lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=C1CCC(c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: DICHJZWSLJFPPU-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 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 253.31, ALogP 2.82, HBA 4, HBD 1, PSA 58.87, QED 0.68
Compound (SMILES): CCCCCCCC/C=C\CCCCCCCC(=O)NC1(/C(N)=N/O)CC1
InChIKey: SQAJCBDQTHDSCM-KTKRTIGZSA-N
Target: Sphingosine kinase 2 (SINGLE PROTEIN), Mus musculus
UniProt: Q9JIA7
Assay: Kinase Assay: Sphingosine kinase activity was assessed as described previously (Kharel, Y., Lee, S., Snyder, A. H., Sheasley-O'Neill, S. L., Morris, M. A., Setiady, Y., Zhu, R., Zigler, M. A., Burcin, T. L., Ley, K., Tung, K. S. K., Engelhard, V. H., Macdonald, T. L. and Lynch, K. R. Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720. J Biological Chemistry 280: 36865-36872 (2005)). High levels of human sphingosine kinase type 1 (mSK1) and mouse sphingosine kinase type 2 (mSK2) were expressed in Sf9 insect cells by infection with cognate baculoviruses. Crude homogenates were incubated with γ-[32P]ATP and 10 micromolar D-erythro-sphingosine in the presence of 100 micromolar concentrations of test compounds for 20 minutes at 37 C. The product, radiolabeled S1P, was isolated by extraction into organic solvents after acidification and displaced by thin layer chromatography.
Measurement: Ki = 50000 nM (pChEMBL 4.300000190734863)
Descriptors: MW 379.59, ALogP 5.42, HBA 3, HBD 3, PSA 87.71, QED 0.08
Compound (SMILES): O=C(O)c1cccc(S(=O)(=O)Nc2ccc(-c3nc4cc(NS(=O)(=O)c5cccc(C(=O)O)c5)ccc4o3)cc2)c1
InChIKey: FJXOTVQQPHYGGK-UHFFFAOYSA-N
Target: HSP60/HSP10 (PROTEIN COMPLEX), Homo sapiens
UniProt: P61604
Assay: Inhibition of human mitochondrial HSP60/HSP10 expressed in Escherichia coli DH5alpha/BL21 (DE3) assessed as inhibition of denatured MDH refolding preincubated for 10 mins followed by ATP addition measured after 40 to 60 mins by fluorescence based assay
Measurement: IC50 = 81000 nM (pChEMBL 4.090000152587891)
Descriptors: MW 593.6, ALogP 4.49, HBA 8, HBD 4, PSA 192.97, QED 0.19
Compound (SMILES): CCN(c1ccccc1)c1c(Cl)nc2ccc(C(O)(c3cccnc3)c3cccc(Cl)c3)cc2c1Cl
InChIKey: MDEOYKJUOUYVQF-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORγt Reporter Assay: The reporter assay was performed by transiently transfecting HEK293T cells with 5 μg of pBIND-RORγt LBD or pBIND-RORγt LBD-AF2 and 5 μg pGL4.31 (Promega Cat no. C935A) using Fugene 6 (Invitrogen Cat no. E2691) at a 1:6 ratio of DNA: Fugene 6 in a T-75 flask in which cells were at least 80% confluent. Twenty four hours after bulk transfection, cells were plated into 96-well plates at 50,000 cells/well in phenol-red free DMEM containing 5% Lipid Reduced FCS and Pen/Strep. Six hours after plating, cells were treated with compounds for 24 hours. Media was removed and cells were lysed with 50 μL 1x Glo Lysis Buffer (Promega). Dual Glo Luciferase Reagent (50 μL/well) was then added and firefly luciferase luminescence was read on an Envision after a ten minute incubation. Finally, Stop and Glo reagent (50 μL/well) was added and renilla luciferase luminescence was read on an Envision after a ten minute incubation.
