lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCNC(=O)Nc1cccc(-n2c([C@H](C)Nc3nc(C)nc(N)n3)nc3cccc(Cl)c3c2=O)c1
InChIKey: NEASEAYTKZMGHT-LBPRGKRZSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET assays for PI3K isoforms: TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site.Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2×Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. Additionally, Compounds 24-164 were added to the assay solution and incubated for 30 minutes at 250 in a similar manner. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3Kα, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 16000 nM (pChEMBL 4.800000190734863)
Descriptors: MW 493.96, ALogP 3.43, HBA 9, HBD 4, PSA 152.74, QED 0.32
Compound (SMILES): COc1cccc(F)c1-c1cc2c(NC(=O)c3ccc(F)c(C)c3)n[nH]c2cn1
InChIKey: JHMTUVWNGMPQPK-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μl of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 394.38, ALogP 4.47, HBA 4, HBD 2, PSA 79.9, QED 0.54
Compound (SMILES): NS(=O)(=O)NCCONc1nonc1/C(=N/O)NCc1ccccc1
InChIKey: RDXKJWRPTBOSBG-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Inhibitory Activity Assay: 1. Materials, Kits and EquipmentsSodium L-ascorbate (Cat: A4034-100G, SIGMA)4-(dimethylamino)benzaldehyde (Cat: 156477-25g, SIGMA)Trichloroacetic acid (Cat: T0699-100ML, SIGMA)L-Tryptophan (Cat: T8941-25G, SIGMA)Methylene blue (Cat: M9140-25G, SIGMA)Potassium dihydrogen phosphate (Cat: 10017618, Sinopharm Chemical Reagent)Disodium hydrogen phosphate (Cat: 20040618, Sinopharm Chemical Reagent)Constant temperature water tank (Cat: DK-8D, Shanghai Jinghong Experimental Equipment)Multifunctional microplate reader (Cat: M5, Molecular Devices)96-well reaction plate (Cat: 3590, costar)IDO1 protease (commercially available)Desktop Microplate Reader: SpectraMax M5 Microplate Reader (Molecular Devices)Test compounds: self-madePositive control agent: INCB024360 (commercially available)2. Reagent Preparation100 mM PBS:100 mM disodium hydrogen phosphate and 100 mM potassium dihydrogen phosphate mixed in a ratio of 3:5, pH 6.5IDO1 assay buffer:100 mM PBS containing 400 μM L-tryptophan, 20 mM ascorbate, 20 μM methylene blue and 1000 U/ml catalase, pH 6.530% trichloroacetic acidddH2 O solution of 30% trichloroacetic acidEhrlich reagent1% (w/v) diluted solution of 4-(dimethylamino) benzaldehyde compoundAll compounds were dissolved with DMSO. During the assay, each compound was diluted to a concentration as needed. The compound of each concentration was added to multi-wells, and the final concentration of DMSO was controlled at 1%.3. Test Methoda.) the reaction mixture was prepared by adding 50 nM IDO1 and the desired concentration of the test compound to 100 μL of IDO1 assay buffer. IDO1 and assay buffer need to be preheated to 37° C.b.) The mixture was reacted in a constant temperature water tank at 37° C. for 30 minutes.c.) 50 μL of 30% trichloroacetic acid was added.d.) The above mixture was reacted in a constant temperature water tank at 52° C. for 30 minutes.e.) The reaction mixture was centrifuged at 12000 g for 10 minutes at room temperature.f.) 100 μL of the obtained supernatant and 100 μL of Ehrlich reagent were mixed.g.) the absorbance at 480 nm was measured using an M5 microplate reader.4. Data AnalysisInhibition rate=(ODpostive−ODsample)/(ODpositive−ODnegative)*100%5. Results and Discussion
Measurement: IC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 371.38, ALogP -0.87, HBA 9, HBD 5, PSA 176.99, QED 0.12
Compound (SMILES): CCCS(=O)(=O)Nc1cc(Nc2nc(Nc3ccc(N4CCN(C)CC4)cc3)ncc2C)ccc1Cl
InChIKey: VYHVASVSRXPKLW-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Alpha Screen Assay: AlphaScreen is a bead-based, non-radioactive Amplified Luminescent Proximity Homogeneous Assay. The details are not described in this patent.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 530.1, ALogP 4.83, HBA 8, HBD 3, PSA 102.49, QED 0.36
Compound (SMILES): COc1cc(C(=O)N2C[C@H](N)C[C@@H](F)C2)cc2nc(-c3cc4ccc(-c5ccc6c(c5)CC(NC(C)=O)C6)nc4n3CC3CC3)n(C)c12
InChIKey: BABGXNDTEONRKP-KSSJMHQNSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 182 nM (pChEMBL 6.739999771118164)
