lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)Nc1cc(-c2cnc(C)c(NS(=O)(=O)CC(F)F)c2)ccn1
InChIKey: ARTRNNGIVOEYSJ-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: Enzyme Assay : 100 nL compounds in DMSO are added to wells of a 384 well microtitre plate (Greiner 780076). At room temperature: 5 ul VPS34 reaction buffer (Invitrogen Assay Buffer Q (diluted 1 in 5 with nanopure water) plus 2 mM DTT and 2 mM MnCl2) containing ATP (20 uM, Promega) and 200 uM PI-PS substrate (Invitrogen PV5122) is added followed immediately by 5 ul VPS34 reaction buffer (as above) containing VPS34 (5 nM, Millennium Protein Sciences Group) and the mixture is incubated with shaking at room temperature for 1 hour. Then 5 ul VPS34 stop-detect mix (as per Invitrogen Adapta Assay kit (PV5009) instructions (contains kinase quench buffer, TR-FRET buffer, Adapta Eu anti-ADP antibody and Alexa Fluor 647 ADP tracer)) is added to quench the reaction. The plates are then incubated for 30 minutes at room temperature with shaking and then read on a BMG PheraStar Plus reader.For the assay methods described above, test compound percent inhibition, at various concentrations, is calculated relative to control (DMSO and EDTA) treated samples. Compound concentration versus percent inhibition curves are fitted to generate IC50 values. One skilled in the art will appreciate that the values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 370.38, ALogP 2.42, HBA 5, HBD 2, PSA 101.05, QED 0.81
Compound (SMILES): Cn1nc2c(c1-c1cc(F)c(F)c(F)c1)C[C@@H]1CCC[C@H]2N1C(=O)c1cn(C)c2nccnc12
InChIKey: SPFPKFQCQBRRBD-SCLBCKFNSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition. Standard conditions for this assay combine 300 nM [Glycerol-1,3-3H]-oleoyl glycerol with human MGL from HeLa cells and test compounds for one hour, after which the reaction is filtered through activated carbon and tritium is measured in the flow through. The test compound concentration in screening mode is 10 uM, while the highest compound concentration in IC50 assays is determined empirically.
Measurement: IC50 = 97.2 nM (pChEMBL 7.010000228881836)
Descriptors: MW 466.47, ALogP 4.08, HBA 6, HBD 0, PSA 68.84, QED 0.42
Compound (SMILES): CCc1nn(CCCOC(=O)c2ccc3c(c2)OCO3)c2c1C(=O)NCC1(CCOCC1)C2
InChIKey: HNTPIFOUXVXQFB-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: Enzyme Activity Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [5380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference compound. Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggers time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 455.51, ALogP 2.5, HBA 8, HBD 1, PSA 100.91, QED 0.53
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(-c3cnc4c(c3)OCO4)nn2-c2ccccc2)[C@H](c2ccnc(F)c2)O1
InChIKey: LFZRUGHTWHWDLG-BWKNWUBXSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: Seven point five μL per well of human TrkA (PV3144, Lifetechnologies, final concentration: 1 nmol/L) suspended in the assay buffer (100 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20 and 1 mmol/L dithiothreitol (DTT)) was applied in a 384 well plate and the plate was pre-incubated for 15 min at room temperature with 0.4 μL of each compound (final concentration: 200 μmol/L-1 pmol/L) dissolved in DMSO. Then, fluorescent substrate (FL-peptide 27, 760424, PerkinElmer, final concentration: 1.5 μmol/L) and ATP (final concentration 500 μmol/L) dissolved in the assay buffer was added in each well. After the incubation of 120 min at 37° C., fifteen μL of termination buffer (100 mmol/L HEPES, 40 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20, 1 mmol/L DTT and 0.16 vol % Coating Reagent 3) was added in each well to stop the enzyme reaction. Fluorescent intensities (FI) of phosphorylated and non-phosphorylated fluorescent substrates were measured by LabChip EZReader II (Caliper LifeSciences, Inc.), and conversion ratio (CR) was calculated by the following formula-1. The CR in the well applied with DMSO alone was used as a negative control and the CR in the well without applying TrkA was used as a positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 561.57, ALogP 3.63, HBA 10, HBD 2, PSA 124.89, QED 0.31
