lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCS(=O)(=O)N1CC(CC#N)(n2cc(-c3c(Cl)cnc4[nH]ccc34)cn2)C1
InChIKey: SIADCNVXYZQNTO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1, JAK2, JAK3 and TYK2 Enzymatic Inhibition Assay: 1. Test Materials    JAK1, JAK2, JAK3 and TYK2 enzymes were purchased from Life Technologies; and substrate GFP-STAT1 was purchased from Life Technologies.2. Test MethodTest compounds, enzymes, the substrate and ATP were diluted to the desired concentrations with the assay buffer (40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1% BSA). The former three were added into a multiple well plate, and incubated at room temperature after being mixed homogeneously. ATP was added to initiate the kinase reaction, and incubation at room temperature was performed. ADP-Glo was added, and incubation at room temperature was performed. The kinase detector reagent was then added, and incubation at room temperature was performed. The luminescence intensity of each test group was detected, and the half maximal inhibitory concentration (IC50) value was calculated.
Measurement: IC50 = 1.14 nM (pChEMBL 8.9399995803833)
Descriptors: MW 404.88, ALogP 2.35, HBA 6, HBD 1, PSA 107.67, QED 0.7
Compound (SMILES): CCOC(=O)c1cn2ccc(NC(=O)C(CCOC)n3cc(OC)c(-c4cc(Cl)ccc4C#N)cc3=O)cc2n1
InChIKey: VLNBINQYXHFUGN-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Biochemical Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions is placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride; 5 mM of calcium chloride; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 564, ALogP 4.09, HBA 10, HBD 1, PSA 136.95, QED 0.28
Compound (SMILES): CCOc1ncc(-c2cccc(N(CC34CCC(c5nc(C(C)(C)C)no5)(CC3)CC4)C(=O)C3CC(OC)C3)c2)cn1
InChIKey: ZRAJJMAHRACFEI-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/mL Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 689 nM (pChEMBL 6.159999847412109)
Descriptors: MW 573.74, ALogP 6.27, HBA 8, HBD 0, PSA 103.47, QED 0.29
Compound (SMILES): Cc1nc2cccc(C#N)c2c(=O)n1CCOc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: MBZSTJIKAVKKEE-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 20.4 nM (pChEMBL 7.690000057220459)
Descriptors: MW 516.97, ALogP 5.28, HBA 8, HBD 1, PSA 118.1, QED 0.33
Compound (SMILES): CNc1cc(Nc2cccn([C@H]3C[C@H](F)C3)c2=O)nc2c(C(=O)N[C@H]3CC[C@H]3O)cnn12
InChIKey: UHJGCIYWKNJVKL-LMRQPLJMSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 441.47, ALogP 1.6, HBA 9, HBD 4, PSA 125.58, QED 0.46
Compound (SMILES): C[C@@H]1OCC2(CCN(c3cnc4c(N5CCNc6cc(-c7ccnnc7)ccc65)n[nH]c4n3)CC2)[C@@H]1N
InChIKey: JVMVJWBENFFNFE-QMHKHESXSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.The inhibition of SHP2 by compounds of the disclosure (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.25 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat #D6567, 100 μM final) was added to the reaction and the conversion of DiFMUP to 6,8-difluoro-7-hydroxyl-4-methyl coumarin (DiFMU) was monitored continuously for 10 minutes with excitation at 355 nm and emission at 460 nm using a microplate reader (PolarStar, BMG).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 498.6, ALogP 2.71, HBA 10, HBD 3, PSA 134, QED 0.39
Compound (SMILES): O=C(N(CC12CCC(c3cc(C(F)(F)F)ccn3)(CC1)CC2)c1cccc(-c2nc(C3CC3)no2)c1)C12CC(F)(C1)C2
InChIKey: VMZWPCHDNNGRSG-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/mL Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 462 nM (pChEMBL 6.329999923706055)
Descriptors: MW 580.63, ALogP 7.55, HBA 5, HBD 0, PSA 72.12, QED 0.27
Compound (SMILES): O=C(c1cnc(NCc2cccnc2)nc1)N1CCn2ccnc2C1
InChIKey: KPKSEHHNXOFTBC-UHFFFAOYSA-N
Target: Pantetheinase (SINGLE PROTEIN), Homo sapiens
UniProt: O95497
