lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C1NCC(c2c(F)cc(-c3ccc(C(F)(F)F)s3)cc2F)N1c1ccc2[nH]cnc2c1
InChIKey: LIGPWNPGTBCPBH-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: QC Inhibition Assay: An inhibition activity assay of QC inhibitors was conducted. See Huang et al., J. Biol. Chem. 2011, 286, 12439-12449. A reaction mixture containing 300 μM of Gln-βNA and 0.2 units of human PAP I was prepared. QC was first incubated with an inhibitor at 25° C. for 30 minutes and the enzyme-inhibitor mixture was then added to the reaction mixture to initiate the cyclization reaction. An IC50 value was obtained by fitting an initial reaction rate versus an inhibitor concentration using KaleidaGraph. A Ki value of the inhibitor was calculated according to an equation IC50=Ki(1+[S]/Km). See Segel, Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, pp. 100-118. New York: John Wiley & Sons, 1993. In this equation, [S] is a substrate (i.e., Gln-βNA) concentration and Km is a Michaelis-Menten constant. The lower the Ki value, the higher the inhibitor's QC inhibition rate.
Measurement: Ki = 101 nM (pChEMBL 7)
Descriptors: MW 464.42, ALogP 5.86, HBA 3, HBD 2, PSA 61.02, QED 0.37
Compound (SMILES): CCn1nc(C(=O)NC[C@]2(O)CC[C@@H](S(C)(=O)=O)CC2)c(Cl)c1-c1ccc(C2(O)CCC(F)(F)CC2)cc1OC(F)F
InChIKey: XVPQQTCIUQXRTP-CKQQTVSASA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 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.
Measurement: Kd = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 640.1, ALogP 4.67, HBA 8, HBD 3, PSA 130.75, QED 0.34
Compound (SMILES): COC(=O)C[C@@H]1COc2cc(O[C@@H]3CCc4c(Oc5ccc6cnn(CCC(C)(C)O)c6c5)ccc(F)c43)ccc21
InChIKey: RSODPAJOHPFJHQ-PIIWDFAUSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Pharmacological Assay: IP1 accumulation measurements using the IPOne assay system 1321N1 cells stably expressing human GPR40 receptor (Euroscreen, Belgium) are seeded 24 h before the assay in white 384-well plates in culture medium containing 10% FCS, 1% Na-Pyruvate and 400 μg/mL G418. IP1 is assayed according to the manufacturer's description (Cisbio Bioassays, France). In brief, the assay is started by substitution of the culture medium by stimulation buffer (Hepes 10 mM, CaCl2 1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM and LiCl 50 mM, pH 7.4). Cells are stimulated for 1 h at 37° C., 5% CO2 by addition of the compounds that are diluted in stimulation buffer containing LiCl. Assays are stopped by adding HTRF-conjugates (IP1-d2 and Anti-IP1 cryptate Tb) and lysis buffer, provided by the manufacturer. After an incubation time of 1 h at room temperature plates are measured using an EnVision, Perkin Elmer. The obtained fluorescence ratios at 665/615 nM are then used to calculate the pEC50 values using Assay Explorer 3.3 Software (Accelrys, Inc.) by interpolation using an IP1 reference curve and subsequent sigmoidal curve fitting allowing for a variable hill slope.
Measurement: EC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 560.62, ALogP 6.23, HBA 8, HBD 1, PSA 92.04, QED 0.24
Compound (SMILES): Fc1ccc2cc(C3CCCN3c3ncnc4[nH]cnc34)c(-c3ccccc3)nc2c1
InChIKey: KABFOXNGHWUHBT-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition Assay: Exemplary compounds of the invention were tested their inhibitory activity or potency against PI3Kδ in 10-dose IC50 mode with 3-fold serial dilution starting at 1 μM. The control compound, PI-103, was tested in the same conditions. Table 4 below summarizes the IC50 values that were collected against PI13Kδ for compounds exemplified in this invention. All IC50 values were reported in unites of nM. An IC50 value less than 0.0508 nM or 0.508 nM, or higher than 1 mM or 10 mM was estimated based on the best curve fitting available. Empty cells indicate no inhibition or compound activity that could not be fit to an IC50 curve.As demonstrated in Table 4 below, many exemplary compounds of formula (I) and formula (II) are potent inhibitors of PI3Kδ. Preferred compounds of the present invention possess an IC50 value for the inhibition of PI3Kδ of less than 10 μM, preferably less than 1 μM, even more preferably less than 0.1 μM, most preferably less than 0.05 μM.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 410.46, ALogP 5.05, HBA 5, HBD 1, PSA 70.59, QED 0.45
Compound (SMILES): N#Cc1cnc2ccc(-c3cccnc3-c3cc(F)ccn3)nn12
InChIKey: GLVMHLFIFCVTSY-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. 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.
