lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCC(CN1CC[C@H](CNC(=O)c2ccc3ccccc3c2)N[C@H](CCCN)C1=O)c1ccccc1
InChIKey: HIXSQRVONZIWDI-KREQFDAOSA-N
Target: Melanocortin receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P33032
Assay: Radioligand Binding Assay: Assessments of compound binding to human MC5R (hMC5R)) by displacement of an 125I-labeled NDP-MSH receptor ligand peptide were performed essentially as described in the data sheets produced by Perkin Elmer to accompany their frozen hMC5R membranes (Perkin Elmer catalog number RBXMC5M400UA). [125I] NDP-MSH: Radiolabeled in House and Purified by HPLC: Na125I (0.5 mCi, 17.4 Ci/mg) was added to 50 μL sodium phosphate (50 mM, pH 7.4) in an eppendorf tube precoated with IODOGEN. After incubation for 10 mins the phosphate buffer containing the iodine was added to NDP-MSH (10 μl at 1 mg/mL) in a separate eppendorf tube. This was incubated for a further 10 mins. The iodinated NDP-MSH was purified by HPLC on a Zorbax SB 300 column using solvent A: 0.05% TFA and solvent B: 90% acetonitrile 0.045% TFA with a linear gradient, 0-67% B over 60 mins. The 125I NDP-MSH eluted at 52 mins after the unlabeled starting material (48 min) and was counted and stored in the freezer. It was used within 48 hrs, as radioactive decay and ligand decomposition resulted in greatly reduced specific binding observed after 72 hrs. Incubation buffer: 25 mM HEPES-KOH (pH 7.0), 1.5 mM CaCl2, 1 mM MgSO4, 0.1 M NaCl, 1 mM 1,10-phenanthroline, and 1 Complete™ protease inhibitor tablet/100 mL (Roche, catalog number 1873580). Perkin Elmer frozen hMC5 membranes: catalog number RBXMC5M400UA, 0.4 mL/vial; 400 microassays/vial, 0.78 mg/mL protein concentration. Vials of frozen membranes were thawed rapidly immediately before use, diluted with binding buffer and vortexed. Resuspended membranes were kept on ice until they were added to the wells of the plate. Assays were performed in 96 well polypropylene plates. Membranes (0.78 μg 40 μL of a 1:40 dilution in incubation buffer) were added to [125I] NDP-MSH (0.84 nM; 2200 Ci/mmol) and test compounds in a total volume of 140 μL. This was incubated for 1 hr at 37° C. Non-specific binding was determined with 3 mM NDP-MSH. Plates were filtered using a Tomtec cell harvester with GF/A filters (Wallac) (presoaked in 0.6% polyethylenimine) and washed three times with 1.0 mL ice-cold wash buffer (the above incubation buffer without 1,10-phenanthroline and Complete™ protease inhibitor tablet). The filters were dried in a 37° C. oven, placed in a sample bag and 5 mL Betaplatescint (Wallac) was added. Prepared filters were counted in cassettes in a Microbeta Trilux (Wallac) for 1 min.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 486.66, ALogP 4.06, HBA 4, HBD 3, PSA 87.46, QED 0.4
Compound (SMILES): COCc1ccc(Cl)c(-c2ccc(Cl)c(C(=O)Nc3cc(C(N)=O)nn3-c3ccccc3)c2)n1
InChIKey: XFFNWUUJMUDZCP-UHFFFAOYSA-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 = 114.87 nM (pChEMBL 6.940000057220459)
Descriptors: MW 496.35, ALogP 4.74, HBA 6, HBD 2, PSA 112.13, QED 0.38
Compound (SMILES): CC(C)(CO)NC(=O)c1ccc(NC(=O)N2Cc3ccncc3C2)cc1
InChIKey: UEYJWRCDFKIIKF-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 50.6 nM (pChEMBL 7.300000190734863)
Descriptors: MW 354.41, ALogP 2.13, HBA 4, HBD 3, PSA 94.56, QED 0.78
Compound (SMILES): COc1ccc(Cc2nc(C(=O)NCc3c(C)cc(N)nc3C)co2)cc1
InChIKey: DCMBPCSGDIXGHI-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 2640 nM (pChEMBL 5.579999923706055)
Descriptors: MW 366.42, ALogP 2.8, HBA 6, HBD 2, PSA 103.27, QED 0.7
Compound (SMILES): CNC(=O)c1c(-c2ccc(C)nc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CO4)cc12
InChIKey: PPKAAKXAYSUNIR-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhbition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 8.5 nM (pChEMBL 8.069999694824219)
