lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CSc1cnc(-c2ccc(NC(=O)Nc3ccc(S(C)(=O)=O)cc3)cc2)nc1N1CCOCC1
InChIKey: IGGFJVBHFGTIRA-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR Kinase Assay: The activity of the compound against mTOR was examined by measuring the incorporation of 33P from [γ-33P]-ATP into 4EBP1. His-tagged, recombinant human 4EBP1 was expressed in E. coli, purified by nickel-nitrilotriacetic acid (Ni-NTA) resins, and stored at −80 °C. Phosphorylation of 4EBP1 by mTOR was assayed in the presence or absence of the compound and performed in a final volume of 25 μl reaction buffer containing 300 ng 4EBP1, 50 ng recombinant mTOR (Invitrogen), 50 mM HEPES (pH 7.5), 1 mM EGTA, 0.01% Polysorbate 20, 10 mM MnCl2, 2.5 mM DTT, 10 μM ATP, and 0.5 μCi [Î³-33P]-ATP (PerkinElmir) for 30 min at 30 °C. The reactions were terminated by adding 3% phosphoric acid. The 33P labeled 4EBP1 was transferred onto UniFilter-96 GF/B plate (PerkinElmer) and quantified by Top Count Microplate Scintillation Counter (PerkinElmer). For primary screening of kinase activity inhibition, each test compound was evaluated at 10 M in duplicate.
Measurement: IC50 = 148.5 nM (pChEMBL 6.829999923706055)
Descriptors: MW 499.62, ALogP 3.75, HBA 8, HBD 2, PSA 113.52, QED 0.49
Compound (SMILES): CCC(=O)NC1CCc2c(-c3ccc(C(F)(F)F)c(F)c3)cncc21
InChIKey: ROSFTULUCXFQOL-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Cellular Enzyme Assay: G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 ug/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 1902.6 nM (pChEMBL 5.71999979019165)
Descriptors: MW 352.33, ALogP 4.42, HBA 2, HBD 1, PSA 41.99, QED 0.83
Compound (SMILES): COc1cccc2c1C=[N+](c1ccc(Br)cc1F)CC2.[Br-]
InChIKey: WFLSKSJEBVBRSZ-UHFFFAOYSA-M
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition of AChE (unknown origin) preincubated for 30 mins followed by substrate addition acetylthiocholineiodide measured after 40 mins by Ellman's method
Measurement: IC50 = 1290 nM (pChEMBL 5.889999866485596)
Descriptors: MW 335.2, ALogP 3.92, HBA 1, HBD 0, PSA 12.24, QED 0.76
Compound (SMILES): NC(=O)[C@@H](c1ccccc1)N(Cc1ccc(Cl)cc1)C(=O)c1ccccc1
InChIKey: XVPURPYMCSUTHD-HXUWFJFHSA-N
Target: Transient receptor potential cation channel subfamily M member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z2W7
Assay: Antagonist activity at human recombinant TRPM8 expressed in HEK293 cells assessed as inhibition of menthol-induced calcium increase by Fluo-4 AM based assay
Measurement: IC50 = 28000 nM (pChEMBL 4.550000190734863)
Descriptors: MW 378.86, ALogP 4.21, HBA 2, HBD 1, PSA 63.4, QED 0.7
Compound (SMILES): O=C(O)c1ccc(Nc2ccc(S(=O)(=O)NCC3CC3)c(C(F)(F)F)c2)c(Cl)c1
InChIKey: NZIXVBDZBUXTGJ-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: Ki = 535 nM (pChEMBL 6.269999980926514)
Descriptors: MW 448.85, ALogP 4.49, HBA 4, HBD 3, PSA 95.5, QED 0.58
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cnc(OC[C@@H]5CN(CC#N)CCO5)c(Cl)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: HIGDQEHUCABLGW-BHVANESWSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 0.281 nM (pChEMBL 9.550000190734863)
Descriptors: MW 899.9, ALogP 7.56, HBA 12, HBD 2, PSA 166.01, QED 0.11
Compound (SMILES): CC(C)CN(Cc1ccc(OCCN2C(=O)CCC2=O)c(F)c1)C(CC(=O)O)c1ccc(Cl)c(Cl)c1
InChIKey: MBNOXTLOPPKGIQ-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Radioligand Binding Assay: The composition of the binding assay buffer is determined in a course of detailed optimization procedure. This resulted in a binding assay buffer constituted by the following components: 25 mM Hepes (pH=7.4), 5 mM MgCl2, 1 mM CaCl2, 100 mM NaCl, supplemented with 0.1% of protease free BSA (as a final concentration). Competition binding assay is performed using 125I-CXCL10 (PerkinElmer, NEX348, specific activity 2200 Ci/mmol) radioligand in a final concentration of 50-70 pM. The nonspecific binding is defined by 150 pM of hr-CXCL10 (R&D Systems, Cat No 266-IP). The total assay volume is equal to 150 ul and contained 1% of DMSO (final concentration). Binding reaction is initiated by adding of membranes (10-20 ug proteins, approximately 5x105 cell equivalents) to the reaction mixture. After 60 minutes of incubation at 25° C. the reaction is terminated by rapid filtration over GF/B glass fibre filters that are pre-soaked with 0.5% polyethyleneimine (Fluka Analytical, P3143) for 1 hour, using a Skatron cell harvester device. Filters then are washed with 8 ml of ice-cold wash buffer (modified binding buffer in which BSA is omitted and the concentration of NaCl is adjusted to 500 mM concentration). The radioactivity retained on the filters is measured by a Wizard 1470 Automatic Gamma counter.
