lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)CC1CCC(c2ccc(-c3ccc4nc(C(=O)Nc5ccc(OC(F)(F)F)cc5)cn4c3)cc2)CC1
InChIKey: LIDUBCTXWWYTGR-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Inhibition Assay: Â Assay buffer [20 mM HEPES (pH 7.4), 100 mM MgCl2 and 0.04% bovine serum albumin (BSA)] containing 500 uM of enzyme substrate (didecanoyl glycerol) and 7.5 uM radiolabeled acyl-CoA substrate ([14C]decanoyl-CoA) was added to each well of a phospholipid-coated radiometric assay plate. A small aliquot of cell lysate having human DGAT1 activity (5 ug/well) obtained in above, of human liver microsomes having human DGAT1 activity (5 ug/well), or of mouse liver microsomes having mouse DGAT1 activity (5 ug/well) was added to the assay plate to start the reaction, and the reaction was allowed to proceed for 60 minutes at 25° C. The reaction was terminated upon the addition of an equal volume (100 uL) of isopropanol. The plates were sealed, incubated overnight and counted the next morning on a liquid scintillation counter and luminometer.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 537.54, ALogP 6.9, HBA 5, HBD 2, PSA 92.93, QED 0.27
Compound (SMILES): CC(C)CN(C(CO)c1ccc(CNC(=O)C(NC(=O)c2ccccc2)C(c2ccccc2)c2cccc3cccnc23)s1)S(=O)(=O)c1ccc2ncsc2c1
InChIKey: UCNMGJDJRFPOCJ-UHFFFAOYSA-N
Target: Gag-Pol polyprotein (SINGLE PROTEIN), Human immunodeficiency virus type 1 group M subtype B (isolate HXB2)(HIV-1)
UniProt: P04585
Assay: Inhibition Assay: The inhibition of WT HIV-1 protease was studied using the reaction of the protease (expressed in Eschericia coli) with a peptide substrate [Val-Ser-Gln-Asn-(betanaphthyl)Ala-Pro-Ile-Val (SEQ ID NO:1)]. Compounds were tested using Procedure A. In Procedure A, the test compound was first preincubated with the enzyme in assay buffer (50 mM sodium acetate, pH 5.5, 100 mM NaCl, and 0.1% BSA) for 30 minutes at room temperature. Substrate was then added to 400 micromolar in a total volume of 80 microliters containing 10 picomolar HIV-1 protease and the reaction was incubated for 1 hour at 30° C. The reaction was quenched with the addition of 120 microliters of 10% phosphoric acid. The product formation was determined after separation of product and substrate on a Zorbax Eclipse XDB-C18 column (Agilent Technologies, Santa Clara, Calif.) connected to an Agilent 1100 high performance liquid chromatography system with fluorescence detection (excitation 270 nanometer and emission 330 nanometer).
Measurement: IC50 = 15.2 nM (pChEMBL 7.820000171661377)
Descriptors: MW 804.03, ALogP 7.53, HBA 9, HBD 3, PSA 141.59, QED 0.1
Compound (SMILES): CC1(CNc2nc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)COC1
InChIKey: MDRQQEXIVHHEJB-WCIBSUBMSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 384.4, ALogP 0.33, HBA 9, HBD 4, PSA 134.57, QED 0.41
Compound (SMILES): Cc1ccccc1NCC1=NCCN1
InChIKey: WOYZHFHQSIRZAK-UHFFFAOYSA-N
Target: Nischarin (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q4G017
Assay: Displacement of [125I]-p-iodoclonidine from I1 receptor imidazoline binding site in Sprague-Dawley rat kidney cell membranes after 30 mins by liquid scintillation counting
Measurement: Ki = 1.995 nM (pChEMBL 8.699999809265137)
Descriptors: MW 189.26, ALogP 1.41, HBA 3, HBD 2, PSA 36.42, QED 0.75
Compound (SMILES): CNC(=O)COC1=CCCC(Nc2ncc(F)c(Nc3ccc(C(F)(F)F)c(Cl)c3)n2)=C1
InChIKey: XBKBSEFUQVSCKW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Biochemical Assay: Several 2,4-pyrimidinediamine compounds were tested for the ability to inhibit Syk kinase catalyzed phosphorylation of a peptide substrate in a biochemical fluorescenced polarization assay with isolated Syk kinase. In this experiment, Compounds were diluted to 1% DMSO in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated Bovine Gamma Globulin). Compound in 1% DMSO (0.2% DMSO final) was mixed with ATP/substrate solution at room temperature. Syk kinase (Upstate, Lake Placid N.Y.) was added to a final reaction volume of 20 uL, and the reaction was incubated for 30 minutes at room temperature. Final enzyme reaction conditions were 20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated Bovine Gamma Globulin, 0.125 ng Syk, 4 uM ATP, 2.5 uM peptide substrate (biotin-EQEDEPEGDYEEVLE-CONH2, SynPep Corporation). EDTA (10 mM final)/anti-phosphotyrosine antibody (1x final)/fluorescent phosphopeptide tracer (0.5x final) was added in FP Dilution Buffer to stop the reaction for a total volume of 40 uL according to manufacturer's instructions (PanVera Corporation) The plate was incubated for 30 minutes in the dark at room temperature. Plates were read on a Polarion fluorescence polarization plate reader (Tecan). Data were converted to amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Assay Kit, Green (PanVera Corporation).
