lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C)C/C=C/C(=O)Nc1ccc(S(=O)(=O)Nc2cccc(Nc3ncc(Cl)c(-c4c[nH]c5ccccc45)n3)c2)cc1
InChIKey: YBVSJBLXFZBKSU-IZZDOVSWSA-N
Target: Mitogen-activated protein kinase kinase kinase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16584
Assay: In Vitro Kinase Assay: In vitro kinase assays with immunoprecipitated proteins (or recombinant CAK complexes) were performed as follows. Kinase reactions were performed in 25 μL of final volume of reaction buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5% glycerol, 25 μM ATP, and 10 μCi of [γ-32P]ATP). Each reaction contained 1 μg of RNAP II. Incubation was at 30° C. for 30-45 min. Reactions were terminated by addition of 5 μL of 5×SDS-PAGE sample buffer and incubated at 95° C. for 5 min. Reactions were analyzed by SDS-PAGE, and the dried gel was exposed to film or PhosphorImager plate.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 602.12, ALogP 5.88, HBA 7, HBD 4, PSA 132.11, QED 0.15
Compound (SMILES): N#CCC1(n2ccc(-c3nc(-c4ccc(O)nc4)cc4ncccc34)n2)CN(C2CC(F)(F)C2)C1
InChIKey: LAJXCUYHMHKKBX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 = 2073 nM (pChEMBL 5.679999828338623)
Descriptors: MW 473.49, ALogP 3.98, HBA 8, HBD 1, PSA 103.75, QED 0.47
Compound (SMILES): CCOP(=O)(OCC)c1nc2ccccc2n([C@H]2C[C@H]3CC[C@@H](C2)N3C23CC4CC(CC2C4)C3)c1=O
InChIKey: CVBWYFBEARUZOC-LJCUTECVSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: In Vitro Kappa-Opioid Receptor Binding Assay: κ-Opioid Receptor Binding Assay Procedures: Membranes from recombinant HEK-293 cells expressing the human kappa opioid receptor (kappa) (cloned in house) were prepared by lysing cells in ice cold hypotonic buffer (2.5 mM MgCl2, 50 mM HEPES, pH 7.4) (10 mL/10 cm dish) followed by homogenization with a tissue grinder/Teflon pestle. Membranes were collected by centrifugation at 30,000×g for 15 min at 4° C. and pellets resuspended in hypotonic buffer to a final concentration of 1-3 mg/mL. Protein concentrations were determined using the BioRad protein assay reagent with bovine serum albumen as a standard. Aliquots of kappa receptor membranes were stored at −80° C. Radioligand dose displacement assays used 0.4-0.8 nM [3H]-U69,593 (NEN; 40 Ci/mmole) with 10-20 μg membrane protein (recombinant kappa opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 L binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 10 μM unlabeled naloxone or U69,593. All reactions were performed in 96-well polypropylene plates for 1 h at a temperature of about 25° C. Binding reactions were determined by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by five filtration washes with 200 μL ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours. Fifty μL/well scintillation cocktail (MicroScint20, Packard) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: Ki = 31.5 nM (pChEMBL 7.5)
Descriptors: MW 511.6, ALogP 5.03, HBA 7, HBD 0, PSA 73.66, QED 0.49
Compound (SMILES): C[C@H](Nc1ccc(S(=O)(=O)Nc2cscn2)cc1Cl)c1ccccc1
InChIKey: NTDFICOOHCFIAB-LBPRGKRZSA-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 = 5851 nM (pChEMBL 5.230000019073486)
Descriptors: MW 393.92, ALogP 4.77, HBA 5, HBD 2, PSA 71.09, QED 0.63
Compound (SMILES): CCOc1ncccc1-c1cc(NCc2cccnc2OC)c(NC2COC2)c(C(C)=N)n1
InChIKey: LWKMAOPQFDSIHC-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1C (SINGLE PROTEIN), Homo sapiens
UniProt: Q14123
Assay: Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 uL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labeled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 hr at room temperature before being terminated through mixing with 20 uL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 hr in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 448.53, ALogP 3.76, HBA 9, HBD 3, PSA 114.27, QED 0.4
Compound (SMILES): C[C@H]1Cn2c(-c3ccc(F)cn3)nnc2-c2nnc(-c3ccc(Cl)cc3Cl)n21
InChIKey: ZRUBBNHLRYYYNP-VIFPVBQESA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Mus musculus
UniProt: Q9Z1M0
Assay: P2X7 FLIPR Assay: 1321N1 cells expressing the recombinant human or rat P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 μl volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250× the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 μL of the compound into 300 μL of assay buffer. A further 3× dilution occurred when transferring 50 μL/well of the compound plate to 100 μL/well in the cell plate. Cells were incubated with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 μL/well of BzATP (final concentration is 250 μM BzATP (human and rat)). The fluorescence change was measured 180 seconds after adding the agonist. Peak fluorescence was plotted as a function of BzATP concentration using Origin 7 software.
