lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CS(=O)(=O)Nc1cccc(-c2cc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)c1
InChIKey: XJGIBEVYLAJXHY-PXNMLYILSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 453.48, ALogP 1.52, HBA 8, HBD 4, PSA 146.59, QED 0.33
Compound (SMILES): Cc1nnc(-c2nc3cccc(-c4cc5c([nH]4)CCNC5=O)c3nc2NC(C)(C)C)o1
InChIKey: SOUGKZTUMVEVDD-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 74.8 nM (pChEMBL 7.130000114440918)
Descriptors: MW 417.47, ALogP 3.48, HBA 7, HBD 3, PSA 121.62, QED 0.47
Compound (SMILES): CCCCCCCCCCCCc1ccc(C(=O)NC2(/C(N)=N/O)CC2)cc1
InChIKey: AYJDBZWRKOTTQB-UHFFFAOYSA-N
Target: Sphingosine kinase 2 (SINGLE PROTEIN), Mus musculus
UniProt: Q9JIA7
Assay: Kinase Assay: Sphingosine kinase activity was assessed as described previously (Kharel, Y., Lee, S., Snyder, A. H., Sheasley-O'Neill, S. L., Morris, M. A., Setiady, Y., Zhu, R., Zigler, M. A., Burcin, T. L., Ley, K., Tung, K. S. K., Engelhard, V. H., Macdonald, T. L. and Lynch, K. R. Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720. J Biological Chemistry 280: 36865-36872 (2005)). High levels of human sphingosine kinase type 1 (mSK1) and mouse sphingosine kinase type 2 (mSK2) were expressed in Sf9 insect cells by infection with cognate baculoviruses. Crude homogenates were incubated with γ-[32P]ATP and 10 micromolar D-erythro-sphingosine in the presence of 100 micromolar concentrations of test compounds for 20 minutes at 37 C. The product, radiolabeled S1P, was isolated by extraction into organic solvents after acidification and displaced by thin layer chromatography.
Measurement: Ki = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 387.57, ALogP 5.16, HBA 3, HBD 3, PSA 87.71, QED 0.14
Compound (SMILES): CC(C)[C@H](NC(=O)C(=O)Nc1c(F)c(F)nc(F)c1F)C(=O)N[C@@H](CC(=O)O)C(=O)COc1c(F)c(F)cc(F)c1F
InChIKey: ZJEAAPUACVGAGQ-FVMDXXJSSA-N
Target: Caspase-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P42574
Assay: Inhibition of recombinant CPP32 (unknown origin) pre-incubated for 60 mins with Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin substrate followed by enzyme addition by fluorescence based assay
Measurement: Ki = 127 nM (pChEMBL 6.900000095367432)
Descriptors: MW 614.4, ALogP 1.88, HBA 7, HBD 4, PSA 163.79, QED 0.13
Compound (SMILES): Cc1cc(Nc2cc(C3CC3)[nH]n2)nc(NCc2cc(-c3ccncc3)no2)n1
InChIKey: BTGVCRAOCZZOKT-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 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 388.44, ALogP 3.79, HBA 8, HBD 3, PSA 117.44, QED 0.44
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2cnccn2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: KYUCAVCNFWUMDG-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 477.53, ALogP 4.03, HBA 9, HBD 0, PSA 91.08, QED 0.34
Compound (SMILES): COc1cc(N2CCC(N3CCN(CCF)CC3)CC2)ccc1Nc1nccc(-c2c(-c3ccc(OC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: XDVUZJSZZMHEEE-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 791.88, ALogP 7.31, HBA 11, HBD 2, PSA 112.39, QED 0.13
Compound (SMILES): CN1CCN(c2ccc(Nc3nccc(-c4cc5c(o4)CCN(S(C)(=O)=O)C5)n3)cc2)CC1
InChIKey: LODYGYIZTLGHEL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Inhibition of GST-tagged recombinant human cytoplasmic JAK3 expressed in baculovirus using TK-substrate-biotin as substrate preincubated for 5 mins followed by substrate addition after 30 mins by HTRF assay
Measurement: IC50 = 204.2 nM (pChEMBL 6.690000057220459)
Descriptors: MW 468.58, ALogP 2.55, HBA 8, HBD 1, PSA 94.81, QED 0.61
Compound (SMILES): O=C1C(N2CCC(c3ccc(O)cc3)C(O)C2)CCN1Cc1ccc(F)cc1
InChIKey: NIPVYFDEJFFCLH-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q00960
Assay: Radioligand Binding Assay: To perform the competition binding assay, thawed membrane homogenate was added to each well of a 96-well plate (20 ug/well). The experimental compounds were serially diluted in 100% DMSO and added to each row of the assay plate to achieve desired compound concentrations, keeping the DMSO concentration in the assay plate at 1.33% of the final reaction volume. Next, 3H Ro 25-6981 (4 nM) was added to the assay plate. After incubation for 1 hr at room temperature, the membrane bound radioligand was harvested on to GF/B filter plates (treated with 0.5% PEI for 1 hr at room temperature). The filter plates were dried at 50° C. for 20 mins, incubated with microscint 20 for 10 minutes and finally, the counts were read on TopCount (Perkin Elmer). Non-specific binding was determined using MK-0657 (the preparation of this compound is described as example 1 in WO 2004 108705 (40 uM).
