lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(c1cnn[nH]1)N1CC2CCC(C1)N2S(=O)(=O)c1cccc(C(F)(F)F)c1
InChIKey: CHGCKMOEOQWWOJ-UHFFFAOYSA-N
Target: Aldo-keto reductase family 1 member C3 (SINGLE PROTEIN), Homo sapiens
UniProt: P42330
Assay: AKR1C3-Inhibitory Activity Assay: The enzyme used was recombinant human AKR1C3 (Aldo-keto reductase family 1 member C3; GenBank Accession No. NM_003739). This was expressed in E. coli as GST (glutathione S transferase) fusion protein and purified by glutathione Sepharose affinity chromatography. The GST was removed by digestion with thrombin and subsequent size exclusion chromatography (Dufort, I., Rheault, P., Huang, X F., Soucy, P., and Luu-The, V., Endocrinology 140, 568-574 (1999)).For the assay, 50 nl of a 100-fold concentrated solution of the test substance in DMSO were pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2.5 μl of a solution of AKR1C3 in assay buffer [50 mM potassium phosphate buffer pH 7, 1 mM DTT, 0.0022% (w/v) Pluronic F-127, 0.01% BSA (w/v) and protease inhibitor cocktail (Complete, EDTA-free Protease Inhibitor Cocktail from Roche)] were added and the mixture was incubated for 15 min to allow pre-binding of the substances to the enzyme prior to the enzyme reaction. The enzyme reaction was then started by addition of 2.5 μl of a solution of NADPH (20 μM→final concentration in 5 μl of assay volume is 10 μM) and Coumberone (0.6 μM→final concentration in 5 μl of assay volume is 0.3 μM) in assay buffer, and the resulting mixture was incubated at 22° C. for the reaction time of typically 90 min. The concentration of the AKR1C3 and the reaction time was adapted to the respective activity of the enzyme preparation and adjusted such that the assay was carried out in the linear range. Typical AKR1C3 concentrations were in the region of 1 nM. The reaction was stopped by addition of 2.5 μl of a stop solution consisting of 3 μM EM-1404 as inhibitor (U.S. Pat. No. 6,541,463) in 50 mM HEPES pH7.5 (3 μM EM-1404→final concentration in 7.5 μl of assay volume is 1 μM). The fluorescence of the Coumberole was then measured at 520 nm (excitation at 380 nm) using a suitable measuring instrument (Pherastar from BMG Labtechnologies). The intensity of the fluorescence was used as a measure of the amount of Coumberole formed and thus of the enzyme activity of AKR1C3. The data were normalized (enzyme reaction without inhibitor=0% inhibition; all other assay components, but no enzyme=100% inhibition). Usually, the test substances were tested on the same microtiter plate at 11 different concentrations in the range from 20 μM to 73 pM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 10.9 nM, 3.1 nM, 0.9 nM, 0.25 nM and 73 pM, the dilution series were prepared prior to the assay on the level of the 100-fold concentrated solution by serial 1:3 dilutions with 100% DMSO) in duplicates for each concentration, and the IC50 values were calculated using a 4-parameter fit.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 415.4, ALogP 1.5, HBA 5, HBD 1, PSA 99.26, QED 0.82
Compound (SMILES): N#Cc1ccc(S(=O)(=O)[C@H]2CN(S(=O)(=O)c3ccc(F)cc3C(F)(F)F)C[C@@]2(O)CO)cc1