Compound (SMILES): COC[C@H](C)NC(=O)O[C@@H]1CC[C@H](c2cc(NC(=O)Cc3ccccc3)[nH]n2)C1
InChIKey: UXZCCJJBACVFJP-BHYGNILZSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 1.29 nM (pChEMBL 8.890000343322754)
Descriptors: MW 400.48, ALogP 2.99, HBA 5, HBD 3, PSA 105.34, QED 0.63
Compound (SMILES): CC[C@H](Nc1cc(C(F)F)c(-c2sc(C(=O)N[C@H]3CC[C@@H]3O)nc2C(=O)N2CCC[C@@H]2C)cn1)C(F)(F)F
InChIKey: ZJRCOGYJBLLVTP-DDWPSWQVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows:    0.065 mg/mL RORγt    60 μM 1,8-ANS    100 mM Hepes, pH 7.0    10 mM NaCl    2.5 mM GSH    0.002% Tween-20
Measurement: Kd = 3.4 nM (pChEMBL 8.470000267028809)
Descriptors: MW 561.58, ALogP 4.77, HBA 7, HBD 3, PSA 107.45, QED 0.4
Compound (SMILES): CNCC(OCc1ccccc1CN1CCN(C)c2nc(SC)ncc2C1=O)c1ccccc1
InChIKey: GXDYPAYAMKRVHY-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay: α2δ-1: Binding Assay to Human α2δ-1 Subunit of Cav2.2 Calcium Channel.Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 477.63, ALogP 3.77, HBA 7, HBD 1, PSA 70.59, QED 0.37
Compound (SMILES): CCOc1nc2c(CS(C)(=O)=O)cc(-c3cc(OC)c(=O)n(C)c3)cc2n1CC1CC1
InChIKey: HVWHLXVJFVUUBU-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BREM was cloned, expressed and purified to homogeneity. Filipakopoulos et al., 468 Nature 1067 (2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotinlabeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at RT, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 445.54, ALogP 2.76, HBA 8, HBD 0, PSA 92.42, QED 0.53
Compound (SMILES): CCCCC(c1ccccc1)n1c(C)cc2c1ccc1nnc(C)n12
InChIKey: MLOVORHOPWVNTN-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 332.45, ALogP 5.08, HBA 4, HBD 0, PSA 35.12, QED 0.51
Compound (SMILES): C[C@@H](C(=O)O)[C@H](C)n1c(=O)[nH]/c(=N\c2ccc3ncccc3c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: AJWDNXVTTUNZHS-CABCVRRESA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 325 nM (pChEMBL 6.489999771118164)
Descriptors: MW 479.92, ALogP 3.1, HBA 7, HBD 2, PSA 122.34, QED 0.44
Compound (SMILES): Cc1nnn(Cc2cc(Cl)ccc2/C=C/C(=O)N2CCC(CCCc3nnc(C)o3)CC2)n1
InChIKey: DORAKWBSJMWNSZ-VQHVLOKHSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: ATX Assay: Reagents LPC (oleoyl (18:1)) was purchased from Avanti Polar Lipids (Alabaster, Ala.) and solubilized in methanol to 20 mM. Amplex Red was obtained from Invitrogen Life Technologies (Paisley, UK) and dissolved in DMSO to 10 mM. Choline oxidase and horseradish peroxidase (HRP) were obtained from Sigma Aldrich (Dorset, UK) and dissolved in HBSS to 20 U/ml and 200 U/ml respectively. All reagents were stored at −20° C. in single use aliquots. All experimental measurements were performed in assay buffer made up immediately prior to use (HBSS, 0.01% BSA essentially fatty acid free).Protein Recombinant human ATX was prepared at Novartis (Basel, CH) in a human embryonic kidney (HEK) cell preparation, and stored in single use aliquots of 26 mg/ml (26 μM) stocks stored at −80° C.Method All experimental measurements were performed in black 384 well polystyrene (low volume, round bottom, Corning (3676)) plates. PerkinElmer EnVision (Fluorescence Intensity/Absorbance Monochromator) or Tecan Infinite 200 PRO series plate reader was used to detect change in fluorescent intensity.Assessing ATX inhibition ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.Data analysis Slope data was exported to Graphpad prism (Graphpad software, San Diego, Calif.)
