lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(NC5CCN(CCCO)CC5)c([N+](=O)[O-])c4)c(Oc4ccc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: UDKSWVJJCHUBEM-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 0.025 nM (pChEMBL 10.600000381469727)
Descriptors: MW 910.54, ALogP 8.69, HBA 11, HBD 4, PSA 173.38, QED 0.06
Compound (SMILES): O=C(Nc1ccc(C(=O)N2CCOC3(CCCNC3)C2)cc1)N1Cc2ccncc2C1
InChIKey: VUWDCMUNOHVZHZ-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 = 318 nM (pChEMBL 6.5)
Descriptors: MW 421.5, ALogP 2.22, HBA 5, HBD 2, PSA 86.8, QED 0.78
Compound (SMILES): CC(C)c1nn(-c2ccc(C(N)=O)c(NC(C)(C)C)c2)c2cccc(-n3cnc(-c4cnn(C)c4)c3)c12
InChIKey: JIGBOAWNDYNSQF-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: AlphaScreen Competitive Assay: The HSP90-binding activity was measured by an AlphaScreen competitive assay system. The purified HSP90 solution was diluted with a binding buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton-X100, 1 mM DTT, 0.1% BSA) and added to a 384-well plate (No. 3673, Corning Incorporated) containing test substances. After reaction at room temperature for 2 hours, biotin-labeled geldanamycin was added to each reaction solution in an amount of 40 nM, followed by reaction for further 1 hour. Detection mix (20 mM HEPES-KOH (pH 7.5), 0.5% BSA, 0.04 mg/mL Nickel Chelate Acceptor beads, 0.04 mg/mL Streptavidin-coated Donor beads) (No. 6760619C, Perkin Elmer, Inc.) was added to each well in the same amount as that of the reaction solution. After reaction in a dark place at room temperature for 1 hour, the fluorescence intensity in each well was measured with a multilabel plate reader, EnVision (Perkin Elmer, Inc.).
Measurement: IC50 = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 496.62, ALogP 5.04, HBA 8, HBD 2, PSA 108.58, QED 0.34
Compound (SMILES): CC(c1nc2c(cnn2C2CCOCC2)c(=O)[nH]1)N1CC(Oc2cccc(C#N)c2)C1
InChIKey: WDCSNTMEYAUKHZ-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of human recombinant PDE9 expressed in insect Sf9 cells using [3H]-cGMP as substrate after 30 mins by scintillation proximity assay
Measurement: IC50 = 24.1 nM (pChEMBL 7.619999885559082)
Descriptors: MW 420.47, ALogP 2.17, HBA 8, HBD 1, PSA 109.06, QED 0.67
Compound (SMILES): CCNC(=O)N1C(=O)N(c2cccc(C(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1
InChIKey: XQLIPAQDVCADES-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Nuetrophil Elastase Acitivty Assay: Various concentrations of the neutrophil elastase inhibitor are incubated with plasma. Subsequently, the enzyme activity is measured using the fluorogenic substrate MeOSuc-Ala-Ala-Pro-Val-AMC (Bachem Cat. No. 1-1270, substrate concentration: 250 uM, pH 7.5, 25 mM TRIS buffer, 250 mM NaCl) in analogous fashion as described for the human neutrophil assay.
