lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(C2=CCN(C(C)C)CC2)ccc1Nc1ncc(Cl)c(Nc2cccnc2S(=O)(=O)C(C)C)n1
InChIKey: XHPGIYLFNMYDEE-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: 1× Kinase Buffer    50 mM HEPES. pH 7.5    0.0015% Brij-35    10 mM MgCl2    2 mM DTT    2) stop buffer    100 mM HEPES, pH 7.5    0.015% Brij-35    0.2% Coating Reagent #3    50 mM EDTAPrepare a Compound Solution to be Tested    Step 2.1 Dilute the compound to 50× of the final desired highest inhibitor concentration in reaction by 100% DMSO. Transfer 100 μl of this compound dilution to a well in a 96-well plate. For example, if desired highest inhibitor concentration is 1 μM, then prepare 50 μM of compound DMSO solution in this step.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 557.12, ALogP 5.7, HBA 9, HBD 2, PSA 109.34, QED 0.34
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)Cc2cccnc2)c2ccccc2N(Cc2c(OC)ccc3c(Br)cccc23)C1=O
InChIKey: MGGITSBKIMQADG-FNZWTVRRSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 630.54, ALogP 4.22, HBA 6, HBD 2, PSA 103.87, QED 0.3
Compound (SMILES): O=S(=O)(Nc1cc(Cl)ccc1SCc1c[nH]cn1)c1cc2ccccc2o1
InChIKey: DBDJFYVRBGLORC-UHFFFAOYSA-N
Target: C-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P41597
Assay: Biological Assay: HEK-Gqi5 cells stably expressing CCR2 were cultured in (DMEM high glucose, 10% FBS, 1% PSA, 400 μg/ml geneticin and 50 μg/ml hygromycin. Appropriate positive control chemokines (MCP-1, MIP1A or RANTES) was used as the positive control agonist for screening compound-induced calcium activity assayed on the FLIPRTetra. The drug plates were prepared in 384-well microplates using the EP3 and the MuItiPROBE robotic liquid handling systems.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 419.92, ALogP 4.9, HBA 5, HBD 2, PSA 87.99, QED 0.43
Compound (SMILES): Cc1nc(C(=O)N[C@H](C)c2ccc(OC(F)(F)F)cc2)cc(=O)[nH]1
InChIKey: FIFOTGXFSFMDHB-MRVPVSSYSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 341.29, ALogP 2.47, HBA 4, HBD 2, PSA 84.08, QED 0.89
Compound (SMILES): COc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)C2(F)CCC2)C[C@@H]1F
InChIKey: DPVZLDSOWMITSW-LSDHHAIUSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 7100 nM (pChEMBL 5.150000095367432)
Descriptors: MW 539.47, ALogP 2.57, HBA 8, HBD 2, PSA 127.74, QED 0.48
Compound (SMILES): Cn1cc(C(=O)Nc2cccc(-c3nnc(Nc4ccc5[nH]nc(F)c5c4)s3)c2)cn1
InChIKey: GADAFEOPHFNEBV-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: Biological Assays for Inhibition of Rho-Associated Protein Kinase: Assays for ROCK inhibition were performed using the following protein constructs: glutathione S-transferase (GST)-tagged human ROCK1 catalytic domain 1-477 from Carna Biosciences (cat #01-109; apparent Km value for ATP is 10 μM) and GST-tagged human ROCK2 catalytic domain 1-553 from Carna Biosciences (Cat#01-110; apparent Km value for ATP is 15 μM). Protein constructs were purified from a baculovirus expression system. The peptide substrate was fluorescent LANCE® Ultra ULight-CREBtide: CKRREILSRRPSYRK (PerkinElmer, # TRF0107-D). Kinase reactions were carried out in in a 10 μL volume in 384-well plates: 50 nM ULight-CREBtide substrate, 2 nM constitutively active ROCK1 or ROCK2 kinase, and test compound in DMSO (or DMSO only for controls) were diluted into assay buffer containing 50 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, and 2 mM DTT such that