lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCN(c1cc(Cl)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)C1CCC(CO)CC1
InChIKey: DEGTXGLOIUINEP-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 460.02, ALogP 4.26, HBA 4, HBD 3, PSA 85.43, QED 0.58
Compound (SMILES): Cc1c(Cc2c(CNCCS(C)(=O)=O)nc3c(N)cc(N4CCOCC4)nn23)cccc1C(F)(F)F
InChIKey: GWFUHMDGIDDKAR-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Binding affinity to human PI3Kalpha (108 to 1068 residues) expressed in mammalian expression system by KINOMEscan assay
Measurement: Kd = 8128 nM (pChEMBL 5.090000152587891)
Descriptors: MW 526.59, ALogP 2.2, HBA 9, HBD 2, PSA 114.85, QED 0.43
Compound (SMILES): COc1cc(C(=O)NC(C)(C)c2ccccc2)ccc1-c1cn[nH]c1
InChIKey: SWFHDLGGWNNIBV-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 129 nM (pChEMBL 6.889999866485596)
Descriptors: MW 335.41, ALogP 3.75, HBA 3, HBD 2, PSA 67.01, QED 0.75
Compound (SMILES): Cn1c(=O)n(CC(C)(C)C)c2ccc(C3=CC4CN(C(=O)CO)CC4C3)nc21
InChIKey: FZOSBJPFKBYPJU-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: [35S]-GTPγS Assay: The stimulation of [35S]-GTPγS binding is a common functional assay to monitor Gαi-coupled receptor in native and recombinant receptor membrane preparation. Membrane from cells stably expressing hmGlu2 CHO-K1 (50 μg) are incubated in a 96 well plate for 1 hour in the presence of GTPγS35 (0.05 nM), GDP (5 μM) and compounds. The reaction is stopped by rapid filtration over Unifilter GF/B plate (Packard, Bioscience, Meriden Conn.) using a 96-well cell harvester (Brandel Gaithersburg, Md.). The filter plates are counted using Topcount counter (Packard, Bioscience, Meriden Conn., USA). When compounds are evaluated as potentiators they are tested in the presence of glutamate (1 μM).
Measurement: EC50 = 2820 nM (pChEMBL 5.550000190734863)
Descriptors: MW 384.48, ALogP 1.64, HBA 6, HBD 1, PSA 80.36, QED 0.87
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(-c4ccccc4)cc3)CC2)C1
InChIKey: DUKGQZXOHHYYOE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 542.65, ALogP 4.72, HBA 7, HBD 1, PSA 106.73, QED 0.33
Compound (SMILES): FC(F)(F)C1(NC[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)CC1
InChIKey: KXXAZUVPEFTPQM-HAQNSBGRSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 378.4, ALogP 3.56, HBA 5, HBD 2, PSA 70.9, QED 0.73
Compound (SMILES): COCCCN1C[C@@H]2CN(C(=O)Cn3cc(NC(=O)c4cnn5cccnc45)c(-c4cc(Cl)ccc4OC(F)F)n3)C[C@@H]2C1
InChIKey: PKRKWOVHSNTKAH-KDURUIRLSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 0.78 nM (pChEMBL 9.109999656677246)
Descriptors: MW 629.07, ALogP 3.53, HBA 10, HBD 1, PSA 119.12, QED 0.25
Compound (SMILES): OC(COc1ccc(C2CCCNC2)cc1)CN1CCc2ccccc2C1
InChIKey: HUPUCRUHTCXPRQ-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 101 nM (pChEMBL 7)
Descriptors: MW 366.51, ALogP 2.95, HBA 4, HBD 2, PSA 44.73, QED 0.83
Compound (SMILES): CNc1nc(-c2nc3ccc(N4CCOCC4)cc3n2Cc2ccccn2)cn2c(C)nnc12
InChIKey: VUUYIKCADVQZHC-UHFFFAOYSA-N
Target: Histone H4 (SINGLE PROTEIN), Homo sapiens
UniProt: P62805
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 uM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 ul of the mix is added to the compound dilutions. Subsequently, 6 ul of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 ug/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer. Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 455.53, ALogP 2.77, HBA 10, HBD 1, PSA 98.29, QED 0.43
Compound (SMILES): O=C(Nc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3ccccc3)ccc2[nH]1)OCC(O)CO
InChIKey: IRVKRNZTIYCKPW-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 488.5, ALogP 2.31, HBA 7, HBD 5, PSA 148.01, QED 0.27
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C(=O)Nc3ncco3)cc2N=C(N)C1
InChIKey: MCEFREGMFDCPIW-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
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 = 45000 nM (pChEMBL 4.349999904632568)
Descriptors: MW 395.46, ALogP 3.35, HBA 6, HBD 2, PSA 113.82, QED 0.75
Compound (SMILES): CN(C)c1ccc(-c2cc(Nc3cc(C4CC4)[nH]n3)nc(NCc3cc(-c4ccccc4)no3)n2)cc1
