lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(Cc1ccccc1Cl)C(=O)N1[C@H]2CC[C@@H]1[C@@H](C(=O)O)N(C(=O)N(c1ccccc1)c1ccccc1)C2
InChIKey: XCUSDOVBIRIUJR-DMDYGQEQSA-N
Target: Type-1 angiotensin II receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30556
Assay: Biological Assay: Measurement of Inhibitory Activity on AT1 Receptor (AT4R)/AT2 Receptor (AT2R)Through the following steps, the inhibitory activity of the compound on AT1R/AT2R (IC50 value) was determined:1) An appropriate amount of 1×TLB (Tag-lite Buffer) was prepared and well mixed for use.2) The compound was diluted by 10 times with ddH2O or DMSO. The compound was then dilute to 4 times of the working concentration with 1×TLB and mixed well for use.3) 8600 nM Tag-lite angiotensin receptor red agonist was diluted to 12 nM (4×Kd) with 1×TLB.4) 5 ml 1×TLB was taken into a 15 ml centrifuge tube.5) After thawing 1 tube of Tb-labeled AT1R/AT2R cells in a 37° C. water bath, the cells were quickly transferred to the 1×TLB in step 4), mixed gently, and centrifuged at 1200 g for 5 minutes at room temperature.6) The supernatant was aspirated gently, and the cells were resuspended and mixed in 2.7 ml 1×TLB, and then placed at room temperature until use.7) 10 μl cells were added to all test wells, and 5 μl 4× working solution of the compound from step 2) was added to the corresponding test wells. 5 μl 4× Tag-lite angiotensin receptor red agonist well diluted in step 3) was added to all test wells.8) After leaving the reaction plate at room temperature for 1 h, data were measured and analyzed using Envision HTRF Reader, and the half inhibitory concentration (IC50) of the compound on AT1R/AT2R was calculated with the GraphPad Prism four-parameter equation.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 533.03, ALogP 5.45, HBA 3, HBD 1, PSA 84.4, QED 0.47
Compound (SMILES): O=C(CO)N1CCC[C@@H](Nc2nc(-c3cnn4ccccc34)nc(OCC(F)F)c2F)C1
InChIKey: YPBDPMATNYYAFH-GFCCVEGCSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In Vitro JAK kinase Assays: The catalytic domains of human JAK1 (aa 850-1154), JAK2 (aa 826-1132), JAK3 (aa 795-1124) and Tyk2 (aa 871-1187) were expressed as N-terminal GST-fusion proteins using a baculovirus expression system and were purchased from Carna Biosciences. The enzymatic activity was assayed using as substrate a biotinylated peptide, poly (GT)-Biotin (CisBio). The peptide concentration in the reactions was 60 nM for JAK1, 20 nM for JAK2, 140 nM for JAK3 and 50 nM for Tyk2. The degree of phosphorylation was detected by TR-FRET (time-resolved fluorescence energy transfer).IC50s of compounds were measured for each kinase in a reaction mixture containing the enzyme, ATP and the peptide in 8 mM MOPS (pH 7.0), 10 mM MgCl2, 0.05% β-mercaptoethanol, 0.45 mg/ml BSA. The ATP concentration in the reactions was 3 μM for JAK1, 0.2 μM for JAK2, 0.6 μM for JAK3 and 1.8 μM for Tyk2. The enzymatic reactions took place for 30 minutes at room temperature. Then, the reactions were stopped with 20 μL of quench detection buffer (50 mM HEPES, 0.5 M KF, EDTA 0.25 M, 0.1% (w/v) BSA, pH 7.5) containing 0.115 μg/mL of anti-phosphoTyr (PT66)-Cryptate (CisBio) and a variable concentration of SA-XL665 (CisBio) to keep the SA-B ratio constant. Incubate for 3 h and read on Victor 2V spectrofluorometer (Perkin Elmer) set to read fluorescence resonance energy transfer.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 450.42, ALogP 1.97, HBA 8, HBD 2, PSA 104.88, QED 0.57
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc3c(c(Nc4cnn(C5CCOCC5)c4)n2)C(=O)NC3)c1
InChIKey: FMFYSVFDXLEMDW-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: EGFR Kinase Inhibition Assay: The inhibitory activity of compounds to be tested on wild-type EGFR kinase (Invitrogen, PV3872) was measured by z-lyte methods.The working concentration of each component in 10 μl of wild-type EGFR kinase reaction system was as follows: 10M ATP, 0.8 ng/μl wild-type EGFR kinase (Invitrogen, PV3872), and 2 μM Tyr04 substrate (Invitrogen, PV3193). After the compounds to be tested were added, the final concentration of DMSO was 2%.10 mM drug stock solutions dissolved at room temperature were gradiently diluted with 4% DMSO in water to a final concentration of 10-0.005 μM. To each well were added 2.5 μl of a solution of the compounds to be tested and 5 μl of a mixture of wild-type EGFR kinase and Tyr04 substrate diluted by a reaction buffer, and then 2.5 μl of ATP was added to initiate the reaction. Reaction buffer instead of ATP were added to C1 wells, no drugs were added to C2 wells, and the phosphorylated substrates were added to C3 wells according to the instruction. After reacting on a shaker at 25° C. for 60 minutes in the dark, 5 μl of Development Reagent B (Invitrogen, diluted with TR-FRET dilution buffer) was added, and reacted on a shaker at room temperature for 60 min. The plate was read in a VictorX5 fluorescent microplate reader (PerkinElmer) and the light absorbance at an excitation wavelength of 405 nm and emission wavelengths of 450 nm and 520 nm was measured (For example, C3520 nm represents the reading of C3 well at 520 nm).
