lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(O)CCc1ccc(Oc2ccc(F)c3c2CC[C@H]3Oc2ccc3c(c2)OC[C@H]3CC(=O)O)c(C#N)c1
InChIKey: UOWWYLPFEMWLKO-NFQMXDRXSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Pharmacological Assay: IP1 accumulation measurements using the IPOne assay system 1321N1 cells stably expressing human GPR40 receptor (Euroscreen, Belgium) are seeded 24 h before the assay in white 384-well plates in culture medium containing 10% FCS, 1% Na-Pyruvate and 400 μg/mL G418. IP1 is assayed according to the manufacturer's description (Cisbio Bioassays, France). In brief, the assay is started by substitution of the culture medium by stimulation buffer (Hepes 10 mM, CaCl2 1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM and LiCl 50 mM, pH 7.4). Cells are stimulated for 1 h at 37° C., 5% CO2 by addition of the compounds that are diluted in stimulation buffer containing LiCl. Assays are stopped by adding HTRF-conjugates (IP1-d2 and Anti-IP1 cryptate Tb) and lysis buffer, provided by the manufacturer. After an incubation time of 1 h at room temperature plates are measured using an EnVision, Perkin Elmer. The obtained fluorescence ratios at 665/615 nM are then used to calculate the pEC50 values using Assay Explorer 3.3 Software (Accelrys, Inc.) by interpolation using an IP1 reference curve and subsequent sigmoidal curve fitting allowing for a variable hill slope.
Measurement: EC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 531.58, ALogP 6.21, HBA 6, HBD 2, PSA 109.01, QED 0.34
Compound (SMILES): O=C1NCc2nc(-c3c(F)cccc3F)cc(Nc3ccc4cc[nH]c4n3)c21
InChIKey: LJSAASODZSKIGQ-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Assay: Tyk2 & JAK2: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 377.35, ALogP 3.89, HBA 4, HBD 3, PSA 82.7, QED 0.51
Compound (SMILES): Cc1ccc(-c2ncccn2)c(C(=O)N2CCC[C@@]2(C)c2nc3cc(F)c(Br)cc3[nH]2)c1
InChIKey: GAKMDUDMVGELOF-DEOSSOPVSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 494.37, ALogP 5.38, HBA 4, HBD 1, PSA 74.77, QED 0.42
Compound (SMILES): O=C(Nc1nc2cccc(-c3ccc(OCc4ccc(N5CCOCC5)nc4)cc3)n2n1)C1CC1
InChIKey: XUQSPYPFYODQAR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Inhibition Assay: Recombinant human JAK1 catalytic domain (amino acids 850-1154; catalog number 08-144) was purchased from Carna Biosciences. 10 ng of JAK1 was incubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Tween-20, 10 mM MgCl2, 2 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 45 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 470.53, ALogP 3.56, HBA 8, HBD 1, PSA 93.88, QED 0.44
Compound (SMILES): CCOC(=O)CCN1CC=C(c2ccn3cc(-c4cc(Cl)c(OC)cc4CO)nc3c2)CC1
InChIKey: DXOHJRLRTHBDNS-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase SUV39H2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H5I1
Assay: Suv 39H2 Assay: Assay buffer (4 μL) was added into all the wells, including test compound and control wells (except for Sinefungin control wells), using the Multidrop Combi. The plate was centrifuged at 1000 rpm for 1 min. Substrate mix (8 μL) containing radiolabelled 3H-SAM (final conc: 100 nM) and H3 Histone Peptide (final conc: 350 nM) was added to all wells using Multidrop Combi and centrifuged at 1000 rpm for 1 min. SUV39H2 (8 μL, final conc: optimized for each lot of the enzyme based on specific activity) was added by using the Multidrop Combi. The assay plate was centrifuged at 1000 rpm for 1 min. Assay buffer was used as background control, and incubated at room temperature for 3 hours. The reaction was stopped with 20 μL of 2.5 mg/mL Streptavidin SPA beads (final conc. 50 μg/well) in buffer using the Multidrop Combi and centrifuged for 1 min. The plate was loaded into the Trilux-Microbeta counter and a delayed read of 10 hours was made. The radioactive signal was measured at 1 min/well. Sinefungin and the internal standard compound were used as tool compounds to determine assay performance.