Descriptors: MW 611.66, ALogP 5.73, HBA 8, HBD 1, PSA 119.56, QED 0.26
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(C#CCC(C)C)cc1C21N=C(N)N(C)C1=O
InChIKey: TVMKDWFWVGMEKF-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 μl OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 μl water containing the desired concentration of test compound and 1% DMSO, 1 μM substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 μl in a 384 well plate. In general, 25 μl of compound dilution were given to the plate followed by the addition of 10 μl of BACE containing OptiMEM diluted 1:2 in water with 0.2% Triton X-100. The reaction was started with the addition of 15 μl substrate in NaOAc buffer. The reaction was incubated at 30° C. in a fluorimeter and the cleavage of the substrate was recorded as kinetic for 60 min. at ex: 530 nm, em: 590 nm. Blank wells containing either no inhibitor or no enzyme were included on each plate.The intensity of fluorescence was regressed against time in order to derive velocities of reaction in all 384 wells. These velocities were used for calculating percent inhibition using an uninhibited control containing 1% DMSO and a fully inhibited control incubations performed in the absence of enzyme. IC50 values were calculated by fitting percent inhibition vs. inhibitor concentration using standard software like GraphPadPrism.
Measurement: IC50 = 138 nM (pChEMBL 6.860000133514404)
Descriptors: MW 393.53, ALogP 3.2, HBA 4, HBD 1, PSA 67.92, QED 0.78
Compound (SMILES): Nc1onc(-c2ccc(Cl)s2)c1C(=O)Nc1ccc(S(N)(=O)=O)cc1
InChIKey: FEMMLRHJJPYAGV-UHFFFAOYSA-N
Target: Carbonic anhydrase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00915
Assay: Inhibition of recombinant human carbonic anhydrase 1 assessed as reduction in CO2 hydration preincubated for 15 mins by stopped flow assay
Measurement: Ki = 402 nM (pChEMBL 6.400000095367432)
Descriptors: MW 398.85, ALogP 2.54, HBA 7, HBD 3, PSA 141.31, QED 0.62
Compound (SMILES): Cn1cc(-c2cc(C(=O)Nc3cccnc3)c3cc(-c4ccccc4)ccc3n2)cn1
InChIKey: NRYOCNWNHYVQIU-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Biotin Assay: A streptavidin-coated plate (Thermo Scientific, NUNC #436014) was washed three times with 300 μL of PBS-0.05% Tween solution. A biotinylated twenty-six amino acid peptide, corresponding to the BH3 domain of Bim, with the sequence biotin-(β)A-D-M-R-P-E-I-W-I-A-Q-E-L-R-R-I-G-D-E-F-N-A-Y-Y-A-R-R-amide (SEQ ID No. 14), hereafter referred to as biotin-Bim, was obtained (Tufts). The biotin-Bim peptide was diluted to 0.018 μg/mL (5 nM) in SuperBlock blocking buffer in PBS (Thermo Scientific, #37515), and 100 μL of this solution was incubated in the streptavidin-coated plate for 2.5 hours while shaking. Separately, a compound of Formula I or Formula II, which had been prepared in a 10 mM stock DMSO solution, was then incubated with 20 nM GST-Mcl-1 fusion protein in PBS. Compounds were tested in six three-fold dilutions ranging from 20 μM to 0.6 μM, 10 μM to 0.3 or 5 μM to 0.15 μM. In addition, Bim peptide (New England Peptide) was utilized as a control and tested in six-fold dilutions ranging from 100 nM to 3.3 nM. The streptavidin-coated plate which had previously been treated with biotin-Bim peptide was then washed three times with 300 μL of PBS-0.05% Tween solution. A solution of GST-MCL-1 and a compound of Formula I or Formula II (100 μL) was then added to the streptavidin-coated plate, with two wells utilized as controls (PBS buffer only, no GST-MCL-1 or biotin-Bim) and two wells as an alternative control (containing GST-MCL-1 but no biotin-Bim), and four three-fold dilutions of DMSO (0.2%, 0.06%, 0.02%, and 0.008%). All wells containing compounds of Formula I or