lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)C(C)(C)Cn1c(=O)[nH]/c(=N\c2ccc(OC(C)C)c(C)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: HNEFEUVSRBUTDF-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 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 515.01, ALogP 3.57, HBA 8, HBD 1, PSA 107.68, QED 0.46
Compound (SMILES): CC(=O)N1CCc2nc(N3CCC(Oc4ccc(F)cc4F)CC3)c(N[C@H]3C[C@@H](O)C3)nc2C1
InChIKey: HRCVXNPXGOAGEG-CALCHBBNSA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: GPR6 In Vitro Assay (A.2): This cell based assay measures the ability of compounds to inhibit the constitutive cAMP activity of GPR6 receptor expressed in CHO-K1 cells. CHO cells were stably expressed with GPR6 receptor, whose expression is controlled by a tetracycline inducable element. The cells were cultured in medium containing F12K, 10% FBS, 1% PennStrep, 200 ug/mL Hygromycin. GPR6 receptor expression was induced for 20 hrs with 2 ug/ml doxycycline (sigma D9891) in growth media. After addition of doxycycline cells were plated at a density of 450-750 cells per well in 96-well half-volume black tissue culture plates (Costar) and placed in an incubator (37°, 5% CO2) for 20 hours prior to cAMP assays. Culture media was removed from cells and they were washed with 50 uL/well of Ringer's Buffer (MgCl2 0.047 mg/mL, NaH2PO4 0.18 mg/mL, Na2HPO4 0.1 mg/mL, KCl 0.34 mg/mL, NaHCO3 1.26 mg/mL, D-glucose 1.8 mg/mL, NaCl 7 mg/mL; pH=7.4). Compounds suspended in DMSO were diluted in Ringer's Buffer.
Measurement: IC50 = 0.0615 nM (pChEMBL 10.210000038146973)
Descriptors: MW 473.52, ALogP 2.64, HBA 7, HBD 2, PSA 90.82, QED 0.69
Compound (SMILES): Cc1c(-c2ccc(N3CCc4cccc(C(=O)Nc5nc6ccccc6s5)c4C3)nc2C(=O)O)cccc1N(C)C(=O)C1CCCCC1
InChIKey: QFDMSUUMMGGQNA-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 659.81, ALogP 7.72, HBA 7, HBD 2, PSA 115.73, QED 0.18
Compound (SMILES): O=C(O)c1cc(F)cc(-c2cccc(Nc3nccc(C(F)(F)F)n3)c2)c1
InChIKey: HGSQMMSBTUAJDB-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 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 377.3, ALogP 4.74, HBA 4, HBD 2, PSA 75.11, QED 0.64
Compound (SMILES): Cc1cc(C)n2nc(-c3ccc(CCCF)cc3)c(CC(=O)N(C)C)c2n1
InChIKey: ZSOLQDPPCUMXOB-UHFFFAOYSA-N
Target: Translocator protein (SINGLE PROTEIN), Rattus norvegicus
UniProt: P16257
Assay: Displacement of [3H]PK11195 from TSPO in rat heart membranes after 15 mins by scintillation counting method
Measurement: Ki = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 368.46, ALogP 3.55, HBA 4, HBD 0, PSA 50.5, QED 0.67
Compound (SMILES): CCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)CNC(=O)[C@H](CC(=O)O)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(C)=O)C(C)C)C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O
InChIKey: AMRSAIWQRBVXGF-MEYXDWQDSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 2386.79
Compound (SMILES): CC(C)OC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3ccccc3)ccc2[nH]1
InChIKey: ZMUGIIMMEYQHNN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 456.5, ALogP 4.37, HBA 5, HBD 3, PSA 107.55, QED 0.41
Compound (SMILES): O=c1c2cn[nH]c2c2ccc(-c3cccc[n+]3[O-])cc2n1CC(F)(F)F
InChIKey: YUEIWBNNOIZLLS-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzymatic Screening Assay: The enzymatic test is a test in two steps.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 360.3, ALogP 2.74, HBA 4, HBD 1, PSA 77.62, QED 0.44
Compound (SMILES): CN(CC(F)F)C(=O)c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1
InChIKey: CAJGPKJTNFNEIY-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 5400 nM (pChEMBL 5.269999980926514)
Descriptors: MW 411.33, ALogP 4.78, HBA 4, HBD 0, PSA 55.32, QED 0.57
Compound (SMILES): N[C@@H]1CC[C@@H](NC(=O)c2cc(NC(=O)c3cc(-c4ccc(F)cn4)c(Cl)cc3Cl)n(-c3ccccc3)n2)C1
InChIKey: GPXJMXXWJOXFNG-IAGOWNOFSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 358 nM (pChEMBL 6.449999809265137)
Descriptors: MW 553.43, ALogP 5.24, HBA 6, HBD 3, PSA 114.93, QED 0.3
Compound (SMILES): CC(C)N1[C@H](c2ccc(Cl)cc2)[C@@H](c2cccc(Cl)c2)C[C@@H](CC(=O)O)S1(=O)=O
InChIKey: CJIWXRJWNIPVBG-QKNQBKEWSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Homogenous Time-Resolved Fluorescence Assay (HTRF1 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1xPBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6-His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.
