lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(c1cc(Cl)ccc1-n1nccn1)N1CCOC[C@H]1Cc1cccc(-n2nccn2)c1
InChIKey: CLOOLVHJLDFMDV-LJQANCHMSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: OX Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 ug/mL G418, 100 U/mL penicillin, 100 ug/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES.
Measurement: IC50 = 199 nM (pChEMBL 6.699999809265137)
Descriptors: MW 449.9, ALogP 2.59, HBA 8, HBD 0, PSA 90.96, QED 0.46
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCC(N2CCC(F)(F)CC2)CC1
InChIKey: GFWDZFDMALWWEW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 460.5, ALogP 4.15, HBA 6, HBD 2, PSA 99.97, QED 0.67
Compound (SMILES): O=C(Nc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3ccccc3)ccc2[nH]1)OCC1CCNCC1
InChIKey: SNFDUCBASQKRTG-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 511.58, ALogP 3.96, HBA 6, HBD 4, PSA 119.58, QED 0.31
Compound (SMILES): C/C(=C\c1cc(F)c(Oc2ccc(S(=O)(=O)NCCOCCOCCOCCNC(=O)OCCOCCOCCOC(=O)NCCOCCOCCOCCNS(=O)(=O)c3ccc(Oc4c(F)cc(/C=C(\C)C(=O)N=C(N)N)cc4F)cc3)cc2)c(F)c1)C(=O)N=C(N)N
InChIKey: QYSJKMSEBSSWTD-CFTRLRGZSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 19.95 nM (pChEMBL 7.699999809265137)
Descriptors: MW 1373.42
Compound (SMILES): Cc1ccc(N(C(=O)c2cc(-c3cc(Cl)ccc3C(=O)N3Cc4ccccc4C[C@H]3CN3CCOCC3)n3c2COCC3)c2ccc(O)cc2)cc1F
InChIKey: VLYLIWNHYCPHAP-XIFFEERXSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 735.26, ALogP 7.37, HBA 7, HBD 1, PSA 87.48, QED 0.19
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CCC(NCCc3ccc(O)cc3)CC2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: ZECMVVWMZPLURH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.15 nM (pChEMBL 8.9399995803833)
Descriptors: MW 665.1, ALogP 4.63, HBA 10, HBD 3, PSA 138.91, QED 0.19
Compound (SMILES): CCN1CCCCC1c1nc(COc2cc3ncnc(Nc4ccc(Cl)c(Cl)c4)c3cc2OC)no1
InChIKey: VYVLFCZOKFCBJQ-UHFFFAOYSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 529.43, ALogP 6.2, HBA 9, HBD 1, PSA 98.43, QED 0.29
Compound (SMILES): CN1CCN(C(=O)Cn2cc(NC(=O)c3cnn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)C[C@H]1CO
InChIKey: VZCCQRUTYQLKDJ-INIZCTEOSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.08 nM (pChEMBL 8.970000267028809)
Descriptors: MW 574.98, ALogP 2.23, HBA 10, HBD 2, PSA 130.12, QED 0.33
Compound (SMILES): COc1cc(OC)cc(N(CC(=O)Nc2cc(C)ccn2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: SSNCGXCONZGQSY-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 509.57, ALogP 4.53, HBA 9, HBD 1, PSA 107.29, QED 0.33
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3cnc4c(c3)CNC4)n2)cnc1N1CC[C@H](F)C1
InChIKey: ZODAGARETRLNST-INIZCTEOSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 402.44, ALogP 2.7, HBA 8, HBD 2, PSA 102.65, QED 0.69
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(NC(=O)CCCCCCC(=O)NO)cc3)cc12)c1ccccc1
