lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cn1cc(-c2cc3nccnc3c(-c3cnn(C4(CC#N)CN(CC(F)(F)F)C4)c3)n2)cn1
InChIKey: YHVGXUJDQWWVRZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% 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 JAK 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% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 169 nM (pChEMBL 6.769999980926514)
Descriptors: MW 453.43, ALogP 2.78, HBA 9, HBD 0, PSA 101.34, QED 0.46
Compound (SMILES): Cn1cnc(-c2c(Cl)csc2NC(=O)Cn2c(=O)ccn3nc(C(F)(F)F)cc23)n1
InChIKey: SJQZGOJOGORHFG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: P45983
Assay: HTRF Assay: (1) Assay buffer #1 with 0.0025% Tween and 1 mM DTT was prepared using JNK buffer stock solution.(2) Assay buffer #2 with 0.025% BSA was prepared using the assay buffer #1.(3) Test compound solution preparation: 5× compound solution was prepared using assay buffer #1 with 5% DMSO. The compound solution (10 μl/well) was added to a 384-well plate (Corning, Cat No. 3654).(4) aJNK1, 2 or 3 preparation: aJNK stock (100 μg/ml) at −80° C. was thawed on ice and an aJNK (1.6 ng/ml) solution was prepared using assay buffer #2. The aJNK solution (20 μl/well) was added to the plate and the plate was shaken. The enzyme was incubated with the compound at RT for 10 min.(5) ATP/substrate solution preparation: ATP and substrate stocks were thawed on ice. 2.5×ATP/substrate (e.g., 25 μM or 2.5 mM ATP/50 nM EPIW-1) was prepared using assay buffer #1. The ATP/substrate (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 15 min. Note: In an exemplary assay, the final ATP concentration was about 1 mM.(6) EDTA preparation: 30 mM EDTA was prepared using 0.5M EDTA stock and assay buffer #1. The EDTA (10 μl/well) was added to the plate to quench the enzyme reaction and the plate was shaken well.(7) Detection reagent preparation: Eu-63 and SA-XL stocks were thawed on ice. 4× Eu-63/SA-XL (2 nM Eu-63/9.4 nM SA-XL) was prepared using JNK detection buffer. The Eu-63/SA-XL solution (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 1 hr before reading the plate on LJL using ratiometric method named HTRF.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 457.83, ALogP 2.66, HBA 9, HBD 1, PSA 99.11, QED 0.51
Compound (SMILES): O=C1NN=C(c2ccc(-c3ccc(F)cc3)c(F)c2)CO1
InChIKey: UGUKSHRHUHJCCM-UHFFFAOYSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13370
Assay: PDE3B Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3B reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3B cell extract from Sf9 cells overexpressing human full length PDE3B (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3B cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:6000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3B activity vs log compound concentration.
Measurement: IC50 = 79 nM (pChEMBL 7.099999904632568)
Descriptors: MW 288.25, ALogP 3.08, HBA 3, HBD 1, PSA 50.69, QED 0.92
Compound (SMILES): Nc1ccc(-c2ccc(F)cc2)cc1NC(=O)N1CCOCC1
InChIKey: ISDWDTRBQFDROY-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Inhibition of Histone Deacetylase Enzymatic Activity Assay: HDAC2: The following trypsin-coupled protocol and Caliper protocol described Herein was used to assay the compounds of the invention. HDAC2-Kinetic IC50 3Hr PreIncubation. Working Stocks: 1xHDAC Assay Buffer: 50 mM HEPES (GIBCO 15630-114), 100mM KCl, 0.001% Tween-20 (Zymed 00-3005), 0.005% BSA (Invitrogen, P2489), pH 7.4, 1.5xHDAC+1.5xTrypsin: 225nM Trypsin(Worthinton)+0.2 ug/ml HDAC2 (BPS Inc) in assay buffer 300x Trypsin stock is made up in HDAC Assay Buffer, aliquoted and stored at -80C. 3xSubstrate: 13.8uM Substrate in assay buffer BPS Enzyme and Substrate amounts (per well).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 315.35, ALogP 2.94, HBA 3, HBD 2, PSA 67.59, QED 0.84