Measurement: IC50 = 650 nM (pChEMBL 6.190000057220459)
Descriptors: MW 456.39, ALogP 5.11, HBA 3, HBD 1, PSA 74.68, QED 0.68
Compound (SMILES): Cc1nn(Cc2ccc(-c3nc4c(F)cccc4[nH]3)cc2F)c(C)c1CC(=O)O
InChIKey: VUKZSJKMPUVLCP-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Binding Assay: The CRTH2 receptor binding assay is performed in a scintillation proximity assay (SPA) format with the radioligand [3H]-PGD2 (Perkin Elmer, NET616000MC). CHO-K1-hCRTH2 cell membranes are again homogenized by passing through a single use needle (Terumo, 23G) and diluted in SPA incubation buffer in suitable concentrations (0.5-10 ug protein/well). The SPA assay is set up in 96 well microtiter plates (Perkin Elmer, CatNo. 6005040) in SPA incubation buffer with a final volume of 200 ul per well and final concentration of 50 mM Tris-HCl, 10 mM MgCl2, 150 mM NaCl, 1 mM EDTA pH 7.4, 0.1% bovine serum albumin). The SPA assay mixture contains 60 ul of the membrane suspension, 80 ul of Wheat Germ Agglutinin coated PVT beads (GE Healthcare, RPNQ-0001, 0.3 mg/well), 40 ul of [3H]-PGD2 diluted in SPA buffer to a final concentration of 1 nM (50 000 dpm) and 20 ul of the test compound (dissolved in dimethylsulfoxid).
Measurement: Ki = 9.7 nM (pChEMBL 8.010000228881836)
Descriptors: MW 396.4, ALogP 4, HBA 4, HBD 2, PSA 83.8, QED 0.53
Compound (SMILES): CCC1CC(=O)NN=C1c1ccc2nc(-c3ccc(OC(C)CO)cc3)oc2c1
InChIKey: UYOASFLFWGQDMV-UHFFFAOYSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 296 nM (pChEMBL 6.53000020980835)
Descriptors: MW 393.44, ALogP 3.5, HBA 6, HBD 2, PSA 96.95, QED 0.67
Compound (SMILES): N#CCC1(n2cc(-c3c(F)cnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)c3ccc(O)cc3F)CC2)C1
InChIKey: MFJNAWBKFSYMLB-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 < 10 nM (pChEMBL 8)
Descriptors: MW 517.54, ALogP 3.64, HBA 7, HBD 2, PSA 114.07, QED 0.42
Compound (SMILES): Cc1ccc(NC(=O)C(=O)c2ccc3c(c2)c2ccccc2n3Cc2ccc(Cl)cc2)cc1
InChIKey: ARAJRAPDNBYFIX-UHFFFAOYSA-N
Target: Pancreatic triacylglycerol lipase (SINGLE PROTEIN), Sus scrofa
UniProt: P00591
Assay: Inhibition of porcine pancreatic lipase assessed as reduction in 4-nitrophenol formation from 4-nitrophenyl butyrate substrate preincubated for 30 mins followed by substrate addition measured after 5 mins by spectrophotometer
Measurement: IC50 = 13530 nM (pChEMBL 4.869999885559082)
Descriptors: MW 452.94, ALogP 6.63, HBA 3, HBD 1, PSA 51.1, QED 0.24
Compound (SMILES): CNC(=O)C(NC(=O)c1cc(OCC(F)(F)F)c(C2CC2)cn1)C(C)(C)C
InChIKey: YWZGEGZSNRFLOJ-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 57.3 nM (pChEMBL 7.239999771118164)
Descriptors: MW 387.4, ALogP 2.79, HBA 4, HBD 2, PSA 80.32, QED 0.79
Compound (SMILES): CC(C)Nc1nc2c(nc1N1CCC(Oc3ccc(F)cc3F)CC1)CCN(C(=O)C1CCCO1)C2
