lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccc(NC(=S)N2CCCC(c3ccccc3-c3cnc(N(C)C)nc3)C2)cc1
InChIKey: AUMIDWVRSLFREM-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: The assay buffer was formulated by removing 120 mL from a 1 L bottle of fresh, sterile dH2O (Mediatech, Herndon, Va.) and adding 100 mL of 10×HBSS that does not contain Ca++ or Mg++ (Gibco, Invitrogen, Grand Island, N.Y.) followed by 20 mL of 1.0 M Hepes, pH 7.3 (Fisher Scientific, BP299-100). The final buffer consisted of 20 mM Hepes, pH 7.3, 1.261 mM CaCl2, 0.493 mM MgCl2, 0.407 mM Mg(SO)4, 5.33 mM KCl, 0.441 mM KH2PO4, 137 mM NaCl, 0.336 mM Na2HPO4 and 0.556 mM D-glucose (Hanks et al., Proc. Soc. Exp. Biol. Med. 71:196 (1949)), and the simple formulation was typically the basic buffer throughout the assay (i.e., all wash and addition steps).
Measurement: IC50 = 2515 nM (pChEMBL 5.599999904632568)
Descriptors: MW 447.61, ALogP 4.79, HBA 5, HBD 1, PSA 53.52, QED 0.56
Compound (SMILES): CCCCNC(=O)C1CCN(Cc2cccc(NC(=O)c3cccc(C(F)(F)F)n3)c2)CC1
InChIKey: CRTPDMOTJUVUTJ-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 462.52, ALogP 4.48, HBA 4, HBD 2, PSA 74.33, QED 0.57
Compound (SMILES): Clc1ccc(Sc2oc(C3CCOCC3)nc2-c2ccc(N3CCOCC3)nc2)cc1
InChIKey: VBWSOXGKUUYYHK-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00519
Assay: FAAH Assay: Enzyme activity was demonstrated in a radioenzymatic test based on measuring the product of hydrolysis (ethanolamine [3]H) of anandamide [ethanolamine 1-.sup.3H] (American Radiolabeled Chemicals; 1 mCi/ml) with FAAH (Life Sciences (1995), 56, 1999-2005 and Journal of Pharmacology and Experimented Therapeutics (1997), 283, 729-734), Analytical. Biochemistry (2003), 318, 270-5. In addition, routine assays were performed monitoring hydrolysis of arachidonyl-7-amino-4-methylcoumarin amide (AAMCA) by following increase in fluorescence upon release of 7-amino 4-methyl coumarin (λEX=355 nm, (λEM=460 nm). Analytical. Biochemistry (2005), 343, 143-51. Assays are performed on either cell lysate fractions prepared as described or in whole cell format employing either the fluorescent substrate AAMCA (Cayman chemical, Ann Arbor, Mich.,) or 3H-anandamide ([ETHANOLAMINE-1-3H] American Radiolabeled Chemicals; 1 mCi/ml). The cell lysate assay is performed in black PerkinElmer OptiPlates-384F.
