lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC1CCCC/C1=C1/SC(N2CCC3(CC2)OCc2ccccc23)=NC1=O
InChIKey: HFSIFWBTJFJCHH-MNDPQUGUSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: competition binding assay: The human V1a receptor was cloned by RT-PCR from total human liver RNA. The coding sequence was subcloned in an expression vector after sequencing to confirm the identity of the amplified sequence. To demonstrate the affinity of the compounds from the present invention to the human V1a receptor binding studies were performed. Cell membranes were prepared from HEK293 cells transiently transfected with the expression vector and grown in 20 liter fermenters with the following protocol.50 g of cells are re-suspended in 30 ml freshly prepared ice cold Lysis buffer (50 mM HEPES, 1 mM EDTA, 10 mM MgCl2 adjusted to pH=7.4+complete cocktail of protease inhibitor (Roche Diagnostics)). Homogenized with Polytron for 1 min and sonicated on ice for 2×2 minutes at 80% intensity (Vibracell sonicator). The preparation is centrifuged 20 min at 500 g at 4° C., the pellet is discarded and the supernatant centrifuged 1 hour at 43,000 g at 4° C. (19,000 rpm). The pellet is re-suspended in 12.5 ml Lysis buffer+12.5 ml Sucrose 20% and homogenized using a Polytron for 1-2 min. The protein concentration is determined by the Bradford method and aliquots are stored at −80° C. until use. For binding studies 60 mg Yttrium silicate SPA beads (Amersham) are mixed with an aliquot of membrane in binding buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM MgCl2) for 15 minutes with mixing. 50 μl of bead/membrane mixture is then added to each well of a 96 well plate, followed by 50 μl of 4 nM 3H-Vasopressin (American Radiolabeled Chemicals). For total binding measurement 100 μl of binding buffer are added to the respective wells, for non-specific binding 100 μl of 8.4 mM cold vasopressin and for compound testing 100 μl of a serial dilution of each compound in 2% DMSO. The plate is incubated 1 h at room temperature, centrifuged 1 min at 1000 g and counted on a Packard Top-Count. Non-specific binding counts are subtracted from each well and data is normalized to the maximum specific binding set at 100%. To calculate an IC 50 the curve is fitted using a non-linear regression model (XLfit) and the Ki is calculated using the Cheng-Prussoff equation.
Measurement: Ki = 1.58 nM (pChEMBL 8.800000190734863)
Descriptors: MW 398.53, ALogP 3.98, HBA 5, HBD 0, PSA 51.13, QED 0.67
Compound (SMILES): CC(C)C[C@](C)(N)COc1ccc(-c2cccc3ncccc23)cc1C1CC1
InChIKey: RQRGSHXEWVXFGK-VWLOTQADSA-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 = 0.07 nM (pChEMBL 10.149999618530273)
Descriptors: MW 374.53, ALogP 5.92, HBA 3, HBD 1, PSA 48.14, QED 0.56
Compound (SMILES): CC(C)(O)c1cnnn1[C@H]1C[C@@H](C(=O)NC2(C(=O)C(N)=O)CCOCC2)N(C(=O)[C@@H](CC2CCCCC2)NC(=O)c2ccc3nccn3c2)C1
InChIKey: ICKUBUINFSWXPE-GVAUOCQISA-N
Target: Serine protease HTRA1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92743
Assay: HTRA1 Assay: Serial dilutions (1/3) from 1000 μM down to 0.051 μM of test compounds were prepared in dimethyl sulfoxide (DMSO). Then 2 μL of solution from each dilution were added to 100 μL of 4 nM full-length human His-HTRA1 in assay buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.25% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate or CHAPS) in white non-binding 96-well plates. The assay solutions were mixed for 5 seconds on a shaker plate and incubated for 10 minutes at room temperature. Mca-H2OPT (Mca-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-Lys(Dnp)-Lys-OH trifluoroacetate salt) (Mca=7-methoxycoumarin-4-acetic acid; Dnp=dinitrophenyl) (5 μM) in 100 μL of assay buffer was added to the assay solutions. The reaction mixture was shaken for 5 seconds on a shaker plate and cleavage of Mca-H2OPT was monitored by spectrofluorometry (SpectraMax M3 by Molecular Devices, CA) for 10 minutes (Exk=330 nm; Emk=420 nm).
