lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=CC(=O)N(CC(C)C)c1cccc(-c2ccc(C(N)=O)c3[nH]ccc23)c1
InChIKey: BJMBVVWJXLIBMA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) : The in-house BTK corresponds to recombinant human catalytic domain (aa 393-659), which was expressed in SF9 cells with an N-terminal his tag and purified by immobilized metal affinity chromatography. BTK was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 0.2 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4 and 0.001 mM ATP. After about 60 mM incubation at room temperature, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 mM at room temperature, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 361.45, ALogP 4.11, HBA 2, HBD 2, PSA 79.19, QED 0.65
Compound (SMILES): CC(C)c1c(-c2cn(CCS(C)(=O)=O)c3ncccc23)[nH]c2ccc(C3CCC(NCC(=O)N(C)C)CC3)nc12
InChIKey: BDVSGLZUPXGFCO-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: 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 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 564.76, ALogP 4.45, HBA 7, HBD 2, PSA 112.98, QED 0.31
Compound (SMILES): Cn1nnn(-c2cc(Nc3ncc(C#N)c(N[C@@H]4C[C@@H]5CCCN5C(C)(C)C4)n3)c(F)cc2OCC(C)(C)O)c1=O
InChIKey: PUZBKIAHRGEHKU-MSOLQXFVSA-N
Target: Interleukin-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P60568
Assay: IL-2 ELISA Assay: Primary T cell stimulation and IL2 ELISA: Human primary T cells (100,000 cells per well) were pre-incubated with or without test compound in Yssel's medium for 1 hour at 37° C. Cells were then stimulated by transferring them to round-bottom 96-well plates pre-coated with 1 μm/ml αCD3 and 5 μg/ml αCD28. For counter assay, cells were instead stimulated by adding 8× stock solutions of PMA and ionomycin in Yssels (for final concentrations of 0.5 ng/ml PMA and 0.1 uM ionomycin, both from Calbiochem). Cells were incubated at 37° C. for 24 hours before 100 μL supernatants were harvested for quantification of IL-2 by ELISA using Human IL-2 Duoset ELISA Kit from R and D Systems, Cat. # DY202E.
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 566.64, ALogP 2.48, HBA 13, HBD 3, PSA 159.04, QED 0.37
Compound (SMILES): OCCc1ccc(Cc2cc(C(F)(F)F)nc(OC3CCCC3)n2)cc1
InChIKey: TYOKGMWVPYYBGB-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Kinetic Assay: Assays are performed in 96-well plates in a total volume of 200 μl/well. Compounds are dissolved in dimethylsulfoxide (DMSO) and added to plates in a volume of 10 μl followed by addition of 165 μl of assay mix. Plates are pre-incubated at 25° C. for 15 min and the reactions are initiated by the addition of 25 μl of cAMP followed by thorough mixing. Reaction rates are measured by monitoring the decrease in fluorescence using excitation at 355 nm and emission at 460 nm for a period of 10 min in a fluorescence plate reader. Initial rates (slopes) are determined from linear portions of the progress curves. Final concentrations of assay components are as follows: 50 mM Tris, pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP), 0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units lactate dehydrogenase, and either 0.5 nM human PDE4D7 or 10 nM human PDE4B1. All data are percent normalized relative to controls and are presented as percent inhibition. An inhibitory concentration 50% (IC50) value is calculated by fitting of a sigmoidal dose response curve. Human PDE4D7 contained a mutation of serine 54 to aspartic acid to mimic activation by cAMP-dependent protein kinase A (PKA). Human PDE4B1 contained a corresponding mutation of serine 133 to aspartic acid to also mimic PKA activation.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 366.38, ALogP 3.94, HBA 4, HBD 1, PSA 55.24, QED 0.84
Compound (SMILES): COC1CN(c2nc3cc(C)c(C)cc3cc2-c2nc(C)no2)CCC1NC1CCCCC1