Measurement: IC50 = 13.7 nM (pChEMBL 7.860000133514404)
Descriptors: MW 416.25, ALogP 4.29, HBA 7, HBD 0, PSA 74.31, QED 0.49
Compound (SMILES): O=C(Nc1ccc(Oc2ccnc3[nH]nc(NC4CCC4)c23)c(F)c1)c1ccnn(-c2ccc(F)cc2)c1=O
InChIKey: ZPEYGVIFDRQGRG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor TYRO3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06418
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 86000 nM (pChEMBL 4.070000171661377)
Descriptors: MW 529.51, ALogP 4.79, HBA 8, HBD 3, PSA 126.82, QED 0.28
Compound (SMILES): CN(C)[C@H](CNC(=O)C[C@@H](c1ccccc1)C1(C(F)(F)F)CC1)Cc1ccc(C(N)=O)cc1
InChIKey: DJYHZMUJSDVUOR-SFTDATJTSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: KOR beta-Arrestin Agonist and Antagonist Assays: KOR β-Arrestin Agonist and Antagonist assays were preformed using the Tango OPRK1-bla U2OS cells from Invitrogen and purchased from ThermoFisher, catalog number K1576. The cells were used according to the manufacturer suggested instructions.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 461.53, ALogP 3.89, HBA 3, HBD 2, PSA 75.43, QED 0.56
Compound (SMILES): C=CC(=O)N1CC(OCCn2c(=O)c(-c3c(Cl)c(OC)cc(OC)c3Cl)cc3cnc(N[C@@H](C)COC)cc32)C1
InChIKey: GKINEURMFPNAJS-INIZCTEOSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition of FGFR2-Dependent Cell Growth: The cell-based effects of FGFR inhibitors were determined by measuring inhibition of FGFR-dependent cell line growth. The cell lines SNU-16 was used for these assays. SNU-16 cells were seeded in a 96-well plate at 5,000 cells per well in RPMI 1640 high glucose medium with 10% fetal bovine serum (FBS. Cells were incubated at 37° C. for 24 hrs. in 5% CO2. Compound dilutions were added to cells starting at a concentration of 30 uM and decreasing in tripling dilutions. The final DMSO concentration was 0.1%. The concentration range was adjusted as needed for compounds of different potencies. The cells treated with compounds were incubated for 72 hrs. at 37° C. in 5% CO2. At the end of the 72 hour incubation period, cell viability was determined using the Cell-titer Glo Luminescence assay from Promega. Percent inhibition of cell growth was calculated as a percentage of untreated cell viability. The percent inhibition was plotted as a function of log compound concentration. The IC50 was then calculated for each compound using Prism software from GraphPad.
