lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCS(=O)(=O)NC(C)c1cc(-c2nc3cc(F)c(F)cc3n2C2CC2)cnn1
InChIKey: JCSUCIVKEUGXRF-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Metalloenzyme Activity: V79-4 cells expressing recombinant andrenodoxin and andrenodoxin reductase with either recombinant human CYP11B2 or CYP11B1 were prepared according to methods previously described (LaSala et al 2009 Anal Bioch 394:56-61). An enzyme enriched microsomal fraction was prepared from cellular lysates and subsequently used as the enzyme source for determining inhibitor IC50s. The substrate Km values were experimentally determined for 11-deoxycorticosterone (CYP11B2 substrate) and 11-deoxycortisol (CYP11B1 substrate). Enzyme assays for inhibitor screening employed CYP11B2 and CYP11B1 enzyme enriched microsomes and were run at the Km of the respective substrates. Products of the enzyme reactions, aldosterone for CYP11B2 or cortisol for CYP11B1, were measured by LC-MS. Assays were run under conditions of less than 20% substrate turnover. Inhibitor IC50s were generated by determining the product formation in the absence or presence of inhibitor at various concentrations. In the absence of the test compound, the product formed (Pt) in each data set was defined as 100% activity. In the absence of enzyme, the product formed (Pb) in each data set was defined as 0% activity. The percent activity in the presence of each inhibitor was calculated according to the following equation: % activity=(P−Pb)/(Pt−Pb), where P=the product formed in the presence of the inhibitor. The IC50 value was defined as the inhibitor concentration causing a 50% decrease in activity relative to the no inhibitor control reaction.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 407.45, ALogP 3.11, HBA 6, HBD 1, PSA 89.77, QED 0.68
Compound (SMILES): Cc1ccc(CN2CCn3nnnc3C2c2ccc(OC(C)C)cc2)o1
InChIKey: JDXPTUYCQABJRJ-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: TR-FRET Assay: In this assay, a GST-tagged human RORγ ligand binding domain (GST-hRORγ-LBD) interacts with a synthetic biotinylated TRAP220 cofactor peptide containing an LXXLL motif (amino acids 631-655 from NP_004765). The ligand-binding domain (LBD) of RORγ is expressed as fusion protein with GST in BL-21 (BL3) cells using the vector pDEST15. Cells are lysed by lysozyme-treatment and sonication, and the fusion proteins purified over glutathione sepharose (Pharmacia) according to the manufacturers instructions. The strong constitutive interaction is disrupted upon binding of functional antagonists. The strength of the interaction is monitored by TR-FRET between streptavidine APC interacting with the biotinylated peptide and Europium-labelled Anti-GST. 6 μl RORγ-LBD-GST solution (final 8.7 nM) in assay buffer (20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl, 0.1% delipidated BSA, 1 mM DTT) are dispensed into black 384 well small volume plates (Greiner #784076). 6 μl TRAP220 peptide (final 400 nM), SA-APC (final 1.6 ng/μl), Eu-anti-GST (final 0.125 ng/μl) in assay buffer and 2 μl test compounds (DMSO stocks prediluted in 20 mM Tris-HCl, pH 6.8, 5 mM MgCl2, 60 mM KCl) are added and the mixture incubated 60 min at RT in the dark before measuring FRET (excitation 337 nm, emission at 615 and 665 nm).
Measurement: IC50 = 24950 nM (pChEMBL 4.599999904632568)
Descriptors: MW 353.43, ALogP 2.97, HBA 7, HBD 0, PSA 69.21, QED 0.7
Compound (SMILES): Cn1nc2c(c1-c1cc(F)c(F)c(F)c1)C[C@@H]1CCC[C@H]2N1C(=O)c1cnc2cc(C(F)(F)F)ccn12
InChIKey: OIFUBLKZFHBACJ-KBXCAEBGSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition. Standard conditions for this assay combine 300 nM [Glycerol-1,3-3H]-oleoyl glycerol with human MGL from HeLa cells and test compounds for one hour, after which the reaction is filtered through activated carbon and tritium is measured in the flow through. The test compound concentration in screening mode is 10 uM, while the highest compound concentration in IC50 assays is determined empirically.