Measurement: IC50 = 57.8 nM (pChEMBL 7.239999771118164)
Descriptors: MW 469.97, ALogP 4.2, HBA 7, HBD 1, PSA 76.3, QED 0.5
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](NS(=O)(=O)N2CC(C)(C#N)C2)C1
InChIKey: DMYHORFMURCVKW-TXEJJXNPSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 375.46, ALogP 0.6, HBA 6, HBD 2, PSA 118.01, QED 0.79
Compound (SMILES): C[C@@H](Cn1c(=O)[nH]/c(=N\c2ccc(OCC(F)F)cc2)n(Cc2ccc(Cl)cc2)c1=O)C(=O)O
InChIKey: YHSATROPGBERGQ-ZDUSSCGKSA-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 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 494.88, ALogP 2.64, HBA 7, HBD 2, PSA 118.68, QED 0.47
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)C5(C#N)CC5)CC4)cc3c2C(C)C)ccn1
InChIKey: TVRCSJUBWREGRU-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 19.7 nM (pChEMBL 7.710000038146973)
Descriptors: MW 426.56, ALogP 5.67, HBA 3, HBD 1, PSA 72.78, QED 0.58
Compound (SMILES): CN(CC1CC1)c1nc(=O)n(-c2ccccc2Cl)c2cc(C3CC3)ccc12
InChIKey: DBIWMXGRVPDYFS-UHFFFAOYSA-N
Target: S-adenosylmethionine synthase isoform type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31153
Assay: Biochemical Assay: Enzyme: MAT2AhMAT2A: 50 nM, Cepter, 10 mg/mL (234 μM), amino acids 1-395Substrates: 500 uM eachReaction time: 1 hourL-methionine Substrate: Alfa Aesar catalog #J61904ATP Substrate: Alfa Aesar cat #J60336Malachite Green Detection Reagent: Millipore Sigma catalog #MAK307-1KTAssay buffer: 50 mM Tris, pH 7.5/50 mM KCl/10 mM MgCl2/0.01% Brij-35/1 mM DTT/0.1% BGGTemperature: 23° C.Total volume: 20 μLControls:0% inhibition control: DMSO100% inhibition control: No enzymeProcedure:5 μL of 3×final concentration test compounds in DMSO or DMSO were transferred to the appropriate wells of a microtiter plate and the plate was centrifuged at 1000 rpm for 1 minute. 5 μL of 3×final concentration MAT2A enzyme in assay buffer or assay buffer alone was transferred to the appropriate wells and the plate was centrifuged at 1000 rpm for 1 minute. The plate was incubated at room temperature for 15 minutes and then 5 μL of 3×the L-methionine and ATP substrate mixture in assay buffer was transferred to all the test wells. The plate was centrifuged at 1000 rpm for 1 minute and then incubated at room temperature for 1 hour. 5 μL of malachite green detection reagent was added to all the test wells and the plate was centrifuged at 1000 rpm for 1 minute and then incubated at room temperature for 30 minutes. The plate was read for absorbance at 620 nm on a plate reader (e.g., Infinite M1000). The high control (DMSO) with high absorbance represents no inhibition of enzymatic reaction while the low control (no enzyme) with low absorbance represents full inhibition of enzymatic reaction.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 379.89, ALogP 4.76, HBA 4, HBD 0, PSA 38.13, QED 0.64
Compound (SMILES): CN1CCN(c2ccc(-c3n[nH]c4ccc(-c5cccc(C(=O)NCc6ccccc6)c5F)nc34)cc2)CC1
InChIKey: IGJOTQAANYCAJP-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech). Ki values were determined.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 520.61, ALogP 5.11, HBA 5, HBD 2, PSA 77.15, QED 0.33
Compound (SMILES): CN(C)C(=O)CNC(=O)C[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1
InChIKey: DNVFTQKCWFAOGH-KRWDZBQOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 25.4 nM (pChEMBL 7.590000152587891)
Descriptors: MW 599.53, ALogP 3.24, HBA 5, HBD 2, PSA 107.02, QED 0.48
Compound (SMILES): O=C(Nc1ccccc1-c1ccc(F)cc1)OC[C@H]1CCCN1