Measurement: IC50 = 303 nM (pChEMBL 6.519999980926514)
Descriptors: MW 539.43, ALogP 5.33, HBA 5, HBD 1, PSA 87.15, QED 0.37
Compound (SMILES): O=C(NCc1c(Cl)cc(Cl)cc1COCCO)C1COc2ncsc21
InChIKey: JFJGBPVPVZWNQH-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: YO-PRO Assay: Cells were grown to confluency in adherent culture at 37° C. in a humidified 5% CO2 incubator (split 1/5 every 3-4 days with DMEM, 10% FCS, 1% Penicillin/Streptomycin, 250 μg/ml Geneticin). Adherent cells were detached by incubation with Trypsine (1 ml per 165 cm2 dish) for 2 minutes, then washed off with 10 ml PBS (without Mg2+ and Ca2+), and resuspended in DMEM, 10% FCS, 1% Penicillin/Streptomycin, no Geneticin. 10,000 cells per well (48 hours before the assay) or 25,000 cells per well (Vi-cell XR (Beckman Coulter) (24 hours before the assay) in 50 μl full medium were seeded on 384-well black-wall, clear bottom plates, that were coated before with 10 μl per well Poly-L-Lysine, incubated for 30-60 minutes at 37° C. and washed once with PBS. Medium was removed from cells and 50 μl of assay buffer containing 0.5 μM YO-PRO-1 was added into the wells. Solutions of antagonist compounds were prepared by serial dilutions of a 10 mM DMSO solution of the antagonist into PBS using a BioMek (Beckman Coulter). Each concentration was performed in duplicate. For IC50 measurements 10 concentration points were measured (10 μM being the highest concentration followed by 9 serial dilution steps 1/3). The cells were incubated with the antagonists of the present invention together with ATP at a final concentration of 250 μM for 90 minutes. During this time period, four time points were taken. Each time point comprised the average of several measurements made within a few seconds. Fluorescence was measured in the FLIPR tetra (Molecular Devices) using the filters appropriate for YO-PRO-1 fluorescence (excitation 485/20, emission 530/25). The FLIPR tetra was equipped with Molecular Devices Screen Works system control software to define and run experimental protocols.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 403.29, ALogP 2.75, HBA 6, HBD 2, PSA 80.68, QED 0.7
Compound (SMILES): CN1CCN(CC(=O)N[C@H]2CC[C@H](Nc3nc(NC4CC4)n4ncc(/C=C5\NC(=O)NC5=O)c4n3)CC2)CC1
InChIKey: QHESEHNFQFGHML-DWBADKDHSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 75 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and the resid
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 537.63, ALogP -0.03, HBA 11, HBD 5, PSA 160.92, QED 0.23
Compound (SMILES): CCN1CCC(CCOc2ncc(-c3nc(C#N)nc4c3ncn4CC3CCOC3)cc2C(F)(F)F)CC1
InChIKey: OYPWTVWOPMIIFZ-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Inhibition Assay: 5 μL of a human cathepsin S enzyme (R&D1183-CY-010) was added to a 96-well plate so as to be 20 ng/well. Next, 10-fold dilution of the test compound (a 10 mM DMSO solution) was serially performed with a buffer solution for assay (a solution of 50 mM sodium methoxide, 250 mM sodium chloride and 5 mM dithiothreitol (DTT), adjusted to pH=4.5 with 1 N hydrochloric acid) six times such that the final concentration becomes 0.1 nM to 10 μM then, 20 μL of each of the solutions was added to the wells (the final DMSO concentration was 0.1%), and 25 μL of synthetic substrate VVR-AMC (3211-V manufactured by PEPTIDE INSTITUTE, INC.) was added thereto such that the final concentration becomes 40 μM, thereby starting a enzyme reaction. A fluorescence intensity (an excitation wavelength: 380 nm and a fluorescent wavelength: 460 nm) was measured at 37° C. for 10 minutes immediately after the start of the reaction using a fluorescence spectrophotometer (SPECTRA MAX GEMINI, manu