InChIKey: BFSZMKLQSRKFTA-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) tri-phosphorylated 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 492.59, ALogP 5.82, HBA 8, HBD 3, PSA 107.79, QED 0.24
Compound (SMILES): CCC(=O)Nc1cc2c(Nc3ccc(Oc4ccc5c(c4)OCO5)cc3)ncnc2cc1OC
InChIKey: FPGRQRNSVWYTAO-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15303
Assay: Irreversible inhibition of GST-tagged ERBB4 (unknown origin) (Gly-259 to Gly-690 residues) expressed in baculovirus infected Sf9 insect cells assessed as reduction in Glu/Tyr copolymer phosphorylation after 6 mins by ELISA
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 458.47, ALogP 5.25, HBA 8, HBD 2, PSA 103.83, QED 0.39
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1ncc(Cl)c(Nc2ccc(N3CCN(C(C)=O)CC3)cc2Cl)n1
InChIKey: GTOSARLOOHHDMV-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 613.55, ALogP 4.62, HBA 9, HBD 2, PSA 106.17, QED 0.4
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3ccc4cc[nH]c4c3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: VGAJKHFCGZSKTP-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 511.59, ALogP 5.53, HBA 7, HBD 2, PSA 107.41, QED 0.32
Compound (SMILES): CN1Cc2cnc3ccc(-c4cnc(N)nc4)nc3c2N(c2cccc(C(F)(F)F)c2)C1=O
InChIKey: NPTPXMTVZDQQPM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR Activity Assay: 1. Preparation of test reagents {circle around (1)} 1x kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 3 mM MnCl, 0.01% Tween-20, 2 mM DTT); {circle around (2)} 4x kinase solution (added mTOR kinase to 1x kinase buffer to provide 4x kinase solution with a final concentration of 2.5 nM); {circle around (3)} 2x substrate solution (added the substrates of 4EBP1 and ATP into 1x kinase buffer to provide 2x substrate solution with 4EBP1 final concentration of 50 nM and ATP final concentration of 10.8 uM); {circle around (4)} 4x test substance solutions (100x test substance solutions with different concentration gradients were prepared using 100% DMSO, and diluted 25-fold with 1x kinase buffer to give 4x test substance solutions with different concentration gradients) {circle around (5)} preparation of test solution (test solution containing EDTA and 4EBP1 phosphorylated antibody was prepared using activity test kit with assay buffer, the final concentration of EDTA was 8 mM, and the final concentration of 4EBP1 phosphorylated antibody was 2 nM) 2. 2.5 uL of 4x test solution was added to a 384-well plate in parallel; 3. added 2.54 uL of 4x kinase solution and incubated for 10 min; 4. then added 5 uL of 2x solutions of 4EBP1 and ATP substrates and incubated for 1 h at room temperature; 5. finally, added 10 uL of test solution to quench the reaction, and read after 60 min; 6. IC50 value was obtained via curve fitting.
Measurement: IC50 = 8.5 nM (pChEMBL 8.069999694824219)
Descriptors: MW 451.41, ALogP 4.39, HBA 6, HBD 1, PSA 101.13, QED 0.49
Compound (SMILES): Cc1cc(Cl)c2c(C(=O)OCC34CCN(CC3)CC4)c[nH]c2c1
InChIKey: DCZGNMUEAXUIHE-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Radioligand Binding Assay: Protocol 1: The assay was conducted following the literature reference Meyer E. M., et al., Analysis of 3-(4-Hydroxy, 2-Methoxybenzylidene) Anabaseine Selectivity and Activity at Human and Rat Alpha-7 Nicotinic Receptors, J. Pharmacol. Exp. Ther., 287: 918-925, 1998. The assay was conducted using the following materials: receptor source: rat brain, radioligand: [125I] α-Bungarotoxin (2200 Ci/mmol) with a final concentration of 1 nM, non-specific determinant: Methyllycaconitine (MLA) [1 μM], reference compound: Methyllycaconitine (MLA), and positive control: Methyllycaconitine (MLA). Incubation Conditions: Reactions were carried out in 20 mM HEPES (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2 and 1.2 mM MgSO4 at 37° C. for 90 minutes. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined by liquid scintillation counting and compared to control values in order to ascertain any interactions of test compound with the nicotinic, ganglionic binding site.
