lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCOC(=O)c1cnc2ccc(OC)cc2c1N1CCN(c2ncccc2C#N)CC1
InChIKey: QYMJYFAIRXKEJM-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of equine serum BuChE preincubated for 5 mins followed by addition of butyrylthiocholine iodide as substrate measured after 2 mins by Ellman's method
Measurement: IC50 = 1900 nM (pChEMBL 5.71999979019165)
Descriptors: MW 417.47, ALogP 3.01, HBA 8, HBD 0, PSA 91.58, QED 0.59
Compound (SMILES): Cc1ccc(C(C)C)c(OC(=O)Nc2cccc(F)c2)c1
InChIKey: NMLLLUWGVMNOCV-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of electric eel AChE using acetylthiocholine iodide as substrate preincubated for 15 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 88520 nM (pChEMBL 4.050000190734863)
Descriptors: MW 287.33, ALogP 4.87, HBA 2, HBD 1, PSA 38.33, QED 0.87
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1cscn1
InChIKey: SHQICENFGOEXMY-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 433.32, ALogP 5.72, HBA 7, HBD 1, PSA 69.16, QED 0.42
Compound (SMILES): C=C[C@H]1CC[C@]23C[C@@H]4[C@H](C)C(=O)[C@H]5O[C@]54[C@@]2(C)C[C@H](O)[C@]13C
InChIKey: QWIJMFGHPKWGBS-UEIIUFPNSA-N
Target: Signal transducer and activator of transcription 1-alpha/beta (SINGLE PROTEIN), Homo sapiens
UniProt: P42224
Assay: Inhibition of Stat1 (unknown origin) expressed in LPS/INF-gamma-stimulated human MONO-MAC-6 cells assessed as reduction in GAS dependent transcription activity measured after 4 hrs by luciferase reporter gene assay
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 302.41, ALogP 2.72, HBA 3, HBD 1, PSA 49.83, QED 0.6
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2ccc(Cl)cc2)C(=O)N[C@@H](Cc2ccccn2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: SJQWETDPGAMVES-JSOSNVBQSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 614.15, ALogP 3.98, HBA 6, HBD 4, PSA 135.44, QED 0.17
Compound (SMILES): N#CCNC[C@H]1CC[C@H](N2CN(CC(F)(F)F)C(=O)c3cnc4[nH]ccc4c32)CC1
InChIKey: WUFVCJVJPCLFKL-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 420.44, ALogP 3.02, HBA 5, HBD 2, PSA 88.05, QED 0.57
Compound (SMILES): Cc1ccc2c(n1)[C@H]([C@@H](C)C(=O)Nc1ccc(Cl)c(-c3nc(C4CC4)no3)c1)CC[C@H]2C
InChIKey: ZUOAPSOEDVMEJO-MUKKUYKPSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of Smo-mediated Hh signalling pathway in mouse Shh Light2 cells by Gli-luciferase reporter gene assay
Measurement: IC50 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 450.97, ALogP 6.23, HBA 5, HBD 1, PSA 80.91, QED 0.5
Compound (SMILES): COc1ccc(Oc2ccc3c(O)c(C(=O)NCCCCC(=O)O)ncc3c2)cc1
InChIKey: CIEXUDARNJDRJD-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 2300 nM (pChEMBL 5.639999866485596)
Descriptors: MW 410.43, ALogP 3.73, HBA 6, HBD 3, PSA 117.98, QED 0.46
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C)C(C)C
InChIKey: AABQDGMXXIRPKT-XGLJNPFSSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 2399.83
Compound (SMILES): Cc1cc(=O)oc2c3c(ccc12)O[C@@H](c1cc(F)c(F)cc1F)[C@@H](N)C3
InChIKey: MXZDPLKXVPVWMV-KXBFYZLASA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: Inhibition of human DPP4 using H-Gly-Pro-AMC peptide substrate assessed as increase in fluorescence for 20 mins by fluorimetric assay
Measurement: IC50 = 50860 nM (pChEMBL 4.289999961853027)
Descriptors: MW 361.32, ALogP 3.52, HBA 4, HBD 1, PSA 65.46, QED 0.53
Compound (SMILES): CC(C)N1CCN(C(=O)OC2CCN(c3cncs3)CC2)CC1
