lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)[C@@H]1CCN1c1nc2ccccc2n([C@H]2C[C@H]3CCC[C@@H](C2)N3[C@H]2C[C@@H]3CCC[C@@H](C3)C2)c1=O
InChIKey: FQAQAGMTMTXLRQ-OGXPWPJRSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: In Vitro Mu-Opioid Receptor Binding Assay: Radioligand binding assays were conducted using freshly thawed membranes expressing human μ-receptors (Perkin Elmer, Shelton, Conn.). Radioligand dose-displacement binding assays for human μ-opioid receptors used 0.2 nM [3H]-diprenorphine (NEN, Boston, Mass.), with 5-20 mg membrane protein/well in a final volume of 500 μL binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 1-2 hr at about 25 °C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard, Meriden, Conn.) presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Brandel, Gaithersburg, Md.) followed by performing three filtration washes with 500 μL of ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Wallac, Turku, Finland) was added (50 μL/well), and plates were counted using a Packard Top-Count for 1 min/well.
Measurement: Ki = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 490.65, ALogP 4.59, HBA 6, HBD 1, PSA 78.67, QED 0.68
Compound (SMILES): Cc1nc2c(C(=O)O)cc(N3CCOCC3)nn2c1Cc1cccc(C(F)(F)F)c1C
InChIKey: DKTMKUHYROTKCJ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Binding affinity to human PI3Kalpha (108 to 1068 residues) expressed in mammalian expression system by KINOMEscan assay
Measurement: Kd = 1349 nM (pChEMBL 5.869999885559082)
Descriptors: MW 434.42, ALogP 3.49, HBA 6, HBD 1, PSA 79.96, QED 0.68
Compound (SMILES): CN1C(=O)CCC1c1cc2[nH]c(-c3ccc(C(F)(F)F)cn3)cc2cc1Oc1ccc(CCC(=O)O)cc1
InChIKey: WPGQTUWWDVWSBZ-UHFFFAOYSA-N
Target: Hexokinase-4 (SINGLE PROTEIN), Homo sapiens
UniProt: P35557
Assay: Glucokinase-Activating Assay: To test the exemplified compounds, the following assay was employed. Recombinant human liver glucokinase was expressed as a FLAG fusion protein in E. coli, and purified on ANTIFLAG M2 AFFINITY GEL (Sigma). The assay was carried out at 30° C. in a 96-well plate. In the plate was distributed 69 ul each of assay buffer (25 mM Hepes Buffer: pH=7.2, 2 mM MgCl2, 1 mM ATP, 0.5 mM TNAD, 1 mM dithiothreitol), to which was added 1 ul of a DMSO solution of the compound or DMSO as control. Then, 20 ul of pre-ice-cooled enzyme mixture (FLAG-GK, 20 U/ml G6PDH) was distributed thereto, to which was added 10 ul of 25 mM glucose as substrate to initiate the reaction (final glucose concentration=2.5 mM). After starting the reaction, the absorbance at 405 nm was measured every 30 seconds for 10 minutes to evaluate the compound based on the initial increase for 5 minutes. FLAG-GK was added so that the increase of absorbance after 5 minutes fell between 0.05 to 0.1 in the presence of 1% DMSO. The OD values of the respective compounds were measured in the respective concentrations, wherein the OD value of DMSO as control is regarded as 100%. From the OD values at the respective concentrations, Emax (%, 2.5 mM Glu) and EC50 (nM, 2.5 mM Glu) were calculated and used as indicators of the GK activation capability of the compounds. According to the above assay, the GK activation capability of the exemplified compounds of the present invention was determined. The following table shows the results. Where different enantiomers of the same compound were tested, two numbers are provided. Where 3 numbers are provided, for example, Ex. #30, data for the racemic form is also shown.
Measurement: EC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 523.51, ALogP 6.35, HBA 4, HBD 2, PSA 95.52, QED 0.29
Compound (SMILES): O=C(NCCN1CCCCC1)[C@@H]1CC[C@@H]2CN1C(=O)N2OS(=O)(=O)O
InChIKey: SWJSAILFIFXFLK-NEPJUHHUSA-N
Target: Beta-lactamase (SINGLE PROTEIN), Klebsiella pneumoniae
UniProt: Q93LQ9
Assay: Spectrophotometric Assay: Enzyme activities were measured in the presence of the test inhibitor in spectrophotometric assay.
