lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(c1ccc(S(=O)(=O)NCc2ccc(F)cc2)cn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: FEQTWGUPNAITJA-GOSISDBHSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z′-LYTE Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE Tyr 6 peptide or Z-LYTE Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100 μM, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 481.56, ALogP 2.69, HBA 7, HBD 2, PSA 107.11, QED 0.42
Compound (SMILES): Cc1cc(/C=C/c2cc3ccc(O)cc3nc2Cl)cc(C)c1O
InChIKey: HMUWEJRDKUXALY-ONEGZZNKSA-N
Target: Transthyretin (SINGLE PROTEIN), Homo sapiens
UniProt: P02766
Assay: Inhibition of TTR V30M mutant (unknown origin) assessed as inhibition of acid-mediated amyloidogenesis by measuring fibril formation preincubated with enzyme followed by amyloidogenesis stimulation by adjusting pH to 4.4 measured after 72 hrs relative to control
Measurement: IC50 = 1630 nM (pChEMBL 5.789999961853027)
Descriptors: MW 325.8, ALogP 5.09, HBA 3, HBD 2, PSA 53.35, QED 0.65
Compound (SMILES): CN(C)c1ccc(Nc2ncc(Br)c(Nc3ccccc3)n2)cc1
InChIKey: XUGPRGXTXMBSIA-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 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 384.28, ALogP 4.79, HBA 5, HBD 2, PSA 53.08, QED 0.66
Compound (SMILES): O=C(O)CCc1ccc2[nH]nc(-c3ccc(F)cc3)c2c1
InChIKey: KECVAQGKMFEIQS-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 10000 nM (pChEMBL 5)
Descriptors: MW 284.29, ALogP 3.39, HBA 2, HBD 2, PSA 65.98, QED 0.77
Compound (SMILES): CSc1cccc(-c2cccc(Nc3nccc(C(F)(F)F)n3)c2)c1
InChIKey: OOTCRJPJEOEEDT-UHFFFAOYSA-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 μM 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 μM final concentration). Final volume of the reaction was 10 μL. 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 μL. The resulting HTRF signal was measured after 30 minutes on an EnVision (Perkin-Elmer) reader using a time-resolved fluorescence protocol.
Measurement: IC50 = 2121 nM (pChEMBL 5.670000076293945)
Descriptors: MW 361.39, ALogP 5.63, HBA 4, HBD 1, PSA 37.81, QED 0.61
Compound (SMILES): CCOc1ccc(C(=O)NCCC(=O)Nc2ccc(C3CCC(CC(=O)O)CC3)cc2)cc1
InChIKey: LAUJJMJFRJSSRD-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Enzyme Inhibition Assay: Human DGAT-1 activity was determined as follows: Assay buffer [20 mM HEPES (pH 7.4), 100 mM MgCl2, 0.04% BSA] containing 500 μM of enzyme substrate (didecanoyl glycerol) and 7.5 μM radiolabeled acyl-CoA substrate ([14C]decanoyl-CoA) was added to each well of a phospholipid FlashPlate (PerkinElmer Life Sciences). A small aliquot of lysate (5 μg/well) or mouse liver microsome (0.1 mg/well) was added to start the reaction, which was allowed to proceed in a 20° C. bath for 1 hour. and maintained overnight at room temperature. Next day, measurement was performed by Wallac 1450 Microbeta Trilux Liquid Scintillation Counter and Luminometer (PerkinElmer Life Science). In the control group, 0.1% DMSO was only added, and for the background value, 0.1% DMSO was only added without the cell fragment having the over-expressed human DGAT1 (5 μg/well) or the liver microsome of mice (0.1 mg/well), which was the same as the control group.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 452.55, ALogP 4.59, HBA 4, HBD 3, PSA 104.73, QED 0.49
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccccc3)n2)CCN(S(=O)(=O)c2cccc(Cl)c2)CC1
InChIKey: RTFTWEZIQMHFQY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF 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 uL of 1.11x enzyme and 1.11x substrate in 1x 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 room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x 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.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 499, ALogP 3.47, HBA 7, HBD 2, PSA 134.11, QED 0.51
Compound (SMILES): N[C@H]1C[C@@H](NC(=O)c2cc(NC(=O)c3cc(-c4ncccc4F)ccc3Cl)n(-c3ccccc3)n2)C1