InChIKey: OFDNGNRGTMQAQN-UHFFFAOYSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: OPRK1 antagonist assay: The cell line for the OPRK1 antagonist assay stably expresses the following elements. The carboxy terminus of the OPRK1 receptor has a 7 amino acid linker, followed by the TEV protease cleavage site and a GAL4-VP16 fusion protein. The cell line also expresses a b-arrestin-2-TEV protease fusion protein and contains a reporter construct consisting of the UAS response element and the b-lactamase (bla) reporter gene. Upon activation of the receptor, g-protein receptor kinase (GRK) phosphorylates specific intracellular residues of OPKR1 and this induces recruitment of B-arrestin2-TEV protease fusion protein. The TEV protease recognizes and cleaves the TEV site, releasing the GAL4-VP16 fusion protein, which then translocates to the nucleus. The GAL4-V16 binds to the UAS element, driving expressing of the b-lactamase gene. B-lactamase expression is detected with the cell permeable, fluorescent substrate, CCF4-AM. This substrate consists of coumarin tethered to fluoroscein via a b-lactam ring. In the absence of b-lactamase, excitation of the dye with 405 nm light results in FRET from the coumarin to fluoroscein and emission of green (525 nm maximum) light. B-lactamase cleavage of the substrate separates the courmarin fluorophore from the fluorscein, and 405 nm excitation results in blue (460 nm maximum) emission. The assay is monitored by the blue/green emission ratio. The antagonist assay is performed by seeding the cells into 384 well plates and incubating them 16-24 hours at 37° C. Test antagonist compounds are added and incubated for 30 minutes at 37° C. Next an EC80 challenge of U-50488 (OKRK1 agonist) is added and the samples are incubated for 4 hours at 37° C., followed by addition of CCF4-AM substrate. The plates are then incubated 2 hours at room temperature in the dark and the blue/green ratio determined on a fluorescent plate reader. See J Biomol Screen April 2009, vol. 14 no. 4, pp 381-394.
Measurement: IC50 = 107 nM (pChEMBL 6.96999979019165)
Descriptors: MW 449.6, ALogP 4.73, HBA 7, HBD 1, PSA 76.31, QED 0.6
Compound (SMILES): CC1OC(=O)NN=C1c1ccc(-c2cnn(C(F)F)c2)c(C(F)(F)F)c1
InChIKey: GYOWSDLEDJOFED-UHFFFAOYSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A (SINGLE PROTEIN), Homo sapiens
UniProt: Q14432
Assay: PDE3A Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3A reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3A cell extract from Sf9 cells overexpressing human full length PDE3A (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3A cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:5000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3A activity vs log compound concentration.
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 374.27, ALogP 3.8, HBA 5, HBD 1, PSA 68.51, QED 0.83
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1C[C@H](COc2ccc3nc(C(C)(F)F)sc3c2F)C1
InChIKey: LIAUAWPACATQTM-DRZSPHRISA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 97 nM (pChEMBL 7.010000228881836)
Descriptors: MW 416.47, ALogP 4.25, HBA 5, HBD 1, PSA 60.45, QED 0.7
Compound (SMILES): COCC1(NC(=O)c2ccc(-n3ncc(-c4ccc(C#N)cc4)c3O)nc2)CCCC1
InChIKey: QFWPGBGTIIWHCH-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 52.5 nM (pChEMBL 7.28000020980835)
Descriptors: MW 417.47, ALogP 3.2, HBA 7, HBD 2, PSA 113.06, QED 0.64
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)NCc2cccc(Cl)c2F)c2ccc(NC(=O)N3Cc4cncnc4C3)cc12
InChIKey: XFFYOFDJSNWDBR-HRFSGMKKSA-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 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 636.06, ALogP 4.23, HBA 7, HBD 2, PSA 129.53, QED 0.29
Compound (SMILES): Cn1ccnc1C(=O)N[C@H](CC1CCCCC1)C(=O)N1C[C@@H](n2nncc2C(C)(C)O)C[C@H]1C(=O)NC(CCCCNC(=O)OCc1ccccc1)C(=O)C(N)=O
InChIKey: OBFJIERBOYOTDF-LBIXZHBDSA-N
Target: Serine protease HTRA1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92743
Assay: Full Length 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 (Exλ=330 nm; Emλ=420 nm). Percent inhibition was calculated by fitting values to a standard mathematical model for determining the dose response curve.
