2, PSA 99.92, QED 0.15
Compound (SMILES): Cc1ccc(CSc2ccc(S(N)(=O)=O)s2)cc1
InChIKey: WEHHDTIKPDFLTK-UHFFFAOYSA-N
Target: Carbonic anhydrase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16790
Assay: Inhibition of human recombinant transmembrane tumor associated carbonic anhydrase 9 preincubated for 10 mins prior to testing by stopped-flow CO2 hydration assay
Measurement: Ki = 10.5 nM (pChEMBL 7.980000019073486)
Descriptors: MW 299.44, ALogP 3, HBA 4, HBD 1, PSA 60.16, QED 0.88
Compound (SMILES): C[C@H](Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1cc(F)c(OCC(F)(F)F)cc1F
InChIKey: AIKWSLRSPJRXMX-IMSIIYSGSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R(−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM. Curve fitting for dose response IC50 determinations was done using a 4-parameter logistic model: y=min+((max−min)/1+(x/IC50)slope).
Measurement: IC50 = 106 nM (pChEMBL 6.96999979019165)
Descriptors: MW 462.38, ALogP 3.58, HBA 7, HBD 2, PSA 96.81, QED 0.61
Compound (SMILES): Nc1ccc(-c2ccccc2)cc1NC(=O)c1ccc2c(cnn2CCN2CCOCC2)c1
InChIKey: ABBMQAYQRRWEKO-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC Enzyme Assay: Compounds for testing were diluted in DMSO to 50 fold the final concentration and a ten point three fold dilution series was made. The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM tris(2-carboxyethyl)phosphine) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer and pre-incubated with the compounds for 24 hours prior to addition of the substrate. The substrate tripeptide substrate 3 (synthesized in house) for each enzyme was equal to the Km as determined by a substrate titration curve. The enzyme and substrate concentrations used are given in Table 2. The substrates were diluted in assay buffer at 6x their final concentration with 0.3 uM sequencing grade trypsin (Sigma). The substrate/trypsin mix was added to the enzyme/compound mix, the plate was shaken for 60 seconds.
Measurement: IC50 = 344 nM (pChEMBL 6.460000038146973)
Descriptors: MW 441.54, ALogP 3.87, HBA 6, HBD 2, PSA 85.41, QED 0.44
Compound (SMILES): CC[C@@H]1CN(c2nnc(Cc3ccccc3)c(C)c2C)CCN1c1cnc(C(C)=O)cn1
InChIKey: YZLNTKQPNGTLIH-OAQYLSRUSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 430.56, ALogP 3.78, HBA 7, HBD 0, PSA 75.11, QED 0.55
Compound (SMILES): Cc1cccc2cc(C(C)Nc3ncnc4[nH]cnc34)n(C3CCC3)c(=O)c12
InChIKey: OVBKKAQIDXMNIF-UHFFFAOYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 374.45, ALogP 3.87, HBA 6, HBD 2, PSA 88.49, QED 0.57
Compound (SMILES): CNC1CCN(CCCN(c2cc(OC)cc(OC)c2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)C1.Cl
InChIKey: ACJGNAWZBBEJBZ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 2754.23 nM (pChEMBL 5.559999942779541)
Descriptors: MW 501.64, ALogP 3.87, HBA 9, HBD 1, PSA 80.57, QED 0.35
Compound (SMILES): CCn1nc(-c2cccc(-c3cc(C(=O)NCCN4CCCCC4)cc(C(=O)NC4CC4)c3)c2)cc(NC(=O)Nc2c(Cl)cncc2Cl)c1=O
InChIKey: PZPNBJPGEXHLCH-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Antagonist activity at adenosine A1 receptor (unknown origin)
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 717.66, ALogP 6.05, HBA 8, HBD 4, PSA 150.35, QED 0.15
Compound (SMILES): N[C@H]1CCCN(c2ccncc2Nc2ncc3ccc(-c4ccccc4F)nn23)C1
InChIKey: KWQWZDJFPORDSD-HNNXBMFYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Enzyme Assay: 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 96-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 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate.