InChIKey: PPEIWLKYAPFJJV-LJQANCHMSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of N-terminal GST-tagged human HER2 (Lys676 to Val1255 residues) expressed in baculovirus expression system using biotinylated FLT3 at Tyr589 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 500.6, ALogP 5.58, HBA 6, HBD 5, PSA 132.03, QED 0.1
Compound (SMILES): CCc1ncc(C(CNC(=O)c2ccccc2Cl)N2CCC(F)(F)CC2)cn1
InChIKey: HYESXFVAJGNDNM-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 408.88, ALogP 3.89, HBA 4, HBD 1, PSA 58.12, QED 0.79
Compound (SMILES): O=C(Nc1ccc(Cl)c(S(=O)(=O)C2CNC2)c1O)Nc1cccc(F)c1Cl
InChIKey: MHTGRXKQMVYVCG-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25025
Assay: Displacement of [125I]IL8 from recombinant human CXCR2 expressed in CHO cell membranes measured after 1 hr by TopCount scintillation counting method
Measurement: IC50 < 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 434.28, ALogP 3.23, HBA 5, HBD 4, PSA 107.53, QED 0.55
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1NC1CCCNC1=O
InChIKey: DXEUSDFBCOEFHQ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The final buffer conditions were as follows: 60 mM Hepes, pH 7.0, 0.05% BSA, 2 mM DTT. Incubations were carried out at RT (22° C.) for 2 h for Pim-1, 1 hour and 30 min for Pim-3, and 45 min for Pim-2. The reaction was stopped by the addition of 3 mM EDTA, and fluorescence was measured by an HTRF® Rubystar microplate reader.
Measurement: IC50 = 85.7 nM (pChEMBL 7.070000171661377)
Descriptors: MW 390.45, ALogP 1.91, HBA 5, HBD 4, PSA 111.8, QED 0.55
Compound (SMILES): COc1cn(C(CC2(C(F)(F)F)CC2)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C#N
InChIKey: PNSIVGGUCOLACI-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 559.93, ALogP 5.66, HBA 6, HBD 2, PSA 121.42, QED 0.37
Compound (SMILES): O=C(Nc1ccc(Oc2ccnc(NC(=O)C3CC3)c2)nc1)Nc1ccc(Cl)c(C(F)(F)F)c1
InChIKey: LDYYUUDPUXWYDF-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition Assay: [Experimental method]: Use ADP-GLO™ assay kit from Promega to evaluate the inhibitory activity of compounds at B-Raf. [Instrument]: PerKinElmer's ENVISION plate reader. [Material]: Optiplate-384 well plate (PerKinElmer), kinase buffer (50 mM Hepes pH7.5, 1 mM EGTA, 2 mM DTT, 10 mM Mg2+, 0.05% BSA), B-Raf kinase (Millipore, catalogue #14-530-K), GST-MEK1 substrate (Carna, catalogue #07-141-10), Super pure ATP (Promega), ADP-GLO™ assay kit (Promega, catalogue # V9102), DMSO (Sigma, catalogue #34869), purified water (Millipore, type: Milli-Q). [Study conditions]: Mix B-Raf kinase (final concentration: 5 nM) and compound (DMSO final concentration: 0.25%) and preincubate at 30° C. for 20 min, then add ATP (final concentration: 6 μM) and reaction substrate (final concentration: 30 nM), React for 2 hrs at 30° C. After reaction, add ADP-GLO™ reagent, react for 40 min at room temperature, then add ADP-GLO™ detecting agent, react for 30 min at room temperature. After that, use ENVSION to measure the fluorescence.