Measurement: Ki = 180 nM (pChEMBL 6.75)
Descriptors: MW 384.45, ALogP 2.48, HBA 4, HBD 2, PSA 64.01, QED 0.85
Compound (SMILES): COc1nn2cc(-c3cccc(C(F)(F)F)c3)nc2s1
InChIKey: VCWQXQODHPDUDF-UHFFFAOYSA-N
Target: Proteinase-activated receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI0
Assay: Antagonist activity at PAR4 in human platelets assessed as inhibition of activating peptide-induced PAC1 binding response preincubated for 20 mins followed by agonist addition for 20 mins by FITC-based flow cytometric analysis
Measurement: IC50 = 36.3 nM (pChEMBL 7.440000057220459)
Descriptors: MW 299.28, ALogP 3.49, HBA 5, HBD 0, PSA 39.42, QED 0.73
Compound (SMILES): Cc1cc(Cl)cc2c(-c3ccccc3)c(C(=O)O)c(N3CCCCC3)nc12
InChIKey: GNZUTXQJDKZNGL-UHFFFAOYSA-N
Target: Fatty acid-binding protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q01469
Assay: Displacement of Bodipy-labeled fatty acid from human N-terminal His6-tagged FABP5 (127 to 132 residues) expressed in Escherichia coli after 30 mins by TR-FRET assay
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 380.88, ALogP 5.55, HBA 3, HBD 1, PSA 53.43, QED 0.65
Compound (SMILES): O=C(O)c1ccc(-n2nc(Sc3c(Cl)cccc3Cl)c3cc(C(=O)N4CCOCC4)ccc32)cc1
InChIKey: QJPLWOYLDJWJRC-UHFFFAOYSA-N
Target: Retinoic acid receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P13631
Assay: Inverse agonist activity at GAL4 DBD-fused RARgamma LBD (unknown origin) expressed in human HG5LN cells after 18 hrs by luciferase reporter gene assay
Measurement: IC50 = 4200 nM (pChEMBL 5.380000114440918)
Descriptors: MW 528.42, ALogP 5.65, HBA 6, HBD 1, PSA 84.66, QED 0.36
Compound (SMILES): CN[C@@H](C)C(=O)N[C@@H](Cc1ccc(NCc2ccc(C(=O)NC3C[C@@H](C(=O)NC4CCCc5ccccc54)N(C(=O)[C@@H](NC(=O)[C@H](C)NC)C(C)(C)C)C3)cc2)cc1)C(=O)N1Cc2ccccc2CC1C(=O)N[C@@H]1CCCc2ccccc21
InChIKey: ZQGQESOASZCZRS-BRUXDLPLSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: AlphaScreen Assay: Assays were performed in white, flat-bottom, 384-well ProxiPlates (Perkin Elmer). The final assay volume was 10 μL prepared from additions of His-BIR2 (124-240/C202A/C213G), Biotinylated SMAC peptide, and test compounds in assay buffer consisting of 25 mM Hepes, 100 mM NaCl, 0.1% BSA, and 5 mM CaCl2. The reaction was incubated at room temperature for 60 minutes. After 60 minutes, 2.5 μL of AlphaScreen detection reagent (Perkin Elmer) was added to the reaction mixture and incubated at room temperature in the dark for 120 minutes. The AlphaScreen signal generated by the reaction was detected on the Envision Plate Reader. Inhibition data were calculated from an AlphaScreen signal generated by the no protein control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay was 50 nM His-BIR2 (124-240/C202A/C213G), 50 nM Biotinylated SMAC peptide, 4 μg/mL AlphaScreen detection reagents, and 0.5% DMSO.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 1127.44
