lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(F)ccc1-c1ccncc1N(C)C(=O)c1cc(Cl)nc(C(F)(F)F)c1
InChIKey: BAGCDFAOGFLZEV-UHFFFAOYSA-N
Target: G-protein coupled bile acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDU6
Assay: cAMP Assay: CHO-dhfr(minus) cells expressing human GPBAR1 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), lx HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. The assay was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaked for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH, Hamburg Germany), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The measured signal at 730 nm has to be corrected for the ruthenium background, the direct excitation of Alexa and the buffer control. The FRET signal is calculated as follows: FRET=T730−Alexa730−P(T645−B645) with P=Ru730−B730/Ru645−B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 439.8, ALogP 5.24, HBA 4, HBD 0, PSA 55.32, QED 0.41
Compound (SMILES): Cc1ncc(C2CCC3(CC2)OCCO3)c(OC[C@@H](C)Cc2ccc3ccccc3n2)n1
InChIKey: YIBMOADFCOOAKN-SFHVURJKSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: 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 phosphodiesterase assays (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 PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60;
Measurement: Ki = 0.73 nM (pChEMBL 9.140000343322754)
Descriptors: MW 433.55, ALogP 4.99, HBA 6, HBD 0, PSA 66.36, QED 0.55
Compound (SMILES): CC(=O)N1CCN(c2cccc(-c3cc(-c4cnn(C)c4)c4c(N)ncnn34)c2F)C(=O)C1(C)C
InChIKey: XWTQWJIHZNVHRD-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 476.52, ALogP 2.49, HBA 8, HBD 1, PSA 114.65, QED 0.49
Compound (SMILES): Cc1c(F)ccc(-n2cc(COc3nccc(C(C)C)n3)nn2)c1F
InChIKey: BIDDYKAHINZFMW-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 1980 nM (pChEMBL 5.699999809265137)
Descriptors: MW 345.35, ALogP 3.35, HBA 6, HBD 0, PSA 65.72, QED 0.71
Compound (SMILES): CC(C)[C@@H]1CC(=O)N(C)c2cc(-c3ccnc(Nc4ccnn4C)c3)cc3nnc1n23
InChIKey: MEMCTQHLJNUYLA-INIZCTEOSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 6.94 nM (pChEMBL 8.15999984741211)
Descriptors: MW 416.49, ALogP 3.37, HBA 8, HBD 1, PSA 93.24, QED 0.55
Compound (SMILES): Cc1cc(NC(=O)C(OC(=O)Cc2ccc(Cl)cc2)c2ccccc2)no1
InChIKey: HVRLYWFJGUCJJA-UHFFFAOYSA-N
Target: Kallikrein-7 (SINGLE PROTEIN), Homo sapiens
UniProt: P49862
Assay: Inhibition Assay: Substrate:(MeO-Suc-Arg-Pro-Tyr-pNA), acetate salt, Peptide international, lot #906841, previously named S-2586Enzyme: Active human KLK7 (SCCE) 0.62 mg/ml in 0.3 M NaCl, 10 mM NaAc, pH 4Buffer: 10 mM NaPhospate, 0.5 M NaCl, pH 7.2150 μl of Substrate (2.5 mM) in Buffer was added to 96 wells plate (F8 Polysorp Unfra, Nunc cat. no. 469957). 10 μl of DMSO was added to the blank and control wells.10 μl of substance in DMSO was added to wells giving final concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, and 0.15625 μM.40 μl of active KLK7 (SCCE) (diluted to 12.5 μg/ml in activity buffer) was added all wells except blank to which activity buffer was added.Immediately after addition of SCCE the plate was transferred to a Spectramax 250 Microplate Reader (Molecular Devices) and enzyme activity (V) was measured as release of pNa measuring absorbance at 405 nm in 37° C. for 15 min with reading every 30 s.The mean value V of each sample was calculated (n=2 for the substances and n=4 for the control). From the values of V the % of total activity was calculated as (Vinhibitor/Vcontrol (no inhibitor))×100.