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 529.57, ALogP 4.32, HBA 9, HBD 0, PSA 101.98, QED 0.43
Compound (SMILES): COCCCn1cc(-c2ccc(-c3c4c(nn3C)CCc3cnc(Nc5ccn(CCN(C)C)n5)nc3-4)cc2)cn1
InChIKey: GOFWFAUOCPWWFW-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: DELFIA Assay: The kinase activity is measured by DELFIA assay (dissociation-enhanced lanthanide fluorescence immunoassay, Perkin Elmer). The cytoplasmic kinase domain of human IGF-1R (amino acids 964-1370) is expressed as a fusion protein with a glutathione-S-transferase tag (IGF-1R-GST) in High Five.TM. Cells (Invitrogen). Enzyme activity is measured in the presence of substances and a control substance. Poly-glutamate-tyrosine peptide (pEY, Sigma Aldrich) and biotinylated pEY (bio-pEY) are used as reaction substrates.10 uL of substance in 25% DMSO are mixed with 30 uL of IGF-1R-GST solution (67 mM HEPES pH 7.4, 15 ug/mL pEY, 1.7 ug/mL bio-pEY, 13.3 mM MgCl2, 3.3 mM dithiothreitol, 0.0033% Brij 35, 2 ng IGF-1R-GST) in 96-well plates. The reactions are started with 10 uL of a 750 uM ATP solution. After 40 min at RT the reactions are stopped with 50 .mu.L of stop solution (250 mM EDTA, 20 mM HEPES pH 7.4). 90 uL from each reaction are transferred.
Measurement: IC50 = 0.25 nM (pChEMBL 9.600000381469727)
Descriptors: MW 552.69, ALogP 4.04, HBA 11, HBD 1, PSA 103.74, QED 0.24
Compound (SMILES): Cc1nc(C)c(-c2csc(Nc3ccc(C(=O)N4C5CCC4CN(CC(F)(F)F)C5)cn3)n2)s1
InChIKey: LORGUYNYACRRRZ-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Antagonist activity at human TRPV4 assessed as inhibition of 4alpha-PDD-induced activation
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 508.6, ALogP 4.87, HBA 8, HBD 1, PSA 74.25, QED 0.53
Compound (SMILES): Cc1cn(-c2ccc3n(c2=O)CCN(C[C@@H]2CC[C@H](c4cc(F)c(C(F)(F)F)c(F)c4)O2)C3=O)cn1
InChIKey: ATYXSHBRZUUWNL-MGPUTAFESA-N
Target: Presenilin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P49768
Assay: Cell-Based Assay: The ability of compounds to modulate production of amyloid beta protein Abeta(1-42) was determined using human WT-APP overexpressing CHO cells. Cells were plated at 22,000 cells/100 uL well in 96 well tissue culture treated, clear plates (Falcon) in DMEM/F12 based medium and incubated for 24 hours at 37° C. Compounds for testing were diluted in 100% DMSO to achieve an eleven points, half log, dose response for IC50 determinations. Compounds were added in fresh medium to achieve 1% final DMSO. Appropriate vehicle or inhibitor controls were added into control wells individually to obtain minimum or maximum inhibition values, respectively, for the assay signal window before the plates were incubated for 24 hours at 37° C. This procedure produces conditioned media in each well which is tested for Abeta(1-42) levels in the ELISA detection step described next. The remaining cell cultures in each well are also tested for cell toxicity as described below.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 508.45, ALogP 4.02, HBA 6, HBD 0, PSA 69.36, QED 0.5
Compound (SMILES): O=C(CN1CCO[C@H](COc2ccccc2)C1)N1CCc2ccccc21
