lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#CCC1(n2ccc(-c3nc(-c4cn[nH]n4)cc4ncccc34)n2)CN(CC(F)(F)F)C1
InChIKey: IMPZSGOWSPHBCV-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 239 nM (pChEMBL 6.619999885559082)
Descriptors: MW 439.41, ALogP 2.77, HBA 8, HBD 1, PSA 112.2, QED 0.51
Compound (SMILES): CC(=O)N1CCC(C(=O)NC[C@@H]2CCc3cc(C(O)(C(F)(F)F)C(F)(F)F)ccc3N2S(=O)(=O)c2ccc(F)cc2)CC1
InChIKey: KSSVQBXNLITGOL-NRFANRHFSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 639.59, ALogP 4.02, HBA 5, HBD 2, PSA 107.02, QED 0.46
Compound (SMILES): Cn1nnc(CF)c1-c1cnc2c3ccc(S(C)(=O)=O)cc3n([C@H](c3ccccc3)C3CCOCC3)c2c1
InChIKey: NWCAGQKBVAOWAH-HHHXNRCGSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: All assay components were dissolved in buffer composition 20 mM Hepes pH 7.5, 150 mM NaCl, 5 mM DTT, 0.005% Tween 20, and 100 ug/ml BSA for BRD4 (1-477 and 44-460). The final concentrations of the bromodomain proteins are 1.6 nM BRD4(44-168), 1 nM BRD4(333-460), and 1 nM BRD4(1-477 or 44-460), and the fluorescent probe molecule is 100 nM, 50 nM, and 7.5 nM respectively. All proteins were biotinylated. A streptavidin labeled with terbium cryptate (Cisbio SA-Tb) was used as detection, and pre-mixed with the bromodomain protein at a final concentration of 0.2 nM. In some instances for BRD4 (44-460), anti-His terbium cryptate was used as a detection. 7.5 nl of dose-responsed test compound or dmso vehicle (0.0375%) was pre-spotted in a black Corning 384 well plate and 10 ul each of bromodomain/detection reagent and fluorescent small molecule solution were added to the plate, and the reaction incubated for 60 min at room temperature. Plates were then read on EnVision plate reader, (λex=340 nm, acceptor λEm=520 nm, and donor λEm=615 nm, LANCE D400 mirror). Time resolved fluorescence intensity measurements were made at both emissions, and the ratio of acceptor/donor was calculated and used for data analysis.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 533.63, ALogP 4.87, HBA 8, HBD 0, PSA 91.9, QED 0.31
Compound (SMILES): CCCCCC(=O)N1CC(F)(F)C[C@H]1C(=O)N(C)[C@H](C(=O)N[C@@H](CCNC(=O)c1cc(F)cc(F)c1)C(=O)NO)C(C)CC
InChIKey: GOEAECKQYGGAJM-KMKZMLIASA-N
Target: Matrix metalloproteinase-9 (SINGLE PROTEIN), Homo sapiens
UniProt: P14780
Assay: Inhibition Assay: MMP-9: Recombinant MMP-9 expressed was obtained from Merck-Millipore (catalog number PF140-5UG, used in examples 1 to 9 and comparative examples) and Enzo (catalog number-SE360-0010, used in examples 10 to 16). DQ gelatin from pig skin fluorescein conjugated (MMP-9 substrate) was obtained from Life Technologies (catalog number E12055).Preparation of the MMP-9 for the activity assay: MMP-9 was provided as a stock solution (0.1 mg/mL). The enzyme was diluted in 1× reaction buffer to a final concentration of 5 μg/mL.Preparation of the DQ gelatin (substrate) solution for the activity assay: the solid form of the substrate was dissolved in water in order to obtain a stock solution of 1 mg/mL.Procedure: The enzymatic assays were performed in 96-well microtiter plate, which allowed simultaneous monitoring of multiple reactions. For each reaction, 84.6 μL of 1× reaction buffer (pH 7.6), 3.4 μL of MMP-9 (final concentration 85 ng/mL) and 2 μL of the corresponding new compound were added to each well. A stock solution of new compound was prepared in DMSO (100 mM), and dilutions were prepared from this stock solution with DMSO. Finally, to the mixture contained in each well 10 μL of the substrate were added (final concentration 50 μg/mL). The reaction was incubated for 4 hours at room temperature in an orbital shaker (100 rpm).