InChIKey: JROYPEPGQNHMNP-ZWKOTPCHSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: FLIPR Assay for hTRPV4 Expressed in BHK Cells: TRPV4 channel activation results in an influx of divalent and monovalent cations including calcium. The resulting changes in intracellular calcium were monitored using a calcium specific fluorescent dye Fluo-4 (MDS Analytical Technologies). BHK/AC9 cells transduced with BacMam virus expressing the human TRPV4 gene at a MOI of 78 were plated in a 384 well poly-D lysine coated plate (15,000 cells/well in 50 μL culture medium containing DMEM/F12 with 15 mM HEPES, 10% FBS, 1% Penicillin-Streptomycin and 1% L-glutamine). Cells were incubated for 24 hours at 37° C. and 5% CO2. Culture medium was then aspirated using a Tecan plate-washer and replaced with 20 μL/well of dye loading buffer: HBSS, 500 μM Brilliant Black (MDS Analytical Technologies), and 2 μM Fluo-4 AM. Dye loaded plates were then incubated in the dark at room temperature for 11.5 hours. 10 μL of test compounds diluted in HBSS (with 1.5 mM Calcium Chloride, 1.5 mM Magnesium Chloride and 10 mM HEPES, pH 7.4)+0.01% Chaps was added to each individual well of the plate, incubated for 10 min at room temperature in the dark and then 10 μL of agonist (N((S)-1-(((R)-1-((2-cyanophenyl)sulfonyl)-3-oxoazepan-4-yl)amino)-4-methyl-1-oxopentan-2-yl)benzo[b]thiophene-2-carboxamide, (Thorneloe et al, Sci. Transl. Med. (2012), 4, 159ra148) (hereinafter: Agonist Compound) was added to have a final concentration equals to the agonist EC80. Calcium signals were measured using FLIPRTETRA (MDS Analytical Technologies) or FLIPR384 (MDS Analytical Technologies) and the inhibition of Agonist Compound-induced calcium signal by the test compound was determined.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 508.47, ALogP 1.29, HBA 7, HBD 2, PSA 135.77, QED 0.58
Compound (SMILES): Cn1c(=O)n(CC2(C)CC2)c2ccc(S(=O)(=O)NC3(C)CC3)cc21
InChIKey: GAYFOPHCTDHHIW-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 μM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61 HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: EC50 = 4040 nM (pChEMBL 5.389999866485596)
Descriptors: MW 349.46, ALogP 1.97, HBA 5, HBD 1, PSA 73.1, QED 0.9
Compound (SMILES): C[C@@H]1CN(c2cccc3c2CN(C(=O)OCc2ccccc2)CC3)CCN1C(=O)Cn1cnc2cccnc21
InChIKey: BXEJBKYTTIEHTN-JOCHJYFZSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A (GenBank: AY242128) coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.038%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 mM, is also used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds. Emission peak values above base level after CXCL10 addition are exported after base line subtraction.
Measurement: IC50 = 7.94 nM (pChEMBL 8.100000381469727)
Descriptors: MW 524.63, ALogP 3.86, HBA 7, HBD 0, PSA 83.8, QED 0.39
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(C(=O)N5CC(N6CC[C@H](O)C6)C5)n4c3)ccc2o1
InChIKey: XAOUBAIYWOQTGH-INIZCTEOSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 418.46, ALogP 1.62, HBA 8, HBD 2, PSA 113.13, QED 0.52
Compound (SMILES): COc1nc2ccc(C(O)(c3cnc(C)n3C)c3cnc(C)n3C)cc2c(Cl)c1CNC(=O)CCC(F)(F)F
InChIKey: KORBVBIJMABUHI-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).In a ThermoFluor experiment where protein stability is monitored as the temperature is steadily increased, an equilibrium binding ligand causes the midpoint of an unfolding transition (Tm) to occur at a higher temperature. The shift in the melting point described as a ΔTm is proportional to the concentration and affinity of the ligand. The compound potency may be compared as a rank order of either ΔTm values at a single compound concentration or in terms of KD values, estimated from concentration response curves.
