lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(Nc1cc(C(=O)N2CCC[C@H]2CO)nn1-c1ccccc1)c1cc(-c2ccccn2)c(F)cc1Cl
InChIKey: DZDIYCUEJAQXKR-SFHVURJKSA-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 = 72.9 nM (pChEMBL 7.139999866485596)
Descriptors: MW 519.96, ALogP 4.58, HBA 6, HBD 2, PSA 100.35, QED 0.39
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cc(Br)ccc3F)ccc2[nH]1
InChIKey: PDNIIKHCAFQJSY-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 511.31, ALogP 4.5, HBA 5, HBD 3, PSA 107.55, QED 0.38
Compound (SMILES): N#Cc1ccc(CN2CCC(c3ncn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: QSKZDSFBRGYQHC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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 = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 356.43, ALogP 3.46, HBA 5, HBD 1, PSA 73.01, QED 0.61
Compound (SMILES): CCC(=O)Nc1cc2c(Nc3ccc4c(c3)CCN4Cc3ccccn3)ncnc2cc1OC
InChIKey: RSJGJVCLGCMCLO-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Irreversible inhibition of GST-tagged ERBB1 (unknown origin) (Met-668 to Ala-1211 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 519 nM (pChEMBL 6.289999961853027)
Descriptors: MW 454.53, ALogP 4.69, HBA 7, HBD 2, PSA 92.27, QED 0.42
Compound (SMILES): CC(C)N(Cc1cccc(-c2nccs2)c1)C(=O)Nc1cnc(C(C)(C)C)nc1
InChIKey: JFQSTEIZOLGXFO-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Kinase Activity Assay: TrkA kinase activity was measured as the ability of the enzyme to phosphorylate a fluorescently labeled peptide substrate. Buffer salts, reagents, and fluorescently labeled peptides were purchased from Carna Biosciences and were of the highest quality available (QSS Assist TRKA_MSA Kit). Enzyme was purchased from Cell Signaling, and was used without further purification. 384-well round bottom assay plates were prepared by the addition of 200 nl of a DMSO solution of compound at various concentrations to final inhibitor concentrations ranging from 100 μM to 0.2 μM. Next, assay buffer (10 μl) containing substrate and ATP were added, followed by addition of 10 μl of enzyme in assay buffer. Final assays concentrations were: [E]=0.37 nM, [S]=1 μM, [ATP]=2 mM. The reactions were incubated at room temperature for 3 hours resulting in approximately 15% substrate phosphorylation and were terminated by the addition of 5 μl of stop buffer.
Measurement: EC50 = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 409.56, ALogP 5.34, HBA 5, HBD 1, PSA 71.01, QED 0.62
Compound (SMILES): CC(C)(O)[C@H](CC1CC1)NC(=O)c1ccc(C2CC2)c(OCC2CC2)n1
InChIKey: ITZSSVBCGHINKH-SFHVURJKSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 113.1 nM (pChEMBL 6.949999809265137)
Descriptors: MW 358.48, ALogP 3.42, HBA 4, HBD 2, PSA 71.45, QED 0.71
Compound (SMILES): C[C@H](c1nc2c(cnn2C2CCCC2)c(=O)[nH]1)N1CCC(c2cccc(C(=O)NC3CCC3)c2)C1
InChIKey: JWZRJXPZKTULEQ-DIAVIDTQSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of PDE9A (unknown origin) using [3H]-cGMP as substrate after 30 mins by microbeta counting method
Measurement: IC50 = 53.3 nM (pChEMBL 7.269999980926514)
Descriptors: MW 474.61, ALogP 4.07, HBA 6, HBD 2, PSA 95.91, QED 0.56
Compound (SMILES): COc1cccc(-c2cccc(OCC(O)CN3CCc4ccccc4C3)c2)c1
InChIKey: VMSAJLLFQIHJIZ-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 101 nM (pChEMBL 7)
Descriptors: MW 389.5, ALogP 4.16, HBA 4, HBD 1, PSA 41.93, QED 0.66
Compound (SMILES): Oc1ccccc1CCCNCc1ccccc1F