Measurement: IC50 = 63.6 nM (pChEMBL 7.199999809265137)
Descriptors: MW 332.83, ALogP 3.77, HBA 3, HBD 1, PSA 45.33, QED 0.87
Compound (SMILES): Cn1nc(-c2ccc(F)cc2)c(-c2ccc3nc(NC(=O)c4ccnc(F)c4)cn3n2)c1-c1ccncc1
InChIKey: RTIGYLOMNNQUDW-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 508.49, ALogP 4.78, HBA 8, HBD 1, PSA 102.89, QED 0.34
Compound (SMILES): CC[C@H](C)[C@@H]1NCCCc2ccccc2/C=C\CNC(=O)[C@H](Cc2cccc(Cl)c2)NC(=O)C2(CCCC2)NC1=O
InChIKey: GPRIWCCKHIWKBV-KIDGYFACSA-N
Target: Growth hormone secretagogue receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92847
Assay: Agonist activity at human GSHR1a transfected in CHOK1 cells co-expressing Galpha16 assessed as increase in Ca2+ release by aequorin assay
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 579.19, ALogP 4.58, HBA 4, HBD 4, PSA 99.33, QED 0.43
Compound (SMILES): O=C(c1cccc(Cl)c1-n1nccn1)N1CCOC[C@H]1Cc1cccc(-n2nccn2)c1
InChIKey: IGNKAYVYFKMOBL-GOSISDBHSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: OX Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 ug/mL G418, 100 U/mL penicillin, 100 ug/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES.
Measurement: IC50 = 435 nM (pChEMBL 6.360000133514404)
Descriptors: MW 449.9, ALogP 2.59, HBA 8, HBD 0, PSA 90.96, QED 0.46
Compound (SMILES): Cc1ccc(C(=O)N[C@@H](CC2CC2)C(N)=O)nc1OCC1CC1
InChIKey: AODJRMWUULMSHS-AWEZNQCLSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 317.39, ALogP 1.56, HBA 4, HBD 2, PSA 94.31, QED 0.76
Compound (SMILES): Cc1nc2ccccc2n1-c1cc(Nc2ccc(OCCN(C)C)cn2)ncn1
InChIKey: JEYSCBUEDROHLD-UHFFFAOYSA-N
Target: Heat shock factor protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00613
Assay: Inhibition of 17AAG-induced HSF1-mediated HSP72 expression in human U2OS cells preincubated for 1 hr followed by 17AAG addition measured after overnight incubation by ArrayScan analysis
Measurement: IC50 = 4280 nM (pChEMBL 5.369999885559082)
Descriptors: MW 389.46, ALogP 3.2, HBA 8, HBD 1, PSA 80.99, QED 0.52
Compound (SMILES): O=C(O)CCC1=CC[C@@H]2[C@@H](/C=C/[C@@H](O)COc3ccccc3)[C@H](O)C[C@@H]2OC1
InChIKey: HTUHVPVHRIFBLU-DCFAJTMGSA-N
Target: Prostaglandin E2 receptor EP3 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43115
Assay: Agonist activity at human EP3 receptor expressed in CHO cells assessed as increase in intracellular calcium level by Fura 2-AM dye based fluorescence assay
Measurement: EC50 = 2700 nM (pChEMBL 5.570000171661377)
Descriptors: MW 388.46, ALogP 2.56, HBA 5, HBD 3, PSA 96.22, QED 0.59
Compound (SMILES): CC[C@H]1C(=O)C[C@@H]2CCN(S(=O)(=O)c3ccc(C)cc3)[C@@H]3CCC[C@H]1[C@@H]23
InChIKey: PZMUSBZBIIMWES-PPVISPMTSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Displacement of [3H]U69593 from KOR (unknown origin) expressed in HEKT cells measured after 90 mins by microbeta scintillation counting method
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 375.53, ALogP 3.79, HBA 3, HBD 0, PSA 54.45, QED 0.81
Compound (SMILES): O=C(CNC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)NCC(F)(F)F
InChIKey: XPNMGPMMJXKVHV-HAQNSBGRSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 409.42, ALogP 2.15, HBA 6, HBD 3, PSA 100, QED 0.58
Compound (SMILES): COc1cc(NC(=O)OCCN(C)[C@H]2CC[C@H](OC(=O)C(O)(c3cccs3)c3cccs3)CC2)c(Cl)cc1CNC[C@H](O)c1ccc(O)c2[nH]c(=O)ccc12