InChIKey: QTIAESZAMKLZSK-IBGZPJMESA-N
Target: 5-hydroxytryptamine receptor 2B (SINGLE PROTEIN), Mus musculus
UniProt: Q02152
Assay: Radioligand Binding Assay: The detailed experimental protocols for the radioligand and functional receptor assays are available on the NIMH PDSP website at http://pdsp.med.unc.edu/UNC-CH %20Protocol %20Book.pdf. A. Serotonin receptors: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6 and 5-HT7. Assay Buffer: Standard Binding Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4) Membrane Fraction Source: Transiently or stably transfected cell lines (e.g., HEK293, COS, CHO, NIH3T3). Protocol adapted from Roth et al. (1986), J. Pharmacol. Exp. Ther., 238(2): 480-485; and Roth et al. (1994), J. Pharmacol. Exp. Ther., 268(3): 1403-1410. Adrenergic Receptors: alpha1A, alpha1B, alpha2A, alpha2B, alpha2C, beta1, beta2, beta3 Assay Buffers: For alpha1 receptors, alpha1 Binding Buffer (20 mM Tris-HCl, 145 mM NaCl, pH 7.4); for alpha2 receptors, alpha2 Binding Buffer (50 mM Tris-HCl, 5 mM MgCl2, pH 7.7); for beta receptors.
Measurement: Ki = 6416 nM (pChEMBL 5.190000057220459)
Descriptors: MW 352.43, ALogP 2.36, HBA 4, HBD 0, PSA 42.01, QED 0.83
Compound (SMILES): N#Cc1c(N)ncnc1N1CCC(c2nc(-c3cccc(S(N)(=O)=O)c3)cn2CCN2CCC2)CC1
InChIKey: LWWDZLGIGGLQBJ-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 > 10000 nM (pChEMBL 5)
Descriptors: MW 507.62, ALogP 1.53, HBA 10, HBD 2, PSA 160.05, QED 0.48
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=N[C@@]2(c2nc(NC(=O)c3ccc(F)cn3)cs2)CO1
InChIKey: QQBRRRLRJBDPNK-YMNVWFMLSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Inhibition of BACE1 in human H4 cells transfected with wild type APP assessed as reduction in soluble portion of APPbeta level in cells incubated for overnight by ELISA
Measurement: IC50 = 23.7 nM (pChEMBL 7.619999885559082)
Descriptors: MW 407.5, ALogP 2.61, HBA 8, HBD 2, PSA 102.49, QED 0.81
Compound (SMILES): COc1nc(OCc2ccc(F)cc2F)c(Cl)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: UGAUEOGHPPFKSL-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 172 nM (pChEMBL 6.760000228881836)
Descriptors: MW 574.95, ALogP 4.69, HBA 9, HBD 1, PSA 125.55, QED 0.28
Compound (SMILES): CC(C)OC(=O)N1CCC(N(C(=O)c2ccc(-c3ccncc3C#N)cc2)C2CC2)CC1
InChIKey: UJOZVMAGDNOBJF-UHFFFAOYSA-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] are 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 β-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37° C. 5% CO2. For the assay, the cells are seeded 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 is aspirated from the wells completely, 10 ul of the test compound are added, the compounds are diluted using 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 room temperature (approx. 20° C.), the cAMP concentrations are determined using the AlphaScreen cAMP Assay Kit (Cat. No. #6760625R from PerkinElmer). 10 ul of Biotin-cAMP (final concentration 1 U/well in lysing buffer (5 mM Hepes (pH 7.4), 0.1% BSA, 0.5% Tween) and 10 uL Bead solution (final concentration 1 U/well in lysing buffer) are added. The plates are incubated for another 2 hours at room temperature. The cAMP concentrations are calculated using a cAMP standard curve from the Alpha Screen Counts. The data analysis is carried out by calculating the EC50 value and the maximum value based on a positive control, using suitable software (Graphpad Prism).