Assay: Enzyme Assay: The test inhibitors were solubilized in DMSO to a stock concentration of 30 mM. On the day of the assay, dose response plates were prepared by diluting the inhibitors in DMSO at compound concentration 200-fold the final in-assay concentration. Intermediate concentrations were prepared by diluting in DMSO in a four-fold series for a total of 11 data points.To prepare a working solution of human vanin-1, the enzyme was diluted to 33.3 pM in the assay buffer consisting of 50 mM Tris-HCl pH=8.0, 50 mM KCl, 0.005% Brij-35 and 1.6 mM cysteamine. To begin the assay 100 nL was transferred from the compound plate to the assay plate. Next, 15 μL of the vanin-1 working solution were transferred to the assay plate. The inhibitor and enzyme were incubated at room temperature for 30 minutes. The enzyme reaction was then initiated by the addition of 5 μL of 200 μM pantetheine 7-amino-4-trifluoromethylcoumarin prepared in assay buffer. The final concentrations in the assay were 25 pM human vanin-1 and 50 uM substrate. The final concentration of DMSO was 0.5%. The assay plates were incubated for 60 minutes and before they were read on a Perkin Elmer EnVision Model 2103 using a 405 nm excitation wavelength and a 510 nm emission wavelength for detection.
Measurement: IC50 = 37.1 nM (pChEMBL 7.429999828338623)
Descriptors: MW 335.37, ALogP 1.34, HBA 7, HBD 1, PSA 88.83, QED 0.77
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](OC)c2ncc(Cl)cn2)nnc1-c1cccnc1
InChIKey: SRJHJXMTYJIIAQ-XOBRGWDASA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 0.21 nM (pChEMBL 9.680000305175781)
Descriptors: MW 546.01, ALogP 3.31, HBA 11, HBD 1, PSA 143.24, QED 0.31
Compound (SMILES): CCCCCCCCCC[C@H](O)C1C=CC2C(=O)N(Cc3ccccn3)C(=O)[C@H]2N1Nc1ccc([N+](=O)[O-])cc1
InChIKey: JXDKTXDCDQXNSG-PAIGMYNKSA-N
Target: Bifunctional polynucleotide phosphatase/kinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q96T60
Assay: Steady-State Fluorescence Assay: Kd values were measured by steady-state fluorescence spectroscopy, by following protein tryptophan fluorescence quenching as a function of ligand concentration, as previously described.
Measurement: Kd = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 549.67, ALogP 5, HBA 8, HBD 2, PSA 128.91, QED 0.1
Compound (SMILES): COC(=O)[C@@H](C)Cn1c(=O)[nH]/c(=N\c2cccc(Br)c2)n(Cc2ccc(C)cc2)c1=O
InChIKey: UBJOUTJBMKHJNV-HNNXBMFYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 64 nM (pChEMBL 7.190000057220459)
Descriptors: MW 487.35, ALogP 2.5, HBA 7, HBD 1, PSA 98.45, QED 0.54
Compound (SMILES): CN1CC[C@@H](NC(=O)c2sc3nccc4c3c2NC(=O)N4c2cccc(-c3ccccc3)c2)C1
InChIKey: UIYWMIHSMGIGNZ-GOSISDBHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TBD: TBD
Measurement: IC50 = 316 nM (pChEMBL 6.5)
Descriptors: MW 469.57, ALogP 5.08, HBA 5, HBD 2, PSA 77.57, QED 0.44
Compound (SMILES): O=C1c2cc(Cl)ccc2-n2c1nc1cnccc1c2=O
InChIKey: LPGMEULGBAUPLT-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Indoleamine-2,3-Dioxygenase 1 Inhibitory Activity: Experimental method: IDO-1 can oxidatively cleave the indole ring of tryptophan to form N-formylkynurenine. Referring to the method in the literature (Eddy W. Yue, et al. J. Med. Chem. 2009, 52, 7364-7367), 20 nM IDO-1, 2 mM D-tryptophan, 20 mM ascorbic acid, 3.5M methyl blue and 0.2 mg/mL antioxidant enzyme were sequentially added to 50 mM potassium phosphate buffer at room temperature. Due to the formation of N-formylkynurenine, the reaction rate was recorded by the increasing absorbance of the solution at 321 nm. IC50 values were calculated using Prism GraphPad software.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 283.67, ALogP 1.98, HBA 5, HBD 0, PSA 64.85, QED 0.49
Compound (SMILES): COc1ccc(CC(=O)N2CCNCC2)cc1Nc1ncc(Cl)c(Nc2ccccc2S(=O)(=O)C(C)C)n1
