lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Nc1ncc(F)cc1-c1ccc(Oc2ncc(NC(=O)Nc3cc(C(F)(F)F)ccc3-c3ccccc3)cn2)cc1
InChIKey: FVWIYWKWDMNSTL-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Activity Assay: On the day before the assay, CellSenser TrkA-NFAT-bla CHO-K1 cells were suspended in an assay medium (Opti-MEM1 Reduced Serum Medium (Invitrogen) containing 0.5% dialysed fetal bovine serum (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin (Invitrogen))) and plated at a density of 2.4x104 cells/40 uL/well in a 96-well clear bottom plate (Corning, Catalogue No.: 3882). In some wells were added only the assay medium at 40 uL/well (Cell-free). On the day of the assay, 10 mM of the present compound (DMSO solution) was distributed in a 96-well plate (Costar, Catalogue No.: 3363) and serially diluted with DMSO with the geometrical ratio of 3. The serial dilutions were diluted with the assay medium to 100-fold to prepare a solution of the present compound with a 10-fold concentration (DMSO concentration: 1%). To the plate where cells were plated was added the present compound at 5 L/well and the plate was incubated in a CO2 incubator with 5% CO2, 95% Air at 37° C. for 30 minutes. For a control and a blank, the assay medium containing 1% DMSO was added at 5 L/well in place of the solution of the present compound. Subsequently the assay medium containing NGF (Mouse 2.5s, Natural, Invitrogen) was added to the plate at 5 L/well (final concentration of NGF: 50 ng/ml) and the plate was incubated in a CO2 incubator with 5% CO2, 95% Air at 37° C. for 5 hours. For the blank group, the assay medium was added in place of NGF at 5 L/well. A reporter assay detection reagent (10 L/well) was added to the plate which was then incubated in the dark at room temperature for 120 minutes. The reporter assay detection reagent was prepared from LiveBLAzer -FRET B/G Loading Kit (Invitrogen). On the Analyst GT (Molecular Devices Japan, K.K.) the wells were irradiated with excitation light at 405 nm and the fluorescence intensities at 460 nm and 530 nm were measured.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 560.51, ALogP 7.38, HBA 6, HBD 3, PSA 115.05, QED 0.19
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4ccc(F)c(C(F)(F)F)c4)cn3CCN3CCCCC3)CC2)c1Cl
InChIKey: KPWXHZUYRNURPQ-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 = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 552.02, ALogP 5.6, HBA 7, HBD 1, PSA 76.1, QED 0.4
Compound (SMILES): Cc1cc(F)ccc1[C@H]1C[C@]2(CC[C@@H]3COCCN32)CCN1C(=O)N(C)[C@H](C)c1cc(C(F)(F)F)cc(C(F)(F)F)c1.Cl
InChIKey: JMESMSYZXUPVFT-SGDKEXRGSA-N
Target: Substance-P receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P25103
Assay: Antagonist at human NK1 receptor expressed in recombinant BacMam virus infected human U2OS cells assessed as inhibition of substance P-induced intracellular calcium release preincubated for 30 mins followed by substance P addition by Fluo-4 calcium dye-based FLIPR assay
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 601.61, ALogP 7.36, HBA 3, HBD 0, PSA 36.02, QED 0.35
Compound (SMILES): CC[C@@H](N[C@@H](C)CC(N)=O)c1ccc(Cl)c(C(=O)c2cc(C)c(N)cn2)c1F
InChIKey: LXICXTMYLDDPBB-XHDPSFHLSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 406.89, ALogP 3.3, HBA 5, HBD 3, PSA 111.1, QED 0.58
Compound (SMILES): C[C@@]1(c2cc(NC(=O)c3ccc(Cl)cn3)ccc2F)CS(=O)(=O)C2(CCCC2)C(=N)N1
InChIKey: VFUXKCUHUHFKCF-FQEVSTJZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3x the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 ul) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol.