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 376.44, ALogP -0.41, HBA 6, HBD 2, PSA 119.49, QED 0.6
Compound (SMILES): O=C(O)c1c[nH]c2cc(Cl)c(-c3ccc(C4CCCCO4)cc3)cc12
InChIKey: GKPYSXKYDOSNMQ-UHFFFAOYSA-N
Target: AMP-activated protein kinase (AMPK) alpha-1/beta-1/gamma-1 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q13131
Assay: Binding affinity to human BAP-tagged AMPK alpha1/beta1/gamma1 by SPR assay
Measurement: Kd = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 355.82, ALogP 5.43, HBA 2, HBD 2, PSA 62.32, QED 0.66
Compound (SMILES): Cc1ccc2c(NCc3ccc(NC(=O)c4ccc(F)cc4)cc3)nc(NCCc3ccccn3)nc2c1
InChIKey: DZXTZSYPFBOUDK-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf22C11 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 408 nM (pChEMBL 6.389999866485596)
Descriptors: MW 506.59, ALogP 5.99, HBA 6, HBD 3, PSA 91.83, QED 0.23
Compound (SMILES): COc1ccc2cccc(OCC(=O)NC[C@H](O)CN3CCc4ccccc4C3)c2n1
InChIKey: UGLJLGAPPPJZLG-FQEVSTJZSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 101 nM (pChEMBL 7)
Descriptors: MW 421.5, ALogP 2.16, HBA 6, HBD 2, PSA 83.92, QED 0.58
Compound (SMILES): CNCCN1CCC(OCc2cccc(-c3ccccc3)c2)CC1
InChIKey: OLFLGFYCUQONAR-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR22
Assay: Inhibition of PRMT8 (unknown origin) assessed as inhibition of methylation activity measured after 3 hrs using [3H]-SAM by scintillation proximity assay
Measurement: IC50 = 573 nM (pChEMBL 6.239999771118164)
Descriptors: MW 324.47, ALogP 3.55, HBA 3, HBD 1, PSA 24.5, QED 0.84
Compound (SMILES): CN(C)c1nc2c(c(Nc3ccc(Cl)c(Cl)c3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: WRPMGFGYLHQODW-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: Functional Assay: The assay was performed as described below, using HEK293 cells transfected with rat TRPV1 (rTRPV1/HEK293). These cells had a geneticin selection marker and were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, penicillin/streptomycin (50 units/mL), and 500 μg/mL geneticin 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 = 2500 nM (pChEMBL 5.599999904632568)
Descriptors: MW 497.35, ALogP 5.61, HBA 6, HBD 1, PSA 57.18, QED 0.51
Compound (SMILES): O=C(c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1)N1CC[C@@H](F)C1
InChIKey: FWLFRXVPNVBXGR-CQSZACIVSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y9
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.8 (Essen, Ann Arbor, Mich.) were quickly thawed and plated at 22,500 cells/well in growth medium [MEM (Invitrogen #11095) with 10% FBS (Invitrogen #10082), 1 mM sodium pyruvate (Invitrogen, #C11360), 10 units/mL penicillin 10 U/mL streptomycin 29.2 ug/mL glutamine ((PSG 1%, Invitrogen #10378), 400 ug/mL zeocin (Invitrogen #R250) in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 60 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA(Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. The depolarizing agent, veratridine (Sigma-Aldrich, St. Louis, Mo.), was made up at 3Ã¿ concentrations in assay buffer containing 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.).