Measurement: IC50 = 95 nM (pChEMBL 7.019999980926514)
Descriptors: MW 691.79, ALogP 0.78, HBA 12, HBD 4, PSA 216.14, QED 0.21
Compound (SMILES): CNC(=O)[C@@H]1[C@@H](Cc2ccnc(N)c2)C(=O)N1C(=O)N1CCC[C@H](c2ccccc2)C1
InChIKey: YMXIORYHKBPTPP-NSHGMRRFSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Enzyme Activity Assay: Method: the test compound was dissolved in 10 mM mother liquor with DMSO, and stored at −80° C. until use. The frozen mother liquor was diluted with DMSO to 2 mM as the initial reaction concentration, and then diluted with DMSO in 4-fold gradient to 9 concentrations as the working solution, 1 μl/well; the preparation of 2× buffer: 200 mM tris-HCl, 400 mM NaCl, 0.04% TWEEN20, pH 7.4; 2 human FXIa protein (hFXIa), the reaction solution was prepared by diluting the FXIa protein (Cat #ab62411) with 2× buffer to the required concentration of the reaction 0.25 ng/μl, 10 μl/well; 2×S-2366 reaction solution was prepared by diluting the S-2366 reaction solution with deionized water to 2 mM, 10 μl/well; the enzyme reaction solution was first added to the 384-well plate, and then the diluted test compound reaction solution was added to the corresponding wells in sequence; the negative control was DMSO solvent; the blank was buffer solution: the plate was centrifuged at 1000 rpm for 1 min at room temperature, and after 30 min of reaction in the dark, S-2366 reaction solution was added to each well; after being shaked and mixed for 30 s, the wells were allowed to react at 37° C.
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 421.5, ALogP 1.78, HBA 5, HBD 2, PSA 108.63, QED 0.73
Compound (SMILES): CCN(CC)C(=O)Oc1nc(-c2ccc(Br)cc2)nc2ccccc12
InChIKey: NLEHRJSZYPOCQC-UHFFFAOYSA-N
Target: Translocator protein (SINGLE PROTEIN), Homo sapiens
UniProt: P30536
Assay: [3H]PK 11195 radio-assay: For the assays described herein systemic injections of kainic in rats were applied and also contusion (Veenman et al. 2002; Soustiel et al. Neuropathol Appl Neurobiol. 2008; Soustiel et al. Exp Neurol. 2008). These models present very well defined cell death in the brain. Cell death in the brain is the major pathological characteristic of progressing neurodegeneration common to brain diseases. As the compounds of the present invention are designed to prevent cell death in the brain as a consequence of disease and progressing brain damage after injury, including behavioral impairments, animal models displaying cell death in the brain are the models of choice for displaying the protective capabilities of the compounds of the invention. For these reasons the R6-2 model for Huntington Disease was chosen to test the effectiveness of Compound 1 and to compare the effectiveness of Compound 1 with those of the compound of U.S. Pat. No. 8,541,428 (Cpd A) and with the classical TSPO ligand PK 11195, as well as other compounds disclosed herein. Compounds 5 and 6 were also tested in the same R6-2 model.