Measurement: Ki = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 464.95, ALogP 3.65, HBA 5, HBD 3, PSA 112.01, QED 0.64
Compound (SMILES): COc1ccncc1-c1ccc2[nH]nc(-c3cc(N4CCN(C(C)C)CC4)ncn3)c2c1
InChIKey: OKWDSKOHFZQLEU-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 1.26 nM (pChEMBL 8.899999618530273)
Descriptors: MW 429.53, ALogP 3.62, HBA 7, HBD 1, PSA 83.06, QED 0.52
Compound (SMILES): O=C(c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1)N1CCc2[nH]cnc2C1
InChIKey: HMUQFYVFFJIGMO-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] 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 90 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. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 1090 nM (pChEMBL 5.960000038146973)
Descriptors: MW 439.4, ALogP 4.36, HBA 5, HBD 1, PSA 84, QED 0.51
Compound (SMILES): COn1c(NC(C)c2ccc(C(F)(F)F)cc2)nc2c(c1=O)CN(C(=O)c1ccc(Cl)c(C)c1)CC2
InChIKey: YUDUDNNKQJKTLW-UHFFFAOYSA-N
Target: Prolactin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P49683
Assay: Inhibition Assay: Receptor-expressing HEK 293 cell membrane fraction was analyzed by modifying the methods of C. J. Langmead et al. (see Langmead C J, Szekeres P G; Chambers J K, Ratcliffe S J, Jones D N C, Hirst W D, et al. Characterization of the binding of [125I]-human prolactin releasing peptide (PrRP) to GPR10, a novel G protein coupled receptor. Br J Pharmacol 2000; 131: 683-688.). To each well of a 96-well plate, 150 μl of assay buffer (20 mM HEPES, 10 mM EDTA, 1 μl/ml protease inhibiting agent cocktail, pH 7.4), 20 μl of cell membrane fraction, 10 μl of test compound and [3H]-PrRP (final concentration 1 nM) were added and incubated at room temperature for 90 minutes. After completion of the reaction, using a cell harvester, the cell-membrane fraction sample product was filtered under aspiration by a glass fiber filter plate (Unifilter; GF/B) previously treated with 0.5% polyethylene imine. The filter was washed with a 50 mM Tris hydrochloric acid buffer (pH 7.4) three times.
Measurement: Ki = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 520.94, ALogP 4.65, HBA 6, HBD 1, PSA 76.46, QED 0.53
Compound (SMILES): CC(C)[C@@H](C)NC(=O)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: ZEMVEISEORCVQS-OAHLLOKOSA-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 = 82.1 nM (pChEMBL 7.090000152587891)
Descriptors: MW 351.45, ALogP 4.01, HBA 2, HBD 2, PSA 61.44, QED 0.87
Compound (SMILES): NCCCCN(Cc1cc2c(cn1)[nH]c1ccccc12)C1CCCc2cccnc21
InChIKey: KFAHIZIXUPRPBG-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Calcium Mobilization Assay: Functional modulation of CXCR4 was determined by calcium mobilization assay using leukemic lymphoid CEM cells, which naturally express high levels of CXCR4. Generally, the assay is carried out as follows: Cells are grown in vitro to confluency. On the day of the assay, cells are removed from the incubator, culture medium removed and replaced with a calcium sensitive dye (Calcium3 assay kit; Molecular Devices, Sunnyvale, Calif.). The cells are allowed to dye load for 45-60 min at 37 °C. in the cell culture incubator and then allowed to equilibrate to room temperature for no more than 15 min before assay. Compound plates were generated containing up to 3% dimethyl sulfoxide in media kept at room temperature. Test compounds were added to the cells at a 1:3 dilution, and calcium mobilization was measured using a Flex Station fluorescence imager (Molecular Devices). This first read was used to determine direct agonist activation of the CXCR4 receptor by the test compounds.