InChIKey: IHTVRUMYBYHNAK-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: LANCE Ultra cAMP Assay: The potency of compounds of the invention as H3 receptor antagonists can be assessed by measuring the blockade of (R)-alpha-methylhistamine-mediated cAMP production utilizing a LANCE Ultra cAMP kit (PE #TRF0263) in CHO cells expressing human H3 receptors (GenBank: BC096840; Strausberg R L et al, Proc. Natl. Acad. Sci. U.S.A. 99(26), 16899-16903; 2002).Protocol:1. Preparation of the stimulation buffer (30 ml): 29.4 ml HBSS (GIBCO #14025), 150 ul 1 M HEPES (GIBCO #15630), 30 ul 500 mM IBMX (CALBIOCHEM #410957) and 400 ul 7.5% BSA (GIBCO #10438-026).2. Preparation of assay plate: Different concentrations of the compounds of the invention (0.01-1000 nM), H3 positive controls and cAMP calibration standards; 3 mM Forskolin (CALBIOCHEM #344270); 5 uM (R)-alpha-methylhistamine (H3 receptor agonist); 1% DMSO (SIGMA #D2650); total volume: 95 nl.3. Preparation of the cell solution: Collect cells with stimulation buffer, final density: 100,000 cells/ml.
Measurement: Ki = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 338.48, ALogP 2.27, HBA 6, HBD 0, PSA 48.91, QED 0.85
Compound (SMILES): COCCNc1cc(NC(=O)N2CCCc3cc(CN4CCOCC4=O)c(C=O)nc32)ncc1C#N
InChIKey: ZTYSXMGILYNNJX-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Biochemical Kinase Assay: Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 ul per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 16 mM MgCl2, 1122 uM ATP, 4 uM peptide (5-Fluo-Ahx-KKKKEEIYFFFG-NH2, Biosyntan GmbH) and 4.5 ul per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 16 mM MgCl2, 6 nM FGFR4 (GST-FGFR4(388-802), produced in-house by expression in insect cells and affinity chromatography). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 ul per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35).
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 493.52, ALogP 1.56, HBA 9, HBD 2, PSA 149.78, QED 0.41
Compound (SMILES): Cc1n[nH]cc1NC(=O)c1cc(NC(=O)c2cc(-c3ncccc3F)c(Cl)cc2Cl)n(-c2ccccc2)n1
InChIKey: ZNGXJZWCPBEKCJ-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 1.51 nM (pChEMBL 8.819999694824219)
Descriptors: MW 550.38, ALogP 5.92, HBA 6, HBD 3, PSA 117.59, QED 0.24
Compound (SMILES): O=C(Cc1nc(N2CCOCC2)cc(=O)[nH]1)N1c2ccccc2CC1CF
InChIKey: UUVJAAZMPNSLBB-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Sandwich Immunoassay: This test is based on measurement of the expression of AKT protein phosphorylated on serine 473 (P-AKT-S473) in the PC3 human prostate carcinoma line, by the technique based on a sandwich immunoassay using the Kit MSD Multi-spot Biomarker Detection of Meso Scale Discovery: phospho-Akt kits (Ser473) whole cell lysate (# K151 CAD) or phospho-Akt (Ser473) / Akt whole cell lysate Total (# K15100D). The primary antibody specific for P-AKT-S473 (Kit # K151 CAD) is coated onto an electrode in each well of 96-well plates of the MSD kit: after adding a protein lysate in each well, the revelation of the signal done by the addition of a secondary antibody labeled with an electrochemiluminescent detection compound. The procedure is described in the kit.On day 1, the PC3 cells are inoculated in 96-well plates (TPP # 92096) at a concentration of 35000 cells / well in 200 uL DMEM medium (DMEM Gibco # 1 1960-044) containing 10% fetal calf serum (FBS Gibco, # 10500-056) and 1% glutamine.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 372.4, ALogP 1.08, HBA 5, HBD 1, PSA 78.53, QED 0.87
Compound (SMILES): COC[C@@H](C)Oc1cc(NC(=O)N2CCCc3cc(CN4C(=O)OC[C@@H]4C)c(C=O)nc32)ncc1C#N
InChIKey: VUCPNMYWKVZATE-JKSUJKDBSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Biochemical Kinase Assay: Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 ul per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 16 mM MgCl2, 1122 uM ATP, 4 uM peptide (5-Fluo-Ahx-KKKKEEIYFFFG-NH2, Biosyntan GmbH) and 4.5 ul per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 16 mM MgCl2, 6 nM FGFR4 (GST-FGFR4(388-802), produced in-house by expression in insect cells and affinity chromatography). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 ul per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35).