Compound (SMILES): C=CC(=O)N1CCC(Oc2cc3c(Nc4cccc(C5CC5)c4)c(C#N)cnc3cc2OC)CC1
InChIKey: LFYFLKNDADLTIG-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 67 nM (pChEMBL 7.170000076293945)
Descriptors: MW 468.56, ALogP 5.29, HBA 6, HBD 1, PSA 87.48, QED 0.47
Compound (SMILES): Cn1nc(Oc2c(Cl)cc(NC(=O)c3noc(=O)[nH]3)cc2Cl)cc(C(C)(C)O)c1=O
InChIKey: MSDWHBYJZBEWSU-UHFFFAOYSA-N
Target: Thyroid hormone receptor alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P10827
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for Thyroid Hormone Receptor Agonist Screening: LanthaScreen TR-FRET Thyroid Receptor alpha Coactivator Assay kit (ThermoFisher) and LanthaScreen TR-FRET Thyroid Receptor beta Coactivator Assay kit (ThermoFisher) were used for agonist compound screening. Compounds in DMSO were diluted using ECHO Liquid Handler (Labcyte Inc.) into 384 plates in 10-point 3-fold series in duplicate (5 micro M final top concentration). Buffer C (ThermoFisher) was added to each well before the 4× mixture of fluorescein-SCR2-2 coactivator (200 nM final concentration), Terbium-labeled anti-GST antibody (2 nM final concentration), and TR alpha-LBD (0.4 nM final concentration) or TR beta-LBD (1.0 nM final concentration) was added. After 2 hour incubation at room temperature in dark, the TR-FRET signal was measured on an EnVision plate reader (PerkinElmer) with excitation at 340 nm and dual emission readout at 495 and 520 nm with the delay time of 100 micro second and the integration time of 200 micro second. The ratio of emission signal at 520 and at 495 was used to calculate EC50 using GraphPad Prism (GraphPad Software). In every batch of compound screening, T3 (L-3,3′,5-Triiodothyronine sodium salt, >95%) (Calbiochem) was used as reference compound. The EC50 of T3 measured were within 3-fold of the reference value provided by the assay kit manufacturer (ThermoFisher Scientific). The Z′ factors measured in every batch of screening using T3 as high percent effect (HPE) control and 0.5% DMSO as zero percent effect (ZPE) control were in the range of 0.5 to 0.8. Compounds' THR-beta selectivity values are derived from T3-selectivity normalized data.
Measurement: EC50 = 212 nM (pChEMBL 6.670000076293945)
Descriptors: MW 456.24, ALogP 2.04, HBA 9, HBD 3, PSA 152.34, QED 0.53
Compound (SMILES): C[C@H]1CCN1c1nc(-c2cnn(C3CCN(CCO)CC3)c2)cc(C(F)(F)F)c1C#N
InChIKey: GRLCTYXHGPVHAY-AWEZNQCLSA-N
Target: Ketohexokinase (SINGLE PROTEIN), Homo sapiens
UniProt: P50053
Assay: KHK Enzyme Activity Assay for Human KHK-C and Human KHK-A: The intrinsic potency for inhibition of KHK C or A activity may be measured using an enzymatic assay which measures the production of FIP. Compounds are prepared in DMSO and tested in a 10-point concentration curve, to create 3-fold serial dilutions of the compounds in a 96-well plate ranging from 20 μM to 1.02 nM. Enzyme is prepared in assay buffer [50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 10 mM potassium chloride, 100 mM magnesium chloride, 2 mM tris(2-carboxyethyl)phosphine (TCEP), 0.01% n-octyl glucoside] and incubated with compounds at RT for 15 min. The reaction is carried out in 100 μL volumes containing substrate concentrations of fructose (250 μM for KHK-C assay and 1.25 mM for KHK-A assay) and ATP (150 μM for both isoforms); which are further incubated at RT for 20 min. The reaction is then halted by the addition of stop buffer; consisting of 0.2% formic acid and 1 μg/ml 13C6-fructose-6-phosphate (13C6-F6P) internal standard. Plates are stored in −20° C. until RapidFire MS analysis.RapidFire MS Analysis for Quantitation of F1PAn Agilent 