lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(-c2ccc(Cl)c(C#N)c2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ncccn3)ccc21
InChIKey: FKDUFFGLDFUQJE-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 561.98, ALogP 4.92, HBA 8, HBD 1, PSA 126.97, QED 0.31
Compound (SMILES): CCNC(=O)c1cc(NC(=O)c2cc(-c3ccc(F)cn3)c(Cl)cc2Cl)n(-c2ccccc2)n1
InChIKey: SRYXUKGCQCUDNZ-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 498.35, ALogP 5.38, HBA 5, HBD 2, PSA 88.91, QED 0.37
Compound (SMILES): O=C(Nc1ccc(C2CCNC2)cc1)c1cn(-c2ccc(OC(F)F)cc2)nn1
InChIKey: ASSNHRVAIIIIMI-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding (mouse TAAR1): 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 μg/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 4μ 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 48'000×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]−(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 0.7 nM, resulting in the binding of approximately 0.5% of the radioligand and a specific binding representing approximately 70% of the total binding. Nonspecific binding was defined as the amount of 3[H]−(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 μM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 μM) in duplicates. The test compounds (20 μl/well) were transferred into a 96 deep well plate (TreffLab), and 180 μl of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 μl of the radioligand 3[H]−(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3×Kd in nM and 500 μl of the membranes (resuspended at 60 μg 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 μl 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 = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 399.4, ALogP 3.2, HBA 6, HBD 2, PSA 81.07, QED 0.67
Compound (SMILES): COC(=O)Nc1nc2ccc(C(=O)c3ccccc3C(=O)Nc3cccc(Br)c3)cc2[nH]1
InChIKey: KWOUWEGXAPHUJW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 493.32, ALogP 4.99, HBA 5, HBD 3, PSA 113.18, QED 0.34
Compound (SMILES): CNC(C)C(=O)Nc1cc(-c2nc(C)nc3c(C)nn(C)c23)cc(NC(=O)c2ccc(Cl)cn2)n1
InChIKey: YCZXNWIFFVKUST-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: DELFIA Assay: R3 domains of human XIAP (covering amino acids 241 to 356; XIAP BIR3) and cIAPl (covering amino acids 256 to 363; cIAPl BIR3) were expressed and purified from Ecoli as GST-fusion proteins. Peptide AVPIAQKSE-Lys(Biotin), representing the N-terminus of mature human SMAC (SMAC peptide), was used as interaction partner in the protein- peptide interaction assay.BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonXlOO) for one hour at room temperature in the presence of inhibitiory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-Nl- antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluoresecence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 493.96, ALogP 2.89, HBA 9, HBD 3, PSA 139.61, QED 0.37
Compound (SMILES): Cc1noc(C)c1-c1ccc2c(c1)C(O)(C1CCCCC1=O)C(=O)N2
InChIKey: ABSZIZKRSYKAPX-UHFFFAOYSA-N
Target: Bromodomain-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25440
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The assay was carried out in binding mixtures of the bromodomain protein, 0-10 μM compounds and tetra-acetylated histone peptide (SGRGAC.KGGAC.KGLGAC.KGGAAC.KRHGSGSK-biotin) in buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 0.1% BSA, 0.05% CHAPS, and detection reagents. The detection reagents, including streptavidin-labeled Tb cryptate and XL665-labeled anti-6×His antibody, were added after binding equilibrium was achieved for protein, compound and peptide. Upon further incubation for 1 hr, the TR-FRET signals were recorded on a BMG PHERAstar FS instrument.