Measurement: IC50 = 713 nM (pChEMBL 6.150000095367432)
Descriptors: MW 631.67, ALogP 2.76, HBA 6, HBD 4, PSA 148.15, QED 0.1
Compound (SMILES): COCC(C)(C)C(=O)N1CCN(c2ccc(-c3cc(-c4cnn(C)c4)cn4ncc(C#N)c34)cn2)CC1
InChIKey: QBHFBEMEGHHMFQ-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 13.9 nM (pChEMBL 7.860000133514404)
Descriptors: MW 498.59, ALogP 2.99, HBA 9, HBD 0, PSA 104.58, QED 0.4
Compound (SMILES): CC(C)n1nnnc1-c1cccc(NC(=O)c2cc(-c3cnn(C4CC4)c3)c(N3CCN(C)CC3)cn2)n1
InChIKey: VGLWQTRQLZMGBI-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay: The ability (IC50) of compounds to inhibit ASK1 kinase activity was determined by HTRF® KinEASE Assay System. ASK1 was purchased from Thermofisher (Catalogue #PV4011), ATP was purchased from Sigma (Catalogue #A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue ##6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 uM STK3 peptide substrate, and 5 nM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 uM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 513.61, ALogP 2.91, HBA 11, HBD 1, PSA 122.78, QED 0.4
Compound (SMILES): Cc1cc2c(cc(NC(N)=O)c3nnc(C)n32)n1Cc1ccccc1
InChIKey: PDDAMENUMPNSRA-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 334.38, ALogP 2.84, HBA 5, HBD 2, PSA 90.24, QED 0.6
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1nc2cc(Cl)ccc2s1)c1ccccc1-n1nccn1
InChIKey: XRCGVDTUCHXOPF-HOTGVXAUSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37° C., 5% CO2. Test compounds (at 10 point half log concentration response curves from 10 uM) were added to cells for 15 minutes prior to the addition of orexin-A to all wells, to achieve a final concentration that produces approximately an 80% maximal response. The IC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 438.94, ALogP 4.29, HBA 7, HBD 2, PSA 84.73, QED 0.49
Compound (SMILES): O=C(c1ccc2c(c1)CCCC2)C1CCCN(C2CCC2)C1
InChIKey: JEHDQSLDGUXOLU-UHFFFAOYSA-N
Target: Lysine-specific demethylase 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NHM5
Assay: KDM2B TR-FRET Assay: Full length KDM2B was cloned, expressed, and purified to homogeneity. Compound inhibition of KDM2B demethylase activity was assessed by monitoring the methylation status of a biotin-H3K36me2 peptide substrate (H2N-RKSAPATGGV(KMe2)KPHRYRPGTV-NTPEGBiot; New England Peptide) in the presence of α-keotglutarate (2-OG) and iron (Fe2+) using the TR-FRET assay technology (Cisbio). Specifically, in a 384 well ProxiPlate KDM2B (5 mM final), ascorbate (500 μM final) and DTT (2 mM final) were combined with the biotin-H3K36me2 peptide substrate (200 nM final), 2-OG (0.3 μM or 6 μM final; Sigma K2010) and Fe2+ (100 μM final; Sigma F1543) in 50 mM HEPES (pH 6.5) and 0.01% Triton-X 100 either in the presence of DMSO (final 0.25% DMSO) or compound dilution series in DMSO and mixed. After a two hour incubation at room temperature, a mixture of EU-anti-H3K36me1 antibody (2 nM final; Cisbio #64CUSKAZ), and Streptavidin-d2 (50 nM final; Cisbio #64CUS000) in 200 mM KF, 200 mM EDTA, 0.1% BSA and 50 mM HEPES (pH 6.5) was added. After 1 hour incubation, the plates were read on an Envision instrument, the readouts were transformed into % inhibition, and IC50 values were generated using a four parameter logistic model (XLFIT5).
