lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cccc(-c2ocnc2C(=O)Nc2c(C)nn(CCOc3nc4ccccc4o3)c2C)c1
InChIKey: KOWMKGDHPXGBPX-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/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 μM 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. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μl/well, incubated for 120 min and finally 10 μl/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 966 nM (pChEMBL 6.010000228881836)
Descriptors: MW 457.49, ALogP 4.94, HBA 8, HBD 1, PSA 108.21, QED 0.37
Compound (SMILES): COc1ccc(-c2cccc(Cl)c2C)cc1C(CC(=O)O)c1ncc(C)s1
InChIKey: LYZXFKKVKREVMP-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature, as substrate then bradykinin carrying an N-terminal® Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) label (JPT Peptide Technologies GmbH; dissolved in assay buffer) was added to the mixture. The final concentration of cathepsin A was 833 ng/ml and the final concentration of labeled bradykinin 2 μM. After incubation for 15 min at room temperature the reaction was stopped by the addition of stop buffer (130 mM 2-(4-(2-hydroxy-ethyl)-piperazin-1-yl)-ethanesulfonic acid, pH 7.4, containing (v/v) 0.013%° Triton X-100, 0.13% Coating Reagent 3 (Caliper Life Sciences), 6.5% DMSO and 20 μM ebelactone B (Sigma, #E0886)).
Measurement: IC50 = 1310 nM (pChEMBL 5.880000114440918)
Descriptors: MW 401.92, ALogP 5.7, HBA 4, HBD 1, PSA 59.42, QED 0.58
Compound (SMILES): C[C@@H](c1nc2c(cnn2C2CCCC2)c(=O)[nH]1)N1CC[C@@H](Oc2ccccc2Cl)C1
InChIKey: DXLLBBOOAFAHQM-GOEBONIOSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of PDE9A (unknown origin) using [3H]-cGMP as substrate after 30 mins by microbeta counting method
Measurement: IC50 = 292 nM (pChEMBL 6.539999961853027)
Descriptors: MW 427.94, ALogP 4.1, HBA 6, HBD 1, PSA 76.04, QED 0.66
Compound (SMILES): CC(C)(C)OC(=O)N1CCC2(CC1)OCCC2Oc1ccc2c(c1)C(C)(C)c1[nH]c3cc(Br)ccc3c1C2=O
InChIKey: FERDMVCRYDEECN-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 19.51 nM (pChEMBL 7.710000038146973)
Descriptors: MW 595.53, ALogP 6.74, HBA 5, HBD 1, PSA 80.86, QED 0.36
Compound (SMILES): Cc1nc(NC(=O)N2CCN(C3CCCC3)C2=O)ccc1Oc1ccnc(-c2cnn(C)c2)c1
InChIKey: XEJIAICXFVLKKE-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: Enzymatic Assay: Activity of unphosphorylated c-FMS kinase (uFMS, Seq. ID no. 1) was determined by following the production of ADP from the FMS kinase reaction with ATP and poly E4Y as substrates through coupling with the pyruvate kinase/lactate dehydrogenase system (e.g., Schindler et al. Science (2000) 289: 1938-1942). In this assay, the oxidation of NADH (thus the decrease at A340 nm) was continuously monitored spectrophometrically. The reaction mixture (100 .mu.L) contained FMS (purchased from Millipore) (10 nM), polyE4Y (1 mg/mL), MgCl.sub.2 (10 mM), pyruvate kinase (4 units), lactate dehydrogenase (0.7 units), phosphoenol pyruvate (1 mM), NADH (0.28 mM) and ATP (500 .mu.M) in 90 mM Tris buffer containing 0.2% octyl-glucoside and 1% DMSO, pH 7.5. The inhibition reaction was started by mixing serial diluted test compound with the above reaction mixture. The absorption at 340 nm was monitored continuously for 4 hours at 30.degree. C. on Synergy 2 plate reader.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 461.53, ALogP 4.19, HBA 7, HBD 1, PSA 105.48, QED 0.61