Measurement: IC50 = 12.83 nM (pChEMBL 7.889999866485596)
Descriptors: MW 457.98, ALogP 5.27, HBA 7, HBD 0, PSA 60.62, QED 0.52
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1ccc(Cl)c(Cl)c1
InChIKey: AWMWLZVPQLGHTC-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 382 nM (pChEMBL 6.420000076293945)
Descriptors: MW 394.3, ALogP 4.5, HBA 3, HBD 3, PSA 84.22, QED 0.46
Compound (SMILES): CC(C)Oc1ccc2[nH]nc(-c3cc(N4C[C@H](O)[C@@H](N(C)C)C4)ncn3)c2c1
InChIKey: QPKGUZJRHPIAKO-ROUUACIJSA-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 = 15.3 nM (pChEMBL 7.820000171661377)
Descriptors: MW 382.47, ALogP 1.92, HBA 7, HBD 2, PSA 90.4, QED 0.7
Compound (SMILES): O=c1ccc2cc(S(=O)(=O)Nc3ccon3)ccc2n1[C@@H]1CC[C@@H](c2cccc(F)c2)C1
InChIKey: VXILAPYYPKTRQF-VQIMIIECSA-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 = 8914 nM (pChEMBL 5.050000190734863)
Descriptors: MW 453.5, ALogP 4.44, HBA 6, HBD 1, PSA 94.2, QED 0.48
Compound (SMILES): Cn1cc(-c2cnc3nnn(C[C@@H]4CN(c5ncc(C6CCN(CCO)CC6)cn5)CCO4)c3n2)cn1
InChIKey: RLRGTRFDYLYKCO-FQEVSTJZSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Lanthanide Fluoro-Immunoassay: The c-Met kinase inhibitory activity was analyzed using dissociation enhanced lanthanide fluoro-immunoassay (DELFIA, Perkin Elmer), which is a kind of time-resolved fluorescence (TRF). 10 mL of the compound prepared in the present invention, as a test compound, was put in a Greiner 96-well V-shaped bottom plate, and a tyrosin kinase buffer (20 mL) mixed with a c-Met enzyme was added thereto, and then the enzyme and the test compound were stirred for 15 minutes, followed by culturing. An ATP solution (10 mL) was added thereto to proceed with a kinase reaction at room temperature for 30 minutes, and then a 50 mM ethylene diamine acetic acid solution (EDTA, 40 mL) was added to stop the reaction. After that, the reaction mixture was transferred to a plate coated with Streptavidin, followed by culturing under shaking. After 2 hours, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). The europium-labeled anti-phosphotyrosine antibody was diluted to 1:2,500, and 100 mL of the diluent was added per well, followed by culturing under shaking. After 1 hour, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). 100 mL of an enhancement solution was added, followed by shaking culturing for 5 minutes, and then the reaction material was read out within a wavelength range of 615/665 nm using a Wallac Envision 2103 device.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 505.59, ALogP 0.49, HBA 13, HBD 1, PSA 136.03, QED 0.38
Compound (SMILES): COc1cc(-c2cc(C)cc(C)c2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: JNVYGBMFKHVIRP-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 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 519.55, ALogP 5.21, HBA 7, HBD 1, PSA 103.43, QED 0.33
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCN(Cc2ccc(-c3ccccc3)cc2)CC1
InChIKey: BWGPRFVNHOJJOH-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Assay: Proteomes (mouse brain membrane fraction or cell lysates) (50 uL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37 C. After 30 min, FP-Rh (1.0 uL, 50 uM in DMSO) was added and the mixture was incubated for another 30 min at 37 C. Reactions were quenched with SDS loading buffer (50 uL-4x) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice. Inhibitors were administered to wild-type C57Bl/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 446.39, ALogP 5.1, HBA 3, HBD 0, PSA 32.78, QED 0.61