Measurement: IC50 = 103 nM (pChEMBL 6.989999771118164)
Descriptors: MW 297.44, ALogP 4.01, HBA 2, HBD 0, PSA 20.31, QED 0.79
Compound (SMILES): O=C(O)CCCOc1c(F)cc(-c2cc(OC3CCC3)cc(Cl)n2)cc1Cl
InChIKey: YUGTVKXDPFGMOQ-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 414.26, ALogP 5.37, HBA 4, HBD 1, PSA 68.65, QED 0.46
Compound (SMILES): CCN(C[C@H]1CC[C@@H](C(F)(F)F)CC1)c1ncnc(NC[C@@]2(O)CCN(CC(N)=O)C[C@@H]2O)c1F
InChIKey: SYFRIFFIVWVYNL-YJXLLHPSSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORgamma Reporter Assay (Gal4): The HEK293 cell line is co-transfected transiently with two plasmids, one with the RORγ ligand-binding domain fused to galactose-responsive transcription factor (Gal4), and the other with the luciferase reporter gene and Gal binding sites (UAS). This construction allows to determine the RORγ activity in a cellular system through the measurement of luminescence.A suspension of RORγ reporter cells was dispensed into plates and cultured 2 h at 37° C. and 5% CO2. Media formulation consisted in DMEM/F-12 medium (Gibco) supplemented with 10% heat inactivated FBS (Sigma-Aldrich), non-essential aminoacids (Sigma-Aldrich), 2 mM Glutamax (Gibco) and 100 U/mL penicillin (Sigma-Aldrich). Dose-response curves with compounds were prepared in 100% DMSO and further diluted 100-fold in culture medium. Compound solutions were added to the plate containing cells (final DMSO concentration of 0.1%) and incubated for 24 h at 37° C. and 5% CO2. Luciferase detection reagent was added to each well, and relative light units (RLUs) were quantified from each assay well using a plate reading luminometer.Values of average RLU±S.D. were computed for all treatment sets, followed by the calculations of percent-reduction of RORγ activity in response to respective test compound. The following formula was used: activity=100*[1−[x test compound/average vehicle where the theoretical minimum reduction (0% reduction). For all experiments, the activity values were plotted versus compound concentrations in one single plot and adjusted to a four-parameter logistic curve to obtain the absolute IC50 value along with the 95% confidence interval. These calculations were performed in excel-fit software using X-204 model curve.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 506.55, ALogP 1.51, HBA 8, HBD 4, PSA 127.84, QED 0.37
Compound (SMILES): CCc1cc(O)c(F)cc1-c1cc2[nH]nc(C(N)=O)c2c(NCc2ccccc2N(C)S(=O)(=O)c2ccccc2)n1
InChIKey: XSERXIPKYAQNID-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 574.64, ALogP 4.57, HBA 7, HBD 4, PSA 154.3, QED 0.2
Compound (SMILES): COc1cc(-c2cc(F)cc(Cl)c2)c(F)cc1-c1cn(C)c(=O)c2cc(S(=O)(=O)Nc3ccon3)ccc12
InChIKey: FBYKUVZRHZVTDP-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to â¿¿10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 557.96, ALogP 5.6, HBA 7, HBD 1, PSA 103.43, QED 0.29
Compound (SMILES): COc1ccc(-n2cc(C(=O)O)c(=O)n(Cc3cccc(C(F)(F)F)c3C)c2=O)cc1OC
InChIKey: QZISHTLWFISWPZ-UHFFFAOYSA-N
Target: Chymase (SINGLE PROTEIN), Homo sapiens
UniProt: P23946
Assay: Enzymatic Assay : The enzyme source used is recombinant human chymase (expressed in HEK293 cells) or chymase purified from hamsters' tongues. The substrate used for chymase is Abz-HPFHL-Lys(Dnp)-NH2. For the assay, 1 μl of a 50-fold concentrated solution of test substance in DMSO, 24 al of enzyme solution (dilution 1:80 000 human or 1:4000 hamster) and 25 al of substrate solution (final concentration 10 μM) in assay buffer (Tris 50 mM (pH 7.5), sodium chloride 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM) are combined in a white 384-hole microtiter plate (Greiner Bio-One, Frickenhausen, Germany). The reaction is incubated at 32 degrees for 60 min and the fluorescence emission at 465 nm after excitation at 340 nm is measured in a fluorescence reader, for example Tecan Ultra (Tecan, Männedorf, Switzerland).