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 384.82, ALogP 4.1, HBA 5, HBD 1, PSA 81.43, QED 0.65
Compound (SMILES): CC[C@@](O)(c1cc(F)c2c(c1)C(=O)N(Cc1ccc(C#N)cc1)C2(O[C@H]1C[C@@H](O)C1)c1ccc(Cl)cc1)C1CCN(C)CC1
InChIKey: RVEVEKZOUQPWSR-JOEBACTPSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: MDM2-p53 Interaction Using a 96-Well Plate Binding Assay (ELISA): The ELISA assay was performed in streptavidin coated plates which were preincubated with 200 μl per well of 1 μg ml−1 biotinylated IP3 peptide. The plates were ready to use for MDM2 binding after washing the plate with PBS.Compounds and control solutions in DMSO aliquoted in 96-well plates were pre-incubated in a final 2.5-5% (v/v) DMSO concentration at room temperature (for example 20° C.) for 20 min with 190 μl aliquots of optimized concentrations of in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4° C. for 90 min. After washing three times with PBS to remove unbound MDM2, each well was incubated at 20° C. for 1 hour with a TBS-Tween (50 mM Tris pH7.5; 150 mM NaCl; 0.05% Tween 20 nonionic detergent) buffered solution of primary mouse monoclonal anti-MDM2 antibody (Ab-5, Calbiochem, used at a 1/10000 or 1/200 dilution depending on the antibody stock solution used), then washed three times with TBS-Tween before incubation for 45 mins at 20° C. with a TBS-Tween buffered solution of a goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (used at 1/20000 or 1/2000 depending on the antibody stock solution). The unbound secondary antibody was removed by washing three times with TBS-Tween. The bound HRP activity was measured by enhanced chemiluminescence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The percentage of MDM2 inhibition at a given concentration is calculated as the [1−(RLU detected in the compound treated sampleRLU negative DMSO control)÷(RLU of DMSO positive and negative controls)]×100 or as the (RLU detected in the compound treated sample÷RLU of DMSO controls)×100. The IC50 was calculated using a plot of % MDM2 inhibition vs concentration and is the average of two or three independent experiments.
Measurement: IC50 = 98000 nM (pChEMBL 4.010000228881836)
Descriptors: MW 618.15, ALogP 5.69, HBA 6, HBD 2, PSA 97.03, QED 0.34
Compound (SMILES): C[C@H](c1ccc(-c2ccc(CO[C@H]3CCC[C@@H]3NC(=O)c3cc(-c4cnn(C)c4)cnc3N)cc2)cc1)N1CCN(C)CC1
InChIKey: MXKYSMZRKIEXAB-KMYQRJGFSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: TBD: TBD
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 593.78, ALogP 4.91, HBA 8, HBD 2, PSA 101.54, QED 0.28
Compound (SMILES): CC1(C)CN[C@@H]1C(=O)N1CCC[C@H]1B(O)O
InChIKey: FWTKUAZIABBHDG-JGVFFNPUSA-N
Target: Dipeptidyl peptidase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q6V1X1
Assay: In Vitro Inhibition Assay: EnzymesRecombinant human DPPIV (R&D Systems, Cat. No. 1180-SE)Recombinant human DPP8 (Enzo Life Sciences, Cat. No. BML-SE527)Recombinant human DPP9 (R&D Systems, Cat. No. 5419-SE)Recombinant human DPPII (R&D Systems, Cat. No. 3438-SE)Recombinant human FAP (R&D Systems, Cat. No. 3715-SE)Recombinant human PREP (R&D Systems, Cat. No. 4308-SE)Assay Buffers25 mM Tris, pH 8.0 (DPPIV and DPP9)50 mM Tris, pH 7.5 (DPP8)25 mM MES, pH 6.0 (DPPII)50 mM Tris, 140 mM NaCl, pH 7.5 (FAP)25 mM Tris, 0.25 M NaCl, pH 7.5 (PREP)Substrates4000× substrate solution (100 mM Gly-Pro-AMC (VWR, Cat. No. 100042-646) in DMSO, DPPIV, DPP8 and DPP9)4000× substrate solution (100 mM Lys-Pro-AMC (Bachem, Cat. No. I-1745) in DMSO, DPPII)100× substrate solution (2.5 mM Z-Gly-Pro-AMC (VWR, Cat. No. I-1145.0050BA) in DMSO, FAP and PREP)General MaterialsCompound96-well black clear-bottom plates (Costar, Cat. No. 3603)InstrumentationPlate shakerMolecular Devices SpectraMax® M2e microplate readerProtocol1. To prepare the compound for the assay, dissolve it in either DMSO or, if cyclization is suspected, in pH 2.0 water (0.01 N HCl) to a final concentration of 100 mM. For pH 2.0 stocks, incubate at room temperature for a minimum of four hours and up to overnight. From this, prepare a 1 mM stock at pH 7.4 in 50 mM Tris. If the inhibitor is insoluble at this concentration, dilute the 100 mM stock 1:10 to 10 mM. Using this stock, prepare a 0.1 mM stock as described above.2. Prepare a dilution plate for the compound stocks to be tested. Add the 0.1 and/or 1 mM stocks prepared previously to row A of a 96-well plate. From this, perform 1:10 serial dilutions into the appropriate assay buffer down the columns as shown below:3. Prepare 20× substrate solution by diluting the DMSO stocks into the appropriate assay buffer.4. Dilute the enzymes into their appropriate assay buffers. The dilution factor is lot dependent and must be determined prior to performing the assay. The final enzyme concentrations should be 0.1, 0.8, 0.4, 0.2, 1.2, and 0.6 nM for DPPIV, 8, 9, II, FAP and PREP respectively. Add 180 μL to each well needed in columns 2-10.5. Add 20 μL of the compound of interest from the dilution plate prepared in step 2 to columns 2-10 of the assay plate where appropriate. Each sample should be tested in triplicate. Allow this to incubate for 10 minutes at room temperature, shaking the plate for the first two minutes.6. Add 10 μL of 20× substrate prepared in step 3 to each well and allow this to incubate for 15 minutes at room temperature, shaking the plate for the first two minutes.