Measurement: Kd = 590 nM (pChEMBL 6.230000019073486)
Descriptors: MW 565, ALogP 4.22, HBA 8, HBD 2, PSA 107.09, QED 0.33
Compound (SMILES): CC1=NN(C)C(Cl)C1S(=O)(=O)Nc1ccc(-c2ncc3c(C)n[nH]c3n2)cc1
InChIKey: HJKCNTIBTLOHJW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Sgk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O00141
Assay: Enzymatic Activity Assay: The compounds were tested for serum and glucocorticoid-regulated kinase 1 (SGK-1) inhibitory activity in a substrate phosphorylation assay designed to measure the ability of the isolated enzyme to catalyze the transfer of phosphate from ATP to serine/threonine residues in a fluorescein-labeled substrate peptide, using recombinant human SGK-1 enzyme produced in a baculovirus system (Biomol, Hamburg, Germany, Cat. No. 4-331). The synthesized fluorescent labeled peptide substrate contained (5(6)-Carboxyfluorescein)-RPRAATF-NH2. The phosphorylated substrate peptide and non-phosphorylated substrate peptide were separated with caliper life science's lab-chip technology based on a micro fluidics method. All fluid flow was established on the chip by applying a vacuum of a few psi to the waste well transporting fluid from various sources through interconnecting channels. Because the phosphoryl group is doubly negatively charged, under the pressure-driven hydrodynamic flow and the voltage-driven flow within the electric field, the fluorescent labeled peptide substrate and its phosphorylation product appear at different times in the detection window to the detection point. Substrate turnover can thus be determined as the ratio of the product peak area and the sum of substrate peak area and product peak area.The enzyme reaction was carried out in a buffer containing 25 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 2 mM MnCl2, 2 mM DTT, and 0.03% bovine serum albumine. The enzyme was pre-incubated with the test compound for 30 min at 24° C. The kinase reaction was initiated by addition of the substrate mixture containing the peptide substrate (final concentration 1 μM) and ATP (final concentration 10 μM). After 60 min incubation at 37° C., the enzyme reaction was terminated by adding a buffer containing 100 mM Hepes (pH 7.4) and 35 mM EDTA.
Measurement: IC50 = 8100 nM (pChEMBL 5.090000152587891)
Descriptors: MW 419.9, ALogP 2.32, HBA 7, HBD 2, PSA 116.23, QED 0.49
Compound (SMILES): CC[C@@H](NC(=O)c1cc(=O)[nH]c(C(C)(C)c2ccc(OC)cc2)n1)c1ccc(OC(F)(F)F)c(F)c1
InChIKey: HYPUVIPZAMSRQG-GOSISDBHSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 507.48, ALogP 5.02, HBA 5, HBD 2, PSA 93.31, QED 0.42
Compound (SMILES): C[C@@H]1CN(C(=O)[C@@](C)(O)C(F)(F)F)Cc2cnn(C(C)(C)C)c21
InChIKey: IAALVZWVURZWMI-YMTOWFKASA-N
Target: [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q15119
Assay: Biochemical Activity Assay: The PDC inactivation assay is performed in Greiner 384-well microtiter plates and is used for high throughput screen. 4 μl of PDHK2 (human, rec, Carna Bioscience, 10 ng/μl-137 nM final concentration) and PDC (isolated from porcine heart, Sigma-Aldrich, 20 mU/ml final concentration) are incubated in the absence or presence of the test compound (10 dilution concentrations) for 30 min at room temperature in kinase buffer (15 mM potassium phosphate buffer, pH 7.0, 60 mM KCl, 1.5 mM DTT, 2.5 mM MgCl2, 0.0125% (w/v) BSA, 0.125% Pluronic F-68). The kinase reaction is started by the addition of 4 l ATP substrate solution (fc 5 μM in kinase buffer). After 30 min incubation at 37° C. 40 μl of PDC reaction solution (100 mM Tris/HCl, pH 7.8, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaF, 0.25 mM Coenzyme A, 5 mM pyruvate, 1 mM NAD, 5 mM DTT, 1 mM thiamine pyrophosphate) is added. The first fluorescence measurement is performed on a Perkin Elmer Envision (Exc 340 nm, Em 450 nm). The reaction is incubated for 45 min at room temperature. Afterwards a second fluorescence measurement is performed and the PDC activity is calculated by the difference between both measurements. As full value for the PDHK2 assay the inhibitor-free PDHK2 reaction is used. The pharmacological zero value used is DCA (Sigma-Aldrich) in a final concentration of 3 mM. The inhibitory values (IC50) were determined using either the program Symyx Assay Explorer® or Condosseo® from GeneData.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 333.35, ALogP 2.4, HBA 4, HBD 1, PSA 58.36, QED 0.86
Compound (SMILES): COc1cc2c(s1)C(=O)N(c1cccc(-c3nncn3C3CC3)n1)C2
InChIKey: BIGXSEPEDGLLOZ-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Kinase Assay: The ASK1 enzymatic assay was run following Promega ASK1 Kinase Enzyme System (Cat #V3881). The kit provides the protocol, enzymes and all reagents necessary to run an assay.The compounds, enzyme, substrate and ATP were diluted in provided assay buffer. The final concentration of the enzyme was 50 nM, substrate (Myelin basic protein) 1 μg/mL and ATP 10 μM. The compound and the enzyme were pre-incubated in a 384-well white solid bottom plate (Greiner, Cat #784075) for 10 minutes. After incubation, the substrate and ATP were added and incubated for an additional 60 minutes. After 60 minutes, ADP-Glo was added and the plate was incubated for another 40 minutes. After 40 minutes, Kinase Detection Reagent was added and the plate was incubated for 45 minutes. After 45 minutes, the plate was read on a Perkin Elmer EnVision using luminescence read (0.5 seconds/well).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 353.41, ALogP 2.91, HBA 7, HBD 0, PSA 73.14, QED 0.72
Compound (SMILES): CCCCc1nc(O)c(-c2nnc(CN(C)C(=O)c3ccncc3)o2)c(O)c1-c1c(OC)cccc1OC
InChIKey: ZAVJXXFBCZDKMH-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyr1)apelin-13) peptide, which was set to 100%.The examples disclosed below were tested in the APJ in vitro assays described above and were found having human APJ cyclic AMP (hcAMP) activity. The EC50 value of each compound is presented at the end of the example description.