InChIKey: ZCOICOYJSZTIHV-OAHLLOKOSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: Binding Assay: Cell membrane proteins (Perkin Elmer) wherein human muscarinic M3 receptor was overexpressed, [3H]-methyl scopolamine and test compounds in various concentration were cultured in 0.2 ml of Tris-HCl buffer at 25° C. for 120 minutes. The same was filtered under suction through glass fiber filter (Whatman GF/B), and then the filter was washed 5 times with 1 ml of Tris-HCl buffer. The radioactivity of [3H]-methyl scopolamine adsorbed on the filter was measured by a liquid scintillation counter. Non-specific binding was evaluated under existence of 5 μM of atropine. Affinity of the compound of the present invention to muscarinic M3 receptor was calculated as the dissociation constant (Ki), which can be calculated from concentration (IC50) of test compounds inhibiting 50% of binding of [3H]-methyl scopolamine (i.e. labeled ligand) according to Cheng and Prusoff [Cheng and Prusoff, Biochem. Pharmacol., 22, 3099, 1973]. In following Table, compounds having stronger binding affinity to human muscarinic M3 receptor have lower dissociation constant (Ki).
Measurement: Ki = 99.7 nM (pChEMBL 7)
Descriptors: MW 314.36, ALogP 3.79, HBA 3, HBD 2, PSA 50.36, QED 0.9
Compound (SMILES): COC(=O)N1c2ccc3c(nc(CCn4cccn4)n3CC(=O)NC3CCOCC3)c2CC[C@@H]1C
InChIKey: JMWZNARCPYKHGB-KRWDZBQOSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: HTRF Biochemical Assay for CBP and BRD4 Activity: The assay was performed in a final volume of 6 μL in assay buffer containing 50 mM Hepes (pH 7.5, (0.5M Hepes, pH 7.5 solution; Teknova H1575)), 0.5 mM GSH, 0.01% BGG (0.22 μM filtered, Sigma, G7516-25G), 0.005% BSA (0.22 μM filtered, EMD Millipore Corporation, 126575) and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO were pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 33 μM to 1.7 nM, top to lowest dose, respectively. 3 of 2× Protein and 3 μL of 2× Peptide Ligand were added to assay plates (pre-stamped with compound). Plates were incubated for varying times at room temperature prior to measuring the signal. TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) was measured on the PHERAstar (BMG, equipped with HTRF optic module [337/520/490]) or on the Envision (PerkinElmer, equipped with the TRF Laser unit, TRF dual mirror D400/D505 and emission filters M520 and M495). Data were reported as percent inhibition compared with control wells based on the following equation: % inh=1−((TR-FRET ratio−AveLow)/(AveHigh−AveLow)) where TR-FRET ratio=(Fluorescence at 520 nm/Fluorescence at 490 nm)*10000), AveLow=average TR-FRET ratio of no enzyme control (n=32), and AveHigh=average TR-FRET ratio of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm. For all assay formats data were reported as percent inhibition compared with control wells based on the following equation: % inh=100*((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured Fluorescence, AveLow=average Fluorescence of no enzyme control (n=32), and AveHigh=average Fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model 205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 480.57, ALogP 2.68, HBA 8, HBD 1, PSA 103.51, QED 0.58
Compound (SMILES): CC(C)c1cc(C(=O)N2Cc3ccccc3C2)c(O)cc1C(C)O
InChIKey: RKFGDFRJEFWGIT-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-beta (SINGLE PROTEIN), Homo sapiens
UniProt: P08238
Assay: Florescence Polarization Assay: Assay was performed in 96-well format in black, flat bottom plates (Santa Cruz Biotechnology) with a final volume of 100 μL. 25 μL of assay buffer (20 mM HEPES, pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 2 mM DTT, 0.1 mg/mL BGG, and 0.01% NP-40) containing 6 nM FITC-GDA (fluorescent tracer, stock in DMSO and diluted in assay buffer) and 50 μL of assay buffer containing 10 nM of either Grp94 or Hsp90α were added to each well. Compounds were tested in triplicate wells (1% DMSO final concentration). For each plate, wells containing buffer only (background), tracer in buffer only (low polarization control), and protein and tracer in buffer with 1% DMSO (highpolarization control) were included. Plates were incubated at 4° C. with rocking for 24 h. Polarization values (in mP units) was measured at 37° C. with an excitation filter at 485 nm and an emission filter at 528 nm. Polarization values were correlated to % tracer bound and compound concentrations. The concentration at which the tracer was 50% displaced by the inhibitor was determined using Graphpad Prism.