the final concentration of DMSO was 0.5%. After a 90 minute incubation at room temperature on a shaker table, the kinase reaction was stopped by addition of 10 mM EDTA, and phosphorylation of the substrate was detected by adding 1 nM LANCE® Ultra Europium-anti-phospho-CREB (ser133) antibody (PerkinElmer, # TRF0200-D) and incubating for 60 minutes on a shaker at room temperature. The flurorescence resonance energy transfer (FRET) signals were read and analyzed on an Envision¿ 2103 Multilabel Reader (Perkin Elmer). The concentration of test compound required to inhibit substrate phosphorylation by 50% (the IC50) was calculated by non-linear regression using GraphPad PRIZM.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 434.46, ALogP 3.95, HBA 8, HBD 3, PSA 113.41, QED 0.39
Compound (SMILES): CNc1ccc(-c2cccc3c2C[C@@H](C)CC(=O)N3)cc1C(=N)C1CC1
InChIKey: ANIBATUKYBCBNA-CYBMUJFWSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: CBP TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer ADO 110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 347.46, ALogP 4.69, HBA 3, HBD 3, PSA 64.98, QED 0.7
Compound (SMILES): [O-][S+]1CCN(c2ccc3nc(-c4ccc(OCc5ccccc5)cc4)[nH]c3n2)CC1
InChIKey: KWLQPUXARLCNIM-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Biochemical Assay : The 6 μL assay reactions are run in Greiner brand black 384-well low volume plates. All reactions contained assay buffer (phosphate buffered saline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken egg white, 1 mM dithiothreitol, 200 nM peptide substrate, 1 μM S-Adenosyl methionine, and 15 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 4 hours at 37° C. Reaction progress was measured using the Transcreener EPIGEN methyltransferase assay (BellBrook Labs, Madison, Wis.) as recommended by the manufacturer. To each reaction 2 μL detection mix were added, containing coupling enzymes, fluorescence polarisation tracer, and AMP antibody. Plates were incubated for 90 minutes before being read on a PerkinElmer EnVision plate reader in fluorescence polarisation mode.
Measurement: IC50 = 570 nM (pChEMBL 6.239999771118164)
Descriptors: MW 418.52, ALogP 3.77, HBA 5, HBD 1, PSA 77.1, QED 0.5
Compound (SMILES): NC(=O)c1cc(-n2nc(C(F)(F)F)c3c2C(=O)N(C2CCN(N4CCOCC4=O)CC2)CC3)ccc1F
InChIKey: BWDWJLCQQJIDKV-UHFFFAOYSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: In Vitro Assay: Factor Xa (human or rat): In the assay for measuring the inhibition of human or rat factor Xa activity, Tris-HCl buffer (50 mM, pH 8.3, 150 mM NaCl) is used. The IC50 is measured by adding dropwise the assay buffer of 50 μL human Factor Xa (Enzyme Research Laboratories, Inc; the final concentration of 8.36 nM) or 50 μL rat Factor Xa (Enzyme Research Laboratories, Inc; the final concentration of 57.5 nM) to the appropriate wells of a Greiner 384 microtiter plate. 2 μL the assay buffer containing 2% (V/V) DMSO (uninhibited control) or various concentrations of the compounds to be tested is diluted in the assay buffer containing 2% (V/V) DMSO, and add the assay buffer of 48 μL substrate S-2222 (Chromogenix; chemical formula: Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA.HCl R═H (50%) and R═CH3 (50%)), the final concentration is 0.172 mM. The assay is performed by pre-incubating the tested compounds with enzyme for 10 min, and then the assay is initiated by adding substrate S-2222 to obtain a final volume of 100 μL.