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 340.38, ALogP 2.86, HBA 5, HBD 2, PSA 92.43, QED 0.88
Compound (SMILES): CC(C)Oc1ccc(Nc2cc(=O)n(C[C@H](C)C(=O)O)c(=O)n2Cc2ccc(Cl)cc2)nc1
InChIKey: KLDNEVAINXHNRD-HNNXBMFYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 in M MgCl2, 5.0 mM CaCl2, 5.6 D-glucose, 2.5 mM probenecid, 1.0% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence inte
Measurement: IC50 = 453 nM (pChEMBL 6.340000152587891)
Descriptors: MW 472.93, ALogP 3.36, HBA 8, HBD 2, PSA 115.45, QED 0.49
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(NCc4ccc(/C=C/C(=O)NO)cc4)cc3)cc12)c1ccccc1
InChIKey: UMCCKWNUMLSPPX-MBNYPGIVSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal GST-fused human EGFR (His672 to Ala1210 residues) expressed in baculovirus expression system using biotinylated PTP1B at Tyr66 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 504.59, ALogP 5.93, HBA 6, HBD 5, PSA 114.96, QED 0.1
Compound (SMILES): CC(c1cccc(NC(=O)c2ccc(C(F)(F)F)cc2)c1)N1CCC(C(=O)NC2CCCCC2)CC1
InChIKey: FDSVZGRJTUGLPU-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 501.59, ALogP 6.18, HBA 3, HBD 2, PSA 61.44, QED 0.5
Compound (SMILES): Cc1cccc(C)c1N1C[C@](C)(C(=O)Nc2cc(C(F)(F)F)cc(C(F)(F)F)c2)[C@H](C)C1=O
InChIKey: AODSSYQJXRAKCV-SZNDQCEHSA-N
Target: Chemerin-like receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99788
Assay: Displacement of [125I]-chemerin from ChemR23 (unknown origin) transfected in mouse BaF3 cells after 3 hrs by scintillation counting method
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 472.43, ALogP 5.97, HBA 2, HBD 1, PSA 49.41, QED 0.56
Compound (SMILES): O=C(Nc1cc2cccnc2cc1N1CCCCC1)c1csc2cncnc12
InChIKey: GGOMKRVCMJCSQQ-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: In vitro IRAK-1 and IRAK-4 Assay: Purified recombinant IRAK-4 protein was incubated with 250 uM synthetic peptide(KKARFSRFAGSSPSQSSMVAR) in 30 ul of kinase buffer including (20 mM MOPS pH7.2, 25 mM beta glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 50 uM ATP, 20 mM MgCl2, 10 uCi gamma-33P, 0.1% BSA) for the indicated time. For purified recombinant IRAK-1 protein kinase assay, 50 uM ATP was used. A 25 ul aliquot of the reaction mixture was transferred on to p81 phosphocellulose squares (Upstate Biotechnology, Lake Placid, N.Y.). The assay squares were washed three times with 0.75% phosphoric acid and once with acetone. Enzyme activity was measured by determining the bound radioactivity by liquid scintillation counting.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 389.48, ALogP 4.48, HBA 6, HBD 1, PSA 71.01, QED 0.56
Compound (SMILES): Cc1cc(C)n2nc(-c3ccc(CCCF)cc3)c(CC(=O)N(C)Cc3cccnc3)c2n1
InChIKey: OPHYTQQKYADXLX-UHFFFAOYSA-N
Target: Translocator protein (SINGLE PROTEIN), Rattus norvegicus
UniProt: P16257
Assay: Displacement of [3H]PK11195 from TSPO in rat heart membranes after 15 mins by scintillation counting method
Measurement: Ki = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 445.54, ALogP 4.51, HBA 5, HBD 0, PSA 63.39, QED 0.4
Compound (SMILES): Cc1nc(N2CC(c3nc4ccccc4n3C)C2)cc(-n2nc(C)nc2C(O)C2CC2)n1
InChIKey: KBFMDGZXCNZBCY-UHFFFAOYSA-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 = 0.36 nM (pChEMBL 9.4399995803833)
Descriptors: MW 430.52, ALogP 2.61, HBA 9, HBD 1, PSA 97.78, QED 0.52
Compound (SMILES): COc1cc(C#N)ccc1N1CCOc2cc(S(=O)(=O)Nc3ccncn3)ccc21
InChIKey: KTTGQIQYWNVMGC-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 910 nM (pChEMBL 6.039999961853027)
Descriptors: MW 423.45, ALogP 2.69, HBA 8, HBD 1, PSA 117.44, QED 0.67
Compound (SMILES): NC1(CO)CCc2cc(-c3nc(-c4ccnc([N+](=O)[O-])c4)no3)ccc2C1
InChIKey: UXWHIQXQKOELNU-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: GTPγS Binding Assay: Test method: wetting membranes by saponin solution, and preparing [35S]-GTPγS in binding solution (4×). The test compound was prepared as 4× of the final concentration which was then performed serial 3× dilution in 100% DMSO. Membranes with high-expressed S1P1 were collected in 5 ml binding buffer, and 50 μL was added to 96-well (containing 5 μg membrane protein with high-expressed S1P1). 25 μL, test compound was added in each well. The compound was incubated with the membranes for 30 min at room temperature, then 25 μL [35S]-GTPγS was added, and the experiment was initiated. The 96-well detection plate was incubated for 1 hour at room temperature, filtered by filtermat B, and washed with washing solution for three times. After the plate was dried for 1 h at 50° C., 8 ml scintillator solution was added and counted using scintillation counter.