Measurement: Kd = 4870 nM (pChEMBL 5.309999942779541)
Descriptors: MW 325.41, ALogP 3.72, HBA 3, HBD 2, PSA 60.77, QED 0.9
Compound (SMILES): Cc1c(Cc2c(CF)nc3c(C(N)=O)cc(N4CCOCC4)nn23)cccc1C(F)(F)F
InChIKey: SPVHGYQLKIPPPO-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00750
Assay: KINOMEscan Enzyme Binding Assays: Kinase enzyme binding affinities of compounds disclosed herein were determined using the KINOMEscan technology performed by DiscoveRx Corporation, San Diego, Calif., USA (www.kinomescan.com).
Measurement: Kd = 10 nM (pChEMBL 8)
Descriptors: MW 451.42, ALogP 3.05, HBA 6, HBD 1, PSA 85.75, QED 0.6
Compound (SMILES): COc1ccc(Cn2cc(C(=O)NCC(=O)O)c(=O)c3ccccc32)cc1
InChIKey: YCBSIGKTKLKJGW-UHFFFAOYSA-N
Target: Atrial natriuretic peptide receptor 3 (SINGLE PROTEIN), Mus musculus
UniProt: P70180
Assay: Vasoreactivity in Rat Mesenteric Arteries In Vitro: Male rats (Sprague-Dawley; 200-250 g) were stunned and killed by cervical dislocation. The mesentery was removed and third-order arteries mounted in an automated tension myograph (Danish Myotechnology). After an equilibration period of 45 min, vessels were normalized and diameter determined. Following normalization, each vessel was contracted repeatedly with the thromboxane A2-mimetic 9,11-dideoxy-11α,9α-epoxymethano-prostaglandin F2α (U46619; 1 μM) until the response was reproducible. The vessels were then washed to restore basal tone before contracting to approximately 50% of the maximum U46619-induced response. Once a stable response to U46619 was achieved, cumulative concentration-response curves were constructed with a variety of NPR-C agonists of the invention at varying concentrations (0.001-30 μM), or vehicle (DMSO). In some studies, concentration-response curves to NPR-C agonists were conducted in the presence of the NPR-C antagonist M372049 (10 μM). Only one curve to any one agonist was constructed in any single tissue.