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 450.45, ALogP 4.83, HBA 4, HBD 1, PSA 93.51, QED 0.72
Compound (SMILES): COC(=O)Nc1nc2ccc(C3(O)c4ccccc4C(=O)N3c3ccccc3)cc2[nH]1
InChIKey: RGVJCTNUQFLEIU-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 414.42, ALogP 3.6, HBA 5, HBD 3, PSA 107.55, QED 0.47
Compound (SMILES): O=C(NCCc1ccc(O)cc1)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: XIDWMGWTMSZTLA-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1: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-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 61.3 nM (pChEMBL 7.210000038146973)
Descriptors: MW 401.47, ALogP 3.91, HBA 3, HBD 3, PSA 81.67, QED 0.61
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2C(c2cccc(S(=O)(=O)NCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCNC(=O)CCOCCOCCOCCOCCOCCOCCOCCOCCOCCC(=O)NCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCNS(=O)(=O)c3cccc(C4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)c2)C1
InChIKey: BONDJRYXWCLHLN-UHFFFAOYSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE-3-mediated Na+ dependent H+ antiport expressed in opossum kidney cells in presence of NHE-1 antagonist EIPA by BCECF-AM dye based fluorescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 2188.31
Compound (SMILES): Cc1cc(Cl)cnc1C(=O)Nc1ccc(F)c([C@]2(CF)C[C@@H](C(F)(F)F)OC(N)=N2)c1
InChIKey: GSHVOYWQLKOCLX-KBXCAEBGSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 462.81, ALogP 4.27, HBA 5, HBD 2, PSA 89.6, QED 0.66
Compound (SMILES): N[C@H]1CCCN(c2ccncc2Nc2ncc3ccc(-c4ncccc4Cl)nn23)C1
InChIKey: KFMIHULBBWHKPZ-AWEZNQCLSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Inhibition of recombinant Pim1 (unknown origin) by electrochemiluminescence assay
Measurement: IC50 = 0.94 nM (pChEMBL 9.029999732971191)
Descriptors: MW 420.91, ALogP 3.51, HBA 8, HBD 2, PSA 97.26, QED 0.52
Compound (SMILES): NC(=O)C12CC3CC(C1)C(NC(=O)c1cccc(NS(=O)(=O)c4cc(Cl)cc(Cl)c4)c1)C(C3)C2
InChIKey: ZHVFMJLSPCXNDW-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: HTRF Assay : The inhibiting activity of 11β-HSD1 derived from microsomal fractions was measured using the HTRF assay (62CO2PEB, Cisbio). Different concentrations of compounds were added to 96-well plates, followed by the addition of TE buffer (20 mM Tris buffer and 5 mM EDTA, pH 6.0) containing 200 μM NADPH (N1630, Sigma) and 160 nM cortisone (C2755, Sigma), The reactions were initiated by the addition of human microsomal fractions (M0317, Sigma), and were allowed to incubate for 2 h at 37° C. Europium (Eu3+) cryptate and XL665-conjugated cortisol were then added to each well and incubated for an additional 2 h at room temperature.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 522.45, ALogP 4.2, HBA 4, HBD 3, PSA 118.36, QED 0.53
Compound (SMILES): COc1cc(/C=C2\SC(=S)N(CCC(=O)O)C2=O)cc(OC)c1
InChIKey: VUXKDLFIXPSUKC-GHXNOFRVSA-N
Target: Taste receptor type 2 member 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYV8
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 5378 nM (pChEMBL 5.269999980926514)
Descriptors: MW 353.42, ALogP 2.38, HBA 6, HBD 1, PSA 76.07, QED 0.62
Compound (SMILES): COc1cc(-c2ccc(Cl)c(C)c2)ccc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: IPKXOBOEJFTCKO-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 = 5053 nM (pChEMBL 5.300000190734863)
Descriptors: MW 521.98, ALogP 5.42, HBA 7, HBD 1, PSA 103.43, QED 0.32
Compound (SMILES): CCC1(N)CCN(c2cccnc2NC(=O)c2nc(-c3ncccc3C(F)(F)F)cnc2N)CC1
InChIKey: SKUHDUHJJACTNK-UHFFFAOYSA-N
Target: Protein kinase C alpha type (SINGLE PROTEIN), Homo sapiens
UniProt: P17252
Assay: In Vitro Inhibition Activity Assay: The compounds of formula I were tested for their activity on different PKC isoforms according to a published method (D. Geiges et al. Biochem. Pharmacol. 1997; 53:865-875) The assay is performed in a 96-well polypropylene microtiterplate (Costar 3794) that has been previously siliconized with Sigmacote (Sigma SL-2). The reaction mixture (50 μL) contains 10 μL of the relevant PKC isozyme together with 25 μL of the PKC inhibitor compound and 15 μL of a mix solution that contains 200 μg/mL protamine sulfate, 10 mM Mg(NO3)2, 10 μM ATP (Boehringer 519987) and 3750 Bq of 33P-ATP (Hartmann Analytic SFC301, 110 TBq/mmol) in 20 mM Tris-buffer pH 7.4+0.1% BSA. Incubation was performed for 15 minutes at 32° C. in a microtiterplate shaking incubator (Biolabo Scientific Instruments). Reaction was stopped by adding 10 μl of 0.5 M Na2EDTA, pH 7.4. 50 μl of mixture are pipetted onto a pre-wetted phosphocellulose paper (Whatmann 3698-915) under gentle pressure.
