lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC[C@@H](N[C@H](C)CC(=O)NCCO)c1ccc(Cl)c(C(=O)c2ccc(N)nc2)c1F
InChIKey: NYWZYWQVRAKWGX-MLGOLLRUSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 430 nM (pChEMBL 6.369999885559082)
Descriptors: MW 436.92, ALogP 2.62, HBA 6, HBD 4, PSA 117.34, QED 0.43
Compound (SMILES): CC(Nc1nccc(N2C(=O)OCC2[C@@H](C)O)n1)c1nc(CCc2ccccc2)no1
InChIKey: LXDWPRMFMLFXHK-NPCAHTBFSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 12400 nM (pChEMBL 4.909999847412109)
Descriptors: MW 424.46, ALogP 2.52, HBA 9, HBD 2, PSA 126.5, QED 0.56
Compound (SMILES): CC[C@@H]1CC(=O)NN=C1c1ccc2nc(-c3ccccc3)oc2c1
InChIKey: CIKXVUUQVXSNHP-GFCCVEGCSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 66 nM (pChEMBL 7.179999828338623)
Descriptors: MW 319.36, ALogP 3.75, HBA 4, HBD 1, PSA 67.49, QED 0.8
Compound (SMILES): CCN[C@H](CC(=O)N1CCN(c2nc(N)c3cc(OC)c(OC)c(F)c3n2)CC1)c1ccc(C#N)cc1
InChIKey: FQQPKJKIBNLDSF-LJQANCHMSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: ELISA Assay: CVF-Bb complex prepared from purified cobra venom factor (1 μM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 507.57, ALogP 2.63, HBA 9, HBD 2, PSA 129.63, QED 0.47
Compound (SMILES): C[C@@H]1c2nnc(-c3cccc(O)n3)n2CCN1C(=O)c1ccc(-c2cccs2)cc1
InChIKey: JSJMFCNSTAYAHE-CQSZACIVSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Aequorin Functional Assay: Changes in intracellular calcium levels are a recognized indicator of G protein-coupled receptor activity. The efficacy of compounds of the invention to inhibit NKA-mediated NK-3 receptor activation was assessed by an in vitro Aequorin functional assay. Chinese Hamster Ovary recombinant cells expressing the human NK3 receptor and a construct that encodes the photoprotein apoaequorin were used for this assay. In the presence of the cofactor coelenterazine, apoaequorin emits a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium. Antagonist Testing: the antagonist activity of compounds of the invention is measured following pre-incubation (3 minutes) of the compound with the cells, followed by addition of the reference agonist (NKA) at a final concentration equivalent to the EC80 (3 nM) and recording of emitted light (FDSS 6000 Hamamatsu) over the subsequent 90-second period. The intensity of the emitted light is integrated using the reader software. Compound antagonist activity is measured based on the inhibition of the luminescence response to the addition of Neurokinin A.Â  Inhibition curves are obtained for compounds of the invention and the concentrations of compounds which inhibit 50% of reference agonist response (IC50) were determined (see results in table 5 below). The IC50 values shown in table 5 indicate that compounds of the invention are potent NK-3 antagonist compounds.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 417.49, ALogP 3.99, HBA 7, HBD 1, PSA 84.14, QED 0.54
Compound (SMILES): C[C@@H](NC[C@H](O)c1ccc(O)c2[nH]c(=O)ccc12)c1cccc(CCNC(=O)c2ccc(N(C)C(=O)CCN3CCC(OC(=O)Nc4ccccc4-c4ccccc4)CC3)cc2)c1
InChIKey: KOHQSQYVBTZTTO-PWOKGZDBSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: Radioligand Binding Assay: Displacement assays for determination of Ki values of test compounds were performed with [3H]-NMS at 1 nM and eleven different test compound concentrations. The test compounds were initially dissolved to a concentration of 400 μM in dilution buffer and then serially diluted 5× with dilution buffer to final concentrations ranging from 10 pM to 100 μM. The order of addition and volumes added to the assay plates were as follows: 25 μL radioligand, 25 μL diluted test compound, and 50 μL membranes. Assay plates were incubated for 6 hours at 37° C. Binding reactions were terminated by rapid filtration over GF/B glass fiber filter plates (PerkinElmer, Inc.) pre-treated in 1% BSA. Filter plates were rinsed three times with wash buffer (10 mM HEPES) to remove unbound radioactivity. The plates were then air-dried and 50 μL Microscint-20 liquid scintillation fluid (PerkinElmer, Inc.) were added to each well. The plates were then counted in a PerkinElmer Topcount liquid scintillation counter (PerkinElmer, Inc.).