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 534.87, ALogP 8.03, HBA 4, HBD 1, PSA 49.25, QED 0.22
Compound (SMILES): CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CO)NC(=O)CN)C(C)C)C(C)C)C(=O)O
InChIKey: DKDNNUOQAQZRAP-POMKDTFGSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Inhibition of C-terminus 10x-His-taged human TFPI-1 (29 to 282 residues) assessed as inhibition of factor 10a using S-2222 substrate incubated for 115 mins by micro-plate reader based assay
Measurement: EC50 = 850 nM (pChEMBL 6.070000171661377)
Descriptors: MW 2589.99
Compound (SMILES): CCOc1cc2ncnc(Nc3ccc4c(cnn4Cc4ccccc4)c3)c2cc1NC(=O)/C=C/CN1CCOCC1
InChIKey: WPKVIBFJECQBCV-RMKNXTFCSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Irreversible inhibition of GST-tagged ERBB2 (unknown origin) (Ile-675 to Val-1256 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 563.66, ALogP 5, HBA 9, HBD 2, PSA 106.43, QED 0.23
Compound (SMILES): CC(C)N1CC2=NNC(=O)c3cccc4[nH]c(c2c34)C1
InChIKey: PZDZBZWWQUDTKX-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: PARP-2 and PARP-3 Enzymatic Assay: PARP-2 and PARP-3 enzymatic assays were conducted using commercial PARP-2/PARP-3 Chemiluminescent Assay Kit (BPS Biosciences) and the protocols with the kits. Briefly, histones were coated in a high binding plate first, and incubated with PARP-2 or PARP-3, and increasing concentrations of compounds for 0.5 h. Then, biotinylated NAD and activated DNA were added to the wells. The biotinylated PARylation product was measured by adding streptavidin-HRP and HRP substrates which produce chemiluminescence.
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 268.32, ALogP 1.84, HBA 3, HBD 2, PSA 60.49, QED 0.83
Compound (SMILES): CC(C)[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)N(C)Cc1ccc(C(=O)Oc2ccc(C(=N)N)cc2F)s1
InChIKey: AEKXZFDUXYLPAD-HNAYVOBHSA-N
Target: Serine protease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P07477
Assay: Inhibition Assay: Using a 96 well plate (#3915, Costar), a test compound (25 μL), 400 mM Tris-HCl buffer (pH 8.0, 25 μL) and 0.5 mg/mL fluorescence enzyme substrate (Gly-Asp-Asp-Asp-Asp-Lys-β-Naphtylamide, 25 μL) were mixed, and recombinant human enteropeptidase (R&D Systems, Inc., 25 μL) was added. Using a fluorescence plate reader fmax (Molecular Devices, Inc.), the reaction rate was measured from the time-course changes at excitation wavelength 320 nm and fluorescence wavelength 405 nm. The Ki value was calculated from the concentration of the test compound, reciprocal of reaction rate, and Km value of the enzyme substrate, and by using Dixon plot.
Measurement: Ki = 0.68 nM (pChEMBL 9.170000076293945)
Descriptors: MW 522.56, ALogP 1.89, HBA 8, HBD 5, PSA 183.11, QED 0.12
Compound (SMILES): O=C(NCc1ccc(NS(=O)(=O)C2CCCC2)cc1)c1cc2cnccc2[nH]1
InChIKey: OMXUTMOTZCFLDJ-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Inhibition of NAMPT in human A2780 cells assessed as decrease in cell viability after 72 hrs by SRB assay
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 398.49, ALogP 3.18, HBA 4, HBD 3, PSA 103.95, QED 0.59
Compound (SMILES): O=S(=O)(NCC1CC1)c1ccc(NC2CN3CCC2CC3)cc1C(F)(F)F
InChIKey: FQXWTIPTBMKIOQ-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: IC50 = 696 nM (pChEMBL 6.159999847412109)
Descriptors: MW 403.47, ALogP 2.9, HBA 4, HBD 2, PSA 61.44, QED 0.77
Compound (SMILES): Cn1cc(-c2cnc3ccc(N(CC4CC4)c4ccccc4)cc3n2)cn1
InChIKey: VDVZOCBAFGDAKS-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 355.45, ALogP 4.58, HBA 5, HBD 0, PSA 46.84, QED 0.53
Compound (SMILES): O=C(NCC(c1cnc(C(F)(F)F)nc1)N1CCC(F)CC1)c1c(Cl)cccc1Cl
InChIKey: QUFQSKVWPKIAIE-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 465.28, ALogP 4.71, HBA 4, HBD 1, PSA 58.12, QED 0.65
Compound (SMILES): CCS(=O)(=O)c1ccc(NCC2CC2)c(-c2cn(C)c(=O)c3[nH]ccc23)c1
InChIKey: ZQRYCQNBELIIBM-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 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 385.49, ALogP 3.15, HBA 5, HBD 2, PSA 83.96, QED 0.68
Compound (SMILES): CN1CCN(c2ccc(-c3ccc4[nH]c5nccc(-c6ccc(-c7ccccc7)cc6)c5c4c3)cc2)CC1