Compound (SMILES): CCC(=O)NC1CCCc2c(-c3ccc4c(c3)CCC(=O)N4C)cncc21
InChIKey: VDEUDSRUMNAXJG-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Cellular Enzyme Assay: The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate. G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 μM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 1020 nM (pChEMBL 5.989999771118164)
Descriptors: MW 363.46, ALogP 3.56, HBA 3, HBD 1, PSA 62.3, QED 0.91
Compound (SMILES): COc1cc(N2CCC(N3CCC(CCS(C)(=O)=O)CC3)CC2)ccc1Nc1nccc(-c2c(-c3ccc(OC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: HHLILBRTRAXHOF-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 850.99, ALogP 7.87, HBA 12, HBD 2, PSA 143.29, QED 0.12
Compound (SMILES): COCCC(C(=O)Nc1ccc2c(=O)n(C)[nH]c2c1)n1cc(OC)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: KYDFSXSNSODXGB-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibition Assay: 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 Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of plasma kallikrein from Kordia (0.6 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 substrate H-Pro-Phe-Arg-AMC dissolved in assay buffer (10 μl 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).
Measurement: IC50 = 8.2 nM (pChEMBL 8.09000015258789)
Descriptors: MW 521.96, ALogP 3.45, HBA 8, HBD 2, PSA 131.14, QED 0.36
Compound (SMILES): COc1cnc(CN2CCC(c3ccc4[nH]c(C(=O)N5CCN(Cc6ccc(C(F)(F)F)cc6)CC5)nc4c3)CC2)cn1
InChIKey: WTVQQXGHNYQSSQ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibitory Assay: Human PIK3CA mutation-positive breast cancer cell line MDA-MB-453 cells were subcutaneously implanted into nude mice, MDA-MB-453 tumor was excised, a mitochondria extraction solution (0.25 M Sucrose, 2 mM EDTA, 10 mM Tris/HCl pH 7.5) of 9 times of tumor weight was added thereto, and crushing was carried out thereon. The reaction mixture was centrifuged at 600×g and 4° C. for 10 minutes to obtain a supernatant, and the supernatant was centrifuged at 14000×g and 4° C. for 10 minutes, thereby obtaining a pellet. The pellet was suspended in 10 mM Tris/HCl pH 7.5 of 5 times of the excised tumor weight, thereby obtaining a human mitochondrial suspension.Next, 25 μl of the human mitochondrial suspension per 1 ml of Complex I activity measurement solution (200 mM potassium phosphate pH 7.6, 0.35% Bovine Serum Albumin (BSA), 60 μM 2,6-dichlorophenol-indophenol, 70 μM decylubiquinone, 1 μM antimycin) was added.