Measurement: EC50 = 49 nM (pChEMBL 7.309999942779541)
Descriptors: MW 432.52, ALogP 4.23, HBA 5, HBD 0, PSA 86.53, QED 0.71
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)c1cnc(N2CCOCC2)c(-c2ccc(CO)nc2)c1
InChIKey: XQHZLUCTIKHAMX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radio ABL1 (64-515) Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 uL of the compound pre-diluted with 10 uL of ATP (20 uM ATP with 0.1 uCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 uL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 mM at room temperature, the reaction was stopped by the addition of 50 uL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3x with 0.5% H3PO4, followed by addition of 30 uL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 474.44, ALogP 3.62, HBA 7, HBD 2, PSA 96.81, QED 0.56
Compound (SMILES): CCN1CCN(c2ccc(Nc3nc(N)c4scc(-c5cnc6ccccc6c5)c4n3)cc2)CC1
InChIKey: ASOQVCNSDQLKCI-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: ULight-LANCE Assay: Full sequence FAK was purchased from Cell Signaling (Catalog No.: 7796). ULight-poly GT (PerkinElmer #TRF0100-D), Eu-anti-phospho-Tyr (PT66) (PerkinElmer #AD0068) and Lance detection buffer (PerkinElmer #CR-97-100) were purchased from PerkinElmer. The kinase solution (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Tween-20) was adjusted to a final concentration of 3 nM by adding FAK diluted to 6 nM (2×) in white 384 OptiPlate, and added with an amount of 5 μL. 4× ULight-poly GT was adjusted to a final concentration of 100 nM. ATP (Sigma #A2383) was adjusted to a final concentration of 10 μM and in 2.5 μL aliquots. The test compound was sequentially diluted at 12 concentrations and treated with an amount of 0.5 μL. After shaking well and allowing to react at room temperature for 60 minutes, 5 μL of ethylenediaminetetraacetic acid (EDTA, final concentration 40 mM) diluted in Lance detection buffer (Lance detection buffer) was added and the mixture was left at room temperature for 5 minutes to stop the reaction. After adding 4× Eu-anti-phospho-Tyr (PT66) phosphorylated antibody diluted to a final concentration of 2 nM in detection buffer with an amount of 5 μL, and reaction was performed at room temperature for 60 minutes. After adjusting to detect time-resolved fluorescence energy transfer (TR-FRET) at excitation wavelength 320 nm and emission wavelength 665 nm, signals were detected using EnVision Multilabel Reader.
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 481.63, ALogP 5.37, HBA 8, HBD 2, PSA 83.2, QED 0.35
Compound (SMILES): CCc1ncc(Cc2ccc3cccc(NS(=O)(=O)CC)c3n2)c2cc(OC)c(OC)cc12
InChIKey: HSWNXHNMEYYLSG-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Diaxonhit Phosphodiesterase Assay: The phosphodiesterase assay was developed using the LANCE cAMP kit (PerkinElmer). The assay buffer contained HBSS with 5 mM HEPES, 0.1% BSA, and 1.5 mM MgCl2, pH 7.4. PDE10A (BPS Bioscience) was used at 200 pg/well (with a specific activity of 3200 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min) and PDE4D3 (BPS Bioscience) was used at 100 pg/well (with a specific activity of 32713 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min). The Biotin-cAMP tracer, supplied in 10 mmol/L Tris-HCl buffered (pH 8.0) salt solution with 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA), and 0.05% sodium azide, is used at a dilution of 1/375. The assay detection mixture contained the LANCE Eu-W8044 labeled streptavidin 1/2250 (supplied in 50 mmol/L Tris-HCl buffer).
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 465.58, ALogP 4.72, HBA 6, HBD 1, PSA 90.41, QED 0.41
Compound (SMILES): CCOc1c(C(=O)N2CCOCC2)nc(-c2ccc(NC(=O)Nc3ccccc3)cc2)nc1N1CCOCC1
InChIKey: OXJLKGYGSDOIIU-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: mTOR Kinase Assay: The activity of the compound against mTOR was examined by measuring the incorporation of 33P from [γ-33P]-ATP into 4EBP1. His-tagged, recombinant human 4EBP1 was expressed in E. coli, purified by nickel-nitrilotriacetic acid (Ni-NTA) resins, and stored at −80 °C. Phosphorylation of 4EBP1 by mTOR was assayed in the presence or absence of the compound and performed in a final volume of 25 μl reaction buffer containing 300 ng 4EBP1, 50 ng recombinant mTOR (Invitrogen), 50 mM HEPES (pH 7.5), 1 mM EGTA, 0.01% Polysorbate 20, 10 mM MnCl2, 2.5 mM DTT, 10 μM ATP, and 0.5 μCi [Î³-33P]-ATP (PerkinElmir) for 30 min at 30 °C. The reactions were terminated by adding 3% phosphoric acid. The 33P labeled 4EBP1 was transferred onto UniFilter-96 GF/B plate (PerkinElmer) and quantified by Top Count Microplate Scintillation Counter (PerkinElmer). For primary screening of kinase activity inhibition, each test compound was evaluated at 10 M in duplicate.