Measurement: IC50 = 6989 nM (pChEMBL 5.159999847412109)
Descriptors: MW 460.5, ALogP 2.88, HBA 9, HBD 4, PSA 135.09, QED 0.4
Compound (SMILES): C=CC(=O)N1CCN(C(=O)c2cc(Sc3cnc(NC(=O)C4CC4)s3)c(C)cc2OC)[C@H](C)C1
InChIKey: SEZAPPISGBESSS-OAHLLOKOSA-N
Target: Tyrosine-protein kinase TXK (SINGLE PROTEIN), Homo sapiens
UniProt: P42681
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 500.65, ALogP 3.82, HBA 7, HBD 1, PSA 91.84, QED 0.58
Compound (SMILES): CCC(=O)Nc1cncc(-c2cc3c(-c4nc5c(-c6cccs6)cccc5[nH]4)n[nH]c3cn2)c1
InChIKey: VHNYODWWMZXVMO-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: TGF-beta 1 Assay: Compound Screening: Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:2, 11-point dose-response curves from 10 μM to 1.87 nM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 384-well clear bottom assay plates (Greiner Bio-One) with appropriate DMSO backfill for a final DMSO concentration of 0.1%. LL29 cells were plated at 1,500 cells/well in 80 μl/well F12 medium supplemented with 1% Fetal Bovine Serum. One hour after addition of the cells, TGF-β1 (Peprotech; 20 ng/mL) was added to the plates to induce fibrosis (ref. 1 and 2 above). Wells treated with TGF-β1 and containing DMSO were used as controls. Cells were incubated at 37° C. and 5% CO2 for 4 days. Following incubation for 4 days, SYTOX green nucleic acid stain (Life Technologies [Thermo Fisher Scientific]) was added to the wells at a final concentration of 1 uM and incubated at room temperature for 30 min. Cells were then fixed using 4% formaldehyde (Electron Microscopy Sciences), washed 3 times with PBS followed by blocking and permeabilization using 3% Bovine Serum Albumin (BSA; Sigma) and 0.3% Triton X-100 (Sigma) in PBS. Cells were then stained with antibody specific to α-smooth muscle actin (aSMA; Abcam) (ref. 1 and 2 above) in 3% Bovine Serum Albumin (BSA; Sigma) and 0.3% Triton X-100 (Sigma) in PBS, and incubated overnight at 4° C. Cells were then washed 3 times with PBS, followed by incubation with Alexa Flor-647 conjugated secondary antibody (Life Technologies [Thermo Fisher Scientific]) and DAPI at room temperature for 1 hour. Cells were then washed 3 times with PBS and plates were sealed for imaging. αSMA staining was imaged by excitation at 630 nm and emission at 665 nm and quantified using the Compartmental Analysis program on the CellInsight CX5 (Thermo Scientific). Dead or apoptotic cells were excluded from analysis based on positive SYTOX green staining. % of total cells positive for αSMA were counted in each well and normalized to the average of 11 wells treated with TGF-β1 on the same plate using Dotmatics' Studies Software. The normalized averages (fold change over untreated) of 3 replicate wells for each compound concentration were used to create dose-responses curves and EC50 values were calculated using non-linear regression curve fit in the Dotmatics' Studies Software.
