lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(C2(O)CCCc3c(-c4ccc(C#N)cc4)cncc32)cc1C
InChIKey: XYHHKKDRQRGTED-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Cellular Enzyme Assay: G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 ug/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 354.45, ALogP 4.81, HBA 3, HBD 1, PSA 56.91, QED 0.72
Compound (SMILES): O=C(N[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1)OCc1ccccc1
InChIKey: FXJPJOOKIQNMQB-WKILWMFISA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 390.45, ALogP 3.56, HBA 6, HBD 2, PSA 97.2, QED 0.56
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)ccn1CCCN1CCOCC1
InChIKey: QQDGNGDGTFIHID-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: IC50 = 8900 nM (pChEMBL 5.050000190734863)
Descriptors: MW 395.43, ALogP 3.79, HBA 4, HBD 1, PSA 46.5, QED 0.81
Compound (SMILES): CC1(C)Cc2nc(C3CCN(c4ncc(OCCO)cn4)CC3)c([C@@H](F)c3ccc(C(F)(F)F)cc3)c(C3CCC(F)(F)CC3)c2[C@@H](O)C1
InChIKey: GLGUWQRRDXFPIY-DHIFEGFHSA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: CETP Inhibition Assay: A recombinant human CETP protein (manufactured by Roar Biomedical Inc.; 4.5 ng), the acceptor lipoprotein described in (2) above (32.5 ug) and 5,5'-dithio-bis-(2-nitrobenzoic acid) (7 mM, 15 ul) were taken in a 96 well plate and the total amount of the mixture was adjusted to 48.5 ul with the PBS solution. The test compound [DMSO solution (concentration: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 uM; 1.5 ul] was added to each well and the mixture was incubated in a thermostatic bath at 37° C. for 60 minutes. The reconstituted HDL (50 ul) described in (1) above was added to each well and the mixture was reacted in a thermostatic bath at 37° C. for 60 minutes. The 96 well plate was moved onto ice and a precipitation reagent [a mixed solution of magnesium chloride (60 mM) and 0.1% dextran sulfate [1/1 (v/v)]; 15 ul] was added to each well, and then the mixture was allowed to stand on ice for 15 minutes.
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 692.75, ALogP 8, HBA 7, HBD 2, PSA 91.6, QED 0.23
Compound (SMILES): NC(=O)[C@@H](c1ccccc1)N(Cc1cccc(F)c1)C(=O)c1ccccc1
InChIKey: LMCMLPSDKRXRRR-HXUWFJFHSA-N
Target: Transient receptor potential cation channel subfamily M member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z2W7
Assay: Antagonist activity at human recombinant TRPM8 expressed in HEK293 cells assessed as inhibition of menthol-induced calcium increase by Fluo-4 AM based assay
Measurement: IC50 = 8100 nM (pChEMBL 5.090000152587891)
Descriptors: MW 362.4, ALogP 3.69, HBA 2, HBD 1, PSA 63.4, QED 0.73
Compound (SMILES): COc1cccc(F)c1C(=O)N[C@H]1CCC[C@@H]1Nc1cnc(C(F)(F)F)cn1
InChIKey: MZNMYXGGGPPBLG-RYUDHWBXSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 = 830 nM (pChEMBL 6.079999923706055)
Descriptors: MW 398.36, ALogP 3.41, HBA 5, HBD 2, PSA 76.14, QED 0.76
Compound (SMILES): CC(=O)c1ccc(OCc2cccc(Oc3cc(-c4nn[nH]n4)ccn3)c2)c(Cl)c1O
InChIKey: VDWCGWUJQRLILT-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Positive allosteric modulation of recombinant human mGlu2 receptor expressed in hamster AV12 cells expressing EAAT1 assessed as potentiation of glutamate-induced intracellular calcium level measured after 24 to 48 hrs by FLIPR assay
Measurement: EC50 = 87.4 nM (pChEMBL 7.059999942779541)
Descriptors: MW 437.84, ALogP 4.19, HBA 8, HBD 2, PSA 123.11, QED 0.41
Compound (SMILES): COc1cc2c(cc1OC)C1Cc3c(OC)ccc(OC)c3CN1CC2
InChIKey: MLYZNZVURPVMPQ-UHFFFAOYSA-N
Target: D(1A) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21728
Assay: Reception Binding Assay: Different concentrations (10^−5 M-10^−11 M) of the compound of the invention and corresponding isotope receptor ligand as well as receptor protein were loaded into the reaction tube and incubated in 30° C. water bath for 60 minutes. The reaction was terminated in a refrigerator. The reaction mixture was put in a Millipore filter (millipore) cell sample collector, filtered through suction filtration using GF/C glass fiber filter paper, and dried. The resulting sample was placed into 0.5 mL tube. 500 μL liquid scintillation fluid was added and intensity of radioactivity was determined by counting. For dopamine D1 receptor: isotope receptor ligands [3H] SCH23390 (85.0 Ci/mmol) (D1-selective, purchased from Amersham Corporation), D1 receptor protein expressed in HEK-293 cells; For D2 dopamine receptor: isotope receptor ligands [3H] Spiperone (77.0 Ci/mmol) (D2-selective, purchased from Amersham Corporation); D2 receptor protein expressed in HEK-293 cells; For 5-HT1A receptor: isotope receptor ligands [3H] 8-OH-DPAT; 5-HT1A receptor protein expressed in HEK-293 cells; For 5-HT2A receptor: isotope receptor ligands [3H]-Ketanserin; 5-HT2A receptor protein expressed in HEK-293 cells.