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 469.98, ALogP 3.65, HBA 8, HBD 0, PSA 102.83, QED 0.46
Compound (SMILES): CC1(C)CC(=O)c2c([nH]c(-c3cc[n+](CCCO)cc3)c2Nc2ccccc2)C1
InChIKey: MBAIKLISIWSXOY-UHFFFAOYSA-O
Target: Mitotic checkpoint serine/threonine-protein kinase BUB1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43683
Assay: Kinase Assay: In a typical assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were tested in duplicate within the same microtiter plate. To this end, 100-fold concentrated compound solutions (in DMSO) were previously prepared by serial dilution (1:3.4) of 2 mM stocks in a clear low volume 384-well source microtiter plate (Greiner Bio-One, Frickenhausen, Germany), from which 50 nl of compounds were transferred into a black low volume test microtiter plate from the same supplier. Subsequently, 2 μL of Bub1 (the final concentration of Bub1 was adjusted depending on the activity of the enzyme lot in order to be within the linear dynamic range of the assay: typically 200 ng/mL were used) in aqueous assay buffer [50 mM Tris/HCl pH 7.5, 10 mM magnesium chloride (MgCl2), 200 mM potassium chloride (KCl), 1.0 mM dithiothreitol (DTT), 0.1 mM sodium ortho-vanadate, 1% (v/v) glycerol, 0.01% (w/v) bovine serum albumine (BSA), 0.005% (v/v) Trition X-100 (Sigma), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added to the compounds in the test plate and the mixture was incubated for 15 min at 22° C. to allow pre-equilibration of the putative enzyme-inhibitor complexes before the start of the kinase reaction, which was initiated by the addition of 3 μL 1.67-fold concentrated solution (in assay buffer) of adenosine-tri-phosphate (ATP, 10 μM final concentration) and peptide substrate (1 μM final concentration). The resulting mixture (5 μL final volume) was incubated at 22° C. during 60 min., and the reaction was stopped by the addition of 5 μL of an aqueous EDTA-solution (50 mM EDTA, in 100 mM HEPES pH 7.5 and 0.2% (w/v) bovine serum albumin) which also contained the TR-FRET detection reagents (0.2 μM streptavidin-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-phosho-Serine antibody [Merck Millipore, cat. #35-002] and 0.4 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no. AD0077, alternatively a Terbium-cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used]). The stopped reaction mixture was further incubated 1 h at 22° C. in order to allow the formation of complexes between peptides and detection reagents.
Measurement: IC50 = 326 nM (pChEMBL 6.489999771118164)
Descriptors: MW 390.51, ALogP 4.25, HBA 3, HBD 3, PSA 69, QED 0.55
Compound (SMILES): N#Cc1cc2c(=O)n(CCOc3ccc(Cl)cc3-c3cc(C(=O)O)cc(F)c3F)cnc2cn1
InChIKey: XZCWTOBOKZFIOY-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 482.83, ALogP 4.04, HBA 7, HBD 1, PSA 118.1, QED 0.44
Compound (SMILES): COc1ccc(-c2ccccc2)cc1NS(=O)(=O)c1c(C)nn(C)c1Cl
InChIKey: ZKIHLDUMYGZXEN-UHFFFAOYSA-N
Target: ATP-citrate synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P53396
Assay: ADP-Glo Assay: Materials and Instruments Used in the ADP-Glo AssayVendor Cat No.Materials ACL Sino Biological 11769-H07BCoANa2 Sigma C3144Potassium Citrate Sigma 89306ATP Promega V915BADP-GloTM Kinase Assay Promega V9102kitHEPES Life Technologies 15630080MgCl2 Sigma M1028Brij 35 detergent Merck 203728DTT Sigma 646563DMSO MP 196055Optiplate-384, White 384- Perkin Elmer 6007290well384 dilution plate Corning 3657Topseal A Perkin Elmer E534196-well plate Nunc 249944InstrumentPlate reader Perkin Elmer Envision 2104Centrifuge Eppendorf 5810RCompounds of the present invention were evaluated in an ADP-GLO assay as follows:a) Dilute cpd 1:3 in succession in DMSO by hand for each cpds for 12 ptsb) Add 0.1 μL diluted cpd solution to assay plate, each dose with 2 replicatesc) Centrifuge 1000 RPM for 1 mind) Add 5 μL ACL working solution to 384-well assay plate, centrifuge 1000 RPM for 1 mine) Incubate at 25° C. for 15 minf) Add 5 μL substrate working solution to initiate reactiong) Final ACL reaction concentrations: 3 nM ACL, 15 μM ATP, 3 μM CoA, 300 μM Citrate, 0.01% Brij35, 4 mM DTT, 1% DMSO;h) Reference final conc: 30 uM starting Conc, 3× dilution, 11+0 points. Test cpd conc: 30/100 uM starting Conc, 3× dilution, 11+0 points.i) Incubate at 25° C. for 60 minj) Add 10 μL ADP Glo reagent, centrifuge 1000 RPM for 1 mink) Incubate at 25° C. for 40 minl) Add 20 μL kinase detection reagent, centrifuge 1000 RPM for 1 minm) Incubate at 25° C. for 40 minn) Read on Envision for US LUM as RLU