Measurement: IC50 = 91.2 nM (pChEMBL 7.039999961853027)
Descriptors: MW 519.45, ALogP 5.46, HBA 5, HBD 0, PSA 55.43, QED 0.26
Compound (SMILES): CC(O)c1cc2nn(-c3ccccc3)c(NC(=O)c3cc(-c4ncccn4)c(C(F)(F)F)cc3F)c2cn1
InChIKey: KFUMLBPRQSNFIS-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Biological Assay: TrkA functional activity was measured using a DiscoverX PathHunter assay. In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK); additionally, Shc1 is fused with a larger fragment, which is called Enzyme Acceptor (EA). Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shc1-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 522.46, ALogP 5.34, HBA 7, HBD 2, PSA 105.82, QED 0.3
Compound (SMILES): O=C(NS(=O)(=O)CC1CC1)c1ccc(COc2cccc(-c3ccc(OCC4CC4)nc3)c2)c(F)c1
InChIKey: HIRARYGHRZFHAE-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: EFS-FRET Assay: This screen is used to determine the effects of compounds on human Nav1.7 channels, utilizing electrical field stimulation (EFS) system in 96-well plate format on FDSS (Hamamatsu Photonics) platform. The changes of membrane potential are monitored with FRET dye pair, DiSBAC2(3) and PTS 18.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 510.59, ALogP 4.73, HBA 6, HBD 1, PSA 94.59, QED 0.4
Compound (SMILES): NS(=O)(=O)c1cccc(-c2ccc(CN3CCCC3CO)cc2)c1-c1nnn[nH]1
InChIKey: ANJHJAVEBWRSSJ-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 14.4 nM (pChEMBL 7.840000152587891)
Descriptors: MW 414.49, ALogP 1.14, HBA 7, HBD 3, PSA 138.09, QED 0.55
Compound (SMILES): CC(=O)N1C[C@@H](C)N(c2cccc(-c3cc(-c4ccnn4CC(F)(F)F)c4c(N)ncnn34)c2)[C@@H](C)C1
InChIKey: QCZXCTRWCRLDFH-IYBDPMFKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 512.54, ALogP 3.85, HBA 8, HBD 1, PSA 97.58, QED 0.45
Compound (SMILES): Cc1nnn(C)c1-c1cnc2c3c(c(C(C)(C)O)nn3C)n([C@@H](c3ncccc3F)C3CCOCC3)c2c1
InChIKey: AOABNCJLYRKHNM-HSZRJFAPSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Binding Assay: Briefly, 20 nL of compounds were transferred to the 384-well plate by Echo® 550 liquid handler (Labcyte, USA), then 5 μL of BRD4(BD1) (Reaction Biology Company, RD-11-157) solution or the assay buffer was added to each well. After incubating at RT for 15 min, 5 μL of the biotinylated H4 derived acetylated peptide (synthesized by GL Biochem (Shanghai) Ltd) and 10 μL of the detection solution (Cisbio Assay) were added to each well. After incubating for 1 h at RT, the HTRF signal was measure at 615 nm and 665 nm using the EnVision Multilabel Plate Reader (PerkinElmer, USA). Results were analyzed with a two-wavelength signal ratio: intensity (665 nm)/intensity (615 nm). The inhibition percentage in the presence of the compound was calculated according to the equation, Percent inhibition=(Max−Signal)/(Max−Min)*100%. Fit the data in GraphPad Prism V5.0 software (San Diego, Calif.) to obtain IC50 values with nonlinear regression analysis using equation, Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC50−X)×HillSlope), where Y stands for inhibition percentage and X stands for compound concentration.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 518.6, ALogP 3.8, HBA 10, HBD 1, PSA 108.7, QED 0.38
Compound (SMILES): O=C(N[C@H](Cc1ccc(-c2cccc(Cl)c2)cc1)C[C@@H](O)C(=O)O)c1cc(-c2ccccc2Cl)on1
InChIKey: BSUFBCUNFHOMLU-HYBUGGRVSA-N
Target: Neprilysin (SINGLE PROTEIN), Homo sapiens
UniProt: P08473
Assay: Enzyme Inhibition Assay: The assays were performed in 384-well white opaque plates at 37° C. using the fluorogenic peptide substrates at a concentration of 10 μM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 μM ZnSO4; ACE: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO4). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 μM of substrate after 20 minutes at 37° C.Test compounds were assayed over the range of concentrations from 10 μM to pM. Test compounds were added to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37° C. by the addition of glacial acetic acid to a final concentration of 3.6% (v/v).