Measurement: EC50 = 455 nM (pChEMBL 6.340000152587891)
Descriptors: MW 465.54, ALogP 5.64, HBA 6, HBD 3, PSA 112.24, QED 0.3
Compound (SMILES): FC(F)(F)c1ccc(COc2ccc(Cl)cc2OC2CCNCC2)o1
InChIKey: HHMUEKPUPRXUSW-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Binding Assay: Receptor binding was performed using membrane fractions prepared from the HEK-293 cell line recombinantly expressing rat 5-HT7 receptors (NCBI accession NM_022938). Compound affinity for the rat 5-HT7 receptor subtype was evaluated by competitive radioligand binding assays using 5-carboxamido[3H]tryptamine ([3H]5-CT) (Amersham Biosciences, cat. 90000403) detection. HitHunter cAMP assays are in-vitro based competitive immunoassays. The assay was performed on the HEK-293 cell line stably transfected with r5-HT7 receptor. Cells were pre-incubated with test compounds for 10 minutes. For antagonist testing, the cells were then challenged with 100 nM 5-CT for 20 minutes. Cells were then lysed and cAMP measured according to manufacturers protocol (Amersham, cat. NET791250UC) or [3H]mesulergine (Amersham, cat. TRK1041). The assay was performed on membranes prepared from HEK-293 cells stably transfected with h5-HT6. Following centrifugation, membranes were resuspended and incubated for 60 min at room temperature with 1.7 nM [3H]LSD in the presence of increasing concentration of test compounds. Nonspecific binding was defined in the presence of 10 μM clozapine (Tocris, cat. TRK1068). Homogenized HEK-293 membranes expressing the human SERT were incubated in 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5 mM KCl with [3H]-citalopram (3 nM) with or without test compounds. Nonspecific binding was determined in the presence of 10 μM fluoxetine. Radioactivity readouts and Ki values were performed as previously described for r5-HT7.
Measurement: Ki = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 375.77, ALogP 4.66, HBA 4, HBD 1, PSA 43.63, QED 0.82
Compound (SMILES): Cc1ccc(C(C)N2CCC(c3ccc4[nH]c(-c5cc(C)c6ncnn6c5)c(C(C)C)c4c3)CC2)nn1
InChIKey: INTQGSOYPDVJFV-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 11.8 nM (pChEMBL 7.929999828338623)
Descriptors: MW 493.66, ALogP 6.35, HBA 6, HBD 1, PSA 75, QED 0.31
Compound (SMILES): O=C(NCCN1CCOCC1)c1n[nH]c2ccc(-c3cccnc3)cc12
InChIKey: BZVVVXRNQBZIHM-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: DYRK1A activity assay: SH-SY5Y cells are cultured in DMEM/F-12 medium supplemented with 15% FBS, Non-essential Amino Acid and Penicillin/Streptamycin. Two days before treatment, cells are seeded onto 96 well plates at 20e5 cells/well.DMSO-resuspended compounds are dispensed to 8 wells as a serial titration from 10 μM to 4.6 nM final and cells are exposed overnight (16-18 h) before harvest. Wells are visualized checked for cell death or change in morphology and supernatants are tested for cytotoxicity by measurement of lactate dehydrogenase release (LDH, CytoToxOne kit, Progema) if necessary. As controls, commercially available DYRK1A inhibitors, Harmine and Indy were shown to have good DYRK1A inhibition in the kinase assay with no CDK1 activity (EC50 18 and 53n M respectively, 6 μM for CDK1) but weak EC50 in the Tau assay: about 10 μM for Harmine and 30 μM for Indy.Cells are lyzed with RIPA buffer complemented with phosphatase and protease inhibitors (Thermo Scientific) then lysates are sonicated and spun down at 12,000 g for 10 min to remove any cellular debris. Lysates are then either directly tested for pSer396 by ELISA (Life Technology, Kit KHB7031) or loaded on NuPage Bis-Tris gels for western blot analysis. Colorimetric detection of ELISA signal is performed by Cytation3 plate reader (Biotek) and the chemoluminecence signal for HRP-linked antibodies used in western blotting is detected using a Carestream Image Station. The same pSer396 antibody is used for detection of pTau in both assays.Blot densitometry for pSer396 and beta-actin are analyzed using ImageJ (NIH) and pSer396/Total Tau ELISA signal was used to plot, draw the curve fitting, and determine each compounds EC50 in Prism (GraphPad).