Compound (SMILES): CC#CCn1c(N2CCC[C@@H](N)C2)nc2c1c(=O)n(Cc1nc(C(F)(F)F)cc3ccccc13)c(=O)n2C
InChIKey: LDSPGQNAAPMWAR-QGZVFWFLSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: DPP-IV Assay: 50 μl substrate solution (AFC; AFC is amido-4-trifluoromethylcoumarin), final concentration 100 μM, were placed in black microtitre plates. 20 μl of assay buffer (final concentrations 50 mM Tris HCl pH 7.8, 50 mM NaCl, 1% DMSO) was pipetted in. The reaction was started by adding 30 μl of solubilised Caco-2 protein (final concentration 0.14 μg of protein per well). The test substances to be investigated were typically added prediluted in 20 μl, and the volume of assay buffer was then reduced accordingly. The reaction was carried out at ambient temperature, incubating for 60 minutes. Then the fluorescence was measured in a Victor 1420 Multilabel Counter, the excitation wavelength being 405 nm and the emission wavelength being 535 nm. Blank readings (corresponding to 0% activity) were obtained in mixtures without any Caco-2 protein (volume replaced by assay buffer), control values (corresponding to 100% activity) were obtained in mixtures with no substance added.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 525.54, ALogP 2.46, HBA 9, HBD 1, PSA 103.97, QED 0.41
Compound (SMILES): CN(C)CC(=O)N1CCC(COc2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(CC5=C(c6ccc(Cl)cc6)CC(C)(C)CC5)CC4)cc3Oc3cnc4[nH]ccc4c3)cc2Cl)CC1
InChIKey: FNMJRIBNMCUQKI-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki < 0.01 nM (pChEMBL 11)
Descriptors: MW 943.01, ALogP 8.75, HBA 10, HBD 2, PSA 140.41, QED 0.11
Compound (SMILES): Cc1c(C(=O)Nc2ccc(Oc3ccnc4c3CNC(=O)N4)c(F)c2)cnn1-c1ccccc1
InChIKey: IEUZMGFDZGWUSU-UHFFFAOYSA-N
Target: Macrophage-stimulating protein receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q04912
Assay: Enzyme Assay: The kinase assay was carried out as 384-well FlashPlate assay. As test plates 384-well streptavidine coated FlashPlate microtitre plates from Perkin Elmer (USA) were used. The components of the kinase reaction were pipetted into the assay plate. 4.5 nM of GST tagged human recombinant RON kinase (Life technologies), 500 nM of biotinylated peptide substrate RDILDREYYSVQQHRH-amide (autophosphorylation site derived peptide substrate, custom-made) and 2 μM of ATP (with 0.5 μCi of <33>P-ATP/well) were incubated in a total volume of 50 μl (50 mM of HEPES, 5 mM of MgCl2, 2 mM of DTT, 0.1% of BSA, 0.01% Igepal®CA630, 1% DMSO, pH 7.5) in the presence or absence of test substance (10 concentrations) at 22 [deg.] C. for 30 min. The reaction was stopped using 50 μl of 200 mM EDTA solution. After incubation for a further 80 min at room temperature, the supernatants were removed by suction, and the wells were washed three times with 100 μl of 0.9% NaCl solution each time.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 458.45, ALogP 4.39, HBA 6, HBD 3, PSA 110.17, QED 0.41
Compound (SMILES): CCn1ccc(Nc2ncc3c(n2)-c2c(nn(C)c2Cc2cccc(-c4cn[nH]c4)c2)CC3)n1
InChIKey: YPFRABRKNDFAON-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: DELFIA Assay: The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five.TM. Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates.10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 .mu.L of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4). 90 uL from each reaction are transferred.
