lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(O)c1cnc(C2=CCN(C(=O)Nc3ccc(C(F)(F)F)cn3)CC2)c(Cl)c1
InChIKey: FECXUIZUFUQTRD-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 ("wash buffer"), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 ("assay buffer"). Plates are then transferred to a FLIPR for assay. The test compound is diluted in assay buffer, and 50 μL of the resultant solution is added to the cell plates and the solution is monitored for two minutes. The final concentration of the test compound is adjusted to range from about 50 picoM to about 3 μM. Agonist buffer (wash buffer titrated with 1N HCl to provide a solution having a pH of 5.5 when mixed 1:1 with assay buffer) (0.1 mL) is then added to each well, and the plates are incubated for 1 additional minute.
Measurement: IC50 = 361.6 nM (pChEMBL 6.440000057220459)
Descriptors: MW 426.83, ALogP 4.52, HBA 4, HBD 2, PSA 78.35, QED 0.76
Compound (SMILES): O[C@H]1[C@@H](O)C[C@@]2(n3cnc4c(NC5CCCCCCC5)nc(Cl)nc43)C[C@@H]12
InChIKey: UVFJMANYMXKZFS-AEVLCDIHSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Radioligand Binding Assay: Radioligand binding assays at A1, A2A, and A3ARs were performed according to the procedures described previously. Each tube in the binding assay contained 100 uL of membrane suspension (20 ug of protein), 50 pt of a stock solution of agonist radioligand, and 50 uL of increasing concentrations of the test ligands in Tris-HCl buffer (50 mM, pH 7.5) containing 10 mM MgCl2. Nonspecific binding was determined using a final concentration of 10 uM NECA, a non-specific agonist, diluted with the buffer.The mixtures were incubated at 25° C. for 60 min. Binding reactions were terminated by filtration through Whatman GF/B filters under a reduced pressure using a MT-24 cell harvester (Brandell, Gaithersburg, Md.). Filters were washed three times with 5 ml of 50 mM ice-cold Tris-HCl buffer (pH 7.5). The radioactive agonists [3H]R-PIA and [3H]CGS21680 were used for the A1 and A2AAR assays, respectively, while [125I]AB-MECA was used for the A3AR assay.
Measurement: Ki = 1530 nM (pChEMBL 5.820000171661377)
Descriptors: MW 391.9, ALogP 2.85, HBA 7, HBD 3, PSA 96.09, QED 0.7
Compound (SMILES): COc1cc(OC)cc(N(C/C=C/c2nccc(OC)n2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: SCXUCUKEHHIEDN-AATRIKPKSA-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 509.57, ALogP 4.7, HBA 10, HBD 0, PSA 100.31, QED 0.28
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3cc4c(cn3)OCn3c-4cc4c(C#N)cccc43)cc12
InChIKey: HQGFCTAGPFLARA-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 621.65, ALogP 5.9, HBA 8, HBD 1, PSA 130.46, QED 0.25
Compound (SMILES): O=C1CCc2cc(OCCCN3CCN(c4cccc5sccc45)CC3)ccc2N1
InChIKey: SVNKBCHDVYMMAY-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Rattus norvegicus
UniProt: P14842
Assay: Serotonin 5-HT2A Receptor Binding Assay: The assay was performed according to the method by Leysen J E et al. (Leysen J E, Niemegeers C J E, Van Nueten J M and Laduron P M. [3H] Ketanserin (R 41 468), a selective 3H-ligand for serotonin 2 receptor binding sites. Mol. Pharmacol., 1982, 21: 301-314). The binding assay was performed using 40 ul of the membrane specimen, 20 ul of [3H]-Ketanserin (final concentration 1 to 3 nM), 20 ul of a test drug and 50 mM Tris-hydrochloric acid buffer (pH 7.4) so that the total amount was 200 ul (final dimethylsulfoxide concentration 1%). The reaction was performed at 37° C. for 20 minutes and terminated by conducting suction filtration with a cell harvester on a glass fiber filter plate.The filter plate made of glass fiber was washed with 50 mM Tris-hydrochloric acid buffer (pH 7.4), and after dried, a microplate liquid scintillation cocktail was added and the radioactivity was measured with a microplate scintillation counter.