Measurement: Ki = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 524.48, ALogP 1.37, HBA 7, HBD 1, PSA 114, QED 0.6
Compound (SMILES): CCCCCN(CCCCC)[C@@H]1CN(S(=O)(=O)c2cn(C)cn2)C[C@H]1c1ncccc1F
InChIKey: CZUAWQRVQUIUAK-TZIWHRDSSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 1000 nM (pChEMBL 6)
Descriptors: MW 465.64, ALogP 3.79, HBA 6, HBD 0, PSA 71.33, QED 0.45
Compound (SMILES): N#Cc1ncc(-c2cnc(NC(=O)C3CC3)cn2)cc1NCC(=O)N1CCO[C@H]1F
InChIKey: LDMSZLXWNHDFRB-IBGZPJMESA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Inhibition Assay: TBK1 inhibition is determined using a 384 well μlate format in buffer containing 20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.01% Brij-L23, 1 mM DTT. Each test compound is prepared in DMSO using 2.5-fold serial dilutions for 11 data points, which are added to the buffer so that each dilution contains 1% DMSO. To each well is added 2 μL of 1 μM 5FAM-DRHDSGLDSMKDE-NH2 (in buffer), 2 μL of diluted test compound (1% DMSO in buffer), and 5 μL of 3 nM TBK1 and 25 μM ATP (in buffer). The reaction mixture is incubated at RT for 60 min, and quenched by adding 20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM EDTA, 0.01% Brij-L23, 1 mM DTT+25 mM EDTA. To quantify the fluorescent-labeled substrate and product following reaction, the test plate is loaded on a Caliper LC-3000, which measures percent of conversion by microfluidic-based separation.
Measurement: IC50 = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 411.4, ALogP 1.28, HBA 8, HBD 2, PSA 133.13, QED 0.68
Compound (SMILES): O=C1NCCN(CCC2CCC(O)CO2)c2c1oc1ccc(C(F)(F)F)cc21
InChIKey: HWXFHRYCCFFRFA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Pim-1 Inhibitory Activity Assay: The calculation of the Pim-1 inhibitory activity of the compound was performed using the following solution according to the protocol attached to ADP-Glo Kinase Assay (cat. V9102, Promega). The purified enzyme of human Pim-1 described above was used. (i) Preparation of Solution. The kinase buffer solution (50 mmol/L HEPES (pH 7.5), 5 mmol/L MgCl2, 1 mmol/L DTT, 0.05% BSA) was prepared by dissolving HEPES (Jena Bioscience), MgCl2 (Sigma-Aldrich), DTT (Sigma-Aldrich) and BSA (Sigma-Aldrich) in purified water. The ATP solution (288 μmol/L) was prepared by dissolving 100 mmol/L ATP (Promega) in the kinase buffer solution. The enzyme/substrate solution (0.2 mmol/L Pim-1, 30 μmol/L Pim2tide) was prepared by dissolving Pim-1 (described above) and Pim2tide (custom synthetic product by GenScript USA, the same product as PIM2tide cat. 12-542 by Millipore) in the kinase buffer solution. The test compound solution (containing 12.5% DMSO) was prepared by dissolving the DMSO solution of the test compound in the kinase buffer solution. The vehicle solution (containing 12.5% DMSO) was prepared by dissolving DMSO in the kinase buffer solution. (ii) Method. To a 384-well assay plate (Corning, 4513), the test compound solution or vehicle solution (control) was added by 1 μL/well, the enzyme/substrate solution or kinase buffer solution (blank) was added by 2 μL/well, and the ATP solution was added by 2 μL/well, and they were mixed. After enzymatically reacting at room temperature for 45 minutes, ADP-Glo Reagent (Promega) was added by 5 μL/well, and they were mixed. After reacting at room temperature for 60 minutes, Kinase Detection Reagent (Promega) was added by 10 μL/well, and they were mixed. After reacting at room temperature for 30 minutes, the luminescence amount of each well was measured for 10 msec with a multi-label plate reader EnVision (PerkinElmer).
Measurement: Ki = 6.22 nM (pChEMBL 8.210000038146973)
Descriptors: MW 398.38, ALogP 2.93, HBA 5, HBD 2, PSA 74.94, QED 0.83
Compound (SMILES): CCCCOc1nc(N)c2nc(O)n(Cc3ccc(Oc4ccc(CNCCOC)cc4)cc3)c2n1
InChIKey: ORCANGUVKGRRQG-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7 Agonist Activity Assay: Engineered human embryonic kidney blue cells (HEK-Blue TLR cells; Invivogen) possessing a human TLR7-secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in a non-selective, culture medium (DMEM high-glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue TLR7 cells were added to each well of a 384-well tissue-culture plate (15,000 cells per well) and incubated 16-18 h at 37° C., 5% CO2. Compounds (100 nl) were dispensed into wells containing the HEK-Blue TLR cells and the treated cells were incubated at 37° C., 5% CO2. After 18 h treatment ten microliters of freshly-prepared Quanti-Blue reagent (Invivogen) was added to each well, incubated for 30 min (37° C., 5% CO2) and SEAP levels measured using an Envision plate reader (OD=620 nm). The half maximal effective concentration values (EC50; compound concentration which induced a response halfway between the assay baseline and maximum) were calculated.