Measurement: IC50 = 102.5 nM (pChEMBL 6.989999771118164)
Descriptors: MW 532.6, ALogP 3.5, HBA 8, HBD 2, PSA 118.15, QED 0.47
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(OCC5(C)COC5)c([N+](=O)[O-])c4)c(Oc4ccc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: BHRRDEZKQXUBAF-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 0.567 nM (pChEMBL 9.25)
Descriptors: MW 854.43, ALogP 8.84, HBA 10, HBD 2, PSA 156.34, QED 0.09
Compound (SMILES): O=C(NC1CCOCC1)C1CC(=O)N(CC(O)CN2CCc3ccccc3C2)C1
InChIKey: KIVYTMOQHAHNJQ-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 > 10000 nM (pChEMBL 5)
Descriptors: MW 401.51, ALogP 0.55, HBA 5, HBD 2, PSA 82.11, QED 0.73
Compound (SMILES): CS(=O)(=O)CCN1CCN(C2CCN(c3ccc(Nc4nccc(-c5c(-c6cccc(C(=O)Nc7c(F)cccc7F)c6)nc6ccccn56)n4)c(OCC(F)(F)F)c3)CC2)CC1
InChIKey: VDXNZNRAXNGSEG-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 889.95, ALogP 7.3, HBA 12, HBD 2, PSA 137.3, QED 0.11
Compound (SMILES): CC(C)Oc1cc(CC(=O)O)ccc1-c1ccc(F)c2c1CN(C(=O)CC1CCOc3ccccc31)CC2
InChIKey: WHORCSLJSQIEIQ-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 517.6, ALogP 5.75, HBA 4, HBD 1, PSA 76.07, QED 0.43
Compound (SMILES): Cc1cc(C(=O)N[C@H](C(=O)N[C@@H](Cc2ccc(F)cc2)C(=O)NNCc2ccc(O)c(O)c2O)C(C)C)no1
InChIKey: IUWBKVURGPJPHY-RXVVDRJESA-N
Target: Genome polyprotein (SINGLE PROTEIN), Coxsackievirus B3 (strain Nancy)
UniProt: P03313
Assay: Inhibition of recombinant Coxsackievirus B3 3C protease expressed in Escherichia coli BL21 (DE3) preincubated for 5 mins followed by addition of NMA-EALFQGPPVK-DNP-rrr-NH2 as substrate measured up to 2 hrs by FRET-based enzyme assay
Measurement: IC50 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 543.55, ALogP 1.54, HBA 9, HBD 7, PSA 186.05, QED 0.14
Compound (SMILES): CC(C)CNc1nc(C#Cc2ccccn2)cn2c(-c3ccc(C(=O)NC4CC4)cc3)cnc12
InChIKey: KMOSUOINFHEGLX-UHFFFAOYSA-N
Target: Dual specificity protein kinase TTK (SINGLE PROTEIN), Homo sapiens
UniProt: P33981
Assay: Mps-1 Kinase Assay: For the assay 50 nL of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 ul of a solution of Mps-1 in assay buffer [0.1 mM sodium- ortho-vanadate, 10 mM MgCl2, 2 mM DTT, 25 mM Hepes pH 7.7, 0.05% BSA, 0.001% Pluronic F-127] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to Mps-1 before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 ul of a solution of 16.7 adenosine-tri-phosphate (ATP, 16.7 uM=> final conc. in the 5 ul assay volume is 10 uM) and peptide substrate (1.67 uM=> final conc. in the 5 ul assay volume is 1 uM) in assay buffer and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of Mps-1 in the assay was adjusted to the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical enzyme concentrations were in the range of about 1 nM (final conc. in the 5 ul assay volume). The reaction was stopped by the addition of 3 ul of a solution of HTRF detection reagents (100 mM Hepes pH 7.4, 0.1% BSA, 40 mM EDTA, 140 nM Streptavidin-XLent [#61GSTXLB, Fa. Cis Biointernational, Marcoule, France], 1.5 nM anti-phospho(Ser/Thr)-Europium-antibody [#AD0180, PerkinElmer LAS, Rodgau-Jugesheim, Germany]. The resulting mixture was incubated 1 h at 22° C. to allow the binding of the phosphorylated peptide to the anti-phospho(Ser/Thr)-Europium-antibody. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Europium-labelled anti-phospho(Ser/Thr) antibody to the Streptavidin-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a Viewlux TR-FRET reader (PerkinElmer LAS, Rodgau-Jugesheim, Germany).