Measurement: Ki = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 421.57, ALogP 4.38, HBA 5, HBD 1, PSA 44.81, QED 0.6
Compound (SMILES): CC1c2nnn(-c3cc[nH]n3)c2CCN1C(=O)c1cccc(C(F)(F)F)c1Cl
InChIKey: LFBDZVSZSDYETC-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 = 14.1 nM (pChEMBL 7.849999904632568)
Descriptors: MW 410.79, ALogP 3.42, HBA 5, HBD 1, PSA 79.7, QED 0.7
Compound (SMILES): Cc1cc(Nc2nccc(C(C)C)n2)cc(-c2cnc(C(C)(O)C3CCC(C(=O)O)CC3)s2)c1
InChIKey: AHAJLJLVEORGRX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45′ at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 480.63, ALogP 5.87, HBA 7, HBD 3, PSA 108.23, QED 0.39
Compound (SMILES): COc1cc(N2CCOCC2)ccc1Nc1nc2c(c(Nc3ccccc3S(=O)(=O)NC3CCCCC3)n1)C1(CC1)CN2
InChIKey: FQZQSAMRLUIZEE-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 2-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q14289
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 605.77, ALogP 4.88, HBA 10, HBD 4, PSA 129.74, QED 0.27
Compound (SMILES): Cc1nc(-c2cccc(CN3C(=O)CCCC3c3cccc4c3OCCO4)c2)cs1
InChIKey: CCTLUOQMQMZQPD-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements: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 g/ml G418, 100 U/ml penicillin, 100 g/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. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 229 nM (pChEMBL 6.639999866485596)
Descriptors: MW 420.53, ALogP 5.14, HBA 5, HBD 0, PSA 51.66, QED 0.59
Compound (SMILES): Cn1c(=O)cc(CCc2cccc(Cl)c2)c2cc(C(c3ccc(Cl)cc3)c3nccs3)ccc21
InChIKey: PWTUFPUVJVRXBD-UHFFFAOYSA-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 505.47, ALogP 7.27, HBA 4, HBD 0, PSA 34.89, QED 0.24
Compound (SMILES): CNc1cc(C(=O)Nc2ccc(C)c(-n3ccn4nc(-c5cccnc5)cc34)c2)cc(C(C)(C)C)n1
InChIKey: SPEYAQZWGXXNKS-UHFFFAOYSA-N
Target: Angiopoietin-1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q02763
Assay: Sandwich ELISA Assay: For measuring the autophosphorylation of the endogenous Tie2 receptor in the cell lysates prepared, a self-prepared anti-Tie2 antibody directed against the N-terminus of the Tie-2 receptor (1.09 mg/ml) and an HRP-coupled anti-phosphotyrosine antibody (Sigma, A4595, clone pY-20) were used. White 96-well ELISA plates (Lumitrac600, Greiner, #655074) were incubated with 100 μl per well of a 1:1000 dilution of the anti-Tie2 antibody in coating buffer [15 mM Na2CO3, 35 mM NaHCO3, pH 9.6] with shaking at 4° C. overnight. The coated ELISA plates were washed three times with 250 μl PBST buffer [0.1% Tween-20 in PBS], the wells were blocked with 250 μl 3% BSA in PBST buffer with shaking at RT for 1-6 h and washed three more times with 250 μl PBST buffer. From the cell lysate plates thawed in the fridge, in each case 100 μl of lysate were transferred into the coated wells of the ELISA plates, and the plates were incubated with shaking at 4° C. overnight. The plates were washed three times with in each case 250 μl of PBST buffer, and 100 μl of a 1:5000 dilution of the anti-phosphotyrosine HRP antibody in 3% Prionex (Calbiochem, #529600) in PBST were then pipetted into each well and the plates were incubated with shaking and protected from light at 4° C. overnight. The plates were washed three times with in each case 250 μl of PBST, and 100 μl of chemiluminescent substrate [BM Chemiluminescence ELISA Substrate (POD) Reagent A and B 1:100, Roche, #1582950] were then pipetted into each well, and after 3 min the plates were measured using a luminescence reader.
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 479.59, ALogP 5.48, HBA 7, HBD 2, PSA 89.14, QED 0.35
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccc6ccccc6c4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: KYLMIDRJSLEDQU-QTDSEMIKSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Binding Assay: Binding assay of certain compounds of this invention to the opioid receptor was determine using radioligand binding assay (screening and dose-displacement).