300 RapidFire automated extraction system (Agilent, Santa Clara, Calif.) with three HPLC quaternary pumps is coupled to an Agilent 6495 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, Calif.) equipped with an electrospray ionization (ESI) interface source. The RapidFire Mass Spec system is equipped with a reusable RapidFire C18 (type C) solid-phase extraction (SPE) cartridge (G9205 Å).Solvent A, used for sample loading and washing, is 6 mM octylamine (Acros Organics 129495000) brought to pH 5.0 using acetic acid. Solvent B, used for sample elution, is 20% water in ACN containing 0.1% formic acid. Samples are sequentially analyzed by aspirating 10 μL onto the collection loop under vacuum directly from multiwell plates. The 10 μL of sample is loaded onto the C18 cartridge and washed using solvent A at a flow rate of 1.25 mL/min for 5000 ms. The retained analytes are then eluted to the mass spectrometer using solvent B at a flow rate of 1.25 mL/min for 5000 ms. The system is re-equilibrated using solvent at flow rate of 1.25 mL/min for 2000 ms.The triple quadrupole mass spectrometer is equipped with an ESI source and analytes are monitored using selected reaction monitoring (SRM) in negative mode [M−H]−. F1P is monitored at m/z 259.02/96.9 and 13C6-fructose-6-phosphate is monitored at m/z 264.99/97. The area ratio values for F1P is calculated using 13C6-fructose-6-phospate as internal standard.
Measurement: IC50 = 3.58 nM (pChEMBL 8.449999809265137)
Descriptors: MW 434.47, ALogP 3.06, HBA 7, HBD 1, PSA 81.21, QED 0.78
Compound (SMILES): CC(C)CC[C@H](O)[C@@H](C)[C@H]1CC[C@H]2[C@@H]3CC[C@H]4C[C@@](C)(O)CC[C@]4(C)[C@H]3CC[C@]12C
InChIKey: HSRUDVSBHVBZAR-XACJTUTOSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: Whole-Cell Patch Clamp: Whole-Cell Patch Clamp of Mammalian Cells (Ionworks Barracuda (IWB)):The whole-cell patch-clamp technique was used to investigate the effects of positive allosteric modulating activity of test compounds on_GlunN1/GluN2A and GluN2B glutamate receptors expressed in mammalian cells.
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 418.71, ALogP 6.83, HBA 2, HBD 2, PSA 40.46, QED 0.51
Compound (SMILES): C=CC(=O)N1CCN(CCn2c(=O)ccc3cnc(Nc4cnn(CCOC(C)C)c4)nc32)[C@@H](CC)C1
InChIKey: WGNFUQJJSPOGCS-QFIPXVFZSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical JAK and Tyk2 Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies. Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 508.63, ALogP 2.27, HBA 10, HBD 1, PSA 110.41, QED 0.39
Compound (SMILES): O=C(NC1CCCNC1)c1ccc(Nc2nc3c(C4=CCN(C(=O)CCC(F)(F)F)CC4)cccn3n2)cc1
InChIKey: KKMFCRLTJUKVKR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.79 nM (pChEMBL 9.100000381469727)
Descriptors: MW 541.58, ALogP 3.91, HBA 7, HBD 3, PSA 103.66, QED 0.42
Compound (SMILES): Cc1c([C@@H](C)Nc2nnc(C)c3cnc(N4CC(O)C4)cc23)cccc1C(F)(F)F
InChIKey: IWNSYDPRACCXER-GFCCVEGCSA-N
Target: Son of sevenless homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07889
Assay: Inhibiting SOS1-Mediated GTP Nucleotide Exchange by Blocking Mant-GDP Exchange: Table 24: This Example illustrates that exemplary compounds of the present invention prevent KRas-mediated GTP nucleotide exchange mediated by SOS1 to inhibit KRas activity thereby inhibiting the generation of the downstream effector pERK. MKN1 cells (15,000/w) or H358 (30,000/w) were seeded in a black clear flat bottom 96-well cell culture plate (Corning, #3904) and incubated at 37° C. overnight. Assay day 1, cells were dosed with compounds of Formula (I) with a 10 μm starting concentration and serially diluted 3× for a total of 9 concentrations. The cells were incubated for approximately 0.5-1 hour with the compounds solubilized in DMSO