lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccc(F)c(-c2cc(N3CCOCC3)nc3c(-c4ccn[nH]4)nccc23)c1
InChIKey: XTZHMKQBVZWEHO-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 103 nM (pChEMBL 6.989999771118164)
Descriptors: MW 405.43, ALogP 3.67, HBA 6, HBD 1, PSA 76.16, QED 0.56
Compound (SMILES): COc1ccccc1C#Cc1cc(-c2csc(NC(=O)c3ccc(CN4CCN(C)CC4)cc3)n2)ccc1C
InChIKey: YINHZEMGJCBRDS-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based in vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 uL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad). The concentration of inhibitor showing 50% inhibition as compared with the control was expressed as IC50 (μM).
Measurement: IC50 = 23000 nM (pChEMBL 4.639999866485596)
Descriptors: MW 536.7, ALogP 5.53, HBA 6, HBD 1, PSA 57.7, QED 0.33
Compound (SMILES): CN1CC=C(c2ccc(N(C)c3ccc4c(c3)OCC[C@H]4CNc3cnccc3C(=O)O)cc2)CC1
InChIKey: MDGUBTPVCAYRES-QFIPXVFZSA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: JMJD2C Assay: The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K9me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO were added to each well of the plate, followed by the addition of 2 μl of 0.9 nM JMJD2C to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K9me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 484.6, ALogP 5.24, HBA 6, HBD 2, PSA 77.93, QED 0.47
Compound (SMILES): O=C(NO)c1cccc2c1CC1(CCN(Cc3cccc(F)c3)C1=O)C2
InChIKey: IQENQDQONVQQEG-UHFFFAOYSA-N
Target: Histone deacetylase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96DB2
Assay: HDAC Inhibition Assays: This example describes in vitro inhibition properties of exemplary HDAC11 inhibitors for various HDACs. HDAC inhibition assays were performed using an electrophoretic mobility shift assay at Nanosyn, Inc. (Santa Clara, Calif.). Full length human recombinant HDAC proteins were expressed in the baculoviral system and purified by affinity chromatography. A human recombinant HDAC3 was co-expressed with nuclear receptor corepressor (Ncor2). The following peptide substrates were used: FAM-RHKK(Ac)-NH2 for HDAC3, HDAC6 and HDAC8; FITC-H3K27(Ac)-NH2 for HDAC1, HDAC2 and HDAC10; FAM-RHKK(tri-fluor-Ac)-NH2 for HDAC4, HDAC5, HDAC7, HDAC9 and HDAC11. Reactions consisting of compound, enzyme, and substrate were performed in reaction buffer (comprised of 100 mM HEPES, pH7.5, 25 mM KCl, 0.1% bovine serum albumin, 0.01% Triton X-100) at 25° C. and quenched by the addition of termination buffer (100 mM HEPES, pH7.5, 0.01% Triton X-100, 0.05% SDS). The fluorescence intensity of the electrophoretically separated de-acetylated product and substrate peptide were measured and analyzed using the LabChip® 3000 microfluidic electrophoresis instrument (Perkin Elmer/Caliper Life Sciences). The IC50 values of inhibitors were determined by fitting the %-inhibition curves with 4 parameter dose-response model using XLfit 4 software (IDBS).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 354.38, ALogP 2.46, HBA 3, HBD 2, PSA 69.64, QED 0.66
Compound (SMILES): CCCCN(CCC#N)C(=O)c1ccc(-c2cn(C)c(=O)c3[nH]c(C(=O)NCC)cc23)c(Oc2c(C)cccc2C)c1
InChIKey: WLAVVBFGQSNHCY-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay (BD1: amino acids K57-E168): Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (μL) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve μL of this solution were added to the assay plate to reach a final volume of 18 μL. The final concentration of 1× assay buffer contains 2% DMSO, 50 μM-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 1080 nM (pChEMBL 5.96999979019165)
Descriptors: MW 567.69, ALogP 5.85, HBA 6, HBD 2, PSA 120.22, QED 0.24
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)Cl)cc1)c1cc(-c2cncnc2)c2c(c1)ncn2CC(F)F
InChIKey: YGROTECFTJMFBU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Biochemical Kinase Assay: ABL1 WT protein (64-515aa) containing an N-terminal His tag was produced by co-expression with YopH in Sf9 insect cells. Cells were harvested by centrifugation and resuspended in 50 mM Tris, 500 mM NaCl, 5 mM β-ME, pH 8.2. Cells were lysed by sonication and clarified by centrifugation. ABL1 was purified by affinity chromatography using a HisTrap column with a wash step in 4% wash buffer (50 mM Tris, 500 mM NaCl, 500 mM imidazole, 5 mM β-ME, pH 8.2) and eluted in a linear gradient of the same buffer. Fractions containing ABL1 were pooled, concentrated and further purified using an ion exchange column washed with 50 mM Tris, pH 8.3 and eluted with a linear gradient of elution buffer (50 mM Tris, 1M NaCl, pH 8.3). Purified protein was stored at -80° C. in 50 mM Tris (pH 8.2), 300 mM NaCl, 1 mM DTT and 20% glycerol.
