lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N=C(N)Nc1ccc(C(=O)Oc2ccc(CC(=O)N(CCC(=O)O)[C@@H](Cc3ccccc3)C(=O)O)c(Cl)c2)cc1
InChIKey: FTYHCWBLLGMQKJ-QHCPKHFHSA-N
Target: Serine protease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P07477
Assay: Inhibition Assay: The compound was dissolved in dimethyl sulfoxide (DMSO), and diluted to an arbitrary concentration (A). A was 100-fold diluted with a buffer (0.1 M Tris (pH 8.0), 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij38) (B). The r-h trypsin was diluted with a buffer to 0.088 ug/mL, and the m-trypsin was diluted with a buffer to 1/50 (C). The dilution ratio of the m-trypsin (1/50) was set to exhibit the same activity as the 0.088 ug/mL r-h trypsin as determined by kinetic analysis. The substrate solution of a substrate for the enzyme reaction, BZiPAR, (Rhodamine Reference Substrate) was diluted with a buffer to 5 umol/L (D). B; 5 uL, C; 5 uL, and D; 10 uL were added to a 384-plate, and incubated at room temperature for 30 minutes. The fluorescent signals were detected with Ex/Em = 497/520 using Tecan Safire Fluorometer. The compound was reviewed from 2500 nM to its 3-fold value, 0.0075 nM, at 12 concentrations, and the inhibitory rate of each compound was calculated.
Measurement: IC50 = 0.42 nM (pChEMBL 9.380000114440918)
Descriptors: MW 567, ALogP 3.41, HBA 6, HBD 5, PSA 183.11, QED 0.09
Compound (SMILES): O=C1CS/C(=N/Nc2nncc3ccccc23)N1c1ccc(Cl)cc1
InChIKey: VXXXFNQITZZUER-OQKWZONESA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR-2 (unknown origin) using poly (Glu, Tyr) 4:1 as substrate after 1 hr by ELISA
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 369.84, ALogP 3.75, HBA 6, HBD 1, PSA 70.48, QED 0.71
Compound (SMILES): CCS(=O)(=O)N(Cc1ccc(F)c(C(F)(F)F)c1)c1sc2ccccc2c1C#N
InChIKey: IWIQCZWVRWQVPG-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 442.46, ALogP 5.29, HBA 4, HBD 0, PSA 61.17, QED 0.5
Compound (SMILES): Cn1c(NC(=O)c2ccc3cc4n(c3c2)CC(C)(C)CNC4=O)nc2ccccc21
InChIKey: MFWROJLDICCFGJ-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15349
Assay: Kinase Glo Plus Assay: Human RSK2 protein, purchased from Invitrogen, is used to measure kinase activity utilizing Kinase Glo Plus (Promega) a homogeneous assay technology, which uses a luciferin-luciferase based ATP detection reagent to quantify residual ATP. The assay is performed using 0.75 nM His-RSK2, 0.75 .mu.M ATP and 1.0 uM S6 Kinase/RSK Substrate Peptide 1 (Upstate, catalog #12-124), in assay buffer consisting of 25 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 uM Na3VO4, 0.5 mM DTT, and 1% DMSO. Solutions of test compounds at various concentrations are prepared by 1:3 fold serial dilution of a 1 mM solution of compound in DMSO. The DMSO solutions are further diluted with assay buffer to a final concentration of DMSO of 5%. The assay is performed in a 384 well, white, non-binding plate (Corning, catalogue #3574). Solutions of test compounds (10 uL) are transferred to a dry assay plate, followed by addition of 20 uL kinase.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 401.47, ALogP 3.55, HBA 5, HBD 2, PSA 80.95, QED 0.54
Compound (SMILES): CC(C)S(=O)(=O)c1ccccc1Nc1nc(NC(=O)N2CCCc3ccc(C=O)nc32)ncc1Cl
InChIKey: TUWHASKAKTUGAI-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Biochemical Kinase Assay: Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 ul per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 16 mM MgCl2, 1122 uM ATP, 4 uM peptide (5-Fluo-Ahx-KKKKEEIYFFFG-NH2, Biosyntan GmbH) and 4.5 ul per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% Tween20, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 16 mM MgCl2, 6 nM FGFR4 (GST-FGFR4(388-802), produced in-house by expression in insect cells and affinity chromatography). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 ul per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35).