Measurement: Ki = 0.1 nM (pChEMBL 10)
Descriptors: MW 851.02, ALogP 7.32, HBA 9, HBD 6, PSA 176.33, QED 0.06
Compound (SMILES): COC(=O)N[C@H](C(=O)NCc1ccc([C@@H](CO)N(Cc2cnon2)S(=O)(=O)c2ccc(N)cc2)s1)C(c1ccccc1)c1ccccc1
InChIKey: YBSFAJGBZFEZBJ-MVSFAKPFSA-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 = 33.8 nM (pChEMBL 7.46999979019165)
Descriptors: MW 690.8, ALogP 3.81, HBA 11, HBD 4, PSA 189.98, QED 0.12
Compound (SMILES): CCN[C@H](CC(=O)N1CCN(c2nc(N)c3cc(OC)c(OC)c(F)c3n2)CC1)c1ccc(F)cc1
InChIKey: KBXQLSIBVLBIHO-GOSISDBHSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: ELISA Assay: CVF-Bb complex prepared from purified cobra venom factor (1 μM), recombinant human complement factor B (expressed in drosophila cells and purified using standard methods) and human complement factor D (expressed in E. Coli, refolded and purified using standard methods). CVF-Bb complex at 3 nM concentration was incubated with test compound at various concentrations for 1 hour at room temperature in PBS pH 7.4 containing 10 mM MgCl2 and 0.05% (w/v) CHAPS. Human complement C3 substrate purified from plasma was added to a final concentration of 1 μM. After 1 hour incubation at room temperature, the enzyme reaction was stopped by addition of a cocktail of concentrated pan-protease inhibitors. The product of the reaction, C3a, was quantified by means of an enzyme-linked-immunosorbent assay.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 500.55, ALogP 2.9, HBA 8, HBD 2, PSA 105.84, QED 0.49
Compound (SMILES): O=C(O[C@H]1CC[C@@H](c2ccc(-c3nc4[nH]c(O[C@@H]5COC6C5OC[C@H]6O)nc4cc3Cl)cc2)CC1)N1CCCC1
InChIKey: ZUPSWAVAKGLOSO-QSFQHBHSSA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13131
Assay: AMPK Activation Assay: In the 384-well plates 1.25 ul of test compound in assay buffer (20 mM HEPES pH 7.0, 0.025% BSA, 15 mM MgCl2, 0.01% Brij) was mixed with 1.25 ul of AMPK and 1.25 ul of the peptide (final concentration of 1 uM) and 1.25 ul of ATP (final concentration of 30 uM), both dissolved in assay buffer. This step was followed by an incubation time of 60 min. Then 5 ul of ADP Glo Reagent was added. This was followed by 40 min of incubation. Then 10 ul of Kinase Detection Reagent was admixed. The plates were sealed and after an incubation period of 30 min, the luminescence signal was measured in an Envision reader. All incubation steps were accomplished at room temperature. Each assay microtiter plate contained wells with vehicle controls instead of compound (0.1% DMSO in water) as reference for the low signal (100% CTL, low signal), and wells with serial dilutions of AMP (final 30 uM) as reference for high signals. The luminescence signal generated was proportional to the ADP concentration produced and was correlated with AMPK activity.