Descriptors: MW 649.77, ALogP 4.79, HBA 8, HBD 2, PSA 120.3, QED 0.26
Compound (SMILES): N#CN1CC(OCc2ccc(-c3cc(C(F)(F)F)cc(C(F)(F)F)c3)cc2)C1
InChIKey: KJHDGIHFLGZUGQ-UHFFFAOYSA-N
Target: N-acylethanolamine-hydrolyzing acid amidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q02083
Assay: Inhibition Assay: In order to have an assay method more conducive to high-throughput screening than those published for measuring the NAE hydrolyzing activity of NAAA, we developed the fluorogenic PEA analog N-(4-methyl coumarin)palmitamide (PAMCA), which is hydrolyzed to fluorescent 7-amino-4-methyl coumarin (AMC) and palmitic acid. For three point concentration inhibition assays with hNAAA the following procedure is used. Purified activated NAAA (final concentration of 0.25 μg/mL) is incubated in assay buffer (100 mM citrate-phosphate buffer, pH 4.5, 3 mM DTT, 0.1% Triton X-100, 0.05% BSA, and 150 mM NaCl) made up to a total volume of 180 μL, followed by addition of the compound dissolved in 10 μL DMSO (along with DMSO neat for the control sample) with the final concentrations for each compound of 100, 10, and 1 μM, in triplicate on a 96 well plate. These samples are allowed to incubate for 15 min at room temperature and then 10 μL of a PAMCA stock solution in DMSO (final PAMCA concentration [5 μM]) was added. After 5 minutes of agitation on a shaking plate, the reaction is allowed to proceed at 37° C. for 120 minutes, with fluorescence readings taken every 10 minutes at a wavelength of 460 nm (using an excitation wavelength of 360 nm) on a Synergy HT Plate Reader using Gen5 software from Bio-Tek. The enzyme activity is calculated by converting the relative fluorescence units to AMC formed, using a standard curve of AMC. For compounds that inhibit hNAAA in range IC50 <1 μM full inhibition curves using eight different concentrations of inhibitor (8 point assay) are generated. The assay procedure used is the same as the three point assay. For ostensibly covalent compounds (as observed by a significant decrease in the slope of a plot of fluorescence vs. time in three point screen) samples are allowed to incubate for 2 hours at 37° C., instead of 15 minutes, before addition of 10 μL of a PAMCA stock solution in DMSO for a final PAMCA concentration of 5 μM. After 5 minutes of agitation on a shaking plate, the reaction is allowed to proceed at 37° C. for 120 minutes Inhibition constants are calculated using pro Fit software (Quantum Soft, Uetikon am See, Switzerland) and a Levenberg-Marquardt algorithm.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 400.32, ALogP 5.07, HBA 3, HBD 0, PSA 36.26, QED 0.53
Compound (SMILES): COc1c(F)cc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)C2CCC(F)CC2)C[C@@H]1F
InChIKey: NNWLZVTUDAUQKO-LCKJDKPRSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 584.52, ALogP 3.95, HBA 7, HBD 2, PSA 114.85, QED 0.44
Compound (SMILES): Nc1nc2c(ncn2[C@@H]2O[C@@H]3CO[P@@](=O)(S)O[C@H]4[C@@H](F)[C@H](n5cnc6c(N)ncnc65)O[C@@H]4CNS(=O)(=O)O[C@H]3[C@@H]2F)c(=O)[nH]1
InChIKey: VESNZZQQTGVRPS-WQHCACNMSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: STING SPA Binding Assay: The assay was run in 1536-well plates in a total volume of 8 μL per well by adding 8 nM [3H]-2′3′-cGAMP and 40 nM biotin-STING protein in assay buffer [25 mM HEPES (Corning 25-060-C1) pH 7.5, 150 mM NaCl (Sigma S5150), 0.5 mg/mL BSA (Gibco 15260-037), 0.001% Tween-20 (Sigma P7949), molecular grade water (Corning 46-000-CM)]. Test compounds (80 nL) were added with an acoustic dispenser (EDC Biosystems) in 100% DMSO for a final assay concentration of 1% DMSO. Plates were centrifuged for 1 min and incubated for 60 min at room temperature. Finally, (2 μL) polystyrene streptavidin SPA beads (PerkinElmer RPNQ0306) were added and plates were sealed and centrifuged for 1 min at room temperature. Plates were dark adapted for 2 h and read on a ViewLux (Perkin Elmer) for 12 min per plate. A saturation binding curve for [3<H]-2′3′cGAMP showed a KD of 3.6±0.3 nM for binding to STING, comparable to reported values for the natural ligand (Zhang et al., Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING (Molecular Cell 2013 51(2):10.1016/j.molcel.2013.05.022.).