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 391.88, ALogP 3.86, HBA 5, HBD 1, PSA 73.22, QED 0.72
Compound (SMILES): Cc1ccc(-n2nc3c(c2NC(=O)CS(C)(=O)=O)C(=O)N[C@@]2(CCc4cc(OC(C)C)ccc42)C3)nc1
InChIKey: AEVDVWXWEQAHSK-SANMLTNESA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: Inhibitory Activity: Solutions of the compounds of the present invention in DMSO were each aliquoted into 0.2-μL portions in a 384-well polystyrene microplate produced by Corning Incorporated, and 5 μL of an enzyme solution prepared with an assay buffer (100 mmol/L phosphate buffer (pH 7.4) containing 2 mmol/L DTT) and 5 μL of a substrate solution (100 mmol/L phosphate buffer (pH 7.4), 30 μmol/L 2-Oleoylglycerol, 10 μmol/L Oleoyl-CoA) were added thereto, and the resultant was stirred and centrifuged, and incubated in a moist chamber at room temperature for 1 hour. After enzymatic reaction, 50 μL of a quenching solution (containing 0.2 μmol/L Diolein-d5, 0.4% formic acid, and 50% isopropanol) containing Internal Standard (IS) was added to terminate the reaction, and the resultant was sealed in a plate produced by Shimadzu GLC Ltd., and then stirred and centrifuged, and measurement was performed by using an electrospray ionization method with a RapidFire360 and Agilent 6550 Q-TOF mass spectrometer. Diolein as a reaction product (P) of 2-Oleoylglycerol as the substrate and an ammonium adduct ion of the IS were detected, and the peak intensity ratio, P/IS, was calculated from the peak heights to evaluate the inhibitory activity. Inhibitory activities with/without addition of enzyme were defined as Control (+)/Control (−), respectively, and the respective % inhibitions were defined as 0% inhibition and 100% inhibition. The inhibitory activity was calculated from formula below with TIBCO Spotfire (produced by TIBCO Software Inc.):Inhibitory activity (%)=[1−{Sample−Control(−)}/{Control(+)−Control(−)}]*100where Sample indicates a peak intensity ratio: P/IS, when the compound of the present invention was added.
Measurement: IC50 = 7.69 nM (pChEMBL 8.109999656677246)
Descriptors: MW 523.62, ALogP 2.47, HBA 8, HBD 2, PSA 132.28, QED 0.51
Compound (SMILES): COc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N[C@H]1CN(C(=O)[C@@](C)(O)C(F)(F)F)CC[C@H]1F
InChIKey: SDCQLSKVRQBCTC-HKZYLEAXSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 25000 nM (pChEMBL 4.599999904632568)
Descriptors: MW 593.46, ALogP 2.38, HBA 9, HBD 3, PSA 147.97, QED 0.38
Compound (SMILES): C[C@H]1C[C@H](n2c(Nc3ccc(OC(F)(F)F)cc3)nc3cc(-c4nnn[nH]4)ccc32)CC(C)(C)C1
InChIKey: FGRWSNMADNLLTB-YOEHRIQHSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 R132H Biochemical Assay: mIDH1 catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption was measured by luminescent readout.The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μl and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μl of detection mix (0.75 μg/ml Luciferase, 0.02 U/ml Oxidoreductase, 4 μg/ml FMN, 2 μl/ml Decanal/Ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity. IC50 values are determined by interpolation from plots of relative luminescence versus inhibitor concentration.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 485.51, ALogP 6.25, HBA 7, HBD 2, PSA 93.54, QED 0.35
Compound (SMILES): C=CC(=O)N1CC(NC(=O)Cn2cc(C(=O)C(=O)OCC)c3cc(Br)ccc32)C1
InChIKey: HSRVJNBCGZGGOL-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay (2 hours): Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 2 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 22000 nM (pChEMBL 4.659999847412109)
Descriptors: MW 462.3, ALogP 1.66, HBA 6, HBD 1, PSA 97.71, QED 0.29
Compound (SMILES): CC(C)c1ccccc1-c1ncc2c(n1)N(Cc1ccc(C(=O)N3CCC(F)(F)C3)cc1)C(=O)C2