Formula II were loaded in duplicate. The plate was incubated for 2 hours at RT, then washed three times with 300 μL of PBS-0.05% Tween solution. Anti-GST HRP (GE Healthcare, #RPN1236) is diluted 1:20,000 in freshly prepared PBS, 0.1% Tween20, and 0.5% BSA was then added to the plate and the plate was incubated for 30 minutes. The plate was then washed five times with 300 μL of PBS-0.05% Tween solution. A solution of color reagents A (stabilized peroxide solution) and B (stabilized chrommogen solution) (R&D Systems, #DY999) is mixed in a 1:1 ratio and added to each well in a quantity of 80 μL. The wells were allowed to develop until control wells containing DMSO are blue (approximately 5-10 minutes). A solution of ELISA stop solution (1M sulfuric acid, 20 μL per well) was then added. Absorbance at 450 nM was read on a Tecan GeniosPro plate reader.
Measurement: IC50 = 7300 nM (pChEMBL 5.139999866485596)
Descriptors: MW 405.46, ALogP 4.95, HBA 5, HBD 1, PSA 72.7, QED 0.46
Compound (SMILES): O=C1NCCc2[nH]c(-c3cccc4ccc(NC5CCOCC5)nc34)cc21
InChIKey: PVPKHHBPGARBMQ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): 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 μM. A reference compound was included on each assay plate in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The final buffer conditions were as follows: 60 mM Hepes, pH 7.0, 0.05% BSA, 2 mM DTT. Incubations were carried out at RT (22° C.) for 2 h for Pim-1, 1 hour and 30 min for Pim-3, and 45 min for Pim-2. The reaction was stopped by the addition of 3 mM EDTA, and fluorescence was measured by an HTRF® Rubystar microplate reader.
Measurement: IC50 = 4.4 nM (pChEMBL 8.359999656677246)
Descriptors: MW 362.43, ALogP 3.11, HBA 4, HBD 3, PSA 79.04, QED 0.67
Compound (SMILES): NC(=O)c1nn(-c2ccc3c(c2)OCO3)c2c1COc1ccc(NC(=O)c3cc(Cl)cnc3Cl)cc1-2
InChIKey: FHPJDNHFYWJZST-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus infected insect cells using biotinylated IkappaBalpha peptide as substrate in presence of [gamma-33P]-ATP after 30 mins by resin-based scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 524.32, ALogP 4.21, HBA 8, HBD 2, PSA 130.59, QED 0.38
Compound (SMILES): O=C(O)COC[C@H]1CC[C@H](COC(=O)N(c2ccccc2)c2ccc(F)cn2)CC1
InChIKey: LQQIFSPLXNASKI-QAQDUYKDSA-N
Target: Prostacyclin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43119
Assay: Agonist activity at human recombinant prostacyclin receptor expressed in CHO-K1 cells assessed as induction of cAMP accumulation incubated for 1 hr by HTRF assay
Measurement: EC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 416.45, ALogP 4.4, HBA 5, HBD 1, PSA 88.96, QED 0.69
Compound (SMILES): CC(C)(O)CCNC(=O)c1cnc(-n2ccc3cc(C#N)cnc32)cc1NC1CCOCC1
InChIKey: MKJWQGQSIXPDIB-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 448.53, ALogP 2.77, HBA 8, HBD 3, PSA 125.09, QED 0.51
Compound (SMILES): COc1ccccc1C1N=C(Nc2nc3ccccc3o2)NC2=C1C(=O)CCC2
InChIKey: RTHIFOVBFAEADB-UHFFFAOYSA-N
Target: Galactokinase (SINGLE PROTEIN), Homo sapiens
UniProt: P51570
Assay: qHTS Assay: Assay details and protocol: The primary assay monitored ATP depletion using Promega's KinaseGlo technology, where ATP levels are measured through luminescence generated from firefly luciferase, a bioluminescent ATP-dependent enzymes. ATP was held at 35 μM, near its reported KM value, and the KM for galactose was determined under the 1536-well assay conditions to be 50-100M (FIG. 1A). As well, the IC50 for a commercially available CD45 inhibitor (N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide) was confirmed (previously found to inhibit GALK (FIG. 1B)). This was used as the positive control for the assay. The assay used 5 nM GALK and a 1 hr incubation time, which gave sufficient signal:background and stability for the HTS.
