lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCC(=O)NC12CCC(c3nc(-c4ccc(C(=O)Nc5cc(C(F)(F)F)ccn5)cc4)c4c(N)nccn34)(CC1)C2
InChIKey: JIHRZOSZENHGGH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Inhibitory Activity Against BTK(wt)/BTK(C481S) (Determination of IC50 Value): The substrate solution was prepared by adding the substrate poly(Glu, Tyr) sodium salt (Sigma Aldrich, St. Louis, Mo.) to the substrate reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT and 1% DMSO) (final substrate concentration in the reaction solution was 0.2 μM). Test compound was formulated into stock solutions at 10 mM concentration with 100% DMSO, and 3-fold serial dilution for 10 doses was performed in a 384-well cyclic olefin copolymer LDV microplate. BTK(wt) or BTK(C481S) kinase (recombinant human full-length protein, histidine tag, expressed in insect cells, Invitrogen, Carlsbad, Calif.) was added to the substrate solution and mixed gently (final BTK concentration in the reaction solution was 8 nM). Test compound in 100% DMSO was then added to the kinase reaction mixture by acoustic liquid transfer technology (Echo 550; nanoliter range) (Labcyte Inc, Sunnyvale, Calif.) and incubated for 20 min at room temperature. 33P-ATP (specific activity 10 μCi/μL) was added to the reaction mixture to initiate the reaction, followed by incubation at room temperature for 2 h. A small portion of the reaction mixture was dropped onto P-81 ion exchange filter paper (Whatman). After washing the unbound phosphate from the filter paper with 0.75% phosphate buffer (three times) and drying, the remaining radioactivity on the filter paper was measured. The kinase activity was expressed as the percentage of the remaining kinase activity in the test sample to the kinase activity in the vehicle (dimethyl sulfoxide) blank control. IC50 values were calculated by curve fitting the data obtained using Prism (GraphPad Software).
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 579.58, ALogP 4.36, HBA 8, HBD 3, PSA 136.53, QED 0.3
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1nc(Nc2cc(C(=O)NOCCO)ccc2OC)ncc1Cl
InChIKey: WWDDDQWQZZTRPH-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P27361
Assay: Omnia Assay: The protocol below describes continuous-read kinase assays to measure potency of compounds against activated ERK1 enzyme. The mechanics of the assay platform are best described by the vendor (Invitrogen, Carlsbad, Calif.) on their website at the following URL: invitrogen.com/site/us/en/home.html.Briefly, a 1.25× stock of ERK1 enzyme (14-439-K) from Millipore (Billerica, Mass.), 5×ATP (AS001A) and ST17-Sox conjugated peptide substrate (KNZ1171C) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT. 10 μL of ATP/ST17-sox peptide substrate mix was combined with 0.5 μL volume of 100% DMSO and serially diluted compounds were prepared in 100% DMSO in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.). Kinase reactions were started with the addition of 40 μL of ERK1 solution and monitored every 71 seconds for 30-240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to 30+ minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (minutes or seconds) and then plotted against inhibitor concentration to estimate AppIC50 from log [Inhibitor] vs Response, Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.).
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 498.93, ALogP 3.4, HBA 9, HBD 5, PSA 146.73, QED 0.15
Compound (SMILES): NC(=O)c1ccn(C(=O)N2CCC3(CCN(Cc4cc(Cl)ccc4C(F)(F)F)C3)CC2)n1
InChIKey: GGBYNCPPPDSAOL-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction for mouse assays; human prefrontal cortex membrane fractions for human assays) (50 mL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh (1.0 mL, 50 mM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software. IC50 data from this assay is shown in Table 1. All compounds in Table 1 were more potent inhibitors of MAGL than FAAH.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 469.9, ALogP 3.61, HBA 5, HBD 1, PSA 84.46, QED 0.74
Compound (SMILES): Nc1c(I)ccc2cc(-n3ccc4ccncc43)ncc12
InChIKey: GXYFXCXEWJOKHC-UHFFFAOYSA-N
Target: Microtubule-associated protein tau (SINGLE PROTEIN), Homo sapiens
UniProt: P10636
Assay: Tau binding assay: For displacement tau binding assay, unlabeled test compounds were dissolved in DMSO at 1 mM. Dilutions of tests compounds to various concentrations were made in 100% DMSO at 1000× final assay concentration and 0.225 μl aliquots were dispensed into assay plates. Brain homogenates were diluted to 0.5 mg/mL from original 30 mg/mL volume in Assay Buffer, and 200 μl were added to the assay plate for a final concentration of 100 μg wet weight/assay tube. [3H]-6 was prepared at 100× final concentration in Assay Buffer plus 20% DMSO and 25 μl was added to the assay plate for final assay concentration of 0.25 nM. The assay plate was incubated at room temperature (25° C.) for 90 minutes. Unbound and bound ligand were separated by filtration of bound onto GF/C filter plates (pre-treated for 30 min with 0.2% polyethyleninamine) using a Packard UniFilter Harvester and washing away unbound with 2.5 ml per well ice cold 5 mM Tris at pH 7.4. Filter plates were dried for 1 hour in a vacuum oven and 50 μl/well MicroScint-20 were added. Plates were counted 1 min per well by Packard Topcount. Total amount of radioligand used in the assay was determined by counting 25 μl of 10× ligand stock. Data was analyzed using Activity Base software to generate 4 p fit of dose responses; and, Ki values were calculated.