Compound (SMILES): O=S(=O)(Nc1nccs1)c1ccc2c(c1)OCCN2c1cccc2cccnc12
InChIKey: FHZKYXNABGEJIH-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
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 = 683 nM (pChEMBL 6.170000076293945)
Descriptors: MW 424.51, ALogP 4.02, HBA 7, HBD 1, PSA 84.42, QED 0.53
Compound (SMILES): CN(CC1(C#N)CCCCC1)C1CCN(C(=O)C2CCN(c3nc(-c4cc(Cl)ccc4OC(F)F)c(NC(=O)c4cnn5cccnc45)s3)CC2)CC1
InChIKey: DRKIVGMYRBBOAA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 766.32, ALogP 6.97, HBA 11, HBD 1, PSA 131.99, QED 0.18
Compound (SMILES): NC/C=C/Cl
InChIKey: ZOGFQDXLOXDFHI-OWOJBTEDSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Bos taurus
UniProt: Q9TTK6
Assay: Inhibition Assay: In the following Examples, 0.9 mL aliquots of a solution of candidate inhibitor in 100 mM potassium phosphate buffer, pH 7.2 was mixed bovine plasma amine oxidase (0.1 mL, about 8 AM) and incubated at 30° C. aerobically. Aliquots (0.1 mL) were periodically withdrawn using disposable calibrated Drummond micropipettes and diluted with 1.0 mL of benzylamine (5 mM in 50 mM sodium phosphate buffer, pH 7.2) in a 1 cm quartz cuvette (1.5 mL volume). The rate of oxidation of benzylamine to benzaldehyde was measured by recording the increase in absorbance at 250 nm for 1 minute and compared to the rate of benzylamine oxidation in a companion control solution of enzyme without inhibitor. The concentration of active BPAO was estimated from the rate of benzylamine oxidation. Inhibitory potency values (IC50 values) and partition values for inactivation of BPAO were measured at various times as in indicated in the Tables.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 91.54, ALogP 0.7, HBA 1, HBD 1, PSA 26.02, QED 0.51
Compound (SMILES): Nc1nn2cccnc2c1C(=O)Nc1cn(C2CCN(CCCCc3ccccc3)CC2)nc1-c1cc(Cl)ccc1OC(F)F
InChIKey: MZVMICFECLTELE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 3.04 nM (pChEMBL 8.520000457763672)
Descriptors: MW 635.12, ALogP 6.34, HBA 9, HBD 2, PSA 115.6, QED 0.16
Compound (SMILES): COc1cc(OCCO)cc(N(CCCN2CCCC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: MJZLZOOCTKZOHS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 79.43 nM (pChEMBL 7.099999904632568)
Descriptors: MW 502.62, ALogP 4.03, HBA 9, HBD 1, PSA 88.77, QED 0.33
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(Nc4cccc(C(=O)O)c4)cc(Nc4cccc(Cl)c4)nc23)N1
InChIKey: WYQDQFWVPFBFQQ-IUXPMGMMSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 489.88, ALogP 3.75, HBA 8, HBD 5, PSA 149.75, QED 0.2
Compound (SMILES): O=C(Nc1ccc(Cl)cc1)Nc1ccc(CCC2CCCNC2)cc1
InChIKey: NRKZUGLLRMIIEM-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 36.5 nM (pChEMBL 7.440000057220459)
Descriptors: MW 357.89, ALogP 4.92, HBA 2, HBD 3, PSA 53.16, QED 0.71
Compound (SMILES): CC#CCn1c(N2CCC[C@@H](N)C2)nc2c1c(=O)n(CC(=O)c1cccc3c1OCCO3)c(=O)n2C
InChIKey: HVJVEWQUSUKIAR-MRXNPFEDSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: DPP-IV Assay: 50 μl substrate solution (AFC; AFC is amido-4-trifluoromethylcoumarin), final concentration 100 μM, were placed in black microtitre plates. 