Measurement: IC50 = 0.37 nM (pChEMBL 9.430000305175781)
Descriptors: MW 451.54, ALogP 3.92, HBA 8, HBD 2, PSA 102.13, QED 0.41
Compound (SMILES): CCS(=O)(=O)c1ccc(OCC2CC2(F)F)c(-c2cn(C)c(=O)c3[nH]c(C(=O)NCC(F)(F)F)cc23)c1
InChIKey: GARLVJYFVUERHV-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 8.5 nM (pChEMBL 8.069999694824219)
Descriptors: MW 547.5, ALogP 3.65, HBA 6, HBD 2, PSA 110.26, QED 0.42
Compound (SMILES): C[C@]1(N2Cc3c[nH]nc3C2)CO[C@H](c2cc(F)ccc2F)[C@@H](N)C1
InChIKey: SNWSUIZYGCLPCO-USXIJHARSA-N
Target: Dipeptidyl peptidase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P27487
Assay: Fluorometric Assay: The utility of the compounds in accordance with the present invention as inhibitors of dipeptidyl peptidase-IV enzyme activity may be demonstrated by methodology known in the art. Inhibition constants are determined as follows. A continuous fluorometric assay is employed with the substrate Gly-Pro-AMC, which is cleaved by DPP-4 to release the fluorescent AMC leaving group. The kinetic parameters that describe this reaction are as follows: Km=50 uM; kcat=75 s-1; kcat/Km=1.5x106 M-1s-1. A typical reaction contains approximately 50 pM enzyme, 50 uM Gly-Pro-AMC, and buffer (100 mM HEPES, pH 7.5, 0.1 mg/ml BSA) in a total reaction volume of 100 uL. Liberation of AMC is monitored continuously in a 96-well plate fluorometer using an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Under these conditions, approximately 0.8 uM AMC is produced in 30 minutes at 25 degrees C.
Measurement: IC50 = 34.5 nM (pChEMBL 7.460000038146973)
Descriptors: MW 334.37, ALogP 2.25, HBA 4, HBD 2, PSA 67.17, QED 0.88
Compound (SMILES): COc1cc(F)ccc1Oc1nc2ccccc2nc1C(=O)Nc1ccc(C#N)cn1
InChIKey: RLWUUPLQCJUVER-UHFFFAOYSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q62968
Assay: Electrophysiology Assay: Patch clamp electrophysiology was used to assess the efficacy and selectivity of sodium channel blockers in dorsal root ganglion neurons. Rat neurons were isolated from the dorsal root ganglions and maintained in culture for 2 to 10 days in the presence of NGF (50 ng/ml) (culture media consisted of NeurobasalA supplemented with B27, glutamine and antibiotics). Small diameter neurons (nociceptors, 8-12 um in diameter) were visually identified and probed with fine tip glass electrodes connected to an amplifier (Axon Instruments). The "voltage clamp" mode was used to assess the compound's IC50 holding the cells at -60 mV. In addition, the "current clamp" mode was employed to test the efficacy of the compounds in blocking action potential generation in response to current injections.
Measurement: IC50 = 2400 nM (pChEMBL 5.619999885559082)
Descriptors: MW 415.38, ALogP 4.09, HBA 7, HBD 1, PSA 110.02, QED 0.52
Compound (SMILES): CC(C)(C(=O)N1CCCCC1)c1ccc2c(c1)nc1n2CCNC1Cc1ccc(C(=N)N)cc1
InChIKey: LGVDAFBCFAMLNA-UHFFFAOYSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Inhibition of human thrombin assessed as reduction in release of free nitroaniline using tosyl-glycyl-prolyl-arginine-4-nitranilide acetate as substrate preincubated for 10 mins followed by substrate addition by spectrophotometer
Measurement: IC50 = 1002 nM (pChEMBL 6)
Descriptors: MW 458.61, ALogP 3.5, HBA 5, HBD 3, PSA 100.03, QED 0.4
Compound (SMILES): COC(=O)Cc1cnc(C2=CCN(C(=O)Nc3ccc(C(F)(F)F)cc3)CC2)c(Cl)c1
InChIKey: WRSIAEQZJVHLOB-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Capsaicin-Based Assay: Two days prior to performing this assay, cells are seeded in poly-D-lysine-coated 96-well clear-bottom black plates (50,000 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 mM CaCl2 and 20 mM HEPES, pH 7.4, and cells are loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final). After one hour, the cells are washed twice with 0.2 mL of wash buffer and resuspended in 0.1 mL of wash buffer. The plates are transferred to a FLIPR for assay. 50 μL of test compound diluted with assay buffer (1× Hank's Balanced Salt Solution containing 1 mM CaCl2 and 20 mM HEPES, pH 7.4) are added to the cell plates and incubated for 2 min. The final concentration of the compound is adjusted to range from about 50 picoM to about 3 μM. Human TRPV1 is activated by the addition of 50 μL of capsaicin (400 nM), and the plates are incubated for an additional 3 min.
