lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@@H]1Cc2c(ccc3[nH]ncc23)[C@@H](c2ncc(NC3CN(CCCF)C3)cc2F)N1CC1(F)CC1
InChIKey: CZXRKQNLGWTZPP-CPJLOUKISA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Binding Assay : hERG (human ether go go-related gene) potassium channels are essential for normal electrical activity in the heart. Arrhythmia can be induced by a blockage of hERG channels by a diverse group of drugs. This side effect is a common reason for drug failure in preclinical safety trials and therefore minimisation of hERG channel blocking activity may be a desirable property for drug candidates.The purpose of the hERG binding assay is to evaluate the effects of test compounds on the voltage-dependent potassium channel encoded by the human ether go go-related gene (hERG) using a constitutively expressing CHO cell line on the Nanion Syncropatch 384PE automated patch clamp system.The assay was conducted as follows with all reagents used at room temperature unless otherwise stated.Reagent Preparations Include:1. Internal IC700 solution used to perfuse the underside of chip (in mM), KF 130, KCl 20, MgCl2 1, EGTA 10 and HEPES 10, (all Sigma-Aldrich; pH 7.2-7.3 using 10 M KOH, 320 mOsm) and supplemented with 25 □M escin.2. External and cell buffer (in mM), NaCl 137, KCl 4, HEPES 10, D-glucose 10, CaCl2 2, MgCl2 1 (pH7.4, NaOH)3. NMDG reference buffer used to establish a stable baseline prior to the addition of test compounds, NaCl 80, KCl 4, CaCl2 2, MgCl2 1, NMDG Cl 60, D-Glucose monohydrate 5, HEPES 10 (pH7.4 NaOH 298 mOsm)4. Seal enhancer used to improve seal quality of cells, NaCl 80, KCl 3, CaCl2 10, HEPES 10, MgCl2 1 (pH7.4 NaOH)
Measurement: IC50 = 29 nM (pChEMBL 7.539999961853027)
Descriptors: MW 484.57, ALogP 4.39, HBA 5, HBD 2, PSA 60.08, QED 0.5
Compound (SMILES): C=CC(=O)N1CCCN(C(=O)c2cc(Sc3cnc(NC(=O)C4CCN(C)CC4)s3)c(C)cc2OC)CC1
InChIKey: WMKUEDUMQQHADK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 557.74, ALogP 3.75, HBA 8, HBD 1, PSA 95.08, QED 0.52
Compound (SMILES): CN1CCN(C2CN(c3cnc4ccc(-c5ccc6oc(N)nc6c5)nc4c3)C2)CC1=O
InChIKey: DAZOFBRNHMHULZ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 429.48, ALogP 1.98, HBA 8, HBD 1, PSA 104.62, QED 0.53
Compound (SMILES): COc1cc(-c2[nH]c3ccc(C4CCN(C5CCOC5)CC4)cc3c2C(C)C)cn2nc(C)nc12
InChIKey: KFDNAYNROXABQC-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
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 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 473.62, ALogP 5.29, HBA 6, HBD 1, PSA 67.68, QED 0.43
Compound (SMILES): CNCCN(c1ccc2ncc(-c3cnn(C)c3)cc2n1)c1cc(OC)cc(OC)c1F
InChIKey: MJHFGTHWKHPARC-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR4 (Enzymatic Assay): In a final reaction volume of 30 μL, FGFR4 (h) (60 ng/ml) was incubated with 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.01% Triton-X-100, 500 nM Btn-Flt3 and 5 μM ATP in the presence of compound (1% DMSO final). After incubation for 60 minutes at room temperature the reaction was stopped with 2.27 nM EU-anti P-Tyr, 7 mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA which was present for 60 minutes at room temperature. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal (ex340 nm. Em 620 nm, em 655 nm) was measured afterwards and results are expressed in RFU (Relative Fluorescence Units). In this assay, the inhibitory effect of different compound concentrations (range 10 μM to 0.1 nM) was determined and used to calculate an IC50 (M) and pIC50 (−log IC50) value.