Measurement: IC50 = 918 nM (pChEMBL 6.039999961853027)
Descriptors: MW 316.3, ALogP 2.86, HBA 6, HBD 0, PSA 79.76, QED 0.57
Compound (SMILES): NS(=O)(=O)OC[C@H]1C[C@@H](Nc2ccnc3cc(-c4ccc(F)c5ccccc45)nn23)[C@H](O)[C@@H]1O
InChIKey: GQLRLNNVZQFNBM-NECJECGFSA-N
Target: Ubiquitin-conjugating enzyme E2 E1 (SINGLE PROTEIN), Homo sapiens
UniProt: P51965
Assay: HTRF Assay : The UAE enzymatic reaction totals 50 μL and contains 50 mmol HEPES (pH 7.5), 0.05% BSA, 2.5 mM MgCl2, 0.1 uM ATP, 8 nM GST-Ubc-2, 35 nM flag-ubiquitin, 1 nM recombinant human UAE or mouse UAE. Compounds for this hUAE 1050 assay are tested at 10 point 3-fold dilution. The top concentration for this assay is 1 μM. Each compound is ordered in duplicate on the same plate. The enzymatic reaction mixture is incubated for 90 minutes at room temperature (24 degrees C.) in a 384 well plate prior to termination with a stop solution (0.1 M HEPES/0.05% Tween20, 20 mmol EDTA, 410 mM KF, 0.5 nM Eu Cryptate anti-FLAG M2-K antibody (Cis-bio International), 8 ug/mL Anti-GST XL-APC (Prozyme)). After incubation for 120 minutes, quantification of the FRET is performed on the Pherostar (BMG).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 487.51, ALogP 1.43, HBA 9, HBD 4, PSA 152.07, QED 0.32
Compound (SMILES): CC(c1ccc2c(c1)OCC2)N1CCC(Oc2ncccn2)CC1
InChIKey: QEBJZHKBYKVRPL-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase Enzyme Inhibition Assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (λemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 325.41, ALogP 3.02, HBA 5, HBD 0, PSA 47.48, QED 0.86
Compound (SMILES): CN(C)c1ccccc1C1CCN(c2nc(C3(C)CCC3)nc3ccc(N(C)CCO)cc23)CC1
InChIKey: XJGHHORLYXVYKH-UHFFFAOYSA-N
Target: Neurotensin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30989
Assay: NTR1 Ca2+ Flux Dose Response: NTSR1 (NTR1) CHO cells are plated in 20 uL of assay media containing Ham's F12 supplemented with 10% fetal bovine serum and 0.4 mg/mL G418 at a concentration of 1.0×106 cells per mL into black, 384-well assay plates with clear bottoms using a Multidrop liquid handler. Assay plates are incubated at 37° C. in 5% CO2. The next day, the assay plates are aspirated to remove growth media and washed once with 20 uL of DPBS. The DPBS is then aspirated from the assay plate and replaced with 25 uL of Fluo-4 NW calcium dye prepared according to the manufacturer's recommendations then the plates are incubated for 1 hour at 37° C. Following the incubation in the presence of dye, the assay is run on a Molecular Devices FlexStation-III using 494 excitation and 516 emission wavelengths set to read for 90 seconds with the addition at 18 seconds of 5 uL of 6× final concentration of test compounds and peptide control diluted in assay media containing 0.1% BSA and no more than 9% DMSO to yield a maximum final DMSO concentration of 1.5%. Percent activation is calculated based on the maximum response minus the minimum value over the time course relative to the neurotensin 1 control peptide at 100 pM. EC50 values were calculated for those compounds tested in 8-point dose dependent response.
