lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(NC1CCN(CCF)CC1)c1n[nH]c2ccc(-c3cncc4ccccc34)cc12
InChIKey: ZYZZARZAWUXKOF-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: DYRK1A activity assay: SH-SY5Y cells are cultured in DMEM/F-12 medium supplemented with 15% FBS, Non-essential Amino Acid and Penicillin/Streptamycin. Two days before treatment, cells are seeded onto 96 well plates at 20e5 cells/well.DMSO-resuspended compounds are dispensed to 8 wells as a serial titration from 10 μM to 4.6 nM final and cells are exposed overnight (16-18 h) before harvest. Wells are visualized checked for cell death or change in morphology and supernatants are tested for cytotoxicity by measurement of lactate dehydrogenase release (LDH, CytoToxOne kit, Progema) if necessary. As controls, commercially available DYRK1A inhibitors, Harmine and Indy were shown to have good DYRK1A inhibition in the kinase assay with no CDK1 activity (EC50 18 and 53n M respectively, 6 μM for CDK1) but weak EC50 in the Tau assay: about 10 μM for Harmine and 30 μM for Indy.Cells are lyzed with RIPA buffer complemented with phosphatase and protease inhibitors (Thermo Scientific) then lysates are sonicated and spun down at 12,000 g for 10 min to remove any cellular debris. Lysates are then either directly tested for pSer396 by ELISA (Life Technology, Kit KHB7031) or loaded on NuPage Bis-Tris gels for western blot analysis. Colorimetric detection of ELISA signal is performed by Cytation3 plate reader (Biotek) and the chemoluminecence signal for HRP-linked antibodies used in western blotting is detected using a Carestream Image Station. The same pSer396 antibody is used for detection of pTau in both assays.Blot densitometry for pSer396 and beta-actin are analyzed using ImageJ (NIH) and pSer396/Total Tau ELISA signal was used to plot, draw the curve fitting, and determine each compounds EC50 in Prism (GraphPad).
Measurement: EC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 417.49, ALogP 3.94, HBA 4, HBD 2, PSA 73.91, QED 0.52
Compound (SMILES): O=C(NCCN1CCOCC1)[C@H]1C[C@]23CCN(CC4CC4)[C@H](Cc4ccc(O)cc42)[C@]3(O)C1
InChIKey: WYFJHNJUNVPTFK-MGRDPADOSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: δ-Opioid Receptor Binding Assay: δ-Opioid Receptor Binding Assay Procedures: δ-Opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 μg membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 μl binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 μM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hour at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 μl ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours. Fifty μl/well scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added and plates are counted in a Packard Top-Count for 1 min/well.δ-Opioid Receptor Binding Data: In certain embodiments, Compounds of the Invention exhibit a Ki (nM) for δ receptors of about 10,000 or more (which, for the purposes of this invention, is interpreted as having no binding to the δ receptors). Certain Compounds of the Invention exhibit a Ki (nM) of about 20,000 or less for δ receptors. In one embodiment, Compounds of the Invention exhibit a Ki (nM) of about 10,000 or less; or of about 9000 or less for δ receptors. In another embodiment, Compounds of the Invention exhibit a Ki (nM) of about 7500 or less; or of about 6500 or less; or of about 5000 or less; or of about 3000 or less; or of about 2500 or less for δ receptors. In another embodiment, Compounds of the Invention exhibit a Ki (nM) of about 1000 or less; or of about 500 or less; or of about 350 or less; or of about 250 or less; or of about 100 or less; or of about 10 or less for δ receptors.