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 351.41, ALogP 1.69, HBA 5, HBD 2, PSA 83.14, QED 0.73
Compound (SMILES): CN([C@H]1CC[C@H](Oc2ccc(-c3ccn[nH]3)nc2)CC1)S(C)(=O)=O
InChIKey: KJFFGYMXNBCZEN-JOCQHMNTSA-N
Target: Cytochrome P450 4A11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02928
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 350.44, ALogP 2.05, HBA 5, HBD 1, PSA 88.18, QED 0.89
Compound (SMILES): C=CC(=O)N1CCC[C@@H](n2nc(C(=O)Nc3nc4cc(F)ccc4o3)c3c(N)ncnc32)C1
InChIKey: XCTFFUYIMFTLML-GFCCVEGCSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: EGFR Inhibitory Activity: With regard to the setting of the conditions for a method for measuring the inhibitory activity of a compound against EGFR kinase activity in vitro, it is described in the consumable reagent supplies price list for LabChip (registered trademark) series of PerkinElmer, Inc. that FL-PEPTIDE 22 corresponds to a substrate peptide for the measurement of EGFR kinase activity. Therefore, a biotinated peptide (biotin-EEPLYWSFPAKKK) was produced by referring to the amino acid sequence of the peptide. The purified recombinant human EGFR protein used in the test was purchased from Carna Biosciences, Inc.With regard to the measurement of the inhibitory activity of the compounds, firstly, the compounds of the present invention were diluted stepwise with dimethyl sulfoxide (DMSO). Subsequently, EGFR protein, a substrate peptide (final concentration was 250 nM), magnesium chloride (final concentration was 10 mM), manganese chloride (final concentration was 10 mM), ATP (final concentration was 1.5 μM), and a DMSO solution of the compound of the present invention (final concentration of DMSO was 2.5%) were added to a buffer solution for kinase reaction (20 mM HEPES (pH 7.5), 2 mM dithiotheitol, 0.01% Triton X-100), and after the solution was incubated for 120 minutes at 25° C., a kinase reaction was carried out. The reaction was terminated by adding EDTA thereto in order to obtain a final concentration of 24 mM. Subsequently, a detection liquid containing Eu-labeled anti-phosphorylated tyrosine antibody PT66 (PerkinElmer, Inc.) and SURELIGHT APC-SA (PerkinElmer, Inc.) was added thereto, and the system was left to stand for 2 hours or longer at room temperature. Finally, the amount of fluorescence upon irradiation of excitation light having a wavelength of 337 nm was measured at two wavelengths of 620 nm and 665 nm, using a PHERAstar FS (BMG Labtech GmbH). The amount of phosphorylation reaction was determined from the ratio of the amounts of fluorescence at the two wavelengths, and the compound concentration at which the phosphorylation reaction could be suppressed in 50% was defined as the IC50 value (nM).
Measurement: IC50 = 84.2 nM (pChEMBL 7.079999923706055)
Descriptors: MW 450.43, ALogP 2.29, HBA 9, HBD 2, PSA 145.06, QED 0.45
Compound (SMILES): CC(C)N1CCC(Oc2cc(C(=O)Nc3cc4cc(-c5cnn(C)c5)ccc4cn3)ccn2)CC1
InChIKey: UUMQTWFSQKEZMP-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: The DYRK1A kinase assa: Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 470.58, ALogP 4.53, HBA 7, HBD 1, PSA 85.17, QED 0.45
Compound (SMILES): Cc1ccc(F)c(NC(=O)Nc2ccc(-c3cc(C(=O)N=S(C)(C)=O)cnc3N)cc2)c1
InChIKey: YEDBBOGEZDFFBL-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS +0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 2.7 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 455.52, ALogP 4.29, HBA 5, HBD 3, PSA 126.54, QED 0.54
Compound (SMILES): Cc1c(-c2cc3cc(NC(=O)O[C@H]4C[C@](C)(O)C4)ncc3c(N)c2F)cnc2c1NCCO2
InChIKey: TULIDBIBOWWIFQ-WXHQJSEUSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: TBD: TBD
Measurement: Ki = 0.253 nM (pChEMBL 9.600000381469727)
Descriptors: MW 453.47, ALogP 3.59, HBA 8, HBD 4, PSA 131.62, QED 0.44
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H](C(=O)N1Cc2ccc(NC(=O)CNC(=O)[C@]3(C)CN(C(=O)CN4C[C@@H](C)NC[C@@H]4CN4CCOC[C@H]4C)c4cc(Cc5ccc(F)cc5)ccc43)cc2[C@H]1C(=O)Nc1c(F)cccc1F)C(C)(C)C
InChIKey: IBZIIVISPQLELC-CEHVYTRKSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assays for XIAP BIR3: For the Bir3 assay, 13.3 nanomolar BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 25.2 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 24) 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Alliophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 0.6 nM and 2.5 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 0.5 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 1065.25
Compound (SMILES): CNCCC(Oc1ccc(CN2CCN(C)c3nc(C)ccc3C2=O)cc1)c1cccs1
InChIKey: PVIBEZAPXVLWAG-UHFFFAOYSA-N
Target: Sodium-dependent noradrenaline transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P23975
Assay: Binding Assay: Human norepinephrine transporter (NET) enriched membranes (5 μg) were incubated with 5 nM of radiolabeled [3H]-Nisoxetin in assay buffer containing 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4.NSB (non specific binding) was measured by adding 10 μM desipramine. The binding of the test compound was measured in five different concentrations. After 60 min incubation at 4° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, 0.9% NaCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.