Measurement: IC50 = 157 nM (pChEMBL 6.800000190734863)
Descriptors: MW 471.84, ALogP 4.77, HBA 6, HBD 3, PSA 88.17, QED 0.5
Compound (SMILES): O=C(NCC(=O)N1CCOCC1)c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1
InChIKey: LKZZJIREYRTGKC-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 703 nM (pChEMBL 6.150000095367432)
Descriptors: MW 460.41, ALogP 3.03, HBA 6, HBD 1, PSA 93.65, QED 0.63
Compound (SMILES): O=C(Nc1ccc(C(=O)N2CC3(CCCO3)C2)cc1)N1Cc2ccc(F)cc2C1
InChIKey: PFHIDYMFWSDPRX-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 = 49.1 nM (pChEMBL 7.309999942779541)
Descriptors: MW 395.43, ALogP 3.38, HBA 3, HBD 1, PSA 61.88, QED 0.85
Compound (SMILES): CC(=O)c1c[nH]c2ncnc(OCC(C)C)c12
InChIKey: KBABZWSSGPHXJR-UHFFFAOYSA-N
Target: TGF-beta-activated kinase 1 and MAP3K7-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15750
Assay: Biological Assay: TAK1-TAB1 Binding Inhibitory Potency: The ability of candidate compounds to interact with TAK1-TAB1 is quantitated by a competitive binding assay using the LanthaScreen technology developed by Life Technologies. This assay is based on the binding of a proprietary, Alexa Fluor 647-labeled, ATP-competitive kinase inhibitor (kinase tracer-236) to the TAK1-TAB1 construct in presence of a europium-conjugated antibody, resulting in a FRET (fluorescence resonance energy transfer) signal. Displacement of the kinase tracer by compound results in a lower emission ratio upon excitation of the europium chelate. Candidate compounds are designed as potential irreversible inhibitors of TAK1-TAB1, capable of ligating to an active site cysteine residue. The time dependent nature of irreversible inhibition is investigated by performing the binding assay with and without a pre-incubation of compound and TAK1-TAB1. An increase in potency in the pre-incubated assay suggests the candidate compound could b of 10 nM TAK1-TAB1, 2 nM Eu-anti-his antibody, and 100 nM kinase tracer-236 using a 384-well plate format. Background signal is defined in the absence of TAK1-TAB1 and uninhibited signal is defined in the presence of vehicle (2% DMSO) alone. Compounds were evaluated in an 11 point dose-response ranging from 20 uM to 0.34 nM. The binding assays are performed under two conditions to evaluate time dependence of inhibition. For the pre-incubation assay, TAK1-TAB1 and Eu-anti-his antibody are preincubated with compound or vehicle for two hours prior to the addition of kinase tracer. The non-preincubated assay is run in which TAK1-TAB1 and Eu-anti-his antibody are added to a mixture of compound and kinase tracer. IC50 values of compounds are determined using a 4 parameter logistical fit of emission ratio as a function of the concentration of compound.