InChIKey: AJZJCRXVQJNSPO-UHFFFAOYSA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: GPR6 In Vitro Assay (A.1): This cell based assay measures the ability of compounds to inhibit the constitutive cAMP activity of GPR6 receptor expressed in CHO-K1 cells. CHO cells were stably expressed with GPR6 receptor, whose expression is controlled by a tetracycline inducable element. The cells were cultured in medium containing F12K, 10% FBS, 1% PennStrep, 200 ug/mL Hygromycin. GPR6 receptor expression was induced for 20 hrs with 1 ug/ml doxycycline (sigma D9891) in growth media. After addition of doxycycline cells were plated at a density of 250-500 cells per well in half-volume black clear bottom plates (Costar) and place in an incubator (37° C, 5% C(O)2) for 20 hours prior to cAMP assays.Culture media was removed from cells and they were washed with 50 uL of Ringer's Buffer (MgCl2 0.047 mg/mL, NaH2PO4 0.18 mg/mL, Na2HPO4 0.1 mg/mL, KCl 0.34 mg/mL, NaHC(O)3 1.26 mg/mL, D-glucose 1.8 mg/mL, NaCl 7 mg/mL; pH=7.4). Compounds suspended in DMSO were diluted in Ringer's Buffer.
Measurement: IC50 = 0.0179 nM (pChEMBL 10.75)
Descriptors: MW 501.58, ALogP 3.69, HBA 7, HBD 1, PSA 79.82, QED 0.65
Compound (SMILES): C[C@H](Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1cccc(OC(F)F)c1
InChIKey: PKRAGBQEVPWLDS-MISXGVKJSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 770 nM (pChEMBL 6.110000133514404)
Descriptors: MW 394.38, ALogP 2.96, HBA 7, HBD 2, PSA 96.81, QED 0.75
Compound (SMILES): CSCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1cnc[nH]1)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]c2ccccc12)NC(=O)[C@H](CCC(N)=O)NC(=O)c1cccc(CNC(=O)CN2CCN(CC(=O)O)CCN(CC(=O)O)CCN(CC(=O)O)CC2)c1)C(C)C)C(N)=O
InChIKey: WHRGCDXABDWFMA-CADXXNFBSA-N
Target: Gastrin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30550
Assay: Displacement of [125I]-BBN from GRPR in human PC3 cells after 1 hr by compuGamma counting or microplate scintillation counting method
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 1459.7
Compound (SMILES): COC(=O)[C@H](C)NP(=O)(Oc1cccc2ccccc12)OC(C)[C@H]1O[C@@H](n2ccc3c(N)ncnc32)[C@](C)(O)[C@@H]1O
InChIKey: SGLVGAVFUPZUBO-DQGDKRCRSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Polymerase Inhibition Assay: The ability of the nucleoside triphosphate derivatives to inhibit the enzymatic activity of the HCV NS5B RNA-dependent RNA polymerase was measured in a radiolabeled nucleotide incorporation assay modified from the method described in International Publication No. WO2002/057287. Briefly, 50 μL reactions containing 20 mM HEPES (pH 7.3), 7.5 mM DTT, 20 units/mL RNasIN, 1 μM each of ATP, GTP, UTP and CTP, 20 μCi/mL [33P]-CTP, 10 mM MgCl2, 60 mM NaCl, 100 μg/ml BSA, 0.021 μM DCoH heteropolymer RNA template and 5 nM NS5B (1b-BKΔ55) enzyme were incubated at room temperature for 1 hour. Assay was terminated by the addition of 500 mM EDTA (50 μL). The reaction mixture was transferred to a Millipore DE81 filter plate and the incorporation of labeled CTP was determined using Packard TopCount.