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 464.4, ALogP 3.09, HBA 7, HBD 1, PSA 99.76, QED 0.6
Compound (SMILES): CNc1nc(N[C@@H](C)c2cc3cc(Cl)ccc3[nH]c2=O)ncc1C#N
InChIKey: JZPKYJWBXABXSU-VIFPVBQESA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 M Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 354.8, ALogP 3.06, HBA 6, HBD 3, PSA 106.49, QED 0.66
Compound (SMILES): CC1=CC(C)=[N+]2C1=Cc1ccc(CCC(=O)NCc3cn(-c4cccc(C(=O)NC(CCCCN)C(=O)COc5c(F)cc(CNC(=O)c6ccc(/N=N/c7ccc(N(C)C)cc7)cc6)cc5F)c4)nn3)n1[B-]2(F)F
InChIKey: RHTHXRANKXCINL-ZOPJMJKSSA-N
Target: Lys-gingipain (SINGLE PROTEIN), Porphyromonas gingivalis (strain ATCC 33277 / DSM 20709 / JCM 12257)
UniProt: B2RLK2
Assay: Inhibition Activity Assay: The capacities of compounds of the present invention to inhibit the activity of lysine gingipain (Kgp) were measured in a fluorogenic assay similar to those described by Barret (Biochemical Journal. 1980, 187(3), 909) using 10 M Z-His-Glu-Lys-MCA as a fluorogenic substrate at 37Â° C. By testing a range of concentrations for each compound, the concentration required to inhibit the activity of lysine gingipain by 500(the âIC50â) was determined.
Measurement: IC50 = 12.1 nM (pChEMBL 7.920000076293945)
Descriptors: MW 1026.91
Compound (SMILES): C[C@@H](NC(=O)[C@@H]1CN(CC2CC2)CC[C@H]1NC(=O)c1cc(-c2ccc(F)cc2F)no1)C1CCC1
InChIKey: JICJJCRHDSRYID-RBQRDFNRSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: Tango CXCR7-bla U2OS cells are detached from culture dishes with 0.05% trypsin-EDTA and collected in growing medium (McCoy's 5A 90% (v/v), dialyzed FCS 10% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), 1 mM sodium pyruvate, P/S 1% (v/v) 50 μg/ml Hygromycin, 100 μg/ml Geneticin, 200 μg/ml Zeocin), spinned down and resuspended in assay medium (McCoy's 5A 90% (v/v), dialyzed FCS 1% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), P/S 1% (v/v)). 10′000 cells per well (in 30 μl) are seeded in a 384 well plate (black-walled, clear bottom). The plate is incubated at 37° C./5% CO2 for 24 hours. Test compounds are dissolved to 10 mM in DMSO and serially diluted in DMSO to 500× of the final concentration for dose response curves. Compounds are then diluted 1:100 in assay medium to 5× of the final concentration. 10 μl/well of diluted compounds are added to the assay plate and incubated for 15 minutes at 37° C. Thereafter CXCL12/SDF1-α is diluted in assay medium to 5× of the final concentration (its EC80 value for receptor activation) and 10 μl/well are added to the assay plate. The agonist leads to activation of the receptor and therefore to b-arrestin recruitment. Compounds acting as antagonists reduce this activation. The plate is incubated for 22 hrs at 37° C. 10 μl/well of detection reagent (LiveBLAzer-FRET BIG (CCF4-AM) substrate) is transferred to the assay plate and the plate is incubated for 2 hours at room temperature protected from light. Fluorescent counts are determined (Scan1: Ex 409/20 nm, Em 460/30 nm, Scan 2: Ex 409/20 nm, Em 530/30 nm).