Measurement: IC50 = 6.6 nM (pChEMBL 8.180000305175781)
Descriptors: MW 591.49, ALogP 4.25, HBA 9, HBD 1, PSA 104.15, QED 0.31
Compound (SMILES): COc1ncccc1-c1nc2c(n1C(C)C)C(c1ccc(Cl)cc1)N(c1cc(OC)c3nnn(C)c3c1)C2=O
InChIKey: OGJPTYDTRCDEJC-UHFFFAOYSA-N
Target: Bromodomain-containing protein 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15059
Assay: In-Vitro Binding Assay: All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 ul per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5ul per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 ul per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (IC50 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 544.02, ALogP 5.23, HBA 9, HBD 0, PSA 100.19, QED 0.29
Compound (SMILES): CSCCC1(CC(C)C(=O)N2CCN(c3cc(Cl)cc(Cl)c3)CC2)NC(=O)NC1=O
InChIKey: QRHNXIGPFHVEIB-UHFFFAOYSA-N
Target: 72 kDa type IV collagenase (SINGLE PROTEIN), Homo sapiens
UniProt: P08253
Assay: In Vitro Assay: MMP2: Protocol 2: The basis for the assay is the cleavage of the substrate 390 MMP FRET substrate I (Anaspec, Catalog n#: AS-27076) by human MMP2 (R&D SYSTEMS INC., Cat #902-MP).For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (45 mM Tris pH 7.5, 9 mM CaCl2, 135 mM NaCl, 0.045% Brij35) containing MMP2 (0.03 ng/L) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate 390 MMP FRET substrate I (10 μL, 2.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 30 min at room temperature (Excitation 485 nm, Emission 530).
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 473.43, ALogP 3, HBA 5, HBD 2, PSA 81.75, QED 0.6
Compound (SMILES): CN(C(=O)C1CCC1)C1CCC(CS(=O)(=O)NNC(=O)c2cc(-c3cn(C)cn3)cc(Cl)c2F)CC1
InChIKey: JMBKFPBDNQRKCW-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95251
Assay: KAT-7 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-7 enzyme (6 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 2 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 120 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 < 10000 nM (pChEMBL 5)
Descriptors: MW 540.06, ALogP 3.26, HBA 6, HBD 2, PSA 113.4, QED 0.5
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CCN(C(=O)CNCCO)CC4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: GDNMFASTFXFQAL-UHFFFAOYSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: 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 = 8256 nM (pChEMBL 5.079999923706055)
Descriptors: MW 490.61, ALogP 2.24, HBA 8, HBD 3, PSA 114.68, QED 0.34
Compound (SMILES): CCc1cc(O)ccc1-c1cc2[nH]ncc2c(N2CCc3c(Cl)cccc3C2)n1
InChIKey: WEEHDGQBNPRIDK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
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 = 111 nM (pChEMBL 6.960000038146973)
Descriptors: MW 404.9, ALogP 5.11, HBA 4, HBD 2, PSA 65.04, QED 0.5
Compound (SMILES): COCC(=O)N[C@@H]1CCC[C@H](C(=O)Nc2cc(-c3cc(F)c4nn(C)c(C(C)C)c4c3)c(Cl)cn2)C1
InChIKey: SVGQRDCSVDIDOX-DOTOQJQBSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: CDK4/CyclinD1 Enzymatic Activity Assay: The inhibitory activity of compounds was evaluated in vitro using TR-FRET assay with white 384-well low volume microplate (Greiner Bio-One). CDK4/Cyclin D1 catalyzed phosphorylation of peptide in the presence and absence of compounds was measured and used in IC50 determination. Recombinant protein complex CDK4/Cyclin D1, expressed from insect cell, was purchased from ProQinase. Testing compounds were dissolved in DMSO at 1 mM and tested in 9-dose IC50 mode. The reaction mixture was prepared by mixing CDK4/CyclinD1 (1 nM final), ULight-4E-BP1 (100 nM final, Perkinelmer, TRF0128-D), and ATP (2 mM final) in assay buffer (20 mM of HEPES pH 7.4, 1 mM of EGTA, 0.05% BSA, 0.005% Tween 20, and 1 mM TCEP). The compound of interest in DMSO was added to each well in 3-fold serial dilution by dispenser (TECAN D300E) to make a 9.9 μL of reaction mixture. After 20 minutes preincubation at room temperature, 0.1 μL MgCl2(10 mM final) was added to initiate the reaction. Following a 45 minutes incubation at 37° C., the reaction was stopped by addition of 2 μL of quenching buffer consisting of Lance detection buffer (Perkinelmer CR97-100C), LANCE Ultra Europium-anti-P-4E-BP1 (Perkinelmer, TRF0216-D), EDTA, and incubate at room temperature for additional 60 minutes in dark. The reaction signal was measured by Envision multimode plate reader (PerkinElmer, 2102-0010).