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 693.57, ALogP -0.73, HBA 18, HBD 5, PSA 268.6, QED 0.13
Compound (SMILES): Cc1ccc(CNC(=O)C2CN(c3ncnn4cc(-c5cnn6c5CCC6)cc34)C2)cc1
InChIKey: RUXFEUIKTSUUCK-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 427.51, ALogP 2.6, HBA 7, HBD 1, PSA 80.35, QED 0.53
Compound (SMILES): Cc1c(OC[C@H]2CN(C(=O)OC(C)(C)C)CCO2)nn(-c2ccccc2)c1NC(=O)NC1c2ccccc2C(C)(C)C[C@H]1O
InChIKey: AZKJEBGTKGYLAQ-UTZIHYNGSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Kinase Binding Assay: TrkA binding activity was determined in a TrkA LanthaScreen Eu Kinase Binding Assay. 5 nM His-tagged recombinant human TrkA (6HIS tagged cytoplasmic domain from Invitrogen, Catalog No. PV3144) was incubated with 4 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat. No. PV5592), 2 nM biotinylated anti-His (Invitrogen Cat. No. PV6090), and 2 nM europium-labeled Streptavidin (Invitrogen Cat. No. PV5899), in buffer (25 mM MOPS, pH 7.5, 5 mM MgCl2, 0.005% Triton X-100). Three fold serial dilutions of compounds of the invention in DMSO were added to a final percentage of 2% DMSO. After 60-minute incubation at 22° C., the reaction was measured using the EnVision mutlimode plate reader (PerkinElmer) via TR-FRET dual wavelength detection at 615 nM and 665 nM. The percent of control was calculated using a ratiometric emission factor.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 605.74, ALogP 5.1, HBA 8, HBD 3, PSA 127.18, QED 0.36
Compound (SMILES): CC(c1ccccc1Cl)N1CC(COc2cccc([C@@H](CC(=O)O)C3CC3)c2)C1
InChIKey: GVNFTLNGZRUALP-XLDIYJRPSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: GRP40 FLIPR Assays: FLIPR (Fluorimetric Imaging Plate Reader, Molecular Devices) assays were performed to measure agonist-induced calcium mobilization of the stable clones. For the FLIPR assay, one day before assay, GPR40/CHO NFAT BLA cells were seeded into black-wall-clear-bottom 384-well plates (Costar) at 1.4×10e4 cells/20 μL medium/well. The cells were incubated with 20 μl/well of the assay buffer (HBSS, 0.1% BSA, 20 mM HEPES, 2.5 mM probenecid, pH 7.4) containing 8 μM fluo-4, AM, 0.08% pluronic acid at room temperature for 100 minutes. Fluorescence output was measured using FLIPR. Compounds were dissolved in DMSO and diluted to desired concentrations with assay buffer. 13.3 μL/well of compound solution was added.The compounds of the present invention, including the compounds in Examples 1-159, have EC50 values less than 2 micromolar (μM) in the FLIPR assay described above
Measurement: EC50 = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 413.95, ALogP 5.38, HBA 3, HBD 1, PSA 49.77, QED 0.6
Compound (SMILES): CCOc1cc(C(=O)N2C3CCC2COC3)ccc1C(=O)NC[C@@H](C)[C@@H]1Cc2ccc(OC)cc2CN1C
InChIKey: XSRSMUJLODCILL-DPLZTYMHSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay: The 6 μL reactions are run in Greiner brand black 384-well low volume assay plates. All reactions contained assay buffer (phosphate buffered saline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken egg white, 1 mM Dithiothreitol, 1 μM peptide substrate, 1 μM S-Adenosyl methionine, and 15 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 4 hours at 37 degree Celsius. Reaction progress was measured using the Transcreener EPIGEN methyltransferase assay (BellBrook Labs, Madison, Wis.) as recommended by the manufacturer. To each reaction 2 μL detection mix were added, containing coupling enzymes, fluorescence polarisation tracer, and AMP antibody. Plates were incubated for 90 minutes before being read on a Perkin Elmer EnVision™ plate reader in fluorescence polarisation mode.
