lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCc1nccn1CCCCCCc1cc(Cl)c(S(=O)(=O)N(c2c(C)nn(C)c2C)C(F)F)c(Cl)c1
InChIKey: GWKJWTPHSUXVCQ-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase (SINGLE PROTEIN), Leishmania major
UniProt: Q4Q5S8
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 562.51, ALogP 6.32, HBA 6, HBD 0, PSA 73.02, QED 0.19
Compound (SMILES): Cc1cccc2cc(Cn3nc(C#C[C@@H](C)O)c4c(N)ncnc43)n(-c3ccccc3Cl)c(=O)c12
InChIKey: MVLVWYIBYUMIEQ-MRXNPFEDSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human 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 484.95, ALogP 3.46, HBA 8, HBD 2, PSA 111.85, QED 0.38
Compound (SMILES): O=C(O)CC[C@H]1CCc2cc(-c3cc(OC4CCC4)ncc3F)ccc2O1
InChIKey: MHJAABXYHDKRBJ-MRXNPFEDSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Agonist activity at human GPR40 expressed in HEK293 cells assessed as increase in IP1 accumulation after 60 mins by HTRF assay
Measurement: EC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 371.41, ALogP 4.38, HBA 4, HBD 1, PSA 68.65, QED 0.82
Compound (SMILES): CCCc1cc(C)[nH]c(=O)c1CNC(=O)c1cc(Cl)cc(N(CC)[C@H]2CC[C@H](N(C)C)CC2)c1C
InChIKey: JSLSZJORGQZIGW-YHBQERECSA-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 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 501.12, ALogP 5.23, HBA 4, HBD 2, PSA 68.44, QED 0.49
Compound (SMILES): CCC(C(=O)Nc1ccc(-c2nnc(C(F)(F)C(F)(F)C(=O)OC)[nH]2)cc1)n1cc(OC)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: VZVPVHDJUOZSAP-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: To determine the factor XIa inhibition of the substances according to the invention, a biochemical test system is used which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and the IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 646.99, ALogP 5.32, HBA 9, HBD 2, PSA 151.99, QED 0.17
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1csc(N(C)C)n1
InChIKey: DGOBVCDUGLUZMU-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
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 476.39, ALogP 5.79, HBA 8, HBD 1, PSA 72.4, QED 0.36
Compound (SMILES): N#Cc1c(-c2cccc(Cl)c2)c2c(=O)[nH]c(=O)[nH]c2[nH]c1=O
InChIKey: OLAIUOJBVWWLOO-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Inhibition of IDO1 in IFNgamma-induced human MDA-MB-231 cells using tryptophan as substrate preincubated for 4 hrs followed by substrate addition for 5 hrs by spectrophotometric method
Measurement: EC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 314.69, ALogP 1.1, HBA 4, HBD 3, PSA 122.37, QED 0.62
Compound (SMILES): FC1(F)CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)CC1
InChIKey: WFOQUQZAIVXBLY-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 970 nM (pChEMBL 6.010000228881836)
Descriptors: MW 374.44, ALogP 3.61, HBA 5, HBD 1, PSA 62.11, QED 0.76
Compound (SMILES): C#CCN1CCCC(CN(CCCOC)S(=O)(=O)c2ccc3ccccc3c2)C1
InChIKey: SJQLNDVTQXMBPO-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Inhibition of human recombinant microsomal MAO-A expressed in baculovirus-infected insect cells using p-tyramine as substrate preincubated for 15 mins followed by substrate addition measured for 20 mins by amplex red assay
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 414.57, ALogP 3.21, HBA 4, HBD 0, PSA 49.85, QED 0.47
Compound (SMILES): CCN(C)C(=O)N[C@H]1CC[C@H](CCN2CCN(c3nsc4ccccc34)CC2)CC1