Measurement: EC50 = 3.95 nM (pChEMBL 8.399999618530273)
Descriptors: MW 455.6, ALogP 1.26, HBA 6, HBD 3, PSA 85.27, QED 0.6
Compound (SMILES): C=CC(=O)N1CCN(CCCc2cn3c(n2)c(-c2c(Cl)c(OC)cc(OC)c2Cl)cc2cnc(NC)cc23)CC1
InChIKey: RUGARJBBFYLJIK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Inhibitory Activities Assay: (1) Preparation of 1×Kinase buffer; (2) Preparation of the gradient concentrations of the compounds: the concentration of the compounds to be tested started from 10 μM, which was 3-fold diluted to prepare 10 dilutions, and then tested in duplicated wells. The compounds were 100-fold serially diluted into 10 dilutions with different final concentrations in 96-well plates, each of which was further diluted with 1×Kinase buffer to prepare an intermediate dilution having a concentration of 5 times the final concentration; (3) adding 5 μL of each of the prepared compound solution into the compound wells of 384-well plates, and each concentration set single well; adding 5 μL of 5% DMSO into negative control wells and positive control wells respectively; (4) preparing a kinase solution having a concentration of 2.5 times the final concentration with 1×kinase buffer; (5) adding 10 μL of kinase solution at a concentration of 2.5 times the final concentration into the compound wells and the positive control wells respectively; and adding 10 μL of 1×Kinase buffer into the negative control wells; (6) centrifuging at 1000 rpm for 30 seconds, and incubating at room temperature for 10 minutes after shaking and mixing evenly; (7) preparing a mixed solution of ATP and Kinase substrate 22 having a concentration of 2.5 times the final concentration with 1×Kinase Buffer; (8) adding 10 μL of the mixed solution of ATP and substrate at a concentration of 2.5 times the final concentration to initiate the reaction; (9) Centrifuging the 384-well plates at 1000 rpm for 30 seconds, and incubating at 28° C. for corresponding time respectively after shaking and mixing; (10) adding 30 μL of stop detection solution to stop the kinase reaction, centrifuging at 1000 rpm for 30 seconds, and shaking and mixing the mixture evenly; (11) reading the conversion rate by Caliper Ezreader II. The dose-effect curve was fitted by the log (inhibitor) vs. response-variable slope of GraphPad Prism 5 with the log value of concentration as the X axis and the percentage inhibition rate as the Y axis, then the IC50 value of each compound on the enzyme activity was obtained.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 583.52, ALogP 5.18, HBA 8, HBD 1, PSA 84.23, QED 0.27
Compound (SMILES): CC(=O)N1CCC(COC(=O)Nc2cc(-c3ccc(Cl)s3)ccc2N)CC1
InChIKey: GBRHYUYYSMUSCE-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Inhibition of Histone Deacetylase Enzymatic Activity Assay: HDAC2: The following trypsin-coupled protocol and Caliper protocol described Herein was used to assay the compounds of the invention. HDAC2-Kinetic IC50 3Hr PreIncubation. Working Stocks: 1xHDAC Assay Buffer: 50 mM HEPES (GIBCO 15630-114), 100mM KCl, 0.001% Tween-20 (Zymed 00-3005), 0.005% BSA (Invitrogen, P2489), pH 7.4, 1.5xHDAC+1.5xTrypsin: 225nM Trypsin(Worthinton)+0.2 ug/ml HDAC2 (BPS Inc) in assay buffer 300x Trypsin stock is made up in HDAC Assay Buffer, aliquoted and stored at -80C. 3xSubstrate: 13.8uM Substrate in assay buffer BPS Enzyme and Substrate amounts (per well).