InChIKey: ODXOPUPUEPJJDN-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition of ALK WT Anaplastic Lymphoma Kinase Activity: The following experiment was performed in order to measure the activity of the N2-(2-methoxyphenyl)pyrimidine derivative represented by formula 1 of the present invention to inhibit anaplastic lymphoma kinase (ALK) activity at enzyme level. To measure the activity to inhibit ALK, each of the compounds prepared in examples listed in table 2 (2 μl) was loaded in a 384 well plate. Then, each of the compounds was mixed with anaplastic lymphoma kinase (ALK) enzyme (1 μl) and biotin conjugated peptide substrate (2 μl), followed by culture for 15 minutes. ATP solution (5 μl) was added thereto, followed by kinase reaction at room temperature for 30 minutes. Streptavidin conjugated XL 665 (5 μl) dissolved in ethylenediaminetetraacetic acid solution and europium (Eu3+) conjugated anti-phosphotyrosine antibody (5 μl) were added to the reaction solution to terminate the reaction. Upon completion of the reaction, one hour culture was performed, followed by analysis using homogeneous time-resolved fluorescence (HTRF, Cisbio). OD615/665 was measured with Wallac Envision 2103. IC50 of each compound was determined by using prism software (Version 5.01, Graphpad).
Measurement: IC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 559.09, ALogP 3.78, HBA 9, HBD 3, PSA 125.55, QED 0.36
Compound (SMILES): Nc1noc2nc(NC3CC3)c(F)cc12
InChIKey: UVQCLCHLERGNRX-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzymatic Assay: Recombinant human IDO1 (rhIDO1) was expressed and purified from cultures of EC538 strain of E. coli transformed with pREP4 and pQE9-IDO plasmid. Reaction mixes were set up in 384-well microplates containing 50 mM phosphate buffer, 10 mM ascorbic, 10 μM methylene blue, 100 μg/mL catalase, 80 μM TRP, 0.01% Tween 20 (v/v) mixed with rhIDO1 (15 μL) at a final concentration of 9 nM in a total volume of 30 μL assay medium. The plates were incubated at 37° C. for 30 min, and the enzymatic reaction was terminated by adding piperidine (200 mM) and heated at 65° C. for 20 min. Fluorescence intensity was read at λex 400 nm and λem 500 nm. Test compounds were dissolved in 100% DMSO and pre-diluted in assay medium prior to adding rhIDO1.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 208.2, ALogP 1.52, HBA 5, HBD 2, PSA 76.97, QED 0.78
Compound (SMILES): C=CC(=O)N1CCC[C@@H](n2nc(-c3ccc(Oc4ccc(Cl)cc4)nc3)c3c(N)ncnc32)C1
InChIKey: POERXYKRYQAMAK-QGZVFWFLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In-Vitro Btk Kinase Inhibitory Activity Assay: The drug was dissolved in DMSO to make a 10 mM (mmol/L) stock solution, and the stock solution was then diluted to a drug solution with 50× test concentrations for later use, wherein the test concentrations were reached through dilution at a 3-fold gradient and were 25 nM (nmol/L), 8.33 nM, 2.78 nM, 0.93 nM, 0.31 nM, 0.10 nM, respectively. 10 μL of the 50× drug stock solution was added to a 96-well plate and then 90 μL of a 1× Kinase Buffer was added and the 96-well plate was shaken for 10 minutes on a shaker. From each well of the 96-well plate, 5 μL of the drug solution was taken and then transferred to a 384-well plate which was provided with 2 duplicate wells.Kinase Reaction:Preparation of a 2.5× Kinase Buffer: an enzyme was added to the 1× kinase base buffer.Prepare a 2.5× oligopeptide solution: FAM-labeled oligopeptide and ATP were added to the 1× kinase base buffer.10 μL of the 2.5× Kinase Buffer was added to the 384-well plate loaded with 5 μL of the drug solution and incubation was then carried out for 10 minutes at room temperature. 10 μL of the 2.5× oligopeptide solution was added to the 384-well plate and incubation was then carried out for 1 hour at 28° C. The reaction was stopped by adding 25 μL of a stop buffer. The readings were recorded and the inhibition rate of the compound on the enzyme was calculated. The IC50 of BTK kinase was calculated by fitting.