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 525.39, ALogP 5.49, HBA 5, HBD 3, PSA 112.66, QED 0.27
Compound (SMILES): CC(C)c1cc(C(=O)Nc2csc([C@]34CO[C@@H](C)C[C@H]3CSC(N)=N4)n2)n(C)n1
InChIKey: NLXQVVUOVUDGPI-ZKTNFTSUSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Cell-Free Assay: Beta-secretase (BACE) is one of the enzymes involved in the generation of the amyloid beta peptide found in the amyloid plaques of Alzheimer's Disease patients. This assay measures the inhibition of the beta-secretase enzyme as it cleaves a non-native peptide.A synthetic APP substrate that can be cleaved by beta-secretase having N-terminal biotin and made fluorescent by the covalent attachment of Oregon Green at the Cys residue is used to assay beta-secretase activity in the presence or absence of the inhibitory compounds. The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a ½ log dose response curve from a top concentration of 100 μM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 μL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37° C. The reaction is stopped with the addition of 30 μL of 1.5 μM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm).
Measurement: IC50 = 10400 nM (pChEMBL 4.980000019073486)
Descriptors: MW 434.59, ALogP 2.93, HBA 9, HBD 2, PSA 107.42, QED 0.77
Compound (SMILES): CCn1c2ccccc2c2cc(-c3nc4cc(C(=O)NS(=O)(=O)Cc5ccc(C(F)(F)F)cc5)ccc4n3CCOC)ccc21
InChIKey: PLCOOUZYMSVRMK-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Test for Antagonism: 4 μl of a cAMP-d2/cell suspension (625000 cells/ml) were added to a test plate containing the substance solutions already initially introduced (0.05 μl; 100% DMSO, concentration range from 0.8 nM-16.5 μM). After a 20-minute preincubation at room temperature (RT), 2 μl of a 3xPGD2 solution (6 nM, in PBS-IBMX) were added and incubated in the presence of the agonist for a further 30 min at RT (volume: ¿6 μl). Subsequently, the reaction was stopped by addition of 2 μl of lysis buffer and incubated for a further 20 min at RT before the actual measurement (volume: ¿8 μl).
Measurement: IC50 = 186 nM (pChEMBL 6.730000019073486)
Descriptors: MW 634.68, ALogP 6.76, HBA 7, HBD 1, PSA 95.22, QED 0.19
Compound (SMILES): COc1cc(CO)cc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(C(N)=O)c4OC)c4ccccc34)ccn2)c1
InChIKey: RXXPIRGHDOIFNF-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: Enzyme Inhibition Assay: Method 2: This method follows the same steps as Method 1 above, but utilises a higher concentration of the p38 MAPKα protein (2.5 uL of 200 ng/mL protein instead of 2.5 uL of 80 ng/mL protein) for mixing with the test compound (tested at either 1 ug/mL, 0.1 ug/mL, 0.01 ug/mL or 0.001 ug/mL). Method 1: The inhibitory activities of test compounds against the p38 MAPKα isoform (MAPK14: Invitrogen) are evaluated indirectly by determining the level of activation\phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPKα protein (80 ng/mL, 2.5 uL) is mixed with the test compound (2.5 uL of either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL or 0.004 ug/mL) for 2 hr at RT. The mix solution (2.5 uL) of the p38a inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 uM; a phosphorylation target for MAPKAP-K2) is then added, then the kinase reaction is initiated by adding ATP (40 uM, 2.5 uL). The mixture is incubated for 1 hr at RT. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Vanoskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 635.72, ALogP 7.32, HBA 8, HBD 5, PSA 157.06, QED 0.11
Compound (SMILES): CS(=O)(=O)c1cccc(N2CCN(CC3(F)CCN(CC(F)(F)F)CC3)[C@H](c3ccccc3)C2)c1
InChIKey: XFSCVGWGYBAUOK-QHCPKHFHSA-N