Measurement: IC50 = 410 nM (pChEMBL 6.389999866485596)
Descriptors: MW 790.92, ALogP 1.79, HBA 13, HBD 5, PSA 245.76, QED 0.09
Compound (SMILES): Cc1nnc(C2CN(c3ccnc(-c4cnc5ccc(C(F)(F)F)cn45)n3)CCN2)[nH]1
InChIKey: UBCMXVZLMOLAEJ-UHFFFAOYSA-N
Target: eIF-2-alpha kinase GCN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P2K8
Assay: Enzyme Inhibition Assay: An assay buffer containing GCN2 kinase and (RS)7 was prepared. 4.7 μL of this stock solution was placed per well of a black, low volume, 384-well microtitre plate (e.g. catalogue number 3676, Corning Inc., NY). To this was added 0.65 μM of DMSO containing serial dilutions of the test compound (typical final concentrations of test compound were 0 to 8 μM). The plate was incubated for 10 minutes at 25° C. prior to the addition of 4.7 μL of ATP stock buffer to initiate the enzyme reaction. The reaction was allowed to proceed for 1 hour at 25° C., prior to the addition of 10 μL detection buffer (consisting of appropriate concentrations of ADP2 antibody and ADP Alexa633 tracer in 1× stop and detect buffer as supplied by BellBrook Labs). The reaction was left to incubate for 1 hour at 25° C., prior to measuring the fluorescence polarisation signal (mP) in each well using a PHERAstar FS reader (BMG Labtech, Germany).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 429.41, ALogP 2.39, HBA 8, HBD 2, PSA 99.92, QED 0.52
Compound (SMILES): CCCNC(=O)[C@@H]1CN(S(=O)(=O)c2cn(C)cn2)C[C@H]1c1ccc(F)cc1
InChIKey: WXKWCOMPXZSLSG-JKSUJKDBSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 1000 nM (pChEMBL 6)
Descriptors: MW 394.47, ALogP 1.49, HBA 5, HBD 1, PSA 84.3, QED 0.8
Compound (SMILES): Cc1cc(C(=O)OC(C)C)c(N2CCC(NCC(C)C)CC2)nn1
InChIKey: KUXIPGBUFUJQSI-UHFFFAOYSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: enzyme assay: Well-known assay for kappa opioid receptor
Measurement: IC50 = 78 nM (pChEMBL 7.110000133514404)
Descriptors: MW 334.46, ALogP 2.56, HBA 6, HBD 1, PSA 67.35, QED 0.81
Compound (SMILES): Cc1cc(C(=O)OCCCc2ccccn2)ccc1N1C(=O)CSC1c1ccc(F)cc1
InChIKey: CFSBLEBYHUSDMT-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: FLIPR Calcium Assay: One day before the assay, CORNING black with clear flat bottom 96-well assay plates were coated with a 0.1 mg/mL Poly-L-Lysine solution. CHO-K1/MOR/Gα15 cells were suspended in the F12 medium and plated at a density of 8×104 cells/well in 200 μL medium. Cells were incubated in a humidified atmosphere of 10% CO2 at 37° C. overnight so as to reach an 80-90% confluent cell monolayer before assay. At the day of assay, 150 μL medium/well was removed from plate. To each well, 50 μL FLIPR calcium assay reagent dissolved in 1× assay buffer (HBSS: KCl 5 mM, KH2PO4 0.3 mM, NaCl 138 mM, NaHCO3 4 mM, Na2HPO4 0.3 mM, d-glucose 5.6 mM, with additional 20 mM HEPES and 13 mM CaCl2, pH 7.4), with 2.5 mM probenecid was added and the plate was incubated at 37° C. for 1 h. Compounds and other reagents were dissolved in the assay buffer. Using a FlexStationIII (Molecular Devices Corp.), the [Ca2+]i fluorescence increases after robotic injections of compounds or other reagents were monitored every 1.52 s interval with excitation wavelength at 485 nm and with emission wavelength at 525 nm. The [Ca2+]i fluorescence was measured up to 90 s after agonist injection. The fluorescence intensity from 6 to 12 wells of cells were averaged and the relative amount of [Ca2+]i release was determined by integrating the AUC of the [Ca2+]i fluorescence averages.