InChIKey: UNTCQFPHUFEWLI-CALCHBBNSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 196 nM (pChEMBL 6.710000038146973)
Descriptors: MW 504.95, ALogP 4.2, HBA 6, HBD 3, PSA 114.93, QED 0.36
Compound (SMILES): O=C(CCCC[C@@H]1SC[C@@H]2NC(=O)N[C@@H]21)NCCOCCOCCOCCn1cc(COc2ccccc2N2CCN(C(=O)c3cc4c(s3)-c3ccccc3S(=O)(=O)C4)CC2)nn1
InChIKey: XQQSPSRXDZJHHI-HLSLCHRCSA-N
Target: Acyl-protein thioesterase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O95372
Assay: Competitive inhibition of HEK293T cells-derived His-tagged APT2 expressed in Escherichia coli BL21(DE3) preincubated for 30 mins followed by substrate addition by resorufin dye based acetate hydrolysis assay
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 923.15, ALogP 3.73, HBA 15, HBD 3, PSA 195.55, QED 0.08
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3cccnc3)ccc2[nH]1
InChIKey: IJPDDEXQDCSGFU-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 429.44, ALogP 2.99, HBA 6, HBD 3, PSA 120.44, QED 0.46
Compound (SMILES): C[C@@H]1CN(c2sc(C(F)(F)F)nc2-c2nc3ccc(N4CCN(C)CC4)cc3[nH]2)CCN1C(=O)Cn1cccn1
InChIKey: XVWKXSFZNTWSCC-QGZVFWFLSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 573.65, ALogP 3.39, HBA 9, HBD 1, PSA 89.42, QED 0.39
Compound (SMILES): CN1C(=O)N(c2cncc(C(F)(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1S(C)(=O)=O
InChIKey: UYOSEYMLVDSLQU-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 = 8.8 nM (pChEMBL 8.0600004196167)
Descriptors: MW 490.46, ALogP 3.61, HBA 6, HBD 0, PSA 111.44, QED 0.65
Compound (SMILES): Cc1ccc(N(C(=O)c2cc(-c3cc(Cl)ccc3C(=O)N3Cc4ccccc4C[C@H]3CN3CCOCC3)n3c2CCCC3)c2ccc(O)cc2)cc1Cl
InChIKey: LHQGKXKEBUSVAN-UMSFTDKQSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 749.74, ALogP 8.69, HBA 6, HBD 1, PSA 78.25, QED 0.18
Compound (SMILES): N#Cc1c(-c2ccc(F)c(Cl)c2)c2c(=O)[nH]c(=O)[nH]c2[nH]c1=O
InChIKey: HVGBDQFMICGFEO-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Inhibition of N-terminus 6xHis-tagged human IDO1 expressed in Escherichia coli M15 using L-tryptophan as substrate preincubated for 1 hr measured after 15 mins in presence of bovine liver and methylene blue by spectrophotometric analysis
Measurement: IC50 = 362 nM (pChEMBL 6.440000057220459)
Descriptors: MW 332.68, ALogP 1.24, HBA 4, HBD 3, PSA 122.37, QED 0.62
Compound (SMILES): Nc1ncnc(N2CCC(N)(CNC(=O)c3ccc(F)cc3F)C2)c1Br
InChIKey: ABTCIXPAKHAJMG-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: Enzyme Assay: P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction.
Measurement: IC50 = 3.3 nM (pChEMBL 8.479999542236328)
Descriptors: MW 427.25, ALogP 1.44, HBA 6, HBD 3, PSA 110.16, QED 0.68
Compound (SMILES): Nc1nc(NCCc2cccc(O)c2)nc2sc(-c3ccco3)nc12
InChIKey: BPJQHNKRHLCRCJ-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Displacement of [3H]DPCPX from human adenosine A1 receptor expressed in CHO cell membranes measured after 90 mins by scintillation counting method
Measurement: Ki = 0.23 nM (pChEMBL 9.640000343322754)
Descriptors: MW 353.41, ALogP 3.29, HBA 8, HBD 3, PSA 110.09, QED 0.5
Compound (SMILES): CN(C)c1cc(NCCCN2CCCC2)c2c3c(onc13)-c1ccccc1C2=O
InChIKey: CILHZSMEBZUMBT-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EHMT2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KQ7
Assay: Inhibition of G9a (unknown origin) using biotinylated-histone H3 (1 to 21 residues)/S-adenosyl-methionine as substrate/methyl donor after 3 hrs by AlphaScreen assay
Measurement: IC50 = 2980 nM (pChEMBL 5.53000020980835)
Descriptors: MW 390.49, ALogP 4, HBA 6, HBD 1, PSA 61.61, QED 0.5
Compound (SMILES): CCOc1ncc(-c2cc(NC(=O)c3ccnc(C(F)(F)F)c3)ccc2C)cc1N1CCOCC1
InChIKey: YEJHEWNOBBCKFK-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 486.49, ALogP 4.96, HBA 6, HBD 1, PSA 76.58, QED 0.53
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1ccccc1)C(=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: JIAUWELFIMYTEU-QPGRNWMBSA-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 = 94 nM (pChEMBL 7.03000020980835)
Descriptors: MW 2505.91