Measurement: IC50 = 3050 nM (pChEMBL 5.519999980926514)
Descriptors: MW 407.92, ALogP 4.46, HBA 5, HBD 2, PSA 84.66, QED 0.73
Compound (SMILES): C=CC(=O)Nc1cccc(C)c1Nc1ncc2cc(-c3cc(C(N)=O)ccc3F)ccc2n1
InChIKey: FNRAYBQHSJCIMJ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR-1 wild type assay: In each well of a 384-well plate, 0.1 ng/ul of wild type FGFR-1 (Carna Biosciences, Inc.) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM CSKtide (5-FAM-KKKKEEIYFFFG-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35 s).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 441.47, ALogP 4.71, HBA 5, HBD 3, PSA 110, QED 0.38
Compound (SMILES): COc1cc(-c2cc(C)c(C)cc2C)c(C#N)cc1-c1nccc2cc(S(=O)(=O)Nc3ccncn3)ccc12
InChIKey: UUUIYGBJQNGORK-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro PX Assay: 293 cells stably transfected with Nav 1.5 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system according the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Cells were held at a holding potential of â¿¿50 mV to inactivate sodium channels. To elicit sodium currents the voltage was changed to â¿¿120 mV to recover a portion of the channels, followed by delivery of test pulses of 20 msec duration to 0 mV, at 0.1 Hz. A single concentration of test compound was applied to cells for a duration of 5 minutes.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 535.63, ALogP 5.97, HBA 7, HBD 1, PSA 117.86, QED 0.28
Compound (SMILES): C[C@@H]1CN(c2cc(F)c(-c3cnc(N4CCOCC4)nc3)cc2NC(=O)c2c[nH]c(=O)cc2C(F)(F)F)C[C@H](C)N1C
InChIKey: CMLDSGMHBCTTQH-CALCHBBNSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 0.323 nM (pChEMBL 9.489999771118164)
Descriptors: MW 589.59, ALogP 3.61, HBA 8, HBD 2, PSA 106.69, QED 0.44
Compound (SMILES): C=CC(=O)Nc1cc(Nc2nc(-c3cnc4c(ccn4C)c3)c3cc[nH]c3n2)c(OC)cc1N(C)CCN(C)C
InChIKey: BCRGWDZMCMRCPL-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition Assay: The inhibitory activities on T790M/L858R double mutant EGFR kinase (Invitrogen, PV4879) and wild-type EGFR kinase (Invitrogen, PV3872) were determined by the z′-lyte assay.The working concentrations of each component in 10 μL T790M/L858R kinase reaction system were: 25 μM ATP, 0.1 ng/μL T790M/L858R kinase, 2 μM Tyr04 substrate (Invitrogen, PV3193). The concentration of DMSO after addition of the compound prepared in the above examples of the present invention (i.e., the compound to be tested) was 2 vol %.The working concentrations of each component in 10 μL wild-type EGER kinase reaction system were: 10 μM ATP, 0.8 ng/μL wild-type EGFR kinase, 2 μM Tyr04 substrate (Invitrogen, PV3193). The concentration of DMSO after addition of the compound to be tested was 2 vol %.10 mM drug stock solution was dissolved at room temperature and diluted to a final concentration of 4-0.002 μM with 8 vol % DMSO solution. 2.5 μL of the solution of the compound to be tested and 5 μL of the mixture of T790M/L858R kinase (or wild-type EGFR kinase) and Tyr04 substrate diluted with the reaction buffer were added to each well. Then 2.5 μl of ATP was added to initiate the reaction. Wherein, ATP was replaced by the reaction buffer in C1 well, no drug was added to C2 well, and the phosphorylated substrate was added to C3 well according to the instructions. The mixture was allowed to react at 25° C. in a shaker in dark for 60 min, 5 μL of Development Reagent B (invitrogen, diluted with TR-FRET dilution buffer) was added and allowed to react at room temperature in the shaker for 60 minutes. The plate was read on a Victor X5 plate reader (PerkinElmer) and the absorbance was measured at excitation wavelengths of 405 nm and emission wavelengths of 450 nm and 520 nm (for example, C3520 nm indicates the absorbance for C3 well at 520 nm)
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 539.64, ALogP 4.39, HBA 9, HBD 3, PSA 116.23, QED 0.22
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CCN(Cc5cccn(C)c5=O)CC4)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: HNRAJKYCHURLAO-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 53.1 nM (pChEMBL 7.28000020980835)
Descriptors: MW 505.59, ALogP 2.69, HBA 10, HBD 0, PSA 100.28, QED 0.36
Compound (SMILES): Cc1cc(C(F)(F)F)cc(O)c1-c1ccc(N[C@@H]2CCCN(CC(C)(C)O)C2)nn1
InChIKey: KVCHBDJNMGDHTP-OAHLLOKOSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: TNF-alpha Secretion Assay: Monocytic THP-1 cells were maintained according to providers' instructions in RPMI media as described above. Undifferentiated cells were plated at 50,000 cells per well in a 384-well flat-bottom cell culture plates (Greiner, #781986), and allowed to rest overnight. Experimental compounds were prepared and added as described above. TNF-α secretion was triggered by the addition of either 1 μg/mL LPS (Sigma, #L4391) or 100 ng/mL Pam3CSK4 (Invivogen, #tlrl-pms) depending on the experiment, and cells were incubated for 3 hours. 10 μL supernatant was removed, and TNF-α levels were monitored using an HTRF assay (CisBio, #62TNFPEC) according to manufacturers' instructions.