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 479.82, ALogP 5.18, HBA 6, HBD 1, PSA 81.93, QED 0.29
Compound (SMILES): Cc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)Nc1cn(Cc2ccc(C(F)(F)F)cc2)cn1
InChIKey: DRHXIIZMKKESLX-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 560.46, ALogP 5.22, HBA 8, HBD 2, PSA 116.02, QED 0.29
Compound (SMILES): Cc1cc2c(cc(NCCN3CCOCC3)c3nnc(C)n32)n1Cc1ccccc1
InChIKey: CGJJQCLDZSLVJH-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 404.52, ALogP 3.09, HBA 7, HBD 1, PSA 59.62, QED 0.54
Compound (SMILES): Cc1nccc(-c2ccc(OC[C@](C)(CC(C)C)NC(=O)OC(C)(C)C)c(C#N)c2)n1
InChIKey: UKAIKIISFDOVNA-DEOSSOPVSA-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 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 424.55, ALogP 5.03, HBA 6, HBD 1, PSA 97.13, QED 0.67
Compound (SMILES): CN1CCN(c2ccc3c(c2)CCCN(C(=O)OCc2ccccc2)C3)CC1
InChIKey: ULLDCBKHRDTZPV-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99720
Assay: TBD: TBD
Measurement: Ki = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 379.5, ALogP 3.52, HBA 4, HBD 0, PSA 36.02, QED 0.82
Compound (SMILES): CC1(C)CC(Oc2nccc3cc(NC(=O)c4ncccc4N)cc(F)c23)CCO1
InChIKey: SMIQZEHYLGGHPQ-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Metabotropic Glutamate Receptor Activity: For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured. Approximately two and a half minutes later, a concentration of mGluR4 orthosteric agonist (e.g. glutamate, L-AP4, or L-SOP) eliciting approximately 20% (EC20) of the maximal agonist response was added to the cells, and the response was measured. Two minutes later, a concentration of mGluR4 agonist (e.g. glutamate, L-AP4, or L-SOP) eliciting 80% (EC80) of the maximal agonist response was added to the cells, and the response was measured. For rat mGluR4/GIRK experiments, a baseline was established for approximately five seconds, disclosed compounds were added, and either an EC20 or EC80 concentration of agonist was added approximately two and one half minutes later.