Measurement: IC50 = 105 nM (pChEMBL 6.980000019073486)
Descriptors: MW 486.56, ALogP 3.75, HBA 5, HBD 2, PSA 87.47, QED 0.59
Compound (SMILES): C[C@H]1CN(C2COC2)CCN1c1cc(NC(=O)Cn2cc(-c3cc(F)c(O)c(C(N)=O)c3)c3c(=O)n(C)cnc32)c(Cl)c(F)n1
InChIKey: RQPMRKHLOYCPHU-AWEZNQCLSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Surface Plasmon Resonance (SPR) Assay: SPR studies were performed using a Biacore™ T200 instrument (GE Health Sciences Inc.). The BCL6 BTB protein used in the FP assay was biotinylated using the site specific biotinylating enzyme BirA, and then cleaved with TEV protease to produce the BCL6 BTB domain (biotin-thrombin-BCL6 amino acids 1-129; 17 kd) that were use in SPR. This protein was stably captured (1000RU) to streptavidin coupled SA chips (BR-1005-31, GE Health Sciences Inc.) according to the manufacture's protocol. Compounds were dissolved in 100% DMSO at 10 mM and 2-fold serial dilutions were done in 100% DMSO. For SPR analysis the serially titrated compounds were diluted 1/20 into buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween-20, 3 mM EDTA) giving a final concentration of 5% DMSO. The Biacore flow rate was set at 100 ul/min. For KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 seconds. Curve fitting and KD calculations were done with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 643.05, ALogP 2.08, HBA 11, HBD 3, PSA 160.84, QED 0.26
Compound (SMILES): Cc1c(-c2c(C)c3c(c(C)c2[C@H](OC(C)(C)C)C(=O)O)CN(C(=O)c2cccc(F)c2)C3)cc(F)c2c1CCCO2
InChIKey: YHHACMRTVOXJRY-PMERELPUSA-N
Target: Human immunodeficiency virus type 1 integrase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q7ZJM1
Assay: Anti-HIV Activity MT4 Assay: Antiviral HIV activity and cytotoxicity values for compounds of the invention from Table 1 were measured in parallel in the HTLV-1 transformed cell line MT-4 based on the method previously described (Hazen et al., 2007, In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus (HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretrovirals and against a panel of protease inhibitor-resistant HIV (Hazen et al., In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus (HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretrovirals and against a panel of protease inhibitor-resistant HIV, Antimicrob. Agents Chemother. 2007, 51: 3147-3154; and Pauwels et al., Sensitive and rapid assay on MT-4 cells for the detection of antiviral compounds against the AIDS virus, J. of Virological Methods 1987, 16: 171-185).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 563.64, ALogP 6.98, HBA 4, HBD 1, PSA 76.07, QED 0.36
Compound (SMILES): Cc1ncc(NC(=O)c2ccnc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)n(C)c(=O)c1
InChIKey: MUEHAHDRQYKWNJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf Activity Assay: Raf and biotinylated Mek, kinase dead, were combined at 2x final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40x of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2xATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 473.46, ALogP 3.26, HBA 7, HBD 1, PSA 89.35, QED 0.63
Compound (SMILES): COc1cnc(C(=O)Nc2ccc(F)c([C@]3(C)CS(=O)(=O)[C@](C)(C4CCC4)C(=N)N3)c2)cn1
InChIKey: YZOGZKNAXGTHLN-FCHUYYIVSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme. Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3Ã¿ the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 ul) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 ul of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 ul in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window. Inhibitor IC50 values are derived from non-linear regression analysis of concentration response curves. Ki values are then calculated from IC50 values using the Cheng-Prusoff equation using a previously determined um value of 8 uM for the QSY7-APPswe-Eu substrate at BACE1. The compounds of Examples 1, 2, and 3 were measured in this assay and each exhibited a Ki value of less than about 5 nM.