Measurement: IC50 = 48516.1 nM (pChEMBL 4.309999942779541)
Descriptors: MW 388.43, ALogP 3.96, HBA 7, HBD 2, PSA 88.75, QED 0.71
Compound (SMILES): Fc1cccc(-c2ncc(-c3cnc(-c4ccccc4)nc3)[nH]2)c1
InChIKey: GEDQDZBPZMITHD-UHFFFAOYSA-N
Target: Hematopoietic prostaglandin D synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O60760
Assay: Fluorescence Polarization Binding Assay (FPBA): This assay essentially is available from Cayman Chemical Company as Catalog item #600007. The test data reported for the aforementioned Examples (H-PGDS FPBA IC.sub.50) were generated using a 96-well, instead of the 384-well, format.Detection analyte and H-PGDS-MBP fusion enzyme were incubated in the presence of reduced glutathione (5 mM) for 60-90 minutes at room temperature and FP was measured using a TECAN SAFIRE 2 plate reader equipped with absorbance, fluorescence, fluorescence polarization and FRET capabilities. Assays were performed in 96-well microtiter plates in 100 .mu.L of total sample volume. Excitation and emission wavelengths appropriate for the employed detection analyte were used.
Measurement: IC50 = 188 nM (pChEMBL 6.730000019073486)
Descriptors: MW 316.34, ALogP 4.34, HBA 3, HBD 1, PSA 54.46, QED 0.61
Compound (SMILES): Cc1cccnc1-n1nnc2c1CCN(C(=O)c1cccc(C(F)(F)F)c1Cl)C2C
InChIKey: ZLYMBLCBXFXHPR-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Radioligand Binding Assay: human or rat P2X7-1321 N1 cells were collected and frozen @−80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl: 10 μl compound (10×)+(b) 40 μl tracer (2.5×)+50 μl membrane (2×). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2009, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM).
Measurement: Ki = 57.5 nM (pChEMBL 7.239999771118164)
Descriptors: MW 435.84, ALogP 4.4, HBA 5, HBD 0, PSA 63.91, QED 0.6
Compound (SMILES): CC(C)OCCNC(=O)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: RBZDDUADIMMOQI-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 51.7 nM (pChEMBL 7.289999961853027)
Descriptors: MW 367.45, ALogP 3.39, HBA 3, HBD 2, PSA 70.67, QED 0.77
Compound (SMILES): O=c1c2cn[nH]c2c2ccc(-c3cccnc3N3CCCNCC3)cc2n1CC(F)(F)F
InChIKey: ZBFZSKYNUAQWCA-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 = 6.08 nM (pChEMBL 8.220000267028809)
Descriptors: MW 442.45, ALogP 3.3, HBA 6, HBD 2, PSA 78.84, QED 0.51
Compound (SMILES): O=c1n(Cc2cnn(-c3ccccc3Cl)c2)nc(-c2ccc(Cl)cc2)n1C[C@H](O)C(F)(F)F
InChIKey: NUELIAFSZWATOT-SFHVURJKSA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Cellular In Vitro Assay: On the day before the assay, the cells are plated out in culture medium (DMEM, 10% FCS, 2 mM glutamine, 10 mM HEPES) in 384-well microtiter plates and kept in a cell incubator (96% humidity, 5% v/v CO2, 37° C.). On the day of the assay, the culture medium is replaced by a Tyrode solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM glucose, 20 mM HEPES), which additionally contains the cofactor coelenterazine (50 μM), and the microtiter plate is then incubated for a further 3-4 hours. The test substances in various concentrations are placed for 10 to 20 minutes in the wells of the microtiter plate before the agonist [Arg8]-vasopressin is added, and the resulting light signal is measured immediately in the luminometer.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 498.29, ALogP 4.18, HBA 7, HBD 1, PSA 77.87, QED 0.43