Measurement: IC50 > 33000 nM (pChEMBL 4.480999946594238)
Descriptors: MW 424.47, ALogP 3.82, HBA 6, HBD 3, PSA 81.51, QED 0.68
Compound (SMILES): C=CC(=O)N1CCN(C(=O)c2cc(Sc3cnc(NC(=O)c4cnn(CCN(C)C)c4)s3)c(C)cc2OC)CC1
InChIKey: AJPQBQFAMSEUAN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase TXK (SINGLE PROTEIN), Homo sapiens
UniProt: P42681
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 583.74, ALogP 3.09, HBA 10, HBD 1, PSA 112.9, QED 0.36
Compound (SMILES): COC(=O)c1ccccc1-c1cnc(N)c(-c2nnc(-c3ccccc3)o2)n1
InChIKey: XOBUKZZGVBPLRL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [γ-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK) (SEQ ID NO:1)).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).
Measurement: Ki = 2550 nM (pChEMBL 5.590000152587891)
Descriptors: MW 373.37, ALogP 3.23, HBA 8, HBD 1, PSA 117.02, QED 0.54
Compound (SMILES): CC(C)CO[C@H]1CCC[C@H]1NC(=O)CN1CCN(c2cccc3c2cnn3-c2ccccc2F)C1=O
InChIKey: JTYPAPSDGPCBLW-BWKNWUBXSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y9
Assay: FLIPR Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.8 (Essen, Ann Arbor, Mich.) were quickly thawed and plated at 22,500 cells/well in growth medium [MEM (Invitrogen #11095) with 10% FBS (Invitrogen #10082), 1 mM sodium pyruvate (Invitrogen, #C11360), 10 units/mL penicillin/10 U/mL streptomycin/29.2 μg/mL glutamine ((PSG 1%, Invitrogen #10378), 400 μg/mL zeocin (Invitrogen #R250) in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 μL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 μM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 μL assay buffer containing 2.5 μM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 60 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR®TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics.