Measurement: Ki = 1.87 nM (pChEMBL 8.729999542236328)
Descriptors: MW 551.73, ALogP 6.29, HBA 5, HBD 0, PSA 40.16, QED 0.33
Compound (SMILES): Nc1ncc(-c2ncccc2OC(F)(F)F)nc1C(=O)Nc1ncccc1N1CCC(N)CC1
InChIKey: ISHUULBGXOVLLF-UHFFFAOYSA-N
Target: Protein kinase C theta type (SINGLE PROTEIN), Homo sapiens
UniProt: Q04759
Assay: Inhibition of PKCtheta (unknown origin) incubated for 15 mins in presence of 33P-ATP by Topcount scintillation counting method
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 474.45, ALogP 2.59, HBA 9, HBD 3, PSA 145.17, QED 0.51
Compound (SMILES): CC(=O)N1CC2CCC(C1)N2C(=O)c1ccc(Nc2nc(-c3sc(C)nc3C)cs2)nc1
InChIKey: RTXZJXPNTJYVIO-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9ERZ8
Assay: Antagonist activity at rat TRPV4 assessed as inhibition of 4alpha-PDD-induced activation
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 468.61, ALogP 3.86, HBA 8, HBD 1, PSA 91.32, QED 0.63
Compound (SMILES): CS(=O)(=O)N1CCC(N2CC(CC#N)(n3cc(-c4c(F)cnc5[nH]ccc45)cn3)C2)CC1
InChIKey: XCIJORQNJVXXGZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of human N-terminal His-tagged JAK2 catalytic domain (828 to 1132 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 10 nM (pChEMBL 8)
Descriptors: MW 457.54, ALogP 1.91, HBA 7, HBD 1, PSA 110.91, QED 0.63
Compound (SMILES): Cc1cccc(CN2C[C@H](CO)[C@@H](O)[C@H](O)C2)c1
InChIKey: KKYOXNWQRVZXFR-MGPQQGTHSA-N
Target: Lysosomal acid glucosylceramidase (SINGLE PROTEIN), Homo sapiens
UniProt: P04062
Assay: Enzyme Assay: To 2.5 μl of supernatant (in 96-well plates) was added 17.5 μl reaction buffer (citrate phosphate buffer, pH 4.5, no Triton X-100), and 50 μl of 4-methyl umbelliferone (4-MU)-labeled substrate, β-glucopyranoside, or a labeled negative controls α-glucopyranoside or α-galacatopyranoside). Plates were incubated at 370 for 1 hour, followed by the addition of 70 μl stop buffer (0.4 M glycine-NaOH, pH 10.6). Activity of GCase was determined by measuring the emission at 460 nm by exciting at 355 nm using a 1 second read time per well (Victor2 multilabel counter-Wallac).
Measurement: IC50 = 12700 nM (pChEMBL 4.900000095367432)
Descriptors: MW 251.33, ALogP 0.14, HBA 4, HBD 3, PSA 63.93, QED 0.71
Compound (SMILES): CNC(=O)c1cccc(Nc2nn(CC3CC3)c3c2CN(C(C)=O)CC3)c1
InChIKey: QEBQWMOQSWEYPS-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 367.45, ALogP 2.3, HBA 5, HBD 2, PSA 79.26, QED 0.85
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCC2CCOCC2)c([N+](=O)[O-])c1)c1ccc(N2CCN(Cc3cc(OCCN4CCOCC4)ccc3-c3ccc(Cl)cc3)CC2)cc1Oc1ccccc1
InChIKey: NSBSBXIPIVARAG-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 27.53 nM (pChEMBL 7.559999942779541)
Descriptors: MW 925.5, ALogP 7.7, HBA 13, HBD 2, PSA 165.05, QED 0.07
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(Cc3ccc(F)cc3F)CC2)C1
InChIKey: PXTAZAMVQUUMPE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of human N-terminal His-tagged JAK2 catalytic domain (828 to 1132 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 > 10 nM (pChEMBL 8)
Descriptors: MW 488.55, ALogP 3.69, HBA 7, HBD 1, PSA 89.66, QED 0.45
Compound (SMILES): N#Cc1ccc(Nc2nncc3ccccc23)cc1
InChIKey: LYKFUIDYYBNHOA-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR-2 (unknown origin) using poly (Glu, Tyr) 4:1 as substrate after 1 hr by ELISA
Measurement: IC50 = 0.43 nM (pChEMBL 9.369999885559082)
Descriptors: MW 246.27, ALogP 3.25, HBA 4, HBD 1, PSA 61.6, QED 0.75
Compound (SMILES): CCCCN(Cc1ccc(C(=O)O)cc1)C(=O)c1ccc(Oc2ccccc2F)cc1
InChIKey: SVNWCVFZMPJBLW-UHFFFAOYSA-N
Target: Lysophosphatidic acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92633