at 37° C. Cells were immediately fixed by adding 50 μL of 4% formaldehyde to all wells in a fume hood and the plates were incubated for 20 minutes at room temperature. The formaldehyde was discarded from the plates and 150 μL of ice-cold methanol was added to permeabilize the cells for 10 minutes at −20° C. The methanol was discarded from each of the plates and any liquid remaining in the plate by tapping the plate against paper towels. Cells were then blocked with 150 μL of Odyssey blocking buffer (LI-COR Biosciences #927-50010) using 0.05% Tween for 1 hour at room temperature on a shaker. The blocking buffer was discarded and 50 μL of primary antibodies pERK (cell signaling Technology #9101L; Rabbit, 1:500) and GapDH (Millipore #MAB34; Mouse, 1:5000) diluted in Odyssey blocking buffer was added. The plates were incubated overnight at 4° C. on a shaker. On Assay Day 2, the primary antibody solution was removed. Each plate was washed 3× times with 150 μL of 1×PBST (PBS+0.1% Tween 20) and incubated with 50 μL of secondary antibodies: Anti-Rabbit (LI-COR Biosciences #926-32211) and Anti-Mouse (LI-COR Biosciences #68070) at 1:800 dilution in Odyssey blocking buffer with Tween at room temperature on a shaker for 2 hours (protected from light). The secondary antibody solution as removed and each plate was washed with PBST 3× times. Any liquid remaining was discarded and the plate was imaged using the Licor Odyssey machine according to the manufacturer's instruction, using a set focus length at 3 mm and both 800 nm and 700 nm filters. The GAPDH normalized scan values for each well were divided by the average of vehicle wells to get the % of pERK inhibition. The IC50 values were then calculated with the Graph pad Prism software.
Measurement: IC50 = 89 nM (pChEMBL 7.050000190734863)
Descriptors: MW 417.44, ALogP 4.01, HBA 6, HBD 2, PSA 74.17, QED 0.67
Compound (SMILES): O=S(=O)(c1ccccc1O)N1CCC(c2c3cc(C(c4ccc(Cl)cc4)c4ccc(Cl)cc4)ccc3nn2C2CC2)CC1
InChIKey: AUENSHQKGCMLFH-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Biological Assay: CB1 and CB2 receptors are Gi-coupled GPCR. Activation of CB1 and CB2 receptors results in a decrease in cAMP production. An inverse agonist of the CB1 or CB2 receptor results in the opposite effect, an increase of cAMP production. The principle of this assay is based on HTRF® technology (Homogeneous Time-Resolved Fluorescence). The method is a competitive immunoassay between native cAMP produced by cells and the cAMP labeled with the fluorophore d2. The tracer binding is quantified by a Mab anti-cAMP labeled with Eu3+TBP-NHS Cryptate (supplied as part of the assay kit). The specific signal (i.e. energy transfer) is inversely proportional to the concentration of cAMP in the standard or sample.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 632.61, ALogP 8.13, HBA 5, HBD 1, PSA 75.43, QED 0.18
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCN(C(=O)NC5CCNC5)CC4)cc3c2C(C)C)cn2ncnc2c1C
InChIKey: RANQGXKGDXSCCL-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 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. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 24.4 nM (pChEMBL 7.610000133514404)
Descriptors: MW 499.66, ALogP 4.87, HBA 5, HBD 3, PSA 90.35, QED 0.37
Compound (SMILES): CN1C(=N)N[C@@]2(CCOc3ccc(NC(=O)c4ccc(OC(F)F)cn4)cc32)CS1(=O)=O
InChIKey: GNEDVTBZOIDBEW-IBGZPJMESA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: BACE-2 Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data is normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values. IC50s are determined by nonlinear regression analysis (sigmoidal dose response, variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent respectively.