Measurement: IC50 = 0.528 nM (pChEMBL 9.279999732971191)
Descriptors: MW 516.02, ALogP 4.81, HBA 6, HBD 2, PSA 98.14, QED 0.47
Compound (SMILES): Nc1ncnc2c1c(-c1cc(O)cc(F)c1)nn2Cc1nc2ccccc2nc1N1CCCC1
InChIKey: BOWYUIBIIJTYEI-UHFFFAOYSA-N
Target: Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00750
Assay: Inhibition Assay: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/ml). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 l 1N HCl followed by 160 μl CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid. The TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated. For each compound, kinase activity is measured at 10-12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 μM). For compounds showing significant activity, IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 456.49, ALogP 3.51, HBA 9, HBD 2, PSA 118.87, QED 0.42
Compound (SMILES): Cc1nc2c(F)cc(-c3nc(Nc4ccc5c(n4)CCN(C[C@H]4CN(C)CCO4)C5)ncc3F)cc2n1C(C)(C)C
InChIKey: PBDNNWWYSHCMCH-OAQYLSRUSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: LANCE Ultra ULight Assay: 1. Take 10 mM stock solution of the test compound, in 96-well compound plate, DMSO was used to dilute the compound to an initial concentration of 100x, then this concentration was used as the first concentration, 3-fold diluted to make 10 serial concentrations; 1 μL each serial dilution was then added to 19 μL 1x reaction buffer to prepare 5x compound for use; 2 μL 5x compound was transferred from 96-well plate to 384-well plate; compound-free control well was added with 2 μL the following liquid: 1x reaction buffer with the addition of 1 μL DMSO; 2 μL 250 mM EDTA was added to the Min control well.2.1x reaction buffer was used to formulate the kinase, substrate and ATP into a 2.5xenzyme/substrate mixture and 2.5xATP solution respectively. In the experiment, the final concentration of CDK4/CycD3 kinase is 0.76 ng/μL, the final concentration of ATP is 8 μM; the final concentration of CDK6/CycD3 kinase is 0.5 ng/μL, the final concentration of ATP is 50M; the final concentration of CDK2/CycA2 kinase is 0.86 ng/μL, the final concentration of ATP is 1 μM; the final concentration of CDK2/CycE1 kinase is 1.016 ng/μL, the final concentration of ATP is 20M; 2.5xenzyme/substrate mixture was added to a 384-well plate, incubated at room temperature for 5 minutes; then added with 2.5xATP solution, reacted at room temperature for 30 minutes.3. LANCE Detection Buffer was used, 1xto prepare 2xLANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) for use. After the enzymatic reaction was continued for 30 minutes, 10 mM EDTA was added to 384-well plate and the mixture was reacted at room temperature for 5 minutes. Then LANCE Ultra Europium-anti-phospho-eIF4E-binding protein 1 (Thr37/46) was added, reacted at room temperature for 1 hour.4. The 384-well plate was placed in HERAEUS Multifuge X1R centrifuge, centrifuged at 2000 rpm for 2 minutes; data were measured on EnVision, 337 nM wavelength laser was used as the excitation light, test at RFU665 nM and RFU615 nM, and RFU665 nM/RFU615 nM x10000 was used as the final data for analysis.