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 233.27, ALogP 2.2, HBA 4, HBD 1, PSA 67.87, QED 0.82
Compound (SMILES): COC(=O)Nc1ccc(S(=O)(=O)C(C)C)c([C@H]2CCCN2C(=O)[C@H](Nc2ccc3c(c2)C(=O)NC3C)c2ccc(OC)c(OC)c2)c1
InChIKey: LAUVCJWPBYKZPC-JOPCKLHNSA-N
Target: Coagulation factor VII/tissue factor (PROTEIN COMPLEX), Homo sapiens
UniProt: P13726
Assay: Inhibition of recombinant human factor-7a/TF using S2288 as substrate measured after 60 mins
Measurement: Ki = 88 nM (pChEMBL 7.059999942779541)
Descriptors: MW 664.78, ALogP 5.39, HBA 9, HBD 3, PSA 152.37, QED 0.26
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1NC1(C)CCC1
InChIKey: MLMNRFXLMUJDHR-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 361.45, ALogP 3.57, HBA 4, HBD 3, PSA 82.7, QED 0.67
Compound (SMILES): CNC(=O)c1c(-c2cnn(CCO)c2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CC4)cc12
InChIKey: WDWXBFITULNFDY-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhibition 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 = 212.3 nM (pChEMBL 6.670000076293945)
Descriptors: MW 628.69, ALogP 4.42, HBA 9, HBD 2, PSA 135.49, QED 0.27
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1nc(Nc2ccc(N3CCN(C(C)=O)CC3)cc2)ncc1Cl
InChIKey: BDOCHZSYLQTMCL-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 579.11, ALogP 3.96, HBA 9, HBD 2, PSA 106.17, QED 0.43
Compound (SMILES): CNc1ccc(CNC(=O)c2cc(NC(=O)c3cc(-c4ccccn4)c(F)cc3Cl)n(-c3ccccc3)n2)cn1
InChIKey: WPQBODQCLNKZEH-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 = 1.77 nM (pChEMBL 8.75)
Descriptors: MW 556, ALogP 5.35, HBA 7, HBD 3, PSA 113.83, QED 0.24
Compound (SMILES): O=C(Cc1nc(N2CCOCC2)cc(=O)[nH]1)N1CC2(CCOCC2)c2ccccc21
InChIKey: XYFUKFPWEIDBQI-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: Sandwich Immunoassay: This test is based on measurement of the expression of AKT protein phosphorylated on serine 473 (P-AKT-S473) in the PC3 human prostate carcinoma line, by the technique based on a sandwich immunoassay using the Kit MSD Multi-spot Biomarker Detection of Meso Scale Discovery: phospho-Akt kits (Ser473) whole cell lysate (# K151 CAD) or phospho-Akt (Ser473) / Akt whole cell lysate Total (# K15100D). The primary antibody specific for P-AKT-S473 (Kit # K151 CAD) is coated onto an electrode in each well of 96-well plates of the MSD kit: after adding a protein lysate in each well, the revelation of the signal done by the addition of a secondary antibody labeled with an electrochemiluminescent detection compound. The procedure is described in the kit.On day 1, the PC3 cells are inoculated in 96-well plates (TPP # 92096) at a concentration of 35000 cells / well in 200 uL DMEM medium (DMEM Gibco # 1 1960-044) containing 10% fetal calf serum (FBS Gibco, # 10500-056) and 1% glutamine.