Measurement: EC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 492.58, ALogP 3.87, HBA 10, HBD 3, PSA 129.57, QED 0.24
Compound (SMILES): COC1(C)CN(c2cnc3c(C(=O)Nc4cnccc4[C@@H]4C[C@H](C)C[C@H](N)C4)ccc(F)c3c2)C1
InChIKey: RQYVFQCCZWEMJR-KSZLIROESA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 477.58, ALogP 4.48, HBA 6, HBD 2, PSA 93.37, QED 0.56
Compound (SMILES): COc1cc(C(=O)NC2CN(C)C2)ccc1Nc1nc(OC2CCCC2)c2c(-c3ccc4nc(C)oc4c3)c[nH]c2n1
InChIKey: AFIVIGSQZUJFLX-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Radioactivity Based Kinase Assay: The kinase activity is measured using a radioactivity based kinase assay, which measures the incorporation of a radioactively labeled phosphate moiety (for example, 33P labeled phosphate) into a substrate, for example, a peptide substrate. Reduced levels of radioactively labeled phosphate incorporation into the substrate indicates inhibition of kinase activity. In another embodiment, the kinase activity is measured using a time-resolved-fluorescence resonance energy transfer (TR-FRET) based kinase assay, which measures loss of fluorescence as a result of substrate phosphorylation, for example, a peptide substrate (see for example Invitrogen Z'-Lyte Assay®). Increased levels of fluorescence indicates inhibition of kinase activity. In another embodiment, the kinase activity is measured using a competitive tracer binding assay (for example, Invitrogen Lanthascreen® Eu binding assay), which measures fluorescence as a result of tracer binding (for example ATP site binding). Reduced fluorescence indicates displacement of tracer binding, which indicates inhibition of kinase activity.
Measurement: IC50 = 4.54 nM (pChEMBL 8.34000015258789)
Descriptors: MW 567.65, ALogP 5.19, HBA 9, HBD 3, PSA 130.43, QED 0.23
Compound (SMILES): Cc1cc(Cl)n(-c2ccc(CN3C(=O)Cc4cnc(-c5cccnc5C(C)C)nc43)cc2)n1
InChIKey: DEFWGJUXKKIVCD-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: HTS Assay: The HTS assay was performed in a final volume of 20 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2) (1M Calcium Chloride solution; Sigma #21114) 1 mM GSH (L-Glutathione reduced; Sigma #G4251), 0.01% Prionex (0.22 μM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at −20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at −20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 μM (DMSO(fc)=0.5%). The final concentration of the enzyme (USP1, construct USP1 (I-785, GG670, 671AA)/UAF1 (I-677)-Flag; Viva) in the assay was 100 μM. Final substrate (Ub-Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 10 μL of 2× enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2×Ub-Rh110 or preincubated with USP1 40 minutes prior to the addition of 10 μL of 2×Ub-Rh110 to compound plates. Plates were incubated stacked for 45 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 458.95, ALogP 4.9, HBA 6, HBD 0, PSA 76.8, QED 0.42
Compound (SMILES): C=CC(=O)N1CC2(CC(n3nc(-c4cnc5c(cnn5C)c4)c(-c4c(Cl)c(C)cc5[nH]ncc45)c3C)C2)C1
InChIKey: JANYTRMYVZEGLC-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Scintillation Proximity Assay (SPA): Assays were run using 384-well plates (781207/Greiner) in which one column was designated as the high signal (no inhibition) control, and contained DMSO with no test compound, and another column was designated as the low signal control (maximal inhibition), and contained no protein. Serial dilutions of compounds to be tested were added to the assay plate (resulting in duplicate 11-point dose response with semi-log compound dilutions from 50 μM to 0.5 nM or from 5 μM to 0.05 nM for the most potent compounds). A 1/20 isotopic dilution of labeled (Compound B) and non-labeled covalent (Compound A) probe was prepared and added to all wells on the plate. The reaction was started by addition of KRasG12C (M1-K169, biotinylated on the N-terminus) to the compounds and incubated for 2 hours with continuous agitation allowing for full modification of KRasG12C with probe or test compounds. Final concentrations in an assay volume of 40 μL were 10 nM KRasG12C, 25 nM radio-ligand and 475 nM unlabelled ligand. The assay buffer contained 20 mM Tris-HCl pH 7.5 (Invitrogen), 150 mM NaCl (Sigma Aldrich), 0.1 mM MgCl2 (Sigma Aldrich), and 0.01% Tween-20 (Sigma Aldrich). Following addition of 50 μL of a 400 μg/ml. suspension of streptavidin-coated YSi beads (Perkin Elmer), plates were incubated for a further 30 min with continuous agitation before reading the plates on a scintillation counter (Topcount NXT 384 (Packard).