Measurement: IC50 = 16.3 nM (pChEMBL 7.789999961853027)
Descriptors: MW 562.67, ALogP 4.66, HBA 9, HBD 1, PSA 84.23, QED 0.38
Compound (SMILES): CCN1CCN(Cc2cc3c4c(cnc3[nH]2)CN(c2c(F)c(OC)cc(OC)c2F)C(=O)N4c2cccc(F)c2)CC1
InChIKey: CKIRXAIIQUVFJU-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Enzymatic Assay: The inhibitor potency of the exemplified compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 30 mM EDTA with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 580.61, ALogP 5.41, HBA 6, HBD 1, PSA 77.17, QED 0.32
Compound (SMILES): N#Cc1ccnc(N2C(=O)[C@H](F)C[C@H]2C(=O)N(c2cccc(F)c2)[C@H](C(=O)NC2CCC(F)(F)CC2)c2ccccc2Cl)c1
InChIKey: JMDFVJCPOQZKCU-OBDYRVMHSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: In Vitro Assay: A test compound is prepared as 10 mM stock in DMSO and diluted to 50× final concentration in DMSO, for a 50 μl reaction mixture. IDH enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutaric acid is measured using a NADPH depletion assay. In the assay the remaining cofactor is measured at the end of the reaction with the addition of a catalytic excess of diaphorase and resazurin, to generate a fluorescent signal in proportion to the amount of NADPH remaining. IDH1-R132 homodimer enzyme is diluted to 0.125 μg/ml in 40 μl of Assay Buffer (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 0.05% BSA, 2 mM b-mercaptoethanol); 1 μl of test compound dilution in DMSO is added and the mixture is incubated for 60 minutes at room temperature. The reaction is started with the addition of 10 μl of Substrate Mix (20 μl NADPH, 5 mM alpha-ketoglutarate, in Assay Buffer) and the mixture is incubated for 90 minutes at room temperature. The reaction is terminated with the addition of 25 μl of Detection Buffer (36 μg/ml diaphorase, 30 mM resazurin, in 1× Assay Buffer), and is incubated for 1 minute before reading on a SpectraMax platereader at Ex544/Em590.Compounds are assayed for their activity against IDH1 R132C following the same assay as above with the following modifications: Assay Buffer is (50 mM potassium phosphate, pH 6.5; 40 mM sodium carbonate, 5 mM MgCl2, 10% glycerol, 2 mM b-mercaptoethanol, and 0.03% BSA). The concentration of NADPH and alpha-ketoglutarate in the Substrate Buffer is 20 μM and 1 mM, respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 628.03, ALogP 5.66, HBA 5, HBD 1, PSA 106.4, QED 0.34
Compound (SMILES): N#Cc1ccnc(N2C(=O)CC[C@H]2C(=O)N(c2cccc3cn[nH]c23)[C@H](C(=O)NC2CC(F)(F)C2)c2ccccc2Cl)c1
InChIKey: JVKSHAPFLQYINL-HOFKKMOUSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: In Vitro Assay: A test compound is prepared as 10 mM stock in DMSO and diluted to 50× final concentration in DMSO, for a 50 μl reaction mixture. IDH enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutaric acid is measured using a NADPH depletion assay. In the assay the remaining cofactor is measured at the end of the reaction with the addition of a catalytic excess of diaphorase and resazurin, to generate a fluorescent signal in proportion to the amount of NADPH remaining. IDH1-R132 homodimer enzyme is diluted to 0.125 μg/ml in 40 μl of Assay Buffer (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 0.05% BSA, 2 mM b-mercaptoethanol); 1 μl of test compound dilution in DMSO is added and the mixture is incubated for 60 minutes at room temperature. The reaction is started with the addition of 10 μl of Substrate Mix (20 μl NADPH, 5 mM alpha-ketoglutarate, in Assay Buffer) and the mixture is incubated for 90 minutes at room temperature. The reaction is terminated with the addition of 25 μl of Detection Buffer (36 μg/ml diaphorase, 30 mM resazurin, in 1× Assay Buffer), and is incubated for 1 minute before reading on a SpectraMax platereader at Ex544/Em590.Compounds are assayed for their activity against IDH1 R132C following the same assay as above with the following modifications: Assay Buffer is (50 mM potassium phosphate, pH 6.5; 40 mM sodium carbonate, 5 mM MgCl2, 10% glycerol, 2 mM b-mercaptoethanol, and 0.03% BSA). The concentration of NADPH and alpha-ketoglutarate in the Substrate Buffer is 20 μM and 1 mM, respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 604.02, ALogP 4.67, HBA 6, HBD 2, PSA 135.08, QED 0.32
Compound (SMILES): CC(N(C)C)C1(c2cccc3ccccc23)CCCCC1
InChIKey: ROFHPDIVEBIITO-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Functional Uptake Assay (hSERT): Inhibition of human serotonin reuptake transporter was assayed using the recombinant human serotonin transporter expressed in HEK-293 cells. HEK-293 cells expressing human serotonin transporter were plated before the assay. Test compound and/or vehicle was preincubated with cells in modified HEPES buffer pH 7.1 or pH 7.4 for 20 minutes at 18 to 25° C. and 65 nM [3H]serotonin was then added for an additional timed incubation period (ten to thirty minutes). Cells with internalized [3H]serotonin were washed and the amount of tritium taken into cells is counted using a liquid scintillation counter to determine [3H]serotonin uptake. Non-specific binding of tritium was measured in a control reaction containing 10 uM fluoxetine, and was subtracted from the counts for assays to correct for non-specific binding of tritium. Reduction of [3H]serotonin uptake by 50 percent or more (50%) relative to an uninhibited control reaction indicates significant inhibitory activity. Compounds were screened at 10, 1, 0.1, 0.01 and 0.001 uM. The reference compound for the assay was fluoxetine, for which the IC50 value of 7.1 nM was obtained in a typical experiment.