Measurement: IC50 = 155 nM (pChEMBL 6.809999942779541)
Descriptors: MW 410.47, ALogP 1.24, HBA 6, HBD 1, PSA 87.76, QED 0.82
Compound (SMILES): O=C(O)c1ccc2[nH]c(-c3ccc(Oc4ccc(F)cc4)cc3)nc2c1
InChIKey: DPMBCATVGWHUTP-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk2 (SINGLE PROTEIN), Homo sapiens
UniProt: O96017
Assay: Inhibition of recombinant human CDS1 using N-terminal biotin-labeled SGLYRSPSMPENLNRPR as substrate in presence of [gamma-33P]-ATP after 3 hrs by scintillation counting method
Measurement: IC50 = 351 nM (pChEMBL 6.460000038146973)
Descriptors: MW 348.33, ALogP 4.86, HBA 3, HBD 2, PSA 75.21, QED 0.55
Compound (SMILES): Cc1c(N(CCCF)S(=O)(=O)c2ccccc2)sc2ccccc12
InChIKey: KZPDHVFSKMAEQH-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 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 363.48, ALogP 4.76, HBA 3, HBD 0, PSA 37.38, QED 0.63
Compound (SMILES): CC(C)Oc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(Cl)c3)cn2CCN2CCCC2)CC1
InChIKey: CIVKYRVMDOHDLK-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 528.08, ALogP 4.98, HBA 8, HBD 1, PSA 85.33, QED 0.44
Compound (SMILES): CN1C2COCC1CC(NC(=O)c1cn(CC(F)(F)F)c3ccccc13)C2
InChIKey: GAVZYULRCFRPLP-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: In Vitro Inhibition of Ca Flux Assay: The 5HT3 antagonist activity of the compounds of the invention was determined by measuring the ability of the compounds to inhibit the calcium flux activity of 3HT3a receptor expressed in HEK-293T cells. HEK-293T cells were transfected with the 5HT3a expression construct using Xtreme Gene 9 (Roche) in 150 mm tissue culture treated plates and incubated for 24 hours at 37 °C. Cells were then split and plated at a density of 60K cells/well in poly-lysine coated, black 96-well plates with clear bottoms (BD BioSciences) and incubated overnight at 37 °C. Growth media was removed and cells loaded with 200 uL calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 5 Assay kit, Molecular Devices) and incubated at 37 °C. for 1 hour. While cells were incubating, the 10× antagonist and agonist/antagonist addition plates were made. For 10× antagonist plate: half log serial dilutions (final concentrations range from 10−7 through 10−10 with the bottom well a negative, no ligand control) were made from test compounds in DMSO at a 1000× concentration and then diluted to 10× in HBSS/20 mM HEPES. For addition plate: 5HT was diluted to 100× in HBSS/20 mM HEPES (final concentration in the assay−216 nM) and 15 uL was added to each well of the addition plate, 15 uL of 10× compound was also added to the addition plate, and finally 120 uL of HBSS/20 mM HEPES (for a total of 150 uL). Cells were then removed from the incubator and equilibrated to room temperature for 10 minutes, then 22.5 uL of 10× test compounds were added in triplicate to the plates and incubated at room temperature for 10 minutes (Tropisetron was used as a positive control in every assay). Test plate and addition plate were loaded into the FlexStation III (Molecular Devices), and using the fluidics, 22.5 uL compound additions were made (at t=~17 seconds), and fluorescence was measured for 90 seconds, reading every 2.2 seconds.
Measurement: IC50 = 0.68 nM (pChEMBL 9.170000076293945)
Descriptors: MW 381.4, ALogP 2.8, HBA 4, HBD 1, PSA 46.5, QED 0.89
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CCCC2=O)c2ccc3ncc(-c4cnn(C(C)C)c4)nc3c2)c1
InChIKey: IXKVURLQMWDRPS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 79.43 nM (pChEMBL 7.099999904632568)
Descriptors: MW 514.63, ALogP 5.24, HBA 8, HBD 0, PSA 85.61, QED 0.29
Compound (SMILES): CC(c1cccc(NC(=O)c2ccccc2)c1)N1CCC(C(=O)NC2CCCCC2)CC1
InChIKey: CBSDCXGDCKHMIJ-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 433.6, ALogP 5.16, HBA 3, HBD 2, PSA 61.44, QED 0.66
Compound (SMILES): C=C(C)C(CC)CN1CC[C@H](CNC(=O)c2ccc(Cl)c(Cl)c2)N[C@H](CCN)C1=O
InChIKey: NCOYGPIFNDMXCO-LQRGHNGLSA-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 < 10000 nM (pChEMBL 5)
Descriptors: MW 455.43, ALogP 3.23, HBA 4, HBD 3, PSA 87.46, QED 0.5
Compound (SMILES): NC(=O)c1nn(-c2ccc(S(N)(=O)=O)cc2)c2c1CCc1ccc(N)cc1-2