Measurement: IC50 = 8.6 nM (pChEMBL 8.069999694824219)
Descriptors: MW 475.94, ALogP 4.27, HBA 8, HBD 1, PSA 112.05, QED 0.43
Compound (SMILES): O=C1NN=C(c2ccc(Cl)c(OC(F)(F)F)c2)CO1
InChIKey: VASSUNOWJBRYCA-UHFFFAOYSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A (SINGLE PROTEIN), Homo sapiens
UniProt: Q14432
Assay: PDE3A Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3A reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3A cell extract from Sf9 cells overexpressing human full length PDE3A (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3A cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:5000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3A activity vs log compound concentration.
Measurement: IC50 = 720 nM (pChEMBL 6.139999866485596)
Descriptors: MW 294.62, ALogP 2.68, HBA 4, HBD 1, PSA 59.92, QED 0.91
Compound (SMILES): COCc1nc(C2CC[C@@H](C)N(C(=O)c3ccccc3-n3nccn3)C2)oc1-c1ccccc1
InChIKey: XQSRAYPXPUZBJP-QSVWIEALSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added.
Measurement: IC50 = 347 nM (pChEMBL 6.460000038146973)
Descriptors: MW 457.53, ALogP 4.48, HBA 7, HBD 0, PSA 86.28, QED 0.42
Compound (SMILES): Nc1n[nH]c2ncc(-c3ccc(CNc4ncc(C(F)(F)F)cc4C(=O)Nc4ccc(F)cc4)cn3)cc12
InChIKey: WJPMPFMFMOSBAE-UHFFFAOYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 522.47, ALogP 5.02, HBA 7, HBD 4, PSA 134.5, QED 0.23
Compound (SMILES): COC(=O)C1CCC(C(=O)Nc2cc3cc(-c4cnn(C)c4)ccc3cn2)CC1
InChIKey: QXGWFGOTMGPLKW-UHFFFAOYSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: The screening assay for Wnt activity: The screening assay for Wnt activity is described as follows. Reporter cell lines can be generated by stably transducing cancer cell lines (e.g., colon cancer) or primary cells (e.g., IEC-6 intestinal cells) with a lentiviral construct that includes a Wnt-responsive promoter driving expression of the firefly luciferase gene.SW480 colon carcinoma cells were transduced with a lentiviral vector expressing luciferase with a human Sp5 promoter consisting of a sequence of eight TCF/LEF binding sites. SW480 cells stably expressing the Sp5-Luc reporter gene and a hygromycin resistance gene were selected by treatment with 150 μg/mL of hygromycin for 7 days. These stably transduced SW480 cells were expanded in cell culture and used for all further screening activities. Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 10-point dose-response curves starting from 10 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 384-well white solid bottom assay plates (Greiner Bio-One) with appropriate DMSO backfill for a final DMSO concentration of 0.1%. For Sp5-Luc reporter gene assays, the cells were plated at 4,000 cells/well in 384-well plates with a DMEM medium containing 1% fetal bovine serum, and 1% Penicillin-Streptomycin and incubated for 36 to 48 hours at 37° C. and 5% CO2. Following incubation, 15 μl of BriteLite Plus luminescence reagent (Perkin Elmer) was added to each well of the 384-well assay plates. The plates were placed on an orbital shaker for 2 min and then luminescence was quantified using the Envision (Perkin Elmer) plate reader. Readings were normalized to DMSO only treated cells, and normalized activities were utilized for EC50 calculations using the dose-response log (inhibitor) vs. response variable slope (four parameters) nonlinear regression feature available in GraphPad Prism 5.0 (or Dotmatics).