Measurement: EC50 = 127 nM (pChEMBL 6.900000095367432)
Descriptors: MW 410.45, ALogP 3.94, HBA 6, HBD 2, PSA 99.36, QED 0.68
Compound (SMILES): C=CC(=O)N1CCCN(C(=O)c2cc(Sc3cnc(NC(=O)C4CC4)s3)cc(C)c2C)C[C@H]1C
InChIKey: YEZJNYHSUDDMPA-MRXNPFEDSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 498.67, ALogP 4.51, HBA 6, HBD 1, PSA 82.61, QED 0.59
Compound (SMILES): C=CCNC1OC(c2cccc3ccccc23)=NC1C#N
InChIKey: HSNRBKHDUAGJTC-UHFFFAOYSA-N
Target: Polyunsaturated fatty acid lipoxygenase ALOX15 (SINGLE PROTEIN), Homo sapiens
UniProt: P16050
Assay: Lipoxygenase UV-Vis Assay: The inhibitor compounds were screened initially using one concentration point on a Perkin-Elmer Lambda 40 UV-Vis spectrometer. The percent inhibition was determined by comparing the enzyme rates of the control (DMSO solvent) and the inhibitor sample by following the formation of the conjugated diene product at 234 nm (ε=25,000 M−1 cm−1). The reactions were initiated by adding either of 40 nM 12-LOX, 40 nM 12/15-LOX, 0.5 μM 15-LOX-2 or 200 nM 5-LOX (ammonium sulfate suspension) to a cuvette with a 2 mL reaction buffer constantly stirred using a magnetic stir bar at room temperature (22° C.). It should be noted that LOX isozymes are often expressed in the inactive demetallated form, so it is best to utilize activity to determine the optimal LOX concentration for the assay (optimal rate of approximately 0.001 abs/sec at 10 μM AA). Reaction buffers used for various lipoxygenase were as follows-25 mM HEPES (pH 7.3), 0.3 mM CaCl2, 0.1 mM EDTA, 0.2 mM ATP, 0.01% Triton X-100, 10 μM AA for the crude, ammonium sulfate precipitated 5-LOX; 25 mM Hepes (pH 8), 0.01% Triton X-100, 10 μM AA for 12-LOX and 25 mM Hepes buffer (pH 7.5), 0.01% Triton X-100, 10 μM AA for 12/15-LOX and 15-LOX-2. The substrate concentration was quantitatively determined by allowing the enzymatic reaction to go to completion in the presence of 15-LOX-2. For the inhibitors that showed more than 50% inhibition at the one point screens (25 μM inhibitor), IC50 values were obtained by determining the enzymatic rate at various inhibitor concentrations and plotted against inhibitor concentration (Approximate range: 0.1 to 25 μM inhibitor), followed by a hyperbolic saturation curve fit (assuming total enzyme concentration [E]<<Ki app, so IC50Ki app). It should be noted that all of the potent inhibitors displayed greater than 80% maximal inhibition, unless stated in the tables. Inhibitors were stored at −20° C. in DMSO.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 277.33, ALogP 2.61, HBA 4, HBD 1, PSA 57.41, QED 0.87
Compound (SMILES): CO[C@H]1[C@@H](C(=O)N[C@@H]2CCCC(C)(C)C2)N(C(=O)Cn2nc(C(N)=O)c3ccccc32)C[C@@H]1F
InChIKey: VNIUXPGGFOMUNC-IEARMKQFSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Complement Factor D Assay: Method 1: Recombinant human factor D (expressed in E. coli and purified using standard methods) at 10 nM concentration is incubated with test compound at various concentrations for 1 hour at room temperature in 0.1 M Hepes buffer, pH 7.5, containing 1 mM MgCl2, 1 M NaCl and 0.05% CHAPS. A synthetic substrate Z-Lys-thiobenzyl and 2,4-dinitrobenzenesulfonyl-fluoresceine are added to final concentrations of 200 μM and 25 μM, respectively. The increase in fluorescence is recorded at excitation of 485 nm and emission at 535 nm in a microplate spectrofluorimeter. IC50 values are calculated from percentage of inhibition of complement factor D-activity as a function of test compound concentration.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 473.55, ALogP 1.78, HBA 6, HBD 2, PSA 119.55, QED 0.66
Compound (SMILES): Cc1noc(C)c1-c1ccc2nc(N3CC(NC(=O)C(C)C)C3)n3c2c1OC[C@@H]3c1ccccn1
InChIKey: CQVUHGMJANIZCA-OAQYLSRUSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 472.55, ALogP 3.65, HBA 8, HBD 1, PSA 98.31, QED 0.47