Measurement: IC50 = 5.01 nM (pChEMBL 8.300000190734863)
Descriptors: MW 436.49, ALogP 3.54, HBA 8, HBD 1, PSA 77.33, QED 0.45
Compound (SMILES): CNC(=O)C(=N)c1c(N)cc(-c2cc(F)c(O)cc2CC(F)(F)F)nc1NCc1cccnc1N(C)S(C)(=O)=O
InChIKey: FIUWLGOGPXZKNM-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: 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 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 = 243 nM (pChEMBL 6.610000133514404)
Descriptors: MW 583.57, ALogP 2.8, HBA 9, HBD 5, PSA 174.39, QED 0.19
Compound (SMILES): CN(c1ccc(O)cc1CNc1nc(-c2cc(F)c(O)cc2CC(F)(F)F)nc2[nH]nc(C(N)=O)c12)S(=O)(=O)c1ccccc1
InChIKey: XFOZFWJYURPVAU-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: 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 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 = 1734 nM (pChEMBL 5.760000228881836)
Descriptors: MW 645.6, ALogP 4.21, HBA 9, HBD 5, PSA 187.42, QED 0.15
Compound (SMILES): C[C@H](Oc1cc(F)c(S(=O)(=O)Nc2nccs2)cc1Cl)c1ccccc1
InChIKey: PLEBXORDNMAGFX-NSHDSACASA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 17527 nM (pChEMBL 4.760000228881836)
Descriptors: MW 412.9, ALogP 4.88, HBA 5, HBD 1, PSA 68.29, QED 0.63
Compound (SMILES): Cc1cc(N2C(=O)c3nc(C4CC4)n(C(C)C)c3C2c2ccc(Cl)cc2)cn(C)c1=O
InChIKey: LMLJGVCFCYGAST-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: AlphaScreen In-Vitro Binding Assay : In order to assess bromodomain selectivity, we set up a binding assay using the bromodomain encoded by the CREBBP gene. Compounds were tested in the CREBBP assay with a similar protocol, however using AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay, Perkin Elmer) as detection readout instead of TR-FRET. The assay was started by stepwise addition of 4.5μl per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 324 nM His-CREBBP(1081-1197) (custom production at Viva Biotech Ltd.)) and 4.5μl per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 120 nM acetyl-histone H4 (AcK 5, 8, 12) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μl per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.02% BSA, 150 mM NaCl, 45 μg/ml Ni-chelate acceptor beads, 45 μg/mIstreptavidin-donor beads) (Perkin Elmer)) were added. After 60 minutes incubation at room temperature, plates were measured in a Perkin Elmer EnVision multilabel reader. IC50 values were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 3400 nM (pChEMBL 5.46999979019165)
Descriptors: MW 436.94, ALogP 4.75, HBA 5, HBD 0, PSA 60.13, QED 0.59
Compound (SMILES): CS(=O)(=O)Nc1cncc(-c2cc3ncccc3c(-c3ccn(C4(CC#N)CN(S(C)(=O)=O)C4)n3)n2)c1
InChIKey: SZHUINHPJUJXEJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 > 10000 nM (pChEMBL 5)
Descriptors: MW 538.62, ALogP 1.81, HBA 10, HBD 1, PSA 163.83, QED 0.37
Compound (SMILES): COc1ccccc1/C=C/[C@@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: MBLXNYWHPRXUGT-OKAAQWNHSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: [35S] GTPgammaS Binding Assays: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Gal5-bla HEK293T cells (EDG3 equivalent S1P3) were added to the compound plate (40 ul/well, final protein 3 ug/well) with MULTIDROP. [35S] GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (Dithiothreitol), 10 uM GDP, 0.1% fatty acid free BSA, and 10 ug/ml Saponin to 0.4 nM. 40 ul of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11 Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 ul) was added to each well for counting on the Packard TOPCOUNT. EC50 is defined as the agonist concentration that corresponds to 50% of the Ymax (maximal response) obtained for each individual compound tested.
