lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nnc2c(NC(C)C)nc(-c3nc4cnc(N5CCOCC5)cc4n3C[C@@H]3CCCO3)cn12
InChIKey: PSVNUTDDCMYBSK-KRWDZBQOSA-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 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM 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. Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (probe molecule JQ1+ with protein/peptide mix) is pipetted.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 477.57, ALogP 2.68, HBA 11, HBD 1, PSA 107.52, QED 0.45
Compound (SMILES): Cc1nn(C)c(C)c1S(=O)(=O)N1CCC(Cc2ccc(Cl)cc2)CC1
InChIKey: ROCOKNRCXBAIJD-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TCW9
Assay: Biological Assay: Prokineticin receptor 1 (PKR1) antagonists may be functionally assessed by measurement of change in intracellular calcium levels induced by Gq mediated increase in inositol triphosphate (IP3) levels. The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro. Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1xHanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1x Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 uM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following excitation at 485 nM. Readings from wells that do not contain antagonist enable percentage inhibition curves to be plotted using 4-parameter fit algorithm and IC50 values are calculated for each test compound.
Measurement: IC50 = 520 nM (pChEMBL 6.28000020980835)
Descriptors: MW 381.93, ALogP 3.33, HBA 4, HBD 0, PSA 55.2, QED 0.82
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(NCc4ccc(Cl)cc4Cl)nc23)N1
InChIKey: SBIPMWPTTMEOBR-RZNTYIFUSA-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 > 1000 nM (pChEMBL 6)
Descriptors: MW 459.3, ALogP 2.8, HBA 8, HBD 4, PSA 125.34, QED 0.33
Compound (SMILES): CCC(C(N)=O)N1CC(c2ccc(Cl)cc2)CC1=O
InChIKey: VJQZFHLIVWLRFO-UHFFFAOYSA-N
Target: Synaptic vesicle glycoprotein 2A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q02563
Assay: Displacement of [3H]-2-[4-(3-azidophenyl)-2-oxo-1-pyrrolidinyl]butanamide from SV2A levetiracetam binding site in Sprague-Dawley rat cerebral cortex membranes after 120 mins by liquid scintillation counting method
Measurement: Ki <= 1000 nM (pChEMBL 6)
Descriptors: MW 280.75, ALogP 1.92, HBA 2, HBD 1, PSA 63.4, QED 0.92
Compound (SMILES): O=C(O)[C@@H]1CCCN1C(=O)c1cccc(COc2ccc(-c3cc(F)c(F)cc3S)cc2)c1
InChIKey: RWMXPRULAYUQHB-QFIPXVFZSA-N
Target: Glycogen [starch] synthase, muscle (SINGLE PROTEIN), Homo sapiens
UniProt: P13807
Assay: Glycogen Synthase Activity Assay: To a polystyrene 96-well plate, a solution containing 30 mM glycylglycine (pH 7.3), 40 mM KCl, 20 mM MgCl2, 9.2% DMSO containing one of the test compounds at various concentrations, and 10 mM glucose-6-phosphate (Sigma-Aldrich Corporation, G7879) was added by 12 uL/well. Next, a substrate solution containing 30 mM glycylglycine (pH 7.3), 4.3 mg/mL of glycogen (Sigma-Aldrich Corporation, G8876), 21.6 mM UDP-glucose (Sigma-Aldrich Corporation, U4625), 21.6 mM phosphoenolpyruvic acid (Sigma-Aldrich Corporation, P0564), and 4.05 mM NADH (Sigma-Aldrich Corporation, N8129) was added by 18 uL/well. Further, an enzyme solution containing 50 mM Tris-HCl (pH 8.0), 27 mM DTT (Nacalai Tesque, Inc., 14128-04), 0.2 mg/mL of bovine serum albumin, 0.17 mg/mL of the glycogen synthase, 1.5 uL of a pyruvate kinase/lactate dehydrogenase solution (Sigma-Aldrich Corporation, P0294) was added by 18 uL/well to prepare a reaction solution. After the reaction solution was incubated (at 30° C. for 25 minutes for Examples 1 to 10, at 37° C. for 20 minutes for Examples 11 to 75), the absorbance at 340 nm was measured using Benchmark Plus (Bio-Rad Laboratories, Inc.).