Measurement: Ki = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 400.28, ALogP 4.9, HBA 4, HBD 0, PSA 55.32, QED 0.63
Compound (SMILES): COc1ccc(Cc2n[nH]c(=O)c3ccccc23)cc1C(=O)N1CCN(c2cnc3[nH]cnc3c2)CC1
InChIKey: AULNAPPLKRYRNF-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: HT Universal Chemiluminescent PARP Assay Kit: HT Universal Chemiluminescent PARP Assay Kit with Histone-coated Strip Wells, commercially available from Trevigen (US), Catalog #: 4676-096-K.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 495.54, ALogP 2.76, HBA 7, HBD 2, PSA 120.1, QED 0.38
Compound (SMILES): CCC(C(=O)Nc1ccc2nn(CC(F)F)cc2c1)n1cc(OC)c(-c2cc(Cl)ccc2-n2cc(Cl)nn2)cc1=O
InChIKey: GTOBEZUWBRGMLK-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: The factor XIa inhibition of the substances according to the invention is determined using a biochemical test system which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions is placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride; 5 mM of calcium chloride; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 602.43, ALogP 5.62, HBA 9, HBD 1, PSA 108.86, QED 0.23
Compound (SMILES): N#Cc1cc(-c2ncnc(Nc3ccc(N4CCN(C=O)CC4)c(F)c3)n2)ccc1O[C@H]1CCOC[C@H]1F
InChIKey: KOJMDEPTVZYEKV-QPPBQGQZSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 4000 nM (pChEMBL 5.400000095367432)
Descriptors: MW 521.53, ALogP 3.08, HBA 9, HBD 1, PSA 116.5, QED 0.47
Compound (SMILES): Cc1ccc(Br)nc1NC(=O)[C@@H]1C[C@@]2(CNC(=O)COCCOCCNC(=O)COCCOCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCCCC(=O)O)C(=O)O)C[C@H]2N1C(=O)Cn1nc(C(N)=O)c2ccc(C(=O)NS(=O)(=O)C3CC3)cc21
InChIKey: JVHDFRPUODQOOJ-QFNKRHIPSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of Complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 1389.43
Compound (SMILES): COC1CN(Cc2nc3cc(Cl)c4c(c3o2)C2(CCCCC2)NC(=O)N4)C1
InChIKey: XGNZFUAGLNUMQL-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 7B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP56
Assay: Enzymatic Assay: PDE7 inhibition was determined by an IMAP TR-FRET assay using PDE7B. The IMAP TR-FRET PDE assay was optimized for concentration of enzyme, FAM-cAMP substrate, reducing agent, DMSO tolerance, and incubation time. First, PDE7 inhibitor compounds in 10 mM DMSO stock were serially diluted in 100% DMSO. Next, into each well of a solid white 1536 well plate (Corning) was dispensed 12.8 pg of N-terminal truncated recombinant human PDE7B enzyme (91-450aa, prep #DBVC-D04614, made in-house by structural biology) in 2.5 μL IMAP BSA reaction buffer (Molecular Devices, Sunnyvale, Calif.) containing 1 mM DTT (Sigma Aldrich.) After a brief centrifugation, 30 nL of serially diluted compounds in DMSO were added by transfer from 1 mM stock using a Kalypsys 1536 Pintool. Plates were incubated for 5 minutes at room temperature before dispensing 1.5 μL of 134 nM 5-carboxy fluorescein (FAM)-labeled cAMP (Molecular Devices, Sunnyvale, Calif.) for a final concentration of 50 nM. After a brief centrifugation, the plates were incubated for 15 minutes at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent/Tb complex (Molecular Devices, Sunnyvale, Calif.) to each well. Plates were incubated 1 hour at room temperature and read on a Viewlux multimode plate reader (Perkin Elmer).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 390.87, ALogP 3.61, HBA 5, HBD 2, PSA 79.63, QED 0.84
Compound (SMILES): CC(C)(Oc1cc(COc2ccc(-c3ccn[nH]3)nc2)ccn1)C(=O)O
InChIKey: UVRIPDCGGUVCPM-UHFFFAOYSA-N
Target: Cytochrome P450 4F2 (SINGLE PROTEIN), Homo sapiens
UniProt: P78329
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 11000 nM (pChEMBL 4.960000038146973)
Descriptors: MW 354.37, ALogP 2.69, HBA 6, HBD 2, PSA 110.22, QED 0.67
Compound (SMILES): CNc1cc(-c2cnc3n([C@@H]4COC[C@H]4OC)cccc2-3)nc2c(C(=O)N[C@@H]3CC[C@@H]3F)cnn12
InChIKey: WXBWQKUBQGTJQN-ONCXSQPRSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 479.52, ALogP 2.56, HBA 9, HBD 2, PSA 107.6, QED 0.44
Compound (SMILES): Cc1cc(F)c(C(=O)NC2CC2)cc1-c1cnn(-c2cnc3ccc(C4CN(C)C4)cn23)c1
InChIKey: GTZILQJNAOSGCQ-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43353
Assay: V9103X ADP-Glo Kinase Assay: In a 384-well plate, test compound diluted in assay buffer (1% DMSO final) is mixed with 8His-RIPK2 FL enzyme (final concentration of 8 nM). After 15 minutes of pre-incubation at RT, ATP dissolved in assay buffer is added (final concentration 5 μM). The mixture is incubated for 60 minutes at 37° C. in a humidified incubator. Then, ADP Glo Reagent is added, followed by a 40 minute incubation at rt. Finally, Kinase Detection Reagent is added and the entire mixture is incubated for 40 min at RT. The luminescence signal is measured with an Envision reader to determine the amount of ADP produced. Assay buffer: 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid), 0.1% BSA (bovine serum albumin), 10 mM MgCl2, 5 mM MnC12, 50 mM KCl, 0.01% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 10 μM Na3VO4, 1 mM DTT (dithiothreitol), pH 7.5 All plates contain wells with vehicle controls instead of compound (1% DMSO) as reference for the high signal (100% CTL (100% of control), high signal), and wells without enzyme as reference for low signal (0% CTL, low signal). The luminescent signal generated is proportional to the ADP produced and is correlated with enzyme activity. The analysis of the data is performed by the calculation of the percentage of ADP production in the presence of the test compound and RIPK2 as compared to the ADP production in the presence of RIPK2 plus 50 μMGefitinib.