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 508.54, ALogP 2.9, HBA 9, HBD 1, PSA 146.98, QED 0.53
Compound (SMILES): COC(=O)[C@@H]1CCCN1C(=O)c1cccc(Nc2c(N[C@H](c3ccc(C)o3)[C@@H]3CCCS3)c(=O)c2=O)c1F
InChIKey: OISJPBYRKOJZRI-HFSMHLIXSA-N
Target: C-X-C chemokine receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25024
Assay: In Vitro Affinity Assay: â¿¿PathHunter HEK293-CXCR2â¿ or â¿¿U2OS hCXCR1 β-arrestinâ¿ cells (DiscoveRx Corporation) were seeded overnight at 10 000 cells/well (384-well format) in 20 μl of Opti MEM I medium. A preincubation with the antagonist or the vehicle for 30 min at 37° C. and 5% CO2 was followed by 60 minutes of stimulation with CXCL8 at 37° C. and 5% CO2. The cells were then placed at ambient temperature for 30 minutes. The PathHunter detection reagent (DiscoveRx Corporation) was added. After incubation for 60 min at ambient temperature, the β-galactosidase induced by the luminescence during the β-arrestin-CXCR2 interaction was measured for 0.3 s in an Envision 2102 Multilabel Reader (PerkinElmer Life and Analytical Sciences).
Measurement: IC50 = 8907 nM (pChEMBL 5.050000190734863)
Descriptors: MW 541.6, ALogP 3.89, HBA 9, HBD 2, PSA 117.95, QED 0.32
Compound (SMILES): O=C(CCCCOc1cc(Oc2ccc(NC(=O)Nc3ccc(Cl)c(C(F)(F)F)c3)cc2)ccn1)NO
InChIKey: CBQXSJJBMYHOPQ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of N-terminal GST-His6-thrombin cleavage site-fused human VEGFR2 (Asp805 to Val1356 residues) expressed in baculovirus expression system using biotinylated gastrin precursor at Tyr87 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 538.91, ALogP 6.24, HBA 6, HBD 4, PSA 121.81, QED 0.14
Compound (SMILES): CC(C)c1cccc(NC(=O)Nc2ccc(-c3cc4ccccc4n3C(N)=O)cc2)c1
InChIKey: QEUJPSURIIRPKP-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 2.7 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.).
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 412.49, ALogP 6, HBA 3, HBD 3, PSA 89.15, QED 0.38
Compound (SMILES): Cc1nc(C)c(-c2csc(Nc3ccc(C(=O)N4[C@@H]5CC[C@H]4CC(=NOCC(C)(C)O)C5)cn3)n2)s1
InChIKey: XXYGNWXZPNKSLD-KDURUIRLSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Antagonist activity at human TRPV4 assessed as inhibition of 4alpha-PDD-induced activation
Measurement: IC50 = 4.4 nM (pChEMBL 8.359999656677246)
Descriptors: MW 526.69, ALogP 4.93, HBA 10, HBD 2, PSA 112.83, QED 0.42
Compound (SMILES): C[C@@]12CC[C@@H](O)C[C@@H]1CC[C@H]1C[C@@]3(C)[C@H](CC[C@@]34CO4)C[C@@H]12
InChIKey: NYSHZNMIKYOREG-FJEFMNTOSA-N
Target: Gamma-aminobutyric acid receptor subunit alpha-4 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P28471
Assay: [35S]-TBPS Displacement Assay: The IC50 values for non-competitive displacers of [35S]-TBPS from the picrotoxin binding site on GABAA receptors.