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 486.5, ALogP 3.49, HBA 8, HBD 3, PSA 135.94, QED 0.5
Compound (SMILES): Cc1cc(Nc2cc(C(F)(F)F)ccn2)nc(-c2ccc(NC3CCN(C(=O)c4cccc5c4-c4ccccc4C5)CC3)nc2)c1
InChIKey: YFPTYPIRHMBRHA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5.
Measurement: IC50 = 141 nM (pChEMBL 6.849999904632568)
Descriptors: MW 620.68, ALogP 7.9, HBA 6, HBD 2, PSA 83.04, QED 0.2
Compound (SMILES): COc1cc(F)cc(-c2nn(C3CCc4ccccc43)c3ncnc(N)c23)c1
InChIKey: AKJDYHXZOAXMDM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of human BTK using KVEKIGEGTYGVVYK as substrate after 20 mins by [gamma-33P]ATP based assay
Measurement: IC50 = 584 nM (pChEMBL 6.230000019073486)
Descriptors: MW 375.41, ALogP 3.76, HBA 6, HBD 1, PSA 78.85, QED 0.59
Compound (SMILES): NC(=O)[C@H](CO)Nc1nc(-c2ccc(Oc3ccc(F)cc3)cc2)c2ccccc2n1
InChIKey: ASBDJSDDFMEYJM-FQEVSTJZSA-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 10xHBSS 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 (flanks 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 = 836 nM (pChEMBL 6.079999923706055)
Descriptors: MW 418.43, ALogP 3.49, HBA 6, HBD 3, PSA 110.36, QED 0.42
Compound (SMILES): O=C(Nc1ccc(C(=O)N2CC3(CCCNC3)C2)cc1)N1Cc2ccncc2C1
InChIKey: ORGGXORLMSGQIE-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 = 577 nM (pChEMBL 6.239999771118164)
Descriptors: MW 391.48, ALogP 2.45, HBA 4, HBD 2, PSA 77.57, QED 0.82
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(CCC3CC3)cc1C21N=C(N)N(Cc2c[nH]cn2)C1=O
InChIKey: QMVCOAFZKAKLLA-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 7.8 nM (pChEMBL 8.109999656677246)
Descriptors: MW 447.58, ALogP 3.44, HBA 5, HBD 2, PSA 96.6, QED 0.71
Compound (SMILES): O=C(CC1CCCO1)NC1CC=C(c2ccc(NC(=O)N3Cc4ccncc4C3)cc2)CC1
InChIKey: ZMMPGBYLZCIUKQ-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 = 6.82 nM (pChEMBL 8.170000076293945)
Descriptors: MW 446.55, ALogP 4.25, HBA 4, HBD 2, PSA 83.56, QED 0.72
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C(=O)Nc3ccc(F)cc3)cc2N=C(N)C1
InChIKey: HCIPFSHBZUQHHC-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Pharmacological Assay: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase.