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 515, ALogP 4.25, HBA 8, HBD 2, PSA 134.25, QED 0.47
Compound (SMILES): CN1CCC2CN(C(=O)Cn3cc(NC(=O)c4cnn5cccnc45)c(-c4cc(Cl)ccc4OC(F)F)n3)CC2C1
InChIKey: NAYKQHDOXYWHIG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 585.02, ALogP 3.51, HBA 9, HBD 1, PSA 109.89, QED 0.35
Compound (SMILES): Cc1c(OCC(F)(F)F)ccc2[nH]cc(C(=O)OCC34CCN(CC3)CC4)c12
InChIKey: FATRSDJWXOSXSG-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Alpha-Bungarotoxin Binding Assay: Protocol 2: Studies were carried out using rat brain P2 membrane. All membrane was obtained from male Sprague-Dawley (SD) rats and collected on phosphate buffer. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent. In an α-Bungarotoxin binding assay, membrane was diluted with assay buffer (50 mM potassium phosphate, 1 mM pH 8.0 EDTA, 0.1 mM PMSF, 0.1% BSA). Test compounds were prepared in 100% DMSO solution. The starting concentration of the test compound was 3 μM, 8 points in 3 fold dilution. Assays were performed in a total volume of 200 μL containing [3H] α-Bungarotoxin (final concentration 0.4 nM), membrane suspension (80 μL containing 150 μg of membrane protein) and 20 μL of test compound. Non-specific binding (NSB) was determined with 1 μM α-Bungarotoxin. After 3 hours incubation (37° C.), the assay solution was transferred to a GF/B plate (Millipore). The assay was terminated by adding 100 μL ice-cold PBS dilution and then rapidly filtering over glass fiber filters presoaked in 0.5% polyethylenimine. The filters were washed 4 times with 200 μL/well of ice-cold PBS. They were then were transferred to the reading plate, and 250 μL of microscine 40 was added. The radioactivity trapped by the filters was determined by liquid scintillation counting MicroBeta.
Measurement: Ki = 229.8 nM (pChEMBL 6.639999866485596)
Descriptors: MW 396.41, ALogP 4.06, HBA 4, HBD 1, PSA 54.56, QED 0.77
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(NCCCn2cccn2)ccc1Oc1ccc(F)cc1F
InChIKey: WFOBHYAOFZSWBL-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method A: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 2.5 mM-797 nM. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 991 nM (pChEMBL 6)
Descriptors: MW 480.52, ALogP 5.22, HBA 7, HBD 1, PSA 70.31, QED 0.32
Compound (SMILES): Cc1ncc(C)n2nc(CCc3ccc4ccc(C(F)(F)F)cc4n3)nc12
InChIKey: HWPNBMFXBLZFQW-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: A PDE10A assay may for example, be performed as follows: The assay is performed in 60 uL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labeled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions are initiated by addition of the cyclic nucleotide substrate, and reactions are allowed to proceed for one hr at room temperature before being terminated through mixing with 15 uL 8 mg/mL yttrium silicate SPA beads (Amersham). The beads are allowed to settle for one hr in the dark before the plates are counted in a Wallac 1450 Microbeta counter.
