lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#Cc1ccc2nc(C3C4Oc5ccc(Oc6ccnc7c6CCC(=O)N7)cc5C43)[nH]c2c1
InChIKey: NPRSDLJOCFPENZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 435.44, ALogP 4.15, HBA 6, HBD 2, PSA 112.92, QED 0.5
Compound (SMILES): COc1ccc2cc(Br)ccc2c1CN1C(=O)[C@@H](NC(=O)[C@H](C)NCCO)C2(CCOCC2)Oc2ccccc21
InChIKey: UFSIJETWBAPLMM-UZTOHYMASA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x 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-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, 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.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 612.52, ALogP 3.54, HBA 7, HBD 3, PSA 109.36, QED 0.36
Compound (SMILES): N#Cc1cccc2c1CN(C(=O)Nc1ccc(NC(=O)CC3CCCC3)cc1)C2
InChIKey: VVBVNKYTYJLUKT-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 = 21.7 nM (pChEMBL 7.659999847412109)
Descriptors: MW 388.47, ALogP 4.62, HBA 3, HBD 2, PSA 85.23, QED 0.8
Compound (SMILES): O=S(=O)(Nc1ncns1)c1cc(F)c(Oc2ccc(Cl)cc2-n2cnc3cncnc32)cc1F
InChIKey: JAQPLQSHWAIPEO-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (In vitro Assay): Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (Nav's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following patch voltage clamp electrophysiology studies were performed on representative compounds of the invention using human embryonic kidney cells (HEK), permanently transfected with an expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit, grown in culture media containing 10% FBS, 1% PSG, and 0.5 mg/mL G418 at 37° C. with 5% CO2. HEK cells used for the electrophysiology (EP) recordings had a passage number of less than 40 for all studies and were used within three days from the time of plating. Nav1.7 and Nav1.5 cDNAs (NM_00297
Measurement: IC50 = 3378.2 nM (pChEMBL 5.46999979019165)
Descriptors: MW 521.92, ALogP 4.19, HBA 10, HBD 1, PSA 124.78, QED 0.35
Compound (SMILES): CC(C)c1ccc(CNC(=O)c2cn3cc(-c4ccc(C5CCC(CC(=O)O)CC5)cc4)ccc3n2)cc1
InChIKey: DMPAXCYZODPHBK-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 509.65, ALogP 6.8, HBA 4, HBD 2, PSA 83.7, QED 0.27
Compound (SMILES): CCS(=O)(=O)N1CCN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)CC1
InChIKey: LLCNZVPYOMHYCZ-WKILWMFISA-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 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 431.57, ALogP 1.85, HBA 7, HBD 1, PSA 99.49, QED 0.66
Compound (SMILES): c1cnc2c(N3CCN(CCCCc4ccc(OCCCN5CCCCCC5)cc4)CC3)cccc2c1
InChIKey: HKPKDTNLXGOYIC-UHFFFAOYSA-N
Target: Histamine H1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35367
Assay: Antagonist activity at human Histamine H1 receptor expressed in CHO cells assessed as inhibition of histamine-induced calcium flux preincubated for 30 mins followed by histamine addition by Fluo-4-AM dye based FLIPR assay
Measurement: Ki = 15.85 nM (pChEMBL 7.800000190734863)
Descriptors: MW 500.73, ALogP 6.02, HBA 5, HBD 0, PSA 31.84, QED 0.3
Compound (SMILES): CC(=O)N1C[C@H]2C[C@@H](C1)N2c1ccc(CN(CC(F)(F)F)S(=O)(=O)Cc2ccccc2)c(F)c1
InChIKey: XHJIUHMDZGTTTD-OYRHEFFESA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Assay: On day of the assay, 100 uL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20x required final concentration in DMSO (15 uL compound+30 uL DMSO). The 20x compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5x the final test concentration in 25% DMSO (10 uL compound+30 uL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 uL volume. For the assay, 10 uL of 5x compound stock solutions in 25% DMSO were added to the assay plate in duplicate.