Measurement: EC50 = 9200 nM (pChEMBL 5.039999961853027)
Descriptors: MW 450.54, ALogP 5.1, HBA 5, HBD 0, PSA 59.5, QED 0.37
Compound (SMILES): Cc1nc(C(=O)Nc2ccc(F)c([C@]3(C)CN(C)S(=O)(=O)C(C)(C)C(=N)N3)c2)c(C)o1
InChIKey: LSHFCSFBNFDSKV-FQEVSTJZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Reagents: Na+-Acetate pH 5.0; 1% Brij-35; Glycerol; Dimethyl Sulfoxide (DMSO); Recombinant human soluble BACE1 catalytic domain (>95% pure); APP Swedish mutant peptide substrate (QSY7-APPswe-Eu): QSY7-EISEVNLDAEFC-Europium-amide (SEQ ID 1).A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide (SEQ ID 1)). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme. Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.
Measurement: Ki = 165 nM (pChEMBL 6.78000020980835)
Descriptors: MW 451.52, ALogP 2.52, HBA 6, HBD 3, PSA 128.39, QED 0.66
Compound (SMILES): Cc1sc2c(c1C)C(c1ccc(Cl)cc1)=N[C@@H](CC(=O)Nc1ccc3c(c1)C1(CC1)NC3=O)c1nnc(C)n1-2
InChIKey: PGJCPLSINKADLZ-QFIPXVFZSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 Biochemical Activity Test: The experiment plate was a plate that contained a gradient concentration of the compound and a corresponding DMSO solution and was prepared with ECHO before the experiment:a) The experiment plate was taken out, and 5 μL/well of solution A (protein solution) was added to columns 2-23 of the experiment plate, then 5 μL/well of 1× assay buffer was added to columns 1 and 24 of the experiment plate, and columns 1 and 24 were used as Min control in the experiment system;b) Centrifugation at 1000 rpm was carried out for 30 seconds;c) The plate was incubated at 23° C. for 20 minutes;d) After 20 minutes of incubation, 5 μL/well of solution B (polypeptide solution) was added to columns 1-24 of the experiment plate;e) Centrifugation at 1000 rpm was carried out for 30 seconds;f) The plate was incubated at 23° C. for 20 minutes;g) After 20 minutes of incubation, 5 μL/well of solution C (test reagent solution) was added to columns 1-24 of the experiment plate;h) Centrifugation at 1000 rpm was carried out for 30 seconds;i) The plate was incubated at 23° C. for 40 minutes;j) The experiment plate was placed on EnVision to read the plate.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 557.08, ALogP 5.56, HBA 7, HBD 2, PSA 101.27, QED 0.34
Compound (SMILES): CCOc1nc(C)c2nc(-c3cncc(F)c3)n(Cc3cnc(Cl)c(F)c3)c2n1
InChIKey: NVNJCBGTUKFFSI-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition of the Activation Assay: As human TLR7 expressing cell line, HEK293 cell line was bought from IMGENEX Corporation (TLR7/NF-ηB/SEAPorter HEK293 cell), which is human embryonic kidney cell line and stably expresses full-length human TLR7 gene and secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NE-ηB response element. The TLR7/NF-ηB/SEAPorter HEK293 cell was cultivated with DMEM containing 10% fetal bovine serum (FBS) and 10 μg/mL blasticidin S at 37° C. in the presence of 5% CO2. The TLR7/NF-ηB/SEAPorter HEK293 cell was seeded into 96-well microtiter plate at 5×104 cell/90 μL/well, and the place was cultivated at 37° C. in a CO2 incubator overnight. Each test compound that was diluted with the medium was added to the wells (10 μL/well), wherein each final concentration was adjusted to 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol/L, or 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μmol/L. After 0.5 hour, R-848 that is TLR7/8 ligand was added to each well (10 μL/well), wherein each final concentration was adjusted to 200 nmol/L. The total volume was adjusted to 110 μL/well, the test samples were incubated in CO2 incubator for 20±1 hours, and then the SEAP activity was measured as activation of TLR7. The SEAP activity was evaluated as follows: p-nitro-phenyl phosphate (pNPP) (Invitrogen) was added to the incubated sample (50 μL/well); after 15 minutes, 4 mol/L sodium hydroxide solution (nacalai tesque) was added thereto (50 μL/well) to quench the reaction; and the absorbance of each sample was measured at 405 nm with microplate reader Elx808 (BioTek). The 50% inhibitory concentration (IC50 value) of each sample compound was calculated based on 100% of the SEAP activity wherein the test sample comprises no sample compound.