Measurement: IC50 = 3850 nM (pChEMBL 5.420000076293945)
Descriptors: MW 405.35, ALogP 4.62, HBA 4, HBD 0, PSA 55.32, QED 0.6
Compound (SMILES): COCCOc1cc2ncnc(Nc3ccc(F)c(Cl)c3)c2cc1NC(=O)CCCCCCC(=O)NO
InChIKey: JYHLYHBEBKVBPC-UHFFFAOYSA-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 533.99, ALogP 4.98, HBA 8, HBD 4, PSA 134.7, QED 0.13
Compound (SMILES): CC1(N)CCN(c2cccnc2NC(=O)c2nc(-c3cc(N4CCOCC4)ccc3C(F)(F)F)cnc2N)CC1
InChIKey: WNEWTOQENXHIHL-UHFFFAOYSA-N
Target: Protein kinase C theta type (SINGLE PROTEIN), Homo sapiens
UniProt: Q04759
Assay: In Vitro Inhibition Activity Assay: The compounds of formula I were tested for their activity on different PKC isoforms according to a published method (D. Geiges et al. Biochem. Pharmacol. 1997; 53:865-875) The assay is performed in a 96-well polypropylene microtiterplate (Costar 3794) that has been previously siliconized with Sigmacote (Sigma SL-2). The reaction mixture (50 μL) contains 10 μL of the relevant PKC isozyme together with 25 μL of the PKC inhibitor compound and 15 μL of a mix solution that contains 200 μg/mL protamine sulfate, 10 mM Mg(NO3)2, 10 μM ATP (Boehringer 519987) and 3750 Bq of 33P-ATP (Hartmann Analytic SFC301, 110 TBq/mmol) in 20 mM Tris-buffer pH 7.4+0.1% BSA. Incubation was performed for 15 minutes at 32° C. in a microtiterplate shaking incubator (Biolabo Scientific Instruments). Reaction was stopped by adding 10 μl of 0.5 M Na2EDTA, pH 7.4. 50 μl of mixture are pipetted onto a pre-wetted phosphocellulose paper (Whatmann 3698-915) under gentle pressure.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 556.59, ALogP 3.55, HBA 9, HBD 3, PSA 135.52, QED 0.43
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3C[C@@H]4C[C@H]3CO4)nc(-c3ccc(NC(=O)NC)c(F)c3)c2C1=O
InChIKey: XXTUPRBURGTSLH-GJZGRUSLSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P27986
Assay: Enzyme Assay: The test uses a lucifeurerin/luciferase system to measure the concentration of ATP and its consumption during the enzymatic reaction. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 μl. To 1 μl of inhibitor in 100% DMSO are added (final concentrations) 50 μM of the substrate PIP2 ((L-α-phosphatidyl-D-myoinositol 4,5-bisphosphate, Echelon 117P-4516-0500), 2 μM of ATP and 1.7 μg/ml of PI3Kα, (p110α/p85α, Invitrogen PV4788) in a buffer of Tris/HCl 50 mM pH 7.5, EGTA 1 mM, MgCl2 10 mM, Chaps 0.03%, 1 mM DTT). After 90 minutes, the reaction is quenched by adding 20 μl/well of KinaseGlo reagent (Promega V6713). After 10 minutes in the dark, the luminescence is read on the PHERAStar microplate reader (reading at 0.8 sec/well).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 484.53, ALogP 2.64, HBA 7, HBD 2, PSA 108.92, QED 0.69
Compound (SMILES): Cn1cc([C@@H](NC(=O)c2cc(F)c3cnc(NC4CCOCC4)nc3c2)c2cccc(F)c2)cn1
InChIKey: JPKBESGDPJRCBB-QHCPKHFHSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an N-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 1 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 478.5, ALogP 3.75, HBA 7, HBD 2, PSA 93.96, QED 0.44
Compound (SMILES): CCCN1CC[C@H]2c3cc4ccccc4nc3[C@H](CC)[C@H]3CCC[C@@H]1[C@@H]32
InChIKey: GHQALVWJILAFPS-VCSLONGPSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Displacement of [3H]DAMGO from human MOR expressed in HEK cells after 90 mins by microbeta scintillation counting method
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 348.53, ALogP 5.73, HBA 2, HBD 0, PSA 16.13, QED 0.7
Compound (SMILES): CC(C)(O)C1CN(c2ncc(C(=O)Nc3ccc(OC(F)(F)F)cc3)cc2-c2cccnc2)C1