Measurement: IC50 = 4500 nM (pChEMBL 5.349999904632568)
Descriptors: MW 226.09
Compound (SMILES): CCc1ccc2c(C)nnc(N[C@H](C)c3ccc(-c4ccccc4CNC)s3)c2c1
InChIKey: ZYZWUGAIMMWVTR-QGZVFWFLSA-N
Target: Son of sevenless homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07889
Assay: Inhibition of Labeled Tracer Ligand: Table 22: This Example illustrates that exemplary compounds of the present invention bind to SOS1 and prevent a labeled tracer ligand from occupying the S0S1 binding site. The ability of a compound of Formula (I) to bind to SOS1 was measured using a HTRF displacement assay. A recombinant human SOS1 polypeptide (corresponding to amino acids 560-1049, expressed in E. Coli with N-terminal His-TEV-AviTag-SOS1 (MW=59.4 kDa) and lanthanide labeled streptavidin (CisBio) was incubated with an exemplary compound of Formula (I) (in a DMSO stock solution) in buffer (25 mM HEPES pH 7.5, 25 mM NaCl, 1 mM DTT, 0.01% Brij 35, 0.02% BSA, 0.1% DMSO). After a 10-15 minute incubation at room temperature, a solution comprised of a custom-made Cy5 labelled tracer and MAb Anti-6HIS Tb cryptate Gold (Cisbio 61HI2TLA) in buffer was added to the solution containing the SOS1 polypeptide and exemplary compound of Formula (I). After a 1-hour incubation at room temperature, the HTRF signal was measured using Clairostar plate reader (BMG Labtech) according to the manufacturer's instructions. Excitation filter EX-TR was used, and emission 1 was detected at 650-610 nm and emission 2 detected at 620-610 nm. The HTRF ratio was calculated using the formula: [emission 1/emission 2]*10000.
Measurement: Ki = 3.74 nM (pChEMBL 8.430000305175781)
Descriptors: MW 416.59, ALogP 6.12, HBA 5, HBD 2, PSA 49.84, QED 0.38
Compound (SMILES): CCN(CC)CCOc1ccc(Nc2cc3c(cn2)cc(-c2c(Cl)cccc2Cl)c(=O)n3CC)nc1
InChIKey: GVHNIJBVVMJQLY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assays: A concentration of a drug that reduces the observed activity of an enzyme by 50%. An IC50 is not a true affinity constant and the specific value determined can vary depending on the particular assay used. Nonetheless the rank order of IC50 for a set of compounds will typically be similar from one assay to another. (The true affinity constant for an inhibitor binding to an enzyme, Ki, can of course also be used for the same purposes as described herein for IC50). With respect to the kinases that are targeted by the compounds disclosed herein, kinase activity can be assayed, without limitation, as consumption of ATP or as incorporation of phosphate into a substrate. For example, the assay used in Example 76 assesses consumption of ATP based on luminescent detection of ADP. As an alternate example, the assay used in Example 78 assesses incorporation of phosphate based on the radiologic detection of 33P-labeled artificial substrates. Myriad other kinase assays are known in the art many of which are commercially available either as a kit or a service and one of skill in the art will recognize their particular usefulness for generating IC50 data.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 526.47, ALogP 6.25, HBA 7, HBD 1, PSA 72.28, QED 0.26
Compound (SMILES): NC(=O)c1cnc(Nc2ccc(OC(F)(F)F)cc2)nc1NC1CCC1
InChIKey: MHGKIMUPXQVGQL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: In Vitro Assay: SYK tyrosine phosphorylation activity is measured using the LANCE Technology developed by Perkin Elmer Life and Analytical Sciences (Boston, Mass.). LANCE refers to homogeneous time resolved fluorometry applications using techniques such as time-resolved fluorescence resonance energy transfer assay (TR-FRET) (see generally for procedures in Perkin Elmer Application NoteHow to Optimize a Tyrosine Kinase Assay Using Time Resolved Fluorescence-Based LANCE Detection, wwww.perkinelmer.com/lifesciences). The assay principle involves detection of a phosphorylated substrate using energy transfer from a phosphospecific europium-labeled antibody to streptavidin-allophycocyanin as an acceptor.To test the ability