Measurement: IC50 = 4632 nM (pChEMBL 5.329999923706055)
Descriptors: MW 281.44, ALogP 4.99, HBA 1, HBD 0, PSA 3.24, QED 0.77
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2C3CC(C3)[C@H](C)C2COc2ccc(F)cc2)n1
InChIKey: BMWCLKVQSHSQLB-ZQWKGRKISA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Scintillation Proximity Assay (SPA) Binding Assay: CHO cells stably transfected with human Orexin type 1 receptors (CHO-hOX1) or HEK-293 cells transiently transfected with human Orexin type 2 receptors (HEK-hOX2) were collected after 16 h induction with 5 mM sodium butyrate. The cell pellets were re-suspended, homogenized in 15 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 2 mM MgCl2, protease inhibitors and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellets were re-suspended in 75 mM Tris/HCl pH=7.5, 1 mM EGTA, 0.3 mM EDTA, 12.5 mM MgCl2, 250 mM Sucrose, protease inhibitors, divided into aliquots and frozen down at −80° C.Compounds of invention were serially diluted in neat DMSO at 100-fold concentrations (1% DMSO final in the assay) and 2 μl/well were plated into 96-well Isoplates (Perkin Elmer).
Measurement: Ki = 6.31 nM (pChEMBL 8.199999809265137)
Descriptors: MW 421.48, ALogP 3.43, HBA 6, HBD 0, PSA 73.14, QED 0.63
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CC(C)N(C(=O)CN5CC6(COC6)C5)C[C@@H]4C)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: BAOYUSPIGNWQIA-XJDOXCRVSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: 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 = 8435 nM (pChEMBL 5.070000171661377)
Descriptors: MW 556.72, ALogP 3.77, HBA 8, HBD 1, PSA 94.89, QED 0.4
Compound (SMILES): CCC(c1cn(-c2ccc(F)cc2F)nn1)c1cc(-c2cnc(C(F)(F)F)nc2)c2c(N)ncnn12
InChIKey: MIXBECUDERLPGI-UHFFFAOYSA-N
Target: Cystic fibrosis transmembrane conductance regulator (SINGLE PROTEIN), Homo sapiens
UniProt: P13569
Assay: Chamber Electrophysiology Assay of CFTR Potentiation in CF Bronchial Epithelial Cells: Primary cystic fibrosis human bronchial epithelial (CF hBE) cells were expanded and cultured according to published methods (Neuberger et al., Ch. 4 of Cystic Fibrosis, Methods in Molecular Biology vol. 741, pp. 39-54 (2011)). Well-differentiated cells (>30 days at air/liquid interface) on Snapwell filters (Corning Costar, cat. no. 3801) were mounted in Ussing chambers (Physiologic Instruments, Inc., San Diego, Calif.). F508del/F508del cultures were assayed at 27° C. and G551D/F508del cells were assayed at 35° C. HEPES buffered physiological saline (composition (in mM): 137 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES Na) was used in both apical and basolateral chambers. Chambers were bubbled with air to promote mixing and the voltage was clamped to zero. Amiloride (30 uM), forskolin (10 uM), test compound (4 increasing concentrations), and CFTRinh-172 (20 uM) were added sequentially with 20-25 minutes between additions. Short-circuit currents were acquired and analyzed using LabScribe2. Test compound responses were scaled relative to responses for DMSO (0%) and the maximal response of a positive control potentiator (100%).