Compound (SMILES): C[C@]12CC[C@@H](NC(=O)C3CC3)CC1=CC[C@@H]1[C@@H]2CC[C@]2(C)C(n3ccnc3)=CC[C@@H]12
InChIKey: OUZRDUUZHNWMHZ-VZDBEYJMSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 405.59, ALogP 5.19, HBA 3, HBD 1, PSA 46.92, QED 0.7
Compound (SMILES): CCCCN(CCCC)C(=O)c1cc(C)n(-c2ccc(N[S+]([O-])c3cccc4ccn(C)c34)cc2C(=O)N2Cc3ccccc3C[C@H]2CO)n1
InChIKey: BTBNZFXFVCHLLK-XNPXXQQWSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Surface plasmon resonance (SPR): Methods for Determining IC50s.The present method includes utility of a Surface plasmon resonance (SPR)- based biosensor (Biacore GE Healthcare, Uppsala, Sweden) to characterize BCL-2 inhibitors.Biacore utilizes the phenomenon of surface plasmon resonance (SPR) to detect and measure binding interactions. In a typical Biacore experiment, one of the interacting molecules (ligand) is immobilized on a flexible dextran matrix while the interacting partner (analyte) is allowed to flow across that surface. A binding interaction results in an increase in mass on the sensor surface and a corresponding direct change in the refractive index of the medium in the vicinity of the sensor surface. Changes in refractive index or signal are recorded in resonance units (R.U.) Signal changes due to association and dissociation of complexes are monitored in a non-invasive manner, continuously and in real-time, the results of which are reported in the form of a sensorgram.
Measurement: IC50 = 1356.79 nM (pChEMBL 5.869999885559082)
Descriptors: MW 694.9, ALogP 6.41, HBA 8, HBD 2, PSA 118.69, QED 0.14
Compound (SMILES): COCCOc1cccc(OC)c1C1CCCC(=O)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: SMUPTAMEIMCAOH-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 2783 nM (pChEMBL 5.550000190734863)
Descriptors: MW 453.46, ALogP 4.87, HBA 5, HBD 0, PSA 57.23, QED 0.51
Compound (SMILES): CN1C(=O)N(c2ccnc(C(F)(F)F)c2)C2=C(C(=O)CC2)C1c1ccc(C#N)cc1C(N)=O
InChIKey: KJLRTRYAAPNXSK-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 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 455.4, ALogP 3.3, HBA 5, HBD 1, PSA 120.39, QED 0.76
Compound (SMILES): CC(C)(C)c1ccc(Nc2ncnc3cc4oc(=O)n(CCOC(=O)CBr)c4cc23)cc1
InChIKey: LRXHBDUMXINFMM-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of His-tagged human HER2 (676 to 1255 residues) expressed in baculovirus after 1 hr by Z-lyte assay
Measurement: IC50 = 6200 nM (pChEMBL 5.210000038146973)
Descriptors: MW 499.37, ALogP 4.52, HBA 8, HBD 1, PSA 99.25, QED 0.31
Compound (SMILES): Cn1ncc(NC(=O)c2nc(-c3c(F)cccc3F)sc2N)c1[C@@H]1C[C@@H](N)[C@@H](F)CCO1
InChIKey: HSTABKBTSDIQRU-BHYNMZESSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K assay: PIM-1, -2, and -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. PIM reactions were carried out in a final volume of 10 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000® (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of −500V, an upstream voltage of −2150V, and a screening pressure of −1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm.
Measurement: Ki = 0.126 nM (pChEMBL 9.899999618530273)
Descriptors: MW 466.49, ALogP 3.17, HBA 8, HBD 3, PSA 121.08, QED 0.54
Compound (SMILES): NS(=O)(=O)c1ccc(OCc2cn(-c3ccccc3)nn2)cc1
InChIKey: SAASQQDVQJBKBR-UHFFFAOYSA-N
Target: Carbonic anhydrase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00915
Assay: Inhibition of human carbonic anhydrase 1 pre-incubated for 15 mins by stopped flow carbon dioxide hydrase assay
Measurement: Ki = 565.6 nM (pChEMBL 6.25)
Descriptors: MW 330.37, ALogP 1.49, HBA 6, HBD 1, PSA 100.1, QED 0.76
Compound (SMILES): CN1C(=N)N[C@]2(c3cc(NC(=O)c4ccc(F)cn4)ccc3F)COC[C@@H]2S1(=O)=O
InChIKey: FEUVBQUAABZEJQ-YJBOKZPZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Homogeneous Time-Resolved FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence. Varying concentrations of inhibitors at 3x the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 u) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO.