Measurement: EC50 = 3407 nM (pChEMBL 5.46999979019165)
Descriptors: MW 392.46, ALogP 3.55, HBA 6, HBD 1, PSA 86.11, QED 0.69
Compound (SMILES): Cn1c(-c2c[nH]nc2C(F)(F)F)nnc1C(C)(C)Oc1c(F)cc(F)cc1F
InChIKey: PXFLBRNOLCGGGK-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P16232
Assay: Inhibition Assay: The procedure for measuring the 11β-HSD1-inhibitory activity is as follows. The enzyme reaction and the measurement were carried out using a 384-well plate. The enzyme was prepared in accordance with Journal of Biological Chemistry, 2001, Vol. 276, p. 21343-21350. The reaction was carried out by adding a test compound at various concentrations to a reaction liquid consisting of a 5 mM phosphate buffer (pH 6.6), 200 nM cortisone, 40 μM reduced nicotinamide adenine dinucleotide phosphate (NADPH), and rat recombinant 11β-HSD1, followed by incubating at room temperature for one hour (10 μl/well). The test compound was prepared by dissolving in dimethyl sulfoxide (DMSO) such that a DMSO concentration reached 1% in the reaction liquid. After the enzyme reaction was completed, the enzyme inhibitory action was measured by detecting cortisol using a homogeneous time-resolved fluorescence (HTRF) method. Each of a d2-labeled cortisol containing 400 μM carbenoxolone and a cryptate-labeled cortisol antibody (CIS Bio International Co., Ltd.) was added at 5 μl/well, followed by incubating at room temperature for 2 hours, and then the fluorescence intensity was measured using a fluorophotometer (trade name: ARVO HTS 1420, Perkin Elmer/Wallac), and the enzyme inhibitory activity was calculated from the fluorescence intensity ratio of two wavelengths (665 nm/620 nm).The measurement results were calculated by averaging the values of 3 wells of the same condition. The ratio when DMSO was added instead of the test compound was taken as 0% and the ratio when 11β-HSD1 was not added was taken as 100%, thereby calculating the 50% inhibition concentration of the test compound as IC50 of the compound inhibitory activity.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 405.3, ALogP 3.96, HBA 5, HBD 1, PSA 68.62, QED 0.67
Compound (SMILES): Cc1cc(CNC(=O)c2cc(Cn3cc[nH]c3=N)cc(-c3cccn3C)c2)ccc1Cl
InChIKey: SCKVLJFPQFKGDA-UHFFFAOYSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: TR-FRET Binding Assay : A Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay that measures the displacement of the 10mer-Thr-FAM probe in response to compound treatment was performed for compounds wherein the IC50 from FPA assay using 10mer-Thr-FAM was below the lower assay IC50 limit 1 nM. Excess 10mer-Thr-FAM probe was utilized with His-tagged WDR5 in conjunction with a commercial anti-His antibody containing a Terbium label. The LanthaScreen Elite Tb-anti-HIS Antibody from ThermoFisher Scientific was used for this purpose. This Th-anti-HIS has an excitation/emission of 340 nm and 490 nm, respectively. The 10mer-Thr-FAM probe when bound to WDR5 will undergo a FRET interaction with the Tb-anti-HIS and emit at 520 nm. The ratio of the 520 and 495 signals are then utilized to generate a dose-response curve to calculate an IC50 value. By virtue of FRET there is little to no background fluorescence interference from 10mer-Thr-FAM probe allowing an excess of the probe to be used permitting an increase in the lower limit of the calculated Ki when testing against highly potent inhibitors with Ki<<1 nM. WDR5-His Tag (A23, residues 24-334) is expressed and purified in our lab in sufficient quantities for screening. 