Measurement: EC50 = 4800 nM (pChEMBL 5.320000171661377)
Descriptors: MW 585.55, ALogP 2.92, HBA 12, HBD 4, PSA 180.28, QED 0.17
Compound (SMILES): CC(=O)c1ccc(Nc2nccc(-c3ccc(Oc4ccccc4N)cc3)n2)cc1
InChIKey: ZGQJIUPIGWPERM-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of IKKbeta (unknown origin)
Measurement: IC50 = 8100 nM (pChEMBL 5.090000152587891)
Descriptors: MW 396.45, ALogP 5.46, HBA 6, HBD 2, PSA 90.13, QED 0.33
Compound (SMILES): Cc1cccc2c1NC(=O)[C@@H](NC(=O)[C@H](CCC(F)(F)F)[C@H](CCC(F)(F)F)C(N)=O)N=C2c1cccc(C(F)(F)F)c1
InChIKey: DFPGVOULJBAUOC-GSHUGGBRSA-N
Target: Neurogenic locus notch homolog protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P46531
Assay: Biological Assay: The pharmacological properties of the compounds of this invention may be confirmed by a number of biological assays.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 624.5, ALogP 5.65, HBA 4, HBD 3, PSA 113.65, QED 0.31
Compound (SMILES): CC[C@@H]1OC(=O)NN=C1N1CCc2nc(-c3ccccc3)oc2C1
InChIKey: HTCXRVMFMCUFKZ-ZDUSSCGKSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 578 nM (pChEMBL 6.239999771118164)
Descriptors: MW 326.36, ALogP 2.53, HBA 6, HBD 1, PSA 79.96, QED 0.92
Compound (SMILES): CNCCN(C)C(=O)c1cc(C#N)ccc1C1C(C(C)=O)=C(C)N(c2cccc(C(F)(F)F)c2)C(=O)N1C
InChIKey: VWEDTLURUVPBKM-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available.The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL, of these compound dilutions were mixed with 10 uL Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL, substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 527.55, ALogP 4.34, HBA 5, HBD 1, PSA 96.75, QED 0.58
Compound (SMILES): CCOc1ccc(C[C@@H](NC(=O)NCc2ccccc2)C(=O)N[C@@H](Cc2ccccc2)C(=O)NCc2ccc(CN)cc2)cc1
InChIKey: HUDMDOPCYOIBRF-JHOUSYSJSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 861 nM (pChEMBL 6.070000171661377)
Descriptors: MW 607.76, ALogP 4, HBA 5, HBD 5, PSA 134.58, QED 0.14
Compound (SMILES): Cn1ncc(C(=O)N2CCC2)c1C(=O)Nc1ccc2nc(N3CCOCC3)nn2c1
InChIKey: GCQBVONOPOFNPX-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 ul contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 ul of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate.
Measurement: IC50 = 23.1 nM (pChEMBL 7.639999866485596)
Descriptors: MW 410.44, ALogP 0.4, HBA 9, HBD 1, PSA 109.89, QED 0.66
Compound (SMILES): Oc1ccc(Cl)cc1/C=N/c1nc(-c2ccccc2)cs1
InChIKey: JBEDXEOJJBSVPS-GIJQJNRQSA-N
Target: Urease (SINGLE PROTEIN), Canavalia ensiformis
UniProt: P07374
Assay: Inhibition Assay: Reaction mixtures consisting of 25 μL (1 unit/well) of Jack bean (Canavalia ensiformis) urease, 55 μL of buffer at pH 6.8, 100 mM of urea, and 5 μL of various concentrations of test compounds were incubated at 30° C. for 15 min in 96-well plates. Subsequently 45 μL phenol reagents (1% w/v phenol and 0.005% w/v sodium nitroprussside), and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% w/v NaOCl) were added to each well. Urease activity through indophenols method was confirmed by the production of ammonia, as described by Weatherburn [12]. After 50 min, the increasing absorbance at 630 nm was measured in a microplate reader SpectraMax M2 (Molecular Devices, CA, USA). All reactions were performed in triplicate in a final volume of 200 μL.