Measurement: IC50 = 301 nM (pChEMBL 6.519999980926514)
Descriptors: MW 499.53, ALogP 3.53, HBA 4, HBD 0, PSA 60.93, QED 0.55
Compound (SMILES): CN1CCC(c2ccc(Nc3ncc(Cl)c(CCc4ccccc4C4(C(N)=O)CC4)n3)cc2)CC1
InChIKey: VDXARSHJPFZWJT-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P35916
Assay: VEGFR3 Biochemical Assay: A biotin labelled peptide is used as substrate (amino acid sequence: Biotin-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2). VEGFR3 cytoplasmic domain (amino acids 798-1298) was purchased as N-terminal GST-fusion protein (the enzyme). The 15 uL assay reactions are run in Greiner brand white 384-well low volume plates. All reactions contained 10 mM HEPES pH 7.4, mM MgCl2, 0.01% (v/v) Tween-20, 50 uM Na3VO4, 0.01% (w/v) albumin from chicken egg white, 1 mM Dithiothreitol, 111 nM peptide substrate, 500 uM ATP, and 3.8 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 90 minutes at 30 degree Celsius.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 490.05, ALogP 4.98, HBA 5, HBD 2, PSA 84.14, QED 0.47
Compound (SMILES): Cc1cc(CN(CC2CC2)C(CC(=O)O)c2ccc(Cl)cc2)ccc1OCCn1c(=O)ccn(C)c1=O
InChIKey: JXPUMVOZGFZTNE-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 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 526.03, ALogP 4.02, HBA 7, HBD 1, PSA 93.77, QED 0.38
Compound (SMILES): C[C@@H](c1nc2ccc(N3CCC[C@@H](C(=O)N4CCCC4)C3)nc2[nH]1)n1cc(Cl)cn1
InChIKey: NOSUIOGIJKVTAO-LSDHHAIUSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Inhibition of DGAT1 (unknown origin)
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 427.94, ALogP 3.26, HBA 6, HBD 1, PSA 82.94, QED 0.69
Compound (SMILES): N#Cc1nc(Nc2cccc([N+](=O)[O-])c2)nc(N2CCNCC2)n1
InChIKey: GVIPJIBLWRMISS-UHFFFAOYSA-N
Target: Cathepsin B (SINGLE PROTEIN), Homo sapiens
UniProt: P07858
Assay: Inhibition of human cathepsin B using Z-Phe-Arg-AMC as substrate after 30 mins by spectromicrofluorimetric method
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 326.32, ALogP 0.8, HBA 9, HBD 2, PSA 132.9, QED 0.62
Compound (SMILES): O=c1[nH]c(N2CCOCC2)nc(NC2CNC2)c1-c1ccc2ccccc2n1
InChIKey: ONSAJXRZYRRXOW-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IMAP technology (Molecular Devices): A 20 ul reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 uM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 ul of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 378.44, ALogP 1.21, HBA 7, HBD 3, PSA 95.17, QED 0.63
Compound (SMILES): COc1ccc(CC2CCOC3(CCN(Cc4ccc(COc5ccc(CCC(=O)O)cc5)cc4)CC3)C2)cc1.Cl
InChIKey: LVEDMCRSHKYPOT-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Agonist activity at human FFA4 receptor expressed in CHO cells assessed as induction of Ca2+ flux by fluo-8 dye based FLIPR assay
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 543.7, ALogP 6.3, HBA 5, HBD 1, PSA 68.23, QED 0.31
Compound (SMILES): CC(C)CN(c1cccc(Cl)c1)S(=O)(=O)c1ccc(-c2ccc(NS(C)(=O)=O)cc2)cc1
InChIKey: OWEZSNBFNMZPTO-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In vitro RORc Ligand Binding Assay: On day of the assay, 100 μL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20× required final concentration in DMSO (15 μL compound + 30 μL DMSO). The 20× compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5× the final test concentration in 25% DMSO (10 μL compound + 30 μL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 μL volume. For the assay, 10 μL of 5× compound stock solutions in 25% DMSO were added to the assay plate in duplicate. Assay plates were 96-well polypropylene V-bottom plates. 10 μL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 μL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 μL of 5 μM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 μL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 μL of 1.5 ug/mL RORc receptor was added to wells (or 40 μL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25 °C. Final concentrations were as follows: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 μM 25-hydroxycholesterol was also present.