Compound (SMILES): NC(=O)c1cccnc1Oc1ccc(CC(=O)Nc2nc3ccc(OC(F)(F)F)cn3n2)cc1F
InChIKey: BENHAPZDHMSBKV-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH) (SEQ ID No. 1). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 93 nM (pChEMBL 7.03000020980835)
Descriptors: MW 490.37, ALogP 3.23, HBA 8, HBD 2, PSA 133.73, QED 0.38
Compound (SMILES): N#Cc1cscc1NC(=O)c1ccc2ccc(N3CC4CCC3CN4)nn12.O=C(O)C(F)(F)F
InChIKey: DTZADWBIHYGWFW-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M (III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 134 nM (pChEMBL 6.869999885559082)
Descriptors: MW 378.46, ALogP 2.46, HBA 7, HBD 2, PSA 85.46, QED 0.73
Compound (SMILES): COc1cccc(-c2cc3c(N4CCN(c5ccccc5)CC4)ncnn3c2)c1
InChIKey: OFHFMDRWBUMSFD-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: KIT D816V assay at Km: 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). Data was normalized to 0% and 100% inhibition controls and the IC50 or EC50 calculated using a 4-parameter fit using GraphPad Prism.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 385.47, ALogP 3.73, HBA 6, HBD 0, PSA 45.9, QED 0.54
Compound (SMILES): CNC(=O)c1cc2c(-c3cc(C(C)(C)O)ccc3Oc3c(C)cc(F)cc3C)cn(C)c(=O)c2s1
InChIKey: PKJWHICQQMEFNO-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Binding Assay (BRD4-1): The bromodomain binding assays were performed by Reaction Biology Corp., Malvern, Pa., USA (www.reactionbiology.com). The BET binding assays were conducted in 384 well microplates in assay buffer (50 mM HEPES-HCl, pH 7.5, 100 mM NaCl, 1 mg/ml BSA, 0.05% CHAPS, and 0.5% DMSO) with compounds added as DMSO stocks at a single concentration or with 10-point dose response titrations. BET protein (20 nM for BRD4) or assay buffer were delivered to the appropriate wells of the microplate. Test compound (20 nM) was then delivered by acoustic technology via a Labcyte Echo550 liquid handler. The microplate was centrifuged for 5 min and pre-incubated for 30 min at RT with gentle shaking. The ligand (histone H4 peptide (1-21) K5/8/12/16Ac-biotin) was delivered and the microplate was again centrifuged for 5 min and allowed to incubate for 30 min at RT with gentle shaking. Donor beads were then added in the absence of light and the microplate was centrifuged and gently shaken. After 5 min, acceptor beads were added in the absence of light and the microplate was centrifuged and gently shaken in the dark for 60 min. The microplate was read using a Perkin Elmer EnSpire Alpha plate reader (λ Ex/λ Em=680/520-620 nm). Percent inhibition was calculated relative to positive and negative controls on a per plate basis.
Measurement: IC50 = 3595 nM (pChEMBL 5.440000057220459)
Descriptors: MW 494.59, ALogP 5.4, HBA 6, HBD 2, PSA 80.56, QED 0.39
Compound (SMILES): Cc1ccc2[nH]c(=O)c(C(=O)/C=C/c3ccc(C#N)cc3)c(-c3ccccc3)c2c1
InChIKey: PHFBVAYTCGKKPB-WYMLVPIESA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Inhibition Assay: Assay buffer (as aforementioned): 100 mM Tris-HCl (pH 7.6), 2 mM DTT, 1 mM CaCl2Final concentrations:100 nM hPAD4 enzyme50 μM (8-fold sub-Km) substrate peptide0.5% DMSOTotal incubation time: 65 mins at 37° C.Stop solution: 40 μl 5% TCA in ACN0.25 μL of compound solution was added to 10 μL of 200 nM PAD4 in assay buffer (100 mM Tris-HCl pH 7.6, 2 mM DTT). After 5 mins, 10 μL of 100 μM of substrate in buffer (100 mM Tris-HCl pH 7.6, 2 mM DTT, 2 mM CaCl2) was added and the reaction incubated for 60 mins at 37° C. The enzymatic reaction was quenched by addition of 40 μl of 5% TCA in ACN (1.7% TCA final concentration) stop solution. Arginine containing substrate and citrulline containing product (+1 Da mass shift) were subjected to solid phase extraction on Agilent RapidFire (RF) 300 system and detected on a coupled, triple quadrupole Agilent 6460 QQQ mass spectrometry (MS) device under application of multiple reaction monitoring (MRM) for quantitation.