Measurement: EC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 519.56, ALogP 4.24, HBA 10, HBD 2, PSA 143.93, QED 0.31
Compound (SMILES): Cc1ccc(NC(=O)C(C)[C@H]2[C@@H]3C[C@@H](n4cnc5cc(F)c(F)cc54)C[C@@H]32)nc1
InChIKey: SCMWMOJMNVVOCN-SPWDSFDRSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: IDO1 Cell-Based Assay: HeLa cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM media containing 10% FBS. Cells (7,000/well) were seeded onto a 384 well plate in 50 μL of media and incubated at 37° C., 5% CO2 overnight. Cell media was aspirated, fresh media containing 10 ng/mL IFNgamma was added, and cells were incubated in absence or presence of various concentrations of test compound (final 0.5% DMSO) for 24 hours. Aliquots of the cell conditioned media were removed from the cell plate, and mixed with an equal volume of 200 mM ZnSO4 to precipitate media containing proteins. Two volumes of acetonitrile were added, mixed, and samples were then centrifuged at 2250 G for 20 minutes at 4° C. Aliquots of the supernatant were diluted 1:10 in 0.1% formic acid containing 3 μM of deuterated Tryptophan as an internal standard.Samples were analyzed via RFMS to quantify N-Formyl Kynurenine (AUC) and L-tryptophan (AUC). A C18 cartridge was used with mobile phases of 0.1% Formic Acid and 80% ACN/0.1% Formic Acid under isocratic conditions. Dose-response curves were analyzed using IC50 regression curve fitting (GeneData Screener). Curves were plotted as percent of control and normalized by high controls without inhibitor (100%), and low controls (0%) containing 1 μM of epacadostat, a potent cell-permeable IDO1 inhibitor. Cell viability was also assessed using the Cell Titer Glo Kit (Promega) following manufacturer recommendation.
Measurement: IC50 = 10.9 nM (pChEMBL 7.960000038146973)
Descriptors: MW 396.44, ALogP 4.49, HBA 4, HBD 1, PSA 59.81, QED 0.71
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)cc1)NC(Cc1ccccc1)C(=O)O
InChIKey: SLIMOPZXCUSPOX-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR: HEK-Gα16 cells stably expressing the human FPR2 receptor was utilized. Cells were plated into 384-well poly-D-lysine coated plates at a density of 18,000 cells per well one day prior to use. The growth media was DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic, 50 μg/ml hygromycin, and 400 μg/ml geneticin. On the day of the experiment, the cells were washed twice with Hank's Balanced Salt Solution supplemented with 20 mM HEPES (HBSS/hepes buffer). The cells were then dye loaded with 2 μM Fluo-4 diluted in the HBSS/Hepes buffer and incubated at 37° C. for 40 minutes. Extracellular dye was removed by washing the cell plates four times prior to placing the plates in the FLIPR (Fluorometric Imaging Plate Reader, Molecular Devices). Ligands were diluted in HBSS/Hepes buffer and prepared in 384-well microplates. Data for Ca+2 responses were obtained in relative fluorescence units.