InChIKey: RCTPWYPPQMZWPM-WGSAOQKQSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Displacement of [3H]spiperone from recombinant human D2L receptor expressed in CHO cell membranes after 60 mins by scintillation counting method
Measurement: Ki = 0.51 nM (pChEMBL 9.289999961853027)
Descriptors: MW 429.63, ALogP 4.03, HBA 5, HBD 1, PSA 51.71, QED 0.75
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)c2ccc(C)cc2)C(=O)N[C@@H](Cc2cccnc2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: QLQUHMZRFARDGM-AJQTZOPKSA-N
Target: Coagulation factor XII (SINGLE PROTEIN), Homo sapiens
UniProt: P00748
Assay: Enzyme Assay: Human serine protease enzymes plasmin, thrombin, trypsin, Factor Xa and Factor XIIa were assayed for enzymatic activity using an appropriate fluorogenic substrate. Protease activity was measured by monitoring the accumulation of liberated fluorescence from the substrate over 5 minutes. The linear rate of fluorescence increase per minute was expressed as percentage (%) activity. The Km for the cleavage of each substrate was determined by standard transformation of the Michaelis-Menten equation.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 593.73, ALogP 3.63, HBA 6, HBD 4, PSA 135.44, QED 0.18
Compound (SMILES): CC1[C@H]2Cc3ccc(O)cc3[C@@]1(C)CCN2[C@@H](C)C1CC1
InChIKey: USUBALPLEUZHID-VCCUOBBTSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Measurement: EC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 285.43, ALogP 3.71, HBA 2, HBD 1, PSA 23.47, QED 0.9
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3cn[nH]c3)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: HVCMZEHRZHHFMA-UHFFFAOYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human 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 462.52, ALogP 3.77, HBA 8, HBD 2, PSA 120.3, QED 0.41
Compound (SMILES): COc1cc(OC)cc(N(Cc2cccc(N(C)C)n2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: ZRDNUCXRIXLMNC-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (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 > 10000 nM (pChEMBL 5)
Descriptors: MW 495.59, ALogP 4.85, HBA 9, HBD 0, PSA 81.43, QED 0.3
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: CTPKPSKOHUBWQJ-ZRICEUNJSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Inhibition of C-terminus 10x-His-taged human TFPI-1 (29 to 282 residues) assessed as inhibition of factor 10a using S-2222 substrate incubated for 115 mins by micro-plate reader based assay
Measurement: EC50 = 1080 nM (pChEMBL 5.96999979019165)
Descriptors: MW 2472.88
Compound (SMILES): Cc1cccc2cc([C@H](C)Nc3nc(Cl)nc4[nH]cnc34)n(-c3ccccc3)c(=O)c12
InChIKey: FHYPPBSZWLGXOQ-AWEZNQCLSA-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 430.9, ALogP 4.79, HBA 6, HBD 2, PSA 88.49, QED 0.4
Compound (SMILES): CCn1ncc(Cl)c1-c1cc(C(=O)N[C@H](CN)Cc2ccc(F)c(F)c2)sc1Cl
InChIKey: IYWMCXIXRQMCTO-NSHDSACASA-N
Target: RAC-gamma serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y243
Assay: Inhibition of full length human AKT3 expressed in Sf9 cells assessed as reduction in substrate phosphorylation using biotin-ahx-ARKRERAYSFGHHA-amide substrate and [gamma-33P]ATP incubated for 40 mins by top count microplate scintillation counting method
Measurement: IC50 <= 25.12 nM (pChEMBL 7.599999904632568)
Descriptors: MW 459.35, ALogP 4.52, HBA 5, HBD 2, PSA 72.94, QED 0.55
Compound (SMILES): CCOc1ccc(-c2nc3ccccn3c2-c2ccnc(Nc3cc(C)c(C4CCN(CCS(C)(=O)=O)CC4)cc3OC)n2)cc1C(=O)Nc1c(F)cccc1F
InChIKey: UZNZGQYTJKDVSH-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 795.91, ALogP 7.67, HBA 11, HBD 2, PSA 140.05, QED 0.12