Measurement: Ki = 7890 nM (pChEMBL 5.099999904632568)
Descriptors: MW 314.8, ALogP 4.92, HBA 4, HBD 1, PSA 45.48, QED 0.7
Compound (SMILES): CC(=O)OCc1n[nH]c(-c2cc(C(=O)N3CC(c4ccc(C#N)cc4)C3)c(C)cc2C)c1C
InChIKey: SWKACKMGPPUJLE-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 851 nM (pChEMBL 6.070000171661377)
Descriptors: MW 442.52, ALogP 4.18, HBA 5, HBD 1, PSA 99.08, QED 0.6
Compound (SMILES): CC1Cc2c(Cl)cccc2N1C(=O)Cc1nc(N2CCOCC2)cc(=O)n1C
InChIKey: XMQJMBYTKNDURO-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Sandwich Immunoassay: This test is based on measurement of the expression of AKT protein phosphorylated on serine 473 (P-AKT-S473) in the PC3 human prostate carcinoma line, by the technique based on a sandwich immunoassay using the Kit MSD Multi-spot Biomarker Detection of Meso Scale Discovery: phospho-Akt kits (Ser473) whole cell lysate (# K151 CAD) or phospho-Akt (Ser473) / Akt whole cell lysate Total (# K15100D). The primary antibody specific for P-AKT-S473 (Kit # K151 CAD) is coated onto an electrode in each well of 96-well plates of the MSD kit: after adding a protein lysate in each well, the revelation of the signal done by the addition of a secondary antibody labeled with an electrochemiluminescent detection compound. The procedure is described in the kit.On day 1, the PC3 cells are inoculated in 96-well plates (TPP # 92096) at a concentration of 35000 cells / well in 200 uL DMEM medium (DMEM Gibco # 1 1960-044) containing 10% fetal calf serum (FBS Gibco, # 10500-056) and 1% glutamine.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 402.88, ALogP 1.79, HBA 6, HBD 0, PSA 67.67, QED 0.78
Compound (SMILES): Cc1nnc(-c2cc(C(=O)N3CC(F)(c4ccc(C#N)cc4)C3)c(C)cc2C2CCC2)[nH]1
InChIKey: QQWSNUCKIMBENB-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 429.5, ALogP 4.55, HBA 4, HBD 1, PSA 85.67, QED 0.66
Compound (SMILES): CC(C)(C)c1ccc(N2C(=O)CC[C@@H]2COCc2ccc(C(=O)O)s2)cc1
InChIKey: BKVIUNNZVOCFGC-MRXNPFEDSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Displacement of [3H]-PGE2 from human EP2 receptor expressed in HEK293 cells at pH 6 after 60 mins
Measurement: Ki = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 387.5, ALogP 4.46, HBA 4, HBD 1, PSA 66.84, QED 0.79
Compound (SMILES): CNC(=O)N1C(=O)N(c2cccc(C(F)(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1[S+](C)[O-]
InChIKey: JOEAZLHYACREHB-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available. The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL of these compound dilutions were mixed with 10 ul Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 516.5, ALogP 4.25, HBA 5, HBD 1, PSA 116.57, QED 0.61
Compound (SMILES): C[C@H]1OC(=O)[C@]2(NC(=O)c3ccc(C#N)cc3)CC(F)(F)[C@@H](C)[C@H](/C=C/c3ccc(-c4ccccc4C#N)cn3)[C@H]12
InChIKey: MLDMOMJXXPHEOW-LPVDQPKTSA-N
Target: Proteinase-activated receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25116
Assay: FLIPR Assay: HEK 293 Cells were grown in media containing DMEM, 10% FBS pen/strep/L-Glutamine and non-essential amino acids. The cells were plated in 384-well PDL coated plates at 12000 cells/well and incubated overnight at 37° C./5% CO2. Media was then removed from the cells, which were then incubated with buffer (Hank's containing HEPES and Chaps) containing FLIPR calcium-5 dye, made with buffer containing probenecid, for 60 minutes at 37° C. Varying concentrations of compound in a final concentration of 5% DMSO were then added to the cells and incubated at 25° C. for 30 minutes. The plates were then added to the FLIPR Tetra and the device added a concentration of a PAR1 selective receptor-activating peptide with the sequence Ala-parafluoroPhe-Arg-Cha-Cit-Try-Nh2 (prepared in water) at a concentration equal to the effective concentration that achieves 80% activation of signaling on the day of the experiment.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 552.58, ALogP 5.53, HBA 6, HBD 1, PSA 115.87, QED 0.42