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 403.47, ALogP 3.6, HBA 7, HBD 2, PSA 84.37, QED 0.54
Compound (SMILES): N#CC1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)CC1
InChIKey: LFSSROFVBXERLJ-WKILWMFISA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 > 1000 nM (pChEMBL 6)
Descriptors: MW 363.47, ALogP 3.11, HBA 6, HBD 1, PSA 85.9, QED 0.77
Compound (SMILES): C/C1=C\C[C@]2(C(C)C)CC[C@@](C)(O2)[C@H](O)CC/C(C)=C/CC1
InChIKey: SSBZLMMXFQMHDP-XAUKRSCVSA-N
Target: Signal transducer and activator of transcription 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P40763
Assay: Inhibition of IFNgamma-induced STAT3 activation in human HeLa cells expressing STAT3 (unknown origin) preincubated for 15 mins followed by IFN-gamma addition measured after 6 hrs by luciferase reporter gene assay
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 306.49, ALogP 5.17, HBA 2, HBD 1, PSA 29.46, QED 0.68
Compound (SMILES): COc1ccc(C(=O)n2c(C3CCCCC3)nc3ccccc32)cc1
InChIKey: ZVMFOTLGDWTGMA-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Displacement of [3H]CP-55940 from human recombinant CB1 receptor expressed in HEK293 cell membranes incubated for 1.5 hrs
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 334.42, ALogP 4.78, HBA 4, HBD 0, PSA 44.12, QED 0.69
Compound (SMILES): Cn1c(SCCCN2CC[C@]3(C[C@@H]3c3ccc(C(F)(F)F)cc3)C2)nnc1-c1ccc(C#N)nc1
InChIKey: LMYXDMLXVWZVJJ-QPPBQGQZSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG transfected in HEK293 cells assessed as reduction in tail current by patch clamp assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 498.58, ALogP 5.13, HBA 7, HBD 0, PSA 70.63, QED 0.33
Compound (SMILES): COc1ccc(-n2nc(C(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc5[nH]nc(C(=O)N(C)C)c5c4)n3)c3ccccc23)cc1
InChIKey: MGVWPOMQHQAMAV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 696.77, ALogP 7.71, HBA 10, HBD 4, PSA 164.21, QED 0.11
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2ccc(C3CC3)c(N3CCCNCC3)n2)n1
InChIKey: JIEGLKQWELHCHA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
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 = 0.046 nM (pChEMBL 10.34000015258789)
Descriptors: MW 426.53, ALogP 3.26, HBA 8, HBD 1, PSA 84.65, QED 0.54
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(NC(=O)c3oc4cc(Cl)ccc4c3C)cc1C21N=C(C)C(N)=N1
InChIKey: HEZJDRFUHMYWFH-HWAYOUJDSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Diluted TR-FRET Assay: TR-FRET AssayThe beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 505.02, ALogP 5.76, HBA 6, HBD 2, PSA 102.21, QED 0.47
Compound (SMILES): O=S(=O)(NCC1CC1)c1ccc(NN2CCCCC2)cc1C(F)(F)F
InChIKey: SFRXOSJENFVHGX-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: Ki = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 377.43, ALogP 3.21, HBA 4, HBD 2, PSA 61.44, QED 0.8
Compound (SMILES): O=C(COc1ccccc1-c1cc[nH]n1)NCC(O)CN1Cc2ccccc2C1
InChIKey: REXFJVZJFKQPPR-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 7)
Descriptors: MW 392.46, ALogP 1.95, HBA 5, HBD 3, PSA 90.48, QED 0.55
Compound (SMILES): CN(C)c1ccc(-c2cc(=O)c3ccc(OCC(=O)NCCCCCCNc4c5c(nc6ccccc46)CCCC5)cc3o2)cc1
InChIKey: RCPICHNPPUOWKZ-UHFFFAOYSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Inhibition of self-mediated amyloid beta (1 to 42) (unknown origin) aggregation after 48 hrs by thioflavin T fluorescence assay
Measurement: IC50 = 9470 nM (pChEMBL 5.019999980926514)
Descriptors: MW 618.78, ALogP 7.12, HBA 7, HBD 2, PSA 96.7, QED 0.14
Compound (SMILES): COc1cc(OC)c(Cl)c(N(CCO)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: BAPFBTJYCKDGNL-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 851.14 nM (pChEMBL 6.070000171661377)