Measurement: IC50 = 83 nM (pChEMBL 7.079999923706055)
Descriptors: MW 593.65, ALogP 4.72, HBA 7, HBD 1, PSA 90.48, QED 0.33
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(O)cc1)nn2Cc1nc2cccc(C#CCCCC(=O)N3CCCC3)c2c(=O)n1Cc1ccccc1Cl
InChIKey: GFPPJADRSAPASM-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition Assay: PI3 kinases catalyse the phosphorylation of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) in the presence of ATP and Mg2+ ions. The PIP3 product can be detected by displacement of biotin-PIP3 from energy transfer complexes consisting of europium labelled anti-GST monoclonal antibody, a GST-tagged Pleckstrin homology (PH) domain, biotinylated PIP3 and streptavidin-allophycocyanin (APC) by the time-resolved fluorescence resonance energy transfer (TR-FRET) (HTRF® PI3K enzyme assay, Millipore). Excitation (330 nm) of europium in the complex results in an energy transfer to the APC and a fluorescent emission at 665 nm although europium itself emits at its characteristic 620 nm. The PIP3 product formed by PI3K activity displaces biotin-PIP3 from the complex and results in a loss of energy transfer (decreasing signal). The compound to be tested was added, at the desired final concentrations, to a mixture of PIP2 substrate and recombinant PI3 kinase α, δ or γ enzymes (Millipore), and the mixture incubated for 2 hr at RT. Following this incubation period, ATP (20 μM) was added to the enzyme/compound/PIP2 substrate mixture and the resulting mixture was incubated for 30 min at RT. A stopping solution containing biotinylated PIP3 and the detection mix containing the GST tagged GRP1 pleckstrin homology (PH) domain and fluorophores were then added and the mixture was incubated at RT for 15-18 hr, prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 673.18, ALogP 5.39, HBA 10, HBD 2, PSA 145.05, QED 0.16
Compound (SMILES): CCC(CC)(NC(=O)c1ccc(C(F)(F)F)c(OCC2CC2)n1)C(=O)O
InChIKey: LHNGTUXRNUJLHN-UHFFFAOYSA-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 374.36, ALogP 3.26, HBA 4, HBD 2, PSA 88.52, QED 0.73
Compound (SMILES): CN(C)C(=O)NC(CCNC(=O)c1nc(C#N)c2cc(Oc3ccccc3)ccc2c1O)C(=O)O
InChIKey: MHZOBNUTUHHAHT-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 11000 nM (pChEMBL 4.960000038146973)
Descriptors: MW 477.48, ALogP 2.45, HBA 7, HBD 4, PSA 164.88, QED 0.38
Compound (SMILES): Cc1ccc(F)c(NC(=O)Nc2cccc(C#Cc3ccc4nccn4n3)c2)c1
InChIKey: HOJNELPFQIDEFF-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: PDGFRbeta Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 2.7 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 385.4, ALogP 4.22, HBA 4, HBD 2, PSA 71.32, QED 0.51
Compound (SMILES): Cc1cc(SCc2nn(-c3ccc(C(F)(F)F)cc3)nc2-c2ccccc2)ccc1OCC(=O)O
InChIKey: YGUIMTQJMJEDCT-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor delta (SINGLE PROTEIN), Mus musculus
UniProt: P35396
Assay: Transactivation of yeast GAL4 DNA binding domain-tagged mouse PPARdelta expressed in HEK293T cells incubated for 20 hrs by luciferase reporter gene assay
Measurement: EC50 <= 10000 nM (pChEMBL 5)
Descriptors: MW 499.51, ALogP 6.02, HBA 6, HBD 1, PSA 77.24, QED 0.3
Compound (SMILES): CC(C(N)=O)c1ccccc1CCc1nc(Nc2cnn(C3CCNCC3)c2)ncc1C(F)(F)F
InChIKey: ZYAMSUYXTXWKGM-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P35916
Assay: VEGFR3 Phospho ELISA Assay: Adult human dermal lymphatic microvascular endothelial cells (HMVEC-dLyAD) (Cat# CC-2810, Lonza) were seeded into clear-bottom, TC treated 12 well plates (Cat #665180, Greiner Bio-One) in EGM-2MV (Cat# CC-3202, Lonza) at 180,000 cells/well (volume 1 mL), and the plates incubated at 37° C. and 5% CO2 for 6 hours. The media was replaced with EBM-2 (Cat # CC-3156, Lonza)+0.1% BSA (Cat# A8412, Sigma) and cells incubated for a further period (overnight at 37° C. and 5% CO2).96 well Maxisorp immuno plates (Cat #439454, Nunc) were coated with 100 uL of Total VEGFR3 capture antibody (Part #841888, Human Total VEGFR3/FLT4 ELISA Kit, Cat # DYC3491, R&D Systems), or Phospho VEGFR3 Capture antibody (Part #841885, Human Phospho VEGFR3/FLT4 ELISA Kit, Cat# DYC2724, R&D Systems). The plates were covered and incubated at room temperature overnight .The coating antibody was flicked out and the plates washed three times with Wash Buffer (Phosphate buffered saline (137 mM NaCl, 2.7 nM KCl, 8.1 nM Na2HPO4, 1.5 mL KH2PO4, pH 7.2-7.4), 0.05% Tween 20). 300 uL of blocking buffer (5% v/v Tween 20, 5% w/v sucrose in PBS) was then added to wells and plate incubated for 2 hours at room temperature.