Measurement: IC50 = 169 nM (pChEMBL 6.769999980926514)
Descriptors: MW 493.05, ALogP 5.23, HBA 4, HBD 1, PSA 83.55, QED 0.47
Compound (SMILES): Cc1cc(C)n(Cc2ccc(Cn3cc(C(=O)NCc4c(C)cc(N)nc4C)cn3)cc2)n1
InChIKey: MAGLWMOZQOEIRW-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 0.134 nM (pChEMBL 9.869999885559082)
Descriptors: MW 443.56, ALogP 3.32, HBA 7, HBD 2, PSA 103.65, QED 0.46
Compound (SMILES): O=C(c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1)N1CCC(O)(C(F)(F)F)C1
InChIKey: JTDKRJSOKDUSEK-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37 °C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30 °C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30 °C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 83.5 nM (pChEMBL 7.079999923706055)
Descriptors: MW 421.37, ALogP 2.47, HBA 5, HBD 1, PSA 65.9, QED 0.74
Compound (SMILES): Cc1nc(C(C)(C)NC(=O)c2ccc(C3CC3)c(OCC3CC3)n2)no1
InChIKey: MXZPEPMKUIRVTC-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 87.1 nM (pChEMBL 7.059999942779541)
Descriptors: MW 356.43, ALogP 3.1, HBA 6, HBD 1, PSA 90.14, QED 0.82
Compound (SMILES): CC1(C)OC(=O)N([C@H]2CC[C@H](c3cnc(N)c(-c4nccc(C(=N)N)n4)c3)CC2)[C@H]1c1ccccc1
InChIKey: JKFAGGDDAYJHJN-BPXKWBHBSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Tankyrase Biochemical Assays: The tankyrase 1 biochemical activity of the compounds was assayed in the following assay buffer (50 mM MOPS pH7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.01% Tween-20, 0.05% BSA, and 1 mM DTT) as follows: 0.25 nM of 6×HIS-tankyrase 1 (1091-1325) was incubated in the presence of compound (DMSO 1.85% final) in a Perkin Elmer 384 well Proxiplate Plus™ (cat. no. 6008289) with 400 nM of NAD for 60 minutes at room temperature. The assay was then stopped with the above assay buffer containing a 0.6 M inhibitor and the following detection components: 0.05 g/mL monoclonal anti-PAR antibody (Trevigen cat. no. 4335-MC-01K-AC) prebound for 60 minutes with 0.63 μg/mL protein G AlphaLisa® acceptor bead (Perkin Elmer cat. no. AL102M) and 5 μg/mL AlphaLisa® nickel chelate donor bead (Perkin Elmer cat. no. AS101M). The assay was incubated for 16 hours at room temperature in the dark and read on a Perkin Elmer Envision® multi label reader using the default program set with laser excitation at 680 nM and emission at 615 nM. The tankyrase 2 biochemical assay was performed using the procedure set forth above with respect to tankyrase 1 except that 4 nM 6×HIS-tankyrase 2 (946-1162) and 250 nM NAD were used.
Measurement: IC50 = 14.3 nM (pChEMBL 7.840000152587891)
Descriptors: MW 485.59, ALogP 4.4, HBA 7, HBD 3, PSA 144.1, QED 0.36
Compound (SMILES): Cc1ncsc1CNc1nc(-n2ncc3ccccc32)nc(N)c1Br
InChIKey: DGFHPCHDAOKRIG-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: [3H] cAMP SPA Assay: The analysis of the activity of the enzyme Phosphodiesterase [3H] cAMP SPA (Perkin Elmer #7090 TRKQ) kit was used for the study of Phosphodiesterase 10. The kit contains:Yttrium scintillation proximity assay (SPA) in beads: yttrium silicate in microspheres resuspended pellet in MiliQ water containing 18 mM zinc sulfate.    10×PDE assay buffer: 500 mM Tris/HCl; 83 mM MgCl2; 17 mM EGTA; pH 7.5.    Tracer [3H] cAMP: substrate for enzymatic reaction.The test is based on the preferential binding of linear nucleotides to the SPA beads in the presence of zinc sulfate, with respect to the binding of cyclic nucleotides, which is almost imperceptible. Therefore, in optimum conditions, the product of the enzymatic reaction binds directly to SPA, and the substrate of the enzyme does not.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 416.31, ALogP 3.54, HBA 8, HBD 2, PSA 94.54, QED 0.53
Compound (SMILES): CCOC(=O)CCCNc1cc(C(c2ccc(Cl)cc2)c2ccc(Cl)cc2)cc2c(NC3CCN(S(=O)(=O)C(F)(F)F)CC3)cc(=O)[nH]c12
InChIKey: RIWCIGOGPPLRIG-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 1124 nM (pChEMBL 5.949999809265137)
Descriptors: MW 739.64, ALogP 7.5, HBA 7, HBD 3, PSA 120.6, QED 0.08