Measurement: IC50 = 864 nM (pChEMBL 6.059999942779541)
Descriptors: MW 453.85, ALogP 4.79, HBA 4, HBD 1, PSA 71.53, QED 0.68
Compound (SMILES): O=C1CN(C(=O)c2ccc(-c3cc(NC4CC4)n4ncc(/C=C5\NC(=O)NC5=O)c4n3)s2)CCN1
InChIKey: VTFWUEWPJBSBPS-AUWJEWJLSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 > 2500 nM (pChEMBL 5.6020002365112305)
Descriptors: MW 492.52, ALogP 0.78, HBA 9, HBD 4, PSA 149.83, QED 0.3
Compound (SMILES): O=C(N[C@H]1CC[C@@H](O)CC1)c1cc(NC(=O)c2cc(-c3ncccc3F)c(F)cc2Cl)n(-c2ccccc2)n1
InChIKey: WDHUBNPJVCIQEU-MAEOIBBWSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 9.71 nM (pChEMBL 8.010000228881836)
Descriptors: MW 551.98, ALogP 5.15, HBA 6, HBD 3, PSA 109.14, QED 0.31
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CN(C2CCC2)CCO1
InChIKey: YJHVTCUKZLXENT-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 489.4, ALogP 5.32, HBA 7, HBD 1, PSA 68.74, QED 0.48
Compound (SMILES): O=C(Nc1ccc(C2CNCCO2)cc1)c1ccn(-c2ccccc2)n1
InChIKey: HGERVBWCKKJBMC-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 348.41, ALogP 2.79, HBA 5, HBD 2, PSA 68.18, QED 0.76
Compound (SMILES): C.COCCCN1CCN(C(=O)N2Cc3c(NC(=O)c4ccccn4)n[nH]c3C2(C)C)[C@@H](C)C1
InChIKey: SRDPXMVCEWFQBD-NTISSMGPSA-N
Target: Protein kinase C beta type (SINGLE PROTEIN), Homo sapiens
UniProt: P05771
Assay: Inhibition Assay: Protein Kinase C beta 2 (PKC beta II) catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the PKC Pseudosubstrate peptide (A->S, RFARKGSLRQKNV). This transfer is coupled to the oxidation of p-NADH through the activities of Pyruvate Kinase (PK) and Lactate Dehydrogenase (LDH). (3-NADH conversion to NAD+ is monitored by the decrease in absorbance at 340 nm (e=6.22 cm-1 mM-1) using a Molecular Devices SPECTRA max PLUS spectrophotometer.A typical assay was carried out on a 96-well, clear microtiter plate in a Molecular Devices spectrophotometer for 20 minutes at 30 C. in 0.1 mL of assay buffer containing 50 mM HEPES, pH 7.4, 5 nM PKC, 23 units of pyruvate kinase, 33 units of lactate dehydrogenase, 0.15 mM peptide, 0.1 mM ATP, 1 mM DTT, 4 mM PEP, 8 mM MgCl2, 0.3 mM NADH, 60 mM CaCl2, 10 mg/mL PS, 50 ng/mL PMA, 7.5% DMSO and from about 10,000 nM to 0.169 nM compound inhibitor.