Measurement: IC50 = 1009 nM (pChEMBL 6)
Descriptors: MW 521.66, ALogP 3.52, HBA 6, HBD 1, PSA 80.34, QED 0.57
Compound (SMILES): COC1CCC(C(=O)N2Cc3cccnc3Nc3ccc(N4C(=O)OC[C@@H]4C(C)C)cc32)CC1
InChIKey: HFPHPGFYQWFXNM-VBVCHWQCSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 464.57, ALogP 4.86, HBA 6, HBD 1, PSA 84, QED 0.7
Compound (SMILES): Cc1nnc2c(NCCCCNC(=O)C(C)(C)C)nc3cc4c(cc3n12)OCCO4
InChIKey: IHDDRPBWLJZKHR-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Evaluation of BRD4 binding inhibition ability: The following experiment was performed to evaluate the ability of [1,2,4]triazolo[4,3-a]quinoxaline derivative of the present invention to inhibit the interaction between BRD4 (BD1+BD2) bromodomain, one of BET protein family, and tetraacetylated histone H4 peptide.The compound was serially diluted at the ratio of 1:5 in assay buffer from 10 mM stock in DMSO (initial concentration: 100 μM) on white OptiPlate-384 (PerkinElmer). A mixture comprising 100 nM GST-BRD4 (BD1+BD2) and 100 nM biotinylated acetyl-histone H4 (Lys5,8,12,16) peptide was prepared in assay buffer (50 mM HEPES pH 7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). After adding 6 μl of the mixture to the diluent, 6 μl of the pre-mixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads (PerkinElmer, 10 μg/ml in assay buffer, respectively) was added thereto. The samples were incubated in the dark at room temperature for 30 minutes (shaking at 300 rpm). Then, the signals were measured with PerkinElmer Envision HTS Multilabel Reader using an alpha screen protocol of PerkinElmer. Each plate contained the negative control in which biotinylated acetyl-histone H4 peptide and GST-BRD4 (BD1+BD2) were replaced by assay buffer. In the case of using the software GraphPad Prism for calculation, the negative control point was input as a low standard value. The positive control (probe molecule l-BET762 containing protein/peptide mixture) proceeded to pipetting. IC50 value was determined by using GraphPad Prism 3.03 software (or an updated version thereof).
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 412.49, ALogP 2.71, HBA 8, HBD 2, PSA 102.67, QED 0.6
Compound (SMILES): O=C(NCc1ncn2ccc(Cl)cc12)c1cnn(CC2CN3C=C(C4CC4)C(F)=CC3N2)c1
InChIKey: KIPUVDFHKKQJSM-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Example compounds were evaluated for inhibition of the human activated kallikrein enzyme in two formats of an assay employing a fluorogenic peptide substrate. In one assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 500 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at RT. After initiation with substrate, reactions were incubated for 10 min at RT and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader. In another assay format, the concentrations of reagents were as follows: 20 mM Tris pH 7.5, 1 mM EDTA, 150 mM sodium chloride, 0.1% PEG-400, 0.1% Triton X-100, 5 pM activated kallikrein enzyme, 300 uM Pro-Phe-Arg-7-amido-4-methylcoumarin substrate. Prior to reaction initiation with substrate, enzyme and inhibitors were preincubated for 30 min at RT. After initiation with substrate, reactions were incubated for 18 h at RT and fluorescence emission at 460 nm from 380 nm excitation measured with a microplate reader.
Measurement: EC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 467.94, ALogP 2.88, HBA 7, HBD 2, PSA 79.49, QED 0.58
Compound (SMILES): COC(=O)c1csc2c(-c3cc(Cl)ccc3OCCn3c(C)nc4cc(C(F)(F)F)cc(C#N)c4c3=O)ccnc12
InChIKey: PPFNEMWSJBWUJF-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 2700 nM (pChEMBL 5.570000171661377)
Descriptors: MW 598.99, ALogP 6.39, HBA 9, HBD 0, PSA 107.1, QED 0.21
Compound (SMILES): CCn1c(CO)nn(-c2ccc3c(=O)n(-c4ccccc4C)cc(N(C)C)c3c2)c1=O
InChIKey: HEBNUUKWUFTOGG-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: In Vitro Assay: DHODH Enzymatic Assay: The following assay is referred to herein as the DHODH Enzymatic Assay. To detect DHODH enzyme activities, dichloroindophenol (DCIP) is added as the final electron acceptor in the assay. DCIP can accept electrons from the reduced coenzyme Q generated in the assay, or from dihydroorotate (DHO) via FMN by binding presumably to the ubiquinone pocket. DCIP solutions are blue, with an intense absorbance around 600 nm, but becomes colorless upon reduction (J. Biol. Chem. (1986) 261, 11386). The assay buffer contained 50 nM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, and 0.1% Triton X-100 in MilliQ water. Substrate consisting of 20 mM DHO, 5 mM CoQ6, and 1 mM DCIP in assay buffer, initiates the reaction. The assay is run in end-point mode by quenching the reaction with the potent DHODH inhibitor brequinar. Absorbance measurements were obtained using the BMG Phera Star plate-reading spectrophotomer. Purified human DHODH was purchased from Proteros (cat. No. PR-0044). Chemicals were purchased from Sigma-Aldrich, Teknova, and Avanti Polar Lipids. Liquid handling was performed using Labcyte Echo and Formulatrix Tempest.