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 304.47, ALogP 4.16, HBA 2, HBD 1, PSA 32.76, QED 0.68
Compound (SMILES): COc1cc(C)cc2cc(-c3c(CN4CCNC(=O)C4)c(CO)n4ncnc(N)c34)sc12
InChIKey: KDZJTYONQQRGIQ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: High ATP Kinase Assay: For the assay, 50 nl of each stock solution of the test compound in DMSO was pipetted into a black, low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany) 2 Î¼l of a solution of the above FGFR-1 fusion protein in aqueous assay buffer [8 mM MOPS pH 7.0, 10 mM magnesium acetate, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumin (BSA), 0.07% (v/v) Tween-20, 0.2 mM EDTA] was added, and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme. Then, the kinase reaction was started by the addition of 3 ul of a solution of adenosine triphosphate (ATP, 3.3 mM; final concentration in the 5 ul assay volume=2 mM) and substrate (0.16 uM; final concentration in the 5 ul assay volume=0.1 uM) in assay buffer, and the resulting mixture was incubated for a reaction time of 15 min at 22° C. The concentration of FGFR-1 fusion protein was adjusted depending on the activity of the enzyme lot.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 452.54, ALogP 1.93, HBA 9, HBD 3, PSA 118.01, QED 0.42
Compound (SMILES): COc1ccc(CC(=O)O)cc1-c1c(F)cc(F)c2c1CN(C(=O)[C@@H]1C[C@H]1c1ccccc1)CC2
InChIKey: NMCQJWHSVGKCDF-VQTJNVASSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 11.2 nM (pChEMBL 7.949999809265137)
Descriptors: MW 477.51, ALogP 4.96, HBA 3, HBD 1, PSA 66.84, QED 0.55
Compound (SMILES): Cc1ccccc1NC(=O)Cn1cc(Cn2c(-c3ccc(Br)cc3)nc(-c3ccccc3)c2-c2ccccc2)nn1
InChIKey: XGLNYNMSVRQSKG-UHFFFAOYSA-N
Target: Oligo-1,6-glucosidase IMA1 (SINGLE PROTEIN), Saccharomyces cerevisiae S288c
UniProt: P53051
Assay: Inhibition of bakers yeast alpha-glucosidase using p-nitrophenyl-alpha-D-glucopyranoside as substrate incubated for 15 mins followed by substrate addition measured after 30 mins by spectrophotometric analysis
Measurement: IC50 = 29270 nM (pChEMBL 4.53000020980835)
Descriptors: MW 603.52, ALogP 7.23, HBA 6, HBD 1, PSA 77.63, QED 0.2
Compound (SMILES): Cn1c(CN(CCCCN)C2CCCc3cccnc32)nnc1-c1ccccc1
InChIKey: IQXADRSWBMCYJU-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Calcium Mobilization Assay: Functional modulation of CXCR4 was determined by calcium mobilization assay using leukemic lymphoid CEM cells, which naturally express high levels of CXCR4. Generally, the assay is carried out as follows: Cells are grown in vitro to confluency. On the day of the assay, cells are removed from the incubator, culture medium removed and replaced with a calcium sensitive dye (Calcium3 assay kit; Molecular Devices, Sunnyvale, Calif.). The cells are allowed to dye load for 45-60 min at 37 °C. in the cell culture incubator and then allowed to equilibrate to room temperature for no more than 15 min before assay. Compound plates were generated containing up to 3% dimethyl sulfoxide in media kept at room temperature. Test compounds were added to the cells at a 1:3 dilution, and calcium mobilization was measured using a Flex Station fluorescence imager (Molecular Devices). This first read was used to determine direct agonist activation of the CXCR4 receptor by the test compounds.