Measurement: Ki = 23.8 nM (pChEMBL 7.619999885559082)
Descriptors: MW 471.61
Compound (SMILES): Cn1ncc(C(=O)N2CCC2)c1C(=O)Nc1ccc2nc(-c3cccnc3)nn2c1
InChIKey: RDMCXMFACXWRIT-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 ul contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 ul of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate.
Measurement: IC50 = 43.6 nM (pChEMBL 7.360000133514404)
Descriptors: MW 402.42, ALogP 1.62, HBA 8, HBD 1, PSA 110.31, QED 0.56
Compound (SMILES): N=C(N)NCCCC[C@H]1C(=O)N(CCc2ccc3ccccc3c2)C[C@H]2SC[C@@H](CCCNC(=N)N)C(=O)N21
InChIKey: UEMPXCVRPBVHGP-TZRRMPRUSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Antagonist activity at wild-type human CXCR4 expressed in COS7 cells co-expressing chimeric galphai assessed as inhibition of CXCL12-induced [3H]inositol-phosphate accumulation measured after 90 mins in presence of Myo-[2-3H]-inositol by scintillation proximity assay
Measurement: EC50 < 100000 nM (pChEMBL 4)
Descriptors: MW 552.75, ALogP 2.03, HBA 5, HBD 6, PSA 164.42, QED 0.13
Compound (SMILES): CC(Nc1nc(NC2CC2)n2ncc(/C=C3\NC(=O)NC3=O)c2n1)c1ccc(-n2ccnc2)cc1
InChIKey: YEOHRZQCEKTFLK-ZDLGFXPLSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 470.5, ALogP 2.24, HBA 10, HBD 4, PSA 143.16, QED 0.24
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCN(Cc2ccnc3[nH]ccc23)CC1
InChIKey: JYHQHPRVUSHKTH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 472.53, ALogP 3.65, HBA 7, HBD 3, PSA 128.65, QED 0.38
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](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: YRIJCQHVMRCARX-VEZNLBEOSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 2420.78
Compound (SMILES): Cc1cc(Nc2cc(C(F)(F)F)ccn2)nc(-c2ccc(NC3CCN(C(=O)c4cccnc4)CC3)nc2)c1
InChIKey: CZPHCJNVDWZGBK-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 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x 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.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 533.56, ALogP 5.72, HBA 7, HBD 2, PSA 95.93, QED 0.33
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(c3)-c3[nH]c5ccccc5c3C(c3ccccc3)O4)cc12
InChIKey: CQGBHDJQXPWBCK-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
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 = 92 nM (pChEMBL 7.039999961853027)
Descriptors: MW 671.75, ALogP 8.29, HBA 5, HBD 2, PSA 104.64, QED 0.19
Compound (SMILES): Clc1ccccc1Nc1nc(Nc2cc(C3CC3)n[nH]2)ncc1Br
InChIKey: YKLIHMPWTZLLMN-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 405.69, ALogP 4.98, HBA 5, HBD 3, PSA 78.52, QED 0.56
Compound (SMILES): CC(C)(NC(=O)c1cccc(-c2cccc(Cl)c2)n1)c1ncco1
InChIKey: MQYFOZSWVASOAR-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.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 99.6 nM (pChEMBL 7)
Descriptors: MW 341.8, ALogP 4.06, HBA 4, HBD 1, PSA 68.02, QED 0.78
Compound (SMILES): COc1ncccc1NC(=O)N1CCC(N2CC(CC#N)(n3cc(-c4ccnc5[nH]ccc45)cn3)C2)CC1
InChIKey: NDQXOJDSSACRIG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 511.59, ALogP 3.45, HBA 8, HBD 2, PSA 127.99, QED 0.41
Compound (SMILES): Cc1ccc(S(=O)(=O)c2ccc(NC(=O)/C=C/c3cccnc3)cc2)cc1Cl