InChIKey: DSCWFQTVXDLKTI-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
Measurement: IC50 = 680 nM (pChEMBL 6.170000076293945)
Descriptors: MW 494.64, ALogP 7.47, HBA 3, HBD 1, PSA 35.16, QED 0.28
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(NC(=O)C(C)c2cc(F)ccc2Cl)ccc1Oc1ccc(F)cc1F
InChIKey: USRFIILCLQIOMW-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method C: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.047 mM to 0.78 nM or 5-fold serial dilution from Assay method A. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of 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) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 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) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 86.4 nM (pChEMBL 7.059999942779541)
Descriptors: MW 556.97, ALogP 7.06, HBA 5, HBD 1, PSA 69.56, QED 0.25
Compound (SMILES): Fc1ccc(N[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: WDSQTCWUUXVUNZ-MQMHXKEQSA-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 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 350.4, ALogP 3.88, HBA 5, HBD 2, PSA 70.9, QED 0.59
Compound (SMILES): CC(OC(=O)Cc1cccc(SCc2ccccc2C(=O)O)c1)c1ccc(C(F)(F)F)cc1
InChIKey: QXOFSEBCNUJJKX-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P37231
Assay: Reporter Gene Assay: Transactivation Assay Expression vectors were prepared by inserting the ligand binding domain cDNA (complementary DNA) of human PPARalpha (amino acid 168-468) and human PPARgamma (amino acid 205-505), 3′ to and in frame with, the yeast GAL4 transcription factor DNA binding domain and the nuclear localization signal from the T-antigen of Polyoma Virus into the mammalian expression vector pSGS (Stratagene). The resulting expression vectors pSGGAL-PPARalpha and pSGGAL-PPARgamma were used in co-transfection experiments together with a modified pGL3 promoter plasmid (Promega) containing five copies of the UAS GAL4 recognition site. 2.5 μg pSGGAL-PPARalpha or pSGGAL-PPARgamma were mixed with 25 μg pGL3p 1×UAS and 22.5 μg pBluescript (Stratagene) in 0.95 mL ice cold PBS containing between 9-12 million U-2 OS (human osteosarcoma) cells. The cell/DNA mixture was incubated on ice for 5 minutes and then divided between two 0.4 cm cuvettes and electroporated at 960 μF, 2
Measurement: EC50 = 3500 nM (pChEMBL 5.460000038146973)
Descriptors: MW 474.5, ALogP 6.54, HBA 4, HBD 1, PSA 63.6, QED 0.3
Compound (SMILES): CCN(CC)CCOC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cccc(Cl)c3F)ccc2[nH]1
InChIKey: SDGGTSSMGYRFDT-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 552.01, ALogP 5.1, HBA 6, HBD 3, PSA 110.79, QED 0.28
Compound (SMILES): Nc1onc(-c2ccc([N+](=O)[O-])cc2)c1C(=O)Nc1ccc(S(N)(=O)=O)cc1
InChIKey: WFALAHLDCCYRAJ-UHFFFAOYSA-N
Target: Carbonic anhydrase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P00918
Assay: Inhibition of recombinant human carbonic anhydrase 2 assessed as reduction in CO2 hydration preincubated for 15 mins by stopped flow assay
Measurement: Ki = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 403.38, ALogP 1.73, HBA 8, HBD 3, PSA 184.45, QED 0.42
Compound (SMILES): CNC(=O)c1c(-c2ccc(OCC(F)(F)F)nc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3C(CO)O4)cc12
InChIKey: QQDQNRRSPUACNU-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 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 725.68, ALogP 5.9, HBA 10, HBD 2, PSA 149.02, QED 0.18
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ccc(OC(F)(F)F)cc1)c1nc2cccc(Cl)c2c(=O)n1-c1cc(F)cc(F)c1
InChIKey: VYUICRDXVBJVPZ-HNNXBMFYSA-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 612.95, ALogP 6.16, HBA 8, HBD 2, PSA 107.95, QED 0.18
Compound (SMILES): Cc1ccc(-c2ncccn2)c(C(=O)N2CCCOC2Cc2cnn(-c3ccc(F)cn3)c2)c1