Measurement: IC50 = 3200 nM (pChEMBL 5.5)
Descriptors: MW 390.54, ALogP 3.5, HBA 6, HBD 1, PSA 72.86, QED 0.6
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(C)(C)C#N)c2)cc1-c1cc(N2CCOCC2)nc(N2CCOCC2)n1
InChIKey: FEJAQUKVYQVYED-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 1.521 nM (pChEMBL 8.819999694824219)
Descriptors: MW 526.64, ALogP 4.18, HBA 8, HBD 1, PSA 103.61, QED 0.51
Compound (SMILES): CCCCCCCCc1ccc(OCC(=O)Cn2ncc3ncccc32)cc1
InChIKey: BKNOWKYEIAGDPA-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97612
Assay: Inhibition of FAAH in rat brain microsomes using N-(2-hydroxyethyl)-4-pyren-1-ylbutanamide as substrate after 60 mins by fluorescence-based reversed phase HPLC analysis
Measurement: IC50 = 93 nM (pChEMBL 7.03000020980835)
Descriptors: MW 379.5, ALogP 4.98, HBA 5, HBD 0, PSA 57.01, QED 0.42
Compound (SMILES): N#Cc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(C(F)(F)F)c3)cn2CC2CCCN2)CC1
InChIKey: BKWJTVGUNLNJRS-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 514.53, ALogP 4.09, HBA 8, HBD 2, PSA 108.68, QED 0.49
Compound (SMILES): CO[C@H]1Cc2ccccc2[C@H]1Nc1ncnc(N[C@@H]2C[C@@H](COS(N)(=O)=O)[C@@H](O)C2)n1
InChIKey: OXHKRQXDLANIMN-QMPXSYISSA-N
Target: NEDD8 activating enzyme (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13564
Assay: Inhibition of recombinant human NAEalpha/N-terminal His-tagged NAEbeta expressed in baculovirus infected insect Sf9 cells using N-terminal FLAG tagged Nedd8 as substrate incubated for 105 mins in presence of N-terminal GST-tagged Ubc12 by FRET assay
Measurement: IC50 <= 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 450.52, ALogP 0.37, HBA 10, HBD 4, PSA 161.58, QED 0.44
Compound (SMILES): CC(C)Oc1cc2c(-c3cc(N4C5CCC4COC5)ncn3)n[nH]c2cc1F
InChIKey: WJMWMDQNTASDCI-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 18.2 nM (pChEMBL 7.739999771118164)
Descriptors: MW 383.43, ALogP 3.31, HBA 6, HBD 1, PSA 76.16, QED 0.74
Compound (SMILES): O=c1[nH]nc(CCC2CCCC2)cc1O
InChIKey: FROLIVDWLCPSEK-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: In Vitro DAAO Enzyme Assay: Human DAAO enzyme was supplied by the Takeda Pharmaceutical Company (Osaka) and each batch was tested and used at concentrations giving comparable levels of activity. The Km of D-Serine was measured for each enzyme batch to maintain consistency; this Km was used in subsequent assays.On the day of the assay compounds were serially diluted in DMSO before being diluted 1:20 with assay buffer (20 mM Tris ph 7.4). A 5 ul portion of assay buffer was added to the wells of a 384 clear base black-walled plate (Corning), 5 ul of diluted compound was then added via automated plate to plate transfer using the Bravo liquid handler (Agilent technologies) followed by 5 ul of human DAAO enzyme and then 5 ul D-Serine 50 mM was added to all but the negative control wells (final concentration of 10 mM). Finally 5 ul Amplex red reagent (Invitrogen) was added to all wells as per manufacturer's protocol. The plate was incubated for 60 minutes in the dark at 25° C., and the fluorescence in each well was measured in the Envision plate reader.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 208.26, ALogP 1.6, HBA 3, HBD 2, PSA 65.98, QED 0.79
Compound (SMILES): CC(C)(C)OC(=O)NCc1nc(-c2nc(C(=O)NCCCCCC(=O)NO)cs2)cs1
InChIKey: CQLJYHNTHMSMLR-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzyme Assay: Enzyme activity of HDAC1,3 is determined using the substrate Ac-Lys-Tyr-Lys(Ac)-AMC while the enzyme activity of HDAC6 is assayed using the substrate Boc-lys(Ac)-AMC. The reaction is carried out in flat-bottom, 96-well, or 384-well microplates. After the substrate was deacetylated by HDACs, the product AMC which was obtained from hydrolysis by trypsin can generate fluorescence signal. Measurement is taken in using an multilabe plate reader a 355 nm excitation filter and a 460 nm emission filter. The initial rate of fluorescence should accurately reflect the rate of product formation and enzyme activity. Sample processing: The sample was dissolved in DMSO and kept at a low temperature. DMSO in the final system is limited under a low concentration which won't affect the enzyme activity.