Compound (SMILES): O=C(O)C(=O)Nc1ccc2nc(NC(=O)c3cc(Br)c(Br)[nH]3)sc2c1
InChIKey: ZDVZBRWEKLWNNU-UHFFFAOYSA-N
Target: DNA gyrase subunit B (SINGLE PROTEIN), Staphylococcus aureus
UniProt: P0A0K8
Assay: Inhibition of Staphylococcus aureus DNA gyrase B ATP binding site assessed as reduction in supercoiling of relaxed pNO1 DNA after 30 mins in presence of biotinylated oligonucleotide by sybrGOLD staining based fluorescence assay
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 488.12, ALogP 3.42, HBA 5, HBD 4, PSA 124.18, QED 0.42
Compound (SMILES): CC(C)Oc1ccc(Nc2nc(=O)n(CC(C)(C)CO)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: HGRPNRICIGWDCD-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 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 490.96, ALogP 3.8, HBA 8, HBD 2, PSA 98.38, QED 0.47
Compound (SMILES): COC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(NS(=O)(=O)O)cc1)c1csc(-c2cnccn2)n1
InChIKey: VXTVFOVFFRVRJC-SFTDATJTSA-N
Target: Receptor-type tyrosine-protein phosphatase beta (SINGLE PROTEIN), Homo sapiens
UniProt: P23467
Assay: Inhibition of recombinant HPTPbeta (unknown origin) preincubated for 10 mins followed by substrate addition measured after 15 mins by DiFMUP probe-based fluorescence assay
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 582.66, ALogP 3.18, HBA 9, HBD 4, PSA 172.5, QED 0.19
Compound (SMILES): Cc1cc(NC(=O)N2CCC(N3CC(CC#N)(n4cc(-c5ncnc6[nH]ccc56)cn4)C3)CC2)n(C)n1
InChIKey: KVMNGYIMVRBSMH-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 499.58, ALogP 2.48, HBA 9, HBD 2, PSA 136.58, QED 0.43
Compound (SMILES): CNC(=O)CN1C(=O)COc2ccc(C)cc21
InChIKey: XGPUHLZHIZACTI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase PLK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53350
Assay: Fluorescent Polarization Binding Assay: The buffer composition was 50 mM Tris, 200 mM NaCl, 2.0 mM DTT, and 0.005% Tween 20 (pH 8.0). Fragment ligated inhibitors and comparison peptides to be tested in the binding assay were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM, compounds were utilized in a range of concentrations from 10 nM to 600 μM. The PLK1 PBD protein (residues 367-603) was obtained from BPS Bioscience Inc. (San Diego, Calif.) and 250 ng was used per sample. The fluorescein-tracer phospho-peptide was used at a final concentration of 100 nM. Each sample was prepared in triplicate in a black 96-well plate (Greiner, Monroe, N.C.). Reactants were added to the plate in darkened conditions to minimize exposure to light. Incubation was carried out at room temperature for 45 minutes. Fluorescence was measured using a DTX 880 Multimode detector plate reader (Beckman Coulter, Brea, Calif.) and Multimode Analysis software (Beckman Coulter).
Measurement: IC50 = 350000 nM (pChEMBL 3.4600000381469727)
Descriptors: MW 234.25, ALogP 0.47, HBA 3, HBD 1, PSA 58.64, QED 0.81
Compound (SMILES): COc1c(Nc2ncc(Cl)c(N[C@H]3[C@@H](C(N)=O)[C@@H]4C=C[C@H]3C4)n2)ccc2c1CCC[C@@H](N1CCOCC1)C2
InChIKey: LIBCMUMCEYECFM-QZYSWDHYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition of human ALK expressed in baculovirus using recombinant GST PLC-gamma as substrate assessed as phosphorylation of the substrate after 15 mins by TRF assay
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 539.08, ALogP 3.55, HBA 8, HBD 3, PSA 114.63, QED 0.36