lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C1Nc2cccc(n2)-c2nncn2CCCCOc2cc(F)c(F)cc21
InChIKey: HOIATHARZXBNTP-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Biochemical Assay: The binding of compounds to ASK1 were determined using DiscoverX's proprietary technology. KINOMEscan is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. The assay is performed by combining three components: DNA-tagged ASK1 kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for ASK1 kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce nonspecific binding. Binding reactions were assembled by combining DNA-tagged ASK1 kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). 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. The compounds were then diluted directly into the assays such that the final concentration of DMSO was 0.9%. All reactions were performed in polypropylene 384-well plate. Each fraction had a final volume of 0.02 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.504 non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR. Most Kds were determined using a compound top concentration=30,000 nM. If the initial Kd determined was <0.5 nM (the lowest concentration tested), the measurement was repeated with a serial dilution starting at a lower top concentration.
Measurement: Kd = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 371.35, ALogP 3.04, HBA 6, HBD 1, PSA 81.93, QED 0.66
Compound (SMILES): O=C(c1cncc(-c2ccnc(Nc3ccnc(C4CC4)n3)c2)c1)N1CC(F)(F)C1
InChIKey: UPSJPZHLSAFVBC-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase catalytic subunit type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NEB9
Assay: VPS34 Enzyme Assays: 100 nL compounds in DMSO are added to wells of a 384 well microtitre plate (Greiner 780076). At room temperature: 5 ul VPS34 reaction buffer (Invitrogen Assay Buffer Q (diluted 1 in 5 with nanopure water) plus 2 mM DTT and 2 mM MnCl2) containing ATP (20 uM, Promega) and 200 uM PI-PS substrate (Invitrogen PV5122) is added followed immediately by 5 ul VPS34 reaction buffer (as above) containing VPS34 (5 nM, Millennium Protein Sciences Group) and the mixture is incubated with shaking at room temperature for 1 hour. Then 5 ul VPS34 stop-detect mix (as per Invitrogen Adapta Assay kit (PV5009) instructions (contains kinase quench buffer, TR-FRET buffer, Adapta Eu anti-ADP antibody and Alexa Fluor 647 ADP tracer)) is added to quench the reaction. The plates are then incubated for 30 minutes at room temperature with shaking and then read on a BMG PheraStar Plus reader.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 408.41, ALogP 3.65, HBA 6, HBD 1, PSA 83.9, QED 0.69
Compound (SMILES): CC1(CN)CCN(c2cnc3c(N4CCCc5cc(C#N)ccc54)n[nH]c3n2)CC1
InChIKey: UMAKLFRWQJMPPB-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.The inhibition of SHP2 by compounds of the disclosure (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.25 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat #D6567, 100 μM final) was added to the reaction and the conversion of DiFMUP to 6,8-difluoro-7-hydroxyl-4-methyl coumarin (DiFMU) was monitored continuously for 10 minutes with excitation at 355 nm and emission at 460 nm using a microplate reader (PolarStar, BMG).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 402.51, ALogP 2.87, HBA 7, HBD 2, PSA 110.75, QED 0.69
Compound (SMILES): CC(=O)N1CCC(c2nc3cnc4[nH]ccc4c3n2[C@H]2CC[C@H](CC#N)CC2)CC1
InChIKey: KYCGHXSOWUNZRN-SAABIXHNSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzymatic Inhibition Assay: 11-point dosing series were made for each compound by serially diluting 1:3 or 1:4 in DMSO, with point 12 being a DMSO control. From the serial dilution plates, sample was transferred to a 384 wells assay plate (#781280, Greiner, Monroe, N.C.) using Labcyte Echo (Sunnyvale, Calif.), or Biosero ATS (San Diego, Calif.). The compounds were tested in duplicate. Column 12 was used for positive controls, and column 24 contained negative controls with no enzyme added. A compound from our internal collection, with inhibitory activity for JAK isoforms, was used as a reference compound. The final concentration of DMSO was ≤0.25% in a 20 μL reaction. Assay conditions for each of the proteins are summarized in Table 3. The enzyme reaction was initiated by the addition of 10 μL of 2× enzyme and