Measurement: EC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 469.51, ALogP 5.19, HBA 4, HBD 2, PSA 66.84, QED 0.49
Compound (SMILES): CCCCCC(O)c1ccc(N2C(=O)CC[C@@H]2COCc2cccc(C(=O)O)c2)cc1
InChIKey: MVXIIKRUEDTRBN-WTQRLHSKSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Displacement of [3H]PGE2 from from human EP4 receptor expressed in HEK293 cells membranes incubated for 60 mins
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 425.53, ALogP 4.71, HBA 4, HBD 2, PSA 87.07, QED 0.51
Compound (SMILES): O=C1NC(=O)/C(=C\c2ccc(OCCCOc3ccccc3Cl)cc2)S1
InChIKey: UIZOSHZUNAOFFH-SFQUDFHCSA-N
Target: CDGSH iron-sulfur domain-containing protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZ45
Assay: Displacement of [3H]rosiglitazone from recombinant human C-terminal His-tagged MitoNEET cytosolic domain (32 to 108 residues) expressed in Escherichia coli BL21 by scintillation proximity assay
Measurement: IC50 = 1399 nM (pChEMBL 5.849999904632568)
Descriptors: MW 389.86, ALogP 4.51, HBA 5, HBD 1, PSA 64.63, QED 0.56
Compound (SMILES): NC(=O)[C@@H](NC(=O)c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1)C1CC1
InChIKey: VZMBXHOIYPJRBB-AWEZNQCLSA-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 = 59.5 nM (pChEMBL 7.21999979019165)
Descriptors: MW 380.4, ALogP 1.32, HBA 5, HBD 2, PSA 97.55, QED 0.71
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)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](CC(=O)O)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
InChIKey: XPZLQXSAQDOJFR-SEFVOVRJSA-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 = 100 nM (pChEMBL 7)
Descriptors: MW 2442.81
Compound (SMILES): CCc1cc(CN(CC2CCCC2)[C@@H](CC(=O)O)c2ccc(Cl)cc2)ccc1OCCN1C(=O)CCC1=O
InChIKey: ZLWWAAACJPQOFS-SANMLTNESA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 541.09, ALogP 5.64, HBA 5, HBD 1, PSA 87.15, QED 0.33
Compound (SMILES): Cc1cc(Nc2cc(N3CCOCC3)nc(NCc3cc(-c4ccccc4)no3)n2)[nH]n1
InChIKey: DKPCJYLKUVGYEQ-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 432.49, ALogP 3.36, HBA 9, HBD 3, PSA 117.02, QED 0.4
Compound (SMILES): N#CCC(=O)N1CC[C@H](C(=O)N2CC[C@](c3ccc(C(OCc4c(F)cccc4F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)C1
InChIKey: XGWAQTHTPKNYLZ-BGOLNKOXSA-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 = 121 nM (pChEMBL 6.920000076293945)
Descriptors: MW 761.66, ALogP 6.3, HBA 6, HBD 0, PSA 107.78, QED 0.19
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3Cl)ncnc2cc1OC[C@@H]1CN2CCC[C@H]2CO1
InChIKey: LTXUQGNDYGUYMC-KBPBESRZSA-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 7.301000118255615)
Descriptors: MW 554.27, ALogP 5.69, HBA 7, HBD 1, PSA 68.74, QED 0.39
Compound (SMILES): O=C1/C(=C/c2ccccc2)Cc2ccc(O)cc21
InChIKey: JEZTWQLYCGBJRP-MDWZMJQESA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Inhibition of recombinant human MAO-A using kynuramine as substrate after 20 mins by fluorescence spectrophotometric analysis
Measurement: IC50 = 440 nM (pChEMBL 6.360000133514404)
Descriptors: MW 236.27, ALogP 3.21, HBA 2, HBD 1, PSA 37.3, QED 0.77
Compound (SMILES): COc1ccc(C(=O)Nc2ccc(C3CCCNC3)cc2)cc1
InChIKey: KBENMJATUUCARX-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 = 48.6 nM (pChEMBL 7.309999942779541)
Descriptors: MW 310.4, ALogP 3.41, HBA 3, HBD 2, PSA 50.36, QED 0.91
Compound (SMILES): O=C(NCCc1ccc(Cl)cc1)c1cccc(Cc2n[nH]c(=O)c3c2CCCC3)c1