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCCC4(CN(C=O)C4)C3)n2)n1
InChIKey: GAFQWVCMOUDTGJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 3.42 nM (pChEMBL 8.470000267028809)
Descriptors: MW 440.51, ALogP 2.64, HBA 8, HBD 0, PSA 92.93, QED 0.45
Compound (SMILES): O=C(NCCc1ccc(F)cc1)[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: VRZLAEVVRQQVDS-WKILWMFISA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 360 nM (pChEMBL 6.440000057220459)
Descriptors: MW 406.46, ALogP 3.38, HBA 5, HBD 2, PSA 87.97, QED 0.53
Compound (SMILES): C[C@H](O)C(=O)N(CCCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)Cc1ccccc1
InChIKey: JBMRKAMTXGNSNR-IODBPXAYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]-diprenorphine from human mu opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 10 nM (pChEMBL 8)
Descriptors: MW 449.6, ALogP 3.3, HBA 4, HBD 2, PSA 86.87, QED 0.62
Compound (SMILES): FC(F)(F)Cn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CCCNCC3)n2)cn1
InChIKey: RLVWGBLSDHEDOU-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.175 nM (pChEMBL 9.760000228881836)
Descriptors: MW 442.45, ALogP 3.04, HBA 8, HBD 1, PSA 76.69, QED 0.52
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(I)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: VKFNBQZRLKDCED-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/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 < 100 nM (pChEMBL 7)
Descriptors: MW 522.35, ALogP 3.98, HBA 7, HBD 1, PSA 91.62, QED 0.36
Compound (SMILES): CN1CCC[C@@H]1COc1ccc(Nc2cc(-n3cnc4ccccc43)ncn2)nc1
InChIKey: JCQSAYNTEFQHGH-MRXNPFEDSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: Inhibition of recombinant full-length human C-terminal His6-tagged CDK9/full-length human cyclin T1 expressed in baculovirus infected insect Sf21 cells using KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC as substrate in presence of [gamma-33P]-ATP after 40 mins by scintillation counting analysis
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 401.47, ALogP 3.43, HBA 8, HBD 1, PSA 80.99, QED 0.53
Compound (SMILES): FC(F)(F)CN(CC1CC1)[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: RYWFFXJBGOPZMK-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 392.43, ALogP 3.91, HBA 5, HBD 1, PSA 62.11, QED 0.72
Compound (SMILES): CC(C)(C)OC(=O)N1CCC2(CC1)CC2CNC(=O)c1cnc2nccn2c1
InChIKey: XQZWWNWHRPOUQF-UHFFFAOYSA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Reversible inhibition of CYP2C9 in human liver microsomes using (S)-warfarin as substrate in presence of NADPH by LC-MS/MS analysis
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 385.47, ALogP 2.5, HBA 6, HBD 1, PSA 88.83, QED 0.88
Compound (SMILES): Cc1cc(-c2ncc(CNC(=O)c3ccc4c(c3)[nH]c3ccccc34)cc2F)ccn1
InChIKey: GOMFFTZBKYVUOE-UHFFFAOYSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of smo-mediated hedgehog signaling pathway in mouse NIH3T3 cells expressing GRE-Luc reporter gene assessed as inhibition of SAG-induced GRE-Luc reporter activity after 48 hrs by luminescence assay
Measurement: IC50 = 71 nM (pChEMBL 7.150000095367432)
Descriptors: MW 410.45, ALogP 5.16, HBA 3, HBD 2, PSA 70.67, QED 0.43
Compound (SMILES): Cc1n[nH]c(C)c1S(=O)(=O)N1CCC(Oc2ccc(Cl)cc2)CC1
InChIKey: QIOQURXCQDYAOJ-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Biological Assay: The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro.Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following to excitation at 485 nM.