Measurement: EC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 422.5, ALogP 4.5, HBA 4, HBD 2, PSA 87.79, QED 0.69
Compound (SMILES): O=C(NC1CCN(Cc2ccc(OC3CCNCC3)c(Br)c2)CC1)c1cnccn1
InChIKey: WGMLIPWHIGZPLL-UHFFFAOYSA-N
Target: Urotensin-2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UKP6
Assay: Displacement of europium-labeled urotensin-2 from human recombinant urotensin-2 receptor expressed in HEK293 cell membranes after 90 mins by filtration based time resolved fluorometric assay
Measurement: IC50 = 610 nM (pChEMBL 6.210000038146973)
Descriptors: MW 474.4, ALogP 2.76, HBA 6, HBD 2, PSA 79.38, QED 0.67
Compound (SMILES): CCCCN(CCCC)C(=O)c1nn(-c2ccc(OCC(=O)O)cc2C(=O)N2Cc3ccccc3C[C@H]2CO)c(C)c1Cl
InChIKey: PCDBCDQIKIBROK-DEOSSOPVSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Surface plasmon resonance (SPR): The SPR assay is configured to examine solution inhibition of BCL-2 binding to peptide derivatized sensor surfaces to generate IC50 values as a measure of inhibitor potency. Solution Inhibition Assay Format: Biacore TM A100 (GE Healthcare, Uppsala, Sweden) was used to conduct all experiments reported herein. Sensor surface preparation and all interaction analyses experiments were performed at 25° C. Reagents were purchased from GE Healthcare. Running buffer containing 10 mM Hepes, pH7.4, 150 mM sodium chloride, 1.25 mM Dithiothreitol, 3% Dimethyl sulfoxide and 0.05% polysorbate 20 were utilized throughout all analyses.Â  Biotinylated BAK, BAD and NOXA peptides were diluted to 10 nM in running buffer and captured onto a sensor surface pre-derivatized with streptavidin (sensor chip SA) to peptide surface densities in the range 50-100 R.U.Â  Peptide captured surfaces were blocked with 50004 PEO2Biotin. A blank detection spot in each flowcell was similarly blocked with PEO2-biotin and served as a reference spot in the competition assay.
Measurement: IC50 = 150.9 nM (pChEMBL 6.820000171661377)
Descriptors: MW 611.14, ALogP 4.9, HBA 7, HBD 2, PSA 125.2, QED 0.28
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cc(Cl)ccc3F)ccc2[nH]1
InChIKey: KLVHHVHPTPOKPQ-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 466.86, ALogP 4.39, HBA 5, HBD 3, PSA 107.55, QED 0.41
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(C#CCO)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: DIZNSMNZOQAXRV-UHFFFAOYSA-N
Target: PI3K p110 beta/p85 alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110beta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 450.5, ALogP 2.72, HBA 8, HBD 2, PSA 111.85, QED 0.41
Compound (SMILES): COC(=O)CCC/C=C\C[C@H]1C(=O)C[C@@H](O)[C@@H]1c1ccc(C(O)C2CCCCC2)cc1
InChIKey: HVQPCSIKQMRYAQ-HBLLDXLXSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Agonist activity at human recombinant EP2 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 428.57, ALogP 4.62, HBA 5, HBD 2, PSA 83.83, QED 0.34
Compound (SMILES): CCCCCC(=O)c1ccc(N2C(=O)CC[C@@H]2CCCc2ccc(C(=O)O)s2)cc1
InChIKey: IIIMTHLYWCQJTA-SFHVURJKSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Displacement of [3H]PGE2 from from human EP4 receptor expressed in HEK293 cells membranes incubated for 60 mins
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 427.57, ALogP 5.73, HBA 4, HBD 1, PSA 74.68, QED 0.37
Compound (SMILES): COc1ccc([C@H](NC(=O)c2ccc3cnc(NC4CCOCC4)cc3n2)c2cnn(C)c2)cc1F
InChIKey: JAUXVEYDNKFQJQ-VWLOTQADSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to a 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 1.86 nM (pChEMBL 8.729999542236328)