InChIKey: WCLXNJUOACLYKE-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 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 421.93, ALogP 3.87, HBA 3, HBD 2, PSA 74.85, QED 0.63
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3cnn(CCO)c3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: ISKFTPLVPSNABN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 506.57, ALogP 3.23, HBA 10, HBD 2, PSA 129.67, QED 0.35
Compound (SMILES): COc1ccc([C@H](CCO)NC(=O)C2(N)CCN(c3ncnc(N)c3Cl)CC2)cc1
InChIKey: BYGZZQHGOYFLGR-HNNXBMFYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: Enzyme Assay: P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction.
Measurement: IC50 = 380 nM (pChEMBL 6.420000076293945)
Descriptors: MW 434.93, ALogP 1.26, HBA 8, HBD 4, PSA 139.62, QED 0.51
Compound (SMILES): Cc1nn(C)c(Cl)c1S(=O)(=O)Nc1ccc(-c2cnc3c(NS(=O)(=O)c4c(C)nn(C)c4Cl)n[nH]c3n2)cc1
InChIKey: IGPTXARXZZFPEN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Sgk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00141
Assay: Enzymatic Activity Assay: The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-1) inhibitory activity in a substrate phosphorylation assay designed to measure the ability of the isolated enzyme to catalyze the transfer of phosphate from ATP to serine/threonine residues in a fluorescein-labeled substrate peptide, using recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol, Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated substrate peptide and non-phosphorylated substrate peptide were separated with caliper life science's lab-chip technology based on a micro fluidics method. All fluid flow was established on the chip by applying a vacuum of a few psi to the waste well transporting fluid from various sources through interconnecting channels. Because the phosphoryl group is doubly negatively charged, under the pressure-driven hydrodynamic flow.
Measurement: IC50 = 1050 nM (pChEMBL 5.980000019073486)
Descriptors: MW 611.5, ALogP 3.01, HBA 11, HBD 3, PSA 182.44, QED 0.25
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CCCC(C#N)C2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: SNPGOAWRSCUFOV-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 = 72.44 nM (pChEMBL 7.139999866485596)
Descriptors: MW 511.63, ALogP 4.81, HBA 9, HBD 0, PSA 92.33, QED 0.32
Compound (SMILES): CN1C(=O)CCC1c1cncc(-c2cnc3c(c2)CCCN3C(N)=O)c1
InChIKey: NKCOXVQAYLNLLJ-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Aldosterone Synthase Inhibition Assay: The aldosterone synthase inhibition assay employs cynomolgus adrenal gland mitochondria as the source of aldosterone synthase (CYP11B2). Mitochondria are prepared from frozen cynomolgus monkey adrenal glands according to Method A described in by J. D. McGarry et al. (Biochem. J., 1983, 214, 21-28), with a final resuspension in the AT buffer described in R. Yamaguchi et al. (Cell Death and Differentiation, 2007, 14, 616-624), frozen as aliquots in liquid nitrogen and stored at -80° C. until use. Assays are performed in 96-well format in a final volume of 60 uL/well, containing 100 mM potassium phosphate, pH 7.4, 1% (v/v) DMSO, and additionally, 2 uM of corticosterone and 6 mg of mitochondrial protein. Reactions are started by the addition of NADPH to 1 mM and allowed to proceed for 60-90 minutes at 37° C. Reactions are terminated by the addition of 60 uL of acetonitrile. One hundred microliters are then transferred to a glass filter plate and centrifuged at 570xg.