Measurement: Ki = 499.2 nM (pChEMBL 6.300000190734863)
Descriptors: MW 475.55, ALogP 2.65, HBA 7, HBD 3, PSA 134.13, QED 0.61
Compound (SMILES): O=C(O)[C@@H]1[C@H]2CC[C@@H](CN1C(=O)N(c1ccccc1)c1ccccc1)N2C(=O)C#Cc1ccccc1
InChIKey: JCKLKLQQHNYRHX-WEWMWRJBSA-N
Target: Type-2 angiotensin II receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P50052
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 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 479.54, ALogP 4.12, HBA 3, HBD 1, PSA 81.16, QED 0.57
Compound (SMILES): Cc1cc(CN2CCN(C(C)C)CC2)ccc1-n1cc(-c2nc(NC3CCN(S(=O)(=O)c4cn(C)cn4)CC3)ncc2Cl)cn1
InChIKey: BEKYPFJQZZGERF-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: CDK2/Cyclin E1 HERE Enzyme Activity Assay: CDK2/Cyclin E1 enzyme activity assays utilize full-length human CDK2 co-expressed as N-terminal GST-tagged protein with FLAG-Cyclin E1 in a baculovirus expression system (Cama Product Number 04-165). Assays are conducted in white 384-well polystyrene plates in a final reaction volume of 8 μL. CDK2/Cyclin E1 (0.25 nM) is incubated with compounds (40 nL serially diluted in DMSO) in the presence of ATP (50 μM or 1 mM) and 50 nM ULight-labeled eIF4E-binding protein 1 (THR37/46) peptide (PerkinElmer) in assay buffer (containing 50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, 0.05 mg/ml BSA, and 0.01% Tween 20) for 60 minutes at room temperature. The reactions are stopped by the addition of EDTA and Europium-labeled anti-phospho-4E-BP1 antibody (PerkinElmer), for a final concentration of 15 mM and 1.5 nM, respectively. HTRF signals are read after 1 hour at room temperature on a PHERAstar FS plate reader (BMG Labtech). Data is analyzed with IDBS XLFit and GraphPad Prism 5.0 software using a three or four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 653.26, ALogP 3.82, HBA 11, HBD 1, PSA 117.31, QED 0.29
Compound (SMILES): CC(C)CN1C(=O)N(CC(=O)NCC(N)=O)C[C@]12CC[C@](c1ccccc1)(N(C)C)CC2
InChIKey: IBWDNMNQUFQKRP-PSWAGMNNSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Human Nociceptin/Orphanin FQ Peptide (hNOP) Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences, Inc. Boston, Mass., USA), and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration, which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd., Buckinghamshire, UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences, Inc. Boston, Mass., USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ß-counter (PerkinElmer Life Sciences/Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]nociceptin-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation, (Cheng and Prusoff, 1973).