Measurement: EC50 = 2043 nM (pChEMBL 5.690000057220459)
Descriptors: MW 569.06, ALogP 4.44, HBA 8, HBD 2, PSA 119.03, QED 0.46
Compound (SMILES): Nc1ccc(S(=O)(=O)Nc2cccc(-c3ccc([C@H]4C[C@@H]4NC4CCC(N)CC4)cc3)c2)cn1
InChIKey: PWPKPNWKBRXCMZ-GBBDAIBUSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Fluorescence-Based (Inhibitor)-Screening Assay: In order to monitor MAO enzymatic activities and/or their inhibition rate by inhibitor(s) of interest, a fluorescence-based (inhibitor)-screening assay was set up. 3-(2-Aminophenyl)-3-oxopropanamine (kynuramine dihydrobromide, Sigma Aldrich), a non fluorescent compound was chosen as a substrate. Kynuramine is a non-specific substrate for both MAO-A and MAO-B activities. While undergoing oxidative deamination by MAO activities, kynuramine is converted into 4-hydroxyquinoline (4-HQ), a resulting fluorescent product.The monoamine oxidase activity was estimated by measuring the conversion of kynuramine into 4-hydroxyquinoline. Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 uL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in duplicate within the same experiment.Briefly, a fixed amount of MAO (0.25 ug for MAO-A and 0.5 ug for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of at least eight 3-fold serial dilutions each. Clorgyline and Deprenyl (Sigma Aldrich) was used as a control for specific inhibition of MAO-A and MAO-B respectively. After leaving the enzyme(s) interacting with the inhibitor, KM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. in the dark. The oxidative deamination of the substrate was stopped by adding 50 uL of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor. The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes. The IC50 values of each inhibitor were calculated with GraphPad Prism Software.
Measurement: IC50 = 100000 nM (pChEMBL 4)
Descriptors: MW 477.63, ALogP 3.85, HBA 6, HBD 4, PSA 123.13, QED 0.41
Compound (SMILES): C[C@@H]1COCCN1c1nc(N2CCO[C@@H](CO)C2)nc2[nH]c(-c3cccc4[nH]ccc34)nc12
InChIKey: QTNDHTFTTXBVPK-HUUCEWRRSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR ATP-Binding Assay: 1. 8-point serial dilutions of compounds (10 mM stock) are performed in 90% DMSO in a 384-well "masterplate" and 50 nL is transferred onto 384-well assay plates (white polystyrene small volume; Matrix/Thermo Scientific Cat. No. #4365). 2. The final volume of the assay is 10 μL and the order of addition is as follows: 50 nL of compounds dilution; 5 μL of a mixture of GST-mTOR and Europium anti-GST antibody with or without the PI3K/mTOR inhibitor P1-103 (3-(4-(4-morpholinyl)pyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl)phenol, Calbiochem); 5 μL of tracer-314. Incubated for 60 minutes at room temperature. TR-FRET measured in Biotek Synergy2 reader at: Excitation 340 nm/emission 665 nm; Excitation 340 nm/emission 620 nm. The assay buffer consists of 50 mM HEPES pH 7.5, 5 mM MgCl2, 1 mM EGTA, 0.01% Pluronic F-127. Tracer-314 (Alexa Fluor® 647-labeled ATP-competitive kinase inhibitor; Cat. No. PV6087), Europium anti-GST antibody (Cat. No PV5594) and N-terminally GST-tagged truncated human mTOR (FRAP1) (Cat. No PV4754) are available from Invitrogen. The following final concentrations are used: 3 nM GST-mTOR; 1 nM Europium anti-GST antibody; +/− 10 μM P1-103; and 10 nM tracer-314. The final concentrations of diluted compounds are 9091; 2730; 910; 273; 91; 27; 9; and 3 nM. The final concentration of DMSO is 0.45%. The following controls are used: High signal: solvent vehicle, GST-mTOR, Eu-anti-GST antibody, tracer-314; Low signal: solvent vehicle, GST-mTOR, Eu-anti-GST antibody, P1-103, tracer-314.