Measurement: IC50 = 52.7 nM (pChEMBL 7.28000020980835)
Descriptors: MW 351.41, ALogP 2.27, HBA 4, HBD 1, PSA 92.42, QED 0.9
Compound (SMILES): CCNC(=O)N1CCN(c2ccnc3cc(Cl)ccc23)CC1
InChIKey: CWGFNQIEHMBOEP-UHFFFAOYSA-N
Target: DNA gyrase subunit B (SINGLE PROTEIN), Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
UniProt: A0QNE0
Assay: Inhibition of Mycobacterium smegmatis 155 6His-tagged DNA gyrase B catalytic domain ATPase activity expressed in Escherichia coli BL21 (DE3) pLysS cells assessed as inorganic phosphate release after 120 mins in presence of ATP by malachite green dye based assay
Measurement: IC50 = 45300 nM (pChEMBL 4.340000152587891)
Descriptors: MW 318.81, ALogP 2.74, HBA 3, HBD 1, PSA 48.47, QED 0.93
Compound (SMILES): C[C@H](NC(=O)CC#N)c1ccc(O[C@@H]2CCN(c3ccc(OCC4CC4)cc3)C2)cc1
InChIKey: BYCPUTJCVIBCAN-MHECFPHRSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 200 μM f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 419.53, ALogP 4.22, HBA 5, HBD 1, PSA 74.59, QED 0.66
Compound (SMILES): COc1cc(-c2cn[nH]c2)ccc1C(=O)NCc1cccc(C(=O)NC2CCN(C)CC2)c1
InChIKey: GPERPWCLYBHCRW-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 1.24 nM (pChEMBL 8.90999984741211)
Descriptors: MW 447.54, ALogP 2.84, HBA 5, HBD 3, PSA 99.35, QED 0.52
Compound (SMILES): NC1=N[C@@](c2cc(Nc3ncccn3)ccc2F)(C(F)F)COC1
InChIKey: FBODVRNZZXUAQO-HNNXBMFYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE-1 Inhibition Assay: Recombinant BACE-1 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 1 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Activity was measured using a final concentration of 3 uM of the fluorescence-quenched substrate Q-C(HSO3)-Ile-Asp-Leu-Ala-Val-Leu-Asp-HN-CH2-CH2-Mca, where Q=2-nitro-5-amino benzoic acid and Mca=7-methoxy-4-coumarinyl acetic acid. Catalytic turnover was monitored in a Spectramax Gemini fluorescence plate reader (Molecular Devices) in black 96-well microplates using excitation/emission wavelength of 325 nm and 400 nm, respectively. Fluorescence increase was followed for 15 min, in 1 minute's intervals.
Measurement: IC50 = 8600 nM (pChEMBL 5.070000171661377)
Descriptors: MW 337.31, ALogP 2.21, HBA 6, HBD 2, PSA 85.42, QED 0.89
Compound (SMILES): C[C@@]1(c2nc(Nc3ncc(F)c4cc(C#N)cnc34)ccc2F)C[C@@H](C(F)(F)F)OC(N)=N1
InChIKey: WFWYGKDLGMHHLO-DJJJIMSYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 188 nM (pChEMBL 6.730000019073486)
Descriptors: MW 463.37, ALogP 3.8, HBA 8, HBD 2, PSA 122.1, QED 0.57
Compound (SMILES): Nc1ncc([C@@H]2CCOC2)nc1-c1ccc(C(=O)N[C@H](CO)c2ccccc2)cc1
InChIKey: NNJVQVNAIFZJPS-UYAOXDASSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
Measurement: IC50 = 4.51 nM (pChEMBL 8.350000381469727)
Descriptors: MW 404.47, ALogP 2.69, HBA 6, HBD 3, PSA 110.36, QED 0.58
Compound (SMILES): O=C(Nc1cccc(Oc2ccc3c(/C=C/c4ccccc4)n[nH]c3c2)c1)c1cccc2[nH]ccc12
InChIKey: RMRGQCNTCGUCGP-NTCAYCPXSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of unphosphorylated human 50 residues truncated cytoplasmic domain of VEGFR2 E990V mutant expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using poly(E4Y1) as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 0.37 nM (pChEMBL 9.430000305175781)
Descriptors: MW 470.53, ALogP 7.26, HBA 3, HBD 3, PSA 82.8, QED 0.24
Compound (SMILES): CN(C)Cc1ccc(CSCCNc2nnc(-c3cccs3)c(-c3cccs3)c2[N+](=O)[O-])o1