Measurement: IC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 399.54, ALogP 4.73, HBA 4, HBD 2, PSA 70.83, QED 0.44
Compound (SMILES): CC(C)(O)c1coc(Cn2ncc(NC(=O)c3ncoc3-c3cccc(Cl)c3)n2)n1
InChIKey: KAVTXLDNQVIPDJ-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Intracellular Calcium Assay: Cells expressing recombinant human ALX receptor and the G-protein Gα16 (HEK293-hALXR-Gα16) were grown to 80% confluency in Growing Medium (GM). Cells were detached from culture dishes with a cell dissociation buffer (Invitrogen, 13151-014), and collected by centrifugation at 1,000 rpm at rt for 5 min in Assay Buffer (AB) (equal parts of Hank's BSS (Gibco, 14065-049) and DMEM without Phenol Red (Gibco, 11880-028)). After 60 min incubation at 37° C. under 5% CO2 in AB supplemented with 1 μM Fluo-4 (AM) (Invitrogen, F14202) and 20 mM HEPES (Gibco, 15630-056), the cells were washed and resuspended in AB. They were then seeded onto 384-well FLIPR assay plates (Greiner, 781091) at 50,000 cells in 70 μl per well and sedimented by centrifugation at 1,000 rpm for 1 min. Stock solutions of test compounds were made up at a concentration of 10 mM in DMSO, and serially diluted in AB to concentrations required for activation dose response curves.
Measurement: EC50 = 17.8 nM (pChEMBL 7.75)
Descriptors: MW 428.84, ALogP 3.1, HBA 9, HBD 2, PSA 132.1, QED 0.48
Compound (SMILES): COCc1cc(Cl)cc(Cl)c1CNC(=O)C1CCCc2ncsc21
InChIKey: DKKJAIAKMMCJIJ-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: YO-PRO Assay: Cells were grown to confluency in adherent culture at 37° C. in a humidified 5% CO2 incubator (split 1/5 every 3-4 days with DMEM, 10% FCS, 1% Penicillin/Streptomycin, 250 μg/ml Geneticin). Adherent cells were detached by incubation with Trypsine (1 ml per 165 cm2 dish) for 2 minutes, then washed off with 10 ml PBS (without Mg2+ and Ca2+), and resuspended in DMEM, 10% FCS, 1% Penicillin/Streptomycin, no Geneticin. 10,000 cells per well (48 hours before the assay) or 25,000 cells per well (Vi-cell XR (Beckman Coulter) (24 hours before the assay) in 50 μl full medium were seeded on 384-well black-wall, clear bottom plates, that were coated before with 10 μl per well Poly-L-Lysine, incubated for 30-60 minutes at 37° C. and washed once with PBS. Medium was removed from cells and 50 μl of assay buffer containing 0.5 μM YO-PRO-1 was added into the wells. Solutions of antagonist compounds were prepared by serial dilutions of a 10 mM DMSO solution of the antagonist into PBS using a BioMek (Beckman Coulter). Each concentration was performed in duplicate. For IC50 measurements 10 concentration points were measured (10 μM being the highest concentration followed by 9 serial dilution steps 1/3). The cells were incubated with the antagonists of the present invention together with ATP at a final concentration of 250 μM for 90 minutes. During this time period, four time points were taken. Each time point comprised the average of several measurements made within a few seconds. Fluorescence was measured in the FLIPR tetra (Molecular Devices) using the filters appropriate for YO-PRO-1 fluorescence (excitation 485/20, emission 530/25). The FLIPR tetra was equipped with Molecular Devices Screen Works system control software to define and run experimental protocols.