Descriptors: MW 490.54, ALogP 3.62, HBA 8, HBD 2, PSA 103.19, QED 0.41
Compound (SMILES): COc1cc(OC)cc(N(CC(NC(C)C)C(C)C)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: NFVFGSNTTFYGGF-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 = 707.95 nM (pChEMBL 6.150000095367432)
Descriptors: MW 488.64, ALogP 5.21, HBA 8, HBD 1, PSA 77.33, QED 0.33
Compound (SMILES): CCc1ccc([C@@H](Oc2ccc3c(cnn3-c3ccc(F)cc3)c2)[C@H](C)NC(=O)C(C)(C)C)cc1
InChIKey: JBTVTEFBQUVIRK-PPHZAIPVSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Agonist activity at glucocorticoid receptor in human ChaGoK1 cells assessed as inhibition of AP1-mediated transcriptional activity by measuring reduction in PMA-stimulated TRE-LacZ activity after 24 hrs in presence of MUG by fluorometric assay
Measurement: IC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 473.59, ALogP 6.4, HBA 4, HBD 1, PSA 56.15, QED 0.34
Compound (SMILES): CCNc1nc(Cl)nc2c1ncn2[C@@]12C[C@H](O)[C@H](O)[C@@H]1C2
InChIKey: UYQIVPRVEBHZPH-FWAJVQJESA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Radioligand Binding Assay: Radioligand binding assays at A1, A2A, and A3ARs were performed according to the procedures described previously. Each tube in the binding assay contained 100 uL of membrane suspension (20 ug of protein), 50 pt of a stock solution of agonist radioligand, and 50 uL of increasing concentrations of the test ligands in Tris-HCl buffer (50 mM, pH 7.5) containing 10 mM MgCl2. Nonspecific binding was determined using a final concentration of 10 uM NECA, a non-specific agonist, diluted with the buffer.The mixtures were incubated at 25° C. for 60 min. Binding reactions were terminated by filtration through Whatman GF/B filters under a reduced pressure using a MT-24 cell harvester (Brandell, Gaithersburg, Md.). Filters were washed three times with 5 ml of 50 mM ice-cold Tris-HCl buffer (pH 7.5). The radioactive agonists [3H]R-PIA and [3H]CGS21680 were used for the A1 and A2AAR assays, respectively, while [125I]AB-MECA was used for the A3AR assay.
Measurement: Ki = 930 nM (pChEMBL 6.03000020980835)
Descriptors: MW 309.76, ALogP 0.75, HBA 7, HBD 3, PSA 96.09, QED 0.72
Compound (SMILES): O=C(c1nnc(CCc2ncc[nH]2)o1)c1nc2ccc(-c3ccccc3)cc2s1
InChIKey: RUXMUNABOCCKNV-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Enzymatic Assay: The enzymatic assay was measured using white, opaque 96-well half area plates. Each well contained 60 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 2 ul of a DMSO solution containing compound of interest. Conditioned media obtained from HT-1080 cells, which were transformed by RAGE technology (Athersys) to overexpress endogenous EL, was added and the reaction was allowed to incubate for 20 min at 37° C. with gentle agitation. The reaction was started by the addition of 20 μL of a 1:4 dilution of vesicles. The final total reaction volume was 100 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 488 nm and an emission of 530 nm. Readings were taken every 20 seconds for 10 min with agitation between each reading.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 401.45, ALogP 4.09, HBA 7, HBD 1, PSA 97.56, QED 0.43
Compound (SMILES): CCC(=O)N[C@@H](C)c1ccc(CN2Cc3ccc(OCC4CC4)cc3C(F)C2)cc1
InChIKey: COMIDLROXGIYLN-KEJDIYNNSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.Each assay microtiter plate contains wells with vehicle instead of compound as controls for the non-inhibited enzyme (100% CTL; HIGH) and wells without acetyl-CoA as controls for non-specific NADH degradation (0% CTL; LOW).