InChIKey: GUNRWPJLOQEWQW-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: HTS Assay: The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 10 μL of 2× enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2×Ub-Rh110 or preincubated with USP1 40 minutes prior to the addition of 10 μL of 2×Ub-Rh110 to compound plates. Plates were incubated stacked for 45 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 476.53, ALogP 4.84, HBA 4, HBD 0, PSA 66.4, QED 0.53
Compound (SMILES): Cc1ccc(Br)nc1NC(=O)[C@@H]1C[C@@]2(C)C[C@H]2N1C(=O)Cc1cccc(-c2nnn[nH]2)c1
InChIKey: ABGLPIANHIZUHA-DMPWYTOCSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of Complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 496.37, ALogP 2.89, HBA 6, HBD 2, PSA 116.76, QED 0.53
Compound (SMILES): O=C(Nc1cc(C2=CCC(NS(=O)(=O)CC3CC3)CC2)c2cc[nH]c2n1)C1CC1
InChIKey: KHZRDTAVIXSOTK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 414.53, ALogP 3.18, HBA 4, HBD 3, PSA 103.95, QED 0.65
Compound (SMILES): COc1nccnc1C(C(N)=O)c1cc(-c2ncnc3cc(N4CCOCC4)ccc23)c(F)cc1F
InChIKey: RKXNBPNEHZAEBV-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: Biochemical Assay: The kinase assay was carried out in streptavidin-coated 348-well microtitre flashplates. To this end, 1.5 ug of DNA-PK/protein complex and 100 ng of biotinylated substrate, such as, for example, PESQEAFADLWKK-biotin-NH2 (biotin-DNA-PK peptide), were incubated for 90 min at room temperature in a total volume of 36.5 ul (34.25 mM HEPES/KOH; 7.85 mM Tris HCl; 68.5 mM KCl; 5 uM ATP; 6.85 mM MgCl2; 0.5 mM EDTA; 0.14 mM EGTA; 0.69 mM DTT; pH 7.4) with 500 ng of DNA from calf thymus, 0.1 uCi of 33P-ATP and 1.8% of DMSO per well with and without the test compound. The reaction was stopped using 50 ul/well of 200 mM EDTA. After incubation for a further 30 min at room temperature, the liquid was removed. Each well was washed three times with 100 ul of 0.9% saline solution. A nonspecific reaction (blank value) was determined using 10 uM of an innate kinase inhibitor. The radioactivity measurement was carried out using a TopCount. IC50 values were calculated in RS1.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 492.49, ALogP 2.83, HBA 8, HBD 1, PSA 116.35, QED 0.44
Compound (SMILES): COC1CN(Cc2ccc(-n3cc(-c4nc(NC5CCN(S(C)(=O)=O)CC5)ncc4C(F)(F)F)nc3C)c(F)c2)C1
InChIKey: MPLLZAJLCSBEDL-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: HTRF Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Carna Product Number 04-165). Assays were conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) was incubated with the compounds of the Examples (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/mL BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions were stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals were read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data was analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 597.64, ALogP 3.46, HBA 9, HBD 1, PSA 105.48, QED 0.39
Compound (SMILES): O=C1Nc2ccccc2[C@]12C[C@H]2c1ccc2c(/C=C/c3ccc(CCN4CCCOC4)cc3)n[nH]c2c1
InChIKey: PCCFPIPMCFEBOP-DAGBDIRBSA-N
Target: Serine/threonine-protein kinase PLK4 (SINGLE PROTEIN), Homo sapiens
UniProt: O00444
Assay: Inhibition Assay: Active PLK4 was purified from an E. coli expression system as an amino terminal GST fusion of residues 1-391 of human PLK4. The protein was purified from clarified cell extracts after induction at 15° C. overnight using glutathione sepharose, gel permeation chromatography, and ion exchange (Resource Q). The resulting protein was dephosphorylated with lambda phosphatase (NEB cat #P0753), and resolved from the phosphatase using gluthione sepharose. The dephosphorylated GST-PLK4 was stored in aliquots at −80° C. until use.PLK4 activity was measured using an indirect ELISA detection system. Dephosphorylated GST-PLK4 (4 nM) was incubated in the presence of 15 μM ATP (Sigma cat #A7699), 50 mM HEPES-Na2+ pH 7.4, 10 mM MgCl2, 0.01% Brij 35 (Sigma cat #03-3170), in a 96 well microtitre plate pre-coated with MBP (Millipore cat #30-011). The reaction was allowed to proceed for 30 minutes, followed by 5 washes of the plate with Wash Buffer (50 mM TRIS-Cl pH 7.4 and 