Measurement: IC50 = 1450 nM (pChEMBL 5.840000152587891)
Descriptors: MW 369.87, ALogP 2.91, HBA 4, HBD 1, PSA 75.29, QED 0.9
Compound (SMILES): Oc1ncn(-c2ccc(-c3c(/C=C/c4ccc5ccccc5n4)nc4c(N5CCOCC5)ccnn34)cc2)n1
InChIKey: INYLSLWNPQZRRW-FMIVXFBMSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 516.57, ALogP 4.24, HBA 10, HBD 1, PSA 106.49, QED 0.36
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)nc(S(C)(=O)=O)n1
InChIKey: BDMRXFODCGLAFM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 1.85 nM (pChEMBL 8.729999542236328)
Descriptors: MW 520.53, ALogP 3.96, HBA 7, HBD 1, PSA 101.49, QED 0.51
Compound (SMILES): CC1(C)CC2=NNC(=O)c3cccc4[nH]c(c2c34)C1
InChIKey: HHHFTURDICHXNW-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H2K2
Assay: Tankyrase-2 Enzymatic Assay: Tankyrase-2 enzymatic assay was conducted using commercial Tankyrase-2 Chemiluminescent Assay Kit (BPS Biosciences) and the protocol with the kit. GST-fused tankyrase-2 (recombinant protein expressed and purified from Bacluovirus) were coated on a GSH-precoated plate first, and incubated with increasing concentrations of compounds for 0.5 h. Then, biotinylated NAD was added to the wells. The biotinylated auto-PARylation product was measured by adding streptavidin-HRP and HRP substrates which produce chemiluminescence.
Measurement: IC50 = 832 nM (pChEMBL 6.079999923706055)
Descriptors: MW 253.31, ALogP 2.59, HBA 2, HBD 2, PSA 57.25, QED 0.74
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(-c3cc(C#N)cc(C#N)c3)cc1C21N=C(C)C(N)=N1
InChIKey: BSTQSBRVJGRBPC-HDVKRFLQSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Diluted TR-FRET Assay: TR-FRET AssayThe beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes.
Measurement: IC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 423.52, ALogP 4.21, HBA 6, HBD 1, PSA 107.55, QED 0.78
Compound (SMILES): COc1cc(OC)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(CCNS(=O)(=O)N(C)C)c4)nc3c2)c1
InChIKey: JCBRKJGZGWDDLQ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (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 > 10000 nM (pChEMBL 5)
Descriptors: MW 551.67, ALogP 3.45, HBA 9, HBD 1, PSA 114.71, QED 0.28
Compound (SMILES): CCc1cc(OCCc2nc(-c3ccc(F)cc3)oc2C)ccc1CCC(=O)O
InChIKey: YFRXOWDDODRUGC-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor delta (SINGLE PROTEIN), Homo sapiens
UniProt: Q03181
Assay: Kinase Activity Assay: After 24 h treatment with the medicament, the cells were lysed with a lysis solution (Cell Culture Lysis buffer, Promega) and centrifuged, and the supernatant was collected. The supernatant was reacted with a fluorescence assay kit (Promega) and counted by a fluorometer (Ascent Fluoroskan FL reader, Thermo Labsystems, Finland), and the relative intensity of luciferase was determined. For the assay of the β-galactosidase activity used in the experiment as the internal reference (the internal reference for calibrating the transfection efficiency), 50 μl of the supernatant was transferred into a fresh microplate, treated with a Promega kit, and the value was read with a microplate reader at a wavelength of 405 nm (Bio-tech Instruments Inc., Winooski, Vt., USA) (Sauerberg, P.; Olsen, G. S.; Jeppesen, L.; Mogensen, J. P. et al., J. Med. Chem., 2007, 50, 1495-1503).