Measurement: Ki = 166 nM (pChEMBL 6.78000020980835)
Descriptors: MW 467.45, ALogP 1.71, HBA 7, HBD 3, PSA 133.71, QED 0.62
Compound (SMILES): CC[C@@H](NC(=O)c1cc(=O)[nH]c(C2CC2)n1)c1ccc(OC(F)(F)F)cc1
InChIKey: QROKVPJKWNQRKZ-CYBMUJFWSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 381.35, ALogP 3.43, HBA 4, HBD 2, PSA 84.08, QED 0.8
Compound (SMILES): COCCS(=O)(=O)N1[C@@H]2CC[C@H]1C[C@H](Nc1nc(Nc3cc(C)[nH]n3)cc3ncccc13)C2
InChIKey: UXKCYKVFLDQKFE-OSYLJGHBSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 471.59, ALogP 2.79, HBA 8, HBD 3, PSA 125.13, QED 0.46
Compound (SMILES): COC(=O)N1Cc2cc(S(=O)(=O)C3CC3)ccc2C1C(=O)Nc1ccc(C(O)(C(F)(F)F)C(F)(F)F)cc1
InChIKey: QFGGZNQAGOPWFT-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: FRET Assay: The assay was run in black 384 well plates (Greiner cat no: 784900). Various concentrations of test ligands in 0.1 microlitres DMSO were dispensed to assay plates using an Echo acoustic dispenser. Two pre-mixes were prepared and incubated for 1 h at room temp in the dark. Pre-mix 1 comprised 100 nM Protein (Biotinylated HN-Avi-MBP-TCS-hRORÎ³ (258-518)) and 60 nM Streptavidin APC in assay buffer, 50 mM MOPS pH7.4, 50 mM KF, 0.003% (w/v) CHAPS, 10 mM DTT and 0.01% (w/v) BSA and pre-mix 2 comprised 160 nM biotinylated SRC-1 peptide (NCOA1-677-700) and 20 nM Europium-W8044 labelled Streptavidin in assay buffer. Five microlitres of pre-mix 2 was dispensed to assay plates containing 0.1 microlitres of test compound and was incubated for 15 minutes prior to adding five microlitres of pre-mix 1. Plates were incubated at room temperature for 1 hour in the dark, prior to reading in a Pherastar multi-mode plate reader using HTRF filter set (ex 320, em 612 and 665). The FRET signal at 665 nm was divided by the signal at 612 nm and multiplied by 10,000 to generate a signal ratio value for each well. The raw data was transformed to % effect using the equation:Compound % effect=100*[(Xâmin)/(maxâmin)],where X represents the normalized value for the compound based on the Min (vehicle) and Max (reference compound) inhibition control.
Measurement: IC50 = 251 nM (pChEMBL 6.599999904632568)
Descriptors: MW 566.48, ALogP 4.2, HBA 6, HBD 2, PSA 113.01, QED 0.53
Compound (SMILES): CC#Cc1cncc(-c2cc([C@]3(C)CC4(CCOCC4)OC(N)=N3)sc2C)c1
InChIKey: KFRODYATXDEEIY-NRFANRHFSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: FP Assay: Compounds are also assessed for BACE1 and Cathepsin D activity using an FP Assay. Compounds to be assessed (e.g. compounds as described in the above examples) are serially diluted 1:3 with DMSO and each concentration point is transferred to a 96 well plate, 6 uL per well, followed by addition of 194 uL of assay buffer (100 mM sodium acetate, pH 4.5 with 0.001% Tween-20). A 10 uL compound sample from this plate is transferred to a well of a 384 well plate and combined with 10 uL per well of BACE1 enzyme (0.3 nM in assay buffer) or Cathepsin D enzyme (1.8 nM in assay buffer) and the plate is incubated at room temperature for 30 minutes. Substrate (0.45 uM for BACE1 or 0.45 nM for Cathepsin D in assay buffer) is added, 10 uL per well, and the plate is incubated at 37° C., 3 hours for BACE1 or 2 hours for Cathepsin D. The reaction is stopped by adding 30 uL per well of cold 1.5 uM Streptavidin Immunopure and incubated for 15 minutes at room temperature and the fluorescent signal determined, excitation 485-20 and Emission 530-25, and the value as a function of compound concentration is used to determine the IC50.