Descriptors: MW 439.9, ALogP 3.83, HBA 8, HBD 1, PSA 85.53, QED 0.47
Compound (SMILES): CN1CCN(c2ccc(Nc3nccc(-c4cc5c(o4)CCN(C(=O)CC#N)C5)n3)cc2)CC1
InChIKey: ZHUHRHZKZBJUHT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of JAK2 in human TF1 cells assessed as suppression of cell growth
Measurement: IC50 = 2338 nM (pChEMBL 5.630000114440918)
Descriptors: MW 457.54, ALogP 3.03, HBA 8, HBD 1, PSA 101.53, QED 0.62
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2cc(OC)nc(OC)n2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: YXSZZTIBRREMPU-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 537.58, ALogP 4.04, HBA 11, HBD 0, PSA 109.54, QED 0.27
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ncccc1F)c1nc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: SYJNHFMRHQCGSB-INIZCTEOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
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 < 100 nM (pChEMBL 7)
Descriptors: MW 511.95, ALogP 4.52, HBA 8, HBD 2, PSA 111.61, QED 0.34
Compound (SMILES): COc1cc(OC)cc(N(c2ccc3ncc(-c4cnn(C)c4)nc3c2)[C@@H](C)CN)c1
InChIKey: SOYCCBHGJYSWNQ-HNNXBMFYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 418.5, ALogP 3.53, HBA 8, HBD 1, PSA 91.32, QED 0.49
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCOC[C@H]2Cc2cccc(-c3ncccn3)c2)c1
InChIKey: JHOLYWCJSQKKLV-OAQYLSRUSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 = 33000 nM (pChEMBL 4.480000019073486)
Descriptors: MW 440.51, ALogP 3.12, HBA 7, HBD 0, PSA 86.03, QED 0.47
Compound (SMILES): Cc1cccc2cc([C@H](C)Nc3ncnc4[nH]ccc34)n(-c3ccccc3)c(=O)c12
InChIKey: RYFJDPSDHUCQPR-INIZCTEOSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 395.47, ALogP 4.74, HBA 5, HBD 2, PSA 75.6, QED 0.46
Compound (SMILES): Cc1c(Cc2ccccc2)nnc(N2CCN(c3cnc(C(=O)NC(C)(C)CO)cn3)[C@H](C)C2)c1C
InChIKey: IIKAHVDOSHVIAM-GOSISDBHSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 489.62, ALogP 2.69, HBA 8, HBD 2, PSA 107.37, QED 0.52
Compound (SMILES): COc1cc(F)ccc1N1CCOc2cc(S(=O)(=O)Nc3nccs3)ccc21
InChIKey: IVDUSXLIMOPNIQ-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 5580 nM (pChEMBL 5.25)
Descriptors: MW 421.48, ALogP 3.62, HBA 7, HBD 1, PSA 80.76, QED 0.68
Compound (SMILES): CCN(CC)c1ccc(-c2csc(COc3cc4ncnc(Nc5ccc(Cl)c(Cl)c5)c4cc3OC)n2)cc1
InChIKey: XCMSAHCDUHXACI-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 580.54, ALogP 8.24, HBA 8, HBD 1, PSA 72.4, QED 0.18
Compound (SMILES): COc1ccc(C[C@@H]2COCCN2C(=O)c2ccccc2-n2nccn2)cc1-c1cnccn1
InChIKey: CZERAVZVKNLXOP-LJQANCHMSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 = 26000 nM (pChEMBL 4.579999923706055)
Descriptors: MW 456.51, ALogP 2.82, HBA 8, HBD 0, PSA 95.26, QED 0.44
Compound (SMILES): CC(CC(N)=O)(NC(=O)c1cc(OCCF)c(C2CC2)cn1)C1CC1
InChIKey: MSUFNCXRNKWDMI-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 349.41, ALogP 2.08, HBA 4, HBD 2, PSA 94.31, QED 0.71
Compound (SMILES): Cn1c(NC(=O)N2CCC(N3CC(CC#N)(n4cc(-c5ncnc6[nH]ccc56)cn4)C3)CC2)nc2ccccc21
InChIKey: LTSCRPSSSDSHSQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In vitro JAK Kinase Assay: The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.)
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 535.62, ALogP 3.33, HBA 9, HBD 2, PSA 136.58, QED 0.35
Compound (SMILES): N#Cc1ccc(CNC(=O)CCOCCN2CCN(c3ncccc3-c3ccc(F)cc3)CC2)cc1
InChIKey: DZHFAWUKQABZMD-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P50406
Assay: Displacement of [3H]-LSD from human 5-HT6 receptor expressed in HEK293 cell membranes after 1 hr by microbeta counting analysis
Measurement: Ki = 1621 nM (pChEMBL 5.789999961853027)
Descriptors: MW 487.58, ALogP 3.6, HBA 6, HBD 1, PSA 81.49, QED 0.44