Measurement: EC50 = 1250 nM (pChEMBL 5.900000095367432)
Descriptors: MW 473.67, ALogP 4.95, HBA 6, HBD 1, PSA 55.73, QED 0.52
Compound (SMILES): CN(C)c1ccc(C#Cc2cc3ccc(F)nc3[nH]2)cc1
InChIKey: XXRXTSVWBPYSMG-UHFFFAOYSA-N
Target: Alpha-synuclein (SINGLE PROTEIN), Homo sapiens
UniProt: P37840
Assay: In Vitro Inhibition Assay: Table 5: Fresh solutions of the test compounds (2.5 mM in DMSO) are diluted in PBS pH7.4, at concentrations between 0.5 nM and 5 μM in the presence of α-syn (200 nM), Aβ1-42 (500 nM) or Tau (200 nM) fibres and ThT 500 nM in a final 2 mL volume. The samples are incubated for 1 h at room temperature (RT) to reach equilibrium. The binding of ThT to α-syn, Aβ1-42 or Tau fibres is measured by fluorescence (excitation: 440 nm, emission: 480 nm). Inhibition of ThT binding by increasing concentrations of each test compound is measured by fluorescence reduction. Due to the partial superposition between the emission spectra of some compounds and ThT, the compounds are also incubated with α-syn, Aβ1-42 or Tau fibers in the absence of ThT. For each concentration, the fluorescence values in the absence of ThT are subtracted from those in the presence of ThT. The results are expressed as a percentage of the maximum fluorescence value of the ThT measured in the absence of a competitor (100% bind).
Measurement: Ki = 14.8 nM (pChEMBL 7.829999923706055)
Descriptors: MW 279.32, ALogP 3.17, HBA 2, HBD 1, PSA 31.92, QED 0.55
Compound (SMILES): N#Cc1cncc(COc2cc(O[C@H]3CCc4c(-c5ccccc5)cccc43)c(Cl)cc2CNCCn2nn[nH]c2=O)c1
InChIKey: HGGCAJPASXXCAN-LJAQVGFWSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayâELISA: Plates were coated with 1 Î¼g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4Â° C. The wells were then blocked with PBS containing 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37Â° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the samples were diluted to 1:5 in dilution medium in the ELISA plates. Human PD-1 and biotin 0.3 Î¼g/mL (ACRO Biosystems) were added and incubated for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was added with PBS containing 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37Â° C. and was washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37Â° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 594.08, ALogP 4.99, HBA 9, HBD 2, PSA 130.74, QED 0.2
Compound (SMILES): COc1cc(-c2cccc(-c3cccc(NC(=O)c4cn(C)c(=O)n(C)c4=O)c3C)c2C)cc(F)c1CN[C@H]1CCOC[C@@H]1O
InChIKey: XYFBAQLUUZZANY-KYJUHHDHSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayELISA: 96 Well plates were coated with 111 g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4° C. The wells were then blocked with 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the compounds were serial diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.31 μg/mL (ACRO Biosystems) were added and incubated for 1 hour at 37° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was performed with 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37° C. then the plates were washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 616.69, ALogP 3.67, HBA 9, HBD 3, PSA 123.82, QED 0.28
Compound (SMILES): CCCCOc1nc(N)c2nc(O)n(Cc3ccc(CNC4CCCC4)nc3)c2n1
InChIKey: ODKGGAOIIBRFIB-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: HEK-Blue TLR7 reporter assay: Engineered human embryonic kidney blue cells (HEK-Blue TLR cells; Invivogen) possessing a human TLR7-secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in a non-selective, culture medium (DMEM high-glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue TLR7 cells were added to each well of a 384-well tissue-culture plate (15,000 cells per well) and incubated 16-18 h at 37° C., 5% CO2. Compounds (100 nl) were dispensed into wells containing the HEK-Blue TLR cells and the treated cells were incubated at 37° C., 5% CO2. After 18 h treatment ten microliters of freshly-prepared Quanti-Blue reagent (Invivogen) was added to each well, incubated for 30 min (37° C., 5% CO2) and SEAP levels measured using an Envision plate reader (OD=620 nm). The half maximal effective concentration values (EC50; compound concentration which induced a response halfway between the assay baseline and maximum) were calculated.