ATP mixture to 10 μL of 2× substrate solution prepared in reaction buffer (50 mM MOPS pH 7.5, 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20). The Tyk2 enzyme was pre-incubated with 2 mM ATP for 30 min prior to the reaction initiation. Immediately after the addition of the enzyme to the reaction mixture, the plate was centrifuged at 1000 rpm for 1 minute and incubated at 25° C. for 45 minutes for JAK 3 and 90 minutes for JAK1, JAK2 and Tyk2. The reaction was quenched by the addition of 20 μL of 0.5% TFA containing 0.15 μM of internal standard peptide using Multidrop Combi reagent dispenser (Thermo Scientific, Waltham, Mass.). Several wells in column 24 were typically used for the product standard curve. After the quench, the assay plate was centrifuged at 3000 rpm for 3 minutes and sealed with pierceable aluminum foil (Cat #06644-001, Agilent) using a PlateLoc (Agilent Technologies, Santa Clara, Calif.). The plates then were transferred on to the RapidFire for the MS analysis. Compound inhibition was assessed by a decrease of the phosphorylated product levels in sample wells compared to the non-inhibited enzyme reaction. The assay conditions for the above assays are shown in Table 3 and the results of Ex. 1-209 as tested in these assays are shown in Table 4.TABLE 3Assay conditions for JAK family enzyme assays* [p], [ATP], [Substrate], [IS],Enzyme nM μM μM nMJAK1-JH1JH2 8.0 12.5 200 100JAK2-JH1JH2 7.0 or 3.6 30 40 100JAK3-JH1JH2 2.0 150 40 100Tyk2-JH1JH2   25 or 14.7 50 200 100*Reaction buffer: 50 mM MOPS, pH 7.5 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.002% Tween-20; IS stands for internal standard peptide; p stands for phospho-peptide (product); Substrate stands for peptide.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 404.52, ALogP 4.28, HBA 5, HBD 1, PSA 90.6, QED 0.71
Compound (SMILES): CN1CCC[C@H]1COC(=O)Nc1ccccc1-c1cc(F)c(F)cc1F
InChIKey: FVBUJFDEFSDKTJ-LBPRGKRZSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: Binding Assay: Cell membrane proteins (Perkin Elmer) wherein human muscarinic M3 receptor was overexpressed, [3H]-methyl scopolamine and test compounds in various concentration were cultured in 0.2 ml of Tris-HCl buffer at 25° C. for 120 minutes. The same was filtered under suction through glass fiber filter (Whatman GF/B), and then the filter was washed 5 times with 1 ml of Tris-HCl buffer. The radioactivity of [3H]-methyl scopolamine adsorbed on the filter was measured by a liquid scintillation counter. Non-specific binding was evaluated under existence of 5 μM of atropine. Affinity of the compound of the present invention to muscarinic M3 receptor was calculated as the dissociation constant (Ki), which can be calculated from concentration (IC50) of test compounds inhibiting 50% of binding of [3H]-methyl scopolamine (i.e. labeled ligand) according to Cheng and Prusoff [Cheng and Prusoff, Biochem. Pharmacol., 22, 3099, 1973]. In following Table, compounds having stronger binding affinity to human muscarinic M3 receptor have lower dissociation constant (Ki).
Measurement: Ki = 16.1 nM (pChEMBL 7.789999961853027)
Descriptors: MW 364.37, ALogP 4.41, HBA 3, HBD 1, PSA 41.57, QED 0.82
Compound (SMILES): Cc1ccc(NC(=O)c2ccnc(N(C)C)c2)cc1-c1cc(N2CCOCC2)c(=O)n(C)c1
InChIKey: JQJFDHIARPZGOT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 447.54, ALogP 2.91, HBA 7, HBD 1, PSA 79.7, QED 0.65
Compound (SMILES): C[C@@H](COCC(=O)OC(C)(C)C)N[C@H]1CC[C@H](Nc2cc(-c3cccc(NCC4(C#N)CCOCC4)n3)c(Cl)cn2)CC1
InChIKey: XNCHAOOIMXRRBN-HJOGWXRNSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: CDK9 Biochemical Assay: To each well of a 96-well plate was added 5× kinase assay buffer with 10 mM DTT (6 μL), 500 μM ATP (1 μL), 5×CDK substrate (10 μL), and water (8 μL). 5 μL of compound were added to the test and positive control groups, while 5 μL of solvent was added to the blank groups. 100 ng of CDK9/CyclinT in 20 μL water was added to the test and positive control groups, while 20 μL of 1× kinase assay buffer was added to the blank groups. The reaction mixtures were incubated at 30° C. for 45 minutes, and 50 μL of Kinase-Glo® Max was added to each well and the plates were shielded from light and incubated for 15 minutes at room temperature. Luminescence was measured on a microplate reader and IC50values were calculated using Prism 9 software.