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 410.53, ALogP 5.09, HBA 3, HBD 1, PSA 41.57, QED 0.66
Compound (SMILES): CC(C)Nc1cc(-n2ncc3cc(C#N)cnc32)ncc1C(=O)NCCC(C)(F)F
InChIKey: TXQDWBPBEVGDTJ-UHFFFAOYSA-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 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 413.43, ALogP 3.28, HBA 7, HBD 2, PSA 108.52, QED 0.62
Compound (SMILES): COCC1CN(c2cc(-c3n[nH]c4ccc(O[C@@H](C)COC)cc34)ncn2)CCO1
InChIKey: BLPHHXZMJVJEAC-LBAUFKAWSA-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 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 413.48, ALogP 2.29, HBA 8, HBD 1, PSA 94.62, QED 0.6
Compound (SMILES): Cc1ccc(-n2ccn3nc(-c4cccnc4)cc23)cc1NC(=O)c1cc(Br)cc(C(C)(C)C)c1
InChIKey: GAKBVNSFOLSGLE-UHFFFAOYSA-N
Target: Angiopoietin-1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q02763
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: For the assay, 50 nl of a 100-fold concentrated solution of the respective test substance in DMSO were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany) 2 μl of a solution of the Tie2 fusion protein in assay buffer [50 mM HEPES/HCl pH 7, mM MgCl2, 2.5 mM MnCl2, 1 mM dithiothreitol, 0.01% (v/v) Nonidet P-40, 0.1% (w/v) bovine serum albumin (BSA), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added, and the mixture was incubated for 15 min to allow pre-binding of the substance to the enzyme prior to the kinase reaction. The kinase reaction was then started by adding 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM→final concentration in 5 μl assay volume=1 mM) and substrate (1.67 μM→final concentration in 5 μl assay volume=1 μM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of the Tie2 fusion protein was adapted to the respective activity of the enzyme and adjusted such that the assay was carried out in the linear range. Typical concentrations were in the range of 30 ng/ml. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [200 nM streptavidin-XL665 and 2 nM PT66-Eu chelate, a europium chelate-labelled anti-phosphotyrosine antibody (Perkin-Elmer); alternatively, it is also possible to use PT66-Tb cryptate (Cisbio Bioassays, Codolet, France)] in aqueous EDTA solution [90 mM EDTA, 0.28% (w/v) bovine serum albumin (BSA) in 50 mM HEPES pH 7.5]. The resulting mixture was incubated at 22° C. for 1 h to allow formation of the complex of the biotinylated phosphorylated substrate and the detection reagents. The amount of phosphorylated substrate was then determined by measuring the resonance energy transfer from PT66-Eu chelate to streptavidin-XL665. To this end, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET measuring instrument (for example Viewlux, Perkin-Elmer).
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 528.45, ALogP 6.81, HBA 5, HBD 1, PSA 64.22, QED 0.28
Compound (SMILES): O[C@H]1CC[C@@H](Nc2ccc3ncc(-c4cccc(OC(F)(F)F)c4)n3n2)CC1
InChIKey: LOMZUAFMSBAHAU-OKILXGFUSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase-Glo Assay: These assays are set up in duplicate 50 ul volumes in white, flat bottom 96 well plates. Inhibitors are added to the solution of 1× kinase buffer, 10 uM ATP, 100 uM Pim-1-specific substrate, 50 ng of active Pim-1 enzyme, and water in serial dilutions ranging from micromolar to nanomolar concentrations. This solution is incubated at 30 degrees Celsius at 360 rpm for two hours. Following the incubation, 50 ul of Kinase-Glo reagent is added to each well, including all positive and negative control wells, and incubated at room temperature for 15 minutes. The plate is then read by the Luminoskan Ascent instrument and the results displayed with the Ascent Software version 2.6.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 392.38, ALogP 4.01, HBA 6, HBD 2, PSA 71.68, QED 0.7
Compound (SMILES): [C-]#[N+]c1ccc(Oc2ccc(-c3nc(C(N)=O)cc([C@@H](O)CO)n3)cc2)cc1
InChIKey: NRTWRTFPDOWKKG-SFHVURJKSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: Assay Buffer: 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 = 965 nM (pChEMBL 6.010000228881836)
Descriptors: MW 376.37, ALogP 2.61, HBA 6, HBD 3, PSA 122.92, QED 0.57