Measurement: EC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 411.51, ALogP 2.77, HBA 9, HBD 3, PSA 124, QED 0.46
Compound (SMILES): O=C(NCc1ccncc1)c1ccc2c(c1)OCc1cnccc1-2
InChIKey: LUFLLWWPNXBABE-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 177 nM (pChEMBL 6.75)
Descriptors: MW 317.35, ALogP 2.97, HBA 4, HBD 1, PSA 64.11, QED 0.81
Compound (SMILES): O=S1(=O)CN(C2CCCC2)CCN1CC(O)Cn1c2ccc(F)cc2c2cc(F)ccc21
InChIKey: MCLXZVRHDMKNLR-UHFFFAOYSA-N
Target: Period circadian protein homolog 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15055
Assay: High-Throughput Circadian Assay: Per2 Assay for Evaluating the Potency of Test Compounds.
Measurement: EC50 = 1498 nM (pChEMBL 5.820000171661377)
Descriptors: MW 463.55, ALogP 3.28, HBA 5, HBD 1, PSA 65.78, QED 0.63
Compound (SMILES): Cc1cnc(C(=O)Nc2cccc(-c3nnnn3C(C)C)n2)cc1-c1ccccn1
InChIKey: MBWCGYMFGLBOTN-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay: The ability (IC50) of compounds to inhibit ASK1 kinase activity was determined by HTRF® KinEASE Assay System. 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 uM STK3 peptide substrate, and 5 nM 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 uM 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.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 400.45, ALogP 3.33, HBA 8, HBD 1, PSA 111.37, QED 0.55
Compound (SMILES): Cc1cn(Cc2c(Cl)ccc(C(=O)N3CC[C@@H](C(=O)O)[C@@H](C)C3)c2Cl)c2c(C)cc(OC(F)(F)F)cc12
InChIKey: OFQPJQDCAUICAI-WMLDXEAASA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 557.4, ALogP 6.69, HBA 4, HBD 1, PSA 71.77, QED 0.38
Compound (SMILES): CC(=O)N1CCc2c(c(-c3c[nH]c4ccccc34)nn2C)C1
InChIKey: LHZWFOVJEWVOSF-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6xHis antibody (6xHis is disclosed as SEQ ID NO: 3) (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 93.5 nM (pChEMBL 7.03000020980835)
Descriptors: MW 294.36, ALogP 2.47, HBA 3, HBD 1, PSA 53.92, QED 0.75
Compound (SMILES): CCOc1ccc2cc(-c3nn(CC4(O)CCNCC4)c4ncnc(N)c34)ccc2n1
InChIKey: JMHFIEVACDXMDP-UHFFFAOYSA-N
Target: Calmodulin-domain protein kinase 1 (SINGLE PROTEIN), Toxoplasma gondii
UniProt: Q9BJF5
Assay: Enzymatic Inhibition Assay: Inhibitors were evaluated in triplicate in eight-point dilutions (3-fold dilutions) during the enzymatic reactions. TgCDPK1 enzymatic inhibition was determined with a coupled luciferase assay (Kinaseglo®). 2.1 nM TgCDPK1 and 20 μM BioSyntide-2 (American Peptide Company, Inc. Sunnyvale, Calif.)) were incubated in 25 μL of buffer containing 1 mM EGTA (pH 7.2), 10 mM MgCl2, 20 mM HEPES, pH 7.5 (KOH), 0.1% BSA, and 2 mM CaCl2. The reaction was initiated with the addition of ATP at a 10 μM final concentration. After incubating at 30° C. for 90 min., changes in ATP concentration were determined by adding Kinaseglo® luciferase reagent (Promega, Madison, Wis.) and measuring luminescence with a MicroBeta2 multi-label plate reader (Perkin Elmer, Waltham, Mass.). Results were converted to percent inhibition, and IC50 values were calculated using nonlinear regression analysis in GraphPad Prism.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 419.49, ALogP 2.14, HBA 9, HBD 3, PSA 124, QED 0.45
Compound (SMILES): Fc1cccc(Cn2cnc3ccc(-c4cc(NC5CC6(CCCNC6)C5)ncc4Cl)cc32)c1
InChIKey: JMPHNRXXSQJZLN-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615665 nm).