Measurement: IC50 = 112 nM (pChEMBL 6.949999809265137)
Descriptors: MW 527.03, ALogP 4.99, HBA 7, HBD 1, PSA 97.52, QED 0.33
Compound (SMILES): COCCS(=O)(=O)C(C(=O)NCC(=O)NC1(c2ncon2)CC1)c1nc2ccc(-c3ccc(F)nc3)cc2s1
InChIKey: MKYZJPGVEUVHIZ-UHFFFAOYSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Biological Activity: Endothelial lipase (EL) and hepatic lipase (HL) activities were measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 571 μL of 29 mM DMPG in a 1:1 mixture of MeOH and CHCl3 with 2000 μL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen in multiple vials then resuspended in 20 mL total volume of 50 mM HEPES pH 8.0 buffer containing 50 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at room temperature for 15 min and then was sonicated 3×4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.11 for the FRET substrate.The enzymatic assay was measured using 384-well white Optiplates. Each well contained 20 μL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 0.25 μL of a DMSO solution containing a compound of interest. EL or HL (10 μL) was added and allowed to incubate with the compound for 30 min at 37° C. The source of EL was conditioned media obtained from HT-1080 cells that were transformed using RAGE technology (Athersys) to overexpress endogenous EL, and HL was partially purified from conditioned media obtained from COS cells overexpressing HL. The reaction was started by the addition of 10 μL of a 1:10 dilution of vesicles. The final total reaction volume was 20.25 μL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. Readings were taken over a period of 60 minutes, and the slope between 300 and 900 secs of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 574.62, ALogP 1.9, HBA 11, HBD 2, PSA 166.27, QED 0.25
Compound (SMILES): C[C@H](N[C@H]1CC[C@H](C(=O)N2Cc3cccnc3Nc3ccc([C@H]4CCCOC4)cc32)CC1)C(F)(F)F
InChIKey: PNMPGHFDMWHLOK-GPHNJDIKSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation.
Measurement: IC50 = 43 nM (pChEMBL 7.369999885559082)
Descriptors: MW 502.58, ALogP 5.67, HBA 5, HBD 2, PSA 66.49, QED 0.56
Compound (SMILES): COc1ccc2cc(C(C(=O)O)C(C)C)ccc2c1
InChIKey: ZVJKEDKTWXLLQE-UHFFFAOYSA-N
Target: Aldo-keto reductase family 1 member C3 (SINGLE PROTEIN), Homo sapiens
UniProt: P42330
Assay: S-Tetralol Oxidation Assay: the inhibitory potency of the individual compounds against the AKR1C isoforms was determined by monitoring the NADP+ dependent oxidation of S-tetralol catalyzed by the AKR1C enzymes using substrate concentrations at their Km values in the presence and absence of varying concentration of the inhibitors (Adeniji, et al., 2012, J Med Chem 55:2311-2323). Reaction systems (200 μL) contained 100 mM potassium phosphate buffer (pH 7.0), 4% DMSO, 200 μM NADP+, a serial dilution of compounds, S-tetralol, and AKR1C enzymes. The concentration of S-tetralol used in the inhibition assays using AKR1C1, 1C2, 1C3, and 1C4 was 5, 22.5, 165, and 25 μM, respectively, which was equal to their Km values in order to make a direct comparison of IC50 values. The concentration of AKR1C1, 1C2, 1C3, and 1C4 was 111, 86, 95, and 552 nM, respectively. Reagents were mixed and incubated at 37° C. for 10 min followed by adding AKR1C enzymes to initiate the reaction. A continuous fluorometric assay (Ex, 340 nm; Em, 460 mM) to measure NADPH formation was conducted at 37° C. for 5 min, and the IC50 value of each compound was calculated. To determine the pattern of inhibition, five fixed concentrations of S-tetralol were used and four different concentrations of inhibitor were used and a gobal fit of the equations for COMP, NONCOMP and UNCOMP to the data was applied using Grafit.