Measurement: IC50 = 11500 nM (pChEMBL 4.940000057220459)
Descriptors: MW 390.44, ALogP 5.27, HBA 3, HBD 1, PSA 73.72, QED 0.38
Compound (SMILES): CC(C)c1cn2cccc2c(N2CC[C@H](NC(=O)c3ncn(-c4ccc(F)cc4)n3)C2)n1
InChIKey: CNAJNEMVBLJYFF-KRWDZBQOSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: Serum Binding Analysis: In order to more properly assess the binding efficacy of the compounds, radioligand binding assays were carried out in the presence of 100% human serum to more accurately reflect the in vivo environment. While observed IC50 values are typically lower than those reported for buffer binding assays, the relevance of the assay for ranking of compound efficacy is enhanced. Target compounds were tested to determine their ability to bind with CXCR7 sites on MCF-7 and/or MDA-MB-435S CXCR7 transfected cells. Efficiency-maxim/zed radioligand binding using filtration protocols as described in Dairaghi D J, et al., HHV8-encoded vMIP-I selectively engages chemokine receptor CCR5. Agonist and antagonist profiles of viral chemokines., J. Biol. Chem. 1999 Jul. 30; 274(31): 21569-74 and Gosling J, et al., Cutting edge: identification of a novel chemokine receptor that binds dendritic cell- and T cell-active chemokines including ELC, SLC, and TECK, J. Immunol. 2000 Mar. 15; 164(6):2851-6 was used.In these assays, MCF-7 and/or MDA-MB-435S cells were interrogated with the target compounds and the ability of these compounds to displace 125 I radiolabeled SDF-1 was assessed using the protocol described in Dairaghi and Gosling. The target compounds were added to the plate to the indicated concentration followed by addition of cells and radiolabeled chemokine (125I SDF-1), both in the following medium (human AB serum with 10 mM HEPES added to stabilize at pH 7.4). All assays were then incubated for 3 hrs at 4° C. with gentle agitation. Following incubation in all binding assays, reactions were aspirated onto PEI-treated GF/B glass filters (Packard) using a cell harvester (Packard) and washed twice (25 mM HEPES, 500 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, adjusted to pH 7.1). Scintillant (MicroScint 10, Packard) was added to the wells, and the filters were counted in a Packard Topcount scintillation counter. Data were analyzed and plotted using GraphPad Prism (GraphPad Software).
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 433.49, ALogP 3.19, HBA 7, HBD 1, PSA 80.35, QED 0.52
Compound (SMILES): Cn1cc(NC(=O)c2cnn3ccc(N4CCNCCC45CC5)nc23)c(C(F)(F)F)n1
InChIKey: QUGSJZJVDZANJF-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 789 nM (pChEMBL 6.099999904632568)
Descriptors: MW 434.43, ALogP 2.07, HBA 8, HBD 2, PSA 92.38, QED 0.66
Compound (SMILES): CCn1c(C(=O)N2CC(F)(F)C2)cc2ccc(-c3nccc(Nc4ccc(-c5cn[nH]c5)cc4)n3)cc21
InChIKey: SIEKPUUOWIHMQN-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Biological Assay: The kinase IC50 was determined by a commercialized CISBIO kinase detection kit, HTRF KinEASE-STK S2 kit (62ST2PEC). ROCK2 (01-119) employed in the reaction was purchased from Carna Biosciences.Before the assay, the following working solutions as needed were formulated with corresponding reagents according to the instruction of the kinase detection kit: 1×kinase buffer, 5×STK-52 substrate working solution (1.5 μM) and 5×ATP working solution (1.5 μM), 5×ROCK2 kinase working solution, 4×Streptavidin-XL665 working solution, and 4×STK-Ab-Cryptate 2 detection solution. Then the assay was performed according to the following procedure.A solution of a compound at a concentration of 10000 nM was prepared with the 1×kinase buffer containing 2.5% DMSO. Gradient dilution of the solution of the compound was performed with the kinase buffer containing DMSO, so as to obtain solutions of a test compound at 9 different concentrations. In addition to wells of test compounds, a positive well (containing all the reagents except the compound) and a negative well (containing all the reagents except the test compound and kinase) were set. Except for the control wells (positive and negative wells), a solution of a test compound (4 μL) was added to each of the reaction wells, and a solution of 2.5% DMSO was added to the control wells. Then the substrate (2 μM, i.e., 2 μL. 5×STK-S2 substrate working solution) was added to each of the reaction wells. The 5×ROCK2 kinase working solution (2 μL, containing 1.4 ng ROCK2 kinase) was added to each of the reaction wells except for the negative well, the volume of which was made up with the 1×kinase buffer (2 μL). The 5×ATP working solution (2 μL) was added to each of the reaction wells, and the mixtures were incubated at room temperature for 2 hours. After the kinase reaction was complete, the 4×Streptavidin-XL665 working solution was added to each of the reaction wells, the solutions were mixed, followed by immediate addition of the 4×STK-Ab-Cryptate 2 detection solution (5 μL), and the mixtures were incubated at room temperature for 1 hour. The fluorescence signal was read on ENVISION (Perkinelmer) (excitation wavelength: 320 nm, and emission wavelength: 665 nm and 615 nm). The inhibitory rate in each well was calculated based on the fluorescence intensity value: ER (Emission Ratio)=(fluorescence intensity at 665 nm/fluorescence intensity at 615 nm); inhibitory rate=(ERpositive−ERtest compound)/(ERpositive−ERnegative)*100%. Curves were plotted and fitted to obtain the median inhibitory concentration (IC50) of each teat compound with the PRISM 5.0 software.