InChIKey: SNXJYSXRLKUBSZ-WBXFWKJNSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: Muscarinic Receptor Binding Assay: The study of binding to human muscarinic M1, M2, M3, M4 and M5 receptors was performed using commercial membranes (Perkin Elmer) prepared from CHO-K1 cells. Radioligand binding experiments were conducted in 96 polypropylene well plates in a total volume of 200 μl. All reagents were dissolved in assay binding buffer (PBS with calcium and magnesium, SIGMA), except compounds that were dissolved in DMSO 100%. Non-specific binding (NSB) was measured in the presence of 1 μM atropine. [3H]-NMS was used as the radioligand at a concentration of 1 nM for M2, M3 and M5 and 0.3 nM for M1 and M4. [3H]-NMS and antagonists were incubated with membranes that express human muscarinic receptors M1, M2, M3, M4 and M5 at concentrations of 8.1, 10, 4.9, 4.5 and 4.9 μg/well, respectively. After an incubation period of two hours with gentle shaking, 150 μl of the reaction mix were transferred to 96 GF/C filter plates (Millipore), previously treated with wash buffer (Tris 50 mM; NaCl 100 mM; pH: 7.4), containing 0.05% PEI (Sigma) during one hour. Bound and free [3H]-NMS were separated by rapid vacuum filtration in a manifold from Millipore and washed four times with ice cold wash buffer. After drying 30 min, 30 μl of OPTIPHASE Supermix were added to each well and radioactivity quantified using a Microbeta microplate scintillation counter.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 811.38, ALogP 6.11, HBA 13, HBD 6, PSA 182.68, QED 0.07
Compound (SMILES): Cc1c(CC(=O)O)c2cccnc2n1Cc1ccc(S(C)(=O)=O)c(Cl)c1
InChIKey: JHVKAZXRIQLDGF-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Antagonist activity against human CRTh2 expressed in CHO-K1 cells assessed as inhibition of PGD2-mediated attenuation of forskolin-induced cAMP accumulation incubated for 60 mins by scintillation counting method
Measurement: IC50 = 95 nM (pChEMBL 7.019999980926514)
Descriptors: MW 392.86, ALogP 3.08, HBA 5, HBD 1, PSA 89.26, QED 0.72
Compound (SMILES): COCCCNc1nc(C(C)(C)C)ncc1C(=O)N(CC(C)C)[C@@H]1CNC[C@H](C(N)=O)C1
InChIKey: UDGOQNBHRYYFSR-SJORKVTESA-N
Target: Renin (SINGLE PROTEIN), Homo sapiens
UniProt: P00797
Assay: Inhibition of recombinant human preprorenin expressed in human 293F cells using angiotensinogen as substrate preincubated for 10 mins followed by substrate addition measured after 30 mins by ELISA
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 448.61, ALogP 1.78, HBA 7, HBD 3, PSA 122.47, QED 0.47
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1noc(C2CCCN(C)C2)n1
InChIKey: WUCSNTVXKXMTBP-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 515.4, ALogP 5.46, HBA 9, HBD 1, PSA 98.43, QED 0.34
Compound (SMILES): Cc1cc(F)c(C(=O)Nc2cccc(-c3nncn3C(C)C)n2)cc1-n1cnc(C2CC2)c1
InChIKey: YIDDLAAKOYYGJG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Inhibition of human ASK1 using myelin basic protein substrate preincubated for 20 mins before [33P]-ATP addition and measured after 2 hrs by filter-binding method
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 445.5, ALogP 4.68, HBA 7, HBD 1, PSA 90.52, QED 0.47
Compound (SMILES): O=C(CCc1ccccc1)N(Cc1nc2c(c(=O)[nH]1)COCC2)Cc1cncs1
InChIKey: SFLOZGZBZKOKIW-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 uL of compound in beta-point serial dilutions with concentrations ranging from 0.0086 to 18.75 uM, 20 nM of purified enzyme, and 250 nM of beta-NAD+ in the 1x Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 uL of 5x quenching solution (20% formic acid and 500 nM [d4]-nicotinamide in water). For the background control wells, 10 uL of the 5x quenching solution per well was added prior to the addition of beta-NAD+.