Measurement: IC50 = 3.75 nM (pChEMBL 8.430000305175781)
Descriptors: MW 613.2, ALogP 5.59, HBA 10, HBD 3, PSA 130.42, QED 0.26
Compound (SMILES): Cc1cccc(C)c1OCCCNCc1cccc(-c2cncnc2)c1
InChIKey: UUJCWXBORMXXGI-UHFFFAOYSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Automated patch clamp (Tonic Block): Test Protocol for Determining the Potency of Compounds on Nav1.2 and Nav1.6 Subtypes of Voltage-Gated Sodium Ion Channels Expressed in Mammalian CellsCell culture procedures: Chinese hamster ovary (CHO) cells were stably transfected with the cDNA sequence of either the human SCN2A or SCN8A genes to create hNav1.2 or hNav1.6 cells respectively. The cell lines were cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum and antibiotics in a tissue incubator at 37° C. under a humidified air/CO2 mixture (95/5 v/v). On the day of the experiment, cells were washed twice with Hank's balanced salt solution, treated with trypsin, and resuspended in fresh culture media at a density of 4−6×106 cells in 20 ml. Immediately before placing in the IonWorks Quattro instrument, cells were washed in Hank's balanced salt solution to remove culture medium and re-suspended in 3 ml of Hank's balanced salt solution. Automated patch clamp recordings of cells using the IonWorks Quattro Instrument: The assay used a 384 microwell plate, which enabled 20 compounds to be studied at four different concentrations in quadruplicate as well as allowing a vehicle control group and a positive control group using mexiletine at 8 different concentrations. Cells were added to the wells of the Population Patch Clamp planar electrode using a 48-channel pipettor in a volume of 6 μL, per well. After 5 minutes incubation at ambient temperature with compounds or vehicle, membrane currents were recorded using the patch clamp amplifier. Block of hNav1.2 or hNav1.6 channels was measured using a stimulus voltage pattern as shown in FIG. 1. The pulse pattern was repeated twice, before and after compound addition and peak current amplitudes were measured at TP1 to measure Tonic Block.Data analysis: Data acquisition and analyses were carried out using the IonWorks Quattro system operation software which uses an algorithm to correct data for leak currents. Concentration-response curves were fitted by non-linear least squares regression allowing calculation of an IC50 value (μM), being the concentration of the compound producing half-maximal inhibition.
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 347.46, ALogP 4.32, HBA 4, HBD 1, PSA 47.04, QED 0.62
Compound (SMILES): COc1ccc(NC(=O)CN2C[C@]3(CC[C@](c4ccccc4)(N(C)C)CC3)N(CC3CCC3)C2=O)nn1
InChIKey: OQYUCZSHDCGLGJ-HNRBIFIRSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Human Nociceptin/Orphanin FQ Peptide (hNOP) Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences, Inc. Boston, Mass., USA), and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration, which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd., Buckinghamshire, UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences, Inc. Boston, Mass., USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]nociceptin-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation, (Cheng and Prusoff, 1973).