InChIKey: PFEXVMJGJVILQY-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 using biotinylated IkappaBalpha peptide as substrate assessed as decrease in [gamma-33P]-ATP incorporation in substrate after 30 mins by scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 383.43, ALogP 0.97, HBA 6, HBD 3, PSA 147.09, QED 0.58
Compound (SMILES): CCOc1cccc(C(=O)Nc2cnn(Cc3c(C)noc3C)c2)c1
InChIKey: RCOWHBHXHQECPR-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 220 nM (pChEMBL 6.659999847412109)
Descriptors: MW 340.38, ALogP 3.19, HBA 6, HBD 1, PSA 82.18, QED 0.75
Compound (SMILES): N#CCC1(n2ccc(-c3ncnc4[nH]ccc34)c2)CN(C2CCN(C(=O)c3ccc(F)c(F)c3F)CC2)C1
InChIKey: LGHGBHSECRINRE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 519.53, ALogP 4.07, HBA 6, HBD 1, PSA 93.84, QED 0.4
Compound (SMILES): N#Cc1ccc(C2C3=C(CCC3=O)N(c3cccc(C(F)F)c3)C(=O)N2C(=O)NCCO)cc1
InChIKey: VILBAOCDDBOTPN-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Nuetrophil Elastase Acitivty Assay: Various concentrations of the neutrophil elastase inhibitor are incubated with plasma. Subsequently, the enzyme activity is measured using the fluorogenic substrate MeOSuc-Ala-Ala-Pro-Val-AMC (Bachem Cat. No. 1-1270, substrate concentration: 250 uM, pH 7.5, 25 mM TRIS buffer, 250 mM NaCl) in analogous fashion as described for the human neutrophil assay.
Measurement: EC50 < 1 nM (pChEMBL 9)
Descriptors: MW 466.44, ALogP 3.8, HBA 5, HBD 2, PSA 113.74, QED 0.7
Compound (SMILES): Nc1ncc(-c2nc(N3CCOCC3)nc3c(CN4CCN(C(=O)CO)CC4)csc23)cn1
InChIKey: YEIOZQMKNMXRFV-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Enzymatic Inhibitory Activity Assay:   1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.  2. Compound was formulated in 100% DMSO in a concentration gradient, deposited to a 384-well plate to make final DMSO concentration of 1%.  3. PI3Kα and PI3Kδ enzymes were diluted to be the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT. Transferred to a 384-well plate and incubated with the compound for a certain time.  4. Substrate was diluted to an optimum concentration with following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, Km ATP. The reaction was performed in a 384-well plate for 1 h at room temperature for PI3Kα and 2 hrs at room temperature for PI3Kδ.  5. Read the conversion rate using Caliper Reader, and calculate the inhibition rate as the average of two tests.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 470.56, ALogP 0.2, HBA 11, HBD 2, PSA 133.83, QED 0.53
Compound (SMILES): CCOCc1nc2c(N)nc3ccccc3c2n1CC(C)(C)OCCN
InChIKey: MMGIMCFVDIHCIY-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Reporter Assay: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells, respectively, (Invivogen, San Diego, Calif., USA) transfected with a SEAP reporter (secreted embryonic alkaline phosphatase) construct were used, in which the reporter expression is regulated by the NF-κB promoter upon stimulation for 24 hr. The reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Calif., USA) at a wavelength of 640 nm.
Measurement: EC50 = 580 nM (pChEMBL 6.239999771118164)
Descriptors: MW 357.46, ALogP 2.46, HBA 7, HBD 2, PSA 101.21, QED 0.64
Compound (SMILES): CCCCCCNC(=O)N[C@H](Cc1ccc(OCC)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCc1ccc(CN)cc1
InChIKey: KRKRLMQLCYJAFZ-AJQTZOPKSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 601.79, ALogP 4.38, HBA 5, HBD 5, PSA 134.58, QED 0.14
Compound (SMILES): CCC(Oc1ccc(OC)c(-c2ccc(F)c3c2CN(C(=O)OCc2ccccc2)CC3)c1)C(=O)O
InChIKey: KTWMOSHUCIZKFR-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 337 nM (pChEMBL 6.46999979019165)
Descriptors: MW 493.53, ALogP 5.44, HBA 5, HBD 1, PSA 85.3, QED 0.45
Compound (SMILES): O=C(N1C[C@@H](O)C=C[C@@H]1Cc1ccccc1)n1ncc(C(O)(c2ccc(F)cc2)c2ccc(F)cc2)n1
InChIKey: WTPKQSZKNFVAOG-RPBOFIJWSA-N
Target: Monoacylglycerol lipase ABHD6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BV23
Assay: Inhibition of human ABHD6 using 2-AG as natural substrate by fluorescence based assay
Measurement: IC50 = 251.19 nM (pChEMBL 6.599999904632568)
Descriptors: MW 502.52, ALogP 3.65, HBA 6, HBD 2, PSA 91.48, QED 0.41
Compound (SMILES): Cn1ncc(C(=O)N2CCCC2)c1C(=O)Nc1ccc2nc(-c3ccccc3)nn2c1
InChIKey: RCQRGNGCXDRYBD-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 ul contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 ul of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate.