Measurement: IC50 = 667 nM (pChEMBL 6.179999828338623)
Descriptors: MW 416.82, ALogP 3.97, HBA 7, HBD 0, PSA 78.61, QED 0.46
Compound (SMILES): Cc1cc2nc(C(CNC(=O)c3c(Cl)cc(-n4cnnc4)cc3Cl)c3ccc(F)cc3)[nH]c2cc1C
InChIKey: ZXOSANLDRROZIG-UHFFFAOYSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Determination of Inhibitory Activity Against Factor IXa: Formation of a clot to stem bleeding at a site of blood vessel injury involves the coordinated activity of a group of plasma proteins that initiate and propagate fibrin formation and subsequently protect fibrin from premature degradation. Factor IX is a key component of the plasma system that forms a fibrin clot at a site of vascular injury. The activity of Factor IXa is measured by monitoring the cleavage of the fluorescent peptide, CH3SO2-D-CHG-Gly-Arg-AFC.AcOH (CHG is cyclohexyl-glycine and AFC is trifluoro aminomethyl coumarin). Factor IXa cleaves the amide bond between Arg and AFC, thereby releasing the AFC fluorophore. The free AFC can be detected with a fluorescence detector at an excitation wavelength of 405 nM and emission wavelength of 510 nM.
Measurement: IC50 = 2.82 nM (pChEMBL 8.550000190734863)
Descriptors: MW 523.4, ALogP 5.77, HBA 5, HBD 2, PSA 88.49, QED 0.3
Compound (SMILES): C=CC(=O)Nc1ccc(Oc2ccc(F)cc2F)c(-c2nn(C)c(=O)cc2OC)c1
InChIKey: BDDWHFPNSOHNHE-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Binding Assay: The bromodomain binding assays were performed by Reaction Biology Corp., Malvern, Pa., USA (www.reactionbiology.com). The BET binding assays were conducted in 384 well microplates in assay buffer (50 mM HEPES-HCl, pH 7.5, 100 mM NaCl, 1 mg/ml BSA, 0.05% CHAPS, and 0.5% DMSO) with compounds added as DMSO stocks at a single concentration or with 10-point dose response titrations. BET protein or assay buffer were delivered to the appropriate wells of the microplate. Test compound was then delivered by acoustic technology via a Labcyte Echo550 liquid handler. The microplate was centrifuged for 5 min and pre-incubated for 30 min at RT with gentle shaking. The ligand (histone H4 peptide (1-21) K5/8/12/16Ac-biotin) was delivered and the microplate was again centrifuged for 5 min and allowed to incubate for 30 min at RT with gentle shaking. Donor beads were then added in the absence of light and the microplate was centrifuged and gently shaken. After 5 min, acceptor beads were added in the absence of light and the microplate was centrifuged and gently shakenin the dark for 60 min. The microplate was read using a Perkin Elmer EnSpire Alpha plate reader (λEx/λEm=680/520-620 nm). Percent inhibition was calculated relative to positive and negative controls on a per plate basis.