Measurement: Ki = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 506.65, ALogP 3.73, HBA 6, HBD 1, PSA 90.9, QED 0.59
Compound (SMILES): COc1cnccc1-c1cn2c(-c3cc(S(C)(=O)=O)ccc3C)cnc2c(N)n1
InChIKey: METOIGQVZGQJNY-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: PI3K-γ:[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).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 409.47, ALogP 2.76, HBA 8, HBD 1, PSA 112.47, QED 0.55
Compound (SMILES): O=C(CN1Cc2ccc(-c3nc(N[C@H]4CCOC[C@@H]4O)ncc3Cl)cc2C1=O)N1CCc2ccccc2C1
InChIKey: FOAWZEYVWILLIG-ZEQRLZLVSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 87 nM (pChEMBL 7.059999942779541)
Descriptors: MW 534.02, ALogP 2.9, HBA 7, HBD 2, PSA 107.89, QED 0.52
Compound (SMILES): CCOc1ccc(S(O)(O)CC)cc1-c1cc(N(C)C)c(=O)n(C)c1
InChIKey: TVVWMCXNTSIYMT-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BRD4 was cloned, expressed and purified to homogeneity. BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 366.48, ALogP 3.65, HBA 6, HBD 2, PSA 74.93, QED 0.82
Compound (SMILES): CCOC(=O)C(C)(C)c1ccc(Nc2nc(-c3c(F)cccc3F)nc3c2C(=O)NC3)cc1
InChIKey: BYKWEBVJSJFCNC-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 & JAK2 Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 452.46, ALogP 4.25, HBA 6, HBD 2, PSA 93.21, QED 0.54
Compound (SMILES): CO[C@@]1(Cc2ccccc2)C[C@H]2CN(CC(=O)c3ccc(O)c(F)n3)C[C@H]2C1
InChIKey: HZOPFPCDLWZHNK-FAQVEIGNSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: FDSS Assay: To each well of the plate, 10 μL test compound, control (MK801) or HHnoCa buffer was added to a final concentration of 10 μM with a final concentration of DMSO of 0.1%. Following 10 minutes pre-incubation in the dark, plates are loaded onto the Hamamatsu FDSS 6000. After collecting baseline fluorescence images, 3 μM glutamate, 3 glycine, and 1 mM Ca2+ in HHnoCa buffer is added to each well, and Ca2+ is recorded for 3 minutes. Data were processed by computing ratio of fluorescence at the end of data collection to baseline fluorescence to assess degree of Ca′ influx inhibition relative to that observed in MK801.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 384.45, ALogP 3.08, HBA 5, HBD 1, PSA 62.66, QED 0.61
Compound (SMILES): O=C(Nc1ccsc1C(F)(F)F)c1ccc2cnc(N3C[C@@H]4CC[C@H]3CN4)nn12
InChIKey: BELKBRCCXCFUPE-QWRGUYRKSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 2271 nM (pChEMBL 5.639999866485596)
Descriptors: MW 422.44, ALogP 3, HBA 7, HBD 2, PSA 74.56, QED 0.68
Compound (SMILES): CN1CCC(F)(CNc2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(CC5=C(C67CC(C(F)F)(C6)C7)CC(C)(C)CC5)CC4)cc3Oc3cnc4[nH]ccc4c3)cc2[N+](=O)[O-])CC1
InChIKey: AVHUOMOOFURFSR-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Binding Assay: Table 1: Binding to Bcl-2 proteins Bcl-2, and Bcl-XL was assessed using the Bcl2scan platform: T7 phage strains displaying BCL2 proteins were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection=0.4) and incubated with shaking at 32° C. until lysis (90-150 minutes). The lysates were centrifuged (5,000×g) and filtered (0.2 μm) to remove cell debris. Streptavidin-coated magnetic beads were treated with biotinylated BIM peptide ligand for 30 minutes at room temperature to generate affinity resins for BCL2 assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining BCL2 proteins, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with one DMSO control point. All compounds for Kd measurements were distributed by acoustic transfer in 100% DMSO. The compounds were then diluted directly into the assays such that the final concentration of DMSO was 0.9%. All reactions performed in polypropylene 384-well plates. Each was a final volume of 0.02 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (lx PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (lx PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes.