Compound (SMILES): CS(=O)(=O)Nc1ccc(-c2c(/C=C/c3ccc4ccccc4n3)nc3c(N4CCOCC4)ccnn23)cc1
InChIKey: FVCUIYWMZSWOIL-OUKQBFOZSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 526.62, ALogP 4.32, HBA 8, HBD 1, PSA 101.72, QED 0.35
Compound (SMILES): CC(CF)Oc1cc(F)ccc1Nc1ncnc2cc(N=S(C)(C)=O)cc(F)c12
InChIKey: PUDCUARKHMDCIS-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: In vitro Kinase Assay: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 424.45, ALogP 4.75, HBA 6, HBD 1, PSA 76.47, QED 0.62
Compound (SMILES): O=c1[nH]nc(Cc2ccc(F)c(CNC3CCOCC3)c2)c2c1CCCC2
InChIKey: NFLHTMCUZUFAJB-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 = 411 nM (pChEMBL 6.389999866485596)
Descriptors: MW 371.46, ALogP 2.65, HBA 4, HBD 2, PSA 67.01, QED 0.85
Compound (SMILES): O=C(COc1cccc(C(=O)N2CCOCC2)c1)NCC(O)CN1CCc2ccccc2C1
InChIKey: VCBCXDCVFJZGTB-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 101 nM (pChEMBL 7)
Descriptors: MW 453.54, ALogP 1.07, HBA 6, HBD 2, PSA 91.34, QED 0.62
Compound (SMILES): CCn1ncc2cc3nc(c21)CCCO[C@@H]([C@@H](C)O)[C@@H](c1ccccc1)NC(=O)N3
InChIKey: ZOCDMCKSAQKHPE-MMACOIQUSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Inhibition of human activated ERK2 assessed as reduction in phosphorylation activity preincubated for 15 mins followed by addition of IMAP peptide as substrate and ATP measured after 60 mins by fluorescence polarization method
Measurement: IC50 = 687.3 nM (pChEMBL 6.159999847412109)
Descriptors: MW 409.49, ALogP 3.03, HBA 6, HBD 3, PSA 101.3, QED 0.62
Compound (SMILES): Cc1c(Cc2cccnc2)nnc(N2CCN(c3cnc(C(C)(C)O)cn3)[C@H](C)C2)c1C
InChIKey: JSKSYTQPNABUFN-MRXNPFEDSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 433.56, ALogP 2.81, HBA 8, HBD 1, PSA 91.16, QED 0.66
Compound (SMILES): O=C(O)CCC(=O)Nc1ccc(-c2cnc(N3CCOCC3)c3nc(CCc4ccc5ccccc5n4)cn23)cc1
InChIKey: ARMKZYCDGHPGEI-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay rPDE10: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi 3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter and results were expressed as cpm. For blank values the enz
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 550.62, ALogP 4.37, HBA 8, HBD 2, PSA 121.95, QED 0.28
Compound (SMILES): CCn1cc(C(=O)NCc2ccc(S(=O)(=NC#N)C(C)C)cc2)c(=O)c(-c2cccc(C(F)(F)F)c2)c1C
InChIKey: QUFKKGIVYVWNAY-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 544.6, ALogP 5.51, HBA 6, HBD 1, PSA 104.32, QED 0.4
Compound (SMILES): CCC(=O)NC1CCCc2c(-c3ccc(F)c(C)c3)cncc21
InChIKey: QZVUORKNBRDTPK-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Cellular Enzyme Assay: G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 ug/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 625 nM (pChEMBL 6.199999809265137)
Descriptors: MW 312.39, ALogP 4.1, HBA 2, HBD 1, PSA 41.99, QED 0.93
Compound (SMILES): C#CC(=O)Nc1cc(-c2cnc3[nH]cc(C)c3c2)ccc1F
InChIKey: IEVQOCMRVHKNMW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: In Vitro Inhibition Assay: ITK and JAK3 Kinase assay procedures:Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μL of 3% phosphoric acid. Volume was increased to 100 μL by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μL 0.8% phosphoric acid and dried for an hour at 60° C. 100 μL scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 293.3, ALogP 3.25, HBA 2, HBD 2, PSA 57.78, QED 0.71
Compound (SMILES): Cc1nnc2c(NC(C)C)nc(-c3nc4cnc(N5CCOC[C@@H]5C)cc4n3Cc3ccccn3)cn12
InChIKey: SJMFHBQDKBQDTQ-KRWDZBQOSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4-H4 Tetraacetylated Peptide Inhibition AlphaScreen: This assay is used to determine whether the compounds inhibit the interaction between the first (BRD4-BD1) or the second (BRD4-BD2) bromodomain of BRD4 and the tetraacetylated histone H4 peptide.Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (probe molecule JQ1+ with protein/peptide mix) is pipetted. Determination of IC50 values are carried out using GraphPad Prism 3.03 software (or updates thereof).