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 476, ALogP 5.88, HBA 5, HBD 2, PSA 54.77, QED 0.38
Compound (SMILES): Cc1nnc2n1-c1ccccc1C(c1ccc(Cl)cc1)=C[C@H]2CC(=O)N1CC(Oc2ccccc2)C1
InChIKey: SHAHTQCXRXCCHI-NRFANRHFSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance energy Transfer (TR-FRET) Assay: For this purpose, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used, which measures the binding between N-terminally His6-tagged BRD4(1) (amino acids 44-168) and a synthetic acetylated histone H4 (Ac-H4) peptide with sequence GRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHGSGSK-biotin. The recombinant BRD4 protein produced in-house according to Filippakopoulos et al., Cell, 2012, 149:214-231 was expressed in E. coli and purified by means of (Ni-NTA) affinity and (Sephadex G-75) size exclusion chromatography. The Ac-H4 peptide can be purchased, for example, from Biosyntan (Berlin, Germany).In the assay, typically 11 different concentrations of each substance (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 analysed as duplicates on the same microtitre plate. For this purpose, 100-fold concentrated solutions in DMSO were prepared by serial dilutions (1:3.4) of a 2 mM stock solution into a clear, 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany). From this, 50 nl were transferred into a black test plate (Greiner Bio-One, Frickenhausen, Germany). The test was started by the addition of 2 μl of a BRD4 solution of 2.5 times the concentration (final concentration typically 10 to 50 nM in the 5 μl of reaction volume) in aqueous assay buffer [50 mM HEPES pH 7.5, 50 mM sodium chloride (NaCl), 0.25 mM CHAPS and 0.05% bovine serum albumin (BSA)] to the substances in the test plate. This was followed by a 10-minute incubation step at 22° C. for the pre-equilibration of putative complexes between BRD4 and the substances. Subsequently, 3 μl of a solution of 1.67 times the concentration (in assay buffer) consisting of Ac-H4 peptide (83.5 nM) and TR-FRET detection reagents [16.7 nM anti-6His-XL665 and 3.34 nM streptavidin cryptate (both from Cisbio Bioassays, Codolet, France), and 668 mM potassium fluoride (KF)] were added.
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 497, ALogP 5.44, HBA 5, HBD 0, PSA 60.25, QED 0.37
Compound (SMILES): CCN1Cc2c(ccnc2N[C@@H](C)c2ccc(-c3cn(C)nn3)cc2)C1=O
InChIKey: WCQOAHJTPLHOEL-ZDUSSCGKSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay for IDH Inhibition: Solution A was prepared by combining 1 M Tris-HCl pH7.5 (300 μL), 5 M NaCl (450 μL), 1 M MgCl2 (150 μL), 1 M DTT (15 μL), 20 mg/mL BSA (37.5 μL), 20 mM NADPH (20 μL), and 0.5 mM α-KG (40 μL), followed by adding deionized water to a final volume of 15 mL.Reaction buffer was prepared by combining 1 M Tris-HCl pH 7.5 (200 μL), 5 M NaCl (300 μL), 1 M MgCl2 (100 μL), 1 M DTT (10 μL) and 20 mg/mL BSA (25 μL), followed by adding deionized water to a final volume of 10 mL.Solution B was prepared by combining the reaction buffer (5 mL) and 192 M IDH1-R132H (5.21 μL) or 107 μM IDH1-R132C (4.67 μL).148 μL Solution A was added into each sample well of a 96-well plate P1, and then 2 μL of the test compound solution at each concentration and 2 μL of DMSO (negative control/blank) were added into individual sample well respectively. 50 μL of Solution B was added into each of the sample wells having test compounds and one sample well having DMSO only, and 50 μL of reaction buffer was added into another sample well having DMSO only, respectively. P1 was put into BioTek Synergy H4 Microplate reader (BioTek Instruments Inc., Winooski, U.S.), and the program was set as follows: 37° C., shake plate 5 seconds, run kinetic mode, total detection time 20 min, detection intervals 40 seconds, detection mode fluorescence (at Excitation 340 nm, Emission 460 nm). The data were exported, the relative enzyme activity and the half-maximal inhibitory concentration (IC50) for the test compound were calculated. Relative enzyme activity was calculated as the absolute value of the slope of the Emission 460 reads of each well plotted against time (which represents the conversion rate of ca-KG to 2-HG through IDH1 modulated utilization of NADPH). The Dose-response curve was drawn and the IC50 was calculated via fitting. The IDH inhibition and selectivity of each test compound can be evaluated according to the effect of the compounds on the IDH enzyme activity.