Measurement: Ki = 246.86 nM (pChEMBL 6.610000133514404)
Descriptors: MW 355.43, ALogP 3.38, HBA 5, HBD 0, PSA 40.16, QED 0.84
Compound (SMILES): CN1CCN(C[C@@H]2Cc3ccccc3CN2C(=O)c2cc3c(cc2-c2cc(C(=O)N(c4ccccc4)c4ccc(O)cc4)c4n2CCCC4)OCO3)CC1=O
InChIKey: BBRCUUBSRCVMLL-XIFFEERXSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
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 = 10 nM (pChEMBL 8)
Descriptors: MW 737.86, ALogP 6.25, HBA 8, HBD 1, PSA 107.79, QED 0.21
Compound (SMILES): COc1cccc2c1N(Cc1ccccc1)C[C@@H](C)N(C(=O)Nc1cccnc1)[C@H]2C
InChIKey: SPPNGNHZLGXNMN-MOPGFXCFSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Channel blocking activity at recombinant human CaV 3.1 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 > 5300 nM (pChEMBL 5.276000022888184)
Descriptors: MW 416.53, ALogP 5.09, HBA 4, HBD 1, PSA 57.7, QED 0.64
Compound (SMILES): N#Cc1c(N)nc(OCc2ccnc(C(=O)NC3CC3)c2)c(C#N)c1-c1ccccc1
InChIKey: WKAHMRMVDYWVPB-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Luciferase Assay: The stock cultures are grown, at 37° C. and under 5% CO2, in DMEM/F12 medium containing 10% FCS (fetal calf serum) and in each case split 1:10 after 2-3 days. Test cultures are seeded in 384-well plates with 2000 cells per well and grown at 37° C. for approx. 48 hours. The medium is then replaced with a physiological sodium chloride solution (130 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 20 mM HEPES, 1 mM magnesium chloride hexahydrate, 5 mM sodium bicarbonate, pH 7.4). The substances to be tested, which are dissolved in DMSO, are pipetted into the test cultures (maximum final concentration of DMSO in the test mixture: 0.5%) in a dilution series of from 5x10-11 M to 3x10-6 M (final concentration). 10 minutes later, forskolin is added to the A1 cells and all the cultures are subsequently incubated at 37° C. for four hours. After that, 35 ul of a solution which is composed of 50% lysis reagent (30 mM disodium hydrogenphosphate, 10% glycerol, 3% TritonX100, 25 mM TrisHCl, 2 mM dithiotreitol (DTT), pH 7.8) and 50% luciferase substrate solution (2.5 mM ATP, 0.5 mM luciferin, 0.1 mM coenzyme A, 10 mM tricine, 1.35 mM magnesium sulfate, 15 mM DTT, pH 7.8) are added to the test cultures, which are shaken for approx. 1 minute and the luciferase activity is measured using a camera system.