Measurement: IC50 = 118 nM (pChEMBL 6.929999828338623)
Descriptors: MW 499.53, ALogP 5.34, HBA 6, HBD 2, PSA 91.73, QED 0.33
Compound (SMILES): C[C@H](Nc1ncnc2ccc(-c3cnc(Cl)c(NS(C)(=O)=O)c3)cc12)c1ccccc1
InChIKey: LPJIZDSOUGHTNB-AWEZNQCLSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Z′-LYTE Biochemical Aassay: Z′-LYTE® Assay Conditions: Test Compounds: The Test Compounds are screened in 1% DMSO (final) in the well. For 10 point titrations, 3-fold serial dilutions are conducted from the starting concentration. Peptide/Kinase Mixtures: All Peptide/Kinase Mixtures are diluted to a 2× working concentration in the appropriate Kinase Buffer. ATP Solution: All ATP Solutions are diluted to a 4× working concentration in Kinase Buffer (50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA). ATP Km apparent is previously determined using a Z′-LYTE® assay. Development Reagent Solution: The Development Reagent is diluted in Development Buffer. 10× Novel PKC Lipid Mix: 2 mg/mL Phosphatidyl Serine, 0.2 mg/mL DAG in 20 mM HEPES, pH 7.4, 0.3% CHAPS. For 5 mL 10× Novel PKC Lipid Mix: 1. Add 10 mgs Phosphatidyl Serine (Avanti Polar Lipids Part #8400032C or 840039C) and 1 mg DAG (Avanti Polar Lipids Part #800811C) to a glass tube. 2. Remove the chloroform from lipid mixture by evaporating to a clear, thin film under a stream of nitrogen. Continuous rotation of the tube, at an angle to ensure maximum surface area of the lipid solution, will promote the thinnest film. 3. Add 5 mLs resuspension buffer, 20 mM HEPES, 0.3% CHAPS, pH 7.4, to the dried lipid mix 4. Heat gently to 50-60° C. for 1-2 minutes and vortex in short intervals until the lipids are dissolved to a clear or slightly hazy solution. The lipids are typically in solution after 2-3 heat/vortex cycles. 5. Cool to room temperature, aliquot into single use volumes and store at −20° C. Assay Protocol: Bar-coded Corning, low volume NBS, black 384-well plate (Corning Cat. #4514) 1. 2.5 μL 4× Test Compound or 100 nL 100× plus 2.4 μL kinase buffer. 2. 5 μL 2× Peptide/Kinase Mixture. 3. 2.5 μL 4× ATP Solution. 4. 30-second plate shake. 5. 60-minute Kinase Reaction incubation at room temperature. 6. 5 μL Development Reagent Solution. 7. 30-second plate shake. 8. 60-minute Development Reaction incubation at room temperature. 9. Read on fluorescence plate reader and analyze the data. In a typical experiment, each data point uses 100 nL 100× test compound in 100% DMSO. Commonly, 100 nL of a 10 μM solution of test compound is used for each experiment, which is equivalent to 1 picomole of test compound. Accordingly, a 10 μM single-point assay uses 100 picomoles of test compound, and a 10-point titration uses about 200 picomoles of test compound 100 picomoles for the initial test and another 100 picomoles for the serial dilution.