Measurement: Ki = 0.512 nM (pChEMBL 9.289999961853027)
Descriptors: MW 386.2, ALogP 3.76, HBA 4, HBD 1, PSA 56.73, QED 0.4
Compound (SMILES): CN(C)[C@H]1C[C@@H](c2cc(Cl)cc(Cl)c2)CC[C@@H]1Oc1cc(F)c(S(=O)(=O)Nc2ccncn2)cc1Cl
InChIKey: JALUEUNLLHKOHL-WVFSVQOHSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In Vitro Assay): Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (NaV's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following voltage clamp electrophysiology studies were performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAs for Nav1.7 (NM_002977) and Nav1.5 (AC137587) were stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents were measured in the whole-cell configuration using Syncropatch 384PE (NanIon Technologies, Germany). 1NPC®-384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10l cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 μl of compounds is added to 40 μl in each well (1:3 dilution of compounds), and after mixing, 20 μl is removed so the volume is retained at 40 μl. After approximately 13 minutes recordings, 20 μl/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.For voltage protocol, an holding potential of −50 mV is applied during the whole experiment. A depolarizing step is applied to −10 mV for 10 ms, followed by a hyperpolarization step to −150 mV for 20 ms to allow channel recovery from inactivation. A second depolarizing step is applied from −150 mV to −10 mV for 10 ms, where currents were measured to derive blocking effects of compounds. Inhibition is determined based on 7.5 min of compound incubation.
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 573.91, ALogP 6.02, HBA 6, HBD 1, PSA 84.42, QED 0.37
Compound (SMILES): COc1cc([C@@H](CO)NC(=O)[C@@H](C)N2Cc3ccc(-c4cc(Nc5cnn(C)n5)ncc4Cl)cc3C2=O)ccn1
InChIKey: CFCZXLBSUSYGHH-IVZQSRNASA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 563.02, ALogP 2.87, HBA 10, HBD 3, PSA 147.39, QED 0.28
Compound (SMILES): COc1ccc(Cc2nc(-c3cnn4c(C5CCNCC5)cc(=O)[nH]c34)no2)cc1F
InChIKey: WQFPQXLSNQVZFY-UHFFFAOYSA-N
Target: Tissue-type plasminogen activator (SINGLE PROTEIN), Homo sapiens
UniProt: P00750
Assay: Plasma-based Clot Lysis Assay: The clot-lysis test system configures the kinetics of clot formation and degradation in vitro and allows quantifying modulation of the process by selected test compounds.The test compounds were dissolved in 1% acetic acid and further complemented with an equal volume of DMSO. The resulting stock solutions were serially diluted in 0.5% acetic acid/50% DMSO. 1 μL aliquots of these solutions were placed into 384 well microplates (Greiner, black, transparent bottom), followed by 30 μL of diluted human citrated plasma (platelet-poor, final concentration: 5%; supplemented with fibrinogen, final concentration: 3 μM; dilution buffer: 20 mM HEPES, 150 mM NaCl, 0.01% Brij (pH 7)). The reactions were started by addition of 20 μL of CaCl2 (final concentration: 10 mM), and tPA (tissue plasminogen activator, final concentration: 0.2 nM) in dilution buffer, followed by an additional volume of 20 μL dilution buffer for improved mixing. The reactions were incubated at 37° C. Clot formation and degradation was monitored spectrophotometrically by kinetic optical density measurements at 405 nm. IC50 values were determined by comparing the resulting time courses with the time course of a blank control reaction.