Measurement: IC50 = 46.5 nM (pChEMBL 7.329999923706055)
Descriptors: MW 419.49, ALogP 2.22, HBA 8, HBD 1, PSA 85.29, QED 0.54
Compound (SMILES): Cn1cnc(S(=O)(=O)N2C[C@H](Nc3cc(OC4CCCC4)ncn3)[C@@H](c3ccc(F)cc3)C2)c1
InChIKey: NORNGILFLQYQBW-UXHICEINSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 486.57, ALogP 2.94, HBA 8, HBD 1, PSA 102.24, QED 0.55
Compound (SMILES): C=CC(=O)N1CCC[C@H](c2nn(-c3ccc(Oc4ccccc4F)cc3)c3c(N)n[nH]c(=O)c23)C1
InChIKey: YVDILMLZOPEJHV-HNNXBMFYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Cell Proliferation Assays: MTS testing kit was purchased from Promega. The DMEM, Fetal bovine serum and Penicillin-Streptomycin were purchased from Gibco. Dimethyl sulfoxide (DMSO) was purchased from Sigma.To investigate whether a compound is able to inhibit the activity of BTK in cells, a mechanism-based assay using DOHH2 (DSMZ catalog#: ACC47) and Mino (ATCC® Number CRL-3000) cells was developed. In this assay, inhibition of BTK was detected by the inhibition of DOHH2 and Mino cell proliferations. DOHH2 or Mino cells were cultured in culture flasks to 40-80% confluence in RPMI-1640 plus 10% fetal bovine serum. C ells were collected and plated onto 96-well plates at desired cell density (DOHH2: 5000 cells/well; Mino: 10000 cells/well). Plates were incubated overnight at 37° C., with 5% CO2 to adhere. Compounds were added to the plates, and the final compound concentrations were 10000, 3333, 1111, 270, 124, 41, 14, 4.6 and 1.5 nM. Plates were placed at 37° C., with 5% CO2 for 120 h (DOHH2) or 72 h (Mino). 20 μl MTS/100 μl medium mixture solution were added to each well and the plates were incubated for exactly 2 hours. The reaction was stopped by adding 25 μl 10% SDS per well. Absorbance at 490 nm and 650 nm (reference wavelength) were measured and IC50 was calculated using GraphPad Prism 5.0.
Measurement: IC50 = 248 nM (pChEMBL 6.610000133514404)
Descriptors: MW 474.5, ALogP 3.51, HBA 7, HBD 2, PSA 119.13, QED 0.43
Compound (SMILES): CCN(CCN(C)c1cc(C#N)ccn1)C(=O)c1ccccc1OC(F)(F)F
InChIKey: KPYXHCOPWPRIJT-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 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 Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 = 58.2 nM (pChEMBL 7.239999771118164)
Descriptors: MW 392.38, ALogP 3.45, HBA 5, HBD 0, PSA 69.46, QED 0.72
Compound (SMILES): CC1(C)CC(=O)c2c([nH]c(-c3cc[n+](CC(F)F)cc3)c2Nc2ccccc2)C1
InChIKey: HZERJSVSXSPPOJ-UHFFFAOYSA-O
Target: Mitotic checkpoint serine/threonine-protein kinase BUB1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43683
Assay: Kinase Assay: In a typical assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were tested in duplicate within the same microtiter plate. To this end, 100-fold concentrated compound solutions (in DMSO) were previously prepared by serial dilution (1:3.4) of 2 mM stocks in a clear low volume 384-well source microtiter plate (Greiner Bio-One, Frickenhausen, Germany), from which 50 nl of compounds were transferred into a black low volume test microtiter plate from the same supplier. Subsequently, 2 μL of Bub1 (the final concentration of Bub1 was adjusted depending on the activity of the enzyme lot in order to be within the linear dynamic range of the assay: typically 200 ng/mL were used) in aqueous assay buffer [50 mM Tris/HCl pH 7.5, 10 mM magnesium chloride (MgCl2), 200 mM potassium chloride (KCl), 1.0 mM dithiothreitol (DTT), 0.1 mM sodium ortho-vanadate, 1% (v/v) glycerol, 0.01% (w/v) bovine serum albumine (BSA), 0.005% (v/v) Trition X-100 (Sigma), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added to the compounds in the test plate and the mixture was incubated for 15 min at 22° C. to allow pre-equilibration of the putative enzyme-inhibitor complexes before the start of the kinase reaction, which was initiated by the addition of 3 μL 1.67-fold