lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@@H]1CN(c2ncc([C@@H](O)CO)cc2F)CCN1C(=O)Nc1nc2ccc(F)cc2s1
InChIKey: GWEKEPTYZIALJB-BZNIZROVSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Heat-based Assay: Briefly, hTRPV1/CHO cells were cultured in growth media in a tissue culture dish at 37° C. in a CO2 incubator. On the day of the assay, culture media were removed and the cells were then detached using 0.05% trypsin at 37° C. with 5% CO2, for 90 s. The detached cells were centrifuged (1000 rpm, 4 min) to remove trypsin-containing supernatant and resuspended in assay buffer (115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2.6H2O, 1.8 mM CaCl2.2H2O, 13.8 mM D-glucose, and 20 mM HEPES). Then, the cells were loaded with 5 uM Fluo-4, a Ca2+ reporter dye, in the presence of 2.5 mM probenecid at 37° C. with 5% CO2, for 45 min. Thereafter, the cells were washed twice with measuring buffer (assay buffer supplemented with 0.1% BSA and 3.2 mM CaCl2) then transferred to a Fast 96-well Reaction Plate (0.1 mL) (Part no. 4346907, MICROAMP, Applied Biosystems, Foster City, Calif.). The cell density was 100,000 cells/24 uL/well. A solution of the compound under test (6 uL/well) was added into each well of the 96-well plate. Thus, the reaction volume per well was 30 uL. The plates were then placed inside an ABI7500 Fast Real-Time PCR instrument (Applied Biosystems) to read fluorescence at different temperatures using 7500 software, version 2.0.2 (Applied Biosystems). The initial temperature was set at 25° C. for 1 min. followed by a temperature ramp to 45° C. in 100 s to deliver heat to cells.
Measurement: IC50 = 18.4 nM (pChEMBL 7.739999771118164)
Descriptors: MW 449.48, ALogP 2.74, HBA 7, HBD 3, PSA 101.82, QED 0.57
Compound (SMILES): C[C@@H](O)CNC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: DDVZGQOSERSQJU-JHJVBQTASA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 320 nM (pChEMBL 6.5)
Descriptors: MW 328.42, ALogP 1.85, HBA 6, HBD 3, PSA 91.13, QED 0.66
Compound (SMILES): COc1ccc(NC(=O)C2=C(c3ccc(C)cc3)C[C@@](c3ccc(OCCCC(F)(F)F)cc3)(C(F)(F)F)NC2=O)cc1
InChIKey: FBBPRPJENPWDPL-GDLZYMKVSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Mus musculus
UniProt: Q921V5
Assay: Inhibition of mouse recombinant MGAT2 expressed in Sf9 cell membrane using 2-oleoylglycerol and oleoyl-CoA as substrates incubated for 10 mins by LCMS assay
Measurement: IC50 > 2050 nM (pChEMBL 5.688000202178955)
Descriptors: MW 606.56, ALogP 7.09, HBA 4, HBD 2, PSA 76.66, QED 0.15
Compound (SMILES): Cn1cc(-c2cc(C(N)=O)nc3cc(CN4CCO[C@@H](C(F)(F)F)C4)ccc23)cn1
InChIKey: PUIAIYDBFPDZPF-GOSISDBHSA-N
Target: Nuclear receptor subfamily 1 group I member 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75469
Assay: Agonist activity at PXR (unknown origin)
Measurement: EC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 419.41, ALogP 2.5, HBA 6, HBD 1, PSA 86.27, QED 0.7
Compound (SMILES): Cc1c(NS(=O)(=O)c2ccc(-c3cccc(CN4CCC4)c3)cc2)c(CC(C)C)nn1C
InChIKey: HQHKSPQNNHMQTF-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Leishmania major
UniProt: Q4Q5S8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 452.62, ALogP 4.6, HBA 5, HBD 1, PSA 67.23, QED 0.54
Compound (SMILES): Cc1ccccc1OC[C@H]1CN(CC(=O)N2c3ccccc3CC2C)CCO1
InChIKey: HHTOEBWSVPCHMS-ROPPNANJSA-N
Target: Alpha-1D adrenergic receptor (SINGLE PROTEIN), Mus musculus
UniProt: P97714
Assay: Radioligand Binding Assay: The detailed experimental protocols for the radioligand and functional receptor assays are available on the NIMH PDSP website at http://pdsp.med.unc.edu/UNC-CH %20Protocol %20Book.pdf. A. Serotonin receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6 and 5-HT7. Assay Buffer: Standard Binding Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4) Membrane Fraction Source: Transiently or stably transfected cell lines (e.g., HEK293, COS, CHO, NIH3T3). Protocol adapted from Roth et al. (1986), J. Pharmacol. Exp. Ther., 238(2): 480-485; and Roth et al. (1994), J. Pharmacol. Exp. Ther., 268(3): 1403-1410. Adrenergic Receptors: alpha1A, alpha1B, alpha2A, alpha2B, alpha2C, beta1, beta2, beta3 Assay Buffers: For alpha1 receptors, alpha1 Binding Buffer (20 mM Tris-HCl, 145 mM NaCl, pH 7.4); for alpha2 receptors, alpha2 Binding Buffer (50 mM Tris-HCl, 5 mM MgCl2, pH 7.7); for beta receptors.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 380.49, ALogP 3.05, HBA 4, HBD 0, PSA 42.01, QED 0.8