Measurement: IC50 = 1120 nM (pChEMBL 5.949999809265137)
Descriptors: MW 395.53, ALogP 3.99, HBA 6, HBD 1, PSA 69.73, QED 0.78
Compound (SMILES): N#Cc1ccc(Cl)cc1-c1cc(=O)n(C(C[C@@H]2CCCCO2)C(=O)Nc2ccc(C(N)=O)nc2)cc1Cl
InChIKey: LNQGJYZTZGLLPG-XNUZUHMRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: The factor XIa inhibition of the substances according to the invention is determined using a biochemical test system which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). 1 μl of the diluted substance solutions is placed into each of the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride; 5 mM of calcium chloride; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 mM of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 540.41, ALogP 4.33, HBA 7, HBD 2, PSA 140.1, QED 0.46
Compound (SMILES): OC(c1ccc(F)c(-c2ncnc3cc(N4CCOCC4)ccc23)c1)c1ncnc2c1CCNC2
InChIKey: CCPJVMRJOCCOET-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Patch Clamp Assay: The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
Measurement: Ki = 12500 nM (pChEMBL 4.900000095367432)
Descriptors: MW 472.52, ALogP 2.79, HBA 8, HBD 2, PSA 96.29, QED 0.47
Compound (SMILES): COc1ccc(Nc2cc(-c3ccccc3OC)ncn2)cc1NC(=O)C(C)(C)C
InChIKey: VIURZHLUCZXIHD-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: In Vitro CDK9/CyclinT Assay: The activity of the compounds described in the present invention can be determined by measuring the phosphorylation of a fluorescently-labeled peptide by human CDK9/CyclinT kinase complex by fluorescent polarization using a commercially available IMAP Screening Express Assay Kit (Molecular devices).Test compounds were diluted in 100% DMSO to 5 mM stock concentration, then further dilutions were made in H2O or 100% DMSO to desirable concentrations.Each reaction consisted of 5 nM enzyme: CDK9/CyclinT (Proqinase cat#0371-0345-1), 400 nM TAMRA-Rbtide (synthetic 15-mer peptide derived from human retinoblastoma tumor suppressor protein labeled with TAMRA dye, Genecust Europe), 12 μM ATP (=Kmapp, Sigma-Aldrich) and kinase buffer: 20 mM MOPS pH 7 (Sigma-Aldrich), 1 mM DTT (Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich), 0.01% Tween 20 (Sigma-Aldrich).For each reaction, 4 or 6 μl containing TAMRA-Rbtide, ATP and kinase buffer were combined with 2 μl diluted compound in H2O or 0.028 μl compound in 100% DMSO. The kinase reaction was started by the addition of 2 μl diluted enzyme. The reaction was allowed to run for 1 hour at room temperature. The reaction was stopped by adding 15 μl IMAP beads (1:400 beads in progressive (100% buffer A) 1× buffer). After an additional 1 hour, fluorescent polarization (Ex: 550-10 nm, Em: 590-10 nm, Dich: 561 nm) was measured using an Analyst GT (Molecular devices).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 406.49, ALogP 4.89, HBA 6, HBD 2, PSA 85.37, QED 0.61
Compound (SMILES): COc1cc(C(=O)NC2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ncc1C#N