Measurement: IC50 = 6.1 nM (pChEMBL 8.210000038146973)
Descriptors: MW 450.55, ALogP 4.15, HBA 6, HBD 2, PSA 84.21, QED 0.43
Compound (SMILES): C#CCOc1cnc(C(=O)Nc2cc(Cl)c(F)c([C@]3(C)C[C@@H](C(F)(F)F)OC(N)=N3)c2)cn1
InChIKey: LVURSRPBGNGMPV-LIRRHRJNSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 < 2 nM (pChEMBL 8.699000358581543)
Descriptors: MW 485.83, ALogP 3.41, HBA 7, HBD 2, PSA 111.72, QED 0.5
Compound (SMILES): O[C@H]1CC[C@H](Nc2ccc3ncc(-c4cccc(C(F)(F)F)c4)n3n2)CC1
InChIKey: OIWKVZIBVNRNOJ-SHTZXODSSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase-Glo Assay: These assays are set up in duplicate 50 ul volumes in white, flat bottom 96 well plates. Inhibitors are added to the solution of 1× kinase buffer, 10 uM ATP, 100 uM Pim-1-specific substrate, 50 ng of active Pim-1 enzyme, and water in serial dilutions ranging from micromolar to nanomolar concentrations. This solution is incubated at 30 degrees Celsius at 360 rpm for two hours. Following the incubation, 50 ul of Kinase-Glo reagent is added to each well, including all positive and negative control wells, and incubated at room temperature for 15 minutes. The plate is then read by the Luminoskan Ascent instrument and the results displayed with the Ascent Software version 2.6.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 376.38, ALogP 4.13, HBA 5, HBD 2, PSA 62.45, QED 0.72
Compound (SMILES): COc1ccc(-c2ccc(CC[C@](C)(C(=O)NO)S(C)(=O)=O)cc2)cn1
InChIKey: HUZCCBJQAUVKSG-GOSISDBHSA-N
Target: UDP-3-O-acyl-N-acetylglucosamine deacetylase (SINGLE PROTEIN), Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG12228)
UniProt: P47205
Assay: Enzyme Assay: IC50 determination in the LpxC enzyme assay was carried out in a similar manner to that described by Malikzay et al in the 2006 Poster, Screening LpxC (UDP-3-O(R-3-hydroxymyristoyl)-GlcNAc deacetylase) using BioTrove Rapid Fire HTS Mass Spectrometry (aNew Lead Discovery and bInflammation and Infectious Disease, cStructural Chemistry, Schering-Plough Research Institute, Kenilworth, N.J. 07033, (BioTrove, Inc. 12 Gill St., Suite 4000, Woburn, Mass. 01801). Briefly, Pseudomonas aeruginosa LpxC enzyme (0.1 nM) purified from E. coli-overexpressing bacteria was incubated at 25° C. in a final volume of 50 ul containing 0.5 uM UDP-3-O(R-3-hydroxydecanoyl)-N-acetylglucosamine, 1 mg/mL BSA, and 50 mM sodium phosphate buffer, pH 8.0 in the presence and absence of inhibitor compound. At the end of 1 hour, 5 ul of 1N HCl was added to stop the enzyme reaction; the plates were centrifuged, and then processed with the BioTrove Rapidfire HTMS Mass Spectrometry System.