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 424.44, ALogP 2.28, HBA 8, HBD 2, PSA 110.34, QED 0.51
Compound (SMILES): C=CC(=O)N1CCCCC(Nc2nc(Nc3ccc(OCCOC)cc3)c3c(c2F)CNC3=O)C1
InChIKey: XTTKZZZHMAQHQG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Lantha Screening Kinase Assay: BTK: The compound was predissolved in 100% DMSO. 10 mM drug stock solution was obtained by dissolution at room temperature and then serially diluted with 8 vol % DMSO solution to a final concentration of 10-0.005 μM. 2.5 μl of a solution of substance to be tested and 2.5 μl of kinase (Invitrogen PV3363) diluted with the reaction buffer were added into each well of 384-well plate (Corning 3676), and then the mixture of Fluososcei-PolyGT (Invitrogen PV3610) substrate and ATP (Invitrogen PV3227) diluted with 5 μl of the reaction buffer were added to initiate the reaction. Among the wells, the kinase in the blank well was replaced with a reaction buffer and the kinase well (Enzyme) was not added with any drug. After shaking at 25° C. for 60 minutes in the dark, 10 μl of Detection Solution (mixture of Invitrogen PV3528 and EDTA, which was diluted with TR-FRET dilution buffer, the working concentration of EDTA was 5 mM, the working concentration of Lanthascreening Tb PY20 antibody was 0.2 nM) was added and shaken at room temperature for 30 minutes. The plates were read on a VictorX5 fluorescent plate reader (PerkinElmer) and the light absorption at an excitation wavelength of 340 nm and emission wavelengths of 500 nm and 520 nm was measured.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 483.54, ALogP 3.21, HBA 7, HBD 3, PSA 104.82, QED 0.37
Compound (SMILES): C[C@](O)(CO)CNC(=O)C[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1
InChIKey: RLUPBJVKCABYOH-LAUBAEHRSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 602.53, ALogP 2.9, HBA 6, HBD 4, PSA 127.17, QED 0.34
Compound (SMILES): Cn1cc(-c2cc(S(C)(=O)=O)ccc2Oc2cccc(NCc3ccccc3C#N)c2)c2cc[nH]c2c1=O
InChIKey: VTUPHGINWICZNI-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay (BD1: amino acids K57-E168): Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (μL) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve μL of this solution were added to the assay plate to reach a final volume of 18 μL. The final concentration of 1× assay buffer contains 2% DMSO, 50 μM-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 524.6, ALogP 5.21, HBA 7, HBD 2, PSA 116.98, QED 0.3
Compound (SMILES): C=C(C)C(=O)c1ccc(Nc2nc(Nc3ccccc3)ncc2F)cc1
InChIKey: OQGGDZLIFOFKSA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Omnia Assay: ITK: This example describes continuous-read kinase assays to measure inherent potency of compound against active forms of ITK enzymes as described in Example 251 above except that the modified ITK-optimized reagent conditions are:[ITK]=10 nM, [ATP]=25 [Y6-Sox]=10 μM (ATP KMapp=33 μM).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 348.38, ALogP 4.86, HBA 5, HBD 2, PSA 66.91, QED 0.49
Compound (SMILES): CSc1cc(C)[nH]c(=O)c1CNC(=O)c1c(C)n([C@H](C)[C@H]2OC[C@@H](N3CCC34COC4)CO2)c2ccccc12
InChIKey: ZSOKQRDTSMDYQM-HIRWGAFWSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: EZH2 biochemical assay (IC50): Compound potencies were assessed through incorporation of 3H-SAM into a biotinylated H3 peptide. Specifically, 30 pM PRC2 containing wt EZH2 (pentameric complex prepared in-house) was pre-incubated with 450 nM SAM, 450 nM 3H-SAM, 2 μM H3K27me3 activating peptide (H2N-RKQLATKAAR(Kme3)SAPATGGVKKP-amide) and compounds (as 10 point duplicate dose response titrations in DMSO, final assay 0.8% DMSO (v/v)) for 3-5 h in 50 mM Tris (pH 8.5), 1 mM DTT, 0.07 mM Brij-35, 0.1% BSA, and 0.8% DMSO in a total volume of 12.5 μl. Reaction was initiated with biotinylated H3 substrate peptide (H2N-RKQLATKAAR(Kme1)SAPATGGVKKP-NTPEGBiot) as a 2 μM stock in 12.5 μl of buffer and allowed to react at room temperature for 18-22 h. Quenching was accomplished by addition of 20 μl of STOP solution (50 mM Tris (pH 8.5), 200 mM EDTA, 2 mM SAH). 35 μl of the quenched solution was transferred to streptavidin coated FlashPlates (PerkinElmer), incubated 1-2 h, washed, and read in a TopCount Reader (PerkinElmer). IC50s were calculated in Genedata Screener using non-linear least square four parameter fits, where the four parameters were IC50, Hill slope, pre-transitional baseline (0% INH), and post-transitional baseline (100% INH).