Measurement: EC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 352.31, ALogP 3.52, HBA 2, HBD 3, PSA 78.43, QED 0.77
Compound (SMILES): CN1C(=O)C(NC(=O)c2ncn(Cc3ccccc3)n2)COc2ccc(C#CC(C)(C)C)cc21
InChIKey: GQRDNZGSZRJDRZ-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: biochemical assay using the ADP-Glo technology: ADP-Glo (Promega, Madison, Wis., USA) reagents were thawed at ambient temperature. Kinase Detection Reagent was prepared by mixing kinase detection buffer with the lyophilized kinase detection substrate.A 500 ml stock volume of 5× Reaction Kinase Buffer was made by mixing 1000 μl of 1M MgCl2, 500 μl of 1M Tris-HCL pH7.4, 0.5 mg/ml (25 mg) of BSA, and 3475 μl of distilled H2O. A 3 ml 2× working stock volume of Reaction Kinase Buffer was made containing a final concentration of 100 μM DTT and 4 mM MnCl2.Components of RIPK1 enzyme (Rigel Pharmaceuticals, South San Francisco, Calif., USA) were thawed on ice. Diluted RIPK1 was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer) to 31 ng/well. A 166 M working stock ATP assay solution was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer).Compounds were serially diluted in DMSO from 250 uM in 4-fold dilutions then diluted 1:5 in 2× Reaction Buffer in a 96 well plate. 1.0 ul of diluted compound was added to a 384 well plate in duplicate. 2 μl of diluted Active RIPK1 was added to 384 well plate (do not add to column1) add 2× rxn buffer to column 1. AKT (Anaspec, Fremont, Calif., USA) at 150 nM was combined with ATP working stock at equal volume and 2 ul/well were added to the 384 well plate. The final reaction volume was 5.0 μl. The plate was quickly centrifuged and the reaction was incubated at 30° C. for 30 minutes. Adding 5 μl of ADP-Glo terminated the reaction. The plate was quickly centrifuged and the reaction was incubated at room temperature for 40 minutes. Kinase Detection Reagent was then added and incubated at room temperature for 30 minutes. The relative light unit (RLU) of kinase reaction was determined by luminescent (Luminescence 0.1 s) using a Wallac Victor2 Luminometer (PerkinElmer, Waltham, Mass., USA).
Measurement: IC50 = 62.4 nM (pChEMBL 7.210000038146973)
Descriptors: MW 457.53, ALogP 2.88, HBA 6, HBD 1, PSA 89.35, QED 0.61
Compound (SMILES): CC(C)N1C(=O)Cc2cc(-c3cncc(-c4ccc(=O)n(C(C)C)c4)c3)ccc21
InChIKey: HZMKNGJNPOBODV-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Luminescence-Based ADPGlo Kinase Activity Assay: Example 53: The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 6 ul. Dose-response curves were generated by incubating 10 nM of each kinase in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 uM Na3VO4, 10 mM ß-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 and 15 uM ATP for 60 min at 32° C. in the presence or absence of compound diluted in DMS. The amount of generated ADP is a measure of kinase activity and is quantified using the ADP-Glo Kinase Assay (Promega) according to manufacturer's instructions. ADP is converted to ATP by adding 3 ul of ADP-Glo Reagent and incubation at 32° C. for 60 min. ATP is subsequently converted into a bioluminescent signal by adding 6 ul luciferase assay reagents (Kinase detection buffer+Kinase Detection Substrate (Promega)) and further incubation at 32° C. for 60 min. For the measurement of luminescence a PHERAstar Multilabel Reader was used at a measurement interval time of 0.1 second (optical module for luminescence measurements in the 230 nm to 750 nm wavelength range). The luminescent signal positively correlates with kinase activity.