Compound (SMILES): C/C=C/c1sc(-c2nccs2)nc1C(=O)NCCCNC(=O)CS
InChIKey: ZGNYXDBIQSAFJW-DUXPYHPUSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzyme Assay: Enzyme activity of HDAC1,3 is determined using the substrate Ac-Lys-Tyr-Lys(Ac)-AMC while the enzyme activity of HDAC6 is assayed using the substrate Boc-lys(Ac)-AMC. The reaction is carried out in flat-bottom, 96-well, or 384-well microplates. After the substrate was deacetylated by HDACs, the product AMC which was obtained from hydrolysis by trypsin can generate fluorescence signal. Measurement is taken in using an multilabe plate reader a 355 nm excitation filter and a 460 nm emission filter. The initial rate of fluorescence should accurately reflect the rate of product formation and enzyme activity. Sample processing: The sample was dissolved in DMSO and kept at a low temperature. DMSO in the final system is limited under a low concentration which won't affect the enzyme activity.
Measurement: IC50 = 937 nM (pChEMBL 6.03000020980835)
Descriptors: MW 382.54, ALogP 2.47, HBA 7, HBD 3, PSA 83.98, QED 0.48
Compound (SMILES): Cc1nn(C)cc1Nc1ncc(Cl)c(CCc2ccccc2C2(C(N)=O)CC2)n1
InChIKey: PXZFWIYLLCBPCU-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P35916
Assay: VEGFR3 Biochemical Assay: A biotin labelled peptide is used as substrate (amino acid sequence: Biotin-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2). VEGFR3 cytoplasmic domain (amino acids 798-1298) was purchased as N-terminal GST-fusion protein (the enzyme). The 15 uL assay reactions are run in Greiner brand white 384-well low volume plates. All reactions contained 10 mM HEPES pH 7.4, mM MgCl2, 0.01% (v/v) Tween-20, 50 uM Na3VO4, 0.01% (w/v) albumin from chicken egg white, 1 mM Dithiothreitol, 111 nM peptide substrate, 500 uM ATP, and 3.8 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 90 minutes at 30 degree Celsius.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 410.91, ALogP 3.22, HBA 6, HBD 2, PSA 98.72, QED 0.62
Compound (SMILES): Cc1ccc(S(=O)(=O)[C@@]2(c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)CCN(Cc3ccccc3)C2)cc1F
InChIKey: ILLRVKROEYFTAD-DEOSSOPVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 3771 nM (pChEMBL 5.420000076293945)
Descriptors: MW 577.54, ALogP 7, HBA 3, HBD 0, PSA 37.38, QED 0.3
Compound (SMILES): CC(C)c1cccc(Nc2nc(=O)n(CCO)c(=O)n2Cc2ccc(Cl)cc2)c1
InChIKey: HHUYYWJOWGZXNS-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 = 347 nM (pChEMBL 6.460000038146973)
Descriptors: MW 414.89, ALogP 2.97, HBA 7, HBD 2, PSA 89.15, QED 0.62
Compound (SMILES): COc1cccc(OC)c1C1CC(C)C(=O)N1Cc1cccc(OC(F)F)n1
InChIKey: NDWXKTBZXNLPRE-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 955 nM (pChEMBL 6.019999980926514)
Descriptors: MW 392.4, ALogP 3.81, HBA 5, HBD 0, PSA 60.89, QED 0.72
Compound (SMILES): COc1ccccc1CN(c1cc2ccccc2s1)S(=O)(=O)c1ccccc1
InChIKey: JSTKACOJNVVZAC-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 = 112 nM (pChEMBL 6.949999809265137)
Descriptors: MW 409.53, ALogP 5.31, HBA 4, HBD 0, PSA 46.61, QED 0.43
Compound (SMILES): COc1ccc(-c2cc3c(N[C@H](C)c4ccccc4F)ncnc3o2)cc1
InChIKey: MHHVDHLBNLIXLU-CYBMUJFWSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human GST-tagged EGFR cytoplasmic domain (668 to 1210 residues) expressed in baculovirus by Z'-LYTE assay