Measurement: IC50 = 206 nM (pChEMBL 6.690000057220459)
Descriptors: MW 413.38, ALogP 3.65, HBA 6, HBD 1, PSA 82.45, QED 0.62
Compound (SMILES): Cc1c(C(=O)CN2C3CCC2[C@@H](O)C3)c2ccc(CCC#N)cc2n1-c1ccc(Cl)cc1
InChIKey: XBIMVGSHAOVKNH-JVPYDEADSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: P54578
Assay: Usp14 Inhibition Assay: Usp14 Inhibition Assay was described in B. H. Lee et al. Nature 2010, 467 (9), 179.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 447.97, ALogP 4.83, HBA 5, HBD 1, PSA 69.26, QED 0.55
Compound (SMILES): Cc1ccc2nc(CCc3nc4c(C)ncc(C)n4n3)cnc2c1C
InChIKey: DUCFFYGNJBJBGR-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The activities of the compounds were determined using the Molecular Devices IMAP PDE Fluorescence Polarization assay using recombinant human PDE-10 enzyme expressed in a baculoviral system. Briefly, 10 μL of a compound (0.2 nM-20 μM) was added to either a 96-well half area black plate or a 384-well black plate along with 10 μL of Fluorescein-labeled cAMP/cGMP substrate as per manufacturer's instructions and 10 μL of PDE enzyme (activity 0.1 U). Following a 40-minute incubation at 37° C., 60 μL of IMAP binding reagent was added. The plate was then read on a Perkin Elmer Victor (480-535 nm). The data was analyzed using Prism Software (GraphPad Inc, San Diego, Calif.).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 332.41, ALogP 3.09, HBA 6, HBD 0, PSA 68.86, QED 0.58
Compound (SMILES): CC(c1ccc2c(c1)OCC2)N1CCN(c2ncnc3c2CS(=N)(=O)C3)CC1
InChIKey: GKDYODZLQJBPQU-UHFFFAOYSA-N
Target: Protein O-GlcNAcase (SINGLE PROTEIN), Homo sapiens
UniProt: O60502
Assay: Human O-GlcNAcase enzyme inhibition assay: 5 μl of the appropriate concentration of a solution of inhibitor in McIlvaine's Buffer (pH 6.5) in 2% DMSO (for a dose response curve calculation) is added into each well of a 384-well plate (Greiner, 781900). Then, 20 nM of His-Tagged hOGA and 10 μM of FL-GlcNAc (Fluorescein mono-beta-D-(2-deoxy-2-N-acetyl) glucopyranoside; Marker Gene Technologies Inc, M1485) were added to the 384-well plate for a final volume of 20 μl. After incubation for 60 min at room temperature, the reaction was terminated by the addition of 10 μL of stop buffer (200 mM glycine, pH 10.75). The level of fluorescence (λexc 485 nm; (Δemm 520 nm) was read on a PHERAstar machine. The amount of fluorescence measured was plotted against the concentration of inhibitor to produce a sigmoidal dose response curve to calculate an IC50. All individual data was corrected by subtraction of the background (Thiamet 3 uM=100% inhibition) whilst 0.5% DMSO was considered as the control value (no inhibition).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 399.52, ALogP 2.36, HBA 7, HBD 1, PSA 82.41, QED 0.85
Compound (SMILES): CN(C)[C@H]1CC[C@H](Nc2ncnc3ccc(-c4cnc5ccoc5c4)cc23)CC1
InChIKey: ZCQVUKNBGUAXJR-IYARVYRRSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: 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 = 109 nM (pChEMBL 6.960000038146973)
Descriptors: MW 387.49, ALogP 4.72, HBA 6, HBD 1, PSA 67.08, QED 0.55
Compound (SMILES): O=c1[nH]c2ccc(Br)cc2n1CCC[C@H]1NCCC[C@@H]1O
InChIKey: YZKCSFRYOWSCMD-OCCSQVGLSA-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 354.25, ALogP 1.99, HBA 4, HBD 3, PSA 70.05, QED 0.79
Compound (SMILES): OC1CCC(n2nc(-c3ccc(N4CCOCC4)cc3)c3cnc(NCC(F)(F)F)nc32)CC1
InChIKey: YLOANLBVLPVTOV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Inhibition Assay: The ectopic expression of Mer receptor tyrosine kinase (Mer) has been identified as a tumor cell survival gene product in Acute Lymphoblastic Leukemia (ALL) cells and a potential cause of ALL chemoresistance. Hence, we investigated whether the development of small molecule Mer inhibitors was possible.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 476.5, ALogP 3.78, HBA 8, HBD 2, PSA 88.33, QED 0.58