InChIKey: YYAWAUZYFOVHQJ-VZUCSPMQSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 8.6 nM (pChEMBL 8.069999694824219)
Descriptors: MW 412.9, ALogP 4.53, HBA 4, HBD 1, PSA 76.13, QED 0.62
Compound (SMILES): O=C(Nc1ccc(C2=CCN(C(=O)c3ccsc3)CC2)cc1)N1Cc2ccccc2C1
InChIKey: BXRUTJDDDOXRTK-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 5.71 nM (pChEMBL 8.239999771118164)
Descriptors: MW 429.55, ALogP 5.23, HBA 3, HBD 1, PSA 52.65, QED 0.62
Compound (SMILES): COc1cc2c(Oc3ccc(NC(=O)c4cn(-c5ccccc5OC)cn4)cc3F)ccnc2cc1OCCCN1CCN(C)CC1
InChIKey: SJSWLZFLXRBNSJ-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Inhibition of c-KIT (unknown origin)
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 640.72, ALogP 5.64, HBA 10, HBD 1, PSA 103.21, QED 0.17
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3cccc(-c4ccc(F)cc4)c3)n2)CC1
InChIKey: ACBAWUXLCITUCM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 518.59, ALogP 5.17, HBA 7, HBD 2, PSA 126.27, QED 0.46
Compound (SMILES): Cc1ccnc(Oc2ccc(-c3cc4c(cc3F)ncc3nc(C)n(C5CCN(C(=O)CO)CC5)c34)c(F)c2)n1
InChIKey: LJLOZWJIGDOOIM-UHFFFAOYSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02750
Assay: Radioactive Binding Assay: The radioactive filter binding assay is standardized using recombinant human activated BRAF (V599E) kinase (Cat No. 14-557) and kinase dead MEK1 (K97R) (Cat No. 14-737) procured from Upstate. The incorporation of 32P into MEK1 (K97R) by BRAF (V599E) is measured with final assay buffer conditions of 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM DTT, 100 mM sucrose, 100 μM sodium orthovanadate, 5 μM ATP and 2 μCi [γ 32P] ATP and 500 mg MEK1 Kinase dead substrate. The enzymatic reaction is stopped after 120 minutes with 8N HCl (hydrochloric acid) and 1 mM ATP. The solution is spotted on P81 filter paper and washed 4 times with 0.75% orthophosphoric acid and lastly with acetone. The dried P81 filter papers are read in a Micro-beta Trilux scintillation counter. The final concentration of DMSO is 1% in the assay. Compounds are screened at 10 μM concentration with pre-incubation of the enzymes in the presence of test compound for 45 minutes. Compounds of the invention were found to be inactive in this assy, e.g. ex. 33 (13% inhibition at 10 uM), ex. 1A (0% inhibition at 10 uM).
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 544.56, ALogP 4.88, HBA 8, HBD 1, PSA 106.26, QED 0.34
Compound (SMILES): Fc1cc(CN2CCC(c3nnn4cnc5[nH]ccc5c34)CC2)ccc1C(F)(F)F
InChIKey: DQTMKPXYLBIZJN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 3.1 nM (pChEMBL 8.510000228881836)
Descriptors: MW 418.4, ALogP 4.14, HBA 5, HBD 1, PSA 62.11, QED 0.51
Compound (SMILES): COc1ccc(CCO[C@@H]2CCCC[C@H]2N2CC[C@H](O)C2)cc1OC.Cl
InChIKey: JMHYCBFEEFHTMK-QQBJDQAASA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: Inhibition of human H1 Na+ channel expressed in HEK cells by patch clamp method
Measurement: IC50 = 25400 nM (pChEMBL 4.590000152587891)
Descriptors: MW 349.47, ALogP 2.64, HBA 5, HBD 1, PSA 51.16, QED 0.82
Compound (SMILES): CSc1nc2c(c(Nc3ccc(C(F)(F)F)cc3Cl)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: PFWKPJYQOCYCOU-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 533.93, ALogP 6.63, HBA 6, HBD 1, PSA 53.94, QED 0.23