InChIKey: XREXIMFORJAGEV-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Antagonistic Activity Assay: The antagonistic activities of the test compounds on human orexin-1 receptor (hOX1R) and orexin-2 receptor (hOX2R) were measured by modifying from the method described in literature (Toshikatsu Okumura et al., Biochemical and Biophysical Research Communications 280, 976-981, 2001). Chinese hamster ovary (CHO) cells forcibly expressing the hOX1R and hOX2R were seeded into a 96 well Black clear bottom plate (Nunc) at 20,000 cells per well, which were cultured in Ham's F-12 medium containing 0.1 mM MEM non-essential amino acids, 0.5 mg/ml G418, 10% fetal bovine serum (all by Invitrogen) for 16 hours under the conditions of 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the medium, 100 uL of 0.5 uM Fluo-4AM ester (Dojin) in an assay buffer (25 mM HEPES (Dojin), Hank's balanced salt solution (Invitrogen), 0.1% bovine serum albumin, 2.5 mM probenecid, 200 ug/ml Amaranth (all by Sigma-Aldrich), pH 7.4) was added and the cells were incubated for 60 minutes at 37° C., 5% CO2. After removing the assay buffer containing fluo-3AM ester, the test compound was dissolved in dimethyl sulfoxide to be 10 mM and diluted with the assay buffer, 150 uL of which was added and incubated for 30 minutes.
Measurement: IC50 = 20.4 nM (pChEMBL 7.690000057220459)
Descriptors: MW 458.5, ALogP 3.6, HBA 7, HBD 0, PSA 86.03, QED 0.45
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)C4CCCC4)cn3n2)c(-c2ccc(F)cc2)n1
InChIKey: BMWJGXAWSPYKAG-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 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 404.45, ALogP 4.06, HBA 6, HBD 1, PSA 77.11, QED 0.56
Compound (SMILES): Nc1ncnc2c1c(-c1cc(O)c(F)c(F)c1)nn2C1Cc2ccccc2C1
InChIKey: VTASVRIYSRXJCL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition of PI3Kdelta (unknown origin) using biotinylated PIP2 as substrate in presence of streptavidin-APC by FRET assay
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 379.37, ALogP 3.4, HBA 6, HBD 2, PSA 89.85, QED 0.56
Compound (SMILES): CN(C)C(=O)C(NC(=O)c1cc(OCC(F)(F)F)c(C2CC2)cn1)C(C)(C)C
InChIKey: UJEAAVOZOGMGNH-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 401.43, ALogP 3.13, HBA 4, HBD 1, PSA 71.53, QED 0.8
Compound (SMILES): CNC(=O)Cn1cc(-c2cnc3ccc(N(CC4CC4)c4cc(OC)cc(OC)c4)cc3n2)cn1
InChIKey: GEEWFYSWIUQFGV-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 472.55, ALogP 3.8, HBA 8, HBD 1, PSA 94.4, QED 0.4
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(-c3cnc(C)s3)cc1C21N=C(N)N(C)C1=O
InChIKey: RAMQFWJJXZGADF-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 410.54, ALogP 3.23, HBA 6, HBD 1, PSA 80.81, QED 0.82
Compound (SMILES): CC(C)Nc1nc2c(-c3cc4c([nH]3)CCNC4=O)cccc2c(=O)n1-c1cccnc1
InChIKey: LWAINAQZGRZKTJ-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 = 16.4 nM (pChEMBL 7.789999961853027)
Descriptors: MW 414.47, ALogP 2.88, HBA 6, HBD 3, PSA 104.7, QED 0.48
Compound (SMILES): CC(CF)(CF)C(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CC1
InChIKey: OTFSNTCKEDKZQS-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 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 463.46, ALogP 2.44, HBA 7, HBD 2, PSA 129.93, QED 0.58
Compound (SMILES): CNC(=O)C1=C(c2ccc(C)cc2)CC(c2ccc(OCCCC(F)(F)F)cc2)(C(F)(F)F)NC1=O
InChIKey: ODHOEYOHFTVHML-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT SPA Assay: MGAT2 enzyme was assayed using membranes isolated from Sf9 cells expressing the recombinant human MGAT2 cDNA with 2-monooleoylglycerol and [3H]-oleoyl-CoA as substrates as described by Seethala et al. [Anal. Biochem., 383(2):144-150 (Dec. 15, 2008)]. Briefly, the assays were conducted in 384-well plates in a total volume of 30 uL at 25° C. In each assay, 200 ng of recombinant human MGAT2 membrane was incubated with 10 uM of 2-monooleoylglycerol and 15 uM of [3H]-oleoyl-CoA in 100 mM potassium phosphate (pH 7.4) for 20 min with various concentrations of compounds delivered in DMSO. The assay was terminated by the addition of 20 ul of Stopping Solution (7.5 mg/ml Yttrium Oxide Polylysine beads, 3.3 mg/ml Fraction V BSA and 200 uM Mercuric chloride in 50 mM HEPES, pH 7.4). The signal was measured 1 h after quenching the reaction using LEADSEEKERSM for 5 minutes. To calculate the degree of inhibition, the zero level of enzyme activity (blank) was defined.