concentrated solution (in assay buffer) of adenosine-tri-phosphate (ATP, 10 μM final concentration) and peptide substrate (1 μM final concentration). The resulting mixture (5 μL final volume) was incubated at 22° C. during 60 min., and the reaction was stopped by the addition of 5 μL of an aqueous EDTA-solution (50 mM EDTA, in 100 mM HEPES pH 7.5 and 0.2% (w/v) bovine serum albumin) which also contained the TR-FRET detection reagents (0.2 μM streptavidin-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-phosho-Serine antibody [Merck Millipore, cat. #35-002] and 0.4 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no. AD0077, alternatively a Terbium-cryptate-labeled anti-mouse IgG antibody from Cisbio Bioassays can be used]). The stopped reaction mixture was further incubated 1 h at 22° C. in order to allow the formation of complexes between peptides and detection reagents.
Measurement: IC50 = 1280 nM (pChEMBL 5.889999866485596)
Descriptors: MW 396.46, ALogP 5.13, HBA 2, HBD 2, PSA 48.77, QED 0.59
Compound (SMILES): COc1cc2[nH]c3nc(-c4cnn(C)c4)ccc3c2cn1
InChIKey: OTFWESDKGNQXGU-UHFFFAOYSA-N
Target: Microtubule-associated protein tau (SINGLE PROTEIN), Homo sapiens
UniProt: P10636
Assay: Radioligand In Vitro Binding Assays on Recombinant and Native Tau Protein: [3H]compound binding to Tau protein methods were adapted from Nobuyuki et al (2013). Biological samples as described above (0.2-50 μg proteins per assays) were incubated for 60 min at 25° C. with [3H]compound in 0.2 ml of PBS containing 0.1% bovine serum albumin (Sigma-Aldrich, Diegem, Belgium). At the end of the incubation period, the protein-bound radioligand was recovered by reduced pressure filtration through GF/F glass fiber filters (GE Healthcare, Diegem, Belgium) pre-soaked in 0.1% polyethyleneimine (Sigma-Aldrich, Diegem, Belgium). Filters were washed with at least 4 times the assay volume of ice-cold PBS (pH 7.4). The entire filtration step did not exceed 10 sec The filters were dried and the radioactivity determined by liquid scintillation. Saturation binding assays were carried out using increasing concentrations of [3H]compound (0.5 to 100 nM). Competition binding experiments were carried out at constant [3H]compounds concentration (15 nM) and increasing concentrations of unlabeled competing compounds (10 concentration data point from 10 μM to 0.1 nM). pIC50 were corrected to pKi according to Cheng and Prusoff (1973). In all experiments, the NSB was defined as the residual binding of [3H]compound observed in the presence of 10 μM T807.
Measurement: IC50 = 39.8 nM (pChEMBL 7.400000095367432)
Descriptors: MW 279.3, ALogP 2.52, HBA 5, HBD 1, PSA 68.62, QED 0.61
Compound (SMILES): COc1ccc(NC(=O)CSc2nc3cc(C#N)ncc3[nH]2)cc1O
InChIKey: LCZDPBVKAJBMBN-UHFFFAOYSA-N
Target: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P22413
Assay: Inhibition Assay: Materials:    Assay Buffer: 1 mM CaCl2, 0.2 mM ZnCl2, 50 mM Tris, pH 9.0    Substrate: 8 mM Thymidine 5′-monophosphate p-nitrophenol ester sodium salt (Sigma Cat # T4510)    Enzyme: 5 ng/μL Recombinant Human ENPP-1 Protein (R&D Cat #6136-EN-010) DMSO    96-well clear assay platesMethods:An eight point serial dilution of drugs was prepared in 10× in assay buffer with the final assay concentrations starting at 10 μM, 3 μM, 1 μM, 0.3 μM . . . 0 μM. A dilution of DMSO was included as a control. The assay plate was set up as follows with each well in duplicate: 81 μL assay buffer+10 μL ENPP1 inhibitor or DMSO+5 μL Substrate+4 μL Enzyme. Both the enzyme and substrate were added to opposite sides of the well to ensure that there was no interaction until all wells had both components. The plate was then centrifuged gently for 10 seconds, followed by an incubation at 37° C. for 45 minutes. The reaction was quantified by measuring absorbance at 405 nm using the Envision.