Measurement: IC50 = 19.7 nM (pChEMBL 7.710000038146973)
Descriptors: MW 491.86, ALogP 5.93, HBA 5, HBD 3, PSA 105.24, QED 0.4
Compound (SMILES): Cc1cc(Cl)cnc1C(=S)Nc1ccc(F)c([C@]2(CF)C[C@@H](C(F)(F)F)OC(N)=N2)c1
InChIKey: KWXZSPGMEBATPQ-KBXCAEBGSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 909 nM (pChEMBL 6.039999961853027)
Descriptors: MW 478.87, ALogP 4.8, HBA 5, HBD 2, PSA 72.53, QED 0.49
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NCc1ccc(C(=O)C(=O)CNC(=O)CN2CCN(CC(=O)O)CCN(CC(=O)O)CCN(CC(=O)O)CC2)cc1)C(C)C)C(N)=O
InChIKey: IFWINQSJXWIZTR-RHYSEJEISA-N
Target: Neuromedin-B receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P24053
Assay: Displacement of [125I]-NMB from NMB receptor in rat C6 cells
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 1501.73
Compound (SMILES): CN(C)Cc1ccc(CSCCNc2nnc(-c3ccccc3)c(-c3ccccc3)c2[N+](=O)[O-])o1
InChIKey: WUSIWPKVWUINMQ-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of equine serum BuChE using butyrylthiocholine as substrate preincubated for 8 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 7050 nM (pChEMBL 5.150000095367432)
Descriptors: MW 489.6, ALogP 5.72, HBA 8, HBD 1, PSA 97.33, QED 0.16
Compound (SMILES): COc1cc(C(=O)NCc2cccc(C(F)(F)F)c2)ccc1-c1cn[nH]c1
InChIKey: LBESRPHKXLREQJ-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 11.5 nM (pChEMBL 7.940000057220459)
Descriptors: MW 375.35, ALogP 4.03, HBA 3, HBD 2, PSA 67.01, QED 0.71
Compound (SMILES): O=C(NO)c1cnc(NC2(c3ccccc3)CCN(C(=S)Nc3ccccc3)CC2)nc1
InChIKey: YTAPUJWMLRAFQT-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: HDAC Enzyme Assay: The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM TCEP) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 uM final concentration were diluted in assay buffer at 6 fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The final substrate concentrations used were 16 uM (HDAC1), 10 uM (HDAC2), 17 uM (HDAC3) and 14 uM (HDAC6). Five ul of compounds and 20 ul of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five u1 of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated.
Measurement: IC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 448.55, ALogP 3.4, HBA 6, HBD 4, PSA 102.41, QED 0.27
Compound (SMILES): Cc1ncc(C(CNC(=O)C2COc3ncccc32)N2CCOCC2)cn1
InChIKey: VUNGWDMBWWWJRH-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: YO-PRO Assay: HEK cells stably expressing P2X7 were used to screen drugs using the YO-PRO1 assay. Cells were grown to confluency in adherent culture at 37° C. in a humidified 5% CO2 incubator (split ⅕ every 3-4 days with DMEM, 10% FCS, 1% Penicillin/Streptomycin, 250 μg/ml Geneticin). Adherent cells were detached by incubation with Trypsine (1 ml per 165 cm2 dish) for 2 minutes, then washed off with 10 ml PBS (without Mg2+ and Ca2+), and resuspended in DMEM, 10% FCS, 1% Penicillin/Streptomycin, no Geneticin. 10,000 cells per well (48 hours before the assay) or 25,000 cells per well (Vi-cell XR (Beckman Coulter) (24 hours before the assay) in 50 μl full medium were seeded on 384-well black-wall, clear bottom plates, that were coated before with 10 μl per well Poly-L-Lysine, incubated for 30-60 minutes at 37° C. and washed once with PBS. Medium was removed from cells and 50 μl of assay buffer containing 0.5 μM YO-PRO-1 was added into the wells. Solutions of antagonist compounds were prepared by serial dilutions of a 10 mM DMSO solution of the antagonist into PBS using a BioMek (Beckman Coulter). Each concentration was performed in duplicate. For IC50 measurements 10 concentration points were measured (10 μM being the highest concentration followed by 9 serial dilution steps ⅓). The cells were incubated with the antagonists of the present invention together with ATP at a final concentration of 250 μM for 90 minutes. During this time period, four time points were taken. Each time point comprised the average of several measurements made within a few seconds. Fluorescence was measured in the FLIPR tetra (Molecular Devices) using the filters appropriate for YO-PRO-1 fluorescence (excitation485/20, emission 530/25).