Measurement: IC50 = 743 nM (pChEMBL 6.130000114440918)
Descriptors: MW 514.47, ALogP 5.19, HBA 3, HBD 2, PSA 67.43, QED 0.3
Compound (SMILES): O=C1CCc2c(Oc3ccc4c(c3)C3C(O4)C3c3nc4c(Cl)cc(C(F)(F)F)cc4[nH]3)ccnc2N1
InChIKey: CMHLENNKQBRTSK-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 512.88, ALogP 5.95, HBA 5, HBD 2, PSA 89.13, QED 0.35
Compound (SMILES): C[C@H](Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1ccc(Cl)c(Cl)c1
InChIKey: NLZRXHNICGFSSB-IMSIIYSGSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 144 nM (pChEMBL 6.840000152587891)
Descriptors: MW 397.26, ALogP 3.66, HBA 6, HBD 2, PSA 87.58, QED 0.8
Compound (SMILES): CNCCN1CCC(Sc2ccccc2)CC1
InChIKey: MKHPOZKKYPKPKO-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96LA8
Assay: Inhibition of human full length PRMT6 (1 to 375 residues) expressed in baculovirus expression system assessed as inhibition of methylation activity preincubated with protein followed by addition of biotin labeled histone H4 (1 to 24) peptide as substrate and [3H]-SAM measured after 3 hrs by scintillation proximity assay
Measurement: IC50 = 488 nM (pChEMBL 6.309999942779541)
Descriptors: MW 250.41, ALogP 2.46, HBA 3, HBD 1, PSA 15.27, QED 0.86
Compound (SMILES): O=C(CCc1ccc(Nc2nccc(Nc3cc(C4CC4)n[nH]3)n2)cc1)NCCCn1ccnc1
InChIKey: KCJQIRDQIKKKFX-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 7.300000190734863)
Descriptors: MW 471.57, ALogP 3.9, HBA 8, HBD 4, PSA 125.44, QED 0.23
Compound (SMILES): Cc1nc(Nc2ncc(C(=O)Nc3c(C)cccc3Cl)s2)cc(N2CCN(CCCCC(=O)NO)CC2)n1
InChIKey: VVVNGZRRKGBVHW-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: Inhibition of human N-terminal GST-tagged full length human Src (Met1 to Leu536 residues) expressed in baculovirus expression system assessed as decrease in biotin signal transduction protein phosphorylation at Tyr160 preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 559.1, ALogP 4, HBA 10, HBD 4, PSA 135.61, QED 0.17
Compound (SMILES): N#CCC1(n2ccc(-c3ncnc4[nH]ccc34)c2)CN(C2CCN(C(=O)c3cc(F)cc(C(F)(F)F)c3)CC2)C1
InChIKey: QJZJHKXDBUEAON-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 551.55, ALogP 4.81, HBA 6, HBD 1, PSA 93.84, QED 0.36
Compound (SMILES): [C-]#[N+]c1ccc(N2CC(OC(=O)N3CCN(C(C)C)CC3)C2)nc1
InChIKey: XIZPDWOPVLJLTD-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: Radioligand Binding Assay: The affinity of compounds of the invention to the H3 receptor can be assessed by measuring displacement of binding of the radioligand [3H]-N-Î±-Methylhistamine (PerkinElmer, #NET1027250UC) to membranes containing human H3 receptors (PerkinElmer, #ES-392-M400UA; GenBank: NM-007232.2; Hill S J et al, International Union of Pharmacology XIII. Classification of histamine receptors, Pharmacol Rev, 49(3), 253-278, 1997).Protocol: 1. Preparation of binding assay buffer (500 ml): 25 ml 1 M Tris-HCl pH 7.5 (Invitrogen, #15567-027), 2.5 ml 1 M MgCl2 (Sigma, #M1028-100ML), 472.5 ml ddH2O. 2. Compound serial dilution: Dilution was performed by BioTek Precision on compound dilution plate. Compound concentrations start at 5 or 10 uM, 10 point dose titrations with 3- or 5-fold serial dilutions. 3. Preparation of 2x membrane solution (25 ml): 1.25 ml human Histamine H3 receptor stock, 23.75 ml assay buffer. 4. Preparation of 2x solution of [3H]-N-Î±-methylhistamine (25 ml).