Measurement: IC50 = 4.3 nM (pChEMBL 8.369999885559082)
Descriptors: MW 385.32, ALogP 4.33, HBA 4, HBD 1, PSA 51.22, QED 0.83
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: SVLKDNYBWMKZHF-OWXMSLSPSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 2431.79
Compound (SMILES): Cc1c(C(=O)Nc2c(C)n(C)n(C3CCCCC3)c2=O)noc1[C@@H]1OCC(C)(C)O[C@H]1C
InChIKey: LVDRKBFVIUBIBB-MGPUTAFESA-N
Target: E3 ubiquitin-protein ligase SMURF1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HCE7
Assay: Inhibition of SMURF1 (unknown origin) in presence of E1/E2/Ub mix measured after 45 mins by europium-labeled streptavidin based TR-FRET assay
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 446.55, ALogP 3.8, HBA 8, HBD 1, PSA 100.52, QED 0.77
Compound (SMILES): CCC[C@H](Nc1nc(N)nc(N)c1Cl)c1nc2cccc(Cl)c2c(=O)n1-c1ccc(N)nc1
InChIKey: RRGONSPDJLJIBH-ZDUSSCGKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 486.37, ALogP 3.58, HBA 10, HBD 4, PSA 163.65, QED 0.32
Compound (SMILES): Cc1ccc(-n2nc(C3CCC(CN)CC3)cc2NC(=O)c2cnn3cccnc23)nc1
InChIKey: DKBHHIWHYHEZFB-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 430.52, ALogP 3.1, HBA 8, HBD 2, PSA 116.02, QED 0.5
Compound (SMILES): COc1ccc(-n2c(SCc3cc(OC)c(OC)c(OC)c3)nc3ccc(C)cc3c2=O)c(OC)c1
InChIKey: QURBHNYJXRPMES-UHFFFAOYSA-N
Target: Dihydrofolate reductase (SINGLE PROTEIN), Bos taurus
UniProt: P00376
Assay: Inhibition of bovine liver DHFR preincubated for 2 mins followed by addition of FH2 as substrate and NADPH
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 508.6, ALogP 5.03, HBA 9, HBD 0, PSA 81.04, QED 0.23
Compound (SMILES): Cc1ccc2nc(N(C)C)nc(N[C@@H](C)c3ccc(NC(=O)c4ccc(Cl)nc4)cc3)c2c1
InChIKey: PQPJKBSPXRPUKP-INIZCTEOSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf33.13 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 1837 nM (pChEMBL 5.739999771118164)
Descriptors: MW 460.97, ALogP 5.48, HBA 6, HBD 2, PSA 83.04, QED 0.37
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCC(NCc2ccccc2O)CC1
InChIKey: VXLQZNBQBRMCHD-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 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 463.52, ALogP 3.31, HBA 8, HBD 4, PSA 141.88, QED 0.38
Compound (SMILES): CC(C)N1CCN(C(=O)OC2CCN(c3cc(CO)ccn3)CC2)CC1
InChIKey: XBTYCJBQLYJSFJ-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: LANCE Ultra cAMP Assay: The potency of compounds of the invention as H3 receptor antagonists can be assessed by measuring the blockade of (R)-alpha-methylhistamine-mediated cAMP production utilizing a LANCE Ultra cAMP kit (PE #TRF0263) in CHO cells expressing human H3 receptors (GenBank: BC096840; Strausberg R L et al, Proc. Natl. Acad. Sci. U.S.A. 99(26), 16899-16903; 2002).Protocol:1. Preparation of the stimulation buffer (30 ml): 29.4 ml HBSS (GIBCO #14025), 150 ul 1 M HEPES (GIBCO #15630), 30 ul 500 mM IBMX (CALBIOCHEM #410957) and 400 ul 7.5% BSA (GIBCO #10438-026).2. Preparation of assay plate: Different concentrations of the compounds of the invention (0.01-1000 nM), H3 positive controls and cAMP calibration standards; 3 mM Forskolin (CALBIOCHEM #344270); 5 uM (R)-alpha-methylhistamine (H3 receptor agonist); 1% DMSO (SIGMA #D2650); total volume: 95 nl.3. Preparation of the cell solution: Collect cells with stimulation buffer, final density: 100,000 cells/ml.