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 387.48, ALogP 4.46, HBA 4, HBD 0, PSA 55.2, QED 0.66
Compound (SMILES): Cc1cc(NC(=O)c2cc([C@@H]3C[C@H]3NCC3CCOCC3)cs2)no1
InChIKey: NEEVDAJZAXQIQT-LSDHHAIUSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Inhibitory Activity Assay: LSD1: A test compound dissolved in DMSO was added by to a reaction solution (50 mM Tris-HCl (pH 8.0), 0.1% BSA, 1 mM DTT) containing LSD1 enzyme, and the mixture was reacted at room temperature for 60 min. Biotin-histone H3 mono methylated K4 peptide solution (NH2-ART(me-K)QTARKSTGGKAPRKQLAGGK(Biotin)-CONH2) was added to start the reaction. After reaction at room temperature for 5 min, 2-PCPA solution was added to terminate the reaction. A detection solution (800 mM potassium fluoride, 0.1% BSA) containing europium-labeled anti-histone H3 antibody (Wako Pure Chemical Industries, Ltd.) and Streptavidin-XL665 (Cisbio) was further added, and the mixture was left standing for 60 min. A time-resolved fluorescence (excitation 320 nm, emission 615 nm, 665 nm) was measured by Envision (PerkinElmer). The LSD1 inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate (%)=(1−(test compound count−blank)÷(control−blank))×100The count of the LSD1 enzyme reaction mixture under compound non-addition conditions is indicated as control, and the count under compound non-addition and LSD1 enzyme non-addition conditions is indicated as blank.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 361.47, ALogP 3.17, HBA 6, HBD 2, PSA 76.39, QED 0.83
Compound (SMILES): Cc1cc(Cn2c(=O)[nH]c3ncc(-c4cc(F)cc(F)c4)cc32)no1
InChIKey: AXXJWYXKURHIPG-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: Standard cell-based calcium flux assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (standard assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 342.31, ALogP 3.01, HBA 5, HBD 1, PSA 76.71, QED 0.62
Compound (SMILES): CN1CCc2nc(Nc3cc(-c4ccc5cc[nH]c5c4)cn4ccnc34)sc2C1
InChIKey: ONWRETPOJYEBFC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Biochemical Assay: A master mix minus Syk enzyme is prepared containing 1X Cell Signaling kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2), 0.5 μM Promega PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, and 0.25% glycerol. A master mix plus Syk enzyme is prepared containing 1X Cell Signaling kinase buffer, 0.5 μM PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, 0.25% glycerol and 0.4 ng/well Syk enzyme. Syk enzyme is purchased from Cell Signaling Technologies, expressed in baculovirus and is an N-terminally GST-tagged full length human wildtype Syk (accession number NM-00377).The Syk protein is purified in one step using glutathione-agarose. The purity of the final protein preparation is assessed by SDS-PAGE and Coomassie staining. A solution of 200 μM ATP is prepared in water and adjusted to pH 7.4 with 1N NaOH. A quantity of 1.25 μL of compounds in 5% DMSO is transferred to a 96-well ½ area Costar polystyrene plate.Compounds are tested singly and with an 11-point dose-responsive curve (starting concentration is 10-1 μM; 1:2 dilution). A quantity of 18.75 μL of master mix minus enzyme (as a negative control) and master mix plus enzyme is transferred to appropriate wells in 96-well ½ area Costar polystyrene plate. 5 μL of 200 μM ATP is added to that mixture in the 96-well ½ area Costar polystyrene plate for final ATP concentration of 40 μM.
Measurement: IC50 = 72000 nM (pChEMBL 4.139999866485596)
Descriptors: MW 400.51, ALogP 4.67, HBA 6, HBD 2, PSA 61.25, QED 0.46
Compound (SMILES): Cc1cc2nc(C(C(=O)NCC(=O)NC3CC3)S(=O)(=O)C(C)C)sc2cc1-c1ccc2c(=O)n(C)ccc2c1
InChIKey: DARDRBXAOOBMQW-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Activity: Endothelial lipase (EL) and hepatic lipase (HL) activities were measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 571 μL of 29 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 2000 μL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen in multiple vials then resuspended in 20 mL total volume of 50 mM HEPES pH 8.0 buffer containing 50 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at room temperature for 15 min and then was sonicated 3×4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.11 for the FRET substrate.The enzymatic assay was measured using 384-well white Optiplates. Each well contained 20 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 0.25 