Measurement: EC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 397.45, ALogP 4.99, HBA 4, HBD 1, PSA 72.56, QED 0.55
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3cccc(O)c3)c3c(N)ncnc32)cc2cccc(F)c2c1=O
InChIKey: JDRCCHYIRIYYTG-UHFFFAOYSA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110alpha/untagged recombinant full length human 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 492.51, ALogP 4.58, HBA 8, HBD 2, PSA 111.85, QED 0.37
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4ccc5oc(N)nc5c4)c4c(N)ncnc43)n(-c3ccccc3)c(=O)c12
InChIKey: OWLYXAGENLRSKD-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 514.55, ALogP 4.46, HBA 10, HBD 2, PSA 143.67, QED 0.35
Compound (SMILES): COc1cc(C(=O)N2CCCC2)cc(N(CCCO)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: XIGQDVFIOYNOFH-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (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 = 6606.93 nM (pChEMBL 5.179999828338623)
Descriptors: MW 486.58, ALogP 3.8, HBA 8, HBD 1, PSA 96.61, QED 0.4
Compound (SMILES): C#CCN(Cc1ccc2cccc(O)c2n1)C(C#N)CC1CCN(Cc2ccccc2)CC1
InChIKey: MFWCFENQSTYMSU-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition of human recombinant AChE expressed in HEK293 cells using acetylthiocholine iodide as substrate preincubated for 30 mins followed by substrate addition measured after 30 mins by Ellman's method
Measurement: IC50 = 3300 nM (pChEMBL 5.480000019073486)
Descriptors: MW 438.58, ALogP 4.57, HBA 5, HBD 1, PSA 63.39, QED 0.52
Compound (SMILES): COc1nc2ccc(C(O)(c3cncn3C)C3CCN(C(C)=O)CC3)cc2c(Cl)c1Cc1ccc(-n2cccn2)cc1
InChIKey: MAJOOHMQVBMIGI-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1 °C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1×=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: Kd = 18 nM (pChEMBL 7.75)
Descriptors: MW 585.11, ALogP 4.9, HBA 8, HBD 1, PSA 98.3, QED 0.29
Compound (SMILES): N#CCC1(n2cc(-c3ccnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)C3CCC(F)(F)CC3)CC2)C1
InChIKey: GQMRHFSVNVKOJI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In vitro JAK Kinase Assay: 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 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 507.59, ALogP 4.17, HBA 6, HBD 1, PSA 93.84, QED 0.56
Compound (SMILES): CCOc1ccc(-c2nc3ccccn3c2-c2ccnc(Nc3ccc(N4CCN(S(C)(=O)=O)CC4)cc3OC)n2)cc1C(=O)Nc1c(F)cccc1F
InChIKey: OFRBTEGWYCEAEP-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 754.82, ALogP 6.22, HBA 11, HBD 2, PSA 143.29, QED 0.16
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(CN4CCN(CCCCC(=O)NO)CC4)cc3)cc12)c1ccccc1
InChIKey: KJTCUAWNUJWJQJ-JOCHJYFZSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of N-terminal GST-His6-thrombin cleavage site-fused human VEGFR2 (Asp805 to Val1356 residues) expressed in baculovirus expression system using biotinylated gastrin precursor at Tyr87 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 527.67, ALogP 4.59, HBA 7, HBD 4, PSA 109.41, QED 0.13
Compound (SMILES): COC(=O)CN(CCCNC(=O)c1cc2nccc(Oc3ccc(NC(=O)Nc4cc(C)ccc4F)c(F)c3)c2s1)CC(=O)OC
InChIKey: SAJGQCIKURPXJS-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17948
Assay: FLIPR (Fluorometric Imaging Plate Reader) Technology Assay: Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of PDGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. NHDF-Ad (Normal human dermal fibroblasts) (Lonza) were seeded in 384-well fibronectin coated black-walled plates overnight at 37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 4 times (ELx405-CW, Bio-Tek) to remove extracellular dye. Test compounds were reconstituted in 100% DMSO and added to the cells to give a final DMSO concentration of 0.1%. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 μM) or at concentrations ranging from 0.001 nM to 10 μM followed by PDGF stimulation (10 ng/mL). Changes in fluorescence at 515 nm were measured simultaneously in all 384 wells using a cooled CCD camera.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 655.68, ALogP 5.09, HBA 10, HBD 3, PSA 148.19, QED 0.13
Compound (SMILES): Cc1c(-c2ccc(C(F)(F)F)cc2)nnc(N2CCN(c3cnc(C(=O)N(c4ccccc4)C(C)C)cn3)[C@H](C)C2)c1C