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccc(C(O)CO)cc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: RTJWYSXNAXKJOC-FEJKDCIJSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Radioligand Dose-Displacement Binding Assay: Delta-opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 ug membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 ul binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 uM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hr at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 ul ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours.
Measurement: Ki = 517.28 nM (pChEMBL 6.289999961853027)
Descriptors: MW 561.72, ALogP 4.16, HBA 7, HBD 2, PSA 80.62, QED 0.45
Compound (SMILES): O=C(NCCN1CCC2(CC1)C(=O)NC[C@@H]2c1ccc(F)cc1)c1cnc2ccccc2c1
InChIKey: BAHMBPJZGYBLIO-JOCHJYFZSA-N
Target: Phospholipase D1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13393
Assay: Inhibition Assay: In one aspect, the compound inhibits PLD activity; that is, a compound can inhibit PLD1 activity and/or PLD2 activity. In a further aspect, the compound inhibits PLD1 response in Calu-1 cells. In a further aspect, the compound inhibits PLD2 response in HEK293gfpPLD2 cells. In a further aspect, the compound inhibits in vitro PLD1 response. In a further aspect, the compound inhibits in vitro PLD2 response.
Measurement: IC50 = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 446.53, ALogP 3.1, HBA 4, HBD 2, PSA 74.33, QED 0.63
Compound (SMILES): O=C([C@@H]1CCCN(c2ccc3nc(C4(c5cccnc5)CC4)[nH]c3n2)C1)N1CCCC1
InChIKey: FEKUNHHIZBHAIU-QGZVFWFLSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: DGAT2 Cell-Based Assay: For evaluation of the effects of DGAT2 inhibitors in a cell-based setting, cryopreserved human hepatocytes (Lot QOC, Celsis, Chicago, Ill.) were thawed and plated onto type I collagen-coated plates according to the manufacturer's instructions. After 24 hours overnight recovery period, the cells were overlayed with media containing 250 ug/ml Matrigel (BD Biosciences, San Jose, Calif.). The following day, media was aspirated and replaced with serum-free Williams Media E (Life Technologies, Grand Island, N.Y.) containing 400 uM sodium dodecanoate (Sigma-Aldrich, St. Louis, Mo.). Forty minutes later, DGAT2 inhibitors (prepared as 100× stocks in 25% DMSO, 75% Williams' Media E) were added to the desired final concentration. All wells contained a selective DGAT1 inhibitor (Example 3, WO2009016462) at a concentration (3 uM) that completely suppressed endogenous DGAT1 activity. After a 20 minute preincubation, 0.2 uCi [1,3-14C]-glycerol (American Radio Chemicals, St. Louis, Mo.) was added to each well and mixed by gentle pipetting prior to a 3 hour incubation. At this point, media was aspirated and the cells were lysed in isopropyl alcohol:tetrahydrofuran (9:1) prior to centrifugation at 3000 rpm for 5 minutes. Radiolabeled lipids were resolved using a 2-solvent system by thin layer chromatography using standard technique (solvent 1 contained ethyl acetate: isopropyl alcohol: chloroform:methanol:0.25% potassium chloride in water (100:100:100:40.2:36.1, v/v/v/v) and solvent 2 contained hexane: diethyl ether: acetic acid (70:27:3, v/v/v)). After separation, radiolabeled lipids were visualized using a Molecular Dynamics' PhosphorImager system. The half maximal inhibitory concentrations (IC50 values) were determined using Graph Pad Prism (GraphPad Software, Inc., La Jolla, Calif.).