Measurement: IC50 = 12500 nM (pChEMBL 4.900000095367432)
Descriptors: MW 362.44, ALogP 3.03, HBA 6, HBD 1, PSA 75.94, QED 0.75
Compound (SMILES): O=C(NCCC1CCN(Cc2ccccc2)CC1)C1(O)CCN(c2cc(F)c(F)c(F)c2)CC1
InChIKey: UFFAQRXBFDMWSO-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 475.56, ALogP 3.85, HBA 4, HBD 2, PSA 55.81, QED 0.6
Compound (SMILES): CC1Sc2nnc(-c3cc(-c4ccccc4)n[nH]3)n2N=C1c1ccc(OC(F)(F)F)cc1
InChIKey: KQTAPLOIKILVDL-UHFFFAOYSA-N
Target: Signal transducer and activator of transcription 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P40763
Assay: Bioassay: Compound samples (in 3-5 mg quantities) were evaluated in a variety of assays. All compound submissions were fully characterized (1H, 13C, IR, FIRMS), satisfied purity criteria (≥95% LC/MC, ELS).
Measurement: IC50 = 24200 nM (pChEMBL 4.619999885559082)
Descriptors: MW 456.45, ALogP 4.98, HBA 7, HBD 1, PSA 80.98, QED 0.47
Compound (SMILES): CC(C)(C(=O)N[C@@H](CCN1CC(CCc2ccc3c(n2)NCCC3)C1)C(=O)O)c1cccnc1
InChIKey: FVNPZCVQDVZNNE-QFIPXVFZSA-N
Target: Integrin alpha-V/beta-6 (PROTEIN COMPLEX), Homo sapiens
UniProt: P06756
Assay: Binding Assay: Microplates were coated with recombinant human integrin αvβ6 (2 ug/ml) in PBS (100 ul/well 25° C., overnight). The coating solution was removed, washed with wash buffer (0.05% Tween 20; 0.5 mM MnCl2; in 1×TBS). Plate was blocked with 200 ul/well of Block Buffer (1% BSA; 5% sucrose; 0.5 mM MnCl2; in 1×TBS) at 37° C. for 2 h. Dilutions of testing compounds and recombinant TGFβ1 LAP (0.67 ug/ml) in binding buffer (0.05% BSA; 2.5% sucrose; 0.5 mM MnCl2; in 1×TBS) were added. The plate was incubated for 2 hours at 25° C., washed, and incubated for 1 hour with Biotin-Anti-hLAP. Bound antibody was detected by peroxidase-conjugated streptavidin. The IC50 values for testing compounds were calculated by a four-parameter logistic regression.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 465.6, ALogP 2.64, HBA 6, HBD 3, PSA 107.45, QED 0.5
Compound (SMILES): CC1(C)CC(=O)C2=C(C1)Nc1n[nH]c(C3CC3)c1C2(C)c1cccc(C#N)c1
InChIKey: VAPVCGAVXYXZNZ-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: Caliper Assay: A solution of 4× inhibitor (5 μL), 4× substrate/ATP-Metal solution (5 μL), and 2× Kinase solution (10 μL) was prepared with assay buffer (20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, pH 7.5) and mixed/incubated in 384 well plates for 1 or 5 hours depending on the kinase being examined, at room temperature. A solution of termination buffer (QuickScout Screening assist MSA; Carna Biosciences) (60 μL) was added to each well. The entire reaction mixture was then applied to a LabChip3000 system (Caliper Life Science) and the product and substrate peptide peaks were separated and quantified. Evaluation of kinase activity was then determined based on ratio of calculated peak heights of product (P) and substrate (S) peptides (P/(P+S)).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 372.47, ALogP 4.53, HBA 4, HBD 2, PSA 81.57, QED 0.81
Compound (SMILES): Cc1nnc(CC(=O)N[C@H]2CC[C@H](CCN3CCc4sc(C5CC(F)(F)C5)nc4C3)CC2)s1
InChIKey: HNRDJYILRSRTOJ-JCNLHEQBSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.6HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.) Radioligand: (final concentration) 2 nM [H]-Methylspiperone ([H���N-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 uM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well) Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.16 nM (pChEMBL 9.800000190734863)
Descriptors: MW 495.67, ALogP 4.48, HBA 7, HBD 1, PSA 71.01, QED 0.61
Compound (SMILES): CN(C)C(=O)/C=C/CC[C@H](NC(=O)C1CCC(F)(F)CC1)C(=O)Nc1cccn(Cc2nc3cccc(OCc4ccc(F)cc4F)c3s2)c1=O
InChIKey: SPPQFYQPRRGSOZ-ALDLVRNQSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 727.78, ALogP 6.04, HBA 8, HBD 2, PSA 122.63, QED 0.13
Compound (SMILES): COc1cncc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(P(C)(C)=O)c4OC)c4ccccc34)ccn2)c1