Measurement: IC50 = 17500 nM (pChEMBL 4.760000228881836)
Descriptors: MW 410.5, ALogP 2.46, HBA 6, HBD 1, PSA 88.18, QED 0.65
Compound (SMILES): NC12CC1CN(c1ncc(C(=O)Nc3ccc(OC(F)(F)Cl)cc3)cc1-c1ccn[nH]1)C2
InChIKey: QSWKBWJSTOMYGX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper Assay: The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 μL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 μM ATP, 4 μM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 μL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 μL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology. Briefly, samples from terminated kinase reactions were applied to the chip. Analytes are transported through the chip by constant buffer flow and the migration of the substrate peptide is monitored by the fluorescence signal of its label. Phosphorylated peptide (product) and unphosphorylated peptide (substrate) are separated in an electric field by their charge/mass ratio.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 460.87, ALogP 3.43, HBA 6, HBD 3, PSA 109.16, QED 0.49
Compound (SMILES): N#Cc1cnc2[nH]cc(C(=O)c3c(F)ccc(NS(=O)(=O)c4ccccc4)c3F)c2c1
InChIKey: LZPYPWMVRFVWBG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 438.42, ALogP 3.74, HBA 5, HBD 2, PSA 115.71, QED 0.46
Compound (SMILES): COc1cccc(-c2ccc3c(c2)[C@@]2(N=C(C)C(N)=N2)[C@]2(CC[C@H](OC)CC2)C3)c1F
InChIKey: MUPBWXCOUHZWEH-WDNCENIBSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: sAPPbeta Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at −140° C. at a concentration of 7.5-9.5×106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 μL cell susp/well. The cell plates were then incubated for 7-24 h at 37° C., 5% CO2. The cell medium was removed, followed by addition of 30 μL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37° C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPβ release. MSD sAPPβ plates were blocked in 1% BSA in Tris wash buffer (40 μL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 μL/well). 20 μL of medium was transferred to the pre-blocked and washed MSD sAPPβ microplates, and the cell plates were further used in an ATP assay to measure cytotoxicity. The MSD plates were incubated with shaking at r.t. for 2 h and the media discarded. 10 μL detection antibody was added (1 nM) per well followed by incubation with shaking at r.t. for 2 h and then discarded. 40 μL Read Buffer was added per well and the plates were read in a SECTOR Imager.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 421.52, ALogP 4.62, HBA 5, HBD 1, PSA 69.2, QED 0.79
Compound (SMILES): Cn1cc(-c2cc(C(=O)NCc3ccncc3)ccc2Oc2ccc(F)cc2F)c2cc[nH]c2c1=O
InChIKey: ZFAUNJLUXLDQOI-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in 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) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 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). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 33.4 nM (pChEMBL 7.480000019073486)
Descriptors: MW 486.48, ALogP 4.93, HBA 5, HBD 2, PSA 89.01, QED 0.36
Compound (SMILES): COC(=O)Nc1nc2ccc(C3(O)c4ccccc4C(=O)N3c3ccc(Cl)c(Cl)c3F)cc2[nH]1
InChIKey: MIYHBUSQLPUGHH-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 501.3, ALogP 5.04, HBA 5, HBD 3, PSA 107.55, QED 0.34
Compound (SMILES): Cc1cc(C)nc(-n2nnc3c2CCN(C(=O)c2cccc(C(F)(F)F)c2Cl)C3C)c1
InChIKey: HBOLBGHPFJCVDU-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 171 nM (pChEMBL 6.769999980926514)
Descriptors: MW 449.86, ALogP 4.71, HBA 5, HBD 0, PSA 63.91, QED 0.57
Compound (SMILES): O=C(Nc1nc(-c2ccncc2)cs1)N[C@H]1CCc2ccccc21
InChIKey: XGVCACISIMVTBC-HNNXBMFYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Inhibition Assay: Attention was focused on exploring, in turn, the SAR around the phenyl ring A, branching and substitution at the benzylic position, urea linkage of 1aa, without varying the [4-(4-pyridinyl)-2-thiazolyl] terminus (FIG. 4). The [4-(4-pyridinyl)-2-thiazolyl] group was believed to act as a hinge-binding moiety with the nitrogen of the pyridyl H-bonding to the back bone NH of the hinge Met156, as seen in the crystal complex of ROCK1 with Fasudil (PDB ID 2ESM) (Jacobs, et al., The structure of dimeric ROCK I reveals the mechanism for ligand selectivity, J Biol Chem, 2006, 281:260-8).All compounds were systematically screened against ROCK1 and ROCK2. IC50 values were systematically determined only for compounds that inhibit 40% of ROCK1 kinase activities at 50 μM.