Target: Oxysterols receptor LXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q13133
Assay: LXR alpha/beta Radioligand Binding Assay: Compounds of the invention were assessed in a competition binding assay where different concentrations of compounds were incubated with the LXR ligand binding domain (LBD) in the presence of radiolabeled LXR ligand [3H]TO901317. The amount of the LXR-LBD that complexed with [3H]T0901317 was measured by scintillation proximity assay (SPA) employing non-specific binding of LXR-LBD to poly-lysine coated Yttrium silicate beads. Partially purified LXR α or β LBD protein (15-45 nM) was incubated at rt for 30 min with 15 nM [3H]TO901317 (25-40 Ci/mmol) and different concentrations of test compounds in 80 μL of phosphate buffered saline (PBS) buffer containing 2.5% DMSO, 1% glycerol, 2 mM EDTA, 2 mM CHAPS and 5 mM DTT in 96-well plates. Poly-lysine SPA beads (50 μg) were added to each well and the total volume was adjusted to 120 μL. The plates were shaken on an orbital shaker for 20 min and then allowed to settle for 10 more min at rt before a brief centrifugation at 2,000 rpm for 1 min. The SPA signal was measured on a MicroBeta® liquid scintillation counter (Perkin Elmer, Waltham, Mass.), and the results were used for calculating IC50 values based on the total binding (DMSO control) and non-specific binding (5 μM of unlabeled TO901317) controls. The Ki values were calculated according to equation 1, where [RL] is the final concentration of [3H]TO901317 in the assay, and the Kd values of 20 nM and 10 nM of TO901317 for LBDs of LXRα and LXRβ, respectively, were determined by direct titration of the radioligand with these proteins.
Measurement: Ki > 3333 nM (pChEMBL 5.4770002365112305)
Descriptors: MW 513.6, ALogP 4.32, HBA 5, HBD 0, PSA 43.86, QED 0.54
Compound (SMILES): CCC(=O)c1ccc(Cl)cc1-c1cc(=O)n(C(Cc2ccccc2)C(=O)Nc2ccc3ncncc3c2)cc1OC
InChIKey: LDYIDSOXDBMFOP-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Biological Assay: Test Example 1: Biological Activity of the Compounds of the Present Invention onThe Inhibition of Factor XIa Detected by Absorption Photometry1. Experimental MaterialsEnzyme: Coagulation Factor XIa protease (Abcam, Art. No ab62411)Substrate: Coagulation Factor XIa specific substrate (HYPHEN1310 med, Art. No. Biophen cs-21(66))Buffer: 100 mM tris-HCl, 200 mM NaCl, 0.02% Tween20, pH 7.42. Experimental Procedure20 mM of test compound dissolved in 100% DMSO was diluted to 200, 20, 2, 0.2, 0.02, 0.002 μM with 100% DMSO; 1 μl of the compound was added to each well in a 384-well plate, blank and control wells were replaced with DMSO. The plate was centrifuged to remove the compound to the bottom. 10 μl (2.5 μg/ml) of FXIa enzyme solution was added to each well, and 10 μl of buffer was added to the blank well. The plate was centrifuged to remove the enzyme solution to the bottom.Finally, 10 μl of 2 mM substrate were added to each well, and the plate was centrifuged to remove the substrate solution to the bottom.The plate was incubated for 10 minutes at 37° C.; and then the absorbance was measured at 405 nm. The absorbance was curve-fitted by graphpad
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 567.05, ALogP 6.14, HBA 7, HBD 1, PSA 103.18, QED 0.22
Compound (SMILES): Cc1cc(N2CCOCC2)ncc1N1C[C@]2(C[C@](c3ccccc3)(N(C)C)C2)NC1=O
InChIKey: CKBIKPDMRFBXJG-PSWAGMNNSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Scintillation Proximity Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences. Inc. Boston, Mass. USA) and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hNOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston, Mass. USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter.