Measurement: IC50 = 47.7 nM (pChEMBL 7.320000171661377)
Descriptors: MW 416.53, ALogP 3.27, HBA 5, HBD 1, PSA 78.01, QED 0.71
Compound (SMILES): CC[C@@](O)(c1cn(Cc2ccc3c(-c4ccc(F)cc4)cc(C(N)=O)nc3c2)cn1)C(F)(F)F
InChIKey: NVHOOOQFDVIAGG-HSZRJFAPSA-N
Target: Metabotropic glutamate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14832
Assay: Inhibition of human mGluR3 by FLIPR assay
Measurement: IC50 = 3500 nM (pChEMBL 5.460000038146973)
Descriptors: MW 472.44, ALogP 4.54, HBA 5, HBD 2, PSA 94.03, QED 0.4
Compound (SMILES): CO[C@]12CC[C@@]3(C[C@@H]1COCc1ccccc1)[C@H]1Cc4ccc(C(=O)O)c5c4[C@@]3(CCN1C)[C@H]2O5
InChIKey: DRGUKZYCXDRQEJ-IPRXQQHWSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Radioligand Dose-Displacement Binding Assay: Delta-opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 ug membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 ul binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 uM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hr at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 ul ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours.
Measurement: Ki = 13416.5 nM (pChEMBL 4.869999885559082)
Descriptors: MW 475.59, ALogP 4.05, HBA 5, HBD 1, PSA 68.23, QED 0.68
Compound (SMILES): Cc1cnc([C@](C)(O)C#Cc2ccc3c(c2)-c2nc(C(N)=O)cn2C2CC3C2)o1
InChIKey: RODIOQSCTKCZKO-BYHBIXDJSA-N
Target: Serine/threonine-protein kinase D1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15139
Assay: Inhibition of full length GST-tagged human PRKD1 expressed in Baculovirus expression system using ATP as substrate incubated for 1 to 2 hrs by transcreener ADP assay based fluorescence correlation spectroscopic analysis
Measurement: Ki < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 388.43, ALogP 2.64, HBA 6, HBD 2, PSA 107.17, QED 0.66
Compound (SMILES): O=C(Nc1ccc([C@H]2CNCCO2)cc1)c1cnn(-c2ccc(OC(F)(F)F)cc2)n1
InChIKey: CVDIRGGTOFSYMF-GOSISDBHSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding (rat TAAR1): HEK-293 cells stably expressing rat TAAR1 were maintained at 37Â° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56Â° C.), penicillin/streptomycin (1%), and 375 Î¼g/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 4Â° C., frozen and stored at â¿¿80Â° 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 4Â° 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 â¿¿80Â° 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 Î¼M unlabeled ligand. All compounds were tested at a broad range of concentrations (10 Î¼M to 10 Î¼M) in duplicates. The test compounds (20 Î¼l/well) were transferred into a 96 deep well plate (TreffLab), and 180 Î¼l of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 Î¼l of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3Ã¿Kd in nM and 500 Î¼l of the membranes (resuspended at 50 Î¼g protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4Â° 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 Î¼l 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 = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 433.39, ALogP 3.08, HBA 7, HBD 2, PSA 90.3, QED 0.64
Compound (SMILES): C[C@@H]1CN(c2ccc(C(F)(F)F)cc2C#N)c2ccc(S(=O)(=O)Nc3nccs3)cc2O1
InChIKey: KTYPXYDRNKHYHU-GFCCVEGCSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 6670 nM (pChEMBL 5.179999828338623)
Descriptors: MW 480.49, ALogP 4.75, HBA 7, HBD 1, PSA 95.32, QED 0.58
Compound (SMILES): Cc1cc(C)nc(-n2nnc3c2CCN(C(=O)c2cccc(C(F)(F)F)c2Cl)[C@@H]3C)n1
InChIKey: VBRHOQZYDPVYSS-GFCCVEGCSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Radioligand Binding: uman or rat P2X7-1321N1 cells were collected and frozen @−80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl:10 μl compound (10x)+(b) 40 μl tracer (2.5×)+50 μl membrane (2x). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2008, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM). The IC50 generated in the binding assay was corrected for tracer concentration and affinity of the tracer to derive at the affinity (Ki) of the test compounds. The data are presented in Tables 2 and 3 under the headings: P2X7 human Ki (μM) and P2X7 rat Ki (μM). Data are analyzed and graphed on Graphpad Prism 5. For analysis, each concentration point is averaged from triplicate values and the averaged values are plotted on Graphpad Prism.