Measurement: EC50 > 625 nM (pChEMBL 6.203999996185303)
Descriptors: MW 457.51, ALogP 4.59, HBA 4, HBD 3, PSA 102.01, QED 0.53
Compound (SMILES): CC(=O)N1CC(Cc2nc3c(C(F)(F)F)cc(C(F)(F)F)cc3[nH]2)C1
InChIKey: ROQLYVJZUATFTQ-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: High-Throughput Cav3.1 T-Type Fluorescent Assay: Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior to use in the FLIPR assay. 100 μL of cells (2.0×106 cell/mL) containing doxycyline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression) were added to each well using a Multidrop (Thermo Scientific, Waltham, Mass.) and were maintained in 5% CO2 incubator at 37° C. On the morning of the assay, cells were transferred to a 5% CO2 incubator at 29° C. Cells were washed with a wash buffer containing (in mM): 118 NaCl, 18.4 HEPES, 11.7 D-glucose, 0.05 CaCl2, 0.5 MgSO4, 1 KCl, and 1.2 KH2PO4, with the pH adjusted to 7.2 with NaOH. 4.4 μM of the fluorescent indicator dye, Fluo-4 (Invitrogen), prepared in pluronic acid (Sigma-Aldrich), were loaded into the wells and incubated for 45 minutes at 29° C. in 5% CO2. Cells were then rinsed with the following low Ca2+ buffer (in mM): 0.34 Na2HPO4, 4.2 NaHCO3, 0.44 KH2PO4, 0.41 MgSO4, 0.49 MgCl2-6H2O, 20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 0.001 CaCl2, with 0.1% BSA and the pH adjusted to 7.2 with NaOH. Concentration-dependent response curves were generated from 5 mM stock solutions prepared in DMSO (Sigma-Aldrich) and diluted in the buffer containing low Ca2+ and incubated for 20 minutes at 29° C. in 5% CO2 Calcium entry was evoked with an addition of (in mM): 0.34 Na2HPO4, 4.2 NaHCO3, 0.44 KH2PO4, 0.41 MgSO4, 0.49 MgCl2-6H2O, 20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 6 CaCl2, with 0.1% BSA and the pH adjusted to 7.2 with NaOH. A change in the Fluo-4 fluorescence signal was assessed using FLIPRTETRA¿ instrument (Molecular Devices, Sunnyvale, Calif.) for 3 minutes following the elevation of extracellular KCl using an illumination wavelength of 470-495 nm with emissions recorded at 515-575 nm.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 365.28, ALogP 3.62, HBA 2, HBD 1, PSA 48.99, QED 0.83
Compound (SMILES): Nc1nnc(CN[C@H]2C[C@@H]2c2ccc(OCc3ccc(Cl)cc3)cc2)o1
InChIKey: JHJUEPUMXHSIIV-SJORKVTESA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Monoamine Oxidase Assays for Determining the Selectivity of the Compounds of the Invention for LSD1: Human recombinant monoamine oxidase proteins MAO-A and MAO-B were purchased from Sigma Aldrich. MAOs catalyze the oxidative deamination of primary, secondary and tertiary amines. In order to monitor MAO enzymatic activities and/or their inhibition rate by inhibitor(s) of interest, a fluorescent-based (inhibitor)-screening assay was set up. 3-(2-Aminophenyl)-3-oxopropanamine (kynuramine dihydrobromide, Sigma Aldrich), a non fluorescent compound was chosen as a substrate. Kynuramine is a non-specific substrate for both MAOs activities. While undergoing oxidative deamination by MAO activities, kynuramine is converted into 4-hydroxyquinoline (4-HQ), a resulting fluorescent product.The monoamine oxidase activity was estimated by measuring the conversion of kynuramine into 4-hydroxyquinoline. Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 μL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in triplicate within the same experiment.Briefly, a fixed amount of MAO (0.25 μg for MAO-A and 0.5 μg for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of various concentrations of inhibitor (e.g., from 0 to 50 μM, depending on the inhibitor strength). Tranylcypromine (Biomol International) was used as a control for inhibition.After leaving the enzyme(s) interacting with the inhibitor, 60 to 90 μM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. in the dark. The oxidative deamination of the substrate was stopped by adding 50 μL (v/v) of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor.The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 370.84, ALogP 3.53, HBA 6, HBD 2, PSA 86.2, QED 0.66
Compound (SMILES): C[C@H]1CN(c2ccc(OC(F)(F)F)cc2)CCN1C(=O)NCCC1CCN(Cc2ccccc2)CC1
InChIKey: WDDVUZDUDNOVJW-NRFANRHFSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 504.6, ALogP 5.11, HBA 4, HBD 1, PSA 48.05, QED 0.57
Compound (SMILES): C=CC(=O)N1CC[C@]2(C1)C[C@@H](n1c(NC(=O)c3ccc(C(F)F)s3)nc3cc(CN[C@@H](C)C(C)(C)C)ccc31)C2
InChIKey: CRYRQUNNVGZTRP-QWWQDABJSA-N
Target: Tyrosine-protein kinase Tec (SINGLE PROTEIN), Homo sapiens
UniProt: P42680