Measurement: EC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 410.44, ALogP 2.94, HBA 7, HBD 2, PSA 137.71, QED 0.64
Compound (SMILES): Nc1ccc(-c2cccs2)cc1NC(=O)c1ccc2nc(N3CCNCC3)ccc2c1
InChIKey: VURDNNVAYZDGDK-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC Enzyme Assay: Compounds for testing were diluted in DMSO to 50 fold the final concentration and a ten point three fold dilution series was made. 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 tris(2-carboxyethyl)phosphine) 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 and pre-incubated with the compounds for 24 hours prior to addition of the substrate. The substrate tripeptide substrate 3 (synthesized in house) for each enzyme was equal to the Km as determined by a substrate titration curve. The enzyme and substrate concentrations used are given in Table 2. The substrates were diluted in assay buffer at 6x their final concentration with 0.3 uM sequencing grade trypsin (Sigma). The substrate/trypsin mix was added to the enzyme/compound mix, the plate was shaken for 60 seconds.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 429.55, ALogP 4.21, HBA 6, HBD 3, PSA 83.28, QED 0.42
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CCc4cc(C#N)ccc4C3)n2)c1
InChIKey: JZRICJXIKFXTAG-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 385.43, ALogP 2.41, HBA 7, HBD 2, PSA 106.83, QED 0.71
Compound (SMILES): Cn1cc(C(CNC(=O)c2cccc(Cl)c2Cl)N2CCC(F)(F)CC2)cn1
InChIKey: AXUDCHXHIHEUEX-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 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 417.29, ALogP 3.93, HBA 4, HBD 1, PSA 50.16, QED 0.8
Compound (SMILES): O=C(NCc1cocn1)c1cc(NC(=O)c2cc(-c3ccccn3)c(F)cc2Cl)n(-c2ccccc2)n1
InChIKey: VSWNLYFBRTUOBK-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 18.2 nM (pChEMBL 7.739999771118164)
Descriptors: MW 516.92, ALogP 4.9, HBA 7, HBD 2, PSA 114.94, QED 0.32
Compound (SMILES): O=c1cc(NC2CCN(S(=O)(=O)c3cccc(F)c3)CC2)c2cc(C(c3ccc(Cl)cc3)c3ccc(Cl)cc3)ccc2[nH]1
InChIKey: XYSBXQKVIGOOPR-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 63 nM (pChEMBL 7.199999809265137)
Descriptors: MW 636.58, ALogP 7.42, HBA 4, HBD 2, PSA 82.27, QED 0.18
Compound (SMILES): Cc1cc(-c2ncc(CNC(=O)c3ccc4c(c3)[nH]c3ccccc34)cc2C)ccn1
InChIKey: YCNUTJIIMNYSHI-UHFFFAOYSA-N
Target: Protein-serine O-palmitoleoyltransferase porcupine (SINGLE PROTEIN), Mus musculus
UniProt: Q9JJJ7
Assay: Inhibition of porcupine in mouse L Wnt3A cells co-cultured with HEK293 cells after 48 hrs by Super-top flash reporter gene assay
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 406.49, ALogP 5.32, HBA 3, HBD 2, PSA 70.67, QED 0.42
Compound (SMILES): COc1cc2c(Nc3ccc(Br)c(Cl)c3)ncnc2cc1O[C@H]1CO[C@H]2[C@@H]1OC[C@@H]2N1CCCCC1
InChIKey: UAUVSHOAYLPSOU-MNPDLOSFSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: Inhibition of recombinant human N-terminal His6-tagged EphB4 kinase domain (E605 to E890 residues) expressed in baculovirus using poly-AEKY as substrate after 150 mins in presence of gamma33P-ATP by liquid scintillation spectrometry
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 575.89, ALogP 5.2, HBA 8, HBD 1, PSA 77.97, QED 0.43
Compound (SMILES): CC(=O)NC(c1nnc(CNC(=O)OC(C)(C)C)o1)c1nc2ccc(-c3ccccc3)cc2s1
InChIKey: JWJILLIBAVQMBD-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Assay: Endothelial lipase activity was measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 285 uL, of 1 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 15 uL, of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen and resuspended in 150 uL, of 50 mM HEPES pH 8.0 buffer containing 100 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at rt for 15 min and then was sonicated 3x4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.05 for the FRET substrate.The enzymatic assay was measured using white, opaque 96-well half area plates. Each well contained 60 uL, of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 2 ul of a DMSO solution containing compound of interest. Conditioned media obtained from HT-1080 cells.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 479.56, ALogP 4.6, HBA 8, HBD 2, PSA 119.24, QED 0.41