Measurement: IC50 = 846 nM (pChEMBL 6.070000171661377)
Descriptors: MW 493.58, ALogP 4.12, HBA 5, HBD 1, PSA 79.7, QED 0.51
Compound (SMILES): Cc1ccc2cc(C(=O)N[C@@H]3C4CCN(CC4)C34CC4)sc2c1C1CC1
InChIKey: IPZJPIUIHUHJMB-HXUWFJFHSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: [3H]BRL 43694 Competition Binding (h-5HT3 Assay: [3H]BRL 43694 competition binding assay was performed under contract by Cerep Poitiers, France following the methods described in Hope, A. G et al., Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells, Brit. J. Pharmacol., (1996) 118: 1237-1245.In brief, Chinese Hamster Ovary (CHO) cells stably expressing human 5-HT3 serotonin receptors, grown to confluence in 175 cm2 flasks. Following aspiration of the culture medium, cells were harvested by mechanical agitation in ice cold PBS containing (in mM): (150 NaCl, 8 K2HPO4, 2 KH2PO4, pH 7.4, 37° C.), centrifuged at 4,000 g for 10 min and subsequently stored as a cell pellet at −80 C. When required, the pellet was thawed and resuspended in ice cold homogenization buffer (Tris 50 mM, EGTA 5.0 mM, phenylmethylsulphonylfluoride 0.1 mM, pH 7.6) and homogenized. The homogenate was centrifuged at 48,000 g for 10 minutes at 40° C. The resulting pellet was resuspended in ice cold binding buffer comprising (in mM): NaCl 140, KCl 2.8, CaCl2 1.0; MgCl2, 2.0; HEPES 10 (pH 7.4) and centrifuged as above. The pellet was resuspended in ice cold binding buffer and the protein concentration was determined by the method of Lowry et al., Protein measurement with the Folin phenol reagent, J. Biol. Chem., (1953) 193, 265-275). The membrane homogenate was adjusted to a protein concentration of approximately 600 mg/mL in binding buffer. Assay tubes were loaded with equal volumes of binding buffer containing [3H]BRL 43694 and test compound and 0.5 mL of membrane homogenate in a total reaction volume of 1 ml. Binding was initiated by the addition of the membrane homogenate and allowed to proceed for 120 min. at room temperature. Bound and free radioligand were separated by the addition of 3 ml of ice-cold binding buffer and immediate vacuum filtration through pre-soaked (0.1% (v/v) polyethyleneimine) Whatman GF/B filters. Filters were washed with a further 2×3 mL applications of binding buffer and counted for radioactivity using a scintillation counter.The results were expressed as a percent inhibition of control specific binding obtained in the presence of the test compounds where Inhibition (%)=100−[(measured specific binding/control specific binding)×100].
Measurement: Ki = 610 nM (pChEMBL 6.210000038146973)
Descriptors: MW 366.53, ALogP 4.44, HBA 3, HBD 1, PSA 32.34, QED 0.87
Compound (SMILES): CCCCCCNCc1ccc(Cn2c(O)nc3c(N)nc(OCCCC)nc32)cn1
InChIKey: ZCPLVONSOMVUEP-UHFFFAOYSA-N
Target: Interleukin-6 (SINGLE PROTEIN), Homo sapiens
UniProt: P05231
Assay: IL-6 Induction assay: Compounds diluted in DMSO were transferred to individual wells of a Matrix Technologies clear, V-bottom 384-well plate using ECHO acoustic liquid handling technology (25 nL per well). Human whole-blood samples (25 uL) were added to each well using a CyBio FeliX liquid handling instrument. The plate was shaken on a plate shaker for three min before incubating the reaction mixtures at 37° C. for 20 h. Basel RPMI 1640 medium (supplemented with L-glutamine) was then added to each well (25 uL per well) prior to liberating plasma from each sample by centrifugation (450×g, 5 min, ambient temperature). Treated plasma samples (3 uL) were subsequently transferred to individual wells of a white, shallow, 384-well ProxiPlate (Perkin Elmer) using the FeliX liquid handling instrument and their interleukin 6 levels were measured using AlphaLISA technology as described by the manufacturer, PerkinElmer. Data analyses software was used to determine compound EC50 values where the baseline was established using average DMSO values and 100% induction established using reference compound values at the highest concentration tested. EC50's can be determined with software such as Graphpad Prism.