InChIKey: JBZKUAZNQCNKEA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper ABL : The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 uL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 uM ATP, 4 uM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 uL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 uL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 472.47, ALogP 4.5, HBA 6, HBD 2, PSA 87.58, QED 0.55
Compound (SMILES): O=C(O)c1ccc(Nc2c(/C=C/c3ccc4ccccc4n3)nc3c(N4CCOCC4)ccnn23)cc1
InChIKey: LEUQHAITIWZSID-VAWYXSNFSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 492.54, ALogP 4.73, HBA 8, HBD 2, PSA 104.88, QED 0.35
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(S(=O)(=O)c3cccc(C#N)c3)CC2)C1
InChIKey: LHNVUHXNJWADTE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of human N-terminal His-tagged JAK2 catalytic domain (828 to 1132 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 527.61, ALogP 2.47, HBA 9, HBD 1, PSA 147.59, QED 0.4
Compound (SMILES): O=C(Nc1nc2ccc(C3(O)c4ccccc4C(=O)N3c3cccc(Cl)c3F)cc2[nH]1)OCCN1CCOCC1
InChIKey: SQKADMZFJFLNRD-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 565.99, ALogP 4.09, HBA 7, HBD 3, PSA 120.02, QED 0.32
Compound (SMILES): CC/C=C(\CC)c1cc(-c2c(C)noc2C)cc2nc(C3CC3)[nH]c12
InChIKey: FIDCRXXEFWLWFD-VGOFMYFVSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 Î¼s read window). Data were normalized based on a positive (2 Î¼M I-BET) and negative (DMSO) controls and IC50values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 65.5 nM (pChEMBL 7.179999828338623)
Descriptors: MW 335.45, ALogP 5.92, HBA 3, HBD 1, PSA 54.71, QED 0.63
Compound (SMILES): Cc1ccc2[nH]ccc2c1-c1nc(C(N)=O)c(-c2ccc(N3CCNC(C)(C)C3)cc2)o1
InChIKey: XSLUZOLUJPDNGT-UHFFFAOYSA-N
Target: Aurora kinase B (SINGLE PROTEIN), Homo sapiens
UniProt: Q96GD4
Assay: Inhibition Assay: Kinase assays were performed at Reaction Biology Corp., Malvern, Pa., USA, using the following general procedure:1) Prepare indicated substrate in freshly prepared Base Reaction Buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO).2) Deliver cofactors (1.5 mM CaCl2, 16 ug/mL Calmodulin, 2 mM MnCl2) to the substrate solution above.3) Deliver indicated kinase into the substrate solution and gently mix4) Deliver varying concentrations of test compound in DMSO into the kinase reaction mixture.5) Deliver 33P-ATP (specific activity 0.01 uCi/uL final) into the reaction mixture to initiate the reaction.6) Incubate kinase reaction for 120 min at room temperature.7) Reactions are spotted onto P81 ion exchange filter paper (Whatman #3698-915)8) Unbound phosphate is removed by washing filters extensively in 0.75% phosphoric acid.9) 33P signal was determined using Typhoon phosphorimagers (GE Healthcare).
Measurement: IC50 = 5.9 nM (pChEMBL 8.229999542236328)
Descriptors: MW 429.52, ALogP 4.09, HBA 5, HBD 3, PSA 100.18, QED 0.45
Compound (SMILES): Cc1csc(C(C)(C)NC(=O)c2cnc(C3CC3)c(OCC3CC3)n2)n1
InChIKey: ZXYRRXDYYVFHPC-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively.
Measurement: EC50 = 11.6 nM (pChEMBL 7.940000057220459)
Descriptors: MW 372.49, ALogP 3.57, HBA 6, HBD 1, PSA 77, QED 0.8
Compound (SMILES): COC(=O)Nc1nc2ccc(C3(O)c4ccccc4C(=O)N3Cc3ccccc3)cc2n1C
InChIKey: XYUPUAMHUFESKM-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 >= 10000 nM (pChEMBL 5)
Descriptors: MW 442.48, ALogP 3.6, HBA 6, HBD 2, PSA 96.69, QED 0.5
Compound (SMILES): CN(C)c1cc(Nc2ccccc2)nc(Nc2ccccc2)n1
InChIKey: ULGGLUFGPKJKNL-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Enzyme Inhibition Assay: The phosphorylation of L-Hse was monitored by coupling the formation of ADP with pyruvate kinase and lactate dehydrogenase (PK/LDH). The resulting oxidation of NADH was monitored at 340 nm by using a SpectraMax plate reader in a 96-well format.