Measurement: IC50 = 620 nM (pChEMBL 6.210000038146973)
Descriptors: MW 367.37, ALogP 1.89, HBA 8, HBD 2, PSA 141.2, QED 0.53
Compound (SMILES): O=C(NC[C@@H]1CCOC1)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: BLZJCPAXCWJANZ-HNNXBMFYSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of CYP3A4 (unknown origin)
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 365.43, ALogP 3, HBA 3, HBD 2, PSA 70.67, QED 0.88
Compound (SMILES): CNC(C)C(=O)Nc1cc(-c2c(C)ncnc2C)cc(NC(=O)c2ccc(Cl)cn2)n1
InChIKey: VMDYIGXFARKGRU-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: DELFIA Assay: R3 domains of human XIAP (covering amino acids 241 to 356; XIAP BIR3) and cIAPl (covering amino acids 256 to 363; cIAPl BIR3) were expressed and purified from Ecoli as GST-fusion proteins. Peptide AVPIAQKSE-Lys(Biotin), representing the N-terminus of mature human SMAC (SMAC peptide), was used as interaction partner in the protein- peptide interaction assay.BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonXlOO) for one hour at room temperature in the presence of inhibitiory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-Nl- antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluoresecence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 315 nM (pChEMBL 6.5)
Descriptors: MW 439.91, ALogP 3, HBA 7, HBD 3, PSA 121.79, QED 0.54
Compound (SMILES): O=C(O)[C@H]1CC[C@H](C(=O)N2CC[C@](c3ccc(C(OCC4CCCC4)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: PLKYVCCAKOPPGQ-ZRLASMASSA-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 = 96 nM (pChEMBL 7.019999980926514)
Descriptors: MW 707.71, ALogP 7.14, HBA 5, HBD 1, PSA 100.98, QED 0.22
Compound (SMILES): CC(C)(C)OC(=O)CNc1nc(NCc2ccc(NC(=O)c3ccc(F)cc3)cc2)c2ccccc2n1
InChIKey: XPVDHKUXDMQEKJ-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf33.13 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 1073 nM (pChEMBL 5.96999979019165)
Descriptors: MW 501.56, ALogP 5.39, HBA 7, HBD 3, PSA 105.24, QED 0.28
Compound (SMILES): CC(C)(O)C(=O)N1CC=C(c2ccc(NC(=O)N3Cc4ccccc4C3)cc2)CC1
InChIKey: NTRBPFHDZXDGKF-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 5.28 nM (pChEMBL 8.279999732971191)
Descriptors: MW 405.5, ALogP 3.62, HBA 3, HBD 2, PSA 72.88, QED 0.82
Compound (SMILES): CC[C@](O)(c1cn(Cc2ccc3c(-c4ccccc4C)cc(C(N)=O)nc3c2)nn1)C(F)(F)F
InChIKey: KULUXECFULRYIY-QHCPKHFHSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 469.47, ALogP 4.11, HBA 6, HBD 2, PSA 106.92, QED 0.44
Compound (SMILES): CC(C)N(C)c1nc2c(c(Nc3ccc(C(F)(F)F)nc3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: QIKNKLZGIIGHNG-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 525.5, ALogP 5.5, HBA 7, HBD 1, PSA 70.07, QED 0.44
Compound (SMILES): CCC(CC)c1cc(C)c(C(=O)N2CCC(c3ccc(C#N)cc3)CC2)cc1-c1nnc(C)[nH]1
InChIKey: YXVNWMKXSIEHNY-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 1210 nM (pChEMBL 5.920000076293945)
Descriptors: MW 455.61, ALogP 5.88, HBA 4, HBD 1, PSA 85.67, QED 0.5
Compound (SMILES): CCC(CC)(NC(=O)c1ccc(C(F)(F)F)c(OCC2CCOCC2)n1)C(=O)OC
InChIKey: XFIIQBLOOXKLBG-UHFFFAOYSA-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 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 432.44, ALogP 3.37, HBA 6, HBD 1, PSA 86.75, QED 0.63
Compound (SMILES): COc1ccc(CN2CC(C)C(c3nn4c(C5CCOCC5)ncc4c(=O)[nH]3)C2)c(Cl)c1