InChIKey: FMDKBRZGISLHJQ-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of electric eel AChE using acetylthiocholine as substrate preincubated for 8 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 501.66, ALogP 5.84, HBA 10, HBD 1, PSA 97.33, QED 0.16
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cn(Cc2ccc3ccc(Cl)cc3n2)nn1
InChIKey: GXNNDHNHDUFBEI-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 421.89, ALogP 3.05, HBA 7, HBD 2, PSA 111.61, QED 0.51
Compound (SMILES): O=C(CN1CCC(C(=O)c2ccc(Cl)cc2)CC1)NCc1nc2c(c(=O)[nH]1)COCC2
InChIKey: HNYALLOQJOASHW-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 444.92, ALogP 1.71, HBA 6, HBD 2, PSA 104.39, QED 0.66
Compound (SMILES): C[C@H]1OC(=O)[C@]2(C(=O)NCCO)CC(F)(F)[C@@H](C)[C@H](/C=C/c3ccc(-c4cccc(F)c4)cn3)C12
InChIKey: MAMOENPXWFQBBC-HSPAPUHMSA-N
Target: Proteinase-activated receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25116
Assay: High Throughput Thrombin Receptor Radioligand Binding Assay: Thrombin receptor antagonists were screened using a modification of the thrombin receptor radioligand binding assay of Ahn et al. (Ahn et al., Mol. Pharmacol., 51:350-356 (1997)). The assay was performed in 96 well Nunc plates (Cat. No. 269620) at a final assay volume of 200 μl. Platelet membranes and [3H]haTRAP were diluted to 0.4 mg/ml and 22.2 nM, respectively, in binding buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.1% BSA). Stock solutions (10 mM in 100% DMSO) of test compounds were further diluted in 100% DMSO. Unless otherwise indicated, 10 μl of diluted compound solutions and 90 μL of radioligand (a final concentration of 10 nM in 5% DMSO) were added to each well, and the reaction was started by the addition of 100 μl of membranes (40 μg protein/well).
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 488.51, ALogP 3.85, HBA 5, HBD 2, PSA 88.52, QED 0.48
Compound (SMILES): NC(=O)[C@H](NC(=O)c1ccc(NC(=O)N2Cc3ccc(F)cc3C2)cc1)c1ccccc1
InChIKey: CFQNWHMLAYNWBI-OAQYLSRUSA-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 = 49.8 nM (pChEMBL 7.300000190734863)
Descriptors: MW 432.46, ALogP 3.33, HBA 3, HBD 3, PSA 104.53, QED 0.58
Compound (SMILES): NC(=O)c1ccc2[nH]c(-c3ccc(S(=O)(=O)NCc4cccs4)cc3)nc2c1
InChIKey: VCVIUBABPKYEOX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk2 (SINGLE PROTEIN), Homo sapiens
UniProt: O96017
Assay: Inhibition of recombinant human CDS1 using N-terminal biotin-labeled SGLYRSPSMPENLNRPR as substrate in presence of [gamma-33P]-adenosine triphosphate after 3 hrs by scintillation counting method
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 412.5, ALogP 2.87, HBA 5, HBD 3, PSA 117.94, QED 0.45
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)nc(NCc4ccc(F)c(C(F)(F)F)c4)nc23)N1
InChIKey: AMDMACCAWNPZCM-NSIKDUERSA-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 > 1000 nM (pChEMBL 6)
Descriptors: MW 476.39, ALogP 2.65, HBA 8, HBD 4, PSA 125.34, QED 0.24
Compound (SMILES): O=C(N[C@H](CO)C1CCCCC1)C1CCCCc2ncsc21
InChIKey: IXCRMSPMULYANL-AWKYBWMHSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: YO-PRO Assay: Cells were grown to confluency in adherent culture at 37° C. in a humidified 5% CO2 incubator (split 1/5 every 3-4 days with DMEM, 10% FCS, 1% Penicillin/Streptomycin, 250 μg/ml Geneticin). Adherent cells were detached by incubation with Trypsine (1 ml per 165 cm2 dish) for 2 minutes, then washed off with 10 ml PBS (without Mg2+ and Ca2+), and resuspended in DMEM, 10% FCS, 1% Penicillin/Streptomycin, no Geneticin. 