20 μl of assay buffer (final concentrations 50 mM Tris HCl pH 7.8, 50 mM NaCl, 1% DMSO) was pipetted in. The reaction was started by adding 30 μl of solubilised Caco-2 protein (final concentration 0.14 μg of protein per well). The test substances to be investigated were typically added prediluted in 20 μl, and the volume of assay buffer was then reduced accordingly. The reaction was carried out at ambient temperature, incubating for 60 minutes. Then the fluorescence was measured in a Victor 1420 Multilabel Counter, the excitation wavelength being 405 nm and the emission wavelength being 535 nm. Blank readings (corresponding to 0% activity) were obtained in mixtures without any Caco-2 protein (volume replaced by assay buffer), control values (corresponding to 100% activity) were obtained in mixtures with no substance added.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 492.54, ALogP 0.5, HBA 11, HBD 1, PSA 126.61, QED 0.4
Compound (SMILES): Cc1cc(Nc2ccnc(Nc3ccc(N4CCOCC4)cc3)n2)no1
InChIKey: RSUGQVWRPJRPSZ-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 352.4, ALogP 3.1, HBA 8, HBD 2, PSA 88.34, QED 0.72
Compound (SMILES): CC[C@H](C)[C@@H]1NCCOc2ccccc2CCCNC(=O)[C@@H](Cc2cccs2)NC(=O)[C@@H](C)N(C)C1=O
InChIKey: HADPWJXOAVRGCK-NNUZDRNGSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Displacement of [125I]-Ghrelin from human GSHR1a transfected in HEK293 cell membranes after 30 mins by microplate scintillation and luminescence counting method
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 528.72, ALogP 2.77, HBA 6, HBD 3, PSA 99.77, QED 0.57
Compound (SMILES): CNC(=O)COc1cccc(Nc2ncc(F)c(Nc3ccc4c(c3)C(N)CCO4)n2)c1
InChIKey: BONRMPSOCBJJJO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Biochemical Assay: Several 2,4-pyrimidinediamine compounds were tested for the ability to inhibit Syk kinase catalyzed phosphorylation of a peptide substrate in a biochemical fluorescenced polarization assay with isolated Syk kinase. In this experiment, Compounds were diluted to 1% DMSO in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated Bovine Gamma Globulin). Compound in 1% DMSO (0.2% DMSO final) was mixed with ATP/substrate solution at room temperature. Syk kinase (Upstate, Lake Placid N.Y.) was added to a final reaction volume of 20 uL, and the reaction was incubated for 30 minutes at room temperature. Final enzyme reaction conditions were 20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated Bovine Gamma Globulin, 0.125 ng Syk, 4 uM ATP, 2.5 uM peptide substrate (biotin-EQEDEPEGDYEEVLE-CONH2, SynPep Corporation). EDTA (10 mM final)/anti-phosphotyrosine antibody (1x final)/fluorescent phosphopeptide tracer (0.5x final) was added in FP Dilution Buffer to stop the reaction for a total volume of 40 uL according to manufacturer's instructions (PanVera Corporation) The plate was incubated for 30 minutes in the dark at room temperature. Plates were read on a Polarion fluorescence polarization plate reader (Tecan). Data were converted to amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Assay Kit, Green (PanVera Corporation).
Measurement: IC50 = 10.7 nM (pChEMBL 7.96999979019165)
Descriptors: MW 438.46, ALogP 3.01, HBA 8, HBD 4, PSA 123.42, QED 0.44
Compound (SMILES): Cc1noc(C)c1-c1cc(S(=O)(=O)NC2CCCC2)c2nc(NCC(F)(F)F)[nH]c2c1
InChIKey: HDARKJYJRLDMQA-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 Î¼s read window). Data were normalized based on a positive (2 Î¼M I-BET) and negative (DMSO) controls and IC50values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 185.62 nM (pChEMBL 6.730000019073486)
Descriptors: MW 457.48, ALogP 4.03, HBA 6, HBD 3, PSA 112.91, QED 0.52
Compound (SMILES): N#CCC1(n2cc(-c3ccnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cnc(C(F)(F)F)cn3)CC2)C1
InChIKey: IWEBPQGVVUAHKX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 535.53, ALogP 3.46, HBA 8, HBD 1, PSA 119.62, QED 0.42