Measurement: Ki = 4.1 nM (pChEMBL 8.390000343322754)
Descriptors: MW 362.47, ALogP 1.71, HBA 6, HBD 1, PSA 69.14, QED 0.88
Compound (SMILES): O=C(O[C@H]1CCNC[C@@H]1F)[C@@H]1CC[C@@H]2CN1C(=O)N2OS(=O)(=O)O
InChIKey: LXEXAEIJAUOEKW-KATARQTJSA-N
Target: Beta-lactamase (SINGLE PROTEIN), Pseudomonas aeruginosa
UniProt: Q8RL90
Assay: Spectrophotometric Assay: Enzyme activities were measured in the presence of the test inhibitor in spectrophotometric assay.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 367.36, ALogP -0.77, HBA 7, HBD 2, PSA 125.48, QED 0.49
Compound (SMILES): CC1CCCN1c1cccc(Nc2cc(-c3cccc(C(=O)NCCN(C)C)c3)nn3ccnc23)n1
InChIKey: DOGIPVLJNCTKRX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition Assay: Assay plates: 96-well MultiScreen 0.65 um filter plates (Millipore Cat. No.: MADVNOB10)Streptavidin coated beads: Streptavidin Sepharose, suspension 5.0 mL, in 50 mM EDTA/PBS diluted (1:100), (Amersham, Cat. No.: 17-5113-01)Compounds: 10 mM in 100% dimethylsulfoxide (DMSO), final conc.: compound 0.003-100 uM in 10% DMSOEnzyme: SYK RPA purified, truncated construct of Spleen Tyrosine Kinase aa 360-635, stock solution 1 mg/mL, MW: 31.2 KDa, final conc.:0.0005 uM. Peptide 1: biotinylated peptide is derived from a naturally occurring phosphor-acceptor consensus sequence (Biotin-EPEGDYEEVLE), special order from QCB, stock solution 20 mM, final conc.: 5.0 uM.ATP: Adenosine-5'-triphosphate 20 mM, (ROCHE Cat. No.: 93202720), final concentration: 20 uM Buffer: HEPES: 2-Hydroxyethyl piperazine-2-ethanesulfonic acid (Sigma, Cat. No.: H-3375)final concentration: 50 mM HEPES pH7.5BSA: Bovine Serum Albumin Fraction V, fatty acid free (Roche Diagnostics GmbH, Cat. No. 9100221).
Measurement: IC50 = 141.44 nM (pChEMBL 6.849999904632568)
Descriptors: MW 484.61, ALogP 3.82, HBA 8, HBD 2, PSA 90.69, QED 0.39
Compound (SMILES): CN1C(=O)N(c2cccc(C(F)(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1C(=O)NC1CCOC1
InChIKey: BLMSFCSVUCRJEV-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 6.9 nM (pChEMBL 8.15999984741211)
Descriptors: MW 524.5, ALogP 4.33, HBA 5, HBD 1, PSA 102.74, QED 0.64
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(CN4CCN(CCCCCC(=O)NO)CC4)cc3)cc12)c1ccccc1
InChIKey: NKQVVPNGDOFUHY-HSZRJFAPSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal GST-fused human EGFR (His672 to Ala1210 residues) expressed in baculovirus expression system using biotinylated PTP1B at Tyr66 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 541.7, ALogP 4.98, HBA 7, HBD 4, PSA 109.41, QED 0.11
Compound (SMILES): COc1ccc(-c2ccc3ncc4c(c3c2)n(C2CCNCC2)c(=O)n4C)cn1
InChIKey: KQZZSMVSVVANOL-UHFFFAOYSA-N
Target: Serine-protein kinase ATM (SINGLE PROTEIN), Homo sapiens
UniProt: Q13315
Assay: Cellular Assay: Assay a) HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 3500 cells/well in 40 μl EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compounds of Formula (I) in 100% DMSO were added to assay plates by acoustic dispensing. After 1 h incubation at 37° C. and 5% CO2 plates (up to 6 at a time) were irradiated using the X-RAD 320 instrument (PXi) with equivalent to Ë¿600 cGy. Plates were returned to the incubator for a further 1 h. Then cells were fixed by adding 20 μl of 3.7% formaldehyde in PBS solution and incubating for 20 minutes at r.t. before being washed with 50 μl/well PBS, using a Biotek EL405 plate washer. Then 20 μ 0.1% Triton X100 in PBS was added and incubated for 20 minutes at r.t., to permeabalise cells. Then the plates were washed once with 50 μl/well PBS, using a Biotek EL405 plate washer. Phospho-ATM Ser1981 antibody (Millipore #MAB3806) was diluted 10000 fold in PBS containing 0.05% polysorbate/Tween and 3% BSA and 20 μl was added to each well and incubated over night at r.t. The next morning plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and then 20 μl of secondary Ab solution, containing 500 fold diluted Alexa Fluor® 488 Goat anti-rabbit IgG (Life Technologies, A11001) and 0.002 mg/ml Hoeschst dye (Life technologies #H-3570), in PBS containing 0.05% polysorbate/Tween and 3% BSA, was added. After 1 h incubation at r.t., the plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and plates were sealed and kept in PBS at 4° C. until read. Plates were read using an ArrayScan VTI instrument, using an XF53 filter with 10× objective. A two laser set up was used to analyse nuclear staining with Hoeschst (405 nm) and secondary antibody staining of pSer1981 (488 nm).