InChIKey: YRIHGAMJWFKQJD-UHFFFAOYSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: Inhibition of MPO chlorination activity (unknown origin) assessed as taurine chloramine formation after 5 mins in presence of H2O2/Cl- by HTS method
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 259.32, ALogP 3.25, HBA 2, HBD 2, PSA 32.26, QED 0.78
Compound (SMILES): CC(C)CC(O)[C@H](C)Nc1cc(-n2ccc3cc(C#N)cnc32)ncc1C(=O)NC[C@@H](F)C(C)(C)O
InChIKey: GXSDTVUVVZPZDW-GWGYHSHPSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 10.9 nM (pChEMBL 7.960000038146973)
Descriptors: MW 496.59, ALogP 3.34, HBA 8, HBD 4, PSA 136.09, QED 0.34
Compound (SMILES): O=C(O)[C@H]1CC[C@H](C(=O)N2CC[C@](c3ccc(-c4c(F)cccc4F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: NZDHORBXJJHDJW-LPBFERMMSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 571.62, ALogP 5.56, HBA 4, HBD 1, PSA 91.75, QED 0.39
Compound (SMILES): CC1CC(C)CC(N2CC[C@H](CNC(=O)c3ccc(Cl)c(Cl)c3)N[C@H](CCN)C2=O)C1
InChIKey: GFFYNOWKGQZKNH-HPIJGSOXSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 469.46, ALogP 3.46, HBA 4, HBD 3, PSA 87.46, QED 0.6
Compound (SMILES): O=C(O)c1ccc(NC(=O)C(Cc2ccccn2)n2ccc(-c3cc(Cl)ccc3C(F)(F)F)cc2=O)cc1
InChIKey: CKFCLGOBAUZLCA-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 541.91, ALogP 5.7, HBA 5, HBD 2, PSA 101.29, QED 0.31
Compound (SMILES): CC(O)CNc1nc(NC2CC2)n2ncc(/C=C3\NC(=O)NC3=O)c2n1
InChIKey: HDCYAPZYFAVEEU-WMZJFQQLSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 358.36, ALogP -0.33, HBA 9, HBD 5, PSA 145.57, QED 0.35
Compound (SMILES): CCNC(=O)c1ccc(-n2cnc3c(NCCC(F)(F)F)cc(Oc4cccc(O)c4)cc32)cc1C
InChIKey: JBDITRAKAZWUNR-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Kinase Assay: For the assay 50 nL of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μl assay volume is 10 μM) and peptide substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 μl assay volume). The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, Perkin Elmer LAS, Rodgau-Jügesheinn, Germany]. The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jügesheinn, Germany).
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 498.51, ALogP 5.95, HBA 6, HBD 3, PSA 88.41, QED 0.28
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)cc(N[C@H]4CC[C@H](NS(=O)(=O)CCC(F)(F)F)CC4)nc23)N1
InChIKey: AQAVJYRZBAYQEK-AAMARJQVSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 556.57, ALogP 2.08, HBA 9, HBD 5, PSA 158.62, QED 0.23
Compound (SMILES): Cc1cccc2cc([C@H](C)Nc3ncnc4ccccc34)n(-c3ccccc3)c(=O)c12
InChIKey: RULMFBNFMMHRPM-SFHVURJKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 406.49, ALogP 5.42, HBA 5, HBD 1, PSA 59.81, QED 0.43
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)cc(N[C@H]4CC[C@H](NC(=O)CCN5CCCC5)CC4)nc23)N1
InChIKey: PGOIHGRYPSJCHC-SOEVKJKVSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 521.63, ALogP 1.81, HBA 9, HBD 5, PSA 144.79, QED 0.25
Compound (SMILES): Cc1ncc(C)n2nc(CCc3nc(N4CCCC4)nn3C3CC3)nc12
InChIKey: UNEZRISWCAWDFE-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Scintillation Proximity Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 μl contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 μl of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate solution (stop reagent). After 1 h under shaking, the plate was centrifuged one minute at 170 g to allow beads to settle. Afterwards, radioactive counts were measured on a Perkin Elmer Top-Count Scintillation plate reader.