Measurement: EC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 366.37, ALogP 1.87, HBA 5, HBD 2, PSA 97.63, QED 0.69
Compound (SMILES): COc1ccnc2[nH]cc(C3CCN(c4nc(OC[C@H]5C[C@H]5C#N)nc(C(=O)N[C@H](C)C(C)(C)O)n4)CC3)c12
InChIKey: FNMIASMCJFGIJW-BPQIPLTHSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL enzyme inhibition (% inhibition, Kiapp and Ki values) by small molecule inhibitors was evaluated using a fluorescence-based microfluidic mobility shift assay. AXL catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-30 (5-FAM-KKKKEEIYFFF-CONH2, CPC Scientific, Sunnyvale, Calif.). 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. Human wild-type receptor tyrosine kinase protein Axl comprising residues 505-811 was produced in-house using the baculoviral expression vector system that incorporated a hexahistidine affinity tag into the protein (LJIC-1916B1.1). The enzyme was preactivated by auto-phosphorylation of 34 uM non-activated enzyme in the presence of 2 mM ATP, 4 mM MgCl2, 50 mM NaCl and 1 mM TCEP in 20 mM HEPES, pH 7.3 at 4° C. for 30 minutes. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 120 μM ATP (ATP Km=70.4 μM), 0.01% Tween-20, 3 μM FL-Peptide-30, and 0.5 nM phosphorylated AXL enzyme in 100 mM HEPES buffer at pH 7.3. 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 30 minutes at 25° C. by the addition of 50 μL of 200 mM EDTA, pH 7.5. The Ki values were determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable. (See, Morrison, J. F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochimica et biophysica acta 185, 269-286; and Murphy, D. J. (2004) Determination of accurate KI values for tight-binding enzyme inhibitors: an in silico study of experimental error and assay design, Analytical biochemistry 327, 61-67.)
Measurement: Ki = 0.032 nM (pChEMBL 10.489999771118164)
Descriptors: MW 534.62, ALogP 2.57, HBA 10, HBD 3, PSA 162.17, QED 0.37
Compound (SMILES): N#Cc1cc(-c2ccnc3[nH]c(-c4cccc(N5CCOCC5)c4)nc23)ccc1OC1CCOCC1
InChIKey: KZSBBJDMPIERRN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assay for TBK1 and IKKs: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKs were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2× Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 25.1 nM (pChEMBL 7.599999904632568)
Descriptors: MW 481.56, ALogP 4.56, HBA 7, HBD 1, PSA 96.29, QED 0.45
Compound (SMILES): C[C@@H]1CN(c2nc3cc(NC(=O)c4c(-c5nc(C(F)F)cs5)cnn4C)ccn3n2)C[C@H](C)O1
InChIKey: BNQLPJHSXJSEGZ-TXEJJXNPSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Phosphodiersterase Evaluation Assay: IMAP TR-FRET Phosphodiersterase Evaluation Assay Kit (Molecular Device) was used for the measurement. Ten μL of a diluted test compound having each concentration and 5 μL of the enzyme PDE10 (BPA Bioscience) that had been diluted to 2 ng/mL with 1×IMAP Reaction Buffer (prepared with 5×included with the kit, 10 mM Tris-HCl, pH=7.2, 10 mM MgCl2, 0.05% NaN3, and 0.1% BSA) containing 0.1% BSA were added to a 384-well plate (Corning). The obtained mixture was then pre-incubated at room temperature for 5 minutes. Thereafter, 5 μL of cAMP substrate solution which was included with the kit and diluted to 400 nM with 1×IMAP Reaction Buffer containing 0.1% BSA, was added to the reaction solution, and the obtained mixture was reacted at room temperature for 60 minutes. Thereafter, 60 μL of an IMAP TR-FRET Binding solution included with the kit was further added to the reaction solution, and the obtained mixture was then left for 3 hours or more. Subsequently, using ARVO SX (PerkinElmer), the fluorescent intensity of Terbium (Emission=490 nm) and TR-FRET (Emission=520 nm) in the reaction solution were measured at an excitation wavelength of 340 nm, so as to calculate the amount of the generated 5′-AMP. The count of a well to which a solvent had been added instead of the test compound was set at 0%, and the count of a well to which the enzyme PDE10 had not been added was set at 100%. Thus, the inhibitory activity of each test compound was calculated.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 488.52, ALogP 3.39, HBA 10, HBD 1, PSA 102.47, QED 0.46
Compound (SMILES): Cc1ccc(NC(=O)c2cc(C(F)(F)F)ccn2)cc1-c1cc(N2CCOCC2)c(OC2CCOCC2)nn1
InChIKey: NRNGEHBPDHSOOR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 543.55, ALogP 4.51, HBA 8, HBD 1, PSA 98.7, QED 0.49
Compound (SMILES): O=C(c1ccc(F)cc1)N1CCn2c(nnc2-c2csc(-c3ccc(Br)cc3)n2)C1
InChIKey: KIKODLIBEQBCIZ-UHFFFAOYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: 3H-SB222200 Binding Competition Assay with Human NK-3 Receptor: The ability of the compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells which express the human NK3 receptor. The membranes were incubated with 5 nM 3H-SB222200_(ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 Mm/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of the compounds of the invention. The amount of tritiated SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentrations of compounds which displaced 50% of bound radioligand (IC50) were determined and then apparent inhibition constant Ki values were calculated by the following equation: Ki=IC50/(1+[L]/KD) where [L] is the concentration of free radioligand and KD is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973).