Assay: Receptor Assay: Engagement of the LPA1 receptor by its ligand, oleoyl-L-Î±-lysophosphatidic acid (LPA), leads to the release of intracellular stores of calcium into the cytoplasm mediated through a Gq-driven increase in inositol triphosphate (IP3) levels. Gq is a heterotrimeric G protein subunit that activates phospholipase C. The ability of test compounds to prevent LPA-driven intracellular release of stored calcium from RH7777 cells expressing human LPA1 receptors was assayed by stimulating the cells with LPA in the presence of various test compounds to measure the test compounds' antagonism, i.e. activity in vitro.Â  Approximately 10,000 cells in normal culture medium (Dulbecco's Modified Eagle Media, 10% Foetal Calf Serum) were dispensed per assay well in a black clear bottom collagen coated 384 well plate (Beckton Dickinson). Cells were allowed to settle for thirty minutes at room temperature before being incubated overnight at 37° C., 5% CO2. Sixteen to twenty hours after dispensing, the cells were loaded with a calcium-sensitive fluorescent dye by replacing the culture medium with assay buffer (1x Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH7.4) containing 1.25 mM probenecid and 1x Calcium 5 Reagent (Molecular Devices). Cells were incubated at 37° C. for one hour to allow for dye uptake.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 421.47, ALogP 5.76, HBA 3, HBD 1, PSA 66.84, QED 0.48
Compound (SMILES): CNC(=O)CC(C)(NC(=O)c1cc(OCC(F)(F)F)c(C2CC2)cn1)C1CC1
InChIKey: OXCWDJBBNIVBSM-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
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×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° C. for 30 min. Compounds were added to a final assay volume of 100 μl 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 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM 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 as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 11.9 nM (pChEMBL 7.920000076293945)
Descriptors: MW 399.41, ALogP 2.93, HBA 4, HBD 2, PSA 80.32, QED 0.7
Compound (SMILES): COc1cc(Nc2nc3cc(C4(OC)c5ccccc5C(=O)N4Cc4ccccc4)ccc3[nH]2)cc(OC)c1
InChIKey: UOGGGAZFNKLMHJ-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 >= 10000 nM (pChEMBL 5)
Descriptors: MW 520.59, ALogP 5.83, HBA 6, HBD 2, PSA 88.71, QED 0.27
Compound (SMILES): O=C(NCC(c1cnc(C(F)(F)F)nc1)N1CCOCC1)c1cccc(C(F)(F)F)c1Cl
InChIKey: MRNXLFVJQDPQCU-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 482.81, ALogP 3.97, HBA 5, HBD 1, PSA 67.35, QED 0.65
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](C)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](C)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](C)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: CUPAMFFXOKTGSX-JPGWIRTJSA-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 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 2327.77
Compound (SMILES): Cc1cc(OCC2CC2)cnc1C(=O)Nc1cc(F)c(F)c([C@]2(C)C[C@@H](C(F)(F)F)OC(N)=N2)c1
InChIKey: ZFVIXMFRDAHNJY-JTSKRJEESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 549 nM (pChEMBL 6.260000228881836)
Descriptors: MW 498.45, ALogP 4.59, HBA 6, HBD 2, PSA 98.83, QED 0.57
Compound (SMILES): c1ccc2ncc(Nc3n[nH]c4cccnc34)cc2c1
InChIKey: CVJFMFGKTFDXQX-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Receptor Activity Assay: The disclosed compounds and compositions can be evaluated for their ability to act as a potentiator of metabotropic glutamate receptor activity, in particular mGluR4 activity, by any suitable known methodology known in the art. For example, Chinese Hamster Ovary (CHO) cells transfected with human mGluR4 or HEK cells co-transfected with rat mGluR4 and the G-protein regulated Inwardly Rectifying Potassium channel (GIRK) were plated in clear bottom assay plates for assay in a Hamamatsu FDSS Fluorometric Plate Reader. The cells were loaded with either a Ca2+-sensitive fluorescent dye or the thallium responsive dye and the plates were washed and placed into a suitable kinetic plate reader. For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured.