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 258.32, ALogP 3.67, HBA 2, HBD 1, PSA 46.53, QED 0.91
Compound (SMILES): N#C[C@H](Cc1ccc(-c2cc3c(cn2)COC32CCN(C3COC3)CC2)cc1F)NC(=O)[C@H]1N[C@@H]2CC[C@H]1C2
InChIKey: VIDWRXWIFCHQDH-HQGMYGGFSA-N
Target: Dipeptidyl peptidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53634
Assay: Inhibition of Human DPPI (Cathepsin C): The recombinant human DPPI was diluted in TAGZyme buffer to 1 U/ml (38.1 μg/ml, respectively), and then activated by mixing in a 1:2 ratio with a Cysteamine aqueous solution (2 mM) and incubating for 5 min at room temperature. Five uL test compound (final concentration 0.1 nM to 100 μM) in aqua bidest 5 (containing 4% DMSO, final DMSO concentration 1%) were mixed with 10 μL of DPPI in MES buffer (final concentration 0.0125 ng/μL) and incubated for 10 min. Then, 5 μL of substrate in MES buffer (final concentration 50 μM) were added. The microtiter plates were then incubated at room temperature for 30 min. Then, the reaction was stopped by adding 10 μL of Gly-Phe-DMK in 10 MES-buffer (final concentration 1 μM). The fluorescence in the wells was determined using a Molecular Devices SpectraMax M5 Fluorescence Reader (Ex 360 nm, Em 460 nm) or an Envision Fluorescence Reader (Ex 355 nm, Em 460 nm).Each assay microtiter plate contained wells with vehicle controls (1% DMSO in bidest+0.075% 15 BSA) as reference for non-inhibited enzyme activity (100% Ctl; high values) and wells with inhibitor (Gly-Phe-DMK, in bidest+1% DMSO+0.075% BSA, final concentration 1 μM) as controls for background fluorescence (0% Ctl; low values). The analysis of the data was performed by calculating the percentage of fluorescence in the presence of test compound in comparison to the fluorescence of the vehicle control after 20 subtracting the background fluorescence using the following formula:(RFU(sample)−RFU(background))*100/(RFU(control)−RFU(background))
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 531.63, ALogP 2.8, HBA 7, HBD 2, PSA 99.51, QED 0.59
Compound (SMILES): COc1cc(-c2cccc(-c3cccc(NC(=O)c4cn(C)c(=O)n(C)c4=O)c3C)c2C)cc(F)c1CNCC(C)(C)C(N)=O
InChIKey: VTDHAMRAYSNYLR-UHFFFAOYSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayELISA: 96 Well plates were coated with 111 g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4° C. The wells were then blocked with 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the compounds were serial diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.31 μg/mL (ACRO Biosystems) were added and incubated for 1 hour at 37° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was performed with 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37° C. then the plates were washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 615.71, ALogP 4.04, HBA 8, HBD 3, PSA 137.45, QED 0.25
Compound (SMILES): CN1CCN(C2CCCN(c3ccn(-c4cc(-c5c(F)cccc5C#N)nc5c4C(=O)NC5)n3)C2)CC1
InChIKey: FSUGIKMGEYMTJV-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 500.58, ALogP 2.4, HBA 8, HBD 1, PSA 93.32, QED 0.59
Compound (SMILES): Cc1ncoc1[C@H](Nc1cc(Cl)c2ncc(C#N)c(Nc3ccc(F)c(Cl)c3)c2c1)C1=CN(C2CCN(C(C)(C)C)CC2)NN1
InChIKey: LQYCXKLMPIURGQ-SSEXGKCCSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 650.59, ALogP 7.17, HBA 10, HBD 4, PSA 117.31, QED 0.16
Compound (SMILES): CCC(=O)NCCCC[C@H](NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CCC(=O)O)NC(C)=O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCC(=O)O)C(=O)NCCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCOCCC(=O)NC[C@@]12C[C@@H](C(=O)Nc3nc(Br)ccc3C)N(C(=O)Cn3nc(C(C)=O)c4cc(-c5cnc(C)nc5)cc(C)c43)[C@@H]1C2
InChIKey: XDVXIAXXVNVAJZ-HNCHSMBISA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 min at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBz and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) is recorded at 30 second intervals for 30 min using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 2506.35
Compound (SMILES): COC1(c2ccnc(C(N)=O)c2)C2CCCC1CN(C1CC[C@H]1C)C2
InChIKey: UWXQVNCJUCOIEQ-ZBLXHBCZSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Competitive Displacement Assay: Membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed one type of the cloned human opioid receptor were incubated with 12 different concentrations of the compound in the presence of 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole or 1 nM [3H]U69,593 in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO and [3H]U69,593. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 μM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. For [3H]naltrindole and [3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for at least 60 min before use.