10'000 cells per well (48 hours before the assay) or 25'000 cells per well (Vi-cell XR (Beckman Coulter) (24 hours before the assay) in 50 μl full medium were seeded on 384-well black-wall, clear bottom plates, that were coated before with 10 μl per well Poly-L-Lysine, incubated for 30-60 minutes at 37° C. and washed once with PBS. Medium was removed from cells and 50 μl of assay buffer containing 0.5 μM YO-PRO-1 was added into the wells. Solutions of antagonist compounds were prepared by serial dilutions of a 10 mM DMSO solution of the antagonist into PBS using a BioMek (Beckman Coulter). Each concentration was performed in duplicate. For IC50 measurements 10 concentration points were measured (10 μM being the highest concentration followed by 9 serial dilution steps 1/3). The cells were incubated with the antagonists of the present invention together with ATP at a final concentration of 250 μM for 90 minutes. During this time period, four time points were taken. Each time point comprised the average of several measurements made within a few seconds. Fluorescence was measured in the FLIPR tetra (Molecular Devices) using the filters appropriate for YO-PRO-1 fluorescence (excitation 485/20, emission 530/25).
Measurement: IC50 = 348 nM (pChEMBL 6.460000038146973)
Descriptors: MW 322.47, ALogP 3.01, HBA 4, HBD 2, PSA 62.22, QED 0.84
Compound (SMILES): O=C(NO)c1cnc(NC2(c3ccccc3)CCN(S(=O)(=O)c3cccc(F)c3)CC2)nc1
InChIKey: TYLUVDVYTNYCKJ-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC Enzyme Assay: The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM TCEP) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 uM final concentration were diluted in assay buffer at 6 fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The final substrate concentrations used were 16 uM (HDAC1), 10 uM (HDAC2), 17 uM (HDAC3) and 14 uM (HDAC6). Five ul of compounds and 20 ul of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five u1 of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated.
Measurement: IC50 = 551 nM (pChEMBL 6.260000228881836)
Descriptors: MW 471.51, ALogP 2.53, HBA 7, HBD 3, PSA 124.52, QED 0.37
Compound (SMILES): Cc1cc(Nc2cc(C(F)(F)F)ccn2)nc(-c2ccc(NC3CCN(C(=O)Cc4ccc5[nH]c(C)c(C)c5c4)CC3)nc2)c1
InChIKey: FBTBRZAIWIFWNS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 613.69, ALogP 7.35, HBA 6, HBD 3, PSA 98.83, QED 0.18
Compound (SMILES): COc1cc(N(Cc2ccc(F)cc2)CC2CC2)cnc1C(=O)C1CC1C(=O)O
InChIKey: YBBVMJBKNSMOJJ-UHFFFAOYSA-N
Target: Leukotriene C4 synthase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16873
Assay: In Vitro hLTC4 Synthase Enzyme Assay (Test A): In the assay, LTC4 synthase catalyses the reaction where the substrate LTA4 is converted to LTC4. Recombinant human LTC4 synthase is expressed in Piccia pastoralis and the purified enzyme is dissolved in 25 mM tris-buffer pH 7.8 supplemented with 0.1 mM glutathione (GSH) and stored at −80 °C. In order to obtain IC50-values for the compounds, the following procedure was used: A volume (24 μL) of 0.25 g/mL LTC4 synthase in 75 mM tris (pH ~8.5), 0.5 mM MgCl2 and 3 mM GSH was preincubated for 10 min with 0.5 μL of compound of interest in DMSO at ten different concentrations, typically in the range 10^−9,5-10^−5 M as well as with DMSO only. The buffer is used as background. Runs are performed in duplicates. The enzymatic reaction is initiated by addition of 0.5 μL LTA4 in diglyme (final assay concentration 8 μM). The reaction is stopped after 1 min by addition of double the reaction volume of a stop solution (MeOH:H2O:acetic acid 70:30:1).