Compound (SMILES): Cc1noc(C)c1Cn1cc(NC(=O)c2ccccc2)cn1
InChIKey: IIEXQVOMUDEHHI-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 2620 nM (pChEMBL 5.579999923706055)
Descriptors: MW 296.33, ALogP 2.79, HBA 5, HBD 1, PSA 72.95, QED 0.8
Compound (SMILES): CC(C)(C)NC(=O)N1CCn2c(c(C(N)=O)c(-c3cccc(C(F)(F)F)c3)c2C2CC2)C1
InChIKey: ZGSKTSCLKIRTQZ-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: In Vitro ATP-33P Casein Assay: "U"-bottomed Falcon plates are prepared by placing 5 uL of solutions of the compounds according to the invention at concentrations of 10, 1, 0.1, 0.01 or 0.001 uM in different wells. The solutions of the compounds according to the invention at these various concentrations are prepared by diluting in a test buffer (Tris 50 mM pH 7.5, MgCl2 10 M, DTT 2 mM and EGTA 1 mM) a stock solution in DMSO at a concentration of 10 mM. Next, 5 uL of dephosphorylated casein are added to a final concentration of 0.2 ug/uL, 20 uL of CK1 epsilon to a final concentration of 3 ng/uL, and 20 uL of ATP-33P to a final concentration of 0.02 uCi/uL mixed with cold ATP (10 uM final approximately 2x106 CPM per well). The final total test volume per well is equal to 50 uL. The "U"-bottomed Falcon test plate mentioned above is vortexed, and then incubated at room temperature for 2 hours. After 2 hours the reaction is stopped by adding an ice-cold solution of 65 uL of cold ATP (2 mM) prepared in test buffer. 100 uL of the reaction mixture are then transferred from the "U"-bottomed Falcon plate into Millipore MAPH filter plates, preimpregnated with 25 uL of ice-cold 100% TCA. The Millipore MAPH filter plates are agitated gently and are left to stand at room temperature for at least 30 minutes to precipitate the proteins. After 30 minutes, the filter plates are sequentially washed and filtered with 2x150 uL of 20% TCA, 2x150 uL of 10% TCA and 2x150 uL of 5% TCA (6 washes in total per plate/900 uL per well). The plates are left to dry overnight at room temperature. Next, 40 uL of Microscint-20 Packard scintillation liquid are added per well and the plates are closed in a leaktight manner. The radiation emitted by each well is then measured for 2 minutes in a TopCount NXT Packard scintillation counter, in which the values of CPM/well are measured.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 448.49, ALogP 4.47, HBA 3, HBD 2, PSA 80.36, QED 0.73
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=N[C@@]2(c2nc(NC(=O)C3(C)CCC3)cs2)CO1
InChIKey: WJELMSNRYNVXCO-DINDLPBHSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a ½ log dose response curve from a top concentration of 100 μM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 μL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37 °C. The reaction is stopped with the addition of 30 μL of 1.5 μM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm).
Measurement: IC50 = 461 nM (pChEMBL 6.340000152587891)
Descriptors: MW 380.54, ALogP 2.95, HBA 7, HBD 2, PSA 89.6, QED 0.84
Compound (SMILES): O=c1ccc2c(OCCCOc3ccc(Cl)cc3)c3ccoc3cc2o1
InChIKey: POFDDBSJPMDKID-UHFFFAOYSA-N
Target: Potassium voltage-gated channel subfamily A member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22001
Assay: Inhibition of Kv1.3 (unknown origin)
Measurement: EC50 = 10 nM (pChEMBL 8)
Descriptors: MW 370.79, ALogP 5.04, HBA 5, HBD 0, PSA 61.81, QED 0.35