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 343.47, ALogP 2.55, HBA 4, HBD 1, PSA 68.45, QED 0.91
Compound (SMILES): Cc1nc(Nc2ncc(F)c(-c3cc(F)c4nc5n(c4c3)C(C)(C)CC5)n2)ccc1CC1CCNCC1
InChIKey: KUQOZDKWHDUTPS-UHFFFAOYSA-N
Target: CDK4/Cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P30281
Assay: Inhibition Assay: In vitro inhibition of CDK (CDK1, CDK4, CDK6) activity by compounds of the present invention was tested by the following method.1. Preparation of kinase reaction buffer I: 5× Reaction Buffer A (Promega; V307A-C) provided in the kit was diluted with a mixture of Milli Q H2O and 0.1M DTT (dithiothreitol) to 4× kinase buffer; then Milli Q H2O was added in a proper proportion, to finally formulate into 1× kinase buffer.Preparation of kinase reaction buffer II: 0.5% DMSO (dimethyl sulfoxide) was added into 1× kinase reaction buffer and uniformly mixed to obtain the title buffer.2. Preparation of kinase solution: 100 ng/μl kinase stock solution was formulated with 1× kinase reaction buffer into kinase solution with the desired concentration for each reaction system.3. Preparation of solution of test compound and LY2835219 as a control:(1) Preparation of Solution of LY2835219 as a Controla. 1 μl of 10 mM standard stock solution was added respectively into 9 μl of kinase reaction buffer I and uniformly mixed; then 90 μl of kinase reaction buffer I was added and uniformly mixed; and then 100 μl of kinase reaction buffer I was added and uniformly mixed, to achieve a final concentration of 50 μM.b. 40 μl of kinase reaction buffer II was added into Well B2-B10 of a 96-well plate; and 50 μl of above-mentioned solution was added into Well B1;c. 10 μl of solution was taken from Well B1, added into Well B2 and uniformly mixed, then 10 μl of dilution was taken from Well B2 and added into Well B3, the serial dilution was continued up to B9, to obtain 5-fold serial dilutions of the reference solution.