10mer-Thr-FAM peptide is used anywhere from 15 to 150 nM depending on the window of sensitivity required. WDR5-His tag protein is used at 2 nM. A source plate is prepared using an Echo Liquid Handler, which distributes the compounds to the assay plate (white, flat-bottom; OptiPlate) in a 10-point, 3-fold dilution schemes with a top concentration of either 5 or 20 μM depending on the anticipated potency of the compounds, in a final volume of 20 μL. A final target (WDR5)/Tb-Ab concentration of 2 nM/1 nM is dispensed from appropriate stock solutions, respectively. The final DMSO concentration in each well of the assay plate is 1% or lower. As before the plate is covered, shielded from light, and incubated for 60 minutes at room temperature with rocking. Anisotropy is then measured on a Biotek Cytation 3 at excitation wavelength of 340 nm, and emission wavelengths of 495 nm and 520 nm. Working buffer conditions (pH 7.0) are similar to that in FPA above. TR-FRET signal is plotted and IC50 and Ki values are calculated in the same manner as the fluorescence polarization anisotropy based competition assays.
Measurement: Ki = 0.35 nM (pChEMBL 9.460000038146973)
Descriptors: MW 433.94, ALogP 4.24, HBA 4, HBD 3, PSA 78.6, QED 0.42
Compound (SMILES): N#CC[C@@]1(n2nc(Nc3ccc4c(c3)CN(C3CCN(CC(F)(F)F)CC3)S4(=O)=O)c3c(=O)[nH]ccc32)CCOC[C@@H]1F
InChIKey: AXLSAJBAFIAJCA-BKMJKUGQSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical HTRF Assay : The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 623.63, ALogP 3.37, HBA 9, HBD 2, PSA 136.35, QED 0.4
Compound (SMILES): Cc1cc(F)ccc1N1C(=O)CSC1c1ccc(F)cc1
InChIKey: WUMZFSYLXLJDHV-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: FLIPR Calcium Assay: One day before the assay, CORNING black with clear flat bottom 96-well assay plates were coated with a 0.1 mg/mL Poly-L-Lysine solution. CHO-K1/MOR/Gα15 cells were suspended in the F12 medium and plated at a density of 8×104 cells/well in 200 μL medium. Cells were incubated in a humidified atmosphere of 10% CO2 at 37° C. overnight so as to reach an 80-90% confluent cell monolayer before assay. At the day of assay, 150 μL medium/well was removed from plate. To each well, 50 μL FLIPR calcium assay reagent dissolved in 1× assay buffer (HBSS: KCl 5 mM, KH2PO4 0.3 mM, NaCl 138 mM, NaHCO3 4 mM, Na2HPO4 0.3 mM, d-glucose 5.6 mM, with additional 20 mM HEPES and 13 mM CaCl2, pH 7.4), with 2.5 mM probenecid was added and the plate was incubated at 37° C. for 1 h. Compounds and other reagents were dissolved in the assay buffer. Using a FlexStationIII (Molecular Devices Corp.), the [Ca2+]i fluorescence increases after robotic injections of compounds or other reagents were monitored every 1.52 s interval with excitation wavelength at 485 nm and with emission wavelength at 525 nm. The [Ca2+]i fluorescence was measured up to 90 s after agonist injection. The fluorescence intensity from 6 to 12 wells of cells were averaged and the relative amount of [Ca2+]i release was determined by integrating the AUC of the [Ca2+]i fluorescence averages.