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 450.85, ALogP 4.11, HBA 6, HBD 0, PSA 76.8, QED 0.59
Compound (SMILES): O=C(c1ccc(-n2nnc3cccnc32)cc1)N(c1ncccc1Cl)[C@@H]1CCCNC1
InChIKey: FDTXHWQFIXYHCL-QGZVFWFLSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of dofetilide binding to human ERG by fluorescence polarization assay
Measurement: Ki = 18100 nM (pChEMBL 4.739999771118164)
Descriptors: MW 433.9, ALogP 3.26, HBA 7, HBD 1, PSA 88.83, QED 0.53
Compound (SMILES): COC[C@H]1CCCN1Cc1ccc(C(=O)NO)cc1
InChIKey: JBIMZYHMXSAQOQ-CYBMUJFWSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: HDAC Activity Assay: HDAC assay is performed using fluorescently-labeled acetylated substrate, which comprises an acetylated lysine side chain. After incubation with HDAC, deacetylation of the substrate sensitizes the substrate such that, in a second step, treatment with the detection enzyme produces a fluorophore. HDACs 1 and 6 were expressed as full length fusion proteins. Purified proteins were incubated with 50 μM fluorescently-labeled acetylated peptide substrate and test compound for 2 hours at RT in HDAC assay buffer containing 50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% DMSO, and 1% BSA. Reactions were terminated by the addition of the Developer after 2 hours, and the development of fluorescence signal, which was relative to the amount of deacetylated peptide, was monitored by time-course measurement of EnVision (PerkinElmer). The HDAC activity was estimated from the slope of time-course measurement of the fluorescence intensity.
Measurement: IC50 = 5590 nM (pChEMBL 5.25)
Descriptors: MW 264.33, ALogP 1.42, HBA 4, HBD 2, PSA 61.8, QED 0.62
Compound (SMILES): CC(Nc1ncc(F)c(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1cc(-c2ccc(Cl)cc2Cl)no1
InChIKey: HJSGEOWEKHAQKS-MVHXDWSJSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+(NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 1460 nM (pChEMBL 5.840000152587891)
Descriptors: MW 482.3, ALogP 4.46, HBA 8, HBD 2, PSA 113.61, QED 0.53
Compound (SMILES): COC[C@@H](Nc1ncnc(N)c1C#Cc1ccc(F)cn1)c1nc2cccc(Cl)c2c(=O)n1-c1cc(F)cc(F)c1
InChIKey: NTQCCUGEADZYGD-HSZRJFAPSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 577.95, ALogP 4.42, HBA 9, HBD 2, PSA 120.84, QED 0.28
Compound (SMILES): CC(C)(C)COC(=O)N1CC[C@@H](n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)[C@H](C#N)C1
InChIKey: WVQQWCGPNVLRFV-RDTXWAMCSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 1231 nM (pChEMBL 5.909999847412109)
Descriptors: MW 442.5, ALogP 3.43, HBA 7, HBD 2, PSA 126.27, QED 0.73
Compound (SMILES): COCCOCCOCCOc1ccc(COC[C@H]2C[C@@]34CC[C@]2(OC)[C@@H]2Oc5c(OC)ccc6c5[C@@]23CCN(CC2CC2)[C@@H]4C6)cc1
InChIKey: DEPQWSCKUDWYLQ-UUBLVUCNSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Radioligand Dose-Displacement Binding Assay: Delta-opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 ug membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 ul binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 uM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hr at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 ul ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours.