Compound (SMILES): O=C(c1cc(Cc2n[nH]c(=O)c3c2NCCC3)ccc1F)N1CCN(C(=O)N2CCCC2)CC1
InChIKey: YKOUEEOVZJIVRL-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 468.53, ALogP 1.83, HBA 5, HBD 2, PSA 101.64, QED 0.72
Compound (SMILES): Cc1nnc2sc(C(=O)NCc3ccc(S(C)(=O)=O)cc3)c(N)c2c1C
InChIKey: BDCMAJVEKVEQNH-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P08485
Assay: Positive allosteric modulation of rat M4 receptor expressed in CHO cells co-expressing Gqui5 by calcium mobilization assay
Measurement: EC50 = 51.3 nM (pChEMBL 7.289999961853027)
Descriptors: MW 390.49, ALogP 2.22, HBA 7, HBD 2, PSA 115.04, QED 0.71
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)c1ccc(C(=O)NCCNC(=O)CN2CCN(CC(=O)O)CCN(CC(=O)O)CCN(CC(=O)O)CC2)cc1)C(C)C)C(N)=O
InChIKey: OCYFHSSIZNBUGV-SEUBIJBUSA-N
Target: Gastrin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30550
Assay: Displacement of [125I]-BBN from GRPR in human PC3 cells after 1 hr by compuGamma counting or microplate scintillation counting method
Measurement: IC50 = 7.5 nM (pChEMBL 8.119999885559082)
Descriptors: MW 1516.75
Compound (SMILES): CC(C)(C)OC(=O)Cn1c2ccccc2c2cc(CN(CCCCN)[C@H]3CCCc4cccnc43)ncc21
InChIKey: AOOHTIPPARZMSX-MHZLTWQESA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Calcium Mobilization Assay: Functional modulation of CXCR4 was determined by calcium mobilization assay using leukemic lymphoid CEM cells, which naturally express high levels of CXCR4. Generally, the assay is carried out as follows: Cells are grown in vitro to confluency. On the day of the assay, cells are removed from the incubator, culture medium removed and replaced with a calcium sensitive dye (Calcium3 assay kit; Molecular Devices, Sunnyvale, Calif.). The cells are allowed to dye load for 45-60 min at 37 °C. in the cell culture incubator and then allowed to equilibrate to room temperature for no more than 15 min before assay. Compound plates were generated containing up to 3% dimethyl sulfoxide in media kept at room temperature. Test compounds were added to the cells at a 1:3 dilution, and calcium mobilization was measured using a Flex Station fluorescence imager (Molecular Devices). This first read was used to determine direct agonist activation of the CXCR4 receptor by the test compounds.
Measurement: IC50 = 949 nM (pChEMBL 6.019999980926514)
Descriptors: MW 513.69, ALogP 5.54, HBA 7, HBD 1, PSA 86.27, QED 0.23
Compound (SMILES): O=C(c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1)N1CCN(c2ccccn2)CC1
InChIKey: CWCCDRQWUHCXRE-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 690 nM (pChEMBL 6.159999847412109)
Descriptors: MW 479.46, ALogP 4.8, HBA 6, HBD 0, PSA 71.45, QED 0.42
Compound (SMILES): Cc1cc(-c2cn(CCNCC3CC3)c(C3CCN(c4ncnc(N)c4C(N)=O)CC3)n2)ccc1F
InChIKey: PIVDYIBVDVOUTC-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 3.3 nM (pChEMBL 8.479999542236328)
Descriptors: MW 492.6, ALogP 2.85, HBA 8, HBD 3, PSA 127.98, QED 0.39
Compound (SMILES): CC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)OC(C)(C)C)C(=O)N[C@@H](Cc1ccccc1)[C@@H](N)CC(=O)N[C@H](C(=O)N[C@H](C(=O)OC)C(C)C)[C@@H](C)CC
InChIKey: OZWVDVFICMDPQD-YUQDWORRSA-N
Target: Cathepsin D (SINGLE PROTEIN), Homo sapiens
UniProt: P07339