InChIKey: PJJXLIVFRBOUIZ-NRFANRHFSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibitory Activity Assay: The method described hereafter is used for determining the inhibitory activity of the present compounds on hERG1. Materials and Instruments(1) FluxOR potassium ion channel assay (F10016, invitrogen)(2) FlexStation3 microplate reader (molecular devices)(3) hERG/HEK293 cell: HEK293 cell line stably expressing the hERG channel transfected by hERG cDNA (NCBI SEQ ID NO. NM-000238(RC215928, origene)).2. Experimental ProcedureExcept for ddH2O, all of the experimental reagents are from FluxOR Potassium Ion Channel Assay Kit and the formulation methods also refer to the kit instructions.(1) Human hERG/HEK293 cell was seeded on PDL (Poly-D-lysine) coated plates at 25000 cells/well on the previous day;(2) After overnight culture, the plate medium was discarded; then according to FluxOR potassium ion channel detection requirements operation, the dye was added at 100 μL/hole, and then incubated for 90 mins at room temperature;(3) The dye was then decanted and 100 μL of assay buffer containing 1004, probenecid were added in each well;(4) 1 μL of compound or DMSO was added to the corresponding wells, shocked for 30 seconds, and incubated for 30 mins at room temperature;(5) The plates were placed in a FlexStation3 microplate reader, and then added with stimulation buffer (K2SO4: Tl2SO4: 1×FluxOR Chloride-free Buffer: ddH2O=2:1:2:5) at 25 μL/well, then the value was read continuously for 5 mins at EX/EM of 490/525 nm immediately; and(6) The IC50 of the present compounds on human hERG ion channel was obtained by data processing software Graphpad.
Measurement: IC50 = 43700 nM (pChEMBL 4.360000133514404)
Descriptors: MW 450.5, ALogP 1.87, HBA 8, HBD 2, PSA 124.78, QED 0.64
Compound (SMILES): CC(C)(C)c1ccc(C(=O)C(C#N)NNc2ccccc2)cc1
InChIKey: XHJCKFFRUFGZEM-UHFFFAOYSA-N
Target: Rap guanine nucleotide exchange factor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8WZA2
Assay: In Vitro Evaluation of EPAC2 Inhibition: From the biological results discussed above, compounds 10, 14-15, 23-24, 26-27, and 31-32 were identified as potent EPAC1 inhibitors with IC50 values lower than 8 μM and more potent than reference compound 1. Therefore, these selected compounds together with hit 1 were further evaluated for their ability to inhibit EPAC2-mediated Rap1b-bGDP exchange activity. As shown in Table 3, the previous hit 1 is 2.5-fold more potent on EPAC2 inhibition than that on EPAC1, with an IC50 value of 4.4 μM. Interestingly, almost all of our selected, newly synthesized analogues exhibit significantly enhanced potency compared to that of 1, except compounds 23 and 24 (FIG. 3). Among these, compound 33 exhibits the best inhibitory activity for EPAC2, with an IC50 value of 1.9 μM (FIG. 3). Compounds such as 14, 26 and 32-33 with IC50 values lower than 4 μM for both EPAC1 and EPAC2 may serve as valuable pharmacological tools to probe the functions of EPAC in diseases or as potential drug candidates for further preclinical development.
Measurement: IC50 = 4400 nM (pChEMBL 5.360000133514404)
Descriptors: MW 307.4, ALogP 3.68, HBA 4, HBD 2, PSA 64.92, QED 0.65
Compound (SMILES): C=CC(=O)N1CCC(Nc2cnc3[nH]cc(C(=O)NCCCC(F)(F)F)c3n2)CC1
InChIKey: XMVHONKSUBHIAR-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.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 JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. 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 = 9.1 nM (pChEMBL 8.039999961853027)
Descriptors: MW 424.43, ALogP 2.62, HBA 5, HBD 3, PSA 103.01, QED 0.47
Compound (SMILES): CC=Cc1cnc(-c2cc(Cl)ccc2F)cc1Nc1ccncc1C(=O)N[C@@H](C)CO
InChIKey: POPIUCWMQJYOGO-AWEZNQCLSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: In Vitro Kinase Assay: Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle.