Measurement: IC50 = 2.03 nM (pChEMBL 8.6899995803833)
Descriptors: MW 352.45, ALogP 2.05, HBA 8, HBD 0, PSA 77.03, QED 0.7
Compound (SMILES): Cc1c(-c2ccc(F)cc2)nnc(N2CCN(c3cnc(C(=O)O)cn3)[C@H](C)C2)c1C
InChIKey: LIOVPPURPDQFMB-CYBMUJFWSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 422.46, ALogP 3.1, HBA 7, HBD 1, PSA 95.34, QED 0.69
Compound (SMILES): CN(C)c1cccc2c(S(=O)(=O)N3CCC[C@H]3C(=O)N[C@@H](Cc3ccc(NC(=O)CCCCNC(=N)N)cc3)C(=O)O)cccc12
InChIKey: NHTSPCQURYFKRF-BDYUSTAISA-N
Target: Integrin alpha-V/beta-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Inhibition of integrin alphaVbeta1 (unknown origin)-mediated CHO cell adhesion to fibronectin preincubated for 15 to 30 mins followed by 60 min incubation for adhesion by crystal violet staining based cell adhesion assay
Measurement: IC50 = 79.43 nM (pChEMBL 7.099999904632568)
Descriptors: MW 651.79, ALogP 2.46, HBA 7, HBD 6, PSA 198.02, QED 0.09
Compound (SMILES): O=C(NCC1(O)COC1)c1cc(NC(=O)c2cc(-c3ncccc3F)c(F)cc2Cl)n(-c2ccccc2)n1
InChIKey: HXCXVGOBKZXGGR-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 = 12.6 nM (pChEMBL 7.900000095367432)
Descriptors: MW 539.93, ALogP 3.61, HBA 7, HBD 3, PSA 118.37, QED 0.33
Compound (SMILES): COCc1nc(CC(=O)Nc2cncc(C(=O)c3cn(C(C)C)c4ccncc34)c2)c(C2CC2)o1
InChIKey: NZDUGNYFTZHHRD-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 181 nM (pChEMBL 6.739999771118164)
Descriptors: MW 473.53, ALogP 4.44, HBA 8, HBD 1, PSA 112.14, QED 0.36
Compound (SMILES): O=C(c1cscn1)N1CCN(C(=O)c2cc(Cc3n[nH]c(=O)c4c3CCCC4)ccc2F)CC1
InChIKey: ICSCMXBFRGCKFU-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 481.55, ALogP 2.43, HBA 6, HBD 1, PSA 99.26, QED 0.62
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=NC2(c2nc(NC(=O)c3cnn(C)c3)cs2)CO1
InChIKey: FLSYKADZRPHBBY-NEYSTMPUSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a ½ log dose response curve from a top concentration of 100 μM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 μL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37 °C. The reaction is stopped with the addition of 30 μL of 1.5 μM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm).
Measurement: IC50 = 3940 nM (pChEMBL 5.409999847412109)
Descriptors: MW 392.51, ALogP 1.81, HBA 9, HBD 2, PSA 107.42, QED 0.82
Compound (SMILES): CC(C)(COc1ncc(-c2ccc(-c3ncc(C(F)(F)F)[nH]3)nc2)cn1)C(=O)O
InChIKey: VWWRHRZXFLXIJY-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: DGAT1 Inhibitory Assay: As a buffer to be used in the enzymatic reaction of DGAT1, 100 mM Tris-HCl (pH 7.4), 200 mM Sucrose, 20 mM MgCl2, 0.125% Bovine Serum Albumin (BSA) were used. To the buffer were added Test compound with a predetermined concentration as well as 15 μM dioleoylglycerol, 5 μM [14C]-palmitoyl-CoA, 100 μg protein/nth DGAT1 highly expressed-expresSF+® microsome, 0.75% acetone, and 1% dimethylsulfoxide, and triglyceride (TG) synthetic reaction was carried out at 30 °C. for 20 minutes with a volume of 100 μL. 90 μL of the reaction solution was added to 810 μL of methanol to stop the reaction. The reaction solution was added to Oasie® μElution plate (available from Waters Corporation), and eluted with 150 μL of a mixed solution of acetonitrile: isopropanol (=2:3). To elute was added 150 μL of MicroScinti™-40 (available from PerkinElmer Inc.), and after thoroughly stirring the mixture, a [14C]-TG amount formed by the reaction was quantitated by measuring the same using TopCount™-NXT (available from PerkinElmer Inc.).