InChIKey: YDFZIUUYKZFPCS-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition Assay: A PDE9 assay may for example, be performed as follows: The assay is performed in 60 uL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labeled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions are initiated by addition of the cyclic nucleotide substrate, and reactions are allowed to proceed for one hr at room temperature before being terminated through mixing with 15 uL 8 mg/mL yttrium silicate SPA beads (Amersham). The beads are allowed to settle for one hr in the dark before the plates are counted in a Wallac 1450 Microbeta counter.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 457.96, ALogP 3.21, HBA 7, HBD 1, PSA 84.75, QED 0.63
Compound (SMILES): Cc1oc(-c2ccccc2)nc1C(=O)NCCC(=O)Nc1ccc(C2CCC(CC(=O)O)CC2)cc1
InChIKey: TWYWMRZINBQYER-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Enzyme Inhibition Assay: Human DGAT-1 activity was determined as follows: Assay buffer [20 mM HEPES (pH 7.4), 100 mM MgCl2, 0.04% BSA] containing 500 μM of enzyme substrate (didecanoyl glycerol) and 7.5 μM radiolabeled acyl-CoA substrate ([14C]decanoyl-CoA) was added to each well of a phospholipid FlashPlate (PerkinElmer Life Sciences). A small aliquot of lysate (5 μg/well) or mouse liver microsome (0.1 mg/well) was added to start the reaction, which was allowed to proceed in a 20° C. bath for 1 hour. and maintained overnight at room temperature. Next day, measurement was performed by Wallac 1450 Microbeta Trilux Liquid Scintillation Counter and Luminometer (PerkinElmer Life Science). In the control group, 0.1% DMSO was only added, and for the background value, 0.1% DMSO was only added without the cell fragment having the over-expressed human DGAT1 (5 μg/well) or the liver microsome of mice (0.1 mg/well), which was the same as the control group.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 489.57, ALogP 5.16, HBA 5, HBD 3, PSA 121.53, QED 0.38
Compound (SMILES): CC(C)Oc1ccc2[nH]nc(-c3cc(N4CCC(O)(C(F)(F)F)CC4)ncn3)c2c1
InChIKey: OXWIUVFQBRVZBT-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 10.5 nM (pChEMBL 7.980000019073486)
Descriptors: MW 421.42, ALogP 3.7, HBA 6, HBD 2, PSA 87.16, QED 0.67
Compound (SMILES): Cn1ncc(NC(=O)c2csc(-c3cccc(Cl)c3F)n2)c1[C@@H]1CC[C@@H](N)[C@@H](F)CO1
InChIKey: LJWZDMYZHCYQGJ-ZENOOKHLSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K 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 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL 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.
Measurement: Ki = 0.064 nM (pChEMBL 10.1899995803833)
Descriptors: MW 467.93, ALogP 4.11, HBA 7, HBD 2, PSA 95.06, QED 0.6
Compound (SMILES): Cn1cc(-c2cc3c(cc2N2CCOCC2)C(C)(C)c2[nH]c4cc(C#N)ccc4c2C3=O)cn1
InChIKey: SYSCKXXCBPVNBG-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 0.81 nM (pChEMBL 9.09000015258789)
Descriptors: MW 451.53, ALogP 4.15, HBA 6, HBD 1, PSA 86.94, QED 0.49
Compound (SMILES): Cc1cc(-n2cnc3c(NCC4CCOCC4)cc(Oc4ccc(Cl)c(F)c4)cc32)ccc1C(=O)NC1CC1
InChIKey: WNRMRNLJHLCNRO-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Kinase Assay: For the assay 50 nL of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Mps-1 in assay buffer [0.1 mM sodium-ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μl assay volume is 10 μM) and peptide substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 μl assay volume). The reaction was stopped by the addition of 3 μl of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, Perkin Elmer LAS, Rodgau-Jügesheinn, Germany]. The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jügesheinn, Germany).