InChIKey: QRUXAUDVXVLKOY-OAQYLSRUSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 589.67, ALogP 6.34, HBA 7, HBD 0, PSA 78.35, QED 0.26
Compound (SMILES): COCCNc1cc(NC(=O)N2CCCc3cc(CO)c(C=O)nc32)ncc1C#N
InChIKey: SYRAVIOIIKVUFT-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Mus musculus
UniProt: Q03142
Assay: Inhibition of FGFR4 in mouse BAF3 cells assessed as reduction in cell viability incubated for 2 days by cell proliferation assay
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 410.43, ALogP 1.7, HBA 8, HBD 3, PSA 140.47, QED 0.46
Compound (SMILES): CCCN(CCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)c1ccc(C(C)(C)C)cc1)C(=O)N[C@@H](CO)C(=O)OC)C(C)C
InChIKey: ITTGNAWNEDCTNG-DUKNTCSXSA-N
Target: Chromobox protein homolog 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HC52
Assay: Antagonist activity at recombinant human N-terminal His-tagged CBX8 chromodomain (8 to 61 residues) expressed in Escherichia coli Rosetta BL21(DE3)pLysS using biotin-labeled H3K9me3 measured after 30 mins by AlphaScreen assay
Measurement: IC50 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 823.09, ALogP 3.79, HBA 9, HBD 6, PSA 195.27, QED 0.07
Compound (SMILES): C=CC(=O)N1CCCC[C@@H](n2c(NC(=O)c3cccc(C(F)(F)F)c3)nc3cccc(C)c32)C1
InChIKey: DVTYPSOMACQPMD-LJQANCHMSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of wild type EGFR phosphorylation in human HaCaT cells incubated for 3 hrs by ELISA method
Measurement: IC50 = 249.6 nM (pChEMBL 6.599999904632568)
Descriptors: MW 470.5, ALogP 5.36, HBA 4, HBD 1, PSA 67.23, QED 0.52
Compound (SMILES): CC(=O)Nc1cc(Oc2ccc(NC(=O)Nc3cc(C(C)(C)C)nn3-c3ccc(C)cc3)c3ccccc23)ccn1
InChIKey: MPFNOCFGLHYSEQ-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay: The enzyme inhibitory activities of compounds disclosed herein were determined by fluorescence resonance energy transfer (FRET) using synthetic peptides labelled with both donor and acceptor fluorophores (Z-LYTE, Invitrogen Ltd., Paisley, UK). Recombinant, phosphorylated p38 MAPKγ (MAPK12: Invitrogen) was diluted in HEPES buffer, mixed with the test compound at the desired final concentrations and incubated for 2 hr at RT. The FRET peptide (2 μM) and ATP (100 μM) were added to the enzyme/compound mixture and incubated for 1 hr. Development reagent (protease) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific). The site-specific protease cleaves non-phosphorylated peptide only and eliminates the FRET signal. Phosphorylation levels of each reaction were calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor) for which high ratios indicate high phosphorylation and low rat
Measurement: IC50 < 18 nM (pChEMBL 7.744999885559082)
Descriptors: MW 548.65, ALogP 7.42, HBA 6, HBD 3, PSA 110.17, QED 0.2
Compound (SMILES): Cc1cccc(NC(=O)Nc2ccc(C#Cc3ccc4nccn4n3)cc2)c1
InChIKey: RUTCUMBDVHNVLK-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: PDGFRbeta: Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 384-well fibronectin coated black-walled plates overnight at 37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (Elx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 uM) or at concentrations ranging from 0.0001 to 10.0 uM followed by VEGF165 stimulation (10 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 384 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples.
Measurement: IC50 = 132 nM (pChEMBL 6.880000114440918)
Descriptors: MW 367.41, ALogP 4.08, HBA 4, HBD 2, PSA 71.32, QED 0.52
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N(C)C)nc(N2CCOCC2)c1
InChIKey: PABZMNZFBMSMPR-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 0.765 nM (pChEMBL 9.119999885559082)
Descriptors: MW 484.52, ALogP 5.23, HBA 5, HBD 1, PSA 57.7, QED 0.54
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1C#N)c1nc2c(Cl)cccc2c(=O)n1-c1cccnc1
InChIKey: FCKCJTUBGNGWLU-JTQLQIEISA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of human full length PI3K p110alpha/p85alpha using phosphatidylinositol 3,4,5-trisphosphate as substrate measured after 30 mins by TR-FRET assay
Measurement: IC50 = 3100 nM (pChEMBL 5.510000228881836)
Descriptors: MW 433.86, ALogP 2.43, HBA 10, HBD 3, PSA 161.42, QED 0.44