Measurement: IC50 = 179 nM (pChEMBL 6.75)
Descriptors: MW 355.38, ALogP 2.27, HBA 7, HBD 3, PSA 123.92, QED 0.6
Compound (SMILES): CCNC(=O)CC1N=C(c2ccc(Cl)cc2)c2cc(OC)ccc2-c2cn(C)c(=O)cc21
InChIKey: XPWOMOKPAAIWAP-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaLISA Assay: Compounds were diluted by step-down dilution method (final concentration of DMSO was 1%) and added to the wells of a 384 well opti plate at desired concentrations. 5 nM BDR4-BD1 enzyme (produced in-house) and 12 nM of biotinylated substrate were added to the wells, covered and incubated at room temperature (RT) for 1 h. At the end of 1 h 250 ng of GSH acceptor beads were added to the well and incubated for 1 h at RT; then 500 ng of streptavidin donor beads were added and incubated again for 1 h at RT. Plates were read in a Pherastar reader at 680 nm excitation and 570 nm emission
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 449.94, ALogP 4.13, HBA 5, HBD 1, PSA 72.69, QED 0.64
Compound (SMILES): CC(C)C[C@@](C)(N)COc1cnc(-c2ccnc(Cl)c2F)cc1C(F)F
InChIKey: SWKIRCKOMQZZFW-GOSISDBHSA-N
Target: AP2-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q2M2I8
Assay: AAK1 Kinase Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2 and ATP) and test compounds in assay buffer (10 mM Tris-HCL pH 7.4, 10 mM MgCl2, 0.01% Tween-20 and 1.0 mM DTT). The reactions were initiated by the combination of bacterially expressed, GST-Xa-hAAK1 with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA buffer to each sample. The reactions were analyzed on the Caliper LabChip 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to EDTA quenched control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assays are ATP, 22 μM; (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2, 1.5 μM; GST-Xa-hAAK1, 3.5 nM; and DMSO, 1.6%. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 1040 nM (pChEMBL 5.980000019073486)
Descriptors: MW 387.83, ALogP 5.02, HBA 4, HBD 1, PSA 61.03, QED 0.68
Compound (SMILES): Cc1noc(C)c1-c1cc(C(N)=O)c2c(c1)c1ccc(NCC3CCCC3)cc1n2CC1CC1
InChIKey: DHLKIDSPPFZBMR-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The binding of compounds to bromodomain BRD4 (44-168), BRD4 (333-460), and BRD4 (1-477 or 44-460) was assessed using a time resolved fluorescent resonance energy transfer binding assay (1), that measures the binding of a fluorescently labeled probe molecule to the bromodomain protein. The bromodomain protein, fluorescent probe molecule (either a biotinylated histone peptide or a fluorescently labeled small molecule), and dose-responsed test compound are incubated together to reach thermodynamic equilibrium. In the absence of a test compound, the bromodomain and small molecule are bound, resulting in a high fluorescent signal. In the presence of a sufficient concentration of inhibitor, this intercation is disrupted resulting in a loss of fluorescent resonance energy transfer.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 456.59, ALogP 6.18, HBA 5, HBD 2, PSA 86.08, QED 0.35
Compound (SMILES): COC1C(=O)C(C)CC/C=C\C=C/C=C(/C)C(NS(C)(=O)=O)C[C@H]2CC[C@H](C)[C@](O)(O2)C(=O)C(=O)N(C)C(C)C(=O)OC([C@H](C)C[C@@H]2CC[C@@H](OC3CCS(=O)(=O)C3)[C@H](OC)C2)CC(=O)C(C)/C=C(/C)C1O
InChIKey: BJRZWTPANBGDFW-HMPFPURJSA-N
Target: Peptidyl-prolyl cis-trans isomerase FKBP1A (SINGLE PROTEIN), Homo sapiens
UniProt: P62942
Assay: SPR Assay to Determine Binding Affinity to FKBP12: Biotinylated avi-FKBP12 was immobilized on a streptavidin chip (Cytiva Series S SA) using a Biacore 8K or 8 k+ (Cytiva). To achieve an immobilization level of 1000 RU, 2 μg/ml biotinylated avi-FKBP12 were injected for 100 sec at a flow rate of 10 μl/min. Rapalogs were diluted in DMSO to 100× working concentration. Each Rapalog was 100-fold diluted in 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20 and a serial dilution prepared (9 concentrations, 3-fold dilutions, 0.08-500 nM). Rapamycin was used as reference sample (9 concentrations, 3-fold dilutions, 0.02-100 nM). The compound dilutions were then injected at 100 uL/min for 120 seconds contact time in sequence with increasing concentrations. Dissociation was monitored for 3600 seconds. 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20, 1% DMSO was used as running buffer. The single-cycle kinetics data were fit to a 1:1 binding model to measure the association rate ka (1/Ms), the dissociation rate kd (1/s) and the affinity Kd (M).