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 371.37, ALogP 4.09, HBA 5, HBD 0, PSA 55.97, QED 0.54
Compound (SMILES): Cn1c(=O)c2c(CC(=O)Nc3nc(-c4ccc(C(F)(F)F)c(F)c4)cs3)nsc2n(C)c1=O
InChIKey: JEPYVPCCTJDATH-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 = 6.74 nM (pChEMBL 8.170000076293945)
Descriptors: MW 499.47, ALogP 3.16, HBA 9, HBD 1, PSA 98.88, QED 0.44
Compound (SMILES): CC(C)Nc1cncc(-c2c[nH]c3ccc(-c4nnc(N)o4)cc23)n1
InChIKey: UIGVFGIYKFGMBS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 1.501 nM (pChEMBL 8.819999694824219)
Descriptors: MW 335.37, ALogP 3.08, HBA 7, HBD 3, PSA 118.54, QED 0.52
Compound (SMILES): Cc1ccc(-c2noc(C3CC(F)(F)C3)n2)cc1NC(=O)c1cnc2ccc(C(C)O)cn12
InChIKey: UYLMYXBJJAZNJX-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 157 nM (pChEMBL 6.800000190734863)
Descriptors: MW 453.45, ALogP 4.51, HBA 7, HBD 2, PSA 105.55, QED 0.46
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2C(c2cccc(S(=O)(=O)NCCOCCOCCNC(=O)C(C)(C)C(=O)NCCOCCOCCNS(=O)(=O)c3cccc(C4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)c2)C1
InChIKey: BFOGCZFQXSLNRQ-UHFFFAOYSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE-3-mediated Na+ dependent H+ antiport expressed in opossum kidney cells in presence of NHE-1 antagonist EIPA by BCECF-AM dye based fluorescence assay
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 1101.01
Compound (SMILES): CC(C)(Nc1ncc(Br)c(Nc2cc(C3CC3)[nH]n2)n1)c1ccccc1
InChIKey: JFNDEGIWUAMVLV-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 413.32, ALogP 4.93, HBA 5, HBD 3, PSA 78.52, QED 0.53
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1C#N)c1nc2cccc(Cl)c2c(=O)n1-c1cncc(Cl)c1
InChIKey: DEGWHLBSFXMEQC-VIFPVBQESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 468.31, ALogP 3.09, HBA 10, HBD 3, PSA 161.42, QED 0.41
Compound (SMILES): c1ccc2nc(OCc3cn4c(-c5ccc(N6CCOCC6)nc5)cnc(N5CCOCC5)c4n3)ccc2c1
InChIKey: TUXJSDBVQINSPH-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay rPDE10: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi 3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter and results were expressed as cpm. For blank values the enz
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 523.6, ALogP 3.59, HBA 10, HBD 0, PSA 90.14, QED 0.33
Compound (SMILES): Cn1ncc2c1CCC[C@@H]2c1nc(-c2ccc(C(=O)Nc3cc(C(F)(F)F)ccn3)cc2)c2c(N)nccn12
InChIKey: GBTGVZYIPISXRS-SFHVURJKSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: LANCE-TR-FRET Assay: Assay Method B: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated "A5" peptide (Biotin-EQEDEPEGDYFEWLE-NH2), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 μL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity).
Measurement: IC50 = 740 nM (pChEMBL 6.130000114440918)
Descriptors: MW 532.53, ALogP 4.85, HBA 8, HBD 2, PSA 116.02, QED 0.34
Compound (SMILES): O=C(c1cccnc1)N1CCc2nc(Nc3ccccc3)ncc2C1
InChIKey: ORCGGUPGDCETGI-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 2.7 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 ul of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 ul of 0-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 ul of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 331.38, ALogP 2.81, HBA 5, HBD 1, PSA 71.01, QED 0.8
Compound (SMILES): O=C(N1C[C@H](O)CC[C@@H]1Cc1ccccc1)n1ncc(C(O)(c2ccc(F)cc2)c2ccc(F)cc2)n1
InChIKey: KJFCLWBWISSVKP-JWQCQUIFSA-N
Target: Monoacylglycerol lipase ABHD6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BV23
Assay: Inhibition of human ABHD6 using 2-AG as natural substrate by fluorescence based assay
Measurement: IC50 = 158.49 nM (pChEMBL 6.800000190734863)
Descriptors: MW 504.54, ALogP 3.88, HBA 6, HBD 2, PSA 91.48, QED 0.43
Compound (SMILES): CCOc1cnc(-c2ccc(NC(=O)Nc3ccc(NS(C)(=O)=O)cc3)cc2)nc1N1CCOCC1
InChIKey: UXAGWDGMSIYELR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR Kinase Assay: The activity of the compound against mTOR was examined by measuring the incorporation of 33P from [γ-33P]-ATP into 4EBP1. His-tagged, recombinant human 4EBP1 was expressed in E. coli, purified by nickel-nitrilotriacetic acid (Ni-NTA) resins, and stored at −80 °C. Phosphorylation of 4EBP1 by mTOR was assayed in the presence or absence of the compound and performed in a final volume of 25 μl reaction buffer containing 300 ng 4EBP1, 50 ng recombinant mTOR (Invitrogen), 50 mM HEPES (pH 7.5), 1 mM EGTA, 0.01% Polysorbate 20, 10 mM MnCl2, 2.5 mM DTT, 10 μM ATP, and 0.5 μCi [Î³-33P]-ATP (PerkinElmir) for 30 min at 30 °C. The reactions were terminated by adding 3% phosphoric acid. The 33P labeled 4EBP1 was transferred onto UniFilter-96 GF/B plate (PerkinElmer) and quantified by Top Count Microplate Scintillation Counter (PerkinElmer). For primary screening of kinase activity inhibition, each test compound was evaluated at 10 M in duplicate.