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 498.6, ALogP 3.33, HBA 11, HBD 1, PSA 111.18, QED 0.38
Compound (SMILES): O=C(Nc1ccc(C(=O)N2CCC(C(F)(F)F)CC2)cc1)N1Cc2ccccc2C1
InChIKey: LMZHAWAYPISDMF-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 86.1 nM (pChEMBL 7.070000171661377)
Descriptors: MW 417.43, ALogP 4.65, HBA 2, HBD 1, PSA 52.65, QED 0.77
Compound (SMILES): O=C(c1c(F)ccc(NS(=O)(=O)c2cccnc2)c1F)c1c[nH]c2nccc(Cl)c12
InChIKey: UGHOYILAXQCULM-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 448.84, ALogP 3.92, HBA 5, HBD 2, PSA 104.81, QED 0.45
Compound (SMILES): CC(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: TVPSBQGRZZNLGN-WNAJCWKFSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 2371.76
Compound (SMILES): COc1cc2c(cc1OC)C(=O)/C(=C/c1ccc(N3CCN(c4ccc(C(F)(F)F)cc4)CC3)cc1)C2
InChIKey: FZPXTPYRSIVANH-RCCKNPSSSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of Electric eel AChE using acetylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition measured after 2 mins by Ellman's method
Measurement: IC50 = 153 nM (pChEMBL 6.820000171661377)
Descriptors: MW 508.54, ALogP 5.87, HBA 5, HBD 0, PSA 42.01, QED 0.4
Compound (SMILES): CCC1c2ccccc2CCN1CC(O)COc1cccc(-c2ccc3ncn(C)c3c2)c1
InChIKey: IFUBQSXECPBBIS-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 101 nM (pChEMBL 7)
Descriptors: MW 441.58, ALogP 4.99, HBA 5, HBD 1, PSA 50.52, QED 0.44
Compound (SMILES): O=S(=O)(Nc1ccc(Oc2ccccc2)cc1)c1cccc2ccccc12
InChIKey: RDRNPFHUNQOZPK-UHFFFAOYSA-N
Target: Progesterone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06401
Assay: Antagonist activity at progesterone receptor in human T47D cells incubated for 24 hrs in presence of P4 by alkaline phosphatase assay
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 375.45, ALogP 5.43, HBA 3, HBD 1, PSA 55.4, QED 0.51
Compound (SMILES): CN(C)c1nc(NCc2ccc(NC(=O)c3ccc(F)cc3)cc2)c2ccc(-c3ccc(C=O)cc3)cc2n1
InChIKey: GTIXVKTURRQJIH-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf33.13 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 2675 nM (pChEMBL 5.570000171661377)
Descriptors: MW 519.58, ALogP 6.18, HBA 6, HBD 2, PSA 87.22, QED 0.24
Compound (SMILES): O=C(Nc1nnc(-c2ccccc2)s1)c1cccc(CCl)c1
InChIKey: JXXVGXULFVEGCR-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Mus musculus
UniProt: P21836
Assay: Uncompetitive inhibition of albino mouse brain AChE using acetylthiocholine iodide as substrate measured up to 2 mins by Ellmans method
Measurement: Ki = 850 nM (pChEMBL 6.070000171661377)
Descriptors: MW 329.81, ALogP 4.2, HBA 4, HBD 1, PSA 54.88, QED 0.73
Compound (SMILES): C[C@H]1COCCN1c1ccc(C(F)(F)F)c(-c2cnc(N)c(C(=O)Nc3ncccc3N3CCC(C)(N)CC3)n2)n1
InChIKey: HTPZZARNXXDSIZ-INIZCTEOSA-N
Target: Protein kinase C alpha type (SINGLE PROTEIN), Homo sapiens
UniProt: P17252
Assay: In Vitro Inhibition Activity Assay: The compounds of formula I were tested for their activity on different PKC isoforms according to a published method (D. Geiges et al. Biochem. Pharmacol. 1997; 53:865-875) The assay is performed in a 96-well polypropylene microtiterplate (Costar 3794) that has been previously siliconized with Sigmacote (Sigma SL-2). The reaction mixture (50 μL) contains 10 μL of the relevant PKC isozyme together with 25 μL of the PKC inhibitor compound and 15 μL of a mix solution that contains 200 μg/mL protamine sulfate, 10 mM Mg(NO3)2, 10 μM ATP (Boehringer 519987) and 3750 Bq of 33P-ATP (Hartmann Analytic SFC301, 110 TBq/mmol) in 20 mM Tris-buffer pH 7.4+0.1% BSA. Incubation was performed for 15 minutes at 32° C. in a microtiterplate shaking incubator (Biolabo Scientific Instruments). Reaction was stopped by adding 10 μl of 0.5 M Na2EDTA, pH 7.4. 50 μl of mixture are pipetted onto a pre-wetted phosphocellulose paper (Whatmann 3698-915) under gentle pressure.