Measurement: Ki = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 450.61, ALogP 4.27, HBA 6, HBD 1, PSA 57.7, QED 0.56
Compound (SMILES): CNCc1cc(OCc2cc(C)ccc2F)n(C(C)C2CCCCC2)n1
InChIKey: MUKFIBLLWQIOSI-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Receptor Binding Assay: 5-HT2C: A solution containing 50 μL of [H]-mesulergine (GE Healthcare) diluted with 50 mmol/L Tris-HCl (pH=7.4) (the final concentration: about 2 nM), 149 μL of 5-HT2C/CHO cell membrane sample (20 μg/well for the amount of protein), and 1 μL of a solution of a test compound dissolved in DMSO or a solvent (DMSO) was reacted at 37° C. for 30 minutes, and then filtered promptly by aspiration under lower pressure with a glass fiber filter coated with a 1% aqueous solution of bovine serum albumin. The filtered substance on the glass fiber filter was washed with 250 μL of 50 mmol/L Tris-HCl (pH=7.4) twice, and then transferred to an ACS-II (Amersham) 4-mL-glass vial. The radioactivity of the filtered substance remained on the filter was measured by liquid scintillation counter. The radioactivity value measured by liquid scintillation counter was deemed to be a receptor binding activity, and the binding inhibition was calculated from the total binding (TB), non-specific binding (NSB), and specific binding (SB) of a test compound according to the following equations.
Measurement: Ki = 3.7 nM (pChEMBL 8.430000305175781)
Descriptors: MW 359.49, ALogP 4.77, HBA 4, HBD 1, PSA 39.08, QED 0.77
Compound (SMILES): CN1CCN(Cc2ccc(N(C)C(=O)Nc3cc(Cl)c(C#N)cn3)nc2C=O)C(=O)C1
InChIKey: HNLOTQJJGQNFHJ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Inhibition Assay: FGFR1: The in vitro activity of FGFR1 was determined by assaying the phosphorylation level of the substrate in the kinase reaction, by means of an HTRF kinase assay kit. The reaction buffer comprised the following components: 5-fold diluted Enzymatic buffer/kinase 5× (main ingredient: 50 mM HEPES, pH 7.0), 5 mM MgCl2, 1 mM DTT; the human recombinant FGFR1 catalytic structural domain protein (amino acids 308-731) was purified by the company itself, diluted with the reaction buffer to a 0.6 ng/μL kinase solution; the substrate reaction solution comprised a biotin labeled tyrosine kinase substrate diluted with the reaction buffer to 400 nM , and 40 μM ATP, and the assay solution comprised an Eu3+ labeled cage-shaped antibody diluted with the assay buffer to 0.125 ng/μL, and 25 nM streptavidin labeled XL665.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 441.88, ALogP 1.76, HBA 7, HBD 1, PSA 122.53, QED 0.7
Compound (SMILES): COc1ccc(F)c(-c2ncc(COc3cccc([C@@H](CC(=O)O)C4CC4)c3)nc2-c2ccc(C(C)C)cc2)c1
InChIKey: NLCAAJNMYUAZQJ-NDEPHWFRSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q8K3T4
Assay: Calcium Flux Assay: Compounds were tested in a calcium flux assay using transfected HEK293 cells stably expressing either human GPR40 or rat GPR40. Human GPR40 expressing cells were cultured in DMEM-High Glucose media supplemented with 10% fetal bovine serum, 1×L-Glutamine, 1× Penicillin/Streptomycin and 500 μg/mL G418. Rat GPR40 expressing cells were cultured in DMEM-High Glucose media supplemented with 10% fetal bovine serum and 1 μg/mL puromycin. Cells were plated into poly-D-lysine coated 384-well plates and cultured overnight in a 37° C. humidified tissue culture incubator under 5% CO2/90% O2 atmosphere. On the day of the experiment, the culture media was replaced with assay buffer (HBSS, 20 mM HEPES, 0.1% BSA) and the cells incubated at 37° C. for 1 h. Calcium-sensitive fluorescent dye (Fluo 8 No-Wash Calcium Dye, ABD Bioquest) was then added and the cells incubated for another 30 min at 37° C. followed by 15 min at room temperature while protected from the light. The cell plate and a plate of diluted compounds of Formula (I) were loaded into a fluorescent plate reader that added compounds onto the cells while measuring the fluorescence intensity of each well. The plate reader recorded fluorescence intensity at 1 second intervals for 8 min and provided the data for analysis in an Excel format.