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 407.35, ALogP 3.44, HBA 6, HBD 2, PSA 113.88, QED 0.64
Compound (SMILES): O=C(Cc1ccc(Cl)cc1Cl)NC1COc2cnc(Cl)cc21
InChIKey: KOLYAMUCGBXOAR-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: In Vitro Assay: Cell line generation was performed in general according to established molecular cloning protocols. Specifically, RNA was extracted from human whole blood using the Qiagen RNeasy kit (Qiagen, CH) according to the manufacturer's instructions. Subsequently cDNA was made (Superscript II, Invitrogen AG, CH) and the human P2X7 gene (genbank ref. BC011913) was amplified with the following primers: ATCGCGGCCGCTCAGTAAGGACTCTTGAAGCCACT and CGCCGCTAGCACCACCATGCCGGCCTGCTGCAGCTGCA. The amplified sequence was subsequently ligated into a pcDNA3.1 (+) NotI, NheI digested plasmid. Human embryonic kidney (HEK) cells (ATCC CRL-1573, Manassas, Va., USA) were transfected with the pcDNA3.1 (+). hP2X7 plasmid using lipofectamine 2000 (Invitrogen AG, CH) according to the manufacturer's instructions. Following a 24 h exposure to DNA, cells were trypsinized and re-seeded at low density in the presence of 250 ug Geneticin. Geneticin resistant cells were then selected during two consecutive rounds of cloning by serial limiting dilution with visual inspection.
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 357.62, ALogP 3.83, HBA 3, HBD 1, PSA 51.22, QED 0.85
Compound (SMILES): CN1CCN(c2ccc(-c3cccc(CN(C)C(=O)CN)c3)cc2)CC1.O=C(O)[C@H](O)[C@@H](O)C(=O)O
InChIKey: SBTBMQLBPVEGMO-LREBCSMRSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Inhibition of human VAP-1 expressed in CHO cells using 14C-benzylamine as substrate preincubated for 20 mins followed by substrate addition measured after 1 hr by scintillation spectrometric analysis
Measurement: IC50 = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 352.48, ALogP 2.02, HBA 4, HBD 1, PSA 52.81, QED 0.89
Compound (SMILES): CC(C)(C)OC(=O)N1CCC2(CC1)CC2CNC(=O)NCc1cccnc1
InChIKey: PIVAZIMYJGDVBC-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Inhibition of human full length C-terminal His6-tagged NAMPT expressed in Escherichia coli Rosetta (DE3) cells using nicotinamide as substrate incubated for 15 mins prior to substrate addition measured after 30 mins in presence of PRPP
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 374.49, ALogP 2.92, HBA 4, HBD 2, PSA 83.56, QED 0.85
Compound (SMILES): CC(C)c1cc(CNc2nc(Nc3cc(C4CC4)[nH]n3)cc(N3CCN(C=O)CC3)n2)on1
InChIKey: LLYCEAVVAGEZLB-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) un-phosphorylated apo-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 451.54, ALogP 2.82, HBA 9, HBD 3, PSA 128.1, QED 0.42
Compound (SMILES): Fc1ccccc1/C=C/c1ccccc1
InChIKey: FMGVTMQBJISCFC-ZHACJKMWSA-N
Target: Nuclear factor erythroid 2-related factor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16236
Assay: Activation of Nrf2 (unknown origin) expressed in human HepG2 cells after 5 hrs by ARE-driven luciferase reporter gene assay