Measurement: IC50 = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 444.51, ALogP 3.56, HBA 6, HBD 1, PSA 67.46, QED 0.51
Compound (SMILES): COCCn1cnc2c(Cl)c(C(=O)Nc3ccc(Cl)cc3)cnc21
InChIKey: GUASIVQSAOTUBB-UHFFFAOYSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Biological Evaluation: The assay was performed using H4 neuroglioma cells and using indirect immunofluorescence detection of the endogenous GR receptor. Fixative: 4% PFA. Permeabilization buffer: 0.3% Triton X-100 in PBS. Primary antibody: 1:200 in 5% BSA block. Secondary antibody: 1:800 in 5% BSA block. HCS Cell Mask: 1:6000 in PBS. DAPI: 100 ng/ml.
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 365.22, ALogP 3.64, HBA 5, HBD 1, PSA 69.04, QED 0.75
Compound (SMILES): CCOc1cc(-c2ccc(N3C[C@H](Oc4cnc(OC)c(F)c4)[C@@H](N)C3)nc2)c2c(C#N)cnn2c1
InChIKey: XMHPDPALNSGILW-VXKWHMMOSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 489.51, ALogP 2.8, HBA 10, HBD 1, PSA 123.82, QED 0.42
Compound (SMILES): Cc1c(-c2ccccc2)nc2ccc(Br)cc2c1C(=O)NC[C@@H](C(=O)O)c1ccc(F)cc1
InChIKey: SJKFCLBGZPLLJU-OAQYLSRUSA-N
Target: Prostaglandin F2-alpha receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43088
Assay: In Vitro Test of Inhibition of Human FP Receptor Activity: For the characterization of test substances in respect of FP antagonism, PGF2α-induced calcium flux in FP-expressing CHEM1 cells (Millipore, HTS093C) was used.3000 cells in 30 μl of full medium [DMEM F12, 10% FCS, 1.35 mM sodium pyruvate, 20 mM HEPES, 4 mM GlutaMAX, 2% sodium bicarbonate, 1% Pen/Strep, 1% 100× non-essential amino acids] are sown per well of a 384 multititre plate (from Greiner, TC plate, black with clear base) and incubated at 33° C., 5% CO2 for 24 hours. Prior to the measurement, the medium is replaced by 30 μl of Fluo-8 AM loading buffer [calcium-free Tyrode (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 4.8 mM NaHCO3, pH 7.4), 2 mM CaCl2, 6.3 mM Probenecid, 5 μM Fluo-8 AM, 0.0112% Pluronic®] and incubated at 37° C., 5% CO2 for 30 minutes. The test substance is prepared in DMSO in various concentrations as a dose-response curve (starting concentration 10 mM, dilution factor 3.16) and prediluted 1:50 with calcium-free Tyrode, 2 mM CaCl2, 0.002% SmartBlock (from CANDOR Bioscience GmbH). 10 μl of the prediluted substance solution are added to the Fluo-8-laden cells and incubated at 37° C., 5% CO2 for 10 minutes. The FP receptor is activated by adding 40 μl of 2 nM (final concentration) PGF2α in calcium-free Tyrode, 2 mM CaCl2, 0.002% SmartBlock, and the calcium flux is determined by measuring the fluorescence at ex. 470 nm/em. 525 nm in a fluorescence measuring instrument (FLIPR Tetra®, Molecular Devices) for 120 seconds.