Measurement: EC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 540.64, ALogP 7.63, HBA 5, HBD 1, PSA 81.54, QED 0.21
Compound (SMILES): Cn1cc(C(=O)Nc2cc(CC(=O)O)cc(-c3nnc(Nc4ccc5[nH]ncc5c4Cl)s3)c2)cn1
InChIKey: QBCWKHAUBKKIGU-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Biological Assays for Inhibition of Rho-Associated Protein Kinase: Assays for ROCK inhibition were performed using the following protein constructs: glutathione S-transferase (GST)-tagged human ROCK1 catalytic domain 1-477 from Carna Biosciences (cat #01-109; apparent Km value for ATP is 10 μM) and GST-tagged human ROCK2 catalytic domain 1-553 from Carna Biosciences (Cat#01-110; apparent Km value for ATP is 15 μM). Protein constructs were purified from a baculovirus expression system. The peptide substrate was fluorescent LANCE® Ultra ULight-CREBtide: CKRREILSRRPSYRK (PerkinElmer, # TRF0107-D). Kinase reactions were carried out in in a 10 μL volume in 384-well plates: 50 nM ULight-CREBtide substrate, 2 nM constitutively active ROCK1 or ROCK2 kinase, and test compound in DMSO (or DMSO only for controls) were diluted into assay buffer containing 50 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, and 2 mM DTT such that the final concentration of DMSO was 0.5%. After a 90 minute incubation at room temperature on a shaker table, the kinase reaction was stopped by addition of 10 mM EDTA, and phosphorylation of the substrate was detected by adding 1 nM LANCE® Ultra Europium-anti-phospho-CREB (ser133) antibody (PerkinElmer, # TRF0200-D) and incubating for 60 minutes on a shaker at room temperature. The flurorescence resonance energy transfer (FRET) signals were read and analyzed on an Envision¿ 2103 Multilabel Reader (Perkin Elmer). The concentration of test compound required to inhibit substrate phosphorylation by 50% (the IC50) was calculated by non-linear regression using GraphPad PRIZM.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 508.95, ALogP 4.09, HBA 9, HBD 4, PSA 150.71, QED 0.26
Compound (SMILES): CC(=O)NCCOc1cc2ncc(-c3ccc(CC(=O)Nc4cc(C(C)(C)C(F)(F)F)on4)c(F)c3)nc2cc1OCCNC(C)=O
InChIKey: LILNWRHOGNBMAS-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005,310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and diluted into the aqueous environment. Kds were determined using an eleven point threefold serial dilutions. DMSO or control compounds were was added to control assays lacking a test compound.
Measurement: Kd < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 660.63, ALogP 4.47, HBA 9, HBD 3, PSA 157.57, QED 0.14
Compound (SMILES): Fc1ccc(-n2cc(CNc3ncc(Cl)cn3)nn2)cc1C(F)F
InChIKey: MDSVIRSWONSGIY-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: NMDA receptor FLIPR 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 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 354.72, ALogP 3.4, HBA 6, HBD 1, PSA 68.52, QED 0.76
Compound (SMILES): COc1cc(-c2cnn(C3CCOCC3)c2)ccc1Nc1cc(NC(=O)C2CC2)nc2cc[nH]c(=O)c12
InChIKey: BVMXUYPAODTXRG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 LanthaScreen JH2-V617F Binding Assay: JAK2 JH2-V617F binding assay utilizes pseudo-kinase domain (JH2, amino-acids 536-812 with 3 surface mutations W659A, W777A, F794H) of human V617F mutant JAK2 expressed as C-terminal His-Avi-tagged, biotinylated protein in a baculovirus expression system (BPS Bioscience, Catalog #79498). The assay was conducted in black 384-well polystyrene plates in a final reaction volume of 20 μL. JAK2 JH2-V617F (0.26 nM) was incubated with compounds (100 nL serially diluted in DMSO) in the presence of 50 nM Fluorescent JAK2-JH2 Tracer (MedChem Express Catalog #HY-102055) and 0.25 nM Streptavidin-Tb cryptate (Cisbio Part #610SATLB) in assay buffer (50 mM Tris, pH=7.5, 10 mM MgCl2, 0.01% Brij-35, 0.1% BSA, 1 mM EGTA, 5% Glycerol and 5 mM DTT). Non-specific binding was accessed in the presence of 2 mM ATP. After incubation for 1 hour at 25° C., LanthaScreen signals were read on a PHERAstar FS plate reader (BMG LABTECH). Data was analyzed with IDBS XLfit and GraphPad Prism 5.0 software using a four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 500.56, ALogP 4.24, HBA 8, HBD 3, PSA 123.16, QED 0.35