Measurement: Ki = 132 nM (pChEMBL 6.880000114440918)
Descriptors: MW 329.4, ALogP 1.98, HBA 5, HBD 0, PSA 53.27, QED 0.79
Compound (SMILES): Cc1ccccc1-n1c(-c2cc(C(=O)N(C)Cc3cccs3)c(O)cc2O)n[nH]c1=O
InChIKey: VAHNSEIURMSJCK-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Protein was prepared by dialysis for 24 hours against 1 liter of 30 mM Hepes pH 7.5, 150mM NaCl and 0.5mM TCEP. The final protein concentration was determined by Bradford Assay. The final protein concentration in the sample cell was 50 uM. Ligand stock solutions of 10 mM in DMSO were diluted to 5uM concentrations with ITC buffer and adjusted to 2% (v/v) DMSO. Both the titrate and titrant solutions were degassed prior to loading the calorimeter cell and injection syringe.The integrated heat data were fit with a one-site binding model using the Origin-7 software provided with the MicroCal VP-ITC. Protein concentration was corrected by titration of a reference compound and normalized for the concentration of each ligand. All ligands bound to N-HSP90 have a stoichiometry of approximately unity.
Measurement: Kd = 5.53 nM (pChEMBL 8.260000228881836)
Descriptors: MW 436.49, ALogP 3.28, HBA 7, HBD 3, PSA 111.45, QED 0.44
Compound (SMILES): COc1cc(OC)cc(N(Cc2ccc(Cl)nc2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: AUFUDTOCLCOVHF-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 486.96, ALogP 5.43, HBA 8, HBD 0, PSA 78.19, QED 0.28
Compound (SMILES): Cn1c(=O)n(CC2CC2(F)F)c2ccc(C3=CC4CCC3CN4C(=O)c3ccon3)nc21
InChIKey: WSMLYHXZMALWCQ-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: [35S]-GTPγS Assay: The stimulation of [35S]-GTPγS binding is a common functional assay to monitor Gαi-coupled receptor in native and recombinant receptor membrane preparation. Membrane from cells stably expressing hmGlu2 CHO-K1 (50 μg) are incubated in a 96 well plate for 1 hour in the presence of GTPγS35 (0.05 nM), GDP (5 μM) and compounds. The reaction is stopped by rapid filtration over Unifilter GF/B plate (Packard, Bioscience, Meriden Conn.) using a 96-well cell harvester (Brandel Gaithersburg, Md.). The filter plates are counted using Topcount counter (Packard, Bioscience, Meriden Conn., USA). When compounds are evaluated as potentiators they are tested in the presence of glutamate (1 μM).
Measurement: EC50 = 26.62 nM (pChEMBL 7.579999923706055)
Descriptors: MW 441.44, ALogP 2.7, HBA 7, HBD 0, PSA 86.16, QED 0.62
Compound (SMILES): c1cc2c(ncn3nnc([C@H]4CC[C@H](CNC5COC5)CC4)c23)[nH]1
InChIKey: SKVYONQIGPDJDD-HAQNSBGRSA-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 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 326.4, ALogP 1.87, HBA 6, HBD 2, PSA 80.13, QED 0.76