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(CCC3CC3)cc1C21N=C(N)N(CC(F)F)C1=O
InChIKey: MUGBBRLBYJHYCQ-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 28.9 nM (pChEMBL 7.539999961853027)
Descriptors: MW 431.53, ALogP 3.78, HBA 4, HBD 1, PSA 67.92, QED 0.75
Compound (SMILES): c1cc(-c2nc3ccc(N4CCC[C@H](c5ncc6n5CCC6)C4)nc3[nH]2)nc(C2CC2)n1
InChIKey: HYSQCOTYMHPUMU-INIZCTEOSA-N
Target: Diacylglycerol O-acyltransferase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96PD7
Assay: In Vitro DGAT2 Assay: For determination of IC50 values, the reactions were carried out in 384-well white Polyplates (Perkin Elmer) in a total volume of 20 uL. To 1 uL of compounds dissolved in 100% DMSO and spotted at the bottom of each well, 5 uL of 0.04% bovine serum albumin (BSA) (fatty acid free, Sigma Aldrich) was added and the mixture was incubated at room temperature for 20 minutes. To this mixture, 10 uL of hDGAT2 membrane fraction (0.01 mg/mL) diluted in 100 mM Hepes-NaOH, pH 7.4, 20 mM MgCl2 containing 200 nM methyl arachidonyl fluorophosphonate (Cayman Chemical; dried from ethyl acetate stock solution under argon gas and dissolved in DMSO as 5 mM stock) was added. After this mixture was preincubated at room temperature for 2 hours, DGAT2 reactions were initiated by the addition of 4 uL of substrates containing 30 uM [1-14C]decanoyl-CoA (custom-synthesized by Perkin Elmer, 50 mCi/mmol) and 125 uM 1,2-didecanoyl-sn-glycerol (Avanti Polar Lipids) dissolved in 12.5% acetone. The reaction mixtures were incubated at room temperature for 40 min and the reactions were stopped by addition of 5 uL of 1% H3PO4. After the addition of 45 uL MicroScint-E (Perkin-Elmer), plates were sealed with Top Seal-A covers (Perkin-Elmer) and phase partitioning of substrates and products was achieved using a HT-91100 microplate orbital shaker (Big Bear Automation, Santa Clara, Calif.). Plates were centrifuged at 2,000×g for 1 min in an Allegra 6R Centrifuge (Beckman Coulter) and then were sealed again with fresh covers before reading in a 1450 Microbeta Wallac Trilux Scintillation Counter (Perkin Elmer). DGAT2 activity was measured by quantifying the generated product [14C]tridecanoylglycerol in the upper organic phase.
Measurement: IC50 = 4240 nM (pChEMBL 5.369999885559082)
Descriptors: MW 426.53, ALogP 3.82, HBA 7, HBD 1, PSA 88.41, QED 0.53
Compound (SMILES): CC(C)(C)OC(=O)N1CCC2(CC1)C[C@H]2CNC(=O)N1Cc2ccncc2C1
InChIKey: DLHHFYXNVIMXSY-KRWDZBQOSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Inhibition of human full length C-terminal His6-tagged NAMPT expressed in Escherichia coli Rosetta (DE3) cells using nicotinamide as substrate incubated for 15 mins prior to substrate addition measured after 30 mins in presence of PRPP
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 386.5, ALogP 3.14, HBA 4, HBD 1, PSA 74.77, QED 0.85
Compound (SMILES): Nc1nn2cccnc2c1C(=O)Nc1cn(C2CCN(CCOCc3ccccc3)CC2)nc1-c1cc(Cl)ccc1OC(F)F
InChIKey: LWILOZMSNVFIKD-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 = 1.25 nM (pChEMBL 8.899999618530273)
Descriptors: MW 637.09, ALogP 5.54, HBA 10, HBD 2, PSA 124.83, QED 0.18
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cnc(C3CC3)c(N3CCC[C@H](N)C3)n2)n1
InChIKey: VPGFPXLEFFWPKW-KRWDZBQOSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.026 nM (pChEMBL 10.59000015258789)
Descriptors: MW 427.52, ALogP 2.78, HBA 9, HBD 1, PSA 111.53, QED 0.53
Compound (SMILES): O=C1NC(=O)/C(=C/c2cnn3c(NC4CC4)cc(-c4ccc(C(=O)N5CCC(O)CC5)s4)nc23)N1
InChIKey: WDEAWKUTKMNXJX-SXGWCWSVSA-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 493.55, ALogP 1.81, HBA 9, HBD 4, PSA 140.96, QED 0.31
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(N(CC2CC2)S(=O)(=O)Cc2ccccc2)ccc1Oc1ccc(F)cc1F
InChIKey: JRSYZDSFRNNVRW-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method C: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.047 mM to 0.78 nM or 5-fold serial dilution from Assay method A. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 1040 nM (pChEMBL 5.980000019073486)
Descriptors: MW 580.65, ALogP 6.27, HBA 6, HBD 0, PSA 77.84, QED 0.21