Measurement: IC50 = 105 nM (pChEMBL 6.980000019073486)
Descriptors: MW 398.43, ALogP 3.55, HBA 5, HBD 1, PSA 79.73, QED 0.65
Compound (SMILES): O=C(O)C1CCN(C(=O)N2CC[C@](c3ccc(C(OCc4c(F)cccc4F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)C1
InChIKey: ZZGZDPKJUDQJEO-NEFVBLPJSA-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 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 738.63, ALogP 6.54, HBA 5, HBD 1, PSA 104.22, QED 0.21
Compound (SMILES): C[C@H](NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](C)NC(=O)C1CCC1)C(N)=O
InChIKey: WHSNAIHMKCUFOW-NHCYSSNCSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Binding Assay: All the FP based experiments were performed in MICROFLUOR 2 Black, "U" Bottom, 96-well Microtiter Plates (ThermoSci.) and FP was measured as mP units in a microplate reader (Tecan Ultra) with excitation at 485 nm and emission at 530 nm. To dilutions of WDR5Δ23 (2.2-0 uM) in 100 ul assay buffer (0.1M Phosphate, 25 mM KCl, 0.01% Triton, pH 6.5) 20 ul of a fixed concentration of the tracer in the assay buffer was added, followed by an addition of 5 ul DMSO to give 125 ul of total volume. Each assay had two controls: blank (without protein and tracer) and tracer only (without protein). The plates were incubated on a shaker at room temperature to reach equilibrium and mP values were measured at the 3 hour time point.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 397.48, ALogP -1.97, HBA 5, HBD 7, PSA 192.29, QED 0.12
Compound (SMILES): CCC[C@@H]1NCCOc2ccccc2CCCNC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@@H](CC(C)C)NC1=O
InChIKey: UPLQIOOHMDDLRG-OYUWMTPXSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 536.72, ALogP 3.14, HBA 5, HBD 4, PSA 108.56, QED 0.45
Compound (SMILES): N#Cc1ccccc1Cc1cccc(C(=O)NCCc2c[nH]c3ccc(Cl)cc23)c1
InChIKey: HBNBPWFISBACEY-UHFFFAOYSA-N
Target: Alpha-synuclein (SINGLE PROTEIN), Homo sapiens
UniProt: P37840
Assay: In Vitro Inhibition Assay: α-Synucleinopathies are characterised by aggregation of α-synuclein in neurons. Aggregation of purified α-synuclein is performed essentially as described by Gerard et al. FASEB. 20(3):524-6 (2006). 20-100 μg purified α-synuclein (Sigma; S7820) at a concentration of about 2.5 μg/mL is incubated in the presence of spermin (250 μM) or paraquat (32 mM) or 6-hydroxydopamine (400 μM) or vehicle in a 384 well plate. Spermin, paraquat and 6-hydroxydopamine promote the α-synuclein aggregation process. Aggregation kinetics is determined by measuring turbidity at 340 nm, every 1-15 minutes for at least one hour.
Measurement: EC50 = 116 nM (pChEMBL 6.940000057220459)
Descriptors: MW 413.91, ALogP 5.26, HBA 2, HBD 2, PSA 68.68, QED 0.45