Measurement: IC50 = 1830 nM (pChEMBL 5.739999771118164)
Descriptors: MW 336.42, ALogP 4.01, HBA 4, HBD 2, PSA 66.91, QED 0.76
Compound (SMILES): Cc1nccn1Cc1ccc(Nc2nccc(-c3ccc(N4CC[C@H](F)C4)c(C#N)n3)n2)cn1
InChIKey: AEPKWENAKIFXHF-KRWDZBQOSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 455.5, ALogP 3.65, HBA 9, HBD 1, PSA 108.44, QED 0.47
Compound (SMILES): Cc1n[nH]c2cnc(C(=O)N3CCC[C@H]3c3ccc(Cl)cc3)cc12
InChIKey: FLEYJCJMMRLIBV-KRWDZBQOSA-N
Target: Cyclin-dependent kinase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: P49336
Assay: Inhibition of CDK8 in human 7dF3 cells preincubated for 2 hrs followed by beta-oestradiol addition measured after 24 hrs by luciferase reporter gene assay
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 340.81, ALogP 3.9, HBA 3, HBD 1, PSA 61.88, QED 0.77
Compound (SMILES): CCOCc1c(-c2cc3cc(C)cc(OC)c3s2)c2c(N)ncnn2c1Cl
InChIKey: NIKUTJUSYMUTDM-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Kinase Assay: For the assay, 50 nl of each stock solution of the test compound in DMSO was pipetted into a black, low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany) 2 ul of a solution of the above FGFR-3 fusion protein in aqueous assay buffer [8 mM MOPS pH 7.0, 10 mM magnesium acetate, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumin (BSA), 0.07% (v/v) Tween-20, 0.2 mM EDTA] was added, and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme. Then, the kinase reaction was started by the addition of 3 ul of a solution of adenosine triphosphate (ATP, 16.7 uM; final concentration in the 5 ul assay volume=10 uM) and substrate (0.8 uM; final concentration in the 5 ul assay volume=0.5 uM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of FGFR-3 fusion protein was adjusted depending on the activity of the enzyme lot.
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 402.91, ALogP 4.7, HBA 7, HBD 1, PSA 74.67, QED 0.52
Compound (SMILES): Fc1cccc(-c2cc3nc4c(c(N5CCNCC5)n3n2)CCCC4)c1
InChIKey: AFYBBCQVYAHRRH-UHFFFAOYSA-N
Target: Beta-glucuronidase (SINGLE PROTEIN), Homo sapiens
UniProt: P08236
Assay: Activity Assay: PNPG Assay: Two ß-glucuronidase activity assays were employed to examine the potency of inhibitors both in vitro and in cell-based studies. An absorbance assay based on the conversion of p-nitrophenyl glucuronide (PNPG) to p-nitrophenol (PNP) (Szasz (1967) Clin Chem 13:752-759), which absorbs at 410 nm, was employed as the primary in vitro assay. A secondary assay involving the conversion of phenolphthalein glucuronide (PheG) to phenolphthalein (Phe), which absorbs at 540 nm, was also employed. Id. Both assays were validated in vitro and in living cells, and the wavelengths monitored did not overlap with absorbance characteristics of putative inhibitors.