Assay: Kinase Inhibition Assay: The in vitro kinase assays were performed at Nanosyn (Santa Clara, Calif.) utilizing microfluidic detection technology. The test compounds were serially pre-diluted in DMSO and added, by the acoustic dispensing (Labcyte 550), directly to 384 well assay plates into 10 uL of a buffer with enzyme (ITK, TXK, BTK or TEC) comprising: 100 mM HEPES, pH7.5, 5 mM MgCl2, 0.1% bovine serum albumin, 1 mM DTT, 0.01% Triton X-100 and the enzyme. Final DMSO concentration was maintained at 1% in all samples, including the controls. The reactions were initiated by addition of ATP (1 mM final concentration) and the fluorescently labeled peptide substrate to a final concentration of 1 uM, and incubated for 3 hours at 25° C. Following incubation, the reactions were quenched by addition of 40 μL of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 50 mM EDTA). Terminated plates were analyzed using Caliper LabChip 3000 microfluidic electrophoresis instrument (Caliper Life Sciences/Perkin Elmer). The enzymatic modification of the peptide substrate (phosphorylation) results in a change of net charge enabling electrophoretic separation of product from substrate. As substrate and product are separated by electrophoresis, two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks was the parameter measured, reflecting enzyme activity. In the presence of inhibitor, the ratio between product and substrate is altered: signal of the product decreases, while the signal of the substrate increases. Activity in each test sample was determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product and S is the peak height of the FAM-cAMP substrate. For each compound, enzyme activity was measured at 12 concentrations spaced by 3× dilution intervals. Negative control samples (0%-inhibition in the absence of inhibitor, DMSO only) and positive control samples (100%-inhibition, in the absence of enzyme or in the presence of control inhibitor) were assembled in replicates of four and were used to calculate %-inhibition values in the presence of compounds. Percent inhibition (Pinh) was determined using the following equation: Pinh=(PSR0%−PSRinh)/(PSR0%−PSR100%)*100, where PSRinh is the product sum ratio in the presence of inhibitor, PSR0% is the product sum ratio in the absence of inhibitor and PSR100% is the product sum ratio in 100%-inhibition control samples. To determine IC50 values, the inhibition curves (Pinh versus inhibitor concentration) were fitted by 4 parameter sigmoid dose-response model using XLfit software (IDBS).
Measurement: IC50 = 49.3 nM (pChEMBL 7.309999942779541)
Descriptors: MW 569.72, ALogP 6.55, HBA 6, HBD 2, PSA 79.26, QED 0.3
Compound (SMILES): Cc1cc(Nc2cc3ncccc3c(N[C@@H]3C[C@H]4CCC[C@@H](C3)N4S(=O)(=O)C3CC3)n2)n[nH]1
InChIKey: ROMQTBSRCDGHLY-OSYLJGHBSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 467.6, ALogP 3.69, HBA 7, HBD 3, PSA 115.9, QED 0.51
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)C[C@H]3CC[C@H](NC(=O)Cc4cccnc4)CC3)c2)c1
InChIKey: FCJNYJFMCJKJAI-RUCARUNLSA-N
Target: Histone acetyltransferase KAT8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7Z6
Assay: KAT-8 Enzyme Assay: Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-8 enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 5 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 = 30000 nM (pChEMBL 4.519999980926514)
Descriptors: MW 563.06, ALogP 2.76, HBA 7, HBD 3, PSA 135.08, QED 0.34
Compound (SMILES): COC1CCN(C(=O)c2ccc3c(c2)CN(c2cccc(-c4nncn4C4CC4)n2)C3=O)CC1
InChIKey: VOBUCXPBTNCKEE-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: Inhibition of LPS-induced TNFalpha in Human PBMCs: Cryopreserved human PBMCs were obtained from AllCells (cat# PB003F). After thawing/dilution protocols using RPMI medium supplemented with 5% FBS (heat inactivated), 100 ul/well of 1×106 cells/ml were plated into 96 well tissue culture plates (Corning). Cells were then pre-incubated for 1 hr at 37° C. in humidified 5% CO2 and 95% air with test compounds diluted in DMSO (final DMSO concentration 0.3%). Each compound was tested at 10 concentrations in duplicate wells. After the pre-incubation, 100 ng/ml LPS (E. Coli; Sigma) in RPMI media with 5% FBS was added for a 6 hr incubation at 37° C. in humidified 5% CO2 and 95% air. Controls on each plate included cells and LPS only, cells and media only (no LPS), and media only. After the 6 hr incubation, plates were centrifuged and the supernatants transferred to a new plate and frozen for subsequent TNFalpha analysis. Human TNFalpha was analyzed by ELISA according to the manufacturer's instructions (BD Sciences, BD OptEIA Cat#550610) and analyzed on a SpectraMax M series (Molecular Devices) microplate reader at OD 450 nm. IC50s were calculated using XLFit4 curve fitting software (IDBS) and a 4-parameter one-site sigmoid dose response fit.