of candidate molecules to inhibit SYK tyrosine phosphorylation activity, molecules are reconstituted in 30% DMSO and serially diluted 1:3 with the final dilution containing DMSO in the absence of the candidate molecule. The final DMSO concentration in the assay is 3%. Kinase assays are performed as a two part reaction. The first reaction is a kinase reaction and which comprises of a candidate molecule, full length active recombinant SYK enzyme (Millipore, CA) and biotin-labeled SYK-specific substrate biotin-DEEDYESP-OH. The second reaction involves termination of the kinase reaction and the simultaneous addition of the detection reagents-europium-labeled anti-phosphotyrosine reagent (Eu-W1024-PY100, Perkin Elmer, Boston, Mass.) and Streptavidin-Allophycocyanin detection reagent (SA-APC, Prozyme, CA). The kinase reaction is performed in a black U-bottom 96-well microtitre plate. The final reaction volume is 50 μL and contains a final concentration of 1 nM active SYK enzyme, 550 nM SYK-substrate, and 100 μM ATP diluted in a buffer containing 50 mM Tris pH 7.5, 5 mM MgCl2, and 1 mM DTT. The reaction is allowed to proceed for 1 hour at room temperature. The quench buffer contains 100 mM Tris pH 7.5, 300 mM NaCl2, 20 mM EDTA, 0.02% Brij35, and 0.5% BSA. The detection reagents are added to the reaction mixture at the following dilutions1:500 for Eu-W1024-PY100 and 1:250 for SA-APC. The kinase reaction is terminated by the addition of 50 μL quench buffer containing the detection reagents. The detection is allowed to proceed for 1 hr at room temperature.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 367.33, ALogP 3.18, HBA 6, HBD 3, PSA 102.16, QED 0.72
Compound (SMILES): Cc1noc(C)c1-c1cnc2c(c1)N(S(=O)(=O)c1ccccc1)CC2CCC(=O)O
InChIKey: MMYCGMGWSTXYTA-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Alphascreen Binding Assay: Binding of compounds of formula (I) with bromodomain 2, 3 4 was assessed using Alphascreen binding assay. The inhibition of the interaction between bromodomain and its acetylated target protein (Filippakopoulos P et al. 2012) was measured quantitatively using recombinant BRD proteins, an acetylated Histone 4 peptide and AlphaScreen technology. In absence of inhibition the BRD protein bound to AlphaScreen nickel chelate acceptor beads can interact with the acetylated Histone 4 peptide which is immobilized by the AlphaScreen Streptavidin coated beads. This interaction brings donor and acceptor beads in proximity. The close proximity allows the singlet oxygen produced by laser excitation of the donor beads to reach the acceptor beads and generate a luminescence signal. BRD inhibitors result in a decrease in the proximity signal through an inhibition of the BRD-acetylated peptide interaction.Recombinant human bromodomains containing the N-terminal bromodomain (BRD2-BD1 (71-194), BRD3-BD1 (24-144) and BRD4-BD1 (44-164)) or dual bromodomains (BRD4-BD12 (1-477), BRD4-BD12 (1-472)) were prepared and purified as described in protein expression and purification session.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 427.48, ALogP 3.51, HBA 6, HBD 1, PSA 113.6, QED 0.64
Compound (SMILES): COc1cnc2c(Nc3cc([C@]4(C)CS(=O)(=O)[C@@](C)(C5CC5)C(N)=N4)c(F)cn3)ncnc2n1
InChIKey: KBHAXEFYGMUHTK-SFTDATJTSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: The compounds of the invention were determined to be potent inhibitors of BACE-1 using the following assay.The following reagents were used in this assay. Na+-Acetate pH 5.0; 1% Brij-35; Glycerol; Dimethyl Sulfoxide (DMSO); Recombinant human soluble BACE-1 catalytic domain (>95% pure); APP Swedish mutant peptide substrate (QSY7-APPswe-Eu): QSY7-EISEVNLDAEFC-Europium-amide.A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE-1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE-1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). 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 BACE-1 enzyme. Inhibition of BACE-1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.