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 503.6, ALogP 5.44, HBA 7, HBD 2, PSA 80.55, QED 0.38
Compound (SMILES): COc1ccc(Cn2c(C)cn3c(C4(C)CCN(C)CC4)ncc3c2=O)cc1
InChIKey: UOMHYPRQAMXYDQ-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P54750
Assay: PDE1 Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 h at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 h in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 1923 nM (pChEMBL 5.71999979019165)
Descriptors: MW 380.49, ALogP 2.84, HBA 6, HBD 0, PSA 51.77, QED 0.7
Compound (SMILES): CO[C@H]1CN(c2ncc(C)cc2-n2ccc(C)n2)CC[C@H]1NC1CCCCC1
InChIKey: WKNTWDLCNNIPKD-CTNGQTDRSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: OPRK1 antagonist assay: The cell line for the OPRK1 antagonist assay stably expresses the following elements. The carboxy terminus of the OPRK1 receptor has a 7 amino acid linker, followed by the TEV protease cleavage site and a GAL4-VP16 fusion protein. The cell line also expresses a b-arrestin-2-TEV protease fusion protein and contains a reporter construct consisting of the UAS response element and the b-lactamase (bla) reporter gene. Upon activation of the receptor, g-protein receptor kinase (GRK) phosphorylates specific intracellular residues of OPKR1 and this induces recruitment of B-arrestin2-TEV protease fusion protein. The TEV protease recognizes and cleaves the TEV site, releasing the GAL4-VP16 fusion protein, which then translocates to the nucleus. The GAL4-V16 binds to the UAS element, driving expressing of the b-lactamase gene. B-lactamase expression is detected with the cell permeable, fluorescent substrate, CCF4-AM. This substrate consists of coumarin tethered to fluoroscein via a b-lactam ring. In the absence of b-lactamase, excitation of the dye with 405 nm light results in FRET from the coumarin to fluoroscein and emission of green (525 nm maximum) light. B-lactamase cleavage of the substrate separates the courmarin fluorophore from the fluorscein, and 405 nm excitation results in blue (460 nm maximum) emission. The assay is monitored by the blue/green emission ratio. The antagonist assay is performed by seeding the cells into 384 well plates and incubating them 16-24 hours at 37° C. Test antagonist compounds are added and incubated for 30 minutes at 37° C. Next an EC80 challenge of U-50488 (OKRK1 agonist) is added and the samples are incubated for 4 hours at 37° C., followed by addition of CCF4-AM substrate. The plates are then incubated 2 hours at room temperature in the dark and the blue/green ratio determined on a fluorescent plate reader. See J Biomol Screen April 2009, vol. 14 no. 4, pp 381-394.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 383.54, ALogP 3.4, HBA 6, HBD 1, PSA 55.21, QED 0.86
Compound (SMILES): CC1CC1C(O)C#Cc1cncc([C@H]2NC(=O)O[C@@H]2c2cccc(F)c2)c1
InChIKey: LJQXOGBJRIRTTJ-BDVDZXALSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: mGluR5 FLIPR Assay: HEK293 (ZF) cells stably transfected with human mGluR5A (pIRES neo) and the rat glutamate-aspartate transporter (GLAST; pIRES puro) are grown in a monolayer culture at 37° C. in 5% CO2 and fed with Minimum Essential Medium (MEM) supplemented with 10% dialysed fetal bovine serum. 24 hours prior to assay, cells are enzymatically dissociated from the culture flask (Trypsin, 0.25%), spun down (1000 rpm, 3 min), resuspended, and plated on Greiner black clear bottomed PDL-coated 384-well plates at a density of 30 thousand cells/well. On the day of the experiment, media is removed from the cell plates and replaced with Molecular Devices Calcium 4 microfluorometric Ca++ sensitive dye in assay buffer (HBSS; Gibco #14025+20 mM HEPES and 250 uM probenacid). Plates are incubated in dye at 37° C. in 5% CO2 for 60 minutes prior to delivery of test compounds in assay buffer. Test compounds are incubated with cells in the presence of dye for 10 minutes prior to being read on the FLIPR platform (Molecular Devices). A Ca++ signal is induced in the assay plates via the delivery of an ¿EC10 concentration of the endogenous agonist 1-glutamate; images are acquired at 1 Hz for 100 seconds post-delivery of agonist stimulus. Positive modulator activity (i.e. the ability of test compounds to increase the Ca++ response to a sub-maximal concentration of agonist) is normalized to a saturating concentration of a known mGluR5 PAM run in each assay plate.
Measurement: EC50 = 129 nM (pChEMBL 6.889999866485596)
Descriptors: MW 366.39, ALogP 3.11, HBA 4, HBD 2, PSA 71.45, QED 0.82
Compound (SMILES): CC(C)(C)OC(=O)N1CC=C(c2cc3c(-c4ccc(OC5CCOCC5)c(C#N)c4)ccnc3[nH]2)CC1
InChIKey: LHPPCLUNEXZVTH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assay for TBK1 and IKKs: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKs were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2× Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 500.6, ALogP 5.68, HBA 6, HBD 1, PSA 100.47, QED 0.49