Measurement: Ki = 1810 nM (pChEMBL 5.739999771118164)
Descriptors: MW 421.55, ALogP 2.75, HBA 5, HBD 1, PSA 60.94, QED 0.82
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(C(F)(F)F)CC(C)/C=C\C[C@H](NC(=O)/C=C/c1cc(Cl)ccc1-n1cnnn1)c1nc-2c[nH]1
InChIKey: CUTHDQZPCLDRBU-ABRCENQTSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa assay : The effectiveness of compounds of the present invention as inhibitors of the coagulation Factors XIa, VIIa, IXa, Xa, XIIa, plasma kallikrein or thrombin, can be determined using a relevant purified serine protease, respectively, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Hydrolysis of the substrate resulted in the release of pNA (para nitroaniline), which was monitored spectrophotometrically by measuring the increase in absorbance at 405 nm, or the release of AMC (amino methylcoumarin), which was monitored spectrofluorometrically by measuring the increase in emission at 460 nm with excitation at 380 nm. A decrease in the rate of absorbance or fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 2171 nM (pChEMBL 5.659999847412109)
Descriptors: MW 656.07, ALogP 6.08, HBA 9, HBD 4, PSA 151.74, QED 0.15
Compound (SMILES): O=C(NCCN(C(=O)c1cc(C(F)(F)F)cc(C(F)(F)F)c1)c1cnccc1-c1ccccc1Cl)OCc1ccccc1
InChIKey: CXESUDHOAPHSDA-UHFFFAOYSA-N
Target: G-protein coupled bile acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDU6
Assay: cAMP Assay: CHO-dhfr(minus) cells expressing human GPBAR1 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), lx 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. The assay was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaked for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH, Hamburg Germany), 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 measured signal at 730 nm has to be corrected for the ruthenium background, the direct excitation of Alexa and the buffer control. 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 = 1488 nM (pChEMBL 5.829999923706055)
Descriptors: MW 621.97, ALogP 8.01, HBA 4, HBD 1, PSA 71.53, QED 0.2
Compound (SMILES): NC1CC(Oc2ncc(Cl)c(-n3cnc(C(F)(F)F)c3)n2)C1
InChIKey: IRDXFULCLWMKQR-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: Inhibition Assay: The synthesized compounds then were tested for inhibition of the kinase activity (IC50) of Mnk1 and/or Mnk2 using the ADP monitoring assay for kinases described in Zegzouti, H.; Zdanovskaia, M.; Hsiao, K.; Goueli, S. A., ADP-Glo: A Bioluminescent and homogeneous ADP monitoring assay for kinases.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 333.7, ALogP 2.2, HBA 6, HBD 1, PSA 78.85, QED 0.93
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@@H](OC)c2ncc(F)cn2)nnc1-c1ccc(C)o1
InChIKey: VQVLQTAZCCCHIN-VBKZILBWSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding.
Measurement: EC50 = 6.8 nM (pChEMBL 8.170000076293945)
Descriptors: MW 532.55, ALogP 3.3, HBA 11, HBD 1, PSA 143.49, QED 0.32
Compound (SMILES): CN1CCC(Nc2nn3c(-c4cccc(OC(F)(F)F)c4)nnc3c3c2N(C)CCO3)CC1
InChIKey: OIOYUXMGVUEQPH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM-1 Biochemical Assay: The biochemical assay to measure PIM-1 activity relies on the ADP Hunter assay kit (DiscoveRx Corp., Cat. #90-0077), that determines the amount of ADP as direct product of the kinase enzyme activity.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 463.46, ALogP 3.02, HBA 9, HBD 1, PSA 80.05, QED 0.63
Compound (SMILES): CNc1cc(F)c(C#Cc2cn([C@@H]3O[C@H](CNS(N)(=O)=O)[C@@H](O)[C@H]3O)c3ncnc(N)c23)c(F)c1
InChIKey: RAAZTGRWNXJQSP-WVSUBDOOSA-N
Target: Carbonic anhydrase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P00918