Measurement: IC50 = 70.4 nM (pChEMBL 7.150000095367432)
Descriptors: MW 512.59, ALogP 3.39, HBA 8, HBD 3, PSA 134.78, QED 0.42
Compound (SMILES): COc1cc(OC)cc(N(CCCc2nccc(OC)n2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: KDMGWBBETGCJDN-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 616.6 nM (pChEMBL 6.210000038146973)
Descriptors: MW 511.59, ALogP 4.62, HBA 10, HBD 0, PSA 100.31, QED 0.27
Compound (SMILES): CSc1ccc(/N=c2\[nH]c(=O)n(CC(C)(C)C(=O)O)c(=O)n2Cc2ccc(Cl)cc2)cc1
InChIKey: CZLXUMOXYGNOSM-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: 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 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 uM Fluo-3-AM solution (pH 7.5) containing 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, 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 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 601 nM (pChEMBL 6.21999979019165)
Descriptors: MW 474.97, ALogP 3.1, HBA 7, HBD 2, PSA 109.45, QED 0.51
Compound (SMILES): COc1cc(NS(=O)(=O)c2ccc(NS(=O)(=O)c3c(C)noc3C)cc2)nc(OC)n1
InChIKey: SSDWSTAYENJABB-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: SPR Competitive Binding Assay: SPR interaction analyses were performed with a Biacore 2000, using Biacore 2000 Control Software v3.2 and BIAevaluation v4.1 analysis software (Biacore) as described in Mol Cancer Ther 7:2621 (2008). For the competitive binding assays and the Ki determination, PtdIns(3,4,5)phosphate-biotin labeled liposomes (Echelon Biosciences) and SA chips were used with increasing concentrations of the compound tested.
Measurement: Kd = 6270 nM (pChEMBL 5.199999809265137)
Descriptors: MW 469.5, ALogP 1.7, HBA 10, HBD 2, PSA 162.61, QED 0.5
Compound (SMILES): N#CC(c1ccc(F)c(C(F)(F)F)c1)N1CCN(c2ncnc(N)c2-c2ccc(F)cc2)CC1
InChIKey: GZBACRMFEDHLMQ-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: Enzyme Assay: P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 474.44, ALogP 4.41, HBA 6, HBD 1, PSA 82.07, QED 0.56
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)c(Cl)c1)N1CC=C(c2ncc(-c3nnc(CCO)o3)cc2Cl)CC1
InChIKey: KLOWQQXEIZVHIP-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 uM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1x Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 (wash buffer), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 uM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1x Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 (assay buffer). Plates are then transferred to a FLIPR for assay.