Measurement: EC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 427.55, ALogP 3.41, HBA 9, HBD 3, PSA 124, QED 0.35
Compound (SMILES): Cc1ccc(F)c(NC(=O)Nc2ccc(-c3cc(B(O)O)cnc3N)cc2)c1
InChIKey: HENUTJSTZJVGEK-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Biochemical Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 2.7 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 380.19
Compound (SMILES): CC(C)(C)OC(=O)N[C@H]1C[C@@H](N(CC(=O)NCc2cccc(Cl)c2F)C(=O)Cn2nc(C(N)=O)c3ccccc32)C1
InChIKey: IMQPBHAEHISFLN-HDICACEKSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Esterolytic Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 587.05, ALogP 3.13, HBA 7, HBD 3, PSA 148.65, QED 0.35
Compound (SMILES): CCOc1nnc(-c2cc(NC(=O)c3ccnc(C(C)(C)C#N)c3)cnc2C)cc1N1CCOC[C@H]1C
InChIKey: MXFQYIBNMAANRS-QGZVFWFLSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 9.3 nM (pChEMBL 8.029999732971191)
Descriptors: MW 501.59, ALogP 3.92, HBA 9, HBD 1, PSA 126.15, QED 0.51
Compound (SMILES): Cn1cnc(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)CC3CCCN(C(=O)NCCO)C3)c2)c1
InChIKey: DACORWVIKLPNEF-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. 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-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 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 < 10000 nM (pChEMBL 5)
Descriptors: MW 516.98, ALogP 0.86, HBA 7, HBD 4, PSA 145.66, QED 0.38
Compound (SMILES): O=C(c1cc(-c2ccco2)on1)N1CC2(COC2)C1
InChIKey: HTTCWZWFULZAQQ-UHFFFAOYSA-N
Target: Stearoyl-CoA desaturase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86SK9
Assay: Compound Profiling Methods: Strains expressing SCD1 or SCD5 as the sole desaturase, the human SCD1 and SCD5 genes were used to evaluate inhibition of SCD1/SCD5 using reduced growth as a surrogate for SCD inhibition. These yeast strains express human SCD1 or SCD5 from a plasmid harbored in a strain in which the yeast OLE1 gene is deleted.All compound profiling experiments were performed using the same basic protocol. Yeast were cultured using standard techniques in complete synthetic media lacking uracil and containing yeast nitrogen base supplemented with 2% (w/v) glucose (SD-Ura) Starter cultures were inoculated in 3 mL SD-Ura media containing 0.01% tween and 0.2 mM palmitoleic and oleic acid. Cultures were incubated overnight in a 30° C. shaker incubator (225 rpm). Saturated morning cultures were centrifuged, washed in SD-Ura media lacking TWEEN-20 and fatty acids, and then diluted 1:20 in fresh SD-Ura media also lacking TWEEN-20 and fatty acids. Cells were grown for 6 h to an  (optical density) of 0.4-0.8 at 30° C. with shaking.Compound stocks (10 mM in 100% DMSO) were arrayed into 384-round well, v-bottom polypropylene plates and diluted according to indicated dilution factors. Compound administration was performed in two separate steps. First, 15 μL of SD-Ura was dispensed into clear 384-well assay plates using a MULTIDROP Combi reagent dispenser. The diluted compound stock plates were then applied to the assay plates using an automated workstation (Perkin Elmer JANUS) outfitted with a 384-pin tool containing slotted pins that deliver 100 nL of compound. The cultures described above were centrifuged and washed with media lacking TWEEN-20 or oleic and palmitoleic acids. Cultures were then resuspended at a 2-fold concentrated  of 0.02 (final  of 0.0.01) in SD-Ura. 15 μL of diluted culture was then dispensed into the pinned assay plate to achieve 30 μL of the 1× culture (0.01) and a top drug concentration of 33.3 μM.After yeast delivery, assay plates were incubated under humidified conditions at 30° C. for 40 h. Yeast growth was monitored by reading the  of each well using a microplate reader (Perkin Elmer EnVision). Data were analyzed as follows. Raw data were processed by background subtracting and converting values to a percent of the nontreated condition for that strain [(EXP-0.035)/(DMSO-0.035)×100%].