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 389.46, ALogP 2.88, HBA 7, HBD 1, PSA 73.97, QED 0.58
Compound (SMILES): CC[C@@H](N[C@@H](C)CO)c1ccc(Cl)c(C(=O)c2cccnc2)c1F
InChIKey: AMNLKVFPQJHGHN-XHDPSFHLSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 350.82, ALogP 3.53, HBA 4, HBD 2, PSA 62.22, QED 0.75
Compound (SMILES): COCCc1nc(C(F)(F)F)c(-c2cc(C(=O)N3CC(c4ccc(C#N)cc4)C3)ccc2C)[nH]1
InChIKey: FWVVSTOCMYAPPJ-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 468.48, ALogP 4.7, HBA 4, HBD 1, PSA 82.01, QED 0.57
Compound (SMILES): Cc1c(Cl)cccc1NC(=O)c1cc(NC(=O)c2ccccc2C(F)(F)F)cc2[nH]c(C3CCCC3)nc12
InChIKey: CXRJHASYZPXNEG-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition Assay: mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC).
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 540.97, ALogP 7.71, HBA 3, HBD 3, PSA 86.88, QED 0.24
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CC(Oc4ccc(Cl)c(C)c4)C3)n2)c1
InChIKey: DWPKMPLLHGTZMC-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 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 424.89, ALogP 3.2, HBA 7, HBD 2, PSA 92.27, QED 0.63
Compound (SMILES): Clc1ccc(Nc2ncc3ccnc(N4CCC5(COC5)C4)c3n2)c(Cl)c1
InChIKey: RWNJNJJDZATFSX-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Enzyme Inhibition Assay: The enzyme reaction (total volume 10 μl) was carried out in black 384-well low volume plates containing full length MPS1 (12.5 nM or 3 nM), fluorescent labelled peptide [known as H236, which has the sequence: 5FAM-DHTGFLTEYVATR-CONH2] (5 μM), ATP(10 μM), either DMSO (1% v/v) or the test compound (in the range 0.25 nM-100 μM in 1% DMSO) and assay buffer (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% BSA, 0.1 mM Orthovandate, 10 μM MgCl2, 1 μM DTT, Roche protease inhibitor). The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μl) containing 20 mM EDTA, 0.05% (v/v) Brij-35, in 0.1M HEPES-buffered saline (Free acid, Sigma, UK). The plate was read on a Caliper EZ reader II (Caliper Life Sciences).
Measurement: IC50 = 94 nM (pChEMBL 7.03000020980835)
Descriptors: MW 402.29, ALogP 4.3, HBA 6, HBD 1, PSA 63.17, QED 0.7
Compound (SMILES): CC(Nc1nc(NC2CC2)n2ncc(/C=C3\NC(=O)NC3=O)c2n1)c1ccccc1Cl
InChIKey: QBYHDBHMDWJNMS-NVNXTCNLSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 438.88, ALogP 2.71, HBA 8, HBD 4, PSA 125.34, QED 0.34
Compound (SMILES): CC(c1ccccc1)N1CCC(c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: SYTKHVAFXFXSLD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 346.44, ALogP 3.55, HBA 5, HBD 1, PSA 62.11, QED 0.62
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@@H]4NC(=O)N3CCNCC3)no2)cc1C#N
InChIKey: HTSHHNZVFQDSQP-QFIPXVFZSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 472.55, ALogP 3.66, HBA 7, HBD 2, PSA 116.31, QED 0.58
Compound (SMILES): O=C(O)COc1ccc(Cl)cc1C1c2scnc2CCN1C(=O)OCC1CCCCC1
InChIKey: SEFFNWAFLMLGTQ-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 μl. First, 25 μl of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 μl Binding-Buffer, 50 μl of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 μl CRTH2 membrane fragments, reaching a final concentration of 20 μg/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 μl of Microscint-40 (Packard) was added to each well and the re
Measurement: IC50 = 9.2 nM (pChEMBL 8.039999961853027)
Descriptors: MW 464.97, ALogP 4.92, HBA 6, HBD 1, PSA 88.96, QED 0.65
Compound (SMILES): CC1(C)c2cc(O)c(-c3nn[nH]n3)cc2C(=O)c2c1[nH]c1cc(Cl)ccc21