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 507.36, ALogP 5.71, HBA 3, HBD 2, PSA 79.29, QED 0.35
Compound (SMILES): CS(=O)(=O)c1nc(-c2cc(N3CCOCC3)nc3c(-c4ccnn4C4CCCCO4)nccc23)cs1
InChIKey: QCZOVYBQLALFPG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 526.64, ALogP 3.56, HBA 11, HBD 0, PSA 112.33, QED 0.39
Compound (SMILES): Cc1c(-c2[nH]c3ccc(C4CCN(C(=O)N5CCN(C(C)C)CC5)CC4)cc3c2C(C)C)cn2ncnc2c1C
InChIKey: RPONUCVWIZRQNX-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 32.8 nM (pChEMBL 7.480000019073486)
Descriptors: MW 541.74, ALogP 5.94, HBA 5, HBD 1, PSA 72.77, QED 0.35
Compound (SMILES): NCC(O)C(=O)Nc1ccc(-c2cn[nH]c2)cc1
InChIKey: SWQBQAADGQUDLB-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Effect Assay: Table 2: Inhibitory effect of each compound synthesized above on Rho-associated protein kinase (ROCK) was tested.Method 1. 10 mM test compound was diluted to 1 mM with DMSO, and then further diluted to 300 nM. Netarsudil (AR-13324) is a commercial drug for decreasing intraocular pressure, and AR-13503 is an active metabolite of AR-13324. 2. The 300 nM test compound mentioned above was serially diluted to obtain test compounds with concentrations of 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM and 0.4 nM, respectively. 3. 1 μL of the serially diluted test compound mentioned above was added to 49 μL of the modified ROCK reaction buffer (0.05 M Trizma® hydrochloride buffer (pH 7.5) containing 0.1 M KCl, 0.01 M MgCl2, 0.1 mM EGTA and 2.25 μg/mL ROCK1) to obtain an experimental group sample. 1 μL of DMSO was taken to add to 49 μL of an adjusted ROCK reaction buffer to obtain a positive control group sample (maximum value=100%). 1 μL of DMSO was taken to add to 49 μL of a buffer (0.05 M Trizma® hydrochloride buffer (pH 7.5) containing 0.1 M KCl, 0.01 M MgCl2 and 0.1 mM EGTA) to obtain a vehicle control group sample (minimum value=0%). 4. 20 μL of the above prepared sample was added to a flat-bottomed 96-well plate, and 20 μL ROCK ATP buffer was added to each well. 5. The 96-well plate mentioned above was placed on an orbital shaker and reacted at room temperature for 90 minutes.6. Next, 40 μL of Kinase-Glo luminescent kinase assay solution (Promega, RV6712) was added to the 96-well plate mentioned above, and the 96-well plate was placed on an orbital shaker and reacted at room temperature for 10 minutes. 7. The luminescence value of each well of the 96-well plate was determined by SpectraMax M5 microplate reader, and the ROCK inhibition rate (%) was calculated by the following formula.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 246.27, ALogP 0.33, HBA 4, HBD 4, PSA 104.03, QED 0.62
Compound (SMILES): Cc1cc([C@H]2CN(C(=O)c3ccc(C(F)(F)F)c(Br)c3)CCO2)n2ncnc2n1
InChIKey: SKCMMPJYCHJZCN-OAHLLOKOSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Enzymatic Assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.PDE2
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 470.25, ALogP 3.43, HBA 6, HBD 0, PSA 72.62, QED 0.57
Compound (SMILES): CS(=O)(=O)Nc1cccc(C(Nc2ncc(-c3nnc(C(F)F)o3)cn2)c2ccc(F)cc2)c1
InChIKey: FAOKXAAIOIOTKM-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzymatic Activity Assay: All enzymatic reactions were conducted in duplicate at room temperature for 17 hours in a 50 μl mixture containing HDAC assay buffer (50 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, 5 μg BSA), an HDAC substrate, an HDAC enzyme, and a test compound. Compound dilution was prepared ten-fold higher than the final concentration of the compounds with 10% DMSO in HDAC assay buffer and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. After enzymatic reactions, 50 μl of HDAC Developer was added to each well and the plate was incubated at room temperature for an additional 20 minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 or Biotek Synergy microplate reader.The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity=(F−Fb)/(Ft−Fb), where F=the fluorescent intensity in the presence of the compound.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 490.47, ALogP 4.18, HBA 8, HBD 2, PSA 122.9, QED 0.38
Compound (SMILES): CCn1cc(C(O)c2cc(-c3ncnc4cc(N5CCOCC5)ccc34)c(F)cc2Cl)cn1