Assay: Enzyme Activity Inhibition: The carbonic anhydrase II enzyme activity inhibition was measured by measuring the esterase activity in which carbonic anhydrase II degrades 4-nitrophenyl acetate (4-NPA) (produced by Sigma-Aldrich). A purified carbonic anhydrase II solution (C6624, produced by Sigma-Aldrich) was diluted with an assay buffer (50 mM Tris-HCl (pH 7.5)) to 100 nM, and 50 μL of the resulting solution was added to a 96-well plate (3695, produced by Costar) containing 40 μL of the test substance. After reaction at room temperature for 10 minutes, 10 μL of a 50 mM 4-NPA solution was added to each well, followed by incubation for 30 minutes at room temperature. The 50 mM 4-NPA solution was prepared by 10-fold dilution of a 500 mM 4-NPA solution, which was prepared at the time of use by dissolving the reaction product in DMSO, with the assay buffer. The absorbance at 405 nm was measured using a microplate reader (SpectraMax 250, produced by Molecular Devices).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 509.5, ALogP -0.85, HBA 10, HBD 6, PSA 190.64, QED 0.24
Compound (SMILES): Cc1nc(C(C)(C)NC(=O)C2CNCCOC2)n2cccc(F)c12
InChIKey: BHUONHWTHYGRRP-UHFFFAOYSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: cAMP Assay: Compounds are dissolved and diluted in DMSO. The final test solution contains 1% DMSO. The cAMP standard (Lance cAMP 384 Kit; PerkinElmer, Cat# AD0264) is prepared in assay buffer (HBSS with 0.1% BSA, 5 mM HEPES, 0.5M IBMX, pH 7.4) containing 1% DMSO and the cAMP standard curve is included at least on one plate. Cells are centrifuged and suspended in assay buffer (incl. 1:100 diluted Alexa antibody).For the assay 5 μl of a cell suspension (approximately 5000 cells/well)¿incl. Alexa antibody (diluted 1:100) are added into a 384 well MTP microtitre plate excepting one row or column (depending on the plate layout), which is reserved for the standard curve. Then 2 μl of compound sample is added as concentration response curve (e.g. le-5M to 6e-10M), usually in triplicates. Each assay contains incubations with vehicle controls instead of compound as controls for non-inhibited cAMP generation (100% CTL; ¿high values¿) and incubations with 1 μM Somatosatin as controls for full inhibition and background (0% CTL; 'low values'). After approximately 10-15 min incubation time 3 μl Forskolin (dissolved in DMSO, final conc. 15 μM) is added. Then the plates are shaken briefly and incubated for 60 min at room temperature. After 60 min 10 μl of the detection mix is added into all wells followed by an additional incubation period of 1 h. The plates are read in a suitable plate reader.The analysis of the data is based on the "ratio" of the time-resolved fluorescence measurements of donor and acceptor fluorophore (Ex: 320 nm; Em1: 665 nm; Em2: 615 nm; ratio 665/615). From this ratio, cAMP concentrations are calculated from standard curve and the EC50 is estimated by least square curve fit program.
Measurement: EC50 = 11.1 nM (pChEMBL 7.960000038146973)
Descriptors: MW 334.4, ALogP 1.37, HBA 5, HBD 2, PSA 67.66, QED 0.89
Compound (SMILES): CN1CCN(c2cccc(Nc3cnc(-c4cnc5ccccn45)c4c3C(=O)NC4)n2)CC1
InChIKey: DINBKMBMJGPECF-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 M), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 Î¼M final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 l/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 440.51, ALogP 2.53, HBA 8, HBD 2, PSA 90.69, QED 0.5
Compound (SMILES): CO[C@H]1CCN(c2ccccc2NC(=O)Nc2cccc(C3CCCN(c4ncnc5[nH]ccc45)C3)c2)C1
InChIKey: ZSCIKAYBSYEVNB-YANBTOMASA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro BTK Kinase Assay: The purpose of the BTK in vitro assay is to determine compound potency against BTK through the measurement of IC50. Compound inhibition is measured after monitoring the amount of phosphorylation of a fluorescein-labeled polyGAT peptide (Invitrogen PV3611) in the presence of active BTK enzyme (Upstate 14-552), ATP, and inhibitor. The BTK kinase reaction was done in a black 96 well plate (costar 3694). For a typical assay, a 24 μL aliquot of a ATP/peptide master mix (final concentration; ATP 10 μM, polyGAT 100 nM) in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 200 μM Na3PO4, 5 mM DTT, 0.01% Triton X-100, and 0.2 mg/ml casein) is added to each well. Next, 1 μL of a 4-fold, 40× compound titration in 100% DMSO solvent is added, followed by adding 15 uL of BTK enzyme mix in 1× kinase buffer (with a final concentration of 0.25 