Measurement: EC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 501.42, ALogP 4.19, HBA 9, HBD 1, PSA 112.7, QED 0.36
Compound (SMILES): CNC(=O)c1ccc(N2CCN(Cc3ccc4c5c(c(=O)[nH]c4c3)CCCN5)CC2)cc1F
InChIKey: XFINPBQKFDIRJH-UHFFFAOYSA-N
Target: Activated CDC42 kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07912
Assay: Inhibition Assay: The tankyrase-1 or tankyrase-2 enzyme inhibitory activities of the compounds of the present invention were assayed in the following manner by use of a kit (cat. 80573 or 80578) purchased from BPS Bioscience.The 96-well plate provided in BPS Bioscience kit was coated with histone and incubated at 4° C. for 16 hours. Then, the plate was washed four times with PBST (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and blocking buffer (provided in BPS Bioscience kit) was added thereto in order to block nonspecific reaction, and was then incubated at 25° C. for 1 hour. After incubation for 1 hour, the plate was washed four times with PBST, and varying concentrations of each of the compounds of the Examples were added to a reaction solution containing tankyrase-1 enzyme (40 ng/well) or tankyrase-2 enzyme (15 ng/well) and an assay mixture, and allowed to react at 25° C. for 1 hour. After 1 hour, each well was washed four times with PBST, and in order to measure the level of ribosylation by PARP enzyme, streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added and allowed to react at 25° C. for 30 minutes. The plate was washed four times with PBST, and then finally an HRP chemiluminescent substrate was added and allowed to react. The level of histone ribosylation formed by each enzyme was quantified using Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., USA).
Measurement: IC50 = 2.86 nM (pChEMBL 8.539999961853027)
Descriptors: MW 449.53, ALogP 2.71, HBA 5, HBD 3, PSA 80.47, QED 0.57
Compound (SMILES): COc1ccc(CN(C)C(=O)N2CC[C@@H](CS(=O)(=O)NNC(=O)c3cc(-c4cn(C)cn4)cc(Cl)c3F)C2)cc1
InChIKey: KVTIPMHUNVUBHF-GOSISDBHSA-N
Target: Histone acetyltransferase KAT7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95251
Assay: KAT-7 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-7 enzyme (6 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 2 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 120 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 < 10000 nM (pChEMBL 5)
Descriptors: MW 593.08, ALogP 3.03, HBA 7, HBD 2, PSA 125.87, QED 0.37
Compound (SMILES): CCOc1nn(CC)cc1Nc1ncc(C)c(-c2c[nH]c3c(NC(=O)[C@@H](C)N4CCN(C)CC4)cccc23)n1
InChIKey: YIPTWCREDSOPJH-LJQANCHMSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Enzymatic Assay: Enzyme inhibition studies were performed using recombinant JAK1 (amino acids 866-1154, Life Technologies, #PV4774, Carlsbad, Calif.), JAK2 (amino acids 831-1132), or JAK3 (amino acids 781-1124) under buffer conditions of 50 mM HEPES pH 7.3, 1 mM DTT, 0.01% Tween® 20, 50 μg/mL BSA, and 10 mM MgCl2. JAK enzyme was expressed as an N-terminal GST fusion in insect cells and purified by glutathione-affinity and size-exclusion chromatographies. Enzymes were assayed both at their respective ATP Km (JAK1: 55 μM, JAK2: 15 μM, JAK3: 3 μM) and the approximated high end of physiological ATP concentration of 5 mM, in the presence of inhibitor dosed at 30, 3, 0.3, 0.03, 0.003 and 0 μM final test concentrations. For JAK1, 6 nM of enzyme (for Km ATP assay) or 4 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (FITC-C6-KKHTDDGYMPMSPGVA-NH2 (SEQ ID NO:1), Intonation, Boston, Mass.). For JAK2, 0.8 nM of enzyme (for Km ATP assay) or 0.3 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). For JAK3, 0.2 nM of enzyme (for Km ATP assay) or 0.1 nM enzyme (for high ATP assay) was incubated with 1.5 μM peptide substrate (5FAM-GEEPLYWSFPAKKK-NH2 (SEQ ID NO:2), Intonation, Boston, Mass.). Phosphorylated and unphosphorylated peptides were separated and quantified by a Caliper LC3000 system (Caliper Life Sciences, MA) for calculating percent inhibition. The results of this assay are shown in Table 16 and indicate that the compounds of Formula (I), (Ia), (Ib) and Table 1 exhibit preferential inhibition of JAK1 over JAK2 (in many cases demonstrating over 100 times selectivity for inhibition of JAK1 vs. JAK2).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 531.67, ALogP 3.87, HBA 9, HBD 3, PSA 116.23, QED 0.3
Compound (SMILES): CNC[C@H](c1ccc2ccccc2c1)[C@H](OCC(=O)N(C)OC)c1ccccc1
InChIKey: QBAGYAXSOMBEGM-ISKFKSNPSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Uptake Assay Protocol: SERT: This protocol was designed to measure inhibition of uptake by the human serotonin transporter. The reagents were human SERT (HEK293F) cells, fluoxetine (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 μL polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1λ HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human serotonin transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.