Measurement: IC50 = 225 nM (pChEMBL 6.650000095367432)
Descriptors: MW 369.43, ALogP 0.85, HBA 7, HBD 1, PSA 89.47, QED 0.84
Compound (SMILES): NC(=O)[C@@H](NC(=O)c1ccc(C2CC2)c(OCC(F)(F)F)n1)c1ccccc1
InChIKey: ONSVWVNIJRHKCX-HNNXBMFYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 393.37, ALogP 2.86, HBA 4, HBD 2, PSA 94.31, QED 0.76
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCCCN2CCCC2)c([N+](=O)[O-])c1)c1ccc(N2CCN(Cc3cc(OCCN4CCOCC4)ccc3-c3ccc(Cl)cc3)CC2)cc1Oc1ccc2[nH]ccc2c1
InChIKey: UDBXCLFGQRGCBQ-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 61.86 nM (pChEMBL 7.210000038146973)
Descriptors: MW 977.58, ALogP 8.24, HBA 13, HBD 3, PSA 174.85, QED 0.04
Compound (SMILES): CC(C)Oc1ccccc1N1CCCN(CCCCOc2ccc3c(c2)NC(=O)CC3)CC1
InChIKey: RJQFZMPQCFFXTK-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Beta-Arrestin Recruitment (Tango) Assay: Recruitment of β-arrestin to agonist-stimulated D2 receptors was performed using a previously described Tango-type assay (Barnea et al., Proc. Natl. Acad. Sci. USA 105:64 (2008)). Briefly, HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase were plated in 15-cm dishes in DMEM containing 10% fetal bovine serum and transfected (via calcium phosphate) with 20 μg of a D2V2-TCS-tTA construct (Barnea et al., Proc. Natl. Acad. Sci. USA 105:64 (2008)). The next day, cells were plated in white, clear-bottom, 384-well plates (Greiner, 10,000 cells/well, 50 μl/well) in DMEM containing 1% dialyzed fetal bovine serum. The following day, the cells were challenged with 10 μl/well of reference agonist (6 μM) or D2 test ligand (6 μM)±reference agonist prepared in HBS, 20 mM HEPES, pH 7.4, 18% DMSO (final ligand concentrations were 1 μM, final DMSO concentration was 3%).
Measurement: EC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 451.61, ALogP 4.73, HBA 5, HBD 1, PSA 54.04, QED 0.56
Compound (SMILES): CCc1cccnc1-n1nnc2c1CCN(C(=O)c1cccc(C(F)(F)F)c1Cl)C2C
InChIKey: RWZPODDHYIFJQX-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 22.3 nM (pChEMBL 7.650000095367432)
Descriptors: MW 449.86, ALogP 4.66, HBA 5, HBD 0, PSA 63.91, QED 0.58
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3cccc(-c4noc(C5CC5)n4)c3)cc2Cl)CS1(=O)=O
InChIKey: NZQWPNSLEOZOGQ-FQEVSTJZSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: HTRF FRET Assay: Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies).
Measurement: Ki = 0.24 nM (pChEMBL 9.619999885559082)
Descriptors: MW 478, ALogP 4.01, HBA 7, HBD 2, PSA 112.18, QED 0.59
Compound (SMILES): O=C(O)C(F)(F)F.O=c1[nH]nc([C@]2(Cc3cccc(Nc4cc[nH]n4)n3)CC[C@@H]([S+]([O-])c3cccc(Cl)c3Cl)CC2)o1
InChIKey: KRVQZGWRVCEHPX-RDEZRPTCSA-N
Target: Aurora kinase B/Inner centromere protein (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9NQS7
Assay: Inhibition of human full-length N-terminal GST-tagged Aurora B/His-tagged INCENP (803 to 916 residues) using kemptide as substrate and [gamma33P]ATP after 45 mins by liquid scintillation counting method
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 533.44, ALogP 4.76, HBA 7, HBD 3, PSA 135.55, QED 0.29
Compound (SMILES): CN1CCN(c2ccc(-c3ccc4[nH]c5nccc(Cl)c5c4c3)cc2)CC1
InChIKey: ZUQQEJBCJAGIIZ-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 376.89, ALogP 4.79, HBA 3, HBD 1, PSA 35.16, QED 0.55
Compound (SMILES): COc1ccc2c(c1)CC1(CCN([C@H]3CCN(Cc4nc5c(c(=O)[nH]4)COCC5)C3=O)CC1)C2=O