Measurement: IC50 > 15000 nM (pChEMBL 4.823999881744385)
Descriptors: MW 305.39, ALogP 4.03, HBA 5, HBD 2, PSA 53.08, QED 0.75
Compound (SMILES): CN(C)C(=O)CNC(=O)c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1
InChIKey: UDJQWHMJZQQMQV-UHFFFAOYSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y9
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.8 (Essen, Ann Arbor, Mich.) were quickly thawed and plated at 22,500 cells/well in growth medium [MEM (Invitrogen #11095) with 10% FBS (Invitrogen #10082), 1 mM sodium pyruvate (Invitrogen, #C11360), 10 units/mL penicillin 10 U/mL streptomycin 29.2 ug/mL glutamine ((PSG 1%, Invitrogen #10378), 400 ug/mL zeocin (Invitrogen #R250) in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 60 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA(Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. The depolarizing agent, veratridine (Sigma-Aldrich, St. Louis, Mo.), was made up at 3Ã¿ concentrations in assay buffer containing 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.).
Measurement: IC50 = 740 nM (pChEMBL 6.130000114440918)
Descriptors: MW 418.38, ALogP 3.26, HBA 5, HBD 1, PSA 84.42, QED 0.69
Compound (SMILES): CN[C@@H](C)C(=O)Nc1ccc2c(n1)CN(C1CCN(C(C)=O)CC1)C(=O)N2c1cccc(Cl)c1
InChIKey: JZAPJOLDXTWWMM-HNNXBMFYSA-N
Target: Baculoviral IAP repeat-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13490
Assay: In Vitro Competitive Displacement Binding Assay: Modified SMAC peptides and compounds were tested for their ability to displace the fluorescent tracer from either XIAP, cIAP-1 or cIAP-2. BIR3 domains of cIAP-1, cIAP-2 and XIAP were incubated with test compounds or SMAC based peptides and their respective peptide probes (Peptide Protein Research) in assay buffer (50 mM Hepes pH 7.5, 0.025% Tween-20, 0.01% BSA, and 1 mM DTT). Positive controls consisted of BIR3 proteins and tracer (no inhibition) and negative controls contained tracer only (100% inhibition). The samples were incubated at room temperature for 1 hr (XIAP and cIAP-2) or 3 hrs (cIAP-1) prior to being read in the BMG Pherastar in Fluorescence Polarization mode (FP 485 nm, 520 nm, 520 nm).
Measurement: IC50 = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 484.99, ALogP 3.37, HBA 5, HBD 2, PSA 97.88, QED 0.68
Compound (SMILES): N#Cc1c(N)ncnc1N1CCC(c2nc(-c3ccsc3)cn2CCN2CCC2)CC1
InChIKey: LYRHYDHWKBCSKX-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 434.57, ALogP 2.95, HBA 9, HBD 1, PSA 99.89, QED 0.64
Compound (SMILES): O=C(NO)c1ccc(Cn2cccn2)cc1
InChIKey: DUGDSZFLAKVUEC-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: HDAC Activity Assay: HDAC assay is performed using fluorescently-labeled acetylated substrate, which comprises an acetylated lysine side chain. After incubation with HDAC, deacetylation of the substrate sensitizes the substrate such that, in a second step, treatment with the detection enzyme produces a fluorophore. HDACs 1 and 6 were expressed as full length fusion proteins. Purified proteins were incubated with 50 μM fluorescently-labeled acetylated peptide substrate and test compound for 2 hours at RT in HDAC assay buffer containing 50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% DMSO, and 1% BSA. Reactions were terminated by the addition of the Developer after 2 hours, and the development of fluorescence signal, which was relative to the amount of deacetylated peptide, was monitored by time-course measurement of EnVision (PerkinElmer). The HDAC activity was estimated from the slope of time-course measurement of the fluorescence intensity.