Measurement: EC50 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 198.24, ALogP 4, HBA 0, HBD 0, PSA 0, QED 0.64
Compound (SMILES): CCCCNC(=O)c1cc(CCC)sc1N
InChIKey: GZOXATNOBGFSRW-UHFFFAOYSA-N
Target: Polyunsaturated fatty acid lipoxygenase ALOX15 (SINGLE PROTEIN), Homo sapiens
UniProt: P16050
Assay: Inhibition of human 15-LOX-1 assessed as reduction in conversion of linoleic acid to 13(S)-HpODE incubated for 10 mins followed by linoleic acid addition by UV-absorbance analysis
Measurement: IC50 >= 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 240.37, ALogP 2.81, HBA 3, HBD 2, PSA 55.12, QED 0.75
Compound (SMILES): CCN1CCC(Nc2cc(N(C)Cc3ccccc3)nn3c(C)nnc23)CC1
InChIKey: QBIPWAYUMTXEPI-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15nM GST-BRD4-BD1 protein (aa 44-168) or 150nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 379.51, ALogP 2.97, HBA 7, HBD 1, PSA 61.59, QED 0.71
Compound (SMILES): Cc1cccc2c1S(=O)(=O)Nc1c-2ccc2cccnc12
InChIKey: XSTZGBWOENAGPE-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 3880 nM (pChEMBL 5.409999847412109)
Descriptors: MW 296.35, ALogP 3.32, HBA 3, HBD 1, PSA 59.06, QED 0.69
Compound (SMILES): O=C(NC1CC=C(c2ccc(NC(=O)N3Cc4ccncc4C3)cc2)CC1)[C@@H]1CCOC1
InChIKey: XEGPHYHQGBJCTD-PSDZMVHGSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 18.6 nM (pChEMBL 7.730000019073486)
Descriptors: MW 432.52, ALogP 3.72, HBA 4, HBD 2, PSA 83.56, QED 0.77
Compound (SMILES): c1ccc(COc2ccc(CN3CCc4ccccc4C3)cc2)cc1
InChIKey: DHGHDLRMOGDQNY-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Anopheles gambiae
UniProt: Q869C3
Assay: Inhibition of Anopheles gambiae recombinant AChE1 expressed in baculovirus infected insect Sf9 cells using acetylthiocholine iodide as substrate measured over 60 secs by Ellman assay
Measurement: IC50 = 2900 nM (pChEMBL 5.539999961853027)
Descriptors: MW 329.44, ALogP 4.82, HBA 2, HBD 0, PSA 12.47, QED 0.66
Compound (SMILES): C=CCOC(=O)C1=C(C)Nc2nc3c(c(N)c2C1c1cccc([N+](=O)[O-])c1)CC(C)(C)CC3
InChIKey: MJVSWROHOTUJRM-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition Assay: Procedure: 48 Ml 1xReaction Buffer was initially added to each compound test well in the 96-well black plate arranged, followed by addition of 2 Ml 100xcompound solution; the test for each concentration was performed in duplicate. To the positive control well, 2 Ml DMSO+48 Ml 1xReaction Buffer were added; while 100 Ml 20 Mm H2O2 working solution was directly added to the positive validation well. To the negative control well, 2 Ml DMSO+98 Ml 1xReaction Buffer were added, and each test was performed in duplicate. 50 Ml 4xAchE application solution was added to each of the compound test wells and the positive control wells. 100 Ml 2xworking solution was added to each of all the wells and mixed until homogenous. The enzymatic reaction was started with the volume of the overall reaction system reaching 200 Ml, which resulted in the final concentrations of the compound solution of 10 umol/L, 2 umol/L, 0.4 umol/L, 0.08 umol/L, 0.016 umol/L, 0.0032 umol/L and 0.064 umol/L.