Measurement: IC50 = 2.75 nM (pChEMBL 8.5600004196167)
Descriptors: MW 552.7, ALogP 3.38, HBA 8, HBD 2, PSA 97.82, QED 0.43
Compound (SMILES): CC1(C)CN(c2ccc(C(=O)N3CCC(N4CCc5ccccc5C4)CC3)cn2)CCO1
InChIKey: PPIZZIWJUGVTDQ-UHFFFAOYSA-N
Target: Alpha-1A adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35348
Assay: In Vitro Assay: Antagonism against the adrenoreceptor α1A was tested using a recombinant human α1A receptor CHO cell line which additionally also recombinantly expresses mtAeq (mitochondrial aequorin). Antagonism against the adrenoreceptor α2A was tested using a recombinant human α2A-Gα16 receptor fusion protein CHO cell line (PerkinElmer Life Sciences) which additionally also recombinantly expresses mtAeq. Antagonism against the adrenoreceptor α2B was tested using a recombinant human α2B receptor CHO cell line (PerkinElmer Life Sciences) which additionally also recombinantly expresses mtAeq. Antagonism against the adrenoreceptor α2C was tested using a recombinant human α2C receptor CHO cell line which additionally also recombinantly expresses a chimaric G protein (Gαqi3) and mtOb (mitochondrial obelin).The cells were cultivated at 37° C. and 5% CO2 in Dulbecco's modified Eagle's Medium/NUT mix F12 with L-glutamine which additionally contains 10% (v/v) inactivated foetal calf serum, 1 mM sodium pyruvate, 0.9 mM sodium bicarbonate, 50 U/ml penicillin, 50 μg/ml streptomycin, 2.5 μg/ml amphotericin B and 1 mg/ml Geneticin. The cells were passaged with enzyme-free Hank's-based cell dissociation buffer. All cell culture reagents used were from Invitrogen (Carlsbad, USA).
Measurement: IC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 434.58, ALogP 3.36, HBA 5, HBD 0, PSA 48.91, QED 0.74
Compound (SMILES): CCOCCOc1ccc(-c2cc(=O)[nH]c(-c3c(F)ccc(CNC(=O)C(C)C)c3Cl)n2)cn1
InChIKey: ZXMWDPUUYWCASI-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 488.95, ALogP 3.98, HBA 6, HBD 2, PSA 106.2, QED 0.42
Compound (SMILES): O=C(/C=C/CN1CCC(CO)CC1)N1CCOc2c1ccc1ncnc(Nc3ccc(OCc4ccccn4)c(Cl)c3)c21
InChIKey: CHLPQPXEVHSEPL-HWKANZROSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of EGFR and HER2 Kinase Activity Assay: The assay for the inhibition of EGFR and HER2 kinase activity by small molecular compounds was carried out using the method as follows:1) Dilution of the compoundsIn a 96-well plate a, the compounds were diluted with DMSO using a 3-fold gradient dilution to form 11 concentrations, the 12th concentration is pure DMSO (as a positive control); and in a new 96-well plate b the above solutions were diluted 25 times with ultrapure water (DMSO concentration is 4%).2) Transferring the compounds to 384-well plateThe compound solutions diluted with ultrapure water in the 96-well plate b above was transferred to the corresponding wells of a 384-well plate in duplicate.3) Addition of 4×kinase solution: 2.5 μl of the above 4× kinase solution was taken using multichannel pipette and added to the corresponding reaction wells of the 384-well plate, mixed well and pre-reacted at room temperature for 5 minutes.4) Addition of 2×substrate/ATP mixed solution: 5 μl of the above 2×substrate/ATP mixed solution was taken using multichannel pipette and added to the corresponding reaction wells of the 384-well plate.5) Negative control: negative control wells were set in the 384-well plate, and 2.5 μl 4×substrate, 2.5 μl 4×enzyme solution, 2.5 μl 1×Kinase Assay Buffer and 2.5 μl ultrapure water containing 4% DMSO were added to each well.6) Mixed by centrifugation and kept at room temperature for 2 hours in the dark.7) Termination of the enzymatic reaction:5 μl of the above 4× stop solution was pipetted to the corresponding wells of the 384-well plate, centrifuged and mixed, and reacted at room temperature for 5 minutes.8) Development reaction:5 μl of the above 4× detection solution