Measurement: Kd = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 905.06, ALogP 8.29, HBA 11, HBD 3, PSA 166.04, QED 0.06
Compound (SMILES): CN1CCNC(C)(c2ccc(Nc3cc(-c4c(F)cccc4F)nc4c3C(=O)NC4)nc2)C1=O
InChIKey: OTZKIUWEGYUJHT-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 & JAK2 Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 464.48, ALogP 2.69, HBA 6, HBD 3, PSA 99.25, QED 0.55
Compound (SMILES): CS(=O)(=O)c1ccc(COC(c2ccc(C3(S(=O)(=O)c4ccc(F)cc4)CCCC3)cc2)(C(F)(F)F)C(F)(F)F)c(Cl)c1
InChIKey: OECJZOZUXBBUGL-UHFFFAOYSA-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 chromotography. 15 ug/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).
Measurement: IC50 = 192 nM (pChEMBL 6.71999979019165)
Descriptors: MW 673.07, ALogP 7.66, HBA 5, HBD 0, PSA 77.51, QED 0.18
Compound (SMILES): CN(C)C(=O)c1cccc(-c2c(Cl)cc(Nc3n[nH]c(N)n3)cc2Cl)c1
InChIKey: UMHCKZCXFYTAAQ-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Pseudotyped HCV particle (HCVpp) reporter assay: Plasmids expressing HCV E1 and E2 envelope proteins of GT1a H77 strain (Proc Natl Acad Sci USA 1997 94:8738-43) or GT1b Con1 strain (Science 1999 285:110-3) were constructed by cloning the nucleic acids encoding the last 60 amino acids of HCV core protein and all of the HCV E1 and E2 proteins into pcDNA3.1(+) vector. Plasmid pVSV-G expressing the glycoprotein G of the vesicular stomatitis virus (VSV G) is from Clontech (cat #631530). The HIV packaging construct expressing the firefly luciferase reporter gene was modified based on the envelope defective pNL.4.3.Luc-Râ¿¿.Eâ¿¿ vector (Virology 1995 206:935-44) by further deleting part of the HIV envelope protein.
Measurement: EC50 = 107 nM (pChEMBL 6.96999979019165)
Descriptors: MW 391.26, ALogP 3.81, HBA 5, HBD 3, PSA 99.93, QED 0.63
Compound (SMILES): Cc1nn(Cc2ccncc2)c2ncc(C(=O)Nc3ccc(C(C)C)cc3)c(Cl)c12
InChIKey: WJJSEJWNCRIVFI-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 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 419.92, ALogP 5.21, HBA 5, HBD 1, PSA 72.7, QED 0.48
Compound (SMILES): C/C(=C\[C@H]1NCCC[C@@H]1O)Cn1cnc2c(C)c(Br)ccc21
InChIKey: PVJVTEMHISZYOW-BGVIPNPZSA-N
Target: Ribose-phosphate pyrophosphokinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P60891
Assay: PRS Enzyme Activity Inhibition Experiment: In order to confirm the biological activities of the compounds prepared in Examples, % inhibition or IC50 values of PRS enzyme (phosphoribosylpyrophosphate synthetase enzyme) activities were calculated.Specifically, the portion corresponding to PRS in cDNA of EPRS was subcloned, and the obtained high-purity PRS protein was purified and used in the experiment. The compounds (1 μM) prepared in Examples were added into the reaction buffer (20 mM KPO4 (pH 7.4), 6 mM MgAc, 5 mM ATP, 400 mg/mL tRNA, 0.5 mM DTT, 20 mCi[3H]proline (1 mCi/mL)) and allowed to react at 37° C. for 5 to 10 minutes. The reaction was terminated with 3M paper that was in advance dried by addition of 5% TCA. The radioactivity was measured using a liquid scintillation counter.% Inhibition and IC50 values of the respective compounds were calculated and analyzed using Microsoft Excel or Sigma Plot 8.0.
Measurement: IC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 364.29, ALogP 3.17, HBA 4, HBD 2, PSA 50.08, QED 0.82
Compound (SMILES): COc1ncnc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](C)c2cnc(C)cn2)nnc1C1CCC1
InChIKey: YUSWNFFDFVVUSZ-KBPBESRZSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager (PerkinElmer, Inc.). All the results presented are means of several independent experiments and analyzed by non-linear regression methods using the commercially available program Prism (GraphPad, San Diego, Calif.) providing the EC50 values.