Measurement: IC50 = 67 nM (pChEMBL 7.170000076293945)
Descriptors: MW 449.52, ALogP 1.92, HBA 8, HBD 3, PSA 115.42, QED 0.43
Compound (SMILES): CC(C)Nc1ncc2c(n1)CN(C(=O)N[C@H](CNc1ncccn1)c1ccc(F)c(Cl)c1)CC2
InChIKey: BVHMQJNXNFDAHI-LJQANCHMSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100x final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 1 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 uM S/T17 peptide substrate and 25 uM ATP (corresponding to the observed Km) for a total reaction volume of 25 uL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 4.43 nM (pChEMBL 8.350000381469727)
Descriptors: MW 484.97, ALogP 3.8, HBA 7, HBD 3, PSA 107.96, QED 0.47
Compound (SMILES): CC(C)(C)OC(=O)[C@H](C1CC1)N1C(=O)[C@@H](CC(=O)O)C[C@H](c2cccc(Cl)c2)[C@H]1c1ccc(Cl)cc1
InChIKey: KJMQFBPHXPWLEW-MPMKUJPUSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Homogenous Time-Resolved Fluorescence Assay (HTRF1 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1xPBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag-TrxA-6-His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 532.46, ALogP 6.26, HBA 4, HBD 1, PSA 83.91, QED 0.43
Compound (SMILES): N[C@@H]1CCN(c2ccc(-c3n[nH]c4cnc(-c5cccnc5)cc34)cn2)C1
InChIKey: IZVRVJUQKJASMV-OAHLLOKOSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 1.02 nM (pChEMBL 8.989999771118164)
Descriptors: MW 357.42, ALogP 2.62, HBA 6, HBD 2, PSA 96.61, QED 0.58
Compound (SMILES): CC(C)C[C@@H](NC(=O)c1ccc(N2CC(F)(F)C2)c(OCC2CC2)n1)c1cccnn1
InChIKey: JNWQBXIJVLIUBQ-GOSISDBHSA-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 = 14.9 nM (pChEMBL 7.829999923706055)
Descriptors: MW 431.49, ALogP 3.63, HBA 6, HBD 1, PSA 80.24, QED 0.65
Compound (SMILES): C[C@@H](NC(=O)Nc1cc2[nH]nc(NS(=O)(=O)C3CNC3)c2cn1)c1ccccc1
InChIKey: ABVUTKMGNDRBEM-LLVKDONJSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 191.1 nM (pChEMBL 6.71999979019165)
Descriptors: MW 415.48, ALogP 1.55, HBA 6, HBD 5, PSA 140.9, QED 0.41
Compound (SMILES): O=C(OC1C2CC3CC(C2)CC1C3)N1CCC[C@@H]1c1nc(-c2ccncc2)cs1
InChIKey: JUTHVZDHPWXUNN-BXDRECAWSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: Scintillation Proximity Assay: Inhibition of microsomal preparations of 11.beta.-HSDl by compounds of the present invention, as described essentially previously (K. Solly, SS Mundt, HJ Zokian, GJ Ding, A. Hermanowski-Vosatka, B. Strulovici, and W. Zheng, High-Throughput Screening of 11-Beta-Hydroxyseroid Dehydrogenase Type 1 in Scintillation Proximity Assay Format. Assay Drug Dev Technol 3 (2005) 377-384) was measured. All reactions were carried out in a clear flexible PET Microbeta plates 96-well at room temperature (PerkinElmer). Assay, substrate solution (50mM HEPES, pH7.4,100mM KCl, 5mM NaCl, 2mM MgCl 2, 2mM NADPH and 160nM [3 H] cortisone (1Ci / mmol)) a 49ul and distribution, and starting from the 0.1mM on to start by combining the semi-log (half-log) multiple of the incremental test compound 1uL in DMSO previously diluted (8 points). After pre-incubation of 10 minutes, incubated human 11-HSD1 overexpress the enzyme containing CHO cells isolated from microsomes solution 50uL.
Measurement: IC50 = 0.91 nM (pChEMBL 9.039999961853027)
Descriptors: MW 409.56, ALogP 5.3, HBA 5, HBD 0, PSA 55.32, QED 0.69
Compound (SMILES): Cn1cc2c(n1)CCC[C@H]2n1nc(-c2ccc(C(=O)Nc3cc(C(F)(F)F)ccn3)cc2)c2c(N)ncnc21
InChIKey: PMTZBZSNTTWACN-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: LANCE-TR-FRET Assay: Assay Method B: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated "A5" peptide (Biotin-EQEDEPEGDYFEWLE-NH2), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 μL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity).