InChIKey: UZRIDTFIIDOBLB-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: Enzyme Binding Assay: Method 1: The inhibitory activities of test compounds against the p38 MAPKα isoform (MAPK14: Invitrogen), are evaluated indirectly by determining the level of activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPKα protein (80 ng/mL, 2.5 μL) is mixed with the test compound (2.5 μL of either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL or 0.004 μg/mL) for 2 hr at RT. The mix solution (2.5 μL) of the p38a inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 μM; a phosphorylation target for MAPKAP-K2) is then added and the kinase reaction is initiated by adding ATP (40 μM, 2.5 μL). The mixture is incubated for 1 hr at RT. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 639.69, ALogP 8.37, HBA 8, HBD 3, PSA 123.7, QED 0.14
Compound (SMILES): C[C@@H](NC(=O)c1ccnc2ccc(-c3ccc(Cl)cc3)cc12)C(=O)N1CC(F)(F)C[C@H]1C#N
InChIKey: IAUWBDOHWFMMLW-KDOFPFPSSA-N
Target: Dipeptidyl peptidase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86TI2
Assay: DPP9 Enzymatic Activity Assay: DPP9 enzymatic activity assay. To assay baseline dipeptidyl peptidase 9 (DPP9) activity, 40 ng of recombinant human DPP9 (rhDPP9) (R&S system, #5419-SE) was incubated with 100 μM of H-Gly-Pro-AMC peptide (BACHEM, #L-1215) in a DDP9 assay buffer (50 mM HEPES, pH 8) for 30 min at 37° C. in 96-well black plates (Nunc, #237108). To assay rhDPP9 activity inhibition by test compounds, test compounds were pre-incubated with the enzyme for 15 min at 37° C. before starting the reaction by substrate addition in 96-well black plates (Nunc, #237108). 7-Amino-4-Methylcoumarin (AMC) release was detected by measuring fluorescence at Ex/Em 380/460 nm using a Multifunction Microplate Reader (Synergy 4, Biotek). All measurements were carried out in triplicate. Val-boroPro, a non-specific prolyl peptidase inhibitor, was used as a positive control.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 468.89, ALogP 4.43, HBA 4, HBD 1, PSA 86.09, QED 0.61
Compound (SMILES): COc1cc(OC)cc(C(Cc2nccn2S(=O)(=O)N(C)C)c2ccc3ncc(-c4cnn(C)c4)nc3n2)c1
InChIKey: FGCIKBFVIMRWHK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR1 (Enzymatic Assay): In a final reaction volume of 30 μL, FGFR1 (h) (25 ng/ml) was incubated with 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1 mM Na3VO4, 0.01% Triton-X-100, 500 nM Btn-Flt3 and 5 μM ATP in the presence of compound (1% DMSO final). After incubation for 60 minutes at room temperature the reaction was stopped with 2.27 nM EU-anti P-Tyr, 7 mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA which was present for 60 minutes at room temperature. Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) signal (ex340 nm. Em 620 nm, em 655 nm) was measured afterwards and results are expressed in RFU (Relative Fluorescence Units). In this assay, the inhibitory effect of different compound concentrations (range 10 μM to 0.1 nM) was determined and used to calculate an IC50 (M) and pIC50 (−log IC50) value.
Measurement: IC50 = 631 nM (pChEMBL 6.199999809265137)
Descriptors: MW 548.63, ALogP 2.67, HBA 11, HBD 0, PSA 130.15, QED 0.27
Compound (SMILES): Cc1cc(C#N)cc2c1cc(Cc1c(Cl)ccc(C(=O)N3CC(N4CCCC4)C3)c1Cl)n2C
InChIKey: UTCLRDMHDQNQHP-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: transient transactivation assay: The primary screen was performed by transient transactivation assays. These cell-based assays were carried out using Cos-7 cells transfected with a chimeric human RORγ-Gal4 receptor expression plasmid and a 5Gal4 pGL3 TK Luc reporter plasmid. Transfections were performed by a chemical agent (Jet PEI). Transfected cells were distributed in 384-wells plates and were allowed to recover for 24 h. The culture medium was then removed and fresh medium containing the compounds to be tested was added (final concentration ranging from 10−4 M to 3 10−10 M). After an overnight incubation, luciferase expression was measured by adding SteadyGlo according to the manufacturer's instructions (Promega). T0901317 at 10−5 M was used as reference. Results were expressed as fold induction compared to basal level or as percentage activity compared to references taken as 100% (basal) or 0% (T0901317). Dose-effect curves and IC50 were calculated using the software Assay Explorer (MDL).