Measurement: Ki = 271 nM (pChEMBL 6.570000171661377)
Descriptors: MW 486.53, ALogP 1.88, HBA 11, HBD 2, PSA 158.23, QED 0.55
Compound (SMILES): Cc1nnc(NC(=O)c2cc(C3CC3NCC3CCOCC3)c3ccccc3c2)s1
InChIKey: NRRVBNCEYIEHLS-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Inhibitory Activity Assay: The MAO-A inhibitory activity evaluation described below followed the protocol of MAO-Glo (registered trademark) Assay of Promega KK.A test compound dissolved in DMSO was added to a reaction solution (100 mM HEPES (pH 7.5), 5% glycerol) containing MAO-A enzyme (Sigma-Aldrich Co. LLC.), and the mixture was reacted at room temperature for 15 min. MAO substrate (Promega KK) was added to start the reaction. After reaction at room temperature for 60 min, Luciferine detection reagent (Promega KK) was added to terminate the reaction. After reaction at room temperature for 20 min with stirring, the luminescence was measured by Envision (PerkinElmer). The MAO-A inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate (%)=(1-(test compound count-blank)/(control-blank))x100The count of the MAO-A enzyme reaction mixture under compound non-addition conditions is indicated as control, and the count under compound non-addition and MAO-A enzyme non-addition conditions is indicated as blank. A concentration necessary for achieving 50% inhibitory rate was taken as IC50 value.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 422.55, ALogP 4.12, HBA 6, HBD 2, PSA 76.14, QED 0.62
Compound (SMILES): CNS(=O)(=O)c1cc(-c2sc(Nc3cccc(N4C(=O)CC[C@H]4CO)n3)nc2C)cc2c1C(=O)N([C@@H](C)C1CC1)C2
InChIKey: IKNJUYUINPOVDE-JXFKEZNVSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzymatic Activity Assay : The activity of recombinant human PI3Kγ ((aa144-1102)-6His) and PI3Kα, β, δ (6-His(p110-p85α)) was determined by measuring the ADP level after phosphorylation of DiC8-PIP2 using a commercially available ADP-Glo kit from Promega. The assay was carried out in white low volume 384 well plates in a final volume of 14 μl at R.T. The assay conditions contained the following: 50 mM Tris buffer pH 7.4, 2.1 mM DTT, 3 mM MgCl2, 0.05% CHAPS, 20 μM ATP, 80 μM DiC8-PIP2 and 1.2 nM PI3Kα, β, γ or 0.6 nM PI3Kδ. Potential inhibitors were made up in DMSO and then diluted in the assay to give a final concentration of not exceeding 1% (v/v) DMSO. A 10-point half-log dilution series of the inhibitors (highest concentration typically 0.1 μM for δ or γ and 33 μM for a or 1) was tested and the pIC50 determined using a 4-parameter logistic equation in a non-linear curve fitting routine. Routinely, inhibitors were pre-incubated with 3 μl of enzyme for 15 min prior to the addition of 2 μl substrate mixture for a further 60 min enzyme reaction. The phosphorylation was stopped with the addition of 3 μl ADP-Glo reagent (stop solution) followed by a 40 min incubation. Prior to detection 6 μl of ADP-Glo Kinase Detection Reagent was added and the plates were read in a micro plate reader using a Luminescence filter. All additions were followed by a short centrifugation step.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 596.74, ALogP 3.41, HBA 9, HBD 3, PSA 144.83, QED 0.34
Compound (SMILES): Cc1cnn(Cc2ccc(Cn3cc(C(=O)NCc4ccc5c(N)nccc5c4)c(CO)n3)cc2)c1
InChIKey: VMIWCYSSHRTVOL-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Sturzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 1.17 nM (pChEMBL 8.930000305175781)
Descriptors: MW 481.56, ALogP 3.04, HBA 8, HBD 3, PSA 123.88, QED 0.31
Compound (SMILES): NC/C(=C\F)COc1ccc(S(=O)(=O)CC23CCC(C(=O)N4CCCC4)(CC2)CC3)cc1
InChIKey: IFENRBXOGNPSBD-XDJHFCHBSA-N
Target: Amine oxidase [copper-containing] 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75106
Assay: SSAO Activity Assay: Amine oxidase activity of recombinant hSSAO, hMAO-A, and hMAO-B isoforms are measured using the MAO-Glo assay kit (Promega, V1402). Ten nL test compounds (with DMSO as vehicle, 0.1% v/v for hSSAO, hMAO-A and hMAO-B) were added into assay plates by Echo, and pre-incubated with 5 μL of enzyme for 30 mins at room temperature before the addition of 5 μL of luminogenic substrate. The substrate concentration is 40 μM for human recombinant SSAO, 40 μM for human recombinant MAO-A, and 4 μM for human recombinant MAO-B. The hSSAO and hMAO-A assays were conducted in the MAO-A reaction buffer in the kit, and the MAO-B assay was conducted in the MAO-B reaction buffer. Oxidation of the substrate was conducted for 1 hr, before the addition of detecting reagent according the manufacture's protocol. The IC50 value of the tested compounds was calculated by fitting the dose response curve using a 4-parameter non-linear regression routine.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 464.6, ALogP 3.61, HBA 5, HBD 1, PSA 89.7, QED 0.63
Compound (SMILES): CC(C)(O)[C@H](F)CNC(=O)c1cnc(Nc2nc3scnc3cc2F)cc1NCC(F)(F)F
InChIKey: VOQGJXFYFCSJMA-CYBMUJFWSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 490.46, ALogP 3.78, HBA 8, HBD 4, PSA 112.06, QED 0.36
Compound (SMILES): O=C(Nc1cc(-c2ccc3ncn(CC4CCOCC4)c3c2)c(Cl)cn1)c1cnc2c(c1)CCCO2
InChIKey: RYJKNNYVMROMSD-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615665 nm).