Measurement: Ki = 3529 nM (pChEMBL 5.449999809265137)
Descriptors: MW 437.43, ALogP 1.01, HBA 6, HBD 3, PSA 124.48, QED 0.66
Compound (SMILES): CCCN(CCC)C(=O)C1=Cc2ccc(C(=O)Nc3cncc(CCN)c3)cc2N=C(N)C1
InChIKey: KCRAPPRPCBOQAK-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Pharmacological Assay: For TLR8 and TLR7 activity testing, HEK-Blue human TLR8 or TLR7 cells (Invivogen, San Diego, Calif., USA) are used, respectively. These cells are designed for studying the stimulation of human TLR8 or TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-κB and AP-1-binding sites. Therefore the reporter expression is regulated by the NF-κB promoter upon stimulation of human TLR8 or TLR7 for 20 hours. The cell culture supernatant SEAP reporter activity was determined using Quanti Blue kit (Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple/blue in the presence of alkaline phosphatase.
Measurement: EC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 448.57, ALogP 3.26, HBA 6, HBD 3, PSA 126.7, QED 0.54
Compound (SMILES): COc1ccc(-c2ccc(C[C@H](NC(=O)[C@@H]3C[C@H](Oc4ccc(-c5ccc(Cl)cc5)cc4)CN3C(=O)OC(C)(C)C)C(=O)NS(=O)(=O)c3ccc(Cl)c([N+](=O)[O-])c3)cc2)cc1
InChIKey: BNBLSEPXLPERMR-LUYPYDKDSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Inhibition of 5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG binding to Mcl-1 (unknown origin) preincubated for 30 mins followed by FAM-labeled peptide addition measured after 20 mins by fluorescence polarization assay
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 889.81, ALogP 8.23, HBA 10, HBD 2, PSA 183.48, QED 0.09
Compound (SMILES): O=C(O)[C@H]1CC[C@H](C(=O)N2CC[C@](c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(Cl)cc3)C2)CC1
InChIKey: OWEBVLUQSSGDRX-UAVUOLJFSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 92 nM (pChEMBL 7.039999961853027)
Descriptors: MW 644, ALogP 6.42, HBA 4, HBD 1, PSA 91.75, QED 0.37
Compound (SMILES): OC(COc1cccc(CN2CCCCC2)c1)CN1CCc2ccccc2C1
InChIKey: SVIVRWZGPFZWJD-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 = 1001 nM (pChEMBL 6)
Descriptors: MW 380.53, ALogP 3.47, HBA 4, HBD 1, PSA 35.94, QED 0.8
Compound (SMILES): CC(O)CNc1cc(Nc2cccc(Cl)c2)nc2c(/C=C3\NC(=O)NC3=O)cnn12
InChIKey: FQKAUDSAIMUPLR-RZNTYIFUSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 = 316.5 nM (pChEMBL 6.5)
Descriptors: MW 427.85, ALogP 2.1, HBA 8, HBD 5, PSA 132.68, QED 0.3
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(C(=O)NO)cc3)cc12)c1ccccc1
InChIKey: HJLXEWSRVJQEGZ-CYBMUJFWSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal GST-fused human EGFR (His672 to Ala1210 residues) expressed in baculovirus expression system using biotinylated PTP1B at Tyr66 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 373.42, ALogP 3.92, HBA 5, HBD 4, PSA 102.93, QED 0.31
Compound (SMILES): O=C(CCc1ccc(Nc2nccc(Nc3cc(C4CC4)n[nH]3)n2)cc1)NCCCN1CCOCC1
InChIKey: CLVKQAUYXOAIRN-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 490.61, ALogP 3.34, HBA 8, HBD 4, PSA 120.09, QED 0.29
Compound (SMILES): CSc1nc(N2CC(c3nc4ccccc4n3C)C2)cc(-n2nc(C)nc2C)n1
InChIKey: ZGSNAIJVRWPEAX-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition is determined using a known PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3):785-795), 2-{4-[pyridin-4-yl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]phenoxymethyl}quinoline succinic acid or 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]quinoline succinic acid (see Schmidt, et al. J Pharmacol Exp Ther (2008) 325:681-690; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3): 785-795). 0% of inhibition is determined by using DMSO (1% final concentrations). A Labcyte Echo 555 (Labcyte, Sunnyvale, Calif.) is used to dispense 200 mL from each well of the titration plate to the 384 well assay plate. A solution of enzyme (1/7000 dilution from aliquots; sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP PDE from Molecular Devices (product # R7506), at a final concentration of 50 nM are made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). The enzyme is added to the assay plates by the addition of 10 μL of enzyme solution to each well, shaken to mix and incubated at room temperature for 60 minutes. The substrate is then added to the assay plates by the addition of 10 μÎ¼L of substrate solution to each well, shaken to mix, and incubated at room temperature for 60 minutes. A binding solution is then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction is stopped by addition of 60 lit of the binding solution to each well of the assay plates and the plates are sealed and shaken for 10 seconds. The plate was incubated at room temperature for at least one hour prior to determining the fluorescence polarization (FP).