Compound (SMILES): C=CC(=O)Nc1ccc(Nc2nc(Nc3cncc(OC)c3)ncc2C(F)(F)F)cc1
InChIKey: MHIWASIZKKCXCN-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Omnia Assay: EGFR (WT) and EGFR (T790M/L858R): The Omnia Assay Protocol for potency assessment against EGFR is performed as described in Example 251 above except that the EGFR-WT- and EGFR T790M/L858R-modified optimized reagent conditions are:[EGFR-WT]=5 nM, [ATP]=15 mM, [Y12-Sox]=5 mM (ATP KMapp˜12 mM); and [EGFR-T790M/L858R]=3 nM, [ATP]=50 mM, [Y12-Sox]=5 mM (ATP KMapp˜45 mM).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 430.39, ALogP 4.51, HBA 7, HBD 3, PSA 101.06, QED 0.47
Compound (SMILES): C=CC(=O)N1CCO[C@@H](COc2ncnc(N)c2-c2ccc(Oc3ccc(C#N)cc3)cc2)C1
InChIKey: FHOXQIJFLYZUQL-OAQYLSRUSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzyme Assay: The following describes a microfluidic, off-chip mobility shift kinase assay used to measure inherent potency of compounds against BTK enzyme. Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent BTK Enzyme Assay IC50. These IC50 values are reported in ranges wherein: A<100 nM, B<1 uM, and C>1 uM.2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM Hepes, pH 7.5, and 10 mM DTT.5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27 C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM Hepes, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorylation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland). Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent PBMC BTK Enzyme Assay IC50.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 457.49, ALogP 3.18, HBA 8, HBD 1, PSA 123.59, QED 0.54
Compound (SMILES): CN1CCN(Cc2ccc(C(=O)Nc3nc(-c4cccc(C#Cc5ccccc5F)c4)cs3)cc2)CC1
InChIKey: CTUPZQPBUUCNEF-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based in vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 uL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad). The concentration of inhibitor showing 50% inhibition as compared with the control was expressed as IC50 (μM).
Measurement: IC50 = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 510.64, ALogP 5.35, HBA 5, HBD 1, PSA 48.47, QED 0.37
Compound (SMILES): CNC(=O)c1cc(C)c(F)c(-c2cnn3cc(-c4cnc(C)o4)c(N[C@H]4CCOC4)nc23)c1
InChIKey: BSWVXMJXEMSCJF-HNNXBMFYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: FGFR Enzymatic Assay: The inhibitor potency of the exemplified compounds was determined in an enzyme discontinuous assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.2 μL was transferred to the wells of a 384-well plate. A 5 μL/well volume of enzyme isoforms of FGFR (-1, -2, -3 wild-type and mutant isoforms, -4) including phosphorylated and un-phosphorylated proteins diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated with inhibitor for 5 to 15 minutes at ambient temperature. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The reaction was initiated by the addition of a 5 μL/well volume containing both biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP in assay buffer. The 10 μL/well reaction concentration of the peptide substrate was 500 nM whereas the ATP concentration was maintained near or below the ATP Km. The ATP Km values were pre-determined in a separate series of experiments. The reaction plate was incubated at 25° C. for 1 hr and the reactions were ended with the addition of 5 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; 45 mM EDTA, 600 nM staurosporin, with Perkin Elmer Lance Reagents at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 10 minutes at ambient temperature before scanning on a PheraStar plate reader (BMG Labtech) instrument.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 450.47, ALogP 3.37, HBA 8, HBD 2, PSA 106.58, QED 0.48