nM). The assay is incubated for 30 minutes before being stopped with 28 μL of a 50 mM EDTA solution. Aliquots (5 μL) of the kinase reaction are transferred to a low volume white 384 well plate (Corning 3674), and 5 μL of a 2× detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20 antibody, Invitrogen PV3552) is added. The plate is covered and incubated for 45 minutes at room temperature. Time resolved fluorescence (TRF) on Molecular Devices M5 (332 nm excitation; 488 nm emission; 518 nm fluorescein emission) is measured. IC50 values are calculated using a four parameter fit with 100% enzyme activity determined from the DMSO control and 0% activity from the EDTA control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 511.63, ALogP 5.21, HBA 6, HBD 3, PSA 98.41, QED 0.33
Compound (SMILES): Cc1c(-c2ccccc2)nc2ccc(Br)cc2c1C(=O)NC[C@@H](CCCC(=O)O)c1ccccc1Cl
InChIKey: ZIDVHLLOHYVUEZ-HXUWFJFHSA-N
Target: Prostaglandin F2-alpha receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43088
Assay: In Vitro Test of Inhibition of Human FP Receptor Activity: For the characterization of test substances in respect of FP antagonism, PGF2α-induced calcium flux in FP-expressing CHEM1 cells (Millipore, HTS093C) was used.3000 cells in 30 μl of full medium [DMEM F12, 10% FCS, 1.35 mM sodium pyruvate, 20 mM HEPES, 4 mM GlutaMAX, 2% sodium bicarbonate, 1% Pen/Strep, 1% 100× non-essential amino acids] are sown per well of a 384 multititre plate (from Greiner, TC plate, black with clear base) and incubated at 33° C., 5% CO2 for 24 hours. Prior to the measurement, the medium is replaced by 30 μl of Fluo-8 AM loading buffer [calcium-free Tyrode (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 4.8 mM NaHCO3, pH 7.4), 2 mM CaCl2, 6.3 mM Probenecid, 5 μM Fluo-8 AM, 0.0112% Pluronic®] and incubated at 37° C., 5% CO2 for 30 minutes. The test substance is prepared in DMSO in various concentrations as a dose-response curve (starting concentration 10 mM, dilution factor 3.16) and prediluted 1:50 with calcium-free Tyrode, 2 mM CaCl2, 0.002% SmartBlock (from CANDOR Bioscience GmbH). 10 μl of the prediluted substance solution are added to the Fluo-8-laden cells and incubated at 37° C., 5% CO2 for 10 minutes. The FP receptor is activated by adding 40 μl of 2 nM (final concentration) PGF2α in calcium-free Tyrode, 2 mM CaCl2, 0.002% SmartBlock, and the calcium flux is determined by measuring the fluorescence at ex. 470 nm/em. 525 nm in a fluorescence measuring instrument (FLIPR Tetra®, Molecular Devices) for 120 seconds.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 565.9, ALogP 7.39, HBA 3, HBD 2, PSA 79.29, QED 0.22
Compound (SMILES): Cn1cnc(S(=O)(=O)N2C[C@@H](Nc3ccc(C(F)(F)F)c(Cl)c3)[C@H](c3ccc(F)cc3)C2)c1
InChIKey: PCSVKHCSLWKVFR-QFBILLFUSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 1000 nM (pChEMBL 6)
Descriptors: MW 502.92, ALogP 4.5, HBA 5, HBD 1, PSA 67.23, QED 0.52
Compound (SMILES): COc1nnc(-c2cc(NC(=O)c3ccnc(C(C)(C)F)c3)ccc2C)cc1N1CCOCC1
InChIKey: REUREILZLZCFOE-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 465.53, ALogP 4.15, HBA 7, HBD 1, PSA 89.47, QED 0.58
Compound (SMILES): COc1cccc(-c2nnc(NS(=O)(=O)CCc3ccc(F)cc3C#N)n2-c2c(OC)ncnc2OC)n1
InChIKey: QERJRSKOFIHDAU-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 540.54, ALogP 2.14, HBA 12, HBD 1, PSA 167.03, QED 0.31
Compound (SMILES): C[C@H]1C[C@H](NC(=O)c2cc(C3CC3)on2)CCN1C(=O)CC(CN)c1ccccc1
InChIKey: IWGIQRJPQZIQBM-JMRRQPBMSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 4666 nM (pChEMBL 5.329999923706055)
Descriptors: MW 410.52, ALogP 2.79, HBA 5, HBD 2, PSA 101.46, QED 0.73
Compound (SMILES): CN(C)CC(=O)N(Cc1ccc(-c2noc(C(F)(F)F)n2)cc1)c1cccc(C(F)(F)F)c1