Measurement: Ki = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 406.52, ALogP 2.89, HBA 9, HBD 0, PSA 77.55, QED 0.38
Compound (SMILES): Cc1cccc(-c2ocnc2C(=O)Nc2cnn(CCOc3cccc(F)c3)c2)c1
InChIKey: DSXDLHOBZOZXGY-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20'000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 °C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM. Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 μM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37 °C. in 5% CO2 followed by equilibration at RT for 30 min before measurement. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μl/well, incubated for 10 min and finally 10 μl/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 931 nM (pChEMBL 6.03000020980835)
Descriptors: MW 406.42, ALogP 4.32, HBA 6, HBD 1, PSA 82.18, QED 0.49
Compound (SMILES): Oc1cccc(/C=N/c2nc(-c3ccccc3)cs2)c1O
InChIKey: BVUCYYIGPMTQPK-RQZCQDPDSA-N
Target: Urease (SINGLE PROTEIN), Canavalia ensiformis
UniProt: P07374
Assay: Inhibition Assay: Reaction mixtures consisting of 25 μL (1 unit/well) of Jack bean (Canavalia ensiformis) urease, 55 μL of buffer at pH 6.8, 100 mM of urea, and 5 μL of various concentrations of test compounds were incubated at 30° C. for 15 min in 96-well plates. Subsequently 45 μL phenol reagents (1% w/v phenol and 0.005% w/v sodium nitroprussside), and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% w/v NaOCl) were added to each well. Urease activity through indophenols method was confirmed by the production of ammonia, as described by Weatherburn [12]. After 50 min, the increasing absorbance at 630 nm was measured in a microplate reader SpectraMax M2 (Molecular Devices, CA, USA). All reactions were performed in triplicate in a final volume of 200 μL.
Measurement: IC50 = 19630 nM (pChEMBL 4.710000038146973)
Descriptors: MW 296.35, ALogP 3.97, HBA 5, HBD 2, PSA 65.71, QED 0.57
Compound (SMILES): COc1ccc2[nH]cc(CC(=O)N3CCN(c4scnc4-c4nc5ccccc5[nH]4)C[C@H]3C)c2c1
InChIKey: BVBNRZTWSSSDKN-MRXNPFEDSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 68 nM (pChEMBL 7.170000076293945)
Descriptors: MW 486.6, ALogP 4.46, HBA 6, HBD 2, PSA 90.14, QED 0.38
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccccc3)n2)CCN(C(=O)OCc2cccnc2)CC1
InChIKey: OIZYDVLEINDKAD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF 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 uL of 1.11x enzyme and 1.11x substrate in 1x 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 room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x 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.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 459.51, ALogP 3.16, HBA 8, HBD 2, PSA 139.16, QED 0.55
Compound (SMILES): N#Cc1cc(-c2cccc(F)c2)ccc1N1CCOc2cc(S(=O)(=O)Nc3nccs3)ccc21
InChIKey: WFAAJGGDSHOXSH-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 144 nM (pChEMBL 6.840000152587891)
Descriptors: MW 492.56, ALogP 5.15, HBA 7, HBD 1, PSA 95.32, QED 0.41
Compound (SMILES): Cn1cc(-c2ccc(F)c(C(F)(F)F)c2)nc1C1CCN(c2ncnc(N)c2-c2ccc(C(F)(F)F)cc2)CC1
InChIKey: JWJLGSTXPQYGNK-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 564.51, ALogP 6.69, HBA 6, HBD 1, PSA 72.86, QED 0.28