Assay: In-Vitro Fluorescence Assay: In order to identify modulators of cathepsin D activity, a continuous enzymatic test was carried out with a synthetic peptide which carries a fluorescent group (MCA=(7-methoxycoumarin-4-yl)acetyl) which is quenched by energy transfer from a Dpn (2,4 dinitrophenyl) group on the same molecule, in Greiner 384-well nb microtitre plates. Cleavage of the peptidic substrate by cathepsin D causes an increase in the fluorescence intensity. In order to determine the efficacy of substances, the time-dependent increase in the fluorescence intensity in the presence of the substance was compared with the time-dependent increase in fluorescence in the absence of substances. The reference substance used was pepstatin A (Sigma-Aldrich). The substrate used was MCAGKPILFFRLK(Dnp)d-R-NH2 (Enzo Life Sciences, Lorrach). The enzyme employed was cathepsin D isolated from the human liver (Sigma-Aldrich) in a final concentration of 1.4 nM. The test was carried out in 100 mM sodium acetate buffer, 1.25% (v/v) of DMSO, 0.25% (w/v) of Chaps, pH 5.5. 2 ul of each substance solution with serially diluted substance concentration were added to in each case 4 ul of cathepsin D solution and incubated at room temperature for 10 min. The reaction was started by addition of 2 ul of substrate solution (final concentration 5 uM). After carrying out a starting-point fluorescence measurement (excitation wavelength 340 nm/emission wavelength 450 nm) using an Envision multilabel reader (Perkin Elmer), the reaction was incubated at room temperature for 60 min. The amount of peptide fragment cleaved off during the reaction time was subsequently measured by determination of the increase in the fluorescence intensity at 450 nm (excitation wavelength 340 nm).
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 732.96, ALogP 3.11, HBA 9, HBD 6, PSA 207.05, QED 0.11
Compound (SMILES): CC(C)Nc1cc(-n2ccc3cc(C#N)cnc32)ncc1C(=O)N[C@H]1CC[C@](O)(c2ccncc2)CC1
InChIKey: PSLZRXATGWSXSD-HLTNAHAZSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 495.59, ALogP 4.07, HBA 8, HBD 3, PSA 128.75, QED 0.37
Compound (SMILES): COc1cc(-c2ccc(C(F)(F)F)cc2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3cccnn3)ccc21
InChIKey: SANFNBRFJGHZEL-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 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 570.52, ALogP 5.42, HBA 7, HBD 1, PSA 103.18, QED 0.27
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CC4CCC(C3)O4)nc(-c3ccc(NC(=O)Nc4cccc(OC)n4)c(F)c3)c2C1=O
InChIKey: IZOARZWDFVBEQV-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 = 495 nM (pChEMBL 6.300000190734863)
Descriptors: MW 591.64, ALogP 4.33, HBA 9, HBD 2, PSA 131.04, QED 0.43
Compound (SMILES): NC(=O)Cc1ccc(Nc2cc(=O)[nH]c3ccc(C(c4ccc(Cl)cc4)c4ccc(Cl)cc4)cc23)cc1
InChIKey: NROPRVDZXGJHLE-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 528.44, ALogP 6.79, HBA 3, HBD 3, PSA 87.98, QED 0.2
Compound (SMILES): Cc1ccc(S(=O)(=O)NC(C(=O)O)[C@H](N)C(=O)O)cc1
InChIKey: QECYIKYYAOOLLH-IENPIDJESA-N
Target: Excitatory amino acid transporter 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P43005
Assay: Inhibition of [3H]-D-Asp uptake at human EAAT3 expressed in HEK293 cells measured after 3 mins by TopCount method
Measurement: IC50 = 1949.84 nM (pChEMBL 5.710000038146973)
Descriptors: MW 302.31, ALogP -0.86, HBA 5, HBD 4, PSA 146.79, QED 0.53
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CC(Oc4c(F)cc(Cl)cc4F)C3)n2)c1
InChIKey: AIUKZFUBRYGVCC-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 = 3160 nM (pChEMBL 5.5)
Descriptors: MW 446.85, ALogP 3.17, HBA 7, HBD 2, PSA 92.27, QED 0.6
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)C3(c4ccc(Cl)cc4)CC3)CC2)C1
InChIKey: MFFGJKOCHMECCI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 541.06, ALogP 4.12, HBA 7, HBD 1, PSA 106.73, QED 0.39