was pipetted into the corresponding wells of the 384-well plate, centrifuged and mixed, and reacted at room temperature for 1 hour.9) The 384-well plate was placed into a microplate reader and the signal was detected using the corresponding program.10) IC50 analysis:Well reading value=10000*EU665 value/EU615 valueInhibition rate=(reading value of positive control well−reading value of experimental well)/(reading value of positive control well−reading value of negative control well)*100%Corresponding IC50s can be calculated by entering the drug concentrations and the corresponding inhibition rates into GraphPad Prism 5.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 601.11, ALogP 4.99, HBA 9, HBD 2, PSA 112.94, QED 0.26
Compound (SMILES): Cc1c(-c2cc3cc(NC(=O)O[C@H]4C[C@H]([C@H](C)O)C4)ncc3c(N)c2F)cnc2c1NCCO2
InChIKey: UXHNAQGLGZVSBL-YDHLFZDLSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBD: TBD
Measurement: Ki < 0.013 nM (pChEMBL 10.88599967956543)
Descriptors: MW 467.5, ALogP 3.84, HBA 8, HBD 4, PSA 131.62, QED 0.43
Compound (SMILES): COc1ccc(C2=Nn3c(nnc3-c3cc(-c4ccccc4)n[nH]3)SC2C)cc1OC
InChIKey: DWHTUMIXHQKPGC-UHFFFAOYSA-N
Target: Signal transducer and activator of transcription 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P40763
Assay: Bioassay: Compound samples (in 3-5 mg quantities) were evaluated in a variety of assays. All compound submissions were fully characterized (1H, 13C, IR, FIRMS), satisfied purity criteria (≥95% LC/MC, ELS).
Measurement: IC50 = 7600 nM (pChEMBL 5.119999885559082)
Descriptors: MW 432.51, ALogP 4.1, HBA 8, HBD 1, PSA 90.21, QED 0.51
Compound (SMILES): Nc1noc2nc(Cl)ccc12
InChIKey: YZSDNYDQYOOJOM-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzymatic Assay: Recombinant human IDO1 (rhIDO1) was expressed and purified from cultures of EC538 strain of E. coli transformed with pREP4 and pQE9-IDO plasmid. Reaction mixes were set up in 384-well microplates containing 50 mM phosphate buffer, 10 mM ascorbic, 10 μM methylene blue, 100 μg/mL catalase, 80 μM TRP, 0.01% Tween 20 (v/v) mixed with rhIDO1 (15 μL) at a final concentration of 9 nM in a total volume of 30 μL assay medium. The plates were incubated at 37° C. for 30 min, and the enzymatic reaction was terminated by adding piperidine (200 mM) and heated at 65° C. for 20 min. Fluorescence intensity was read at λex 400 nm and λem 500 nm. Test compounds were dissolved in 100% DMSO and pre-diluted in assay medium prior to adding rhIDO1.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 169.57, ALogP 1.46, HBA 4, HBD 1, PSA 64.94, QED 0.61
Compound (SMILES): Cc1cc(C)nc(NC(=S)N2CCC(C(=O)N3CCCCC3)CC2)c1
InChIKey: ODAOKFCCIOZGKF-UHFFFAOYSA-N
Target: D-3-phosphoglycerate dehydrogenase (SINGLE PROTEIN), Homo sapiens
UniProt: O43175
Assay: Enzyme Assay: PHGDH assay buffer contained 50 mM TEA pH 8.0, 10 mM MgCl2, 0.05% BSA, and 0.01% Tween-20. PHGDH enzyme buffer consisted of assay buffer with 20 nM PHGDH and 0.2 mg/mL diaphorase. PHGDH substrate buffer contained 0.3 mM NAD 1.25 mM glutamate, 0.1 mM 3-phosphoglycerate, 0.2 mM resazurin, 1 μM PSAT1, and 1 μM PSPH. qHTS was performed in 1536-well plates dispensed with a BioRAPTR FRD. Each well contained equal volumes of substrate buffer and assay buffer. Plates were read at 0 minutes and 20 minutes at room temperature with a ViewLux uHTS Microplate Imager (PerkinElmer). Follow-up assays were performed in black 384-well plates (Greiner) in 20 μL of enzyme buffer to which compounds were added in dose-response with an HP D300 digital dispenser (Hewlett-Packard), followed by addition of 20 μL of substrate buffer. Plates were incubated at room temperature (25° C.) and read at 0 and 20 minutes with a Spectramax M5 plate reader (Molecular Devices) in fluorescence intensity mode with a λex=550 nm and λem=600 nm (emission cutoff=590 nm).