Measurement: IC50 = 1055 nM (pChEMBL 5.980000019073486)
Descriptors: MW 448.52, ALogP 4.65, HBA 7, HBD 2, PSA 120.38, QED 0.3
Compound (SMILES): CC(NC(=O)c1ccc(NC(=O)N2Cc3cccnc3C2)cc1)C1(C)CC1
InChIKey: FXFDUCDFYZIJOL-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 61.3 nM (pChEMBL 7.210000038146973)
Descriptors: MW 364.45, ALogP 3.55, HBA 3, HBD 2, PSA 74.33, QED 0.87
Compound (SMILES): Nc1ncnc2c1c(I)nn2Cc1cc2cccc(Cl)c2c(=O)n1C1CC1
InChIKey: KONDPISCYGNXRE-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 492.71, ALogP 3.36, HBA 7, HBD 1, PSA 91.62, QED 0.44
Compound (SMILES): c1ccc(Nc2nc(NCCc3ccncc3)nc(Nc3ccccc3)n2)cc1
InChIKey: FWGBWQZTIHKDLG-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9ULD8
Assay: Inhibition of BEC1 (unknown origin) expressed in dihydrofolate reductase-deficient CHO cells assessed as decrease in KCl-induced intracellular 86Rb ion release after 20 mins
Measurement: IC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 383.46, ALogP 4.41, HBA 7, HBD 3, PSA 87.65, QED 0.42
Compound (SMILES): CN1CCN(c2ccc(C(=O)Nc3n[nH]c4cc(OCc5cccc(Oc6ccccc6)c5)ccc34)cc2)CC1
InChIKey: ZEMPBQAQWWJUJQ-UHFFFAOYSA-N
Target: Receptor-type tyrosine-protein kinase FLT3 (SINGLE PROTEIN), Homo sapiens
UniProt: P36888
Assay: Biochemical Assay: The assay was performed using FLT3 cytoplasmic domain product and purified in house as GST fused protein. The FLT3 protein (1 microM) was pre activated with 800 microM ATP for 1 hour at 28° C in order to obtain a linear kinetic. Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 4 mM MgCl2, 1 mM DTT, 10 microM Na3VO4, and 0.2 mg/mL BSA. The FLT3 kinase assay was run with a final pre activated enzyme concentration of 2 nM, in the presence of 254 microM ATP (residual ATP from KIT pre activation step is negligible), 8 nM 33P-γ-ATP and 55 microM of substrate BioDB n*24 (Aminoacidic sequence: GGKKKVSRSGLYRSPSMPENLNRPR- SEQ ID NO: 1).
Measurement: IC50 = 1171 nM (pChEMBL 5.929999828338623)
Descriptors: MW 533.63, ALogP 5.94, HBA 6, HBD 2, PSA 82.72, QED 0.26
Compound (SMILES): CC(C(N)=O)c1ccccc1CCc1nc(Nc2cn[nH]c2)ncc1Cl
InChIKey: RKVTUBMTXZINFU-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P35916
Assay: VEGFR3 Biochemical Assay: A biotin labelled peptide is used as substrate (amino acid sequence: Biotin-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2). VEGFR3 cytoplasmic domain (amino acids 798-1298) was purchased as N-terminal GST-fusion protein (the enzyme). The 15 uL assay reactions are run in Greiner brand white 384-well low volume plates. All reactions contained 10 mM HEPES pH 7.4, mM MgCl2, 0.01% (v/v) Tween-20, 50 uM Na3VO4, 0.01% (w/v) albumin from chicken egg white, 1 mM Dithiothreitol, 111 nM peptide substrate, 500 uM ATP, and 3.8 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 90 minutes at 30 degree Celsius.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 370.84, ALogP 2.97, HBA 5, HBD 3, PSA 109.58, QED 0.59
Compound (SMILES): COc1cc(C(=O)N2CC[C@H]2c2nc(-c3ccccc3OC(F)(F)F)no2)c(-n2nccn2)cc1F
InChIKey: FLEPWMSNJHLEST-HNNXBMFYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Intracellular Calcium Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 μL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μL/well, incubated for 120 min and finally 10 μL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 1420 nM (pChEMBL 5.849999904632568)
Descriptors: MW 504.4, ALogP 3.95, HBA 9, HBD 0, PSA 108.4, QED 0.36