Measurement: IC50 = 510 nM (pChEMBL 6.289999961853027)
Descriptors: MW 503.99, ALogP 5.15, HBA 7, HBD 1, PSA 91.16, QED 0.4
Compound (SMILES): CC(C)C[C@@H](N)c1nc2cc(-c3ccncc3F)ccc2[nH]1
InChIKey: UIOIVMMOGBZAHU-CQSZACIVSA-N
Target: AP2-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q2M2I8
Assay: AAK1 Kinase Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2 and ATP) and test compounds in assay buffer (10 mM Tris-HCL pH 7.4, 10 mM MgCl2, 0.01% Tween-20 and 1.0 mM DTT). The reactions were initiated by the combination of bacterially expressed, GST-Xa-hAAK1 with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA buffer to each sample. The reactions were analyzed on the Caliper LabChip 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to EDTA quenched control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assays are ATP, 22 μM; (5-FAM)-Aha-KEEQSQITSQVTGQIGWR-NH2, 1.5 μM; GST-Xa-hAAK1, 3.5 nM; and DMSO, 1.6%. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 298.37, ALogP 3.81, HBA 3, HBD 2, PSA 67.59, QED 0.77
Compound (SMILES): CSC[C@H](N)[C@](O)(Cc1cc(CCCC2CC2)c(C)cn1)C(=O)O
InChIKey: VLBZWEZIKXOCHS-FUHWJXTLSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Rattus norvegicus
UniProt: P97629
Assay: Inhibition Assay: The inventors have assumed that inhibition of nocturnal activity of placental leucine aminopeptidase (P-LAP), i.e. aminopeptidase that cleaves AVP, would maintain and/or increase an endogenous AVP level to enhance the antidiuretic effect, which would contribute to a decreased number of nocturnal voids, and have extensively studied compounds which inhibit P-LAP.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 352.5, ALogP 2.17, HBA 5, HBD 3, PSA 96.44, QED 0.6
Compound (SMILES): CC(C)n1cnc2cc(-c3ccc4c(c3)N([C@H]3C[C@@H](N5CC6CCC5CC6)C3)C(=O)C4(C)C)nc(Nc3ccncc3F)c21
InChIKey: YVJZEBBSEQQMNE-BCCZULTASA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Enzymatic Activity Assay: Recombinant HPK1 kinase domain produced via baculovirus infection of insect cells was obtained from Proteros (Proteros Biostructures #PR-0322) and was pre-activated in the presence of 2 mM ATP (Sigma-Aldrich, cat #GE27-2056-01) and 2 mM magnesium chloride for 16 hours at 4° C. The protein reaction mixture was then loaded to a desalting column (Thermo Fisher Scientific, Cat #89889) to remove excess ATP. HPK1 was eluted with buffer containing 20 mM Tris (2-Amino-2-(hydroxymethyl)propane-1,3-diol) pH 8.0, 150 mM NaCl, 2 mM dithiothreitol and 5% glycerol, and was frozen at −80° C. for later use. HPK1 dual phosphorylation was confirmed by mass spectrometry.Ten nanoliters of test compounds dissolved in DMSO at various concentrations were dispensed into a 384-well ProxiPlate (PerkinElmer #6008289). Five microliters of a solution of recombinant HPK1 diluted in HPK1 kinase assay buffer (50 mM BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], pH 7.0; 10 mM magnesium chloride; 0.01% Triton X-100; 1 mM dithiothreitol; 0.01% bovine serum albumin; 0.1 mM sodium orthovanadate) was added to the compound-containing plate and was incubated for 15 minutes at 25° C. Five microliters of a mixture of ATP (Sigma-Aldrich #A6559) and peptide substrate STK S1 (Cisbio #61ST1BLC) diluted in HPK1 kinase assay buffer was then added to start the reaction. Final concentrations were 0.15 nM for HPK1, 10 μM for ATP, and 1 μM for the STK S1 peptide substrate. The reaction mixture was incubated at 25° C. for 3 hours and was stopped with the addition of 10 μl of an EDTA (Ethylenediaminetetraacetic acid)-containing detection buffer (Cisbio #62SDBRDF) supplemented with Europium cryptate-labeled anti-phospho-serine/threonine antibodies (Cisbio #62ST1PEJ) and XL665-labeled streptavidin (Cisbio #610SAXLG). The mixture was incubated for 16 hours at room temperature and peptide phosphorylation was measured by time-resolved fluorescence energy transfer (665 nm/620 nm) on an Envision plate reader (PerkinElmer).