Measurement: EC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 488.57, ALogP 2.37, HBA 11, HBD 1, PSA 146.9, QED 0.48
Compound (SMILES): COc1cc2c(cc1OCCCF)CCN1C[C@@H](CC(C)(C)C)N(C)CC21
InChIKey: ZBADGAJHEJIBOU-DUSLRRAJSA-N
Target: Synaptic vesicular amine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q05940
Assay: Inhibitory Activity Assay: In a total volume of 0.15 mL in low-binding 96-well plates (Corning #3605), twelve concentrations of test compound were competed against 10 nM 3H-dihydrotetrabenezine (American Radiolabeled Chemicals, Kd 2.6 nM) on rat forebrain homogenate (100 μg membrane protein per well) or human platelet homogenate (15 μg membrane protein per well) in VMAT2 binding buffer (Dulbecco's phosphate buffered saline, 1 mM EDTA, pH 7.4). Following incubation at 25° C. for 90 minutes, bound radioligand was collected by rapid filtration onto GF/B glass fiber filters using a Unifilter-96 Harvester (PerkinElmer). Filter plates were pre-treated with 0.1% polyethylenimine and allowed to dry overnight, and following harvesting the filter plates were washed with 800 μl VMAT2 binding buffer. Bound radioligand was quantified by scintillation counting using a Topcount NXT (PerkinElmer).
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 378.53, ALogP 4.08, HBA 4, HBD 0, PSA 24.94, QED 0.7
Compound (SMILES): CC(C)(C)[C@H](c1cc(-c2cc(F)ccc2F)cn1Cc1ccccc1)N(CC[C@H](N)C(=O)N[C@H](CCC(=O)O)C(=O)O)C(=O)CO
InChIKey: YNAWYQUAVSBSDH-MWSYKCOMSA-N
Target: Kinesin-like protein KIF11 (SINGLE PROTEIN), Homo sapiens
UniProt: P52732
Assay: Inhibition Assay: The motor domain of the human kinesin spindle protein KSP/Eg5 (tebu-bio/Cytoskeleton Inc, No. 027EG01-XL) was incubated in a concentration of 10 nM with microtubuli (bovine or porcine, tebu-bio/Cytoskeleton Inc) stabilized with 50 μg/ml taxol (Sigma No. T7191-5MG) for 5 min at RT in 15 mM PIPES, pH 6.8 (5 mM MgCl2 and 10 mM DTT, Sigma). The freshly prepared mixture was aliquoted into a 384 MTP (Greiner bio-one REF 781096). The inhibitors to be examined at concentrations of 1.0×10-6 M to 1.0×10-13 M and ATP (final concentration 500 μM, Sigma) were then added. Incubation was at RT for 2 h. ATPase activity was detected by detecting the inorganic phosphate formed using malachite green (Biomol). After addition of the reagent, the assay was incubated at RT for 50 min prior to detection of the absorption at a wavelength of 620 nm. The positive controls used were monastrol (Sigma, M8515-1 mg) and ispinesib (AdooQ Bioscience A10486). The individual data of the dose-activity curve are eight-fold determinations.
Measurement: IC50 = 2.91 nM (pChEMBL 8.539999961853027)
Descriptors: MW 642.7, ALogP 3.54, HBA 7, HBD 5, PSA 175.19, QED 0.17
Compound (SMILES): Cc1cc(-c2cc(NS(C)(=O)=O)c3nc(C(F)(F)F)n(CC4CC4)c3c2)cn(C)c1=O
InChIKey: ZKFVJZHIQVBDKB-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BREM was cloned, expressed and purified to homogeneity. Filipakopoulos et al., 468 Nature 1067 (2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotinlabeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at RT, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 454.47, ALogP 3.51, HBA 6, HBD 1, PSA 85.99, QED 0.64
Compound (SMILES): Cc1cnc([C@H]2CC(Br)=NO2)cc1Nc1cccc(C(F)(F)F)c1
InChIKey: SJVNPAVEMZJBKB-CQSZACIVSA-N
Target: Transcriptional enhancer factor TEF-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28347
Assay: TEAD Inhibition Assay: TEAD inhibition can be assayed using Hippo Pathway TEAD ReporterMCF7 Cell Line (BPS Bioscience, Catalog #: 60618).