Measurement: Ki = 1983.26 nM (pChEMBL 5.699999809265137)
Descriptors: MW 663.85, ALogP 5.19, HBA 9, HBD 0, PSA 77.08, QED 0.2
Compound (SMILES): CC1C2Cc3ccc(O)cc3[C@]1(C)CCN2CC1(C)CC1
InChIKey: CHPZBNWSVQPLCP-GFEPPQMASA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Measurement: EC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 285.43, ALogP 3.72, HBA 2, HBD 1, PSA 23.47, QED 0.9
Compound (SMILES): COC(=O)N[C@@H](C(=O)N1CCC[C@H]1c1nc2ccc(-c3ccc4cc(-c5cnc([C@H]6CCCN6C(=O)[C@H](NC(=O)OC)[C@@H](C)OC)[nH]5)ccc4c3)cc2[nH]1)C(C)C
InChIKey: SPEHMKGCCXRFKM-WXXZOMQASA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Transient Replicon Assay: In a transient set-up, a Huh-7 lunet hepatoma cell line was transiently transfected with an autonomously replicating RNA encoding a bi-cistronic expression construct. This construct comprises a firefly luciferase reporter gene preceding the NS3-NS5B subgenomic region of HCV (genotype 1a H77 or 1b Con1). Translation of the HCV subgenomic region is mediated by an internal ribosome entry site of encephalomyocarditis virus. The construct is furthermore flanked by 5' and 3' untranslated regions of HCV (genotype 1a H77 or 1b Con 1, respectively), which allow for replication of the RNA. In addition to the wild-type constructs, site-directed mutations were introduced into the transient HCV genotype 1b replicon in the gene encoding for the non-structural protein 5A (NS5A). More precisely, amino acid residues 28, 30, 31 and 93 in NS5A were independently altered. Cells were plated in 384 well plates in the presence of test and control compounds, which were added in various concentrations. Following an incubation of two days, replication of the HCV subgenomic replicon RNA was measured by assaying luciferase activity (using standard luciferase assay substrates and reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager). HCV subgenomic replicon containing cells in the control cultures have high luciferase expression in the absence of any inhibitor. The inhibitory activity of the compound was monitored, enabling a dose-response curve for each test compound.
Measurement: EC50 = 0.021 nM (pChEMBL 10.680000305175781)
Descriptors: MW 778.91, ALogP 6.24, HBA 9, HBD 4, PSA 183.87, QED 0.12
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1noc(Cc2ccccc2)n1
InChIKey: NBOLFMVBIZMHBG-JYRZLJSNSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+(NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 > 25000 nM (pChEMBL 4.6020002365112305)
Descriptors: MW 410.43, ALogP 2.33, HBA 9, HBD 2, PSA 126.5, QED 0.6
Compound (SMILES): Cc1nc(C2C[C@H]2c2nc3ccccc3n2C)cc(-n2nc(C)nc2C(O)C(F)(F)F)n1
InChIKey: AZVXNJWTMXGGSL-YNAXMUEKSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. A Labcyte Echo 555 (Labcyte, Sunnyvale, Calif.) is used to dispense 200 nL from each well of the titration plate to the 384 well assay plate. A solution of enzyme (1/1600 dilution from aliquots; sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP PDE from Molecular Devices (product # R7506), at a final concentration of 50 nM are made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). The enzyme and the substrate are then added to the assay plates in two consecutive additions of 10 μL, and then shaken to mix. The reaction is allowed to proceed at room temperature for 30 minutes. A binding solution is then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction is stopped by addition of 60 μL of the binding solution to each well of the assay plates and the plates are sealed and shaken for 10 seconds. The plate was incubated at room temperature for at least one hour prior to determining the fluorescence polarization (FP). The parallel and perpendicular fluorescence of each well of the plate was measured using a Perkin Elmer EnVision™ plate reader (Waltham, Mass.).