Measurement: IC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 487.53, ALogP 3.73, HBA 7, HBD 3, PSA 110.75, QED 0.45
Compound (SMILES): CCOC(=O)C1c2ncn(-c3ccccc3)c2CCN1C(=O)c1cccc(Cl)c1Cl
InChIKey: WSQUUPXORCPRSA-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 = 179.9 nM (pChEMBL 6.75)
Descriptors: MW 444.32, ALogP 4.48, HBA 5, HBD 0, PSA 64.43, QED 0.56
Compound (SMILES): O=C(Nc1ccccc1OC(F)(F)F)Nc1ccc(Cl)c(S(=O)(=O)CC2CCNC2)c1O
InChIKey: UCTMKTXPZVSONA-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25025
Assay: Displacement of [125I]IL8 from recombinant human CXCR2 expressed in CHO cell membranes measured after 1 hr by TopCount scintillation counting method
Measurement: IC50 < 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 493.89, ALogP 3.97, HBA 6, HBD 4, PSA 116.76, QED 0.45
Compound (SMILES): CC(C)C[C@@H](CNc1ccc([N+](=O)[O-])c2nonc12)NC(=O)c1ccc(N2CCCC2)c(OCC2CC2)n1
InChIKey: LRXMNXYYCJCBQH-SFHVURJKSA-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 523.59, ALogP 4.17, HBA 10, HBD 2, PSA 148.55, QED 0.26
Compound (SMILES): N#Cc1cc(C(F)(F)F)ccc1N1CCOc2cc(S(=O)(=O)Nc3nccs3)c(F)cc21
InChIKey: YYWBVRDWEWYDOV-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 183 nM (pChEMBL 6.739999771118164)
Descriptors: MW 484.46, ALogP 4.5, HBA 7, HBD 1, PSA 95.32, QED 0.55
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2CCOC[C@H]2Cc2cccc(-c3cccnn3)c2)c1
InChIKey: QZXHCJASGVSEHH-OAQYLSRUSA-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 = 2380 nM (pChEMBL 5.619999885559082)
Descriptors: MW 440.51, ALogP 3.12, HBA 7, HBD 0, PSA 86.03, QED 0.47
Compound (SMILES): CN1C(=O)CCc2cc(-c3cncc(COc4cccc(F)n4)c3)ccc21
InChIKey: FVICHURAHFZIPV-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: G402-Based Assay: Herein we identified the use of the G-402 cell line as a host cell to ectopically express (transiently or stably) enzymes of the CYP11 family. Specifically we developed stable G-402 cells expressing ectopically human CYP11B1, human CYP11B2, human CYP11A1, cynmolgus CYP11B1 or cynomolgus CYP11B2 enzyme activity. Importantly the identified cell line G-402 expresses co-factors (adrenodoxin and adrenodoxin reductase) important for the activity of the CYP11 family and no relevant enzyme activity of the CYP11 family (in comparison to H295R cells) was detected in these cells. Therefore the G-402 cell line is uniquely suited as a host cell for the ectopic expression of enzymes from the CYP11 family.Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 7.5 nM (pChEMBL 8.119999885559082)
Descriptors: MW 363.39, ALogP 3.77, HBA 4, HBD 0, PSA 55.32, QED 0.66
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCC(Nc2ccc(Cl)cc2)CC1
InChIKey: ZNFRGYYRNYBBDH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 466.95, ALogP 5.18, HBA 6, HBD 3, PSA 108.76, QED 0.44
Compound (SMILES): OC(c1ccc(Cl)cc1)(c1ccc2nccc(/C=C/c3ccccc3)c2c1)c1cccs1
InChIKey: PHGPWHPEFFHWNO-CMDGGOBGSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 453.99, ALogP 7.4, HBA 3, HBD 1, PSA 33.12, QED 0.3
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1COc2ccccc2N(Cc2cc(=O)n(C)c3ccc(F)cc23)C1=O
InChIKey: PMDXADUCFFKNBG-KSSFIOAISA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 452.49, ALogP 1.7, HBA 6, HBD 2, PSA 92.67, QED 0.61