InChIKey: WLHWLWPETFPYGR-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 0.456 nM (pChEMBL 9.34000015258789)
Descriptors: MW 379.81, ALogP 3.58, HBA 5, HBD 3, PSA 107.55, QED 0.47
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3cc(F)c(F)c(F)c3)CC2)C1
InChIKey: UZQKOMLXZWMLBI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 520.52, ALogP 3.47, HBA 7, HBD 1, PSA 106.73, QED 0.4
Compound (SMILES): Cc1ncc(C(CNC(=O)C2CCOc3ncsc32)N2CCOCC2)cn1
InChIKey: LZNHMGVDSMIJFY-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: YO-PRO Assay: Cells were grown to confluency in adherent culture at 37° C. in a humidified 5% CO2 incubator (split 1/5 every 3-4 days with DMEM, 10% FCS, 1% Penicillin/Streptomycin, 250 μg/ml Geneticin). Adherent cells were detached by incubation with Trypsine (1 ml per 165 cm2 dish) for 2 minutes, then washed off with 10 ml PBS (without Mg2+ and Ca2+), and resuspended in DMEM, 10% FCS, 1% Penicillin/Streptomycin, no Geneticin. 10'000 cells per well (48 hours before the assay) or 25'000 cells per well (Vi-cell XR (Beckman Coulter) (24 hours before the assay) in 50 μl full medium were seeded on 384-well black-wall, clear bottom plates, that were coated before with 10 μl per well Poly-L-Lysine, incubated for 30-60 minutes at 37° C. and washed once with PBS. Medium was removed from cells and 50 μl of assay buffer containing 0.5 μM YO-PRO-1 was added into the wells. Solutions of antagonist compounds were prepared by serial dilutions of a 10 mM DMSO solution of the antagonist into PBS using a BioMek (Beckman Coulter). Each concentration was performed in duplicate. For IC50 measurements 10 concentration points were measured (10 μM being the highest concentration followed by 9 serial dilution steps 1/3). The cells were incubated with the antagonists of the present invention together with ATP at a final concentration of 250 μM for 90 minutes. During this time period, four time points were taken. Each time point comprised the average of several measurements made within a few seconds. Fluorescence was measured in the FLIPR tetra (Molecular Devices) using the filters appropriate for YO-PRO-1 fluorescence (excitation 485/20, emission 530/25).
Measurement: IC50 = 1430 nM (pChEMBL 5.840000152587891)
Descriptors: MW 389.48, ALogP 1.3, HBA 8, HBD 1, PSA 89.47, QED 0.82
Compound (SMILES): CS(=O)(=O)c1ccc(Oc2ccc(Nc3ccc(C(=O)O)cn3)cc2CN2CCCC2=O)cn1
InChIKey: ISVQLFKEZJXRNG-UHFFFAOYSA-N
Target: Hexokinase-4 (SINGLE PROTEIN), Mus musculus
UniProt: P52792
Assay: Activation Assay: The final assay volume was 200 uL and the final buffer conditions used in this assay were as follows: 25 mM HEPES, 5 mM glucose, 1 mM ATP, 2 mM MgCl2, 1 mM NAD, 1 mM DTT, 8.5 U/mL G6PDH, 100 nM glucokinase, and 25 mM KCl. The buffer pH was 7.1. The mixture A was first made containing KCl, MgCl2, DTT and Glucose in HEPES buffer. The mixture B was made containing NAD and ATP. The test compound in DMSO solution, the mixture A, the mixture B and G6PDH were first mixed in a 96-well plate. Glucokinase was then added to initiate the reaction, and the absorbance at 340 nm was monitored every 5 mins. The activity of glucokinase was represented by the initial generation rate of glucose-6-phosphate, which was calculated by drawing the slope values from the absorbance changing curve at 340 nm vs. the time points. Compounds of this invention have an EC50 value as measured using the assay described herein of less than 50 μM.
Measurement: EC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 482.52, ALogP 3.24, HBA 8, HBD 2, PSA 138.79, QED 0.5