Measurement: Ki = 0.92 nM (pChEMBL 9.039999961853027)
Descriptors: MW 443.43, ALogP 3.43, HBA 8, HBD 1, PSA 94.54, QED 0.52
Compound (SMILES): NC1CCC(N[C@H]2C[C@@H]2c2ccc(OCc3cccc(N4CCNCC4)c3)cc2)CC1
InChIKey: TVYIZPPTJYFBQU-FJJJVWAOSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biological Assay: The compounds of the invention can be tested for their ability to inhibit LSD1. The ability of the compounds of the invention to inhibit LSD1 can be tested as follows. Human recombinant LSD1 protein was purchased from BPS Bioscience Inc (catalog reference number 50100: human recombinant LSD1, GenBank accession no. NM_015013, amino acids 158-end with N-terminal GST tag, MW: 103 kDa). In order to monitor LSD1 enzymatic activity and/or its inhibition rate by our inhibitor(s) of interest, di-methylated H3-K4 peptide (Anaspec) was chosen as a substrate. The demethylase activity was estimated, under aerobic conditions, by measuring the release of H2O2 produced during the catalytic process, using the Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen).Briefly, a fixed amount of LSD1 was incubated on ice for 15 minutes, in the absence and/or in the presence of at least eight 3-fold serial dilutions of the respective inhibitor (e.g., from 0 to 75 uM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition. Within the experiment, each concentration of inhibitor was tested in duplicate. After leaving the enzyme interacting with the inhibitor, KM of di-methylated H3-K4 peptide was added to each reaction and the experiment was left for 30 minutes at 37° C. in the dark. The enzymatic reactions were set up in a 50 mM sodium phosphate, pH 7.4 buffer. At the end of the incubation, Amplex Red reagent and horseradish peroxidase (HPR) solution were added to the reaction according to the recommendations provided by the supplier (Invitrogen), and left to incubate for 5 extra minutes at room temperature in the dark. A 1 uM H2O2 solution was used as a control of the kit efficiency. The conversion of the Amplex Red reagent to resorufin due to the presence of H2O2 in the assay, was monitored by fluorescence (excitation at 540 nm, emission at 590 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure level of H2O2 produced in the absence and/or in the presence of inhibitor. The maximum demethylase activity of LSD1 was obtained in the absence of inhibitor and corrected for background fluorescence in the absence of LSD1. The IC50 value of each inhibitor was calculated with GraphPad Prism Software.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 420.6, ALogP 3.39, HBA 5, HBD 3, PSA 62.55, QED 0.64
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(C(F)(F)F)cc2)[C@@H]3C)n1
InChIKey: MJAOQGKUYGSDSK-SECBINFHSA-N
Target: Substance-P receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25103
Assay: Binding Assay: The affinity of compounds of the invention for the NK-1 receptor was evaluated in CHO recombinant cells which express the human NK-1 receptor. Membrane suspensions were prepared from these cells. The following radioligand: [3H] substance P (PerkinElmer Cat#NET111520) was used in this assay. Binding assays were performed in a 50 mM Tris/5 mM MnCl2/150 mM NaCl/0.1% BSA at pH 7.4. Binding assays consisted of 25 μl of membrane suspension (approximately 5 μg of protein/well in a 96 well plate), 50 μl of compound or reference ligand (Substance P) at increasing concentrations (diluted in assay buffer) and 2 nM [3H] substance P. The plate was incubated 60 min at 25° C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in 0.5% PEI for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard).
Measurement: Ki > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 408.41, ALogP 3.34, HBA 7, HBD 0, PSA 76.8, QED 0.65
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccncc2NC1=O
InChIKey: XYICNNPOQLZANR-GYCJOSAFSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 522.98, ALogP 6.19, HBA 4, HBD 1, PSA 75.19, QED 0.41
Compound (SMILES): O[C@H](CN[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)c1ccccc1
InChIKey: RJVYQTHPYZENAN-JFIYKMOQSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 376.46, ALogP 2.96, HBA 6, HBD 3, PSA 91.13, QED 0.5
Compound (SMILES): COC(=O)N[C@H](C(=O)N1CCC[C@H]1c1ncc(-c2ccc3cc(-c4ccc5nc([C@@H]6CCCN6C(=O)[C@@H](NC(=O)OC)[C@@H](C)OC)[nH]c5c4)ccc3c2)[nH]1)C(C)C
InChIKey: QCDPCGCVNMYPHK-CVSAGBRJSA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Transient Replicon Assay: In a transient set-up, a Huh-7 lunet hepatoma cell line was transiently transfected with an autonomously replicating RNA encoding a bi-cistronic expression construct. This construct comprises a firefly luciferase reporter gene preceding the NS3-NS5B subgenomic region of HCV (genotype 1a H77 or 1b Con1). Translation of the HCV subgenomic region is mediated by an internal ribosome entry site of encephalomyocarditis virus. The construct is furthermore flanked by 5' and 3' untranslated regions of HCV (genotype 1a H77 or 1b Con 1, respectively), which allow for replication of the RNA. In addition to the wild-type constructs, site-directed mutations were introduced into the transient HCV genotype 1b replicon in the gene encoding for the non-structural protein 5A (NS5A). More precisely, amino acid residues 28, 30, 31 and 93 in NS5A were independently altered. Cells were plated in 384 well plates in the presence of test and control compounds, which were added in various concentrations. Following an incubation of two days, replication of the HCV subgenomic replicon RNA was measured by assaying luciferase activity (using standard luciferase assay substrates and reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager). HCV subgenomic replicon containing cells in the control cultures have high luciferase expression in the absence of any inhibitor. The inhibitory activity of the compound was monitored, enabling a dose-response curve for each test compound.