Compound (SMILES): FC(F)Oc1cc(-c2cnn(Cc3cn[nH]c3)c2)ccc1Cl
InChIKey: WQIJWPZIOIFLHO-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: Antagonist activity at human NR2B expressed in HEK293 cells co-expressing NRI assessed as inhibition of glutamate/glycine-induced Ca2+ flux measured after 90 secs
Measurement: IC50 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 324.72, ALogP 3.58, HBA 4, HBD 1, PSA 55.73, QED 0.78
Compound (SMILES): O=C(NC[C@@H]1CCNC1)c1cc(NC(=O)c2cc(-c3ncccc3F)ccc2Cl)n(-c2ccccc2)n1
InChIKey: RNSHFANCHUEVLU-QGZVFWFLSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 1360 nM (pChEMBL 5.869999885559082)
Descriptors: MW 518.98, ALogP 4.32, HBA 6, HBD 3, PSA 100.94, QED 0.34
Compound (SMILES): NC(c1ccc(-c2cccc(Nc3nccc(C(F)(F)F)n3)c2)cc1)C(F)(F)F
InChIKey: XHALKFXCGPWDMN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 172.7 nM (pChEMBL 6.760000228881836)
Descriptors: MW 412.34, ALogP 5.47, HBA 4, HBD 2, PSA 63.83, QED 0.56
Compound (SMILES): CC#C[C@@H](CC(=O)O)c1ccc(OCCOc2cccc(Cl)c2)cc1
InChIKey: LBEBVHDEHJNPCA-INIZCTEOSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q8K3T4
Assay: Agonist activity at rat GPR40 expressed in HEK293 cells assessed as increase in intracellular Ca2+ flux by Fluo-4-AM dye based FLIPR assay
Measurement: EC50 = 10 nM (pChEMBL 8)
Descriptors: MW 358.82, ALogP 4.38, HBA 3, HBD 1, PSA 55.76, QED 0.56
Compound (SMILES): O=C(NCc1ccc([S+]([O-])c2cc(F)cc(F)c2)cn1)c1ccc2nccn2c1
InChIKey: DFEUKEQPQVSNCZ-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT Enzymatic Reaction. The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 pM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 36.5 nM (pChEMBL 7.440000057220459)
Descriptors: MW 412.42, ALogP 3.1, HBA 5, HBD 1, PSA 82.35, QED 0.51
Compound (SMILES): CC#Cc1cncc(-c2ccc3c(c2)[C@@]2(N=C(C)C(N)=N2)[C@@]2(C3)C[C@@H](C)[C@H](OC)[C@@H](C)C2)c1
InChIKey: HBTQOOAUHHGLEO-CBGRAPDISA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Quench Assay: The assay was performed in the presence of OptiMEM (supernatant collected over 24 h and cleared from cellular debris by centrifugation) containing the ectodomain of BACE1, 25 μl water containing the desired 2-fold concentration of test compound and 2% DMSO, 1 μM substrate peptide, 20 mM NaOAc, pH 4.4, and 0.04% Triton-X100 in a total assay volume of 50 μl in a 384 well plate. In general, 25 μl of compound dilution were added to the plate followed by the addition of 10 μl of BACE1 containing OptiMEM diluted 1:10 in water with 0.2% Triton X-100. The reaction was started with the addition of 15 μl substrate in NaOAc buffer. The reaction was incubated at rt (dark) in an Envision® multilabel reader (Perkin Elmer) and the cleavage of the substrate was recorded as kinetic for 60 min at ex: 485 nm, em: 538 nm. Blank wells containing no enzyme were included on each plate.
Measurement: IC50 = 8.2 nM (pChEMBL 8.09000015258789)
Descriptors: MW 440.59, ALogP 4.73, HBA 5, HBD 1, PSA 72.86, QED 0.69
Compound (SMILES): CNC(=O)c1cccc(-c2ccc([C@@H](O)[C@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]3O)cc2)c1
InChIKey: KOOGCRKRQNOZBW-QONVVCGCSA-N
Target: Type 1 fimbrin D-mannose specific adhesin (SINGLE PROTEIN), Escherichia coli K-12
UniProt: P08191
Assay: Inhibition of Escherichia coli UTI89 FimH-mediated biofilm formation after 48 hrs by crystal violet staining-based assay
Measurement: IC50 = 900 nM (pChEMBL 6.050000190734863)
Descriptors: MW 403.43, ALogP -0.41, HBA 7, HBD 6, PSA 139.48, QED 0.4
Compound (SMILES): CN(C(=O)c1cocn1)c1cccc(OCC(O)CN2CCc3ccccc3C2)c1
InChIKey: BVAFCFBOFWENGT-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 10001 nM (pChEMBL 5)
Descriptors: MW 407.47, ALogP 2.75, HBA 6, HBD 1, PSA 79.04, QED 0.65
Compound (SMILES): COCC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CC1