Measurement: Ki = 4.4 nM (pChEMBL 8.359999656677246)
Descriptors: MW 392.5, ALogP 3.92, HBA 4, HBD 1, PSA 50.8, QED 0.56
Compound (SMILES): Clc1cccc(CCOc2ccc(Cl)cc2OC2CNC2)c1
InChIKey: WJADBJDMYMVZGL-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P50406
Assay: Binding Assay: Receptor binding was performed using membrane fractions prepared from the HEK-293 cell line recombinantly expressing rat 5-HT7 receptors (NCBI accession NM_022938). Compound affinity for the rat 5-HT7 receptor subtype was evaluated by competitive radioligand binding assays using 5-carboxamido[3H]tryptamine ([3H]5-CT) (Amersham Biosciences, cat. 90000403) detection. HitHunter cAMP assays are in-vitro based competitive immunoassays. The assay was performed on the HEK-293 cell line stably transfected with r5-HT7 receptor. Cells were pre-incubated with test compounds for 10 minutes. For antagonist testing, the cells were then challenged with 100 nM 5-CT for 20 minutes. Cells were then lysed and cAMP measured according to manufacturers protocol (Amersham, cat. NET791250UC) or [3H]mesulergine (Amersham, cat. TRK1041). The assay was performed on membranes prepared from HEK-293 cells stably transfected with h5-HT6. Following centrifugation, membranes were resuspended and incubated for 60 min at room temperature with 1.7 nM [3H]LSD in the presence of increasing concentration of test compounds. Nonspecific binding was defined in the presence of 10 μM clozapine (Tocris, cat. TRK1068). Homogenized HEK-293 membranes expressing the human SERT were incubated in 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5 mM KCl with [3H]-citalopram (3 nM) with or without test compounds. Nonspecific binding was determined in the presence of 10 μM fluoxetine. Radioactivity readouts and Ki values were performed as previously described for r5-HT7.
Measurement: Ki = 177 nM (pChEMBL 6.75)
Descriptors: MW 338.23, ALogP 3.97, HBA 3, HBD 1, PSA 30.49, QED 0.86
Compound (SMILES): C[C@H]1CN(C(=O)CCC2(C)NC(=O)NC2=O)CCN1c1cccc(F)c1
InChIKey: NZHFWSTVWWJDKQ-RSXQAXDFSA-N
Target: Disintegrin and metalloproteinase domain-containing protein 17 (SINGLE PROTEIN), Homo sapiens
UniProt: P78536
Assay: In Vitro Assay: TACE:The basis for the assay is the cleavage of the substrate 5FAM-LAQAVRSSSRK-5TAMRA (SEQ ID No 3) (Anaspec, custom 34891) by human TACE (R&D SYSTEMS INC., Cat #930-ADB).For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (25 mM Tris pH8.0, 2.5 μM ZnCl2, 0.01% CHAPS) containing TACE (0.05 ng/μL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate 5FAM-LAQAVRSSSRK-5TAMRA (5 L, 5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 75 min at room temperature (Excitation 485 nm, Emission 530).
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 362.41, ALogP 1.24, HBA 4, HBD 2, PSA 81.75, QED 0.79