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 363.39, ALogP 5.21, HBA 5, HBD 1, PSA 60.18, QED 0.53
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cn(Cc2cccc(C#N)c2)nn1
InChIKey: RFUVSHLZUAOYPH-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 361.41, ALogP 1.72, HBA 7, HBD 2, PSA 122.51, QED 0.71
Compound (SMILES): COc1cc(-c2cccc(C)c2F)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: AEFAQHPEHNORQY-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 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 523.52, ALogP 5.04, HBA 7, HBD 1, PSA 103.43, QED 0.33
Compound (SMILES): Cc1nc(C)n(CC(=O)Nc2cncc(C(=O)c3cn(C(C)C)c4ccncc34)c2)n1
InChIKey: YRHMMZORCPZXJQ-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 9800 nM (pChEMBL 5.010000228881836)
Descriptors: MW 417.47, ALogP 3.09, HBA 8, HBD 1, PSA 107.59, QED 0.48
Compound (SMILES): CCOC(=O)N1CCC(N(C(=O)c2ccc(-c3cnco3)cc2)C2CC2)CC1
InChIKey: UMJCMKGKDSYCEC-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] are 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 β-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37° C. 5% CO2. For the assay, the cells are seeded 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 is aspirated from the wells completely, 10 ul of the test compound are added, the compounds are diluted using 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 room temperature (approx. 20° C.), the cAMP concentrations are determined using the AlphaScreen cAMP Assay Kit (Cat. No. #6760625R from PerkinElmer). 10 ul of Biotin-cAMP (final concentration 1 U/well in lysing buffer (5 mM Hepes (pH 7.4), 0.1% BSA, 0.5% Tween) and 10 uL Bead solution (final concentration 1 U/well in lysing buffer) are added. The plates are incubated for another 2 hours at room temperature. The cAMP concentrations are calculated using a cAMP standard curve from the Alpha Screen Counts. The data analysis is carried out by calculating the EC50 value and the maximum value based on a positive control, using suitable software (Graphpad Prism).
Measurement: EC50 = 222 nM (pChEMBL 6.650000095367432)
Descriptors: MW 383.45, ALogP 3.57, HBA 5, HBD 0, PSA 75.88, QED 0.79
Compound (SMILES): O=C(Nc1ccc(-c2ncc(OCCN3CCOCC3)c(N3CCOCC3)n2)cc1)Nc1ccc(N2CCOCC2=O)cc1
InChIKey: ZFHHQBNWBRJBBL-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 = 42.3 nM (pChEMBL 7.369999885559082)
Descriptors: MW 603.68, ALogP 2.7, HBA 10, HBD 2, PSA 130.62, QED 0.38
Compound (SMILES): FCCn1cc(-c2cc3c(-c4cccc(N5CCCNCC5)n4)n[nH]c3cn2)cn1
InChIKey: DLZIZKARHRLIHL-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 = 0.014 nM (pChEMBL 10.850000381469727)
Descriptors: MW 406.47, ALogP 2.65, HBA 7, HBD 2, PSA 87.55, QED 0.53
Compound (SMILES): C[C@H](n1cnnc1-c1nc(NC(=O)c2cc(-n3cnc(C4CC4)c3)c(N3CCN(C4CC4)CC3)cn2)cs1)C(F)(F)F
InChIKey: DLDZDZXEDKRYGO-INIZCTEOSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Apoptosis Signal-Regulating Kinase Assay: The assay measures the phosphorylation level of a biotinylated peptide substrate by the ASK1 kinase using HTRF detection. This is a competitive, time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, based on HTRF® KinEASE™-STK manual from Cisbio. Test compound, 1 μM STK3 peptide substrate, 4 nM of ASK1 kinase were incubated with 10 mM MOP buffer, pH. 7.0 containing 10 mM Mg-acetate, 0.025% NP-40, 1 mM DTT, 0.05% BSA and 1.5% glycerol for 30 minutes then 