Measurement: Ki = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 443.59, ALogP 1.75, HBA 4, HBD 2, PSA 98.98, QED 0.64
Compound (SMILES): N#Cc1cncc(COc2cc(O[C@H]3CCc4c(-c5ccccc5F)cccc43)c(Cl)cc2CNCCC(=O)N[C@@H](C(=O)O)c2c[nH]cn2)c1
InChIKey: DUUSUFIJNOEEFT-LBHUVFDKSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayâELISA: Plates were coated with 1 Î¼g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4Â° C. The wells were then blocked with PBS containing 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37Â° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the samples were diluted to 1:5 in dilution medium in the ELISA plates. Human PD-1 and biotin 0.3 Î¼g/mL (ACRO Biosystems) were added and incubated for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was added with PBS containing 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37Â° C. and was washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37Â° C. then washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37Â° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 695.15, ALogP 6.2, HBA 8, HBD 4, PSA 162.25, QED 0.1
Compound (SMILES): c1cnc2c(c1)OC[C@H](c1ccc(CN3CCCOCC3)cc1)O2
InChIKey: ITUJIFICDMKWCG-GOSISDBHSA-N
Target: Leukotriene A-4 hydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: P09960
Assay: Enzyme Assay: The compounds of the invention are assessed for the ability to interact with human LTA4 hydrolase in an enzymatic assay that measures the ability of the enzyme to cleave the peptide bond of arginyl-aminomethylcoumarin (Arg-AMC). LTA4H Enzyme (1 nM final), Arg-AMC substrate (50 μM final), and compound are combined in a reaction buffer (50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.5% bovine serum albumin) at room temperature for 1 h. The formation of product is assessed by measuring the fluorescence of aminomethylcoumarin product (excitation wavelength 380 nm/emission wavelength 460 nm).
Measurement: IC50 = 0.21 nM (pChEMBL 9.680000305175781)
Descriptors: MW 326.4, ALogP 2.82, HBA 5, HBD 0, PSA 43.82, QED 0.87
Compound (SMILES): CC(C)S(=O)(=O)c1c(Cl)ccc(NC(=O)N[C@@H]2CCCc3ccoc32)c1O
InChIKey: PREOQRAKGIZOEI-CQSZACIVSA-N
Target: C-X-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25025
Assay: Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 412.9, ALogP 4.02, HBA 5, HBD 3, PSA 108.64, QED 0.66
Compound (SMILES): CCc1cccc([C@@H](C)[C@H](NS(=O)(=O)c2ccc(Cl)cc2OC)c2n[nH]c(=O)o2)c1C
InChIKey: UMJCXNQRGMBWAP-YJYMSZOUSA-N
Target: Ribonucleotide reductase (PROTEIN COMPLEX), Homo sapiens
UniProt: P23921
Assay: Human RNR Inhibition Effect: The inhibitory activity against the ribonucleotide reduction reaction (hereinafter referred to as RNR reaction) of the test compound was determined by measuring the formation of deoxycytidine diphosphate (hereinafter referred to as dCDP) from cytidine diphosphate (hereinafter referred to as CDP) by the following method.Human M1 subunit and human M2 subunit (mutant lacking amino terminal 59 amino acids), which are fused a histidine tag at the amino terminus, are overexpressed in Escherichia coli and are solubilized after collection, and histidine tagged human M1 and M2 proteins were purified on a nickel chelate column.For measuring the inhibitory activity of the test compound against the RNR reaction, the method described in the document [CANCER RESEARCH 64, 1-6, 2004] was referred to.