Measurement: IC50 = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 533.52, ALogP 4.4, HBA 9, HBD 2, PSA 129.43, QED 0.35
Compound (SMILES): CC[C@@H](N[C@H](C)CC(N)=O)c1ccc(OC)c(C(=O)c2ccc(C#N)cc2)c1F
InChIKey: QZSUFAVKQWNRCM-CXAGYDPISA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: FRET-Based Assay: The HCV NS3 protease functions have been extensively studied and are considered as potential targets for antiviral therapy: see for example the many references listed in the introductory section of this application. Therefore, the activity of the compounds of the invention as anti-HCV agents was assessed using a full length HCV NS3 protease.The protease activity of the full length NS3/4a was measured using a FRET-based assay utilizing a peptide substrate derived from the NS4A/B cleavage site (Anaspec) and labelled at one end with a quencher (QXL520) and at the other with a fluorophore (5-FAMsp). NS3/4a (produced in-house by literature methods) was incubated with test compounds and peptide substrate in 50 mM Tris pH8, 20 mM DTT, 1% CHAPS, 10% glycerol and 5% DMSO. The reaction was followed by monitoring the change in fluorescence on a Molecular Devices Gemini plate reader for 30 minutes at room temperature.
Measurement: IC50 = 730 nM (pChEMBL 6.139999866485596)
Descriptors: MW 397.45, ALogP 3.24, HBA 5, HBD 2, PSA 105.21, QED 0.63
Compound (SMILES): CCNc1n[nH]c2cc(NC(=O)N[C@H](c3ccccc3)[C@H]3CCCO3)ncc12
InChIKey: UZDJMBVYGMDPGV-SJLPKXTDSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 359 nM (pChEMBL 6.449999809265137)
Descriptors: MW 380.45, ALogP 3.43, HBA 5, HBD 4, PSA 103.96, QED 0.52
Compound (SMILES): O=c1[nH]c(N2CCOCC2)nc(N[C@@H]2CCCNC2)c1-c1nc(-c2cccc(C(F)(F)F)c2)cs1
InChIKey: TZDMICBCMITENF-MRXNPFEDSA-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 = 141 nM (pChEMBL 6.849999904632568)
Descriptors: MW 506.55, ALogP 3.58, HBA 8, HBD 3, PSA 95.17, QED 0.49
Compound (SMILES): Cc1cc(N=S2(=O)CCCC2)cc2ncnc(Nc3ccc(F)cc3O[C@H](C)C(=O)N3CCC(=O)CC3)c12
InChIKey: ZHWAWNJEBSDLJI-GOSISDBHSA-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 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 539.63, ALogP 4.67, HBA 8, HBD 1, PSA 113.85, QED 0.48
Compound (SMILES): Cc1cnc(Nc2cnn(C)c2)nc1CCc1ccccc1[C@@H](C)C(N)=O
InChIKey: JRSJILWBSZUJGY-CQSZACIVSA-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 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 364.45, ALogP 2.64, HBA 6, HBD 2, PSA 98.72, QED 0.67
Compound (SMILES): CCNC(=O)C(C)c1ccc(O[C@@H]2CCN(c3ccnc(N(C)C4CC4)n3)C2)cc1
InChIKey: MQTXGWVAWOXXGO-OTOKDRCRSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Malonyl CoA formation by acetyl CoA carboxylases is stoichometrically linked to the consumption of ATP. ACC2 activity is measured in a NADH-linked kinetic method measuring ADP generated during the ACC reaction using a coupled lactate dehydrogenase/pyruvate kinase reaction. Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetylCoenzyme A (both 2000 f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 575 nM (pChEMBL 6.239999771118164)
Descriptors: MW 409.53, ALogP 2.97, HBA 6, HBD 1, PSA 70.59, QED 0.72
Compound (SMILES): Cc1ccc(Cc2cc([C@@H]3O[C@H](CO)[C@@H](O)[C@H](O)[C@H]3O)c3c(c2C)OCO3)cc1
InChIKey: UPXRNMVSQVCWSW-YRIDSSQKSA-N
Target: Sodium/glucose cotransporter 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31639