Measurement: IC50 = 0.237 nM (pChEMBL 9.619999885559082)
Descriptors: MW 378.45, ALogP 2, HBA 6, HBD 2, PSA 105.59, QED 0.56
Compound (SMILES): CN1CCN(C(=O)c2ccc(Nc3ncc(Br)c(Nc4cc(C5CC5)n[nH]4)n3)cc2)CC1
InChIKey: HPRGJQZHCAPWLK-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 497.4, ALogP 3.71, HBA 7, HBD 3, PSA 102.07, QED 0.48
Compound (SMILES): FC(F)(F)c1cccc(Nc2ccnc(Nc3ccccc3)n2)c1
InChIKey: JSIPMFNPSRHGKA-UHFFFAOYSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Enzyme Inhibition Assay: The phosphorylation of L-Hse was monitored by coupling the formation of ADP with pyruvate kinase and lactate dehydrogenase (PK/LDH). The resulting oxidation of NADH was monitored at 340 nm by using a SpectraMax plate reader in a 96-well format.
Measurement: IC50 = 14100 nM (pChEMBL 4.849999904632568)
Descriptors: MW 330.31, ALogP 4.98, HBA 4, HBD 2, PSA 49.84, QED 0.71
Compound (SMILES): CS(=O)(=O)Nc1ccc(S(=O)(=O)N(CCC(F)(F)F)c2sc3ccccc3c2Cl)cc1
InChIKey: DYGBUMGUVBKPJE-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 25.9 nM (pChEMBL 7.590000152587891)
Descriptors: MW 512.98, ALogP 5.07, HBA 5, HBD 1, PSA 83.55, QED 0.48
Compound (SMILES): CN[C@@H](C)C(=O)Nc1ccc2c(n1)CN(C(C)C(C)C)C(=O)N2c1cccc(Cl)c1
InChIKey: WWPGRSUZWJAQMC-MLCCFXAWSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: In Vitro Competitive Displacement Binding Assay: Modified SMAC peptides and compounds were tested for their ability to displace the fluorescent tracer from either XIAP, cIAP-1 or cIAP-2. BIR3 domains of cIAP-1, cIAP-2 and XIAP were incubated with test compounds or SMAC based peptides and their respective peptide probes (Peptide Protein Research) in assay buffer (50 mM Hepes pH 7.5, 0.025% Tween-20, 0.01% BSA, and 1 mM DTT). Positive controls consisted of BIR3 proteins and tracer (no inhibition) and negative controls contained tracer only (100% inhibition). The samples were incubated at room temperature for 1 hr (XIAP and cIAP-2) or 3 hrs (cIAP-1) prior to being read in the BMG Pherastar in Fluorescence Polarization mode (FP 485 nm, 520 nm, 520 nm).
Measurement: IC50 = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 429.95, ALogP 4.4, HBA 4, HBD 2, PSA 77.57, QED 0.71
Compound (SMILES): Nc1cc(CNC(=O)c2cc(NC(=O)c3cc(-c4ccccn4)c(Cl)cc3Cl)n(-c3ccccc3)n2)n[nH]1
InChIKey: BMHUJIIYUFKBSY-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 20.7 nM (pChEMBL 7.679999828338623)
Descriptors: MW 547.41, ALogP 4.73, HBA 7, HBD 4, PSA 143.61, QED 0.23
Compound (SMILES): O=C(c1ccc(F)cc1)N1CCC(F)(F)C(Oc2ccc3ccccc3n2)C1
InChIKey: VKQHUBNARZWDMS-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Displacement of (2S)-N-(2-pyrrol-1-ylphenyl)-1-[2-[1-(tritritiomethyl)benzimidazol-2-yl]sulfanylacetyl]pyrrolidine-2-carboxamide from human OX2 receptor expressed on CHO cell membrane measured after 3 hrs by TopCount method
Measurement: Ki > 4379 nM (pChEMBL 5.359000205993652)
Descriptors: MW 386.37, ALogP 4.3, HBA 3, HBD 0, PSA 42.43, QED 0.67
Compound (SMILES): Cc1ccnc(C)c1-c1cc(-c2c(C)noc2C)cc2[nH]cnc12
InChIKey: PNNSPWUDQDPRGS-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 Î¼s read window). Data were normalized based on a positive (2 Î¼M I-BET) and negative (DMSO) controls and IC50values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 481 nM (pChEMBL 6.320000171661377)
Descriptors: MW 318.38, ALogP 4.51, HBA 4, HBD 1, PSA 67.6, QED 0.59