Measurement: IC50 = 60.8 nM (pChEMBL 7.21999979019165)
Descriptors: MW 528.32, ALogP 5.31, HBA 6, HBD 2, PSA 104.38, QED 0.47
Compound (SMILES): CC1(C)C2CCC1(C)[C@@H](NC(=O)[C@H](CC1CCCCC1)NC(=O)N[C@@H](CCCCN)C(=O)O)C2
InChIKey: AUFLFYNRNWZZKB-SONHOIRQSA-N
Target: Carboxypeptidase B2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96IY4
Assay: Inhibition of activated human plasma TAFI incubated for 15 mins by chromogenic assay
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 478.68, ALogP 3.54, HBA 4, HBD 5, PSA 133.55, QED 0.29
Compound (SMILES): COc1ccc(Cc2c(O[C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)n[nH]c2C(F)(F)F)cc1
InChIKey: SPPQJVPPWKBTGA-MPZWGZMNSA-N
Target: Sodium/glucose cotransporter 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31639
Assay: Inhibition of human SGLT2 expressed in COS7 cells assessed as reduction in uptake of methyl-alpha-D-(U-14C)glucopyranoside incubated for 2 hrs by microplate scintillation counting method
Measurement: IC50 = 415 nM (pChEMBL 6.380000114440918)
Descriptors: MW 434.37, ALogP 0.21, HBA 8, HBD 5, PSA 137.29, QED 0.44
Compound (SMILES): CC1(C)C(=O)N(c2ccc(C#N)c(C(F)(F)F)c2)C(=S)N1c1ccc(C(=O)NCCCCCCn2cccc(O)c2=O)c(F)c1
InChIKey: NRMDHQRFKSFABW-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Inhibition of HDAC2 (unknown origin) assessed as inhibition of fluorogenic peptide deacetylation
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 643.66, ALogP 5.49, HBA 7, HBD 2, PSA 118.67, QED 0.17
Compound (SMILES): CC(C)c1cc(-c2cn(CCN3CCC3)c(C3CCN(c4ncnc(N)c4Cl)CC3)n2)ccn1
InChIKey: BBTVEWXIIOMQJE-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 481.05, ALogP 4.18, HBA 8, HBD 1, PSA 88.99, QED 0.54
Compound (SMILES): NS(=O)(=O)OC[C@H]1O[C@@H](n2cnc3c(NCCN4CCOCC4)ncnc32)[C@H](O)[C@@H]1O
InChIKey: NJGVOJZDTRPSHG-XNIJJKJLSA-N
Target: NEDD8-activating enzyme E1 catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TBC4
Assay: Inhibition of recombinant human untagged NAEalpha/N-terminal His-tagged NAEbeta expressed in Bac-to-Bac expression system using FLAG-tagged NEDD8 as substrate after 90 mins in presence of GST-tagged Ubc12 by HTRF assay
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 459.49, ALogP -2.59, HBA 13, HBD 4, PSA 187.18, QED 0.32
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)C3CCCC3)CC2)C1
InChIKey: HPACOSDHEUMMSF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 458.57, ALogP 2.93, HBA 7, HBD 1, PSA 106.73, QED 0.63
Compound (SMILES): CCN(C(=O)Oc1ccc(F)cc1)[C@@H]1CN(C(=O)C2CCN(c3ccc(C#N)cn3)CC2)C[C@H]1c1ccc(Cl)cc1
InChIKey: MJJYDSNIHQTGEG-WUFINQPMSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: [3H]SR142801 Competition Binding Assay: hNK3 receptor binding experiment were performed using [3H]SR142801 (Catalog No. TRK1035, specific activity: 74.0 Ci/mmol, Amersham, GE Healthcare UK limited, Buckinghamshire, UK) and membrane isolated from HEK293 cells transiently expressing recombinant human NK3 receptor. After thawing, the membrane homogenates were centrifuged at 48,000×g for 10 mM at 4° C., the pellets were resuspended in the 50 mM Tris-HCl, 4 mM MnCl2, 1 μM phosphoramidon, 0.1% BSA binding buffer at pH 7.4 to a final assay concentration of 5 μg protein/well. For inhibition experiments, membranes were incubated with [3H]SR142801 at a concentration equal to KD value of radioligand and 10 concentrations of the inhibitory compound (0.0003-10 μM) (in a total reaction volume of 500 μl) for 75 min at room temperature (RT). At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% PEI+0.3% BSA, Packard BioScience, Meriden, Conn.) with a Filtermate 196 harvester (Packard BioScience) and washed 4 times with ice-cold 50 mM Tris-HCl, pH 7.4 buffer. Nonspecific binding was measured in the presence of 10 μM SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 μl of microscint 40 (Canberra Packard S.A., Zürich, Switzerland) and shaking for 1 h.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 576.07, ALogP 5.48, HBA 6, HBD 0, PSA 89.77, QED 0.39
Compound (SMILES): O=[N+]([O-])c1cccc(C2Nc3ccc(Cl)cc3C3C=CCC32)c1
InChIKey: QTCBVCMEJTUCSW-UHFFFAOYSA-N
Target: Serine protease hepsin (SINGLE PROTEIN), Homo sapiens
UniProt: P05981
Assay: High-Throughput Screening Assay: To identify novel inhibitors of hepsin, the Chembridge DIVERSet 10,000 compound high-diversity library was screened using an assay based on the cleavage of the chromogenic peptide. In addition, the NINDS II library of 1040 compounds (MicroSource Discovery) was screened to identify hepsin inhibitors among established drugs and known bioactive molecules. Screens were performed in a 96-well format at a final compound concentration of 2 uM. To minimize false positives, positions 1 and 12 of each row contained DMSO/buffer controls. As a measure of reproducibility, the Z' score for this assay was 0.78 (Zhang et al., 1999 J Biomol Screen 4:67).