Measurement: Ki < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 484.35, ALogP 4.63, HBA 6, HBD 0, PSA 63.91, QED 0.43
Compound (SMILES): CC(C)(O)c1cnc2cc(OCc3cccc(Cl)c3)cnc2c1
InChIKey: HIGSFBHYANKCKN-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Enzymatic Assay: The MAO enzymatic assay was performed according to the fluorometric method described by Matsumoto and colleagues (Matsumoto et al., Clin. Biochem. 1985, 18, 126-129) with the following modifications. Human recombinant MAO-A and MAO-B expressed in insect cells were used. For both assays, test compound and/or vehicle were preincubated with purified enzyme in phosphate buffer pH 7.4 for 15 minutes at 37° C. The reaction was initiated by addition of 50 μm kynuramine. Following a 60 minute incubation period, the reaction was terminated by the addition of 6 N NaOH. The amount of 4-hydroxyquinoline that formed was determined by spectrofluorimetrically at 325 nm/465 nm.
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 328.8, ALogP 4.09, HBA 4, HBD 1, PSA 55.24, QED 0.78
Compound (SMILES): O=C(O)c1c(O)c(=O)n(Cc2cnn(Cc3ncc4ccccn34)c2)c2ccsc12
InChIKey: FMNMDXDRHRXYKW-UHFFFAOYSA-N
Target: Flap endonuclease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P39748
Assay: Enzymatic IC50 Assays: FEN1, EXO1 or XPG enzyme was incubated with compound or vehicle (DMSO) and the FAM-labeled DNA oligomer substrate in a microtiter plate. The stop buffer contains EDTA to stop the enzymatic reaction. The plate is read for fluorescence intensity. The high control (DMSO) with high fluorescence intensity represents no inhibition of enzymatic reaction while the low control (10 μM) with low fluorescence intensity represents full inhibition of enzymatic reaction.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 421.44, ALogP 2.41, HBA 9, HBD 2, PSA 114.65, QED 0.45
Compound (SMILES): COc1cc2nccc(Oc3ccc(NC(=O)c4c[nH]c(=O)[nH]c4=O)cc3F)c2cc1OC
InChIKey: FRPFVCADSKMWNW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Kinase Assay: The ability of compounds to inhibit the kinase activity of recombinant human baculovirus-expressed AXL was measured by homogeneous TRF (HTRF) using Cisbio's KinEASE assay system in white 384-well Optiplates. Assay buffer contained 1 mM DTT, 2 mM MnCl2, 2% DMSO, 50 nM supplement enzymatic buffer, and 1× enzymatic buffer. A 2× concentration of tyrosine kinase (TK) substrate-biotin/ATP mixture made in assay buffer was added to plates at 10 μL/well using the Multidrop Combi (Thermo Fisher Scientific, Waltham, Mass.). The final concentrations were 0.3 μM TK substrate-biotin, and 1.3 μM ATP. Compounds (100 nL), diluted in 100% DMSO on the Biomek FX, (Beckman Coulter, Inc., Brea, Calif.), were transferred to the assay plates using the Biomek FX pintool (2.5% final DMSO in assay). A 2× concentration (final=12 ng/mL) of GST-AXL (diluted in assay buffer) was added to plates at 10 uL/well using the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 30 minutes. A 4× stock of Streptavidin-XL665 (final=18.8 nM) and a 1:100 diluted stock of TK antibody-cryptate were made in HTRF detection buffer and mixed together just prior to adding 20 μL/well on the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 1 hour. The fluorescence of the resulting solution was measured using the PerkinElmer EnVision 2102 multi-label plate reader (PerkinElmer, Waltham, Mass.) with an excitation wavelength of 337 nm (laser) and emission wavelengths of 590 and 665 nm. Raw data was expressed as the ratio of 665/590×10,000.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 452.4, ALogP 2.81, HBA 7, HBD 3, PSA 135.4, QED 0.41