InChIKey: QQVCKAJSGSGZCZ-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: Biochemical Assay: The kinase assay was carried out in streptavidin-coated 348-well microtitre flashplates. To this end, 1.5 ug of DNA-PK/protein complex and 100 ng of biotinylated substrate, such as, for example, PESQEAFADLWKK-biotin-NH2 (biotin-DNA-PK peptide), were incubated for 90 min at room temperature in a total volume of 36.5 ul (34.25 mM HEPES/KOH; 7.85 mM Tris HCl; 68.5 mM KCl; 5 uM ATP; 6.85 mM MgCl2; 0.5 mM EDTA; 0.14 mM EGTA; 0.69 mM DTT; pH 7.4) with 500 ng of DNA from calf thymus, 0.1 uCi of 33P-ATP and 1.8% of DMSO per well with and without the test compound. The reaction was stopped using 50 ul/well of 200 mM EDTA. After incubation for a further 30 min at room temperature, the liquid was removed. Each well was washed three times with 100 ul of 0.9% saline solution. A nonspecific reaction (blank value) was determined using 10 uM of an innate kinase inhibitor. The radioactivity measurement was carried out using a TopCount. IC50 values were calculated in RS1.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 467.93, ALogP 4.22, HBA 7, HBD 1, PSA 76.3, QED 0.47
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)NCc2cccc(Cl)c2F)c2cc(C(=O)C[S+](C)[O-])ccc12
InChIKey: PSLKTDUJUWPRMK-PCGGDDIDSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Enzyme Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 578.04, ALogP 3.45, HBA 6, HBD 1, PSA 111.54, QED 0.31
Compound (SMILES): CC(C)(O)c1ccc2c(c1)[C@H]1O[C@@H]2c2ccc(C(=O)NCc3ccc(C(=N)N)cc3)cc21
InChIKey: IFLAJNOVITVTGC-PKTZIBPZSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Plasma Kallikrein Inhibition Assay: The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
Measurement: IC50 = 35.5 nM (pChEMBL 7.449999809265137)
Descriptors: MW 427.5, ALogP 3.65, HBA 4, HBD 4, PSA 108.43, QED 0.37
Compound (SMILES): CC(C)NC(=O)N1CCC(CC#N)(N2CC(CNC3CC3c3ccccc3)C2)CC1
InChIKey: GZYXUPOSMJNNNG-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 histone demethylase biochemical assay: LANCE LSD1/KDM1A demethylase assay10 μL 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 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 μM 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 μL 1× 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). Compounds having an IC50 of 1 μM or less were considered active.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 409.58, ALogP 2.93, HBA 4, HBD 2, PSA 71.4, QED 0.73
Compound (SMILES): CCNC(=O)c1cc2c(-c3cc(C4(O)CC=CC4)cnc3Oc3c(C)cccc3C)cn(C)c(=O)c2[nH]1
InChIKey: OEGYHIJISSQZMD-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus#6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe.The final concentration of 1× assay mixture contained 2% DMSO, 12 nM His tagged BRD4 (BDI_K57-E168) and 100 nM probe or 4 nM His tagged BRD4 (BDII_E352-M457) and 30 nM probe, and 1 nM Europium-conjugated anti-His-tag antibody, and compound concentrations in the range of: 49.02 μM-0.61 nM or 0.98 μM-0.15 nM.After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 215 nM (pChEMBL 6.670000076293945)
Descriptors: MW 498.58, ALogP 4.63, HBA 6, HBD 3, PSA 109.24, QED 0.34
Compound (SMILES): Cn1c(=O)n(CC2CC2)c2ccc(S(=O)(=O)NC3(C)CC3)cc21
InChIKey: QQRHOJQCOMIPFU-UHFFFAOYSA-N
Target: Poly(ADP-ribose) glycohydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: Q86W56
Assay: PARG Assay: PARG In vitro assays were conducted in a total volume of 15 ul in a standard 384 well format. 5 ul of Human Full Length PARG (Produced internally by Astra Zeneca), used at a final reaction concentration of 80 μM, was added to 5 ul of Ribosylated PARP substrate (also produced internally by Astra Zeneca) at final reaction concentration of 4.5 nM in assay buffer (50 mM Tris pH7.4, 0.1 mg/ml BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at room temperature for 10 minutes and then 5 ul detection reagent was added. Detection Reagent consists of 42 nM MAb Anti-6HIS XL665 (CisBio: 61 HISXLB) and 2.25 nM Streptavidin Europium Cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM respectively), in a detection buffer of 50 mM Tris pH7.4, BSA at 0.1 mg/ml and KF at 100 mM. Following incubation at room temperature for 60 minutes in the dark, TR-FRET signal was measured at Ex 340 and Em 665 and Em 620. A ratio was calculated as Em665/EM620×104 for each well and used to calculate percent inhibition for test compounds.