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 593.75, ALogP 6.99, HBA 7, HBD 1, PSA 79.18, QED 0.26
Compound (SMILES): O=C(NCc1cc(-c2ccc(C(F)(F)F)nc2)ncn1)[C@@H]1C[C@@H](F)CN1S(=O)(=O)c1ccc(Cl)s1
InChIKey: ZQDXHKPYPKVBFP-DOMZBBRYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay: IC50s (effective concentration) of compounds on the human and rat TRPA1 channels were determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37C, and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes. at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 20 minutes at room temperature prior to adding agonist. Following this incubation, EC80 concentration of cinnamaldehyde (75 uM for human TRPA1 and 45 uM for rat TRPA1) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 549.96, ALogP 3.69, HBA 7, HBD 1, PSA 105.15, QED 0.47
Compound (SMILES): Cc1ccc(NC(=O)c2cc3cc(Br)cnc3n(Cc3ccc(F)cc3)c2=O)cc1
InChIKey: CGJPACGPCJFGLC-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: CB1 and CB2 Receptor Binding Assay: CB1R binding protocol involves the use of the same solution buffer used for both incubation and washing reaction (Tris-HCl, 50 mM; EDTA, 2.5 mM; MgCl2, 2.5 mM; BSA, 0.5 mg/mL at pH 7.4), 0.4 nM for [3H]CP-55,940, test compounds (concentrations from 0.001 to 10 μM), and finally 8 μg/sample membrane in a total volume of 200 μL. CB2R binding assays were carried out with two different buffers: incubation buffer (Tris-HCl, 50 mM; MgCl2, 5 mM; CaCl2 1 mM; BSA, 0.2% at pH 7.4) and washing buffer (Tris-HCl, 50 mM; NaCl 500 mM; BSA, 0.1% at pH 7.4). The assay mixture contained incubation buffer, 0.4 nM [3H]CP-55,940, test substances (concentrations from 0.001 to 10 μM), and 4 μg/sample membrane in a total assay volume of 200 μL. Assays were performed in duplicate and incubated for 120 mM at 37° C. After the incubation, the assay mixture is filtered through 96 GF/C filter plates (Perkin Elmer #6005174) using Perkin Elmer Filtermate Harvester, and then washed four times with ice-cold washing buffer.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 466.31, ALogP 4.91, HBA 4, HBD 1, PSA 63.99, QED 0.47
Compound (SMILES): C[C@@H](C1CC1)N1Cc2cc(-c3cnc4c(N)ncnn34)cc(C(O)(CO)C(F)(F)F)c2C1=O
InChIKey: VZMDCALWDLYZOV-SMNKDYLDSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 ��L. 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 PI3K�� 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 ��M PIP2, 2 ��M ATP, 0.5 ��Ci [��-33P]ATP, 3.4 nM PI3K��. Reactions were incubated for 120 min and terminated by the addition of 40 ��L 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 (PerkinElmer).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 462.43, ALogP 1.87, HBA 8, HBD 3, PSA 129.87, QED 0.53
Compound (SMILES): Cc1nnc(-c2cc(-c3cc(S(=O)(=O)NCC(C)(C)O)ccc3C)cnc2N)s1
InChIKey: OVCMBXRXDHSOSA-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Kinase Glo Luminescent Kinase Assay (Kglo) for PI 3-Kinase Alpha (A), PI 3-Kinase Beta (B), Vps34 (C), PI 4-Kinase Beta (D): The luminescence-based ATP detection reagent KinaseGlo was obtained from Promega, (Cat. No. V6714, Lot No. 236161) through Catalys, Wallisellen, Switzerland. L-alpha-phosphatidylinositol (PI, liver, bovine) was obtained from Avanti Polar Lipid (Cat. No. 840042C, Lot#LPI-274), phosphatidylinositol-4,5-bisphosphate (PIP(4,5)2) was obtained from Avanti Polar Lipid (Cat. No. 840046X). L-α-phosphatidylserine (PS) was obtained from Avanti Polar Lipid (Cat. No. 840032C), n-octylglucoside from Avanti Polar Lipid (Cat. No. 10634425001). Luminescence is a well established readout to determine ATP concentrations and can thus be used to follow the activity of many kinases regardless of their substrate. The Kinase Glo Luminescent Kinase Assay (Promega, Madison, Wis., USA) is a homogeneous HTS method of measuring kinase activity by quantifying the amount of ATP remaining in solution following a kinase reaction.50 nL of compound dilutions were dispensed onto black 384-well low volume Non Binding Styrene (NBS) plates (Costar Cat. No. NBS#3676). L-α-phosphatidylinositol (PI), provided as 10 mg/ml solution in methanol, was transferred into a glass tube and dried under a nitrogen beam. It was then resuspended in 3% OctylGlucoside (1-O-n-octyl-beta-D-glucopyranoside) by vortexing and stored at 4° C. 5 μl of a mix of PI/OctylGlucoside with the PI 3-kinase alpha and PI 3-kinase beta subtypes, or Vps34 or PI 4-kinase beta were added. Kinase reactions were started by the addition of 5 μl of an ATP-mix containing in a final volume 10 μl 10 mM TRIS-HCl pH 7.5, 3 mM MgCl2, 50 mM NaCl, 0.05% CHAPS, 1 mM DTT and 1 μM ATP at room temperature. Reactions were stopped with 10 μl of KinaseGlo and plates were read 10 mins later in a Synergy2 reader using an integration time of 0.1 seconds per well. 2.5 μM of NVP-BGT226 (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was added to the assay plates to generate the 100% inhibition of the kinase reaction, and the 0% inhibition was given by the solvent vehicle (90% DMSO in water). (1-(3-(trifluoromethyl)-4-(piperazin-1-yl)phenyl)-8-(6-methoxypyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-one) was used as a reference compound and included in all assay plates in the form of 16 dilution points in duplicate.IC50 values of the percentage inhibition of each compound at 8 concentrations (10, 3.0, 1.0, 0.3, 0.1, 0.030, 0.010 and 0.003 μM) n=2 were derived by fitting a sigmoidal dose-response curve to a plot of assay readout over inhibitor concentration as described. All fits were performed with the program XLfit4 (ID Business Solutions, Guildford, UK).