Measurement: IC50 = 121 nM (pChEMBL 6.920000076293945)
Descriptors: MW 469.59, ALogP 3.09, HBA 9, HBD 4, PSA 142.54, QED 0.24
Compound (SMILES): CCCn1cc(S(=O)(=O)c2ccc(CNC(=O)c3cc4ccncc4s3)nc2)cn1
InChIKey: FYIPERXOWGWKMC-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT Enzymatic Reaction. The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 pM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 441.54, ALogP 3.06, HBA 8, HBD 1, PSA 106.84, QED 0.47
Compound (SMILES): CCNC(=S)Nc1cc(N2CCN(C)CC2)ncn1
InChIKey: URKZPWMTSCUXPE-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P37136
Assay: Inhibition of rat cortex AChE using acetylthiocholine iodide as substrate measured after 20 mins by Ellman's method
Measurement: IC50 = 3260 nM (pChEMBL 5.489999771118164)
Descriptors: MW 280.4, ALogP 0.53, HBA 5, HBD 2, PSA 56.32, QED 0.78
Compound (SMILES): COc1cccc(OC)c1C1CCCCC(=O)N1Cc1ccc2nc(C)sc2c1
InChIKey: NWFKASOEMQBZIN-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements: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 g/ml G418, 100 U/ml penicillin, 100 g/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 M 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.
Measurement: IC50 = 155 nM (pChEMBL 6.809999942779541)
Descriptors: MW 410.54, ALogP 5.27, HBA 5, HBD 0, PSA 51.66, QED 0.58
Compound (SMILES): COc1cc(OC)cc(N(CC2(O)CCNCC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: JNTQLKRLUHCITH-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 absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 741.31 nM (pChEMBL 6.130000114440918)
Descriptors: MW 474.57, ALogP 3.3, HBA 9, HBD 2, PSA 97.56, QED 0.42
Compound (SMILES): CCC1CC(=O)NN=C1c1ccc2nc(-c3ccc(OCCN4CCCC4=O)cc3)oc2c1
InChIKey: SMGVAGZBYCNKFH-UHFFFAOYSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 1032 nM (pChEMBL 5.989999771118164)
Descriptors: MW 446.51, ALogP 3.75, HBA 6, HBD 1, PSA 97.03, QED 0.6
Compound (SMILES): C[C@@H]1CN(Cc2ccc(F)c(F)c2)c2ccccc2[C@H](C)N1C(=O)Nc1cccnc1
InChIKey: FESPTXQLNNHGLK-SJORKVTESA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Channel blocking activity at recombinant human CaV 3.1 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 422.48, ALogP 5.36, HBA 3, HBD 1, PSA 48.47, QED 0.62
Compound (SMILES): COc1nc(N)nc2c1ncn2C1O[C@H](COP(=O)(N[C@@H](C)C(=O)OCC(C)(C)C)Oc2ccccc2)[C@@H](O)[C@]12CCO2
InChIKey: QBBUGVSYQKSNSK-ZPROQFFVSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: HCV Replicon Assay: The HCV NS5B reaction was performed in a 20 μL mixture containing varying concentrations of the test compound, 1 μM of all four natural ribonucleotides, [α-32P]UTP, 20 ng/μL of genotype 1b (−) IRES RNA template, 1 unit/μL of SUPERase·In (Ambion, Austin, Tex.), 40 ng/μL of wild type or S282T NS5B Genotype 1b, 1 mM MgCl2, 0.75 mM MnCl2, and 2 mM DTT in 50 mM Hepes buffer (pH 7.5). The reaction was quenched by