Compound (SMILES): O=C(O)c1c[nH]c2ccc(-c3ccc(-c4c(O)cccc4O)cc3)cc12
InChIKey: GQAXJYFQZNZNIM-UHFFFAOYSA-N
Target: 5'-AMP-activated protein kinase subunit beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y478
Assay: Activation of human recombinant AMPK beta1 expressed in African green monkey COS7 cells or baculovirus infected insect cells assessed as increase in biotinylated AAC (1 to 120 residues) peptide phosphorylation at Ser-79 residue measured after 60 mins by Alphascreen assay
Measurement: EC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 345.35, ALogP 4.61, HBA 3, HBD 4, PSA 93.55, QED 0.44
Compound (SMILES): N[C@H](C(=O)O)C(NS(=O)(=O)c1cccc(Cl)c1)C(=O)O
InChIKey: UKRLHWGNMLLOIW-JAMMHHFISA-N
Target: Excitatory amino acid transporter 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P43005
Assay: Inhibition of [3H]-D-Asp uptake at human EAAT3 expressed in HEK293 cells measured after 3 mins by TopCount method
Measurement: IC50 = 12000 nM (pChEMBL 4.920000076293945)
Descriptors: MW 322.73, ALogP -0.52, HBA 5, HBD 4, PSA 146.79, QED 0.55
Compound (SMILES): COc1cc(/C=C/C(=O)CC(=O)/C=C/c2ccc3cc[nH]c3c2)ccc1O
InChIKey: SIABTKWBOMYBGS-FCXRPNKRSA-N
Target: Microtubule-associated protein tau (SINGLE PROTEIN), Homo sapiens
UniProt: P10636
Assay: Inhibition of recombinant human TAU three-repeat microtubule-binding domain aggregation expressed in Escherichia coli after 16 hrs by thioflavin T fluorescence assay
Measurement: IC50 = 440 nM (pChEMBL 6.360000133514404)
Descriptors: MW 361.4, ALogP 4.14, HBA 4, HBD 2, PSA 79.39, QED 0.49
Compound (SMILES): COC(=O)Nc1nc2ccc(C3(O)c4ccccc4C(=O)N3c3cccc(OC)c3F)cc2[nH]1
InChIKey: VETJYUNLKSAQFW-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 462.44, ALogP 3.74, HBA 6, HBD 3, PSA 116.78, QED 0.43
Compound (SMILES): Cc1c([C@@H](O)CN2CCC3CN(C(=O)c4ccc(-n5cnnn5)cn4)CC32)ccc2c1COC2=O
InChIKey: GNVGXWQIEWEACZ-KGSCVUAUSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Electrophysiology Assay: Block of Kir1.1 (ROMK1) currents was examined by whole cell voltage clamp (Hamill et. al. Pfluegers Archives 391:85-100 (1981)) using the IonWorks Quattro automated electrophysiology platform (Molecular Devices, Sunnyvale, Calif.). Chinese hamster ovary cells stably expressing Kir1.1 channels were maintained in T-75 flasks in cell culture media in a humidified 10% CO2 incubator at 37° C. Prior to an experiment, Kir1.1 expression was induced by overnight incubation with 1 mM sodium butyrate. On the day of the experiment, cells were dissociated with 2.5 ml of Versene (Invitrogen 15040-066) for approximately 6 minutes at 37° C. and suspended in 10 ml of bath solution containing (in mM): 150 NaCl, 10 KCl, 2.7 CaCl2, 0.5 MgCl2, 5 HEPES, pH 7.4. After centrifugation, the cell pellet was resuspended in approximately 4.0 ml of bath solution and placed in the IonWorks instrument. The intracellular solution consisted of (in mM): 80 K gluconate, 40 KCl, 20 KF, 3.2 MgCl2, 3 EGTA, 5 Hepes, pH 7.4. Electrical access to the cytoplasm was achieved by perforation in 0.13 mg/ml amphotericin B for 4 minutes. Amphotericin B (Sigma A-4888) was prepared as a 40 mg/ml solution in DMSO.