Assay: SGLT2 Inhibition Assay: For sodium-dependent glucose transport assay, cells expressing hSGLT2 were seeded into a 96-well culture plate at a density of 5×104 cells/well in RPMI medium 1640 containing 10% fetal bovine serum. The cells were used 1 day after plating. They were incubated in pretreatment buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2, pH 7.4) at 37 °C. for 10 min. They were then incubated in uptake buffer (10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM 14C-nonlabeled AMG pH 7.4) containing 14C-labeled AMG (8 μM) and the inventive compound or dimethyl sulfoxide (DMSO) vehicle at 37 °C. for 2 h. Cells were washed twice with washing buffer (pretreatment buffer containing 10 mM AMG at room temperature) and then the radioactivity was measured using a liquid scintillation counter.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 402.44, ALogP 1.14, HBA 7, HBD 4, PSA 108.61, QED 0.61
Compound (SMILES): C[C@@H](NC(=O)c1ccc(-c2cn[nH]c2)c(OC(F)F)c1)c1ccc(F)cc1
InChIKey: MRSCRUMYAVOXFF-LLVKDONJSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 22.5 nM (pChEMBL 7.650000095367432)
Descriptors: MW 375.35, ALogP 4.31, HBA 3, HBD 2, PSA 67.01, QED 0.67
Compound (SMILES): CN1CCC(COC(=O)N2CC[C@@H](n3cc(C(N)=O)c(Nc4ccc(F)cc4)n3)[C@H](C#N)C2)CC1
InChIKey: JFLXPNCMPSZGDJ-DYESRHJHSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 483.55, ALogP 2.73, HBA 8, HBD 2, PSA 129.51, QED 0.65
Compound (SMILES): COCCOc1cc(C(=O)Nc2cccc(C(F)(F)F)c2)ccc1-c1nc([C@@H]2CC[C@H]3CCC(=O)N3C2)n2ccnc(N)c12
InChIKey: SWPJNJVPWVHTPQ-KNQAVFIVSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 uM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 uL) was dispensed, followed by the addition of 7.5 uL of 1x kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/uL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 uL 1x kinase buffer containing 8 uM biotinylated "A5" peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ ID NO: 1), and 100 uM ATP. The final reaction in each well of 10 uL consists of 100 pM hBTK, 2 uM biotin-A5-peptide, and 25 uM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1x quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 608.62, ALogP 5.14, HBA 8, HBD 2, PSA 124.08, QED 0.27
Compound (SMILES): CCOC(=O)[C@H]1CC[C@](O)(c2ncc(-c3cc(Nc4nccc(C(F)(F)F)n4)cc(-c4cn[nH]c4)c3)s2)C[C@H]1C
InChIKey: HTELRNAOPAGXNG-VPDXNNENSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSYK fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-SYK (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45' at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5, 40 mM EDTA, 0.01% tween 20. Final volume of the quenching solution was 10 uL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 162.1 nM (pChEMBL 6.789999961853027)
Descriptors: MW 572.61, ALogP 5.94, HBA 9, HBD 3, PSA 125.91, QED 0.23
Compound (SMILES): CC(F)(F)Cn1cc(Nc2cc(-n3ncc4cc(Cl)cnc43)ncc2C(=O)NC[C@@H](F)C(C)(C)O)cn1
InChIKey: YCWXXNCLYYNMMU-GOSISDBHSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 536.95, ALogP 3.9, HBA 9, HBD 3, PSA 122.78, QED 0.3
Compound (SMILES): COC(=O)N[C@H](C(=O)Nc1n[nH]c2cc(NC(=O)N[C@H](C)c3ccc(F)cc3)ncc12)C1CCOCC1
InChIKey: RAAGGYWADVUGCS-XCLFUZPHSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 365 nM (pChEMBL 6.440000057220459)
Descriptors: MW 513.53, ALogP 3.07, HBA 7, HBD 5, PSA 159.36, QED 0.32
Compound (SMILES): CC(C)(C)c1cccc(-c2c(N[C@@H]3CCCNC3)nc(N3CCOCC3)[nH]c2=O)n1
InChIKey: HBIKXAABRSPKCO-OAHLLOKOSA-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 = 537 nM (pChEMBL 6.269999980926514)
Descriptors: MW 412.54, ALogP 2.13, HBA 7, HBD 3, PSA 95.17, QED 0.71
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N(C)Cc2ncccn2)n2c1COCC2)c1ccccc1
InChIKey: DBAUBEJYLNWCES-LJQANCHMSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 2700 nM (pChEMBL 5.570000171661377)
Descriptors: MW 433.51, ALogP 2.96, HBA 6, HBD 1, PSA 89.35, QED 0.62