InChIKey: QJZZATMXSBBZQC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 415.43, ALogP 1.14, HBA 8, HBD 2, PSA 139.16, QED 0.65
Compound (SMILES): CS(=O)(=O)NC1CCC(c2cnc(N)c(-c3ccc(C(=O)N[C@H](CO)c4cccc(Cl)c4)c(F)c3)n2)CC1
InChIKey: OISIMDWQCVCCDF-LHMWJSETSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
Measurement: IC50 = 0.26 nM (pChEMBL 9.59000015258789)
Descriptors: MW 562.07, ALogP 3.56, HBA 7, HBD 4, PSA 147.3, QED 0.33
Compound (SMILES): CC(C)(C)c1ccc(C(c2ccccc2)N2CCN(C(=O)CC(c3ccccc3)c3ccccc3)CC2)cc1
InChIKey: KJKQQWDHZCWYRN-UHFFFAOYSA-N
Target: Voltage-dependent N-type calcium channel subunit alpha-1B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q02294
Assay: Antagonist activity at rat alpha1B N-type calcium channel expressed in HEK293 cells assessed as 0.067 Hz current frequency dependent channel blocking for 2 mins in presence of Ba2+ by whole-cell patch clamp assay
Measurement: IC50 = 370 nM (pChEMBL 6.429999828338623)
Descriptors: MW 516.73, ALogP 7.44, HBA 2, HBD 0, PSA 23.55, QED 0.25
Compound (SMILES): Cc1nsc(-c2nnc3n2CCN(C(=O)c2ccc(Cl)c(F)c2)C3C)n1
InChIKey: WVMPHKVLGBDWHJ-UHFFFAOYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Functional Assay: Chinese Hamster Ovary recombinant cells expressing the human NK-3 receptor and a construct that encodes the photoprotein apoaequorin were used for this assay. The antagonist activity of compounds of the invention is measured following pre-incubation (3 minutes) of the compound (at various concentrations) with the cells, followed by addition of the reference agonist (NKA) at a final concentration equivalent to the EC80 (3 nM) and recording of emitted light (FDSS 6000 Hamamatsu) over the subsequent 90-second period. The intensity of the emitted light is integrated using the reader software. Compound antagonist activity is measured based on the concentration-dependent inhibition of the luminescence response to the addition of Neurokinin A.
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 392.85, ALogP 3.11, HBA 7, HBD 0, PSA 76.8, QED 0.67
Compound (SMILES): O=C(N[C@H]1CC[C@@H](c2ccc(NC(=O)N3Cc4ccncc4C3)cc2)CC1)C1CCCCC1
InChIKey: QSTJEBKANMTSIG-DQMBHMMVSA-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 = 4.71 nM (pChEMBL 8.329999923706055)
Descriptors: MW 446.6, ALogP 5.35, HBA 3, HBD 2, PSA 74.33, QED 0.66
Compound (SMILES): O=C(c1cc(Cc2n[nH]c(=O)c3c2CCCC3)ccc1F)N1CCN(C(=O)N2CCOCC2)CC1
InChIKey: HCCAPOWPOYHXAW-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 483.54, ALogP 1.59, HBA 5, HBD 1, PSA 98.84, QED 0.72
Compound (SMILES): COc1cc(OC)cc(N(CCO)c2ccc3ncc(-c4cnn(CCCN)c4)nc3c2)c1.Cl
InChIKey: USEJTVZDKKLZFE-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 absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 128.82 nM (pChEMBL 6.889999866485596)
Descriptors: MW 448.53, ALogP 2.99, HBA 9, HBD 2, PSA 111.55, QED 0.38
Compound (SMILES): CNc1ncc(-c2ccnc(Nc3ccc(NC4CCN(C)CC4)nc3)n2)cc1C#N
InChIKey: NJEDMUVUHDHHJU-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
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 < 1000 nM (pChEMBL 6)
Descriptors: MW 415.51, ALogP 3.1, HBA 9, HBD 3, PSA 114.68, QED 0.56
Compound (SMILES): N#Cc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(C(F)(F)F)c3)cn2CCN2CCCCC2)CC1
InChIKey: IKNUWQGXRGMLOY-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 542.59, ALogP 4.82, HBA 8, HBD 1, PSA 99.89, QED 0.45
Compound (SMILES): O=C(O)CCc1cc2cc(-c3cc(C(=O)C4CC4)ccc3F)ccc2o1
InChIKey: ZBFAQUJBQJNPBX-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Agonist activity at human GPR120 expressed in CHOK1 cells measured after 60 mins by IP1-HTRF assay
Measurement: EC50 = 97 nM (pChEMBL 7.010000228881836)
Descriptors: MW 352.36, ALogP 4.85, HBA 3, HBD 1, PSA 67.51, QED 0.64