Measurement: IC50 = 10900 nM (pChEMBL 4.960000038146973)
Descriptors: MW 440.91, ALogP 4.82, HBA 5, HBD 3, PSA 87.14, QED 0.49
Compound (SMILES): CCS(=O)(=O)c1ccc(CNC(=O)c2cnc3c(c2)CN(CC2CCC(F)(F)CC2)[C@H]3C(C)C)nc1
InChIKey: NUAIJYRORBTTPY-DEOSSOPVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ROR gamma t 5xRORE Assay in Jurkat Cells (Assay 2): Compounds of the present invention were tested for ROR gamma inverse agonist activity in a cell-based, transcriptional activity assay. Secreted Nanoluc® luciferase was used as a reporter for transcriptional activity of the full-length ROR gamma t in Jurkat cells (ATCC, Cat. # TIB-152). A reporter plasmid was constructed by inserting 5 repeats of the ROR Response Element (RORE) AAAGTAGGTCA (SEQ ID NO:1) into a commercially available promoterless plasmid pNL1.3[secNluc] (Promega, Cat. # N1021) using KpnI and HindIII restriction sites. The expression plasmid for ROR gamma t was purchased (Geneocopoeia, Cat. # EX-T6988-M02). Jurkat cells (30 million cells) were transfected with 11 μg of EX-T6988-M02 and 26 μg of the reporter plasmid in OptiMEM® media using Lipofectamine LTX and Plus reagents (Life Technologies, Cat. #15338-100). After 5-6 hrs of incubation at 37° C./5% CO2, the cells were collected, resuspended in phenol-red free RPMI media containing 10% (v/v) delipidated FBS (Hyclone, Cat. # SH30855.03) and dispensed into 96-well clear bottom tissue culture plates (CoStar, Cat. #3603), at 80,000 cells per well. Tested compounds were added to the cells in the same media (final concentration of DMSO was 0.1% (v/v)), and the plates were incubated at 37° C./5% CO2 for 16-18 hrs. Luciferase activity in the conditioned supernatants was determined with NanoGlo assay reagents (Promega, Cat.# N1130). Percent inhibition values were calculated based on the fully inhibited and non-inhibited (DMSO) controls, and the values were regressed against concentrations of the tested compounds to derive IC50 values using a four-parameter non-linear fitting model.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 520.65, ALogP 4.54, HBA 6, HBD 1, PSA 92.26, QED 0.55
Compound (SMILES): CCOc1ccc(S(=O)(=O)N2C[C@@H](C)[C@@H](n3cc(CC(=O)O)c4ccc(-c5ncco5)cc43)C2)cc1
InChIKey: AWQCJXQAHCPIIO-OSPHWJPCSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Receptor Binding Inhibition Assay: Cell membrane was prepared from CRTH2-expressing K562 cell. The cell membrane (20 μg/well) was mixed with reaction buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2). Then, [3H]-PGD2 was added to the mixture and incubated for 60 min at room temperature. The concentration of [3H]-PGD2 was determined as equal to Kd calculated from Scatchard plot. Then, the mixture was filtrated with glass paper immediately. The filter paper was washed several times and radio activity on the filter was measured. Specific binding was defined as difference between total and non-specific binding (radio activity with 10 μM PGD2). Ki values for each compound were calculated from displacement curve.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 509.58, ALogP 4.2, HBA 7, HBD 1, PSA 114.87, QED 0.38
Compound (SMILES): O=C(Nc1cnn(Cc2ccccc2)c1)c1cc(-c2ccncc2)on1
InChIKey: NJGBDSGCWOKYIM-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86SK9
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 = 7000 nM (pChEMBL 5.159999847412109)
Descriptors: MW 345.36, ALogP 3.23, HBA 6, HBD 1, PSA 85.84, QED 0.6