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 458.52, ALogP 3.09, HBA 7, HBD 0, PSA 93.45, QED 0.58
Compound (SMILES): CCC(c1cnn(-c2ccc(F)cc2F)c1)c1c(C#N)c(-c2cnc(C(F)(F)F)nc2)c2c(N)ncnn12
InChIKey: LJEPJUHRGHAJKD-UHFFFAOYSA-N
Target: Cystic fibrosis transmembrane conductance regulator (SINGLE PROTEIN), Homo sapiens
UniProt: P13569
Assay: FRT Ion Flux Assay of F508del CFTR Potentiation: Fischer rat thyroid (FRT) cell lines stably expressing recombinant F508del V470 CFTR and halide-sensitive yellow fluorescent protein (Pedemonte et al., J. Clin. Invest. 115(9) 2564-71 (2005)) were seeded at 25,000 cells/well in 50 uL/well of culture medium into black-walled, clear bottom tissue-culture-treated 384-well plates (Corning, cat. no. 3712). After one day, the cells were pre-incubated at 27° C./5% CO2 for 16-24 hours. The cells were then washed with dPBS and treated with forskolin (20 uM) and test compound for 30 min by addition of 20 uL of compound dilution buffer (dPBS containing forskolin and test compound). Plates were loaded into FLIPR384 fluorescence imaging plate reader (Molecular Devices). After an initial fluorescence reading, iodide buffer (25 uL) (composition (in mM): 137 NaI, 1.5 K2PO4, 8.1 NaH2PO4, 2.7 KCl, 0.5 MgCl2, 1 CaCl2) was added and a second fluorescence reading was made after approximately 21 seconds. Data treatment involved division of the second fluorescence reading by the initial fluorescence reading, then scaling of the resulting normalized endpoint fluorescence with respect to the responses for DMSO (0%) and a positive control potentiator (100%).
Measurement: EC50 = 13.3 nM (pChEMBL 7.880000114440918)
Descriptors: MW 525.45, ALogP 4.66, HBA 9, HBD 1, PSA 123.6, QED 0.33
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCc1csc(C2CCCN2)n1
InChIKey: UJKCNZDTMJJLEP-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 502.43, ALogP 6.15, HBA 8, HBD 2, PSA 81.19, QED 0.31
Compound (SMILES): CCN1CC(CC#N)(n2ccc(-c3nc(-c4cc(N)n(C)n4)cc4ncccc34)n2)C1
InChIKey: OPXDUYVUJWZXNF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 > 10000 nM (pChEMBL 5)
Descriptors: MW 413.49, ALogP 2.42, HBA 9, HBD 1, PSA 114.47, QED 0.53
Compound (SMILES): C=CC(=O)N1CCN(CCn2c(=O)ccc3cnc(Nc4ccc(N5CCN(C)CC5)cc4)nc32)CC1
InChIKey: KRYAMCLSIFVPLO-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
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 502.62, ALogP 1.62, HBA 9, HBD 1, PSA 89.84, QED 0.49
Compound (SMILES): CC(C)N1CCOc2c(F)cc(-c3nc(Nc4ccc(N5CCN(C(=O)CN(C)C)CC5)cn4)ncc3F)cc21
InChIKey: YCOFHJMLYTXYAJ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Activity Assay: PI3Kδ: Briefly, this assay was conducted in buffer (Tris-HCl 40 mM (pH7.5), Orthovanadate 3 mM, MgCl2 20 mM, DTT 2 mM, CHAPS 0.05%, DMSO 1%). PI3Kδ kinase was added to the reaction solution and mixed gently. The test compounds in 100% DMSO (at specific solutions) were mixed with the kinase reaction mixture to achieve the final compounds at pre-defined concentrations (e.g., range—0.5 nM to 100 μM) by Acoustic technology (Echo550; nanoliter range). After incubating for 10 min at room temp, ATP was added into the reaction mixture to initiate the reaction followed by a 30-min incubation at 30° C. After quenching the reaction with ADP-Glo reagent, the plates were incubated for 40 min. The Detection Mixture was added, and the plate was incubated for an additional 30 min. At the end of incubation, luminescence was measured. For data analysis, the luminescence was converted into μM ADP production based on ADP standard curves.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 552.63, ALogP 3.38, HBA 9, HBD 1, PSA 89.96, QED 0.47
Compound (SMILES): CS(=N)(=O)c1ccc(Nc2ncc3cc4n(c3n2)C2(CCCCC2)C(=O)NN4)cc1
InChIKey: WHDHBZVTAJDTDO-UHFFFAOYSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P30281