Measurement: EC50 = 3800 nM (pChEMBL 5.420000076293945)
Descriptors: MW 305.35, ALogP 4.05, HBA 2, HBD 0, PSA 20.31, QED 0.83
Compound (SMILES): COC(=O)CCn1c(=O)[nH]/c(=N\c2ccc(Oc3ccccn3)c(F)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: BPMLFIMUNBQLTG-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 525.92, ALogP 3.16, HBA 9, HBD 1, PSA 120.57, QED 0.35
Compound (SMILES): O=C1CCC(N2Cc3c(NC(=O)Nc4ccccc4OC(=O)CCC(=O)OCCN4CCN(CCNC=C5C(=O)CC(c6ccccc6)CC5=O)CC4)cccc3C2=O)C(=O)N1
InChIKey: YZGXROPEUJUSLL-MXNGAVTRSA-N
Target: Microtubule-associated protein 1 light chain 3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZQ8
Assay: Assay Targeting LC3B: By constructing a prokaryotic expression system, the LC3B protein was successfully expressed and purified, and a preliminary screening and verification platform was established using fluorescence polarization experiments to determine the activity of purchased and synthesized small compound libraries. The recombinant protein GST-LC3B (final concentration 180 nM) (SEQ ID NO: 1) and N-terminal FITC-labeled peptide (SEQ ID NO: 2, final concentration 18 nM) were placed in the FP buffer (50 mM HEPES pH 7.5, 0.1 mg/ml BSA and 1 mM DTT), to which a compound serially diluted with the FP buffer was added. Then the mixture was incubated at 25° C. in the dark. The fluorescence polarization value (PerkinElmer Envision, emission wavelength 480 nm; absorption wavelength 535 nm) was monitored, and the IC50 value was calculated using the GraphPad Prism 6.0 program.
Measurement: IC50 = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 847.93, ALogP 3.13, HBA 13, HBD 4, PSA 212.86, QED 0.04
Compound (SMILES): O=C(NC1CC(=O)N(c2ccc(CN3CCCCC3)cc2)C1)c1nnc(CCc2ccco2)s1
InChIKey: PPTXDUSMYKQDEK-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Quanta Red Assay: Measuring ATX activity using an enzyme coupled Quanta Red assay (Thermo Scientific Pierce Protein Research Products, Product #15159) was determined as follows. 8 μL human recombinant ATX (final concentration 0.8 μg/mL) in 1× Assay buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM CaCl2 was added to an opaque black flat-bottom 384-well plate (Corning, #3575) containing 2 μL test compound in 40% DMSO (4% final DMSO concentration). 10 μL of Quanta Red, Horseradish peroxidase (HRP), Choline Oxidase (CO), Rac-1-Palmitoyl-glycero-3-phosphocholine solution (final concentration 1:250 for Quanta Red, 0.5units/ml HRP, 0.5units/ml CO, 15 μM Rac-1-Palmitoyl-glycero-3-phosphocholine) in 1× assay buffer (as described previously) was added to each well to start the reaction and the plate was incubated at room temperature for 2 hours. The reaction was stopped after 2 hours with a 20 μL addition of Quanta Red Stop solution (1:20 dilution in distilled water). The above-described mixture with DMSO alone was used as a positive control whereas that with DMSO alone without ATX was taken as a negative control. For each test compound, ten concentrations were measured covering a range of 6.1 nM to 120 μM to determine IC50 values. The top concentration was decreased to 1.2 μM when a test compound's IC50 value was evaluated in low nanomolar range. Fluorescence was determined in a BMG Labtech Pherastar plus plate reader (λ emission=540 nm, λ excitation=590 nm). Data were analysed using Excel fit software.
Measurement: IC50 = 766 nM (pChEMBL 6.119999885559082)
Descriptors: MW 479.61, ALogP 3.44, HBA 7, HBD 1, PSA 91.57, QED 0.53
Compound (SMILES): Cn1cnc(S(=O)(=O)N2C[C@@H](C(=O)c3ccc(Cl)c(F)c3)[C@H](c3ccc(F)cc3)C2)c1
InChIKey: SVQXSPKLUQRVKA-DLBZAZTESA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 465.91, ALogP 3.64, HBA 5, HBD 0, PSA 72.27, QED 0.54