Measurement: EC50 = 4995 nM (pChEMBL 5.300000190734863)
Descriptors: MW 335.43, ALogP 1.58, HBA 5, HBD 1, PSA 73.1, QED 0.9
Compound (SMILES): C[C@]1(c2ccc(F)cc2)CN(C(=O)NCc2ccccc2)C[C@H]1c1ccc(C(F)(C(F)(F)F)C(F)(F)F)cc1
InChIKey: GLVVPWJDHUSGLA-UKILVPOCSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: SPA Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 μg/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat # G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined.
Measurement: IC50 = 133 nM (pChEMBL 6.880000114440918)
Descriptors: MW 556.5, ALogP 7.38, HBA 1, HBD 1, PSA 32.34, QED 0.33
Compound (SMILES): CC(C)N1CCN(CC(C)(O)Cn2c3ccc(F)cc3c3cc(F)ccc32)S(=O)(=O)C1
InChIKey: RBAOHULVMWLPHD-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 = 3092 nM (pChEMBL 5.510000228881836)
Descriptors: MW 451.54, ALogP 3.14, HBA 5, HBD 1, PSA 65.78, QED 0.65
Compound (SMILES): Cc1cc(Nc2nc(N[C@H]3CC[C@H](N4CCCCC4)CC3)c3sccc3n2)sn1
InChIKey: NUANULAIWXHYDG-WKILWMFISA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Activity Inhibition Assay: IRAK4 kinase (purchased from Life Technologies, Cat. No.: PR5612U) was diluted to 2 folds of the final concentration (the final concentration is 0.76 ng/μL) with reaction buffer (40 mM Tris-HCl, pH 7.5; 20 mM MgCl2; 0.1 mg/mL BSA; 1 mM DTT), and added to a 384-well plate at 5 μL/well. The test drugs were 10-fold serial diluted from 10 μM to set six concentration points and added to the test wells of the 384-well plate at 2.5 μL/well. After incubation for 10 min at 25° C., 10 μM of ATP and 0.1 μg/L of an enzyme reaction substrate, myelin basic protein MBP (purchased from SignalChem, Cat. No.: M42-51N) were added at 2.5 L/well, and reacted at 25° C. for 60 minutes. After the reaction was completed, the kinase activity assay was performed using the ADP-Glo kinase assay kit (purchased from Promega, Cat. No.: V9102) according to the manufacture's instruction, i.e., 10 μL of ADP-Glo reaction reagent was first added and reacted at 25° C. for 40 minutes, 10 μL of the solution to be tested was taken and mixed with 10 μL of ADP-Glo detection reagent, and reacted at 25° C. for 30 minutes.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 428.63, ALogP 5.41, HBA 8, HBD 2, PSA 65.97, QED 0.57
Compound (SMILES): COc1cnc2c(C(=O)CN3CCCCC3)c(C)n(-c3ccc(C#N)cc3)c2c1
InChIKey: ODLBQQUCFVDONT-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: P54578
Assay: USP14 Inhibition Assay: Using previously described methodology [B. H. Lee et al. Nature 2010, 467 (9), 179, the contents of which are expressly incorporated by reference herein], select compounds described herein were found to inhibit USP14.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 388.47, ALogP 3.88, HBA 6, HBD 0, PSA 71.15, QED 0.62