Measurement: EC50 < 0.017 nM (pChEMBL 10.770000457763672)
Descriptors: MW 778.91, ALogP 6.24, HBA 9, HBD 4, PSA 183.87, QED 0.12
Compound (SMILES): CO[C@]12CC[C@@]3(C[C@@H]1COCc1ccccc1)[C@H]1Cc4ccc(Br)c5c4[C@@]3(CCN1CC1CC1)[C@H]2O5
InChIKey: ZQPMWEQJZNJYEY-VTPACTEHSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Measurement: EC50 = 266.83 nM (pChEMBL 6.570000171661377)
Descriptors: MW 550.54, ALogP 5.89, HBA 4, HBD 0, PSA 30.93, QED 0.43
Compound (SMILES): Nc1ncc(-c2ccncc2)cc1-c1ccc(C(=O)N[C@H](CO)c2ccccc2)c(F)c1
InChIKey: CWJSKMKBLVAHGN-HSZRJFAPSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: AlphaScreen Assay: 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.).
Measurement: IC50 = 0.166 nM (pChEMBL 9.779999732971191)
Descriptors: MW 428.47, ALogP 4, HBA 5, HBD 3, PSA 101.13, QED 0.43
Compound (SMILES): COC(=O)N[C@H](C(=O)N1CCC[C@H]1c1ncc(-c2ccc3cc(-c4ccc5nc([C@@H]6CCCN6C(=O)[C@@H](NC(=O)OC)[C@@H](C)OC)[nH]c5c4)ccc3c2)[nH]1)[C@@H](C)OC
InChIKey: LWKDNBZBQYGISG-OYDNEJTASA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Transient Replicon Assay: In a transient set-up, a Huh-7 lunet hepatoma cell line was transiently transfected with an autonomously replicating RNA encoding a bi-cistronic expression construct. This construct comprises a firefly luciferase reporter gene preceding the NS3-NS5B subgenomic region of HCV (genotype 1a H77 or 1b Con1). Translation of the HCV subgenomic region is mediated by an internal ribosome entry site of encephalomyocarditis virus. The construct is furthermore flanked by 5' and 3' untranslated regions of HCV (genotype 1a H77 or 1b Con 1, respectively), which allow for replication of the RNA. In addition to the wild-type constructs, site-directed mutations were introduced into the transient HCV genotype 1b replicon in the gene encoding for the non-structural protein 5A (NS5A). More precisely, amino acid residues 28, 30, 31 and 93 in NS5A were independently altered. Cells were plated in 384 well plates in the presence of test and control compounds, which were added in various concentrations. Following an incubation of two days, replication of the HCV subgenomic replicon RNA was measured by assaying luciferase activity (using standard luciferase assay substrates and reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager). HCV subgenomic replicon containing cells in the control cultures have high luciferase expression in the absence of any inhibitor. The inhibitory activity of the compound was monitored, enabling a dose-response curve for each test compound.
Measurement: EC50 = 0.02 nM (pChEMBL 10.699999809265137)
Descriptors: MW 794.91, ALogP 5.62, HBA 10, HBD 4, PSA 193.1, QED 0.12
Compound (SMILES): Cc1ccc(-n2ncc3cnc(-c4cnn(CC(F)(F)F)c4)cc32)nc1N1CCNC[C@@H](O)C1
InChIKey: LMYCHBCZOYRSRO-QGZVFWFLSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.127 nM (pChEMBL 9.899999618530273)
Descriptors: MW 472.48, ALogP 2.32, HBA 9, HBD 2, PSA 96.92, QED 0.47