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 291 nM (pChEMBL 6.539999961853027)
Descriptors: MW 425.52, ALogP 3.33, HBA 8, HBD 4, PSA 124.79, QED 0.51
Compound (SMILES): [C-]#[N+]c1cn(CC2CCOCC2)nc1-c1cc2c(cn1)CN(c1c(F)c(OC)cc(OC)c1F)C(=O)C21CC1
InChIKey: FCRIZVHJKRHMNK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 535.55, ALogP 4.8, HBA 7, HBD 0, PSA 83.07, QED 0.42
Compound (SMILES): Cn1cc(-c2cn3nccc3c(-c3cnn(C4(C)CCCC4)c3)n2)cn1
InChIKey: KRTPHJWJOAAJKP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK3 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK3 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 10 μM Omnia® Y7 peptide (Catalog # IVGN KNZ3071C, Invitrogen Corporation, Carlsbad, Calif.) and 2 nM JAK3 in a total volume of 25 μL. JAK3 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4080).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 347.43, ALogP 3.28, HBA 7, HBD 0, PSA 65.83, QED 0.57
Compound (SMILES): CN1CCN(CC(=O)Nc2cc(C(=O)Nc3nnc(-c4cccc(Cl)c4)s3)ccc2OC(F)(F)F)CC1
InChIKey: DZKLRJPRYPLYLJ-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: HEK FOP Assay: HEK293 cells were cotransfected with the FOP control vector and pcDNA3. The FOP vector is identical to the TOP construct, but it contains instead of functional TCF elements a randomized, non-functional sequence
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 554.98, ALogP 4.2, HBA 8, HBD 2, PSA 99.69, QED 0.45
Compound (SMILES): O=C(NC[C@@H](O)C1CCCCC1)[C@H]1CCN(c2ncnc3[nH]ccc23)CC12CC2
InChIKey: YURIZDASFIBLEQ-QZTJIDSGSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Kinase Assays: Human baculovirus-expressed JAK1, 2, 3 and TYK2 were purchased from Carna Biosciences, Inc. All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag.Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay using the TK substrate-Biotin from the Cisbio HTRFKinEASE TK kit. First, 2 μl of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) is added to a plate containing 1 μl prediluted compound (final assay concentration DMSO: 0.75%). Then, 5 μl kinase-ATP mix (prepared in kinase buffer) is added to the wells and the plates are incubated at RT for 20-30 min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng TYK2. Thereafter, the kinase reaction is stopped by adding 4 μl detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates are incubated overnight in the dark. The HTRF signal is read using an Envision plate reader.
Measurement: EC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 397.52, ALogP 2.62, HBA 5, HBD 3, PSA 94.14, QED 0.72
Compound (SMILES): CN1CCN(Cc2ccc(-c3nc4ccc(C(=O)NO)cc4[nH]3)cc2)CC1
InChIKey: CVLLNANRQSBRJP-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: In vitro enzymatic HDACs assays: All Examples were tested in HDAC1, 2, 3, 6 and 8 in vitro enzymatic assays. The assay principle is well known (Hauser et al. 2009, Bradner et al. 2010) and all necessary reagents like enzymes, substrates, developer and reference compounds are commercially available (see e.g. BPS Biosciences http://www.bpsbioscience.com/). A series of dilutions of the compounds were prepared with a top concentration of 200 μM (2 μM for HDAC6). The enzymatic reactions were conducted in a mixture containing assay buffer, bovine serum albumin, HDAC substrate, and a test compound. After enzymatic reaction, developer was added and after an additional incubation time, fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. A selection of the Examples were tested in a full HDAC panel containing HDAC1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 in vitro enzymatic assays. The tests were performed as above except that a series of dilutions of the compounds were prepared with a top concentration of 10 μM for all HDACs.
Measurement: IC50 = 250 nM (pChEMBL 6.599999904632568)
Descriptors: MW 365.44, ALogP 2.1, HBA 5, HBD 3, PSA 84.49, QED 0.49