Compound (SMILES): C=CC(=O)N1CCC(Nc2cnc3[nH]cc(C(=O)N[C@@H]4C[C@H]4C(F)(F)F)c3n2)CC1
InChIKey: LQLNGEMDEZXPBE-CHWSQXEVSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: JAK Caliper Enzyme Assay: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 4 μM ATP and 1 μM peptide substrate. The JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition 1 nM JAK3 enzyme and were incubated at room temperature 75 minutes for JAK3. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 422.41, ALogP 2.23, HBA 5, HBD 3, PSA 103.01, QED 0.64
Compound (SMILES): CN(C)[C@@H]1CCCN(Cc2ccc(C(=O)N[C@@H]3[C@H]4Oc5ccc(Oc6ccnc7c6CCC(=O)N7)cc5[C@@H]34)cc2C(F)(F)F)C1
InChIKey: DRWSMZPVRXENHA-KRUKBSHNSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: TBD: TBD
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 621.66, ALogP 4.96, HBA 7, HBD 2, PSA 96.03, QED 0.39
Compound (SMILES): CN(c1ccc2c(c1)C(=O)N(C1CCC(=O)NC1=O)C2=O)[C@H]1CCCC[C@@H]1N
InChIKey: UNCMECUBEPXFGV-KSCSMHSMSA-N
Target: Zinc finger protein Helios (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UKS7
Assay: Prolabel Quantification of IKZF1, IKZF2, or GSPT1 Protein Levels in 293GT Cells: The Prolabel system from DiscoverX was used to develop high-throughput and quantitative assays to measure changes in IKZF1, IKZF2, and GSPT1 protein levels in response to compounds. The prolabel tag is derived from the alpha fragment of beta galactosidase and has the following protein sequence: mssnslavvlqrrdwenpgvtqlnrlaahppfaswrnseeartdrpsqqlrslnge. The complementary fragment of beta-galactosidase (from DiscoverX), is added to the prolabel tag to form an active beta galactosidase enzyme whose activity can be precisely measured. In this way, the levels of a fusion protein with the prolabel tag can be quantified in cell lysates.Lentiviral vectors, based on the Invitrogen pLenti6.2/V5 DEST backbone, were constructed that placed the prolabel tag upstream of IKZF1, IKZF2, or GSPT1 and expressed the fusion protein from a CMV promoter.To ensure moderate and consistent expression of the prolabel fusion proteins across all cells in the population, stable cell lines were constructed from cells expressing a single copy of the construct. Lentivirus packaged with the constructs was made using the Virapower kit from Invitrogen. Strongly adherent 293GT cell, GripTite 293 MSR cells from Thermo Fisher Scientific (Catalog number: R79507), were infected with the virus at low multiplicity of infection and selected by 5 μg/mL blasticidin for 2 weeks.
Measurement: EC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 384.44, ALogP 0.79, HBA 6, HBD 2, PSA 112.81, QED 0.75
Compound (SMILES): CN(C(=O)C1CCN(c2ccc(OC(F)(F)F)cc2)CC1)C1CCN(C2CCN(Cc3ccccc3)CC2)C1
InChIKey: AUTDNNJECLIKKT-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 544.66, ALogP 5, HBA 5, HBD 0, PSA 39.26, QED 0.49
Compound (SMILES): Cc1cccc([C@@H](CO)NC(=O)CN2C(=O)c3cc(-c4nc(Nc5cnn(C)n5)ncc4Cl)ccc3C2C)c1
InChIKey: DXPPJWCUBNDWMQ-VXNXSFHZSA-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 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 547.02, ALogP 3.34, HBA 9, HBD 3, PSA 138.16, QED 0.31
Compound (SMILES): CC[C@H](NC(=O)C1(C)CN(c2ncnn3cc(C4=CCN(C(=O)OC)CC4)cc23)C1)c1ccc(Cl)cc1
InChIKey: SNHGPIABTGDWPI-QFIPXVFZSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit Wild Type Assay: In each well of a 384-well plate, 0.2 ng/ul final (2 nM) of wild type Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 400 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 523.04, ALogP 4.33, HBA 7, HBD 1, PSA 92.07, QED 0.52