Measurement: EC50 = 961 nM (pChEMBL 6.019999980926514)
Descriptors: MW 261.29, ALogP 3.25, HBA 4, HBD 2, PSA 66.49, QED 0.58
Compound (SMILES): COc1cc(C(=O)NCc2cccc(Cl)c2Cl)ccc1-c1cn[nH]c1
InChIKey: ZQYPIHNDLBYJOW-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 2.53 nM (pChEMBL 8.600000381469727)
Descriptors: MW 376.24, ALogP 4.32, HBA 3, HBD 2, PSA 67.01, QED 0.7
Compound (SMILES): COc1cc(OC)cc(N(CC(O)CN2CCC(F)(F)C2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: JWLSSKVRBZFBNN-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 524.57, ALogP 3.89, HBA 9, HBD 1, PSA 88.77, QED 0.35
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1)nn2C[C@@H]1CCCN1C(=O)/C=C/CN1CCOCC1
InChIKey: CZCUSNQZHNYXNJ-LKYAVPRHSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: HotSpot kinase assay: IC50s were determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 μM ATP.). For enzyme inhibition assays, compounds were tested in range of ten concentrations from 10 uM to 0.0005 uM using purified enzymes and the Hotspot kinase assay. Reaction conditions were 1 uM ATP, one hour incubation with inhibitor, and kinase activity detected using 33-ATP phosphorylation of an appropriately selected peptide substrate.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 539.64, ALogP 3.75, HBA 9, HBD 1, PSA 111.63, QED 0.34
Compound (SMILES): COc1nc(C2CC2)c(Cl)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: BUIXSRQQTIRZDD-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 17250 nM (pChEMBL 4.760000228881836)
Descriptors: MW 472.91, ALogP 3.71, HBA 8, HBD 1, PSA 116.32, QED 0.45
Compound (SMILES): N[C@@H]1[C@H]2CN(c3ncc(C(=O)Nc4ccc(OC(F)(F)F)cc4)cc3-c3cncnc3)C[C@@H]12
InChIKey: UJSDGMRXYFUBBD-BWTSREIZSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper ABL : The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 uL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 uM ATP, 4 uM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 uL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 uL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 456.43, ALogP 3.08, HBA 7, HBD 2, PSA 106.26, QED 0.61
Compound (SMILES): C[C@@H]1CN(CC2CCCC2)c2ccccc2[C@H](C)N1C(=O)Nc1cccnc1
InChIKey: YWXMMYXHDBSBEQ-MSOLQXFVSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Channel blocking activity at recombinant human CaV 3.2 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 62 nM (pChEMBL 7.210000038146973)
Descriptors: MW 378.52, ALogP 5.08, HBA 3, HBD 1, PSA 48.47, QED 0.81
Compound (SMILES): O=C1C(N2CCC(c3ccc(O)cc3)C(F)(F)C2)CCN1Cc1ccc(C(F)(F)F)cc1
InChIKey: SVPPSJNIPGZMHE-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 = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 454.44, ALogP 4.64, HBA 3, HBD 1, PSA 43.78, QED 0.69
Compound (SMILES): CCc1ccc(/N=C2\S/C(=C\c3ccnc(NC(=O)CCC(=O)O)c3)C(=O)N2C)cc1
InChIKey: AIYAAHSLCORTFA-QFLUIHSUSA-N
Target: Aurora kinase B/Inner centromere protein (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9NQS7
Assay: Inhibition of Aurora-B/INCENP (unknown origin) using biotinylated STK2 substrate incubated for 30 mins by HTRF assay
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 438.51, ALogP 3.68, HBA 6, HBD 2, PSA 111.96, QED 0.64
Compound (SMILES): CN1Cc2cnc3ccc(-c4cnc5[nH]ccc5c4)nc3c2N(c2cccc(C(F)(F)F)c2)C1=O
InChIKey: BBEVGQNDTQLVSC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR Activity Assay: 1. Preparation of test reagents {circle around (1)} 1x kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 3 mM MnCl, 0.01% Tween-20, 2 mM DTT); {circle around (2)} 4x kinase solution (added mTOR kinase to 1x kinase buffer to provide 4x kinase solution with a final concentration of 2.5 nM); {circle around (3)} 2x substrate solution (added the substrates of 4EBP1 and ATP into 1x kinase buffer to provide 2x substrate solution with 4EBP1 final concentration of 50 nM and ATP final concentration of 10.8 uM); {circle around (4)} 4x test substance solutions (100x test substance solutions with different concentration gradients were prepared using 100% DMSO, and diluted 25-fold with 1x kinase buffer to give 4x test substance solutions with different concentration gradients) {circle around (5)} preparation of test solution (test solution containing EDTA and 4EBP1 phosphorylated antibody was prepared using activity test kit with assay buffer, the final concentration of EDTA was 8 mM, and the final concentration of 4EBP1 phosphorylated antibody was 2 nM) 2. 2.5 uL of 4x test solution was added to a 384-well plate in parallel; 3. added 2.54 uL of 4x kinase solution and incubated for 10 min; 4. then added 5 uL of 2x solutions of 4EBP1 and ATP substrates and incubated for 1 h at room temperature; 5. finally, added 10 uL of test solution to quench the reaction, and read after 60 min; 6. IC50 value was obtained via curve fitting.