Measurement: IC50 = 0.32 nM (pChEMBL 9.489999771118164)
Descriptors: MW 571.61, ALogP 3.33, HBA 10, HBD 3, PSA 148.41, QED 0.42
Compound (SMILES): CCN1CN([C@H]2CC[C@H](CNS(=O)(=O)CC(F)(F)F)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: LFSAJENJLBQMME-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 473.52, ALogP 2.84, HBA 5, HBD 2, PSA 98.4, QED 0.67
Compound (SMILES): CCc1cc2cnc(Nc3ccc(N4CCC(O)CC4)cn3)nc2n(C2CCCC2)c1=O
InChIKey: YDCBBXNPMFCEGD-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Inhibition of Cdk4/Cyclin D (unknown origin) using retinoblastoma protein substrate and [gamma32P]ATP incubated for 15 mins by beta plate counter based assay
Measurement: IC50 = 135 nM (pChEMBL 6.869999885559082)
Descriptors: MW 434.54, ALogP 3.57, HBA 8, HBD 2, PSA 96.17, QED 0.63
Compound (SMILES): Cc1ccc(C(C)C)cc1Nc1ncc(Br)c(Nc2cc(C3CC3)n[nH]2)n1
InChIKey: UIDZGGSFPDKPPA-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 427.35, ALogP 5.76, HBA 5, HBD 3, PSA 78.52, QED 0.47
Compound (SMILES): COc1ccc(NC(=O)c2ccc(NC(=O)N3Cc4cccnc4C3)cc2)cn1
InChIKey: WTEIHJLLBLJTCX-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 = 19.2 nM (pChEMBL 7.71999979019165)
Descriptors: MW 389.42, ALogP 3.29, HBA 5, HBD 2, PSA 96.45, QED 0.71
Compound (SMILES): CN(C)CCCNC(=O)COc1ccc2c(c1)C(C)(C)c1oc3ccccc3c1C2=O
InChIKey: CUEIVRPDMMWTES-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 3120.3 nM (pChEMBL 5.510000228881836)
Descriptors: MW 420.51, ALogP 3.75, HBA 5, HBD 1, PSA 71.78, QED 0.59
Compound (SMILES): C[C@]1(O)CC[C@H](Nc2ccc3nnc(-c4cccc(C(F)(F)F)c4)n3c2)CC1
InChIKey: IWQQAFVEJVPPRE-RHDGDCLCSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase-Glo Assay: These assays are set up in duplicate 50 ul volumes in white, flat bottom 96 well plates. Inhibitors are added to the solution of 1× kinase buffer, 10 uM ATP, 100 uM Pim-1-specific substrate, 50 ng of active Pim-1 enzyme, and water in serial dilutions ranging from micromolar to nanomolar concentrations. This solution is incubated at 30 degrees Celsius at 360 rpm for two hours. Following the incubation, 50 ul of Kinase-Glo reagent is added to each well, including all positive and negative control wells, and incubated at room temperature for 15 minutes. The plate is then read by the Luminoskan Ascent instrument and the results displayed with the Ascent Software version 2.6.
Measurement: IC50 = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 390.41, ALogP 4.52, HBA 5, HBD 2, PSA 62.45, QED 0.69
Compound (SMILES): CCOc1cc(C2CCN(CCS(C)(=O)=O)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: VWWDKFOSAHCQHN-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 751.86, ALogP 7.36, HBA 10, HBD 2, PSA 130.82, QED 0.13