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 481.43, ALogP 5.18, HBA 4, HBD 0, PSA 52.27, QED 0.52
Compound (SMILES): NC1(c2ccc(-c3nc4ccc(NC(=O)C(F)(F)F)nn4c3-c3ccccc3)cc2)CCC1
InChIKey: SCASZVGHWLKNJB-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Kinase Assay: For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of Akt1 in assay buffer [50 mM TRIS/HCl pH 7.5, 5 mM MgCl2, 1 mM dithiothreitol, 0.02% (v/v) Triton X-100 (Sigma)] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of adenosine-tri-phosphate (ATP, 16.7 μM=>final conc. in the 5 μl assay volume is 10 μM) and substrate (1.67 μM=>final conc. in the 5 μl assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Akt1 in the assay was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 0.05 ng/μl (final conc. in the 5 μl assay volume). The reaction was stopped by the addition of 5 μl of a solution of HTRF detection reagents (200 nM streptavidine-XL665 [Cisbio] and 1.5 nM anti-phosho-Serine antibody [Millipore, cat. #35-001] and 0.75 nM LANCE Eu-W 1024 labeled anti-mouse IgG antibody [Perkin Elmer]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES/NaOH pH 7.5). The resulting mixture was incubated 1 h at 22° C. to allow the binding of the biotinylated phosphorylated peptide to the streptavidine-XL665 and the antibodies. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the anti-mouse-IgG-Eu-Chelate to the streptavidine-XL665. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a HTRF reader, e.g. a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer).
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 451.45, ALogP 4.9, HBA 5, HBD 2, PSA 85.31, QED 0.46
Compound (SMILES): C/C=C/C(=O)N1CC(N(C)CCn2c(=O)c(-c3c(Cl)c(OC)cc(OC)c3Cl)cc3cnc(NC)nc32)C1
InChIKey: RBNKCLPELCJMAS-VOTSOKGWSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Caliper-Based Kinase Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of FGFR family (FGFR1, FGFR2, FGFR3, FGFR4) kinase activity of a compound of Formula (III). Serial dilutions of test compounds were incubated with either human recombinant FGFR1 (0.5 nM), FGFR2 (0.1 nM, FGFR3 (0.9 nM), or FGFR4 (2 nM), ATP (FGFR1: 100 μM; FGFR2: 75 μM; FGFR3: 120 μM; FGFR4: 250 μM) and a phosphoacceptor peptide substrate FAM-KKKKEEIYFFF-CONH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper LabChip 3000. Percent inhibition was calculated for each compound dilution and the concentration that produced 50% inhibition was calculated.
Measurement: IC50 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 561.47, ALogP 3.54, HBA 9, HBD 1, PSA 101.82, QED 0.4
Compound (SMILES): CC(NC(=O)c1c(N)nn2cccnc12)c1cc(Cl)c2cn[nH]c2c1N1CCC(S(=O)(=O)N(C)C)CC1
InChIKey: UMAPLNWBQIZJTK-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: PI3K-gamma Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer). IC50 determination was performed by fitting the curve of percent of the solvent control activity versus the log of the inhibitor concentration using the GraphPad Prism 6.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 546.06, ALogP 2.19, HBA 9, HBD 3, PSA 154.61, QED 0.33
Compound (SMILES): CCOc1c(C(C)n2nc(C)c3c(N)ncnc32)cc(Cl)c(F)c1C1COC(=O)N1
InChIKey: UCYDWQYLQRHHCX-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay: [γ-33P]ATP (10 mCi/mL) was purchased from PerkinElmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kδ (p110δ/p85α) was purchased from Millipore (Bedford, Mass.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from SigmaAldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from GE healthcare life sciences (Piscataway, N.J.).The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 μL Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ-33P] ATP, 4 nM PI31(6. Reactions were incubated for 210 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1800 rpm for 10 minutes, the radioactivity of the product was determined by scintillation counting on Topcount (PerkinElmer).
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 434.86, ALogP 3.3, HBA 8, HBD 2, PSA 117.18, QED 0.63