adding 80 μL of stop solution (12.5 mM EDTA, 2.25 M NaCl, and 225 mM sodium citrate) after incubating at 27° C. for 30 minutes. The radioactive RNA products were separated from unreacted substrates by passing the quenched reaction mixture through a Hybond N+ membrane (GE Healthcare, Piscataway, N.J.) using a dot-blot apparatus. The RNA products were retained on the membrane and the free nucleotides were washed out. The membrane was washed 4 times with a solution containing 0.6 M NaCl and 60 mM sodium citrate. After rinsing the membrane with water followed by ethanol, the membrane was exposed to a phosphorscreen and the products were visualized and quantified using a phosphorimager.
Measurement: EC50 = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 620.6, ALogP 2.61, HBA 14, HBD 3, PSA 191.4, QED 0.21
Compound (SMILES): Cc1c(-c2ncnc3[nH]c(C4=CCN(C(=O)N(C)C)CC4)cc23)ccc(F)c1NC(=O)N1CC(OC(C)(C)C)C1
InChIKey: WMTYXEKVXXLDOO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Biochemical Enzyme Assay: The inhibitory activity of the present compounds against Btk was assessed in a biochemical enzyme assay. Assay plates in 384 well format were prepared with 8-point serial dilutions for the test compounds on a Thermo CatX workstation equipped with a Innovadyne Nanodrop Express. The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 ul per well of peptide/ATP-solution (4 uM FITC-Ahx-TSELKKVVALYDYMPMNAND-NH2, 164 uM ATP) in kinase buffer (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 18 mM MgCl2, 1 mM MnCl2) and 4.5 ul per well of enzyme solution (6.4 nM full-length human recombinant BTK) in kinase buffer. Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 ul per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Kinase reactions were analyzed on a Caliper LC3000 workstation by separating phosphorylated and unphosphorylated peptides and kinase activities were calculated from the amounts of newly formed phospho-peptide. Inhibition data were calculated by comparison to control reactions without enzyme (100% inhibition) and without inhibitors (0% inhibition).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 549.65, ALogP 4.87, HBA 5, HBD 2, PSA 106.69, QED 0.48
Compound (SMILES): CCOc1cc(C(C)C(=O)O)ccc1-c1ccc(F)c2c1CN(C(=O)OCc1ccccc1)CC2
InChIKey: NJZPENHOXDSUBG-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 11.3 nM (pChEMBL 7.949999809265137)
Descriptors: MW 477.53, ALogP 5.77, HBA 4, HBD 1, PSA 76.07, QED 0.47
Compound (SMILES): O=C(Nc1ccc(C2CCNC2)cc1)OC(c1ccc(Cl)cc1)C(F)(F)F
InChIKey: CXJJKPDRQHNXTN-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 398.81, ALogP 5.27, HBA 3, HBD 2, PSA 50.36, QED 0.74
Compound (SMILES): COc1ccc(-n2nc(C3CCN(S(=O)(=O)C(C)C)CC3)cc2NC(=O)c2cnn3cccnc23)cc1
InChIKey: RGXWFCOWYDFJRU-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 523.62, ALogP 3.09, HBA 9, HBD 1, PSA 123.72, QED 0.39