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 360.53, ALogP 3.12, HBA 3, HBD 1, PSA 48.47, QED 0.82
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3c(F)c(F)cc(F)c3F)cc1F)nn2C1CCCC(N2C(=O)C=CC2=O)C1
InChIKey: SOZGNAMCSJWJFO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: HTRF Assay: The enzyme reaction mixture of BTK wild type standard HTRF assay contained 1 nM BTK wild type, 1 μM biotin-TK1 peptide, and 30 μM ATP in a buffer. The enzyme reaction were carried out at room temperature for 60 minutes. 5 μl of 0.2 M EDTA were added to quench the reaction and then the inhibitors (5 μl) were added at final concentrations of 2 nM antibody and 62.5 nM XL665. The plates were incubated at room temperature for 60 minutes and then read in the Envision plate reader. The readouts were transformed into inhibition rate % by the equation of (Min Ratio)/(Max-Min)*100%. Hence the 1050 data of test compounds were generated by using four parameters curve fitting.
Measurement: IC50 = 593 nM (pChEMBL 6.230000019073486)
Descriptors: MW 570.48, ALogP 4.97, HBA 8, HBD 1, PSA 116.23, QED 0.2
Compound (SMILES): C[C@H](C(=O)N[C@H](CO)c1cc(O)cc(F)c1)N1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O
InChIKey: KKLYRZBXJQXIJG-OYLFLEFRSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 570.02, ALogP 3.43, HBA 8, HBD 4, PSA 136.91, QED 0.32
Compound (SMILES): O=C(O)[C@H](CCN(CCCCc1ccc2c(n1)NCCC2)C1CC(F)(F)C1)Nc1ncnc2ccccc12
InChIKey: IEIQVKZZQPWGHH-DEOSSOPVSA-N
Target: Integrin alpha-5/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P18564
Assay: Binding Assay: Microplates were coated with recombinant human integrin αVβ6 (2 μg/mL) in PBS (100 μL/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). The plate was blocked with 200 μL/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 μg/mL) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 524.62, ALogP 4.76, HBA 7, HBD 3, PSA 103.27, QED 0.29
Compound (SMILES): CC(C)=Cc1cc(S(=O)(=O)N2CCCC2)ccc1N(c1ccnc(N)n1)C(C)C
InChIKey: HLFJCWKBSKZNNM-UHFFFAOYSA-N
Target: Histamine H4 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3N8
Assay: Binding Assay on Recombinant Human Histamine H4 Receptor: Receptor binding was assessed using isolated plasma membranes from SKNMC neuroblastoma cell lines stably expressing recombinant human H4 receptor. To isolate plasma membrane enriched in recombinant human H4 receptor, cells were collected from 100 culture dishes (150 mm) in PBS-EDTA. The cells were pellet by centrifugation (800 g) and resuspended in 5×TE buffer (50 mM Tris/5 mM EDTA). The cells were then homogenized and centrifuged at 1000 g for 10 min. The supernatant was collected and centrifuged for an additional 26000 g for 30 min. The supernatant was removed and the pellet was gently resuspended in 30 ml of 5×TE buffer. Protein concentration was then determined using Bradford protein assay (Bradford, M. M., Anal. Biochem., 1976, 72: 248-254). Isolated cell membranes were incubated with 2×KD (10 nM), [3H] histamine (Specific activity: 23 Ci/mmol), with or without test compounds for 45 min at 25° C. Nonspecific binding was defined with 100 μM cold histamine. Ki values were calculated based on an experimentally determined appropriate KD values according to Cheng and Prusoff (Biochem. Pharmacol. 1973, 22(23):3099-3108). Membranes were harvested by rapid filtration using the 96 well Brandel system or a cell harvester using a Whatman GF/C filter or filter plates treated with 0.5% polyethylenimine (PEI), and washed 4 times with ice-cold 50 mM Tris/5 mM EDTA buffer. Filters were then dried, mixed with scintillant and radioactive counts were determined.
Measurement: Ki = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 415.56, ALogP 3.81, HBA 6, HBD 1, PSA 92.42, QED 0.77
Compound (SMILES): Cn1cc(-c2ccc3c(c2OCC2CCC2)n(CCN2CCN(S(C)(=O)=O)CC2)c(=O)n3C)c2cc[nH]c2c1=O
InChIKey: SBOJEZDUTMGDOM-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 5.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 568.7, ALogP 1.94, HBA 9, HBD 1, PSA 114.57, QED 0.35
Compound (SMILES): COCCN1CCC(c2nnc(-c3cc(Cl)c(N)c4c3OCCC4)o2)CC1
InChIKey: GTUCZTYJFXBSTJ-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13639
Assay: Cell-Based Assay: A stable CHO cell line expressing recombinant human 5-HT4 receptor and pCRE-Luc reporter system was used for cell-based assay. The assay offers a non-radioactive based approach to determine binding of a compound to GPCRs. In this specific assay, the level of intracellular cyclic AMP which is modulated by activation or inhibition of the receptor is measured. The recombinant cells harbor luciferase reporter gene under the control of cAMP response element.The above cells were grown in 96 well clear bottom white plates in Hams F12 medium containing 10% fetal bovine serum (FBS). Prior to the addition of compounds or standard agonist, cells were serum starved overnight. Increasing concentrations of test compounds were added in OptiMEM medium to the cells. The incubation was continued at 37° C. in CO2 incubator for 4 hours. Medium was removed and cells were washed with phosphate buffered saline.