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 549.05, ALogP 6.65, HBA 6, HBD 2, PSA 77.41, QED 0.26
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(NCCOc3ccc(Cl)cc3)n2)c1
InChIKey: KHVHBKRNIATKAH-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 10360 nM (pChEMBL 4.989999771118164)
Descriptors: MW 398.85, ALogP 3.12, HBA 7, HBD 3, PSA 101.06, QED 0.5
Compound (SMILES): CC(=O)N1CCC(Nc2ncc(C(=O)NO)cn2)(c2ccccc2)CC1
InChIKey: OEZVATBVQFFKOU-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 = 346 nM (pChEMBL 6.460000038146973)
Descriptors: MW 355.4, ALogP 1.55, HBA 6, HBD 3, PSA 107.45, QED 0.57
Compound (SMILES): CNC1=N[C@@H]2[C@H](F)[C@H](O)[C@@H]([C@H](C)O)O[C@@H]2S1
InChIKey: DYOCXHLLDLBZHU-ANBNMOMCSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Fluorescence-Based Assay: Enzymatic reactions were carried out in a reaction containing 50 mM NaH2PO4, 100 mM NaCl and 0.1% BSA (pH 7.0) using 2 mM 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide dihydrate (Sigma M2133) dissolved in ddH2O, as a substrate. The amount of purified human O-GlcNAcase enzyme used in the reaction was 0.7 nM. Test compound of varying concentrations was added to the enzyme prior to initiation of the reaction. The reaction was performed at room temperature in a 96-well plate and was initiated with the addition of substrate. The production of fluorescent product was measured every 60 sec for 45 min with a Tecan Infinite M200 plate-reader with excitation at 355 nM and emission detected at 460 nM, with 4-Methylumbelliferone (Sigma M1381) used to produce a standard curve.
Measurement: Ki = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 250.29, ALogP -0.52, HBA 6, HBD 3, PSA 74.08, QED 0.58
Compound (SMILES): COc1cc(C)cc2cc(-c3c(CNC(C)=O)c(CNC(C)=O)n4ncnc(N)c34)sc12
InChIKey: LNCJTAGAWGYDHX-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: High ATP Kinase Assay: For the assay, 50 nl of each stock solution of the test compound in DMSO was pipetted into a black, low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany) 2 Î¼l of a solution of the above FGFR-1 fusion protein in aqueous assay buffer [8 mM MOPS pH 7.0, 10 mM magnesium acetate, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumin (BSA), 0.07% (v/v) Tween-20, 0.2 mM EDTA] was added, and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme. Then, the kinase reaction was started by the addition of 3 ul of a solution of adenosine triphosphate (ATP, 3.3 mM; final concentration in the 5 ul assay volume=2 mM) and substrate (0.16 uM; final concentration in the 5 ul assay volume=0.1 uM) in assay buffer, and the resulting mixture was incubated for a reaction time of 15 min at 22° C. The concentration of FGFR-1 fusion protein was adjusted depending on the activity of the enzyme lot.
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 452.54, ALogP 2.78, HBA 8, HBD 3, PSA 123.64, QED 0.41
Compound (SMILES): CC1(C)CN(C2CCOCC2)C(=O)c2c(nc(N3CC4CCC(C3)O4)nc2-c2ccc(NC(=O)Nc3ccon3)c(F)c2)O1
InChIKey: OTHOAKOLOMZDHI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The test detects the phosphorylation of the substrate GFP-4EBP1 using a terbium-labelled antibody that is specific for the phospho-Thr46 epitope. Binding of the Tb-antibody to the phosphorylated GFP peptide allows the transfer of energy from Tb, excited at 340 nm, to GFP with an increase in the fluorescence emission of the GFP at 520 nm. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 ul. To 1 ul of inhibitor in 100% DMSO are added (final concentrations) 400 nM of GFP-4EBP1 (Invitrogen PV4759), 8 uM of ATP, 200 ng/ml of enzyme (human recombinant aa mTOR 1360-2549, Invitrogen PV4753) in a buffer of HEPES 50 mM pH 7.5, EGTA 1 mM, MnCl2 10 mM, BSA 0.1 mg/ml, glycerol 0.5%, NV-10 0.1 mg/ml, DTT 2 mM. The mixture is incubated for 30 minutes at room temperature. 10 ul of solution are taken up and the reaction is quenched by adding 10 ul of Tb-antibody solution (Invitrogen PV4757)/EDTA.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 607.64, ALogP 4.07, HBA 10, HBD 2, PSA 144.18, QED 0.44