Measurement: IC50 = 850 nM (pChEMBL 6.070000171661377)
Descriptors: MW 475.51, ALogP 0.92, HBA 10, HBD 1, PSA 126.57, QED 0.54
Compound (SMILES): CNc1nc2ccc(F)cc2n1-c1nc2c(c(C(C)(C)S(C)(=O)=O)n1)OC[C@@H]1COCCN21
InChIKey: MAKGGNQMSGBLFS-ZDUSSCGKSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: ADP Hunter Plus Assay: The kinase activity was measured by using the commercial ADP Hunter Plus assay available from DiscoveRx (#90-0083), which is a homogeneous assay to measure the accumulation of ADP, a universal product of kinase activity. The enzyme, DNA-PK was purchased from Promega (#V5811). The assay was done following the manufacturer recommendations with slight modifications: Mainly the kinase buffer was replace by 15 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM NaCl, 1 mM EGTA, 0.1 mg/mL BGG, 0.02% Tween 20. The DNA-PK was assayed in a titration experiment to determine the optimal protein concentration for the inhibition assay. To calculate the 1050 of the Compounds, serial 1:3 dilutions of the compounds were added to the enzyme at a fixed concentration (2 U/uL). The enzyme was preincubated with the inhibitor and 200 uM DNA substrate and then ATP was added to a final 75 uM concentration. Reaction was carried out for 1 hour at 37° C. Reagent A and B were sequentially added to the wells and plates were incubated for 30 min at RT. Fluorescence counts were read in a EnVision instrument (Perkin Elmer) with the recommended settings (550 and 590 nm as excitation and emission wavelengths, respectively). Values were normalized against the control activity included for each enzyme (i.e., 100% DNA_PK kinase activity, without compound). These values were plotted against the inhibitor concentration and were fit to a sigmoid dose-response curve by using the model sigmoidal Four-Parameter Logistc implement for Activity base - software.
Measurement: IC50 = 3110 nM (pChEMBL 5.510000228881836)
Descriptors: MW 476.53, ALogP 1.87, HBA 10, HBD 1, PSA 111.47, QED 0.6
Compound (SMILES): NC(=O)C1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)CC1
InChIKey: SXKVHEDIBVBTOH-HDJSIYSDSA-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 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 381.48, ALogP 2.08, HBA 6, HBD 2, PSA 105.2, QED 0.72
Compound (SMILES): CCCCN(CCCC)C(=O)c1cc(C)n(-c2ccc(N[S+]([O-])c3ccc(Oc4ccccc4C)cc3)cc2C(=O)N2Cc3ccccc3C[C@H]2CO)n1
InChIKey: LUKSLVKCSAKMOO-XYMJDGAWSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Surface plasmon resonance (SPR): Methods for Determining IC50s.The present method includes utility of a Surface plasmon resonance (SPR)- based biosensor (Biacore GE Healthcare, Uppsala, Sweden) to characterize BCL-2 inhibitors.Biacore utilizes the phenomenon of surface plasmon resonance (SPR) to detect and measure binding interactions. In a typical Biacore experiment, one of the interacting molecules (ligand) is immobilized on a flexible dextran matrix while the interacting partner (analyte) is allowed to flow across that surface. A binding interaction results in an increase in mass on the sensor surface and a corresponding direct change in the refractive index of the medium in the vicinity of the sensor surface. Changes in refractive index or signal are recorded in resonance units (R.U.) Signal changes due to association and dissociation of complexes are monitored in a non-invasive manner, continuously and in real-time, the results of which are reported in the form of a sensorgram.
Measurement: IC50 = 3349.66 nM (pChEMBL 5.46999979019165)
Descriptors: MW 747.96, ALogP 8.02, HBA 8, HBD 2, PSA 122.99, QED 0.1
Compound (SMILES): CC1(C)CN(c2ccc3cccc(Cl)c3n2)CC[C@H]1C(=O)N[C@H]1CC[C@@H](CO)CC1
InChIKey: FSHAZAGZDCHQMH-UHOSZYNNSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition of mPGES-1 in human A549 cells assessed as reduction in recombinant human interleukin-1 beta-induced PGE2 production preincubated for 30 mins measured after 18 hrs by ELISA
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 429.99, ALogP 4.41, HBA 4, HBD 2, PSA 65.46, QED 0.76
Compound (SMILES): CC(C)(CNC(=O)c1nc(C#N)c2cc(Oc3ccc(F)cc3Cl)ccc2c1O)C(=O)O
InChIKey: NDEWXUUQBMZATN-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 5550 nM (pChEMBL 5.260000228881836)
Descriptors: MW 457.85, ALogP 4.24, HBA 6, HBD 3, PSA 132.54, QED 0.5