Measurement: IC50 > 45000 nM (pChEMBL 4.3470001220703125)
Descriptors: MW 260.25, ALogP 1.41, HBA 5, HBD 0, PSA 68.71, QED 0.82
Compound (SMILES): CCOc1cc(-c2ccc(N3CCC(COC)(Cc4ccccc4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: MYIZCTXJNZVGSF-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 1086 nM (pChEMBL 5.960000038146973)
Descriptors: MW 481.6, ALogP 5.14, HBA 7, HBD 0, PSA 75.68, QED 0.35
Compound (SMILES): COc1cc(NC2CCN(c3cc(F)c(-c4cn[nH]c4)cc3F)C2=O)ccc1F
InChIKey: AAWYZLOIEGYNNO-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assays: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH (SEQ ID NO: 1)). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 1150 nM (pChEMBL 5.940000057220459)
Descriptors: MW 402.38, ALogP 3.72, HBA 4, HBD 2, PSA 70.25, QED 0.68
Compound (SMILES): CC(C)(O)COc1cc(-c2ccc(N3CCC(C)(NC(=O)C4CC4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: ILNDLZBXHMGMOQ-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 488.59, ALogP 3.3, HBA 8, HBD 2, PSA 115.78, QED 0.52
Compound (SMILES): CN1CCN(c2ccc(-c3n[nH]c4cnc(-c5c(F)cccc5Cl)nc34)cc2)CC1
InChIKey: QVACMUBOOYJHNN-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech). Ki values were determined.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 422.9, ALogP 4.23, HBA 5, HBD 1, PSA 60.94, QED 0.53
Compound (SMILES): CN(C)c1ccc(C#Cc2cc3ncccc3[nH]2)cc1
InChIKey: FIQQLONWDXAKPQ-UHFFFAOYSA-N
Target: Microtubule-associated protein tau (SINGLE PROTEIN), Homo sapiens
UniProt: P10636
Assay: In Vitro Inhibition Assay: Table 5: Fresh solutions of the test compounds (2.5 mM in DMSO) are diluted in PBS pH7.4, at concentrations between 0.5 nM and 5 μM in the presence of α-syn (200 nM), Aβ1-42 (500 nM) or Tau (200 nM) fibres and ThT 500 nM in a final 2 mL volume. The samples are incubated for 1 h at room temperature (RT) to reach equilibrium. The binding of ThT to α-syn, Aβ1-42 or Tau fibres is measured by fluorescence (excitation: 440 nm, emission: 480 nm). Inhibition of ThT binding by increasing concentrations of each test compound is measured by fluorescence reduction. Due to the partial superposition between the emission spectra of some compounds and ThT, the compounds are also incubated with α-syn, Aβ1-42 or Tau fibers in the absence of ThT. For each concentration, the fluorescence values in the absence of ThT are subtracted from those in the presence of ThT. The results are expressed as a percentage of the maximum fluorescence value of the ThT measured in the absence of a competitor (100% bind).