Compound (SMILES): Cc1cccc(NC(=O)Nc2ccc(Oc3ccnc(-c4cc(C(=O)N=S(C)(=O)CCCC(=O)O)c[nH]4)c3)cc2)c1
InChIKey: CDVOVVMOSZWTNG-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: FLIPR Assay: Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 96-well fibronectin coated black-walled plates overnight at 37.degree. C./5% CO.sub.2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37.degree. C. Cells were washed 4 times (Original Cell Wash, Labsystems) to remove extracellular dye. Test compounds were reconstituted in 100% DMSO and added to the cells to give a final DMSO concentration of 0.1%. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 .mu·M) or at concentrations ranging from 0.01 to 10.0 .mu·M followed by VEGF stimulation (5 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 96 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples. IC.sub.50 values for test compounds were calculated from % inhibition of VEGF stimulated responses in the absence of inhibitor.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 575.65, ALogP 5.92, HBA 6, HBD 4, PSA 162.84, QED 0.18
Compound (SMILES): COc1cc2c(cn1)C(=O)N(CCN1CCN(c3nsc4ccccc34)CC1)CC2
InChIKey: FOQOXYFYHFUCMT-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Binding Activity Assay: Binding affinity of the present compound for human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor was measured by the following procedures.CHO cell membrane fraction in which human 5-HT1A receptor, human 5-HT2A receptor, and human D2 receptor were expressed was purchased from PerkinElmer, Inc. In a test for evaluating binding affinity, a test compound dissolved in dimethylsulfoxide (DMSO) and each receptor membrane sample diluted in buffer were mixed with [3H]8-OH-DPAT, [3H]Ketanserin, or [3H]Spiperone (all purchased from PerkinElmer, Inc.) for 5-HT1A receptor, 5-HT2A receptor, and D2 receptor, respectively. Each mixture was incubated at room temperature for 60 minutes. Then, the mixture was added quickly on a glassfiber filter plate (Multiscreen FB, Millipore, Inc.) coated with 0.3% aqueous polyethylenimine, and vacuum-filtered. Radioactivity remaining on the filter was measured with a liquid scintillation counter (PerkinElmer, Inc.). Binding inhibition rate was calculated from the following formula.
Measurement: Ki = 0.03 nM (pChEMBL 10.520000457763672)
Descriptors: MW 423.54, ALogP 2.52, HBA 7, HBD 0, PSA 61.8, QED 0.63
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CCC(=O)Nc1ccc(S(C)(=O)=O)cn1)C(=O)N2CC1CCC1
InChIKey: YAVIRWXVHJDMHB-RNPORBBMSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Receptor Binding Assay: MOP: The hMOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl (pH 7.4) supplemented with 0.052 mg/ml bovine serum albumin (Sigma-Aldrich Co. St. Louis. Mo.). The final assay volume (250 μl/well) included 1 nM of [N-allyl-2.3-3H]naloxone as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 25 μM unlabelled naloxone for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 90 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac. Turku. Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]naloxone-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation.