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 400.2, ALogP 5.32, HBA 4, HBD 1, PSA 46.51, QED 0.76
Compound (SMILES): CC(N=[N+]=[N-])c1ccc(C(F)(F)F)cc1
InChIKey: LLNUOERZVCLBMV-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Fluorescence Polarization (FP): In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.). Cells were harvested 48 hours post-transfection and lysed by sonication (setting 3, 10×5 sec pulses) in a buffer containing 20 mM HEPES pH 7.4, 1 mM EDTA and Complete Protease Inhibitor Cocktail Tablets (Roche, Indianapolis, Ind.). Lysate was collected by centrifugation at 75,000×g for 20 minutes at 4° C. and supernatant utilized for evaluation of PDE2 activity. The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to examine the effects of phosphodiesterase inhibitors (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition is determined using a known PDE2 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described below, such as Bay 60-7550 (Ki-0.2 nM) at 1 μM concentration for 100% inhibition. Bay 60-7550 was obtained from Axxora via Fisher Scientific (cat# ALX-270-421-M025/cat# NC9314773). Put another way, any compound with Ki of 0.2 to about 2 nM could be used at 1 to 10 μM. 0% of inhibition is determined by using DMSO (1% final concentrations).
Measurement: Ki = 8.4 nM (pChEMBL 8.079999923706055)
Descriptors: MW 215.18, ALogP 4.08, HBA 1, HBD 0, PSA 48.76, QED 0.41
Compound (SMILES): CN(C1COC2(CCN(C(=O)OC(C(F)(F)F)C(F)(F)F)CC2)C1)S(=O)(=O)CC1CCC1
InChIKey: WGPUFTSDDKZSSV-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: MAGL Enzymatic Assay (0 minute): Assessment of MAGL inhibition utilizes human recombinant Monoacylglycerol Lipase and the fluorogenic substrate 7-hydroxycoumarinyl arachidonate (7-HCA, Biomol ST-502). 400 nL of a test compound at decreasing concentration (ranging from 150 μM down to 1.5 nM) was spotted into a 384-well back plate (PerkinElmer, 6007279) using a Labcyte Echo, followed by addition of 10 μL of MAGL enzyme in assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100 and 25% glycerin). An equal volume of 7-HCA in assay buffer with 10% DMSO was added either immediately (T=0 min) or after a 30 minute incubation (T=30 min) to initiate the reaction. The final concentration of MAGL enzyme was 88 μM and 7-HCA substrate was 5 μM. After these dilutions, the final concentration of the test compound ranged from 3 μM to 0.03 nM. The reaction was allowed to progress for 60 minutes, after which the plate was read at an Ex/Em of 340/465. Percent inhibitions were calculated based on control wells containing no compound (0% inhibition) and a control compound (e.g., a MAGL inhibitor whose activity is known or was previously reported in the literature, such as one with about 100% inhibition). IC50 values were generated based on a four parameter fit model using ABASE software from IDBS. See e.g., Wang, Y. et al., A Fluorescence-Based Assay for Monoacylglycerol Lipase Compatible with Inhibitor Screening, Assay and Drug Development Technologies, 2008, Vol. 6 (3) pp 387-393 (reporting an assay for measuring MAGL activity).To measure MAGL inactivation, the same protocol for the (T=0 min) MAGL inhibition IC50 assay was performed with data collected every minute to acquire enzyme progress curves at decreasing concentrations of compound. Kobs values were calculated from this data and kinact/Kl ratios were determined from a plot of Kobs values vs. compound concentrations.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 496.47, ALogP 3.3, HBA 5, HBD 0, PSA 76.15, QED 0.55
Compound (SMILES): COc1cc(-c2cn(C)c(=O)c3cnccc23)cc(OC)c1CN1CCC(OC2CCN(C(=O)CCOCCOc3ccc4c(c3)C(=O)N(C3CCC(=O)NC3=O)C4=O)C2)CC1
InChIKey: KVUAMLFHOOXWSA-UHFFFAOYSA-N