Measurement: IC50 = 17.5 nM (pChEMBL 7.760000228881836)
Descriptors: MW 474.45, ALogP 5.9, HBA 4, HBD 1, PSA 78.01, QED 0.35
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(NC(=O)Cc2ccc(Cl)cc2F)ccc1Oc1ccc(F)cc1F
InChIKey: AFSMWBGDSQECTB-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method B: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.47 mM to 7.8 nM. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 77.7 nM (pChEMBL 7.110000133514404)
Descriptors: MW 542.94, ALogP 6.5, HBA 5, HBD 1, PSA 69.56, QED 0.28
Compound (SMILES): CCN(Cc1cccnc1)C(=O)Cn1cc(NC(=O)c2cnn3cccnc23)c(-c2cc(Cl)ccc2OC(F)F)n1
InChIKey: YZOAWYJKBHMXJU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.36 nM (pChEMBL 9.4399995803833)
Descriptors: MW 580.98, ALogP 4.54, HBA 9, HBD 1, PSA 119.54, QED 0.26
Compound (SMILES): O=C1C(=O)c2nn(-c3ccccc3)nc2-c2ccccc21
InChIKey: KDKORSNHTOHNMW-UHFFFAOYSA-N
Target: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WIS7
Assay: Inhibition Assay: Mtb PDH (Lpd+DlaT+AceE) is provided by Dr. Bryk Ruslana. The assay was performed in a manner similar to that described in Bryk et al., Biochemistry (2010) 49:1616-1627 and modified for an online robotics screening system.A solution containing PDH (Lpd=15 μM; DlaT=30 μM; AceE=15 μM) was diluted 2-fold in 100 mM of potassium phosphate buffer (pH 7.0) for the assay. Another reaction solution containing 50 mM potassium phosphate buffer (pH 7.0), 200 μM TPP, 2 mM Pyruvate, 200 μM CoA, 1 mM MgCl2, 1 mM NAD+ was prepared. 5 μL/well of PDH solution was dispensed into 1536-well black plates. Next, 50 nL of each test compound (1 mM dissolved in DMSO) was added into each well. After 30 minutes of incubation, 5 μL/well of reaction solution was added. At the 30th minutes of the reaction, the florescence signal (excitation 360 nm and emission 460 nm) was obtained by Viewlux reader (PerkinElmer).
Measurement: IC50 < 40 nM (pChEMBL 7.3979997634887695)
Descriptors: MW 275.27, ALogP 2.31, HBA 5, HBD 0, PSA 64.85, QED 0.64
Compound (SMILES): C(=C/c1n[nH]c2cc(Sc3ccccc3)ccc12)\c1ccccc1
InChIKey: DKHVKMQAOLEJKJ-SDNWHVSQSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of human 50 residues truncated cytoplasmic domain of phosphorylated VEGFR2 E990V mutant expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using poly(E4Y1) as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 328.44, ALogP 5.88, HBA 2, HBD 1, PSA 28.68, QED 0.51
Compound (SMILES): O=C(c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1)N1CCC12COC2
InChIKey: CVROSPZRAIHVTA-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×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 °C. for 30 min. Compounds were added to a final assay volume of 100 μl 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 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM 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 as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 365.38, ALogP 1.94, HBA 5, HBD 0, PSA 54.9, QED 0.8
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cccc(OC)c3)ccc2[nH]1
InChIKey: XUVBCCAHCFQSCS-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 444.45, ALogP 3.6, HBA 6, HBD 3, PSA 116.78, QED 0.44