Compound (SMILES): CC(C)(C)Oc1ccc(-c2cc3c(N4CC(C(=O)NCc5ccc(Cl)cc5)C4)ncnn3c2)cc1
InChIKey: VPZCOGGZDCVEPJ-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit D816V Assay: In each well of a 384-well plate, 0.04 ng/ul (0.5 nM) of D816V 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 15 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 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 490.01, ALogP 4.98, HBA 6, HBD 1, PSA 71.76, QED 0.41
Compound (SMILES): Cn1cc(-c2cccc(-c3ncc(-c4cnn(C)c4)c(NC4CCC4)n3)c2)cn1
InChIKey: IQMFQXFGETWXJL-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Enzymatic Assay: IRAK4 is a human purified recombinant enzyme (His-TEV-IRAK1 (194-712). IRAK4 hydrolyses ATP, autophosphorylates and phosphorylates a Serine/Threonine generic peptidic substrate (STK: 61ST1BLC from CisBio International based in Bagnols/Cèze FR).Measurement of IRAK-4 inhibition was performed in streptavidin coated 384well FlashPlate (PerkinElmer #SMP410A). His-TEV-IRAK4 (20 ng/well), ATP (2 μM, [33P]ATP 0.25 Ci/well), STK1-biotin peptide (300 nM) and compounds in DMSO (range of concentrations from 20 M to 1 nM) or controls (2% DMSO) were incubated for 3 hours at 30° C. in assay buffer: Hepes pH7.0 50 mM, Fatty acid-free BSA 0.1%, Dithiothreitol DTT 2 mM, MgCl2 10 mM, EGTA 0.5 mM, Tween-20 0.01%, MnCl2 5 mM.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 385.48, ALogP 3.91, HBA 7, HBD 1, PSA 73.45, QED 0.56
Compound (SMILES): CCC(c1ccc(C2CC2)cc1)N1C[C@H](C)N(c2nc(=O)n(C)c3ccc(C#N)nc23)C[C@H]1CC
InChIKey: JCMBFPCVVCXWKC-HYECRXSHSA-N
Target: Diacylglycerol kinase zeta (SINGLE PROTEIN), Homo sapiens
UniProt: Q13574
Assay: In Vitro DGK Inhibition Assay: The reactions were carried out in 50 mM MOPS pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 μM CaCl2, and 1 mM DTT (assay buffer). The reactions using a detergent/lipid micelle substrate also contained 50 mM octyl B-D-glucopyranoside. The lipid substrate concentrations were 11 mM PS and 1 mM DAG for the detergent/lipid micelle reactions. The lipid substrate concentrations were 2 mM PS, 0.25 mM DAG, and 2.75 mM PC for the extruded liposome reactions (5 mM total lipid). The reactions were carried out in 150 μM ATP. The enzyme concentrations for the DGKα and DGKζ were 5 nM. The compound inhibition studies were carried out as follows: 25 nL (ADPGLO assay) or 50 nL (LIPGLO assay) droplets of each test compound (top concentration 10 mM with 11 point, 3-fold dilution series for each compound) solubilized in DMSO were transferred to wells of a white 1536 well plate (Corning 3725). A 5 mL enzyme/lipid substrate solution at 2× final reaction concentration was prepared by combining 2.5 mL 4× enzyme solution (20 nM DGKα or DGKζ (prepared as described below) in assay buffer) and 2.5 mL of either 4× liposome or 4× detergent/lipid micelle solution (compositions described below) and incubated at room temperature for 10 minutes. Next, 1 μL 2× enzyme/lipid substrate solution was added to wells containing the test compound and reactions were initiated with the addition of 1 μL 300 uM ATP. The reactions were allowed to proceed for 2 hr (ADPGLO assay) or 1 hr (LIPGLO assay), after which 2 μL Glo Reagent (Promega V9101) was added and incubated for 40 minutes. Next, 4 μL Kinase Detection Reagent was added and incubated for 30 minutes. Luminescence was recorded using an EnVision microplate reader. The percent inhibition was calculated from the ATP conversion generated by no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition.