Measurement: IC50 < 2 nM (pChEMBL 8.699000358581543)
Descriptors: MW 453.96, ALogP 4.89, HBA 6, HBD 2, PSA 96.87, QED 0.4
Compound (SMILES): COc1cc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc(C(=O)N[C@@H]2CN(C(=O)C(C(F)(F)F)C(F)(F)F)C[C@@H]2F)c1OC
InChIKey: VJDIKEYYESCOCN-QWHCGFSZSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 94000 nM (pChEMBL 4.03000020980835)
Descriptors: MW 646.44, ALogP 4.03, HBA 8, HBD 2, PSA 124.08, QED 0.39
Compound (SMILES): Cc1cccc(C(=O)N2CCC[C@@H](Oc3ccc(C(F)(F)F)cn3)[C@@H]2C)c1-n1nccn1
InChIKey: XIYPJKVOOSGFDY-MAUKXSAKSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Receptor Radioligand Binding Studies: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat #SH30022), 10% FBS, 1×Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1×Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat #SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 445.45, ALogP 4.06, HBA 6, HBD 0, PSA 73.14, QED 0.61
Compound (SMILES): CC(C)n1cc(-c2cnc(N)c(-c3cc(-c4ccc(CNC5CC5)cc4)no3)n2)ccc1=O
InChIKey: YBUCANGYLKKOBK-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 μM [γ-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1 M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHNOB50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 442.52, ALogP 4.04, HBA 8, HBD 2, PSA 111.86, QED 0.44
Compound (SMILES): C[C@@H](CC(=O)N1CCN(c2ccc(C#N)cn2)CC1)OC[C@@H]1CCCN1c1cn[nH]c(=O)c1C(F)(F)F
InChIKey: ASVGZTBYKZSPHF-WMZOPIPTSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 519.53, ALogP 2.17, HBA 8, HBD 1, PSA 118.45, QED 0.59
Compound (SMILES): CC(C)(COc1nccc2ccccc12)NC(=O)C1CNCCOC1
InChIKey: QNTDFVOIXJCYFO-UHFFFAOYSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: cAMP Assay: Compounds are dissolved and diluted in DMSO. The final test solution contains 1% DMSO. The cAMP standard (Lance cAMP 384 Kit; PerkinElmer, Cat# AD0264) is prepared in assay buffer (HBSS with 0.1% BSA, 5 mM HEPES, 0.5M IBMX, pH 7.4) containing 1% DMSO and the cAMP standard curve is included at least on one plate. Cells are centrifuged and suspended in assay buffer (incl. 1:100 diluted Alexa antibody).For the assay 5 μl of a cell suspension (approximately 5000 cells/well)¿incl. Alexa antibody (diluted 1:100) are added into a 384 well MTP microtitre plate excepting one row or column (depending on the plate layout), which is reserved for the standard curve. Then 2 μl of compound sample is added as concentration response curve (e.g. le-5M to 6e-10M), usually in triplicates. Each assay contains incubations with vehicle controls instead of compound as controls for non-inhibited cAMP generation (100% CTL; ¿high values¿) and incubations with 1 μM Somatosatin as controls for full inhibition and background (0% CTL; 'low values'). After approximately 10-15 min incubation time 3 μl Forskolin (dissolved in DMSO, final conc. 15 μM) is added. Then the plates are shaken briefly and incubated for 60 min at room temperature. After 60 min 10 μl of the detection mix is added into all wells followed by an additional incubation period of 1 h. The plates are read in a suitable plate reader.The analysis of the data is based on the "ratio" of the time-resolved fluorescence measurements of donor and acceptor fluorophore (Ex: 320 nm; Em1: 665 nm; Em2: 615 nm; ratio 665/615). From this ratio, cAMP concentrations are calculated from standard curve and the EC50 is estimated by least square curve fit program.
Measurement: EC50 = 393 nM (pChEMBL 6.409999847412109)
Descriptors: MW 343.43, ALogP 1.74, HBA 5, HBD 2, PSA 72.48, QED 0.87