Measurement: EC50 = 15.6 nM (pChEMBL 7.809999942779541)
Descriptors: MW 392.89, ALogP 3.12, HBA 7, HBD 1, PSA 86.64, QED 0.78
Compound (SMILES): COCCOCCOCCOCCOCCOCCOCCOc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)C)nn4-c4ccc(C)cc4)c4ccccc34)n2)cc(OC)c1
InChIKey: GOMLJGVTZFYKJG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 12 (SINGLE PROTEIN), Homo sapiens
UniProt: P53778
Assay: Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against p38MAPKγ (MAPK12: Invitrogen), are evaluated in a similar fashion to that described hereinabove. The enzyme (800 ng/mL, 2.5 μL) is incubated with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solution (2.5 μL, 400 μM) is then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, Thermo Scientific).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 938.09, ALogP 8.56, HBA 16, HBD 3, PSA 189.06, QED 0.04
Compound (SMILES): COc1cccc(-c2nnc(NS(=O)(=O)[C@@H](C)[C@@H](OC(C)C)c3ncc(C)cn3)n2[C@@H](C)[C@H]2CCCO2)n1
InChIKey: WQMLZVQRMWRLGM-GWOFKNCZSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager.
Measurement: EC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 545.67, ALogP 3.48, HBA 11, HBD 1, PSA 143.24, QED 0.38
Compound (SMILES): CCOC(=O)C1=CN(C(=O)c2cc3ccccc3s2)CC(C)(C)c2cn[nH]c21
InChIKey: MKHOTVHNGSTQLF-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: FXR Agonist Assay: Starting from 3.33 mM of compound in DMSO solution, a 10-point 3-fold serial dilution was made by diluting 5 μL of compound into 10 μL of DMSO. The serially diluted compound was then diluted 1:33 into DMEM. This medium was then diluted ten-fold into the culture medium with the cells (10 μL/well). All concentration points are assayed in duplicate. Plates were incubated at 37° C. for 20 hours. After the incubation, 20 μL of culture medium were removed from each well and mixed with 50 μL of assay solution (Pierce Gaussia Luciferase Flash Assay Kit). The luminescence was measured immediately after addition of the Luc substrate with an Envision microplate reader. The raw data was uploaded to CDD and dose-response curves were generated using the Levenberg-Marquardt algorithm integrated into CDD. A negative control DMSO is included on each plate and used to normalize the data with the CDD built-in normalization function.
Measurement: EC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 395.48, ALogP 3.96, HBA 5, HBD 1, PSA 75.29, QED 0.68
Compound (SMILES): CC(C)(COc1ccc(-c2ccc(-c3ncc(-c4ccccc4)[nH]3)cn2)cc1)C(=O)O
InChIKey: VPXXJXQAIYCGPR-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Measurement of DGAT1 Inhibitory Activity: As a buffer used for the enzymatic reaction of DGAT1, 100 mM Tris-HCl (pH7.4), 200 mM Sucrose, and 20 mM MgCl2, 0.125% Bovin Serum Albumin (BSA) were used. To this buffer, a test compound with predetermined concentration of test compound, 15 μM dioleylglycerol, 5 μM [14C]-palmitoyl-CoA, 100 μg-protein/mL, highly DGAT1-expressing expresSF+® microsome, 0.75% acetone, and 1% dimethylsulfoxide were added, and a triglyceride (TG) synthesis reaction in a volume of 100 μL was carried out at 30° C. for 20 minutes. 90 μL of the reaction solution was added to 810 μL of methanol to cease the reaction. The reaction solution was added to Oasis® μ Elution plate (Waters) and eluted with 150 μL of mixture of acetonitrile:isopropanol (=2:3). 150 μL of MicroScinti-40 (Perkin-Elmer Corp.) were added to the eluted solution and the mixture was sufficiently stirred, and an amount of [14C]-TG produced in the reaction was determined by measuring using TopCount-NXT (Perkin-Elmer Corp.).The inhibitory ratio was calculated by the following equation.Inhibitory ratio (%)=(1−(TG amount when the test compound was added−blank TG amount)/(control TG amount−blank TG amount))×100Here, a count of [14C]-TG in the solution where the reaction was carried out without adding the test compound was regarded control TG amount, and a count of [14C]-TG in the solution to which the test compound and highly DGAT1 expressing expresSF+® microsome were not added was regarded as blank TG amount. Further, a concentration of test compound required to inhibit the synthesis of [14C]-TG by 50% (IC50 value) was calculated by Prism 5.01 (GraphPad Softwear).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 413.48, ALogP 5.3, HBA 4, HBD 2, PSA 88.1, QED 0.43