Measurement: EC50 = 811 nM (pChEMBL 6.090000152587891)
Descriptors: MW 351.43, ALogP 2.82, HBA 5, HBD 1, PSA 45.46, QED 0.77
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(Cc2ccccn2)CCO1
InChIKey: KOHLHBQFFZUSET-UHFFFAOYSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: Inhibition of recombinant human N-terminal His6-tagged EphA2 (N598 to R890 residues) expressed in baculovirus using poly-EY as substrate after 180 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 526.42, ALogP 5.36, HBA 8, HBD 1, PSA 81.63, QED 0.33
Compound (SMILES): O=C(N[C@@H](Cc1ccc(F)cc1)C(=O)C(=O)N1CCC(O)CC1)c1cc2cc(Cl)ccc2[nH]1
InChIKey: VCKKIPCWUZKXJQ-FQEVSTJZSA-N
Target: Glycogen phosphorylase, muscle form (SINGLE PROTEIN), Oryctolagus cuniculus
UniProt: P00489
Assay: Inhibition of rabbit muscle glycogen phosphorylase b using 10 mM of glucose-1-phosphate as substrate preincubated for 60 mins followed by substrate addition in presence of glycogen
Measurement: IC50 = 178 nM (pChEMBL 6.75)
Descriptors: MW 471.92, ALogP 2.85, HBA 4, HBD 3, PSA 102.5, QED 0.48
Compound (SMILES): CCC(=O)Nc1nc2ccc(C3(O)c4ccccc4C(=O)N3c3ccc(F)cc3)cc2[nH]1
InChIKey: OROSVFNNWMXDQP-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 430.44, ALogP 3.9, HBA 4, HBD 3, PSA 98.32, QED 0.46
Compound (SMILES): COc1ccc(/C=C/C(=O)NCCC2CCN(Cc3ccccc3)CC2)c(OC)c1
InChIKey: AHWHBOQIIOPNCM-PKNBQFBNSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition of human recombinant AChE using acetylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition measured for 5 mins by Ellman's method
Measurement: IC50 = 630 nM (pChEMBL 6.199999809265137)
Descriptors: MW 408.54, ALogP 4.14, HBA 4, HBD 1, PSA 50.8, QED 0.63
Compound (SMILES): c1ccc2c(N3CCC4(CCCC4)CC3)cncc2c1
InChIKey: RFERWVRPLUDUIY-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition of human CYP11B1-8C7 expressed in chinese hamster V79 cells using 11-deoxycortisol as substrate preincubated for 1 hr followed by substrate addition measured for 3 hrs by HTRF assay
Measurement: IC50 = 1674 nM (pChEMBL 5.78000020980835)
Descriptors: MW 266.39, ALogP 4.4, HBA 2, HBD 0, PSA 16.13, QED 0.76
Compound (SMILES): CNC(=O)N=S(C)(=O)Cc1cccc(Nc2ncc(F)c(-c3ccc(F)cc3OC)n2)c1
InChIKey: SOSTXYOXUQYPHG-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: CDK2/CycE Kinase Assay: Recombinant fusion proteins of GST and human CDK2 and of GST and human CycE, expressed in insect cells (Sf9) and purified by Glutathion-Sepharose affinity chromatography, were purchased from ProQinase GmbH (Freiburg, Germany). As substrate for the kinase reaction biotinylated peptide biotin-Ttds-YISPLKSPYKISEG (C-terminus in amid form) was used which can be purchased e.g. form the company JERINI Peptide Technologies (Berlin, Germany)For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 ul of a solution of CDK2/CycE in aqueous assay buffer [50 mM Tris/HCl pH 8.0, 10 mM MgCl2, 1.0 mM dithiothreitol, 0.1 mM sodium ortho-vanadate, 0.01% (v/v) Nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min at 22 C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction.
Measurement: IC50 = 430 nM (pChEMBL 6.369999885559082)
Descriptors: MW 461.49, ALogP 4.11, HBA 6, HBD 2, PSA 105.57, QED 0.57
Compound (SMILES): CN(C)[C@H]1c2cc3ccn(S(=O)(=O)c4ccccc4)c3cc2[C@H]1CCO
InChIKey: OCKYRBSJWIYEBM-OXQOHEQNSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]-methyl-spiperone from human dopamine D3 receptor expressed in CHO cell membranes after 1 hr by liquid scintillation counting
Measurement: Ki = 398.11 nM (pChEMBL 6.400000095367432)
Descriptors: MW 370.47, ALogP 2.96, HBA 5, HBD 1, PSA 62.54, QED 0.75