Measurement: IC50 = 1750 nM (pChEMBL 5.760000228881836)
Descriptors: MW 433.56, ALogP 2.52, HBA 8, HBD 3, PSA 131.09, QED 0.54
Compound (SMILES): N#Cc1ccc(N2CCN(Cc3cc(F)c4c5c(c(=O)[nH]c4c3)CCCN5)CC2)cc1C(F)(F)F
InChIKey: MLCFDNDRFBGJAZ-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Inhibition Assay: The PARP-1 enzyme inhibitory activities of the compounds of the present invention were assayed in the following manner by use of a kit (cat. 80551) purchased from BPS Bioscience.The 96-well plate provided in BPS Bioscience kit was coated with histone and incubated at 4° C. for 16 hours. Then, the plate was washed four times with PBST (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and blocking buffer (provided in BPS Bioscience kit) was added thereto in order to block nonspecific reaction, and was then incubated at 25° C. for 1 hour. After incubation for 1 hour, the plate was washed four times with PBST, and varying concentrations of each of the compounds of the Examples were added to a reaction solution containing PARP-1 enzyme (50 ng/well), an assay mixture and activated DNA, and allowed to react at 25° C. for 1 hour. After 1 hour, each well was washed four times with PBST, and in order to measure the level of ribosylation by PARP enzyme, streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added and allowed to react at 25° C. for 30 minutes. The plate was washed four times with PBST, and then finally an HRP chemiluminescent substrate was added and allowed to react. The level of histone ribosylation formed by each enzyme was quantified using Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., USA).
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 485.49, ALogP 4.24, HBA 5, HBD 2, PSA 75.16, QED 0.54
Compound (SMILES): CC(C)(C)NC(=O)CN1[C@@H]2CC[C@H]1C[C@H](CC(=O)Nc1cccc(Cl)c1)C2
InChIKey: IDYJBJPAOZYLAH-GJWQZFMSSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Activity Assay: The HEK293 cells having human Cav3.2 stably expressed therein were cultured at 37° C. in Alpha-MEM, to which 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), and G418 (250 g/mL) had been added. The cells were suspended in the culture liquid and were inoculated onto a 96-well plate. Subsequently, the cells were cultured for 48 hours. The culture liquid was removed, and the liquid was changed to S-MEM, to which 5% (v/v) FBS, calcium chloride (0.5 mmol/L), L-glutamine (2 mmol/L), L-alanine (8.9 ng/mL), L-asparagine (13.2 ng/mL), L-aspartic acid (1.33 ng/mL), L-glutamic acid (14.7 ng/mL), glycine (7.5 ng/mL), L-proline (11.5 ng/mL, L-serine (10.5 ng/mL), penicillin (100 U/mL), and streptomycin (100 μg/mL) had been added. The cells were cultured for another 24 hours. The culture liquid was removed again, and the cells were washed with an assay buffer (140 mmol/L sodium chloride, 5 mmol/L potassium chloride, 0.5 mmol/L magnesium chloride, 0.5 mmol/L calcium chloride, 10 mmol/L glucose, 0.4 mmol/L magnesium sulfate, 10 mmol/L HEPES, and 250 mol/L sulfinpyrazone, pH 7.4) that had been kept warm at 37° C. Subsequently, an assay buffer prepared by dissolving Fura2-AM, which is a fluorescent Ca2+ indicator, to a concentration of 5 μM, was added to the cells, and the system was incubated for 30 minutes at 37° C. The assay buffer having Fura2 dissolved therein was removed, and the cells were washed with the assay buffer. Subsequently, an assay buffer prepared by adding a test compound was added to the cells, and the system was incubated for 15 minutes. The plate was mounted on a fluorescence analyzer (FLEX STATION II, Molecular Devices, LLC), and the baseline was measured for 20 seconds. Subsequently, any change in the intracellular calcium concentration that was induced when an assay buffer prepared by adding 100 mmol/L calcium chloride was added to the cells, was measured (excited at 340 nm or 380 nm, and detected at 510 nm).
Measurement: IC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 391.94, ALogP 3.83, HBA 3, HBD 2, PSA 61.44, QED 0.8