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 465.96, ALogP 3.72, HBA 6, HBD 2, PSA 114.29, QED 0.52
Compound (SMILES): Nc1ncnc2c1C(=O)N(c1ccc3c(ccn3C3COC3)c1)CCO2
InChIKey: QPDRVZIQNRAIPV-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Lipid Extraction (LE) Assay: For inhibition of triacylglycerol product formation, 11 uL reactions were run in white Polyplate-384 (PerkinElmer6007300) starting with a 30 minute pre-reaction incubation of 5 uL of 2.2× enzyme and 1 uL of 100% DMSO containing test compound or control compound, {4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]cyclohexyl}acetic acid. Some assays were performed with inclusion of didecanoylglycerol in the pre-reaction incubation of test compounds and enzyme. Reactions were initiated after 30 minute pre-reaction incubation via addition of 5 uL of 2.2× substrate. Final reaction conditions consisted of 50 mM HEPES pH 7.5, 2 mM MgCl2, 1 mM CHAPS, 25 uM didecanoylglycerol, 0.5 uM decanoyl-CoA, 0.3 nCi/uL [14C]-decanoyl-CoA or 0.5 nCi/uL [3H]-decanoyl-CoA, 0.05-4 ug/mL microsomal protein, and 1% DMSO. Following 60 minute reaction incubation, reactions were stopped with 40 uL of 45% isopropanol and 50 mM sodium carbonate in water and mixed. Extraction of tridecanoylglycerol product was accomplished via addition of 30 uL Microscint-E (Perkin Elmer) and 2 hours of incubation (sealed). Plates were read on a Microbeta Microplate reader Inhibition was normalized to controls containing DMSO or 10 uM {4-[4-(4-amino-7,7-dimethyl-7H-pyrimido[4,5-b][1,4]oxazin-6-yl)phenyl]cyclohexyl}acetic acid.
Measurement: IC50 = 39.1 nM (pChEMBL 7.409999847412109)
Descriptors: MW 351.37, ALogP 1.62, HBA 7, HBD 1, PSA 95.5, QED 0.75
Compound (SMILES): CC(C)(C)C(=O)Nc1cn2nc(-c3c(-c4ccc(F)cc4)ncn3CCO)ccc2n1
InChIKey: BYASUXNLIPQMHW-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 8.98 nM (pChEMBL 8.050000190734863)
Descriptors: MW 422.46, ALogP 3.38, HBA 7, HBD 2, PSA 97.34, QED 0.51
Compound (SMILES): O=C(CN1C(=O)C2(CCN(C(=O)c3ccc4[nH]ncc4n3)CC2)c2c(Br)cccc21)N1CCC(F)(F)C1
InChIKey: KCZXNJAGFOBAQR-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: In Vitro Binding Assay for DDR1: An in vitro binding competition assay was performed to evaluate the effect of the compounds of present invention on DDR1 protein.Binding Competition Assay:This assay is based on the intracellular domain of the DDR1 protein which contains the kinase active site. The recombinant protein additionally carries a GST-tag that can be recognized by an Eu-labeled anti-GST antibody. A tracer compound binding to the active site is labeled with a dye so that a FRET donor acceptor pair can be formed. Excitation energy absorbed by the Europium complex (350 nm flash light or pulsed laser) is transferred to a suitable fluorescent dye, if it is in close proximity. Compounds binding competitively with the tracer molecule will displace the bound tracer molecules and reduce the FRET signal in a dose dependent manner. Due to the long lifetime of the Eu excited state, the emission of the donor and the acceptor can be measured in time-gated mode such that most of the intrinsic fluorescence contributions have already decayed. This results in high sensitivity, excellent reproducibility and high data quality. This sensitive detection method enables protein concentrations below 20 nM.Protein, tracer and labeled antibody were obtained from commercial sources. The assay was performed in 384 low volume microtiter-plates with a final volume of 15 al. Dose response curves were generated from 16 compound dilutions in DMSO as solvent compound dilutions, a solution containing protein and labeled antibody, and a solution containing the tracer which is added in the last step. The fluorescence of donor and acceptor were then measured after one hour incubation at room temperature. Every assay run was quality-controlled with dose response curves for two reference compounds.
Measurement: IC50 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 573.4, ALogP 3.11, HBA 5, HBD 1, PSA 102.5, QED 0.52
Compound (SMILES): COc1cccc(-c2ccc(Nc3nn(C)c4c3CN(C(C)=O)CC4)cc2)c1
InChIKey: FRIOEGXJPBCNAX-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 376.46, ALogP 3.74, HBA 5, HBD 1, PSA 59.39, QED 0.75