Measurement: Kd < 0.3 nM (pChEMBL 9.52299976348877)
Descriptors: MW 1055.36
Compound (SMILES): OCCc1n[nH]c2cc(-c3cc(Nc4ccc(N5CCOCC5)cn4)c4nccn4c3)ccc12
InChIKey: JLQXRJUDGNOZIR-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 = 17000 nM (pChEMBL 4.769999980926514)
Descriptors: MW 455.52, ALogP 3.39, HBA 8, HBD 3, PSA 103.6, QED 0.36
Compound (SMILES): C[C@H](NC(=O)c1nc(C(=O)N2CC(F)(F)C[C@@H]2C)c(-c2cnc(NC3(C)CCC3)cc2C(F)(F)F)s1)C(C)(C)O
InChIKey: PPGSSVKOTGAMDS-KBPBESRZSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows:    0.065 mg/mL RORγt    60 μM 1,8-ANS    100 mM Hepes, pH 7.0    10 mM NaCl    2.5 mM GSH    0.002% Tween-20
Measurement: Kd = 0.43 nM (pChEMBL 9.369999885559082)
Descriptors: MW 589.63, ALogP 5.34, HBA 7, HBD 3, PSA 107.45, QED 0.38
Compound (SMILES): NC1CCN(c2c(-c3cc(F)cc(F)c3)cnc3ccc(-c4c(O)c(F)cc(F)c4F)cc23)CC1
InChIKey: HZAYODUPKJUWDH-UHFFFAOYSA-N
Target: Somatostatin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P30874
Assay: cAMP Assay : Four days prior to the assay, 5,000 Chinese hamster ovary cells (CHO-K1, ATCC #CCL-61) stably expressing the human SSTR2 are plated in each well of a 96-well tissue culture-treated plate in Ham's F12 growth media (ThermoFisher #10-080-CM) supplemented with 10% donor bovine serum (Gemini Bio-Products #100-506), 100 U/mL penicillin; 100 ug/mL streptomycin; 2 mM L-glutamine (Gemini Bio-Products #400-110) and 0.2 mg/mL hygromycin B (GoldBio #31282-04-9). The cells are cultured at 37° C., 5% CO2 and 95% humidity. On the day of the assay, the media is aspirated and the cells are treated with 50 μL of 1.6 μM NKH477 (Sigma #N3290) plus various dilutions of compounds of the invention in assay buffer [1× Hank's Balanced Salt Solution (ThermoFisher #SH3058802), 0.5 mM HEPES pH 7.4, 0.1% bovine serum albumin, 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX, VWR #200002-790)]. The cells are incubated for 20 minutes at 37° C. (the final concentration of the compounds of the invention are typically 0-10,000 nM). The cells are treated with 50 μL of lysis buffer (HRTF cAMP kit, Cisbio). The lysate is transferred to 384-well plates and cAMP detection and visualization antibodies are added and incubated for 1-24 hours at room temperature. The time-resolved fluorescent signal is read with a Tecan M1000Pro multiplate reader. The intracellular cAMP concentrations are calculated by regression to a standard curve and are plotted vs. the concentration of the compounds of the invention and the EC50 of the compounds are calculated using standard methods. All data manipulations are in GraphPad Prism v6.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 485.46, ALogP 5.9, HBA 4, HBD 2, PSA 62.38, QED 0.28
Compound (SMILES): CN1CCC[C@@H](COc2nc(-c3ccc(C#N)cc3)c(-c3cc(F)c(CO)c(F)c3)n3cncc23)C1
InChIKey: CONBDUZCCZKAFJ-GOSISDBHSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Demethylase Biochemical Assay: LANCE LSD1/KDM1A demethylase assay¿10 uL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 uL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 uL of assay buffer containing 0.4 uM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO:1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 uL 1x LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 489.53, ALogP 4.43, HBA 7, HBD 1, PSA 86.68, QED 0.43