InChIKey: NNEJNQOEVVTDEY-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzymatic Assay: HDAC1 and 6 reagents: FLAG-tagged HDACs 1 and 6 were prepared in-house by protein expression in HEK293F cells followed by anti-FLAG affinity purification. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 137 mM NaCl [Sigma, catalog #S5150, 5M stock], 2.7 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], and 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock]. In addition to the above buffer ingredients, TCEP [CalBiochem, catalog #580561, 500 mM stock] was added at a final concentration of 0.5 mM to the buffer for the HDAC6 assays. HDAC1, 2, 3, and 6 enzymes were run at the final concentrations of 0.3 nM, 1.5 nM, 0.3 nM, and 1.333 nM, respectively. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-104], used to evaluate enzyme activity, was added at the final concentrations of 20 uM, 40 uM, 20 uM, and 2.5 uM for HDACs 1, 2, 3, and 6. To enable detection of the signal, Developer [BioMol Research Laboratories, catalog #KI-105] was added at a 1:250 dilution to the stop solution, which also included 10 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 472.39, ALogP 4.87, HBA 5, HBD 0, PSA 62.47, QED 0.48
Compound (SMILES): CC(C)c1cccc(Cl)c1COc1ccc2c(c1)OCC21CCN(CCCC(=O)O)CC1
InChIKey: CDRQLTIDTBIFBT-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H228
Assay: In Vitro Functional Activity (Agonism) on Human S1P5 Receptors: The CHO-human-S1P5-Aeqorin assay was bought from Euroscreen, Brussels (Euroscreen, Technical dossier, Human Lysophospholid S1P5 (Edg8) receptor, DNA clone and CHO AequoScreen™ recombinant cell-line, catalog no: ES-593-A, September 2006). Human-S1P5-Aequorin cells express mitochondrial targeted apo-Aequorin. Cells have to be loaded with coelanterazine, in order to reconstitute active Aequorin. After binding of agonists to the human S1P5 receptor the intracellular calcium concentration increases and binding of calcium to the apo-Aequorin/coelenterazine complex leads to an oxidation reaction of coelenterazine, which results in the production of apo-Aequorin, coelenteramide, CO2 and light (□max 469 nm). This luminescent response is dependent on the agonist concentration. Luminescence is measured using the MicroBeta Jet (Perkin Elmer). Agonistic effects of compounds are expressed as pEC50. Compounds were tested at a 10 points half log concentration range, and 3 independent experiments were performed in single point's measurements.
Measurement: EC50 = 126 nM (pChEMBL 6.900000095367432)
Descriptors: MW 458, ALogP 5.63, HBA 4, HBD 1, PSA 59, QED 0.56
Compound (SMILES): CC#CC(=O)N1CCC[C@H]1c1nc(-c2ccc(C(=O)Nc3cc(OC)ccn3)cc2)c2c(N)nccn12
InChIKey: SYDQZIKNKGLECH-FQEVSTJZSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity: Btk enzyme (His-Btk (Millipore catalog #14-552), is diluted to 0.4 U/mL in KR buffer (10 mM Tris-HCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% NaN3, 1 mM DTT, 2 mM MnCl2, pH 7.2).Serial dilution log 10 from 2 mM to 63.2 nM of test compounds are made in 100% DMSO. The dilutions in DMSO are then diluted 50-fold in KR-buffer. Final compound concentration range in the assay from 10 μM to 0.316 nM.5 μL/well of test compound in KR buffer (final DMSO concentration in the assay is 1%) is mixed with 5 μl/well of 0.4 U/mL Btk enzyme (final concentration in the assay is 0.1 U/mL). Test compounds and Btk enzyme are pre-incubated 60 minutes at room temperature, before adding 5 μL/well of 200 nM Fluorescin labeled substrate peptide (Blk/Lyntide substrate, e.g. #R7188/#R7233, Molecular Devices) in KR-buffer. Final peptide substrate concentration in assay is 50 nM. The kinase assay is started by adding 5 μL/well of 20 μM ATP in KR-buffer (final ATP concentration is 5 μM ATP, Km ATP in Btk IMAP assay). Following incubation for 2 h at room temperature the enzyme reaction is stopped by adding 40 μL/well IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol using 75% 1× buffer A and 25% 1× buffer B with 1:600 Progressive Binding Solution). After 60 min incubation at room temperature in the dark the FP signal is read. Fluorescence at 535 nm is measured using parallel and perpendicular filters to determine differences in rotation due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmPi) of the controls with and without ATP. EC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 495.54, ALogP 3.32, HBA 8, HBD 2, PSA 127.74, QED 0.41