Measurement: IC50 = 10.9 nM (pChEMBL 7.960000038146973)
Descriptors: MW 217.23, ALogP 1.05, HBA 4, HBD 2, PSA 67.15, QED 0.6
Compound (SMILES): Cc1cncc(-c2cc3c(cnn3-c3cccc(N4CCNCC4)n3)nn2)n1
InChIKey: SJECHZVGJJSCJC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.71 nM (pChEMBL 9.149999618530273)
Descriptors: MW 373.42, ALogP 1.39, HBA 9, HBD 1, PSA 97.54, QED 0.58
Compound (SMILES): CCOC(=O)CNC1CCN(C(=O)Cn2cc(NC(=O)c3cnn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)CC1
InChIKey: UNLQAMCMAYMLED-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 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 631.04, ALogP 3.24, HBA 11, HBD 2, PSA 144.98, QED 0.24
Compound (SMILES): c1cc(Nc2ccon2)nc(Nc2ccc(N3CCOCC3)cc2)n1
InChIKey: XDBOLBOLMUGCDZ-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 >= 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 338.37, ALogP 2.79, HBA 8, HBD 2, PSA 88.34, QED 0.73
Compound (SMILES): CCOc1cnc(-c2c[nH]c3ccc(-c4nnc(N)o4)cc23)cn1
InChIKey: VIJAADBVHYURKM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: 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 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 324.24 nM (pChEMBL 6.489999771118164)
Descriptors: MW 322.33, ALogP 2.66, HBA 7, HBD 2, PSA 115.74, QED 0.59
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)cc1)N1CC=C(c2ncc(CO)cc2Cl)CC1
InChIKey: HERAWSIIQJRSRE-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 ("wash buffer"), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 ("assay buffer"). Plates are then transferred to a FLIPR for assay. The test compound is diluted in assay buffer, and 50 μL of the resultant solution is added to the cell plates and the solution is monitored for two minutes. The final concentration of the test compound is adjusted to range from about 50 picoM to about 3 μM. Agonist buffer (wash buffer titrated with 1N HCl to provide a solution having a pH of 5.5 when mixed 1:1 with assay buffer) (0.1 mL) is then added to each well, and the plates are incubated for 1 additional minute.
Measurement: IC50 = 41.7 nM (pChEMBL 7.380000114440918)
Descriptors: MW 411.81, ALogP 4.57, HBA 3, HBD 2, PSA 65.46, QED 0.78
Compound (SMILES): N#CCn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CCCNCC3)n2)cn1
InChIKey: NDXJCTYWGYPGPM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 1.49 nM (pChEMBL 8.829999923706055)
Descriptors: MW 399.46, ALogP 2, HBA 9, HBD 1, PSA 100.48, QED 0.56
Compound (SMILES): COc1nccnc1Nc1ccc(F)c([C@]2(C(F)F)COCC(N)=N2)c1
InChIKey: FOXRQWMEUJAVSI-INIZCTEOSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: BACE-2 Inhibition Assay: Recombinant BACE-2 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 1 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Activity was measured using a final concentration of 3 uM of the fluorescence-quenched substrate Q-C(HSO3)-Ile-Asp-Leu-Ala-Val-Leu-Asp-HN-CH2-CH2-Mca, where Q=2-nitro-5-amino benzoic acid and Mca=7-methoxy-4-coumarinyl acetic acid. Catalytic turnover was monitored in a Spectramax Gemini fluorescence plate reader (Molecular Devices) in black 96-well microplates using excitation/emission wavelength of 325 nm and 400 nm, respectively. Fluorescence increase was followed for 15 min, in 1 minute's intervals.
Measurement: IC50 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 367.33, ALogP 2.22, HBA 7, HBD 2, PSA 94.65, QED 0.84
Compound (SMILES): O=C(N[C@H](CO)c1ccccn1)c1ccc(NC(=O)N2Cc3ccc(F)cc3C2)cc1
InChIKey: WLUODVVHPABVSO-OAQYLSRUSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2: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-1287128.0. 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 = 10.4 nM (pChEMBL 7.980000019073486)
Descriptors: MW 420.44, ALogP 3.23, HBA 4, HBD 3, PSA 94.56, QED 0.59
Compound (SMILES): CCCc1nn(C)c2c(=O)[nH]c(-c3cc(S(=O)(=O)N4CCN(c5ncc(C(=O)NO)cn5)CC4)ccc3OCC)nc12
InChIKey: WNMRXNCXRGFIHO-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition of full length recombinant human C-terminal His/Flag-tagged HDAC1 expressed in baculovirus infected Sf9 cells using fluorogenic HDAC substrate after 30 mins by fluorescence analysis
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 597.66, ALogP 1.09, HBA 12, HBD 3, PSA 188.53, QED 0.19
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)CCCCC(=O)N2C[C@H](NC(=O)[C@H](C)NC)C(=O)N(Cc3c(OC)ccc4cc(Br)ccc34)c3ccccc32)c2ccccc2N(Cc2c(OC)ccc3cc(Br)ccc23)C1=O
InChIKey: XEUPNOJSSNQTHX-DYGKRUFJSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6× 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-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, 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.