Measurement: IC50 = 430 nM (pChEMBL 6.369999885559082)
Descriptors: MW 470.62, ALogP 4.52, HBA 7, HBD 0, PSA 78.05, QED 0.53
Compound (SMILES): CC(C)(O)C(O)CSc1nonc1/C(=N/O)Nc1ccc(F)c(Cl)c1
InChIKey: IUXNKJYDZMPGEO-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 uM delta aminolevulinic acid for 48 h at 16° C. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 uM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 uM) or buffer alone (30 uM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at RT for 30 min. Afterwards, 10 uL of 400 uM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at RT for 60 min and reactions were quenched by addition of 10 uL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 h or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 min at 1000 ug. The resulting fluorescence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 79.1 nM (pChEMBL 7.099999904632568)
Descriptors: MW 390.82, ALogP 2.33, HBA 8, HBD 4, PSA 124, QED 0.19
Compound (SMILES): Cc1cc(S(=O)(=O)NCCOCCOCCNC(=O)NCCCCNC(=O)NCCOCCOCCNS(=O)(=O)c2ccc(O[C@H]3c4cc(Cl)cc(C#N)c4C[C@@H]3N3CCC[C@@H](N(C)C)C3)c(C)c2)ccc1O[C@H]1c2cc(Cl)cc(C#N)c2C[C@@H]1N1CCC[C@@H](N(C)C)C1
InChIKey: CQYYERZXUMICCO-IDGSHISQSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Cell-Based Assay of NHE-3 Activity (Persistent Inhibition): The ability of compounds to inhibit human and rat NHE-3-mediated Na+ dependent H+ antiport after application and washout was measured using a modification of the pH sensitive dye method described above. PS120 fibroblasts stably expressing human NHE3 and NHERF2 were obtained from Mark Donowitz (Baltimore, Md.). Opossum kidney (OK) cells were obtained from the ATCC and propagated per their instructions. The rat NHE-3 gene was introduced into OK cells via electroporation, and cells were seeded into 96 well plates and grown overnight. Medium was aspirated from the wells, cells were washed once with NaCl-HEPES buffer (100 mM NaCl, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4), then overlayed with NaCl-HEPES buffer containing 0-30 μM test compound. After a 60 min incubation at room temperature, the test drug containing buffer was aspirated from the cells. Following aspiration, cells were washed once with NaCl-HEPES buffer without drug, then incubated for 30 min at 37° C. with NH4Cl-HEPES buffer (20 mM NH4Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) containing 5 μM BCECF-AM. Cells were washed once with Ammonium free, Na+-free HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and incubated in the same buffer for 10 minutes at room temperature to lower intracellular pH. NHE-3-mediated recovery of neutral intracellular pH was initiated (10 min after compound washout) by addition of Na-HEPES buffer. For the rat NHE3 assay, the Na-HEPES buffer contained 0.4 ethyl isopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity that does not inhibit NHE-3). Changes in intracellular pH were monitored using a FLIPR Tetra® (Molecular Devices, Sunnyvale, Calif.) by excitation at λex 439 to 505 nm, and measuring BCECF fluorescence at λem 538 nm. The initial rate of the fluorescence ratio change was used as a measure of NHE-mediated Na+/H+ activity, and reported as the change in fluorescence ratio per minute. Initial rates were plotted as the average of 2 or more replicates, and pIC50 values were estimated using GraphPad Prism.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 1380.57
Compound (SMILES): COc1cccc(-c2nnc(NS(=O)(=O)[C@@H](C)[C@@H](OC)c3ncc(Cl)cn3)n2-c2c(OC)cccc2OC)n1
InChIKey: RINUMDKUZBWYGM-LHSJRXKWSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 576.04, ALogP 3.32, HBA 12, HBD 1, PSA 152.47, QED 0.28
Compound (SMILES): COCCOCCOCCOc1cc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(C(=O)NC5COC5)c4OC)c4ccccc34)ccn2)cc(OC)c1
InChIKey: UMXBELQMJUPJRY-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 μL) is incubated with the test compound (either 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL, or 0.001 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5 μL, 800 μM for c-Src, and 60 μM ATP for Syk) are then added to the enzymes/compound mixtures and the mixture incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 823.94, ALogP 7.92, HBA 12, HBD 4, PSA 168.99, QED 0.06
Compound (SMILES): COC1CN(C2CCC(c3ccc4[nH]c(-c5cn6ncnc6c(C)c5C)c(C(C)C)c4n3)CC2)C1
InChIKey: NZYDEZBAGMNAGF-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
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 = 3.3 nM (pChEMBL 8.479999542236328)
Descriptors: MW 472.64, ALogP 5.37, HBA 6, HBD 1, PSA 71.34, QED 0.42