0.2% Tween 20), and incubation for 30 minutes with a 1:3000 dilution of primary antibody (Cell Signaling cat #9381). The plate was washed 5 times with Wash Buffer, incubated for 30 minutes in the presence of secondary antibody coupled to horse radish peroxidase (BioRad cat #1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and incubated in the presence of TMB substrate (Sigma cat #T0440). The colourimetric reaction was allowed to continue for 5 minutes, followed by addition of stop solution (0.5 N sulphuric acid), and quantified by detection at 450 nm with either a monochromatic or filter based plate reader (Molecular Devices M5 or Beckman DTX880, respectively).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 490.61, ALogP 5.33, HBA 4, HBD 2, PSA 70.25, QED 0.38
Compound (SMILES): CC1(C)C(=N)N[C@](C)(c2nc(Nc3cccc(Cl)c3F)ccc2F)CS1(=O)=O
InChIKey: ZKWCJXOVBHYDOX-SFHVURJKSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 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 3Ã¿ 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, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 ul of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 ul in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window. Inhibitor IC50 values are derived from non-linear regression analysis of concentration response curves. Ki values are then calculated from IC50 values using the Cheng-Prusoff equation using a previously determined um value of 8 uM for the QSY7-APPswe-Eu substrate at BACE1. The compounds of Examples 1, 2, and 3 were measured in this assay and each exhibited a Ki value of less than about 5 nM.
Measurement: Ki = 3198 nM (pChEMBL 5.5)
Descriptors: MW 428.89, ALogP 3.75, HBA 5, HBD 3, PSA 94.94, QED 0.69
Compound (SMILES): Cn1nc2c(c1-c1cc(F)c(F)c(F)c1)C[C@@H]1CCC[C@H]2N1C(=O)c1cnc2ncccn12
InChIKey: NHBPHKYINLMHPO-SUMWQHHRSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition. Standard conditions for this assay combine 300 nM [Glycerol-1,3-3H]-oleoyl glycerol with human MGL from HeLa cells and test compounds for one hour, after which the reaction is filtered through activated carbon and tritium is measured in the flow through. The test compound concentration in screening mode is 10 uM, while the highest compound concentration in IC50 assays is determined empirically.
Measurement: IC50 = 221 nM (pChEMBL 6.659999847412109)
Descriptors: MW 452.44, ALogP 3.84, HBA 6, HBD 0, PSA 68.32, QED 0.43
Compound (SMILES): CC(C)(O)[C@H](F)CN1Cc2cc(NC(=O)c3cnn4cccnc34)c(N3CCC([C@@H](O)C(F)F)CC3)cc2C1=O
InChIKey: GHALLSIHBGBCLC-FGZHOGPDSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 560.58, ALogP 2.89, HBA 8, HBD 3, PSA 123.3, QED 0.39
Compound (SMILES): COC1(c2ccnc(C(N)=O)c2)C2CCCC1CN(Cc1cnc[nH]1)C2
InChIKey: PKDLVSTWEFLSNG-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Competitive Displacement Assay: Membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed one type of the cloned human opioid receptor were incubated with 12 different concentrations of the compound in the presence of 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole or 1 nM [3H]U69,593 in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO and [3H]U69,593. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 μM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. For [3H]naltrindole and [3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for at least 60 min before use.
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 355.44, ALogP 1.68, HBA 5, HBD 2, PSA 97.13, QED 0.85
Compound (SMILES): O=C(NC1CCOCC1)c1cn(Cc2ccccc2F)c(Nc2ccc(Oc3cccc(F)n3)cc2)nc1=O
InChIKey: LORCYRYAWQHGOH-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 3000 cells/well and cultured in the medium (7.0% fetal bovine serum, 7.0% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 1.37 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH7.5), and each well was added with 20 μL of this buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 384 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound.
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 533.54, ALogP 4.41, HBA 8, HBD 2, PSA 107.37, QED 0.32