Measurement: IC50 = 5959 nM (pChEMBL 5.21999979019165)
Descriptors: MW 1132.95
Compound (SMILES): Cc1nn(-c2ccccc2)cc1CN1CCN(C(=O)OC(C(F)(F)F)C(F)(F)F)CC1
InChIKey: QIMGORSOWHPBSK-UHFFFAOYSA-N
Target: Platelet-activating factor acetylhydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q13093
Assay: Inhibition of PLA2G7 derived from human PC3 cell lysates preincubated for 30 mins followed by JW912 addition after 30 mins by gel-based ABPP assay
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 450.38, ALogP 3.93, HBA 5, HBD 0, PSA 50.6, QED 0.66
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3ccco3)ccc2[nH]1
InChIKey: JXJRYRLOPAJKKZ-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 418.41, ALogP 3.18, HBA 6, HBD 3, PSA 120.69, QED 0.47
Compound (SMILES): c1cncc(-c2nc(NC3CC4CCC3C4)c3ccccc3n2)c1
InChIKey: FPKKVYLMXWIWLV-UHFFFAOYSA-N
Target: Lysosomal acid glucosylceramidase (SINGLE PROTEIN), Homo sapiens
UniProt: P04062
Assay: High Throughput Screening (HTS) by Fluorescence Polarization (pH = 7.2): The enzyme in GCase enzyme activity buffer (25 μL/well) was added to a 384-well black plate. The fluorescent probe 3 (25 nL/well, 50 nM final concentration) was transferred to a 384-well black plate using a Labcyte Echo 550 Liquid Handler system. Compounds in DMSO stock solution (50 nL) were transferred to the plate. The plate was shaken at room temperature in dark for 20 min. The final concentration was 19.5 nM to 10 μM, 10 concentrations, 2 times dilution. The fluorescence polarization was measured in a Molecular Devices Analyst GT with Ex=535 nm and Em=580 nm, G Factor=1.05. To the recombinant wild type enzyme (22 μM, 95 μL, 1 equiv) in 0.1 M phosphate buffer (pH 7.2) was added 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(4-(4-(2,4-dimethylpyrrolo [1,2-a]pyrimidine-8-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)propanoate (4) in DMSO (0.89 mM, 5 μL, 2 equiv) in one portion, and was vortexed for 5 sec immediately. At indicated time points, the reaction solution (2 μL) was sampled and diluted (1:3125 dilution) into the assay buffer (50 mM citric acid, 176 mM K2HPO4, and 0.01% Tween-20 at pH 5.9). After 2 h the reaction solution was dialyzed three times with 0.1 M phosphate buffer (pH 7.2). The enzyme was adjusted to the same concentration and sampled for activity. The dilution solutions were assayed with three substrates, resorufin substrate, 4-MU substrate, and natural substrate using the methods described in the examples above without adding compounds.
Measurement: IC50 = 355 nM (pChEMBL 6.449999809265137)
Descriptors: MW 316.41, ALogP 4.29, HBA 4, HBD 1, PSA 50.7, QED 0.78
Compound (SMILES): O=C(C[S+]([O-])C(c1ccc(F)cc1)c1ccc(F)cc1)N1CCN(Cc2ccc(F)cc2)CC1
InChIKey: WHIDTYLNLLBBBW-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Rattus norvegicus
UniProt: P31652
Assay: Displacement of [3H]citalopram from SERT in Sprague-Dawley rat brain stem membranes incubated for 60 mins by radioligand binding assay
Measurement: Ki = 23600 nM (pChEMBL 4.630000114440918)
Descriptors: MW 486.56, ALogP 4.29, HBA 3, HBD 0, PSA 46.61, QED 0.47