μL of a DMSO solution containing a compound of interest. EL or HL (10 μL) was added and allowed to incubate with the compound for 30 min at 37° C. The source of EL was conditioned media obtained from HT-1080 cells that were transformed using RAGE technology (Athersys) to overexpress endogenous EL, and HL was partially purified from conditioned media obtained from COS cells overexpressing HL. The reaction was started by the addition of 10 μL of a 1:10 dilution of vesicles. The final total reaction volume was 20.25 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Readings were taken over a period of 60 minutes, and the slope between 300 and 900 secs of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 566.71, ALogP 3.38, HBA 8, HBD 2, PSA 127.23, QED 0.34
Compound (SMILES): CNc1cc(-c2cnc3n([C@H]4COC[C@@H]4O)cccc2-3)nc2c(C(=O)N[C@@H]3CCC3(F)F)cnn12
InChIKey: BVRVYAPTYQKMIF-OKZBNKHCSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 483.48, ALogP 2.2, HBA 9, HBD 3, PSA 118.6, QED 0.4
Compound (SMILES): CCN(CC)CCOc1ccc(Nc2nc(N3CCC(CC#N)CC3)nc(C=N)c2C(N)=O)cc1
InChIKey: WSMPCKSTTVSVQF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition of Enzymatic Syk Kinase Activity: 2.5 μL of 10% DMSO or compound in 10% DMSO was added to the wells in a 96 well V-bottom plate. Optimized concentration of in-house Syk enzyme (different batches of Syk (356-635) kinase domain) were used at optimized concentrations) ranging from 0.035 ng to 7.5 ng/reaction diluted in assay buffer was added to a total volume of 12.5 μL). Compound and protein were incubated for 30 minutes at room temperature on a plate shaker. Ten μL of a substrate mix containing 100 μM ATP (0.25 μL), γ-P32-ATP (0.1 μL; 10 μCi/μL), pG4T (0.25 μL; 10 mg/mL) and 1× assay buffer (9.4 μL) was added to all the wells. Samples were incubated at 30° C. for 10 minutes after mixing. The reaction was stopped by the addition of 8N HCl (13 μL) containing 100 mM ATP. Thirty μL of sample was transferred to the center of a 2×2 cm2 Whatman P81 chromatography paper. After allowing the sample to dry for one minute, the assay squares were washed 3 times for 5 minutes each in ortho-phosphoric acid (0.5%) and once in acetone. Assay squares were dried for 15 minutes in a 30° C. oven and transferred to 96 well optiplate. Microscint-O reagent (100 μL, Perkin Elmer) was added to each well, the plate was sealed with Topseal-A microplates and incubated for 10 minutes at room temperature at very low speed on rocker and the plate was read in the Topcount NXL instrument.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 478.6, ALogP 3.17, HBA 9, HBD 3, PSA 144.25, QED 0.39
Compound (SMILES): C/C(=C1/SC(N2CCC3(CC2)OCc2ccccc23)=NC1=O)c1c(C)noc1C
InChIKey: NGOZZZJWUIFBFK-UYRXBGFRSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: competition binding assay: The human V1a receptor was cloned by RT-PCR from total human liver RNA. The coding sequence was subcloned in an expression vector after sequencing to confirm the identity of the amplified sequence. To demonstrate the affinity of the compounds from the present invention to the human V1a receptor binding studies were performed. Cell membranes were prepared from HEK293 cells transiently transfected with the expression vector and grown in 20 liter fermenters with the following protocol.50 g of cells are re-suspended in 30 ml freshly prepared ice cold Lysis buffer (50 mM HEPES, 1 mM EDTA, 10 mM MgCl2 adjusted to pH=7.4+complete cocktail of protease inhibitor (Roche Diagnostics)). Homogenized with Polytron for 1 min and sonicated on ice for 2×2 minutes at 80% intensity (Vibracell sonicator). The preparation is centrifuged 20 min at 500 g at 4° C., the pellet is discarded and the supernatant centrifuged 1 hour at 43,000 g at 4° C. (19,000 rpm). The pellet is re-suspended in 12.5 ml Lysis buffer+12.5 ml Sucrose 20% and homogenized using a Polytron for 1-2 min. The protein concentration is determined by the Bradford method and aliquots are stored at −80° C. until use. For binding studies 60 mg Yttrium silicate SPA beads (Amersham) are mixed with an aliquot of membrane in binding buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM MgCl2) for 15 minutes with mixing. 