Measurement: IC50 = 980 nM (pChEMBL 6.010000228881836)
Descriptors: MW 326.78, ALogP 5.07, HBA 3, HBD 1, PSA 55.17, QED 0.48
Compound (SMILES): Cc1cccnc1-n1c(Cn2nc(-c3cc(O)cc(F)c3)c3c(N)ncnc32)cc2ccccc2c1=O
InChIKey: JOWSQZZQBWNGOC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 493.5, ALogP 3.98, HBA 9, HBD 2, PSA 124.74, QED 0.38
Compound (SMILES): CC(C)CC(=O)N1CCc2c(C(=O)N3CCC(c4ccc(F)c(F)c4C(F)(F)F)CC3)n[nH]c2C1
InChIKey: OIOXFKNTAAPPQB-UHFFFAOYSA-N
Target: Retinol-binding protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P02753
Assay: Scintillation Proximity Binding Assay: Untagged human RBP4 purified from urine of tubular proteinuria patients was purchased from Fitzgerald Industries International. It was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit from Pierce following the manufacturer's recommendations. Binding experiments were performed in 96-well plates (OptiPlate, PerkinElmer) in a final assay volume of 100 ul per well in SPA buffer (IX PBS, pH 7.4, 1 mM EDTA, 0.1% BSA, 0.5% CHAPS). The reaction mix contained 10 nM 3H-Retinol (48.7 Ci/mmol; PerkinElmer), 0.3 mg/well Streptavidin-PVT beads, 50 nM biotinylated RBP4 and a test compound. Nonspecific binding was determined in the presence of 20 uM of unlabeled retinol. The reaction mix was assembled in the dark under dim red light. The plates were sealed with clear tape (TopSeal-A: 96-well microplate, PerkinElmer), wrapped in the aluminum foil, and allowed to equilibrate 6 hours at room temperature followed by overnight incubation at +4° C. Radiocounts were measured using a TopCount NXT counter (Packard Instrument Company).
Measurement: IC50 = 15.05 nM (pChEMBL 7.820000171661377)
Descriptors: MW 498.5, ALogP 4.66, HBA 3, HBD 1, PSA 69.3, QED 0.62
Compound (SMILES): NC(=O)c1cccc(-c2n[nH]c3cnc(-c4cccnc4)cc23)c1
InChIKey: FVCNCURYFWVWRH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 307 nM (pChEMBL 6.510000228881836)
Descriptors: MW 315.34, ALogP 2.79, HBA 4, HBD 2, PSA 97.55, QED 0.61
Compound (SMILES): O=C(CC1([C@H]2COC[C@@H](C3CC3)N2S(=O)(=O)c2ccc(Cl)cc2)CC1)N1CC2CCC(C1)N2
InChIKey: ADNLECGZTDQUQX-IJZHQTRZSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of recombinant human CYP3A4 expressed in baculovirus infected insect cells using fluorescent substrate
Measurement: IC50 = 9900 nM (pChEMBL 5)
Descriptors: MW 494.06, ALogP 2.64, HBA 5, HBD 1, PSA 78.95, QED 0.66
Compound (SMILES): CCOc1ccnc(-c2cnc(N)c(C(=O)Nc3ncccc3N3CCC(C)(N)CC3)n2)c1F
InChIKey: GAOWZWXUASHKMF-UHFFFAOYSA-N
Target: Protein kinase C alpha type (SINGLE PROTEIN), Homo sapiens
UniProt: P17252