InChIKey: FMURKSCWRQGCFO-NRFANRHFSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 uL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 uM, 20 nM of purified enzyme, and 250 uM of p-NAD+ in the 1x Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 uL of 5x quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 uL of the 5x quenching solution per well was added prior to the addition of p-NAD+. The % Inhibition was calculated as: (Control-Sample)/(Control-Background)*100. "Control" is the average value of 8 wells without compound; and "Background" is the average of 8 wells mixed with 5x quenching solution measured prior to initiation of the reaction.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 478.55, ALogP 1.47, HBA 7, HBD 1, PSA 104.83, QED 0.71
Compound (SMILES): CC(c1ccccc1-c1ccc(Cl)cc1)N1CCN(c2ccc(C(=O)NS(=O)(=O)c3ccc(NCC4CCOCC4)c([N+](=O)[O-])c3)c(Oc3ccc4[nH]ccc4c3)c2)CC1
InChIKey: DOIRGIGOPKQZNI-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 849.41, ALogP 9.03, HBA 10, HBD 3, PSA 159.14, QED 0.08
Compound (SMILES): NCCOc1c(-c2ccc(Br)cc2)[nH]c(=O)c2cc(F)ccc12
InChIKey: IZPURWNXWPTMHH-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: Biochemical Assay: The identification of compounds capable of binding several PARP proteins is carried out through a screening method including the steps of  a) providing a reaction mixture containing:  b) comparing the polarization signal generated in the absence of the test compound with the one generated in the presence of different concentrations of the test compound, and  c) evaluating the ability of the test compound to displace the compound of formula (IP) as defined above indicated from a decreased fluorescence polarization level.The polarization signal can be measured, e.g., by a plate reader such as the Saphire2 (Tecan).
Measurement: Kd = 1030 nM (pChEMBL 5.989999771118164)
Descriptors: MW 377.21, ALogP 3.43, HBA 3, HBD 2, PSA 68.11, QED 0.73
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2cc(C#N)ccn2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: WJQLXQXBPJJDTC-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 501.55, ALogP 4.5, HBA 9, HBD 0, PSA 101.98, QED 0.31
Compound (SMILES): Cc1c(-c2c(F)cc(C(N)=O)c3[nH]c4c(c23)CC[C@@H](C(C)(C)O)C4)cccc1-n1c(=O)c2cccc(F)c2n(C)c1=O
InChIKey: ZRYMMWAJAFUANM-MRXNPFEDSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of BTK in human Ramos-B cells assessed as suppression of BCR/anti-IgG-stimulated Ca2+ flux after 1 hr incubation in dark by FLIPR1 assay
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 572.61, ALogP 4.4, HBA 6, HBD 3, PSA 123.11, QED 0.3
Compound (SMILES): O=C(CN1CCC(c2n[nH]c3cc(F)ccc23)CC1)NCc1nc2c(c(=O)[nH]1)COCC2
InChIKey: RZPNNTCBQSMUAY-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H2K2
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 440.48, ALogP 1.35, HBA 6, HBD 3, PSA 116, QED 0.55
Compound (SMILES): O=C(O)/C=C/c1ccc2cccc(N3CCN(CCc4ccc(OCCCN5CCCCCC5)cc4)CC3)c2n1
InChIKey: NFCYQBPNSWBYEF-DTQAZKPQSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: Antagonist activity at human Histamine H3 receptor expressed in CHO cell membranes assessed as inhibition of histamine-induced [35S]GTPgammaS binding after 2 to 6 hrs by scintillation proximity assay
Measurement: Ki = 1.585 nM (pChEMBL 8.800000190734863)
Descriptors: MW 542.72, ALogP 5.34, HBA 6, HBD 1, PSA 69.14, QED 0.26