Target: Bromodomain-containing protein 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H8M2
Assay: Bromodomain TR-FRET Competition Binding Assay: A mixture of biotinylated-ligand and SureLight Allophycocyanin-Streptavidin (APC-SA, PerkinElmer AD0201) in 50 mM HEPES (pH 7.4), 50 mM NaCl, 1 mM TCEP (pH 7), 0.01% (v/v) Tween-20, 0.01% (w/v) bovine serum albumin was added to a white 384-well PerkinElmer Proxiplate Plus plate. DMSO or 3-fold serially diluted compounds were then added to the Proxiplate followed by addition of Flag-BRD9. After a 10-minute incubation at room temperature, Eu-W1024 anti-FLAG (PerkinElmer, AD0273) was added. The final reaction miture that contained 3.75 nM biotinylated ligand, 3 nM Flag-BRD9, 7.5 nM SureLight Allophycocyanin-Streptavidin, and 0.2 nM Eu-W1024 anti-FLAG was incubated at room temperature for 90 minutes.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 850.93, ALogP 3.09, HBA 14, HBD 1, PSA 188.14, QED 0.14
Compound (SMILES): Cc1ncc(-c2cnc(NCc3cccc4c3CCO4)n3cnnc23)cn1
InChIKey: HYWSXOLNEXHKPD-UHFFFAOYSA-N
Target: Polycomb protein EED (SINGLE PROTEIN), Homo sapiens
UniProt: O75530
Assay: EED-H3K27Me3 Peptide Competition Binding Assay by AlphaScreen: To assess the compounds potency in the EED-H3K27Me3 competition binding assay, compounds were serially diluted 3-fold in DMSO to obtain a total of twelve concentrations. Then compounds at each concentration (75 nL of each) were transferred by Mosquito into a 384-well Perkin Elmer ProxiPlate 384 plus plates. 8 uL of solutions containing 30 nM EED (1-441)-His protein and 15 nM biotin-H3K27Me3 (19-33) peptide in the buffer (25 mM HEPES, pH 8, 0.02% Tween-20, 0.5% BSA) were added to the wells and then incubated with compound for 20 min. AlphaScreen detection beads mix was prepared immediately before use by mixing nickel chelate acceptor beads and streptavidin donor beads in a 1:1 ratio (Perkin Elmer, Product No. 6760619C/M/R) into the buffer described above. Then 4 μL of detection beads mix was added to the plate and incubate in the dark at the rt for 1 h. The final concentration of donor and acceptor beads was 10 μg/mL for each. Plates were read on EnVision (PerkinElmer) using the AlphaScreen setting adapted for optimal signal detection with a 615 nm filter, after sample excitation at 680 nm. The emission signal at 615 nm was used to quantify compounds inhibition. AlphaScreen signals were normalized based on the reading coming from the positive (maximum signal control) and negative controls (minimum signal control) to give percentage of activities left. The data were then fit to a dose response equation using the program Helios (Novartis) to get the IC50 values. Helios is a Novartis in-house assay data analysis software using the methods described by Normolle, D. P., Statistics in Medicine, 12:2025-2042 (1993); Formenko, I. et al, Computer Methods and Programs in Biomedicine, 82, 31-37 (2006); Sebaugh, J. L., Pharmaceutical Statistics, 10:128-134 (2011); Kelly, C. et al., Biometrics, 46(4):1071-1085 (1990); and Kahm, M. et al., Journal of Statistical Software, 33(7): (2010) (grofit: Fitting Biological Growth Curves with R, pages 1-21, available at http://www.jstatsoft.org/).Each compound was counterscreened to determine if it interfered with the AlphaScreen beads. Compounds were diluted as described in the preceding section, and the assay was performed by adding 12 μL of 10 nM biotin-miniPEG-His6 peptide in the above buffer and incubating for 20 min at rt prior to addition of the beads to 10 μg/mL each. The plates were then incubated for 1 h at rt in dark before being read on EnVison.
Measurement: IC50 = 19.5 nM (pChEMBL 7.710000038146973)
Descriptors: MW 359.39, ALogP 2.44, HBA 8, HBD 1, PSA 90.12, QED 0.6