Compound (SMILES): Cn1cnc2c(c(-c3cc(Cl)c(O)c(C(N)=O)c3)cn2CC(=O)Nc2cc(N3CC4CC3CO4)ncc2Cl)c1=O
InChIKey: NEDWDNSTIAJHBL-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Surface Plasmon Resonance (SPR) Assay: SPR studies were performed using a Biacore™ T200 instrument (GE Health Sciences Inc.). The BCL6 BTB protein used in the FP assay was biotinylated using the site specific biotinylating enzyme BirA, and then cleaved with TEV protease to produce the BCL6 BTB domain (biotin-thrombin-BCL6 amino acids 1-129; 17 kd) that were use in SPR. This protein was stably captured (1000RU) to streptavidin coupled SA chips (BR-1005-31, GE Health Sciences Inc.) according to the manufacture's protocol. Compounds were dissolved in 100% DMSO at 10 mM and 2-fold serial dilutions were done in 100% DMSO. For SPR analysis the serially titrated compounds were diluted 1/20 into buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween-20, 3 mM EDTA) giving a final concentration of 5% DMSO. The Biacore flow rate was set at 100 ul/min. For KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 seconds. Curve fitting and KD calculations were done with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 33.9 nM (pChEMBL 7.46999979019165)
Descriptors: MW 584.42, ALogP 2.52, HBA 10, HBD 3, PSA 157.6, QED 0.31
Compound (SMILES): CC(C)n1cc(S(=O)(=O)c2ccc(NC(=O)NCc3ccncc3)cc2)cn1
InChIKey: LYCUWFRAFXSTKG-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Enzyme Assay and NMN Detection: NAMPT reaction was performed in 50 mM HEPES, pH 7.5, containing 5 mM MgCl2, 0.1% Prionex, 0.005% Tween 20, and 1 mM TCEP at room temperature in Greiner 384-well black polypropylene plates. The concentration of substrates and enzymes in the final assay were as follows: ATP (120 uM), nicotinamide (5 uM), phosphoribosylpyrophosphate (6.25 uM), NAMPT enzyme (16 nM), pyrophosphatase (0.04 U/ml) in the final reaction volume 6 uL. Following 2 h incubation the reaction was stopped by addition 2.5 ul 20% acetophenone in dimethyl sulfoxide (DMSO) and 2.5 ul 2 MKOH. Plates were centrifuges and added with 10.5 ul 88% formic acid. The fluorescence (ex380/em460) was measured following another short incubation and 30 min incubation. The increased concentration of NMN was determined from a calibration curve obtained at the time of experiment.
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 399.48, ALogP 3.01, HBA 6, HBD 2, PSA 105.98, QED 0.66
Compound (SMILES): COC(=O)c1c2n(c3ccc(C)cc13)C[C@@](NC(=O)c1ccc(-n3cnnc3)cc1Cl)(c1cccc(F)c1)CC2
InChIKey: WFDJJTKAABNDAV-SSEXGKCCSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: TBD: TBD
Measurement: IC50 = 4.58 nM (pChEMBL 8.34000015258789)
Descriptors: MW 558.01, ALogP 5.38, HBA 7, HBD 1, PSA 91.04, QED 0.29
Compound (SMILES): CC(C)c1c(-c2cc(NCc3ccccc3)c3ncnn3c2)[nH]c2ccc(C3CCNCC3)cc12
InChIKey: PGWLRVKJBTTYHK-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 464.62, ALogP 6.08, HBA 5, HBD 3, PSA 70.04, QED 0.28
Compound (SMILES): Cc1c(Cl)cccc1-c1noc([C@@H]2CCCN2C(=O)c2cccnc2-c2ccccn2)n1
InChIKey: SDZYVJRZDHHLBX-FQEVSTJZSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 1040 nM (pChEMBL 5.980000019073486)
Descriptors: MW 445.91, ALogP 5.13, HBA 6, HBD 0, PSA 85.01, QED 0.43