Compound (SMILES): CC(C)OC(=O)c1ccc(C#Cc2c(N)ncnc2N[C@@H](C)c2nc3cccc(Cl)c3c(=O)n2-c2ccccc2)nc1
InChIKey: DBWKOVCXXRYQHM-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 580.05, ALogP 4.94, HBA 10, HBD 2, PSA 137.91, QED 0.21
Compound (SMILES): CCN1CCCCC1c1nc(COc2cc3ncnc(Nc4ccc(Cl)c(Cl)c4)c3cc2OC)cs1
InChIKey: WVXLPNDNCDLCQW-UHFFFAOYSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 10500 nM (pChEMBL 4.980000019073486)
Descriptors: MW 544.51, ALogP 7.27, HBA 8, HBD 1, PSA 72.4, QED 0.25
Compound (SMILES): CC(=O)N1CCc2c(C(=O)N3CCC(c4cccc(F)c4Cl)CC3)n[nH]c2C1
InChIKey: UZSKQBUZGPQZTH-UHFFFAOYSA-N
Target: Retinol-binding protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P02753
Assay: TR-FRET Assay: TR-FRET assay for retinol-induced RBP4-TTR interaction Binding of a desired RBP4 antagonist displaces retinol and induces hindrance for RBP4-TTR interaction resulting in the decreased FRET signal (FIG. 7). Bacterially expressed MBP-RBP4 and untagged TTR were used in this assay. For the use in the TR-FRET assay the maltose binding protein (MBP)-tagged human RBP4 fragment (amino acids 19-201) was expressed in the Gold(DE3)pLysS E. coli strain (Stratagene) using the pMAL-c4x vector. Following cell lysis, recombinant RBP4 was purified from the soluble fraction using the ACTA FPLC system (GE Healthcare) equipped with the 5-ml the MBP Trap HP column. Human untagged TTR was purchased from Calbiochem. Untagged TTR was labeled directly with Eu3+ Cryptate-NHS using the HTRF Cryptate Labeling kit from CisBio following the manufacturer's recommendations. HTRF assay was performed in white low volume 384 well plates (Greiner-Bio) in a final assay volume of 16 ul per well. The reaction buffer contained 10 mM Tris-HCl pH 7.5, 1 mM DTT, 0.05% NP-40, 0.05% Prionex, 6% glycerol, and 400 mM KF. Each reaction contained 60 nM MBP-RBP4 and 2 nM TTR-Eu along with 26.7 nM of anti-MBP antibody conjugated with d2 (Cisbio). Titration of test compounds in this assay was conducted in the presence of 1 uM retinol. All reactions were assembled in the dark under dim red light and incubated overnight at +4° C. wrapped in aluminum foil. TR-FRET signal was measured in the SpectraMax M5e Multimode Plate Reader (Molecular Device). Fluorescence was excited at 337 nm and two readings per well were taken: Reading 1 for time-gated energy transfer from Eu(K) to d2 (337 nm excitation, 668 nm emission, counting delay 75 microseconds, counting window 100 microseconds) and Reading 2 for Eu(K) time-gated fluorescence (337 nm excitation, 620 nm emission, counting delay 400 microseconds, counting window 400 microseconds).
Measurement: IC50 = 23.73 nM (pChEMBL 7.619999885559082)
Descriptors: MW 404.87, ALogP 3.13, HBA 3, HBD 1, PSA 69.3, QED 0.84
Compound (SMILES): CC1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccc(C#N)cc2NC1=O
InChIKey: BKSCSZWESYMLPD-KKFPZRRJSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: In Vitro Assay: 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 = 27.87 nM (pChEMBL 7.550000190734863)
Descriptors: MW 547.01, ALogP 6.67, HBA 4, HBD 1, PSA 86.09, QED 0.36
Compound (SMILES): CN[C@H](C(=O)N[C@@H]1C(=O)N(Cc2c(OC)ccc3cc(Br)ccc23)c2ccc(C)cc2O[C@H]1C)[C@H](C)O
InChIKey: LKIFTWKEGXUQGG-FRSFCCSCSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 27170 nM (pChEMBL 4.570000171661377)
Descriptors: MW 570.48, ALogP 3.69, HBA 6, HBD 3, PSA 100.13, QED 0.4