100 μM ATP was added to start the kinase reaction and incubated for 3 hr. Peptide antibody labeled with 1× Eu3+ Cryptate buffer containing 10 mM EDTA and 125 nM Streptavidin XL665 were added to stop the reaction and phosphorylated peptide substrate was detected using Envision 2103 Multilabeled reader from PerkinElmer. The fluorescence was measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm was calculated for each well. The resulting TR-FRET level (a ratio of 665 nm/615 nm) was proportional to the phosphorylation level. Under these assay conditions, the degree of phosphorylation of peptide substrate was linear with time and concentration for the enzyme.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 598.66, ALogP 4.52, HBA 11, HBD 1, PSA 109.89, QED 0.32
Compound (SMILES): Cc1cnc(CNC(=O)c2ccc(NC(=O)N3Cc4ccccc4C3)cc2)o1
InChIKey: WAINGHMRPZCBEP-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 62.7 nM (pChEMBL 7.199999809265137)
Descriptors: MW 376.42, ALogP 3.46, HBA 4, HBD 2, PSA 87.47, QED 0.73
Compound (SMILES): Cn1cc(-c2cnc(N)c(C(=O)Nc3ccncc3)c2)cn1
InChIKey: VSGIYMAWHNSRDN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 : The kinase assay is performed as 384-well Flashplate assay assay (for e.g. Topcount measurement. 0.6 nM TANK binding kinase (TBK1), 800 nM biotinylated MELK-derived peptide (Biotin-Ah-Ah-AKPKGNKDYHLQTCCGSLAYRRR) SEQ ID NO: 2 and 10 uM ATP (spiked with 0.25 uCi 33P-ATP/well) are incubated in a total volume of 50 ul (10 mM MOPS, 10 mM Mg-acetat, 0.1 mM EGTA, 1 mM DTT, 0.02% Brij35, 0.1% BSA, pH 7.5) with or without test compound for 120 Min at 30° C. The reaction is stopped with 25 ul 200 mM EDTA. After 30 Min at room temperature the liquid is removed and each well washed thrice with 100 ul 0.9% sodium chloride solution.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 294.32, ALogP 1.71, HBA 6, HBD 2, PSA 98.72, QED 0.77
Compound (SMILES): O=C(CC(c1ccccc1)c1ccccc1)N1CCN(C2c3ccccc3-c3ccccc32)CC1
InChIKey: SGOBOOGKVDCWAY-UHFFFAOYSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1G (SINGLE PROTEIN), Homo sapiens
UniProt: O43497
Assay: Inhibition of human alpha1G T-type calcium channel expressed in HEK293 cells assessed as current dependent channel blocking at -110 mV holding potential with pre-pulse at -100 mV for 1 sec prior to test pulse at -40 mV for 50 ms by whole cell patch clamp assay
Measurement: IC50 = 371 nM (pChEMBL 6.429999828338623)
Descriptors: MW 458.61, ALogP 6.12, HBA 2, HBD 0, PSA 23.55, QED 0.36
Compound (SMILES): CN(c1ccc(F)cn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: WRKNUXRARNRWJC-GFCCVEGCSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z′-LYTE Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE Tyr 6 peptide or Z-LYTE Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100 μM, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 312.35, ALogP 2.21, HBA 5, HBD 1, PSA 60.94, QED 0.8
Compound (SMILES): O=C(Nc1ccc(F)c(C(F)(F)F)c1)c1cc2nc(Nc3c(F)cccc3Cl)[nH]c2c2c1OCCO2
InChIKey: FHKJUWSZNJHMSD-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition of mPGES-1 in human whole blood assessed as reduction in LPS-induced PGE2 production preincubated followed by LPS stimulation for 20 to 24 hrs
Measurement: IC50 = 1127 nM (pChEMBL 5.949999809265137)
Descriptors: MW 524.83, ALogP 6.28, HBA 5, HBD 3, PSA 88.27, QED 0.27