Measurement: Ki = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 567.76, ALogP 4.12, HBA 6, HBD 1, PSA 102.92, QED 0.49
Compound (SMILES): CN(C)C1CN(C(=O)C2CCc3c(-c4ccc(C(=O)O)cc4F)nn(C(=O)c4c(Cl)cccc4C4(C(F)(F)F)CC4)c3C2)C1
InChIKey: NYSJYBZYTHJVTD-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 mg/ml bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per ml in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 ml SF9 lysate per 15 ml of diluted RORγ-LBD.Compounds to be tested were injected to the 384-well assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, CA).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-SPSSHSSLTERHKILHRLLQEGSP) (SEQ ID NO:1) and APC-conjugated streptavidin (final concentrations 100 nM and 8 nM respectively) were also added to each well.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 633.04, ALogP 5.2, HBA 6, HBD 1, PSA 95.74, QED 0.38
Compound (SMILES): Cn1cc(-c2cccc(-c3cc(C(F)(F)F)c(-c4cnn(C5CCOCC5)c4)cn3)c2)cn1
InChIKey: KPNWQXHBZBTLOB-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P51617
Assay: Enzymatic Assay: IRAK1 is a human purified recombinant enzyme (His-TEV-IRAK1 (194-712)). In this assay, IRAK-1 hydrolyses ATP and autophosphorylates. Measurement of IRAK-1 inhibition was performed in streptavidin coated 384well FlashPlate (PerkinElmer #SMP410A).His-TEV-IRAK-1 (15 ng/well), ATP (1 μM, [33P]ATP 0.25 μCi/well) and compounds in DMSO (range of concentrations from 20 M to 1 nM) or controls (2% DMSO) were incubated for 3 hours at 30° C. in assay buffer: Hepes pH7.0 50 mM, Fatty acid-free BSA 0.1%, Dithiothreitol DTT 2 mM, MgCl2 10 mM, EGTA 0.5 mM, Triton-X-100 0.01%. Kinase reaction was stopped by addition of EDTA. Supernatant was discarded, plates were washed three times with 150 mM NaCl and radioactivity was then measured in a Microbeta Trilux reader.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 453.47, ALogP 5.38, HBA 6, HBD 0, PSA 57.76, QED 0.42
Compound (SMILES): CC(C)N(C)C/C=C/C(=O)N1CCC[C@@H](n2nc(C(=O)Nc3nc4cc(Cl)ccc4o3)c3c(N)ncnc32)C1
InChIKey: ONUKEVKZBUDRTK-QQOXCAACSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK Inhibitory Activity: With regard to the setting of the conditions for a method for measuring the inhibitory activity of a compound against BTK kinase activity in vitro, it is described in the consumable reagent supplies price list for LabChip (registered trademark) series of PerkinElmer, Inc. that FL-PEPTIDE 2 corresponds to a substrate peptide for the measurement of BTK kinase activity. Therefore, FL-PEPTIDE 2 was used as a substrate. The purified recombinant human BTK protein used in the test was purchased from Carna Biosciences, Inc.With regard to the measurement of the inhibitory activity of the compounds, firstly, the compounds of the present invention were diluted stepwise with dimethyl sulfoxide (DMSO). Subsequently, BTK protein, a substrate peptide (final concentration was 1 μM), magnesium chloride (final concentration was 10 mM), ATP (final concentration was 45 μM), and a DMSO solution of the compounds of the present invention (final concentration of DMSO was 5%) were added to a buffer solution for kinase reaction (20 mM HEPES (pH 7.5), 2 mM dithiotheitol, 0.01% Triton X-100), and after the solution was incubated for 40 minutes at 25° C., a kinase reaction was carried out. The reaction was terminated by adding EDTA thereto in order to obtain a final concentration of 30 mM. Finally, a substrate peptide that was not phosphorylated (S) and a phosphorylated peptide (P) were separated and detected by microchannel capillary electrophoresis using a LabChip EZ Reader II (PerkinElmer, Inc.). The amounts of phosphorylation reaction were determined from the respective peak heights of S and P, and the compound concentration at which the phosphorylation reaction could be suppressed in 50% was defined as the IC50 value (nM).
Measurement: IC50 = 0.788 nM (pChEMBL 9.100000381469727)
Descriptors: MW 552.04, ALogP 3.52, HBA 10, HBD 2, PSA 148.3, QED 0.33