50 μl of bead/membrane mixture is then added to each well of a 96 well plate, followed by 50 μl of 4 nM 3H-Vasopressin (American Radiolabeled Chemicals). For total binding measurement 100 μl of binding buffer are added to the respective wells, for non-specific binding 100 μl of 8.4 mM cold vasopressin and for compound testing 100 μl of a serial dilution of each compound in 2% DMSO. The plate is incubated 1 h at room temperature, centrifuged 1 min at 1000 g and counted on a Packard Top-Count. Non-specific binding counts are subtracted from each well and data is normalized to the maximum specific binding set at 100%. To calculate an IC 50 the curve is fitted using a non-linear regression model (XLfit) and the Ki is calculated using the Cheng-Prussoff equation.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 409.51, ALogP 4.17, HBA 6, HBD 0, PSA 67.93, QED 0.66
Compound (SMILES): O[C@@H]1[C@H](O)CS[C@H]1c1cnc2c(NCc3ccncn3)nccn12
InChIKey: ZMAQNVIROOLBCL-UPJWGTAASA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Assay for Binding Affinity for Adenosine Receptor A3: HEK-293 cell membrane homogenates (32 μg protein), in which A3 adenosine receptors were expressed, are incubated for 120 min at 22° C. with 0.15 nM [1251]AB-MECA in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA and 2 UI/ml ADA. Nonspecific binding is determined in the presence of 1 μM IB-MECA. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is IB-MECA, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 344.4, ALogP 0.64, HBA 9, HBD 3, PSA 108.46, QED 0.63
Compound (SMILES): [C-]#[N+]c1ccc(-c2nc(N3CCC(N)CC3)n(C)c(=O)c2-c2ccc(OCCO)cc2)cc1F
InChIKey: GHWRPIYAKKDPKS-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: In Vitro Enzyme Inhibition Assay: This assay determines the ability of a test compound to inhibit LSD1 demethylase activity. E. coli expressed full-length human LSD1 (Accession number O60341) was purchased from Active Motif (Cat#31334).The enzymatic assay of LSD1 activity is based on Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The inhibitory properties of compounds to LSD1 were determined in 384-well plate format under the following reaction conditions: 0.1-0.5 nM LSD1, 50 nM H3K4me1-biotin labeled peptide (Anaspec cat #64355), 2 μM FAD in assay buffer of 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody (PerkinElmer) in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer (PerkinElmer) to final concentration of 12.5 nM and 0.25 nM respectively.The assay reaction was performed according to the following procedure: 2 μL of the mixture of 150 nM H3K4me1-biotin labeled peptide with 2 μL of 11-point serial diluted test compound in 3% DMSO were added to each well of plate, followed by the addition of 2 μL of 0.3 nM LSD1 and 6 μM of FAD to initiate the reaction. The reaction mixture was then incubated at room temperature for one hour, and terminated by the addition of 6 μL of 1.8 mM 2-PCPA in LANCE detection buffer containing 25 nM Phycolink Streptavidin-allophycocyanin and 0.5 nM Europium-anti-unmodified H3K4 antibody. Enzymatic reaction is terminated within 15 minutes if 0.5 LSD1 enzyme is used in the plate. Plates were read by EnVision Multilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 463.51, ALogP 3.1, HBA 7, HBD 2, PSA 97.97, QED 0.55
Compound (SMILES): COc1ccc2nc([C@@H]3CCCN3C(=O)c3cnc(C)nc3-c3cccc(Cl)c3)[nH]c2c1
InChIKey: UUPVTEWSICLUSQ-NRFANRHFSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.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 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M 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.
Measurement: IC50 = 369 nM (pChEMBL 6.429999828338623)
Descriptors: MW 447.93, ALogP 4.97, HBA 5, HBD 1, PSA 84, QED 0.48
Compound (SMILES): COc1cc2c(cc1C(=O)Nc1cccc(-c3nnnn3[C@H](C)CO)n1)CN(C(=O)C(C)(C)CO)CC2
InChIKey: HUEFKJLEERUJCB-OAHLLOKOSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay : ASK1 was purchased from Thermofisher (Catalogue #PV4011), ATP was purchased from Sigma (Catalogue #A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue ##6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 μM STK3 peptide substrate, and 5 μM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES Ph 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 μM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well. The resulting TR-FRET is proportional to the phosphorylation level. Staurosporine was used as the positive control. IC50 was determined by Xlfit 5.3.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 509.57, ALogP 1.45, HBA 10, HBD 3, PSA 155.59, QED 0.41