Measurement: Ki = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 261.33, ALogP 3.03, HBA 2, HBD 1, PSA 31.92, QED 0.68
Compound (SMILES): C=CC(=O)N1CCOc2ccc(Nc3nc(Nc4ccc(N(C)C(=O)C(F)(F)F)nc4)ncc3C(C)=O)cc21
InChIKey: JJADLSVURKFRPJ-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Omnia Assay: EGFR (WT) and EGFR (T790M/L858R): The Omnia Assay Protocol for potency assessment against EGFR is performed as described in Example 251 above except that the EGFR-WT- and EGFR T790M/L858R-modified optimized reagent conditions are:[EGFR-WT]=5 nM, [ATP]=15 mM, [Y12-Sox]=5 mM (ATP KMapp˜12 mM); and [EGFR-T790M/L858R]=3 nM, [ATP]=50 mM, [Y12-Sox]=5 mM (ATP KMapp˜45 mM).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 541.49, ALogP 4, HBA 9, HBD 2, PSA 129.65, QED 0.34
Compound (SMILES): COc1cccc(-c2[nH]c(C3(C)CCN(c4ncnc5[nH]c(-c6ccc(NS(C)(=O)=O)cc6)cc45)CC3)nc2C)c1
InChIKey: CSILAYADHFIJDS-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Biochemical Assay: The c-kit mutant D816V (or kinase domain thereof) is an active kinase in AlphaScreen. IC50 values are determined with respect to inhibition of c-Kit mutant D816V kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested were dissolved in DMSO to a concentration of 30 mM. These were diluted 30 ml into 130 ml of DMSO (4 mM) and 1 μl was added to an assay plate. These were then serially diluted 1:3 (50 μl to 100 ml DMSO) for a total of 8 points. Plates were prepared such that each kinase reaction is 30 ml in 1× kinase buffer (25 mM HEPES, pH 7.2, 8 mM MgCl2, 2 mM MnCl2, 50 mM NaCl, 0.01% Brij, 1 mM DTT, 0.01% BSA), 5% DMSO and 10 μM ATP. Substrate was 30 nM biotin-(E4Y)10 (EMD Millipore, Cat#12-440). C-kit mutant D816V kinase was at 0.75 ng per sample. After incubation of the kinase reaction for 30 minutes at room temperature, 5 μl of donor beads (Streptavidin coated beads (Perkin Elmer Life Science) final concentration 7.5 μg/ml) in stop buffer (25 mM Hepes pH 7.5, 100 mM EDTA, 0.01% BSA) was added, the sample was mixed and incubated for 30 minutes at room temperature before adding 5 ml of acceptor beads (PY30 coated beads (Perkin Elmer Life Science) final concentration 7.5 μg/ml) in stop buffer.
Measurement: Ki = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 571.71, ALogP 5.26, HBA 7, HBD 3, PSA 128.89, QED 0.24
Compound (SMILES): COc1cccc(CC(=O)N2CCc3cccc(CCc4ccc(C(=O)O)cc4)c32)c1
InChIKey: YTBOANLSJHKTHF-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: FLIPR Assay: Cells were seeded at a density of 5×104 cells per well in Biocoat® Poly-D-lysine-coated black-wall, clear-bottom 96-well plates (Becton-Dickinson) and allowed to attach overnight in an incubator at 37° C. Cells were then washed two times with HBSS-HEPES buffer (Hanks Balanced Salt Solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using a Denley Cellwash plate washer (Labsystems). After 45 minutes of dye-loading in the dark, using the calcium-sensitive dye Fluo-4 AM at a final concentration of 2 mM, plates were washed four times with HBSS-HEPES buffer to remove excess dye leaving 100 ml in each well. Plates were re-equilibrated to 37° C. for a few minutes.Cells were excited with an Argon laser at 488 nm, and emission was measured through a 510-570 nm bandwidth emission filter (FLIPR™, Molecular Devices, Sunnyvale, Calif.). Drug solution was added in a 50 μl volume to each well to give the desired final concentration. The peak increase in fluorescence intensity was recorded for each well. On each plate, four wells each served as negative (HBSS-HEPES buffer) and positive controls (standard agonists: BW245C (hDP); PGE2 (hEP1; hEP2/Gqs5; hEP3A/Gqi5; hEP4/Gqs5); PGF2a (hFP); carbacyclin (hIP); U-46619 (hTP), depending on receptor). The peak fluorescence change in each drug-containing well was then expressed relative to the controls.
Measurement: Ki = 449 nM (pChEMBL 6.349999904632568)
Descriptors: MW 415.49, ALogP 4.31, HBA 3, HBD 1, PSA 66.84, QED 0.62
Compound (SMILES): C[C@@H]1CC(=O)Nc2cccc(-c3cn(C)c4ccccc34)c2O1
InChIKey: HVMOCLOBKVZTJL-GFCCVEGCSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 8072 nM (pChEMBL 5.090000152587891)
Descriptors: MW 306.37, ALogP 3.95, HBA 3, HBD 1, PSA 43.26, QED 0.74