Assay: In Vitro Inhibition Activity Assay: The compounds of formula I were tested for their activity on different PKC isoforms according to a published method (D. Geiges et al. Biochem. Pharmacol. 1997; 53:865-875) The assay is performed in a 96-well polypropylene microtiterplate (Costar 3794) that has been previously siliconized with Sigmacote (Sigma SL-2). The reaction mixture (50 μL) contains 10 μL of the relevant PKC isozyme together with 25 μL of the PKC inhibitor compound and 15 μL of a mix solution that contains 200 μg/mL protamine sulfate, 10 mM Mg(NO3)2, 10 μM ATP (Boehringer 519987) and 3750 Bq of 33P-ATP (Hartmann Analytic SFC301, 110 TBq/mmol) in 20 mM Tris-buffer pH 7.4+0.1% BSA. Incubation was performed for 15 minutes at 32° C. in a microtiterplate shaking incubator (Biolabo Scientific Instruments). Reaction was stopped by adding 10 μl of 0.5 M Na2EDTA, pH 7.4. 50 μl of mixture are pipetted onto a pre-wetted phosphocellulose paper (Whatmann 3698-915) under gentle pressure.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 466.52, ALogP 2.62, HBA 9, HBD 3, PSA 145.17, QED 0.5
Compound (SMILES): COc1ccc(C(=O)NCCCCCC(=O)Nc2ccccc2N)cc1
InChIKey: MKNIPDRRTRTMNQ-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 355.44, ALogP 3.21, HBA 4, HBD 3, PSA 93.45, QED 0.48
Compound (SMILES): COCC1CN(c2cc(Nc3nccc(C(F)(F)F)n3)cc(-c3ccccc3)c2)CCO1
InChIKey: XRHNOGUCOODBMC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 μM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 μM final concentration). Final volume of the reaction was 10 μL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 78.7 nM (pChEMBL 7.099999904632568)
Descriptors: MW 444.46, ALogP 4.76, HBA 6, HBD 1, PSA 59.51, QED 0.59
Compound (SMILES): COc1cc(-c2cccc(OC(C)C)c2)ccc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3cc(C)ncn3)ccc21
InChIKey: HDHZDOAUCBIXOW-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 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 -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 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 556.64, ALogP 5.35, HBA 8, HBD 1, PSA 112.41, QED 0.27
Compound (SMILES): COc1ccc(C(C)(C)C)cc1-c1cccc(C(CC(=O)O)c2nc(C)c(C)s2)c1
InChIKey: LEUGOCPTHUIKAO-UHFFFAOYSA-N
Target: Lysosomal protective protein (SINGLE PROTEIN), Homo sapiens
UniProt: P10619
Assay: Inhibition Assay: Recombinant human cathepsin A (residues 29-480, with a C-terminal 10-His tag; R&D Systems, #1049-SE) was proteolytically activated with recombinant human cathepsin L (R&D Systems, #952-CY). Briefly, cathepsin A was incubated at 10 ug/ml with cathepsin L at 1 ug/ml in activation buffer (25 mM 2-(morpholin-4-yl)-ethanesulfonic acid (MES), pH 6.0, containing 5 mM dithiothreitol (DTT)) for 15 min at 37° C. Cathepsin L activity was then stopped by the addition of the cysteine protease inhibitor E-64 (N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamide; Sigma-Aldrich, # E3132; dissolved in activation buffer/DMSO) to a final concentration of 10 uM.The activated cathepsin A was diluted in assay buffer (25 mM MES, pH 5.5, containing 5 mM DTT) and mixed with the test compound (dissolved in assay buffer containing (v/v) 3% DMSO) or, in the control experiments, with the vehicle in a multiple assay plate. After incubation for 15 min at room temperature.
Measurement: IC50 = 530 nM (pChEMBL 6.28000020980835)
Descriptors: MW 423.58, ALogP 6.34, HBA 4, HBD 1, PSA 59.42, QED 0.5