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 415.46, ALogP 2.62, HBA 7, HBD 1, PSA 97.42, QED 0.55
Compound (SMILES): OCCNC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: VVLWVRFOUHKKKW-HAQNSBGRSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 314.39, ALogP 1.46, HBA 6, HBD 3, PSA 91.13, QED 0.62
Compound (SMILES): COc1cc(-c2c[nH]c3ccc(-c4nnc(N)s4)cc23)nc(OC(C)C)n1
InChIKey: AZPAUNKZBWYQME-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 15.41 nM (pChEMBL 7.809999942779541)
Descriptors: MW 382.45, ALogP 3.52, HBA 8, HBD 2, PSA 111.83, QED 0.54
Compound (SMILES): COc1cc(CNCc2ccc3c(c2)CCO3)ccc1OCCN1C(=O)CCC1=O
InChIKey: WXVMUGNWNLQHNN-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 290 nM (pChEMBL 6.539999961853027)
Descriptors: MW 410.47, ALogP 2.45, HBA 6, HBD 1, PSA 77.1, QED 0.64
Compound (SMILES): Cn1cc(-c2cc(NS(=O)(=O)CC(F)(F)F)ccc2Oc2ccccc2)c2cc[nH]c2c1=O
InChIKey: GWLFFSSEFAMDAQ-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 477.46, ALogP 4.63, HBA 5, HBD 2, PSA 93.19, QED 0.42
Compound (SMILES): CC(C)(C)c1ccc(NC(=O)c2cccc(Cc3n[nH]c(=O)c4c3CCCC4)c2)cc1
InChIKey: BXOMZRSQQUMQIJ-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 363 nM (pChEMBL 6.440000057220459)
Descriptors: MW 415.54, ALogP 4.79, HBA 3, HBD 2, PSA 74.85, QED 0.64
Compound (SMILES): CCN(C(=O)Oc1ccc(F)cc1)[C@@H]1CN(C(=O)C2CCN(c3ccc(C)cn3)CC2)C[C@H]1c1ccc(Cl)c(F)c1
InChIKey: NJYSVQSHFLGGGI-LBNVMWSVSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: [3H]SR142801 Competition Binding Assay: hNK3 receptor binding experiment were performed using [3H]SR142801 (Catalog No. TRK1035, specific activity: 74.0 Ci/mmol, Amersham, GE Healthcare UK limited, Buckinghamshire, UK) and membrane isolated from HEK293 cells transiently expressing recombinant human NK3 receptor. After thawing, the membrane homogenates were centrifuged at 48,000×g for 10 mM at 4° C., the pellets were resuspended in the 50 mM Tris-HCl, 4 mM MnCl2, 1 μM phosphoramidon, 0.1% BSA binding buffer at pH 7.4 to a final assay concentration of 5 μg protein/well. For inhibition experiments, membranes were incubated with [3H]SR142801 at a concentration equal to KD value of radioligand and 10 concentrations of the inhibitory compound (0.0003-10 μM) (in a total reaction volume of 500 μl) for 75 min at room temperature (RT). At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% PEI+0.3% BSA, Packard BioScience, Meriden, Conn.) with a Filtermate 196 harvester (Packard BioScience) and washed 4 times with ice-cold 50 mM Tris-HCl, pH 7.4 buffer. Nonspecific binding was measured in the presence of 10 μM SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 μl of microscint 40 (Canberra Packard S.A., Zürich, Switzerland) and shaking for 1 h.
Measurement: Ki = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 583.08, ALogP 6.05, HBA 5, HBD 0, PSA 65.98, QED 0.35